Horticultural Reviews, Volume 38

HORTICULTURAL REVIEWS Volume 38

Horticultural Reviews is sponsored by: American Society of Horticultural Science International Society for Horticultural Science

Editorial Board, Volume 38 Harry S. Paris Hilde Nybom Dan Cantliffe

HORTICULTURAL REVIEWS Volume 38

edited by

Jules Janick Purdue University

Copyright Ó 2011 by Wiley-Blackwell. All rights reserved. Published by John Wiley & Sons, Inc., Hoboken, New Jersey Published simultaneously in Canada Wiley-Blackwell is an imprint of John Wiley & Sons, formed by the merger of Wiley’s global Scientific, Technical, and Medical business with Blackwell Publishing. 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 Section 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, Inc., 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400, fax 978-750-4470, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, 201-748-6011, fax 201-748-6008, or online at http://www.wiley.com/go/permission. Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. For general information on our other products and services or for technical support, please contact our Customer Care Department within the United States at 877-762-2974, outside the United States at 317-572-3993 or fax 317- 572-4002. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic formats. For more information about Wiley products, visit our web site at www.wiley.com. Library of Congress Cataloging-in-Publication Data ISBN 978-0-470-64470-6 (cloth) ISSN 0163-7851 Printed in the United States of America oBook ISBN: 978-0-470-87236-9 ePDF ISBN: 978-0-470-87237-6 10 9 8 7 6 5 4 3 2 1

Contents

Contributors Dedication: Maxine M. Thompson

ix xiii

Kim Hummer

1. Biology and Physiology of Vines

1

Kevin C. Vaughn and Andrew J. Bowling I. Introduction II. Evolution and Taxonomic Distribution of Vines III. Classification of Vines IV. How Is Touch Perceived? V. Light Perception and Finding a Support VI. Genetic Approaches VII. Summary and Conclusions Literature Cited

2. Origin and Dissemination of Almond

1 3 3 13 15 16 16 18

23

Thomas M. Gradziel I. Introduction II. Classification III. Wild Badam IV. Cultivated Greek Nut V. Old World Dissemination VI. New World Dissemination VII. Global Commerce VIII. Evolving Requisites for Commercial Almond IX. Conclusions Literature Cited

23 24 32 39 42 59 61 66 71 73

v

vi

CONTENTS

3. Factors Affecting Extra-Virgin Olive Oil Composition

83

Paolo Inglese, Franco Famiani, Fabio Galvano, Maurizio Servili, Sonia Esposto, and Stephania Urbani I. The Concept of Olive Oil Quality II. EVOO Composition and Nutritional Properties III. Sources of Variability of EVOO Composition and Properties IV. Agronomical and Environmental Factors Affecting EVOO Composition and Quality V. Technological Factors Affecting EVOO Composition and Quality VI. Summary and Conclusions Literature Cited

4. Quality and Yield Responses of Deciduous Fruits to Reduce Irrigation

84 86 95 97 121 130 132

149

M. H. Behboudian, J. Marsal, J. Girona, and G. Lopez I. Introduction II. Definitions III. Stone Fruits IV. Pome Fruits V. Conclusions and Future Prospects Literature Cited

5. Hot Water Treatments of Fruits and Vegetables for Postharvest Storage

150 151 152 166 180 181

191

Elazar Fallik I. Introduction II. Hot Water Technologies III. Combination Treatments IV. Hot Water Treatments and Fresh Cut V. Summary and Conclusions Literature Cited

192 193 197 204 205 206

CONTENTS

6. Promotion of Adventitious Root Formation of Difficult-to-Root Hardwood Tree Species

vii

213

Paula M. Pijut, Keith E. Woeste, and Charles H. Michler I. Introduction II. Genetics and Physiology of Adventitious Root Formation III. Controllable Factors That Affect Rooting of Cuttings IV. Case Study of Horticultural versus Forest Tree Species Literature Cited

7. Water and Nutrient Management in the Production of Container-Grown Ornamentals

215 218 227 239 243

253

John C. Majsztrik, Andrew G. Ristvey, and John D. Lea-Cox I. Introduction II. Soilless Substrates III. Nutrients IV. Water V. Conclusions Literature Cited

8. World Vegetable Industry: Production, Breeding, Trends

254 258 261 273 286 288

299

Jo~ ao Silva Dias and Edward J. Ryder I. Introduction II. The Worldwide Vegetable Industry III. Vegetable Production Strategies IV. Vegetable Breeding V. Summary and Conclusions Literature Cited

300 303 319 324 345 348

9. Regulation of Anthocyanin Accumulation in Apple Peel

357

Adriana Telias, James M. Bradeen, James J. Luby, Emily E. Hoover, and Andrew C. Allen I. Introduction II. Apple Peel Color

358 358

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CONTENTS

III. Genetic Control of Anthocyanin Accumulation IV. Factors Affecting Anthocyanin Accumulation V. Mechanisms Affecting Anthocyanin Accumulation Patterns VI. Conclusions Literature Cited

363 370 376 383 384

Subject Index

393

Cumulative Subject Index

395

Cumulative Contributor Index

423

Contributors

Andrew C. Allan Plant and Food Research, Mt Albert Research Centre, Private Bag 92169, Auckland, New Zealand M.H. Behboudian Institute of Natural Resources (INR 433), Massey University, Palmerston North, New Zealand Andrew J. Bowling Dow AgroSciences, 9330 Zionsville Rd., Indianapolis, IN 46268, USA James M. Bradeen Department of Plant Pathology, University of Minnesota, 1991 Upper Buford Circle, St. Paul, MN 55108, USA Jo~ ao Silva Dias Technical University of Lisbon, Instituto Superior de Agronomia, Tapada da Ajuda, 1349-017 Lisbon, Portugal Sonia Esposto Dipartimento di Scienze Economico-estimative e degli Alimenti, Sezione di Tecnologie e Biotecnologie degli Alimenti, Universit a degli Studi di Perugia 06121, Perugia, Italy Elazar Fallik Agricultural Research Organization, The Volcani Center, Department of Postharvest Science of Fresh Produce, P.O.Box 6, Bet Dagan 50250, Israel Franco Famiani Dipartimento di Scienze Agrarie e Ambientali, Universit a degli Studi di Perugia, Borgo XX Giugno, 74, 06121, Perugia, Italy Fabio Galvano Dipartimento di Chimica Biologica, Chimica Medica e Biologia Molecolare, Universita degli Studi di Catania, 95125 Catania, Italy J. Girona Irrigation Technology, Institut de Recerca i Tecnologia Agroaliment aries, 191 Av. Alcalde Rovira Roure, E-25198 Lleida, Spain Thomas M. Gradziel Department of Plant Sciences, University of California, Davis, CA 95616, USA Emily E. Hoover Department of Horticultural Science, University of Minnesota, 305A Alderman Hall, 1970 Folwell Avenue, St. Paul, MN 55108, USA Kim Hummer USDA ARS National Clonal Germplasm Repository, Corvallis, OR 97333, USA Paolo Inglese Dipartimento di Colture Arboree, Universit a degli Studi di Palermo, Viale delle Scienze, 90128, Palermo, Italy John D. Lea-Cox Department of Plant Science and Landscape Architecture, 2120 Plant Sciences Building, University of Maryland, College Park, MD 20742, USA

ix

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CONTRIBUTORS

G. Lopez Irrigation Technology, Institut de Recerca i Tecnologia Agroaliment aries, 191 Av. Alcalde Rovira Roure, E-25198 Lleida, Spain James J. Luby Department of Horticultural Science, University of Minnesota, 305A Alderman Hall, 1970 Folwell Avenue, St. Paul, MN 55108, USA John C. Majsztrik Department of Plant Science and Landscape Architecture, 2120 Plant Sciences Building, University of Maryland, College Park, MD 20742, USA J. Marsal Irrigation Technology, Institut de Recerca i Tecnologia Agroaliment aries, 191 Av. Alcalde Rovira Roure, E-25198 Lleida, Spain Charles H. Michler USDA Forest Service, Northern Research Station, Hardwood Tree Improvement and Regeneration Center, Purdue University, 715 West State Street, West Lafayette, IN 47907, USA Paula M. Pijut USDA Forest Service, Northern Research Station, Hardwood Tree Improvement and Regeneration Center, Purdue University, 715 West State Street, West Lafayette, IN 47907, USA Andrew G. Ristvey Wye Research and Education Center, 124 Wye Narrows Drive, Queenstown, MD 21658, USA Edward J. Ryder 77 Paseo Hermoso, Salinas, CA 93908, USA Maurizio Servili Dipartimento di Scienze Economico-estimative e degli Alimenti, Sezione di Tecnologie e Biotecnologie degli Alimenti, Universit a degli Studi di Perugia 06121, Perugia, Italy Adriana Telias Department of Plant Pathology, University of Minnesota, St. Paul, MN 55108, USA Stefania Urbani Dipartimento di Scienze Economico-estimative e degli Alimenti, Sezione di Tecnologie e Biotecnologie degli Alimenti, Universit a degli Studi di Perugia 06121, Perugia, Italy Kevin C. Vaughn USDA-ARS-SWSRU, PO Box 350, Stoneville, MS 38776, USA Keith E. Woeste USDA Forest Service, Northern Research Station, Hardwood Tree Improvement and Regeneration Center, Purdue University, 715 West State Street, West Lafayette, IN 47907, USA

Maxine M. Thompson

Dedication: Maxine M. Thompson Volume 38 of Horticultural Reviews is dedicated to Dr. Maxine M. Thompson, world-renowned horticulturist and plant explorer. Maxine was born on November 3, 1926, in Bloomington, Illinois. After a few years in Illinois and Minnesota, her family moved to Pasadena, California, where she was raised. She received an Associate of Arts degree from Pasadena Junior College in 1945, a BS in Plant Science in 1948, an MS in Horticulture (Pomology) in 1951, and a PhD in Genetics in 1960, all from the University of California–Davis. While in graduate school, she married Harry S. Thompson, a student in the veterinary college, and had two children, Michael and Laurie. From 1960 to 1964, Dr. Thompson took a position as a part-time junior specialist in the Viticulture Department at the University of California–Davis while simultaneously caring for her young children. In 1964, she accepted a position as assistant professor in biology at Wisconsin State College-Oshkosh, where she taught general botany, cytology, and genetics. In 1965, she moved to Corvallis, Oregon, where she had a series of temporary appointments in the Department of Botany and the Department of Horticulture. In 1969, she became Assistant Professor of Horticulture, the first woman to be appointed to a tenuretrack position in that department. Her major research activities involved fruit breeding and genetics of hazelnut and sweet cherry, floral biology, pollination, fruit set, and cytological studies of fruit and nut species. Her teaching responsibilities included undergraduate classes in general botany and fruit systematics, and graduate classes in plant genetics, pollination, and fruit set. During her assignment at Oregon State University, Dr. Thompson was an excellent mentor to graduate students. She provided a friendly face, generous use of laboratory equipment, and helpful advice whenever students visited her fourth-floor lab. In difficult times she spoke up for those who faced the complexities of balancing a professional career while managing a young family. Dr. Thompson has had a fascination with wild and cultivated plant variation. This interest was born in a freshman general botany class and has expanded over many years to her current project, breeding blue honeysuckle, Lonicera caerulea L. Dr. Thompson was one of the xiii

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DEDICATION

founding scientists for the first clonal genebank in the U.S. National Plant Germplasm System (NPGS) to be established in Corvallis, Oregon, in 1981. For many years she participated on the Technical Committees for the National Clonal Germplasm Repository in Corvallis, and the Western Regional Plant Introduction Station, Pullman, Washington. Her international genetic resources activities began with consultancies with the Food and Agriculture Organization of the United Nations in 1982. She was hired to assess underutilized fruits and nuts in six southeastern Asian countries (India, Nepal, Thailand, Malaysia, Indonesia, and the Philippines). She was assigned to a second consultancy that assessed fruit and nut genetic resources in Pakistan. Her objective was to recommend plant collection expeditions and design a plan for clonal genebanks in that country. In 1986, she retired from her faculty position at Oregon State to embark on a series of U.S. Department of Agriculture–sponsored international plant explorations for fruit and nut genetic resources. Her first trip, in 1987, was a six-month expedition to the mountains of northern Pakistan, a region adjacent to Central Asia and rich in diversity of fruit and nut species. Next, she accompanied Dr. Calvin Sperling, USDA Plant Explorer, to Uzbekistan, Tajikistan, and Kazakhstan to collect apricots, cherries, peaches, plums, and apples. That same year she and Dr. Jim Ballington traveled to Ecuador to collect Rubus, Vaccinium, and other members of the Ericaceae with potential ornamental value. In 1992, she led an expedition to the southwest of the People’s Republic of China to collect blackberries and raspberries in Guizhou Province. She then traveled to Kyrgystan in 1994 to collect walnuts. In 1996, she returned to the People’s Republic of China, this time to the northeast, leading the expedition to collect small fruit germplasm in Jilin and Heilongjian provinces with collaborators Chad Finn and Joseph Postman. Her final two expeditions occurred in 1998, to eastern Siberia, Russia, and in 2000, to Hokkaido, Japan, to obtain blue honeysuckle. As a result of her plantcollecting expeditions, Dr. Thompson has donated 645 accessions (seeds and plants) to the U.S. National Plant Germplasm System. In 1997, Dr. Thompson was honored with the Crop Science Society’s Frank M. Meyer Medal for Plant Genetic Resources, and, in 2000, with the American Pomological Society’s Wilder Medal. The high caliber of Dr. Thompson’s science continues to be recognized. Fruit breeders and students of pomology study her Rubus cytogenetics manuscripts as seminal. Her manuscripts on the floral biology and nondormancy of hazelnut continue to be frequently cited. Her research into incompatibility in hazelnuts provided techniques for standard tests now used by several generations of nut breeders. The

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Oregon State University’s hazelnut breeding program released three main crop cultivars (‘Willamette,’ ‘Lewis’, and ‘Clark’), four pollinizers with complete resistance to eastern filbert blight (VR 4-31, VR 11-27, VR 20-11, and VR 23-18), and one red-leafed ornamental (‘Rosita’). Each of these resulted from crosses made by Dr. Thompson. ‘Lewis’ is now the most widely planted cultivar in Oregon, and demand far exceeds what nurseries can propagate. Since her final plant-collecting expedition in 2000, Dr. Thompson embarked on the breeding of blue honeysuckle, which is called “haskap” in Hokkaido. For the past decade she obtained plant material from Russia and Japan, and has been making crosses between subspecies, planting out thousands of seedlings, and selecting improved genotypes suited to the Willamette Valley, Oregon. Now retired, she is unsalaried in this task but continues the program, supplemented with small research grants out of her love and devotion to horticulture. Maxine’s fierce independence and sharp scientific mind have led to a remarkable horticultural career. Her work spanned a score of years when our society’s concept of women in the workplace greatly changed. When she began, women worked very hard to be noticed professionally and were offered lower salaries than were received by male counterparts. Those of us who have come along since then take for granted that our abilities are considered on an equal par with other qualified individuals. The outstanding work of women like Maxine Thompson has brought about this change. Dr. Maxine M. Thompson—geneticist, horticulturalist, professor, world explorer, and mentor—continues to inspire us. Kim E. Hummer USDA ARS National Clonal Germplasm Repository Corvallis, Oregon

Plate 9.1. ‘Royal Gala’ apple epidermal skin cells, showing distinctive, anthocyaninfilled vacuoles. Cells were peeled from red sectors of the fruit (a, c) or yellow sectors (b), and viewed at 100 magnification (a) or 400 (b, c).

Plate 9.2. The two different types of fruit peel pigment patterns in ‘Honeycrisp’ apple. Distribution of anthocyanin in apple peels of blushed (a) and striped (b) fruits of ‘Honeycrisp’.

Plate 9.3. Apple cultivars that carry the R6-MdMYB10 allele accumulate anthocyanin throughout the plant. The R6 mutation causes ectopic anthocyanin accumulation independent of other signals, such as light and tissue specificity. Pictures of 91.136—progeny of Redfield OP—have red foliage (a), petals (b), fruitlets (c), and fruit flesh (d). Source: Courtesy of the P&FR Pipfruit Breeding Program.

1 Biology and Physiology of Vines Kevin C. Vaughn and Andrew J. Bowling USDA-ARS-SWSRU P.O. Box 350 Stoneville, MS 38776, USA

I. INTRODUCTION II. EVOLUTION AND TAXONOMIC DISTRIBUTION OF VINES III. CLASSIFICATION OF VINES A. Tendrils B. Twining Vines C. Adventitious Root Formation IV. HOW IS TOUCH PERCEIVED? V. LIGHT PERCEPTION AND FINDING A SUPPORT VI. GENETIC APPROACHES VII. SUMMARY AND CONCLUSIONS ACKNOWLEDGMENTS LITERATURE CITED

I. INTRODUCTION Vines have long fascinated botanists. Charles Darwin (1875) dedicated an entire volume to this subject, as he painstakingly documented the movement patterns of these intriguing plants. Curiously, since this rather auspicious scientific beginning, vines have received relatively much less attention from botanists than other plant groups. However, recently there has been a greatly increased interest in this area on the part of plant scientists from many disciplines (Isnard and Silk 2009). In this review, we bring together some historical perspective on the study of vines with more recent physiological and structural investigations on this fascinating group of plants.

Horticultural Reviews, Volume 38 Edited by Jules Janick Copyright
2011 Wiley-Blackwell. 1

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K. C. VAUGHN AND A. J. BOWLING

Plants that twine, or use tendrils or holdfasts to climb other plants or objects, have been traditionally classified as vines (Penfound 1966), although a looser classification would include any plant that receives its vertical support from an external object (Gartner 1991). A group of plants sometimes included with vines are trailing plants or scramblers that have lax stems and often possess prickles or other epidermal appendages that allow for the plant to crawl over other plants or ascend above them (Bowling et al. 2008). These lax plants share with the vines the role of “structural parasite,” essentially using the upright mass of another plant to support the vine, in lieu of increasing the woodiness of its own tissue to support its mass. Some ground-hugging, prostrate plants that produce long strands, such as Vinca major, are also sometimes called vines, even though they lack the ability to climb. In this review we use the term “vine” in its more restrictive sense to mean only those plants that truly ascend other objects, either by twining or with tendrils or holdfasts Vines are an important component of the flora, with over 9,000 species in just the New World (Gentry 1991). The relative percentage of vine species increases with decreasing latitude. In the tropics, woody vines, known as lianas, occupy a greater percentage of the biome than do vines in temperate climates, partly because the water-conducting anatomy of vines is more suited to a frost-free environment than to more temperate climates (Sperry et al. 1987). Commercially important foodcrop vines include grapes, cucurbit species (cucumber, squash, and pumpkin), peas, hops, and sweetpotato. Wild-type soybeans and more ancient cultivars from China are vines, although most of this growth habit has been suppressed in modern cultivars that exhibit shorter and stronger internodes, leading to plants better suited to cultivation and mechanical harvesting practices. Ornamental vines abound, with wisteria, jasmine, morning glory species, sweet peas, English and Irish ivies, and black-eyed Susan vine being some of the more commonly encountered species. The ability of vines to act as structural parasites has made them formidable weeds as well. Morning glories, dodders (Cuscuta spp.), and redvine (Brunnichia ovata) are pernicious weeds of row crops, and kudzu blankets thousands of hectares of rights-of-way and forests in the southeastern United States. Many weedy vines are tolerant of traditional weed-control methods (Stone et al. 2005). Poison ivy and poison oak (Gartner 1991) are widespread and caused untold numbers of painful skin blistering every year. Two garden plants that have now escaped into the wild, Japanese honeysuckle and Irish ivy, have invaded forest ecosystems. It is likely that the weedy vines will be even more prevalent as a consequence of global carbon dioxide (CO2) enrichment as vines seem to be more vigorous under enriched CO2 conditions (Granadas and Korner

1. BIOLOGY AND PHYSIOLOGY OF VINES

3

2002). Thus, vines touch us both as important food and ornamental plants as well as important agricultural and rights of way pests.

II. EVOLUTION AND TAXONOMIC DISTRIBUTION OF VINES The vines (or morphological processes that allow a plant to produce either tendrils or vines) must have arisen several times in evolution. For example, the Hartford climbing fern and Japanese climbing fern occur as rare twining examples in the extant pteridophytes that otherwise have few climbing members. However, it is likely that more extensive pteridophyte vines were once in existence, as examples of scrambling, rootclimbing, tendril-climbing, and twining pteridophytes have been found in the fossil records (Krings et al. 2003). Thus, the genetic capacity to grow as a vine existed in some of the earliest land plants. Likewise, within the monocots, vines are relatively rare, although the tendrilbearing group of weeds known as greenbriers (Smilax spp.) and the twining Gloriosa lily would both be classified as vines. Climbing palms would be more like the scramblers, as they neither twine nor use tendrils, but instead use hooklike appendages born on whiplike rachis leaf extensions (Putz 1990). Because they do not truly twine or coil, they are much less effective at climbing than true vines. Due to the more ephemeral nature of the attachment, they frequently fall from their supports. However, climbing palms are fairly well equipped to climb using a variety of mechanisms (Isnard and Rowe 2008a,b); thus they are an interesting evolutionary parallel with the true vines described elsewhere in this review. In dicots, vines are widespread, although they tend to occur more prominently in some groups than others. The Convolvulaceae and Cucurbitaceae both have large numbers of species with this habit of growth. Fabaceae has many members with these characteristics (e.g., peas), although a number of species in this large group are not vines. Despite the taxonomic diversity of the species involved, each seems to use common mechanisms by it is able to climb. Gianoli (2004) examined the impact of having the ability to climb on the evolution of a plant group by comparing vines with nonclimbing sister groups. In 38 of the 48 comparisons, the vines group showed greater species diversification than in the nonclimbing sister groups. These data indicate that climbing constitutes a “key innovation” allowing these groups to succeed where the nonclimbing ones did not. With the ability to ascend over would-be competitors, plants that climb would have immediate advantages over their neighbors.

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K. C. VAUGHN AND A. J. BOWLING

III. CLASSIFICATION OF VINES Bowling and Vaughn (2009) and Penfound (1966) have classified vines by the methods used to climb. Basically there are three groups: those that use (1) tendrils for climbing, (2) twining stems, and (3) sticky adventitious roots. Some mechanisms of growth are common to all three groups whereas others are unique to a given group. A. Tendrils The ability of tendrils to allow vines to climb is truly an anatomical wonder. In vine species with tendrils, these relatively small, almost ephemeral structures can be used to support a vine weighing many times their mass. Tendrils are produced rapidly within the apical meristem of the plant and elongate maximally before coiling (Fig. 1.1). As the stem circumnutates, the tendrils touch potential surfaces about which to climb. When the tendril reaches sufficient maturity, stimulation of the touch response sets in place a series of morphological changes so that the tendril begins to coil around the object. In some cases, specific touchsensitive cells are noted on the tendril surface (Engelberth et al. 1995), although in other species, no such unique cells are noted. In redvine

Fig. 1.1. Redvine tendrils coiling about a dowel rod. Besides the coiling of the tendril to affix itself to the dowel rod, groups of cells have formed extensions where adhesives are produced to secure the tendril to the support.

1. BIOLOGY AND PHYSIOLOGY OF VINES

5

140

mean length in mm

120 100 80 60 40 adhesive pad 20

coiling

0 1

2

3

4

5

6

7

8

Days of emergence Fig. 1.2. Graph of the development of the redvine tendril. Note that coiling does not occur until the tendril has elongated maximally and that adhesive pad development follows a successful coiling.

(Brunnichia ovata), the ability to coil commences as soon as 5 days after initiation of the tendril. In some species, in addition to coiling of the tendril about an object, a group of epidermal cells elongate, form a padlike structure, and secrete an adhesive. (A line diagram of the changes in redvine tendril development is shown in Fig. 1.2.) Shortly after coiling is complete, the tendrils begin a programmed cell death or senescence. Oftentimes these final stages involve the breaking of vacuoles, and a general tanning of the structures occurs due to the free mixing of phenols released from the vacuoles with plastidic polyphenol oxidase. The dried and tanned tendril is highly rigid and relatively impervious to the elements and unpalatable to herbivores. Tendrils are either highly modified stems or leaves or, in the case of plants such as redvine, formed from both stem and leaves, all possessing the ability to coil. So-called leaf-climbers, such as those vines in the Rannuculaceae, are classified with tendrils as they are anatomically similar to tendrils but produce an expanded lamina that tendrils do not. In grapes, tendrils are actually gibberellin-inhibited inflorescences (Boss and Thomas 2002). In our previous study of tendril diversity (Bowling and Vaughn 2009), we grouped tendrils into three groups: (1) those capable of coiling in any direction, (2) those that coil in one direction only, and (3) those that have adhesive properties. Our investigations of these groups have found both similarities and differences in their internal anatomy. Redvine is our most well-studied tendril and is capable of coiling in many directions (Meloche et al. 2007; Bowling and Vaughn 2009).

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K. C. VAUGHN AND A. J. BOWLING

Fig. 1.3. Scanning electron micrograph of a free-hand cross-section from a coiled redvine tendril piece showing a thick layer of fiber cells 3 to 4 cells deep from the epidermis. These are gelatinous (G) fibers.

Redvine tendrils emerge and elongate for about 5 days before coiling is possible (Fig. 1.2). In that time period, the most salient change in tendril anatomy is the formation of a group of fiber cells, as illustrated in the scanning electron micrograph in Fig. 1.3, These cells are easily detected with the LM10 and 11 antibodies that recognize xylans (Fig. 1.4). At day 3, only the xylem elements react, but, by day 4, a second group of cells reacts, and by day 5, these new cells react strongly, forming a cylinder of cells 5 to 6 cell layers deep in the cortical cytoplasm (Figs. 1.3 and 1.4). Thus, the appearance of these fibers paralleled the appearance of the ability of the tendril to coil. Further investigations revealed that these fiber cells had an inner layer with immunolabeling characteristics similar to those in gelatinous (G) fibers of trees (Bowling and Vaughn 2008a). To this point, most reported instances of G fibers were limited to woody specimens, although there have been reports of G fibers in some herbaceous plants (Tomlinson 2008), where they perform similar sorts of movement/change in already existing tissue. The G layer itself is critical in this process, as the rare tendril that fails to coil produces the fibers but no G layers (Meloche et al. 2007).

1. BIOLOGY AND PHYSIOLOGY OF VINES

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Fig. 1.4. Redvine tendril cross-sections probed with the LM10 antibody that recognizes xylans. At day 3 after emergence, only the xylem elements (arrowheads) react with this antibody. However, at day 4 a new group of fiber cells (f) begins to react, and by day 5 these have increased to form a 4- to 6-cell layer in the cortex of the tendril.

Although the exact mechanism by which G fibers cause the coiling motion is not well understood, our best model of the sorts of interactions that must occur in G fibers (Bowling and Vaughn 2009) is presented here. The G layer of the G fiber contains cellulose enriched in an assortment of mucilaginous polysaccharides such as arabinans, rhamnogalacturonans, and other pectins (Bowling and Vaughn 2008, 2009; Meloche et al. 2007). This is in sharp contrast to the constituents of the secondary wall layers, S1 and S2, of the fiber, which are composed primarily of cellulose, xyloglucans, and xylans (Meloche et al. 2007; Bowling and Vaughn 2008a, 2009). The G layer has the capacity to become highly hydrated, whereas the other layers do not. Upon drying of the G layer, shrinkage of the layer exerts pressure on the S1 and S2 layers. This pressure manifests itself in the net bending of the tissue to accommodate this pressure. Although cortical microtubules can elicit shape changes in

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forming cells, G fibers can induce shape changes in cells already formed, especially woody tissue that would be impossible to change otherwise. Tendrils that coil in just one direction and tendrils with adhesive tips both have G fibers, but the distribution and position differ from those in tendrils that coil in any direction (Meloche et al. 2007; Bowling and Vaughn 2009). In species that coil in one predetermined direction, such as the cucurbits, the G fibers are distributed in a plate along the inner surface of the coil. These were first described by Borzi (1901) as a “Bianconi plate”; later Kaiser et al. (1993) remarked that their occurrence seeming to parallel the occurrence of coiling and that they could be lignified. Bowling and Vaughn (2009) established that they were G fibers but had a more limited distribution than those that coiled freely in all directions. Tendrils with adhesive tips such as Virginia creeper (Vaughn and Bowling 2008b) and Boston ivy (Endress and Thompson 1976, 1977) attach to a structural host not by coiling about the object but by firmly attaching to a substrate by way of adhesive tips. This strategy allows supports to be climbed that would be difficult using coiled tendrils, such as flat walls. Bowling and Vaughn (2008b) determined that the adhesive at the tip in Virginia creeper occurs as a pad and is a complex mixture of polysaccharides, consisting of debranched rhamnogalacturonan (RG) I, homogalacturonans, arabinogalactans, and callose. Some of the polysaccharides may have been generated by the loss of these components from the primary wall, which also serves to loosen these walls. Such loosening of walls makes the walls more amenable to shape changes so that the tendril tip can conform to the surface to be attached. In addition, individual components of the adhesive seem to play different roles. Callose encased in the mucilagenous pectins has the ability to spread in the presence of moisture, as demonstrated in cell plates (Vaughn et al. 1996). Thus, the pectins and other mucilaginous molecules are spread across the surface of the structure to be attached, filling in the gaps. Arabinans and arabinogalactan proteins (AGPs) appear to be an even more mobile component of the adhesive, filling in spaces between the papillate epidermal cells and even moving into small cracks in the structure that is attached. Coiling of the adhesive tendrils occurs after the adhesive end has established contact with a support. In these tendrils, the G fibers are located in a cylinder, very close to the center of the tendril. This position of the G fibers might set up a smaller radius of coiling and thus allow the coiling to proceed but without causing as much torsion to the tendril (Bowling and Vaughn 2009). Moreover, the tendrils of Virginia creeper often are coiled in one direction for half of the tendril length and in the opposite direction for the remainder. This

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coiling reversal phenomenon, similar to what is observed in coiled phone cords, allows the dramatic shortening of a tissue but without as much tension to either ends of the coil. Thus, these adhesive tendrils have a free-coiling mechanism that draws the vine closer to the support, similar to other tendrils. An examination of the related Parthenocissus stricta in which there was no adhesive tip but rather the tendrils coiled in all directions indicated that the G fibers distributed much the same as in redvine, between the vascular tissue and the epidermis (K. Vaughn, unpubl.). Although not quite cause and effect, the distribution of the G fibers appears to have a marked effect on the kinds of tendril coiling possible. Although tendrils with adhesive tips use the adhesive as their primary mechanism for attaching to an object, many other species that use coiled tendrils also produce an adhesive. In the case of redvine, adhesive production occurs after coiling has occurred (Fig. 1.2). Groups of papillate epidermal cells elongate and form a pad, similar to the adhesive tip of the adhesive tendrils. These cells also conform to the shape of the object to which they are attached and secrete an adhesive. Although we have not performed as comprehensive an analyses on the adhesive in the pads, many of the same components found in Virginia creeper tendril adhesive are also found in the adhesive on the pads of homogalacturonans, de-branched RG I, and arabinans (A. Bowling and K. Vaughn, unpublished). Twining stems that we have observed do not produce similar structures involved in mucilage production, with the exception of dodder at sites where haustoria attach to the host (Vaughn 2002). This product is pectinaceous but much simpler in composition and structure than those in the tendrils. Essentially, it is an external version of middle lamellae, enriched in highly deesterified homogalacturonans. B. Twining Vines Twining vines are probably the most familiar vines. All members of the Convolvulaceae (including the Cuscutaceae), the largest group of vines, utilize this method for climbing. Twining stems circumnutate rapidly and widely, much more so than nonclimbing plants. A large circumference and rapid circumnutation rate (> 50 Hz) allows them to “hunt” for sites about which to climb. Because of this fact, leaf development is retarded in twining vines compared to nonvines, thereby facilitating movement of the meristem (French 1977; French and Fisher 1977). Once the vine has found suitable mechanical support, two new phases of activity occur. During initial phases, the stem can be removed from the support, will remain supple, and will return to hunting for a new

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support. However, if the vine remains in contact with the support for a sufficient length of time, the twining stem becomes fixed in this position, and the vine is unable to easily find a new support if the support is removed. Scanning electron microscopy reveals twisted cell morphology in older twining stems but not in younger ones that straighten as they are cut and placed in fixative (K. Vaughn, unpublished observations). Twining stems appear to elongate close to maximally and then start twining, much as do the tendrils when they are coiling. The two phases of growth of twining vine stems are also manifested as differences in anatomy. Sher et al. (2001) documented an increase in two types of fiber cells (cells that react positively with the lignin stain phloroglucinol) as the vine begins to twine about a support. Xylary fibers, located near the xylem elements, and a second class of fibers, distributed in the cortex, both increased as the twining stem secured itself to a support. Moreover, the numbers of both types of fibers increased with increasing diameter of the support up to a point, after which the vine could no longer twine about the support. Thus, at least in the case of morning glory, there seemed to be a limit to the size of support that the vine could no longer climb, probably because the vine could not achieve sufficient torsion to do so. Often vines respond to this inability to grow up a large-diameter support by twining about themselves (Fig. 1.5; Bowling and Vaughn 2009). Darwin (1876) also reported several examples of this phenomenon, such as Solanum dulcrama being able to twine around

Fig. 1.5. Ivy leaf morning glory plants challenged with two sorts of support. The plant on the left has been challenged with a relatively thin support and easily ascends the structure. The plant on the right is unable to climb the 4.5-cm-diameter dowel rod, and the vine twines about itself.

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3-mm supports but not 5- or 6-mm ones. Frictional components also have a role in the ability of a vine to coil about a support, so that roughness of the support and extent and nature of trichomes on the stem surface might have critical roles in climbing success (Silk and Holbrook 2005). Bowling and Vaughn 2009 probed twining stems with the same series of antibodies as used to probe tendrils. Xylary fibers, xylem elements, and fibers in the cortex all reacted with antibodies to xylan (LM10 and 11), but only the fibers in the cortex were immunolabeled with antibodies to arabinans, RG I, and galactans as were the G fibers in tendrils. These data indicate that in twining vine stems, two fiber types, the G fibers in the cortex and xylary fibers surrounding the xylem elements, appear to be involved in twining. Bowling and Vaughn (2009) interpreted these results that the G fibers would be involved with twining, whereas the xylary fibers would be involved in stiffening the stem. This is a reasonable explanation for the data, as xylary fibers occur in many other plants and do not initiate any twining behavior. However, because both fiber types increase in response to twining, it could be that both are involved in the twining process. Isnard et al. (2009) added several new potential players to the twining of stems. These authors recognized that a “squeezing force” was required for the climbing stem to clasp supports and found that both the stiffening of the stem and the expansion of stipules at the leaf base were involved in the generation of the squeezing force to stabilize the stem against gravity. It is possible that the xylary fibers found in vines could be involved in this stiffening response whereas the G fibers would be able to cause the actual coiling of the stem as well as generating some of the squeezing force. This would explain why both kinds of fiber cells increase in twining stems. An Arabidopsis mutant with a severe twisting to the stem to the left (anticlockwise; “lefty” mutant; Thitamadee et al. 2002) has been likened to the condition of twining stems (Hussey 2002). In this mutant, a change in cortical microtubule organization results in cells that are twisted or leaning. The stem is more spiraled than that of the wild type. However, despite the mutation, the lefty mutant of Arabidopsis does not twine; it only has a twisted phenotype. Moreover, the phenotype of these cells is not the same as a vine. In both tendrils and twining stems, the stem elongates maximally and the cells are not twisted at this point but rather are aligned in vertical columns. Only when the stem is fixed in the twined position do the cells exhibit a spiraled morphology, but even then the appearance of the cells is different from that in lefty. Microtubules are effective agents for determining the shape of primary and secondary cell walls. However, after the walls are already produced, G fibers appear to be principal components that elicit structural change in righting tree

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branches, movement of leaves, and contractile roots (Tomlinson 2003; Fisher 2008). Although a majority of twining vines formed a right-handed (clockwise) coil, some species coil to the left (anticlockwise). In a survey of vines, Edwards et al. (2007) found that a majority of species (92%) coiled to the right only, but for species exhibiting right or left coiling, the incidence of coiling was random. The greatest percentage of vines coiling to the left (33%) were in Eucalypt forests in Australia, perhaps indicating a mild Coriolis effect that favors movement to the right in the Northern Hemisphere and movement to the left in the Southern Hemisphere. However, even in Australia and other points south of the equator, the majority of species coiled to the right. Thus, an overriding mechanism establishes the direction of twining and cannot be changed by external forces, such as direction of the sun or Coriolis effects. If we can interpret from the Arabidopsis mutants that microtubules exert a role in the spiraling of the stem, then it might be possible that there are changes in microtubule orientation that might influence handedness of the coiling direction. C. Adventitious Root Formation A third class of climbing plants includes those species that use adventitious roots to attach to a support. The most familiar of these is the English ivy (Hedera helix). English ivy produces stems that have adventitious roots at each node. When the ivy is growing across a horizontal surface on soil, the adventitious roots penetrate the soil, serving to anchor the vine as well as to provide nutrients. However, when the vine climbs a tree or other support, the roots do not expand as much and produce a close tuft of material, with the mass of roots connected together in an adhesive/mucilage complex that is involved in securing the plant to the support. This adhesive, like the one in the adhesive tendrils, is extremely effective at securing the plant to the support, as anyone who has tried to remove ivies from a house or tree can attest. Although, to our knowledge, no studies have been conducted on the nature of the ivy adhesive, Groot et al. 2003 investigated that topic in the dwarf climbing fig (Ficus pumila) using histochemical techniques. These authors concluded that the adventitious roots excreted a mucilaginous compound, probably pectins and other acidic mucopolysaccharides, based on reactivities to histochemical stains. Ivy and several vines that utilize this mechanism of vining growth also are dimorphic in their life history. For much of their lives, ivies produce vines that are incapable of producing flowers and fruits. This is the

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“juvenile” stage of the plant. Growing the plant on the ground or in pots without support tends to keep the clone in this juvenile state. At some stage, generally as the plant reaches the top of a support, the morphology of the plant changes radically. The stems become sharply thicker and woody, and the plant may then produce flowers and then fruit. This is the adult or shrub stage. Poison ivy and poison oak go through similar morphological changes (Gartner 1991) morphing from vining forms to more shrub- or treelike forms. This inability to produce flowers and fruit in the vine stage of the plant may be due to simple anatomy as vines tend to produce flowers/fruit in leaf axils and branches, but rarely (or never) produce a terminal inflorescence such as in composites, indicating that a different sort of meristem may need to be produced in order for the plant to flower. Because both vine and shrub phases of the life cycle may be maintained as individuals if plants are propagated from a given morph, potentially comparisons could be made of gene expression between the two forms. Gartner (1991) compared shrub and vine morphs of the poison oak plant and found that the shrub versions could ascend to greater heights when unsupported, but the vine phase could only achieve a much more limited height unless supported. The vines never produced the extra, lignified tissue required to achieve an upright habit. When growth of the supported vine was compared to the shrub, the vine phase was much more vigorous in terms of biomass than the shrub form. Interestingly, most of the English ivies now propagated are derived from a single cultivar called ‘Pittsburgh’ that has relatively short nodes that root easily. This mutation not only made this a wonderful pot plant but also seems to have reduced the ability of plants descended from ‘Pittsburgh’ to become “adult” or shrublike and thus rarely produce fruit (K. Vaughn, unpubl. observations). ‘Pittsburgh’ is highly mutable, and hundreds of ivy cultivars have been registered that trace back to this cultivar, so most of the available commercial clones currently have this same habit. Thus, it is unlikely that use of any of the more modern cultivars of ivy would have any environmental impact, because their spread is solely through the growth of the vine, not by seeds. Moreover, many of these cultivars do not grow well under field or woodland conditions unless under intense cultivation.

IV. HOW IS TOUCH PERCEIVED? Tendrils are one of the most touch-sensitive of all plant organs, with as little as a 0.25 mg thread drawn across a tendril being able to evoke a coiling (Simons 1992). Darwin (1876) reported a coiling response in the

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1- to 5-mg range. Amazingly, the plant is able to discriminate between true touch stimuli and water droplets from rain, so that no coiling happens in the absence of a support (Jaffe and Galston 1968). Stimuli must be applied along the surface of the tendril, and a rough surface is required to initiate the stimulus response (Haberlandt 1904 cited in Engelberth et al. 1995). The most studied systems in terms of touch response are the tendrils of Bryonia by the Weiler group (Kaiser et al. 1993; Engelberth et al. 1995; Engelberth 2003) and of pea (Pisum sativum) (Jaffe and Galston 1968). In Bryonia, touch perception occurs in epidermal cells with “tactile blebs,” 4 mm outgrowths of the cell raised slightly above the cell surface. In the bleb, the wall makes ingrowths, and the cytoplasm surrounding the ingrowth contains a high concentration of organelles, chiefly mitochondria, Golgi, and smooth endoplasmic reticulum (ER). This area is also enriched in two cytoskeletal elements, microfilaments and microtubules. The blebs are also a site of calcium accumulation (Engelberth et al. 1995). Many other touch-sensitive processes in plants are also under calcium control with calmodulin being one of the proteins downregulated in response to touch (Braam 2003). However, many other vines respond to touch stimulation without the presence of tactile blebs, indicating that other epidermal cells may have this ability to serve as the site of touch stimulation. This may allow greater flexibility in terms of coiling positions than the more preprogrammed touch response noted in Bryonia. The response to touch stimulation can be amazingly quick. Passiflora gracilis and P. sicyoides coil within 20 to 30 seconds, and Cyclanthera pedata coiling may start as early as 30 seconds after touch stimulation (Simons 1992). In redvine, we have shown that the ability to respond to touch stimulation does not occur until G fibers have been produced, indicating their role in the process, but the series of events that causes them to start the coiling process is not well understood (i.e., they might be the motor for the coiling, but what turns on the motor?). The careful studies of Weiler and colleagues (Weiler et al. 1993, 1994; Engelberth et al. 1995; Engelberth, 2003) have demonstrated the role of jasmonates (Turner et al. 2002) in the process of tendril coiling in Bryonia, as the exogenous application of these compounds can themselves induce tendril coiling. Engelberth (2003) produced an elaborate model to describe the interactions that could be involved, including auxin, cortical microtubules, calcium, an oxidative burst, and soluble sugars. The jasmonate pathway does not appear to be a universal inducer of the tendrilcoiling process, however, as pea tendrils do not respond to exogenous application of jasmonate (Engelberth et al. 2001). It is possible that indole3-acetic acid (IAA) and other auxins might have a role in this process.

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Although twining stems have been less investigated than tendrils in the touch response, they, too, seem to use jasmonates as part of their touch response (Gianoli and Molina-Montenegro 2005; Atala and Gianoli 2008). Leaf damage and other processes that enhance jasmonate production in the plant also induce twining.

V. LIGHT PERCEPTION AND FINDING A SUPPORT How does a vine find a support? Certainly, one aspect is random touching of the shoot tip as the vine circumnutates, but are there other mechanisms not involving the random touch? One of the most interesting studies involves Monstera (Strong and Ray 1975). Unlike most species that grow toward light, Monstera grows toward darkness caused by a tree’s (or other support’s) shadow. This negative phototropism is called skototropism. However, when the vine reaches the support, the plant changes in morphology to facilitate climbing, and leaf and stem development are altered in anticipation of being able toconduct photosynthesis, indicating that successfully encountering a support triggers a new growth form. A very interesting and simple study showed how a single species responded to variation in colors of the support (Price and Wilcut 2007). These authors compared the ability of morning glory plants to grow toward poles of different colors and toward maize plants. Interestingly, morning glory made very few vines on black stakes (only 17%), whereas 75% of the plants found green and yellow stakes and made vines. Blue and red stakes were intermediate between these extremes. Maize plants were the most successful supports with a 92% success rate. These data indicate that there may be more than simply sensing shadows made by prospective supporting objects to direct a vine’s growth path, as the black poles should have been equally if not more successful supports than the other colors. The parasitic weed dodder is one of the more interesting vines, as it has no true roots (Sherman et al. 2008), with only tiny leaves and an extremely reduced amount of chlorophyll (Sherman et al. 1999). With only a limited supply of its own resources and little ability to acquire more on its own, quickly locating a suitable host plant is paramount to its survival (Dawson et al. 1994). Recent work by Runyon et al. (2006) has shown that dodder responds to volatile cues from prospective hosts, even discriminating between potentially successful hosts such as impatiens or tomato and a nonsuccessful host such as wheat. One of the volatiles from wheat actually repelled the dodder. Because most grasses would also represent poor choices for a support for any vine (due to lack of strong stems to support the vine), it might be interesting to perform the

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same set of experiments with nonparasitic species, especially those in the related Convolvulaceae. VI. GENETIC APPROACHES With the advent of molecular biology tools, other questions may be answered that were technically intractable only a few years ago. Some of these go back to simple mutant analysis, such as in the tendril-less mutant of pea (Hofer et al. 2009). In those lines, the leaflets that normally would be tendrils developed expanded lamina as in a normal leaflet. Tendrils are found throughout the Fabae and Cicerae clades of legumes, and these species also had homologues of the Tl gene. The authors concluded that tendrils arose as a semidominant mutation in a tendrilless progenitor legume and that tendrils provided a selective advantage to plants possessing this trait. Homologues of Tl exist in other species as well, and mutations in these homologies (LMl1) also influence lamina expansion (Saddic et al. 2006). It is likely that reducing lamina expansion is only one factor that must be induced in order to produce tendrils or to convert stems from a normal nontwining habit to a twining one. In common bean, genotypes with both bush-type habit and climbing forms may be found, and initial studies indicated that the inheritance was monogenic for climbing versus bush types (Coyne 1967). However, later authors identified other characteristics and environmental effects on this phenotype (Bliss 1971). To investigate climbing behavior using molecular techniques, Checa and Blair (2008) crossed an aggressive, indeterminate climbing bean with an indeterminate bush bean and developed recombinant inbred lines. By using quantitative trait loci (QTL) technology, they could identify 7 loci for plant height, 9 for climbing ability, 6 for internode length, and 1 for branch number that correlated with ability to vine. Interestingly, most of these loci were in a single linkage group, which may have lead to the concept in initial studies of a relatively simple inheritance for the climbing character, as the linked genes would have inherited nearly as a unit. It will be interesting to identify the genes responsible for each of these characters and to determine if homologues of these genes exist elsewhere, especially in nonclimbing plants. VII. SUMMARY AND CONCLUSIONS It is perhaps appropriate in the Darwin Bicentennial Year (2009) that research on one of Darwin’s pet projects, the climbing plants, has

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aroused renewed interest in this beguiling group of plants (Isnard and Silk 2009). For many years just a handful of workers labored to study vines and tendrils (Jaffe and Galston 1968). We have seen notable progress in a few areas related to vines, although much still needs to be investigated and integrated before a complete picture may be obtained. Indeed, Thomas Andrew Knight’s 1812 comments on the lack of experimental work on vines is still somewhat appropriate today: “The motions of the tendrils of plants, and the efforts they apparently make to approach and attach themselves to contiguous objects, have been supposed by many naturalists to originate in some degrees of sensation or perception; and though other naturalists have rejected this hypothesis, few, or no experiments have been made by them to ascertain with what propriety the various motions of tendrils, of different kinds, can be attributed to peculiarity of organization, and the operation of external causes.” Structural studies have established the presence of two potential players in the coiling of tendrils and twining of vines. In the tendrils, G fibers are highly correlated with ability of the tendril to coil (Meloche et al. 2007) and are present in certain locations and distributions that are consistent with the type of coiling behavior (Bowling and Vaughn 2009). In the twining stems, both G fibers and xylary fibers occur; although their distribution and occurrence are not as tightly correlated with the twining as they are with coiling in tendrils, they are found consistently (Bowling and Vaughn 2009). The studies of Isnard et al. (2009) indicate that other anatomical parameters, such as the expansion of stipules at the base of leaves, are involved in generating a “squeezing force” that is required for the vine to stay wrapped to the support. This might also explain the role of the xylary fibers in vines; the presence of these fibers should result in substantial stem stiffening and contribute to the squeezing force. Now that we have some basic understanding of the process of coiling, these systems are well suited for some genetic investigations. For example, G fibers are produced rapidly from day 3 to day 5 in redvine tendrils (Fig. 1.3); comparisons of mRNA (such as subtractive hybridizations) of these stages might allow us to examine the genes that are involved in induction of coiling. For example, we know that xylans are increased dramatically as a result of G fiber production (Fig. 1.3), making this a system to study xylan biosynthesis as well. Mutations that affect the ability of vines to twine and tendrils to coil will be increasingly useful tools in understanding the genetic roots of the ability to be a vine. Since the pioneering work of Weiler and colleagues (1993) on the roles of hormones in the coiling of tendrils, relatively little work on the physiology of vines has taken place. It is crucial to unraveling the

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mechanisms by which tendrils coil and vines twine to understand the cascade of events from the touch stimulation to the actual coiling takes place. The model of Engelberth (1995) is a step toward integrating the data from physiological, biochemical, and structural investigations into a holistic perspective of the coiling process. A more global perspective is also needed. In the United States, the change to glyphosate-resistant crops has resulted in a shift in the weed species as well, with vines now a much greater percentage of weed problems than before this technology was used in such a widespread manner. The shift in weeds may be exacerbated by the concomitant change in CO2 enrichment, which also favors vines over nonvines. In the tropics, the enhanced growth of lianas may have even greater impact on an already threatened tropical rain forest. Dealing with these problems will be a major problem for eco-physiologists for the foreseeable future. It is hoped that some of the laboratory studies of vines will give these workers clues regarding approaches that may be taken to solve these daunting problems.

ACKNOWLEDGMENTS Andrew Bowling was supported by an ARS Research Associate Position to Kevin C. Vaughn. Some of this work was initiated by Christopher Meloche, who was also funded by an ARS Research Associate Position. Thanks to the many colleagues throughout the country who supplied plant material or insights into this project and Ernest Delfosse, former National Program Leader for Weeds, who supported these investigations.

LITERATURE CITED Atala, C., and E. Gianoli. 2008. Induced twining in Convolvulaceae climbing plants in response to leaf damage. Botany 86:595–602. Bliss, F.A. 1971. Inheritance of growth habit and time of flowering in beans Phaseolus vulgaris L. _J. Am. Soc. Hort. Sci. 93:715–717. Borzi, A. 1901. Anatomia dell-appraro sensomotore dei cirri delle cucurbitacee. Atti revev Accad. Lincei, ser. V, Rc.Cl. Sciencia for Materiales Naturales 10:395–400. Bowling, A.J., and K.C. Vaughn. 2008a. Immunocytochemical characterization of tension wood: Gelatinous fibers contain more than just cellulose. Am. J. Bot. 95:655–663. Bowling, A.J., and K.C. Vaughn. 2008b. Structural and immunocytochemical characterization of the adhesive tendril of Virginia creeper (Parthenocissus quinquefolia [L.] Planch.). Protoplasma 232:153–163. Bowling, A.J., and K.C. Vaughn. 2009. Gelatinous fibers are widespread in coiling tendrils and twining vines. Am. J. Bot. 96:719–727.

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Bowling, A.J., B.H. Maxwell, and K.C. Vaughn. 2008. Unusual trichome structure and composition in mericarps of bedstraw (Galium aparine L.). Protoplasma 233: 223–230. Braam, J. 2004. In touch: Plant responses to mechanical stimuli. New Phytologist 165:373–389. Checa, O.E., and M.W. Blair. 2008. Mapping QTL for climbing ability and component traits in common bean (Phaseolus vulgaris L.). Mol. Breed. 22:201–215. Coyne, D.P. 1967. Photoperiodism: Inheritance and linkage in Phaseolus vulgaris. J. Hered. 58:313–314. Darwin, C. 1875. The movements and habits of climbing plants. Henry Murray, London. Dawson, J.H., L.H. Musselman, P. Wolswinkel, and I. Dorr. 1994. Biology and control of Cuscuta. Rev. Weed Sci. 6:265–317. Edwards, W., A.T. Moles, and P. Franks. 2007. The global trend in plant twining direction. Global Ecol. Biogeography 16:795–800. Endress, A.G., and W.W. Thompson. 1976. Ultrastructural and cytochemical studies on the developing adhesive disc of Boston ivy tendrils. Protoplasma 88:315–331. Endress, A.G., and W.W. Thompson. 1977. Adhesion of the Boston ivy tendril. Can J. Bot. 55:918–924. Engelberth, J. 2003. Mechanosensing and signal transduction in tendrils. Adv. Space Res. 32:1611–1619. Engelberth J., G. Wanner, B. Groth, and E.W. Weiler. 1995. Functional anatomy of the mechanoreceptor cells in tendrils of Bryonia dioica Jacq. Planta 196:539–550. Fisher, J.B. 2008. Anatomy of axis contraction in seedlings from a fire prone habitat. Am. J. Bot. 95:1337–1348. French, J.C. 1977. Growth relationships of leaves and internodes in viny angiosperms with different modes of attachment. Am. J. Bot. 64:292–304. French, J.C., and J.B. Fisher. 1977. A comparison of meristems and unequal growth internodes in viny monocotyledons and dicotyledons. Am. J. Bot. 64:24–32. Gartner, B.L. 1991. Structural stability and architecture of vines vs. shrubs of poison oak, Toxicodendron diversilobum. Ecology 72:2005–2015. Gentry, A.H. 1991. The distribution and evolution of climbing plants. p. 3–49. In: F.E. Putz and H.A. Mooney (eds.), The biology of vines. Cambridge Press, New York. Gianoli, E. 2004. Evolution of a climbing habit promotes diversification in flowering plants. Proc. Royal Soc. B: Biol. Sci. 271:2011–2015. Gianoli, E., and M.A. Molina-Montenegro. 2005. Leaf damage induces twining in a climbing plant. New Phytologist 167:385–390. Granados, J., and C. Korner. 2002. In deep shade, elevated CO2 increases the vigor of tropical climbing plants. Global Change Biol. 8:1109–1117. Groot, E.P., E.J. Sweeney, and T.L. Rost. 2003. Development of the adhesive pad on climbing fig (Ficus pumila) stems from clusters of adventitious roots. Plant & Soil 248:85–96. Hofer, J., L. Turner, C. Moreau, M. Ambrose, P. Isaac, S. Butcher, J. Weller, A. Dupin, M. Dalmais, C. LeSignor, A. Bendahmane, and N. Ellis. 2009. Tendril-less regulates tendril formation in pea leaves. Plant Cell 21:420–428. Hussey, P.J. 2002. Microtubules do the twist. Nature 417:128–129. Isnard, S., and N.P. Rowe. 2008a. Mechanical role of leaf sheath in rattans. New Phytologist 177:643–652. Isnard, S., and N.P. Rowe. 2008b. The climbing habit in palms: Biomechanics of the cirrus and flagellum. Am. J. Bot. 95:1538–1547. Isnard, S., and W.K. Silk. 2009. Moving with climbing plant from Darwin’s time into the 21st century. Am. J. Bot. 96:1205–1221.

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Isnard, S., A.R. Cobb, N.M. Holbrook, M. Zwieniecki, and J. Dumais. 2009. Tensioning the helix: A mechanism for force generation in twining plants. Proc. Royal Soc. B Biol. Sci. 276:2643–2650. Jaffe, M.J., and A.W. Galston. 1968. The physiology of tendrils. Annu. Rev. Plant Physiol. 19:417–434. Kaiser, I., J. Engelberth, B. Groth, and E.W. Weiler. 1993. Touch- and methyl jasmonate– induced lignification in tendrils of Bryonia dioica Jacq. Botanica Acta 107:24–29. Knight, T.A. 1812. On the motion of the tendrils of plants. Read before the Royal Society, May 4, 1812. p. 164–168, In: Anon. A selection from the physiological and horticultural papers published in the Transactions of the Royal and Horticultural Societies by the late Thomas Andrew Knight, Esq., President of the Horticultural Society of London, etc. etc. to which is prefixed a sketch of his life. Published in 1841. (Facsimili edition, ASHS Press, 2001.) Krings, M., H. Kerp, T.N. Taylor, and E. Taylor. 2003. How Paleozoic vines and lianas got off the ground: On scrambling and climbing carboniferous-early Permian pteridosperms. Bot. Rev. 69:204–224. Meloche, C.G., J.P. Knox, and K.C. Vaughn. 2007. A cortical band of gelatinous fibers causes the coiling of redvine tendrils: A model based upon cytochemical and immunocytochemical studies. Planta 225:485–498. Pendfound, W. 1966. The roles of vines in plant communities. Advan. Front. Plant Sci. 17:187–192. Price, A.J., and J.W. Wilcut. 2007. Response of ivyleaf morning glory (Ipomoea hederacea) to neighboring plants and objects. Weed Tech. 21:922–927. Putz, F.E. 1990. Growth habit and trellis requirements of climbing palms (Calamus sp.) in North-eastern Queensland. Aust. J. Bot. 38:603–608. Runyon, J.B., M.C. Mescher, and C.M. Morales. 2006. Volatile chemical cues guide host location and host selection by parasitic plants. Science 313:1964–1967. Saddic, L.A., B.R. Huverman, S. Bezhani, Y.H. Su, C.M. Winter, C.S. Kwon, R.P. Collum, and D. Wagner. 2006. The LEAFY target LMl1 is a meristem identity regulator and acts together with LEAFY to regulate expression of CAULIFLOWER. Development 133:1673–1682. Sher, J.M., N.M. Holbrook, and W.K. Silk. 2001. Temporal and spatial patterns of twining force and lignification of stems of Ipomoea purpurea. Planta 213:192–198. Sherman, T.D., W.T. Pettigrew, and K.C. Vaughn. 1999. Structural and immunological characterization of the Cuscuta pentagona chloroplast. Plant Cell Physiol. 40:592–603. Sherman, T.D., A.J. Bowling, T.W. Barger, and K.C. Vaughn. 2008. The vestigial root of dodder (Cuscuta pentagona) seedlings. Int. J. Plant Sci. 169:998–1012. Silk, W.K., and N.M. Holbrook. 2005. The importance of frictional interactions in maintaining the stability of the twining habit. Am. J. Bot. 92:1820–1826. Silk, W.K., and M. Hunnard. 1991. Axial forces and normally distributed loads in twining stems of morning glory. J. Biomechanics 24:599–606. Simons, P. 1992. The action plant. Blackwell, Oxford, UK. Sperry, J.S., N.M. Holbrook, M.H. Zimmerman, and M.T. Tyree. 1987. Spring filling of xylem vessels in wild grapevine. Plant Physiol. 83:414–417. Stone, A.E., T.F. Peeper, and J.P. Kelly. 2005. Efficacy an acceptance of herbicides for field bindweed (Convolvulus arvensis) control. Weed Tech. 19:148–153. Strong, D.R.J., and T.S.J. Ray. 1975. Host tree location behavior of a tropical vine (Monstrera gigantea) by skototropism. Science 190:804–806. Thitamadee, S., K. Tuchihara, and T. Hashimoto. 2002. Microtubule basis for left-handed helical growth in Arabidopsis. Nature 417:193–196.

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Tomlinson, P.B. 2003. Development of gelatinous (reaction) fibers in stems of Gnetum gnemon (Gnetales). Am. J. Bot. 90:965–972. Turner, J.G., C. Ellis, and A. Devoto. 2002. The jasmonate signal pathway. Plant Cell Suppl. S153–164. Vaughn, K.C. 2002. Attachment of the parasitic weed dodder to the host. Protoplasma 219:227–237. Vaughn, K.C., J.C. Hoffman, M.G. Hahn, and L.A. Staehelin. 1996. The herbicide dichbenil disrupts cell plate formation: Immunogold characterization. Protoplasma 194:117–132. Weiler, E.W. 1993. Octadecanoid-derived signaling molecules involved in touch perception in a higher plant. Botanica Acta 106:2–4. Weiler, E.W., T. Albrecht, B. Groth, Z.Q. Xia, B. Luxem, H. Lis, L. Andert, and P. Spengler. 1993. Evidence for the involvement in of jasmonates and their octadecanoid precursors in the tendril coiling response of Bryonia dioica. Phytochemistry 32:591–600.

2 Origin and Dissemination of Almond Thomas M. Gradziel Department of Plant Sciences University of California Davis, CA 95616, USA I. INTRODUCTION II. CLASSIFICATION A. Botanical B. Horticultural III. WILD BADAM IV. CULTIVATED GREEK NUT V. OLD WORLD DISSEMINATION A. Asiatic Stage B. Mediterranean Stage VI. NEW WORLD DISSEMINATION VII. GLOBAL COMMERCE A. Production B. Consumption VIII. EVOLVING REQUISITES FOR COMMERCIAL ALMOND A. Mediterranean B. California C. Eastern Europe and Asia IX. CONCLUSIONS ACKNOWLEDGMENTS LITERATURE CITED

I. INTRODUCTION The almond differs from the other Prunus crops both botanically and horticulturally, and the combinations of these differences have had widespread consequences on its role in human history and the role of humans in its dissemination. Botanically, the consumed part is a nut rather than a fruit, representing a durable propagation source for Horticultural Reviews, Volume 38 Edited by Jules Janick Copyright
2011 Wiley-Blackwell. 23

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expanding plantings as well as a concentrated, desirable, and relatively nonperishable food item, which made it a readily fungible commodity even in ancient times. The wild almonds traded and consumed by early human communities were represented by over 30 species of diverse quality, morphology, and geographic origin. Early dissemination of this genetically diverse commodity followed the trade routes of emerging civilizations from Central Asia westward to the Mediterranean. Almond’s widespread desirability and easy transportability appear to have made it an important commodity in prehistoric trade in Asia, North Africa, and Europe, apparently leading to the establishment of an evolving market standard as well as a new species: the cultivated sweet almond, or “Greek nut,” [Prunus dulcis (Mill.) D. A. Webb L, syn. Prunus amygdalus Batsch., Amygdalus communis L., Amygdalus dulcis Mill.] possibly selected by prehistoric societies from an interspecific hybridization. The subsequent, rapid reverse dissemination of these Greek nuts from the early Greek and Persian civilizations eastward into centers of almond’s origin and beyond, including India and China, is an indication of the extent of global commerce at this time. Also disseminated was a rich folklore and associated culinary practices based on almond’s unique horticultural characteristics of very early flowering and associated traits, allowing it to thrive under harsh arid environments yet produce a sweet and symmetrical amygdaloidal-shape kernel. The widespread adoption of the commercially more desirable Greek nut would displace previously utilized almond species, inevitably resulting in a loss of germplasm and trait diversity. The current globalization of trade is promoting even greater uniformity in commerce, leading to further reductions in genetic, ecological, and culinary diversity.

II. CLASSIFICATION A. Botanical As with other Prunus crops, almond fruit is classed botanically as a drupe with a pubescent exocarp (skin), a fleshy but thin mesocarp (hull) and a distinct hardened endocarp (shell) (Fig. 2.1). In almond, however, the mesocarp undergoes only limited enlargement during development, becoming dry and leathery and dehiscing at maturity. The mature endocarp ranges from hard to soft and papery. The tree, while relatively slow growing, can survive for 100 years or more, reaching heights exceeding 20 m. Almond’s outlier status within the Prunus, however, has also confounded its botanical classification. Presently, the most

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Fig. 2.1.

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Nut, flower, shoot, and fruit of cultivated almond, Prunus dulcis.

widely accepted scientific name, Prunus dulcis (from Latin dulcis, for “sweet”), acknowledges its taxonomic affinities with other Prunus based on similar morphology, molecular-genetic relatedness, and reported hybridization with peach, apricot, and some plums. Because it was the first to be proposed in the literature, Prunus dulcis has superseded the scientific name Prunus amygdalus (amygdalus is Latin for “almond”) commonly still found in the European literature. In its Central Asian center of origin and diversity, the taxonomic experts most familiar with almond species in their native ecosystems have preferred to classify them in a separate genus, Amygdalus communis (Browicz and Zohary 1996), arguing that their evolution of specialized botanical structures and development patterns in these often extreme environments justify a separate genus. Conversely, the molecular genetic structure and composition of almond is very similar to peach, suggesting that both belong to the same species (Mart
ınez-Go´mez et al. 2007). This view is further supported by absence of any formidable barriers to their hybridization and subsequent gene introgression (Gradziel et al. 2001; Mart
ınez-Go´mez et al. 2003). Cultivated almond also readily intercrosses with wild almond relatives (Fig. 2.2), which represent a wide range of morphological and

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Fig. 2.2. Leaf and nut morphology of parent species (top) and hybrids with cultivated almond P. dulcis (bottom). Leaf and nut typical of cultivated almond at right.

developmental forms as found throughout western and central Asia (Komorov et al. 1941; Browicz and Zohary 1972; Grasselly, 1976a; Grasselly and Crossa-Reynaud 1980; Denisov 1988; Kester et al. 1991; Browicz 1996). Some of the more than 30 species described by botanists may represent subspecies or ecotypes within a broad collection of genotypes adapted to the wide range of ecological niches in the deserts, steppes, and mountains of central Asia. A classification into five sections was proposed by the German botanist Spach (1843) (Grasselly and Crossa-Raynaud 1980; Kester et al. 1991) (Table 2.1). More recently, Browicz (1974) separated almond species into two subgroups: Amygdalus (leaves conduplicate in bud and 20 to 30 or more stamens) and Dodecandra (leaves convolvulate in bud and fewer than 17 stamens). The most northeasterly group located in western China and Mongolia includes P. mongolica, P. pedunulata, and P. tangutica (P. dehiscens), the latter probably in Section Chamaeamygdalus. The remainder occupies a more or less contiguous area in west-entral Asia (Fig. 2.3). Those with the

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Table 2.1. Botanical relationship of Prunus species in subgenus Amygdalus. Almond Group Section Euamygdalus Spach Prunus dulcis (Miller) D.A.Webb P. bucharica Korshinsky P. communis (L) Archangeli P. fenzliania Fritsch P. kuramica Korchinsky P. orientalis (Mill.), syn. P. argentea (Lam) P. kotschyi [Boissier and Hohenm. (Nab,) and Rehd.] P. korschinskii Hand-Mazz. P. webbii (Spach) Vieh. P. zabulica Serafimov Section Spartioides Spach P. scoparia Spach P. spartioides Spach P. arabica Olivier P. glauca Browicz Section Lycioides Spach P. spinosissima Franchet P. turcomanica Lincz. Section Chamamygdalus Spach P. nana (Stock) P. ledebouriana Schle. P. petunnikowi Lits. P. tangutica Batal.(syn. P. dehiscens) Koehne Peach Group P. persica (L.) Batsch. P. mira Koehne P. davidiana (Carriere) Fransch.

most northern range include species in Section Chamaeamygdalus and extend from the Balkan Peninsula to the Altai Mountains. The most southern and xerophytic groups includes species in the Spartiodes Section, which developed leafless, slender shoots, and the Lyciodes (Dodecandra) Section, which are very dwarfed and thorny. A third section (Euamygdalus) resembles cultivated almonds and includes many species extending from central Asia to southern Jordan and parts of Europe. The cultivated almond as well as most almond species express gametophytic self-incompatibity, although self-compatibility is present in some P. bucharica and P. webbii populations. Gametophytic incompatibility prevents self-fertilization (Socias i Company 1992), favors crosspollination (Weinbaum et al. 1985), and maintains genetic variability within seedling populations (Socias i Company and Felipe 1992). This

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Fig. 2.3. Physical map of Eurasia showing distribution of wild almond species of the subgenus Euamygdalus (dashed outlines) and major ancient trade routes.

trait would have contributed to almond’s extensive genetic diversity, which insured wide adaptation and wide distribution of these species in the wild. The chromosome number of Prunus dulcis (P. amygdalus), P. fenzliana, P. nana (P. tenella), P. bucharica, P. kotschyi, P. scoparia, and peach (P. persica) is 2n ¼ 16 (Darlington and Ammal 1945; Grasselly 1977), which is the same as many other Prunus. The taxonomic closeness of almond with peach (Fig. 2.4) led Watkins (1979) to suggest that both originated from the same primitive species but evolved separately following the mountain upheavals of the Central Asian massif approximately 10 million years ago. Thus peach evolved in the East, spread over several regions of China, in a more humid and uniform climate and at lower elevations, whereas almond evolved in the West, in arid steppes, deserts, and mountainous regions, under severe and erratic conditions. The often highly variable nature of these environments may have encouraged almond’s evolution toward self-incompatibility as it would enforce outbreeding and so promote greater genetic diversity to cope with changing environments.

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Fig. 2.4. Peach and almond stones, of natural size. viewed edgeways. 1. Common English Peach. 2. Double, crimson-flowered Chinese Peach. 3. Chinese Honey Peach. 4. English Almond. 5. Barcelona Almond. 6. Malaga Almond. 7. Soft-shelled French Almond. 8. Smyrna. Source: From Darwin 1868.

The fruit of different almond species and cultivars vary in size, shape, pubescence, shape and retention of the pistil remnants and suture line, all of which can be useful in identification. The pattern by which “splitting” occurs in the hull also differs and can be described by specific classes. Wood (1925) showed four basic types: (1) ventral split, opening on one side; (2) ventral and dorsal split; (3) four-way split; and (4) dorsal split. The thickness and weight of the mature hull may also differ significantly. Some hulls are thin and dry and constitute only a small

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portion of the entire fruit. Others are thick and fleshy and provide a relatively large proportion of the final weight. In California, hulls of cultivated almond are used for livestock feed, and the food value is better with larger hulls (Aguilar et al. 1984). Shell hardness is the most important characteristic of the endocarp and is associated with the total amount of lignin deposited during nut development. Shelling proportion (dry weight of kernel/weight of inshell nut) is used to obtain a quantitative measure of shell hardness and is utilized in commercial activities to calculate final yield of kernels from whole nuts. Shelling percentage has an exponential relationship to hardness but is subject to considerable variation due to nongenetic factors. Markings on the outer shell are unique and identify specific species as well as cultivars. Within Prunus dulcis, the markings or openings (pores) tend to be mostly circular, though sometimes elongated and sometimes a mixture of both. Among species, pores may be large or small, many or few. Other species have smooth and thin shells (as in P. bucharica) or are distinctly grooved (scribed) as in P. kuramica (Fig. 2.2). The shell consists of an outer and an inner layer separated by channels through which vascular tissues develop (Fig. 2.1). As the hull dehisces and separates from the nut, the outer layer may remain attached to the hull and separate from the inner shell layer. The latter type is associated with high shelling percentages but often poor seal. Kernel size is established during the first growth phase of nut development in the spring and is completed by late spring. There is a strong environmental and seasonal component on size, including crop load, vigor of tree, soil moisture, and environment. Crop density has a strong inverse relationship to average size. Kernel mass is determined during the last accumulative phase of almond nut growth and increases continuously until maturity (Labavitch 1978). Native almond species predominantly have bitter kernels because of high levels of the glucoside amygdalin, which hydrolyzes to benzaldehyde and cyanide when exposed to the enzyme emulsin (Conn 1980; S
anchez-P
erez 2008). Both substrate and enzyme are present in the seed and come together when the cells are injured. This trait has adaptive value in the wild by discouraging seed predation by birds and rodents but would discourage human consumption since the cyanide would first have to be processed out using heating, grinding, or leaching practices similar to those used by hunter-gatherer societies to detoxify certain wild roots and legumes (Alexander-Essers 1994). Plants producing sweet almond kernels have appeared from mutations and subsequent seedling segregation within various almond species, including Prunus bucharica, as well as other Prunus, such as peach and apricot (Prunus

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armeniaca L.) (Bailey and Hough 1975). Seed bitterness is determined by the genotype of maternal parent and not the embryo. Bitterness is inherited as a simple recessive (ss) in cultivated almond (Heppner 1923, 1926; Dicenta and Garcia 1993; Dicenta et al. 2007). Sweetness is dominant, transmitted from homozygous (SS) parents as 100% sweet and segregates from heterozygous seed parents (Ss) at a ratio of either 3 sweet:1 bitter (if both parents heterozygous) or 1 sweet:1 bitter (if the heterozygote is pollinated by a bitter (ss) almond tree (Spiegel-Roy and Kochba 1974). Trace amounts of amygdalin in genetically sweetkernel trees may account for flavor differences and the distinct flavor characteristics claimed for some Mediterranean-produced almonds (Horoschah 1971; Socias i Company et al. 2007). B. Horticultural Horticulturally, almonds are classified as a nut, in which the edible seed or kernel is the commercial product. The kernel includes an embryo surrounded by the pellicle, which is derived from the seed coats and remnants of nucellus and endosperm (Fig. 2.1). The almond is the earliest deciduous fruit and nut tree to bloom in spring due to its relatively low winter chilling requirement and quick response to warm growing temperatures in the spring (Tabuenca et al. 1972; DiGrandiHoffman et al. 1994). In nature, the almond growth cycle is well adapted to a Mediterranean or desert climate where plants are dormant during winter precipitations and associated low temperatures. Blooming and vegetative growth occur in late winter or early spring when temperatures become mild (Tabuenca et al. 1972; Egea et al. 2003). Growth ceases in late spring as the moisture in the ground is depleted and a dormant condition develops during the hot, dry, rainless summer (Denisov 1988). When almonds are irrigated during the growing season; however, growth continues through the spring and summer, and production can be increased many times over that of nonirrigated almond trees (Asai et al. 1994; Micke 1994) Almond trees require some winter chilling, and many cultivars do not grow well in areas of little or no winter chilling. The early-flowering habit of almond also made it very susceptible to spring frost in more temperate growing regions and limited its plantings to more moderate, almost subtropical climates. In addition, a general susceptibility of the almond foliage to fungal diseases limited tree survival to those regions free from appreciable summer rainfall. Excessive moisture in the root zone is also deleterious (Kester and Grasselly 1987) and can result in tree losses due to “crown rot,” or asphyxia.

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Consequently, the range of almond production has been limited to areas with relatively mild winters. Fall freezing is a hazard because almonds tend to respond to warm temperature and accessible moisture even in late fall by growing and delaying the normal acquisition of hardiness. During the winter when trees are fully dormant, cambium and buds reportedly can withstand temperatures of 2 C (Grasselly and CrossaRenaud 1980). Flower buds may be injured by temperatures of 15 to 20 C, particularly in late winter after the normal rest period has been fulfilled (Cociu 1985; Ristevski 1992).

III. WILD BADAM The kernels of a geographically wide range of Asian almond species represented a nutritious, compact, and relatively nonperishable food source that is also appetizing even when eaten in quantity or over a period of time. These qualities combined with its presence throughout the range of early plant domestication by humans ensured that it was among the first tree crops to be domesticated, probably during the third millennium BCE (Spiegel-Roy 1976). The natural range of the various almond species from northwestern China to the northern Indus Valley in the East, to Mesopotamia and southern Europe in the West (Fig. 2.2), overlapped areas important in the transition of humans from hunter-gatherers to more permanent settlements. These cradles of civilization were also inherently cradles of plant domestication, which undoubtedly involved selection within the numerous wild almonds. The edible kernels of wild almonds and related species were thus important food staples from ancient times. Stone tools used for the apparent cracking of almond shells supports the harvesting of wild almonds in northern Israel by our human ancestors as early as 780,000 years ago (Goren-Inbar et al. 2002; Martinoli and Jacomet 2004; Weiss et al. 2004). Around 11,000 BCE, almonds, pistachios, and lentils were being utilized at Franchthi cave in southern Greece, indicating that the farming of legumes and nuts preceded that of grain in Greece and possibly the rest of Asia Minor (Hansen and Renfrew 1978; Farrand 1999). In addition to wild almond, kernels of wild apricots, plums, and possibly peaches, also present in various western and Central Asian ecosystems, would have been consumed regardless of fruit quality, as they are to this day. The term badam, which when used alone refers to almond in a wide range of Asian languages (Turkish, Persian, Arabic [either badam or loz], Urdu, Hindi, Punjabi, Telugu Kashmiri, Kannada, Marathi, Gujarati, Tamil, and Chinese [either

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badam, bwa-dam or xı`ng r
en—the latter also referring to apricot seed]) can also refer to the edible kernels of other Prunus; for example, tao’ze badam refers to peach kernel in western China and khasta badam to apricot kernel (sometimes called “poor man’s almond”) in India. Thus, in the absence of well-preserved endocarp remnants for species identification, it can be difficult to know which Prunus species was present in archaeological reports. Other distinguishing characteristics of almond species, however, are the symmetrical amygdaloidal (from amygdala, or amugdalh) kernel shape (versus the more ovate peach and apricot kernel) and a flowering time among the earliest of all temperate trees and in which the flowers emerge well before the leaves. This explosion of life toward the end of an otherwise barren winter apparently captured the imagination of ancient observers as it does now, contributing to a rich and varied folklore. Almonds are mentioned in the earliest Sumerian culinary texts in a list of banquet menu items (Rosengarten 1984). Biblical references to the almond show it was common in Palestine (where it can bloom as early as January) by at least 1700 BCE (Goor and Nurock 1968; Janick 2007). A reference to almonds in the book of Genesis 43:11 documents its high value: “their father Israel said unto them, if it must be so now, do this; take the best foods in the land in your vessels, and carry down to the man a present, a little balm, and a little honey, spices, and myrrh, nuts and almonds.” The man in this case was the governor of Egypt, from whom the Israelites were soliciting food aid in their time of famine, suggesting that almonds were also valued in Egypt but possibly not grown there. The or shaqed, which has its roots in an Hebrew name for almond is ancient Semitic term, as seen in the Akkadian sˇiqdu and Ugaritic thaqid as well as in old Ethiopic language. Shaqed may also be translated as “watchful,” symbolizing God’s watchfulness over his people; as in Jeremiah 1:11–12, “And the word of the Lord came to me, saying “Jeremiah, what do you see?’ And I said, ‘I see an almond branch.’ Then the Lord said to me, ‘You have seen well, for I am watching over my word to perform it.’” An early biblical reference, Numbers 17: 8, describes how the staffs of the 12 princes of Israel were placed into the Tabernacle after the Exodus. Only the staff of Aaron of the house of Levy, which was almond, flowered (Fig. 2.5). This was interpreted as a sign of divine favor to Aaron, of God’s watchfulness over him and his descendants (Rosengarten 1984). According to tradition, the staff of Aaron bore sweet kernels on one side and bitter kernels on the other, symbolizing sustenance if the Israelites followed the Lord but bitterness if they were to forsake of the Lord. (Although an almond staff could flower if cut just prior to bloom and even continue to flower for many days if placed in

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Fig. 2.5. Flowering of the almond staff of Aaron at the Jewish Tabernacle during the Exodus. Source: Rosengarten 1984.

water, the cut branch would soon collapse well before fruit development.) The almond blossom also supplied a symbolic model for the menorah or ark that stood in the Holy Temple: “Three cups, shaped like almond blossoms, were on one branch, with a knob and a flower; and three cups, shaped like almond blossoms, were on the other. . . on the candlestick itself were four cups, shaped like almond blossoms, with its knobs and flowers” (Exodus 25:33–34; 37:19–20). That the golden candlesticks for the Tabernacle should have almond-shape bowls may explain why ornamental pieces of crystals attached to candlesticks sometimes are still called almonds. Interestingly, in areas of Pakistan, western China, and India, the prayer cap, or topi, is often adorned with paisleylike patterns said to represent almond blossoms (Fig. 2.6). In Arabia and other Muslim areas, almond’s early flowering on leafless branches is seen as a symbol of hope (Rosengarten 1984). The earlyflowering habit of almond appears also to have made it symbolic for watchfulness or insight in ancient Greek mythology (Fig. 2.7). The symbolism derived from the Mycenaean Bronze Age (ca. 1200 BCE) myth of Phyllis of Thrace who grieved so much when the her lover Demophon did not return from Troy that the gods transformed her into an almond tree, thereafter called Phylla by the Greeks. Upon Demophon’s return, he embraced the tree, which burst into blossom. A more ominous interpretation of almond bloom (perhaps alluding to the rather sudden whitening of a man’s hair as he approaches old age and death), is given in Ecclesiastes 12:5: “when men are afraid of heights and of dangers in the

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Fig. 2.6. A prayer cap, or topi, adorned on top with paisley-like patterns said to represent single almond blossoms.

Fig. 2.7. Greek vase from 450 BCE showing the Oracle at Delphi holding almond branch in right hand. Source: www.talariaenterprises.com.

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streets; when the almond tree blossoms and the grasshopper drags himself along and desire no longer is stirred. Then man goes to his eternal home and mourners go about the streets.” A similarly ominous significance appeared to be held by the Bronze Age Phrygian culture of Anatolia, in present-day Turkey, whose rites of worship to their main goddess Cybele were so savage that they were eventually banned. According to legend, the almond tree sprang from the blood of Cybele and played prominent roles in the creation of lesser gods. Besides symbolizing Cybele for her devotees, the almond was also regarded as the Father of Everything, according to Rawlinson (1917). The symbolic importance of almond in these early cultures may have referred to its dramatic, life-reaffirming early bloom or to the amygdaloidal kernel shape. The almond shape symbolized the female genitalia in the East, while in Europe it often represented the womb. In early Christian art, Christ is sometimes shown surrounded by almonds or an almond-shape mandorla (from the Italian mandorlo, for “almond”) representing the womb (Fig. 2.8). In other parts of Europe, particularly central Italy, the almond symbolized the Virgin Mary. Consequently, almond nuts often have been associated with fertility. Romans showered newlyweds with almonds as a fertility charm. An old Romanian contraceptive measure was to carry roasted almonds on the person, perhaps because the roasting counteracted the nuts’ traditional powers on fertility. It is still a modern European custom to give female guests at weddings a bag of five sugared almonds representing children, happiness, romance, good health, and fortune (Fig. 2.9). In Greece, almond cookies remain a popular wedding food. In Britain, their traditional Mothering Sunday Simnel cake is covered with almond paste in a possible reference to motherhood. The traditional almond paste and royal icing of British wedding cakes symbolizes the intermingled sweetness and bittersweetness of the couple’s new life together. Even in ancient China, almond’s amygdaloidal shape was considered a symbol of female beauty as well as enduring sadness (perhaps because the symmetrical amygdalus or mandorla is the product of overlapping circles or because it refers to the inevitable bitterness present in the occasional bitter kernels). In the wild, almond species usually produce cyanogenic and bitter seed; however, individual trees producing sweet and edible nuts have been reported in native populations of many almond and related species (Vavilov 1930; Denisov 1988; Werner and Crellar, 1997). Although relatively rare, these individuals can be readily identified by consumption of their seed by native rodents and birds. Human gatherers from early times to present would mark such trees for recurrent annual

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Fig. 2.8. A medieval Christian artistic convention in which an almond-shaped area, or mandorla, surrounds Jesus. Source: http://en.wikipedia.org/wiki/Aureola.

harvest, as has been documented in present-day hunter-gatherer groups in Asia and Africa (Alexander-Essers 1994). The mutation for sweet kernel is expressed in all seed of the parent tree and, unlike most other cyanogenic plants, including apricot and peach, is dominant in almond (Werner and Crellar 1997; Dicenta et al. 2007; Negri et al. 2008). Consequently, not only will all seed in a selected tree have sweet kernels, but the majority of seedlings derived from those seeds will also have sweet kernels. Domestication of sweet kernel genotypes would have been advanced by the propagation of these selected individuals, either through the germination and growth of harvested seed or by relatively simple propagation techniques, such as rooted cuttings developed during these prehistoric times, or through the weeding out of bitter almonds within a wild grove and the promotion of greater growth of the selected sweet types (Zohary and Spiegel-Roy 1975). In addition, birds of

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Fig. 2.9. A bomboniere, or Italian party favor, containing five symbolic almonds. Note the almond flower design in this example.

certain species, such as the western scrub-jay, Aphelocoma californica, will systematically bury or cache single sweet almond seeds at several thousand distinct sites for later retrieval and consumption (Pravosudova et al. 2006). It is not unusual for many of these seeds to germinate and, if the site is appropriate, to grow into productive trees. Toxins in bitter seeds can be removed through various basic processing methods. Bitter almonds possess the almond’s nutritional quality and long storage life as well as a natural protection against undesired feeding by mammals, birds, and insects. Nutritionally, almond represents a compact, readily stored, and high-energy food (Table 2.2). Both the edible, immature fruit and the mature kernel contain the amino acid linolenic acid, which is essential but not naturally synthesized in humans. Recent studies have also indicated that moderate consumption of almond kernels can suppress hunger. An exceptional postharvest stability of almond kernels of over 2 years, when stored dry and in shell, would further contribute to almonds value as an easily transportable, high-quality food source in early human transmigrations and commerce. The same traits would also facilitate the establishment of widely dispersed almond stands through the accidental or deliberate planting of seed.

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Table 2.2. Nutrient composition of the almond kernel per 100 g fresh weight of edible portion. Nutrient

Value

Energy Protein Carbohydrate Fiber, total dietary Glucose Starch Calcium Magnesium Phosporus Potassium Sodium Folate, total Vitamin E Saturated fatty acids Monounsaturated fatty acids Polyunsaturated fatty acid

578 kcal 21.26 g 19.74 g 11.8 g 4.54 g 0.73 g 248 mg 275 mg 474 mg 728 mg 1 mg 29 mcg 25.87 mg 3.88 g 32.16 g 12.21 g

Source: Adapted from Socias i Company et al. 2007.

IV. CULTIVATED GREEK NUT Although the kernel of native almond and even apricot and peach species continue to be harvested today much as they were in antiquity, evolving market factors, particularly during the great flourishing of trade associated with the Achaemenian Dynasty of Persia (559–334 BCE), resulted in a market standard that appears to have become widely known in ancient times as the Greek nut and in more modern times as the cultivated sweet almond. Several hypotheses have been advanced concerning its origin. Russian scientists Kovalev and Kostina (1935) suggested that the cultivated almond emerged by selection from within the species listed initially as Amygdalus communis L., whose range may have extended across Iran and eastern Turkey into Syria, Lebanon, and Jordan (Browitz 1974). Two natural sweet-kerneled A. communis populations have been reported on the slopes of the Kopet Dagh Mountains in central Asia between present-day Iran and Tadjikistan and on the slopes of the Tian Shan Mountains between Uzbekistan and western China (Vavilov 1930; Denisov 1988) (Fig. 2.3). This species was reported to be adapted to mild winter and dry hot summer conditions by traits of low chilling, early bloom, rapid early shoot growth, deep penetrating root systems, and high tolerance to summer heat and drought. Its phenotypic range closely resembles that of present-day cultivated almonds.

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Another hypothesis originated by Evreinoff (1958) is that the cultivated almond arose by hybridization among P. fenzliana and possibly P. bucharica, P. kuramica, and other species. This view, which has recently been supported by molecular analysis (Zeinalabedini et al. 2009), holds that the cultivated almond originated through human intervention and is not a natural species. Grasselly (1976b) reported that P. kuramica, whose range includes Afghanistan and northern Pakistan, somewhat resembles cultivated almonds and coexists with it in many farming areas. P. kuramica grows on the more xerophytic sites, P. dulcis being a mesophytic species. Different almond species, particularly those of the same section, cross readily, and considerable natural hybridization between cultivated almond and nearby wild species takes place (Grasselly 1976; Denisov 1988). Introgressive hybridization and exchange of genes thus can take place whenever ranges overlap. Many of the more than 30 named almond species may not be true species but products of such interspecific hybridization events. The previously described Kopet Dagh and Tian Shan sweet-kerneled populations are proposed to be more recent feral populations of cultivated almonds or their natural hybrids (Browicz and Zohary 1996). Amygdalus communis thus represents feral populations of P. dulcis (A. dulcis) rather than a native species. Furthermore, as almond cultivation expanded, new hybridizations/introgression would have occurred, as with the wild species P. webbii (Spach) Vierh. (Godini 1977; Socias i Company 2002) and cultivated almond populations found along the northern shore of the Mediterranean sea from Greece and the Balkans to Spain and Portugal. Godini (2000) showed that P. webbii from the Italian region of Puglia was self-compatible and that it probably contributed this trait to local cultivated almond landraces since many almond orchards were made up of seedling plantings during prehistoric times to the present. Molecular analysis recently has shown this proposed interspecific introgression of self-compatibility from wild P. webbii to cultivated P. dulcis to be correct (Certal et al. 2002; Mart
ınez-Go´mez et al. 2007). Zohary and Hopf (1993) proposed that the area of initial domestication was the eastern part of the Mediterranean basin. However, the wild populations and species found in the eastern Mediterranean appear genetically more distant from the cultivated almond than the wild populations and species of the Caucasus and Zagros mountains of eastern Asia Minor and Persia (Sorkheh et al. 2007). Both regions, however, were then part of the Achaemenian Dynasty of Persia (559–334 BCE), which actively encouraged both commercial and cultural exchange among its diverse regions (Fig. 2.10). The Achaemenian kings,

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Fig. 2.10. Map of the Achaemenian Empire at about 500 BCE showing overlap with both time and locations of documented early almond plantings (see text).

including both Cyrus the Great (ca. 600–530 BCE) and Darius the Great (ca. 549–486 BCE), took a special interest in plant collection and cultivation. The Spartan mercenary Lysander, who joined the Achaemenian Persian king Cyrus the Younger in 401 BCE, reported to Xenophon of Athens how the Persian kings excelled not only in war but also in creating protected gardens, or pairidaeza, of plants, especially fruitbearing trees, collected during their foreign expeditions. Xenophon (who was a supporter and chronicler of Socrates) went on to write, in his Oeconomicus (Economics [399 BCE]) “The Great King . . . in all the districts he resides and visits . . . takes great care that there are ‘paradises’ (from Greek form paradeisos) as they call them, full of all the beautiful things the soil will produce” (Eduljee 2009). One of the earliest pairidaeza may have been the mythical Hanging Gardens of Babylon, purportedly built by King Nebuchadnezzar (605–562 BCE) to placate his homesick Median wife, Amytis, by copying the lush gardens from her childhood home on the slopes of the Zagros Mountains. Several wild almonds are common species in the Zagros Mountains, suggesting their possible inclusion in the Hanging Gardens. One of these species, P. fenzliana, from which cultivated almond was probably derived, also may have been present at an even earlier paradeisos; recent archaeological research has identified an area within the native habitat of P. fenzliana in the Caucasus Mountains as a possible site of the mythical Garden of Eden (Eduljee 2009).

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V. OLD WORLD DISSEMINATION It is known that the introduction of cultivated almond in the eastern Mediterranean area took place by the second millennium BCE, because cultivated almond remains have been found in the tomb of Tutankhamen, who was buried in 1323 BCE (Zohary and Hopf 1993). Almond cultivation appears to have existed in Greece long before the creation of the Greek myths to explain its incorporation into them (Graves 1955), and there is evidence of extensive almond trade in the eastern Mediterranean in the fourth century BCE (Cerd
a, 1973) and possibly much earlier (Hansen and Renfrew 1978; Farrand 1999). The wide dissemination of modern almond and its cultivation has been separated into four phases: Asiatic, Mediterranean, Californian, and Southern Hemisphere (Kester et al. 1991). Often concurrent with the spread of cultivated almond is a dissemination of a surprisingly similar folklore, including medical and culinary uses, suggesting that the spread was through well established and interconnected trade routes (Albala 2009). A. Asiatic Stage The Asiatic stage included the initial domestication and the subsequent spread throughout central and southwestern Asia. The Greek naturalist Theophrastus described almond, which he called amugdalai in his treatise on the history of plants about 300 BCE. During the early Roman expansion, Marcus Porcius Cato (ca. 236–149 BCE) referred to almond as the “Greek nut,” suggesting its dissemination via Greece. Pliny (23–79), in his Natural History, also listed almond as prima omnium, or “first of all.” Within a few hundred years, the range of known almond cultivation includes the regions now known as Turkey (Ayfer 1975; Dokuzogus 1975), Iran (Grigorian 1976), Syria (Thompson 1983), Israel (Spiegel-Roy 1976), and east to the Xinjiang Province of China (Gustaffson et al. 1988), northern Pakistan (Thompson et al. 1989), and northwest India (Singh and Uppal 1977; Singh et al. 1977). In Kashgar, in Xinjiang Province, cultivated almonds were reported to originate from Central Asia across the Tian Shan Mountains to the west (Gustaffson et al. 1988). Kashgar is on the old Silk Road connecting China to India and the West, as were most other sites of eventual domestication. The extent and sophistication of this prehistoric trade was recently documented with the discovery of the wreck of the Kyrnenia, dating from around 350 BCE, in which both vessel and cargo remain remarkable intact (Fig. 2.11). In addition to cultivated almonds, the ship was carrying amphorae of wine and olive oil, grain millstones, coins, and iron blooms.

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Fig. 2.11. The Kyrenia II replica of the almond-carrying Greek cargo vessel sunk off Cyprus about 350 BCE (top). Proposed last route of the Kyrenia suggesting extensive commerce in the region (lower left). Samples of the 10,000 almonds found with the Kyrenia wreckage (lower right).

Using presumed and sometimes known origins for this cargo, the vessel’s probable trading route was deduced, showing that an advanced commerce had already been well established by this time (Albala 2009). Such archaeological finds, as well as parallels in the associated folklore, support a central role of prehistoric Greco-Persian culture and commerce in advancing the cultivation, utilization, and dissemination of the modern almond. As a traded commodity, the range of almond extended to the edge of the known world (Fig. 2.3). In far eastern China, the almond initially was described in the ancient text Yu yan tsa tsu as a flat peach from Persia. ‘The meat is bitter and acrid, and cannot be chewed; the interior of the kernel, however, is sweet, and is highly prized in the Western Regions and all other countries” (Albala 2009). The Persian name for almonds, badam, or in Old Persian, vadam, became the Old Chinese p’o-tam or bwa-dam. It also entered Tibetan as ba-dam. A later document, Pen ts’ap kan mu by Li Si-sen,

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identifies the origin of almonds as the land of the Mohammedans and states that they occur everywhere west of Gansu, the province bordering Xinjiang (literally, “New Frontier”). Because the more temperate and humid climate of eastern China limits almond cultivation, it was imported, especially in the T’ang Dynasty (618–907), a period of rich cultural exchange between East and West. Fragments of Chinese pharmacopoeias survive, including seventh-century works by Meng Shen, which mention almonds and a number of western foods grown in imperial gardens (Simoons 1991). Later, the Hsin T’ang shu (new T’ang history) describes plants grown in Western Asia, including almonds, grapes, and figs from Persia (Albala 2009). In the 16th century, Li Shizen in his Bencao Gangmu (a classification of materia medica) reports: “It comes from the lands of Hui people and is now in all the lands of the west. . .. The tree is like an apricot but its leaves are smaller; the fruit is pointed and small, the flesh thin. Its kernel is like a plumstone, the skin is thin and the almond is sweet and nice. It is eaten for tea, its taste is similar to that of the hazelnut. The people in the west consider it a local specialty” (M
etaili
e 2001). In India, where almonds can be cultivated in the more Mediterranean climates of the northern provinces of Jammu and Himachal Pradesh, descriptions of almond and its often medical and culinary uses appeared during the same period and also suggest a Greco-Persian origin (Peregrine and Melvin 2003). Almonds in Hindi are called badam, which comes from the Sanskrit vatama, in turn from Persian badam or old Persian vadam. As early as the third century BCE (around the time of voyages of the Kyrnenia), the classic Indian medical texts Caraka Samhit a and Susruta Samhit a characterized sweet almond (v^ at^ ama or b^ ad^ ama) as heavy, hot in potency, unctuous, sweet, strength promoting, alleviator of Vata (wind/spirit/air), nourishing, aphrodisiac, and aggravator of Kapha (phlegm) as well as Pitta (bile). Although the Caraka Samhit a and Susruta Samhit a, ancient Indian Ayurvedic texts on internal medicine, are an early source of medical understanding believed to be independent of ancient Greece, Albala (2009) has recently pointed out the strong similarities between medical (and culinary) uses of almond as initially described in Greece with subsequent uses in Persia and its trading partners, including India and China. He proposes the apparent Greek origin of much of the traditional folklore associated with cultivated almond is evidence for a Greek origin or major role in the dissemination of the cultivated form of almond (P. dulcis). The ancient Greek physician Hippocrates (ca. 460–370 BCE), considered the father of western medicine, founded the humoral, or Unani, doctrine

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of medicine. Unani medicine is based on the concepts of the four humors: phlegm (balgham), blood (dam), yellow bile (safr a’), and black bile (saud a’). (The word “Unani” refers to Ionian Greek, meaning the Greek-populated west coast of Asia Minor, in what is now Turkey. Thus the term “Greek” during this period of history refers not only to the Greek islands but much of the eastern Mediterranean, which was then also strongly networked with Persian culture [Fig. 2.10].) Hippocrates was among the first to record the medical uses of almonds. He reports: “Almonds are burning but nutritious; burning because they are oily, and nutritious because they are fleshy” (Jones 1967). In the Greek system of humoral physiology, this means that almonds would have been categorized as a hot and dry food, one that stimulates choler or energy in the body. Diocles of Carystus, a follower of Hippocrates, adds: “almonds are nourishing and good for the bowels, and are moreover, calorific because they contain some of the properties of millet. The green are less unwholesome than the dry, the soaked than the unsoaked, the roasted than the raw” (Bottero 2004). Soaking almonds would activate seed digestive enzymes and facilitate removal of the bitter brown seedcoat. Roasting makes them more easily digested. Although green almonds would have been unknown outside their area of cultivation, soaking or blanching was a common practice in both Indian and Chinese medicine (Achaya 1998). Medical philosophies differed, but the goal appears to be the same for each: to reduce their tendency to heat the body (Albala, 2009). Because of its classification as a hot food, which scours the body’s passages, almond was recommended by Hippocrates and his followers to relieve coughs, for weight gain, and as an aphrodisiac (Albala 2009). The Persian Al-Qanun fil-tibb (canon of Avicenna) (980–1037) became the standard medical text in medieval Europe and was the primary means of Greek humoral medicine reaching Arabia and Asia, particularly in India (where the canon is still the principal authority for Unani medicine) (Achaya 1998; Bottero 2004). Almond’s medical values were described in a manner very consistent with the earlier view of Hippocrates: “Almonds are more slowly digested and thus less likely to convert to choler,” and “sweet almonds comfort coughs and spitting of blood. . . they open clogs of the liver and spleen on account of their bitterness. They even open clogs occurring in the extremities of the veins and if eaten fresh with the peel, clean humidity in the stomach” (Albala 2009). The Yin-Shan Cheng-Yao dietary by Szu-Hui, of the Chinese Mongol era, dating from 25 AD, also describe almonds in a similar fashion: “Almonds control coughing and bring down ch’i. They disperse impeded pressing of the chest and abdomen” (Buell et al. 2000). The Greco-Persian view of health as a balance of humors thus finds its

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parallel in the Chinese idea of ch’I, or the balancing yin and yang forces, which can also be described as cold and hot. In the Indian Ayurvedic medical system, almonds, in addition to the previously listed attributes of cough relief and aphrodisiac, are also classified as a hot food. In Ayurvedic medicine, as in Greek medicine, health consists of a balance of elemental forces, or doshas (which are not exactly humors but energy principles that regulate physiological functions). Almonds are said to enhance the kapha dosha, which maintains the structural integrity of the body, but they also suppress an excess of the vata dosha, which is the principle of movement and transportation in the body. Thus, low weight gain in a thin overactive body in which nourishment dissipates quickly can be treated with a regimen including almonds (Albala 2009). In the associated Rasayana approach to health and longevity, almonds provide vital energy, or ojas, a belief comparable to the Chinese concept of ch’i and the Greek concept of pneuma (Albala 2009). Almond kernels, in addition to being a high-quality food source, are also source of high-quality oil. The oil, which can constitute more than 50% of the kernel by weight, is primarily composed of oleic acid (approximately 65%) and linoleic acid (approximately 30%), which results in good flavor and nutritional value as well as stability in storage (Abdallah et al. 1998). These qualities encouraged its use as a base for various ointments in both ancient and modern cultures. The most extensive use of almonds in Ayurveda is in the form of oil, used for various skin ailments and to warm the body in massage and other therapies. Almond oil is used in various vata disorders, chronic constipation, dry cough, semen disorders, leucorrhoea, and dysmenorrhoea. It is a good aphrodisiac, galactogogue, and health tonic (Albala 2009). In addition to the similar medicinal uses of almond in disparate Asian cultures, there were also a number of culinary dishes that appear to have a Greco-Persian origin. An ancient Baghdad cookbook, the Kitab alTabikh, records a number of sweet almond recipes, among which are lauzinaj, faludhaj, and samal wa-aqras (Bottero 2004; Albala 2009). (The Arabic word for almond is lauz or loz, whose occasional reference in the Old Testament documents its use in antiquity). The lauzinaj recipe begins with finely pounded sugar and pounded almonds, kneaded together with rosewater. This is essentially marzipan, and it also comes with various flavorings, such as camphor or musk (Albala 2009). These are ancestors of a number of dishes of India, such as badam barfi, halwa, and similar almond sweets found throughout central Asia and as far as China. The term “lozenge” is also derived from the Arabic lauz, perhaps in reference to its almond shape (Albala 2009).

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B. Mediterranean Stage The westward dissemination of cultivated almond into the Mediterranean had two stages. Almonds were first brought into the Peloponnesian peninsula and Greek isles (Stylianides 1976), becoming well established by 300 to 400 BCE (Fig. 2.3). Gradually almonds were introduced to all adapted areas of the Mediterranean, including Italy, southern France, Spain, Portugal, North Africa, and the Madeira Islands. These introductions may have come from the early ocean trading Phoenicians of Asia Minor (Egea and Garcia 1975) and/or from the Greeks while establishing colonies in Sicily, Europe, and North Africa, and from other merchant groups as extensive trade routes were well established by this time. Cultivation typically was limited to within 50 miles of the Mediterranean coast, extending onto the slopes of river valleys and the interior plateau of Spain. Further introductions came from about 500 to 600 with the conquest of North Africa by the Arabs; they brought almonds into Tunisia (Jaquani 1976) and Morocco (Laghezali 1985) and then into Spain and Portugal (Egea and Garcia 1975). One of the ancient Silk Road caravan routes also crossed north-central Africa, through Timbuktu into Morocco, thus representing an even earlier route of possible dissemination to North Africa and western Europe (Evreinoff 1952, 1958). Remnants of such pre-Arabic introductions may exist today in the diverse germplasm only now being documented in the geographically isolated Atlas Mountains in Morocco and Tunisia (Laghezali 1985; Lansari et al. 1994). Archaeological studies at the site of the Mount Vesuvius eruption, which occurred in the year 79, indicate that almond was a common food of the Campenians of southern Italy by the first century, although almonds appear to be well known much earlier in Rome’s history, as they were described by Marcus Porcius Cato as the “Greek nut” as early as 200 BCE. In Latin, almond was called amandola, derived from the Greek amugdalh (amingdola) (cf. amygdala). The Latin amandola appears to be the root for the term used for almond in Italian (mandorla), German (mandel), Swedish (mandel), Russian (mindal), Croatian (mandula), French (amande), Spanish (almendra), and Portuguese (amendoa). Interestingly, in the etymologically more isolated Romani language, almond is migdala and so probably derived from the Greek. It has been widely postulated that the “al” prefix, as in the Spanish almendra, and English almond, resulted from its fusion of the Arabic article “al” with the Latin amandola (and dropping the initial “a,” as done with the Italian mandorla), which represents an etymological and cultural legacy of the 800-year control of Spain by the Arabic-speaking Moors.

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There is no doubt that Roman knowledge of almond was strongly influenced by the Greeks. Pliny the Elder (23–79), the ancient Roman nobleman and historian, reiterated earlier Greek ideas that almonds act as a diuretic and emmenagogue. They provoke sleep and sharpen the appetite and are also useful against headache and fever, the latter presumably from their purging qualities to drive out the fever rather than to counteract or cool it (Albala 2009). Galen of Pergamum (129–200), a highly influential Greek physician serving several Roman emperors, believed that although bitter almonds were not very astringent, they cleansed and attenuated and thus purged, and “act towards the expectoration of moist matter from the lungs and chest. The very bitter cut through thick and viscous matter. But they are also oily, so not as useful for purging the stomach, and bitter almonds unlike the sweet afford little nourishment for the body” (Grant 2000). Rosengarten (1984) describes an almond-based cold cream used by fashionable French women in the 17th century. This ‘Ninon de Lenclos’ ointment contained 4 ounces (112 g) of almond oil, 3 ounces of hog’s lard (104 g), and 1 ounce (28 g) of spermaceti (a waxy component of sperm whale oil). These ingredients were melted, lemon juice was added, the mixture was stirred until cool and then scented with rose water. Almond oil continues to be an important oil base for cosmetics and other pharmaceuticals to the present. Additional medicinal uses of almond ointment included the treatment of pattern baldness and as a soothing ointment for burns. A continuing practice in parts of Asia is the application of almond oil to the heads of babies, as it is believed to promote their subsequent intelligence. Salves containing concentrations of oleic and related fatty acids may also help control of head lice (Socias i Company et al. 2007), which might further explain the application of almond oil to the scalps of children. While bitter almonds and almond oil were used primarily as medicine, sweet almonds and oil of bitter almond (an alcohol-based extract of bitter almond essence, primarily benzaldehyde) were used as both food and medicine. Both also found culinary uses. One of the oldest surviving Roman cookbooks, Apicius (fourth or fifth century CE), describes a dish called apothermum, which is boiled wheat with pine nuts and almonds that have been soaked, skinned, and whitened with silver chalk (creta argentaria) to which is added raisins and raisin wine, with pepper over the top (Bottero 2004). As pointed out by Albala (2009), this recipe bears a striking resemblance to a rice dish in Persian cookery. In turn it has found its way to India and China and ultimately to England and the West as rice pudding. A similar soup made with ground almond, rice, and sugar was used for sore throats in the Ch’ing court in China (1644–1911)

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(Simoons 1991). In modern China, a popular dish to combat colds and sore throats is made with apricot kernels powdered and mixed with rice congee (Simoons 1991). A traditional Christmas dessert in Sweden and parts of northern Germany is cinnamon-flavored rice pudding into which a single almond has been hidden; the one who finds the almond will have good fortune for a year. A renaissance in European cuisine that involved spices as well as almond and other foods from Asia appears to have occurred from the 10th through the 13th centuries following the spread of Islam. Evidence for even earlier almond plantings in France can be found in the charter granted by King Chilperic II (716) and in an edict of Charlemagne in 812 (Rosengarten 1984). An inventory of the household goods of the Queen of France in 1372 listed only 20 pounds of sugar but included 500 pounds of almonds. Almonds appear to have been relatively inexpensive and generally available to the emerging middle class even in areas such as England, where the climate would make a local production more difficult. Rosengarten (1984) estimates an average price of approximately 2 to 3 pence per pound between the years 1259 and 1400. The almond trade appeared substantial by the 14th century with Venice becoming an important center of commerce. To capitalize on this extensive commerce, the Knights Templar levied tithes on almonds, honey, and sesame seed in 1441. The Arab conquest of North Africa and Spain introduced Middle Eastern cuisine, which included almond and sugar-based confections similar to the previously described marzipan as well as more staple fare such as harisa, a popular dish of 10th-century Baghdad, which consisted of meat and vegetables served with a sauce thickened with powdered almonds (Rosengarten 1984). Similarly, almond milk, which could be used as a milk substitute for direct consumption or in cooking, was made by soaking in water pulverized almonds from which the outer seed coat had first been removed. Almond milk became a more frequent ingredient in Mediterranean cuisine than in the Middle East. For example, the grand medieval European dish blancmanger was typically chicken stewed in almond milk. The comparable and popular Persian dish isfidhabaj (white stew) used almond milk in some recipes though many recipes contained no reference to almonds at all. Religious and medical views have played roles in the emergence of almond in European cuisine. For example, almond milk could be conveniently substituted for dairy milk, whose consumption was forbidden on Fridays by the Roman Catholic Church. In addition, physicians from the 12th to the 16th century continued the belief that the nature of the food consumed was a critical component of

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health. Echoing ancient Greek beliefs, foods such as almonds, chicken, and rice were viewed as moderately warm and moist in their characteristics and so compatible with the healthy human body. The prevailing view among the upper class was that such moderate foods were more ideally suited for consumption and had the added value of moderating foods perceived as being too hot (ginger), too cold (many vegetables), too dry (turnip), and too wet (watermelon) (Albala 2009). Even more extensive culinary uses of almond were eventually brought to Europe by the returning Crusaders, many of whom had developed a preference for the more exotic Middle Eastern cuisine compared to the blander European diet of the time. As an example, a recipe from the Forme of Cury, dating back to 1390 (http://homecooking.about.com/od/foodhistory/a/almondhistory.htm), uses ground almonds in a gravy for oysters that is still fashionable. A rich folklore, including extensive medical and culinary uses of almond, also can also be found in Spain, particularly southern Iberia, which was under Moorish control the longest. The origin of this folklore is often uncertain, though during the Dark Ages, the Muslim Arab kingdoms were the main repository and disseminator of the earlier Greek and Roman knowledge. Paella, the national dish of Spain, or at least the Valencia region, has its equivalent in the Persian dish of dan-pukhtak also known as biryani, with almonds often being included in both (Albala 2009). A bit of mythology often credited to Aristotle (but probably not disseminated by the abstinent Arab Muslims) was this proposal of Roman author Pliny the Elder: “It is said that if five bitter almonds are taken by a person before sitting down to drink, he will be proof against inebriation.” Plutarch (ca. 42 BCE–37 CE) likewise says that Drusus, brother of Tiberius, who was a prodigious drinker, used almonds this way. The logic here is that the almond, due to its bitterness and diuretic properties, speeds the alcohol through the system before it has a chance to send vapors up to the head (which was believed to cause inebriation). The Herbal of John Gerard (1597) states that “five or six (almonds) being taken fasting do keepe a man from being drunke” and may have become the forerunner of the cocktail nut of today (Rosengarten 1984). Purported protection from inebriation may have also contributed to the previously cited popularity of almonds in weddings. In traditional Greek weddings, slightly bitter sugar-coated almonds called koufeta are placed in little bags in odd numbers and served on a silver tray. Odd numbers are indivisible, symbolizing how the newlyweds will share everything and remain undivided. Tradition holds that if an unmarried woman puts the almonds under her pillow, she will dream of her future husband.

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Five (often sugar-coated or “Jordan”) almonds signifying five wishes (health, wealth, happiness, fertility, and longevity) are common in traditional Italian weddings and other special occasions. These almonds decorate each place setting as favors, tucked into pretty boxes or tulle bags called bomboniere that are often personalized with the couple’s names and date (Fig. 2.9). A different and new type of favor is the favor cake or Torta Bomboniera, which is made using little boxes forming one or more tier of a cake. Inside each box are the sugared almonds and a card printed with the data of ceremony. Sugar-coated almonds are also traditional in Middle Eastern weddings and are considered by some to be aphrodisiacs. In southern Europe, almonds also symbolized good luck as well as long life and happiness. The heartening capability of a dormant almond shoot in winter to quickly flower when cut and brought into warmer temperatures is frequently alluded to in European art and literature from medieval times to the present (Figs. 2.12, 2.13, 2.14). In the legend of Tannhauser, made famous by Richard Wagner’s 1845 opera, the knight was informed by the pope that he was as likely to have his sins forgiven as the pope’s staff was likely to bloom (Rosengarten 1984). The staff, which was made of almond, did indeed bloom but, tragically, Tannhauser died

Fig. 2.12. Lithographic reproduction of an almond twig from the 1517 Herbal by Johanes Niger. Source: D. Avanzato and I. Vasssallo 2006.

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Fig. 2.13. Almond Blossom by Vincent Van Gogh (1853–1890) for his newborn nephew as a symbol of a budding life.

before his pardon arrived. The almond’s seemingly magical flowering ability also may have contributed to the preference for a wand made from almond wood by professional magicians during the Middle Ages as well as the use of almonds in Tuscany for making dowsing rods for the divination of underground water or other hidden items of value (reminiscent of the almond branch of the Oracle at Delphi (Fig. 2.7)). Almond was also well suited for cultivation in the Mediterranean climate with its traditional low-input moderate-return dry-land cropping systems. Easy propagation by seed, rapid development of the tree, and adaptation to marginal soil, summer drought and heat, combined with its food value made the almond well adapted to the subsistence form of agriculture predominating in early Mediterranean culture. High root tolerance to drought but high susceptibility to excessive soil moisture placed almonds in a mixed culture system with olives, carob, and other drought-tolerant crops. Almonds were usually found on welldrained slopes at higher elevations of valleys to escape shoot damage from frosts and root damage from excessive soil water. A traditional culture system evolved that minimized inputs of labor, fertilizers, and use of supplementary water. Some trees producing bitter nuts were tolerated in local seedling orchards since bitter seed were mixed in

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Fig. 2.14. Gathering Almond Blossoms (1916) by John William Waterhouse.

small quantities with sweet kernel to produce the distinctive amaretto flavor. Because sweetness is the dominant trait, genes for bitterness may still be present in sweet-kernel trees, allowing the occasional recombination of bitter seedling trees. The Italian naturalist Scribonius Largus documented this distinction in the first century CE by naming bitter almonds Amygdali amari. Bitter forms of the cultivated almond sometimes are classified as Prunus dulcis (Mill.) D. A. Webb var. amara (DC.) Buchheim (Fig. 2.15). Bitter almonds are also commercially cultivated for their oil, which is used to extract benzaldehyde for the amaretto essence, known to bakers and chefs as almond extract or oil of bitter almond but known to many consumers as the cherry flavor in cherry colas, drinks, and confectionaries. The presence of excessive trees producing bitter or otherwise undesirable seeds in segregating seedling populations would be an incentive to use the undesired bitter trees as

54

T. M. GRADZIEL

Fig. 2.15. Bitter almond (Amygdalus communis L. var. amara (syn. P. dulcis var. amara) from 1926 Textbook of Pharmacognosy by T.C. Denston showing similar morphologies to cultivated sweet almond despite distinct botanical classification.

rootstocks for topworking with scions from the more desirable trees. Grafting was widely used in antiquity and is mentioned in writings of Hippocrates, Theophrastus, Cato, Varro, Columella, and Pliny. Two thousand years of continuous almond culture in the Mediterranean basin concentrated production to specific regions where well-defined seedling ecotypes and local cultivars evolved (Grasselly 1972, 1976b; Grasselly and Crossa-Raynaud 1980). These highly selected regional

2. ORIGIN AND DISSEMINATION OF ALMOND

55

cultivars and landraces represent a rich genetic, horticultural, and culinary diversity (Tables 2.3 and 2.4). Dryland, seedling orchards remained the major almond culture system in the Mediterranean region for centuries, persisting to recent times in such areas as Sicily, Sardinia, Majorca, the Madeira Islands, Greece, Turkey, and Morocco. By the middle of the 19th century, cultivars of specific Mediterranean countries were recognized by nurseries by name (Fig. 2.16) and were introduced into California as early as 1843. By the turn of the 20th century, most of the almond countries of the Mediterranean region and the United States had identified “local cultivars” that were seedling selections which, while representing the germplasm of the region, were often of unknown origin. Italy became a major almond-growing area, and many cultivars have been described in Puglia (Fanelli 1939; Grasselly and Crossa-Raynaud 1980), Sicily (Bianca 1872; Spina 1958), and Sardinia (Milella 1959; Agabbio et al. 1984; Chessa and Pala 1985). Puglian cultivars were predominantly late blooming, had hard shells with a kernel/nut ratio of 25% to 30%, with high percentages of double kernels. Sicilian cultivars tended to be early blooming, very hard shells, and more or less round in shape. Several Italian cultivars were also selfcompatible. Spain has a long history of almond culture with almonds being grown in most provinces (Felipe 1976; Gardner and Lee 1979). Principal concentrations are in coastal provinces (Tarragona, Valencia) along the Mediterranean coast (Murcia) and the Ebro valley (Lerida), although the range extends to the southeastern border with Portugal and into the interior (Murua et al. 1993). ‘Marcona’ and ‘Desmayo Largueta’, both of unknown origin, have been the most widely grown cultivars. Both have very hard shells, are highly productive, and have well-recognized eating and cooking qualities such that they are marketed by name. Many other named cultivars of local origin (Vargas 1975; Felipe 1984; Garcia et al. 1985, 1988) have evolved from localized ecological niches that occur in different valleys extending inland from the Mediterranean coast. France is an old almond-growing country with areas in the southern Rhone valley and the surrounding foothills (Grasselly and CrossaRaynaud 1980). Cultivars originating in Provence and the foothills of the Alps tend to be late blooming. Cultivars associated with the Languedoc region nearer the center of the Rhone valley (‘Ai’, ‘Princesse’, ‘Languedoc’, ‘Pistache’, ‘Ardechoise’, ‘Fournat de Brezenaud’, ‘Rabasse’) show a range of tree and nut characteristics from hard to soft shelled and large to small kernels. Portugal has two principal areas of production, the Algarve region in the south and the Alto Duoro in the north. Each region has cultivars

56

T. M. GRADZIEL

Table 2.3. Characteristics of important new almond cultivars from selected breeding programs. Cultivar

Description1

SPAIN CITA of Arago´n (Zaragoza) Blanquerna ‘Genco’ OP, SC, mid-blooming, hard shell, large kernel of excellent quality, early ripening Cambra ‘Ferragnes’  ‘Tuono’, SC, late blooming, hard shell, medium ripening Felisia ‘Titan’  ‘Tuono’, SC, very late blooming, medium-hard shell, small kernel, very low alternance, early–medium ripening Belona ‘Blanquerna’  ‘Belle d’Aurons’, SC, late blooming, hard shell, large kernel with an outstanding composition, medium ripening Soleta ‘Blanquerna’  ‘Belle d’Aurons’, SC, late blooming, large kernel with an outstanding performance when roasted, medium–late ripening Mard
ıa ‘Felisia’  ‘Bertina’, SC, extremely late blooming, disease tolerant, early–medium ripening CEBAS - CSIC (Murcia) Anton˜eta ‘Ferragnes’  ‘Tuono’, SC, late blooming, hard shell, high vigor, spreading with dense, very early ripening Marta ‘Ferragnes’  ‘Tuono’, SC, hard shell, high vigor, upright, late blooming, early ripening Penta S5133  ‘Lauranne’, SC, extremely late blooming, hard shell, intermediate vigor and branching, early ripening Tardona S5133  R1000, SC, extremely late blooming, hard shell, small kernel, intermediate vigor with dense branching, medium ripening IRTA - Mas de Bover (Reus) Constant
ı (‘Ferragnes’  ‘Ferraduel’) OP, SC, late blooming, mid ripening, vigorous, mid branching Marinada ‘Lauranne’  ‘Glorieta’, SC, very late blooming, mid-ripening, midvigor, mid-branching Tarraco (‘Ferralise’  ‘Tuono’)  Anxaneta, SI, very late blooming, midripening, mid-vigor, large kernel, mid-branching Vairo (‘Primorskij’  ‘Cristomorto)  ‘Lauranne, SC, late bloomig, early ripening, high vigor, mid-branching FRANCE INRA (Avignon) Lauranne Steliette Mandaline ISRAEL Shefa

‘Ferragnes’  ‘Tuono’, SC, medium-hard shell, medium vigor, late blooming, early–medium ripening, some double kernels ‘Ferragnes’  ‘Tuono’, SC, semi-hard shell, medium vigor, late blooming, early ripening, some double kernels ‘Ferralise’  ‘Tuono’, SC, late blooming, medium ripening, hard shell, medium to upright growth ‘Tuono’  local cross, SI, vigorous, early blooming, highly adapted to Israeli conditions, soft shell, large kernel, early ripening

2. ORIGIN AND DISSEMINATION OF ALMOND Table 2.3. Cultivar

57

(Continued). Description1

UNITED STATES University of California (Davis) Avalon Probably ‘Nonpareil’ OP, SI, medium kernel, early blooming, paper shell, harvest approx. 8 days after ‘Nonpareil’ Kahl Chance seedling in a ‘Nonpareil’, ‘Davey’, and ‘Mission’ planting, SI, mid- blooming, large kernel, semi-soft shell, harvest 14 days after ‘Nonpareil’ Morley ‘Mission’  late-blooming almond seedling, SI, late blooming, medium kernel, semi-soft shell Savanna ‘Nonpareil’  late-blooming almond seedling, SI, late blooming (2 weeks after ‘Nonpareil’), large kernel, semi-soft shell, harvest 14 days after ‘Nonpareil’ Sweetheart SB3,54–39E [{‘Lukens Honey’ peach  ‘Mission’}  ‘Nonpareil’]  Sel 25–26. SC, mid-blooming, large ‘Marcona’-type kernel, harvest approx. 10 d after ‘Nonpareil’, semi-soft shell, high kernel oil and roasting quality, resistant to postharvest worm damage Winters ‘3-1’ (‘Peerless’  ‘Harpereil’)  ‘6-27’ (‘Nonpareil’  ‘Jordanollo’), SI, early blooming, large Carmel-type kernel, paper shell, good bloom overlap with early ‘Nonpareil’ bloom, harvest 3 weeks after ‘Nonpareil’ 1

OP: open pollinated; SC: self-compatible; SI: self-incompatible.

specific to the region (Grasselly and Crossa-Raynaud 1980). Similar situations exist in other traditional almond-growing countries, such as Tunisia, Greece, and Canary Islands. The traditional almond production system of the Mediterranean and Asian regions, however, began to fail in the 1940s, and the French almond industry essentially went out of production by the 1950s (Grasselly and Crossa-Raynaud 1980). By the 1970s, production could not keep pace with the world demand for almond products because the most productive land was used for high-value crops such as peach and grape. Italy, historically the leading almond producer of the world, experienced a sharp decline in production (Federation Italiani 1973; Godini 1977; Bacarella 1993) and began to import almonds (Bacarella et al. 1991). Spain expanded its area and production but the scarcity of productive irrigated land has made production increases relatively modest (Abdelwahed and Albisu 1993; Murua 1993; Murua et al. 1993). In addition, supplemental honeybee pollination is not widely practiced (Felipe and Socias y Company 1992; Godini 1977b; Godini et al. 1992), significantly reducing final yield potential. Declines in production relative to other higher-value tree crops also took place in

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T. M. GRADZIEL

Table 2.4. Characteristics of the new rootstocks for almond from selected breeding programs. Rootstock

Description

SPAIN CITA de Arago´n (Zaragoza) Felinem ‘Garfi’ almond  ‘Nemared’ peach, red leaves, easy propagation, nematode resistant, good vigor, adapted to replanting and to poor and calcareous soils Garnem ‘Garfi’ almond  ‘Nemared’ peach, red leaves, easy propagation, nematode resistant, good vigor, adapted to replanting and to poor and calcareous soils Monegro ‘Garfi’ almond  ‘Nemared’ peach, red leaves, easy propagation, nematode resistant, good vigor, adapted to replanting and to poor and calcareous soils EE Aula Dei - CSIC (Zaragoza) Adafuel Natural hybrid selection (probably ’Marcona’ seedling), easy propagation, very vigorous, adapted to calcareous soils Adarcias Natural hybrid selection, easy propagation, low vigor, adapted to calcareous soils ITALY University of Pisa Sirio ‘INRA GF 557’ OP, low vigor, poor vegetative propagation, good root system UNITED STATES California Atlas Interspecific cross to Prunus blireiana, vigorous, upright Hansen 536 Almond  peach hybrid, vigorous, deep rooting, resistant to drought Nickels Almond  peach hybrid, vigorous, deep rooting, resistant to drought, soil fungi Marianna M40 P. cerasifera  P. munsoniana, improved anchorage, fewer suckers Viking Interspecies cross to P. blireiana, vigorous, upright, tolerant wet soils

the Asiatic regions of cultivation, where in many areas almonds continue to be grown under conditions similar to those used thousands of years ago. The genetic, horticultural, and culinary diversity that initially made these selections highly adapted to their production regions proved inconsistent with the new kernel ideotype of an increasingly global and so increasingly standardized market. Similarly, attempts to extend the range of cultivated almond into continental areas, such as Yugoslavia (Ristevski 1992), southern Russia (Richter 1972), Romania (Cociu 1985), Bulgaria (Serafimov 1975, 1976), and Hungary, have met with only partial success. This has been due to almond’s susceptibility to winter cold and spring frosts as well as flower and foliar disease, which is exacerbated by summer rains in these regions.

2. ORIGIN AND DISSEMINATION OF ALMOND

59

Fig. 2.16. Illustrations representing different heirloom almond varieties from the Genoa region of Italy.

VI. NEW WORLD DISSEMINATION Almond dissemination to the New World followed early colonization by European and Asian settlers, eventually resulting in commercial plantings in North and South America, Australia, and South Africa. Successful cultivation typically occurred only after a series of failures as early settlers, not recognizing the degree of almond’s vulnerability to winter cold, spring frosts, and summer rains, tested different growing regions and germplasm until suitable combinations were found. Almond’s introduction into California, which began as an extension of the Mediterranean culture utilizing a limited range of European germplasm (Wood 1925) is representative of the New World stage from 1850 to the present. California production, however, inevitably broke from the “traditional” methods of almond growing utilized in the rest of the world. Key adaptations included: (1) selection of specific vegetatively propagated cultivars and rootstocks to maximize production; (2) standardization of markets based on cultivar; (3) selection and optimization of growing sites; and (4) development of new cultural and management techniques, including increased mechanization, agrochemical inputs, and supplemental pollination. The impact of these changes has been to maximize yield and to promote modern industrial and marketing techniques (Kester et al. 1991; Micke 1994). Commercial cultivars introduced to California from the Languedoc area of southern France from 1850 to 1900 included ‘Princess’, ‘Languedoc’, ‘Gros Tendre’, ‘Sultana’,

60

T. M. GRADZIEL

and others that ultimately provided the germplasm from which the California almond industry evolved (Wood 1925). Originally these were grown primarily in solid plantings and considered to be “shy-bearing” (because almond is self-sterile and needs pollenizers), “nonadapted,” and “susceptible to frost and disease” (Chappelow 1893; Dargitz 1910; Wickson 1910). The eventual combination of highly adapted and rootstocks and multiple interplanted cultivars, the use of honeybee hives to maximize cross-pollination at flowering (Thorp and Roper 1994), favorable soil and climate, and abundant water and effective management has resulted in the highest productivity in the world and the domination of world markets (Kester et al. 1991; Micke 1994). Yields per hectare continues to show upward trends with yield as high as 4,500 kg/ha not unusual. An important initial step toward maximizing output was the (1900 to 1925) selection of four cross-compatible cultivars (‘Nonpareil’, ‘Mission’, ‘Ne Plus Ultra’, and ‘Peerless’) that established the basic industry cultivar pattern in the orchard and the marketplace. Shifts to more productive soil areas, changes in management,and the change to peach rootstocks brought about changes (1925 to 1955) that resulted in a shift from what was initially a subsistence enterprise to a major, dedicated agricultural industry. This change reflected the contributions from many sources, including research and extension from the University of California, Davis, a progressive nursery industry, innovative growers and industry leaders, and government policies that promoted the development of irrigation and marketing. In the period 1955 to 1965, the management patterns and world economic and marketing trends began to change and the industry entered an explosive growth period. Expansion occurred into all areas of the central valleys of California, increasing fivefold in area and tenfold in overall production. As a result, California production came to dominate world production (Fig. 2.17). Almond cultivars and cultural management methods were introduced to Australia, Chile, and Argentina from California and different Mediterranean areas in the early to mid- 19th century. In Australia, chance seedlings from this material resulted in selection of cultivars that have became more or less standard, such as ‘Chellastan’, ‘Johnston’, ‘White Brandis’, ‘Bruce’, and ‘Boxendale’ (Quinn 1928). More recently, the list has been supplemented by new introductions from California. South Africa grows limited almonds following introduction about the turn of the 20th century. The principal cultivars are from California, but a local selection, known as ‘Britz’, was important in establishing the early industry (Davis 1928).

2. ORIGIN AND DISSEMINATION OF ALMOND

California

627315.0

Spain Australia

67993.1 26535.0

Turkey

15512.8

Greece

14015.9

Italy

12020.1

Chile

6622.4

China

1632.9

India

997.9

Fig. 2.17. 2008.

61

Commercial almond production (million tonnes) in 2007–2008. Source: ABC

VII. GLOBAL COMMERCE A. Production Global commercial production was approximately 772,468 million tonnes (Mt) in 2007–2008. California accounted for approximately 80% with a 2008 production of 627,315 Mt (Fig. 2.17) from 261,076 ha (bearing). Spain, the second leading country, produced approximately 8% of world production but utilizes a cultivated area of over 436,500 ha (Murua et al. 1993). The remaining world production comes from about 20 countries including Australia (3%), Turkey (2%), Greece (2%), Italy (1.5%), Chile (1%), China (0.2%), and India (0.1%) (ABC 2008). Limited almond production for both local and export markets also occur in other areas of the Mediterranean, including Portugal, Morocco, Tunisia, Algeria; areas of the Balkan Peninsula, including Bulgaria, Romania, and Hungary; and central and southwestern Asia, including Syria, Iraq, Israel, Iran, Ukraine, Tajikistan, Uzbekistan, Afghanistan, and Pakistan. Almond growing in much of this area is in a relatively archaic state, although modern production areas exist in Portugal and in some parts of Ukraine and Iran. Despite falling production in most traditional almond producers, global production actually has more than doubled in the last

62

T. M. GRADZIEL

Almonds

$1,899

Wine

$736

Dairy

$604

Cotton

$554

Grapes

$499

Walnuts

$365

Oranges

$359

Pistachios

$287

Processing tomatoes

$286

Strawberries

$273 $0

$200 $400

$600 $800 $1,000 $1,200 $1,400 $1,600 $1,800 $2,000

Fig. 2.18. Value (in millions of US dollars) of major California export crops in 2006–2007. Source: ABC 2008.

20 years from a 1998 crop of 354,259.3 Mt. Most of the increase in production has occurred in California, where a fivefold increase in crop area combined with a more than doubling in yields per hectare resulted in an almost tenfold increase in production over the last 30 years. This combination of increased plantings coupled with increased cropping efficiency has made almonds the largest export crop for California (Fig. 2.18) and has resulted in expanded research on production efficiency as well as phytonutrient value and culinary uses of almond (ABC 2008). B. Consumption Consistent with its Mediterranean and Asian culinary origins, almond continues to show great versatility with its distinctive culinary heritage, as with marzipan paste (see Fig. 2.19) in addition to its more traditional role as a convenient and nutritious snack nut (Table 2.5). At 0.6 kg, the per capita consumption of almonds in the United States has increased by over 20% over the last four years. Similar increased demand has occurred in traditional European and Asian markets (Fig. 2.20). Expanding markets have increased the food and even industrial utilizations of

2. ORIGIN AND DISSEMINATION OF ALMOND

63

Fig. 2.19. Almond paste (left), which is commonly called marzipan but known by regional names throughout Europe, Asia, and North Africa, is a confection consisting primarily of sugar and almond meal. The incorporation of bitter almonds, which constitute approximately 5% of the total weight, gives it its distinctive flavor. It is often made into sweets or glazes for cakes, or used as a cake ingredient, as in st€ ollen. In addition, it can be consumed directly, in many regions after being shaped into small figures as a traditional treat for New Year’s Day (right).

almond (such as hand creams) well beyond its traditional use. As in the past, the perceived culinary and medicinal value of almonds remains a driving force for market expansion. Recent medical studies have documented health benefits from almond consumption in a range of areas, including protection from cancer, obesity, and heart disease (see recent review by Socias y Company et al. 2007). The widespread availability and low cost of almonds relative to other nut crops have also encouraged expanded use (Fig. 2.21), either in combination or as replacements for other nuts in confectioneries, cereals, and baked goods. A highly developed international marketing system has emerged to distribute the billion-dollar-per-year almond crop (Alston et al. 1993; ABC 2008). In addition to ensuring product quality and availability, these advanced marketing systems are also important in ensuring food safety, particularly the prevention of product contamination by fungal toxins such as aflatoxin or microbial contamination as with Salmonella spp. (IPM 1995). Global market requirements for product consistency, however, have moved beyond food safety and food quality concerns. The increasing sophistication of culinary and food service uses are progressively demanding more standardized kernel type for industrialized handling, as in slicing and slivering (Table 2.5). Even in the making of food products emphasizing the natural amaretto almond flavor, processors often prefer using a more consistently bland cultivar as the basic nut to which they can add either limited bitter nuts or almond extract as needed. In California, ‘Nonpareil’ (Fig. 2.22) has become the cultivar

64

Table 2.5.

T. M. GRADZIEL

Commercial almond products and applications.

Common products

Benefits

Applications

Natural almonds, whole and whole & broken

Provides color contrast to lighter foods; adds visual appeal nutritional value and texture; stronger flavor than blanched products Increases almond recognition; adds flavor, visual appeal, and nutritional value; provides texture contrast Adds almond flavor and characteristics, nutritional value, and visual appeal Complementary flavor, high quality, garnishing, visual contrast and nutritional value

Roasting, flavoring, snack foods; complementary ingredients for confectionery, cake, bread, cookies and cooking, etc. Cake, bread and cooking garnish; cereal additive ingredients.

Natural or blanched sliced almonds Natural or blanched diced almonds Blanched almonds, whole & broken

Blanched slivered almonds

Adds crunchy, complementary flavor, and nutritional value

Natural or blanched almond meal

Adds color, flavor, richness and nutritional value; fat replacement and binding agent Strengthen flavor and color and nutritional value

Roasted almonds

Oil

Hull

Stability, low rancidity, high oleic acid content and nutritional value High carbohydrates and nutrients, adds flavor

Cake and confectionery fillings; additive ingredients for cooking Ingredients for mixed dried fruits and nuts retail packing, and blanched manufactured products; cookie and cake garnish Ingredients for cake, cookie, bread, snack, and cereals; additive ingredients for cooking Cake and confectionery fillings; ingredients for fortified breads and cereals Fillings and garnish for dairy products (e.g., ice cream); ingredients for energy and chocolate bars. Base for cosmetics, ointments, skin creams, and other pharmaceuticals Dairy cattle feed

most widely planted, partly because it satisfies most of these market needs for consistency combined with a relatively long and elliptical kernel, which further facilitates its processing. Approximately 37% of the current California crop area is planted to ‘Nonpareil’ with an additional 15% planted to do similar market type ‘Carmel’. The remaining cultivars are primarily planted as pollenizers to ensure high fruit set. Thus, the anticipated introduction of self-fruitful ‘Nonpareil’-type cultivars in the next decades would further and probably dramatically

2. ORIGIN AND DISSEMINATION OF ALMOND

Eastern Europe 4% Canada/Mexico 6%

65

Other 3%

Middle East 10%

Asia 23%

Fig. 2.20.

Western Europe 54%

California export markets in 2007–2008.

Hazelnuts 1%

All Others 1%

Pecans 9% Pistachios 10%

Walnuts 13%

Almonds 66%

Fig. 2.21. World commercial use of various tree nuts by proportion (2007–2008).

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T. M. GRADZIEL

Fig. 2.22. ‘Nonpareil’-type paper shell and uniform, amygdaloidal kernel, which is becoming the market standard in world commerce.

promote greater crop uniformity and compatibility with expanding international markets. These global market pressures toward more uniformity in the almond crop would consequently lead to continued and perhaps accelerated loss of native genetic diversity and thus genetic options against emerging pests and diseases. Considerable losses in germplasm have already been documented in traditional Mediterranean and Asian growing areas as new market-orientated cultivars are brought in to replace the traditional, locally adapted cultivars and landraces established over hundreds to thousands of years.

VIII. EVOLVING REQUISITES FOR COMMERCIAL ALMOND Recognizing the genetic, environmental, and management deficiencies of the traditional almond industries, most almond-producing countries are pursuing almond research with emphasis on germplasm evaluation and cultivar and rootstock improvement. These activities followed a pattern that included: (1) surveys of local cultivars and seedling populations within the traditional almond growing areas; (2) establishment of

2. ORIGIN AND DISSEMINATION OF ALMOND

67

cultivar collections and test planting to evaluate local and introduced almond cultivars; (3) establishment of germplasm collections for maintenance of local and introduced cultivated almond as well as related species; and (4) development of controlled breeding methods to incorporate desirable new traits into locally adapted material and so generate new cultivars and rootstocks to optimize regional performance. Some traits, such as self-fruitfulness and certain disease resistances, were not readily available in cultivated almond and required introgression from related species. Crosses of Prunus dulcis with other almond species in Sections Euamygdalus and Spartiodes are generally easily accomplished (Denisov 1982, 1988, 1989; Kester et al. 1991; Gradziel et al. 2001). Hybridization with Section Lycioides is somewhat more difficult and even more so with Chameamydalus and Leptopus (Denisov et al. 1983; Denisov 1988; Chepinoga 1990). Crosses with peach (Prunus persica) can be made easily, although some types of sterility may occur in the progeny (Ryabov 1969; Kester and Gradziel 1996). Crosses with plum are possible but difficult (Grasselly et al. 1992). Crosses with apricot are very difficult but have been reported (Jones 1968). The extent of new germplasm incorporation, as well as general breeding approach, however, varies by region (Table 2.6). A. Mediterranean Modern cultivar improvement had its start with the beginning of cultivar collections at Bordeaux in 1951 and in 1961 at Nimes under the direction of Dr. Charles Grasselly (1972). A survey of the germplasm potential throughout the Mediterranean region was followed by collecting trips to Iran, the Soviet Union and Afghanistan (Grasselly and Crossa-Raynaud 1980), and 450 accessions from 10 different countries were collected, evaluated and described. This study provided the basis of the concept of local ecotypes in different almond-growing districts (CrossaRaynaud 1977, 1981), and the collection is the base of an almond Germplasm Repository at Montfavet, France. Controlled crosses begun in 1961 in France produced valuable inheritance data (Grasselly 1972) and resulted in new cultivars and rootstocks. Initial objectives were to combine late bloom, high production, and improved nut and kernel quality. The first releases (Grasselly 1975) from this program, ‘Ferragnes’ and ‘Ferraduel’, were cross-compatible and when planted in combination shifted the blooming date approximately 2 weeks later than prevailing cultivars. This combination quickly became the basis for new orchard plantings in France and the rest of the Mediterranean. These cultivars arose from crossing representatives of two ecotypes,

68

Table 2.6.

T. M. GRADZIEL

Objectives of modern almond improvement programs.

Problem

Trait

Objective

Country

Spring frosts

Late bloom; blossom resistance

Avoid early and late spring frosts

Winter freezing

Hardy buds and wood

Low winter chilling

Low chilling requirements

Avoid loss of dormant flower buds; avoid tree damage Grow in subtropical area

Moisture stress

Drought tolerance

USA, Ukraine, France, Greece, Spain, Italy, Romania, Turkey, Bulgaria Ukraine, Romania, Bulgaria Israel, Tunisia, Australia, Morocco Spain, Italy

Lack of or reduced bee populations Disease and pests

Self-fertility

High management costs Difficult harvest and handling

Resistance to fungus and bacterial diseases, and insects Virus and viruslike organisms Modified tree size, shape, branching, growth habit Optimize time of maturity, ease and completeness of nut removal, hulling Shell character—very hard shell —semi-soft to soft

Inconsistent yields

—well sealed Improve tree: precocity, productivity, regularity of bearing Nut quality

Grow with deficient irrigation Eliminate or reduce need for bee pollinizers Eliminate or reduce need for chemical sprays “Clean” propagation sources Efficient orchard management; adjust orchard density; pruning; shaking Extend harvest period; efficient and complete harvest Kernel protection and storage; prevent worm infestation Higher shelling percentages Kernel protection Early production; high yield, no alternate bearing Increased kernel yield and reduced damage

France, Greece, Tunisia, Italy, Spain, USA USA, France, Spain, Italy, Turkey USA, Spain, Italy USA, Spain

All programs

Spain, France, Italy USA, Ukraine, France USA, all programs All programs

All programs

2. ORIGIN AND DISSEMINATION OF ALMOND

69

‘Cristomorto’, an Italian cultivar from Puglia, which was late blooming due to a high chilling requirement for bloom, and ‘Ai’, a cultivar from France that was late blooming due to a high heat requirement. Later disease resistance from the French cultivars ‘Ardechoise’ and ‘Mandaline’ were incorporated into the program as well as late bloom from ‘Tardy Nonpareil’ (Grasselly 1978; Duval 1999). Almond improvement programs were developed at Zaragoza, Spain, by A. Felipe (Felipe and Socias i Company 1977a, 1985, 1992); at Reus, Spain, by F. VargasGarcia (1975a,b), Barraquer and Vargas-Garcia (1975), and Vargas and Romero (1993); and at Murcia, Spain, by Egea and Garcia (1975), Garcia et al. (1988), and Dicenta et al. (1993). Genetic improvement programs were initiated in Tunisia by Jaquani (1976) and El Gharbi (1977) and in 1970 at Rome, Italy, by F. Monastra (Monastra and Fideghelli, 1977; Monastra et al. 1982, 1985). Almond improvement programs were initiated in Greece in 1960 by D. Stylianides (1977). Israel began a crossing program in 1966 that involved early bloom, low chilling, and improved nut and kernel quality (Spiegel-Roy and Kochba 1977). Parental material included ‘Marcona’ crossed with local cultivars ‘Greek’ and ‘Poria’ (Spiegel-Roy 1985). Rootstock surveys of local seedling populations resulted in discovery of nematode-resistant almond clones, known as the ‘Alnem’ series (Kochba and SpiegelRoy 1975). Turkey began a program of selection among seedling populations for late bloom and frost resistance for interior Turkey (Ayfer 1975) and in southwest Turkey for late bloom, adaptation, and improved quality (Dokuzogus and Gulcan 1973, 1977; Dokuzogus 1975; Gulcan 1977). Crossing programs were begun in 1990 (Gulcan et al. 1992). Surveys of local germplasm also occurred in Turkey (Dokuzogus 1975; Kuden et al., 1993), Sicily (Barbera et al. 1988), and Sardinia (Agabbio et al. 1984; Chessa and Pala 1985). Evaluations of unique North African germplasm in the local seedling populations were carried out in Morocco (Barbeau and El Baudami 1977; Laghezali 1985; Loussart et al. 1989). These populations are considered to have good potential for germplasm exploration (Lansari et al. 1994). In virtually all of these areas, destruction of many local groves and so loss of traditional germplasm has already occurred. The Groupe de Recherche et d’Etude Mediterraneen Pour l’Amandier (GREMPA) was formed in 1974. This group organized almond researchers into an informal association whose purposes were to provide a forum to exchange information and plant materials and to coordinate breeding and testing efforts (Crossa-Raynaud 1975; Socias i Company 1998).

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B. California Beginning in the mid-1950s, the major source of new cultivars was the population of almond seedlings found throughout California either along roadsides and near commercial orchards or in orchards from unbudded almond seedling rootstock “escapes.” Since 1957, many selections from this source were patented and introduced by individual growers as commercial cultivars through commercial nurseries (Brooks and Olmo 1997). Most, including ‘Merced’, ‘Price’, ‘Carmel’, and ‘Fritz’, were used to provide cross-pollination to ‘Nonpareil’. Others, such as ‘Thompson’ and ‘Livingston’, were later blooming and were combined with ‘Texas’. A cooperative breeding program initiated in 1923 at Davis, California, between the U.S. Department of Agriculture (USDA) and the University of California was an outgrowth of early pollination and variety evaluation studies (Wood 1938). This program was separated in 1948. The USDA program was continued until 1975. The University of California program has continued and resulted in the release of a number of cultivars and rootstocks (Kester et al. 1996).

C. Eastern Europe and Asia After California, the second oldest continuous breeding program for almond has been at the Nikitski Botanical Garden at Yalta, Crimea, in the former Soviet Union (now Ukraine) under the direction of A.A. Richter (Richter 1969, 1972) and Yadrov (1993). These began with the research of N. Vavilov (1930) and were based on extensive species and cultivar collections. Other programs have been in progress in the Asiatic republics (Denisov 1988). The primary objectives have been to develop hardiness to winter cold and spring frosts and to investigate the breeding potential of wild almond species. Hardy, late-blooming cultivars were introduced for commercial production in the southern Soviet Union during the 1950s (Denisov, 1988), into Bulgaria in 1956 (Serafimoov 1975, 1976), and later into other Mediterranean countries (Guerriero et al. 1974; Grasselly and CrossaRaynaud 1980; Garcia et al. 1988; Vargas and Romero 1988). Additional almond cultivars have been introduced by Yadrov in a continuation of this program. Research has been carried out by other Asiatic republics including Turkmenistan, Tadjististan (Mizgireva 1973), and Uzbekistan (Komarov et al. 1941). This research involves populations of the Kopet Dagh populations of P. communis (Denisov 1977a,b; Saparov 1978) and many wild species (Denisov 1982; Eremin and Denisov 1984; Chepinoga 1990).

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In Romania, selection of local cultivars has resulted in improved commercial plantings, and a breeding program has introduced new cultivars (Cociu 1981). The foothill districts of northern India, including the Kashmir, provinces of Jammu and Himachal Pradesh (Uppal 1977a,b; Singh et al. 1977; Singh and Uppal 1977) as well as northeastern Pakistan (Thompson et al. 1990) have historically had many seedling almond orchards. Studies of promising seedlings and introduced almond cultivars have been undertaken (Kumar et al. 1989; Kumar and Uppal 1990). Several almond species and commercial cultivars exist in the Xinjiang Province of western China (Gustafson et al. 1989) near the ancient cities of Kashgar and Sache on the ancient Silk Route. A program of almond improvement has been carried out by Professor Zhu Jing Lin at the Xinjiang Academy of Forestry since 1970 with the objectives of cold hardiness and dwarfing rootstocks.

IX. CONCLUSIONS The wide dispersion of almond and its wild relatives in the often severe environments from Central Asia to the Mediterranean was possible largely because of the genetic and associated developmental/physiological diversity promoted by this typically self-sterile yet interspecies fruitful genus. Even today, evidence of species mixing of the traditional Asian badam is evident in many rural bazaars by the presence of shell patterning characteristic of other species (Fig. 2.2). The cultivated Greek nut, which appears to have gained wide prominence in prehistoric Greco-Persian commerce, may well have originated from an interspecies cross with P. fenzliana and owes its existence and eventual commercial ascendancy to human selection. Gene transfer between wild and cultivated almond continued to be important in the early dissemination of this crop, as evidenced in the transfer of self-compatibility from wild P. webbii to the old cultivated landraces in southern Italy. ‘Tuono’, an old Italian cultivar selected from these landraces, is currently the most important source for self-compatibility in Mediterranean and Asian breeding programs (Table 2.3). However, despite its historical wide acceptance throughout the Mediterranean, ‘Tuono’ and many of the regional heirloom cultivars are largely being replaced by new orchards designed to maximize production (Fig. 2.23) of a more globally standardized market quality. Regionally adapted (to both local culture and cultivation) cultivars are also being lost as traditional plantings are converted to other crops or to other consequences of modern

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Fig. 2.23. High-input, high-yield monoculture cropping system typical of 2007 California production.

development. Similar “progress” is leading to the loss of native germplasm throughout the Asian and Mediterranean centers of origin and diversity (Ledig 1992). The fragile nature of many of these wild almond ecosystems (e.g., the dependency on a minimum spring snowmelt for summer drought survival) make them particularly vulnerable to a range of environmental perturbations from human enterprises to global warming. Concurrent with and contributing to germplasm loss is the rapid expansion of almond cultivation to satisfy an escalating global demand. The extensive commodity standardization associated with these complex, international markets has been largely achieved by dependence on a few standard cultivar types, as with ‘Nonpareil’/‘Carmel’ type in California (Fig. 2.22), which is also increasingly grown in many international plantings. And with a 2007–2008 California production of over 225,000 Mt, the 5% substandard-size nuts that might normally be considered rejects can effectively compete with markets normally utilizing smaller, lower-value nuts. Even when found to be poorly adapted locally,

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these market standards often are used as breeding parents to combine desired market quality with local adaptability (Tables 2.3 and 2.6). For example, virtually all of the other commercially important California cultivars are progeny of ‘Nonpareil’ by ‘Mission’ crosses (Kester et al. 1991, 1996), with the few exceptions being cultivars developed by university programs (Table 2.3). Recent history has demonstrated the dangers of too great a dependence on a limited germplasm, including significant economic losses from disease, pest, and climate change (Tanksley and McCouch 1997). A similar danger also exists in the loss of the extensive medical and culinary legacy of the current diversity of almond germplasm at a time when we are just beginning to understand their unique contributions. Inevitably, the modern marketing folklore that uniformity is good may be found to be myth.

ACKNOWLEDGMENTS The author gratefully acknowledges the helpful review and editorial assistance of Federico Dicenta, Jules Janick, Pedro Mart
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Murua, J.R. 1994. Yield model for Spanish almonds. Acta Hort. 373:325–332. Murua, J.R., H. Carman, and J. Alston. 1993. California leads Spain in almond production, exports to world. Calif. Agr. 47(6):11–14. Negri, P., D. Bassi, E. Magnanini, M. Rizzo, and F. Bartolozzi. 2008. Bitterness inheritance in apricot (P. armeniaca L) seeds. Tree Genet Genomes 4:767–776. Peregrine, P.N., and E. Melvin. 2003. Encyclopedia of prehistory. Vol. 8: South and southwest Asia. Princeton Univ. Press, Princeton, NJ. Pravosudova V.V., and S.R. de Kortb 2006. Is the Western Scrub-Jay (Aphelocoma californica) really an underdog among food-caching corvids when it comes to hippocampal volume and food caching propensity? Brain Behav. Evol. 67:1–9. Quinn, N.R. 1928. Almond culture in south Australia. Dept. of Agr. South Australia Bul. 367. Rawlinson, G. 1917. The history, geography and antiquities of Chaldaea, Assyria, Babylon, Media, Persia, Parthia, and Sassanian or New Persian Empire. Vol. 3. Oxford Univ. Press, Oxford. Richter, A. A. 1972. L’Amandier. Academie les Sciences Agricoles. Jard. Bot. de Nikitski, Yalta. (Quoted by Grasselly and Crossa-Raynaud, 1980.) Ristevski, B. 1992. Damages on almond trees from late spring frosts in Macedonia. In: Amelioration genetiques de deux especes de fruits secs mediterraneens: l’amandier et le pistachier. 8th Colloque, Nimes, France 1990. Com. de Com. Euro. Brussels. p. 137–138. Rosengarten, F. Jr. 1984. The book of edible nuts. Walker and Co., New York. Ryabov, I.N. 1969. Experiments on the self-pollination of some interspecific hybrids of common peach and common almond with Persica mira (Koehne) Kost. et Kov. (In Russian, trans. by R. Socias i Company, 1974). Byull. Gos. Nikit. Bot. Sad. 3(10):24–28. S
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Wood, M.N. 1938. Two new varieties of almond: The Jordanolo and Harpareil. USDA Cir. 542:1–12. Yadrov, A. 1994. Main results of research on almond. Acta Hort. 373:75–82. Zeinalabedini M., V. Grigorian, M. Torchi, M. Khayam-Nekoui, K. Majourhat, F. Dicenta, and P. Mart
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3 Factors Affecting Extra-Virgin Olive Oil Composition Paolo Inglese Dipartimento di Colture Arboree Universit
a degli Studi di Palermo Viale delle Scienze 90128, Palermo, Italy Franco Famiani Dipartimento di Scienze Agrarie e Ambientali Universit
a degli Studi di Perugia Borgo XX Giugno 06121, Perugia, Italy Fabio Galvano Dipartimento di Chimica Biologica Chimica Medica e Biologia Molecolare Universit
a degli Studi di Catania 95125, Catania, Italy Maurizio Servili, Sonia Esposto, and Stefania Urbani Dipartimento di Scienze Economico-estimative e degli Alimenti Sezione di Tecnologie e Biotecnologie degli Alimenti Universit
a degli Studi di Perugia 06121, Perugia, Italy

ABBREVIATIONS I. THE CONCEPT OF OLIVE OIL QUALITY II. EVOO COMPOSITION AND NUTRITIONAL PROPERTIES III. SOURCES OF VARIABILITY OF EVOO COMPOSITION AND PROPERTIES

Horticultural Reviews, Volume 38 Edited by Jules Janick Copyright
2011 Wiley-Blackwell. 83

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IV. AGRONOMICAL AND ENVIRONMENTAL FACTORS AFFECTING EVOO COMPOSITION AND QUALITY A. Genotype B. Growing Area and Seasonal Conditions C. Tree Water Status D. Productivity and Alternate Bearing E. Orchard Management 1. Cultivation Method 2. Training System and Pruning 3. Fertilization and Soil Management 4. Pest and Disease Control F. Fruit Ripening and Harvest 1. Ripening 2. Harvest Time and Production Objectives 3. Harvesting Systems V. TECHNOLOGICAL FACTORS AFFECTING EVOO COMPOSITION AND QUALITY A. Olive Fruit Storage B. Olive Fruit Crushing C. Olive Paste Malaxation D. EVOO Extraction Systems E. EVOO Storage VI. SUMMARY AND CONCLUSIONS LITERATURE CITED

ABBREVIATIONS CVD EVOO HDL LDLA LOX MUFA VOO PDO POD PPO

cardiovascular disease extra-virgin olive oil high-density lipoprotein low-density lipoprotein lypoxygenase monounsaturated fatty acid virgin olive oil protected designation of origin peroxidase polyphenyloxidase

Uva pendet in vitibus, et oliva in arboribus. . . et nec uva vinum est, nec oliva oleum, ante pressuram. [Grape droops in grapevine and olives in the olive tree. . . but neither grapes are wine, nor olives are oil, until they are pressed.] —Agostino Enarrationes in Psamos 83, 1, 22, 16–20

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I. THE CONCEPT OF OLIVE OIL QUALITY The oil of the fruit of the olive (Olea europaea L.) is the cornerstone of the Mediterranean diet (Keys 1980). This product represents a heritage at the very center of the civilization, history, religion, and economy of all countries surrounding the Mediterranean Sea, which Braudel (1986) referred to as the “sea of the olive trees.” In antiquity, the principal use of olive oil was for illumination. Although oil lamps are no longer used, olive oil is still important for nonfood uses, such as cosmetics and body care. However, as a result of the extraordinary nutraceutical properties and alimentary value of extra-virgin olive oil (Keys 1980; Bendini et al. 2007), consumption and trading is increasing worldwide, even in countries with relatively low production, such as Argentina, Australia, Chile, New Zealand, South Africa, and the United States. According to the international regulations and trade standards of the International Olive Council (COI/T.15/NC no. 3/Rev. 3, Nov. 2008), olive oil is the oil obtained solely from the fruit of the olive tree, to the exclusion of oils obtained using solvents or re-esterification processes, and of any mixture with oils of other kinds. Before pressing the olives to extract the oil, the Romans, upon a very slight pressure, obtained from the fruit a liquid of watery consistency dark in color, bitter to the taste, called amurca (amo´rgh by the Greeks), which was used as a manure as well as for various purposes in the domestic and agricultural economy. Nowadays, among other designations, we can distinguish extra-virgin olive oil and virgin olive oil. The extra-virgin olive oil (EVOO), which is a marketable class of oil extracted from the olive fruit using only mechanical extraction process, can be consumed directly as crude oil, without any additional physical or chemical treatments, other than washing, decantation, centrifugation, and filtration. EVOO must be relatively low in free acidity (lower that 0.8% expressed in oleic acid), with low peroxide number (lower that 20 meq O2/kg). Virgin olive oil (VOO) is a second marketable class of olive oil extracted using the same mechanical process but characterized by higher free acidity (between 0.8% and 2.0%). Up to now, the marketable quality of EVOO has not included parameters that are important in determining the health and sensory characteristics of the oil. In fact, several markers, such as phenolic and tocopherols composition, are not considered to define the EVOO marketable class. In the last few decades, many aspects related to EVOO organoleptic properties and nutritional quality have been intensely investigated, with the ultimate goal of distinguishing the product and increasing its commercial value. EVOO composition and properties greatly change with the peculiar characteristics of the different geno-

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types (cultivar) and their interaction with environmental conditions, orchard management, and oil extraction technologies. As a result of this differentiation of EVOO quality, the awareness and the preferences of new and traditional consumers are rapidly changing in relation to its different uses in cooking and gastronomy. At the present time, EVOO is perceived as a functional food rather than as a simple dressing for salads or cooking oil. Different reviews have been published on the flavor and volatile components of olive oil (Kiritsakis 1998; Kalua et al., 2007) as well as on its functional properties (Newmark 1997; Fito et al. 2007) and on the relation between olive oil extraction methods and its final quality (Uceda et al. 2006). This chapter presents and discusses the sources of variability of olive oil composition and quality, such as genotype, environment, cultural, and technological factors, with particular emphasis given to aspects that may play an important role in the further development of the olive oil industry.

II. EVOO COMPOSITION AND NUTRITIONAL PROPERTIES EVOO is the main source of edible lipids and one of the staple foods of the Mediterranean diet. In comparison to other vegetable oils, EVOO must be considered a unique dietary food due to its fatty acid composition, such as the prevalent presence of the monounsaturated oleic acid (C18:1) and its hydrophilic compound content, such as phenolic alcohols and acids, flavonoids, lignans, and secoiridoids. Evidence from an increasing number of scientific studies attribute the health properties of EVOO to its peculiar fatty acid composition, which confers consistent beneficial effects on human health, particularly in the prevention of cardiovascular diseases (Keys 1980). The fatty acid fraction accounts for not less than 98% of the oil components and is characterized by a relative low level of polyunsaturated fatty acids and a high level of monounsaturated fatty acids (MUFA). Triglycerides account for 98% to 99% of total fatty acid composition, with diglycerides accounting for 1% to 1.5% and monoglycerides less than 1%. Almost 99% of the fatty acid fraction is composed of: saturated palmitic (C16:0; 7.5–20%) and stearic fatty acids (C18:0; 0.5–5.0%); monounsaturated palmitoleic (C16:1; 0.3–3.5%) and oleic fatty acids (C18:1; 55.0–83.0%); and polyunsaturated linoleic (C18:2; 3.5–21.0%) and linolenic fatty acids (C18:3; 0–1.0%) that are not synthesized by humans (Montedoro et al. 2003). Total MUFA content of EVOO is 70–80 g/100 g, a value much higher than those of vegetable oils, such as canola (59 g/100 g), peanut (46 g/100 g), sunflower (32 g/100 g), corn (29 g/ 100 g), soybean (24 g/100 g), and safflower (14 g/100 g) (Nicklas et al. 2004).

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In contrast to all other vegetable oils, which are obtained from seeds, EVOO is obtained from a fruit (drupe). EVOO contains a pool of compounds that although quantitatively of minor importance (about 2% of EVOO’s weight) show important biological properties. Indeed, EVOO contains more than 230 chemical compounds, including the triterpene hydrocarbon squalene, the phytosterol b-sitosterol, and a pool of antioxidant polyphenols such as tyrosol, hydroxytyrosol, secoiridoids, and lignans that importantly contribute to its distinctive flavor due to the typical pungent and bitter taste properties but also to its chemical stability in terms of both shelf life and resistance to lipid oxidation during cooking (Servili et al. 2009a). Phenols of EVOO belong to different chemical classes, such as phenolic acids, phenolic alcohols, flavonoids, secoiridoids, and lignans. Secoiridoids, which include aglycon derivatives from oleuropein, demethyloleuropein, and ligstroside, found exclusively in the Oleaceae, are the most abundant phenolic antioxidants of EVOO (Servili et al. 2004). The concentration of secoiridoides and lignans in EVOO is highly variable as it largely depends on agronomic factors, fruit ripening stage at harvest, and oil extraction techniques. Eventually, the total phenols concentration of an EVOO may range from 20 to 900 mg kg
1 (Montedoro e Garofolo 1984; Servili et al. 2007a,b). The natural antioxidants of EVOO are lipophilic and hydrophilic polyphenols and carotenes (Boskou 1996). Carotenes, which include lutein as the main component, violaxanthin, and b-carotene, can be found in small amounts in EVOO, with concentrations, expressed as total carotenes, between 5 and 24 mg kg
1 (Servili et al. 2004). The lipophilic phenols including tocopherols and tocotrienols that do not occur in EVOO can be found in other vegetable oils. In EVOO, more than 90% of total tocopherols are represented by a-tocopherol, which shows high variation according to soil and climatic conditions and agronomic factors, such as area of origin, cultivar, and fruit ripening stage (Servili et al. 2009b). EVOO contains different classes of hydrophilic phenols (Table 3.1, Figs. 3.1, 3.2) (Boskou 1996; Shahidi 1996). Phenolic acids, represented by caffeic, vanillic, syringic, p-coumaric, o-coumaric, protocatechuic, sinapic, p-hydroxybenzoic, and gallic acid, were the first group of phenols discovered in EVOO (Montedoro et al. 1992a; Tsimidou et al. 1996; Servili et al. 2004). Phenolic alcohols include (3,4-dihydroxyphenyl) ethanol (3,4-DHPEA) and (p-hydroxyphenyl) ethanol (pHPEA). Their concentration is generally low in fresh oils but increases during oil storage (Montedoro et al. 1992a) as a result of the hydrolysis of EVOO secoiridoids, such as 3,4-DHPEA-EDA, p-HPEA-EDA, and 3,4DHPEA-EA, which contain 3,4-DHPEA and p-HPEA in their molecular structures (Fig. 3.1) (Brenes et al. 2001). Flavonoids, such as luteolin

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Table 3.1.

P. INGLESE ET AL.

Phenolic composition of an olive fruit.

Anthocyanins Cyanidin-3-glucoside Cyanidin-3-rutinoside Cyanidin-3-caffeyglucoside Cyanidin-3-caffeylrutinoside Delphinidin 3-rhamosylglucoside-7-xyloside Flavonols Quercetin-3-rutinoside Flavones Luteolin-7-glucoside Luteolin-5-glucoside Apigenin-7-glucoside Phenolic acids Clorogenic acid Caffeic acid p-Hidroxybenzoic acid Protocatechuic acid Vanillic acid Syringic acid p-Cumaric acid o-Cumaric acid Ferulic acid Sinapic acid Benzoic acid Cinnamic acid Gallic acid

Phenolic alcohols (3,4 Dihydroxiphenil) ethanol (3,4-DHPEA) (p-Hydroxyphenyl) ethanol (p-HPEA) Secoiridoids Oleuropein Demethyloleuropein Ligstroside N€ uzhenide

Lignans ( þ )-1-Acetoxypinoresinol ( þ )-Pinoresinol Hydroxycinnamic acid derivatives Verbascoside

Source: Servili et al. 2004, 2009a.

and apigenin, were also reported as phenolic components of EVOO (Rovellini et al. 1997). The lignans include ( þ )-1-acetoxypinoresinol and ( þ )-1-pinoresinol (Fig. 3.2) (Brenes et al. 2000; Owen et al. 2000). Technological parameters of oil extraction process have a marginal impact on their concentration (Servili et al. 2004). Other phenolic compounds, including oleuropein glucoside (with a range of 5–60 mg kg
1), were found in EVOO as minor components. Secoiridoids are the main compounds of EVOO and include the dialdehydic form of decarboxymethyl elenolic acid linked to 3,4-DHPEA or p-HPEA (3,4-DHPEA-EDA or p-HPEA-EDA), an isomer of oleuropein aglycon (3,4-DHPEA-EA), and the ligstroside aglycon (p-HPEA-EA) (Montedoro et al. 1992a,b, 1993; Angerosa et al. 1996a; Owen et al. 2000) (Fig. 3.1). These substances, aglycon derivatives of secoiridoid glucosides contained in the olive fruit, originate during oil mechanical extraction

3. FACTORS AFFECTING EXTRA-VIRGIN OLIVE OIL COMPOSITION 2' 2' 3'

6' O

HO 4' 5'

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10

8'

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5'

4

6

7'

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DIALDIALDEHYDIC FORM OF DECARBOXYMETHYL ELENOLICACID LINKED TO p-HPEA (p-HPEA-EDA) = OLEOCHANTAL

LIGSTROSIDE AGLYCON (p-HPEA-EA)

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OLEUROPEIN AGLYCON (3,4-DHPEA-EA)

Fig. 3.1. Chemical structures of secoiridoids derivatives and phenyl alcohols of EVOO. Source: Servili et al. 2004, 2009a.

OH

OH 5'

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Fig. 3.2. Chemical structures of lignans found in olives and in EVOO. Source: Servili et al. 2004, 2009a.

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process by the enzymatic hydrolysis of oleuropein, demethyloleuropein, and ligstroside, catalyzed by the endogenous b-glucosidase (Montedoro et al. 2002; Gonz
alez-Pombo et al. 2008). High amount of phenols also can be found in olive fruit, mainly in the pulp, where the total phenols can range between 1% and 3% of fresh pulp weight (Garrido et al. 1997). Oleuropein, demethyloleuropein, ligstroside, and n€ uzhenide are the most abundant secoiridoid glucosides in the olive fruit (Gariboldi et al. 1986; Garrido et al. 1997; Servili et al. 1999b) (Table 3.1), which also contains, as main phenolic compounds, the verbascoside in two isomeric forms (Ryan and Robards 1998; Servili et al. 1999a). Sensory properties of EVOO are largely affected by phenolic content, which is responsible for its “bitter” and “pungent” flavor (Morales et al. 2000). Volatile compounds are responsible for EVOO aroma and flavor. More than 180 volatile compounds have been described in EVOO (Angerosa et al. 2004). It has been demonstrated that the relationship between the “fruitiness” note in olive oil and the presence of aldehydes and C5–C6 saturated and unsaturated alcohols originate from the enzymes involved in the lipoxygenase pathway during olive crushing and following malaxation (slowly mixing of the olive paste after crushing) (Morales et al. 1999; Angerosa et al. 2004). The use of the statistical sensory wheel as an appropriate method to relate volatile compounds and sensory data was clearly demonstrated, and the aroma notes of 32 virgin oil samples from three Mediterranean countries corresponded well to olive oil volatile compounds (Aparicio et al. 1996). The volatile fraction of EVOO is mainly composed by carbonyl compounds, alcohols, esters, and hydrocarbons (Flath et al. 1973; Angerosa 2000). However, the typical aroma of an EVOO rises from several volatile compounds, which are responsible for fragrances described by these attributes: “fruity,” “cut grass,” “tomato leaf,” “tomato,” “artichoke,” “walnut husk,” “apple,” or other fruits. The C6 and C5 substances, especially C6 linear unsaturated and saturated aldheydes and alcohols, represent the most important fraction of the volatile compounds that were associated to several EVOO sensory notes, such as “fruity,” “cut grass,” and “tomato leaf” (Angerosa et al. 2004; Servili et al. 2009b). From a quantitative point of view, C6 and C5 compounds (Vick and Zimmermann 1987; Hatanaka 1993; Angerosa et al. 1998a; Aparicio and Morales 1998), in particular C6 linear unsaturated and saturated aldehydes, are the most important volatile substances of high-quality EVOOs, whereas other neo-formation volatile compounds, namely C7–C11 monounsaturated aldehydes (Solinas et al. 1987, 1988), C6–C10 dienals (Aparicio et al. 2000), C5 branched aldehydes and alcohols (Angerosa et al. 1996b), and some C8 ketones (Angerosa et al. 1999a), reach high concentrations in the aroma of EVOOs affected by organoleptic defects.

3. FACTORS AFFECTING EXTRA-VIRGIN OLIVE OIL COMPOSITION

ADH

LA

hexanal LOX

91

AAT hexan-1-ol

hexyl acetate

HPL 13-hydroperoxides

Isomerase

ADH (E)-2-hexenal

(E)-2-hexen-1-ol

(Z)-3-hexenal

LnA

ADH

AAT (Z)-3-hexen-1-ol

13-alkoxy radical

(Z)-3-hexenyl acetate

pentene radical

pentene dimers

2-penten-1-ol 1-penten-3-ol

2-pentenal 1-penten-3-one

Fig. 3.3. Lipoxygenase (LOX) pathways involved in the production of EVOO C6 and C5 volatile compounds. Source: Angerosa et al. 2004; Servili et al. 2009b.

C6 and C5 compounds are produced from polyunsaturated fatty acids by the lipoxygenase (LOX) pathway, and their concentrations depend on the level and the activity of each enzyme involved (Montedoro and Garofolo, 1984; Angerosa and Di Giacinto 1995; Di Giovacchino and Serraiocco 1995; Morales et al. 1996; Aparicio and Morales 1998; Angerosa et al. 1998a; Morales et al. 1999; Angerosa et al. 2001; Angerosa and Basti 2003; Angerosa et al. 2004). The LOX pathway (Fig. 3.3) starts with the production of 9- and 13hydroperoxides of linoleic (LA) and linolenic (LnA) acids mediated by LOX. Very specific hydroperoxide lyases (HPL) catalyze the subsequent cleavage of 13-hydroperoxides and lead to C6 aldehydes, whose unsaturated (E)-3-hexenal can isomerize from Z-3 to the more stable E-2 form. The mediation of alcohol dehydrogenase (ADH) reduces C6 aldehydes to corresponding alcohols, which can produce esters because of the catalytic activity of alcohol acetyl transferases (AAT) (Vick and Zimmermann 1987; Hatanaka 1993). A collateral byway of the LOX pathway (Fig. 3.3) is active when the substrate is LnA. LOX would also catalyze the formation of stabilized 1,3-pentene radicals. These compounds could dimerize leading to C10 hydrocarbons (known as pentene dimers) or couple with a hydroxy radical present in the medium producing C5 alcohols, which can be enzymatically oxidated to corresponding C5 carbonyl compounds (Angerosa et al. 1998b).

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LOX pathway

Homolytic cleavage of I3-hydroperoxides

Conversion of amino acids

Virgin Olive Oil Volatile Compounds

Sugar fermentation

Fatty acid metabolism

Autoxidation

Fig. 3.4. The enzymatic and chemical paths involved in the production of EVOO volatile compounds. Source: Angerosa et al. 2004; Servili et al. 2009b.

The pathways involved in the EVOO aroma production are shown in Fig. 3.4. (The size of arrows gives an idea of the importance of each path). In high-quality EVOOs, the LOX pathway predominates, while in oils with sensory defects, other pathways occur that result in disagreeable aromas (Solinas et al. 1987; Solinas et al. 1988; Angerosa et al. 1996b; Aparicio et al. 2000; Morales et al. 2000; Garcıa-Gonzales and Aparicio 2002a,b; Morales et al. 2005). The volatile fraction represents an important marker for the genetic and geographical origin of EVOO (Angerosa et al. 2004; Servili et al. 2009b). Clinical and epidemiological evidence of the health properties of EVOO are constantly increasing. As a result, a plethora of reviews are available emphasizing the ability of macro- and micro-components of EVOO to reduce the risk of a number of chronic degenerative maladies, such as cardiovascular diseases (CVD) (Covas 2008; Huang and Sumpio 2008), atherosclerosis (Covas 2008), obesity and metabolic syndrome (Soriguer et al. 2007; Babio et al. 2009), Parkinson’s disease (Perez-Jimenez et al. 2005), Alzheimer’s disease (Pasinetti and Eberstein 2008), some types of cancer (Escrich et al. 2007; La Vecchia 2009), insulin sensitivity and diabetes (Schr€ oder 2007; Tierney and Roche 2007), nonalcoholic fatty liver disease (Assy et al. 2009), and inflammatory diseases (Patrick and Uzick 2001; Sales et al. 2009). The ability of EVOO to reduce the risk of CVD has to be linked primarily to a series of beneficial health effects on the atherosclerotic and thrombotic pathways, including lipid oxidation, hemostasis, plate-

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let aggregation, coagulation, and fibrinolysis. Both oleic acid and the polyphenols seem to exert antiatherosclerotic effects jointly. Plasma lipoproteins are carriers of plasmatic cholesterol, and their ratio is a fundamental factor in the onset and development of atherosclerosis and CVD. Due to their high susceptibility to oxidation, low-density lipoproteins (LDL) are a well-acknowledged risk factor of CVD, as their oxidation is a crucial step in the progress of atherogenic process. In contrast, high-density lipoproteins (HDL) are a protective factor, due to their ability to remove cholesterol from arteries and carry it to the liver. For this reason, the HDL/LDL ratio is considered a reliable marker of CVD risk. Oleic acid and, particularly, EVOO antioxidant polyphenols are efficacious in reducing the susceptibility of LDL to oxidation (Cicerale et al. 2009). Besides, EVOO is able to reduce the circulating levels of LDL and to increase those of HDL. However, EVOO beneficial effects are not exclusively linked to the plasma lipoprotein balance but also to the reduction of plasma triglycerides and total cholesterol and to the positive modulation of endothelial and platelet function (Cicerale et al. 2009). EVOO consumption also provides protection against the risk of some types of cancer. Recently, La Vecchia (2009) reviewed literature data obtained from a series of case control studies conducted in Italy and comprising over 20,000 cases affecting 20 cancer sites. The author concluded that olive oil consumption is associated with reductions in the risk of breast and colorectal cancer as well as of upper digestive tract neoplasms (i.e., oral/pharyngeal and laryngeal neoplasms, and esophageal cancer). Additional evidence arises from a number of laboratory studies suggesting that minor components of EVOO have the ability to mitigate the initiation, promotion, and progression of the multistage carcinogenesis process. Interestingly, the anticarcinogenic properties of EVOO polyphenols are not related per se to their antioxidant ability but rather to the capacity of polyphenols to induce cell differentiation, inhibit cell cycle progression, and exert antiproliferative effects (Eschrich et al. 2007; Corona et al. 2009). Squalene is the first minor compound of EVOO indicated as a potential anticancer agent (Newmark 1997). Animal and in vitro studies put forward a protective mechanism likely due to the remarkable squalene ability to inhibit the activity of beta-hydroxy-beta-methylglutaryl-CoA reductase, leading to reduced farnesyl pyrophosphate availability for prenylation of the ras oncogene (Soutirodis 2003; Owen et al. 2004). However, it must be emphasized that clinical confirmations of the anticarcinogenic properties of squalene are still lacking (Sotiroudis and Kyrtopoulos 2008). Numerous cell culture studies highlighted the ability of EVOO to modulate the expression and activity of some oncogenes, playing

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a crucial role in the initiation and progress of tumorigenesis and metastasis. In this regard, the modulation of HER2, an oncogene dramatically overexpressed in breast cancer cells, was suggested as the protective mechanism induced by oleoeuroperin aglycone (Menendez et al. 2008). Besides the modulation of oncogenes, in human promyelocytic HL60 leukemia cells EVOO polyphenols appear able to reduce cell growth and to promote apoptosis, thus inhibiting cell proliferation (Fabiani et al. 2006). Antiproliferative properties of hydroxytyrosol, through inhibition of ERK1/2 and cyclin D1, have been also reported in human colon adenocarcinoma cells (Corona et al. 2009). It is a fact that, analogously to what was mentioned earlier for squalene, the EVOO polyphenol anticancer properties have been observed exclusively in laboratory studies; thus clinical confirmation is needed. It is remarkable that the anticancer properties of EVOO are not attributable solely to polyphenols. Indeed, as accurately described by Menendez and Lupu (2006), the regulation of the amount and/or activity of diverse transcription factors is the underlying mechanism by which oleic acid can interact with the human genome. Mounting epidemiological evidence indicates a favorable effect of EVOO on obesity, type II diabetes, and metabolic syndrome (Schr€ oder 2007). De Ferranti and Mozaffarian (2008) efficaciously defined the vicious circle linking obesity, oxidative stress, inflammation, and metabolic disorders as the perfect storm. Hence, it is conceivable that the putative protection provided by EVOO is due to its antioxidant and antinflammatory properties. Adipose tissue produces a number of proinflammatory factors, such as tumor necrosis factor (TNF-a), interleukin (IL) 6, and IL-1, which have been demonstrated to play an important role in the onset of the major obesity-related comorbidities. Vassiliou et al. (2009) reported that oleic acid can counteract the negative effects of inflammatory cytokines by reversing their inhibitory effect in insulin production. In a human study, Jimenez-Go´mez et al. (2009) observed that consumption of an olive oil–based meal elicits low postprandial expression of proinflammatory cytokine. Beauchamp et al. (2005) attributed to the EVOO dialdehydic form of deacetoxy-ligstroside aglycone (also called oleocanthal) the ability to inhibit the cyclooxygenase enzymes COX-1 and COX-2, thus exerting a pharmacological anti-inflammatory action comparable to that of the structurally similar drug ibuprofen. Hydroxytyrosol was also attributed of anti-inflammatory properties by reducing TNF-alpha and COX-2 (Zhang et al. 2009). Like other fat sources, EVOO is an energy-dense food. However, epidemiological studies have demonstrated that EVOO consumption is not associated with increased body weight (Schr€ oder 2007). A physio-

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logical explanation of why EVOO consumption is less prone to promote weight gain could be that oleic acid is oxidized more easily than saturated fatty acids. Moreover, administration of EVOO promoted postprandial fat oxidation and diet-induced thermogenesis in abdominally obese women (Soares et al. 2004). Recent data from an intervention trial revealed that increases in dietary palmitic acid decreased fat oxidation and daily energy expenditure, whereas oleic acid had the opposite effect (Kien et al. 2005). Also in this regard, polyphenols seem to contribute importantly. Indeed, additional evidence arises from laboratory study reporting that EVOO polyphenols upregulate in rats the expression and the activity of uncoupling proteins (Rodrıguez et al. 2002; Oi-Kano et al. 2007, 2008), which are able to increase heat production in brown adipose tissue and muscles. EVOO composition and nutritional properties have also been correlated with genotype and geographical origin (Galvano et al. 2007; Mineo et al. 2007). III. SOURCES OF VARIABILITY OF EVOO COMPOSITION AND PROPERTIES Vetustas oleo taedium adfert, no item ut vino, plurimumque aetatis annuo est. [Aging affects oil but more than wine and it can last for no more than one year.] —Pliny the Elder, Naturalis Historiae XV, 7

Oil accumulation in olive fruits starts toward the end of the pit-hardening stage and becomes very rapid from 9 to 17 weeks after fruit set (Tombesi 1994). The basic pattern of oil accumulation is linear during most of the fruit development period and until peel color breakage; however, the oil accumulation pattern may change considerably under limiting growing contitions, such as major water stress or highly competitive fruit growth (Lavee and Wodner 1991, 2004; Tombesi 1994). Oil content and percent yield in the olive fruit is genetically determined and depend on cultural and environmental conditions (Lavee and Wodner 1991). The oil accumulation rate pattern also depends on genotype (Lavee and Wodner 1991; Fiorino e Ottanelli 2004), seasonal environmental conditions and tree water status (Lavee and Wodner 1991), and sink-source relationships related to the amount of crop load (Barone et al. 1994; Lavee and Wodner 2004). At full black fruit ripening stage, the relative oil content in the mesocarp is not yield or fruit size dependent, but differences may exist in the oil accumulation rate pattern (Lavee and Wodner 2004). The amount of oil produced by each

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fruit of the same cultivar is regulated by the size of the mesocarp (Inglese et al. 1996; Lavee and Wodner 2004). Differences in fruit size between cultivars often have no relation to the relative yield and the absolute oil content in the fruit. During the fruit maturation process, a number of physical and biochemical changes occur within the pericarp. The rate of these changes varies with the cultivar and the growing conditions and include the change in peel color, fruit water content, and the profiles of the fatty acid fraction, phenols, tocopherols, chlorophylls, and volatile compounds. All these changes account for large differences in the sensory profile as well as in the oxidative stability and nutritional value of the oil (Baccouri et al. 2006). Olive oil composition in fruits of any specific cultivar results from a very complex multivariate interaction between the genotypic potential and the environmental, agronomic, and technological factors that characterize fruit growth and ripening as well as oil extraction and storage (Montedoro and Garofolo 1984; Lavee and Wodner 1991). In terms of relative content, the individual components of the fatty acid fraction as well as the minor components may range independently and depending on factors that are not always interrelated. This creates a “field of indetermination” (Fiorino and Nizzi Grifi 1991) that makes it difficult to find markers to consistently identify the geographical area of origin and the genetic inheritance of any EVOO. However, the analytical and sensory profiles of most of the EVOOs produced by the most important cultivars worldwide have been largely described, and an acceptable level of probability can be reached using combined information on fatty acid composition and minor compound, analyzed with specific statistical analysis (Mannina et al. 2003). The range of variability of the individual fatty acid content in EVOO of different genotypes appears similar to the variation induced, within a genotype, by factors such as fruit ripening stage at harvest (Fiorino and Ottanelli 2004) or seasonal environmental conditions during fruit growth and ripening (Lombardo et al. 2008; Ripa et al. 2008). Use of the fatty acid fraction as a potential marker of the geographical and genetic origin of olive oil has been proposed, whether in terms of absolute content of its individual components or considering the relative oleic/linoleic or oleic/palmitic þ linoleic ratios (Fiorino and Alessandri 1996; Bongi 2004; D’Imperio et al. 2007). Indeed, fruit characteristics vary with location and between years in the same orchard. Even the fruit population in the olive tree is highly variable in terms of size, flesh/pit ratio, ripening, and oil accumulation rate pattern, due to the prolonged bloom and fruit set period, within-tree different source-sink relationships and environmental (i.e., light availability)

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conditions (Lavee and Wodner 2004). However, the range of variability of pomological characteristics does not necessarily imply a similar range in oil relative and absolute yield and composition (Lavee and Wodner 2004) since fruit growth and the oil accumulation rate patterns are only partially interrelated (Lavee and Wodner 1991). From this point of view, all factors affecting the genotypic fruit growth potential (size and flesh/pit ratio) may not play a similar role on oil composition. However, the fruit ripening stage at harvest time is one of the main sources of variability of the composition of the EVOO fatty acid fraction and, even more, of phenolic and volatile compounds. This means that all the agronomic (crop load, irrigation, pruning) and environmental factors (temperatures, soil water content) that influence the oil accumulation rate pattern and the nature of fruit ripening also account for the seasonal and geographical variability of the composition and properties of the EVOO of a specific genotype (Fiorino and Nizzi Grifi 1991; Lavee and Wodner 1991, 2004; Gucci and Servili 2006; D’Imperio et al. 2007; Lombardo et al. 2008). Pliny the Elder, in his Naturalis Historiae more than 20 centuries ago, distinguished the value and the quality of the olive oil according to fruit ripening stage at harvest as follows: oleum ex albis ulivis or oleum acerbum (oil from very clear unripe fruits), oleum viride (greenish oil), oleum maturum (mature oil), and oleum caducum (defective oil). The finest oil was considered the one obtained by pressing fruits before they were fully ripe. Cato, in his De agri cultura, stated: Quam acerbissimus olea oleum facies tam oleum optimum erit (the earlier the ripening stage of the olives the better the oil which results from them). Columella, in his De re rustica, recommends the grower to harvest oleum viride, because of the considerable yield and the high price.

IV. AGRONOMICAL AND ENVIRONMENTAL FACTORS AFFECTING EVOO COMPOSITION AND QUALITY A. Genotype The statement that EVOO quality is genotype dependent is a clear and well-known concept that now appears self-evident. However, many factors may overlap and override genetic potential of a single cultivar. Furthermore, most of the time, commercial olive oil is based on a blend of different cultivars. The great diffusion of Protected Designation of Origin (PDO) for olive oils, based on Regulation EEC 2081/92 and 2082/92 in the European Union countries, is based on the definition

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and regulation of cultivar- and environment-specific analytical and sensory profiles. There are more than 30 PDOs in Italy alone. Olive oil PDO is based on the assumption that EVOO chemical and sensory profile is dependent on the interaction between the genetic potential of the cultivar and the growing conditions, which should result in a typical and consistent phenotypic expression. Genotype clearly affects the evolution of the fatty acid, phenolic, and volatile fraction as well as oil color (Angerosa et al. 2004; Fiorino and Ottanelli 2004; Servili et al. 2004; Lombardo et al. 2008) (Tables 3.2, 3.3; Fig. 3.5). Olive cultivars differ in seasonal crop load, fruit size, flesh/pit ratio, and ripening pattern as well as in their adaptive response to water stress or high temperatures during fruit growth and ripening. The seasonal or environmental variability of the olive oil composition of a given cultivar depends on its phenotypic stability (Lavee and Wodner 1991; Salvador et al. 2001; Sweeney et al. 2002; Fiorino e Ottanelli 2004; D’Imperio et al. 2007; Lombardo et al. 2008). More than 1,200 cultivars are cultivated worldwide, but 3 cultivars cover 63% of Spanish production, 24 cultivars account for 58% of the olive-cultivated area in Italy, 3 cultivars cover more than 90% of the olive area in Greece, Tunisia, and Portugal, and a single cultivar represents 97% of the cultivated area in Morocco (Inglese and Famiani 2008). Many cultivars have only a very local diffusion (Inglese and Famiani 2008). Differences in the linoleic acid content up to 500% have been measured between cultivars or seedlings in the same season or for oils of the same genotype coming from different growing sites, while differences in the oleic content accounted up to 42% (between environments) and 57% (between cultivars) (Fiorino and Ottanelli 2004; Lombardo et al. 2008). Leo´n et al. (2004a) reported wide ranges of variation for all the fatty acids on progenies deriving from crossing programs that were as large or even larger than the ranges reported from the evaluation of olive cultivar collections. The fatty acids profile of an EVOO is genotype dependent in terms of unsaturated/saturated fatty acids ratio or, among the unsaturated ones, in terms of the monounsatured/polyunsatured ratio (Cucurachi 1965; Gouveia 1997; Stefanoudakii et al. 1999a). The application of statistical methods, such as the multivariate analysis of variance (MANOVA), principal component analysis (PCA), and the linear discriminant analysis (LDA), has demonstrated the usefulness of fatty acids analysis to group monovarietal EVOO (Mannina et al. 2003; D’Imperio et al. 2007). The ultraviolet (UV), ultraviolet-visible (UV-VIS), and near infrared (NIR) absorption spectroscopy covering a 200–1,700 nm spectral range, associated with LDA and PCA showed significant correlations with

99

Argentina Argentina Italy Argentina Italy Argentina Italy Argentina Italy Argentina Italy Argentina Italy Argentina Italy Argentina Italy

Arbequina Biancolilla

Source: Mannina et al. 2001.

Peranzana

Leccino

Kalamata

Frantoio

I-77

Coratina

Cerasuola

Origin

Cultivar

20.66 16.31 11.61 13.75 9.86 16.29 12.36 15.34 9.82 17.19 12.34 12.93 9.87 17.39 13.23 18.16 12.27

C16:0 3.69 1.81 0.52 0.51 0.22 0.67 0.51 0.91 0.50 1.65 1.01 1.46 0.61 1.16 1.25 1.79 0.80

C16:1 0.04 0.11 0.12 0.05 0.02 0.05 0.08 0.05 0.05 0.01 0.01 0.04 0.01 0.05 0.01 0.02 0.07

C17:0 0.20 0.19 0.20 0.07 0.03 0.08 0.05 0.08 0.12 0.09 0.02 0.13 0.56 0.09 0.09 0.07 0.11

C17:1 1.53 1.80 2.23 1.87 2.54 1.77 2.1 1.52 1.58 1.63 1.65 1.78 1.52 1.71 1.53 2.21 1.86

C18:0 53.39 70.47 74.10 70.98 76.83 71.50 75.43 70.52 80.54 63.55 75.77 65.79 78.95 68.45 77.96 62.57 76.45

C18:1

Fatty acid composition (%)

18.72 7.34 9.81 10.84 9.34 7.99 7.94 9.54 5.82 14.03 8.04 16.04 6.56 9.19 4.54 13.08 7.21

C18:2 1.16 1.12 0.69 1.12 0.51 1.27 0.72 1.45 0.70 1.23 0.55 1.33 0.72 1.43 0.68 1.37 0.58

C18:3

0.29 0.37 0.39 0.37 0.36 0.37 0.31 0.32 0.32 0.28 0.29 0.22 0.40 0.33 0.28 0.36 0.33

C20:0

Table 3.2. Fatty acid composition of extra-virgin olive oils obtained from several olive cultivars cultivated in the Catamarca region (Argentina) and in Italy.

0.22 0.31 0.31 0.41 0.34 0.35 0.33 0.25 0.39 0.31 0.29 0.29 0.52 0.25 0.33 0.32 0.28

C20:1

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Table 3.3. Concentrations of phenolic compounds (mg/kg) in extra-virgin olive oils of different Italian olive cultivars.z Cultivar

Phenolic compounds

Coratina

3,4-DHPEAy p-HPEA 3,4-DHPEA-EDA p-HPEA-EDA 3,4-DHPEA-EA Total polyphenols

1.96  0.30 2.08  1.79 1.38  1.42 0.89  0.99 0.87  0.65 0.82  0.91 382.4  138.2 340.0  26.3 154.0  26.1 193.2  65.2 99.8  61.2 89.8  7.8 177.5  92.6 157.1  84.5 84.1  103.0 755.9  153.1 599.9  67.1 330.1  27.3

Moraiolo

Frantoio

Carolea

Leccino

2.70  2.03 0.72  1.11 268.0  11.4 189.6  89.7 134.5  56.3 595.5  106.5

7.94  1.10 12.3  1.6 67.6  15.5 12.5  6.2 47.2  15.0 147.5  22.5

z

Data represent the mean  sd of 10 samples. Olives were harvested at the industrial ripening stage and malaxed at 30 C for 60 min and extracted by pressure on lab scale. y The concentrations of hydrophilic phenols were evaluated by HPLC. Source: Servili et al. 2004.

individual fatty acid contents, such as the oleic, the palmitic, and the total content of palmitic þ stearic (Mignani et al. 2006). DNA analysis of the oil also discriminated monovarietal EVOOs (Cresti et al. 1996). Research of appropriate methods and markers (Lain et al. 2004; 200 Total chlorophyll content (ppm)

180 160 140 120 100 80 60 40 0

Borgiona Correggiolo Dolce Agogia Frantoio Leccino Moraiolo Nebbia Nostrale R. Orbetana Picciolo Raia Raio San Felice Tendellone Vocio

20

Cultivar Fig. 3.5. Total chlorophyll content in extra-virgin olive oils of different cultivars, in central Italy (Umbria region). Bars ¼ SE. Source: Pannelli et al. 2000.

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Marmiroli et al. 2004) is still ongoing. However, no attempts have been made to define the genotype origin of blended EVOOs on an analytical basis. Due to its variability, also in terms of analytical methodology, the phenol fraction is largely ignored by PDO protocols, which usually indicate very low thresholds (100 ppm) if any of polyphenols, generally less than the genetic potential of most Italian cultivars (Table 3.3). Phenol content and composition vary greatly within genotype and among genotypes, with environmental conditions such as water shortage, and particularly with fruit ripening stage at harvest, extraction technologies. Nevertheless, the phenolic composition in terms of the relation between secoiridoids and lignans has been proposed as a potential marker of the genetic origin of an EVOO (Servili et al. 2004). Indeed, the variability of the EVOO phenolic profile, among other factors, can be related to the genetic potential (Briante et al. 2002) in terms of range of variation of total or individual content of polyphenols (Table 3.3) (Lo Curto et al. 2001). The sensory analysis made by a panel of experts, which is essential in the evaluation of EVOO, is a powerful tool for the discrimination of the genetic inheritance of monovarietal and blended EVOOs in terms of aroma and taste. The flavor notes of “fruitness,” “green,” “herbaceous,” “sweet,” “bitter,” “pungent,” as well as “apple,” “almond,” “artichoke,” or “tomato” may characterize different EVOOs, particularly in relation to their use in gastronomy (Panneli and Alfei 2008). As a matter of fact, the application of the panel test for the analysis of the genetic origin of EVOOS is very often associated with the promotion of nutritional and sensory value in oil marketing, Cross-breeding programs have been carried out with oil quality being considered one of the most important objectives. These programs made it possible to obtain new interesting genotypes in terms of oil quality (Guerin et al. 2000; Bellini et al. 2002; Leo´n et al. 2004b, 2008; Baccouri et al. 2007a; Manai et al. 2008; Ripa et al. 2008). Hybridization provided a wider range of variability than the original parents in several cases (Leo´n et al. 2008). Information on comparison of oil content and composition among commercial olive cultivars (Ola europaea L. var. europaea), oleaster (Olea europaea L. var. Sylvestris Brot.), and Olea europaea L. subsp. cuspidata (Wall. ex G. Don) Cif is also available (Hannachi et al. 2009). Olive cultivars had a higher oil content than fruits of oleaster and subsp. cuspidata from Kenya, but subsp. cuspidata from Pakistan also showed a high oil content (Gulfraz et al. 2009; Hannachi et al. 2009). Oil from subsp. cuspidata from Kenya had a relatively low oleic acid content

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(Hannachi et al. 2009), whereas subsp. cuspidata oils from Pakistan had a fatty acid composition comparable to that of oils obtained from commercial olive cultivars, although with a high linolenic acid content (> 1%) (Gulfraz et al. 2009). The fatty acid composition of oils from oleasters is variable, and in some cases they gave oils with less saturated fatty acids and higher oleic acid contents compared to those obtained from commercial cultivars, such as ‘Chetoui’, ‘Chemlali’, and ‘Gerboui’ (Hannachi et al. 2009). Evaluation and selection of wild olives on the basis of fatty acid composition, chlorophylls, carotenoids, tocopherols, phenolic and volatile compounds show a wide variability in chemical and aroma characteristics of oleaster virgin olive oils, with aromatic profiles distinctively different from those of European and Tunisian commercial oils (Baccouri et al. 2007b, 2008a). B. Growing Area and Seasonal Conditions Lavee and Wodner 1991 have shown that the genetic control of oil accumulation rate and pattern acts via cultivar-environment interactions. However, seasonal and environmental conditions also affect both the fatty acid and the insaponifiable fraction of the EVOOs. If the olives are healthy and processed soon after harvesting (within 24 hr), environmental conditions do not appear to have any substantial influence on the free acidity, peroxide number, and UV absorbencies of the oil, which are normally within the values that allow the classification of the oils as EVOOs (Pannelli et al. 1990a; Ripa et al. 2008). The earliest investigations indicate that latitude and altitude modify the relative proportions of unsaturated and saturated fatty acids (Frezzotti 1934). Higher contents of oleic acid and, consequently, an increase of the unsaturated/saturated fatty acid ratio move from the warmer areas in southern Italy to the cooler ones in northern Italy (latitude effects) or from the lower altitudes to the higher ones (altitude effects). This response has been observed analyzing oils collected from the main areas of olive cultivation in Italy (Vitagliano et al. 1961) or from the same cultivars (‘Frantoio’ and ‘Coratina’) cultivated in areas at different latitudes (Lotti et al. 1982). Lombardo et al. (2008), with different genotypes grown in the same orchard, and Ripa et al. (2008), with a high number of genotypes cultivated in three different areas in Italy (Basilicata, Calabria, and Umbria regions), determined the relationships between temperature and fatty acid composition and demonstrated that season-dependent and site-dependent fluctuations in the fatty acid compositions were related to average degree-days accumulated until harvest (Ripa et al. 2008), or from pit hardening to harvesting

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(Lombardo et al. 2008). In warm seasons and areas, oils had lower contents of oleic acid, which were associated with higher contents of palmitic and/or linoleic acids (Lombardo et al. 2008; Ripa et al. 2008). Linolenic acid can also be higher under warm conditions (Lombardo et al. 2008) (Table 3.4). A similar behavior can be seen in previous studies that compared the fatty acid composition of oils obtained from the same cultivars cultivated in environments characterized by different temperature regimes, such as the hot region of Catamarca (Argentina), and Italy (Mannina et al. 2001), Tuscany (Italy), Saudi Arabia, and Australia (Fiorino 2005), or areas at different altitudes in Chania, Greece (Mousa et al. 1996), or in Andalusia, Spain (Paz Aguilera et al. 2005). In addition, in cultivar comparative studies carried out in an arid region in Tunisia, most oils showed a decrease in oleic acid percentage and an increase in palmitic and linoleic acid percentages as compared to those from their original sites (Zarrouk et al. 2009). In Australia, oils originating from southern cooler areas had significantly higher oleic acid and lower polyunsaturated and saturated fatty acids (Ganz et al. 2002). Sweeney et al. (2002) showed higher levels of oleic acid in oils from southern latitudes of Australia. However, it is not possible to correlate specific environmental thresholds with consistent genotype behavior in terms of EVOO fatty acid composition. The environmental effect on fatty acid composition changes, indeed, with the different genotype-environment combinations. As a matter of fact, EVOOs from the ‘Carolea’ and ‘Canino’ cultivars showed no significant site variations for their fatty acid compositions (Montedoro et al. 2003), whereas comparisons of the fatty Table 3.4. Annual variation of the main fatty acids in extra-virgin olive oils extracted from 68 olive cultivars cultivated in southern Italy. The warmest and the coldest years were 2003 and 2005, respectively. C 16 : 0z

C 16 : 1

C 18 : 0

Year

Mean CV

Mean CV

Mean CV

2001 2002 2003 2004 2005 General mean

15.8 14.7 15.4 13.4 13.1 14.5

1.5 1.3 1.8 1.5 1.7 1.6

1.3 1.9 1.8 1.7 1.1 1.6

z

16.6 18.8 13.2 14.4 14.9 17.1y

41.9 44.0 43.0 39.8 42.3 43.7y

27.5 33.0 32.0 31.0 58.0 41.5y

C 18 : 1z

C 18 : 2z

C 18 : 3

Mean CV

Mean CV

Mean CV

68.4 70.1 67.9 72.0 73.8 70.4

11.6 10.5 11.3 9.7 9.0 10.4

0.69 0.74 0.87x 0.71 0.55x 0.71

7.5 8.0 7.7 6.1 6.4 7.9y

32.4 34.1 33.2 35.3 38.5 35.7y

26.2 23.9 26.2 23.1 24.0 30.1y

All data in the columns are statistically different at P  0.05. The general coefficient of variation (CV) is calculated on the overall data. x In the column of C 18:3, only the data with x are statistically different for P  0.05. Source: Lombardo et al. 2008. y

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acid compositions of oils obtained in the hot region of Catamarca, Argentina, with those of oils of the same cultivars produced in Italy showed that the oils produced in Argentina had a lower content of oleic acid and higher contents of palmitic, linoleic, and linolenic acids. These variations were, however, cultivar dependent, with 5% to 8% reductions in the oleic acid content in the ‘Biancolilla’, ‘Cerasuola’, and ‘Coratina’ cultivars, and 16% to 18% reductions in ‘Frantoio’ and ‘Peranzana’ (Mannina et al. 2001) (Table 3.2). Comparison of the fatty acid compositions of the oils of the ‘Frantoio’ and ‘Coratina’ in Tuscany (Italy), Saudi Arabia, and Australia showed larger variations in the oils of ‘Frantoio’ than ‘Coratina’ (Fiorino 2005). The fatty acid composition of ‘Koroneiki’ EVOO did not change in relation to growing site (Greece and Tunisia), whereas when ‘Sigoise’ was cultivated in Tunisia, it provided an oil with lower oleic acid and higher palmitic and linoleic acids than is normally obtained in Algeria (Mahjoub Haddada et al. 2007; Zarrouk et al. 2009). In a relatively hot year, Lombardo et al. (2008) reported that the oils of the most numerous group of cultivars had a decrease in oleic acid that was mainly compensated for by an increase in palmitic acid. The oils of another group of cultivars showed a decrease in oleic acid that was mainly compensated for by an increase in polyunsatured fatty acids (linoleic and linolenic acids). The oils of a very few cultivars showed no significant variations in the monounsaturated fatty acid contents (palmitoleic and oleic), whereas there was an increase in saturated fatty acids and a decrease in polyunsaturated fatty acids. Apparently, cultivars that originated in northern environments have higher phenotypic instability, in terms of fatty acid composition, than cultivars that originated in southern environments (Lombardo et al. 2008). The variations in fatty acid composition induced by the environment can be so large as to affect the commercial suitability of the oil as defined by the laws of the European Union (Reg. EC No 702/2007) and of the trade standards of the International Olive Council (COI/T.15/NC no. 3/Rev. 3— Nov. 2008). For example, in the Catamarca region of Argentina, which is characterized by high temperatures during the development and ripening of the olives, ‘Arbequina’ produced an oil with 53.4% oleic acid and 1.2% linolenic acid (Mannina et al. 2001); these percentages are, respectively, lower and higher than those established by trade standards of the International Olive Council (IOC) and the rules of the European Union (Table 3.2). Moreover, oils of ‘Biancolilla, ‘Cerasuola’, ‘Coratina’, ‘I-7’7, ‘Frantoio’, ‘Kalamata’, ‘Leccino’, and ‘Peranzana’ had percentages of linolenic acid greater than 1%, which is the maximum value allowed by the above-cited rules for all the categories of olive oils.

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High rainfall reduced EVOO polyphenol content (Pannelli et al. 1994). The role of temperatures on polyphenols content is controversial, depending on genotype and environmental conditions. In a study carried out in different Italian regions, a negative relationship between temperature and the amount of total polyphenols in the oils was observed (Ripa et al. 2008), whereas the total polyphenol content of ‘Casaliva’, but not ‘Leccino’, increased with the degree-days accumulated from August to October in the cool area of northern Italy (Tura et al. 2008). Moreover, in some studies on the effects of altitude, which is a factor that affects thermal regime, in some cases (Chania, Greece) polyphenols content decreased with altitude (Osman et al. 1994; Mousa et al. 1996), whereas in other cases, the effects of altitude was unclear (Paz Aguilera et al. 2005). As far as the contents and composition of volatile compounds in olive oils are concerned, the effects of rainfall and temperature are not unambiguous and depend on environment and interaction with genotype. In a study conducted in central Italy, rainfall negatively correlated with hexanal and isobutyl-acetate contents and positively correlated with the other compounds in the head-space of the oil (Pannelli et al. 1994). In a northern and relatively rainy area of Italy, no significant effects of temperature on total volatile compounds in oils of the cultivar ‘Leccino’ were observed, whereas positive relationships were seen between the cumulated degree-days in the period August to October in oils of the cultivar ‘Casaliva’ (Tura et al. 2008). Oils of ‘Biancolilla’, ‘Carpellese’, and ‘Racioppella’ produced in a warm coastal area were less fruity, bitter, pungent, and sweeter than those produced in the fresh hilly areas where these cultivars are traditionally grown (Di Vaio et al. 2006). Temperature and rainfall can also have indirect effects on oil quality, as they can affect fruit ripening pattern. Earlier olive ripening occurs in years with low rainfall and warm temperatures (Pannelli et al. 1996; Di Vaio et al. 2006). There is no clear information on the influence of soil type on EVOO composition, although some specific relationships between the qualitative parameters of an oil and the soil characteristics have been reported (Angerosa et al. 1996a; Ranalli et al. 1997). The oil of the ‘Moraiolo’ obtained in a stony soil had a higher polyphenol content and oxidation stability than that obtained in a clay soil; these effects were mainly attributed to the lower water availability in the stony soils, with respect to the clay ones (Pannelli et al. 1990a; Servili et al. 1990). In Sardinia, soil differences appeared not to have a strong influence on ‘Bosana’ oil quality (Deidda et al. 1994).

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Altitude affects the tocopherol content of the oils. At lower altitudes, higher amounts of tocopherols were observed in several studies (Osman et al. 1994; Mousa et al. 1996; Paz Aguilera et al. 2005). Altitude was also able to affect the chlorophyll content, oxidation stability and quantity of hydrocarbons, triterpenic alcohols, and sterols of olive oils (Ferreiro and Aparicio, 1992; Osman et al. 1994; Mousa et al. 1996; Paz Aguilera et al. 2005). In the coldest areas in which olive trees are grown, the fruits, and consequently the oils extracted from them, can be damaged by freezing temperatures during the ripening period. In an oil extracted within 24 hr of the occurrence of freezing temperatures, reductions were shown for the chlorophyll, carotenoid, and total polyphenol contents and the oxidation stability and bitterness index (K 225), whereas no significant negative effects were seen on free acidity, peroxide number, ultraviolet absorbency (K 270), or a-tocopherol (Morello´ et al. 2003). Oil from frosted olives also showed significant changes in the concentrations of several phenolic compounds and reductions in the bitter and pungent notes, and the onset of sensory defects that prevent the oil from being marketable as EVOO (Morello´ et al. 2003). The variability of the effects of environmental factors, which sometimes is rather large, also arises from interaction effects with other factors, such as genotype and ripening stage of the fruit (Tous and Romero ; Pannelli et al. 1996). C. Tree Water Status In the Mediterranean area, olive trees traditionally have been grown under rainfed conditions and with limited water resources during the fruit developmental stages, since most of rains occur during the winter period. Complementary irrigation, distributed during critical stages of fruit growth, particularly during mesocarp development and oil accumulation, increases fruit size, flesh-to-pit ratio, and oil yield per hectare, although the percent of oil in an individual fruit may decline because of a proportionally larger increase in the water content in the fruit and a minor efficiency in oil physical extraction (Spiegel 1955; Vitagliano 1969; Lavee et al. 1990; Lavee and Wodner 1991; Goldhamer et al., 1994; D’Andria et al. 2002; Berenguer et al. 2006). The nature of fruit ripening and the rate pattern of oil accumulation are also significantly affected by water availability (Vitagliano 1969; Lavee and Wodner 1991). Indeed, the fruit is the organ most sensitive to water stress, and severe water shortage, lasting up to the third stage of the fruit development period, may result in shriveling of the fruits, advanced and rapid ripening, early and

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pronounced preharvest fruit drop, and, eventually, a complete or temporary arrest of the oil metabolism, to such an extent that its final accumulation could be sharply reduced and the metabolism of individual components may also be affected (Lavee 1986; Dettori et al. 1990; Dettori and Russo 1993; Inglese et al. 1996; Gucci and Servili 2006). Under irrigated intensive growing conditions, oil accumulation is linear during most of the fruits’ growing period. The degree of diversity of the oil accumulation from linearity in a single cultivar could serve as a partial index of water stress and cultivar sensitivity. The water deficit that occurs during the first stage of fruit growth determines a significant reduction of mesocarp cells dimension, which is only partially recovered during the subsequent developmental stages, even if the tree is regularly watered (Rapoport et al. 2004). Complementary irrigations, however, even with low volumes, distributed during the cell extension in the mesocarp result in an increase of fresh and dry fruit weight, flesh percent, and oil content (Lavee et al. 1990; Lavee and Wodner 1991; Dettori and Russo 1993; Motilva et al. 2000; Gucci et al. 2004; Servili et al. 2007a). The effect of water availability on the relative growth of the endocarp and the mesocarp is controversial, since it also depends on other factors, such as crop load (Barone et al. 1994; Inglese et al. 1999), irrigation strategy, and fruit growth potential defined by the genotype (Lavee et al. 1990; Lavee and Wodner 1991, 2004; Inglese et al. 1996; Rapoport et al. 2004; Gucci and Servili 2006). The prolonged duration of the second stage of fruit growth could be related to water deficit (Lavee 1986; Inglese et al. 1996). The effects of water availability on EVOO composition and quality have been investigated under a wide range of environmental conditions, with different genotypes and water deficit levels. Water shortage affects fruit ripening pattern, and differences of EVOO composition might be both a direct effect of water stress on oil accumulation pattern and single component metabolism or on the frequency at harvest of populations of fruits highly differentiated in terms of ripening stage. These differences increase with time because fruits from nonirrigated trees mature earlier and faster than those on irrigated trees (Inglese et al. 1996). Nevertheless, it is generally agreed that the oils coming from trees under a severe water deficit (25% restitution of actual evapotranspiration [Etc]), or fully irrigated (100% restitution of ETc) comply with the international merceological standards of high-quality EVOOs, with differences that, in most cases, concern the phenolic fraction and the sensory parameters. There is no evidence of significant effects of water shortage on oil acidity, peroxide number, and spectrophotometric indexes, while the fatty acid composition changes slightly with tree irrigation. However,

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fluctuations of oleic acid and stearic acid occur inconsistently among years, cultivars, and irrigation treatments (Inglese et al. 1996; Berenguer et al. 2006; Gucci e Servili 2006; Gomez-Rico et al. 2007; Servili et al. 2007a). Berenguer et al. (2006) report a significant and consistent decrease of stearic fatty acid from 15% ETc to 107% ETc, while palmitoleic, linoleic, and linolenic fatty acid levels significantly increased with irrigation only in one year out of two. In any case, the range of variations of the fatty acid composition do not account for any significant change of the physical and nutraceutical properties of the oil (Faci et al. 2002; Tovar et al. 2002). Tree water status, however, has a consistent effect on the phenolic fraction and the volatile compounds of the oil, and on its sensory properties (Inglese et al. 1996; Gucci et al. 2004; Berenguer et al. 2006; Gucci and Servili 2006; Servili et al. 2007a). Most studies report a decrease of total polyphenol content and oxidation stability as the amount of supplied water increases (Patumi et al. 2002; Berenguer et al. 2006; Gucci and Servili 2006; Gomez-Rico et al. 2007). The effect is clear at low (66% ETc) (D’Andria et al. 2002) and high (25% ETc) water stress level (Motilva et al. 2002). Nevertheless, some studies report no effect of tree water status on phenolic composition and total content or an increase of total phenols, particularly during the early stages of fruit ripening, in irrigated trees compared to rainfed ones (Dettori and Russo 1993; Inglese et al. 1996). Reasons for such differences may lie on the overlap of crop load, fruit ripening rate pattern, and water regime effects (Gucci and Servili 2006). The reduction of polyphenol content in the oil of irrigated trees could be a consequence either of a greater dilution of hydrosoluble compounds during oil extraction or reduced activity of the enzymes responsible for phenolic compound synthesis, such as L-phenylalanine ammonya-lyase, whose activity is greater under water stress conditions (Tovar et al. 2002; Gomez-Rico et al. 2007; Servili et al. 2007a). Total phenol content decreased with fruit ripening from 1,700 to 900 mg/kg and from 1,080 to 650, respectively, for EVOO from rainfed or irrigated olive trees (Gomez-Rico et al. 2007). Tree water status affects the composition of the phenol fraction; a greater concentration of secoiridoids and aglycon derivatives of oleuropein together with a reduction of tyrosol and hydrossid tyrosol content has been measured in nonirrigated olive trees (Servili et al. 2007a). Berenguer et al. (2006) report significant but inconsistent through the years variation of total and individual sterol contents in oil from 15% to 107% ETc treatments. Tree water status has a marked effect on volatile compounds and EVOO sensory properties (Servili et al. 2007a). Rainfed or poorly irrigated olive trees produce oils characterized by marked notes of pungency

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and bitterness, which decrease with irrigation (Patumi et al. 1999; Tovar et al. 2002; Berenguer et al. 2004; Gucci et al. 2004; Gucci e Servili 2006; Servili et al. 2007). Fruitiness, herbaceous, and floral flavors may also decrease with increasing water availability, although variations may not be always consistent (Berenguer et al. 2006; Servili et al. 2007a). These variations may be related to a reduction of some compounds of the lypoxygenase pathway, such as C5 and C6 saturated and unsaturated aldehydes, alcohols, and esters (Servili et al. 2007a). Very few studies have examined the effect of saline water on oil quality (Gucci and Tattini, 1997). Cresti et al. (1994) reported that salinity increased aliphatic and triterpenic alcohol contents and the linoleic/oleic and linoleic/linolenic acid ratios, while Royo et al. (2005) reported a decrease of the aliphatic alcohols and palmitoleic acid with salinity in ‘Arbequina’ oils. In Israel, 48 olive oils from the years 2002 to 2004 were compared and graded; oils produced under high evaporation and saline irrigation did not differ significantly from most other oils produced from rainfed and freshwater-irrigated orchards (Dag et al. 2008a). No significant differences were found between saline- and control-water-irrigated ‘Barnea’ trees in terms of olive oil basic quality parameters, such as free fatty acids, peroxide value, and fatty acid profile. However, the saline treatments increased the levels of certain antioxidant components (polyphenols and vitamin E) in the oil extracted from the olives as compared with the control (Wiesman et al. 2004). Eventually, rainfed olive trees produce EVOO with strong taste, with a clear pungent and bitter flavor, while oils from irrigated trees are more aromatic and sweet, with a sharp reduction of bitterness and pungency. Result obtained on ‘Leccino’ indicate that an appropriate use of irrigation (qualitative irrigation) may modulate EVOO sensory properties, taking into account the different evolution of the fruit ripening pattern (Gucci e Servili 2006). D. Productivity and Alternate Bearing The olive tree shows a typical alternate bearing behavior with a frequency and intensity regulated by the genotype and the growing conditions. The olive tree may show “on” and “off” years on single branches within a tree, single trees within an orchard, single orchards within the same location or between different geographical area. The intense competition for assimilates and water between fruits during the earliest stages of their development is primarly responsible for the regulation of crop load (Lavee 1986). It has been reported (Vitagliano 1969; Zucconi et al. 1978;

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Shulman and Lavee 1979; Lavee 1986; Lavee and Wodner 1991, 2004) that not only final fruit size and flesh-pit ratio but also the nature of fruit growth and ripening and both rate pattern and final oil accumulation in the mesocarp depend on crop load. In the case of table olive, fruit thinning becomes essential to regulate fruit growth and final commercial size (Barone et al. 1994; Inglese et al. 1999; Lavee and Wodner 2004). In trees with a light crop load, oil accumulation and ripening rate pattern are fastened; fruit ripening occurs earlier and is more concentrated than in heavily cropping trees (Barone et al. 1994; Gucci 2006). Eventually, the oil content in the single fruit may be reduced by heavy crop loads, due to a consistent reduction of the mesocarp size and a slower oil accumulation rate than in lightly cropping trees (Barone et al. 1994; Lavee and Wodner 2004; Gucci and Servili 2006). The influence of crop load on oil quality is less clear, and it may occur only in case of very heavy reductions of crop load (> 50%), with effects on fatty acid composition and polyphenol content (Barone et al. 1994; Gucci e Servili 2006) (Fig. 3.6). The effect of harvest date, hence the different degrees of fruit ripening at picking, is much stronger than the effect of crop load in determining the variability of oil composition (Maestro Dur an, 1990), and differences in oil compositon between trees with heavy or light crop loads may largely depend on the different time course 300

Polyhenol content (ppm)

250

200

150

100 30 Oct.

14 Nov.

29 Nov.

13 Dec.

9 Jan.

Harvest date Fig. 3.6. Changes during olive ripening of total polyphenol content of oils obtained from fruit produced from trees with full load (FL ¼ ), partially reduced load (70% FL ¼ ~), and halved load (50% FL ¼ &). Analysis of variance showed significant effects for P < 0.01 of date of harvesting, fruit load, and their interaction. Source: Barone et al. 1994.

.

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of fruit ripening and uniformity of fruit ripening stage of the pending fruits. Considering the evolution of single oil components, fruits from lightly cropping trees show a more rapid metabolism and accumulation rate, which eventually result in a higher total palmitic and linoleic acid and polyphenol content than in heavily cropping trees (Barone et al. 1994). Crop load has no effect on peroxides and sensory parameters of EVOOs (Gucci e Servili 2006). The effect of crop density and different source-sink relationships on fruit growth and ripening rate pattern can be measured even at a single fruiting branchlet (Inglese et al. 1999). This means that tree management in terms of training system, pruning, and fruit thinning may account for the uniformity of fruit growth and ripening and, ultimately, for the final oil accumulation and composition. Optimum harvest time to maximize oil yield and quality changes, then, with tree crop load; in trees with a light crop load, it occurs early and lasts for a short period, resulting in rapid changes of the degree of ripening of pending fruits. The timing and the speed of harvest are therefore crucial in trees with a light yield, while the picking season of heavily loaded trees, with a slower fruit ripening pattern, is later and more extended. Provided these differences of the fruit ripening pattern are taken into account in determining the optimum harvest time, large differences in tree crop load have only a limited effect on EVOO composition (Barone et al. 1994). E. Orchard Management 1. Cultivation Method. The organic farming of olive groves has spread through all of the most important olive-growing areas and particularly in Italy, where it covers about 10% of the total area for olives. Despite this, few studies have been carried out to determine the influence that organic farming has on the characteristics of the oil produced. In a one-year study on the cultivar ‘Picual’, Gutierrez et al. (1999) showed that the EVOO obtained with organic farming had lower free acidity, peroxide number, and linoleic acid content and higher organoleptic score, oxidation stability, oleic acid, a-tocopherol, total polyphenol, ortho-diphenol, and D5-avenasterol contents. Perri et al. (2002) and Ninfali et al. 2008, comparing the qualitative characteristics of EVOOs of ‘Coratina’ and ‘Ogliarola Salentina’, and ‘Frantoio’ and ‘Leccino’ obtained by organic and integrated or conventional methods, found inconsistent results with a large season- and genotype- dependent variability. The sensory analyses showed only slight differences in a few aromatic notes. In Spain, the oil of the ‘Hojiblanca’ obtained with

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integrated farming had higher levels of sterols and tocopherols than those from conventional farming while the free acidity, peroxide number, ultraviolet absorbencies, acid composition, polyphenol content, and sensory traits were not affected by the cultivation system (Cayuela et al. 2006). The inconstant results obtained in the comparison of the organic, integrated, and conventional cultivation systems indicate that the effects of the cultivation method on the quality of the oil is likely dependent on the interaction with the other factors that affect oil quality (genotype, environment, and season). 2. Training System and Pruning. Comparisons of different training systems (monocone, Y-shape, and vase) in young trees of the cultivars ‘Frantoio’, ‘Leccino’, ‘Maurino’, ‘Moraiolo’, and ‘Nostrale di Rigali’ showed no significant differences in terms of free acidity, peroxide number, total polyphenol and chlorophyll contents, fatty acid composition, and oxidation stability (Preziosi et al. 1994; Palliotti et al. 1999). At the end of the 20th century a new concept of olive orchard arose, based on high-density plantations (1,100–2,050 trees/ha), forming hedgerows, and the use of straddle harvesting machines to collect the fruit (superintensive or high-density hedgerow orchards). Until now, the cultivars most used in these groves are the low vigor and compact cultivars such as ‘Arbequina’, ‘Arbosana’, and ‘Koroneiki’ and their clones, along with new cultivars such as ‘Urano’, ‘FS-17’, and ‘Askal’; the evaluation of suitability of some traditional cultivars (‘Picual’, ‘Leccino’, ‘Barnea’, ‘Souri’, ‘Picholine’, ‘Chemlali’, ‘Chetoui’, ‘Picholine Marocaine’) to this cultivation system is in progress (Dag et al. 2006; Godini et al. 2006; Larbi et al. 2006; Leo´n et al. 2006; Pastor et al. 2006; De la Rosa et al. 2007; Tous et al. 2007). There is no experimental evidence of oil quality with this new cultivation system as compared to the traditional growing systems. The oils obtained with high-density hedgerow orchards had physicochemical characteristics (free acidity, peroxide number, ultraviolet absorbencies) within the ranges established for extra-virgin olive oil by the European Union (Reg. EC No 702/ 2007) and the International Olive Council (COI/T.15NC no 3/Rev. 3— Nov. 2008) (Berenguer et al. 2006; Allalout et al. 2009). The main cultivars used for high-density hedgerow orchards showed differences in contents of fatty acids, pigments, phenolic compounds, tocopherols, and oxidative stability (Larbi et al. 2006; De la Rosa et al. 2007; Allalout et al. 2009). An interaction is suggested between the main cultivars used for this cultivation system and the environment in determining fatty acid composition and total phenol amounts (De la Rosa et al. 2007; Allalout et al. 2009).

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Fruit location within the canopy position can be considered a source of variability for the composition of the oil, resulting from large differences in light environments within the canopy. Oils of ‘Frantoio’ and ‘Leccino’ obtained from fruits that developed under optimal and low-light availability showed no differences in terms of free acidity, peroxide number, and fatty acid composition, whereas those obtained under optimal light availability had higher total polyphenol and chlorophyll contents and, in the case of ‘Frantoio’, greater fruity, bitter, and spicy tastes (Proietti et al. 2009). This supports the importance of a balanced pruning to allow light diffusion within the canopy, even though no light thresholds for oil quality have been defined. 3. Fertilization and Soil Management. The little information available on the effects of fertilization on the qualitative characteristics of olive oils relates primarily to nitrogen and is in some cases contradictory. Uceda Ojeda (1985) reported a positive correlation between administration of nitrogen and the levels of oleic and stearic acids in the oil, while a lack of nitrogen resulted in an increase in the palmitic and linoleic acid contents. Cimato et al. (1994) showed an increase in the total polyphenol and tocopherol contents in the oil obtained from ‘Frantoio’ and ‘Moraiolo’ as a result of foliar treatments with urea. These effects were explained as a result of the delay in the ripening of the fruits on treated plants, caused by the greater vegetative activity induced by the nitrogen. In a study carried out in Portugal on ‘Carrasquenha’, Simo˜es et al. (2002) suggested that high levels of nitrogen fertilization had a negative influence on the content of saturated fatty acids of the oil. Fern andez-Escobar et al. (2006), supplying nitrogen to plants of ‘Picual’ where the leaf nitrogen status was always above the limit for deficiency even in the nonfertilized control trees, showed that overfertilized trees produced oils with lower total polyphenol content, oxidation stability, and compounds responsible for the bitter taste and higher contents in tocopherols, in particular a-tocopherol. No effects were found on the carotenoid and chlorophyll concentrations and fatty acid composition. In the oil of ‘Manzanilla de Sevilla’, with an increase in the supply of a complex fertilizer (4 : 1 : 3 of N-P-K) through fertigation, Morales-Sillero et al. (2006) found doserelated reductions in total polyphenol content, oxidation stability, and monounsaturated to polyunsaturated fatty acid ratio. Eventually, it seems that high availability/excess of nutrients, especially nitrogen, may result in a significant worsening of the oil quality. Very little information is available on the influence of soil management on oil quality. Differences in the sensory profile were noticed for ‘Carolea’

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EVOOs, although there were no differences in the panel test scores (Briccoli Bati et al. 2002). 4. Pest and Disease Control. Attacks of olive fly (Bactrocera oleae) that are not controlled can cause serious damage to oil production and quality. Quantitative damage is due to premature olive drop and loss of part of the olive pulp caused by the larvae. Qualitative damage is due to the galleries produced by the larvae (rupture of tissue and presence of oxygen) where fungi (decay) and bacteria can also develop. The greatest amount of damage has been observed when there is a large number of olives with mature larvae and galleries with exit holes (Zunin et al. 1992). Increasing intensity of infestation corresponds to a progressive worsening of oil quality, because of an increase in free acidity, peroxide number, and UV absorbencies and a decrease in oxidation stability and polyphenol content (Parlati et al. 1990; Perri et al. 1996; Go´mez-Caravaca et al. 2008; Tamendjari et al. 2009). Fly attack results in the loss of phenols, o-diphenols, and, in particular, some secoiridoid derivates (Go´mez-Caravaca et al. 2008). When the percentage of infected olives increases, phenols decreases and stability of resulting oils is compromised, probably because of an increase of polyphenoloxidase activity caused by larval damages and by the presence of exit holes that expose the olive pulp to oxygen (Tamendjari et al. 2004). Attack of olive fly causes a reduction in the chlorophyll and carotenoid contents as the degree of infestation increases (Tamendjari et al. 2004). The fatty acid composition is quite stable, but in case of massive attacks, levels might be altered (Parlati et al. 1990; Zunin et al. 1992; Perri et al. 1996; Pereira et al. 2004; Tamendjari et al. 2004). Fly attack causes a reduction of the total amount of volatile compounds, attributable mainly to a decreased concentration of trans2-hexenal, and a quicker flattening of fruity, pungent, and bitter attributes of the oil during olive ripening (Tamendjari et al. 2004, 2009); moreover, it can cause the onset of sensory defects, such as fusty, winy, and grubby sensations (Angerosa et al. 1992; Tamendjari et al. 2009). The quicker reduction of the positive bitterness and pungent attributes are caused by the losses in phenolic compounds under the effects of fly attack. Postharvest storage of olives presents more problems in infected olives that have worse qualitative characteristics at harvest time (Kyriakidis and Dourou 2002). During oil storage, the sensory characteristics worsen more rapidly in oils extracted from infected olives (Esposito et al. 2004). In some cases, olive fly attack caused a reduction of the total b-sitosterol content below the level legally required for olive oil (Zunin et al. 1992).

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Olive anthracnose caused by Colletotrichum spp. affects olive production and oil quality. As the olive infestation increases, there is a parallel worsening of oil quality extracted from attacked fruits. This disease causes an increase in peroxide number and free acidity and a decrease in the oxidative stability and polyphenol and a-tocopherol contents and, in some cases, with infestations of 15% to 20% and 40% to 45%, the oil quality was beyond the limits to be marketed as EVOO or as VOO due to too-high free acidity (> 0.8% and 2%, respectively) (Iannotta et al. 1999; Mincione et al. 2004; Carvalho et al. 2008). Fatty acid composition is little affected by anthracnose whereas sometimes the changes induced in individual and total amount of sterol contents can cause problems in respecting the international trade standards for olive oils (i.e., total b-sitosterol < 93% and total sterols < 1,000 mg/kg, which are the minimum values established by the trade standards of IOC and UE rules, Reg. EC No 702/2007; Iannotta et al. 1999; Mincione et al. 2004). Anthracnose attack increases the content of aldehydes, such as heptaldehyde, octyl aldehyde, and nonanal (Runcio et al. 2008). Colletotrichum spp. also affect the aliphatic and terpenic alcohols and wax contents (Mincione et al. 2004). Olive rot caused by Camosporium dalmatica does not greatly affect free acidity and peroxide number. Oils can be classified as EVOO even when 100% of the olives were attacked, but an increase in the percentage of olive infection corresponds to a decrease in total phenols and oxidative stability (Iannotta et al. 1999). Because of the great negative effects of disease and insect predation on oil, these biotic stresses must be carefully controlled to obtain a high-quality EVOO, especially in environments or seasons that are favorable to them and with sensitive cultivars. In case of late attacks by the olive fly, earlier fruit harvesting and rapid milling are important to avoid or reduce its deleterious effect on oil quality, especially in organic olive orchards, where the control of olive fly could be more difficult. F. Fruit Ripening and Harvest 1. Ripening. During fruit ripening, a number of changes take place both in the fruit and in the oil. Olives show a reduction in the resistance to detachment, which results in falling of the more mature fruits (fruit drop), and firmmess of the pulp and a progressive increase in the pigmentation of the skin (the epicarp) and, later, of the pulp (the mesocarp), starting from the outside layers; moreover, they complete

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their growth and accumulate oil (Pannelli et al. 1990a; Famiani et al. 2002; Tombesi et al. 2009) (Table 3.5). The quantity of olive oil per tree is based on number of fruits, fruit weight, oil content, and fruit drop (Zucconi et al. 1978; Famiani et al. 2002). Generally, total quantity of oil from olives on the canopy decreases when fruit drop exceeds 5–10% of total olive production (Famiani et al. 2002; Tombesi et al. 2006). During fruit ripening, oleic acid, and sometimes linoleic acid, tends to increase, while stearic acid and especially palmitic acid tend to decrease, with a consequent increase in the ratio of unsaturated to saturated fatty acids (Servili et al. 1990; Fiorino and Nizzi Grifi 1991; Di Matteo et al. 1992; Fiorino and Alessandri 1996; Tombesi et al. 2009) (Table 3.6). However, the fatty acid composition does not always show significant variations, particularly in the period in which the harvesting is effectively carried out, which is shorter than the entire ripening period (Famiani et al. 2002). In some cases, a different pattern of fatty acid modifications was observed. In ‘Cornicabra’, during fruit ripening, a reduction in oleic acid and an increase in stearic and linoleic acid

Table 3.5. Changes in fruit characteristics and fruit drop during fruit ripening determined in olives collected from an intensive olive grove in central Italy. Cultivar Harvest time

Dry weight (g fruit
1)

Oil content (g fruit
1)

Detachment force (N)

Pigmentation (0–4)z

Fruit dropy (%)

Frantoio Begin-Nov. End-Nov. Mid-Dec.

1.062 ax 1.148 b 1.150 b

0.368 a 0.468 b 0.471 b

5.60 c 3.98 b 3.60 a

0.7 a 1.0 b 1.4 c

6.8 a 11.1 b 16.7 c

Leccino Begin-Nov. End-Nov. Mid-Dec.

0.972 a 1.041 ab 1.058 b

0.348 a 0.428 ab 0.445 b

5.77 b 4.41 ab 3.82 a

2.5 a 3.1 b 3.5 c

4.5 a 5.3 a 11.6 b

Maurino Begin-Nov End-Nov. Mid-Dec.

0.698 a 0.729 ab 0.740 b

0.276 a 0.330 b 0.349 b

4.63 b 3.95 ab 3.10 a

2.6 a 2.8 b 3.1 c

2.8 a 4.8 b 12.7 c

z

0 ¼ green; 1 ¼ pigmentation on less than 50% of fruit surface; 2 ¼ pigmentation on more than 50% of fruit surface; 3 ¼ pigmentation on 100% of fruit surface; 4 ¼ pigmentation on 100% of fruit surface and on the pulp. y Expressed as percentage of total olive production. x For each parameter and cultivar, means followed by different letters are significantly different at P  0.05. Source: Famiani et al. 2002.

117

2.52 a

2.27 a 2.44 a 2.42 a 2.49 a

C 18 : 0 A B BC C

76.31 C

72.99 74.47 75.35 76.19

C 18 : 1

135 a 203 a 260 a 137 a

148 a

109 a

Esters

390 a

985 b 831 b 758 b 653 ab

Aldehydes b b b ab

215 a

621 596 589 484

Trans-2-hexenal

Aromatic composition (I.U. n  103)

0.95 a

a a a a

303 b 352 b 291 ab 131 a

Alcohols

11.37 A

1.02 1.08 1.06 0.95

C 16 : 1

C BC B AB 11.8 A

57.5 39.4 27.1 20.8

203 a

253 ab 310 b 276 b 226 a

Total polyphenols (ppm)

Total chlorophyll (ppm)

1.34 B 1.05 A 0.99 A 0.95 A 0.94 A

a a a a

C 18 : 3

7.91 a

8.28 7.87 8.06 7.78

C 18 : 2

a a a a

7.6 A

9.1 A 12.5 AB 13.6 B 12.0 AB

Induction time (h)

9.76 a

8.90 9.64 9.58 9.97

O/L

A A B B 6.22 B

5.12 5.45 5.92 6.10

U/S

y

In each column, for each parameter, means followed by different letters are significantly different at P  0.05 and P  0.01 (capital letters). I.U. ¼ Integration unit. O/L ¼ oleic/linoleic ratio; U/S ¼ unsaturated/saturated fatty acids ratio. Source: Servili et al. 1990.

Green 50% of surface 100% of surface 100% of surface, 50% of pulp 100% of surface, 100% of pulp

14.09 13.10 12.11 11.63

Green 50% of surface 100% of surface 100% of surface, 50% of pulp 100% of surface, 100% of pulp

C B A A

C 16 : 0

Fruit pigmentation

Fatty acid composition (%)

Table 3.6. Influence of fruit ripening stage (expressed as fruit pigmentation) on fatty acid and aromatic compound compositions, chlorophyll and total polyphenol contents and oxidation stability (induction time—Swift test with Rancimat apparatus) of the olive oil. Data represent the average values of 6 cultivars (‘Frantoio’, ‘Leccino’, ‘San Felice’, ‘Tendellone’, ‘Nostrale di Rigali’ and ‘Moraiolo’) cultivated in central Italy.y

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contents occurred (Salvador et al. 2001). In Tunisia, recent studies of fruit ripening showed reductions in oleic acid and increases in linoleic acid, and palmitic acid decreased in some cultivars and increased in others (Baccouri et al. 2008b; Sakouhi et al. 2008). These results might have arisen due to the occurrence of high temperatures during the ripening period. The peroxide number of oil obtained from healthy olives often decreases through the ripening period. This is related to a reduction in the lipoxygenase enzyme activity (Guti errez et al. 1999; Salvador et al. 2001; Baccouri et al. 2008b). Ripening also has large effects on the sensory and nutritional characteristics of olive oils, affecting the contents of volatile substances, phenolic compounds, and pigments (Angerosa et al. 2004; Servili et al. 2004). The contents of volatile compounds is greatest in the initial phases of the fruit surface pigmentation, with particular reference to the components involved in the lipoxygenase pathway, such as aldehydes and saturated and unsaturated C5 and C6 alcohols and, in particular, trans-2-hexenal (Solinas et al. 1987; Montedoro et al. 2003; Angerosa et al. 2004) (Table 3.6). These then decrease because of a lower activity of the enzymes involved in their synthesis, with the consequent reduction in the intensity of the fruity aroma that can be smelled/tasted, in particular the “green” sensory notes (Montedoro and Garofolo, 1984; Solinas et al. 1987; Angerosa and Basti 2001; Montedoro et al. 2003; Angerosa et al. 2004). In very mature fruits (with pigmentation that includes the mesocarp), there is a marked inactivation of the endogenous enzyme activities of the lipoxygenase pathway, with a consequent strong decrease in the volatile compounds in the oil obtained from these fruits (Servili et al. 1990; Angerosa et al. 2004). The total content of the phenolic compounds increases during the early stages of fruit maturation and then decreases more or less rapidly with the intensification of the pigmentation of the epicarp and the mesocarp, according to the cultivar ripening pattern (Table 3.6). The lowest concentrations are seen in oils obtained from very mature olives (Servili et al. 1990; Brenes et al. 1999; Uceda et al. 1999; Salvador et al. 2001; Gimeno et al. 2002; Montedoro et al. 2003; Panaro et al. 2003; Servili et al. 2004; Baccouri et al. 2008b). The hydrophilic phenolic compounds are exclusively present in virgin olive oil, and they are of great importance: As well as being antioxidants, they contribute to the sensory characteristics of the oil, as they are responsible for the bitter and spicy tastes. Indeed, the analytical index of ‘bitterness’ (K225) that is considered in some studies also tends to decrease during ripening (Garcıa et al. 1996). During the development of the fruits, there are also

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variations in the ratios between the individual phenols (Salvador et al. 2001; Briante et al. 2002). As has been shown in fruits of ‘Arbequina’, ‘Morrut’, and ‘Farga’, the concentrations of the aglyconic derivatives of oleuropeine decrease with the beginning of pigmentation, and this coincides with an increase in the phenolic alcohols, such as hydroxytirosol and tirosol (Morello` et al. 2005). The different equilibrium between phenolic compounds toward the most simple forms appears to be connected with a greater activity of the glucosidases and of the esterases during the first stages of fruit ripening (Briante et al. 2002). Generally, tocopherols show a decrease in their content during ripening (Solinas 1990; Di Matteo et al. 1992; Garcıa et al. 1996; Gimeno et al. 2002). Nevertheless, some results show no changes in a-tocopherol or g-tocopherol content (Garcıa et al. 1996; Beltran et al. 2005; Sakouhi et al. 2008). During ripening, there are variations in oxidation stability of the oils that are known to depend on the antioxidant content (phenolic compounds, tocopherols, carotenoids) and fatty acid composition. Generally, there is an increase in the initial phases of ripening and then a decrease or, in some cases, a constant reduction (Servili et al. 1990; Garcıa et al. 1996; Panaro et al. 2003; Baccouri et al. 2008b) (Table 3.6). The content in pigments, such as chlorophylls and carotenoids, decreases during ripening (Servili et al. 1990; Guitierrez et al. 1999; Salvador et al. 2001; Gimeno et al. 2002; Montedoro et al. 2003; Beltran et al. 2005; Baccouri et al. 2008b) (Table 3.6). As ripening proceeds, the oils go from a very green color with intense green-fruity (grassy), bitter, and spicy flavors that are often not very well balanced, to green or green/yellow oils with these various flavors very evident and well balanced, to yellow/green to yellow oils that tend to be organoleptically flat (very weak fruity, bitter, and spicy hints) with an overall sweetness. In general, the score attributed by the panel test tends to decrease in the latest stages of ripening, sometimes to a great extent (Garcıa et al. 1996; Salvador et al. 2001). However, it is worth considering that for the organoleptic characteristics, there is no unique reference of quality, because the best typology is that which best satisfies the section of the market being targeted and the gastronomic use of the oil. In general, oil quality variations during fruit ripening correlate with the level of fruit pigmentation, although with a certain genotypic variability (Servili et al. 1990). In very ripe olives, free acidity and oxidation level of the oil can increase (Pannelli and Montedoro 1988; Pannelli et al. 1990a; Dugo et al. 2004). In very mature fruits, the decrease in pulp texture can

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determine conditions such as marking and bruising during harvesting, transport, and later conservation that allow the breaking up of the cellular compartments, putting the oil in contact with enzymes that can cause endogenous oxidative or hydrolytic processes, particularly if the temperature is high. 2. Harvest Time and Production Objectives. As time of harvest can have very important effects on the quantity and quality of the final product, its choice needs to be made according to production objectives (Famiani et al. 2005). By correctly modifying the harvesting period, it is possible to produce various different types of product: “novel” early oil by early harvest to get to the market at the beginning of the oil season, with a product that is well characterized in terms of color (green) and intensity of green fruity, bitter, and spicy flavors; PDO (Protected Designation of Origin) or PGI (Protected Geographical Indication) oil by harvesting olives at the ripening level that allows one to obtain an oil with qualitative characteristics that satisfy the production rules that must be respected for this kind of product; “typical” or “differentiated” oil by harvesting the olives when the composition of the oil obtained allows the typical or differential characteristics to be accentuated (e.g., particular organoleptic flavors and/or higher levels of antioxidant compounds that provide nutritional value to the oil); standard extra-virgin oil, by harvesting the olives when they have the maximum amounts of oil and, at the same time, the extracted oil satisfies the commercial standards of that type of product; “sweet” oil, ideal for delicate dishes and often preferred by consumers who are not used to olive oil, by harvesting olives at a relatively late stage. It is important to note that the search for a particular oil quality can result in a decrease in the quantity of product obtained. Numerous studies have been carried out to determine the optimal period for mechanical harvesting in different environments in relation to the quantity and quality of the oil (Famiani et al. 1993, 2002; Panaro et al. 2003; Dugo et al. 2004; Tombesi et al. 2006, 2009). In most cases, the best results, in terms of oil quality, are obtained by harvesting the fruits when their pigmentation is limited to the epicarp (surface pigmentation). 3. Harvesting Systems. Fruit damage needs to be minimized in harvesting systems as it can reduce oil quality especially if olives are not processed immediately. Mechanical beaters can result in some damage to the fruit, but trunk shakers do not generally cause damage. Hand-held

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121

harvesting machines do not produce excessive damages if used correctly (Tombesi et al. 1996). In central Italy (Umbria region), the use of hand-held machines and trunk shakers had no negative consequence on the oil quality of ‘Leccino’ and ‘Moraiolo’ in terms of free acidity, peroxide number, ultraviolet absorbencies, fatty acid composition, oxidation stability, and contents in polyphenols and chlorophylls (Pannelli et al. 1990b; Tombesi et al. 1996), while in Sicily, mechanical harvesting of ‘Biancolilla’, ‘Cerasuola’, ‘Nocellara of Belice’, and ‘Tonda Iblea’ was associated with reduced free acidity and increased tocopherol content (Dugo et al. 2004). In Israel, hand-held machine harvesting resulted in an increase of free acidity and peroxide number and a reduction of polyphenol content as compared to oils obtained from manual harvest (Dag et al. 2008b). Olives from irrigated trees appeared to have a higher sensitivity to mechanical wounding that was associated with reduced oil quality (Dag et al. 2008b). Trunk shakers did not completely remove all the olives on the trees, but the fruits that remained on the trees, being generally located in the lower and more shaded parts of the canopy, did not reduce quality because the oil extracted from these fruits have lower total polyphenols (Pannelli et al. 1990b). Mechanical harvesting, particularly with trunk shakers, greatly improves harvesting productivity and makes it possible to concentrate the harvest in the period that is optimal for production objectives. Moreover, the greater productivity provided by mechanical harvesting has positive effects on oil quality by providing sufficient product required by the processing system and avoiding or reducing the need for storage. High-density hedgerow orchards are harvested using straddle harvesters able to remove 90% of fruits, regardless of fruit size, position in the canopy, and ripening stage (Tous et al. 2007). These machines make it possible to concentrate harvesting in a very short time.

V. TECHNOLOGICAL FACTORS AFFECTING EVOO COMPOSITION AND QUALITY Since the occurrence of EVOO hydrophilic phenols and volatile compounds is directly related to the activities of various endogenous enzymes of olive fruit, their concentration in the oil is strongly affected by the extraction conditions. All the steps of EVOO mechanical extraction process may influence its volatile and phenolic composition, but the storage condition of the fruit before the EVOO extraction and crushing

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and malaxation can be considered the most critical point (Capella et al. 1997; Caponio et al. 1999; Angerosa et al. 2004; Servili et al. 2004; Servili et al. 2002b; Servili et al. 2009a,b). A. Olive Fruit Storage The storage of olives in unsuitable conditions, in sacks or in piles, has heavy negative repercussions on the sensory quality of the resulting oils. Microorganisms produce different metabolites that give rise to different sensory defects related to the decrease in concentrations of compounds from the LOX cascade (Angerosa et al. 1996a). Microorganism development is promoted by the temperature reached in the pile and humidity. Clostridia and Pseudomonas develop, producing branched aldehydes, branched alcohols, and their corresponding acids (Solinas et al. 1987; Solinas et al. 1988; Angerosa et al. 1996b); concentrations in a few days overreach the threshold levels for the perception of “fusty” defects. If temperatures are high, the growth of yeasts can produce considerable ethanol and ethyl acetate, leading to the onset of the “winey” defect. The possible presence of Acetobacter is responsible for the “vinegary” defect because of the production of acetic acid Angerosa et al. 1996a). If fruit storage lasts several days, mold may develop, generally Penicillium and Aspergillus (Marsilio and Spotti 1997), whose enzymes interfere with those of the olive fruit involved in the LOX pathway (Kaminski et al., 1974; Wurzenberger and Grosch 1984; B€ orjesson et al. 1993). Mold invasion not only can cause complete rotting of fruits but can reduce production of C6 and C8 compounds (Angerosa et al. 1999b). Storage temperatures of about 5 C in air considerably reduce fungal growth, so olives could be stored for at least 30 days at that temperature without great changes in the sensory quality of the resulting oil (Kiritsakis et al. 1998). Olive storage also has a strong effect in the phenolic degradation in the fruit before mechanical extraction. As consequence, EVOO after olive storage shows lower amount of phenols in comparison to fresh fruits (Angerosa et al. 2004; Servili et al. 2004, 2009a). B. Olive Fruit Crushing Crushing and malaxation are critical points in the mechanical extraction process that affect phenolic and volatile compositions of EVOO. In fact, the main hydrophilic phenols of EVOO, such as

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123

secoiridoid aglycons, originate during this phase by the hydrolysis of oleuropein, demethyloleuropein, and ligstroside, and catalyzed by endogenous b-glucosidases. During malaxation, the concentration of secoiridoid aglycons and phenolic alcohols decreases in olive pastes and in the relative oils, with increasing temperature and process time. The impact of crushing in EVOO phenolic and volatile compounds can be related to the different distribution of endogenous oxidoreductases and of phenolic compounds in the constitutive parts of the olive fruit (pulp, stone, and seed). The peroxidase (POD), in combination with the polyphenoloxidase (PPO), is the main endogenous oxidoreductase responsible for the phenolic oxidation during the process, and it occurs in high amounts in the olive seed. The phenolic compounds, on the contrary, are largely concentrated in the pulp while stone and seed contain little quantities of these substances (Servili et al. 2004, 2007b). Consquently, the crushing methods that reduce seed tissue degradation, such as the stoning process or mild seed crushers, limit the release of POD in the pastes. This prevents the oxidation of hydrophilic phenols during malaxation and improves their concentration in the EVOO (Table 3.7) (Servili et al. 1999a, 2004, 2007). Crushing operative conditions also affect the volatile composition of EVOO (Table 3.8). Almost all volatile compounds responsible for the flavor of high-quality EVOOs arise at the moment of olive pulp tissue disruption. Thus the effectiveness of crushing plays an important role in their production. The use of a hammer mill crusher, or other crushers that produce a more violent grinding of pulp tissues, causes an increase of the olive paste temperature and a reduction of HPL activity. The use of new crushers, such as blade crushers, improves the concentration of volatile compounds, especially hexanal, trans-2-hexenal, and C6 esters, leading to a positive increase of the intensity of “cut grass” and “floral” notes (Table 3.8) (Angerosa et al. 2004). Several researchers have shown that olive stoning during EVOO mechanical extraction process increases the phenolic concentration in EVOO (Angerosa et al. 1999a; Lavelli and Bondesan 2005; Mulinacci et al. 2005) and, at the same time, modifies the composition of volatile compounds produced by the LOX pathway. This increases the concentration of volatile substances related to the “green” sensory notes (Table 3.8) (Servili et al. 2007b). These results are particularly important because they demonstrate that the enzymes involved in the LOX pathway have a different activity in the pulp and in the seed (Table 3.8) (Servili et al., 2007b).

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Table 3.7. Effect of different crushers and the stoning process on EVOOs phenolic compositionz (mg/kg) of ‘Franotio’ cultivar.

Phenolic compounds

Hammer crusher

Pre-crusher þ blade crusher

Crusher with low turns number

Stoned

I Ripening stage (pigmentation index 0.95)y 3,4 DHPEA p-HPEA 3–4 DHPEA-EDA p-HPEA-EDA ( þ )-1-acetoxypinoresinol ( þ )-pynoresinol 3–4 DHPEA-EA Sum of the phenolic compounds

1.0  0.1 11.2  0.7 71.8  1.9 54.3  0.8 31.0  1.7 9.8  0.2 76.0  1.1 255.1  2.4

0.5  0.0 12.8  0.4 78.8  2.3 58.7  0.9 41.9  1.4 11.9  0.2 93.6  2.1 298.2  3.3

2.2  0.0 16.0  0.2 89.6  1.6 60.1  0.8 39.4  0.8 13.8  0.3 98.5  3.0 319.6  3.5

8.0  0.2 19.8  0.3 98.8  2.9 55.4  0.9 43.7  1.2 15.3  0.1 94.3  3.1 335.3  4.4

II Ripening stage (pigmentation index 1.49) 3,4 DHPEA p-HPEA 3–4 DHPEA-EDA p-HPEA-EDA ( þ )-1-acetoxypinoresinol ( þ )-pynoresinol 3–4 DHPEA-EA Sum of the phenolic compounds

3.4  0.1 8.4  0.3 35.7  1.2 32.2  1.5 26.3  1.0 6.6  0.4 54.8  1.9 167.5  2.8

1.1  0.0 13.4  0.7 40.8  2.2 39.2  0.6 29.1  0.4 7.6  0.2 54.6  0.3 185.7  2.5

1.1  0.3 13.8  0.3 44.0  1.9 45.2  0.9 31.4  0.2 8.7  0.6 58.3  1.1 202.4  2.5

4.2  0.1 7.5  0.3 51.6  4.2 40.5  3.7 30.9  0.9 8.3  0.6 65.8  4.3 208.7  6.9

z

Phenolic content is the mean value  SD of three independent experiments. Pigmentation index was determined according to Pannelli et al. (1994). Source: upubl. data. y

C. Olive Paste Malaxation The distribution of phenols between the oil and the water phase, as related to their liposolubility, is not the only mechanism involved in the reduction of the EVOO phenolic concentration during malaxation. The oxidative reactions catalyzed by endogenous oxidoreductases such as PPO and POD, which promote the phenolic oxidation in the pastes during this step, strongly affect their concentration in the oily phase. The enzyme inhibition obtained by the reduction of the O2 concentration in the covered malaxers improves the concentration of hydrophilic phenols in the olive pastes and in the corresponding EVOO. In this context, the O2 control during malaxation can be considered a new technological parameter that, in combination with the traditional ones (time and temperature of the process), can be used to optimize the EVOO phenolic and volatile concentrations (Tables 3.9 and 3.10) (Servili et al. 2004,

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Table 3.8. Effect of different crushers and the stoning process on the volatile compositionz (mg/kg) of ‘Frantoio’ EVOO. Volatile compounds

Hammer crusher

Pre-crusher þ blade crusher

Crusher with low turns number

Stoned

Aldehydes Pentanal 236.5  4.0 273.4  2.1 17.9  1.0 35.7  2.7 Hexanal 280  2.9 511.4  35.7 553.7  0.3 748.4  27.9 2-Hexenal (E) 43600.6  327.0 44718.9  208 39811.6  587.0 27866.0  705.5 2,4-Hexadienal (E,E) 19.4  0.1 42  3.5 341.6  14.4 256.5  12.1 Alcohols 1-Pentanol 2-Penten-1-ol (E) 1-Penten-3-ol 1-Hexanol 3-Hexen-1-ol (Z) 3-Hexen-1-ol (E)

167.0  5.2 166.011.3 960.3  53.2 1788  57.0 88.4  22.2 22.2  0.2

94.5  4.7 91.4  5.1 899  43.3 2152  74.0 103.6  10.1 20.2  0.1

23.3  0.7 52.4  3.5 522  49.2 512  41.0 49.2  2.3 9.9  0.2

20.8  1.7 12.8  2.1 114.5  4.6 405  18.4 163.7  8.0 21.5  2.3

z Data are the mean values of three independent experiments,  SD. Source: unpubl. data.

2008, 2009b). The time of exposure of olive pastes to the air contact (TEOPAC) was studied as process parameter to regulate the averaged concentration of O2 in the paste and as consequence the phenolic amount in the EVOO (Servili et al. 2003a, 2003b). The natural production of inert gas, such as CO2, due to the olive cell metabolism during malaxation may be combined with the use of nitrogen or argon to reduce the O2 contact with the olive pastes during malaxation (Parenti et al. 2006a, 2006b; Servili et al. 2008). The application of new technologies, such as the EVOO mechanical extraction from destoned pastes, improves the oil phenolic concentration, confirming the relationships between the control of oxidative reactions during extraction process of the EVOO and its phenolic content. Since the POD is highly concentrated in the olive seed, the removal of the stones before the malaxation partially removes the POD activity in the pastes and reduces the enzymatic degradation of the phenols in the oils during this phase, thus increasing their concentration in the EVOO and its oxidative stability (Servili et al. 2007b, 2009b). The oxidative reactions occurred in the pastes during the malaxation explains the relationships between EVOO phenolic concentration and malaxing temperatures (Servili et al. 2004, 2009a,b). The O2 present in the pastes during the malaxation activate POD and PPO that oxidize phenolic compounds according to temperature and, as a consequence, reduce their concentration in EVOOs obtained by pastes malaxed at high

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Table 3.9. Phenolic compositionz (mg/kg) of EVOOs obtained after malaxation in different initial atmosphere compositions. Initial O2 partial pressure in the malaxer chamber headspace (kPa) Phenolic compounds

0a

30b

50

100

Ogliarola cv. 3,4-DHPEA 1.0  0.02a p-HPEA 3.1  0.03a 3,4-DHPEA-EDA 247.7  1.9a p-HPEA-EDA 126.4  0.4a (+)-1-Acetoxypinoresinol 21.0  0.4a (+)-Pinoresinol 6.8  0.07a 3,4-DHPEA-EA 212.2  0.1a

0.8  0.05b 0.6  0.004c 0.8  0.01d 3.1  0.7a 4.1  0.001b 4.2  0.03b 235.2  5.5b 117.8  0.8c 118.1  0.03c 118.6  5.9b 86.3  0.3c 85.4  0.62c 25.4  1.5b 22.3  0.3ac 24.1  0.09bc 7.6  0.3b 7.0  0.04a 7.1  0.03a 186.4  4.8b 100.9  1.1c 98.19  0.2c Coratina cv.

3,4-DHPEA 6.8  0.7a 3.2  0.8b 4.4  0.7b p-HPEA 10.0  1.1a 5.9  0.5bc 7.8  0.9b 3,4-DHPEA-EDA 478.9  16.2a 437.7  14.3b 343.1  11.5c p-HPEA-EDA 144.2  1.8a 135.3  1.59b 126.2  1.4c (+)-1-Acetoxypinoresinol 30.8  0.94a 25.8  2.8b 29.2  0.4ab (+)-Pinoresinol 8.1  0.03ab 8.0  0.04a 8.6  0.4b 3,4-DHPEA-EA 475.6  13.9a 361.9  14.1b 339.2  6.9b

1.4  0.2c 4.4  0.4c 229.9  9.2d 125.1  3.1c 27.1  0.5ab 7.9  0.1a 170.6  2.3c

a

Saturated with N2. Corresponding to the air composition. z Data are the mean values of three independent experiments  standard deviation. Values in each row having different letters (a–d) are significantly different from one another (p < 0.01). Source: Servili et al. 2008. b

temperatures. The traditional malaxers, which contain high amounts of O2 dissolved in the paste during the process due to the air contact represent a classical example of the relationship between high temperatures and EVOO phenolic loss. Low amounts of O2, on the contrary, inhibit the oxidative reaction of phenols during the malaxation; in this case, their concentration in the EVOO increases with high temperatures, because of a higher solubility of such substances in the EVOOs (Servili et al. 2008, 2009a,b). Interactions between polysaccharides and phenolic compounds in the olive pastes may also be involved in the loss of such substances during the process of malaxation. Polysaccharides may link the phenols, thus reducing their release in the oil during crushing and malaxation. In this regard, it has been shown that the use of technical enzymatic preparations containing cell wall–degrading enzymes

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Table 3.10. Volatile compositionz (mg/kg) of EVOOs obtained after malaxation in different initial atmosphere compositions. Initial O2 partial pressure in the malaxer chamber headspace (kPa) Volatile compounds

0

30b

50

100

Ogliarola cv. Aldehydes 2-Pentenal (E) Hexanal 2-Hexenal (E) Alcohols 1-Pentanol 2-Penten-1-ol (E) 1-Penten-3-ol 1-Hexanol 3-Hexen-1-ol (E) 3-Hexen-1-ol (Z) 2-Hexen-1-ol (E)

291.5  31.8ab 343.0  31.1a 247.5  11.7b 269.5  13.5b 939.5  9.2a 1546.0  200.8b 1011.5  27.6a 1499.5  16.3b 43645.0  912.2a 39130.0  1054.7b 37315.0  233.3b 38170.0  1258.7b 28.5  2.1a 55.5  3.5a 567.0  17a 8357.0  102.6a 35.0  1.2a 286.5  4.9a 7662.5  75.7a

128.0  6.8b 63.0  4.6a 871.0  4.7b 9699.0  106.1b 41.0  3.5a 434.0  20.6b 8616.0  87.9b

122.5  3.5b 158.0  1.4c 50.5  9.2ab 38.5  6.4b 690.0  1.4c 809.5  3.5d 11660.0  99c 13675.0  63.6d 47.5  2.1b 61.5  2.1c 341.0  11.3c 400.5  7.8d 9355.0  353.6c 9780.0  60.8c

Coratina cv. Aldehydes 2-Pentenal (E) Hexanal 2-Hexenal (E) Alcohols 1-Pentanol 2-Penten-1-ol (E) 1-Penten-3-ol 1-Hexanol 3-Hexen-1-ol (E) 3-Hexen-1-ol (Z) 2-Hexen-1-ol (E)

548.5  16.3ab 509.7  5.8b 636.7  17.9c 613.0  51.2ac 1187.0  9.9a 1624.3  30bc 1532.1  27.3b 1744.0  121.2c 51565.0  827.3a 52900.0  565.7ab 54340.5  355.7b 53920.0  332.1b 40.0  5.7a 87.5  0.7a 890.0  2.8a 2326.0  49.5a 25.5  0.7ab 561.0  4.2a 3654.5  30.4a

54.3  5b 67.0  0.2b 820.0  1.2b 3694.2  2b 31.6  3.8a 513.6  9.6b 5905.0  321b

39.4  5a 105.8  5.7c 1093.5  33.7c 1788.0  57.2c 20.0  1.9b 486.3  11.1b 3350.1  80.5a

48.0  3.2ab 105.0  8.3c 1185.0  91.2c 2170.0  123.1a 21.0  1.9b 498.0  31.2b 4185.0  35.6c

a

Saturated with N2. Corresponding to the air composition. z Data are the mean values of three independent experiments  standard deviation. Values in each row having different letters (a–d) are significantly different from one another (p < 0.01). Source: Servili et al. 2008. b

during the malaxation can improve the EVOO phenolic concentration (Ranalli and De Mattia 1997; Vierhus et al. 2001). Furthermore, it has been observed that the enzymes involved in the LOX pathway remain active during the malaxation process since the concentration of volatile compounds increases in the pastes (Esposto et al. 2008; Servili et al. 2009a). The analysis of the composition of EVOOs produced from traditional and stoned olive pastes confirms that

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the amounts of C6 unsaturated aldehydes were higher in “stoned” EVOOs than in the “traditional” ones because of the presence of the seed responsible of the C6 alcohols increase during the traditional process (Servili et al. 2007b). Time and temperature of malaxation affect the volatile profile and therefore the sensory characteristics of the resulting EVOOs (Angerosa et al. 2004; Servili et al. 2009a,b). The increase of C6 and C5 carbonyl compounds, especially of hexanal, which, due to its low odor threshold, represents an important contributor to the olive oil flavor, is the main effect of the malaxation time, whereas high temperatures of malaxation promote a fall of esters and cis-3-hexen-1-ol and an accumulation of hexan-1-ol and trans-2-hexen-1-ol, both considered by some authors as eliciting odor not completely agreeable (Angerosa et al. 2004; Servili et al. 2009b). In addition, high temperatures in the malaxation step make active the amino acid conversion pathway with production of considerable amounts of 2-methyl-butanal and 3-methyl-butanal, but without accumulation of corresponding alcohols correlated with the “fusty” defect. The sensory analysis of the relative EVOOs highlights a weakening of typical “green” attributes with the prolonging of malaxation time and of all sensory notes with high temperatures during the malaxation (Angerosa et al. 2004; Servili et al. 2009b). D. EVOO Extraction Systems Extraction systems, such as pressure and centrifugation, play an important role in the EVOO phenolic and volatile composition. The dilution water added to the olive pastes during the centrifugation modifies the distribution of hydrophilic phenols between oil and water, enhancing their loss through the water phase. Several studies have been carried out to compare the traditional three-phase decanter with the new two-phase decanter (Di Giovacchino et al. 1994; Montedoro 1996; Ranalli and Angerosa 1996; Stefanoudakii et al. 1999b; Servili et al. 2002b). Similar results were obtained, using Spanish and Greek cultivars, by other authors (Di Giovacchino et al. 2001; Garcia et al. 2001). An increased concentration of phenols was also observed in EVOOs extracted by threephase decanters at low water addition as compared to the traditional three-phase centrifuges (Table 3.11) (Amirante et al. 2001). The increase of the EVOO phenols concentration was observed according to the reduction of water addition used during the mechanical extraction process by three- and two-phase decanters (Montedoro 1996). Other results obtained using two typical Italian cultivars, such as ‘Coratina’ and ‘Ogliarola’, provide evidence that higher concentrations

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Table 3.11. Effect of the water reduction during centrifugation on EVOOs phenolic composition (mg/kg)z.

Phenolic compounds 3,4-DHPEA p-HPEA Vanillic acid Caffeic acid 3,4-DHPEA-EDA p-HPEA-EDA Lignans 3,4-DHPEA-EA Total polyphenols Induction time [h]

Coratina cv. Two phases 0.9  0.02a 3.7  0.07a 0.4  0.01a 0.2  0.01a 522.2  13.5a 78.2  0.5a 38.4  0.1a 351.7  11.0a 673.0  4.0a 17.8  0.1a

Three phases 0.6  0.08b 2.3  0.08b 0.2  0.01b 0.1  0.02b 427.2  13.8b 67.3  2.5b 35.6  1.1b 244.9  13.6b 585.0  7.0b 15.5  0.2b

Ogliarola cv. Two phases 0.7  0.1a 3.3  0.10a 0.3  0.01a 0.1  0.01a 30.1  1.0a 21.0  0.8a 48.0  3.4a 68.0  6.0a 304.0  5.0a 5.2  0.1a

Three phases 0.5  0.1a 4.2  0.1b 0.1  0.05b 0.2  0.03b 18.5  0.6b 22.4  0.3a 46.7  5.7a 52.0  3.1b 263.0  4.0b 4.6  0.1b

z

Data are the mean values  SD of three independent experiments  standard deviation. Values in each row within cvs. having different letters are significantly different from one another (p < 0.01). Source: Servili et al. 2002.

of hydrophilic phenols in EVOOs were obtained using two-phase decanters as compared to the traditional three-phase centrifuges (De Stefano et al. 1999; Servili et al. 2002a). E. EVOO Storage During storage, the phenolic composition of the EVOO is modified by the endogenous enzymatic activities contained in the cloudy phase. These enzymes may reduce the “pungent” and “bitter” sensory notes, the intensity of which is strictly to the aglicon secoiridoids’ content and, at the same time, can cause olfactive and taste defects. The oil filtration, partially removing water and enzymes from EVOOs, allows the stabilization of EVOO phenolic content during storage (Montedoro et al. 2005). The olive oil profile changes during storage because of the simultaneous drastic reduction of compounds from LOX pathway and the neoformation of volatile compounds responsible for some common defects known as “rancid,” “cucumber,” and “muddy sediment” (Morales and Aparicio 1997, Angerosa et al. 2004; Servili et al. 2009b). Among saturated aldehydes, nonanal and above all hexanal increase in parallel to the oxidation process, but this last cannot be considered a useful marker of oxidation since it is also present in the aroma of high-quality EVOOs (Angerosa et al. 2004; Servili et al. 2009b). Furthermore, the presence of sediment consequent to unfiltered olive oil decantation

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during its storage can determine, under suitable temperature conditions, the production of unpleasant compounds responsible for the typical “muddy sediment” defect due to the fermentation that produces compounds, likely of the butyric kind (Angerosa et al. 2004; Servili et al. 2009b).

VI. SUMMARY AND CONCLUSIONS The composition of olive oil results from a multivariate interaction in which genotype-, environment-, and agronomic-dependent factors are involved. The genotype may control genetic traits accounting for the rate pattern of fruit growth, oil accumulation in mesocarp cells, and fruit ripening, while the genotype  environment interaction changes the rate of fruit growth, oil accumulation, and fruit ripening pattern. The latter accounts for large changes in oil composition and sensory features. The influence of genotype is linked to differences in the fruit growth and ripening pattern, although all those factors that may have an influence on fruit size, flesh/pit ratio, and relative growth rate have a lower and more erratic influence on the olive oil composition. The genotype is the primary source of sensory differences. This has been proven for most of the cultivars, giving them a specific role in gastronomy. The influence on olive oil composition of environmental factors, such as temperatures during fruit growth and ripening or water availability, may also be a function of changes in the fruit growth and ripening patterns and of the oil accumulation rate pattern. Some facts have been generally recognized, such as the changes of saturated versus unsaturated fatty acids ratio in relation to temperature and latitude or the progressive reduction of polyphenols content in the oil along with fruit ripening or with the increase of water availability. Crop load influences the fruit ripening rate pattern and the rate pattern of oil accumulation, and this may account for differences in oil composition related to polyphenols and fatty acid content. From a historical point of view, the recognition of EVOO geographical and genetic origin was not a major cultural and commercial issue of olive oil production and trading, as it always has been for wine production. The multivarietal composition of the traditional groves, due to the biology of flowering and pollination of the species, the highly differentiated orchard systems and fruit harvesting periods and methods, as well as the different oil extraction technologies and storage systems, and, eventually, the historical separation between the olive farmers and the oil mill industry, together made it very difficult to establish effective

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policies endorsing the geographical and genetic origin of the EVOO as a cultural and commercial value recognized by consumers. At present, even at the legislative level, the EVOO origin has become a central issue in the marketing strategies, particularly for those Mediterranean countries with a wide olive germplasm and rich gastronomy. However, provenience-related differences of EVOO quality were claimed even by Pliny the Elder during the Roman age, and modern research activity has made it possible to appreciate the large variability of EVOO composition and properties. Nevertheless, it is difficult to understand the interrelation of the different sources of variation and to apply appropriate strategies for a consistent and clear EVOO production and identification. This is the greatest paradox of EVOO production: typical cultivar-related sensory properties are largely unknown or unrecognizable by consumers and still are underutilized in marketing strategies. More information is needed on the genotype and environment influence on fruit ripening and EVOO components evolution in order to regulate the ripening process and standardize the characteristics that make any EVOO unique and typical for its geographical and genetic origin. On the basis of our understanding of the technological factors affecting EVOO quality, we suggest the following approach to improve this rapidly expanding industry: 1. Improve management of oil mechanical extraction processes for working olive pastes with and without olive stones. 2. Improve the malaxation efficiency through technological coadiuvants (inert materials and enzymatic preparations) to increase coalescence and, as a consequence, the recovery of oil and phenolic compounds. 3. Apply technologies, in malaxation particularly, to improve the efficiency of thermal exchange. 4. Control and regulate oxygen percentage in contact with the olive paste during the malaxation to optimize the phenolic content as well as the flavor of EVOO. The olive oil consumption has increased, worldwide, from 1990 to 2008 by 70%, moving from 1,666 Mt to 2875 Mt. Spain, Italy, and Greece account for 77% of the olive oil production and for 65% of its consumption. Per capita consumption ranges from 25 l year
1 in Greece to 15 l year
1 in Spain and Italy, 5.5 l year
1 for the European Community, and 0.6 l year
1 for the United States. The United States is the fourth largest market, and the oil consumption has increased, in the same

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period, three times (88 Mt in 1990 and 250 Mt in 2008) (IOC 2008). This increase in oil use has been accompanied by a general decrease of its price at world level. This creates large problems for those countries such as Italy, which have high labor costs, a very traditional industry based on small crop areas of single farmers, and thousands of artisan mills (5 t day
1). Spain and the New World producers are focusing on highdensity orchards and large mills, which makes it possible to reduce costs. Different strategies in Spain and Italy are, then, related to the structure of their olive oil industry. For instance, 66% of the olive farms in Italy are dedicated to self-consumption, and the variability of cultivars and environments explain the great number of PDO in Italy (37, which means 43% of PDO dedicated to olive in the European Union). Hundreds of new brands appear in the market every year, and consumers are likely to expect a large opportunities to choose high-quality oils with different levels of price. It is hoped that the role of genotype (cultivar) and cultivar  environment interaction will be more and more appreciated by consumers and that the fraudulent marketing of inferior products will be eliminated. LITERATURE CITED Allalout, A., D. Krichene, K. Methenni, A. Taamalli, I. Oueslati, D. Daoud, and M. Zarrouk. 2009. Characterization of virgin olive oil from super intensive Spanish and Greek varieties grown in northern Tunisia. Sci. Hortic. 120(1):77–83. Amirante, P., P. Catalano, R. Amirante, G. Montel, G. Dugo, V. Lo Turco, L. Baccioni, D. Fazio, A. Mattei, and F. Marotta. 2001. Estrazione da paste denocciolate. Olivo Olio 43:48–55. Angerosa, F. 2000. Sensory quality of olive oils. p. 355–392. In: J. Harwood,and R. Aparicio (eds.), Handbook of olive oil. Analysis and properties. Aspen Publ., Gaithersburg, MD. Angerosa, F., and C. Basti. 2001. Olive oil volatile compounds from the lipoxygenase pathway in relation to fruit ripeness. Ital. J. Food Sci. 13:421–428. Angerosa, F., and C. Basti. 2003. The volatile composition of samples from the blend of monovarietal olive oils and from the processing of mixtures of olive fruits. Eur. J. Lipid Sci. Technol. 105:327–332. Angerosa, F., C. Basti, R. Vito, and B. Lanza. 1999a. Effect of fruit stone removal on the production of virgin olive oil volatile compounds. Food Chem. 67:295–299. Angerosa, F., L. Camera, N. D’Alessandro, and G. Mellerio. 1998a. Characterization of seven new hydrocarbon compounds present in the aroma of virgin olive oils. J. Agr. Food Chem. 46:648–653. Angerosa, F., N. D’Alessandro, and F. Corana. 1996a. Characterization of phenolic and secoiridoid aglycons present in virgin olive oil by gas chromatography-chemical ionization mass spectrometry. J. Chromatogr. 736:195–203. Angerosa, F., N. D’Alessandro, C. Basti, and R. Vito. 1998b. Biogeneration of volatile compounds in virgin olive oil: Their evolution in relation to malaxation time. J. Agr. Food Chem. 46:2940–2944.

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4 Quality and Yield Responses of Deciduous Fruits to Reduced Irrigation M. H. Behboudian Institute of Natural Resources (INR 433) Massey University Palmerston North, New Zealand J. Marsal, J. Girona, and G. Lopez Irrigation Technology Institut de Recerca i Tecnologia Agroaliment
aries 191 Av. Alcalde Rovira Roure E-25198 Lleida, Spain

ABBREVIATIONS I. INTRODUCTION II. DEFINITIONS III. STONE FRUITS A. Peach 1. Vegetative and Reproductive Characteristics 2. Regulated Deficit Irrigation 3. Whole-Season Deficit Irrigation 4. Partial Rootzone Drying B. Apricot C. Cherry D. Prune and Plum IV. POME FRUITS A. Apple 1. Yield 2. Fruit Quality 3. Partial Rootzone Drying B. Pear V. CONCLUSIONS AND FUTURE PROSPECTS ACKNOWLEDGMENTS LITERATURE CITED Horticultural Reviews, Volume 38 Edited by Jules Janick Copyright
2011 Wiley-Blackwell 149

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ABBREVIATIONS CCL CI DAFB DI ED ET FF IEC LCL LD NI PRD RDI RDI-I RDI-II RDI-III RDI-PH RDI-I þ II RDI-II þ PH RWC SPI TSS

commercial crop load conventional irrigation, commercial irrigation days after full bloom deficit irrigation early deficit evapotranspiration fruit firmness internal ethylene concentration light crop load late deficit no irrigation partial rootzone drying regulated deficit irrigation RDI applied at Stage I of fruit development RDI applied at Stage II of fruit development RDI applied at Stage III of fruit development RDI applied at postharvest period RDI applied during stages I and II of fruit growth RDI applied at Stage II þ postharvest period relative water content starch pattern index total soluble solids

I. INTRODUCTION Water supplies are becoming limited worldwide, and they will not be sufficient to meet the growing demands by 2025 (Postel 1998). Although the irrigation water use varies from country to country, on the world scale, irrigation consumes at least 85% of all the water used (van Schilfgaarde 1994). Even a small saving of water in irrigation could therefore make substantial amounts available for other purposes. Reduced irrigation will be necessary in water-limited regions. However, saving of water in irrigation should not compromise yield and quality of agricultural products. This is especially true for deciduous fruit crops whose quality will command consumers’ preference. This chapter addresses quality and yield responses of deciduous fruits to the existing methods of reduced irrigation. Although these methods have been applied since early 1980s, no specific review has been published on

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this topic in the widely used horticultural publications. The topic has had limited coverage as a part of other general reviews by Behboudian and Mills (1997) and by Naor (2006). The fruit quality aspect has been addressed, to a more limited extent than here, as a book chapter by Behboudian et al. (2005). Quality and yield responses of deciduous fruits to reduced irrigation may vary according to the selected reduced irrigation technique. Growth pattern of the crop can also play a significant role in the yield and quality responses to reduced irrigation. We have therefore considered it necessary to distinguish among different species of deciduous fruit trees and different existing reduced irrigation techniques. This chapter emphasizes the most important stone fruits (peach, apricot, cherry, prune, and plum) and pome fruits (apple and pear) under different reduced irrigation techniques. Differences in fruit growth habits of species have necessitated using different formats of presentation, especially for peach and apple. Differences in cultivar, tree age, climate, and cultural practices also make it difficult to explain contradictory results from various experiments, but we have attempted to do so whenever possible.

II. DEFINITIONS Reduced irrigation in this chapter alludes to different versions of deficit irrigation (DI) and to partial rootzone drying (PRD). DI is a system of managing soil water supply to impose periods of predetermined plant or soil water deficit that can result in some economic benefit (Behboudian and Mills 1997). It involves giving less water to the plant than the prevailing evapotranspiration (ET) demand at selected times during the growing season. The term regulated deficit irrigation (RDI) is normally used to denote DI of trees early in the season before rapid fruit growth starts. Late-season RDI refers to the application of RDI late in the season but before harvest, with a duration depending on the purpose, species, and environmental conditions. This term has been used by some authors and will be specified in each case. The concept of RDI was initially studied in Australia on peach (Chalmers et al. 1981) and was used as a management strategy to control vigor in high-density plantings of ‘Golden Queen’ peach (Chalmers et al. 1981) and ‘Bartlett’ pear (Mitchell et al. 1984). In these experiments, controlled water deficit was established in the tree during rapid shoot or slow fruit growth. Trees were initially irrigated at a rate lower than the ET with water being made available to the plant just as the fruit started the rapid growth phase. Application of RDI had positive effects on fruit growth and on final yield.

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In this chapter, we use the terms RDI and DI interchangeably, as done in a previous review (Behboudian and Mills 1997) and also by a large number of authors cited here. PRD was introduced in Australia by Dry and Loveys (1998) and was applied to grapevine (Vitis vinifera). It involves watering only a part of the rootzone at each irrigation episode, leaving the other part to dry to a predetermined level of soil moisture. The next irrigation will be for the previously unirrigated part of the rootzone. The physiological basis of PRD is well described in the literature, including Dry and Loveys (1998). Briefly, roots in drying soil produce some chemical messages (e.g., abscisic acid) that will be transported to the shoot causing partial closure of stomata. Transpiration will then decrease and so does photosynthesis but to a lower extent. Plant water potential will expectedly equilibrate with the wetter part of the rootzone (Hsiao 1990), the irrigated part, and therefore plant water potential will presumably be higher than classical DI where the same amount of water is applied to the entire rootzone.

III. STONE FRUITS The effects of reduced irrigation are reviewed on peach, cherry, apricot, prune, and plum. A. Peach Peach (Prunus persica) is grown both for the fresh market and for the processing industry. Yield and fruit quality determine the economic value of peach production. Yield is mainly related to crop load, fruit size at harvest, and the weight of marketable fruit. In the processing market, growers are usually paid in terms of total yield. They therefore manage their orchards to obtain the maximum yield for the highest return. There is general agreement that total yield and fruit size in peach trees increase with more irrigation water (Gregory 1988). For this reason, commercial peach orchards usually are managed under full irrigation. However, better quality is not necessarily achieved with applying more water (Fereres and Soriano 2007). Fruit quality is increasingly important, and fruit growers may expect receiving premium price for high-quality produce. This fact offers the opportunity of applying deficit irrigation in peach orchards. Moreover, the vegetative and reproductive characteristics of peach trees permit application of DI during specific periods of the growing season that are less sensitive to water stress without adverse effects on the yield. Hence before describing the effects of DI on yield and

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quality, it is important to describe the vegetative and reproductive characteristics of peach trees. 1. Vegetative and Reproductive Characteristics. In peach trees, as well as in other stone fruit trees, the growing season starts with the bloom. Flowers set fruit shortly after full bloom and before leaves are fully expanded. Peach fruit growth consists of three distinct developmental stages (Chalmers and van den Ende 1975): (1) reproductive cell division and initial fruit growth (Stage I); (2) intermediate stage of slow growth and pit hardening (Stage II); and (3) final period of rapid fruit growth and high accumulation of fresh and dry weight that concludes with fruit maturity and ripening (Stage III). In mid- to late-maturing cultivars, the most shoot extension occurs during Stages I and II. In early-maturing peach cultivars, usually there is a second important flux of shoot extension after harvest. No significant shoot growth has been reported during Stage III unless trees are defruited. 2. Regulated Deficit Irrigation. RDI in peach trees is reviewed here during Stage I (RDI-I), Stage II (RDI-II), Stage III (RDI-III), and postharvest (RDI-PH). Combined RDI during Stage I and II (RDI-I þ II) and combined RDI during Stage II and postharvest (RDI-II þ PH) is also covered. Stage I of Fruit Development (RDI-I). Water stress during flowering and fruit set may inhibit fertilization (Hsiao 1993), reduce fruit set, and increase fruit abscission (Powell 1974). However, when RDI is applied some days after the completion of flowering and fruit set, RDI-I trees could have the same yield as conventionally irrigated (CI) trees with a considerable reduction in vegetative growth and saving of water. For example, Li et al. (1989) applied RDI-I to ‘Merrill Sundance’ peach from 50 days after full bloom (DAFB) until the onset of Stage II in the semihumid climate of Valence, Rhone Valley, France. DI did not reduce yield and resulted in water saving of 553 m3ha1. However, another study on the effects of RDI-I reported substantial negative effects on fruit growth (Girona et al. 2004). In this case, RDI-I was applied to young ‘Groc de l’Escola’ peach trees in the semiarid climate of Catalonia, Spain. This finding may indicate that RDI-I could be more suitable for peach orchards growing in semihumid climates with low evaporative demand and adequate precipitation during Stage I. Effect of RDI-I on fruit quality parameters has been mostly negative (Li et al. 1989) (Table 4.1). Stage II of Fruit Development (RDI-II). Application of RDI-II to ‘Golden Queen’ peach led to reduced tree vigor, increased yield, and

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Table 4.1. Summary of the most important effects of reduced irrigation on peach quality in terms of fruit firmness (FF), skin color, total soluble solids (TSS), titratable acidity (TA), TSS/TA ratio, and pH of the juice. Maturity indices Strategy

Irrigation water applied (%)

RDI, Stage I Li et al. 1989 0 Li et al. 1989 0 RDI, Stage II Li et al. 1989 40 Li et al. 1989 34 Gelly et al. 2003 35 Gelly et al. 2004 35 Domingo et al. 2007 32 Lopez et al. 2008 0 RDI, Stage III Li et al. 1989 43 Li et al. 1989 34 Besset et al. 2001 64 Besset et al. 2001 23 Mechlia et al. 2002 88 Domingo et al. 2007 73 Mercier et al. 2009 60 Mercier et al. 2009 40 RDI, Stage I þ II Li et al. 1989 0 þ 40 Li et al. 1989 0 þ 34 Mechlia et al. 2002 90 Buendıa et al. 2008 25 þ 25 RDI, postharvest Gelly et al. 2003 35 Gelly et al. 2004 35 RDI, Stage II þ postharvest Gelly et al. 2003 35 þ 35 Gelly et al. 2004 35 þ 35 DI Stage I þ II þ III Li et al. 1989 0 þ 40 þ 43 Li et al. 1989 0 þ 34 þ 34 Crisosto et al. 1994 50 þ 75 þ 50 Mechlia et al. 2002 77 þ 77 þ 77 50 þ 50 þ 50 O’Connell et al. 2006

FF

Skin color

Chemical parameters TSS

TA

TSS/TA

ns   ns

ns þ

pH

ns ns

ns  ns ns

þ þ ns

ns ns þ þ þ ns

ns

ns ns ns

þ þ  þ þ þ ns þ

ns

ns

ns ns ns ns

ns

ns

ns ns

þ ns

þ ns

ns ns

ns ns

ns 

þ þ

þ ns

ns 

ns þ

ns

þ ns þ þ þ

ns

ns 

ns

ns

ns

() indicates a significant decrease in the selected parameter in response to deficit irrigation. ( þ ) indicates a significant increase in the selected parameter in response to deficit irrigation. (ns) indicates no significant effect of deficit irrigation in the selected parameter.

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compensatory fruit growth after reestablishment of full irrigation (Chalmers et al. 1981; Mitchell and Chalmers 1982). The compensatory fruit growth was attributed to active osmotic adjustment during deficit (Chalmers 1989). No report is available on osmotic adjustment in deficitirrigated peach but it has been demonstrated in the DI fruit of ‘Nijisseiki’ Asian pear by Behboudian et al. (1994) and in ‘Braeburn’ apple fruit by Mills et al. (1997). RDI-II has been explored in semiarid and arid climates with the aim of conserving irrigation water. Although no negative effects on yield have been observed (Williamson and Coston 1990; Boland et al. 1993, 2000; Gelly et al. 2003, 2004; Girona et al. 2003, 2005), in general, RDI-II studies were not able to reproduce the increases of yield reported by Chalmers et al. (1981) and by Mitchell and Chalmers (1982). This discrepancy may be associated with the duration and degree of water stress (Johnson and Handley 2000). RDI-II will produce different results depending on the way trees undergo water stress and the way they recover from water stress. In order to obtain yield benefit, RDI-II should be applied some time before the initiation of Stage II, as done by Mitchell and Chalmers (1982). This will ensure that trees are under water stress at the onset of Stage II, increasing the effectiveness of RDI in reducing vegetative growth. It is important that trees recover from water stress before Stage III. This may be easy to accomplish in shallow soils, such as those used by Chalmers et al. (1981) and by Mitchell and Chalmers (1982). It is more difficult to do so in deeper soils (Girona et al. 1993). Soil depth has played a significant role in some experiments in which negative influences on yield were observed following RDI-II (Goldhamer et al. 2002; Lopez et al. 2008). When RDI-II is applied in deep soils, it may be difficult to refill the soil profile at the beginning of Stage III, which is sensitive to water stress (Li et al. 1989; Berman and DeJong 1996; Genard and Huguet 1996; Besset et al. 2001; Girona et al. 2004). If some level of water stress is still present during Stage III, fruit may not achieve its potential size, and negative effects on yield could be observed (Goldhamer et al. 2002; Lopez et al. 2008). Crop load is another factor influencing fruit growth recovery following RDI-II. Tree water status at high crop load has been reported to be more sensitive to irrigation restrictions during Stage III than at low crop loads (Berman and DeJong 1996; Naor et al. 1999, 2001). A significant negative correlation has been found between crop load and soil water content at harvest following RDI-II (Lopez et al. 2008). This may indicate that the amount of water available for fruit growth at high crop load after reestablishing full irrigation was below the threshold required for optimum fruit growth. This finding explains the observed limitations in yield for RDI-II trees under heavy crop loads (Lopez et al. 2008). It may also explain the

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positive effects of RDI in Chalmers et al. (1981), in which low crop loads of about 34 fruit per tree may have facilitated water stress recovery during Stage III. Another justification for the application of RDI-II is a potential improvement in fruit quality. Significant improvements in fruit quality were measured following RDI-II in ‘Andross’ peach growing in semiarid climate and deep soil conditions of Catalonia, Spain (Gelly et al. 2003, 2004). In the latter experiment, fruit under moderate levels of water stress during Stage II had significantly higher total soluble solids (TSS) than CI fruit both at harvest and after cold storage. The average values (MPa) of midday stem water potential (Ystem) for DI and CI were, respectively, 1.1 and 0.6. TSS increased from 11 Brix to 12 Brix under RDI while no effect on titratable acidity (TA) was observed. The TSS/TA ratio therefore increased (Gelly et al. 2004), improving fruit quality, as this ratio is closely related to consumer acceptance of peach (Crisosto and Crisosto 2005). The increment in TSS following RDI-II was explained by a passive concentration of sugars after partial fruit dehydration; this finding was later supported in a review by Naor (2006a). Therefore, if partial fruit dehydration does not occur in response to RDIII, the positive effects on TSS accumulation may not be realized. This may be the case in RDI-II studies in which no obvious effect on TSS were observed (Table 4.1) (Li et al. 1989). It is difficult to draw conclusions about the effects of RDI-II on TA. Results on peach fruit acidity in response to RDI are inconsistent. In some studies water stress was associated with lower fruit acidity (Gelly et al. 2004; Domingo et al. 2007) while in others no significant effect was reported (Gelly et al. 2003; Lopez et al. 2008). Advances in fruit maturity have been observed following RDI-II. ‘Andross’ peach under RDI-II showed higher red coloration than CI fruit (Gelly et al. 2003, 2004). Part of the enhancement in red coloration has been attributed to an indirect effect of water stress via a reduction in vegetative growth (Naor 2006a). Since water stress reduces vegetative growth, light environment within the tree crown may improve fruit coloration eventually (Marini et al. 1991; Lewallen and Marini 2003; Dani 2007). Effect of RDI-II on fruit skin color may also vary with cultivar. While in ‘Andross’ a positive effect on fruit skin color was observed in response to RDI-II (Gelly et al. 2003, 2004), no significant effects were observed in ‘O’Henry’ (Lopez et al. 2008). For this reason, other maturity indices not related to cultivar or light environment should be evaluated. This may include fruit ethylene production and firmness. Advanced fruit maturity in response to RDI-II was observed by evaluating fruit ethylene production, which was higher in fruit under RDI (Gelly

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et al. 2003). However, this finding has not been always confirmed with fruit firmness (FF) data. For the same irrigation treatments in the same commercial orchard, FF response to RDI-II was inconsistent between the data of Gelly et al. (2003), showing no significant effect on FF, and those of Gelly et al. (2004), showing an increase of firmness with RDI. Other studies have shown no effects of RDI-II on fruit firmness (Domingo et al. 2007; Lopez et al. 2008). Information presented so far indicates that RDI-II fruit will either mature earlier than CI fruit or at least at the same time, but not later. Considering the overall results of RDI-II, it seems feasible to impose moderate RDI-II in high-density peach orchards. Besides saving water and maintaining the yield, some improvements in fruit quality parameters will be expected especially if a certain degree of water stress is still present during Stage III. Stage III of Fruit Development (RDI-III). Since peach accumulates a high percentage of total fruit weight during Stage III and almost 85% of final fruit weight is water, many studies indicate that any reduction in irrigation water during Stage III will be associated with limitations in fruit growth and yield (e.g., Mercier et al. 2009). Yet deficit irrigation during Stage III might be used judiciously in an attempt to improve fruit quality. Improvements in fruit quality following RDI-III could be in terms of increases in TSS (Table 4.1) (Li et al. 1989; Besset et al. 2001; Mechlia et al. 2002; Domingo et al. 2007; Mercier et al. 2009). Considering the effects of RDI-III on TSS, Li et al. (1989) concluded that RDI in Stage III could improve peach’s taste quality, which is especially of interest for markets in which moderate size is preferred. RDI-III could also be of interest in peach cultivars with extra-large fruit, in which a reduction in fruit size at harvest does not diminish market value (Li et al. 1989). Other positive effects on fruit quality following RDI-III may be advancement in fruit maturity (Mercier et al. 2009), reduction in fruit weight loss during cold storage (Mercier et al. 2009), and longer storage life (Li et al. 1989). These positive effects were obtained under moderate or mild water stress. For example, in the experiment of Mercier et al. (2009), midday Ystem values were 1.55 MPa. If more severe water stress occurs during Stage III, these effects might not be realized. In an early study with peach, fruit growth on trees without irrigation in Stage III tended to be arrested, flavor was astringent, and fruit skin often lacked red pigmentation (Proebsting and Middleton 1980). In this study, midday Yleaf in nonirrigated trees ranged between 3.0 and 4.0 MPa. Regarding fruit size, many studies (e.g., Li et al. 1989; Naor et al. 1999, 2001; Girona et al. 2004; Lopez et al. 2006, 2007a; Marsal et al. 2006; Naor 2006b) have shown that

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fruit weight at harvest under severe water stress is reduced in comparison with CI fruit. RDI-III is not recommended as a tool for improving fruit quality in peach, but it might become inevitable in water-limited areas. Postharvest RDI (RDI-PH). The effect of RDI applied after fruit harvest would be on fruit yield and quality in the following season. RDI-PH is especially popular in early-maturing cultivars because it offers the opportunity of saving more water during the longer postharvest period than in the preharvest period and does not interfere with fruit growth in the same season. In some studies, RDI-PH had no negative effect on the subsequent yield (Johnson et al. 1992; Gelly et al. 2003, 2004; Dichio et al. 2007). In others, however, RDI-PH decreased crop yield in the subsequent season because water stress negatively influenced flowering and fruit set (Larson et al. 1988; Girona et al. 2003; Naor et al. 2005; Marsal et al. 2008). The negative effects of RDI-PH on subsequent yield were discussed in a review of the effects of DI at different phenological stages (Naor 2006). Naor warned of the risks of applying too much water stress late in the season and mentioned two potential carry-over side effects on the subsequent yield: (1) a reduction in flowering and (2) a reduction in fruit set. Reductions in fruit set in peach trees can be partly explained by a limitation placed on the accumulation of carbohydrate reserves in response to water stress applied during the postharvest period of the previous season (Lopez et al. 2007b). Another disadvantage of RDI-PH is the substantial increase of fruit disorders, such as double fruits (Johnson et al. 1992; Naor et al. 2005), deep sutures (Handley and Johnson 2000; Johnson and Phene 2008), and external split pits (Johnson and Phene 2008). The latter authors suggested that the best strategy for avoiding double fruits was withholding water during June–July period (in California) and then applying full irrigation during August–September. Positive long-term effects of RDIPH on fruit quality have been published. In the case of ‘Andross’ peach, RDI-PH increased fruit TSS in the following season (Gelly et al. 2003, 2004). This may be due to the reduction in crop load in response to RDIPH. Low crop load has been associated with increases in TSS (Crisosto et al. 1997; Giacalone et al. 2002; Walsh et al. 2007). Combined RDI during Stage I and II of Fruit Development (RDI-I þ II). This topic has been explored by different authors (Li et al. 1989; Mechlia et al. 2002). In general, RDI-I þ II did not produce any result different from application of either RDI-I or RDI-II. Therefore, RDI-I þ II may be used to obtain greater reduction in vegetative shoot extension and higher

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water savings in mature peach orchards. Any positive effect of RDI-I þ II on fruit quality will be brought about by water stress in Stage II. But as explained in the RDI-II section, the positive effects may not be always manifested. In a recent study (Buendia et al. 2008), RDI-I þ II trees were watered with 25% of ET during Stages I and II and with 100% during Stage III. No effect was observed on fruit’s TSS, TA, juice pH, FF, and color (Table 4.1). However, significant effects of DI on peach antioxidant concentration were measured. RDI-I þ II fruit had lower vitamin C and carotenoids in the peel of the fruit than CI fruit, whereas increases in anthocyanins and procyanidins were observed (Buendia et al. 2008). The authors suggested that the effects of water stress on the antioxidant compounds of peach could be explained by a high sunlight exposure as a consequence of a reduction in vegetative growth. Combined RDI During Stage II and Postharvest (RDI-II þ PH). To save more water and further reduce vegetative growth in early-maturing cultivars, RDI could be combined during Stage II and postharvest, but this idea has received little attention in the literature. Two studies reported similar results to those observed when RDI was individually applied during Stage II or during postharvest (Gelly et al. 2003; Gelly et al. 2004). RDI-II þ PH increased TSS and TSS/TA ratio. It advanced fruit maturity in terms of an increase in ethylene production, reduction in FF, and redder fruit color. 3. Whole-Season Deficit Irrigation. Whole-season deficit irrigation has been applied to impose periods of predetermined tree or soil water deficit throughout the fruit growing season. The imposed levels of deficit may be either constant during the season (Mechlia et al. 2002; O’Connell et al. 2006) or may vary according to the phenological stage of the fruit (Li et al. 1989; Crisosto et al. 1994) (Table 4.1). In general, whole-season DI may be applied to obtain a combination of the positive effects realized by individual applications of RDI-I, RDI-II, and RDI-III, but especially when certain improvements in fruit quality parameters, such as higher TSS, are desired. Crisosto et al. (1994) applied combined reduced irrigation to ‘O’Henry’ peach by watering the trees to 50% of CI early in the season and then by 75% of CI later in the season. This had no effect on fruit flesh firmness, percent of red surface, and pH of the fruit juice. It reduced average fruit size at harvest and increased TSS (Table 4.1). Increases in TSS in DI fruit were, respectively, by 1.6 and 0.5 Brix in the first and second year of the experiment. Crisosto et al. (1994) argued that the smaller size in DI fruit would be preferred by consumers because of the higher TSS. This argument was based on a previous study (Parker

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et al. 1991) in which it was found that peaches with a higher TSS had a higher retail value in the California peach market. This was also confirmed by Mechlia et al. (2002) who found higher fruit TSS under DI in ‘Carnival’, a late peach cultivar. These authors suggested that increases of fruit TSS in response to DI may compensate for the loss of yield, which was decreased by 10% in comparison to CI. The increase in TSS in response to DI was by 1 Brix but statistically significant (Mechlia et al. 2002). The increases in TSS in most studies has been by 1 Brix. This improvement has been considered, by the latter authors, large enough to compensate for potential yield losses in response to deficit irrigation. 4. Partial Rootzone Drying. Two reports are available on PRD in peach. Goldhamer et al. (2002) reported no significant physiological or horticultural differences between PRD and classical DI. However, Abrisqueta et al. (2008) reported higher root length density values in PRD trees than in whole-season RDI trees. They did not report on fruit yield or quality. B. Apricot Although DI in apricot (Prunus armeniaca) has received less research attention than in peach, there is grower acceptance of using DI after harvest. Postharvest is usually the preferred period for applying DI in apricot because it is longer than the preharvest period. For example, for ‘Bu´lida’ apricot grown in southeast Spain, the postharvest period (from fruit harvest until the beginning of leaf fall) can last from early June until late November, whereas the whole preharvest irrigation season (from leaf emergence until harvest) lasts only from mid-March until early June (Perez-Pastor et al. 2004). Postharvest DI in apricot therefore offers the opportunity of reducing excessive vegetative growth and saving more water than preharvest DI without any possible negative impact on the crop in the same season. Moreover, higher levels of water deficit can be imposed during postharvest than during preharvest due to the higher evaporative demand of the atmosphere during the former period (Torrecillas et al. 2000). In the last study, performed in southern Spain, mild levels of water stress were observed when irrigation was withheld from early March until early May (midday Yleaf  1.0 MPa) and from early May until early June Y(leaf  1.28 MPa). However, during postharvest, midday Yleaf values were about 1.92 and 2.42 MPa when DI was applied, respectively, from early June to mid-July and from mid-July to mid-September. Long-term effects on yield and fruit quality may be realized in the season following postharvest DI (Uriu 1963), and these should be taken

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into account by fruit growers. Long-term effects of postharvest DI on crop yield are related mainly to early reproductive events (flowering and fruit set). Flower buds in stone fruits initiate just before the end of shoot enlargement phase and will continue to form and develop throughout postharvest (Flore 1994). In the spring, full bloom and fruit set will occur before leaves are fully expanded (Perez-Pastor et al. 2004). Therefore, the postharvest water status of the tree may influence flower bud formation and have a possible negative effect on crop load in the following season. Moreover, fruit set in the spring will be affected by the carbohydrate reserve of the tree that had been accumulated during the previous growing season (Loescher et al. 1990; Lopez et al. 2007). This accumulation may be affected by the water status of the tree in each season, especially postharvest water status (Esparza et al. 2001). Another factor that may negatively affect subsequent crop yield is pollen germination capacity (Ruiz-Sanchez et al. 1999). Floral biology of ‘Bu´lida’ apricot trees was studied in response to postharvest DI. It was found that a lower percentage of pollen germinated from trees subjected to postharvest water stress from 6 weeks after harvest (Yleaf  2.75 MPa). This reduction was not observed when similar or even lower values of midday Yleaf (2.95 MPa) were reached if DI were applied later in the season (from 2 months after harvest until leaf senescence). This helped identify critical postharvest periods for DI management. It was recommended that sufficient water should be applied immediately after harvest to obtain an adequate crop yield in the following season (Ruiz-Sanchez et al. 1999). This critical period was later confirmed by Torrecillas et al. (2000). Although postharvest is usually the preferred period for applying DI in apricot, the pattern of vegetative and fruit growth allows the application of DI at other phenological stages. Apricot, similar to other stone fruits, has a double sigmoid pattern of fruit growth. In ‘Bu´lida’, when fruit growth slows significantly and the first sigmoid phase is completed, almost 85% of vegetative growth has been realized (Perez-Pastor et al. 2006). Vegetative growth is then completed by the time fruit growth starts the second increase in weight (Stage III) (Perez-Pastor et al. 2006). This pattern provides an opportunity for the application of DI during Stage II of fruit development. This has been investigated by different authors with two main objectives: (1) vigor control in the absence of dwarfing rootstocks (Arzani et al. 2000a,b), and (2) irrigation water saving in water-limited areas, such as southeast Spain (Ruiz-Sanchez et al. 1999; Torrecillas et al. 2000; Perez-Pastor et al. 2009). When DI was applied with the objective of reducing excessive vegetative growth in New Zealand (Arzani et al. 2000a,b), ‘Sundrop’ apricot trees did not

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receive any irrigation during Stages I and II while full irrigation was applied during Stage III. DI had no negative effects on yield and reduced vegetative growth during late Stage I. The authors suggested that irrigation during Stage II may be of questionable value in areas with low temperatures, high rainfall, or deep soils (Arzani et al. 2000a). A different conclusion for irrigation at Stage II was reached by researchers in Murcia (southeast Spain). Murcia is an important apricot-producing area and is characterized by low seasonal rainfall and high evaporative demand. Different publications are available on the responses of ‘Bu´lida’ apricot to various DI strategies applied in a commercial orchard from 1994 to 1999 showing that DI is a useful technique in water-limited areas (Ruiz-Sanchez et al. 2000; Torrecillas et al. 2000; Perez-Pastor et al. 2009). To save water, Perez-Pastor et al. (2009) applied different combinations of DI during preharvest and postharvest periods. The following treatments were evaluated: CI receiving 100% of crop ET, whole-season DI receiving 50% of CI (DI-50), and a DI treatment that received 100% of CI during the critical periods (Stage III and 2 months after harvest) and either 25% of CI (DI-25) or 40% of CI (DI-40) during other periods. DI-50 had lower yield than CI, indicating that DI applied during the whole season negatively affects apricot productivity due to a reduction in current fruit growth. Predawn Yleaf of lower than 0.5 MPa should not be allowed to develop when applying DI during the whole season (PerezPastor et al. 2009). DI-25 had lower yield than CI while DI-40 had similar yield to CI. Trees should be irrigated with more than 25% of crop ET during the noncritical periods to obtain satisfactory yields (Perez-Pastor et al. 2009). Most DI research on apricot has focused on the yield. However, fruit quality can also be affected (Torrecillas et al. 2000; Perez-Pastor et al. 2007). In the experiment of Perez-Pastor et al. (2009) just described, these fruit parameters were the same between CI and DI: diameter, weight, and firmness. TSS and TA were higher in the DI fruit than CI fruit, but the latter fruit were redder. Torrecillas et al. (2000) evaluated different apricot quality parameters at harvest in an experiment where irrigation was withheld from ‘Bu´lida’ apricot at different fruit development stages or in combination of these stages. Different DI periods were: at flowering and fruit set, Stages I þ II, Stage III, from 6 weeks after harvest, and from 8 weeks after harvest. CI received 100% of crop ET throughout the season. Only DI during Stage III had negative effects on diameter, volume, and weight of the fruit. But fruit skin color was more intense compared with other treatments. This may indicate an advancement in fruit maturity in response to DI just before harvest. No significant

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differences in TSS, TA, and pH of juice were observed among the different treatments. These results were somewhat different from those obtained in a recent study, also on ‘Bu´lida’, for the effects of DI on fruit quality (Perez-Pastor et al. 2007). The latter authors evaluated the effects of three DI treatments on fruit quality at harvest, during cold storage, and for some days after removal from cold storage. The three irrigation treatments were: (1) CI irrigated at 100% of ET, (2) DI irrigated at 50% of ET during the whole season (DI-50), and (3) DI irrigated at 100% ET during critical periods (Stage III and early postharvest) and 25% of ET during the rest of the growing season (DI-25). Some benefits from DI were observed in terms of increased TSS and fruit firmness both at harvest and during cold storage. The DI-25 fruit had reduced weight loss in storage and less fungal attack after removal from storage. To summarize for apricot, DI can be applied during the fruit growing season and postharvest period with adequate irrigation in two critical periods of Stage III and early postharvest. Besides saving of water, combined DI treatments at different stages of fruit development will lead to a reduction in excessive vegetative growth without negatively affecting the yield and some likelihood of improving fruit quality. C. Cherry Quality in sweet cherry (Prunus avium) is mainly based on the external appearance of skin integrity and color. Firmness is also an important component of fruit quality (Toivonen et al. 2004). Cherry tends to soften fast after harvest because of skin water loss and cell wall relaxation with maturity. Although sweet cherry has been studied for yield responses to DI (Proebsting et al. 1981), there is a paucity of information on proper irrigation management. Cherry orchard irrigation requirements can be approximated from Allen et al. (1998), but specific data on cherry crop coefficients obtained from orchard lysimeters are lacking in the literature. Therefore, irrigation requirements have to be estimated, and the concept of water deficit has to be assessed critically. Part of the reason for the paucity of information on responses of cherry to DI is that cherry, in terms of global economic value, is less important than other deciduous fruit crops, such as apple and peach. One study dealt with sustained DI on ‘Ziraat’ sweet cherry grown under a continental Mediterranean climate in Turkey (Cigdem et al. 2008). The authors found no significant changes in TSS, sugar content, or acidity as a result of reducing irrigation to 50% or to 25% of CI throughout the season. However, firmness significantly increased in DI fruit compared to CI fruit (Cigdem et al. 2008). No data on tree water status was reported in this study;

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consequently, further discussion on whether water stress was effectively occurring in the tree is not feasible. The timing of DI is important in cherry for various reasons. There is grower acceptance of using postharvest DI because it decreases tree internal shading and controls excessive vigor following harvest. Water deficit before harvest is not acceptable because besides reducing fruit growth and fruit final size, it can also increase cracking if stress is relieved during ripening (Sekse 1995). DI is currently applied in the absence of research-based recommendations. From the few research reports published, it can be seen that under certain conditions, postharvest DI can negatively affect cherry quality. Marsal et al. (2009) applied postharvest DI to ‘New Star’ sweet cherry grown in the semiarid climate of Catalonia, Spain. They found some negative effects on the quality of fruit produced in the next season, but fruit yield was not affected. These negative effects were mainly apparent after 9 days of cold storage and were characterized by slight reductions in firmness and in TSS in fruit from the most stressed trees. Fruit maturity in the next season was advanced, based on fruit skin color, by at least 1 week. Such advancement can be economically relevant because there is a market premium for early production of cherry. A central issue in the use of postharvest DI is the possibility of applying excessive water stress and negatively influencing fruit set and crop load in the next season, as has been reported for peach (Girona et al. 2003) and almond (Goldhamer and Viveros 2000). The implication for cherry is important because low crop load increases fruit-soluble solids and firmness (Roper and Loescher 1987; Whiting and Lang 2004; Neilsen et al. 2007; Marsal et al. 2010). In a recent study on ‘Summit’ cherry Marsal et al. (2010) applied postharvest DI, receiving 50% of water given to CI, and noticed reduced fruit set and crop load in the following season. No negative effect on fruit quality in terms of TSS and firmness was observed at harvest and after 9 days in cold storage. There was a tendency for advanced maturity in fruit harvested from the most stressed trees (Marsal et al. 2010). In light of the available information, it seems that cherry quality responds to DI, but more research is needed before reliable assessments can be made. For instance, when comparing the data from the two reports by Marsal et al. (2009, 2010) there seems to be a cultivar response to postharvest DI in terms of TSS and firmness values. The quality of ‘New Star’ seemed to be more sensitive to postharvest DI than that of ‘Summit’ after 9 days in cold storage. The available information for cherry indicates that DI is not feasible before harvest but could be applied cautiously after harvest in view of possible negative effects on fruit quality in the next season.

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D. Prune and Plum Prune (Prunus domestica) and plum (P. salicina) are closely related species. Because of their genetic proximity and limited data available for each, they are discussed together here. Prune can be consumed fresh or dried, but dried consumption is of more economic importance worldwide. Plum is mainly consumed fresh. In both cases, an important aspect regarding irrigation that will affect fruit quality for both species is fruit cracking, similar to sweet cherry. DI in both species can be applied pre- or postharvest. However, most of the information available is on preharvest DI. There is almost no information on whole-season DI in prune or in plum. In the rainfed treatment of Battilani (2004), seasonal water deficit produced an increase in fruit TSS and firmness with no change in acidity compared to CI. However, fruit size in the largest category (> 60 mm) was reduced in the rainfed treatment with no significant difference in the yield between treatments. This study was carried out in the subhumid climate of Po Valley, Italy, with annual low water requirements. Plant water status was not measured in this experiment. The effects of preharvest water deficit on fruit cracking were studied by Uriu et al. (1962) and Milad and Shackel (1992). Those authors noticed the susceptibility of prune to cracking when deficit irrigation was applied and released before harvest. A solution was proposed in further studies, which indicated that fruit cracking can be avoided if the recovery from stress is not induced rapidly (Lampinen et al. 2001). In the last study, irrigation rates in the DI treatment were not increased to CI level until after harvest and DI treatments were aimed at not inducing a midday stem water potential of lower than 2.0 MPa (Lampinen et al. 2001). DI fruit had lower hydration ratio (fruit fresh weight/fruit dry weight) but did not have a lower dry mass accumulation than CI fruit. No detrimental effect was observed on fruit yield and quality because production was aimed at dried prune. The reduction in fruit fresh weight was a preferred outcome because of the lower energy cost of fruit drying (Lampinen et al. 1995, 2001). Another study on the effect of DI on fruit quality is by Naor et al. (2004). They reported that in ‘Black Amber’ plum growing in a semiarid environment, a significant increase in TSS of up to 2% occurred for a DI treatment that was irrigated at less than 40% of crop ET during Stage III of fruit development. There were no changes in acidity and in firmness. According to Crisosto et al. (2004), an increase in TSS with no decrease in acidity would be perceived positively by consumers. Naor et al. (2004) further described an increase in red pulp in DI fruit, but no differences in visual marketable attributes were found between irrigation treatments

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even after cold storage. However, DI fruit size and yield were smaller than those of CI. We are not aware of any published research in refereed journals on the effect of postharvest DI on plum and prune. IV. POME FRUITS A. Apple There are numerous literature reports on the effect of reduced irrigation on the yield and fruit quality of apple (Malus  domestica). Here we discuss the key and new references covering both aspects. We cover the yield aspect first followed by fruit quality. The presentation format here had to be different from that of peach because, unlike peach, apple does not have distinct stages of fruit growth, and a large part of vegetative and reproductive growth overlap during the growing season (Forshey et al. 1983). 1. Yield. In deciduous fruits, including apple, marketable yield is dependent on fruit number, size, and quality. Fruit number depends on the number of initiated flowers and on the final fruit set. In apple, floral initiation occurs in early summer (Westwood 1992). DI at this time may decrease return bloom, as demonstrated for ‘Braeburn’ apple in New Zealand (Kilili et al. 1996a; Behboudian et al. 1998). In these experiments, fruit thinning was still needed and yield was not affected. Decreased yield by reduced irrigation will quite possibly be due to a reduction in fruit size, explored next. Fruit size in apple is an important quality attribute and yield component. Citing numerous examples from the literature, Leib et al. (2006) indicated that RDI cannot have the same positive effects on apple as it had on peach, pear, and olive because of a reduction of yield and fruit size in apple. However, in their own 3-year experiment, carried out on ‘Fuji’ apple using DI and PRD in the semiarid climate of Washington State, fruit size was the same among the three treatments in every year and so was the yield except in Year 2, when it was lower in DI than in CI and PRD. The authors did not give a clear reason for the similarity in fruit size among the three treatments. They did provide soil water potential values at three depths with DI having generally lower values than CI and PRD. The latter generally had lower values than CI. The authors cited Green and Clothier (1999), who had shown that apple tree quickly adjusts root water uptake in response to changing soil water by increasing uptake from the wet part of the rootzone while reducing uptake from the drying part. Leib et al. (2006) did not provide information on plant

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water status to clarify whether a possible adjustment in root water uptake would have been reflected in tree water status. Talluto et al. (2008) did a 2-year experiment on ‘Pink Lady’ apple in central Sicily. In Year 1 trees on MM106 rootstock were used; in Year 2 those growing on M.9 and MM106 were employed. In Year 1 they applied CI, DI, and PRD. In Year 2 they applied CI and PRD. DI received 50% of water given to CI. Soil water potential was more negative in DI than in CI. The drying side of PRD also had more negative soil water potential than that of CI for most of the irrigation season. The authors presented data on leaf relative water content (RWC) on two occasions in each year. In all the occasions the treatments had the same RWC values except for the first measurement in Year 1, when DI had lower values than the other treatments. In Year 1 fruit size in DI was lower than the other two treatments, and the yield was lower in DI and PRD than in CI but not significantly. Crop load can modify the effect of DI on fruit size in apple. Mpelasoka et al. (2001a) applied whole-season DI in New Zealand to ‘Braeburn’ apple with commercial crop load (CCL) and with light crop load (LCL). They presented midday leaf water potential data for 101, 171, and 185 DAFB. For these days the values (MPa) for CI under CCL were, respectively, 1.5, 1.5, and 1.4. The corresponding values for DI were significantly lower: 1.9, 2.0, and 2.1. Under the LCL, the CI values were the same as under CCL. The corresponding DI values were, respectively, 1.8, 1.9, and 1.9. The authors found that for the LCL, mean fruit weight at harvest was similar between CI and DI. For CCL, mean fruit weight was lower in DI than in CI. The same conclusions were reached for the yield. The authors suggested that interactions between irrigation and crop load on fruit size and yield could be due to effects on fruit water relations and photosynthesis. Naor et al. (2008) reached a similar conclusion about the crop load effect for two sites in the semiarid climate of Israel. They applied three levels of irrigation to ‘Golden Delicious’ having two different crop loads. They applied 1, 3, and 7 mm of water per day in both sites; crop loads (fruit per tree) were 100 and 300 in one site and 50 and 150 in the other. The authors were also interested in finding whether the threshold of midday stem water potential was transferable among apple orchards within a region. For each crop load, more irrigation resulted in higher yield and larger fruit. In both orchards mean fruit weight decreased with decreasing irrigation rate and with increasing crop load. Mean fruit weight was significantly correlated with midday stem water potential. The difference in fruit weight between trees bearing a high crop load and trees bearing a lower crop load increased with increasing midday stem water potential. The results of Naor et al. (2008) are in general agreement with those of Mpelasoka

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et al. (2001a). The former authors recommended that when growers are forced to use DI, they could apply fruit thinning to minimize the reduction in fruit size. Naor et al. (2008) also concluded that a midday stem water potential of 2.5 MPa was the threshold value at which apple stomata closed, and this value would be transferable within orchards in the same region. We conclude that fruit size in apple is decreased with application of DI, especially if more severe water stress develops. A lower crop load could mitigate the response. 2. Fruit Quality. The important fruit quality attributes, other than size, which was mentioned earlier, that may be affected by reduced irrigation are discussed next. Firmness. Fruit firmness is important in apple and is strongly influenced by fruit maturity, with firmness decreasing as the fruit ripens (Kingston 1991). Firmness is also influenced by fruit size (Ebel et al. 1993), with smaller fruit being generally firmer than larger fruit due to a higher cellular density. Treatments that alter fruit size may therefore alter firmness. In the early literature, information on the influence of soil moisture on fruit firmness was conflicting. Drake et al. (1981) indicated that apple slices were softer from trees supplied with less water. Mills et al. (1994) observed a reduction in firmness in ‘Braeburn’ apple from trees with reduced water status. These findings indicate an increased maturity in fruit from drier treatments. In contrast, other researchers in the earlier literature had shown that apples from nonirrigated plots were firmer than those from irrigated plots (GuelfatReich et al. 1974; Assaf et al. 1975; Guelfat-Reich and Ben-Arie 1979). Assaf et al. 1975 indicated that fruit from trees under water deficit were smaller than those from CI trees, which may account for the observed increase in fruit firmness. Reduced firmness of fruit from well-irrigated trees may be the result of an inflation of cell size and an increase in the fragility of cell walls (Guelfat-Reich and Ben-Arie 1979). More conclusive results can be found in recent literature. In the 3-year study of Leib et al. (2006) with ‘Fuji’ apple, it was shown that firmness in the DI fruit was higher than that of the CI fruit for each of the 3 years, both at harvest and after 14 days from harvest. In an experiment on ‘Braeburn’ in New Zealand, it was shown that firmness was higher in fruit for which irrigation was withheld either late in the season or during the entire season (Kilili et al. 1996b). Mpelasoka et al. (2000) applied early-season and late-season DI to ‘Braeburn’ apple in New Zealand and divided the fruit into three size categories of small, medium, and large. They measured flesh firmness at harvest and after 12 weeks of storage at 0 C

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followed by 7 days at 20 C. They found that both DI treatments had a significantly higher fruit firmness than CI treatment for all the size categories both at harvest and after storage. For ‘Pink Lady’ apple, two studies have shown that fruit firmness was not affected by deficit irrigation. One study was in central Sicily (Talluto et al. 2008) and the other in Victoria, Australia (O’Connell and Goodwin 2007a). Firmness in ‘Pink Lady’ does not seem to respond to DI. Possible cultivar differences notwithstanding, our general conclusion is that apple fruit firmness increases in response to DI. Color. Red color in apple is based on anthocyanins and flavonols and is stimulated by light and cool temperatures (Lancaster 1992) and by carbohydrate levels of fruit at preharvest (Westwood 1992). Mills et al. (1994) found enhanced red color in ‘Braeburn’ apple with a lowered water status. They suggested that this may have been due to the advanced accumulation of sugars measured in these fruit, as sucrose plays an important role in anthocyanin development. The effect of reduced irrigation on enhancing the red color in ‘Braeburn’ apple in New Zealand was also reported by Kilili et al. (1996a,b). However, this was not confirmed for the same cultivar undergoing DI in New Zealand (Mpelasoka et al. 2001b). In the latter case, red color was not fully developed in ‘Braeburn’ commercial orchards either. This indicates the overriding effects of other environmental factors, such as high night temperatures occurring during this experiment, on fruit color development. For both years of their experiment on ‘Pink Lady’ apple in Australia, O’Connell and Goodwin (2007a) did not find any effect of DI on fruit skin redness and greenness. The same conclusion was again reached for ‘Pink Lady’ in the 2-year study of Talluto et al. (2008) in Sicily. We suggest that reduced irrigation has the potential of enhancing color in the apple fruit when other environmental conditions are conducive to full development of the desired color. Otherwise these other prevailing environmental factors will override the potential of DI in enhancing the fruit color. The color development of both red and yellow apple cultivars is dependent on light reaching the fruit (Lancaster 1992). A reduction in vegetative growth under DI may allow better light penetration into the canopy. However, we are not aware of any study that has correlated light penetration with apple color under reduced irrigation. Another possible reason for DI effects on color is that DI reduces the level of N in the apple fruit (Mills et al. 1994). A reduction in fruit N concentration may play an important role in the development of desirable color attributes in DI fruit. However, direct experimental data are not available to substantiate this.

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Total Soluble Solids. TSS and acidity have a marked influence on the sensory quality of the apple fruit (Ackermann et al. 1992). Numerous authors report a significant increase in apple fruit TSS under DI, as reviewed by Naor (2006a). A more recent example is for the 3-year study of Leib et al. (2006) with ‘Fuji’ apple, where it was shown that TSS in the DI fruit was higher than that of the CI fruit for each of the 3 years both at harvest and after 14 days from the harvest. The 2-year study of O’Connell and Goodwin (2007a) on ‘Pink Lady’ in Victoria, Australia, showed that TSS was markedly, but not significantly, higher in DI fruit than CI fruit for each of the 2 years. The values ( Brix) for Year 1 in DI and CI were, respectively, 17.6 and 16.9 (LSD¼ 1.0). The corresponding values for Year 2 were 16.2 and 15.2 (LSD¼ 1.1). The results of Talluto et al. (2008) for ‘Pink Lady’ in Sicily showed a similarity in TSS values between DI and CI treatments. While there might be a cultivar effect in this respect, literature results are convincing that DI fruit usually have a higher level of TSS than CI fruit. This could be a result of increasing dry matter concentration of the DI fruit (Naor 2006a) and a higher conversion of starch to sugars under water stress (Kramer 1983). Soluble Sugars. The four major soluble sugars in the apple fruit are sucrose, glucose, fructose, and sorbitol, and their concentrations normally rise with DI, as exemplified for ‘Braeburn’ apple reported by Mills et al. (1994), Kilili et al. (1996a), and Mpelasoka et al. (2000). Mpelasoka et al. (2001c) also reported an increase in total sugar concentration at harvest in DI ‘Braeburn’ fruit. Marlow and Loescher (1984) cited some early literature linking high sorbitol concentration to the incidence of water core, an important apple disorder. However, in none of the studies just mentioned was an incidence of water core reported. The increase in sugar concentration is a positive contribution of DI to improvement of fruit quality in apple. Titratable Acidity. Citing the literature, Behboudian and Mills (1997) concluded that DI effects on apple TA were not conclusive. The same conclusion can be reached now based on further information. In the experiment of Kilili et al. (1996a) on ‘Braeburn’ apple in New Zealand, it was shown that TA, in terms of % malic acid, was the same in CI fruit and the fruit for which irrigation was withheld early in the season, late in the season, and during the entire season (Kilili et al. 1996a). Mpelasoka and Behboudian (2002) applied whole-season DI in New Zealand to ‘Braeburn’ apple with a commercial crop load and with a light crop load. They found that TA was not affected either by irrigation or by crop load. Mpelasoka et al. (2001b) had reached the same conclusion in a

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similar experiment with trees grown in lysimeters. In their 3-year experiment on ‘Fuji’ apple, Leib et al. (2006) found that TA was higher in CI fruit than DI fruit in Year 1, and the opposite was the case in Year 2. They did not measure TA in Year 3. In the 2-year study of Talluto et al. (2008) on ‘Pink Lady’, it was shown that TA in the first year was lower in the DI fruit than the CI fruit whereas in the second year there was no difference in TA between the treatments. Considering that in various experiments there has been no consistent effect of DI on apple TA for different cultivars under various environments, we suggest that further measurement of TA is not feasible in DI studies unless there is an interest in the actual values of TA for certain cultivars. Aroma Volatiles. Aroma volatiles are important quality attributes for apple, and there is not enough information on how their production is affected by DI. Behboudian et al. (1998) applied early deficit (ED) and late deficit (LD) irrigation in ‘Braeburn’ growing in lysimeters in New Zealand. Both soil water content and midday leaf water potential were lower in ED and LD than in CI. The authors found that the LD fruit had higher concentrations of aroma volatiles than the CI fruit at harvest and after cold storage of 12 weeks. For most volatile compounds, the LD fruit had higher concentrations than the ED fruit. Ethyl-2-methyl butanoate, which is considered an important volatile in the apple fruit (Flath et al. 1967), was detectable only in the CI fruit at harvest. But it was present in all the treatments after storage, being higher in LD fruit than in CI and ED fruit. Ester 2-methyl butyl acetate, which is considered to be characteristics of red cultivars and may be an indicator of ripening (Mattheis et al. 1991), was higher in LD fruit than in CI and ED fruit. Mpelasoka and Behboudian (2002) applied whole-season DI in New Zealand to ‘Braeburn’ apple with CCL and with LCL. The volumetric soil water content and midday values of leaf water potential were always lower in DI than in CI irrespective of the crop load. The authors reported that the proportion of certain volatile compounds produced by the fruit is more important that the total volatile concentration, which may not necessarily reflect the highest aroma. They therefore used the concept of “odor units” used extensively in applied flavor research (Frijters 1979). They found that the total concentration of aroma volatiles and total odor units in the fruit juice was the same at harvest in DI and CI fruit. They measured these parameters again on Days 2, 6, and 14 for fruit left at 20 C. Total concentration of aroma volatiles was higher in DI fruit than CI fruit on Day 6 and total odor units was higher in DI fruit on Days 6 and 14. These parameters were higher, but not significantly, in DI fruit than CI fruit on all other days. The authors also measured these parameters on

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Days 1 and 7 on fruit that had been taken out of cold storage after 17 weeks. The total concentration of aroma volatiles was higher in DI fruit on Days 1 and 7 and significantly so for Day 1. Total odor units were higher for DI fruit on both days. The authors concluded that DI enhanced volatile production during ripening and after cold storage. Mpelasoka and Behboudian (2002) also cited literature indicating that increased production of aroma volatiles coincides with an increase in ethylene production, with production decreasing as fruit senesces. This relationship was demonstrated in their own experiment. Considering the limited amount of information available in the literature, we conclude that the aroma volatile concentration of the apple fruit will improve by reduced irrigation. Fruit Mineral Concentration. Behboudian and Mills (1997) reviewed the literature on the importance of the contribution of mineral elements to fruit quality especially the effects of low Ca levels in the development of bitter pit, water core, and cork spot. The authors also mentioned the negative effects of high N in increasing the incidence of rot in apple. Mills et al. (1994) found that both Ca and N decreased in the apple fruit under DI. But this has not been consistent in some other studies. Calcium is transported in the transpiration stream (Mengel and Kirkby 1987), and therefore a reduction in plant transpiration will result in a reduction of Ca transport within the plant. However, with reduced plant water status, the vegetative growth is suppressed and fruit may therefore be preferentially supplied with Ca under drier conditions (Raese 1985). For apple there are examples of no difference in the concentration of fruit mineral nutrients between CI and DI treatments. Nakajima et al. (2004) reported that there were no differences in the concentrations of N, P, Mg2 þ , Ca2 þ , and K þ in leaf and fruit, between well-watered and reduced-irrigated ‘Pacific Rose’ TM apple. Kilili et al. (1996a) found that there were no differences in the concentration of Ca2 þ and N between CI and DI in ‘Braeburn’ fruit when water was withheld at various times during the growing season. Mpelasoka et al. (2000) confirmed the same for Mg2 þ , Ca2 þ , and K þ in ‘Braeburn’ fruit grown in Marlborough, New Zealand. Mineral deficiency should not be an expected outcome in trees undergoing DI because of the reduced demand for nutrients and sufficient nutrients normally being supplied through fertigation. Disorders. Fruit disorders, present at harvest or developed in storage, are among the most important aspects of fruit quality. Both increases and decreases of disorders for the apple fruit undergoing DI have been reported. Examples of lower incidence of disorders are for bitter pit

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(Guelfat-Reich et al. 1974; L€ otter et al. 1985; Irving and Drost 1987), scald (Guelfat-Reich et al. 1974; L€ otter et al. 1985), and water core (L€ otter et al. 1985). Goode et al. (1975) reported increased cracking and russetting in apple fruit grown under reduced irrigation. Irving and Drost (1987) reported a greater incidence of apple fruit cracking under early-season DI. Opara et al. (1997) indicated that fluctuations in soil moisture and especially a sudden increase late in the season, particularly after a dry period, could split the skin of various fruits. L€ otter et al. (1985) reported an increase in sunburned apple fruit from DI treatments. O’Connell and Goodwin (2007) reported that in each of their 2-year study with ‘Pink Lady’ apple, incidence of fruit disorders (sunburn, russet, misshape, markings, frost damage) was not affected by DI. It is difficult to arrive at a general conclusion here. It is prudent to assume that the effect of DI on incidence of disorders would depend on the nature of the disorder, severity, and duration of water stress. Fruit Maturity. The fruit quality attributes of size, flesh firmness, color, and TSS that could also be used as maturity indices were discussed earlier. In this section we emphasize two criteria of maturity affected by reduced irrigation: internal ethylene concentration (IEC) and starch pattern index (SPI). Ebel et al. (1993) used early-season RDI on ‘Delicious’ apple and found that the IEC increased earlier in the RDI fruit than in the CI fruit. Starch degradation was delayed in the RDI fruit, but starch content during storage was the same between RDI and CI fruit. The authors concluded that RDI levels severe enough to reduce fruit growth may advance fruit maturity and alter quality at harvest. The studies cited in this section are all on ‘Braeburn’ apple in different experiments carried out in New Zealand. Kilili et al. (1996b) applied early-deficit (ED) and late-deficit (LD) irrigation as well as no-irrigation (NI) for the entire season. They reported that LD and NI fruit had more advanced maturity and earlier ripening than CI and ED fruit in terms of higher IEC, more yellow skin background color, and higher TSS. Mpelasoka et al. (2001d) applied ED and LD treatments and had three harvests. In all harvests, the fruit SPI was higher in LD than in ED and CI with the last two treatments being the same. The fruit IEC was the same among the treatments in the first two harvests. But it was the highest in LD in the last harvest with ED and CI being the same. Mpelasoka and Behboudian (2002) applied whole-season DI and measured the fruit that were kept for 14 days at 20 C on Days 2, 6, and 14. The SPI was higher in CI than DI on Day 2, and it was the same on Days 6 and 14. IEC was higher in DI fruit than CI fruit on Days 6 and 14. Kingston (1991) indicated that of paramount importance in determining fruit quality is the maturity of

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fruit for eating, commonly termed commercial maturity. Despite the occasional evidence to the contrary as cited in this paragraph, it can be concluded that maturity is advanced in DI fruit compared to CI fruit, based on the existing literature reports. Storage Behavior. In addition to fruit quality at harvest, it is also important to consider the effect of reduced irrigation on fruit storage behavior and to explore whether any improvement in fruit quality at harvest is also maintained during the storage and shelf life of the fruit. Kilili et al. (1996) found that the following properties of DI fruit for ‘Braeburn’ were maintained either in cold storage or on the shelf. Fruit that were not irrigated late in the growing season or for the entire growing season in New Zealand maintained redder color than control, by having significantly lower hue angle in cold storage for 7 weeks and on the shelf for 16 days. The DI fruit that had a higher TSS and flesh firmness than CI fruit at harvest maintained these differences during 12 weeks of cold storage. Mpelasoka et al. (2001d) found a similar result for ‘Braeburn’ apple undergoing DI in early, late, and entire season. The DI fruit had higher TSS and flesh firmness than CI fruit at harvest and these differences were maintained during cold storage of 12 weeks in one experiment and 17 weeks in another. In yet another experiment on ‘Braeburn’ apple undergoing early- and late-season DI in New Zealand, Mpelasoka et al. (2000) found that TSS was higher in DI fruit than CI fruit, and this difference was maintained after 12 weeks of cold storage followed by 7 days at 20 C. TAwas also higher in DI fruit than CI fruit at harvest, but the difference was not maintained in storage. Glucose, fructose, and total sugars that were higher in DI fruit at harvest showed the same differences with CI fruit after the storage conditions of 12 weeks at 0 C followed by 7 days at 20 C. In a whole-season DI experiment on ‘Braeburn’ that involved two levels of crop load, Mpelasoka and Behboudian (2002) found that DI and CI fruit had similar maturity at harvest (in terms of IEC and SPI) but DI fruit became more advanced in maturity during cold storage of 17 weeks. DI enhanced volatile production during ripening and after cold storage. Firmness and TSS were higher in DI fruit at harvest, but the increased firmness was lost during the subsequent storage. The existing information is therefore indicative that, in general, the quality attributes that are improved in the DI apple fruit at harvest are maintained during the subsequent cold storage and for fruit on the shelf. Postharvest weight loss is a major cause of deterioration in storage, and in apple, a loss of more than 6% of the harvest weight results in an unattractive and shriveled appearance (Hatfield and Knee 1988). The susceptibility of the fruit to water loss is therefore an important

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factor to be considered along with other quality attributes. Information on this aspect is limited to studies in New Zealand on ‘Braeburn’. Kilili et al. (1996b) showed that withholding of water late in the growing season or for the entire season resulted in a decrease in weight loss of ‘Braeburn’ fruit over 9 weeks in cold storage and also over 14 days at room temperature of 20 C following the cold storage. This was confirmed for ‘Braeburn’ fruit by Mpelasoka et al. (2000), who applied early and late DI and found that weight loss of DI fruit was significantly less than the CI fruit while in cold storage for 12 weeks as well as during the following 7 days at 20 C. The difference in weight loss could be due to differences in the structure and/or composition of the skin or the epicuticular waxes covering the skin. Cuticle modification by deficit irrigation has been reported by Crisosto et al. (1994), who attributed a lower rate of water loss for peach grown under DI to a thicker cuticle and a higher density of trichomes on the skin surface. Mpelasoka et al. (2000) and Kilili et al. (1996b) presented data showing that dry matter concentration in the DI fruit was higher than CI fruit, but they did not give this as one possible reason for DI fruit having less weight loss in storage than CI fruit. This could have been one plausible reason. Research is needed for apple to identify the mechanisms of lower weight loss in storage for deficitirrigated fruit. 3. Partial Rootzone Drying. More research has been done on PRD in apple than in peach. The first two studies reviewed here were carried out in the humid climate of New Zealand. Zegbe et al. (2008) applied PRD to ‘Pacific Rose’TM apple in a 2-year experiment. In Year 1, the PRD treatment was watered only on the same side of the tree row for the entire season. In Year 2, PRD watering was alternated between the two sides of the row and the drying side was watered when the volumetric soil water content ranged between 0.18 and 0.22 m3m3. For both years, the volumetric soil water content was higher in CI than in the drying side of the PRD treatment. PRD and CI fruit had similar quality attributes at harvest and after storage except that PRD fruit had lower weight loss in storage in Year 1 and a lower firmness after storage in Year 2. The similar fruit quality attributes were fruit size, TSS, and fruit skin color. The preharvest parameters and yield components of this research were reported elsewhere (Zegbe and Behboudian 2008) indicating a similarity between the two treatments in the gross yield. Van Hooijdonk et al. (2007) applied three irrigation treatments to ‘Pacific Rose’ TM: CI, PRD, and NI. Leaf water potential was similar between PRD and CI and that of NI became lower than CI later in the season. Fruit yield and most of the quality attributes were similar among the treatments at harvest including

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concentrations of sugars and aroma volatiles. However, PRD and NI fruit were firmer and had less weight loss in storage than CI fruit. In another publication based on this work, van Hooijdonk et al. (2004) reported that there were no differences among the treatments in these fruit quality attributes: size, color, TSS, and fruit water content. In the 3-year study of Leib et al. (2006) with ‘Fuji’ apple in semiarid Washington State, United States, it was shown that fruit size was the same among the three treatments of CI, DI, and PRD for the 3 years. Yield was lower in DI than the other treatments in Year 2 and was the same in the other years. Firmness and TSS were generally higher in the PRD fruit than CI fruit, and there were no consistent differences among the treatments for fruit titratable acidity and starch pattern index. O’Connell and Goodwin (2007a) applied DI and PRD to ‘Pink Lady’ apple in Victoria, Australia, for 2 years. PRD had lower leaf water potential than CI but these fruit parameters were the same between the two treatments: yield, fruit size, TSS, firmness, and skin color. We have so far alluded to the study of Talluto et al. (2008) in relation to their DI treatment applied in central Sicily. Here we emphasize their PRD treatment. They did two experiments on ‘Pink Lady’ apple. In Experiment 1, trees on MM106 were used; in Experiment 2, those growing on M.9 and MM106 were employed. In Experiment 1, they applied CI, DI, and PRD. In Experiment 2, they applied CI and PRD. In Experiment 1, fruit size and titratable acidity were lower in DI than in CI and PRD. However, these fruit attributes were the same among the three treatments: yield, TSS, water content, flesh firmness, starch pattern index, and peel color. In Experiment 2, irrespective of the rootstock, there were no differences between CI and PRD in these fruit attributes: yield, size, peel color, water content, starch pattern index, TSS, and titratable acidity. Another report is by Lo Bianco et al. (2008) for ‘Pink Lady’ apple grown on M.9 and MM106 undergoing PRD in central Sicily. The authors measured the aroma volatiles separately on the fruit flesh and on the peel. They found a complex array of aroma volatiles with more being produced by the peel. Their overall conclusion was that PRD increases the aroma volatiles of the fruit flesh, but it decreases them in the peel. The concentration of aroma volatile of the whole fruit could then decrease as a result. But considering the relative contribution, in terms of weight, from flesh and from the peel, the volatile concentration of the entire fruit was improved for fruit on M.9 but it decreased in fruit growing on more vigorous MM106. They attributed this rootstock difference to the different levels of water stress being perceived. Their overall conclusion was that the combination of less vigorous rootstocks and PRD could improve the aroma concentration of the apple fruit. One more report is by Fallahi

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et al. (2008), who applied five irrigation treatments to ‘Autumn Rose Fuji’ apple in Parma, Idaho, United States. There were two PRD treatments. One was applied using sprinkle irrigation and resulted in smaller fruit size and higher fruit TSS than the fully irrigated control. The other PRD treatment was applied using drip irrigation and resulted in more advanced fruit maturity in terms of lower firmness and a higher starch degradation pattern compared to the control. The reports that are so far available are indicative that PRD does not affect the apple yield and fruit quality to the extent that DI does. Apparently the higher water status of the PRD trees than the DI trees is not conducive to profound effects on yield and quality. Research in New Zealand (e.g., Mills et al. 1996; Mpelasoka et al. 2000, 2001c) has shown that ‘Braeburn’ apple fruit quality starts being affected when the midday leaf water potential is between 1.5 and 2.5 MPa. This is likely to happen by the application of DI but not by PRD. This range of leaf water potential was not reached in any of the PRD studies cited here from New Zealand, and studies done elsewhere used other indicators of tree water status. A summary of DI and PRD effects on apple yield and quality is presented in Table 4.2. B. Pear Pear trees are not drought resistant, and commercial production in areas with dry seasons depends on irrigation. The use of DI during certain periods of growing season will have implications for flowering in the following season and vigor control during the same season. There are two main cultivated species of Pyrus: P. communis (European pear), which is grown in Europe, America, South Africa, and Oceania; and P. pyrifolia (synonymous with P. serotina) (sand pear or nashi), which is traditionally grown in Asia. Because data for both species are scant, we will discuss the effects of reduced irrigation on both species in the same section. The pear fruit has two growing stages. Stage I corresponds to the initial slow-growth phase and Stage II corresponds to the rapid-growth phase until harvest (Bain 1961). Depending on the cultivar, pear fruit can be kept in cold storage for several months. Therefore, fruit may be prone to physiological disorders during storage. Although the effects of reduced irrigation on pear fruit quality have received little attention, it can be said that water stress during the fruit growing season increases fruit sugar concentration, acidity, and firmness (Cabral et al. 1995). In addition, it seems that water deficit can reduce incidence of fruit disorders such as alfalfa greening and cork spot, and this has been related to lower ratio of N/Ca in reduced-irrigated fruit

178

Decreased Decreased Increased Improved increased Increased No effects Increased

No effects Not conclusive Enhanced

Improved Reduced

Mineral nutrients Disorders Maturity

Storability Wt loss in storage

Effect

Yield Size Firmness Color Total soluble solids Soluble sugars Titratable acidity Aroma volatiles

Fruit parameter

Mpelasoka and Behboudian 2002 Mpelasoka et al. 2000

Talluto et al. 2008 Talluto et al. 2008 Mpelasoka et al. 2000 Kilili et al. 1996 Leib et al. 2006 Mpelasoka et al. 2001c Mpelasoka et al. 2001b Mpelasoka and Behboudian 2002 Mpelasoka et al. 2000 O’Connell and Goodwin 2007a Mpelasoka et al. 2001d

Reference

Deficit irrigation (DI)

Improved Reduced

No effect Not studied No effect

No effect No effect Increased No effect Increased No effect No effect Increased

Effect

Nakajima et al. 2004 — van Hooijdonk et al., 2007 van Hooijdink et al. 2007 Zegbe et al. 2008 van Hooijdonk et al., 2007

Leib et al. 2006 Leib et al. 2006 Talluto et al. 2008 Talluto et al. 2008 Fallahi et al. 2008 van Hooijdonk et al. 2007 Talluto et al. 2008 Lo Bianco et al. 2008

Reference

Partial rootzone drying (PRD)

Table 4.2. Effects of classical deficit irrigation (water applied to the whole rootzone) and partial rootzone drying (water applied to one part of the rootzone at a time) on yield and quality attributes of the apple fruit.

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(Brun et al. 1985). Research has also indicated that quality can be affected depending on the period DI is applied. This has been reported for ‘Nijisseiki’ Asian pear (Behboudian and Lawes 1994). In general, water stress during Stage I seems to have negative implications for fruit quality while stress during Stage II could have positive effects on some quality attributes. For example, water stress during Stage I significantly increased the incidence of postharvest flesh spot decay in Asian pear while TSS and fruit size were not different from CI fruit (Behboudian and Lawes 1994). In the latter study, water stress occurring during Stage II decreased the incidence of postharvest flesh spot decay, but this was at the cost of fruit size being smaller than fully irrigated trees. Marsal et al. (2000) found that RDI applied at Stage I to the European pear cultivar of ‘Williams’ negatively affected fruit quality in terms of lower TSS, firmness, size, and yield. RDI slightly advanced fruit maturity. The study of Behboudian and Lawes (1994) and Marsal et al. (2000) were carried out on trees in containers, and a moderate level of water stress was applied during Stage I. However, under field conditions, it is not always easy to reach moderate levels of water stress during Stage I with midday stem water potential not becoming lower than 1.4 MPa. Stage I normally follows a wet dormant season in temperate and Mediterranean areas. Perhaps for this reason in field studies dealing with DI during Stage I, no clear impact on fruit quality has been found at harvest (Asın et al. 2007). A different situation can be met during Stage II. Warm weather normally coincides with this stage, and water stress can easily develop in DI trees, as reported by Marsal et al. (2002). O’Connell and Goodwin (2007) applied whole-season PRD to ‘Williams’ and found higher water stress levels during Stage II than during Stage I. Their experiment will be described in more details below. Ramos et al. (1994) applied DI by irrigating ‘Barlett’ European pear at 65% of ET and found significant increases in TSS and acidity with no changes in fruit firmness and storage scald symptoms. However, fruit size was reduced. Other studies reported decreases in fruit firmness as a result of water stress during Stage II, suggesting an advancement in ripening (Raese et al. 1982). Inconsistency between experiments with regard to fruit firmness may be related to the fact that smaller fruits tend to be firmer than larger fruits; this size effect under deficit irrigation may correlate negatively with any ripening advancement. PRD has been assessed for pear in a 2-year study in Goulburn Valley, Australia, by O’Connell and Goodwin (2007b). They used a mature micro-irrigated orchard of ‘Williams Bon Chretein’ (‘Bartlett’) and applied these treatments: CI receiving 100% of crop ET on both sides of the tree row, PRD100 receiving the same on one side of the tree row and

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alternating every 14 days, and PRD50 receiving 50% of CI on one side of the tree row and alternating every 14 days. Midday leaf water potential and midday stem water potential in CI and PRD100 were similar, both being higher than those of PRD50. PRD100 was similar to CI in terms of fruit yield, size, TSS, and FF. PRD50 showed symptoms of severe water stress and had a high fruit drop and reduced fruit size and yield (by 16% to 28% of CI). The authors recommended that PRD50 should not be considered for Goulburn Valley. There are large gaps of knowledge in the effect of DI on pear fruit quality. For instance, there is no information on the effect of postharvest water stress on pear fruit quality in the following season. Also, information on cultivar differences in fruit quality in response to DI is limited.

V. CONCLUSIONS AND FUTURE PROSPECTS The effects of reduced irrigation on most of the fruit quality attributes were deemed controversial in earlier literature. However, there is now sufficient information to reach some firm conclusions. For example, one negative effect of reduced irrigation would be smaller fruit size, hence industry’s hesitation in using DI. However, reduced fruit size is a problem only if DI is too severe and too long. This is also of less concern for large-fruited cultivars and for fruit destined for processing. Other than possible reduced size, we cannot firmly identify any other fruit quality attribute where the judicious application of reduced irrigation has had a consistently negative effect. We conclude that sufficient progress has been made in irrigation research in providing information for the judicious application of reduced irrigation. DI seems feasible for peach if applied in Stage II of fruit development. Yield will be maintained and fruit quality improved (in terms of redder fruit with higher TSS) especially if a mild measure of water stress is carried over to Stage III. Moderate DI in Stage III could improve fruit quality and compensate for possible yield reduction. Whole-season DI can reduce yield and increase fruit TSS, which has been deemed as a satisfactory compensation for yield loss. Postharvest DI should be applied cautiously in view of possible fruit disorders, such as double fruits, occurring in the following season. For apricot, DI can be applied during the fruit growing season and postharvest period if adequate irrigation could be applied in two critical periods of Stage III and early postharvest. Besides saving of water, combined DI treatments at different stages of fruit growth will check vegetative growth without negatively affecting the yield and has some likelihood of improving fruit quality.

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For apple and pear, more quality benefits seem to be realized through the application of DI late in the season than earlier in the season. Lateseason DI might maintain yield and improve these fruit quality attributes in apple: firmness, color, concentrations of TSS, soluble sugars, and aroma volatiles, enhanced maturity, better storability, and reduced weight loss in storage. DI, if applied properly, might not have any negative effects on fruit acidity, mineral nutrient concentration, and incidence of disorders. Among the stone fruits covered in this chapter, information is especially limited for cherry, prune, and plum, and research is needed on these species. Further research is needed on fruit quality of apricot and peach addressing how aroma volatiles are being affected by the application of DI. More information is needed on storage weight loss for peach. Apple has received the highest attention in research among the crops covered here. However, gaps of knowledge exist, especially in how the aroma volatile profile is affected by DI and the mechanisms involved. The paucity of information on pear is surprising, considering the fact that it was one of the first species studied under RDI. Two areas of research require special attention. The first is sensory evaluation of DI fruit quality, which is of primary importance in marketing. The other is further evaluation of PRD for its effectiveness in various climates and its possible effects on yield and quality. The results published so far, for the crops reviewed here, are indicative of effectiveness of PRD in saving water, maintaining yield, and possibly improving fruit quality. For both PRD and classical DI, research results should be transferable, but comparisons between studies are better made if a standard water status indicator is used. Plant-based water status indicators are better linked with fruit quality. We suggest that midday stem water potential is a suitable parameter to monitor. ACKNOWLEDGMENTS We are grateful to Dr. Tessa Mills, Institute of Plant and Food Research in New Zealand, for her critical comments on the manuscript. LITERATURE CITED Abrisqueta, J.M., O. Mounzer, S. Alvarez, W. Conejero, Y. Garcia-Orellana, L.M. Tapia, J. Vera, I. Abrisqueta, and M.C. Ruiz-Sanchez. 2008. Root dynamics of peach trees submitted to partial rootzone drying and continuous deficit irrigation. Agr. Water Manag. 95:959–967.

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Lopez, G., J. Girona, and J. Marsal. 2007b. Response of winter root starch concentration to severe water stress and fruit load and its subsequent effects on early peach fruit development. Tree Physiol. 27:1619–1626. Lopez, G., M. Mata, A. Arbones, J.R. Solans, J. Girona, and J. Marsal. 2006. Mitigation of effects of extreme drought during stage III of peach fruit development by summer pruning and fruit thinning. Tree Physiol. 26:469–477. L€ otter, J. De V., D.J. Beukes, and H.W. Weber. 1985. Growth and quality of apples as affected by different irrigation treatments. J. Hort. Sci. 60:181–192. Marini, R.P., D. Sowers, and M.C. Marini. 1991. Peach fruit quality is affected by shade during final swell of fruit growth. J. Am. Soc. Hort. Sci. 116:383–389. Marlow, G.C., and W.H. Loescher. 1984. Water core. Hort. Rev. 6:189–251. Marsal, J., G. Lopez, A. Arbones, M. Mata, X. Vallverdu, and J. Girona. 2009. Influence of postharvest deficit irrigation and preharvest fruit thinning on sweet cherry (cv. New Star) fruit firmness and quality. J. Hort. Sci. Biotech. 84:273–278. Marsal, J., G. Lopez, J. del Campo, M. Mata, A. Arbones, and J. Girona. 2010. Postharvest regulated deficit irrigation in ‘Summit’ sweet cherry: fruit yield and quality in the following season. Irrig. Sci. 28:181–189. Marsal, J., G. Lopez, and J. Girona. 2008. Recent advances in regulated deficit irrigation (RDI) in woody perennials and future perspectives. Acta Hort. 792:429–439. Marsal, J., G. Lopez, M. Mata, and J. Girona. 2006. Branch removal and defruiting for the amelioration of water stress effects on fruit growth during Stage III of peach fruit development. Scientia Hort. 108:55–60. Marsal, J., M. Mata, A. Arbones, J. Rufat, and J. Girona. 2002. Regulated deficit irrigation and rectification of irrigation scheduling in young pear trees: an evaluation based on vegetative and productive response. Eur. J. Agron. 17:111–122. Marsal, J., H.F. Rapoport, T. Manrique, and J. Girona. 2000. Pear fruit growth under regulated deficit irrigation in container-grown trees. Scientia Hort. 85:243–259. Mattheis, J.P., J.K. Fellman, and P.M. Chen. 1991. Changes in headspace volatiles during physiological development of Bisbee Delicious apple fruit. J. Agr. Food Chem. 39:1902–1906. Mechlia, N.B., M. Ghrab, R. Zitouna, M.B. Mimoun, and M. Masmoudi. 2002. Cumulative effect over five years of deficit irrigation on peach yield and quality. Acta Hort. 592:301–307. Mengel, K., and E.A. Kirkby. 1987. Principles of plant nutrition. 4th ed. International Potash Institute, Worblaufen-Bern, Switzerland. Mercier, V., C. Bussi, F. Lescourret, and M. Genard. 2009. Effects of different irrigation regimes applied during the final stage of rapid fruit growth on an early maturing peach cultivar. Irrig. Sci. 27:297–306. Milad, R.E., and K.A. Shackel. 1992. Water relations of fruit end cracking in French prune (Prunus domestica L. cv. French). J. Am. Soc. Hort. Sci. 117:824–828. Mills, T.M., M.H. Behboudian, and B.E. Clothier. 1996. Water relations, growth, and the composition of ‘Braeburn’ apple fruit under deficit irrigation. J. Am. Soc. Hort. Sci. 121:286–291. Mills, T.M., M.H. Behboudian, and B.E. Clothier. 1997. The diurnal and seasonal water relations, and composition, of ‘Braeburn’ apple fruit under reduced plant water status. Plant Sci. 126:145–154. Mills, T.M., M.H. Behboudian, P.Y. Tan, and B.E. Clothier. 1994. Plant water status and fruit quality in ‘Braeburn’ apples. HortScience 29:1274–1278. Mitchell, P.D., and D.J. Chalmers. 1982. The effect of reduced water-supply on peach-tree growth and yields. J. Am. Soc. Hort. Sci. 107:853–856.

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Mitchell, P.D., P.H. Jerie, and D.J. Chalmers. 1984. The effects of regulated water deficit on pear tree growth, flowering, fruit growth and yield. J. Am. Soc. Hort. Sci. 109:604–606. Mpelasoka, B. S., and M.H. Behboudian. 2002. Production of aroma volatiles in response to deficit irrigation and to crop load in relation to fruit maturity for ‘Braeburn’ apple. Postharvest Biol. Technol. 24:1–11. Mpelasoka, B. S., M.H. Behboudian, J. Dixon, S.M. Neal, and H.W. Caspari. 2000. Improvement of fruit quality and storage potential of ‘Braeburn’ apple through deficit irrigation. J. Hort. Sci. Biotech. 75:615–621. Mpelasoka, B. S., M.H. Behboudian, and S. Ganesh. 2001c. Fruit quality attributes and their interrelationships of ‘Braeburn’ apple in response to deficit irrigation and to crop load. Gartenbauwissenschaft 66:247–253. Mpelasoka, B. S., M.H. Behboudian, and S.R. Green. 2001b. Water use, yield and fruit quality of lysimeter-grown apple trees: Responses to deficit irrigation and to crop load. Irrig. Sci. 20:107–113. Mpelasoka, B. S., M.H. Behboudian, and T.M. Mills. 2001a. Water relations, photosynthesis, growth, yield and fruit size of ‘Braeburn’ apple: Responses to deficit irrigation and to crop load. J. Hort. Sci. Biotech. 76:150–156. Mpelasoka, B. S., M.H. Behboudian, and T.M. Mills. 2001d. Effects of deficit irrigation on fruit maturity and quality of ‘Braeburn’ apple. Scientia Hort. 90:279–290. Nakajima, H., M.H. Behboudian, M. Greven, and J.A. Zegbe-Domınguez. 2004. Mineral contents of grape, olive, apple, and tomato under reduced irrigation. J. Plant Nutr. Soil Sci. 167:91–92. Naor, A. 2006a. Irrigation scheduling and evaluation of tree water status in deciduous orchards. Hort. Rev. 32:111–165. Naor, A. 2006b. Irrigation scheduling of peach—deficit irrigation at different phenological stages and water stress assessment. Acta Hort. 713:339–349. Naor, A., H. Hupert, Y. Greenblat, M. Peres, A. Kaufman, and I. Klein. 2001. The response of nectarine fruit size and midday stem water potential to irrigation level in Stage III and crop load. J. Am. Soc. Hort. Sci. 126:140–143. Naor, A., I. Klein, H. Hupert, Y. Grinblat, M. Peres, and A. Kaufman. 1999. Water stress and crop level interactions in relation to nectarine yield, fruit size distribution, and water potentials. J. Am. Soc. Hort. Sci. 124:189–193. Naor, A., S. Naschitz, M. Peres, and Y. Gal. 2008. Responses of apple fruit size to tree water status and crop load. Tree Physiol. 28:1255–1261. Naor, A., M. Peres, Y. Greenblat, Y. Gal, and R. Ben Arie. 2004. Effects of preharvest irrigation regime and crop level on yield, fruit size distribution and fruit quality of field-grown ‘Black Amber’ Japanese plum. J. Hort. Sci. Biotech. 79: 281–288. Naor, A., R. Stern, M. Peres, Y. Greenblat, Y. Gal, and M.A. Flaishman. 2005. Timing and severity of postharvest water stress affect following-year productivity and fruit quality of field-grown ‘Snow Queen’ nectarine. J. Am. Soc. Hort. Sci. 130:806–812. Neilsen, G., F. Kappel, and D. Neilsen. 2007. Fertigation and crop load affect yield, nutrition, and fruit quality of ‘Lapins’ sweet cherry on Gisela 5 rootstock. HortScience 42:1456–1462. O’Connell, M.G., and I. Goodwin. 2007a. Responses of ‘Pink Lady’ apple to deficit irrigation and partial rootzone drying: physiology, growth, yield, and fruit quality. Austral. J. Agr. Res. 58:1068–1076. O’Connell, M. G., and I. Goodwin. 2007b. Water stress and reduced fruit size in microirrigated pear trees under deficit partial rootzone drying. Austral. J. Agr. Res. 58:670–679.

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O’Connell, M.G., I. Goodwin, and G.M. Dunn. 2006. Towards a better understanding of crop water requirement in orchards: A case study from the Goulburn Valley. Austral. J. Expt. Agr. 46:405–412. Opara, L. U., C.J. Studman, and N.H. Banks. 1997. Fruit skin splitting and cracking. Hort. Rev. 19:217–262. Parker, D., D. Ziberman, and K. Moulton. 1991. How quality relates to price in California fresh peaches. Calif. Agr. 45 (2):14–16. Perez-Pastor, A., R. Domingo, P.A. Nortes, J.P. Perez-Abellan, A. Torrecillas, and M.C. RuizSanchez. 2006. Growth pattern of ‘Bulida’ apricot trees in Mediterranean conditions. Acta Hort. 717:59–62. Perez-Pastor, A., R. Domingo, A. Torrecillas, and M. Ruiz-Sanchez. 2009. Response of apricot trees to deficit irrigation strategies. Irrig. Sci. 27:231–242. Perez-Pastor, A., M.C. Ruiz-Sanchez, R. Domingo, and A. Torrecillas. 2004. Growth and phenological stages of Builida apricot trees in south-east Spain. Agronomie 24:93–100. Perez-Pastor, A., M.C. Ruiz-Sanchez, J.A. Martinez, P.A. Nortes, F. Artes, and R. Domingo. 2007. Effect of deficit irrigation on apricot fruit quality at harvest and during storage. J. Sci. Food Agr. 87:2409–2415. Postel, S. 1998. Water for food production: will there be enough in 2025? BioScience 48:629–637. Powell, D.B.B. 1974. Some effects of water stress in late spring on apple trees. J. Hort. Sci. 49:257–272. Proebsting, E.L., and J.E. Middleton. 1980. The behavior of peach and pear trees under extreme drought stress. J. Am. Soc. Hor. Sci. 105:380–385. Proebsting, E.L., J.E. Middleton, and M.O. Mahan. 1981. Performance of bearing cherry and prune trees under very low irrigation rates. J. Am. Soc. Hort. Sci. 106:243–246. Raese, J.T. 1985. Nutrition practices to improve quality in d’Anjou pears discussed. Goodfruit Grower 36:42–44. Raese, J.T., C.A. Brun, and E.J. Seeley. 1982. Effect of irrigation regimes and supplemental nitrogen on alfalfa greening, cork spot and fruit quality of ‘d’Anjou’ pears. HortScience 17:666–668. Ramos, D.E., S.A. Weinbaum, K.A. Shackel, L.J. Shcwankl, E.J. Mitcham, F.G. Mitchell, R. G. Snyder, G. Mayer, and G. McGourty. 1994. Influence of tree water status and canopy position on fruit size and quality of Bartlett pears. Acta Hort. 367:192–200. Roper, T.R., and W.H. Loescher. 1987. Relationship between leaf area per fruit and fruit quality in ‘Bing’ sweet cherry. HortScience 22:1273–1276. Ruiz-Sanchez, M.C., J. Egea, R. Galego, and A. Torrecillas. 1999. Floral biology of ‘Bulida´ apricot trees subjected to postharvest drought stress. Ann. Appl. Biol. 135:523–528. Ruiz-Sanchez, M.C., A. Torrecillas, A. Perez-Pastor, and R. Domingo. 2000. Regulated deficit irrigation in apricot trees. Acta Hort. 537 (2):759–766. Sekse, L. 1995. Cuticular fracturing in fruits of sweet cherry (Prunus avium L) resulting from changing soil water contents. J. Hort. Sci. 63:135–141. Talluto, G., V. Farina, G. Volpe, and R. Lo Bianco. 2008. Effects of partial rootzone drying and rootstock vigour on growth and fruit quality of ‘Pink Lady’ apple trees in Mediterranean environments. Austral. J. Agr. Res. 59:785–794. Toivonen, P.T.A., F. Kappel, S. Stan, D.L. McKenzie, and R. Hocking. 2004. Firmness, respiration, and weight loss of ‘Bing’, ‘Lapins’ and ‘Sweetheart’ cherries in relation to fruit maturity and susceptibility to surface pitting. HortScience 39:1066–1069. Torrecillas, A., R. Domingo, R. Galego, and M.C. Ruiz-Sanchez. 2000. Apricot tree response to withholding irrigation at different phenological periods. Scientia Hort. 85:201–215.

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Uriu, K. 1963. Effect of postharvest soil moisture depletion on subsequent yield of apricots. Proc. Am. Soc. Hort. Sci. 84:93–97. Uriu, K., C.J. Hansen, and J.J. Smith. 1962. The cracking of prunes in relation to irrigation. J. Am. Soc. Hort. Sci. 80:211–219. van Hooijdonk, B.M., K. Dorji, and M.H. Behboudian. 2004. Responses of ‘Pacific Rose apple to partial rootzone drying and to deficit irrigation. Eur. J. Hort. Sci. 69:104–110. van Hooijdonk, B.M., K. Dorji, and M.H. Behboudian. 2007. Fruit quality of ‘Pacific Rose apple grown under partial rootzone drying and deficit irrigation. J. Food Agr. Environ. 5:173–178. van Schilfgaarde, J. 1994. Irrigation—a blessing or a curse. Agr. Water Manag. 25:203–219. Walsh, K.B., R.L. Long, and S.G. Middleton. 2007. Use of near infra-red spectroscopy in evaluation of source-sink manipulation to increase the soluble sugar content of stonefruit. J. Hort. Sci. Biotech. 82:316–322. Westwood, M.N. 1992. Temperate zone pomology: physiology and culture. 3rd ed. Timber Press, Portland, OR. Whiting, M.D., and A.G. Lang. 2004. ‘Bing’ sweet cherry on the dwarfing rootstock ‘Gisela 5’: Thinning affects fruit quality and vegetative growth but not CO2 exchange. J. Am. Soc. Hort. Sci. 129:407–415. Williamson, J.G., and D.C. Coston. 1990. Planting method and irrigation rate influence vegetative and reproductive growth of peach planted at high density. J. Am. Soc. Hort. Sci. 115:207–212. Zegbe, J.A., and M.H. Behboudian. 2008. Plant water status, CO2 assimilation, yield, and fruit quality of ‘Pacific Rose’ apple under partial rootzone drying. Adv. Hort. Sci. 22:27–32. Zegbe, J.A., M.H. Behboudian, B.E. Clothier, and A. Lang. 2008. Postharvest performance of ‘Pacific Rose apple grown under partial rootzone drying. HortScience 43:952–954.

5 Hot Water Treatments of Fruits and Vegetables for Postharvest Storage Elazar Fallik Agricultural Research Organization The Volcani Center Department of Postharvest Science of Fresh Produce Bet Dagan 50250, Israel

ABBREVIATIONS I. INTRODUCTION II. HOT WATER TECHNOLOGIES A. Hot Water Dips B. Hot Water Rinsing and Brushing III. COMBINATION TREATMENTS A. Hot Water Treatment and Fungicides B. Hot Water and Modified Atmosphere Packaging C. Hot Water Treatment and Biocontrol D. Hot Water Treatment and Ethanol E. Hot Water Treatments with Other Combinations IV. HOT WATER TREATMENTS AND FRESH CUT V. SUMMARY AND CONCLUSIONS ACKNOWLEDGMENT LITERATURE CITED

ABBREVIATIONS 1-MCP AZX CA FLU HWD HWRB

1-methyl cyclopropene azoxystrobin controlled atmosphere fludioxonil hot water dip hot water rinsing and brushing

Horticultural Reviews, Volume 38 Edited by Jules Janick Copyright
2011 Wiley-Blackwell. 191

192

IMZ LDPE MAP NaClO RF SBC SC TBZ TM VHT

E. FALLIK

imazalil low-density polyethylene modified atmosphere packaging sodium hypochlorite radio frequency sodium bicarbonate sodium carbonate thiabendazole thiophanate methyl vapor heat treatment

I. INTRODUCTION Since the dawn of history, fresh fruits and vegetables have been part of the human diet. Although fruits and vegetables have always provided variety in the diet, through differences in color, shape, taste, aroma, and texture (Kays 1997), their full nutritional importance has been recognized only in recent times. Advances in agronomic, processing, preservation, packaging, shipping, and marketing technologies on a global scale have enabled the fresh fruit and vegetable industry to supply consumers with a wide range of high-quality produce year-round. However, economic losses caused by postharvest pathogens can be high, and avoidable losses between the farm gate and consumer should be reduced. Fresh fruits and vegetables, because of the added costs of harvesting and handling, increase in value several times as they are moved from the field to the consumer. To decrease our dependency on chemical control for ensuring the quality of harvested produce, various methods to control postharvest decay are being developed. Thermal treatments have been very effective in controlling pathogens that are the main causes of prestorage and postharvest decay development (BarkaiGolan 2001; Vicente et al. 2002). Prestorage heat treatments to control decay development during storage and marketing are often applied for a relatively short time (minutes), because the target decay-causing agents are found on the surface of the fruit or vegetable or in the first few cell layers under its skin (Barkai-Golan 2001). Heat treatments against decay-causing agents can be applied to the fresh-harvested produce by several means: hot water dipping, vapor heat, dry hot air (Lurie 2008), very short hot water rinsing and brushing (Fallik 2004), and thermal heat treatment using radio frequency (RF) energy to control, mainly, pests in agricultural commodities (Tang et al. 2000). This chapter summarizes the information accumulated since the beginning of the

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millennium on hot water treatments, applied either directly or in combination with other means to control rot development on fruits, vegetables or flowers.

II. HOT WATER TECHNOLOGIES There are three basic technologies for hot water treatment: (1) the batch system (Fallik 2007); (2) the continuous system for short- to mediumterm treatments (minutes to hours) (Tsang et al. 1995), both of which are used for hot water dips (HWD); and (3) hot water rinsing and brushing system (HWRB) for a very short-term treatments (seconds) (Fallik 2004). A. Hot Water Dips The batch system is the most commonly used commercial technology in many packinghouses. In this technology, the freshly harvested produce is loaded into baskets, which are then lowered into the hot water immersion tank, where the fruits or vegetables remain at the prescribed temperature for a certain length of time before being taken out, usually by means of an overhead hoist. In the continuous system, the fresh produce is placed on a moving conveyor belt, which submerges them slowly in the hot water tank. The belt speed is monitored by a special gear to ensure that the fresh produce is submerged for the required length of time. The main components in these two systems are: (1) an insulated treatment tank of several hundred liters’ capacity; (2) a heat-exchange unit operated by gas, diesel, or electricity; (3) a pump to circulate the water to ensure uniform water temperatures throughout the treatment process and to prevent formation of cool pockets during treatment; and (4) temperature sensors to control and monitor the water temperature during treatment (Tsang et al. 1995). An inexpensive hot water immersion system can be assembled easily, and there is little difficulty in making it mobile (Sharp et al. 1991). Fresh tropical, subtropical, citrus, and deciduous fruits, and fresh vegetable produce, as well as organic fresh produce, can benefit from hot water dipping or immersion. Postharvest rots are among the most serious problems in commercialization of ‘Prata’ banana, but they can be controlled by immersing the fruit in hot water: the combinations of 50
C for 6 and 12 min, 53
C for 9 min and 56
C for 3 min, enabled appropriate decay control for preservation of postharvest quality (Nolasco et al. 2008). Mango fruits cv. ‘Namdokmai’ at the mature-green stage were dipped in water at 55
C or ambient temperature (control) for 5 min;

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HWD remarkably delayed the onset of disease infection, reduced the number of infected fruits, and lowered the severity of infection. At the end of storage, HWD-treated fruits still had lower disease severity than unheated fruits (Benitez et al. 2006). The most effective HWD for inhibiting decay development in litchi fruit was 1 min at 52
C; a hot water spray was equally as effective as the HWD. Comparisons with other studies indicated that a hot water spray was about half as effective as a hot benomyl dip (Olesen et al. 2004). Rodriguez et al. (2005) reported that dipping cactus fruit (Opuntia ficus indica) for 3 min at 52
C significantly reduced fungal development and improved the visual quality of the fruit after subsequent prolonged storage. Subjecting pineapples that had been artificially inoculated with Thielaviopsis black rot to an HWD treatment at 54
C for 3 min inhibited decay development after 3 weeks of storage at 10
C and 2 days at ambient temperature of 28
C (Wijeratnam et al. 2005). ‘Big-Top’ nectarine fruits were subjected to HWD treatments at 45
, 50
, or 55
C for 2 or 3 min after harvest and were then kept at 0
C for 45 days and at 20
C for an additional 4 days to determine the effects of HWD treatments on their storage and shelf life; the treatment at 45
C for 2 min reduced fungal decay development during storage and shelf life, while the higher temperatures caused damage (Candir et al. 2009). Mature-green plums (Prunus salicina cv. ‘Friar’) were treated in water at 40
C for 40 min, 45
C for 35 min, 50
C for 30 min, or 55
C for 25 min and then stored for 35 days at 0
C followed by ripening at 20
to 25
C for 9 days; decay symptoms were retarded by dipping at 45
C and 50
C, for 35 and 30 min, respectively, and fruit quality remained acceptable for the market (Abu-Kpawoh et al. 2002). Dipping organically grown apples in hot water at 53
C for 2 min controlled decay development caused by Gloeosporium spp. during 5 to 6 months in storage (Trierweiler et al. 2003). For the reduction of postharvest decay on organic apples, treatment at 53
C for 3 min was recommended (Maxin et al. 2005). Nafussi et al. (2001) reported that an HWD for 2 min at 52
C to 53
C inhibited decay development on lemons that had been artificially inoculated with Penicillium digitatum. ‘Satsuma’ mandarins (Citrus unshiu) of an early-harvesting cultivar, ‘Gungchun’, were treated by HWD at 52
C for 2 min, 55
C for 1 min, or 60
C for 20 s and then stored at 5
C for 3 weeks and subsequently held at 18
C for 1 week. The development of stem-end rots, moldy decay, and black rots was manifestly lower in fruit that had been heat-treated for 20 s at 60
C than in untreated controls (Hong et al. 2007). Postharvest HWDs of organically grown strawberries, at 55
C and 60
C for 30 s significantly reduced decay incidence to 3.4% and 2.7%, respectively, compared with 28% in the control (Karabulut et al. 2004a).

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A 1-min exposure of rock melon to 60
C was the optimum time/temperature combination for controlling decay development caused by Fusarium spp., Alternaria spp., and Colletotrichum spp. after 3-week storage at 5
C (McDonald et al. 2005). Dipping of bell peppers (Capsicum annuum) at 45
or 53
C for 15 or 4 min, respectively, prior to storage at 8
C markedly reduced the incidence of fungal infections (GonzalezAguilar et al. 2000). Dipping red ginger flowers (Alpinia purpurata) at 49
C or 50
C for 12 or 15 min, respectively, extended postharvest vase life by killing most pests and saprophytic molds (Jaroenkit and Paull 2003). Hot water treatment of chestnut at 70
C and 80
C did not control fungal growth, but treatment at 90
C for 10 min reduced the infection level by 95% (Panagou et al. 2005). HWDs were found to be very useful as quarantine treatments. Using hot water baths at 50
C for 10 min and at 54
C for 6 min as a quarantine treatment against codling moths (Cydia pomonella) on sweet cherries may be feasible if the fruits are transported by air at 5
C for 2 days, but not if they are carried by sea at 0
C for 14 days (Feng et al. 2003). Immersion of litchi fruits in water at 49
C for 20 min followed by hydrocooling in water at ambient temperature for 20 min was tested as a quarantine treatment against potential infestations of Hawaiian litchi with eggs or larvae of Mediterranean fruit fly, Ceratitis capitata and oriental fruit fly, Bactrocera dorsalis (Armstrong and Follett 2007). The treatment provided probit 9 (99.9968% mortality) quarantine security against eggs and first instars without affecting fruit quality. B. Hot Water Rinsing and Brushing In contrast to hot water immersion, a technology based on a brief hot water rinsing and brushing (HWRB) for simultaneous cleaning and disinfestation of fresh produce was first introduced commercially in 1996 (Fallik et al. 1996). Under this technology, immediately after harvest and before storage, fruits and vegetables are rinsed and brushed at temperatures of 48
C to 62
C for 15 to 25 s, depending on the commodity. To meet the high standards of the food processing industry, all the components of the HWRB machine are made from stainless steel. A speed-adjustable conveyor belt is connected to the simultaneous cleaning and disinfecting stage, and its speed controls the rate at which produce is exposed to brushing and rinsing. The machine contains 22 to 24 parallel cylindrical revolving brushes, all driven by a single motor. After moving on the conveyor belt, the produce is transported on 8 to 10 medium-soft brushes while being prewashed by nonrecycled pressurized tap water at an ambient temperature of 20
C to 25
C for about 10 s.

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The produce then progresses to a further 8 to10 brushes while being rinsed with pressurized hot water from nozzles that can be positioned at predetermined angles relative to the produce. The fruits or vegetables then move to a set of 4 softer brushes that remove water droplets and then into drying tunnel, where the remaining excess water is removed by a forced air stream from several fans (Fallik 2004). The first report on HWRB technology was published in 1996 by Fallik et al. (1996). Treating sweet bell peppers by HWRB at 55
C for about 12 s significantly reduced decay incidence compared with untreated fruits while maintaining fruit quality (Fallik et al. 1999). The optimal HWRB treatment to reduce decay development on ‘Galia’-type netted melon fruit was 59
C for 15 s (Fallik et al. 2000). Recently, Gal et al. (2006) reported that treating ‘Galia’-type melon with HWRB prior to 1-methylcyclopropene (1-MCP) treatment at a concentration of 300 nL/L significantly reduced decay incidence, compared with HWRB alone. Treating pink tomatoes with HWRB at 52
C for 15 s or dipping the fruit in water at 52
C for 1 min significantly reduced decay development caused by Botrytis cinerea after 3 weeks in storage at 2
C or 12
C followed by an additional 5 days at 20
C (Ilic et al. 2001), and kumquat benefited from HWRB at 55
C for 20 s (Ben-Yehoshua et al. 2000). Deciduous fruits also were found to benefit from HWRB treatments. Treating apples with HWRB at 55
C for 15 s significantly reduced decay development in P. expansum-inoculated apple fruits after 4 weeks at 20
C or in naturally infected apples after prolonged storage of 4 months at 1
C plus 10 days at 20
C (Fallik et al. 2001). Peaches and nectarines inoculated with Monilinia fructicola and then treated with HWRB at 55
C or 60
C for 20 s exhibited decay reductions of 70% and 80%, respectively (Karabulut et al. 2002). HWRB at 55
C or 58
C for 30 s reduced the percentage of apples showing typical symptoms of white rot and also reduced the number of lesions on each fruit (Oster et al. 2006). Treatment of newly harvested ‘d’Anjou’ pears (Pyrus communis) with a washing system comprising a high-pressure spray of warm water at 30
C, wetting agent, and rotating soft brushes was significantly effective in removing surface pests and controlling decay, without causing internal or external damage to the fruit (Bai et al. 2006). Treating organic citrus by HWRB at 62
C for 20 s significantly reduced P. digitatum decay development (Porat et al. 2000a). A 20-s HWRB treatment at 59
C or 62
C reduced decay in ‘Star Ruby’ grapefruit that had been artificially inoculated with P. digitatum by 52% and 70%, respectively, whereas HWRB at 53
C or 56
C was ineffective (Porat et al. 2000b). Both hot drench brushing at 56
C or 60
C for 10 s and HWD at 52
C for 2 min reduced decay incidence on ‘Oroblanco’ fruit

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(Citrus grandis  C. paradisi) after 2 weeks at 1
C, followed by 12 to 13 weeks at 11
C and 1 additional week at 20
C (Rodov et al. 2000). Green mold incidence caused by P. digitatum on ‘Eureka’ lemons (Citrus limon) and ‘Valencia’ oranges, after storage for 3 weeks at 12.8
C, was significantly reduced by HWRB treatment at 62.8
C for 30 s, from 97.9% and 98% on untreated fruits to 14.5% and 9.4%, respectively (Smilanick et al. 2003). Treatments of cactus pear (Opuntia ficus-indica) at 60
C for 30 s or 65
C for 20 s reduced decay incidence by 86% to 91% without affecting fruit quality (Dimitris et al. 2005). HWRB technology has been found useful for the fresh-cut industry. Treating melons with HWRB at 75
C for 20 s significantly reduced total microbial counts by 4 logs reduction, compared with a 2.5 log reduction in fruits that were HWRB-treated at 58
C and a 1.5 log reduction in those treated with chlorine at 150 mL/L, 4 days after treatment. Although HWRB at 75
C for 20 s severely damaged the fruit peel if the fruit was subsequently left in storage, none of the HWRB treatments affected the taste, aroma, color, or firmness of the flesh used for fresh cuts (Fallik et al. 2007).

III. COMBINATION TREATMENTS A. Hot Water Treatment and Fungicides Fungi are usually the primary agents in the spoilage of fresh produce, and control can be exercised by the application of fungicides at dose rates that do not harm the produce or the consumer of the produce. Very few fungicides have been approved by government agencies for application on fresh-harvested produce; therefore, a combination of a fungicide at a low dosage with heat treatment can be more effective than a fungicide or heat treatment alone without being harmful to the consumers. Postharvest diseases of mango fruit (Mangifera indica) cause economic losses during storage but can be controlled by chemical, physical, or biological methods. ‘Kensington Pride’ mango fruits were dipped in hot water at 52
C for 10 min; dipped in prochloraz (0.55 mL/L) at ambient temperature for 5 min; or dipped in hot prochloraz (0.55 ml/L) at 52
C for 5 min, dipped in carbendazim (2 mL/L) for 5 min, and dipped in hot carbendazim (2 mL/L) at 52
C for 5 min. Hot water dipping or fungicide treatments (at ambient or at a high temperature) reduced incidence and severity of postharvest diseases, and fruit quality was not substantially affected by any of the treatments (Dang et al. 2008). Hot water treatment at 52
C for 10 min combined with the fungicide bavistin at 0.1 mL/L was found to be the best in controlling the incidence of

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anthracnose and stem-end rot in ‘Kesar’ mango fruits, whereas the untreated (control) fruits were found to have been infected with C. gloeosporioides and Diploidia natalensis. It was also found that shelf life of mango fruits could be extended for more than 28 days when they received hot water treatment coupled with fungicides and were stored in a cool chamber (Waskar 2005). The use of heated fungicide can significantly reduce residue levels in stored fresh produce if reduced concentrations of fungicides are used. The residue levels of fludioxonil (FLU) were determined in pear cultivars ‘Precoce di Fiorano’, ‘Coscia’, and ‘Spadona Estiva’ after a 2-min dip in an aqueous mixture of FLU containing 300 or 100 mg/L of active ingredient at 20
C or 50
C (Schirra et al. 2008a). Treatment with FLU at 300 mg/L at 20
C resulted in residue levels similar to those from treatment with FLU at 100 mg/L at 50
C in ‘Coscia’ fruits but in significantly lower levels in ‘Precoce di Fiorano’ and ‘Spadona Estiva’ pears. Treatments with heated FLU were more effective than those with unheated FLU in controlling blue and gray mold decay in these pears. Green mold caused by P. digitatum and sour rot, caused by Geotrichum citri-aurantii on lemons, were effectively controlled when potassium sorbate (KS) was added to heated imazalil (IMZ) or thiabendazole (TBZ) solutions at 50
C. Heat, but not KS, increased residue levels of all the fungicides in oranges (Smilanick et al. 2008). Complete control of decay development on ‘Star Ruby’ grapefruit (Citrus paradisi Macf.) was achieved when fruits were dipped in the fungicide azoxystrobin (AZX) at 50
C for 3 min (Schirra et al. 2002b). Exotic fruits were found to benefit from treatment with hot water and fungicides. The storage response of ‘Gialla’ cactus pears (Opuntia ficusindica) was investigated over 6 weeks at 6
C, followed by an additional week at 20
C, after a 3-min dip treatment with TBZ at 1000 mg/L at 20
C or with TBZ at 150 mg/L at 52
C (Schirra et al. 2002a); the latter treatment was much more effective, providing 89% control of decay without affecting fruit quality. Banana (Musa acuminata) is very susceptible to anthracrose, crown rot, and blossom end rot at the postharvest stage. Dipping the fruits in water at 50
C for 5 min combined with prochloraz at 250 mg/L reduced disease incidence (Hassan et al. 2004). A combination of HWRB and fungicides was found very effective in controlling decay development and also enabled reduction of fungicide concentrations (Fallik 2004). Depending on the cultivar, HWRB at 48
C to 62
C for 15 s to 20 s, combined with prochloraz at 225 mg/mL, was found to be the most effective treatment for control of Alternaria alternata in mango fruit (Prusky et al. 1999, 2002). Application of a combination of HWRB for 15 s to 20 s, followed by spraying with 50 mM HCl,

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effectively controlled Alternaria rot in stored mango fruits. Similar HWRB treatments followed by spraying with increasing concentrations of prochloraz at 45 to 900 mg/L in 50 mM HCl were as effective as treatment with the acid alone in preventing Alternaria rot development (Prusky et al. 2006). Treating litchi at 55
C for 20 s significantly reduced the amount of prochloraz needed to control decay development after 5 weeks of storage followed by a marketing simulation period (Lichter et al. 2000). B. Hot Water and Modified Atmosphere Packaging Modified atmosphere packaging (MAP) has become an important technology for the preservation of fruits and vegetables. In MAP, the gaseous environment [oxygen, carbon dioxide (CO2), and nitrogen (N2)] in the package is modified to create an optimal mixture to extend the shelf life and maintain the quality of the commodity. The fact that gas modifications are only fungistatic or bacteriostatic has encouraged producers to use additional preservation technology to enhance or support safety and to retard spoilage (Rosnes et al. 2007). Optimal conditions for the improvement of storability of ‘Fuyu’ persimmon fruits in MAP were obtained with treatment for 10 to 15 min at 48
C, 5 min at 50
C, and 20 s at 57
C to 60
C. However, when the oxygen concentration in MAP storage was low, the fruit samples treated by HWD showed enhanced incidence of pitted blotch browning; therefore, it is necessary to design a MAP that maintains a high oxygen concentration (Lee et al. 2008). The effects of hot water treatment at 54
C for 5 min, combined with MAP, on the storage and fruit quality of ‘Alona’ and ‘Naomi’ cherry tomatoes (Solanum lycopersicum, syn. Lycopersicon esculentum) were investigated by Akbudak et al. (2007). At the end of storage, HWD combined with MAP produced better results than MAP alone for both cultivars. Prior to packaging with low-density polyethylene (LDPE) film (0.02 mm in thickness), tomatoes were immersed in hot water at 42.5
C for 30 min. Control tomatoes were not treated and were stored for 2 weeks at 10
C and then for 3 days at 20
C, without packaging. The use of HWD combined with MAP reduced decay and maintained fruit quality (Suparlan 2003). Nectarines and peaches (Prunus persica) were hot water treated at 46
C for 25 min, sealed in thin polyethylene bags, and stored at 0
C for 1 and 2 weeks. Hot water combined with MAP storage resulted in decay reduction and maintained good fruit quality after 1 week of postharvest handling (Malakou and Nanos 2005).

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C. Hot Water Treatment and Biocontrol Microbial biocontrol agents have shown great potential as an alternative to synthetic fungicides for the control of postharvest decay of fruits and vegetables. Utilization of antagonist microorganisms appears to be a promising technology; some antagonist-based products are commercially available, and others are currently at various stages of development (Janisiewicz and Korsten 2002). At present there are two commercial products available for postharvest use, each having a very small market share among the technologies used to manage postharvest diseases. Biosave (Pseudomonas syringae, EcoScience Corp., Worcester MA, USA) is registered in the United States and is used mostly for the control of diseases of sweetpotato and potato (Stockwell and Stack 2007); Shemer (Metschnikowia fructicola, AgroGreen Ltd., Ashdod, Israel) is registered in Israel and is used commercially for the control of storage diseases of sweetpotato and carrot (Blachinsky et al. 2007; Droby et al. 2009). Both hot water at 46
C for 10 to 20 min and the biocontrol agent Rhodotorula glutinis, as a stand-alone treatment, reduced the incidence of blue mold decay on pear fruit, but complete control was not achieved by either treatment alone. However, a combination of hot water treatment and R. glutinis, which was applied immediately after heat treatment, completely controlled decay of inoculated fruit (Zhang et al. 2008). Pseudomonas isolates were evaluated as biocontrol agents, alone and in combination with 3% sodium bicarbonate (SBC) treatments at 24
C and 45
C on artificially inoculated ‘Thomson’ navel oranges. Fluorescent Pseudomonas spp. isolates in combination with a treatment with hot SBC formed a practical alternative or complement to fungicides for postharvest control of green mold on oranges (Zamani et al. 2008). Zhang et al. (2007) examined the potential of using an antagonistic yeast, Cryptococcus laurentii, alone or which applied after a brief HWDs at 55
C for 30 s (combined treatment) for the control of postharvest Rhizopus rot and natural infections of strawberries and found this combination very effective against Rhizopus rot; and none of the treatments impaired fruit quality. Obagwu and Korsten (2003) reported that treatment with a biocontrol agent following hot water treatment at 45
C for 2 min was as effective as the fungicide treatment, which gave 100% control of both green and blue molds on artificially inoculated ‘Valencia’ and ‘Shamouti’ oranges. De Costa and Erabadupitiya (2005) found that hot water treatment at 50
C for 3 min prior to treatment with the bacterial antagonist Burkholderia cepacia gave more effective control of anthracnose, crown rot, and blossom end rot in banana than either of the treatments used individually.

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D. Hot Water Treatment and Ethanol Ethanol occurs naturally in fruits and many other food products, and its toxic properties with respect to fungal pathogens have been studied (Margosan et al. 1997). In an in vitro test, spores of the fungi were exposed to solutions containing up to 30% (v/v) ethanol at 25
C or 50
C for 30 s, and their survival was then determined by germination on semisolid media. Combinations of ethanol and heat were synergistic: Control of spores of these fungi could be accomplished with much lower temperatures and ethanol concentrations when the two means were combined than when either was used alone. Botrytis cinerea and Alternaria alternata were less resistant to the combination than Aspergillus niger or Rhizopus stolonifer (Gabler et al. 2004). Table grapes form an important component of the fruit diet in many countries. Grapes are consumed raw with minimal washing and recently have also been introduced into several ready-to-eat fruit salads. Similar to other fresh produce and salads, grapes might be vulnerable to contamination by food-borne pathogens in the field or during harvest. The incidence of gray mold among grape berries that were untreated or immersed in 35% (v/v) ethanol at 25
C or 50
C for 1 min was 78.7, 26.2, and 3.4 berries/kg, respectively, after 1 month of storage at 0.5
C followed by 2 days at 25
C (Gabler et al. 2005). Immersion in 10% ethanol at 50
C, 55
C, or 60
C for 30 or 60 s significantly reduced the number of grape berries that developed decay after storage for 30 days at 1
C (Karabulut et al. 2004c). The control of postharvest diseases of sweet cherry by treatment with ethanol and hot water was evaluated by Karabulut et al. (2004b). The most effective treatment was immersion in 10% ethanol at 60
C. Treatments with 20%, 30%, 40%, or 50% ethanol or with water at 55
C or 60
C significantly reduced natural fungal populations on the surface of fruit in all of the experiments. Addition of 10% ethanol to water significantly increased the efficacy of water in reducing the fungal populations at elevated temperatures. None of these treatments caused surface injuries to the fruit or adversely affected stem color.

E. Hot Water Treatments with Other Combinations The combination of any type of physical, chemical, or environmentally friendly chemical methods, used to inhibit physiological and/or pathological deterioration of fresh-harvested produce, is called a physicochemical treatment. Better synergistic effects in controlling

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postharvest decay-causing agents can be achieved by combining several means. Using hot water at 52
C for 3 min, in combination with wax and TBZ fungicide minimized postharvest decay in fruits of two orange cultivars, especially that caused by Penicillium molds (Ansari and Feridoon 2008). Two approaches—fungicide applications to trees before harvest and drenching fruits after harvest—were evaluated as ways to minimize postharvest green mold caused by P. digitatum, particularly among fruit subjected to ethylene gas after harvest, a practice termed “degreening” that eliminates the green rind color (Smilanick et al. 2006). Preharvest applications of thiophanate methyl (TM) controlled postharvest green mold consistently. Application of 372 mg/L TBZ to harvested fruit in bins before degreening was also very effective, and its effectiveness was enhanced by mild heating at 41
C for 1 min, adding 3% SBC, and immersing the fruits in the solution rather than drenching them. The efficacy of TBZ in controlling postharvest decay of citrus fruit caused by P. digitatum can be enhanced by combining it with SBC and/or heat treatment: The combination of SBC and TBZ at 400 mg/L with mild heating at 40
C generally gave better control of green mold than the combined treatment at 20
C. Efficacy of TBZ was also improved when it was applied at a reduced rate of 200 mg/L at 50
C: It significantly suppressed green mold caused by a TBZ-sensitive isolate of P. digitatum and effectively controlled a TBZ-resistant isolate (Schirra et al. 2008b). The use of 2-min dips in 3% aqueous solutions of sodium carbonate (SC) or SBC at 40
C followed by application of the biocontrol agent Pantoea agglomerans was found to be a very promising alternative to the use of conventional fungicides to control green mold in citrus packinghouses (Usall et al. 2008). Combinations of several types of heat treatments were evaluated. Erkan et al. (2005a) assessed the effects of postharvest hot water treatments at 53
C for 3 or 6 min, or at 48
C for 12 min, and curing treatments at 53
C for 1 or 6 h, or at 48
C for 6 h, on decay of ‘Clementine’ mandarins (Citrus reticulata). They also examined treatments of ‘Valencia’ oranges, which were either dipped in hot water at 53
C for 3 or 6 min or at 48
C for 12 min, or cured at 53
C for 1 or 6 h or at 48
C for 12 h (Erkan et al. 2005b). All the fruit samples were stored at 4
C for 6 months following the treatments; it was found that both HWD and curing treatments reduced decay compared with that of the untreated controls. Treatments of ‘Star Ruby’ grapefruit with a combination of HWRB at 62
C for 20 s, SBC at 2% (w/v) and Candida oleophila yeast cells at 108 cells/mL, either 24 h after artificial inoculation with P. digitatum or following natural infection, reduced decay development by 89% to 90% from the level in untreated control fruit (Porat et al. 2002).

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Integrated treatments that combined hot water immersion at 55
C for 30 min, UV-C irradiation at 4.98 kJ/m2, and essential oil at 0.8 mL/cm3 for 2 weeks were more promising than the separate treatments for controlling decay caused by Fusarium oxysporum f. sp. gladioli in gladiolus corms that were stored for 4 and 12 weeks (Sharma and Tripathi 2008). To increase the storage shelf life of ‘Delicious’ and ‘Golden Delicious’ apples, they were exposed to UV-C irradiation for 0, 5, and 15 min at dose rates of 1.435  10–4 W/cm2, and 4 treatments with hot water containing 4% CaCl2: control, 25
C for 10 min, 38
C for 5 min, and 54
C for 1 min (Hemmaty et al. 2007). The results showed that UV-C irradiation and dipping fruits in hot water increased the storage life and improved fruit quality factors of ‘Delicious’ and ‘Golden Delicious’ apples at the end of cold storage. The modes of action and effectiveness of treatments with hot water and iodine were evaluated, and the treatments were integrated with systemic acquired resistance as alternatives to fungicide for controlling postharvest rots of melon (Cucumis melo). Dipping melons in hot iodine at 55
C was as effective as the commercial fungicide guazatine at 500 mg/L. Treating melons with hot iodine at 30 mg/L increased storage life and maintained fruit quality to similar levels to those achieved with fungicide treatment. Treating field plants with benzothiadiazole 2 weeks before harvest reduced storage rots of melons as a result of the induction of systemic acquired resistance. Good control of postharvest rots was obtained by integrating a postharvest dip treatment with iodine in hot water and field treatment to induce systemic acquired resistance: The total reduction of rots achieved by benzothiadiazole treatment in the field followed by a postharvest dip in hot iodine was much greater than that achieved by treating dipped noninduced fruit with a commercial fungicide (Bokshi et al. 2007). Mangoes were heated by: dipping in hot water at 55
C for 5 min (HWD55), vapor heat treatment (VHT) to raise the core temperature to 46.5
C for 10 min, dipping in water at 38
C for 1 h before VHT (HWD38 þ VHT), or dipping in water at room temperature (control). Disease incidence on fruit treated with HWD55, VHT, and HWD38 þ VHT was reduced to 0.24%, 0.26%, and 0.14%, respectively, compared with the control (Sopee et al. 2005). Combination of 1-MCP at 300 nL/L with hot water treatment at 46
C for 5 min extended the shelf life of ‘Keitt’ mango fruits by 5 days (Osuna-Garcia et al. 2007). Preharvest treatments with the biocontrol agent Epiccocum nigrum, followed by postharvest HWDs at 60
C for 20 s plus 1% SBC significantly reduced Monilinia rot on nectarine and peach by over 70%, compared

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with that on fruits that received postharvest physicochemical treatment without preharvest treatment (Mari et al. 2007). The efficacy of HWDs at 63
C for 12 s, biological control with Pichia guilliermondii, and controlled atmospheres (CA) containing CO2 at 15 kPa, alone and in combinations, were evaluated with harvested strawberry fruits for 5 and 14 days followed by 2 days at 20
C (Wszelaki and Mitcham 2003). Fruits treated with the combination of heat, biocontrol, and CA exhibited significantly less decay than those in all of the other treatments.

IV. HOT WATER TREATMENTS AND FRESH CUT PRODUCTS Fresh-cut fruit products for both retail and food service use have increasingly appeared in the marketplace recently. In the coming years, it is commonly believed that the fresh-cut fruit industry will have unprecedented growth. The U.S. Department of Agriculture and Food and Drug Administration definitions of “fresh” and “minimally processed” fruits and vegetables imply that fresh-cut (precut) products have been freshly cut, washed, packaged and maintained with refrigeration. The International Fresh-Cut Produce Association defines a fresh-cut product as fruits or vegetables that have been trimmed and/or peeled and/or cut into a 100%-usable product that is bagged or prepackaged for offering to consumers. Microbial decay can be a major cause of spoilage of fresh-cut produce; it may affect fresh-cut fruit much more rapidly than vegetable products because of the high levels of sugars found in most fruits. However, the acidity of fruit tissue usually helps to suppress bacterial growth, but not that of yeast and molds (Brackett 1994). Shredded green papaya was dipped in 0.5% calcium chloride solutions at 25
C or 40
C after which the shreds were stored at 4
C for 10 days. The results indicate that calcium chloride could maintain the quality and prolong shelf life of shredded papaya, especially when the higher dipping temperature was used (Kakaew et al. 2007). The effect of the disinfectant sodium hypochlorite (NaClO), with or without mild heat (50
C) and fumaric acid on native bacteria and the food-borne pathogens Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella typhimurium DT104 attached to iceberg lettuce leaves was examined (Kondo et al. 2006). The combination of NaClO at 200 mg/L and mild heat treatment at 50
C for 1 min reduced the pathogen populations by 94% to 98% (i.e., by 1.2 to 1.7 log reduction) without increasing browning. A study was conducted by Fan et al. (2006) to investigate the feasibility of using hot water treatment in combination with low-dose

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irradiation to reduce native microbial populations without impairing the quality of fresh-cut cantaloupe. Whole cantaloupes were washed in tap water at 20
C or 76
C for 3 min, and fresh-cut cubes were prepared from the washed fruit and packaged in clamshell containers. Half the samples were exposed to 0.5 kGy of gamma radiation. The hot water surface pasteurization reduced the microflora population on the surface of whole fruits by 3.3 log reduction, resulting in a lower microbial load on the fresh-cut cubes than on cubes cut from fruits treated with cold water. Cubes prepared from untreated fruits and irradiated to an absorbed dose of 0.5 kGy exhibited a low microbial load similar to that of cubes prepared from hot water–treated fruit. The combination of the two treatments was able to further reduce the microflora population (Fan et al. 2006). Slices of Chinese water chestnut, cv. Guilin, were immersed in boiling water for 30 s, placed in film-wrapped trays, and then stored at 4
C for up to 12 days, after which no microbial growth was detected and the quality of the fresh-cut product was maintained (Peng and Jiang 2004).

V. SUMMARY AND CONCLUSIONS Fresh fruits and vegetables have always been a part of the human diet. Since the beginning of crop cultivation, producers and distributors have been concerned about losses. At present, international trade in fruits and vegetables around the world is severely constrained by quarantine and phytosanitation barriers erected to prevent the spread of fungal and bacterial diseases as well as insects in fresh and fresh-cut produce. These barriers can be removed only when there is an effective treatment for use on fresh produce after harvest. Heat treatments such as hot water dips, vapor heat, dry heat or curing, and hot water rinsing and brushing, alone or in combination with modified or controlled atmosphere storage, biocontrol agents, ethanol, bicarbonate salts, low dosages of fungicides, and irradiation have been found to control postharvest fungal decay development without leaving residues. In developed countries, the average losses caused by postharvest decay incidence on fruits, vegetables, and ornamentals range from 5% to more than 20%, depending on the genetic background of the commodity, the climate, and preharvest and postharvest practices; and in developing countries, these losses may rise to 50%. Fungicides have been the main agent used to control decay development on freshly harvested produce during storage and marketing for more than five decades, but their use has become increasingly restricted because of

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perceived harmful effects on human health and the environment, declining effectiveness due to development of resistance in major postharvest pathogens, and increasing difficulties in developing and registering new effective fungicides. Prestorage heat treatments are addressed by a wide range of research into postharvest decay control; there are many avenues that merit further exploration. Hot water treatments, such as as HWDs or HWRB, are relatively simple techniques—economical, safe from rapid temperature changes, and applicable to many potential postharvest problems. These treatments, alone or in combination with modified atmosphere packaging, biocontrol agents, ethanol, bicarbonate salts, and even fungicides at low dosages, have been found in many cases to be more effective than traditional fungicides in controlling decay-causing agents. These combined treatments can also provide better residual protection. However, a better understanding of the physiology, pathology, biochemistry, and molecular biology of heat-treated fresh-harvested produce will enable the development of more precise and effective combinations of hot water treatments with other control agents, compounds, and technologies. These simple technologies against quarantine insects, hot water dips and cold treatments, should be explored with regard to a broader range of organic crops or freshly harvested commodities destined for sale as minimally processed products, thereby reducing our current extensive reliance on pesticides and enhancing our protection of the environment. ACKNOWLEDGMENT This is a contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel No. 559/09. LITERATURE CITED Abu-Kpawoh, J.C., Y.F. Xi, Y.Z. Zhang, and Y.F. Jin. 2002. Polyamine accumulation following hot-water dips influences chilling injury and decay in ‘Friar’ plum fruit. J. Food Sci. 67:2649–2653. Akbudak, B., N. Akbudak, V. Seniz, and A. Eris. 2007. Sequential treatments of hot water and modified atmosphere packaging in cherry tomatoes. J. Food Qual. 30:896–910. Armstrong, J.W., and P.A. Follett. 2007. Hot-water immersion quarantine treatment against Mediterranean fruit fly and oriental fruit fly (Diptera: Tephritidae) eggs and larvae in litchi and longan fruit exported from Hawaii. J. Econ. Entomol. 100:1091–1097. Ansari, N.A., and H. Feridoon. 2008. Postharvest application of hot water, fungicide and waxing on the shelf life of Valencia and the local orange cv. Siavarz. Acta Hort. 768:271–277.

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Bai, J.H., E.A. Mielke, P.M. Chen, R.A. Spotts, M. Serdani, J.D. Hansen, and L.G. Neven. 2006. Effect of high-pressure hot-water washing treatment on fruit quality, insects, and disease in apples and pears Part I. System description and the effect on fruit quality of ‘d’Anjou’ pears. Postharvest Biol. Technol. 40:207–215. Barkai-Golan, R. 2001. Postharvest diseases of fruits and vegetables. Development and control. Elsevier. Amsterdam, The Netherlands. Benitez, M.M., A.L. Acedo, P. Jitareerat, and S. Kanlayanarat. 2006. Induction of disease resistance in mango fruit by postharvest heat treatment. Acta Hort. 712:785–792. Ben-Yehoshua, S., J. Peretz, V. Rodov, B. Nafussi, O. Yekutieli, A. Wiseblum, and R. Regev. 2000. Postharvest application of hot water treatment in citrus fruits: The road from the laboratory to the packing-house. Acta Hort. 518:19–23. Blachinsky, D., J. Antonov, A. Bercovitz, B. Elad, K. Feldman, A. Husid, M. Lazare, N. Marcov, I. Shamai, M. Keren-Zur, and S. Droby. 2007. Commercial applications of “Shemer” for the control of pre- and postharvest diseases. IOBCWPRS Bul. 30: 75–78. Bokshi, A.I., S.C. Morris, and R. McConchie. 2007. Environmentally-safe control of postharvest diseases of melons (Cucumis melo) by integrating heat treatment, safe chemicals, and systemic acquired resistance. New Zealand J. Crop Hort. Sci. 35:179–186. Brackett, R.E. 1994. Microbiological spoilage and pathogens in minimally processed refrigerated fruits and vegetables. p. 269–312. In: R.C. Wiley (ed.), Minimally processed refrigerated fruits and vegetables. Chapman and Hall, London. Candir, E.E., F. Temizyurek, and A.E. Ozdemir. 2009. The effects of hot water dip treatments on the cold storage of Big Top nectarines. J. Appl. Bot. Food Qual. —Angewandte Bot. 82:136–140. Dang, K.T.H., Z. Singh, and E.E. Swinny. 2008. Impact of postharvest disease control methods and cold storage on volatiles, color development and fruit quality in ripe ‘Kensington Pride’ mangoes. J. Agr. Food Chem. 56:10667–10674. De Costa, D.M., and H.R.U.T. Erabadupitiya. 2005. An integrated method to control postharvest diseases of banana using a member of the Burkholderia cepacia complex. Postharvest Biol. Technol. 36:31–39. Dimitris, L., N. Pompodakis, E. Markellou, and S.M. Lionakis. 2005. Storage response of cactus pear fruit following hot water brushing. Postharvest Biol. Technol. 38:145–151. Droby, S., M. Wisniewski, D. Macarisinb, and C. Wilson. 2009. Twenty years of postharvest biocontrol research: Is it time for a new paradigm? Postharvest Biol. Technol. 52:137–145. Erkan. M., M. Pekmezci, I. Karasahin, and H. Uslu. 2005a. Reducing chilling injury and decay in stored ‘Clementine’ mandarins with hot water and curing treatments. Eur. J. Hort. Sci. 70:183–188. Erkan, M., M. Pekmezci, and C.Y. Wang. 2005b. Hot water and curing treatments reduce chilling injury and maintain post-harvest quality of ‘Valencia’ oranges. Intl. J. Food Sci. Technol. 40:91–96. Fallik, E. 2004. Prestorage hot water treatments (immersion, rinsing and brushing). Postharvest Biol. Technol. 32:125–134. Fallik, E. 2007. Heat treatments to control fungal disease. p. 21–38. In R. Troncoso-Rojas and M.E. Tiznado-Hernandez (Eds.), Recent advances in postharvest technologies to control fungal diseases in fruits and vegetables. Research Signpost. Kerala, India. Fallik, E., Y. Aharoni, A. Copel, V. Rodov, S. Tuvia-Alkalai, B. Horev, O. Yekutieli, A. Wiseblum, and R. Regev. 2000. A short hot water rinse reduces postharvest losses of ‘Galia’ melon. Plant Pathol. 49:333–338.

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Fallik, E., S. Grinberg, S. Alkalai, O. Yekutieli, A. Wiseblum, R. Regev, H. Beres, and E. Bar-Lev. 1996. A unique method for simultaneously cleaning and disinfecting sweet pepper using hot water wash and brushes. (In Hebrew with an English summary.) Gan Sadeh Vameshek 10:38–42. Fallik, E., S. Grinberg, S. Alkalai, O. Yekutieli, A. Wiseblum, R. Regev, H. Beres, and E. Bar-Lev. 1999. A unique rapid hot water treatment to improve storage quality of sweet pepper. Postharvest Biol. Technol. 15:25–32. Fallik, E., V. Rodov, B, Horev, S. Sela, S. Alkalai-Tuvia, and Y. Vinokur. 2007. Hot water rinsing and brushing technology for the fresh-cut industry. Acta Hort. 746:229–235. Fallik, E., S. Tuvia-Alkalai, X. Feng, and S. Lurie. 2001. Ripening characterization and decay development of stored apples after a short prestorage hot water rinsing and brushing. Innovative Food Sci. Emerging Technol. 2:127–132. Fan, X.T., B.A. Annous, K.J.B. Sokorai, A. Burke, and J.P. Mattheis. 2006. Combination of hot-water surface pasteurization of whole fruit and low-dose gamma irradiation of fresh-cut cantaloupe. J. Food Prot. 69:912–919. Feng, X., J.D. Hansen, B. Biasi, J. Tang, and E.J. Mitcham. 2003. Use of hot water treatment to control codling moths in harvested California ‘Bing’ sweet cherries. Postharvest Biol. Technol. 31:41–49. Gabler, F.M., M.F. Mansour, J.L. Smilanick, and B.E. Mackey. 2004. Survival of spores of Rhizopus stolonifer, Aspergillus niger, Botrytis cinerea and Alternaria alternata after exposure to ethanol solutions at various temperatures. J. Appl. Microbiol. 96:1354–1360. Gabler, F.M., J.L. Smilanick, J.M. Ghosoph, and D.A. Margosan. 2005. Impact of postharvest hot water or ethanol treatment of table grapes on gray mold incidence, quality, and ethanol content. Plant Dis. 89:309–316. Gal, S., S. Alkalai-Tuvia, Y. Elkind, and E. Fallik. 2006. Influence of different concentrations of 1-methylcyclopropene and times of exposure on the quality of ‘Galia’-type melon harvested at different stages of maturity. J. Hort. Sci. Biol. 81:975–982. Gonzalez-Aguilar, G.A., L. Gayosso, R. Cruz, J. Fortiz, R. Baez, and C.Y. Wang. 2000. Polyamines induced by hot water treatments reduce chilling injury and decay in pepper fruit. Postharvest Biol. Technol. 18:19–26. Hassan, M.K., W.A. Shipton, R. Coventry, and C. Gardiner. 2004. Extension of banana shelf life. Australasian Plant Pathol. 33:305–308. Hemmaty, S., N. Moallemi, and L. Naseri. 2007. Effect of UV-C radiation and hot water on the calcium content and postharvest quality of apples. Spanish J. Agr. Res. 5:557–566. Hong, S.I., H.H. Lee, and D. Kim. 2007. Effects of hot water treatment on the storage stability of satsuma mandarin as a postharvest decay control. Postharvest Biol. Technol. 43:271–279. Ilic, Z., Y. Polevaya, S. Tuvia-Alkalai, A. Copel, and E. Fallik. 2001. A short prestorage hot water rinse and brushing reduces decay development in tomato, while maintaining its quality. Tropical Agr. Res. Ext. 4:1–6. Janisiewicz, W.J., and L. Korsten, 2002. Biological control of postharvest diseases of fruits. Annu. Rev. Phytopathol. 40:411–441. Jaroenkit, T., and R.E. Paull. 2003. Postharvest handling of Heliconia, red ginger, and birdof-paradise. HortTechnology 13:259–266. Kakaew, P., H. Nimitkeatkai, V. Srilaong, and S. Kanlayanarat. 2007. Effects of CaCl2 dips and heat treatments on quality and shelf-life of shredded green papaya. Acta Hort. 746:335–341. Karabulut, O.A., U. Arslan, and G. Kuruoglu. 2004a. Control of postharvest diseases of organically grown strawberry with preharvest applications of some food additives and postharvest hot water dips J. Phytopathol. 152:224–228.

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Karabulut, O.A., U. Arslan, G. Kuruoglu, and T. Ozgenc. 2004b. Control of postharvest diseases of sweet cherry with ethanol and hot water. J. Phytopathol. 152:298–303. Karabulut, O.A., L. Cohen, B. Weiss, A. Daus, S. Lurie, and S. Droby. 2002. Control of brown rot and blue mold of peach and nectarine by short hot water brushing and yeast antagonists. Postharvest Biol. Technol. 24:103–111. Karabulut, O.A., F.M. Gabler, M. Mansour, and J.L. Smilanick. 2004c. Postharvest ethanol and hot water treatments of table grapes to control gray mold. Postharvest Biol. Technol. 34:169–177. Kays, S.J. 1997. Postharvest physiology of perishable plant products. 2nd ed. AVI Books, Van Nostrand Reinhold, New York. Kondo, N., M. Murata, and K. Isshiki. 2006. Efficiency of sodium hypochlorite, fumaric acid, and mild heat in killing native microflora and Escherichia coli O157:H7, Salmonella typhimurium DT104, and Staphylococcus aureus attached to fresh-cut lettuce. J. Food Prot. 69:323–329. Lee, Y.J., Y.H. Park, J.S. Kang, Y.W. Choi, and B.G. Son. 2008. Storability improvement of ‘Fuyu’ persimmon fruit by the combination of hot-water dipping and MAP. Korean J. Hort. Sci. Technol. 26:34–41. Lichter, A., O. Dvir, I. Rot, M. Akerman, R. Regev, A. Wiseblum, E. Fallik, G. Zauberman, and Y. Fuchs. 2000. Hot water brushing: An alternative method to SO2 fumigation for color retention of litchi fruits. Postharvest Biol. Technol. 18:235–244. Lurie, S. 2008. Heat treatment for enhancing postharvest quality. p. 246–259. In: P. Paliyath, D.P. Murr, A.V. Handa and S. Lurie (eds.), Postharvest biology and technology of fruits, vegetables, and flowers. Wiley-Blackwell, Ames, IA. Malakou, A., and G.D. Nanos. 2005. A combination of hot water treatment and modified atmosphere packaging maintains quality of advanced maturity ‘Caldesi 2000’ nectarines and ‘Royal Glory’ peaches. Postharvest Biol. Technol. 38:106–114. Margosan, D.A., J.L. Smilanick, G.F. Simmons, and D.J. Henson. 1997. Combination of hot water and ethanol to control postharvest decay of peaches and nectarines. Plant Dis. 81:1405–1409. Mari, M., R. Torres, L. Casalini, N. Lamarca, J.F. Mandrin, J. Lichou, I. Larena, M.A. De Cal, P. Melgarejo, and J. Usall. 2007. Control of post-harvest brown rot on nectarine by Epicoccum nigrum and physico-chemical treatments. J. Sci. Food Agric. 87:1271–1277. Maxin, P., S. Huyskens-Keil, K. Klopp, and G. Ebert. 2005. Control of postharvest decay in organic grown apples by hot water treatment. Acta Hort. 682:2153–2157. McDonald, K.L., M.R. McConchie, A. Bokshi, and S.C. Morris. 2005. Heat treatment: A natural way to inhibit postharvest diseased in rockmelon. Acta Hort. 682:2029–2033. Nafussi, B., S. Ben-Yehoshua, V. Rodov, J. Peretz, B.K. Ozer, and G. D’Hallewin. 2001. Mode of action of hot-water dip in reducing decay of lemon fruit. J. Agr. Food Chem. 49:107–113. Nolasco, C.D., L.C.C. Salomao, P.R. Cecon, C.H. Bruckner, and A. Rocha. 2008. Postharvest quality of ‘Prata’ banana as affected by hot water treatment. Ciencia Agrotecnol. 32:1575–1581. Obagwu, J., and L. Korsten. 2003. Integrated control of citrus green and blue molds using Bacillus subtilis in combination with sodium bicarbonate or hot water. Postharvest Biol. Technol. 28:187–194. Olesen, T., N.L. Nacey, N. Wiltshire, and S. O’Brien. 2004. Hot water treatments for the control of rots on harvested litchi (Litchi chinensis Sonn.) fruit. Postharvest Biol. Technol. 32:135–146.

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6 Promotion of Adventitious Root Formation of Difficult-to-Root Hardwood Tree Species Paula M. Pijut, Keith E. Woeste, and Charles H. Michler USDA Forest Service Northern Research Station Hardwood Tree Improvement and Regeneration Center Purdue University 715 West State Street West Lafayette, IN 47907, USA

ABBREVIATIONS I. INTRODUCTION A. Adventitious Root Formation B. Types of Root Formation in Stem Cuttings II. GENETICS AND PHYSIOLOGY OF ADVENTITIOUS ROOT FORMATION A. Herbaceous and Woody Models of Root Formation 1. Poplar as a Model System 2. Herbaceous Models of Phase Change B. Crucial Elements: Auxin and Competent Tissue 1. Operational Model of ARF 2. Role of Wounding and Mobilization of Resources 3. Genomic Analysis Reveals Molecular Details 4. Cell Building 5. Auxin and Cytokinin 6. Light and Auxin 7. MicroRNAs C. Regulation of Radial Pattern Formation D. Juvenility and Maturation 1. Methylation as a Model 2. Epigenetic Regulation of Phase Change

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III. CONTROLLABLE FACTORS THAT AFFECT ROOTING OF CUTTINGS A. Type of Stem Cutting and Season of Collection 1. Hardwood 2. Semi-Hardwood 3. Softwood B. Stock Plant Maturation and Manipulation 1. Juvenile ! Adolescent ! Mature 2. Pruning, Hedging, Forcing, or Serial Grafting 3. Light, Etiolation, Banding, or Shading C. Treatment of Cuttings D. Rooting Medium E. Greenhouse Parameters IV. CASE STUDY OF HORTICULTURAL VERSUS FOREST TREE SPECIES A. Horticultural Species B. Forest Species V. CONCLUDING REMARKS ACKNOWLEDGMENTS LITERATURE CITED

ABBREVIATIONS ABA AGO1 ARF ARF17 EST IAA IBA JA K-IBA LRF NAA NO PCR PIN QTL Rol SA SAM SCL SCR SHR WRC

abscisic acid ARGONAUTE1 adventitious root formation auxin response factor 17 expressed sequence tags indole-3-acetic acid indole-3-butyric acid jasmonate indole-3-butyric acid-potassium salt lateral root formation naphthalene acetic acid nitric oxide polymerase chain reaction proteinase inhibitor quantitative trait loci root loci salicylic acid S-adenosylmethionine synthase SCARECROW-LIKE SCARECROW SHORT-ROOT wound-related compounds

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I. INTRODUCTION North American hardwood tree species, such as alder (Alnus spp.), ash (Fraxinus spp.), basswood (Tilia spp.), beech (Fagus spp.), birch (Betula spp.), black cherry (Prunus serotina), black walnut (Juglans nigra), black willow (Salix nigra), elm (Ulmus spp.), hackberry (Celtis occidentalis), hard maple (Acer spp.), hickory (Carya spp.), oak (Quercus spp.), pecan (Carya spp.), sassafras (Sassafras albidium), sweetgum (Liquidambar styraciflua), sycamore (Platanus spp.), and yellow poplar (Liriodendron tulipifera), are important resources for the forest products industry worldwide and to the international trade of lumber and logs. The economic market for these tree species can be very high. Many of these hardwood species are also planted in the urban landscape, plantations, or orchards for seed or nut production. Timber, sawlog, and veneer log production of many of these fine hardwood species provides material for the manufacture of residential and commercial structures and furnishings and numerous specialty products, as contrasted with other hardwoods used for fuel or pulp (Pijut et al. 2007). Hardwood trees are also important for ecological reasons, such as wildlife habitat, mast production, riparian buffers, windbreaks, erosion control, watershed protection, agroforestry, conservation, land reclamation, native woodland restoration, and aesthetics (MacGowan 2003; Jacobs 2006). Many economically and ecologically important hardwood tree species have a low genetic or physiological capacity for adventitious root formation and are considered recalcitrant to routine, commercial-scale vegetative propagation via rooted cuttings. Why this phenomenon exists for many tree species (such as ash, beech, oak, and walnut) and not for others is not fully understood. Propagation of forest or urban tree planting stock by rooted cuttings can: circumvent problems with seed viability, germination, and storage; help overcome complex dormancy issues; shorten the time to flowering or encourage consistent flowering; maintain superior genotypes; and contribute to the genetic uniformity of tree plantations (Macdonald 1986). Clonal reproduction (resulting in progeny genetically identical to the original plant material) via adventitious rooting that can be easily adapted for many species, genotypes, or cultivars will allow for the production of clones of elite, pest-, or diseaseresistant trees or genetically improved trees (e.g., high-value wood) for planting and breeding programs. Some disadvantages associated with clonal reproduction and adventitious root formation can be less branched roots, more horizontal orientation, poor adventitious root distribution around the stem, or too few roots (D. Struve, person. commun.). In a tree improvement or breeding program, the genetic gains

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(additive and nonadditive) associated with clonal reproduction can be substantially greater than those associated with seed- or seedling-based forestry production systems (Zobel and Talbert 1984; Ritchie 1994). A. Adventitious Root Formation Adventitious root formation (ARF) as used in this review can be distinguished from lateral root formation (LRF), as the development of roots on excised aerial plant parts (shoots or stems), or from an unusual point of origin on the plant (old roots that have undergone secondary growth) (Esau 1977). Hence, LRF can be defined as the development of roots in a typical succession from the primary tap root of a tree and its laterals still in the early stage of growth. The fundamental mechanism(s) that trigger or regulate the initiation and development of adventitious roots on stem cuttings from woody species is a complex physiological, genetic, and environmental process and is still largely unknown. The endogenous origin and development (division of parenchyma cells) of adventitious roots close to the vascular tissue closely resemble the process of LRF (Esau 1977). De Klerk et al. (1999) summarized the successive phases in rooting of apple microcuttings as dedifferentiation, induction, outgrowth in the stem, and outgrowth from the stem. The initial phase, dedifferentiation, was the activation of cells (to become competent) by wounding related compounds and auxin. The induction phase was the initiation of cell division (to become committed) where auxin stimulates the formation of root meristemoids. During outgrowth in the stem phase, meristemoids develop into typical dome-shape root primordia (to become determined), and at this stage auxin (exogenously applied) then becomes inhibitory. Root primordia elongate and develop during the differentiation phase and finally grow out of the stem during the last phase of the adventitious rooting process. B. Types of Root Formation in Stem Cuttings There are two major types of root formation in stem cuttings: preformed and wound induced. Preformed or latent root initials are present during stem development and lie dormant until stem cuttings are made and placed in the proper environmental conditions favorable for further development and emergence as adventitious roots (Hartmann et al. 2002). Examples include willow (Salix) and poplar (Populus). Root initials often are established by the end of the season in the current year’s wood and will develop from cuttings made the following season.

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Species with preformed root initials generally root easily from stem cuttings (Hartmann et al. 2002). Wound-induced roots are the major type of ARF in stem cuttings. Once the stem (or shoot) is removed from the plant (wounding), a series of wound responses occur and de novo adventitious root regeneration proceeds (Hartmann et al. 2002). At the wounded sites, sealing off of the wound (protection from desiccation and pathogens) occurs by the production of suberized, protective cells. Cells begin to divide, and a layer of parenchyma cells (callus) then forms at the wound site. The use of auxin during adventitious rooting enhances the formation of callus in addition to inducing the formation of roots. Cells in the vicinity of the vascular cambium and phloem (near the source of hormones and carbohydrates) begin to divide and initiate adventitious roots (Hartmann et al. 2002). The developmental phases that occur in the stem cutting follow the stages described previously for apple microcuttings. The maturation-related loss of adventitious rooting competence in hardwood tree species is a major limiting factor to the clonal propagation of these woody species. Successful vegetative propagation via ARF in cuttings of hardwood tree species can be achieved if numerous factors are considered carefully. This chapter concentrates on what is known or largely unknown about the genetics and physiological basis of ARF, various factors that affect rooting of cuttings from some valuable North American hardwood tree species, individual case studies of horticultural versus forestry species, and future challenges and opportunities in understanding ARF of woody species. A comprehensive review of the literature was not possible, but we hope that we have captured or highlighted many important species and research studies over the last 12 years (1997–2009). The Rol (root loci) genes rolA, rolB, rolC, and rolD are plant oncogenes carried on root-inducing plasmids of the plant pathogen Agrobacterium rhizogenes. Natural infection of plants by A. rhizogenes causes hairyroot disease characterized by a massive growth of adventitious roots at the site of infection. Transformation of species with A. rhizogenes or with its rol genes produce hairy roots from which adventitious shoots (whole plants) can be regenerated, but typically with the characteristic hairyroot phenotype. The phenotypic alterations caused by the expression of these genes vary in degree but can include reduced apical dominance in both stems and roots, dwarfism, shortened internodes, abundant lateral branching, wrinkled and wider leaves, smaller leaves, adventitious root production, altered flowering and morphology, and reduced pollen and seed production (Welander and Zhu 2006). Each rol gene or combination of rol genes produces specific developmental alterations in transformed

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plants, but the biochemical functions of rol genes are not yet fully understood. The altered phenotypes can be advantageous in some ornamental and horticultural crop species (Casanova et al. 2005), but may not prove very advantageous in the genetic improvement and clonal propagation of hardwood tree species for timber, sawlog, and veneer log production. For example, silver birch (Betula pendula) plants transformed with rolC and rolD genes or all the rol genes were significantly shorter, had smaller leaves, and had a bushy growth habit (Piispanen et al. 2003). Therefore, rol genes in the context of producing adventitious roots with hardwood tree cuttings will not be addressed in this chapter.

II. GENETICS AND PHYSIOLOGY OF ADVENTITIOUS ROOT FORMATION A. Herbaceous and Woody Models of Root Formation Progress understanding the fundamental biology of ARF in woody plants has been slow, and recent important advances have been based primarily on investigations using model systems such as Arabidopsis. Because of the advantages of herbaceous models and because of the importance of LRF, research on LRF outpaces research into ARF (which is most important in woody perennials), even in species where both types of root formation can occur or be induced. While research into LRF potentially informs ARF (Butler and Gallagher 1998; Brinker et al. 2004), the full extent to which these two processes overlap has emerged only recently. It remains to be seen if it will be possible to translate what is learned about LRF and ARF in herbaceous species to practical use in woody species. Caution is warranted because it is unlikely that the biology of ARF is univocal in plants, and it is likely that some woody species will differ substantially biologically (De Klerk et al. 1999; Sanchez et al. 2007; Negi et al. 2008). There has been some progress in understanding ARF in woody species, and the apple ‘Jork 9’ is probably the closest to a woody model for ARF (Butler and Gallagher 1998; Sedira et al. 2007). 1. Poplar as a Model System. Poplar might also be useful as a model, since its genome was fully sequenced and a variety of Populus genomics tools are available. Comparison of poplar clones or mutants that are difficult to root would be an important step in the exploitation of this species for basic research in ARF (Haissig et al. 1992). Zhang et al. (2009) exploited an approach based on mapping root extension–related

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quantitative trait loci (QTL) in a pseudo–test cross between the difficultto-root P. deltoides (eastern cottonwood) and easily rooted P. euroamericana. Mean root length and total number of roots were found to be under strong genetic control, and the traits were strongly correlated with each other. They identified five QTLs related to root growth trajectory; four of the QTLs were derived from P. euroamericana and one from P. deltoides. By matching their findings with QTL and gene expression studies in P. trichocarpa, they hoped to identify conserved genomic regions affecting root formation that can be used for improvement. 2. Herbaceous Models of Phase Change. After germination, flowering plants develop through additional distinct stages or phases, including juvenile, mature (or adult), and reproductive phases (Willmann and Poethig 2005). The shift from juvenile to mature phases is economically significant to plant propagators because, in many species, it is difficult or impossible to induce adventitious roots from mature tissues. Phase change from mature to reproductive is economically important in both herbaceous annuals and woody perennials, but it differs in an important way for the two groups. Herbaceous plants typically make a once-in-alifetime decision to flower; perennials develop in a pattern of seasonal vegetative and floral development that repeats over many years (Hsu et al. 2006). The ability of meristems to switch between vegetative and floral phase may be regulated by a small number of genes that also affect growth habit and life span (Melzer et al. 2008). Phase change from juvenile to mature can be observed and studied in a number of tractable herbaceous model systems, including Arabidopsis and maize (Willmann and Poethig 2005). The same herbaceous models may also provide insight into the practical problem of rejuvenating mature tissues (Orkwiszewski and Poethig 2000). B. Crucial Elements: Auxin and Competent Tissue The two indispensible factors in nearly every example of ARF were auxin (De Klerk et al. 1999) and a tissue that was predisposed to initiate roots (Haissig et al. 1992), since mature and juvenile tissues from the same source plant often have completely different responses to auxin treatment (Geneve and Kester 1991; Vielba et al. 2008). The list of environmental and endogenous factors shown to influence ARF includes nearly everything that can affect plant growth (e.g., hormones, light quantity, light quality, oxygen, carbon dioxide, nitric oxide [NO], free radicals, relative humidity, pH of the growth media, physical structure of the growth media, antioxidants, wounding, polyamines,

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P. M. PIJUT, K. E. WOESTE, AND C. H. MICHLER

and concentration and types of nutrients in the media, etc.). For most of these factors, the biological role in ARF is uncertain; some may enhance ARF simply by keeping shoots or microshoots healthy while the process of ARF takes place. Others, such as polyamines, may act more directly by affecting the production or distribution of endogenous factors, such as auxin (Castillo and Casas Martinez 2008; Naija et al. 2009); or, as in the case of NO, by acting as an intermediary in auxin signaling (Pagnusset et al. 2003). Thus, the most parsimonious explanation for the action of all other factors shown to influence ARF is that they interact with, enhance, or suppress the action of auxin or that they “juvenilize” the shoot. The physiology and molecular genetics of ARF can thus be reduced to the genetics (of the parent plant material) and physiology of auxin (regulation) and of maturation, the former subject being better researched and understood than the latter. Li et al. (2009) has reviewed exogenous and exogenous factors in ARF signaling and auxin response genes. 1. Operational Model of ARF. The developmental stages of ARF in apple were summarized by De Klerk et al. (1999). The first phase (0–24 hr after indole-3-butyric acid [IBA] treatment) involves dedifferentiation of shoot tissues, and was marked by the accumulation of starch grains and wound-related compounds (WRCs). The second phase, induction (24–96 hr), was marked by the initiation of cell division, usually at about 48 hr, and the degradation of starch grains; at the end of this phase, meristemoids of about 30 cells can be observed. Meristemoid cells were typically small with relatively large nuclei and dense cytoplasm. During the second phase, hormone treatment with cytokinins or treatment with salicylic acid (SA) inhibited ARF. In Petunia, the first macroscopic anatomical events associated with ARF could be observed 72 hr after treatment with IBA (Ahkami et al. 2009). The third phase was root outgrowth within the stem (96–120 hr). By this time, auxin was no longer required and can even become inhibitory. Typical dome-shape primordia can be observed, and sensitivity to cytokinins and SA decreases. In the final phase, after 120 h, new roots have grown through the epidermis of the stem. 2. Role of Wounding and Mobilization of Resources. The model by De Klerk et al. (1999), while reductionist and not generalizable to all species (Brinker et al. 2004), can be useful for integrating disparate observations. For example, the role of WRC early in ARF was highlighted by the association between ARF and ethylene, abscisic acid (ABA), jasmonate (JA), NO, peroxide, peroxidases, and other WRCs during the dedifferentiation and initiation in phases (De Klerk et al. 1999; De Klerk 2002;

6. PROMOTION OF ADVENTITIOUS ROOT FORMATION

221

Pagnussat et al. 2002; McDonald and Wynne 2003; Husen 2008; Tartoura 2008; Ahkami et al. 2009). Although wounding is inescapable in the production of woody cuttings, the induction of WRCs may be more than incidental to subsequent ARF. Brinker et al. (2004) suggested that a defense barrier may be initiated during the early stages of ARF. In Petunia, wounding (at the time when cuttings were collected) set off a predictable cascade of JA signaling and subsequent activation of both JA responsive and JA biosynthetic genes (Ahkami et al. 2008). The jasmonate and salicylate pathways were thought to act in concert to regulate downstream defense responses (Schenk et al. 2000). A separate physiological role for JA in the establishment of a tissue competent for ARF is described below. Ethylene, a hormone associated with wounding in many species, was shown to both enhance and inhibit rooting (Biondi et al. 1990; Mori et al. 2008; Negi et al. 2008). The significance of this observation was complicated, and perhaps explained by the fact that auxin itself stimulates production of ethylene in at least some plants (Biondi et al. 1990; Woeste et al. 1999), and that ethylene can up-regulate auxin biosynthesis to affect root development (Swarup et al. 2007). The coordination of cell repair, DNA replication, cell division, and cell elongation processes necessary for ARF requires the investment of considerable energy and structural carbohydrates (Ahkami et al. 2008; Husen 2008), and the production of proteins (Hutchison et al. 1999). In Petunia, Ahkami et al. (2008) found that sugars, starch, and the concentration of enzymes in the pentose phosphate and glycolytic pathway were maximal after meristem formation. This, according to the authors, indicated more of a supportive role of root growth rather than initiation of ARF. This conclusion was at least conceptually in agreement with the findings of Li and Leung (2000), Takahashi et al. (2003), and Ruedell et al. (2008). Ahkami et al. (2008) suggested that wounding induces JA, which in turn activates enzymes that degrade sucrose in the apoplast to hexoses. Sucrose was transported for use in cell repair and cell division, establishing the wounded tissue as a sink. The importance of transporters in the early phases of ARF was also a significant finding of Kohler et al. (2003). 3. Genomic Analysis Reveals Molecular Details. The capacity to describe changes in gene expression during plant development has been considerably enhanced by the use of transcript profiling methods, such as differential messenger RNA display, microarray, and quantitative realtime polymerase chain reaction (PCR) technologies. Although transcript abundance is at best an indirect assessment of physiological activity, changes in transcript abundance that correspond with cellular processes

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P. M. PIJUT, K. E. WOESTE, AND C. H. MICHLER

can shed light on the regulation of development and reveal similarities that underlie apparently unrelated developmental events. Microarrays in particular can produce a large amount of data, but it can be difficult to determine which of the observed changes in transcript abundance are most important (i.e. up- or down-regulations of some genes may shed light on the process of tissue building, but may not be specifically related to ARF). The use of new techniques to isolate specific cells, the use of certain controls, and the analysis of tissues from early phases of ARF can help avoid pitfalls (Schnable et al. 2004). Experiments that include genotypic differences (comparing hard-to-root and easy-to-root genotypes of the same species, and comparing wild-type plants with nearisogenic mutants) or differences related to maturation (juvenile versus mature tissues from the same plant) also add power to analyses of messenger RNA abundance (Haissig et al. 1992). The most complete microarray analyses of ARF to date were published by Brinker et al. (2004) for Pinus contorta and by Kohler et al. (2003) for poplar. Lindroth et al. (2001a,b) had previously identified PSTAIRE CDC2, a gene putatively involved in cell division competence, and two apparently root-specific S-adenosylmethionine synthase (SAM) genes (the key regulatory enzyme of ethylene biosynthesis for many species) associated with ARF. By using a microarray of 2,178 cDNAs, they identified 220 genes that were differentially expressed during root development, with most of the genes (121) differentially expressed within 3 days of wounding and initial auxin treatment. Transcriptional profiling of the first 3 days after auxin treatment showed an increase in expression of genes needed for protein synthesis and decreases in genes for protein degradation; the opposite pattern appeared later as roots formed and elongated. They found that an ATP-binding cassette (ABC) transporter, a gene typically repressed by auxin, was up-regulated during the phase when meristems were being formed, possibly indicating that auxin was actively transported to the site of meristem formation during this phase. Kohler et al. (2003) generated 7,013 expressed sequence tags (ESTs) from the adventitious roots of hybrid cottonwood at various stages of development, focusing on aquaporins and transporters that were differentially expressed during the time course of ARF. They found that several were regulated in a stage-specific manner, but made no explicit connection between their data and current models of ARF. 4. Cell Building. Coordinated DNA replication and cell division was clearly necessary for the development of new meristems (Sedira et al. 2007). Brinker et al. (2004) identified a member of the PSTAIRE class of cyclin-dependent kinases, CDC2, which was up-regulated

6. PROMOTION OF ADVENTITIOUS ROOT FORMATION

223

during ARF in Pinus contorta. Cyclins and cyclin-dependent kinases such as CDC2 cooperate to regulate cell cycle progression. Brinker et al. (2004) speculated that CDC2 may have a role in establishing cell division competence in preparation for organogenesis. In this respect, the cyclin CycB1 may prove an important marker of cell division since it was expressed early in the pericycle in cells that will develop into lateral roots (Beeckman et al. 2001) and adventitious roots (Ahkami et al. 2009), but the gene itself was not expressed in primary roots (Porceddu et al. 1999). In the same paper where they describe the regulation of CDC2 in association with ARF in Pinus contorta, Brinker et al. (2004) also identified the up-regulation of a PINHEAD-ZWILLE–like gene closely related to a gene from Arabidopsis, ARGONAUTE1 (AGO1), that was first identified as essential for normal leaf development (Bohmert et al. 1998). Since its discovery, AGO1 has emerged as a critical factor regulating development in plants and animals (Peters and Meister 2007). 5. Auxin and Cytokinin. In the model of ARF just described, cytokinins strongly inhibited auxin-induced organogenesis in the second phase, from 24–48 hr after IBA treatment. It has been demonstrated by several researchers, however, that cytokinin can increase ARF in woody plants (Huetteman and Preece 1993; Ricci et al. 2005; Van Staden et al. 2007). The explanation for this apparent paradox is almost certainly found in the details of the interplay of auxin and cytokinins. Laplaze et al. (2007) and Pernisova et al. (2009) have shown that cytokinins play an integral role in auxin-induced organogenesis. In the model proposed by Laplaze et al. (2007), cytokinins interfere with polar auxin transport, the establishment of an auxin gradient, and the subsequent coordination of cellcycle progression and cell type respecification that produces organized lateral root primordia. Pernisova et al. (2009) were able to demonstrate that cytokinin was necessary for modulating the organogenic signal of auxin. They showed that auxin-induced organogenic development was accompanied by endogenous cytokinin production and the localized induction of cytokinin signaling pathways. In agreement with Laplaze et al. (2007), they found that cytokinin inhibits proteinase inhibitor (PIN) auxin efflux carriers, and the inhibition was independent of ethylene effects, but they concluded that endogenous cytokinin was necessary for producing auxin differentials that are required for de novo organogenesis. The interaction between cytokinin and PIN auxin carriers may be tissue and PIN gene specific, so species-specific details may yet emerge. 6. Light and Auxin. For at least some species, light quality and quantity clearly influence ARF (Fett-Neto et al. 2001; Wynne and McDonald 2002;

224

P. M. PIJUT, K. E. WOESTE, AND C. H. MICHLER

Takahasi et al. 2003; Fuernkranz et al. 2006; Pinker et al. 2008; Ruedell et al. 2008). The effects of light on ARF may be mediated through several downstream pathways, but the best evidence documented to date may be the interaction of GH3 class II family genes and AGO1, mentioned earlier. AGO1 mutants (ago1) were defective in light-regulated hypocotyl elongation and ARF, but not LRF, indicating that a properly functioning AGO1 was necessary for auxin homeostasis and for processes specific to ARF (Sorin et al. 2005). AGO1 was a critical element in micro-RNAmediated regulation of gene silencing, because AGO1 was a principal component of RNA-induced silencing complex (Hammond et al. 2001). AGO1 has been shown to assist in the regulation of ARF by influencing the expression of AUXIN RESPONSE FACTOR 17 (ARF17) and through ARF17, GH3 genes (Sorin et al. 2005). GH3 genes were one of three major classes of auxin early response genes that can be regulated by both light and auxin (Hsieh et al. 2000), and the accumulation of GH3 was positively correlated with ARF (Sorin et al. 2005). One possible mechanism for this observation was that GH3 can adenylate hormones, including indole-3-acetic acid (IAA). 7. MicroRNAs. As mentioned previously, the GH3 class of auxin early response genes was probably regulated by ARF17. ARF17, in turn, appears to be negatively regulated by microRNAs MIR160 and MIR167 working in concert with AGO1. These two microRNAs also positively regulate the auxin response factors ARF6 and ARF8 (Gutierrez et al. 2008), possibly setting up a system where ARF17 and ARF6/8 are maintained in a dynamic balance to modulate auxin homeostasis appropriate for local cell fate determination. Mutations in two other auxin response factors, NPH4/ARF7 and ARF19, also led to loss of ARF (Wilmoth et al. 2005). Auxin response factors such as ARF6/8 were regulated by light, and can regulate other auxin response factors at both the transcriptional and the posttranscriptional level by affecting the maturation of MIR160 and MIR167 (Gutierrez et al. 2008). A second microRNA, MIR164, responds to auxin induction and participates in the regulation of NAM/ATAF/CUC (NAC) domain transcription factor proteins. MIR164 interacts with NAC1 mRNA to down-regulate auxin signals related to LRF (Guo et al. 2005), providing a mechanism of homeostatic balance and preventing overproliferation of roots. C. Regulation of Radial Pattern Formation A second group of regulatory molecules implicated in ARF in both conifers and Arabidopsis were the members of the GRAS family of genes,

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225

specifically SCARECROW-LIKE (SCL) and SHORT-ROOT (SHR) (Hochholdinger and Zimmerman 2008). Both SCL and SHR act within the first 24 h after auxin treatment, during dedifferentiation but before cell division. In Arabidopsis, these genes have been shown to regulate radial pattern formation, are required when ground tissues such as the endodermis and cortex must be separated by asymmetric cell divisions, and to function in endodermal cell fate specification (Miyashima et al. 2009). SHR moves from the stele outward where it induces SCR (SCARECROW) expression. SCR can enhance its own expression and interact with SHR to activate additional downstream targets (Hochholdinger and Zimmerman 2008). In Pinus radiata, PrSCL1 was induced by exogenous auxin, but expression of PrSHR was auxin independent (Gutierrez et al. 2008). A similar pattern of SCL induction was observed in chestnut (Castanea sativa), although root primordia do not originate from the same location in Pinus and Castanea (S
anchez et al. 2007), possibly indicating that the roles of key genes were conserved even when the spatial or temporal details of ARF were not. Vielba et al. (2008) reported that SCL was induced by auxin treatment even in explants of mature stems of chestnut, indicating that the block in root formation in mature explants may occur downstream of GRAS gene interactions. The SHR/SCR pathway apparently regulates root pattern formation independently of the previously mentioned ARGONAUTE1regulated pathway (Shunsuke et al. 2009). D. Juvenility and Maturation 1. Methylation as a Model. Although auxin signaling (Lau et al. 2008) and the role of auxin in the regulation of ARF are beginning to be unraveled, the regulation of maturation and phase change is more complex. Specifically, what are the cellular and biochemical hallmarks of maturation and phase change, and how does maturation reconfigure the process of auxin response such that ARF is inhibited in mature tissues? As in the regulation of auxin responses, the genetics of phase change probably will require research using tractable herbaceous models (Poethig 1990; Hunter et al. 2006). A prevalent theory of maturation is that it reflects changes in DNA methylation. By comparing the DNA methylation of samples from juvenile and mature chestnut cuttings, Hasbun et al. (2007) found that aging implied a progressive increase of methylated 5-deoxycytidines (5-dmC), although flowering shoots showed a slight decrease in methylation compared to mature vegetative shoots. Baurens et al. (2004) found the opposite trend to prevail in microshoots of Acacia mangium; their results indicated that DNA from

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shoots with juvenile leaf morphology was more methylated than DNA from shoots with mature leaves. They also identified three sites with C5mCGG modification that were exclusive to juvenile samples and three similarly modified sites exclusive to mature samples. In maize, it was found that the methylation patterns at a locus called purple-plant1, which codes a transcription factor that modifies anthocyanin biosynthesis, were affected by vegetative phase. Methylation of an allele of the purple-plant1 gene called Pl-Blotched increased during the juvenile-toadult transition, was maximal in mature leaves, and its methylation state was reset each generation (Hoekenga et al. 2000). Irish and McMurray (2006) investigated the same gene using an in vitro system, and found that rejuvenated shoot apices were hypomethylated, indicating that reversion of phase was associated with loss of methylation. 2. Epigenetic Regulation of Phase Change. The connection between phase change and epigenetic gene regulation was further solidified when it was determined that a number of vegetative phase change mutants in Arabidopsis were also implicated in the genesis of small RNAs (19- 24-nt RNAs that include both microRNAs [miRNA] and short interfering RNA [siRNA]). Trans-acting siRNAs (ta-siRNA) were a subclass of siRNA (Willmann and Poethig 2005). It appears now that small RNAs have an important role in determining meristem boundaries and in meristem initiation (Chuck et al. 2009), perhaps pointing to the connection between the ability to form adventitious roots and developmental phase change. This connection was reinforced by the observation that tasiRNAs appear to regulate the maturation of auxin response factor mRNA, and this function appears to be conserved across diverse lineages (Axtell et al. 2007). In maize, levels of the ta-siRNA that target auxin response factor genes decreased in some mutants that were defective in meristem maintenance and the siRNA pathway; mutations in the same genes in Arabidopsis were defective in phase change (Chuck et al. 2009). Studies of the developmental defects associated with the misexpression of MIR156 microRNA in Arabidopsis and MIR172 of maize permitted Chuck et al. (2009) to suggest a converse regulatory relationship between MIR156 and MIR172. When the level of MIR156 was high (during the juvenile phase), the level of MIR172 was low; vice versa during the mature phase. Although the most tractable herbaceous models provide hints of connections between maturity of vegetative tissues, auxin metabolism, and organogenesis, the genetic and physiological connections between phase change and competence to form adventitious roots remains as an opportunity for exploration.

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III. CONTROLLABLE FACTORS THAT AFFECT ROOTING OF CUTTINGS A. Type of Stem Cutting and Season of Collection 1. Hardwood. Hardwood (dormant; leafless) stem cuttings are obtained from the previous season’s growth. Leaves can be removed without peeling the bark from the cutting, if cuttings are taken late in the growing season. Cuttings are usually prepared in late fall, winter, or early spring. Healthy stems free of disease or damage with at least two nodes are collected. Hardwood cuttings are not readily perishable but are prone to desiccation, and can be stored for several months and still retain high ARF potential. Prior to sticking, cuttings should be kept cool, out of direct sunlight, or can be stored for a few days to months (0  C) if not stuck the same day. Successful rooting of hardwood stem cuttings of forest tree species usually requires wounding (basal cut just below a node), the use of an auxin treatment to the basal end of the cutting, warm soil temperature (bottom heat), cool ambient air temperature, light, and intermittent mist or fog when propagated in the greenhouse (Table 6.1). Pijut and Moore (2002) reported hardwood stem cuttings (cut to 20–23 cm in length) of 5- and 6-year-old Juglans cinerea rooted (9%) when treated with a basal dip (10–15 s) in 29 mM indole-3-butyric acidpotassium salt (K-IBA), placed in a moist medium (perlite:peat), under intermittent mist (15 s every 18 min), with bottom heat (27  C), 12 h of supplementary lighting (60 mmol m2 s1), and a greenhouse temperature of 22  2  C. Swamy et al. (2002) reported hardwood stem cuttings (cut to 17–19 cm in length) of 2- and 15-year-old Robinia pseudoacacia rooted (40% or 50%) when treated with a basal dip (24 hr) in 1.3 mM naphthalene acetic acid (NAA) or 3.7 mM IBA, placed in a sterilized medium (sand:vermiculite), under intermittent mist (10 s every 30 min), with bottom heat (25  C), 10 h of normal light (50 mE cm2 s1), and a greenhouse temperature of 25  C. 2. Semi-Hardwood. Semi-hardwood (greenwood) stem cuttings are usually prepared in late summer or early fall. Stems with partially matured wood (lower region of the stem becoming lignified) are collected. Semi-hardwood cuttings are prone to desiccation and wilting; therefore, these should be collected in the cool, early morning when leaves and stems are turgid and should be stuck as soon as possible. Prior to insertion in media, cuttings should be kept cool, moist, and out of direct sunlight. Successful rooting of semi-hardwood stem cuttings, much like hardwood cuttings, usually requires wounding, the use of an auxin

228

Forced softwood shoots Softwood; greenhouse plants Softwood in July

A. saccharum (Caddo)

A. saccharum ‘Legacy’ A. saccharum

Acer saccharum

8-yr-old; during rapid shoot elongation and shoot lignification Eight mature trees; softwood

Forced softwood shoots Juvenile; softwood

A. rubrum ‘Autumn Flame’ A. rubrum ‘Franskred’, Semi-hardwood in ‘Red Sunset’ June A. rubrum ‘Bowhall’, Softwood; semi‘Franksred’ hardwood A. rubrum ‘Franskred’ Not stated

A. rubrum ‘Autumn Flame’

Acer rubrum

Age of tree or cutting type

Mist; wounding; strong-lite high porosity mix

Perlite or pumice; subirrigation Perlite or pumice; subirrigation Fog; peat:perlite: sand; shading Perlite; subirrigation temperature Mist; perlie: vermiculite Fog; peat:perlite: sand; shading Mist: peat: vermiculite; phenology

Mist; perlie: vermiculite Perlite; subirrigation temperature

Propagation parameters

<30

25–75

Stim-Root #2

12.3 or 24.6 IBA; 13.4 or 26.9 NAA; IBA þ NAA

0–13

15

31–80

27–87

poor

49.2 IBA

Dip’N Grow solutions 4.9 IBA

Rooting (%)

poor

69

Hormodin 3

Dip’N Grow solutions Dip’N Grow solutions 24.6 IBA

59

4.9 IBA in talc

Treatment of cuttingsa

Alsup et al., 2004

Tousignant et al. 2003

Zaczek et al. 1997

Henry and Preece 1997

Regan and Henderson 1999 Regan and Henderson 1999 Zaczek et al. 1997, 1999, 2000 Owen et al. 2003

Zhang et al. 1997

Henry and Preece 1997

References

Recent advances (1997–2009) in vegetative propagation of some important North American hardwood tree species.

Tree species

Table 6.1.

229

Q. alba, Q. palustris

Quercus alba, Q. bicolor, Q. macrocarpa, Q. Rubra

Platanus occidentalis Prunus serotina

Liquidambar styraciflua

Juglans cinerea

Fraxinus americana

Fagus grandifolia

Castanea dentata

Betula spp.

A. saccharum

4-yr-old;18-yr-old; softwood

2- and 9-yr-old; during rapid shoot elongation until end of shoot lignification Juvenile; greenhouse; field Forced softwood shoots Shoots from exposed roots Semi-woody epicormic sprouts 5- to 6-yr-old; hardwood; softwood 3- to 5-yr-old; softwood; severely pruned Hardwood 9-yr-old; hardwood; softwood 45-day-old; 3-yr-old; softwood Etiolation; banding; girdling; agrobacterium rhizogenes; peat: perlite; mist; stoolbeds Fog; peat:perlite: sand; shading

Hot-beds in winter Mist; peat:perlite

Mist; peat:perlite; bottom heat; pruning Mist; peat:perlite; fertilization

Mist; wounding; bottom heat Mist

Mist; wounding; bottom heat Mist; perlite

Mist; peat: vermiculite; light; etiolation; phenology

49.2 IBA

no PGRs 0–74 IBA; 0–62 K-IBA 39.4 IBA talc

Hormodin 2

9.8 IBA þ 5.4 NAA Dip’N Grow solutions 0–74 IBA; 0–62 K-IBA

Dip’N Grow; 4.9 IBA 20.7 K-IBA

Stim-Root ; IBA; NAA

0–30

10–48

>80 50–54

76–86

6.3–88

>80

25

3

24–100

70–96

(Continued)

Zaczek et al. 1997

Ferrini and Bassuk 2002

Arene et al. 2002 Pijut and Espinosa, 2004

Rieckermann et al. 1999

Van Sambeek and Preece 1999 Pijut and Moore 2002; Pijut, 2004

Barnes 2003

Preece et al. 2001

Barnes 2002

Richer et al. 2003, 2004

230

8-yr-old

2- to 6-yr-old

8-yr-old

9-yr-old

8-yr-old

Q. bicolor

Q. bicolor, Q. macrocarpa

Q. bicolor, Q. macrocarpa

Q. bicolor, Q. macrocarpa

Q. bicolor, Q. macrocarpa

Q. bicolor

10- to 15-yr-old; softwood; forced; stumps; hedges 3- to 5-yr-old

Age of tree or cutting type

Q. bicolor

Tree species

Table 6.1. (Continued).

Mist; perlite; light; banding; etiolation; age; severe cutback Mist; perlite; light; banding; etiolation; severe cutback Peat:perlite; etiolation; age; solvents; shoot position; modified container layering Field & air layering; severe cutback; etiolation Mist; perlite; light; banding; etiolation; severe cutback Perlite; mist; light; severe; cutback; etiolation; banding; stem anatomy

Mist; pro-mix bx



Propagation parameters

29.5 IBA

29.5

49.2 IBA basal paint

39.4–49.2 IBA

29.5

29.5 IBA

41.4 K-IBA

Treatment of cuttingsa

0–85

6–81

62–83

0–100

0–79

88–91

0.6–2.5

Rooting (%)

Amissah et al. 2008

Amissah and Bassuk 2007

Amissah and Bassuk 2005

Amissah and Bassuk 2004

Amissah and Bassuk 2007

Amissah and Bassuk 2007

Fishel et al. 2003

References

231

Juvenile; semihardwood 20-yr-old; semihardwood Mature; grafted plants; softwood 2-yr-old grafts; semi-hardwood 10- to 15-yr-old; softwood; forced; stumps; hedges Juvenile; mature; serial grafting 2- and 15-yr-old; softwood; hardwood

Q. nigra

41.4 K-IBA

49.2 IBA

49.2 IBA

49.2 IBA

49.2 IBA

39.4 IBA basal paint

Fog; peat:perlite: 59.0 IBA coarse sand Mist; sand: 1.3–4.0 NAA; vermiculite; age; 1.2–3.7 IBA bottom heat; season

Peat:perlite; severe cutback; incandescent light; age; root restriction & pruning; modified stoolbed; etiolation Fog; peat:perlite: sand; shading Fog; peat:perlite: sand; shading Fog; peat:perlite: coarse sand Fog; peat:perlite: sand; shading Mist; pro-mix bx

35–83

55–91

15–43

33–48

9–67

10–40

20–77

24–100

Swamy et al. 2002a,b

Zaczek et al. 2006

Fishel et al. 2003

Zaczek and Steiner 1997; Zaczek 1999 Zaczek et al. 1999, 2000

Zaczek et al. 1999, 2000

Zaczek et al. 1999, 2000

Hawver and Bassuk 2000

Concentration of auxins tested reported in millimolar (mM) unless indicated otherwise; Indole-3-butyric-acid (IBA); Indole-3-butyric acidpotassium salt (K-IBA); Naphthalene acetic acid (NAA); Plant growth regulators (PGRs); Dip’N Grow: http://www.dipngrow.com/; Hormodin : http://www.ohp.com/; Stim Root : http://www.plant-prod.ca/index_e.php.

a

Robinia pseudoacacia

Q. rubra

Q. rubra

Q. rubra

Q. rubra

Q. palustris

3- to 5-yr-old

Q. bicolor, Q. macrocarpa, Q. palustris

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treatment, ambient light or shading, and intermittent mist or fog when propagated in the greenhouse (Table 6.1). Leaves on semi-hardwood stem cuttings are usually trimmed to reduce the leaf surface area, in order to lower transpirational water loss, reduce leaf rotting, and save space in the propagation house. Regan and Henderson (1999) reported semi-hardwood cuttings of Acer rubrum ‘Franksred’ and ‘Red Sunset’ desiccated quickly when collected in June in Oregon and rooted poorly in a perlite medium when cuttings received a 10 s dip in Dip’N Grow (49.2 mM IBA and 26.9 mM NAA) with subirrigation. Semiwoody leafy cuttings of Fraxinus americana obtained from epicormic sprouts forced from mature branch segments rooted (>80%) when treated with Dip’N Grow solutions (1:24 or 1:99) (Van Sambeek and Preece 1999). Semi-hardwood oak and maple shoots were rooted [Quercus nigra (20–77%), Q. palustris (10–40%), Q. rubra (33–48%), and A. rubrum ‘Bowhall’(53–80%)], when treated with 49.2 mM IBA (oaks) and 24.6 mM IBA (maple), placed in a moist medium of peat:perlite:sand, maintained under low irradiance (93% shading), and intermittent cool fog (Zaczek et al. 1999, 2000). Oak shoots had all but the uppermost three leaves removed, whereas maple shoots were pruned to single-node cuttings. Semi-hardwood cuttings of J. cinerea rooted (11–46%) when treated with 29 or 62 mM K-IBA and 34 or 74 mM IBA (Pijut and Moore 2002). All but two leaflets were removed from these butternut cuttings, and rooting data were collected after 5–6 weeks. Acer saccharum semi-hardwood cuttings collected at 0800, treated with Stim-Root #2 (19.7 mM IBA), and placed in a peat:vermiculite medium under mist rooted 65–75% after 12 weeks (Tousignant et al. 2003). 3. Softwood. Softwood stem cuttings are obtained from the current season’s soft, succulent, new growth. Cuttings are usually prepared in late spring through early summer, during the period of active growth, but before extensive stem lignification. Several weeks exist where softwood cuttings can be collected and rooted, but the best time period needs to be determined for each individual species. Softwood cuttings generally root easier than other types of cuttings and have the highest rooting potential. Softwood cuttings are extremely perishable, stress easily, desiccate quickly, and therefore should be collected in the early-morning hours and kept moist, cool, and turgid at all times. Softwood cuttings need to be stuck as soon as possible the same day as collected. Successful rooting of softwood stem cuttings of forest tree species usually requires some sort of stock plant manipulation, certain cutting length, wounding or auxin treatment, the use of various rooting substrates, and several

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different greenhouse parameters, such as for light and air temperature, shading, mist, fog, humidity, or fertilization. In summary, there is a large environmental affect on ARF in cutting propagation. Leaves on cuttings are usually reduced in size to lower transpiration rates when propagated in the greenhouse. Cutting yield is the important factor in cutting propagation (i.e., the percentage of plantable cuttings obtained from the propagation system). A large factor in cutting yield is overwintering success. Cuttings from many taxa can be rooted, but few can be successfully overwintered and outplanted. Table 6.1 illustrates recent advances in vegetative propagation of several difficult-to-root hardwood tree species using softwood cuttings. A few will be highlighted here and in the next sections dealing with other factors that affect rooting of cuttings, since no one protocol works best for all species or genotypes. Zaczek et al. (1997) found that adventitious rooting of maple and oak cuttings was influenced by shade and auxin treatment. Softwood cuttings of A. rubrum ‘Bowhall’, ‘Franskred’, and ‘Red Sunset’ treated with 24.6 mM IBA rooted 67%, 87%, and 87%, respectively, when inserted in a peat:perlite:sand medium with intermittent cool fog and kept under 91% shade (control, 83% shade). Softwood oak cuttings (Q. alba and Q. palustris) only rooted at 30% and 23%, respectively, when treated with 49.2 mM IBA under the same fog and shade conditions (Zaczek et al. 1997). Softwood cuttings from nine Caddo sugar maples (10- to 30year-old) exhibited variability in rooting percentages among individual trees, but the type and concentration of auxin used made no significant difference (Alsup et al. 2004). Rooting was <30% when 13–46 cm cuttings (bottom leaves removed and wounded on each side of the base of the cutting) were treated with IBA, NAA, or IBA plus NAA and placed in a high-porosity rooting medium under mist with natural photoperiod (Alsup et al. 2004). Betula (B. alleghaniensis, B. lenta, and B. nigra) tip cuttings with two to four leaves rooted 87–100% when collected in late spring to early summer and treated with Dip’N Grow (1:10) or 4.9 mM IBA (Barnes 2002). Early-season softwood cuttings were effective for vegetative propagation from 5- and 6-year-old J. cinerea trees when treated with 62 mM K-IBA (77% rooting) or 74 mM IBA (88% rooting) (Pijut and Moore 2002). Butternut rooted cuttings survived overwintering in cold storage and acclimatization to the field (Pijut 2004). Sweetgum (Liquidambar styraciflua) succulent sprouts, from severely pruned 3- to 5-year-old stock plants previously established in the field from rooted cuttings, rooted well when treated with Hormodin 2 (14.8 mM IBA) (Rieckermann et al. 1999); 86% of all cuttings rooted, 76% survived, and 58% were deemed plantable when terminal and subterminal cuttings, with the lower leaves removed (leaving at least two leaves) and the

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remaining lobes of leaves cut in half, were placed in a peat: perlite medium under 55% shade with intermittent mist for 10 weeks (Rieckermann et al. 1999). B. Stock Plant Maturation and Manipulation 1. Juvenile ! Adolescent ! Mature. The maturation-related loss of adventitious rooting competence in hardwood trees becomes a major limiting factor in clonal forestry, since many desirable traits or characteristics (e.g., wood quality or seed production) are not expressed until the tree matures. Therefore, in these already difficult-to-root species, ARF from cuttings declines with the chronological age of the tree; cuttings from seedling (juvenile) trees root more readily than cuttings from mature (reproductive) trees. Phase change (juvenile ! adolescent ! mature) results from the expression of certain genes at specific times in the life cycle of a plant, or in response to environmental or internal signals (Hartmann et al. 2002). The length of the juvenile period in hardwood trees can range from 5 to 40 years, therefore making ARF in cuttings from selected mature trees extremely difficult and timely. A juvenile ! mature gradient (“cone of juvenility”) also exists in juvenile (seedling) trees, making ARF from cuttings variable (Hartmann et al. 2002). The area near the base of the tree is considered the oldest chronologically, but youngest in terms of ontogenetic age (Hartman et al. 1997). The stems and branches, therefore, are oldest in maturity (capable of reproduction), but youngest in chronological age. Therefore, rooting potential decreases with increasing distance from the ground. 2. Pruning, Hedging, Forcing, or Serial Grafting. Several horticultural or forestry-related practices or techniques can be utilized to help promote physiological juvenility (improve ARF from cuttings) in difficultto-root tree species. Pruning or hedging is the process of cutting back the original stock plant from which cutting material is taken, in order to produce new physiologically invigorated (juvenile) shoots or stump sprouts that root more readily (Macdonald 1986; Hackett 1988; Howard 1994). Pijut and Moore (2002) reported that it was necessary to prune back 2 years of growth after hardwood stem cutting collection of 5- to 6-year-old J. cinerea, in order to encourage sprouting of softwood stems for collection and rooting (77–88%). This same protocol was also important to encourage sprouting of softwood stems for collection and rooting (50–54%) from 9-year-old Prunus serotina field trees (Pijut and Espinosa 2004). Amissah and Bassuk (2005) reported significantly higher rooting percentages (77% for Q. bicolor and 70% for Q. macrocarpa) in

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layered propagules arising from 8-year-old field trees cut back to a 0.04 m stump above the soil surface. Forcing shoots from latent buds formed during primary growth, but remaining dormant throughout secondary growth under the bark (Hartmann et al. 2002), of dormant trees cut into sections and placed in a greenhouse, has been used successfully for cutting propagation of maple, white ash, and oak. Red maple stem sections (>50%) produced shoots, and softwood cuttings taken from these sections rooted at 60%, compared to shoots produced (20%) on sugar maple stem sections with rooting at 15% (Henry and Preece 1997). However, there may be unintended consequences; the genotypes that readily produce shoots from latent buds are the genotypes that provide the most cuttings. Therefore, if these good “shoot producing” individuals are propagated, one will produce genotypes that have a high potential for forming undesirable shoots (D. Struve, person. commun.). Semiwoody epicormic sprouts forced from branch segments of white ash were rooted (>80%) and field planted, but cuttings of forced black walnut or white oak did not root (Van Sambeek and Preece 1999). Semi-hardwood cuttings from 10- to 15-year-old forced northern red oak rooted on average 40%, but the main boles exhibited a vertical gradient in the number of shoots produced for cutting propagation (Fishel et al. 2003). Serial grafting of mature scion wood (with subsequent sequential regrafting) from difficult-to-root species onto seedling (juvenile) rootstock has improved the ARF of cuttings from these grafted stock plants of several woody species (Howard 1994; Hartmann et al. 2002). Zaczek et al. (2006) performed serial grafting over three consecutive years in order to study the effects of ontogeny, genotype, and grafting on rooting performance of northern red oak cuttings. Grafting tended to increase rooting and the number of roots per cutting, but the effect was not progressive with increasing serial grafting (Zaczek et al. 2006). Rootstock maturation did not significantly affect rooting potential. From the results, the authors hypothesized that northern red oak buds are predetermined in the developmental fate relative to rooting parameters and are only minimally influenced by serial grafting (Zaczek et al. 2006). 3. Light, Etiolation, Banding, or Shading. Light or the exclusion of light can be a major factor that influences the physiological and anatomical status of the stock plant (photosynthesis, levels of endogenous hormones, carbohydrate levels, and cell formation and development (root primordia, lignification, etc.), and subsequent ARF of cuttings collected

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from these plants (Davis et al. 1988; Davis and Haissig 1994; Hartmann et al. 2002). Reducing light levels (irradiance), modifying light quality (red and far-red), and the use of supplemental lighting to extend the photoperiod (fluorescent, metal halide, or high-pressure sodium) on stock plants prior to cutting collection are several ways to increase the rooting potential of cuttings (Moe and Anderson 1988; Howard 1994; Hartmann et al. 2002). Etiolation (forcing new shoot growth under conditions of heavy shade), banding (excluding light from that portion of the stem that is to become the cutting base), and shading (reduced light) have been shown to improve the rooting potential of cuttings taken from treated stock plants (Maynard and Bassuk 1988; Howard 1994; Hartmann et al. 2002). Three- to 5-year-old oak seedlings (Q. bicolor, Q. macrocarpa, and Q. palustris) cut back to a 2- to 4-cm stem stump and subjected to 98% light exclusion produced etiolated shoots that were then subjected to an IBA treatment (basal paint), and modified stoolbed technique (Hawver and Bassuk 2000). Etiolation used in conjunction with this modified stoolbed technique improved ARF of all three oak species from 29%, 52%, and 25% to 64%, 83%, and 100%, respectively (Hawver and Bassuk 2000). A 30-min application of far-red light or incandescent light at 22:30 hr every evening encouraged ARF from these stock plants (Hawver and Bassuk 2000). Amissah and Bassuk (2004), using this same etiolation plus container layering technique, evaluated the use of gibberellin (GA4 þ 7) on stock plant budbreak, and also evaluated the effect of stock plant age, shoot origin, and IBA solvents on ARF in Q. bicolor and Q. macrocarpa. GA4 þ 7 at 500 mg l1 applied every fourth day increased budbreak in stock plants, but stock plant age (2- to 6-year-old) had no negative effect on ARF (Amissah and Bassuk 2004). ARF was greater (36%) in shoots from stock plants cut back to a 3–4 cm stump above the soil and etiolated, compared to shoots from intact tall plants (1.8%) of Q. bicolor (Amissah and Bassuk 2004). Rooting percentages were highest when IBA was dissolved in less toxic solvents, such as 50% or 100% acetone or 98% ethanol (Amissah and Bassuk 2004). Amissah and Bassuk (2007) investigated the effect of light and etiolation with or without stem banding on ARF in Q. bicolor and Q. macrocarpa cuttings. Light and stem banding had no significant influence on ARF using stock plants grown in the greenhouse, whereas etiolation enhanced rooting in field-grown cuttings (Amissah and Bassuk 2007). Amissah et al. (2008) analyzed the relationship of stem anatomy to ARF in stem cuttings of Q. bicolor and Q. macrocarpa using etiolation and age of the stem at time of cutting as a pretreatment. The overall conclusion was that the difference in rooting was more related to the ease of adventitious

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root initiation rather than to restrictions on root emergence (Amissah et al. 2008). Partial or total light exclusion (blanching, etiolation, and a combination of both) on 9-year-old A. saccharum trees did not stimulate ARF from cuttings (Richer et al. 2003, 2004). Maynard and Bassuk 1996 also came to this same conclusion (i.e., it is cell competency, not anatomical barriers) when they studied the effects of etiolation, shading, and banding on shoot development of Carpinus betulus L. fastigiata cutting propagation. C. Treatment of Cuttings The length of stem cuttings collected for ARF varies from 7.5 to 76 cm, with at least two or more nodes, depending on the type of cutting (hardwood, semi-hardwood, or softwood) and species (Hartmann et al. 2002). Spethmann (2007) reported improved rooting success, survival, and further growth when long cuttings (50–150 cm) were used to propagate roses and several tree species. All cuttings were treated with 24.6 mM IBA, stuck in a peat:sand (3:1) mixture (pH 4.5), and rooted in a high-pressure fog system (Spethmann 2007). Differences also exist in rooting success when lateral, terminal, flowering, or vegetative shoots were used in a clonal propagation system (Rieckermann et al. 1999; Hartmann et al. 2002). Wounding of stem cuttings occurs when the material is first collected from the stock plant, but additional basal stem wounding can be beneficial to rooting success with certain woody species (Macdonald 1986; Hartmann et al. 2002). Many plant growth regulators, such as auxins, cytokinins, ethylene, abscisic acid, gibberellins, polyamines, and brassinosteroids, to name a few, influence ARF in cuttings (Macdonald 1986; Davis et al. 1988; Davis and Haissig 1994; Hartmann et al. 2002), but auxins usually have the greatest effect on ARF. Various concentrations (depending on the species and type of cutting) of the auxins IAA, IBA, or NAA are most commonly employed in ARF studies of North American hardwood tree species (Table 6.1). These auxins can be applied singly or in combination as a basal dip or soak, in talc or lanolin paste, or as a basal paint. A recent review by Blythe et al. (2007) discusses the methods of auxin application in cutting propagation over the last 70 years; therefore, it will not be discussed further in this chapter. Auxin application as a foliar spray has recently been reported to work well for rooting several woody plant species (Drahn 2007). Commercial formulations, solvents, and carriers used in auxin application can be reviewed in Hartmann et al. (2002).

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D. Rooting Medium No one rooting medium works best for ARF in cuttings of North American hardwood tree species. Typically, peat or sphagnum moss, perlite, vermiculite, or sand was used singly or in various ratios (Table 6.1). Peat is usually included because of its large total pore space and its ability to hold water. Perlite, vermiculite, or sand improved drainage of the rooting medium. Occasionally commercial soil mixes have been used successfully for ARF in cuttings. Regardless of the medium used, it is important to steam sterilize the rooting medium before use in order to rid it of any pathogens that could be detrimental to the cuttings.

E. Greenhouse Parameters Greenhouse parameters can be an important factor in the success or failure of ARF in cuttings. Intermittent mist or fog systems are regularly employed to control water loss of leaves. Intermittent mist systems minimize water vapor in the leaves by lowering the leaf-to-air vapor pressure gradient slowing down leaf transpiration, and also reduce leaf temperature (Loach, 1988a; Hartmann et al. 2002). Fog systems maximize water vapor in the air by raising the ambient humidity (Hartmann et al. 2002). Unlike mist, the very fine water droplets from a fog system remain suspended in the air longer, do not condense on the leaf surface of the cutting, and can minimize physiological stress of leafy cuttings of some woody species (Mateja et al. 2007). Greenhouse air temperatures of approximately 21  C to 27  C (day) and approximately 15  C (night) are usually employed for rooting cuttings of most temperate species (Hartmann et al. 2002), but higher day and night temperatures are also successfully employed in some greenhouses that are maintaining more than propagation benches. The temperature of the rooting medium can also be a crucial factor. Bottom heat (usually 18  C to 25  C), by the use of heating pads or circulating hot water tubing or pipes below the rooting medium, was employed when rooting hardwood cuttings, but has also been used successfully with other types of cuttings (Loach, 1988; Hartmann et al. 2002). Irradiance (relative amount of light), light duration (length of the photoperiod), and light quality (wavelength) can also influence ARF of cuttings (Hartmann et al. 2002). Light is necessary for photosynthesis of leafy cuttings, but each of these parameters must be determined to fit the particular species being propagated. Light or the absence of light has been studied extensively during in vitro rooting (plant tissue culture), but unfortunately, many times this important greenhouse parameter was

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not mentioned in a research report. Zaczek et al. (1997, 1999, 2000) reported that the duration of exposure (0–119 days) to low solar irradiance (93% shade) during the rooting phase was beneficial to several oak and maple taxa that are considered difficult to root. Mineral nutrition of cuttings during the rooting phase may also be an important factor in ARF in cuttings, but optimal nutrition levels for many North American hardwood tree species have yet to be determined (Rieckermann et al. 1999). It is difficult to determine the effect of applied fertilization during the rooting process on root primordia initiation versus root primordia elongation (Hartmann et al. 2002).

IV. CASE STUDY OF HORTICULTURAL VERSUS FOREST TREE SPECIES In the past, many horticultural and forest tree species were grafted as a result of either recalcitrance to vegetative propagation by rooted cuttings or because of the desire to impart attributes of a particular rootstock to the scion, mostly in the case of the horticultural trees. With some species, because of delayed graft incompatibility issues that started to plague the commercial nursery industry or because of the desire to have a particular cultivar grown on its own roots, research was pursued in the past several decades to improve rooted cutting success. We will use a case study to explore recent successes and continued difficulties with two forest species, Quercus rubra and Acer rubrum in comparison to the horticultural species Malus spp. and Pyrus communis. A. Horticultural Species The breakthroughs in improvement of rooted cutting success of Malus spp. and Pyrus communis came when researchers started testing greenwood cuttings taken early in the growing season, increasing the length of cuttings compared to what had been traditionally employed (Spethmann 2007), and providing shading to the base of the cutting. Even with the best treatments, both Malus and Pyrus cultivars still vary in rooting percentage. Currently, Malus spp. are generally propagated in May or early June as softwood cuttings, although cuttings taken later in the season can be successfully rooted, but much less so than early softwood cuttings (Dirr and Heuser 1987). In general, 12 to 49 mM IBA was used as the rooting stimulant with the 10–16 cm cuttings placed in peat:perlite under mist. Savaci et al. (2007) found etiolation of hardwood cuttings improved rooting success, and after biochemical analysis the

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improvement was found to be associated with a decrease in chlorophyll a and b, carotenoids, and anthocyanin. Pyrus communis was also vegetatively propagated as leafy softwood cuttings, and IBA was usually supplied at 39–49 mM. In various studies, the addition of heat to the cutting as a prepropagation treatment, altered day-length, controlled environments, fertilizer, shading, and other root promoting chemicals have been tested. In addition, research on hardwood cuttings continues. Barbosa et al. (2007) found with 10 mM IBA, rooting was the best (83%) in growth chamber conditions with short days (8 h), 90% relative humidity, and temperature at 25  C. Mbabu and Spethmann (2005) studied the effect of slow-release fertilizer, substrate pH, and cutting length on rooting success of various P. communis cultivars. The highest rooting percentage occurred at pH 5.7 with 2 kg m3 of Plantacote Mix 4 M. In their study, the length of cutting did not make a difference in rooting. Baraldi et al. 1993 were able to shed light on the rooting differences between cultivars when they compared easy- and difficult-to-root cultivars of P. communis. They found that the easy-toroot cultivar ‘Conference’ was able to metabolize IBA to IAA faster and at a higher rate than the difficult cultivar ‘Doyenne d’Hiver’. Wang (1991) found shading the base of Pyrus ‘PB10030’ rootstock microcuttings increased rooting. In addition, phloroglucinol (0.125–1.0 mM) along with darkness for 5 days promoted rooting. Turovskaya (1988) found that with two rootstocks, rooting was reduced by 50% when leaves on the cuttings were not left intact. Hardwood cuttings were possible to root, but required a heat treatment in storage for 30 days or more before placing the cuttings in outdoor beds. Barbosa et al. (2007) had some success with rooting hardwood cuttings (25 cm) of ‘Limeira’ pear in the greenhouse and growth chamber after treatment with 10–30 mM IBA. El-Shazly and El-Sabrout (1994) studied hardwood cuttings of ‘Le Conte’ pear taken throughout the season and found with 20 mM IBA cuttings taken in April (in the Middle East) had the highest rooting percentage (30%). B. Forest Species In contrast to the two horticultural species just discussed, physiologically mature Quercus rubra is nearly impossible to root from either softwood or hardwood cuttings, and the most success has occurred with using material with juvenile physiology. In comparison, much more progress has been made with Acer rubrum to the point that rooted cutting propagation is now a routine commercial practice. Although not thoroughly studied, the difference in success with these two species may well be the variation in the length of the juvenile period for that species.

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Red oak has a longer juvenile period than red maple, so one would think that there is additional time where red oak could be propagated (D. Struve, person. commun.). Most red maples are from rooted microcuttings. In studies with both species, Zaczek et al. (1999) theorized that low irradiance (7%) during fog-humidified propagation could improve rooting success of semi-hardwood cuttings. The treatment significantly increased both the percentage rooting and number of roots per cutting of Q. rubra as well as A. rubrum. Later, rejuvenation was attempted by grafting cuttings with juvenile to mature physiology on rootstocks, before attempting to root cuttings from those grafts (Zaczek et al. 2006). Researchers found that Q. rubra cuttings were only marginally affected by grafting, and those cuttings were predetermined to respond to rooting whether cuttings had been grafted or not. Earlier Zaczek and Steiner (1997) had found that cuttings from shoots stimulated to grow from axillary buds on grafted plants had a lower percentage rooting (25%) than cuttings with buds that were not stimulated to elongate (66.7%). Ferrini and Bassuk (2002) found that rooting decreased with the increasing age of the mother plants, which had been seen as early as 1939 (Thimann and Delisle 1939). Sanchez et al. (1996) confirmed the importance of juvenility by demonstrating good rooting capacity of shoots derived from basal epicormic sprouts that had been placed in vitro. In one of the earliest reports of cutting propagation of A. rubrum, it was found that anthocyanin in leaves was the most important factor that led to root initiation over other factors, such as continuous light, 16-h photoperiod, IBA dips, or application of sucrose or riboflavin (Bachelard and Stowe 1962). Even earlier, the use of IBA as a necessary rooting treatment to the cut ends of softwood cuttings was established in the literature (Afanasiev 1939). Struve and Arnold (1986) found IBA to be the superior auxin source for rooting of A. rubrum when it was compared to K-IBA, N-phenyl-indole-3-butyramide (NP-IBA), phenyl indole-3-thiobutyrate (P-ITB), and phenol indole-3-butyrate (P-IBA), although the aryl esters improved rooting quality. Most of the recent work has been performed with particular horticultural cultivars of A. rubrum, but the particular cultivars will not be the focus of this section. In addition, despite the use of cultivars, these studies still shed light on rooted cutting propagation for this species. The work that will be discussed has focused on use of subirrigation, various substrate temperatures, and fertilization in addition to cutting types and seasonal effects. In a study by Owen et al. (2003), A. rubrum cuttings were subjected to subirrigation. In addition, three auxin concentrations were evaluated

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along with three substrate temperatures. With the intermediate substrate temperature (23  C), root length (156 mm) and rooting percentage (69%) increased, although the 20  C treatment resulted in the greatest number of cuttings that rooted (15). Henry and Preece (1997) forced softwood shoots from excised stem sections of A. rubrum, and those shoots had a 60% rooting percentage. Zaczek et al. (1997) rooted A. rubrum under 91% and 97% shade, and those cuttings resulted in faster rooting, higher number of roots per cutting, and higher percentage rooting. Zhang et al. (1997) studied transpiration rate, survival, and rooting of unmisted A. rubrum softwood cuttings. The highest rooting percentage (74%) occurred at the lowest medium temperature treatment (24  C). The addition of subirrigation improved rooting percentage. Zhang and Graves (1995) added nitrogen fertilizer (3.6 and 7.2 mol N m3) to subirrigation, and rooting was improved (95%) over subirrigation alone. Without subirrigation, fertilizer did not have an effect on rooting (Lane and Still 1984). Wilkins et al. (1995) tested rooting of single-node stem cuttings. Cuttings taken in May and dipped in 3 or 8 g kg1 IBA supplied as Hormodin No. 2 or 3 had a range of rooting percentage from 22% to 100%, depending on the genotype. Dehgan et al. (1989) studied the effect of season on A. rubrum semi-hardwood cuttings. They found that cuttings taken in May and July–September had the highest rooting percentage (up to 88%). Even after numerous attempts to root cuttings of Q. rubra by manipulating the pre- and posttreatment physiology of the material used for cuttings, for the most part, the species is still recalcitrant to vegetative propagation by traditional rooted cutting methods. However, improvements in rooting success with Malus spp., P. communis, and A. rubrum continue to be made while new knowledge is being gained about the underlying biochemical mechanisms that are leading to those improvements. For the most part, except for particular cultivars, published rooting procedures can guide commercial applications of these technologies.

V. CONCLUDING REMARKS For many North American fine hardwood species, treatments to enhance adventitious rooting that manipulate environmental factors, rooting medium, and exogenous chemical stimuli have been extensively explored, and probably only minor improvements in ARF would be gained by expending much more effort in this line of research. The fundamental factors that influence ARF and the cofactors that subtly

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influence the endogenous or exogenous effects have been identified. What remains to be understood are the biochemical underpinnings of the observed biology of ARF. An improved understanding of the regulation of auxin metabolism, transport, and action, and the regulation of tissue differentiation and maturation probably will be the most important and productive areas of ARF research in the future. Model plant systems, including rooting mutants within a species with varied rooting capacity, will be useful tools for gaining insight into the genetic basis for ARF. Comparisons of gene regulation between juvenile and mature phenotypes within the same species might also provide some useful insight into inhibition of rooting that occurs with the transition to maturity. Until recently, the costs associated with large-scale analyses of the genes, proteins, and metabolites shown to affect plant development have prohibited their application to all but a few model species. The advent of new (pyro)-sequencing technologies and metabolomics has placed these types of analyses within reach of woody-plant researchers. Molecular tools and genetic data are available now for us to begin to identify genes and (epi)genetic regulators that are influencing ARF in fine hardwoods. Once we increase our understanding of the underlying genetic controls, we can begin to develop novel means of overcoming this inhibition.

ACKNOWLEDGMENTS The authors would like to thank Drs. Brian Maynard and Daniel K. Struve for critical review of this manuscript. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that also may be suitable. LITERATURE CITED Afanasiev, M. 1939. Effect of indolebutyric acid on rooting of greenwood cuttings of some deciduous forest trees. J. For. 37:37–41. Ahkami, A.H., S. Lischewski, K.-T. Haensch, S. Porfirova, J. Hofmann, H. Rolletschek, M. Melzer, P. Franken, B. Hause, U. Druege, and M.R. Hajirezaei. 2009. Molecular physiology of adventitious root formation in Petunia hybrida cuttings: Involvement of wound response and primary metabolism. New Phytol. 181:613–625. Alsup, C.M., J.C. Cole, and P.L. Claypool. 2004. Stem cuttings from Caddo sugar maple trees differ in their rooting potential. Acta Hort. 630:263–269.

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Sorin, C., J.D. Bussell, I. Camus, K. Ljung, M. Kowalczyk, G. Geiss, H. McKhann, C. Garcion, H. Vaucheret, G. Sandberg, and C. Bellini. 2005. Auxin and light control of adventitious rooting in Arabidopsis require ARGONAUTE. Plant Cell 17:1343–1359. Spethmann, W. 2007. Increase of rooting success and further shoot growth by long cuttings of woody plants. Propag. Ornam. Plants 7(3):160–166. Struve, D.K., and M.A. Arnold. 1986. Aryl esters of IBA increase rooted cutting quality of red maple ‘Red Sunset’ softwood cuttings. HortScience 21(6):1392–1393. Swamy, S.L., S. Puri, and K. Kanwar. 2002a. Propagation of Robinia pseudoacacia Linn. and Grewia optiva Drummond from rooted stem cuttings. Agroforest. Syst. 55:231–237. Swamy, S.L., S. Puri, and A.K. Singh. 2002b. Effect of auxins (IBA and NAA) and season on rooting of juvenile and mature hardwood cuttings of Robinia pseudoacacia and Grewia optiva. New For. 23:143–157. Swarup, R., P. Perry, D. Hagenbeek, D. Van Der Straeten, G.T.S. Beemster, G. Sandberg, R. Bhalerao, K. Ljung, and M.J. Bennett. 2007. Ethylene up-regulates auxin biosynthesis in Arabidopsis seedlings to enhance inhibition of root cell elongation. Plant Cell 19:2186–2196. Takahashi, F., K. Sato-Nara, K. Kobayashi, M. Suzuki, and H. Suzuki. 2003. Sugar-induced adventitious roots in Arabidopsis seedlings. J. Plant Res. 116:83–91. Tartoura, K.A.H. 2008. Involvement of nitric oxide in ABA-induced regulation of adventitious root formation in Vigna radiata (L.) Wilczek cuttings. p. 69. In: Book of abstracts, 5th Intl. Symposium on Adventitious Root Formation: From cell fate flexibility to root meristem determination and biomass formation. June 16–20, 2008. Madrid, Spain. www. rooting2008.fgua.es/docs/ABSTRACTS_BOOK.pdf. Thimann, K.V., and A.L. Delisle. 1939. The vegetative propagation of difficult plants. J. Arnold Arbor. 20:116–36. Tousignant, D., C. Richer, J.-A. Rioux, N. Brassard, and J.-P. Mottard. 2003. Vegetative propagation of sugar maple: Relating stem water content and terminal bud developmental stage to adventitious rooting of stem cuttings. Can. J. Plant Sci. 83:859–867. Turovskaya, N.I. 1988. Softwood grafting of apples and pears. Sadov. Vino. 8:19–21. Van Sambeek, J.W., and J.E. Preece. 1999. Forcing environment affects epicormic sprout production from branch segments for vegetative propagation of adult hardwoods. p. 272–273. In: J.W. Stringerand D.L. Loftis (eds.), Proc. 12th Central Hardwood For. Conf., Gen. Tech. Rep. SRS-24, Asheville, NC, USDA For. Serv., South. Res.Stn. Van Staden, J., E. Zazimalova, M.A. Hall, and E.F. George. 2007. Plant growth regulators II: Cytokinins, their analogues and antagonists. p. 205–226. In: Plant propagation by tissue culture, Vol. 1: The background. 3rd ed., E.F. George, M.A. Hall, G-J. De Klerk (eds.), Springer, New York. Vielba, J.M., E. Ferro, D. Abarca, C. Diaz-Sala, and C. Sanchez. 2008. Expression profile of chestnut genes in relation to rooting and auxin homeostasis. p. 149. In: Book of abstracts, 5th Intl. Symposium on Adventitious Root Formation: From cell fate flexibility to root meristem determination and biomass formation. June 16–20, 2008, Madrid, Spain. www. rooting2008.fgua.es/docs/ABSTRACTS_BOOK.pdf. Wang, Q.C. 1991. Factors affecting rooting of microcuttings of the pear rootstock PB10030. Sci. Hort. 45(3–4):209–213. Welander, M., and Zhu, L.H. 2006. Rol genes: molecular biology, physiology, morphology, breeding uses. p. 79–97. In: J. Janick (ed.). Plant breeding reviews, Vol. 26, Wiley, Hoboken, NJ. Willmann, M.R., and R.S. Poethig. 2005. Time to grow up: The temporal role of smallRNAs in plants. Curr. Opin. Plant Biol. 8:548–552.

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7 Water and Nutrient Management in the Production of Container-Grown Ornamentals John C. Majsztrik, Andrew G. Ristvey, and John D. Lea-Cox Department of Plant Science and Landscape Architecture University of Maryland College Park, MD 20742, USA

ABBREVIATIONS I. INTRODUCTION A. The Changing Nature of Ornamental Production B. Economic Impact and Trends C. Scope of the Review D. Overview of the Issues II. SOILLESS SUBSTRATES A. Key Physical and Chemical Properties B. Sustainable Supply and Amendments III. NUTRIENTS A. Historical Context B. Quantifying Plant Nutrient Requirements C. Nutrient Applications and Methods D. Nutrient Uptake Efficiency E. Denitrification F. Water and Nutrients IV. WATER A. Global Issues B. Water Rights C. Water Quality D. Irrigation Systems E. Increasing Water Application Efficiency 1. Matching Plant Water Needs to Irrigation 2. Irrigation Management 3. Reducing Leaching and Runoff 4. Precision Applications Horticultural Reviews, Volume 38 Edited by Jules Janick Copyright
2011 Wiley-Blackwell. 253

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F. Intercepting Runoff: Capture and Recycling 1. Closed Systems 2. Open Systems 3. Vegetated Buffers 4. Wetland Remediation 5. Containment Basin Design and Stormwater Runoff V. CONCLUSIONS LITERATURE CITED

ABBREVIATIONS BMP CRF EC EPA LF SRF TDR TMDL VWC

best management practice controlled release fertilizer electrical conductivity Environmental Protection Agency leaching fraction slow-release fertilizer time domain reflectometry total maximum daily load volumetric water content

I. INTRODUCTION A. The Changing Nature of Ornamental Production During the past 25 years, there has been a shift in how ornamental plants are produced in the United States. Before the energy crisis of the early 1970s, the production of ornamental plants was largely focused on maximizing plant growth rate using extensive systems, given that the cost of most inputs was relatively low. Containerized production signaled a shift in the efficiency of ornamental production both in nursery and greenhouse environments, primarily driven by the increasing cost of resources. This trend has continued until today, with over 50% of plants now being grown in containers (U.S. Dept. Agr. 2007). In this way, the increased cost of production has been counter-balanced by increased productivity per unit area. This shift to containerization has resulted in much greater numbers of plants being grown per unit area (plant density), since they can be shifted from close-packed spacing to more open spacing when light interception limits growth (plant canopies interact). Highdensity production has therefore become the dominant method of growing most types of plants in both greenhouse and container-nursery operations. There has also been a cross-over shift within traditional field

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production to pot-in-pot operations, where large plants are grown in 27 to 175 L containers. In the greenhouse industry, net profit is generally driven by turnover (profit per unit area) whereas in recent years the greatest net profits in the nursery industry have been realized by larger-sized plant material, together with sales of new (and unusual) plant introductions. B. Economic Impact and Trends The nursery and greenhouse industry ranks sixth in U.S. market value of agricultural products sold and is in the top five market values of agricultural products sold for 34 states (U.S. Dept. Agr. 2009). Sales revenues have steadily increased at an average of 2.4% per year from $10.7 billion in 1987 to $14.7 billion in 2004, expanding even during recessionary periods (Hall et al. 2006). Nationally, the cost of production in the container and greenhouse industry has increased faster than the cost per unit of plant being sold in recent years (Jerardo 2006). This increase in cost is due to several factors, primarily the increased cost of labor. Increases in productivity may have been gained by more automation (with high initial costs) and/or by reducing the number of person-hours in the operation (Hodges et al. 1997, 1998; Hall et al. 2006; Hodges and Haydu 2006). Additionally, the cost of fertilizer has doubled in the last 5 years, mainly due to the increasing cost of natural gas, further reducing profit margins (Huang 2009). Retail market pressures have also depressed price increases. For example, Hall et al. (2006) noted that mass market retailers and other buyers have added additional ‘‘pseudo-grades and standards,’’ based on plant-to-pot ratios, which have forced growers either to conform to these standards, accept a lower price, or sell to another buyer. Mass merchandising stores also demand a more consistent supply, forcing growers to hold more inventory, which requires larger production areas or increased efficiency. The net effect of these issues has forced many smaller and even medium-size operations to reduce costs and/or produce more plants per unit time to maintain profits, or to close due to competitive pressures in local areas (Hall et al. 2006). Consequently, many growers are very focused on increasing system production efficiency to significantly reduce expensive inputs, including fertilizer, growth regulators, and labor and irrigation costs, without compromising plant growth or health. C. Scope of the Review Together with shifts in the ornamental market and more intensive production practices over the past 30 or so years, there has been a

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growing recognition that intensive plant production operations are having a significant impact on the local environment. Starting in the early 1980s, a number of nursery researchers recognized that many best management practices could be implemented that would improve resource use efficiency, increase productivity, and reduce the overall environmental impacts of those practices (Wright and Niemiera 1987; Yeager et al. 1997; Fain et al. 2000; Bilderback 2001; Yeager et al. 2007). This culminated in the development of the first set of Best Management Practice (BMP) guidelines for the nursery industry (Yeager et al. 1997), which was significantly reviewed and updated by Yeager et al. (2007). Water and nutrient management are inextricably linked in the production of plants in containers, since soilless substrates such as peat, pine bark, and a large variety of organic and inorganic amendments are used. The goals of this chapter are to look at the changes and increases in efficiency of water and nutrient practices in containerized production over the past three decades and focus on topics that need further attention. To our knowledge, there has never been a substantive review of the literature on water and nutrient management, which complements other reviews, such as that of Wright and Niemiera (1987), which focused primarily on the nutrient requirements, application and timing, and the measurement of nutritional levels in the container production of woody ornamentals. We also refer readers to the latest editions of comprehensive textbooks on growing media (Handreck and Black 2002) and soilless culture (Raviv and Lieth 2008), and the greenhouse management textbooks by Hanan (1998) and Nelson (2008). D. Overview of the Issues Water issues, specifically involving irrigation scheduling, surface and groundwater water supply, and runoff water quality, including nutrient, herbicide, pesticide, and pathogen-related issues, are topics of major concern even in areas where rainfall is relatively abundant. Drought, salinity, urban competition for surface and groundwater reserves, and increasing legislation at federal, state, and county levels are all increasing the need for ornamental crop producers to manage water resources more effectively (Fernandez et al. 2009). Legislation regarding water use and/or water quality has been implemented in California, Delaware, Florida, Maryland, Michigan, North Carolina, Oregon, and Texas (Fernandez et al. 2009). Optimizing the management of water and nutrients offers unique challenges and opportunities for nursery and greenhouse operations across the United States and in many other parts of the world.

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With limited root volumes in containers, one or more daily irrigations—and often daily or weekly fertilizations using soluble fertilizers, or seasonal applications of slow-release fertilizers (SRFs)—are required to optimize plant growth (Lea-Cox et al. 2001a). Irrigation of ornamental crops in containers, therefore, tends to be excessive, and water and nutrient use is known to be very inefficient (Bauerle et al. 2002; Bilderback 2002; Ristvey et al. 2004; Ross and LeaCox 2004). Over half of the irrigation water used by intensive container nurseries is applied by overhead sprinkler systems (Beeson et al. 2004). Commercial nurseries commonly apply high irrigation rates of 25 mm
day1 that can produce leaching fractions (volume leached as a percentage of volume applied to the container surface area) as high as 110%. This can generate from 18,000 to 90,000 L of runoff per hectare per day (Huett 1997). It is known that improving irrigation efficiency can directly improve nutrient efficiency because the volume of water leaving production beds is reduced (Ristvey 2004) and, therefore, the carrier for nitrogen (N) and phosphorus (P) leaching and transport is reduced (Bilderback 2002; Ristvey 2004; Ristvey et al. 2004, 2007; Bilderback and Lea-Cox 2005). In 1994, Niemiera wrote a prescient chapter that provided an integrated view of water, nutrient, and substrate management, with a focus on increasing resource efficiency and maximizing growth and productivity. Niemiera linked irrigation management with leaching fractions and nutrient leaching and gave specific recommendations for monitoring production areas and reducing the environmental impact of current management practices (Niemiera 1994). Nursery and greenhouse operations can employ any number and combination of best management practices (BMPs) to increase the efficiency of water and nutrient management (Yeager et al. 2007). Best management practices can be defined as schedules of activities, prohibitions, maintenance procedures, and structural or other management practices found to be the most effective and practical to prevent or reduce the discharge of pollutants (Yeager et al. 2007). The goal of any BMP is to increase the economic efficiency of plant production while reducing the impact of production practices on the environment. Usually several BMPs can be used to achieve the same purpose, with the grower typically deciding which is best based on the particular situation and needs (Lea-Cox et al. 2001a). The problem is that published BMP recommendations are necessarily general and are designed to give commonsense guidelines for nurseries to improve irrigation and nutrient management rather than provide information on specific practices. More specific BMPs reflect current scientific knowledge and, if used in combination, have been shown to demonstrably

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reduce the impact of excessive water and nutrient applications to nursery and greenhouse operations, for example, cyclic irrigation (Beeson and Haydu 1995; Tyler et al. 1996a,b), calcined clay amendments (Owen 2006; Owen et al. 2008), and recycling and remediation of containment water (Taylor et al. 2005; Vymazal 2007; White 2007). For a more extensive list of irrigation best management practices, see Environmental Protection Agency (1993), Waskom (1994), Fain et al. (1999), Fain et al. (2000), and Mostaghimi et al. (2001). In 2001, Lea-Cox, Ross, and Teffeau developed the first comprehensive water and nutrient management planning process for nursery and greenhouse systems in the United States. This process was developed in response to the Maryland Water Quality Improvement Act of 1998, a set of regulations that were intended to reduce nonpoint nutrient loading from agricultural operations into the Chesapeake Bay. Until that time, it was not clear how nursery and greenhouse growers should account for the primary nutrients (N, P, K) that were applied to the large number (typically > 250 species) of plants grown by individual operations at any one time. To date, over 350 nutrient management plans have been written by nursery and greenhouse operations in Maryland, and many operations have now implemented the site-specific best management practices that resulted from that planning process (Lea-Cox and Ross 2007).

II. SOILLESS SUBSTRATES When containers were first used to grow ornamental plants, many growers quickly found that the use of native soils in containers led to the development of a ‘‘perched’’ water table at the base of the container, primarily because the small particle size of most soils impedes water drainage from the container. Poor drainage led to aeration and disease issues in container production, which spurred the development of the John Innes Composts, the University of California (UC) mixes, and the Cornell peat-lite mixes for container culture from the 1950s to 1970s (Hanan 1998). The development of these and many other soilless substrates has led to the majority of plants being grown in containers, ranging from small volume (< 4L) greenhouse containers to midsize (4–28 L) containers for perennial production, to large (28–175 L) containers for tree production. Soilless substrates are commonly selected for their low weight, relative low cost, and favorable chemical and physical characteristics (Wright and Niemiera 1987; Raviv and Lieth 2008).

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A. Key Physical and Chemical Properties Since there is no ideal substrate with universal availability, substrate mixtures are often a compromise to achieve optimal growth for a relatively broad range of species. Physical properties, such as bulk density, air-filled porosity, water holding capacity, particle size, cation and anion exchange capacities, pH, wettability after drying, and longevity are important considerations when choosing a substrate (Raviv and Lieth 2008). The particle size fraction and component ratios of the substrate largely determine the physical (Argo 1998b) and chemical (Argo 1998a) properties of the substrate. However, container shape and geometry do play an important interactive role in the retention of water and nutrients in the root zone (Argo 1998a). The porous nature of soilless substrate often results in high potential for leaching of water and nutrients if irrigation scheduling and management are not given proper attention. Efficient fertilization and irrigation practices are therefore predicated by knowledge of substrate physical properties and container capacity. Soilless substrates generally have a much higher percent of organic material, with higher air-filled porosity but lower anion and cation exchange capacities in comparison to most soils (Bilderback et al. 2007). It is this lower anion and cation exchange capacity that causes N, P, and other nutrients to leach easily from soilless media. The addition of clay, humus, peat, composted pine bark, composted sawdust, or vermiculite as a substrate amendment can greatly increase the cation exchange capacity, which decreases the leaching of cations such as H þ , Al3 þ , Ca2 þ , Mg2 þ , K þ , NH4 þ , and Na þ (Handreck and Black 2002). Typical amendments to increase anion exchange capacity are calcined clays, attapulgite, and shale, which can bind negative ions such as PO43, HPO42, H2PO4, SO42, and NO3 but, in general, most soilless substrates have a low anion exchange capacity (Handreck and Black 2002).

B. Sustainable Supply and Amendments There is an exhaustive literature on soilless substrates for use in the nursery and greenhouse industry (Raviv and Lieth 2008). The most recent research can be found in the Proceedings of the International Symposium of Growing Media (Carlile and Coules 2009). A few recent developments should be noted. Locally available and sustainable substrates and amendments are playing an increasingly important role in what growers choose for soilless substrates, as the cost of basic materials

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and transport increases. Apart from cost, the major concerns for many growers is the continuity of supply, the uniformity and longevity of the substrate in the container, and any added costs associated with a change in practice that is required to grow quality plant material. In the United States, hammer-milled fresh pine tree material is being actively researched as a substrate, especially in the South, where clean-chip residual, a by-product of the pulp industry, is an abundant and inexpensive alternative to pine bark substrates (Boyer et al. 2008). Similarly, whole pine tree substrates have recently been extensively tested for use in nursery production (Jackson et al. 2009). Although plants have been shown to grow well in milled fresh pine, in order to get the same growth rate, plants require higher fertilizer amounts compared with pine bark and peat, presumably to overcome nitrogen fixation by the substrate (Wright and Latimer 2007; Wright et al. 2008). One potential use for milled fresh pine is as a rooting substrate, since rooted cuttings typically do not receive fertilization (Bilderback and Lorscheider 1995). Additional economic research is needed to determine whether the total cost involved with using milled fresh pine substrates outweighs the cost of the substrate it is replacing. Coir, a by-product of the coconut industry, is also being studied extensively as an alternative to peat moss, especially in Europe, where consumers are demanding peat-free substrates. Some studies have shown plant growth equivalent to that in peat substrate controls, while others show reduced growth above 50% coir; this could be a result of N immobilization by microorganisms due to the high carbon : nitrogen (C : N) ratio or due to high porosity inducing mild water stress (Holman et al. 2005). Amendment with gypsum is recommended to increase calcium and sulfur, which are below recommended levels in coir (Handreck and Black 2002). Rice hulls and peanut shells also have the potential to be used to increase aeration and decrease weight up to 25% by volume, although they decompose relatively quickly and should not be used for crop cycles longer than 12 weeks (Ingram et al. 2003). Peanut shells should be heat pasteurized to kill nematodes, and extra N is recommended. Rice hulls are high in Mn, so pH should not be allowed to fall below 5 to avoid Mn toxicity (Raviv and Lieth 2008). Grape marc, which consists of grape skins, stems and seeds, can be composted and added up to 20% by volume, but the compost is very saline if it is not leached before incorporation; it also contains high levels of Ca and low levels of Mg, Cu, and Zn (Handreck and Black 2002). Bagasse, the fibrous material left after juice extraction from sugarcane, is a material that can be used as an amendment, but since it breaks down quickly like hardwood barks, it should be used only in relatively low volumes for

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short-rotation crops (Ingram et al. 2003). Composted municipal solid waste, animal manure, and sewage sludge also have the potential to be used as substrate amendments from 20% to 50% by volume, but there is a potential for batch-to-batch variability and heavy metal toxicity, so testing before incorporation and pH adjustment and monitoring is recommended (Handreck and Black 2002). A benefit of some compost-amended substrates is that they have been shown to suppress soilborne diseases in containers such as root rot (Hoitink et al. 2003). There are other substrate amendments that a grower can consider for a variety of applications. Calcined clays are heat-expanded clays that are resistant to chemical and physical degradation and have noncapillary pore spaces and a high cation exchange capacity (Ingram et al. 2003). Although calcined clay is more expensive than other amendments, it has the potential to reduce nutrient runoff of both N and P in container operations (White et al. 2006). Owen et al. (2007) found that pine bark: sand substrate amended with calcined clay decreased N and P leaching by 39% and 34%, respectively, and reduced water use by 15% compared with controls grown without calcined clay. Wulpak is a pelleted byproduct of wool manufacturing, which contains no manure or compost but does contain significant plant-available nutrients and is recommended as a container mulch to act as a starter fertilizer and suppress weeds, moss, and algae (Bilderback and Neal 2004). As current soilless substrates and amendments become scarcer or more expensive to ship, growers will likely look to alternative, locally available substrates and amendments to maintain profitability.

III. NUTRIENTS A. Historical Context The advent of the Haber-Bosch process in the early 20th century allowed for the abundant production of fertilizers, which along with advances in crop breeding and new pesticides fueled the agricultural (‘‘green’’) revolution starting in the mid-1940s. During the subsequent 60-plus years, fertilizers have been used extensively to maintain or increase plant production at increasing levels of intensity, as land values increase and economic returns for agricultural products have declined in real terms. Nitrogen fixed by humans is now thought to exceed the amount of N produced by all natural terrestrial N fixation combined (Zapata 2008). Worldwide fertilizer use has been increasing at a rate of 5% a year since the 1940s, with agriculture using an estimated 86% of the total N used by

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humans (Jordan and Weller 1996). With increased fertilizer use, eutrophication and aquatic degradation has been seen over several decades from application of N and P fertilizers, wastewater treatment outflows, and other anthropogenic sources, which has led to the degradation of a number of watersheds in the United States, including the Chesapeake Bay (Cerco and Noel 2004; Fisher et al. 2006; Howarth and Marino 2006) and the Gulf of Mexico (Mitsch et al. 2001; Livingston 2007). In the 1970s, the prevailing thought was that phosphorus and carbon were the main drivers of eutrophication in lakes and rivers (Howarth and Marino 2006). Since that time, it has become clear that P and N are the two main nutrients involved in eutrophication of surface waters. Current aquatic research agrees that freshwater ecosystems are typically P limited, but more research is needed to establish the generality of N limitation in marine ecosystems (Hecky and Kilham 1988; Correll 1999). For this reason, we need to focus on optimizing the application and conservation of water and nutrients to minimize N and P runoff from production systems. B. Quantifying Plant Nutrient Requirements Since an effective nutrient management strategy is dependent on the interactions among substrate use, fertilization, and irrigation strategies, few holistic research data sets exist for container-production systems, which constitute the highest-risk nursery and greenhouse production situations (Lea-Cox and Ross 2001). This is especially true for P data. There is a real need to rigorously quantify nutrient use, leaching, and loss from intensive plant-production systems. The first step in reducing nutrient use in containerized production is obtaining a better understanding of plant nutrient requirements over the course of the growing cycle. Although there are exhaustive numbers of journal articles that deal with optimizing plant growth with increasing nutrient addition, very few studies in the ornamental literature quantify nutrient application, leaching, and plant uptake efficiency to understand the interaction of these variables and optimize efficiency. While more research is needed to quantify the water and nutrient requirements for many plant species, information from a few well-studied species can be used as a basis for current irrigation and nutrient application recommendations. Ku and Hershey (1997a,b) were among the first to publish a complete N and P budgetary study for poinsettia. They found that marketable poinsettias could be produced with N rates of 210 mg
L1 and a leaching fraction (LF) < 0.2 (20%), compared with rates of 300 mg
L1 and leaching fraction of 0.4 (40%). In this study, up

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to 51% and 75% of the N applied was found in the leachate with 0.2 and 0.4 LF respectively (Ku and Hershey 1997a). This is notable, since a LF of 15% is considered a best management practice (Yeager et al., 2007), although the practice of leaching only when electrical conductivity (EC) levels rise to 1.5–2.0 ds
m1 could further reduce nutrient runoff. Cabrera (2003) found higher N uptake efficiencies at lower nutrient concentrations from 15 to 300 mg N
L1 for Ilex opaca ‘Hedgeholly’ and Lagerstroemia  ‘Tonto’, and optimum shoot biomass and leaf area at 60 mg N
L1, suggesting that current rates of fertilizer application may be in excess of plant needs. Rose (1999) suggested that current nitrogen recommendations for many field-grown plants, which vary between 48 and 287 kg N
ha1, far exceed plant recommendations for agricultural crops; these rates are based on a goal of maximum growth rate rather than a goal of maximizing nutrient uptake efficiency and minimizing nutrient loss. For example, Ristvey et al. (2007) compared three different N and P levels and found that azalea (Rhododendron ‘Karen’) uptake efficiency was maximized at 100 mg N and 5 mg P per plant per week (Table 7.1), which are similar to the recommended rates of 40 to 80 mg N and 15 to 30 mg P per plant per week recommended by Dole and Wilkins (1999), assuming 0.25 L per plant per week of a 315 ppm solution of 21N-7P-7K. Table 7.1 highlights the efficiency gains possible when nutrient application matches plant need, with a more than threefold higher efficiency for N and more than fivefold increase for P compared to the most inefficient application rates. Nutrient application above minimally sufficient levels may lead to higher plant uptake amount but also causes higher levels of leachate and decreased uptake efficiency, with no significant increase in growth. Ristvey et al. (2007) concluded that N loss could be reduced from 1,100 to 326 kg N
ha1, a nearly threefold decrease in potential N loss to the environment, by reducing N application rates from 250 mg N per week to 100 mg N per week due to increased nutrient uptake efficiency and lower loading rates. In this particular case, even greater reductions in P loading could be achieved by decreasing P application rates to levels more attuned to actual plant requirements (100 kg N
ha1 versus 22 kg N
ha1) (Ristvey 2004). C. Nutrient Applications and Methods In general, greenhouse fertility programs commonly utilize high levels of nutrients applied in soluble form via irrigation, and total applications of N can vary from several hundred to several thousand kilograms hectare1 year1, depending on such factors as container size, plant density, and fertilizer rate and frequency. However, in recent years, there

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N leachate (mg)

324.9  34.1 117.1  16.5 b 276.3  19.6 152.3  17.6 a 313.4  37.6 147.6  7.9 a 251.3  60.8 35.6  7.5 c 222.2  22.1 44.2  2.3 c 242.2  44.4 41.9  8.8 c 5.3  0.8 d 84.0  13.3 82.8  20.2 3.6  0.8 d 73.9  12.7 3.8  0.4 d

Plant N uptake (mg) 14.4 12.3 13.9 27.9 24.7 26.9 37.3 36.8 13.0

N uptake efficiency%

P Leachate (mg)

31.5  6.8 8.3  1.3 b 21.8  0.9 2.4  0.2 c 12.4  3.6 2.0  0.8 c 33.7  9.1 13.8  4.5 a 22.1  2.3 2.7  0.9 c 17.6  2.3 1.5  0.5 c 24.3  3.5 16.3  1.3 a 18.0  5.1 5.1  1.4 b c 13.0  4.2 2.5  0.9 c

Plant P uptake (mg) 14.0 48.5 — 15.0 49.2 — 10.8 40.1 —

P uptake efficiency (%) 18.71  1.86 a 17.86  0.44a 16.85  1.64 a 16.77  2.58 a 15.69  0.87 ab 17.39  1.37 a 11.60  0.88 bc 9.88  1.30 c 11.50  0.85 bc

Shoot dry mass (g)

4.72  0.56 b 5.04  0.21 ab 4.80  0.62 b 5.72  0.66 ab 5.33  0.43 ab 5.82  0.23 ab 6.12  0.42 a 6.37  0.34 a 5.81  0.36 a

Root dry mass (g)

0.25 0.28 0.28 0.34 0.34 0.33 0.53 0.64 0.51

Root/shoot ratio

z Initial plant N content was 104.0 mg N, and initial plant P content was 19.0 mg. Initial substrate N and P was 7.4 mg and 28.6 mg. N and P uptake efficiency is the percentage of applied nutrient that was taken up after 11 applications. Standard errors are in parentheses (n ¼ 5). Lowercase letters indicate significant differences (LSD at P ¼ 0.05) between treatments. Source: From Ristvey 2004.

N250: P25 N250: P5 N250: P0 N100: P25 N100: P5 N100: P0 N25: P25 N25: P5 N25: P0

Treatment (mg N and P week1)

Table 7.1. Nitrogen and phosphorus uptake efficiency for azalea Rhododendron var. ‘Karen’ during a 12-week experimental period. Plants were deficit irrigated twice a week, then leached the day before treatments were applied at 250 ml per container. Plant nutrient uptake (N and P) is the accumulation of nutrient from initial to final harvest.z

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has been a marked reduction in the type and quantity of fertilizers used, primarily due to cost and environmental concerns (Lea-Cox and Ross 2001). Greenhouse and intensive liner production operations typically apply fertilizer through irrigation lines (fertigation) to meet crop nutrient requirements. Fertilizers typically are applied with every irrigation (constant feed) or every few days with higher rates. One of the main benefits of fertigation is that nutrients can be adjusted easily to meet current plant growth requirements. In addition, when plant shoot:root ratios are low, irrigation frequencies may be reduced, which can also reduce nutrient leaching losses, depending on management. Although nutrient application rates are typically high in intensive greenhouse operations, interception efficiencies are also high, with 90% or more of fertigation typically reaching the root zone using boom or low-volume irrigation systems. Since greenhouses are covered year-round, fertilizer leaching is due primarily to poor irrigation scheduling (frequency and duration of events). Greenhouse operations typically are considered low risk for nutrient leaching into surface waters, since they are covered structures, but nutrient leaching to groundwater can be a concern, with nutrient concentrations building up in the soil over time (Cabrera et al. 1993). Many field nursery operations apply only conventional fertilization a few times per year as a side-band application, but many newer operations fertigate in-ground plant material through drip irrigation, as it is typically more cost effective and efficient in terms of optimizing growth rates. Rates of nutrient application are typically very low in field operations. From writing and reviewing a number of nutrient management plans, we have found that annual rates are typically between 33 to 56 kg N per ha and 0 to 14 kg P per ha, depending on soil analyses, in Maryland (Majsztrik unpubl. data). Rooting volumes are larger in field production than in container or greenhouse operations, and nutrients typically move through soils at a slower rate than in soilless substrates, increasing nutrient interception and possibly increasing nutrient uptake efficiency. The main risk for nutrient runoff with field operations is from overland flow, which can carry dissolved or undissolved fertilizers and sediment-bound P, if proper irrigation and sediment reduction best management practices are not followed. There is also a risk of N leaching below the root zone during periods of high rainfall. Container operations typically apply controlled-release fertilizers (CRFs) either incorporated into the substrate before planting or top-dressed on the substrate surface, although some operations still incorporate conventional fertilizers in the substrate. Some container

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production operations also fertigate, particularly when larger-size containers are used, usually in combination with microsprinkler or drip irrigation (e.g., pot-in-pot operations). Many container-nursery operations have adopted the use of CRFs in recent years due to nutrient runoff concerns, and since customized blends have increased the ease of application and use, for species with different growth rates and nutritional requirements. Most people recognize that the term ‘‘controlledrelease’’ is a misnomer, since release rates of encapsulated fertilizers are based on temperature (Huett and Gogel 2000; Du et al. 2006), which often does not correlate with plant nutrient uptake requirements. Nutrient requirements are typically highest in fall and early spring months when temperatures (and fertilizer release) are relatively low. Many therefore refer to these fertilizers as ‘‘slow-release’’ fertilizers (SRFs). Many research studies have been done on the use of SRF formulations over the past three decades (Maynard and Lorenz 1979; Wright and Niemiera 1991; Ruter 1992; Catanzaro et al. 1998; Huett and Gogel 2000; Blythe et al. 2002; Merhaut et al. 2006; Broschat and Moore 2007). Typically, release rates for SRFs are highest during summer months, when plants are not growing as fast as during spring, and when irrigation requirements are higher. This can create the potential for high nutrient release rates, resulting either in salt buildup in the container and/or high nutrient leaching losses. In nursery settings, there have been reports of SRF release times being cut in half due to high temperatures (Huett and Gogel 2000). For every 10 C increase over optimum release temperature (21 C), SRFs have been shown to have increased release rates from 15% up to 200%, while temperatures below optimal show inconsistent release patterns (Merhaut et al. 2006). Since container operations are open to rainfall during the growing season, there is the potential for nutrient leaching into surface water during rainfall events or with overapplication of irrigated water. Several experiments have looked at the effect of top-dressing as opposed to incorporation of fertilizer into a substrate to improve nutrient uptake efficiency and reduce nutrient loss. Broschat 2005 found that at constant temperature of 23 C, surface-applied fertilizer was released slower than incorporated SRF, hypothesizing that surface applied SRF dried out between irrigation cycles, increasing longevity. Warren et al. (2001) showed that NutricoteTM had 105% higher nitrate and 33% higher P losses, while MeisterTM had 258% higher nitrate losses and 88% higher P losses when SRF was incorporated versus surface applied. Based on this information, surface application of SRF should prolong the release time of nutrients in container operations, but surface application requires more labor, and there is a higher environmental and production

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cost if fertilizer prills are lost due to plants blowing over under windy conditions or during handling or shipping. D. Nutrient Uptake Efficiency Nutrient use efficiency is based on: (1) plant uptake efficiency, (2) metabolic assimilation into roots and shoots, and (3) utilization (or remobilization) efficiency (Baligar et al. 2001). Nutrient use efficiency is one way to gauge the impact that different fertilization techniques have on plant growth. Since (2) and (3) are due largely to plant genotype, nutrient uptake efficiency is the only variable that we have much control over with fertilizer applications or timing. A number of studies have showed that plants have a higher nutrient uptake efficiency at lower levels of nutrient application and that nutrient uptake efficiency decreases significantly with increasing rates of nutrient addition (Cabrera et al. 1995; Kent and Reed 1996; Lea-Cox and Syvertsen 1996; Ku and Hershey 1997a; Cabrera 2003; Ristvey et al. 2007). For example, Cabrera (2003) found that for Lagerstroemia  ‘Tonto’ and Ilex opaca ‘Hedgeholly’ nitrogen uptake efficiency was maximal at 30 mg N
L1 at 49.6% and 48.0% respectively and lowest at 300 mg N
L1 at 7.9% and 6.5% respectively. Weekly uptake efficiency of N in Rhododendron ‘Karen’ azalea under different N and P rates in the greenhouse over 12 weeks was highest at 25 mg N per plant per week, while P uptake efficiency was highest at 5 mg P per plant per week (Ristvey et al. 2007; Table 7.1). As previously noted, P application and use efficiency is of particular concern. There is widespread belief in the ornamental industry that P fertilization stimulates root growth over shoot growth. In a review of root:shoot ratios in trees, Harris (1992) cited seven examples of books or manuals on plant care that either stated or implied that increasing P rates promoted root growth and increasing N fertilization promotes shoot growth. The belief that P fertilization preferentially stimulates root growth over shoot growth persists in the industry, since fertilizers with low N:P ratios still are used extensively (Williams and Nelson 1996; Hansen and Lynch 1998). However, there are few definitive data in the literature to support this contention. There are some data to show that P-starved roots grow and branch more profusely when P is added to their environment (Drew and Saker 1978), but there is no evidence to indicate that the addition of higher levels of P increases either root or shoot growth rates above that of minimally P-sufficient plants. Since consumers focus on the top part of the plant, growers in the nursery and greenhouse industry tend to focus on maximizing shoot growth. In the nursery industry, this leads to the need

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to remove top growth or apply growth regulators to produce acceptable plants, which can be expensive. If growers were better able to adjust fertilizer rates to control growth, they could save both on labor costs and improving resource efficiency, by not having to prune the nutrients stored primarily in leaves (shoot growth). Ristvey (2004) showed that for azalea, root:shoot ratios were significantly higher at lower nutrient rates, which lessens the stress on the roots to supply adequate water and nutrients because of reduced shoot volume (see Table 7.1). Lower root:shoot ratios are also likely to result in higher survival rates after planting. Baligar et al. 2001 stated that estimates of overall P uptake efficiency in agricultural systems are less than 10%, based on actual plant need. Currently, P fertilization in many nursery and greenhouse operations is likely in excess of plant requirements, resulting in low uptake efficiencies and increasing the potential for P runoff. Table 7.1 shows that P uptake efficiency for Rhododendron L. ‘Karen’ azalea was optimized at 5 mg P per plant per week, much lower than the rate applied by most nursery and greenhouse operations. Tyler et al. (1996a) recovered 50% to 80% of applied P in the leachate, substrate, and plant, and found P uptake efficiencies to be between 17% and 25% in a field study examining leaching fractions and SRF rates on growth in containerized cotoneaster plants. Other research on woody perennial species has focused on the effect of P on plant growth (especially roots) and the appropriate levels of P fertilization to reduce P loss into the environment (Lynch et al. 1991; Broch et al. 1998; Hansen and Lynch 1998). However, there have been few integrated studies of P fertilization in containernursery production systems. In general, we need to reevaluate our nutrient application recommendations in light of resource use efficiency and the cost of potential remediation measures, since it is often more cost effective to reduce system inputs. For example, recommended rates of nitrogen for greenhouse-grown azaleas (2200 kg
ha1; Chen et al. 2001) were reported to be 10 times greater than recommended levels for corn, with P levels often an order of magnitude larger than plant requirements (Ristvey et al. 2007). There are a variety of recommendations for timing fertilizer applications, with most suggesting application during early spring (before budbreak), which is usually a poor time to apply fertilizer (Rose 1999). In agronomic environments, applied fertilizer uptakes typically are reported to be 50% or lower for N, less than 10% for P, and about 40% for K (Baligar et al. 2001), which strongly suggests that we are doing a poor job at timing application rates and/or optimizing conditions for uptake. Plant nutrient uptake efficiencies are especially low in irrigated production environments, but certain practices can increase uptake efficiency. Low nutrient

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uptake efficiencies for container-grown plants are due to a combination of practices, including high application rates of soluble fertilizers, large leaching volumes, small root volumes, and using soilless substrates that have low nutrient-retention abilities (Ingram et al. 1991; Handreck and Black 2002). Runoff volumes and N uptake efficiencies for azalea (Rhododendron ‘Karen’) and holly (Ilex cornuta  regosa ‘China Girl) were calculated during a 3-year container-production study that was open to rainfall (Ristvey 2004). The amount of runoff was significantly lower for drip irrigation compared to overhead across all treatments. In this study, time domain reflectometry (TDR) controlled irrigation applied less water compared to cyclic control (303.3 L per plant versus 397.6 L per plant for drip, and 881.9 L per plant versus 1811.1 L per plant for overhead). The benefits of TDR and drip irrigation become apparent when N runoff is taken into account in Fig. 7.1. Nitrogen runoff was reduced 53% for overhead TDR compared to cyclic overhead and 82% for TDR controlled drip compared to overhead cyclic irrigation over the study period, with

70

Nitrogen Runoff / Leachate (g)

60

50 ppm Holly Drip Holly Overhead Holly Drip/TDR Holly Overhead/TDR 53% Reduction

50

82% Reduction

40 30 20

100 ppm 75 ppm

10 0 Oct

Nov

2001

Jun

Jul

Aug

Sep

Oct

Nov

2002

Fig. 7.1. Mean cumulative runoff of N from holly (Ilex cornuta  regosa cv. ‘China Girl’) under cyclic and TDR-controlled drip and overhead irrigation on a per-plant basis over a 14-month period between September 2001 and November 2002. Three different soluble N rates were applied as shown, representing a ‘‘worst-case’’ application scenario. Source: Ristvey 2004.

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similar reductions seen for P (Ristvey 2004). Plant N uptake efficiencies were higher when SRF and fertigation was used compared to fertigation alone. Overhead fertigation, especially on spaced containers, is inefficient because of the low interception efficiencies typical of spaced containers, which varied from 19.4% to 63.5% of the applied fertigation reaching the container substrate. There is a potential for significant increases in N and P uptake efficiency, considering that only 15.9% to 35.4% of the N applied was taken up by the plants, which corresponds to uptake efficiencies reported by others (Cabrera 2003; Scagel et al. 2007). Other studies in the literature illustrate the dynamics of nutrient uptake, which can help hone fertilization strategies. Studying ‘Royalty’ roses, Cabrera et al. (1995) found that total annual N uptake was 16.8 g per plant, with N uptake rate changing up to fivefold during a cycle of flower growth peaking at maximum flower shoot elongation and lowest at maximum vegetative shoot elongation. Nutrient cycles also have been noted in other woody ornamentals, including Ilex crenata and Euonymus japonica, where nutrient uptake is highest after a flush of foliage (Cabrera et al. 1995). Evidence suggests that this cyclical uptake is due to root growth and soil exploration, which then allows for shoot growth (Bilderback et al. 1999; Rose 1999). Rose (1999) reported that for the fruit trees and container-grown ornamentals studied, nutrient uptake and biomass accumulation are linked, so the highest nutrient concentrations are needed during times of active growth (e.g., late spring and late summer), with minimal growth during budbreak, leaf abscission, and the hottest times of the year. Based on this information, it appears that most current fertilizer practices in containerized production are not timed to match plant nutrient uptake requirements. Plants that are fertigated often are supplied with a constant rate of nutrient applied at regular intervals, which is rarely changed during the production cycle. Encapsulated fertilizers often have high initial nutrient release rates from broken prills or initial incorporation in the substrate, which are then dependent on temperature and water availability for release patterns. Slow-release fertilizers often release maximally during hot weather, which is the time when the growth rate and nutrient uptake of plants are limited by those same high temperatures. Typical fertilization recommendations vary greatly between 48 to 287 kg per ha per year, based on research from the 1950s to 1970s, when maximum growth response was the primary production goal (Rose 1999). It is clear from this and other research that nutrient recommendations should be based on a species’ requirements, growth rate, time of year, and the type of fertilizer used rather than on generic recommendations.

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E. Denitrification Denitrification is the process by which bacteria in the soil break down nitrogen oxides (NO3 and NO2) in the soil or substrate (as an electron acceptor), into N2O or N2. Although denitrification has been studied in detail in agronomic settings with soils, there appears to be little knowledge of this subject in the ornamental literature or its impact on container-production practices. It is important to understand the consequences of denitrification and the role that it plays in nitrogen uptake efficiency. Several conditions are required for denitrification to occur regardless of whether it occurs in soils or soilless substrates. In order for denitrification to occur, there needs to be a carbon source (electron donor), a nitrogen source (electron acceptor), anoxic or low oxygen conditions (<0.2 mg/L), moderate temperatures, and microbe availability. Under high oxygen conditions, bacteria preferentially use oxygen as an electron acceptor to produce energy. Under anoxic conditions, facultative anaerobic bacteria use nitrate, instead of oxygen (which has a higher energy yield), for electron transfer, which produces N2O or N2, and energy for the bacteria (Agner 2003). Terrestrial soils are reported to contribute 22% of the total global loss of N through denitrification, with freshwater systems contributing an additional 20% (Seitzinger et al. 2006). Although denitrification is beneficial when excess nutrients are removed from water and soils from anthropogenic overapplication, denitrification within the root zone can decrease the available N supply for plants, requiring additional N applications. Understanding the conditions in which denitrification occurs is a first step to reducing N loss. Ristvey (2004) concluded that N loss could be exacerbated by current container-nursery production practices, with high-volume and frequency (often daily) irrigation applications. When nutrient budgets are reported in the literature, often a moderate to high percentage of N cannot be accounted for in the total budget, even with 15N studies (Lea-Cox et al. 2001b). This N loss typically is attributed to denitrification. Cabrera (2003) reported 23% to 41% of the N could not be accounted for with N concentrations from 15 to 300 mg
L1, regardless of concentration for Ilex opaca ‘Hedgeholly’ and Lagerstroemia  ‘Tonto’ in a 9-month container study. Nieder et al. (1989) reviewed a number of in situ denitrification experiments in field soils and found a range of 5 kg per ha to 60 kg per ha N losses using 15 N labeled compounds and N loss rates of 0.7 to 233 kg per ha using the acetylene inhibition method over a variety of soil conditions. In a greenhouse study, Agner (2003) found that denitrification rates for Pelargonium zonale and Euphorbia pulcherrima increased up to 36 hr

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after flood irrigation at a maximum rate of 30 mg
pot1
hr1 in a peat substrate, with denitrification continuing until air volume reached 30%, using acetylene inhibition. It was also reported that 8-week-old Pelargonium zonale plants had higher denitrification rates compared with 4-week-old plants, presumably because of higher carbon exudates from a larger root system (Agner 2003). Since carbon (C) is the initial electron donor, it is essential for denitrification. Prade (1988) found that roots of wheat (Triticum vulgare ‘Kolibri’) significantly increased denitrification rates in containers with roots versus containers without roots at air-filled porosities below 10%. It is thought that root turnover and root exudates have the potential to supply the soluble C necessary for denitrification in soils, with organic components also supplying C in soilless substrates. Since the nitrate (NO3) or nitrite (NO2) forms of N can be used for denitrification, any N that is supplied to the plant has the potential to be used for denitrification (Daum and Schenk 1996). Ammonium (NH4þ ) ions can also be converted via nitrification into NO2 and used in the denitrification process (Groffman et al. 2006). The higher the rate of N applied, the higher the potential for denitrification in the substrate. Low oxygen conditions typically are brought about by two different mechanisms in soilless substrates, saturated conditions in the container and root respiration decreasing oxygen levels. Overapplication of irrigation water can quickly bring about anoxic conditions in containerized plants, especially in smaller containers, which have a larger proportion of the volume subject to a perched water table. Water can provide suitable conditions for microbial growth, restrict O2 supply to microsites, release available C and N through drying and wetting cycles, and provide a diffusion medium for substrates and products (Aulakh et al. 1992). Although soilless substrates tend to have a high air-filled porosity, applying too much or too frequent irrigation may increase the rate of denitrification. Aulakh (1992) reports that denitrification is negligible below 60% of water-holding capacity, at least for mineral soils. From this information, denitrification could reasonably be presumed to be a major loss mechanism for N from containerized production systems, yet there is very little information on denitrification in the ornamental literature. This lack of information should be addressed, given the magnitude of potential N loss from production systems. Denitrification inhibitors, which can be mixed with fertilizers or potting media, are a relatively low-cost way of reducing denitrification rates; however, they have been shown to have limited ability to control denitrification in soilless substrates (Goh and Haynes 1977). It is likely that the highest reductions in denitrification rates will be seen with a

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combination of denitrification inhibitors, moderate rates of slow-release fertilizers, and better irrigation practices. F. Water and Nutrients There is a direct connection between the movement of water and nutrients at the micro scale in the root zone and at the macro scale in container-production operations. Our common goal is to increase the efficiency of water and nutrient applications to reduce the potential for nutrient leaching and runoff with surface water movement and therefore to avoid expensive containment and remediation measures. Water is the main transport mechanism of nutrients both into the plant and the surrounding environment (Ross et al. 2002). Nutrients that are not taken up by the plant, adsorbed by the substrate, or utilized by microorganisms have the potential to leach into groundwater and/or runoff. The interaction of enhanced nutrient levels can be directly associated with algal blooms of surface water, eutrophication, and other environmental problems. Excessive surface water erosion is also able to transport nutrients that are soil-bound (i.e., phosphorus) in sediments, when proper safeguards (e.g., buffer strips and sediment ponds) are not used. The primary goal of irrigation water applications in greenhouse and container operations is to apply the precise amount of water that meets the immediate water requirement of the plant without significant leaching, thereby maintaining available nutrients in the rootzone and maximizing the time for uptake. This is much easier to control in greenhouse operations, which are not influenced by rainfall, compared to container and field nursery operations, which receive rainfall (uncontrolled leaching events) for at least part of the year.

IV. WATER A. Global Issues Over the past 50 years, the demand for freshwater in many states has been increasing (Hutson et al. 2004) while the quality of both surface and groundwater has been decreasing due to pollution from both point and nonpoint sources (Secchi et al. 2007). N, P, and other organic pollutants such as pesticides and herbicides are being found at increasing concentrations in groundwater under agricultural and other areas (Guimera 1998). Recent studies have also found that N (Mahvi et al. 2005) and P (McCobb et al. 2003; Fuchs et al. 2009) that have been bound by soils are

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now entering streams and estuaries. Even though steps are being taken to reduce nutrient and sediment input into surface waters, those effects may be reduced by the additional N and P that continues to move through the soil environment to surface waters (Fisher et al. 2006). Now and into the future, regulators and scientists face the difficult task of determining what proportion of bioavailable nutrients in a particular watershed are from recent applications as opposed to historical applications of nutrients and other pollutants. As the demand for water and the cost of purification increase, the cost of freshwater resources will increase and the availability will likely decrease to individual operations. Population growth in the 20th century increased by a factor of 3 while water withdrawals increased by a factor of 7 during the same time period, with little hope of these rates slowing in the near future (Agarwal et al. 2000). Increased water demand is compounded by a migration of the world population to cities and areas close to shorelines, where freshwater withdrawals may be complicated by saltwater intrusion. These factors will make water conservation an important issue not only for the nursery and greenhouse industry but for many aspects of human life in the near future. B. Water Rights Water rights in the United States are dichotomous, handled differently in the eastern and western parts of the country. In the West, which is mostly arid or semiarid, water rights typically are based on priority, which specify the amount of water that a state or user may take or how much must be left for the remaining users. Thirteen of the 22 agreements that exist were made before 1953 (Dellapenna 2007). Western states typically use appropriative rights, giving priority for water use to the person or entity that has been there the longest, as long as it does not negatively impact the remaining users (Dellapenna 2000). The difficulty with appropriative rights is in determining what constitutes a negative impact on another user. In the eastern states, water compacts began in the mid20th century, mainly between states. These compacts did not focus as much on allocation issues; instead, states agreed to reduce pollution and cooperate on other ecological issues. These agreements were designed for agencies to consult and share information, but these agencies typically had little or no authority to make significant changes in water allocations (Dellapenna 2007). Around the world, 268 major river systems drain almost 50% of the world’s land area and serve 40% of the world’s population (Dellapenna 2007). Most of these river systems are shared by two or more countries, which could lead to conflicts over this shared resource (Phelps 2007).

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Additionally, conflicts have arisen over freshwater resources that form boundaries between states in the United States (Poff et al. 2003). Water conflicts in the United States have been occurring for the past 80 or more years and are likely to increase as fresh water resources become more limited due to human population growth and urban development (Cox 2007; Davis 2007; Dellapenna 2007; Phelps 2007). Currently, the majority of conflicts involving water have dealt with surface waters. It is likely that as surface waters become more taxed, conflicts will arise over transboundary aquifers, which currently are relatively free of conflict (Dellapenna 2007). This is especially true in ‘‘fossil’’ aquifers, such as the Ogallala aquifer, which underlies parts of Colorado, Kansas, Nebraska, New Mexico, Oklahoma, South Dakota, Texas, and Wyoming; such aquifers are not renewable in the near future, due to poor recharge (Torell et al. 1990; Draper 2007). Most field (in-soil) producers of ornamental trees and shrubs use irrigation water at some point during production, since growth rates likely will be reduced by water stress at some point during long production cycles (Fernandez et al. 2009). Many field producers use lowvolume (drip) irrigation, and many also use this system to deliver soluble fertilizers during the growing season. While supplemental irrigation is at times advantageous in field production, it is essential for the container-production of ornamental plants. Container substrates need to be well drained primarily for disease prevention, but the container volume limits the amount of water that can be stored. This dichotomy results in frequent applications of irrigation water, with large volumes typically being used on a daily basis. In a recent survey, over 75% of nursery crops in 17 states were grown in containers and use irrigation (U.S. Dept. Agr. 2009). In Florida, container nurseries annually apply 140 to 300 cm of irrigation per year, in addition to the 100 to 150 cm of average annual rainfall (Fernandez et al. 2009). Container nurseries in Alabama were estimated to have used 37 to 49 billion L of water in 1985 (Fare et al. 1992), and container nursery production in that state has almost tripled since 1987 (U.S. Dept. Agr. 1994, 2004). Opinions from 12 scientists, growers, and nursery organization leaders were summarized by Beeson et al. (2004) and highlight many of the water issues facing ornamental production producers. Increased water demand, decreased availability, and increased costs associated with water purification and delivery will force growers to limit water use, either by increasing their efficiency or installing containment basins for recycling, especially in coastal and arid regions. Principal solutions suggested implementing effective management and BMP techniques such as microirrigation for larger containers (>28 L) and recycling of irrigation water used for overhead irrigation (Beeson et al. 2004). Future areas of research such as outdoor

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subirrigation, plant-based sensor technology, and container or production area modifications were discussed as possible solutions. However, the authors concluded that no one technology or management plan would provide all benefits, and site-specific decisions must be tailored to the individual operation and their needs. C. Water Quality Humans have had major impacts on a variety of earth systems and notably on freshwater supplies around the world. Eutrophication caused by nutrient pollution of rivers also has major impacts on estuaries and marine ecosystems, particularly in the Puget Sound, the Gulf of Mexico, and the mid-Atlantic states (Livingston 2007). In recent years, various environmental groups in the United States have been putting increasing pressure on the Environmental Protection Agency (EPA) to enforce largely ignored parts of the federal Clean Water Act of 1972, which would impose strict total maximum daily load (TMDL) limits for both point and nonpoint sources of pollution in all watersheds and bodies of water listed on the impaired waters list (Lea-Cox et al. 2002). The nursery and greenhouse industry faces additional difficulties compared to traditional agriculture, in that the nutrient requirements of many species being grown is not known, crop production times can vary from weeks to years, a variety of production systems exist that have differing impacts, and different nutrient and irrigation practices are used. Due to these variables, writing water and nutrient management plans requires a risk assessment approach, which has the advantage of allowing the grower to design and implement site-specific best management practices for the entire operation (Lea-Cox et al. 2001a). Frequent irrigation in combination with high fertilizer and pesticide use can lead to significant losses of agricultural chemicals in runoff water that transports agricultural chemicals to containment structures and/or off-site into groundwater or surface water (Camper et al. 1994; Briggs et al. 1998; Briggs et al. 2002; Cabrera 2005). Irrigation water management is the key to nutrient management in ornamental crop production and reducing the impact of runoff water on local water resources (Tyler et al. 1996b; Lea-Cox et al. 2001a; Ullah and Zinati 2006). Increasing substrate anion and cation exchange capacity—for example, through the use of aluminum or various clay amendments—can help to reduce leaching of nutrients from soilless substrates (Williams and Nelson 1996; Owen et al. 2008). However, the recycling of runoff water raises other management issues for growers, primarily in the form of disease pressure (Hong and Moorman 2005) and salinity management. Emerging

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constraints on water use and quality mean that the ornamental industry needs to find ways to manage water without detracting from production schedules and crop health and quality. The quality and quantity of water required by an operation is largely dependent on the irrigation system used. Typically, the more precise the application of water, the higher the quality of that water must be in regard to particulate matter and water quality (e.g., pH, dissolved salts, alkalinity), to avoid problems. Higher-volume overhead irrigation systems generally require less filtration compared to boom, microsprinkler, or drip systems. Although the latter systems use smaller water volumes compared to overhead systems, they may require additional filtration and better irrigation system design for precise water applications to individual plants. The reuse of water from the operation, either from rainfall or from recaptured surface water, is another important concern for water quality. This water may contain pesticides, herbicides, fertilizer, pathogens, and particulate matter, which need to be treated and filtered before reapplication of this recaptured irrigation water. D. Irrigation Systems The amount and quality of irrigation water that is applied varies greatly depending on plant needs, water source, and system design. The irrigation system used by an operation will have a large impact on the amount of water used over a given time. The design of an irrigation system is governed largely by the size and type of plants grown, the size of the operation, water availability, and cost. Overhead irrigation has the lowest cost to install and maintain but also tends to have the lowest interception efficiency, requiring more water to irrigate the same number and size of plants compared to microirrigation or subirrigation. Besides a low initial cost, the other main benefit of overhead irrigation is the flexibility that this system allows. Plants of almost any size can be placed under overhead irrigation with few labor hours required to move and relocate emitters. Regardless of whether water is purchased from a public utility company, pumped from groundwater or surface water resources, or collected and recycled, most likely energy is required to pump the water from the storage area to where it is applied to the plants. By increasing the efficiency of the system, the cost of pumping this water can be greatly reduced. Increasing the efficiency of water application allows an operation to irrigate more plants, or to irrigate the same number of plants with less, thereby conserving water. The design, implementation, and maintenance of irrigation systems are critical factors for determining the

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overall efficiency of water applications. Although we do not have the space to go into the details of system design and maintenance, we will summarize the current state of irrigation systems. A variety of references are available for more information (EPA 1993; Waskom 1994; Fain et al. 2000; Bilderback and Lorscheider 2007a,b; Ross 2008c). There are several major disadvantages to overhead irrigation. Interception efficiencies are typically low, ranging from 20% or less for containers that are spread one container diameter apart, to about 90% for tightly spaced containers (Mathers et al. 2005). Overhead irrigation also requires high pressure (1.4–5.5 bar) and large volumes of water (1.9–45 L/min) per impact sprinkler, or up to 3,800 L/min for large center-pivot or traveling guns (Bilderback and Lorscheider 2007b; Ross 2008c). Impact sprinklers inherently apply more water closer to the sprinkler head, requiring double overlap of sprinklers to ensure application uniformity. In addition, up to 25% to 30% of irrigation water typically is lost to evaporation in the air, or on leaf or container surfaces, before the water reaches plant roots, with higher percentages possible during hot dry days (Ross 2008c). Overhead sprinklers also are impacted by wind and changes in system pressure, which can affect uniformity. Operators also typically irrigate a block of plants until the driest pots are at container capacity, leading to a high leaching fraction, especially if sprinklers have a low coefficient of uniformity. Avariety of microirrigation systems (i.e., drip and microsprinkler) can be used effectively in most nursery and greenhouse operations, including in-ground and pot-in-pot production systems. These systems apply water at or near the rootzone, unlike overhead irrigation systems. Water is directed to the substrate of a plant, with higher interception efficiency, so a greater proportion of applied water enters the root zone, with higher uniformity. Compared to overhead systems, microirrigation uses lower water pressure and water volume and has lower pumping (horsepower and energy) requirements. Microirrigation is particularly useful with plants that have high leaf densities or an architecture that makes it difficult for the water to get to the substrate, or with large containers that are spaced farther apart and have low interception efficiencies with overhead irrigation. High-value crops also benefit from the increased uniformity of microirrigation systems, as do operations that have limited water availability. Microirrigation also lends itself well to automation, reducing labor costs associated with irrigation scheduling. However, these microirrigation systems are not economical for most nursery producers using container sizes below 20 L volume, since these plants typically are spaced and moved at least twice per year, if not more often (to consolidate blocks during production and after

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sale). Time and labor costs therefore dominate decisions in these smallcontainer nursery systems. Additional disadvantages of microirrigation systems are higher initial infrastructure costs and the increased time to place emitters and check for proper functioning. With coarse porous substrates such as pine bark, spray stakes should be used for adequate container wetting, since capillary action in the substrate is not able to sufficiently wet all portions of the substrate profile using a drip emitter, due to low lateral capillary movement (Handreck and Black 2002). Filtration requirements for microirrigation systems are also higher, typically requiring 150- to 200-mesh filtration, compared to overhead irrigation, which may require a filtration system upgrade before a microirrigation system can be used (Ross 2008c). Regardless of the type of irrigation used, system maintenance is important for proper functioning. It is important to routinely check for clogged or broken emitters, system uniformity, orifice wear, pump function, and the like (Ross 2008b). Dosing equipment for fertilizers and other chemicals should also be maintained and checked periodically to be sure they are functioning properly. E. Increasing Water Application Efficiency 1. Matching Plant Water Needs to Irrigation. Irrigation is generally a small portion of the overall cost of producing a plant, a fact that has the potential to lead to the overapplication of water out of concern for the plants experiencing drought stress. Overapplication of irrigation water can lead to nutrient leaching, surface water runoff, erosion, and root diseases. Water conservation is typically a concern only for growers during drought or when greater production acreages are planned, when water is a limiting factor. Increasing irrigation efficiency can have a large impact on an operation by decreasing peak water demand, which can help conserve current and future water resources. In container production environments, irrigation scheduling and water management are critical factors in plant growth and salability. Growers usually are constrained by the amount of water that is available for irrigation (Beeson et al. 2004; Mathers et al. 2005). Restrictions may be in the form of permitted withdrawals, well capacity, total production area, or irrigation system design. Some of these constraints, such as system design, can be changed, while others (e.g., permitted daily volumes) are not under a grower’s control. Unfortunately, currently we do not have very efficient methods to determine plant irrigation requirements for different species on a daily basis (Lea-Cox et al. 2009b). Most growers typically base irrigation decisions on substrate appearance, cumulative

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knowledge, and experience that integrates recent weather and irrigation events and using subtle plant indicators (such as changes in leaf reflectivity) instead of quantifying water use data. These more subjective scheduling methods typically lead to overapplication of irrigation water, since the economic cost (risk) of reduced plant growth is much higher than the cost of water. 2. Irrigation Management. In most woody ornamental nurseries, irrigation has changed little since the 1960s (Beeson 2005). Beeson and Knox (1991) found that for two azalea cultivars (Rhododendron spp. ‘Duc du Rohan’ and ‘George L. Taber’) and Pittosporum tobira ‘Laura Lee’, as the plants grew, their architecture decreased interception efficiencies to an average of 37% for three different container sizes. The authors recommended keeping containers closely spaced and transplanting into a larger container when foliage begins to touch. The dilemma that this recommendation raises is that it requires a large amount of labor and resources, and most growers typically do not transplant until doing so becomes an economically viable decision, based on market demands for certain container sizes and sales. If a plant is under water stress, its growth potential has already been greatly decreased (Caron et al. 2005). The goal of irrigation management is to supply a plant with enough water to fill the container to its waterholding capacity and satisfy total plant water requirements before the next irrigation cycle but not to apply too much water so that nutrients are leached from the substrate. Irrigation timing is therefore critical to achieve these goals without inducing water stress and a reduction in growth. Warren and Bilderback (2004) found that irrigating Cotoneaster dammeri ‘Skogholm’ 3 times per day (12, 3, and 6 PM) produced plants with 57% and 69% higher dry weights compared to the control, which was irrigated at 3, 5, and 7 AM in pine bark, at leaching fractions of 0.4 and 0.15, respectively. Cyclic irrigation also has the potential of significantly reducing the volume of irrigation water applied. Tyler et al. (1996) found that by applying 2, 3 or 6 cycles, 1 hour apart each, compared to the control, which had 1 continuous cycle using a pine bark:sand media, application efficiency increased 38% over the control. This coincides with a study by Lamack and Niemiera (1993) using spray stakes; the authors found a 24% reduction in irrigation requirements compared to a noncyclic control. Both studies also documented a decrease in nutrient loss from the substrate using cyclic irrigation. 3. Reducing Leaching and Runoff. One of the most beneficial ways to keep nutrients in the rootzone is by reducing leaching and runoff. This

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benefits the plant by making more of the fertilizer available for a longer amount of time, which increases uptake efficiency. The grower benefits by irrigating less frequently and reducing water and energy use. Few quantitative irrigation versus nutrient leaching data exist for open container-nursery systems, which constitute the highest-risk nursery and greenhouse production situation (Lea-Cox and Ross 2001). This is especially true for phosphorus. In a 3-year study of irrigation and nutrient application rates to container-grown azalea and holly, Ristvey (2004) estimated N loading (runoff) rates to be 274 to 513 kg
ha1 (11-L containers) to 1050 to 2260 kg
ha1 N (19-L containers), depending on irrigation type and fertilization strategy. Similarly, Ristvey 2004 estimated P loading rates from 65 to 119 kg
ha1 P to 98 to 210 kg
ha1 P (11-L and 19-L containers, respectively) under the same management conditions. Cumulative N loading was further reduced by the use of drip irrigation and sensor-controlled irrigation (Fig. 7.1), with similar reductions seen for P loading (see Ristvey 2004 for P data). As mentioned previously, Ristvey (2004) showed that time domain reflectometry (TDR) to control irrigation could reduce overhead N runoff by 53% compared to overhead cyclic and by 82% for TDR controlled drip compared to overhead cyclic irrigation (Fig. 7.1). Precision application, whether it controls irrigation volume, timing, or interception efficiency, has the ability to significantly reduce nutrient leaching if used correctly. Reducing nutrient leaching by efficient water management practices is probably the easiest best management practice to implement, but often growers do not recognize the inefficiency of their current practices. 4. Precision Applications. Applying the correct volume of water at the correct time to maintain optimal growth rates is one of the most fundamental aspects of growing plants but also one of the most difficult, since both plant water use and environmental conditions are constantly changing. Irrigation scheduling decisions often are based on intuition or an integration of tangible factors (pot weight, plant condition and size, time from prior irrigation, and environmental conditions) rather than actual plant water use, which is difficult to determine quickly and accurately. Tools to measure soil moisture such as tensiometers and gypsum blocks are either not accurate or are management intensive in porous soilless substrates. Other methods, such as load balances, are useful in greenhouse production but do not lend themselves for use in open production systems, where recalibration for incremental plant growth and daily water use can be cumbersome (Jones 2008). Computer programs to estimate daily plant evapotranspiration, based on plant models, have been shown to be useful, especially in agronomic crops, but

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are imprecise in ornamental nurseries because of the varying water use of the many different species grown, often in mixed blocks. Beeson (2005) published a model for making irrigation decisions for woody ornamentals but notes that average canopy area and percent closure would need to be calculated periodically based on canopy closure and growth, with different models required based on plant water use. The author also notes that application uniformity and canopy structure will affect irrigation scheduling and are not included in the current model (Beeson 2005). Several recent advances in irrigation technology could help growers to better understand plant irrigation needs and reduce water requirements in the future. Burnett and van Iersel (2008) used capacitance sensors, a technology similar to TDR mentioned earlier, to grow Gaura lindheimeri ‘Siskiyou Pink’ (gaura) and Phlox paniculata ‘David’ (garden phlox) for 5 weeks at a variety of volumetric water contents from 0.10 to 0.45 m3
m3 (10%–45% volumetric water content, VWC), applying between 3.8 L and 53 L of water, that resulted 0 to 7.74 L of leachate per pot. They found that significant leaching occurred above 40 % VWC. Above 25% VWC, plants were marketable, but Gaura plants grown below 25% VWC were generally shorter, with fewer branches and lower dry weight (Burnett and van Iersel 2008). In a greenhouse experiment comparing soil moisture sensors to standard practice for ‘Mini penny’ hydrangea, sensor irrigation used 87,960 L per bay to irrigate 4 bays, compared to 504,630 L per bay for 3 bays during the same 11-week period, with less variability in soil moisture and less leaching in sensor-controlled plots compared to control plants (van Iersel et al. 2009). From these articles and current ongoing research, it is clear that capacitance sensors have the potential to provide information and irrigation control in the plant root zone, where water stress is experienced by the plant. Wireless sensor networks have made major advances in recent years to help understand and meet plant irrigation needs. The use of capacitance sensors has the potential to provide accurate real-time information to schedule and remotely control irrigation applications at a relatively low cost (Lea-Cox et al. 2009a,b). Although many challenges remain in determining precision water applications for specific crops, these sensors have been shown to be very precise in both soils (Lea-Cox et al. 2009b) and soilless substrates (Arguedas et al. 2007; Kang and van Iersel 2009). Additionally, these wireless sensor systems allow for the measurement of other environmental variables, such as soil temperature, air temperature and relative humidity, rainfall, irrigation volumes, photosynthetically active radiation, and wind speed and direction. Having a suite of soil moisture and environmental data at the

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microclimatic scale in the production area will allow ornamental producers to make precise real-time irrigation and nutrient management decisions (Lea-Cox et al. 2009b). Further development of specific crop models that integrate all of this environmental data (Kim and van Iersel 2009) will allow for the development of more predictive management tools in the future (Lea-Cox et al. 2009b). As costs for this technology continue to decrease, these systems should be able to pay for themselves in water, electricity, and increased growth in a relatively short period of time. With high-value crops, this payback period could be a short as a single crop. It is clear that the industry as a whole can employ a number of existing and emerging irrigation management practices and technologies not only to mitigate environmental impacts but potentially to increase profits.

F. Intercepting Runoff: Capture and Recycling There are many ways to reduce the effects of nutrients and sediment loading to surface water, but each method has different costs, advantages, and disadvantages associated with it. Unfortunately, there is limited information on the costs associated with implementing BMPs and on the relationship between the cost and the benefit of those BMPs in terms of environmental and production benefits. Veith (2002) developed a program that begins to address this complicated task of balancing environmental benefits with cost of implementation. 1. Closed Systems. Several different types of structures can be used to capture irrigation runoff and rainfall either for reuse on the operation or for storage to reduce the volume of runoff reaching surface waters. Ebb-and-flood systems are used almost exclusively in greenhouse systems to irrigate and recycle water and nutrients with very little loss, both as extensive floor systems or ebb-and-flood bench systems. In its simplest form, this system uses two tanks and a series of pumps and pipes (Nelson 2008). Water or nutrient solution is pumped into a contained growing area from below, filling it to a certain depth. This water saturates the substrate by capillary action, and after a certain period of time, the water is drained into a second tank, where it can be filtered and adjusted as needed (pH, fertilizer, etc.) before being pumped back into the first tank. The main benefits of this system are that it is closed and that the fertilizer and water remain in the system until they can be utilized by the plants, which reduces both water and nutrient cost. The major drawbacks of this system is that it requires substantial monitoring, usually by a computer-controlled system, and there is a higher potential for disease

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buildup if the water is not filtered properly. Additional research is required to optimize fertilizer concentrations for ebb-and-flood systems (Kent and Reed 1996; Cox 2001; Pennisi et al. 2003). 2. Open Systems. Capturing surface water runoff through the use of containment ponds is an effective yet expensive method for sediment and nutrient abatement, if land areas are available and groundwater tables are not too high. It has the advantage that significant volumes of runoff water and nutrients can be returned to production areas for repeated use, especially where rainfall or water supplies are limited. Collection basins ideally are located at the lowest point in an operation to collect the maximum amount of irrigation and stormwater runoff. Minimum containment should be sized to capture at least 90% of the runoff volume from an irrigation event, with a buffer capacity to hold an additional 13 mm of rainfall (Yeager 2008). Water typically is conveyed to ponds by lined or grassed drainage ditches or via underground pipes for maximum efficiency. Lined ditches and pipes will have faster flow rates compared to unlined ditches, so it is important that these structures have areas where suspended sediment can drop out of water, such as sediment ponds or rock structures (riprap) to slow water velocity before the water enters the containment pond. The purpose of a sediment pond is to allow suspended sediment to settle out of the water column so the soil and any nutrients (particularly P) that it contains are retained before being discharged or recycled. Water from a containment pond can then be used to irrigate crops, or it may be discharged to surface waters after the suspended particles have settled. Containment basins are also valuable for N removal by aquatic plants and bacterial denitrification. Containment basins and sediment ponds are becoming increasingly popular because of tighter water and nutrient management regulations and the increased cost and decrease in availability of water. The main issues with recycling runoff from production areas in an operation is the potential for recycling pesticides, herbicides, and pathogens back into the operation (Hong and Moorman 2005). Typically, growers reduce pathogen loads by treating captured water using filtration, chlorination, or ultraviolet (UV) light. Injection of fungicides also can be used to reduce pathogen loading, although there is a higher cost compared with chlorine injection and the possibility of resistance to fungicides developing with prolonged use (Hong et al. 2003). There has been some research to suggest that containment basin design and the placement of pump intake can reduce disease pressure (Hong and Moorman 2005; Ghimire et al. 2009; Kong et al. 2009). Hong and

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Moorman (2005) conclude that disease management is affected by these factors: (1) quality of the water to be treated; (2) quantity of water to be treated over a given time; (3) allowable changes in water quality due to treatment; (4) pathogen population reduction for crop protection; (5) susceptibility of the crop to specific pathogens; (6) cultural practices being used; and (7) economic resources together with level of experience and time required for control measure. Hong and Moorman (2005) report that there are a number of treatment methods available, but many have not been tested at the operational scale. Although additional research is needed in the area of pesticide and pathogen management, it is clear from many existing operations that recycling from catchment basins is an effective and efficient way to control nutrient and sediment runoff and to increase the resource use efficiency of an operation. Another option that currently is being tested for use in the nursery and greenhouse industry is reclaimed water from wastewater treatment plants (Yeager et al. 2009). This postconsumer water goes through a series of filtration and treatment steps before it is transported to growers. Reclaimed water costs about half that of potable fresh water, although costs vary based on a number of conditions, such as condition of water to be treated, posttreated water requirements, quantity, and transport distance (Yeager et al. 2009). There are several uses for reclaimed water, including aquifer recharge, nursery and greenhouse use, and agricultural use. 3. Vegetated Buffers. Vegetated buffer areas are probably the most costeffective primary method for sediment and nutrient abatement in open production systems, for both field and container-nursery operations. A disadvantage is that unless they are used in association with containment ponds, there is no recovery of water and nutrients for reuse. Vegetated buffers can consist of various structures, including grassed or vegetated ditches, swales, and buffer strips. Various grass and/or tree species can be used to slow the velocity of surface water runoff from production areas, to allow for sediment removal and infiltration of water, denitrification of N, the uptake of nutrients, and the denaturing of herbicides and pesticides. Although a variety of buffer widths are recommended in the literature (Osborne and Kovacic 1993; Wenger 1999; Dosskey 2002; Todd 2002), a 15-m is width is often used for regulatory purposes. This width could be considered relatively arbitrary, since the efficacy of a vegetated buffer depends on many factors including volume (rate) of runoff handled per minute, the infiltration capacity of the soil, slope, the plant species used, the buffer condition, and the amount of sediment/nutrients to be removed (Wenger 1999).

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4. Wetland Remediation. Sediment, nutrients, and other pollutants can be removed using either constructed or existing wetlands to treat runoff leaving an operation before it reaches surface waters. A number of articles have extolled the benefits of wetlands for N removal (Vymazal 2007; Blankenberg et al. 2008; Lin et al. 2008; Polomski et al. 2009). In general, wetland systems can be very effective at removing N, if sized correctly for the volume and concentration of N loading. Constructed wetlands, however, are not very efficient at removing soluble reactive P, except during peak wetland plant growth, although the addition of clay in the form of calcined clay or crushed brick after wetland treatment has been shown to significantly reduce soluble reactive P (White et al. 2006; Owen et al. 2008). Unfortunately, since most aquatic systems are considered P limited, as discussed earlier, constructed wetlands without secondary treatment such as that described by White et al. (2006) are usually not sufficient to remediate nutrient runoff from nurseries, particularly during early spring, when biological activity in wetlands is limited by temperature and nutrient loading rates are relatively high. However, there is no doubt that wetland systems are excellent polishing stems for posttreatment of water after containment and slow release. 5. Containment Basin Design and Stormwater Runoff. There are few resources available for growers to understand the regional requirements for efficient containment basin design to ensure that these structures have adequate volume and functionality to meet the requirements for pathogen and nutrient management and recycling. This lack of information has been met in part by a series of learning modules (Hong 2008; Ross 2008a; Yeager 2008) in a knowledge center developed to provide information directly to growers on substrate, water, nutrient, and crop health management (Lea-Cox et al. 2008). However, containment basin and stormwater bypass design specifications are complex and often site-specific. A greater effort should be placed on the development of more guidelines and resources for growers, especially since these types of designs often has to be approved and permitted by local Natural Resource Conservation Service and other environmental agency staff in specific states.

V. CONCLUSIONS A number of changes have occurred in the production of containergrown ornamental crops during the past 30 years that have helped the industry grow and become more economically competitive. Water constraints due to growing demand, reduced supply, and changing weather

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patterns now and in the future have the potential to further restrict irrigation use by the nursery and greenhouse industry. This provides a challenge to the industry to reduce water volumes using a variety of current management practices and new technologies. Concerns about nutrient runoff and infiltration should be met with additional research and better recommendations to reduce the potential for nutrient runoff from an operation. We need to develop a better understanding of plant nutrient requirements, better technology to assess rootzone conditions, and better fertilizers or practices that are able to match plant nutrient requirements during the growing season. Several additional areas of research would be beneficial to the container nursery and greenhouse industry. With concerns over substrate availability, shipping costs, and the effect of substrates on nutrient and water use, progress in the development of alternative, sustainable soilless substrates and amendments is vital for the industry. Increasing the anion exchange capacity by using a variety of soil amendments is important for N and P retention in soilless substrates. Additional research is needed to gain a better understanding of plant nutrient requirements for a variety of plant species to be used as models for nutrient application recommendations. New formulations of slow-release fertilizers are needed that match both the timing and amount of nutrient based on actual plant growth requirements instead of release rates being based on temperature. Liquid and solid fertilizer recommendations also need to be based not only on growth but also on reducing the environmental impact of production. Better rates and timing of fertilizer applications have the potential to reduce leaching losses in both containers and field situations. Much more research is needed to thoroughly investigate denitrification in soilless substrates, as there is evidence that this is a substantial loss mechanism, both in field soils and in soilless substrates. It is important both to understand this loss mechanism and to determine ways to reduce its impact to increase N uptake efficiency. This is an area where significant gains in efficiency are possible since both nutrient and irrigation reductions could decrease denitrification rates. We need to develop and implement irrigation scheduling based on actual plant water use instead of the subjective methods typically used by most growers today. Computer- and sensor-driven irrigation management and control technology represents a significant advance over current irrigation technologies (Lea-Cox et al. 2009a,b). The integration of plant water use models such as those developed by Beeson (2005), Bowden et al. (2005), Wang et al. (2008), and Kim and van Iersel (2009) with this technology will allow us to develop much better predictive tools for growers in the near future. There is also a need for research to

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better understand new technologies that can sense real-time electrical conductivity in the root zone, which can be used to more effectively manage nutrient applications (Arguedas et al. 2009). Additional research into container nursery pond design and nonchemical approaches to pathogen management in closed and open recycling systems is a vital area for continued research. Since recycling is likely to become more popular as growers face increased restrictions for nutrient runoff, and as water becomes more limited, additional research will help ensure crop health in the future. In order for growers to make informed decisions about changing practices, accurate information on the cost of BMP implementation with the corresponding financial and/or environmental benefit would be beneficial. Local or regional BMP cost and benefit guides would enable growers to make informed decisions on how best to mitigate their impact on the environment. LITERATURE CITED Agarwal, A., M.S. delos Angeles, R. Bhatia, I. Cheret, S. Davila-Poblete, M. Falkenmark, F. Gonzalez Villarreal, T. Jønch-Clausen, M.A. Kadi, J. Kindler, J. Rees, P. Roberts, P. Rogers, M. Solanes, and A. Wright. 2000. Integrated water resources management. Global Water Partnership Technical Advisory Committee (TAC), Background Paper No. 4. Global Water Partnership. Denmark 71. Online at www.cepis.opsoms.org/ bvsarg/i/fulltext/tac4/tac4.pdf. Agner, H. 2003. Denitrification in cultures of potted ornamental plants. Ph.D. thesis. Plant Sciences. Univ. Hannover. Hannover, Germany. Argo, W.R. 1998a. Root medium chemical properties. HortTechnology 8:486–494. Argo, W.R. 1998b. Root medium physical properties. HortTechnology 8:481–485. Arguedas, F.R., J.D. Lea-Cox, and A.G. Ristvey. 2007. Characterizing air and water content of soilless substrates to optimize root growth. Comb. Proc. Intl. Plant Prop. Soc. 57:701–708. Arguedas, F.R., J.D. Lea-Cox, and A.G. Ristvey. 2009. Real-time measurement of electrical conductivity in soilless substrates. SNA Research Conference. Atlanta, Georgia. S. Nursery Assocc. 54:216–220. Aulakh, M.S., J.W. Doran, and A.R. Mosier. 1992. Soil denitrification-significance, measurement, and effects of management. Adv. Soil Sci. 18:1–57. Baligar, V.C., N.K. Fageria, and Z.L. He. 2001. Nutrient use efficiency in plants. Commun. Soil Sci. Plant Anal. 32:921–950. Bauerle, W.L., C.J. Post, M.F. McLeod, J.B. Dudley, and J.E. Toler. 2002. Measurement and modeling of the transpiration of a temperate red maple container nursery. Agr. Forest Meteorology 114:45–57. Beeson, R.C., Jr. 2005. Modeling irrigation requirements for landscape ornamentals. HortTechnology 15:6–23. Beeson, R.C., Jr., M.A. Arnold, T.E. Bilderback, B. Bolusky, S. Chandler, H.M. Gramling, J.D. Lea-Cox, J.R. Harris, P.J. Klinger, H.M. Mathers, J.M. Ruter, and T.H. Yeager. 2004. Strategic vision of container nursery irrigation in the next ten years. J. Env. Hort. 22:113–115.

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Tyler, H.H., S.L. Warren, and T.E. Bilderback. 1996b. Reduced leaching fractions improve irrigation use efficiency and nutrient efficacy. J. Env. Hort. 14:199–204. U.S. Dept. Agr. 1994. 1992 Census of agriculture. U.S. Dept. Agr. Washington, DC. U.S. Dept. Agr. 2004. 2002 Census of agriculture. National Agricultural Statistics Serv. Washington, DC. AC-02-A-51. U.S. Dept. Agr. 2007. Nursery crops 2006 summary. National Agricultural Statistics Serv. Washington, DC. Sp Cr 6–3 (07). U.S. Dept. Agr. 2009. 2007 Census of agriculture. National Agricultural Statistics Serv. Washington, D.C. Ullah, S., and G.M. Zinati. 2006. Denitrification and nitrous oxide emissions from riparian forest soils exposed to prolonged nitrogen runoff. Biogeochemistry 81:253–267. van Iersel, M., R.M. Seymour, M. Chappell, F. Watson, and S.K. Dove. 2009. Soil moisture sensor-based irrigation reduces water use and nutrient leaching in a commercial nursery. SNA Research Conference. Atlanta, GA. S. Nursery Assoc. 54:17–19. Veith, T.L. 2002. Agricultural BMP placement for cost-effective pollution control at the watershed level. Biological Systems Engineering. Ph.D. thesis, Virginia Polytechnic Inst. and State Univ. Blacksburg, VA. Vymazal, J. 2007. Removal of nutrients in various types of constructed wetlands. Sci. Total Env. 380:48–65. Wang, Y., W.L. Bauerle, and R.F. Reynolds. 2008. Predicting growth of deciduous tree species in response to water stress: FVS-BGC model evaluation and application. Ecol. Modeling 217:139–147. Warren, S.L., and T.E. Bilderback. 2004. Irrigation timing: Effect on plant growth, photosynthesis, water-use efficiency, and substrate temperature. Acta Hort. 644:29–37. Warren, S.L., T.E. Bilderback, and H.H. Kraus. 2001. Method of fertilizer application affects nutrient losses of controlled-release fertilizer. Acta Hort. 548:349–355. Waskom, R.M. 1994. Best management practices for irrigation management. Colorado State Univ. Coop. Ext. Bul. XCM-173. Wenger, S. 1999. A review of the scientific literature on riparian buffer width, extent, and vegetation. Office of Public Service and Outreach. Institute of Ecology. University of Georgia, Athens. White, S. 2007. Constructed wetlands for the landscape nursery industry: Nutrient assimilation and removal. Environmental toxicology. Ph.D. thesis, Clemson Univ. Clemson, SC. White, S.A., M.D. Taylor, T. Whitwell, and S.J. Klaine. 2006. Managing phosphorus in simulated constructed wetlands. S. Nursery Assoc. Res. Conf. S. Nursery Assoc. 51:496–500. Williams, K.A., and P.V. Nelson. 1996. Modifying a soilless root medium with aluminum influences phosphorus retention and chrysanthemum growth. HortScience 31:381–384. Wright, R., and J. Latimer. 2007. Grinding pine logs to use as a container substrate. Greenhouse Product News 17:32–37. Wright, R.D., B.E. Jackson, J.F. Browder, and J.G. Latimer. 2008. Growth of chrysanthemum in a pine tree substrate requires additional fertilizer. HortTechnology 18:111–115. Wright, R.D., and A.X. Niemiera. 1987. Nutrition of container-grown woody nursery crops. Hort Rev. 9:75–101. Wright, R.D. and A.X. Niemiera. 1991. Timing of Osmocote reapplication on growth of ‘Helleri’ holly. Proc. S. Nursery Assoc. Res. Conf. 36:74–76. Yeager, T., C. Larsen, J. von Merveldt, and T. Irani. 2009. Use of reclaimed water for irrigation in container nurseries. Environmental Hort. Dept., Florida Cooperative Ext. Serv., Univ. Florida. ENH1119, Gainesville, FL.

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8 World Vegetable Industry: Production, Breeding, Trends Jo~ ao Silva Dias Technical University of Lisbon Instituto Superior de Agronomia Tapada da Ajuda 1349–017 Lisbon, Portugal Edward J. Ryder U.S. Department of Agriculture Agricultural Research Service Salinas, CA 93905, USA Present Address: 77 Paseo Hermoso, Salinas, CA 93908, USA

I. INTRODUCTION II. THE WORLDWIDE VEGETABLE INDUSTRY A. World Importance of Vegetables B. Vegetable Marketing C. Vegetable Migration D. Processed Vegetables III. VEGETABLE PRODUCTION STRATEGIES A. Social Importance B. Multiple Cropping, Urban Production IV. VEGETABLE BREEDING A. Objectives B. Goals and Techniques C. Prospects for Poor Growers and Marginal Land Production D. Improvement through Genetic Modification E. Future of Breeding and Diversity V. SUMMARY AND CONCLUSIONS LITERATURE CITED

Horticultural Reviews, Volume 38 Edited by Jules Janick Copyright
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I. INTRODUCTION Farming and plant breeding have been closely associated since the early days when crops were first domesticated. Plant breeders play a key role in determining what we eat, since the cultivars they develop begin the dietary food chain. Every food product, including vegetables, that we see on the market has benefited from plant breeding. Without understanding the science, early farmers saved the seed from the best portion of their crop each season. Over the years they selected the traits that they liked best and were most useful for the crop, transforming and domesticating the vegetable species they grew. In the 18th and 19th centuries the Vilmorin-Andrieux family, owner of the first commercial seed company in the world, played an important role in a number of theoretical and technical advances in commercial vegetable breeding, such as producing the first vegetable seed catalog for horticulturists, developing the principles of genealogical breeding programs, improving seed quality through cross-breeding initiatives, and creating disease-resistant and hybrid cultivars of vegetables (Gayon and Zallen 1998). In 1856 Louis Vilmorin published ‘‘Note on the creation of a new race of beetroot and considerations on heredity in plants’’ establishing the theoretical groundwork for the modern vegetable breeding industry. The first suggestion to exploit hybrid vigor, or heterosis, in vegetables was made by Hayes and Jones (1916) for cucumber. Commercial hybridization of vegetable species began in the United States in the middle 1920s with sweet corn, followed by onions in the 1940s. Since that time, private breeding companies have been placing more and more emphasis on the development of vegetable hybrids, and many species of vegetables have been bred as hybrid cultivars for the marketplace. Hybrids allow breeders to express heterotic effects by combining the best horticultural traits and multiple disease and stress resistances. Furthermore, if the parents are homozygous, the hybrids will be uniform, an increasingly important trait in commercial vegetable market production. In the 1970s breeders’ rights protection has been provided through UPOV (International Union for the Protection of New Cultivars of Plants), which coordinates an international common legal regime for plant cultivar protection. In addition, in the United States, plant breeding companies can take advantage of the U.S. utility patent law to protect not only the cultivar itself but all of the plant’s parts. The creation of vegetable hybrid cultivars requires homozygous inbred parental lines, which provide a natural protection of plant breeders’ rights without legal recourse and ensure a market for seed companies. Such product protection options have presented a business incentive to corporations to

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invest in the seed industry, leading to an enormous increase in private research and development (R&D), insuring strong competition in the marketplace among the major seed companies. The majority of current vegetable cultivars sold nowadays are proprietary products developed by private R&D. A significant consequence of this increase in private R&D has been a reduction in public breeding programs. As a result, the cost for R&D to develop new cultivars is shifting from publicly supported research programs to customers of the major seed companies. No practical breeding program can succeed without large numbers of lines (genotypes) to evaluate, select, recombine, and inbreed (fix genetically). This effort must be organized, so valid conclusions can be reached and decisions made. Scientists, breeders, support people, and facilities, budgets, and good management are requirements to assure success in the vegetable seed business. Science must be state of the art to maximize success in a competitive business environment. Since the continued need for fundamental breeding research is critical to sustain development of new technology and expansion of the knowledge base that supports cultivar development, competition among proprietary cultivars results in owner-companies striving to do the best possible research to develop their own products and to compete on genetic and physiological quality of vegetable seed in the marketplace. Reasonable profit margins are necessary to pay back the R&D costs to the owner and to fund future research on developing even better vegetable cultivars to stay competitive. There is considerable genetic variation within the various vegetable species, which can be exploited in the development of superior proprietary cultivars. The consequences of this dynamic situation mean that relatively short-lived cultivars are replaced by either the owner of the cultivar or a competitor seed company. This intense competition means constantly improved and more sophisticated cultivars for the vegetable industry. Seed companies are in the business of manipulating genes to improve plant cultivar performance for a profit. The success of the research is judged by the success of the product in making a reasonable profit. The research must improve economic performance starting with the seed production costs and include the grower-shipper/processor and the end user. If any link in this sequence of events is weak or broken, the new cultivar likely will fail. Biotechnology is a new, and potentially powerful, tool that has been added by all the major seed corporations to their vegetable breeding research programs. It can augment and/or accelerate conventional cultivar development programs through time saved, better products, and more genetic uniformity, or achieve results not possible by conventional

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breeding. The first commercially grown genetically modified crop was Flavr Savr tomato by Calgene in 1994, where the tomato fruit was made more resistant to rotting by adding an antisense gene that interferes with the production of the enzyme polygalacturonase (Kramer and Redenbaugh 1994). This tomato was deemed to have a long shelf life. However, it proved later to have a very short ‘‘market shelf life,’’ since the cultivar was considered inferior by growers and was rapidly withdrawn from the market. Plant genetic engineers learned an essential lesson: the importance of cooperation with breeders. New vegetable cultivars must be tested for performance in all markets before sales. In 2008 the global vegetable seed market was estimated at US$3.5 billion with these shares of vegetables: solanaceous (30%), cucurbits (21%), roots and bulbs (16%), brassicas (13%), large seed (13%), leafy and other (7%) (Monsanto 2009). In the last 8 years, global commercial vegetable seed sales had an annual growth rate of 5.8%. With the increase in world population and consumption and the advent of a high degree of added value through biotechnology, the global market of vegetable seeds is expected to expand in future years. Food availability and population growth are related. When increases in population exceed increases in food supply, poverty and starvation rapidly ensue. There are now over 6.5 billion human beings inhabiting this planet, and it has been projected that worldwide population growth may exceed 70 million annually over the next 40 years. The population is expected to reach approximately 9.5 billion by 2050, when approximately 90% of the global population will reside in Asia, Africa, and Latin America. Today 840 million people in the developing countries suffer from malnutrition and 1.3 billion are affected by poverty and food insecurity. Food security is not only about solving the urgency in the short term; it must also address the long-term issue of poverty alleviation and economic growth. Greater investment in agriculture, including breeding and cultivar development, more effective development aid, and reforms to trade and domestic policies are all part of the solution. As worldwide health awareness increases and household income grows, an increasing global demand for vegetables is expected. At the same time, available arable land and a suitable water supply is lessening, so energies should be directed to enhance vegetable productivity and quality to face these real challenges in the society. Also, biodiversity is considered essential for food security and nutrition. This chapter discusses the importance of plant breeding as key to modernizing the production, marketing and trading of vegetables, and alleviating some of the social obstacles to achieving worldwide food security for people at all levels of farming and consumption.

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II. THE WORLDWIDE VEGETABLE INDUSTRY A. World Importance of Vegetables Vegetables make up a major portion of the diet of humans in many parts of the world and play a significant role in human nutrition, especially as sources of vitamins (C, A, B1, B6, B9, E), minerals, dietary fiber, and phytochemicals (Quebedeaux and Eisa 1990; Craig and Beck 1999; Wargovich 2000; Liua et al. 2001). Vegetables in the daily diet have been strongly associated with overall good health, improvement of gastrointestinal health and vision, and reduced risk of some forms of cancer, heart disease, stroke, diabetes, and other chronic diseases (Prior and Cao 2000; Southon 2000; Hyson 2002; Goldberg 2003). Low vegetable intake, in unbalanced diets, has been estimated to cause about 31% of ischemic heart disease and 11% of stroke worldwide. The International Agency for Research on Cancer (IARC) estimates that the preventable percentage of cancer due to such diets ranges from 5% to 12% for all cancers and 20% to 30% for upper gastrointestinal tract cancers. According to the 2007 World Health Report, unbalanced diets with low vegetable intake and low consumption of complex carbohydrates and dietary fiber (Slavin 2001) are estimated to cause some 2.7 million deaths each year, and were among the top 10 risk factors contributing to mortality. Some phytochemicals of vegetables are strong antioxidants and are thought to reduce the risk of chronic disease by protecting against free radical damage, by modifying metabolic activation and detoxification of carcinogens, or even by influencing processes that alter the course of tumor cells (Southon 2000; Wargovich 2000). Women in particular benefit from good vegetable nutrition, particularly during later pregnancy and lactation. The interplay of the different micronutrients and antioxidants found in vegetables has important health impacts, explaining, for instance, the higher birth weight of children in India when mothers consumed higher rates of green leafy vegetables and fruits during pregnancy (Rao et al. 2001). ‘‘Hidden hunger,’’ or micronutrient deficiency, is a pernicious problem around the world that is caused by a lack of vitamins and minerals, such as vitamin A, iodine, and iron in the human diet and affects the health of between 2 and 3.5 billion people worldwide. Nutrition is both a quantity and a quality issue, and vegetables in all their many forms can bring a much-needed measure of balance back to diets. For example, mungbean (Vigna radiata), either eaten as whole pod grains or grown to produce bean sprouts, is an important source of iron for women and children throughout South Asia. Bitter gourd

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(Momordica charantia) has antidiabetic properties and can be used to ameliorate the effects of type 2 diabetes. Diet is the primary therapy for this type of diabetes, and bitter gourd is particularly critical when pharmaceuticals are not available, as happens in a great part of the developing world. Because of their nutritional importance, vegetables have been recognized within the nutrition and medical communities, and there is a general sense that the promotion of a greater consumption of vegetables will improve health. There is also an increasing awareness among the general public of the advantages of diets rich in vegetables to ensure an adequate intake of most micronutrients, dietary fibers, and phytochemicals that promote health. According to statistics from the Food and Agriculture Organization of the United Nations (FAO), the production of vegetables in the world in 2007 was almost 900 million tonnes (FAO 2009). Asia produced 74.7% of the world’s vegetables (671 million t) on 72.8% of the world’s vegetable production area (52.7 million ha). China has always been a large contributor to world vegetable production and currently produces over 50% of the world’s vegetables, which translates to 313 kg per capita. China’s liberalization of trade practices in the 1980s, after the government allocated land on a family basis and eliminated fixed procurement and retail prices for vegetables, has contributed to the increased importance of vegetable production. Vegetable production during the period 1965 to 2005 grew at an average annual rate of 5.6% in China and 3.5% in other developing countries in Asia. For example, in recent years, large areas in Shandong Province (China) have been converted from grain to vegetable production to supply inexpensive vegetables for consumers in nearby Japan. As a result, China has replaced the United Sates as the number -ne supplier of fresh and frozen vegetables to Japan (USDA 2002). In other developing countries of southern Asia, with a mid-subtropical climate favorable for vegetable production, the per capita production is only 80 kg. India is the second largest producer of vegetables in the world but at almost a sixfold lower level than China. Worldwide the area of arable land devoted to vegetables is expanding at 2.8% annually, higher than fruits (1.75%), oil crops (1.47%), root crops (0.44%), and pulses (0.39%) and at the expense of cereals (–0.45%) and fiber crops (–1.82%) (FAO 2009). The share of cultivated areas allocated to vegetables in 2007 was high in east and southeast Asia (around 10%) and in China (around 20%) but low in south Asia (5%). Although the cultivated area is largest in China, growers in other regions of Asia and in the Pacific have also found it profitable to expand production of vegetables. In contrast, the production of vegetables in Africa is lagging far behind the worldwide average. Annually only about 50 kg of vegetables per

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capita are produced in Africa, which is less than half of the average production on the other continents of the world. In marginal areas of vegetable production, subsistence production predominates, often with inefficient farming methods. Additionally in these areas, vegetable production is hampered by a limited supply of inputs and a lack of access to markets. Therefore, productivity and income are low, and per capita production is declining, resulting in food insecurity. The worldwide consumption and importance of vegetables in the diet is difficult to estimate owing to scant production statistics. Even where crop reporting services are an integral part of the agricultural infrastructure, information is available on only a small percentage of the vegetable crops grown. The consumption and caloric contribution of vegetables to the diet varies widely with geographical region, nationality, local customs, and cuisine. China is the largest consumer of vegetables in the world. Vegetables make up about 35% of per capita food consumption in that nation, a much higher share than the world average (Gale 2002). Besides India, other southern Asian countries such as Bangladesh, Cambodia, North and South Vietnam, Laos, and the Philippines are also high producers and great consumers of vegetables. For example, vegetables comprise 40% of the Bangladeshi diet (Rich 2008). Many vegetables are consumed near where they are produced, especially in China, India, and other Asian countries. The per capita consumption of vegetables in Asia has increased from 41 kg to 141 kg between 1975 and 2003 (FAO 2009), particularly in China, where the per capita consumption has increased from 43 kg (1975) to 154 kg (2003). Near the Arctic Circle, due to isolation and unfavorable climate conditions, vegetables make up only a small part of the caloric intake of Aleuts and Eskimos (Bell and Heller 1978; Nobmann et al. 1992). In Africa, the per capita consumption of vegetables lags far behind the worldwide average with less than the Food and Agriculture Organization/World Food Organization (FAO/WFO) recommended minimum uptake of 200 g of vegetables/day (73 kg/year). In contrast, orange, yellow, or white flesh sweet potatoes often comprise up to 90% of the daily diet of Murapin highlanders in Papua New Guinea (Sinnett 1975). Rapid growth in mean per capita incomes in developed countries during the 1990s enabled consumers to purchase a broader range of relatively expensive vegetable commodities, such as off-season produce, relatively new or renewed vegetables, and organic produce. Higher incomes of consumers in developed countries have also raised the demand for other attributes, such as better-quality vegetables and more variety in the daily menu. In developing countries, consumption and domestic vegetable markets are also expanding because of an emerging

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educated middle class with increasing incomes. China, India, and Indonesia, countries with the largest populations, have large emerging middle classes, which impact the demand for high-value vegetables (Senauer and Goetz 2003). Also, a great and growing number of urban consumers—in the markets of Hanoi, for example—are demanding safe vegetables (Figui e 2004). Increasingly, consumers in developed countries are also concerned about the quality and safety of their food as well as the social and the environmental conditions under which it is produced. This trend, in turn, has led to the increased importance of organic vegetables and labeled brands (Reardon et al. 1999; Yussefi and Willer 2003). Most of the total organic arable land for vegetables (213,453 ha) is located in Europe (74%), followed by North America (20%) and Latin America (3%), but represents only 0.04% of the total area of vegetables in the world (Willer and Kilcher 2009). Organic vegetable trade from developing to developed countries is currently growing at over 20% per year (Raynolds 2004) but represents only 3% of total vegetable production (Willer and Kilcher 2009). Desire for year-round availability and increased diversity, and growing health awareness, have also been important reasons for increased consumption of vegetables in developed countries. For example, the dietary benefit of fresh produce is the major reason for the 25% increase in fresh vegetable consumption in the United States during the period from 1977 to 1999 (Regmi and Gehlar 2001). Factors such as increased participation by women in the labor market have created demand for processed, readyto-eat convenience vegetable products, however. A world vegetable survey showed that 392 vegetable crops are cultivated worldwide, representing 70 families and 225 genera (Kays and Dias 1995, 1996); noncultivated species, lower organisms (e.g., fungi), most trees and woody shrubs, and plants grown in or gathered from salt water were excluded. Vegetable crops, of which the leaves or young leafy shoots are consumed, were the most common group of vegetables utilized (53% of the total), followed by vegetable fruits (15%). Belowground crop vegetable organs ranked as follows in frequency of use: roots > tubers > rhizomes > corms > stolons, and together comprised 17% of the total number. In many vegetable crops, more than one part is used. Most of the vegetables are marketed fresh with only a small proportion processed. Of these marketed vegetables, only 67 (17%) have attracted great breeding attention by international seed companies, due to their large area of production and substantial consumption; 52 (13%) were considered minor, and 85 (22%) were considered rare. In Guangxi Province (China), one of the largest vegetable production areas in

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the world, only 33 of the 229 total cultivated vegetable species, in 56 families, were considered major: Chinese cabbage and pak-choy–type vegetables (19.1%), comprising both pekinensis and chinensis groups; leafy vegetables (12.7%) (lettuce, spinach, leafy mustards, and celery); cucurbit vegetables (11.4%) (muskmelon, bitter gourd, cucumber, and pumpkin); root and tuberous vegetables (11.2%) (radishes, carrots, sweet potato, taro, ginger, and Chinese yam); cole crops (10.5%) (head cabbage types, Chinese kale, and broccoli); solanaceous fruit vegetables (9%) (tomato, bell pepper, and eggplant); leguminous vegetables (5.5%) (French or green bean, pea, and mungbean); bulb vegetables (3.9%) (garlic, onion, and leek); and aquatic vegetables (3%) (watercress, water spinach, lotus root and Chinese water chestnut [chufa, Eleocharis dulcis]). B. Vegetable Marketing Vegetables typically are perishable products that are of specific high value and that usually are sold through specialized markets. Currently more than 60% of the vegetables produced in the world are sold by vegetable growers to wholesale dealers or huge supermarket chains. Relatively few growers sell their product at retail prices to consumers in farm markets. Globally the horticultural product markets are still dominated by a large number of wholesalers or middlemen, which means not only that the producers have a lower profit but also the consumer often does not have access to lower-priced vegetables. Globally growers receive only 30% or less of the retail price. This situation is a serious problem for growers. Domestic and international markets for vegetables are changing rapidly all over the world, partially fueled by the spread of supermarkets. Consumers increasingly purchase their vegetables and other foods in large convenience stores such as super- and hypermarkets. The proliferation of supermarkets in developed and developing countries creates both challenges and opportunities for vegetable producers. Indeed, supermarkets may contribute to a higher demand for horticultural products and increase expectations for quality, safety, and presentation while simultaneously excluding small growers from participating in procurement and contracts. The importance of supermarkets is growing especially fast in the Southeast Asia region. Supermarkets are expanding in China 30% to 40% per year, 2–3 times faster than in other developing countries. According to the U.S. Department of Agriculture, China could support at least 9,100 hypermarkets (Bi et al. 2004). In China, growth of modern retailing markets is spreading from the early-growth areas of Guangzhou, Shanghai, and Beijing to inner western regions. This is

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already having a profound effect on the way that vegetables are produced in the country. In Indonesia, Malaysia, and Thailand, supermarkets account for 33% of the market; in Taiwan and the Philippines, the figure is closer to 63% (Weinberger and Lumpkin 2005). In Indonesia, the local retail supermarket chain Ramayana is growing at 25% a year (Manalili and Tumlos 2004). The share of fresh vegetables sold through supermarkets in some countries and in metropolitan areas of Asia such as Malaysia, Bangkok (Thailand), Manila (the Philippines), South Korea, and China is respectively 35%, 30%, 15%, 11%, and 10%, respectively (Shepherd 2005). In Vietnam, where modernization of food retailing is only about 10 years old, and where wet markets are still the major source of food shopping, the supermarket’s share of vegetable products increased from around 0.5% in 2000 to around 40% in 2007. The Metro supermarket chain has announced programs of assistance and consulting for 4,000 growers and suppliers to help them to upgrade the quality, marketability, and competitiveness of their products. Outside of Southeast Asia, supermarket sales in Kenya are growing at 18% a year. They are expected to become the dominant urban food retailers by 2011 (Neven et al. 2006). The growing importance of supermarket outlets has implications of its own regarding methods of procurement and quality standards. Supermarkets in the cities bring quality to the shelves. Vegetables are well packed and presented, providing scope for premium quality as well as novelty items. The difficulties that growers can experience is reflected in fairly rapid declines in the numbers of growers involved, as companies tend to delist suppliers who do not meet expectations in terms of volume, quality, and timely delivery. In Malaysia, for example, the Giant supermarket chain had 200 vegetable suppliers in 2001 but just 30 in 2003. In Thailand, similar changes have been seen following the introduction of a distribution center for the TOPS supermarket chain (Shepherd 2005). Small-scale growers can find it difficult to produce and deliver according to the supermarket chain’s standards (Boselie et al. 2003; Reardon et al. 2003; Weatherspoon and Reardon 2003). In Brazil, the Wal-Mart supermarket chain has about 1,500 associated growers producing vegetables, of which 90% are large producers and only 10% are small producers. The standards of quality, safety, and presentation make it difficult for the small producers to compete. In Portugal and other European countries, the situation is similar. The market is getting more refined in terms of quality and yield expectations, and there is a clear demand for excellent hybrid vegetable cultivars. Success for vegetable growers will depend on their ability to access diverse markets and respond promptly to changes in market conditions. Growers grow

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vegetables for immediate marketing, and their produce is subject to competition to decide the prices. Hence they cannot compromise the quality of the seeds they use for fear of rejection of their produce. They buy the best seeds, mainly hybrids, and frequently try new products to remain successful. The seed companies in turn get instant response and success if they develop new promising hybrids. Price of seeds is a more critical factor in marginal vegetable areas, where capital and spending input regimes are low, but is less important where high yields can be obtained and the grower’s produce can be sold profitably. Providing that the benefits of the hybrid seed are understood, the price of seeds is less important than other factors, such as availability of capital, confidence in the produce market, and ability to buy other inputs, such as fertilizers and pesticides. Worldwide, the vegetable industry is now in a transition period, changing from expanding quantity to increasing quality and efficiency. This is being achieved by the introduction of innovative production technology and improvement of market competitiveness. The grower has more vegetable crop production knowledge and is adopting more convenient management techniques and cultural practices, such as irrigation and fertigation, protected cultivation, and transplant technology as well as the use of better hybrid vegetable cultivars. It is expected that the assurance of ‘‘safe’’ vegetable products will become increasingly important. Food safety legislation in the European Union and in the United States is introducing increasingly stricter standards. In general, vegetable production requires more plant protection products per hectare. Because of their cultivation intensity, vegetables suffer from many biological stresses, including pests, diseases, and weeds. Due to the diversity within and between plant families, their pest loads are far more varied and complex compared to field crops. Considerably fewer resources have been directed at improving their production and pest and disease management options compared to field crops such as rice, wheat, and maize (Lumpkin et al. 2005). Since vegetables are high-value commodities with high ‘‘cosmetic’’ standards, the main method for controlling pests, diseases, and weeds has been the use of pesticides. Vegetables often are consumed in fresh form, so pesticide residue and biological contamination in them is a serious issue. Vegetables account for the major share of the global pesticide market. Almost 25 kg/ha of active pesticide substances are used on average in vegetable production in the European Union (OECD 1997). Vegetable production accounts for less than 1% of the United States crop area but 14% of its total pesticide use (Osteen 2003). In the case of insecticides, nearly 20% of the worldwide US$8.1 billion annual

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insecticide products are applied to vegetables (Krattiger 1997). Insecticides are regularly applied to control a complex of insects that cause damage by feeding directly on the plant or by transmitting pathogens that harm plants. Studies on pesticide levels in vegetables in some developing and developed countries found that the pesticide residues sometimes exceeded tolerance limits (Mansour 2004; Ferreira 2009). For example, in India, a survey of pesticide residues in vegetable crops taken at the farm gate and in markets from 1999 to 2003 confirmed that of the 3,043 samples, two-thirds were found to have pesticide residues. These were within accepted tolerances; 9% contained residues above the minimum recommended levels (Choudhary and Gaur 2009). The increase of residues in vegetables is a major concern to consumers, and there are some concerns about the ability of small growers to meet exacting quality and safety standards of the commercial sector. Pesticide residues often are attributed to the failure of growers to restrain application before harvesting and the use of prohibited pesticides. Residues affect the health of growers and consumers and contaminate the environment Also, the presence of pesticide residues may restrict trade opportunities. In South Asia, pest and disease vectors of eggplant, tomato, and legumes, notably eggplant fruit and shoot borer, cotton bollworm, root knot nematode, white fly, and legume pod-borer, have been identified as the major targets of pesticide use and abuse with frequent and excessive applications of pesticide. However, eggplant fruit and shoot borer pheromone traps and net houses developed by the Asian Vegetable Research and Development Center (AVRDC) have helped reduce pesticide application significantly. The promotion of integrated and biological pest control is expanding worldwide. The use of improved vegetable cultivars with resistance or tolerance to diseases and pests also can contribute to the reduction of pesticide applications and pesticide residues. Transgenic, or genetically modified (GM), vegetables also offer unique opportunities for controlling insects and the pathogens they transmit. They have provided some successes. Growers in the United States have benefited from having GM squash cultivars resistant to zucchini yellow mosaic virus (ZYMV), watermelon mosaic virus (WMV), and cucumber mosaic virus (CMV). These cultivars were deregulated and commercialized in 1996 (Medley 1994; Tricoli et al. 1995; Acord 1996). Also in the United States, Bt sweet corn has proven effective for control of some lepidopterous species and continues to be accepted in the fresh market. In India, genetically transformed Bt eggplant will be introduced by late 2009 by Malarashtra Hybrid Seeds Company Limited (Mahyco) in order to reduce pesticide use. Biotechnology products will be successful if clear advantages and safety are

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demonstrated to consumers. Countries vary in their market standards of acceptance of GM products. Consumers also want more vegetable diversification and a continuous supply. Vegetables are purchased based partly on eye appeal, which means that the development of desire to consume increases market demand. Diversification also tends to increase consumption. Product differentiation, including new or renewed product introductions, is still a key strategy for expanding sales in vegetable markets. For example, the fresh tomato category has been differentiated to more than 10 offerings (beefsteak tomato, Roma-type tomato, vine-ripe tomato, cocktail tomatoes on vine, tiny-plum tomatoes, mini-plum tomatoes, red cherry tomatoes on vine, attractive yellow and orange cherry tomatoes, mini San Marzano–type tomatoes, teardrop or pear-shaped tomatoes, super or premium taste tomatoes). The introduction of specialty fresh baby leaf vegetable salads and fresh-cut products has opened new opportunities for domestic producers. The increased production of baby leaf vegetables in the world is intended to increase desire among elite consumers and is also an excellent way of supplying micronutrients. For example, baby leaf curly kale, as well as other dark green leafy vegetables, is a rich source of lutein and zeaxanthin carotenoids and, when cooked, contains seven times as much vitamin A as cooked broccoli and provides more calcium per 100 g than milk, yogurt, cooked broccoli, or cooked spinach. Until recently, kale was not a particularly popular vegetable in Europe (except Portugal), but as a baby leaf vegetable with 5 leaves, it is now accepted by many European consumers. An example of diversification in peas is the pea shoot. It is a nutritious leaf vegetable with high levels of vitamin C, folic acid, and vitamin A. To exploit such opportunities, it is important to continue research and to disseminate information regarding the benefits of vegetables, develop new improved vegetable cultivars and processed products, evaluate the economic opportunities and the market scope of these new products, and identify marketing trends and alternatives. Increasingly consumers also demand processed, ready-to-eat vegetables in developed countries as well as developing countries. The growth rate in retail sales of processed vegetables in several Asian countries over the past years has been tremendous. For example, the total percentage of retail sales of processed vegetables during the period from 2001 to 2005 in Vietnam, China, Indonesia, South Korea, Thailand, India, and the Philippines was 11.2%, 9.6%, 7.8%, 6.8%, 6.1%, 6.0%, and 5.1% respectively (FAO 2007). The food processing industry has also improved dramatically over the past 30 years in transportation technology, sanitation packaging, storage, and product and packaging appeal. Taken together, these changes have

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sharply increased the value-added share of vegetable processing and distribution, opening up new marketing opportunities. C. Vegetable Migration The introduction and trade of crops and seeds from one region to another has been continuous throughout history due to the migration of people, conquests, discoveries, and development of commercial trade routes. The ‘‘Silk Route,’’ for example, was responsible for many plant exchanges and trades between the West and China. As people immigrated to new countries, they carried not only languages, religions, and traditional customs but foods and seeds. Vegetables, in particular, are attractive candidates for introduction into a new environment, as most vegetables tend to be fairly short-season crops and, as a consequence, lend themselves for cultivation in many diverse areas. Thus, the tropical tomato has been transformed to a temperate annual. Superior vegetable crops and cultivars often are assimilated into the cuisine of the indigenous populations. Worldwide there has been, and continues to be, substantial emigration of peoples from a diverse range of countries. In western Europe and in the United States, immigration has had a pronounced effect on the vegetables consumed. For example, in the 1960s, southern Europeans immigrated to work in the northwestern European industrial zone, bringing with them their distinct consumer behavior patterns and vegetable preferences. They were responsible for the introduction of broccoli, eggplant, pepper, and fennel (Smith 1989). Portuguese immigrants carried tree kales and tronchuda cabbages to Brazil in the 16th century and to France and Germany in the 1960s. The introduction and popularity of a small number of new vegetable crops in some countries have resulted in a steady increase in their utilization. For example, Chinese cabbage and pak-choy were novelties in much of the United States in the 1970s but are now widely consumed due to the immigration of Asiatic people. Likewise, since the 1970s, sweet corn consumption has increased in several European countries. Nowadays many cities in the United States are excellent examples of increasing ethnic diversity and its impact on the vegetables available. Ethnic markets sprang up followed by shopping malls catering to Chinese, Korean, Vietnamese, or Hispanic populations. Vegetables such as Thai eggplant, tindora, parval, cactus leaves, and others are now readily found in ethnic American markets. New vegetables also have moved into traditional markets, as superior crops are accepted by the general public, greatly enriching the diversity of vegetables grown and consumed.

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As a result of the increased frequency of world travel, a substantially greater variety of vegetable crops is available worldwide in many local markets. However, the number of new crops that become mainstream vegetables remains relatively small. Examples are rocket (Eruca vesicaria syn. E. sativa), lamb’s lettuce (Valeriana olitoria), physalis (Physalis peruviana, P. pruinosa, P. ixocarpa), and pepino (Solanum muricatum). The number of crops that can be utilized within a local area will depend on the ethnic diversity of the location, affluence of the population, production and marketing constraints, and other factors. Globalization also has now become increasingly important among vegetable producers, as indicated by the recent massive increases in international trade of fresh and processed horticultural crops. Worldwide the total volume traded in vegetables has increased fivefold from 1965 to 2005 (FAO 2007). The European Union (EU), followed by North America and Japan, is the world’s most important fresh vegetable import region. A large share of these imports comes from Africa, Latin America, and the Caribbean and from China, each contributing roughly one-tenth to overall vegetable exports. While more than half of global vegetable production takes place in Asia and in the Pacific, only about 12% of the total value of exported horticultural products is generated there (FAO 2007). Because of the large share that Asia and the Pacific has in global population, domestic markets and their requirements are likely to continue to be more important in the near future than the attraction of export markets. In Asia, China, Thailand, and the Philippines are the largest net exporters of vegetables, while Singapore, the Republic of Korea, and Malaysia are the largest net importers of vegetables (FAO 2007). In sub-Saharan Africa, Kenya and Coˆte d’Ivoire are two of the most important vegetable producers for export. Many other agricultural commodities face stagnation and declining world prices, but vegetable exports have grown dramatically. In many African countries, export of fresh vegetables has become a bright spot in an otherwise dim agrarian landscape (Dolan et al. 1999; Dolan and Humphrey 2000). There are several reasons for the rapid increase in vegetable trade over the past decade. In the 1990s, many countries, particularly in Africa and Latin America, changed their trade policies from protectionist to more openness, economic liberalization, and export diversification. This opened the way for several new trade agreements that facilitated trade in high-value agricultural products, such as vegetables. For example, liberalization under the North American Free Trade Agreement (NAFTA) underpinned the rapid growth in United States of agricultural imports from Canada and Mexico (Putnam and Allshouse 2001). In the European Union, horticultural products also have

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benefited from preferential trade access. For instance, imports of fresh vegetables into the European Union rose by 150% between 1989 and 1997, with almost three-quarters of the value of these exports coming from sub-Saharan Africa. The majority of exports come from countries with preferred trade status under the Lom e Convention. For instance, in 2000, Kenya was the single largest supplier of green beans to the European Union, followed by Ethiopia, South Africa, and Switzerland. Kenya captured 53% of the total traded value. Coˆte d’Ivoire was the second largest supplier of green onions and shallots to the European Union, after New Zealand, capturing 16% of the total traded value (FAO 2005). The increase in total volume of vegetables traded worldwide has been dramatic. Still, compared to overall exports of agricultural products, the importance of vegetable exports remains minor, comprising less than 10% of the total value. However, in recent years, the share has been rising and is projected to continue to rise faster than other agricultural products. During the 1990s, the value of fresh and processed vegetables imported by the European Union surpassed all other categories (Dolan et al. 1999). Growth in these commodities is also linked to changing trends in consumer preference and food retailing. In this situation, many vegetable growers are eager to produce value-added horticultural crops as compared to field crops and to obtain higher yields of high-quality products. International supermarket chains and large processors are becoming the main buyers of exported fresh vegetables (Reardon et al. 2003), and small-scale growers worldwide need to be trained and organized to meet the challenge of supplying these international players. The major constraints against the participation of small-scale growers in international vegetable exports are the increasing attention that food quality and safety are receiving in food trade and an expansion in the number of nontariff measures that developed countries apply to horticultural products (Henson and Loader 2001; Dinham 2003; Henson et al. 2005). Vegetables belong to the class of food items most frequently affected by sanitary and phytosanitary measures (Unnevehr 2000). Sanitary issues refer to ensuring a safe food supply for consumers, while phytosanitary issues concern the protection of domestic crops from imported pests and diseases. The Sanitary and Phytosanitary Agreement (SPS) of the World Trade Organization specifies that countries can pursue their own levels of food safety standards. However, SPS issues sometimes are used as a protectionist tool against imports since multilateral trade agreements have reduced the ability to protect domestic production with tariffs and quotas (Cerrex 2003; Henson and Loader 2001). SPS regulations may be the most important barrier to international trade in fresh vegetables

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(Unnevehr 2000). Thus exporters from less developed countries must be provided training opportunities and information access on how to produce and supply safe products to developed countries. Traceability, phytosanitary, infrastructure, and productivity issues will continue to be a barrier for participation in the vegetable trade for most of the developing world. Application of agricultural chemicals is often poorly regulated, and industrial pollutants are common hazards in the soil, water, and air of developing countries. In the future, the inability of these countries to meet increasingly strict phytosanitary and traceability requirements for food products will constrict exports to developed countries. Small-scale growers and processors in developing countries thus will have to learn to supply safe products with traceability labels, if their participation in global trade is to continue and expand. Technologies for safe and environmentally friendly vegetable production as well as capacity building should therefore gain particular attention for training to enable small-scale growers to participate in vegetable production for international markets. Horticultural production, particularly in Mediterranean, subtropical, and hot-wet tropical environments, is severely constrained by postharvest losses, which have been estimated as 15%, depending on the crop and season (Kader 2003; Genova et al. 2006a, 2006b). Vegetables often are highly perishable, restricting the ability of producers to store them to cope with price fluctuations. Reducing postharvest losses would make diversification into vegetable production less risky and more attractive. Postharvest-related quality losses also reduce opportunities for export and export revenues. Improved vegetable cultivars subject to fewer postharvest losses can help improve this situation. Vietnam, for instance, has experienced declining export revenues for vegetables in the last decade. This decline was attributed to low quality of export goods, due to poor storage and postharvest technologies (Anon. 2004; Genova et al. 2006c). Competitive participation in international markets requires relatively sophisticated marketing, information, and transportation networks as well as improved cultivars, quality control, product standardization, and, for some future markets, traceability. Other improvements required include pre- and postharvest processing technologies as well as market information systems that include prices, information on seasonality, constraints in postharvest handling, and technological opportunities. D. Processed Vegetables As most vegetables are perishable by nature, consumption shortly after harvest is the best guarantee for optimal quality. However, in most cases

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there is a time lag between harvest and consumption. Climate-controlled storage and distribution often is required to prolong the quality of fresh vegetables, even though in the end deterioration is inevitable. Processing of vegetables provides a firmer answer to increasing shelf life and involves methods such as drying, canning, salting, pickling, sweetening, and freezing. Processing usually enables vegetables to be stored for longer periods and distributed over longer distances without the need for expensive climate-controlled conditions. Processing is also necessary to provide consumers with seasonal vegetables year-round; at the same time, processed vegetables provide convenience in the kitchen of the consumer or professional foodservice companies. Also, the food processing industry is a major purchaser of partly processed vegetables that are used for further processing into end consumer products. Worldwide the three most important processed vegetables, in terms of production, are tomato, sweet corn, and snap bean. The three most valuable are tomato, sweet corn, and cucumber for pickles, accounting for almost 80% of the total value when combined. More of the tomato crop is processed than any other vegetable type. Almost one-quarter of the global fresh tomato production (estimated at 125 million t) is processed into concentrates, pastes, and juices, or is diced or peeled, and much is used in the food industry as an ingredient of soups, sauces, and pizzas. The United States, in particular the state of California, is the world’s largest producer of processed tomato products accounting for 35% of global production. In the United States, the two most important vegetables for processing are tomato and sweet corn, for all three of the production categories: area harvested, total production, and value. The rapid emergence of China in this industry within a short time span is striking. Over the past seven years, China has increased its global production share from 4% to 16% and is now a serious rival to exporters from the United States and Europe. A wide range of vegetables (e.g., beans, peas, carrot, cauliflower, sweet corn, spinach) is preserved by deep freezing. The United States leads the world with over 2.2 million t for seven commodities (sweet corn, green beans, peas, spinach, broccoli, and asparagus) in 2008 (NASS 2009). Belgium was second with a production of 900,000 t. Production is surprisingly concentrated in a small area in west Flanders and processed by a dozen mostly family-owned companies. The majority of this Belgian frozen vegetable production is exported. Including reexports, Belgium accounts for 27% of global exports (OEITFL 2009). Canned vegetables, packed in cans, glass, or cartons, and including tomato, beans, peas, sweet corn, asparagus, cabbage, carrot, artichoke,

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and a few others, are another major part of the vegetable processing industry. China is the major producer and exporter of canned vegetables. United States is the other great producer, and France and the Netherlands are the leading players in Europe. Each vegetable type has its specific leading producers and exporters. For instance, the export of canned sweet corn is dominated by the United States, France, Thailand, and Hungary. Precut, fresh vegetables (lightly processed) are usually not included in the definition of processed vegetables, but the procedure is an important one. It requires appropriate packaging, such as plastic wrap, which is important to keep the cut vegetables fresh. The United States leads in the light processing industry, with estimated sales of US$13 billion in 2008 in both retail and foodservice (including minor sales of precut fruits), which is double the amount in 2000. Brands play an important role in this segment. For example, the brands of Dole and Fresh Express together accounted for 88% of the U.S. packaged salad market in 2004. Vitacress is Europe’s leading grower and packer of watercress, rocket, baby leaf salads, and other specialty vegetables. There are major differences in requirements for growers supplying either fresh market or processing facilities. The processing industry requires a guarantee for large volumes of vegetables, so contracts tend to be established with growers on volumes to be delivered usually with minimum prices set. For processing purposes, specific cultivars of vegetables may be required, and seeds often are supplied by the processing industry to its contracted growers. It is clear that in these cases, the main focus of growers is aimed at achieving crops that meet the contract demands in terms of quantity, quality, and time of delivery. The processing industry comprises a wide range of activities in the transformation and preservation of vegetables. Basically, B2B (businessto-business) activities, where processed vegetables are ingredients for other vegetable and food processing industries, can be distinguished from the production of end consumer products, although individual companies may undertake both activities. End consumer product manufacturers include the very large food multinationals, such as Unilever, Heinz, Del Monte and Bonduelle, as well as smaller specialized companies, like the Belgian frozen vegetable companies or U.S. tomato processors. For the large food multinationals, vegetables are part of a wide range of food products, except for the French-based Bonduelle, which is a dedicated vegetable company. These companies focus mainly on the marketing of their products, so they can be considered ‘‘brand

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managers.’’ The actual processing of vegetables can be done in-house, but outsourcing to contract processors is common practice as well. Increased food retail power of super- and hypermarkets puts pressure on branded vegetable companies in two ways: growth of private labels at the expense of branded products and the reduction of the number of brands on the supermarket shelf. Production under private labels is a much-discussed topic among branded vegetable processors, which often find a range of private labels alongside their branded products. The smaller brand producers in particular have a tough fight on their hands competing with the major brands for supermarket shelf space. Vegetable processors producing either private labels or working as subcontractors or product suppliers are strongly driven by cost/price ratios. Optimization of the processing process is a key priority, with a strong focus on maximizing factory capacity and a continuous supply of raw material. Packaging material, such as metal for cans or glass for jars, is a major expense. When the cost of these materials rise (due to rising energy costs and high demand), the impact on processors may be significant. Producers of processed vegetables with a long shelf life, in particular the canning industry, are encountering stagnating consumption in highincome regions. (Canned vegetables traditionally are eaten by older and/or poorer consumers.) As a result, processors and others try to leverage the increasing consumer preference for vegetables that are freshly processed, including precut, cleaned, prepacked or in the form of meal components ready to heat or eat. Examples are the diversification into fresh vegetables by French processor Bonduelle or the major acquisition of U.S.-based fresh salad producer Fresh Express by Chiquita. Despite low-volume growth levels, frozen vegetable products appeal to consumers, as their nutritional values are close to those of fresh vegetables, and they can be stored for fairly long periods. Mixtures, meal components, or complete meals seem to offer better growth prospects than basic frozen vegetables in terms of value. Processed vegetables— fresh, canned, or frozen—are also widely used in the foodservice industry, which takes an increasing share of the consumer’s food dollar. Convenience and health products are still largely unexplored for most processors. New processing and packaging technology enables fresh vegetables to be presented in a convenient format (e.g., increasing the shelf life of fresh precut vegetables) while maintaining aspects of health, safety, and freshness. For example, juices are common products within the fruit sector but are still in their infancy within the vegetable sector. Providing customers with innovative combinations of products and services is key to the processing industry’s search for value-added activities.

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III. VEGETABLE PRODUCTION STRATEGIES A. Social Importance Commercial vegetable production is a high-input and labor-intensive activity that needs a large labor force from cultivation to processing. Furthermore, many vegetable crops require careful monitoring of plant health and careful attention to weed control, irrigation, fertilization, harvest timing, and handling. Since many of these activities cannot be mechanized, there may be very limited economies of scale in production of crops that require high labor inputs. The production of vegetables offers opportunities for poverty reduction and for overcoming food insecurity by creating and providing employment, because it is more labor intensive than the production of field crops. Vegetable production often requires two to four times as much labor as the production of cereal crops. In Kenya, the production of snow peas and French beans, the two most widely grown export vegetables, require 600 and 500 labor days per ha, respectively (Dolan 2002). In Mexico, the vegetable sector accounts for more than 20% of the total labor days within the agricultural sector. Shifting from cereal production to vegetable production therefore generates additional employment opportunities, which generates greater incomes for poor households. Diversification of vegetable production can affect both the structure and the level of employment. But where labor is scarce, availability of hired labor actually may be a limiting factor to vegetable production. Labor demands also arise in the postharvest sector, since transport, packing, sorting, grading, and cleaning are all labor-intensive activities (Weinberger and Genova 2005). Generally, the vegetable export industry also generates substantial employment. Many work tasks, such as chopping, washing, labeling, and bar coding, are increasingly being transferred to developing countries and are providing many new jobs, particularly among the unskilled segment of the workforce. Usually employment increases are on farms owned by the major exporters and on independent large farms producing for these exporters under contract (Maertens 2006). Often these workers are landless women who have few other opportunities for earning an income (McCulloch and Ota 2002). Women in particular have been able to capitalize on these new labor market opportunities. The increasing feminization of vegetable production worldwide is well documented. In a study conducted by Gill (2001) in Punjab (India) during the mid-1990s, around three-quarters of all workers in the vegetable production sector were hired labor, and female hired labor accounted for 49%. Further, female labor in vegetable

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production accounted for 58% of total labor hours. In tomato production alone, female labor accounted for almost 60% of the total labor hours. In Africa, Asia, and Latin America, high-value vegetable exports are female- intensive industries, with women dominating most aspects of production and processing. In Chile, Ecuador, Guatemala, Kenya, Mexico, South Africa, and Zimbabwe, evidence suggests that women occupy at least 50% or more of the employment in these export vegetable industries (Dolan and Sorby 2003). Men move out of the sector more quickly than women, and women are becoming the preferred labor type for many employers (Gill 2001; Singh 2003). Production of vegetables is attractive for many poor farmers worldwide because it is profitable. Studies show that growers engaged in the production of vegetables often earn higher net farm incomes than farmers who are engaged in the production of cereal crops only, even though vegetable production is greater risk and requires better, more innovative management. A study in Kenya that sampled small-holder vegetable growers who produce for export found that net farm incomes were five times higher per family member compared to small holders who did not grow vegetable products (McCulloch and Ota 2002). Lumpkin et al. (2005) has shown that the per capita farm income of vegetable growers is up to five times higher than for cereal producers in Asia. Maertens (2006) estimates that households participating in export vegetable production in Senegal earn incomes that are 120% higher than the average household income in the region. The relative profitability of vegetable crops compared to cereals has been shown to be a determining factor for crop diversification into vegetable production in India (Joshi et al. 2003). Other studies from Southeast and South Asia also frequently show higher average net farm incomes per household member among vegetable producers. In these countries, vegetable production is most profitable compared to rice production in terms of cropping days, since the growing period for vegetables is usually shorter than for rice. Profitability is also high in terms of cropped area. Thus, the production of vegetables has a comparative advantage under conditions where arable land is scarce and labor abundant, the typical situation in many countries of South and Southeast Asia. Here, diversification into vegetables may be a more viable strategy than in regions where labor and access to inputs are the limiting factors. In these areas, the average size of landholding is among the lowest in the world, and transportation infrastructure has seen dramatic improvements. The production of vegetable crops is generally more knowledge and capital intense than cereal crops and is often riskier, because these crops are much more costly to produce per hectare than traditional crops and

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because yields and prices are more variable than for field crops (Key and Runsten 1999). Small-scale and poor growers thus must be supported by an enabling institutional environment, such as access to credit and capital, and must be provided with access to market price information. Still, vegetable production is usually more lucrative compared to field crops. Vegetables have high value-added and income-generation potential, and due to a relatively smaller scale, compared to grain production and livestock, their production is a desirable alternate. Vegetable production activity is booming in many regions and countries with favorable mild climates. The development of rural markets is considered to be a prerequisite for poor agrarian regions. Growth in vegetable production can contribute to the development of supporting and secondary industries through several means. Due to the high perishability of vegetables, their growers usually sell a share of the crop. The cash income thus earned can spur the demand for inputs and services. Market integration of producers of vegetables has been shown to be higher than that of field crop producers. For instance, in Bangladesh, farmers on average sell 96% of their vegetable products but only 19% of their cereal output (Weinberger and Genova 2005). The same pattern is reported for other countries in Southeast Asia and East Africa. This phenomenon is consistent across income groups, although wealthier farmers usually sell a larger share of their production. Minot (2002) found that vegetable production in Vietnam is highly commercialized, with about 70% of vegetable farmers selling their output. Minot compared the degree of commercialization for wealthy and poor growers. The market integration of the highest-income growers is higher at 75%, while the lowest-income growers sell only 56% of their output to the market. In a study performed in Tanzania by Weinberger and Msuya (2004), there is an interesting difference between selling exotic and traditional landrace vegetables. While 95% of the lowest-income growers sell at least some of their produce, compared to 100% of the highest-income farmers, the pattern is reversed for traditional vegetables. Here, 95% of poor growers sell their output, while only 87% of growers in the highest income bracket sell their output of traditional landrace vegetables. Since vegetable producers usually are better integrated into markets than field crop producers, the production of vegetable crops contributes to commercialization of the entire rural economy, which is characterized by increased trade and marketing. Studies show that commercialization of vegetables stimulates and benefits the rural economy and contributes to the growth and development process through generation of employment and increasing agricultural productivity (Pingali and Rosegrant 1995; Braun 1995).

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B. Multiple Cropping, Urban Production Some activities, such as seeding, transplanting, and greenhouse production, are quite specific to commercial vegetable production, and they also have influence in the acquisition of improved vegetable seed. Throughout the world, in recent years there has been a rapid growth of the transplant industries, and more and more vegetable growers are purchasing nursery-grown vegetable transplants rather than sowing seeds or growing transplants for their own uses (Cantliffe 2009). This industry requires improved cultivars and high-quality seed. Thus, in the developed countries and even in some developing countries, seed companies are selling more and more seeds to transplant growers rather than to individual growers. International trade of vegetable transplants, mostly grafted ones, has been steadily increasing over the world (Kubota 2008; Lee 2008). For example, the United States imports grafted seedlings produced in Mexico or Canada; north European countries import grafted and/or plug vegetable seedlings from southern European countries or Morocco; and Korea exports grafted transplants to Japan. Use of improved and healthy seeds is vital for grafting. For this reason, dry heat treatment has been used extensively to eradicate seed-borne diseases, mainly in cucurbits and solanaceous crops. Protected cultivation of fresh and high-quality vegetables has increased in many vegetable-producing regions in temperate-zone countries to satisfy the strong need for a year-round supply of fresh vegetables. The yield may be doubled or even tripled by growing vegetables under protected environments as compared to field growing. Since both costs and returns of protected cultivation are higher, growers always use improved and high-quality seeds and usually buy their seedlings and transplants (or grafted seedlings) from certified seedling/transplant nursery growers. The market prices of certain vegetables show large changes depending on the time of the year, thus making protected cultivation more and more attractive to growers with small farms. The expansion of protected cultivation areas and improved productivity associated with open-air production of vegetables has led to year-round availability of fresh vegetables in the market at reasonable prices. There is no longer a vegetable ‘‘off’’ season. Multiple cropping of vegetables is also very common in the major vegetable production areas. Triple and even quadruple cropping per year is routinely practiced in Mediterranean and mid-subtropical regions. In some parts of Asia in the paddy rice fields, short-season vegetables or overwintering vegetables are grown prior to or after rice cultivation. In these situations, growers also preferred intact or grafted maximum-size

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healthy transplants, and they are buying more and more from certified seedling/transplant growers. Crop production in urban and periurban environments is also important and attractive, especially for highly perishable and delicate products, such as vegetables. Estimates indicate that about 800 million growers are actively engaged in urban vegetable production and that about 200 million provide food for the market (Smit et al. 1996; FAO 1999). These urban growers produce 15% to 20% of the world’s vegetables for urban consumers (Armar-Klemesu et al. 2000). Periurban production of vegetables, in close proximity to urban centers, is also important for nourishing city populations. Generally, urban and periurban vegetable production plays an important role for household food security mainly in huge cities of developed and developing countries. For some people, urban agriculture is a strategy adopted by households whose monetary incomes are not adequate to purchase sufficient food. For others, mainly in developed countries, it is a way of enjoying horticultural activities, clean air, and home-grown vegetables. Urban and periurban areas are the origin of 24% of fruit-type vegetables, 50% of crucifers, and up to 100% of leafy vegetables in Hanoi (An et al. 2003) and of 60% of total vegetable production in Shanghai (Cai and Zhangen 2000) and Dakar, Senegal (Mbaye and Moustier 2000). In Havana, urban agriculture has emerged as a response to the food crisis. Over 26,000 small gardens cover 2,439 ha in Havana producing 25,000 t of food each year (Novo and Murphy 2000). Another aspect of periurban production is often overlooked, particularly for low-input or organic production. Urban and periurban vegetable production faces opposition from local authorities in many places. The main reason is the various risks associated with production close to urban centers, which include contamination of crops with pathogenic organisms through irrigation with polluted water, inadequately treated wastewater, or organic solid wastes; phytotoxins and human diseases associated with unsanitary postharvest activities; contamination of crops and/or drinking water by residues of agrochemicals; and contamination of crops by uptake of heavy metals from contaminated soils, air, or water (Lock and Veenhuizen 2001; Alam et al. 2003; Midmore and Jansen 2003; Mansour 2004). Recently there has been a boom in organic horticulture in the developed countries. Most of the total arable land for organically grown vegetables (213,453 ha) is located in Europe (74%) followed by North America (20%) and Latin America (3%) (Willer and Kilcher 2009). Although organic producers are limited in number, they are a new market for seed companies. Usually the organic growers use landraces

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or open-pollinated cultivars that are less expensive and sometimes have unique quality factors; tomatoes are often better tasting yet have poor storage life. Furthermore hybrids are considered antithetical to the organic ethos. The advantages of using open-pollinated cultivars are that seeds are cheap, can be produced on farm, and often provide reliable performance with little inputs from the growers. However, some of the open-pollinated cultivars do have a number of disadvantages, including poor uniformity and low yields. The promotion of organic horticulture could provide a niche for small-scale growers provided they achieve consistent production.

IV. VEGETABLE BREEDING The impact of vegetable breeding on vegetable production is dependent on the complex relationships involving the growers, the cultivars available to them, and the developers of those cultivars. Vegetable growers consist of commercial producers with varying size landholdings ranging from moderately small farms to very large ones, and subsistence farmers with small farms often on marginal lands. The subsistence farmers are usually also poor. Several types of cultivars are available. The least sophisticated in terms of the method of development are landraces, also known as local varieties. Modern cultivars consist of those developed by crossing and selection alone, those developed by crossing and selection but with specific important improvements often obtained from crosses with wild species or by transgenic methods, and F1 hybrids between desirable inbred lines. The developers of landraces are usually the farmers themselves, and are obtained by repeated simple selection procedures generation after generation. Improved cultivars and hybrids are created either by public sector breeders or seed companies. The consequences of these relationships may be quite profound for the growers at each level, the seed producers, the availability of food worldwide, and the future of crop diversity and sustainability. Therefore, it is worthwhile to examine vegetable breeding and its connections to assess our future expectations. A. Objectives Vegetable breeding has to address and satisfy the needs of both the consumer and the grower. The general objectives for growers are good yield, disease and pest resistance, uniformity, and abiotic stress resistance. Objectives for consumers are quality, appearance, shelf life, taste,

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and nutritional value. Quality in vegetable crops, in contrast to field crops, is often more important than yield. For growers to survive, cultivars must be accepted by the market. Thus, color, appearance, taste, and shape are usually more important than productivity. For example, tomatoes to be used either fresh or in processing must have distinct quality characteristics. Fresh tomatoes must have acceptable flavor, color, texture, and other taste parameters to satisfy consumer demands and handling requirements. Processing tomatoes, however, must have intrinsic rheological characteristics that make them suitable for various processing applications, such as juice, ketchup, or sauce production. Traditional breeding requires the selection of a tomato genotype or a related wild species that has a desirable trait, such as early ripening or disease resistance, and crossing it with another tomato cultivar that has a good genetic background. The desired result is an earlier-ripening tomato that makes it to the market sooner or cultivars that resist pathogen attack. In this way, several thousands of tomato cultivars have been developed over the years. The final goal of vegetable breeding programs is to release new cultivars having elite combinations of many desirable horticultural characteristics. Plant breeding for improved taste, convenience, and consumer appeal has already contributed to increased per capita vegetable consumption with the development of products such as baby carrots, yellow and orange peppers, cherry and pear tomatoes, nonbitter cucumbers, mild-tasting eggplants, seedless watermelons, and lettuces with different colors, textures, and flavors for baby leaf and precut salads. Other important objectives of vegetable breeding are disease and pest resistances. Since the early days of the 20th century, traditional breeding for disease resistance in vegetables has been a major method for controlling plant diseases. Cultivars that are resistant or tolerant to one or a few specific pathogens are already available for many vegetable crops. Resistant hybrids with multiple resistances to several pathogens exist and currently are used in vegetable production. For example, in tomato, the genetic control of pathogens is a very useful practice, and most resistances are monogenic and dominant. So far, tomato breeding has resulted in cultivars with resistance to at least 15 pathogens, although with varying stability and level of expression (Grube et al. 2000). Tomato cultivars with some resistance to fungi or oomycetes (Alternaria alternata f. sp. lycopersici, Cladosporium fulvum, Fusarium oxysporum f. sp. lycopersici, Fusarium oxysporum f. sp. radicis-lycopersici, Phytophthora infestans, Pyrenochaeta lycopersici, Verticillium dahliae), bacteria (Corynebacterium michiganense, Pseudomonas solanacearum, Pseudomonas syringae pv. tomato), virus (beet curly top

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hybrigeminivirus, tomato mottle bigeminivirus, tomato spotted wilt tospovirus, and several variants of the tomato yellow leaf curl bigeminivirus), and nematodes (Meloidogyne spp.) are available (Lacerrot, 1996). Many open-pollinated cultivars of tomato currently cultivated possess genetic resistance to 3 or 4 pathogens. With the increasing use of F1 hybrids it is possible to use cultivars combining from 4 up to 6 resistances (Grube et al. 2000). Pest resistance is essential in vegetable production but is marginal in vegetable breeding research. Few vegetable cultivars are resistant to insects. Resistance may be unstable due to genetic variants of the insect that are able to overcome that source of resistance. Depending on the complexity of the interaction between the pest and the vegetable plant, plant resistance may break down rapidly or be long-lived. Insects, including aphids, whiteflies, thrips, and leafhoppers, are also very important in vegetables because they vector many viruses. Viruses can substantially reduce production and quality and are becoming increasingly problematic worldwide due to the absence of virus-resistant germplasm for many important vegetable crops. Aphid-vectored viruses are particularly problematic because many are transmitted in a noncirculative and nonpersistent manner (Zitter et al. 1996; Gonsalves 1998). This means that a very short time—that is, a few seconds or minutes—is sufficient for aphids to acquire virus particles when probing on infected plants. A similarly short time period is enough for aphids to release virus particles when probing on healthy plants. The primary injury caused by aphid-vectored viruses arises not from direct feeding damage by the aphids but from their ability to allow the virus to enter the plant and initiate the disease. A successful application of biotechnology has been the development of vegetable cultivars that resist insect-transmitted viruses as well as cultivars that directly resist insect feeding or development. Bt potato cultivars expressing resistance to Colorado potato beetle (Leptinotarsa decemlineata) and aphids associated with potato virus Y and potato leaf roll virus were approved for sale in the United States in 1995. These cultivars were marketed and sold under the trade names NewLeaf , NewLeafY, and NewLeafPlus (Thomas et al. 1997), until potato processors, concerned about consumer resistance and loss of market share in Europe and Japan, suspended contracts for Bt potatoes with growers in 2000 (Grafius and Douches 2008). Because of this consumer concern, Bt potato cultivars were taken off the market in 2000. Two other Bt vegetable crops are under development: Bt eggplant targeted for control of eggplant fruit and shoot borer, and Bt crucifer vegetables targeted against diamondback moth.

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The economic return of investment in breeding for disease and pest resistance may be low because it is dispersed among many different vegetable crop types. Also, resistant cultivars compete directly with nonresistant ones that may still be used by growers with minimum problems. Therefore, disease resistance is most important when the disease is a limiting factor in production and is especially important for many virus diseases. The high interest in and the increasing present demand for breeding for disease and pest resistance is related to a generalized interest in releasing ‘‘environmentally friendly’’ vegetable cultivars requiring little or no use of pesticides. Breeding for postharvest traits, mainly transport quality, shelf life, and cosmetic exigencies, is of increasing importance in vegetables. For example, in tomato, textural properties of fruits are important contributors to the overall quality for the fresh market and to the properties of products processed from tomatoes (Barrett et al. 1998). Because cell wall disassembly in ripening fruit contributes to fruit texture, modification of cell wall proteins and enzymatic activity during ripening can impact cell wall polysaccharide metabolism and influence texture. Lettuce and other leafy vegetables used for salads deteriorate rapidly following harvest, requiring a considerable investment of effort to maintain quality and shelf life of cut material. Harvesting increases respiration, stimulating deterioration, with increases in the synthesis of phenylalanine ammonia lyase and phenolic compounds, such as chlorogenic acid, which cause tissue browning (Kang and Saltveit 2003). Consequently, delaying leaf senescence is an important target for breeding of leafy vegetables. Also in lettuce, breeding efforts have targeted tipburn, marginal browning, and rib discoloration, which detract from overall appearance (Ryder 1999). Vegetable products with good transport quality, better shelf life, and good appearance will be preferred by traders and also by consumers. Since vegetables are rich in vitamins, minerals, and other micronutrients and therefore are vital for health, breeding objectives should include improving their nutritional value. Historically vegetable breeders have applied selection pressure to traits related to agronomic performance, particularly yield and quality, because these are the traits important to the producer. Rarely have growers been paid for nutritional factors, so there have not been economic incentives to pay much attention to these traits. However, consumers are becoming more aware of these traits. Vegetable breeding for nutritional quality was not mentioned as a primary goal in plant breeding textbooks through the mid-20th century (e.g., Hayes and Garber 1927; Allard 1960; Fehr 1987). However,

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vegetable breeding efforts targeting improved micronutrient content and composition had begun in the 1940s and 1950s with research describing the inheritance and development of tomato breeding stocks and lines high in provitamin A carotenoids and vitamin C (Lincoln et al. 1943; Lincoln and Porter 1950; Tomes et al. 1953). Lincoln et al. (1943) noted a fourfold variation in vitamin C among commercial cultivars and up to 1,194 ppm in red-fruited tomato interspecific crosses with Solanum pimpinellifolium. Similar research leading to the development of darker orange, and consequently high provitamin A, carrots began in the 1970s (Gabelman and Peters 1979). Yellow core color occurs only in older openpollinated carrot cultivars since uniform orange storage root color has been a trait of interest in carrot for over a century (Simon 2000). Similar studies were made in squash, where rapid gains in carotenoid content have been made with phenotypic selection for orange color versus green and cream (Sudhakar et al. 2002). Genetic improvement to increase levels of specific micronutrients has been pursued in several other vegetables, such as melon, spinach, sweetpotato, potato, lettuce, broccoli, pepper, watermelon, collard, kale, peas, and bean. This field of study is relatively new, and also complex because of mineral interactions with each other and numerous other compounds in the soil and in the plant (Frossard et al. 2000). There is usually a large environmental effect, when the component is present in tiny amounts, such as for some micronutrients and phytochemicals. Success in vegetable breeding for higher vitamin and mineral content must consider not only substance concentration but also organic components in plants that can be abundant and either reduce or increase bioavailability (Frossard et al. 2000). With these numerous considerations, breeding vegetable plants for improved nutritional value is a complicated goal that needs expertise in many disciplines, such as plant breeding, nutrition, and soil science. When a vegetable compound (micronutrient or phytochemical) is found to be important for human health, and growers, vegetable markets, and seed companies can capitalize on the value of the compound, there may be an opportunity for vegetable breeders to increase the amount of this compound. Breeders can be successful in reaching this goal if the vegetable crop contains genetic variability for the compound, if selection is effective without detrimental pleiotropic effects, and if there is an easy method to measure the compound. Enhanced nutritional content would add value for poor, malnourished populations. Breeding for provitamin A carotenoids, iron, and zinc is of keen interest as a strategy to alleviate nutrient deficiencies in developing countries (Khush 2002; King 2002; Carvalho et al. 2006;

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Graham et al. 2007; Hotz and McClafferty 2007). An example is the ‘‘golden tomato,’’ which contains three to six times more provitamin A carotenoids than standard tomatoes. Developed at AVRDC with conventional breeding techniques, these improved nutritionally rich tomato lines could help prevent many children of developing countries from going blind, since vegetarians and populations with limited access to animal products depend on provitamin A carotenoids for vitamin A. One golden tomato can provide a person’s full daily vitamin A requirements. Tomato fruit and its processed products are the principal dietary sources of carotenoids such as lycopene. Lycopene is a potent antioxidant with the potential to prevent epithelial cancers and improve general human health. Therefore, there is considerable interest in elevating the levels of carotenoids in tomato fruit and thereby improving the nutritional quality of the crop (Fraser et al. 2002; Mehta et al. 2002). The B gene from Solanum hirsutum shifts tomato carotenoid accumulation from lycopene almost entirely to b-carotene and results in orange fruit color (Premachandra 1986). This consequently dramatically increases the provitamin A carotenoid content. b-carotene content of commercial cultivars, as mentioned, is of interest (Markovick et al. 2002), and several high-b-carotene orange cherry tomato breeding lines have been bred (Stommel et al. 2005). Rainbow carrots, which are super sweet and crunchy, have multipigmented roots that naturally contain several antioxidants, such as lycopene, lutein, and anthocyanin. Similarly, yellow sweet potatoes are much more nutritious than white ones since they are high in provitamin A carotenoids. Unfortunately, the popularity of white-fleshed sweetpotato cultivars in many tropical regions may complicate the acceptance of more nutritious orange ones. Recent studies across a range of Andean potato cultivars show wide variation in calcium, iron, and zinc content (Andre et al. 2007) as well as anthocyanins (Brown et al. 2005; Reyes et al. 2005) due to the existence of red-, blue-, and purple-fleshed potatoes. In order to have impact for its nutrient content, a vegetable must be appealing to consumers. Sensory appeal, including color, is an attribute important to consumers when selecting many vegetables. Enhanced pigmentation of carrot, potato, tomato, and pepper, for example, is considered a quality factor (Stevens 1986; Simon 2000). In peppers, carotenoid content of green, yellow, orange, and particularly red peppers can be relatively high. Selection for high pigment is an important goal because these carotenoids are important for visual appeal in many markets but until now did not have any specific nutritional impact (Biacs et al. 1993; Hanson et al. 2004). In the past, yellow or orange tomatoes could not compete with red tomatoes because they were

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unfamiliar to consumers; now they are commercialized and are challenging the market. This fact and recent development of orange-colored cauliflower (Dickson et al. 1988) and orange-flesh cucumbers (Simon and Navazio 1997) reflect a new direction in vegetable breeding: nutritional quality. The white versions of these two vegetables lack carotenoids present in high enough concentration to alter their appearance. As nutritional quality becomes a more common breeding goal, and the novelty of unusual colors brings added value to seed companies and vegetable growers, unusual colors will quite certainly become more available and perhaps more widely consumed. But consumer requirements for quality—appearance, shelf life, and taste—must be met. Breeding to increase consumer appeal by improving convenience and the quality factors of a moderately nutritious crop often can be a more effective approach to increase intake of shortfall nutrients (Simon et al. 2009). Nutritional quality identifiable by the consumer and available at a moderate price might induce increased consumption and thus confer an important marketing incentive for breeding activity. B. Goals and Techniques International seed companies are interested mainly in the breeding and production of vegetable seeds with high commercial value. Traditional vegetable landraces have largely been neglected by seed companies, policy makers, and researchers. But while their production often takes place under low-input conditions, they contribute substantially to household food and livelihood security, particularly for small resource-poor farmers (Cavendish 2000; Weinberger and Msuya 2004). For example, in Africa, traditional vegetable landraces constitute an important source of micronutrients, contributing between 30% and 50% of iron and vitamin A consumed, respectively, in poor households (Gockowski et al. 2003; Weinberger and Msuya 2004). Most vegetables are propagated by seed. Vegetatively propagated vegetables include potato, sweetpotato, artichoke, Jerusalem artichoke, taro, common ginger, cassava, yam, horseradish, water chestnut, and Japanese artichoke (Kays and Dias 1996). At present, breeding of vegetables is largely in the hands of international seed companies. Due to the great diversity of vegetable crops, it is uneconomical to carry out breeding efforts on all of them; thus, only crops with large markets have received attention by these companies. Examples include solanaceous fruits (tomato, pepper, eggplant), cucurbits (cucumber, melon, watermelon, squash), and crucifers (broccoli, cauliflower, cabbage, Brussels sprouts, turnip, Chinese cabbage, pak-choy, radish).

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Breeding companies have striven more and more to bring hybrid seeds onto the market. Worldwide the share of hybrid seed is increasing at a fast pace of 8% to 10% annually in most of the vegetables. More than twothirds of the 5,000 nonhybrid vegetable cultivars available in 1984 seed catalogs from North America have been dropped during the last 25 years. A hybrid is produced by crossing two carefully selected parent lines to produce seed combining the best characteristics of each. Hybrids often exhibit higher productivity and vigor than open-pollinated cultivars due to heterosis. The superior characters of hybrid plants, unlike that of open-pollinated cultivars, cannot be maintained by growers who save their seeds for the next growing season since the uniformity, vigor, and overall performance of the hybrid is lost during seed multiplication. Therefore, growers are obliged to buy seeds from the seed companies every growing season if they want to compete in the marketplace. Many growers have been skeptical about the cost of hybrid seeds but have found that they give excellent returns. The main reasons that influence the growers’ decisions to adopt improved or hybrid vegetable seeds are the ability of the product to meet the market demand for high productivity, uniformity, resistance or tolerance to diseases or pests, better response to costly inputs, high quality, and storability. Hybrid vegetable cultivars are used increasingly for large-scale intensive production because they provide increased marketable product for commercial growers and thus add to commercial incentives for the seed companies. Open-pollinated cultivars are derived by repeated selection of superior plants from within the same line and are genetically uniform for appearance traits. In naturally self-pollinated plants such as tomato, lettuce, and legumes, inbreeding leads to homozygosity. It has generally been found that self-pollinating vegetables such as tomato, after they have become stabilized, do not substantially change their genetic constitution (Kerr 1969). Thus it is easy to maintain the purity of selfpollinated vegetable crops by removing occasional variants. In the case of cross-pollinated plants such as cucurbits, cole crops, sweet corn, beet, and spinach, uniformity can be achieved by rigorous selection and confining natural crossing within the selected population. Isolation distances of at least 1,500 m between different cultivars of the same species are recommended for all cross-pollinated crops that are to be harvested for seed. Hybrid vegetable technology has made significant impact on most vegetable crops in developed countries, but a major limitation to vegetable production in many developing countries is the unavailability of high-quality seeds. Hybrid seed production is a high-level technology

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and cost-intensive venture. Only well-organized seed companies with good scientific manpower and well-equipped research facilities can afford hybrid seed production. The public sector in developing countries frequently does not have sufficient capacity to supply adequate quantities of good-quality vegetable seed to poor growers. At present, there are few private sector seed companies adapting cultivars to local environments, especially in the poorer countries (Rohrbach et al. 2003). Growers themselves often produce seeds of locally preferred or traditional landraces, as the individual markets are too small and private companies lack interest in open-pollinated cultivars (Weinberger and Msuya 2004). Without proper seed production, processing technology, quality assurance, and management supervision, locally produced seeds often are contaminated by seed-transmitted viruses and other disease organisms, and are genetically diverse. Lack of proper storage facilities and an effective monitoring mechanism often leads to low or uncertain seed viability and vigor. Moreover, low capital resources and poor market information discourage the development of seed-related agribusinesses. Seed quality and treatment are keys to product quality, and there is a need for upgrading quality control laboratories to meet international standards. The global seed trade is now dominated by international corporations, whose vast economic power has effectively marginalized the roles of public sector plant breeding and local, small-scale seed companies. Thirty years ago there were thousands of seed companies in the world, most of which were small and family owned. Today, there are many fewer, and the top six global seed companies control almost 50% of the commercial seed trade. Some of these companies belong to worldwide corporations that are also involved with pesticides and biotechnology. The vegetables attracting the most breeding attention vary considerably between small and huge seed companies/corporations. Small seed companies have a tendency to specialize in a few vegetable crops. In large international companies, the breeding activity is more diverse but is concentrated on the more economically important crops. In these companies, molecular methods have become integral to many commercial vegetable breeding programs (Dias 1989). The aim of molecular breeding is to supplement conventional methods with faster and more efficient breeding through marker-assisted selection (MAS) and/or marker-assisted backcrossing (MAB). Molecular markers that are closely linked to a trait of interest may be identified and applied in gene pyramiding, facilitating introgression of desirable traits into cultivars, early selection, and so on. For more complex traits conferred by polygenes, quantitative trait loci (QTL) analysis is carried out. The decision to

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implement molecular markers as a selection tool is based on the fact that the cost of molecular marker analyses has steadily decreased due to the development of new, less expensive marker technologies and by the implementation of automation for marker detection. Thus, for phenotypes that are simply inherited, screening with markers is often less expensive, and may be more precise, than screening for the phenotype in the field. QTL analysis is more complex and therefore may be more expensive. Markers bring additional value when they can be used to accelerate the development of new improved vegetable cultivars. This may occur when phenotypes can be assessed only in specific seasons (since markers can be done anytime), or by improving the efficiency of selecting a recurrent parent for backcrossing or for selecting multilocus genotypes. For example, in southern India, molecular-based tools were used to identify strains of the tomato leaf curl virus (ToLCV) and to select genes of resistance from wild tomatoes that were then bred into cultivated lines. ToLCV-resistant lines were developed that produce twice the yield of the most popular cultivars in the region after only a few years (Hanson et al. 2000). Tomato resistant to geminivirus were also developed with the help of the molecular markers. Sequence characterized amplified region (SCAR) and cleaved amplified polymorphic sequences (CAPS) markers linked to the beta gene in tomato have been developed for MAS (Zhang and Stommel 2001). MAS for higher provitamin A carotenoids content is under way in muskmelons. Recently developed markers for watermelon lycopene b-cyclase allows selection differentiating lutein yellow carotenoid plants from lycopene redfruited plants early in development (Bang et al. 2007). In vitro haploidization techniques, such as anther and microspore culture and in vitro gynogenesis, are also used by many seed company breeders to accelerate the production of homozygous inbred parental lines for new hybrid vegetable cultivars. For example, Dias (2001, 2003) described successful in vitro microspore culture of broccoli and an improvement in the existing cole protocols, to make this technique available for the purpose of routine breeding, in 10 different broccoli genotypes. This protocol is now used by several seed companies worldwide to produce doubled haploid lines for pure inbred progenitors to obtain and ensure uniformity in hybrid cultivars. In onion, commercial breeding lines have been derived from open-pollinated populations with high levels of genetic variation. The development of doubled haploids by in vitro gynogenesis to produce inbred onion lines also is currently under way for hybrid cultivars with much higher levels of uniformity (Bohanec 2002; Sidhu et al. 2004). Developmental cycles of commercial hybrid cultivars range between 5 and 12 years, and breeding lines

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necessary for creating hybrid seed must be refined for specific markets. Therefore, these important R&D investments make commercial vegetable breeding expensive. Genetically modified (GM) vegetables recently have been introduced in the market and more are expected to be licensed for sale. Virus-resistant transgenic plants are particularly valuable if no genetic sources of resistance have been identified or if host resistance is difficult to transfer due to genetic incompatibility or links to undesired traits. In such cases, engineered resistance may be the only viable option to develop virusresistant cultivars. However, vegetables are considered minor crops and traditionally have had fewer resources channeled to them compared to field crops. While it is becoming less expensive to create GM crops for pest management, developing a marketable product and a regulatory package remains costly. Development and regulatory costs can be recouped more readily if the product is grown on an extensive area, as would be done with field crops, but which is not generally the case for individual vegetable crops. For example, the large agricultural biotechnology companies have for the most part abandoned the development of GM vegetable crops because of the high costs associated with product development and deregulation. For vegetables, there are many cultivars of the same crop, and the expected life of a particular cultivar can be quite limited. Introducing a GM trait into a breeding program can be complicated and cost prohibitive, especially in crops where backcrossing is difficult or impossible (e.g., potato). In most countries, deregulation of a GM trait is event specific. For many vegetable crops, it is not possible to develop a single GM event that can be converted into many different cultivars of a single or closely related group of vegetable species via conventional breeding. For example, Brassica contains about 40 closely related commercialized crops, including cabbage, cauliflower, broccoli, Brussels sprouts, turnip, Chinese cabbage, pak-choy, various mustards, swede, and vegetable rape (Kays and Dias 1995, 1996). No single parent exists that can be used to backcross the transgene into the many different types of Brassica botanical varieties. Individual events would have to be developed for many of the crop types, and deregulation of more than one event for a single protein is problematic for most business models. For the few transgenic vegetable crops that are being developed, novel or unconventional strategies have been employed to bring the crops to market based generally on private and public partnerships, in which the private sector would focus on selling hybrids to higher- end producers while the public sector would focus on low-resource growers. Active international trade and overseas vegetable seed production by contract is common in many countries. Each multinational company

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vies to provide better vegetable seeds to compete with domestic seed producers. In China, whose seed market is estimated to be valued at more than US$1.4 billion, the increased recognition of new and high-yielding hybrid cultivars has encouraged the local development of a large number of vegetable seed producers and distributors. Four types of vegetable seed producers were established: public seed companies, research institutes, foreign seed companies, and local seed companies. Private seed companies have been expanding rapidly in recent years, and there are now thousands of small firms. Some companies have started to breed their own cultivars and establish marketing networks. They play a strong role in the Chinese vegetable seed industry. About 60 foreign seed companies have opened branch companies or stations in China. Most of them not only sell their vegetable seeds but also have established breeding stations. In other southeastern Asian countries, such as India, Indonesia, Vietnam, and Malaysia, the percentage of hybrid vegetable cultivars is lower than in China, so a large expansion of seed companies has not yet occurred. However, India liberalized vegetable seed import policies in 1988, and the ‘‘New Policy on Seed Development’’ was instituted to augment productivity and output quality. These events stimulated major growth in the industry and attracted more investment in the seed business from domestic and international seed companies. Consequently the estimated area under vegetable hybrids has gone up from 192,100 ha in 1993–1994 to 416,013 ha in 1999–2000. The seed replacement by hybrids during the period from 2001 to 2006 in the main vegetables was: tomato (99.3%), eggplant (63.4%), chili (83.7%), peppers (95%), cauliflower (86.4%), cabbage (100%), radish (96.5%), onion (87.3%), okra (92.4%), muskmelon (71%), watermelon (89.2%), cucumber (72.6%), and gourds (73.5%) (ICRP 2007). The shares of vegetable hybrids have this distribution: private (64%), imported (33%), and public (3%). Today the hybrid vegetable seed industry in India is estimated to be worth US$40 million and the open-pollinated industry to be around US$118 million (MAGV 2008). While there has been rapid growth in the seed markets of developing countries due to a shift away from farm-saved seed, the seed markets in developed countries, particularly those of Europe and Japan, are stagnant. In Europe and the United States, the seed industry has been concentrated and is largely in the hands of large corporations; many small firms are closing. Vegetable breeding strategy and targets are dependent on market trends. Successful breeders anticipate changes in the market by developing new cultivars that are ready to be released to the growers when their demand increases. Therefore, it will be interesting to see how breeding companies react to changes in vegetable consumption and to

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evaluate the potential influence that the vegetable market and growing systems may have on breeding targets and priorities. C. Prospects for Poor Growers and Marginal Land Production What are the prospects for nearly half of the world’s vegetable growers who are poor and cannot afford to buy hybrid seed every growing season? These growers produce 15% to 20% of the world’s vegetables, and they directly feed almost 1 billion people in Asia, Latin America, and Africa. Capital and risk factors are the key constraints that limit the adoption of improved vegetable cultivars by small-scale and poor growers, because these vegetables generally are much more costly to produce per hectare than traditional landrace cultivars (Key and Runsten 1999; Ali and Hau 2001; Ali 2002), and most growers require credit to finance their production. While landraces usually are cultivated using a level of input intensity appropriate to the financial resources available within a household, improved vegetable cultivars often require an intensive input regime, including large labor inputs for planting and harvest that cannot be met with family labor alone (Weinberger and Genova 2005). For smallscale and poor growers, improved vegetable cultivars also tend to be riskier than landraces, since the higher costs associated with seeds and production impose a greater income risk. Small-scale growers may have lower production costs with landraces, because they achieve adequate yields with fewer inputs. In addition, the profits from improved cultivars or hybrids tend to vary because yields are often higher but prices fluctuate. From another perspective, variable prices and yields increase the variability in market supply (Key and Runsten 1999). The lack of capital available to small-scale and poor growers denies them the opportunity to invest in vegetable production inputs. Without collateral help, these growers are usually unable to secure a loan from a bank or money lender. For those who can get a loan, rates are often unmanageably high, with strict penalties for late repayments. Similarly, a lack of education, resources, skill training, and support prevent these growers from using improved cultivars and then from generating a stable income from their production. In addition, governments usually do not regulate the price of vegetable crops or even provide market information, unlike for field crops. Improving market information systems for vegetable crops and facilitating farmers’ access to credit are essential components of a strategy to enable poor growers to grow improved vegetable cultivars and to overcome the insecurity of their food supplies. The problem of food insecurity in this situation, like that of poverty, thus frequently is traceable to macroeconomic conditions and market failures

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due to actions of exploitative intermediaries, including landowners, moneylenders, and traders. A major obstacle to success in vegetable production is the shortage of affordable credit. In some cases, vegetable growers must pay high interest rates of 15% to 25% per 100 days. Desperate for cash, subsistence growers are forced to sell their crops immediately after the harvest to middlemen or their creditors at unfavorable prices. Credit facilities and other inputs must be part of these vegetable production systems, so that the use of improved vegetable cultivars can help subsistence vegetable growers to overcome their poverty and food insecurity. The benefits from the use of improved cultivars are shown by a project supported by the United States Agency for International Development (USAID) and conducted by AVRDC from 1991 to 2000 in two districts in Bangladesh (Jessore and Savar), with the aim of overcoming constraints in vegetable production (Weinberger and Genova 2005). The two districts were selected because they have large vegetable production areas. In Savar, 75% of all agricultural land is in vegetable production, while in Jessore, the share is 50%. Technological interventions included germplasm evaluation and cultivar development for many vegetables, off-season production technologies, and grafting technologies for tomato and watermelon to control soil-borne diseases (Kubota 2008; Lee 2008). Between 1996 and 2000, after cultivar development and the introduction of some new facilities, rural infrastructures, and extension services, vegetable production grew at an average annual rate of 5.4%. In a survey of 300 growers, the adoption of improved cultivars (42%) and hybrid seed (30%) were the most responsible for this increase in vegetable production of all the technologies used. Among 27 vegetable crops propagated by seed there were 5 (19%) where the only change in vegetable production was the adoption of improved cultivars or hybrid cultivar seeds; in 14 (52%) there was the additional adoption of simple cultural practices, such as row sowing and fertilization. Tomato grafting and other more sophisticated practices were not implemented. Of the hybrid cultivars used, 92% were of cross-pollinated species in which hybrids are important for uniformity. Vegetatively propagated vegetables were not included in the activities. Eager to increase their production, the majority of the growers (91%), regardless of farmer type, invested in some new vegetable technology over the last 5 years of the project. The average proportion of growers who adopted an improved technology was 43%, and the average adoption rate among all technologies was 31%. Improved or hybrid vegetable cultivars were 72% responsible for the increase of vegetable production, since it was easier and cheaper for the growers to buy improved cultivar seeds than to adopt other technologies.

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This fact highlights the importance of improved cultivars and the need to invest in cultivar improvement research, since it requires fewer behavioral changes compared to adopting new crop management practices. Small-scale growers, particularly small landowners, tend to be late adopters of new technologies due to skepticism about the cost of improved and hybrid seeds and wariness about capital and risk constraints, as compared to larger-scale growers with large cultivated areas (Collins 1995). Increased vegetable production has also resulted in important employment benefits for the community, such as new employment opportunities, substitution of family labor by hired labor, and increased wages. The average hired-labor man-days in the cultivation of vegetables were 170 per ha (excluding labor from contracted companies), half of the total labor requirements. Likewise, the total value added in wages was approximately US$400 per ha, 7.5 times higher than valued added through employment in rice. In particular, small-scale growers benefited from additional employment opportunities. In small-landowning households cultivating small areas, more than 50% of men and 16% of women sought employment outside their own farm. One-third of these employment opportunities were vegetable related. These off-farm employment activities were mainly at the production and postharvest levels since the vegetable processing sector is not yet fully developed in Bangladesh. Local support industries also have benefited from the expansion of vegetable cultivation both on the input and output side. A higher degree of input commercialization was observed for vegetables as compared to cereals and included all inputs such as seed, inorganic fertilizers, pesticides, farm manure, plastic, mesh netting, and bamboo poles. In general, a higher share of vegetable output was sold on markets as compared to the production of cereals. Vegetable growers were highly integrated into markets, selling a large share of their products and retaining a small portion for home consumption. This was true for both small-scale and large-scale growers. Since supermarkets continue to play a minor role in Bangladesh, most of the vegetable produce was sold either in the local markets or to wholesalers. In general, the survey project found that vegetable production has contributed to widespread welfare improvement and poverty alleviation in Bangladesh. While nearly all communities agreed that they were benefiting from increased vegetable production (in terms of enhanced consumption, enhanced investment, saving opportunities, or increased welfare), the grower-level data also showed that larger-scale growers have been able to capitalize more. On average, 90.3% of households experienced an improvement in their lives over the past 5 years, but

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large-scale growers reported greater increases in their well-being. The study has also shown that more impact still can be expected, particularly if agro-technology industries develop further. However, the availability of cheap, high-quality vegetable seed may be restricted and a major impediment to progress. Similar projects were implemented by AVRDC in other Asia-Pacific countries and in Africa, and the results were similar: Farmers receive more income from vegetables per hectare than from grain crops, and efficient vegetable production using cheap improved seeds contributes to poverty reduction and less food insecurity. These projects show that improved vegetable cultivars could benefit poor growers and landless laborers by increasing both production and employment. It could benefit the rural and urban poor through growth in the rural and urban nonfarm economies and by making available food that is high in nutrients. D. Improvement through Genetic Modification Another means for improving vegetable cultivars is through GM technology. Two successful examples are GM squash and eggplant cultivars. In the United States, squash yield losses due to viruses often range from 20% to 80% in summer squash (Cucurbita pepo) with a reported US$2.6 million economic loss in the state of Georgia in 1997 (Gianessi et al. 2002). Three of the most important viruses affecting squash production are zucchini yellow mosaic virus (ZYMV), watermelon mosaic virus (WMV), and cucumber mosaic virus (CMV) (Zitter et al. 1996). No summer squash cultivar with satisfactory resistance to CMV, ZYMV, and WMV has yet been developed by conventional breeding (Gaba et al. 2004). Control of squash viruses has focused on cultural practices, including delayed transplanting relative to aphid flights, use of reflective film mulch to repel aphids, and application of stylet oil to reduce virus transmission, in combination with insecticides to reduce aphid vector populations (Perring et al. 1999). In the state of Georgia, it is estimated that 10 applications of stylet oils and insecticides are made routinely to control aphids and, hence, limit virus incidence and transmission (Gianessi et al. 2002). Two lines of squash expressing the CP gene of ZYMV, WMV, and CMV were deregulated and commercialized in 1996 (Medley 1994). Subsequently, many squash types and cultivars have been developed, using crosses and backcrosses with the two initially deregulated lines. This material is highly resistant to infection by one, two, or all three of the target viruses (Clough and Hamm 1995; Fuchs and Gonsalves 1995; Ochoa et al. 1995; Tricoli et al. 1995; Fuchs et al. 1998; Schultheis and Walters 1998). The adoption of virus-resistant squash

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cultivars has steadily increased in the United States since 1996. In 2005, the adoption rate was estimated at 12% (approximately 3,100 ha) across the country with the highest rates in New Jersey (25%), Florida (22%), Georgia (20%), South Carolina (20%), and Tennessee (20%) (Shankula 2006). Virus-resistant transgenic squash has allowed growers to achieve yields comparable to those obtained in the absence of viruses with a net benefit of US$22 million in 2005 (Shankula 2006). Engineered resistance was the only practical approach to development of cultivars with multiple sources of resistance to CMV, ZYMV, and WMV in those markets where GM squash is allowed. Another example is GM eggplant (aubergine) in India. Eggplant is a popular vegetable crop grown in many countries throughout the subtropics, tropics, and Mediterranean area (since it requires a relatively long season of warm weather to give good yields) on a total of 2 million ha in 2007 (Choudhary and Gaur 2009) for a total production of 32 million t. Asia contributed 91.5% of the world production (FAO 2009). India produces 8 to 9 million t, one-quarter of the global production, which makes India the second largest producer in the world after China. Eggplant, commonly known as brinjal in India (550,000 ha) and Bangladesh (64,208 ha), is the most popular vegetable grown in the Philippines (20,000 ha). The crop is often considered a ‘‘poor man’s vegetable’’ and is cultivated mainly on small family farms. A total of 1.4 million small marginal and resource-poor growers grow eggplant in all eight vegetable growing zones of India. Eggplant is an important source of nutrition and cash income for many resource-poor growers, since they transplant it from nurseries at different times of the year to produce two or three crops, each of 150 to 180 days’ duration. Growers start harvesting fruits at about 60 days after planting and continue the harvest for 90 to 120 days, thereby providing a steady supply of food for the family and a stable income. Eggplant was one of the first vegetable crops adopted by growers in India to be used as hybrids. Hybrids occupy more than 50% of the eggplant-planted area, the balance being planted with open-pollinated cultivars. Eggplant is an annual plant. It is attacked by a number of insects (including thrips, cotton leafhopper, jassids and aphids); however, the most damaging is the eggplant fruit and shoot borer (Leucinodes orbonalis), which is considered the most serious and destructive pest of eggplant in Asia and Africa (Palada et al. 2006). Infestation is caused by adults migrating from neighboring fields, from eggplant seedlings, or from previously grown eggplants in the same planting area. Damage from L. orbonalis starts at the nursery stage and continues after crop transplanting until harvest. Losses have been estimated to be 54% to 70% in

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India and Bangladesh and up to 50% in the Philippines (Choudhary and Gaur 2009). Recommended insect pest management practices include the prompt manual removal of wilted shoots, trapping male moths using pheromones to prevent mating, ensuring regular crop rotation, and using nylon net barriers. These methods, however, are not widely adopted by growers because of time and resource constraints or lack of awareness. There are no known eggplant cultivars resistant to the fruit and shoot borer, so the use of insecticide sprays continues to be the most common control method used by growers. The borer is vulnerable to sprays only for a few hours before they bore into the plant. Therefore, growers in India spray insecticides as many as 40 to 80 times over a 7-month cropping season (AVRDC 2001; Choudhary and Gaur 2009). Growers may even spray every other day, particularly during the fruiting stage, which contributes to the presence of pesticide residues. But despite the application of many insecticides, the eggplant fruits sold in the Indian market are still of inferior quality, infested with borer larvae (Choudhary and Gaur 2009). A survey of pesticide use in Central Luzon in the Philippines indicates that growers there spray up to 56 times with insecticides during a crop season to protect their eggplant crops against the borer (Palada et al. 2006). The decision of growers to spray is influenced more by subjective assessment of visual presence of the insect rather than guided by the more objective science-based methodology of threshold levels. This reliance on visual assessment leads to gross overspraying with insecticides, higher insecticide residues, and unnecessary increase in the growers’ exposure to insecticides. On average, 4.6 kg of active ingredient of insecticide per hectare per season is applied on eggplant (Choudhary and Gaur 2009). Such pesticide use, besides being detrimental to the environment and human health, also increases the cost of production, making this humble vegetable expensive for poor consumers. In Asia, chemical spraying for this insect accounts for 24% of the total cost of production (Choudary and Gaur 2009). Intensive use of improperly applied insecticides raises serious concerns for environmental and human health. A study conducted in the Jessore district of Bangladesh found that ‘‘98% of farmers felt sickness and more than 3% were hospitalized due to various complexities related to pesticide use’’ (AVRDC 2003). It is expected that Bt eggplant cultivars will reduce pesticide applications and will contribute to poverty reduction and to overcoming food insecurity. E. Future of Breeding and Diversity About one-half (52%) of the total number of vegetables cultivated in the world receive commercial breeding attention by seed companies.

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Of those, only 17% are in large-scale breeding programs, fostering a need for serious attention to maintenance of vegetable crop biodiversity. There has been a severe decline in the vegetable cultivar genetic base, as evidenced by the significant reduction, especially within the last 50 years, in the number and range of vegetable cultivars grown. During this period, vegetable genetic diversity has been eroding all over the world, and vegetable genetic resources are disappearing, on a global scale, at an unprecedented rate of 1.5% to 2% per annum. Widespread adoption of simplified vegetable systems with low genetic diversity carries a variety of risks, including food insecurity. In the short term, such systems risk potential crop failure. In the longer term, they encourage the reduction of the broad genetic base that contributes to high yields, quality traits, disease and pest resistance, and the like. This compromises the future genetic health of vegetables. Especially prominent among the ‘‘enemies’’ of genetic diversity are the commercial markets and economic social pressures that have practiced breeding methods that promote uniformity, encouraging extensive cultivation of preferred improved and hybrid vegetable cultivars with insufficient diversity. In addition, globalization has stimulated the consolidation of vegetable seed companies into huge corporations and the decline of small seed companies that serve local and regional markets. In consequence, some vegetable breeding programs have been merged or eliminated to reduce costs. Thus, fewer and fewer companies/corporations are making critical decisions about the vegetable research agenda and the future of vegetables worldwide. Inevitably, two things will happen. There will be fewer vegetable breeders in the future, and growers will be dependent on a narrower genetic background that could contribute in the near future to food insecurity for poor growers and consumers. Also, with the advent of genetic engineering, the huge seed corporations are also assuming ownership of a vast array of living organisms and biological processes. Of equal concern are expanded uses of legal mechanisms, such as patents and plant breeder’s rights, that are removing vegetable plant germplasm from general public use (Ryder 2005). Intellectual property rights for plants was intended as a defensive mechanism to prevent the loss of invented cultivars to competitors. However, with the more stringent enforcement of plant breeding rights, and particularly with the application of the utility patent law in the United States to protect all forms of an innovation, this has become an offensive weapon to stifle competition and inhibit the flow of germplasm and information. This can have serious implications for the future conservation of vegetable genetic resources and for world food security.

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Some landraces and old open-pollinated cultivars of vegetables have existed for long periods outside the commercial and professional plant breeding circles, kept alive within communities by succeeding generations of seed savers. Unfortunately, there are fewer and fewer active seed savers among the millions of vegetable growers, due to the demand of commercial markets and the professionalization of the sector. This is an additional threat to genetic diversity. Continued survival of landraces and open-pollinated cultivars of vegetables depends largely on popular interest and initiative as well as preservation in gene banks. We should be alerted and concerned about the loss of biodiversity in vegetables and about its impact on food security. Vegetable growers have an important role in conserving and using vegetable biodiversity. The future of world food security depends not just on stored vegetable genes but also on the people who use and maintain crop genetic diversity on a daily basis. In the long run, the conservation of plant genetic diversity depends not only on a small number of institutional plant breeders and seed banks but also on the vast number of growers who select, improve, and use vegetable diversity, especially in marginal farming environments. That is why we should be also alerted and particularly alarmed by the current trend to use improved and hybrid vegetable cultivars exclusively. Growers do not just save seeds; they are plant breeders who are constantly adapting their vegetable crops to specific farming conditions and needs. For many generations, vegetable growers have been selecting seeds and adapting their plants for local use. This genetic biodiversity is the key to maintaining and improving the world’s food security and nutrition. No plant breeder or genetic engineer starts from scratch when developing a new cultivar of tomato, pepper, cabbage, or lettuce. They all build on the accumulated success of generations of growers who have selected and improved vegetable seeds for thousands of years. If poor small-scale growers in marginal areas stop saving seeds, we will lose genetic diversity. Growers will lose the means to select and adapt vegetable crops to their unique farming conditions, which are characterized by low external inputs. Hybrid seed technology is designed to prevent growers from saving seed from their harvest, thus forcing them to return to the commercial seed market every year. Hybrid vegetable seeds alone, and used globally, can be a dead-end to biodiversity. If growers abandon completely their traditional vegetable landraces in the process of adopting only hybrids, crop genetic diversity achieved over centuries will be lost forever. Many agronomic benefits will be lost to worldwide growers and thus to consumers.

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The exclusive adoption of hybrid cultivars in marginal areas may restrict the vegetable-producing capacity of growers. It will also destroy biodiversity, and it may contribute in the long term to food insecurity. A study by Daunay et al. 1997 points out that the release of F1 hybrids (in Europe and some Asian countries, e.g., China and Japan) displaying higher productivity, but with poor phenotypic variability, has contributed to the losses of eggplant landraces, thus inevitably leading to genetic erosion of S. melongena. Moreover, some African cultivated eggplants have been lost following social, economic, and political changes (Lester et al. 1990). Therefore, the cultivated eggplant has been considered a priority vegetable species for the preservation of genetic resources since 1977. Several studies have been carried out in Asia and Africa (Lester et al. 1990; Collonnier et al. 2001; Gousset et al. 2005), and collections have been built up (Bettencourt and Konopka 1990), particularly in China (Mao et al. 2008). Fortunately, in some developed countries, new independent seed companies, offering unique collections of regionally adapted landrace vegetable cultivars, have recently emerged. Furthermore vegetable hobbyist groups, mainly from organic horticulture, are thriving and maintaining old vegetable landraces in organizations known as seed savers. In this way traditional landraces are being restored to native growers and to urban and periurban growers. Some of these traditional landraces display combinations of traits that make them especially responsive to local or regional conditions, or are well suited to particular growing methods, such as those used in organic horticulture or low-externalinput systems, or are tolerant to local pests and diseases or other stresses and constraints. Organic growers who seek to grow full cycle, or seed to seed, are also working to ensure the continued availability of organically grown seeds. There are also considerable ongoing efforts by national governments and international organizations to preserve plant vegetable germplasm in gene banks. This is a valuable but static approach, as further evolutionary changes and improvements will not occur until the seeds are planted and selection takes place. It is also an activity that relies heavily on continued political stability and support, including sustained governmental funding. Active and positive connections between the private breeding sector and large-scale gene banks are required to avoid possible conflict involving breeders’ rights and gene preservation. The diversity of crop species will be promoted by the maintenance of crop gene banks by governments and nongovernmental organizations, the continued use of diverse sources by plant breeders, especially in the public sector, and the use of local cultivars and landraces by farmers.

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V. SUMMARY AND CONCLUSIONS Domestic and international vegetable markets are changing rapidly, and a variety of factors, such as supermarkets and improvements in transportation and refrigeration, have largely contributed to this development. Direct investment from global retailers also has had an impact on the structure of retailing. Trade liberalization has impacted the increasing importance of exports, which are increasing for high-value vegetable crops and processed foods alike. Increasing urbanization, with increasing incomes mainly of growing middle classes in most parts of the world, requires large quantities of vegetables. These may be produced locally or at great distances from where they are consumed, with effects on vegetable postharvest processing and value-added activities. In addition, vegetable postharvest processing for added value has become important. Processing provides the means to increase shelf life of vegetables; at the same time, processed vegetables provide convenience in the kitchen of the consumer or professional foodservice companies. Increased food retail power of supermarkets puts pressure on branded vegetable companies in two ways: growth of private labels at the expense of branded products, and the reduction of the number of brands on the supermarket shelf. Producers of processed vegetables with a long shelf life, in particular the canning industry, are encountering stagnating consumption in high-income regions. The areas of convenience and health products provide new opportunities and are still large unexplored by most processors. The standards for participation in high-value vegetable markets have increased, in both developed and developing countries. Supply chains are increasingly complex, undergoing rapid changes, and often based on strong vertical integration. Informed policies and a conducive regulatory environment will increase the incentives for agents in the supply chain to accept the produce of small-scale growers as inputs and improve the growers’ capacity to meet the product attributes required in a rapidly modernizing agricultural marketplace. The participation of small-scale growers in dynamic vegetable markets for higher-value vegetables is a major challenge. Participation requires, particularly in developing countries, a set of institutional changes, training, and credit facilities to allow farmers to compete in increasingly competitive global markets that demand safe, uniform, and high-quality produce. Increasing urbanization and the needs of growing cities to feed their populations will require also more attention to urban and periurban vegetable production. Among the obstacles to participation in modern markets is the fact that yield improvements in vegetables have been lower than in cereals.

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Concerted actions are required to raise the average yields of vegetable crops. Yields will increase by focusing on inherent productivity and on maintaining yield stability. More emphasis will have to be placed on the development of modern cultivars, using the natural vigor of hybrids to fight stresses of disease, heat, and drought. Molecular tools will be useful for selecting resistance genes and increasing quality, nutritional value, and yields. These traits plus food safety will be important aspects of future breeding efforts. Overall, there is great genetic and phenotypic diversity for types and amounts of micronutrients in the various vegetables. Consequently, there is a good potential for increasing micronutrient content and thus enriching the diet of the average consumer. More research is needed with the goals of providing benefit to poor and malnourished populations. Creation of vegetable hybrids is a key means toward the development of modern cultivars. Hybrid seed production is a high-technology and a cost-intensive venture. Only well-organized seed companies with good scientific manpower and well-equipped research facilities can afford seed production. Due to globalization, today most vegetable breeding research and cultivar development in the world is conducted in and funded by the private sector, mainly by huge multinational seed companies. Public vegetable breeders and public sector cultivar development are disappearing worldwide. This means in general that there will be fewer decision-making centers for vegetable breeding and cultivar development. The focus is now on relatively few major vegetables produced worldwide, to the detriment of all other cultivated vegetables. It is imperative that national governments and policy makers, as part of a social duty, invest in breeding research and cultivar development of traditional open-pollinated cultivars and in the minor and so-called forgotten vegetables. More investments in this area will mean less expensive seed for growers to choose from and increased preservation of vegetable biodiversity. The accomplishment of this goal may require new approaches to vegetable breeding research and development by both the public and the private sector. Until recently, vegetable breeding research and development that targets small-scale and poor vegetable growers has been undertaken largely by public sector institutions and national agricultural research institutes. However, the capacity to undertake the work depended mainly on national or international funding and expertise. The work has been limited by the capacity of these institutions to pay for it. As a result, horticultural advancement has varied enormously among countries and even within regions in developed and developing countries. In the area of plant breeding, the process of producing improved cultivars

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is slow, and it requires long-term sustained commitment that may not fit the continuing changes in the national and international politics of research funding. The application of biotechnology promises acceleration of some aspects of plant breeding, but the adoption of more advanced technologies significantly raises the cost of research at a time when investment funding has diminished. Public plant breeding remains a key component of vegetable research systems worldwide, especially in developing countries. However, the increasing presence of private sector breeding and a decrease in national and international support makes it difficult for the public sector to continue operating in the traditional manner. Declining funding for public vegetable breeding, coupled with the rapid increase of vegetable production and consumption and an urbanizing population, has created a difficult situation. More public sector vegetable breeders are needed worldwide to select and to produce nonhybrid cultivars of the minor vegetables. This will benefit small-scale growers and will safeguard biodiversity and food security in developing countries. While the maintenance of vigorous public sector breeding programs in areas where private companies are not interested in providing low-cost cultivars is highly desirable, an additional approach to maximize vegetable and horticultural research input would be the development of global programs with public–private partnerships. The public sector may support portions of vegetable and horticultural R&D that are not attractive to the private sector, feed improved breeding lines and systems to the private sector for exploitation in regions where the private sector is active, and nurture private sector development in regions where it is lacking. Many in the public and private sectors support such a complementary approach to overcome poverty and malnutrition in developing countries. The GM eggplant in India is an excellent working example of a model philanthropic public/private sector partnership. Bt eggplant technology has been generously donated by its private sector developer, Mahyco, to public sector institutes in India, Bangladesh, and the Philippines for incorporation in open-pollinated cultivars of eggplant for the use of farmers, and especially small resource-poor farmers. The development and regulation of Bt eggplant in India also will serve the common regulatory needs in Bangladesh and the Philippines and generally will facilitate the regional harmonization of biotech vegetable crop use in Asia. It also can serve as a model to facilitate regional harmonization in other developing countries in Asia, the Pacific, sub-Sahara African, or Latin America, where the need for simplified, responsible, and appropriate regulation is great. It is the key barrier that denies developing

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countries timely access to the significant benefits that biotech vegetable crops offer. This sharing of knowledge and of experience with the regulatory process could greatly simplify and lighten the regulatory burden by eliminating duplication of the significant effort already expended by India, thereby contributing to the important goal of harmonizing regulations among countries. Many current vegetable breeding efforts remain underfunded and disorganized. There is a great need for a more focused, coordinated approach to efficiently utilize funding, share expertise, and continue progress in horticultural technologies and programs. Vegetable production can help the poor escape poverty and malnutrition in the 21st century, but only if enough investments are made to improve and sustain the breeding and productivity of vegetable crops. Policy makers and donors have to turn their attention to enhanced funding for the vegetable and horticultural sector, allowing growers to compete with their products on a world market increasingly determined by market quality standards and phytosanitary concerns and regulations. Only then will the silent vegetable and horticultural revolution currently under way benefit a significant portion of the world’s poor nations, growers, and landless laborers and enable us to overcome poverty and food insecurity. In summary, we must ensure that society will continue to benefit from the vital contribution that plant breeding offers, using both conventional and biotechnological tools, because improved and hybrid vegetable cultivars are, and will continue to be, the most effective, environmentally safe, and sustainable way to ensure global food security in the future. LITERATURE CITED Acord, B.D. 1996. Availability of determination of non-regulated status for a squash line genetically engineered for virus resistance. Federal Register 61:33484–33485. Allard, R.W. 1960. Principles of plant breeding. Wiley, New York. Alam, M.G.M., E.T. Snow, and A. Tanaka. 2003. Arsenic and heavy metal contamination of vegetables grown in Samta village, Bangladesh. Sci. Total Environ. 308:83–96. Ali, M. 2002. The vegetable sector in Indochina: A synthesis. The vegetable sector in Indochina countries: Farm and household perspectives on poverty alleviation. AVRDCARC, Bangkok. Ali, M., and V.T.B. Hau. 2001. Vegetables in Bangladesh. Tech Bul. 25. AVRDC, Shanhua, Tainan, Taiwan. An, H.B., L.N.D. Thinh, D. Dam, N.V. Nam, L.T. Hang, and T.Q. Thoai. 2003. Spatial and institutional organization of vegetable markets in Hanoi. Report Project No. 00005600 ‘‘Sustainable Development of Peri-Urban Agriculture in South-East Asia,’’ funded by Ministry of Foreign Affairs of France. RIFAV, Hanoi.

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9 Regulation of Anthocyanin Accumulation in Apple Peel Adriana Telias, James M. Bradeen, James J. Luby, and Emily E. Hoover University of Minnesota St. Paul, MN 55108, USA Andrew C. Allan Plant and Food Research Mt Albert Research Centre Private Bag 92169 Auckland, New Zealand I. INTRODUCTION II. APPLE PEEL COLOR A. Marketing Importance B. Apple Flavonoids C. Anthocyanin Accumulation D. Color Measurements III. GENETIC CONTROL OF ANTHOCYANIN ACCUMULATION A. Inheritance B. Biosynthetic Genes C. Regulatory Genes IV. FACTORS AFFECTING ANTHOCYANIN ACCUMULATION IN APPLE A. Light B. Temperature C. Mineral Nutrition D. Carbohydrate Availability E. Growth Regulators F. Orchard Management Practices V. MECHANISMS AFFECTING ANTHOCYANIN ACCUMULATION PATTERNS IN APPLE PEELS A. Chimeras B. Cytosine Methylation

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C. Transposable Elements D. Other Mechanisms VI. CONCLUSIONS LITERATURE CITED

I. INTRODUCTION Apple production is of great importance in the United States, with 4.2 million tonnes (t) of production in 2007 and a value equivalent to US$2.6 billion. World production of apple in 2007 was 64 million t (FAO 2009). A key quality attribute of apple fruit is its peel color, which affects consumer preferences and is also associated with the fruit’s nutritional value due to the antioxidant properties of anthocyanins, the pigments determining red peel color. Pigment accumulation in apple fruit can be affected by environmental conditions and fruit production practices. In recent years, significant progress has been made in understanding the genetic regulation of anthocyanin accumulation in apple. Here we summarize the current state of knowledge regarding the regulation of anthocyanin accumulation in apple peel. II. APPLE PEEL COLOR A. Marketing Importance Apple peel color is an important factor determining apple market acceptance. In general, red apples are preferred, particularly well-colored bright red types (Saure 1990). However, very dark, purplish black red apples are losing favor. Furthermore, consumer preferences vary from country to country and region to region (Cliff et al. 2002). New Zealand consumers prefer striped apples; consumers from Nova Scotia, Canada, favor blushed apples; while consumers in British Columbia, Canada, were more accepting of a range of apple types. Panelists in Lleida, Spain, on the contrary, did not show a preference for peel appearance when presented with eight ‘Gala’ strains with varying pigmentation (Iglesias et al. 2008). Peel color is one of the main traits enabling cultivar discrimination, and there is increasing interest in breeding materials with altered color. In New Zealand (Chagne et al. 2007) and Europe, researchers are working toward the development of a red-fleshed apple cultivar. B. Apple Flavonoids Flavonoids, including anthocyanins, flavones, and flavonols, are polyphenolic plant secondary metabolites. Anthocyanins, the main red

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pigments in apple flowers and peels, are important in attracting insects and birds for pollination and fruit-eating animals for seed dispersal. Many apples are red leaved as well. Flavones and flavonols, although not visible to the human eye, may attract insects that see farther into the ultraviolet (UV) range of the spectrum to flowers containing them, and can also protect cells from excessive UV-B radiation. Additional functions of flavonoids include a role in nitrogen fixation, auxin transport, and plant defense (Ronald et al. 1994; Taiz and Zeiger 2002). Total polyphenolic content and levels of individual compounds were shown to vary significantly in apple (Tsao et al. 2003). Peels have, in general, a much higher concentration of polyphenols than flesh. The predominant groups of polyphenolics in apple peel and flesh are the colorless procyanidin, followed by the colorless or yellow quercetin glycosides in the peel and hydroxycinnamic acid esters in the flesh (Tsao et al. 2003). The main anthocyanin identified in apple peel is cyanidin 3-galactoside, while the cyanidin 3-glucoside level is very low (Honda et al. 2002; Kondo et al. 2002; Tsao and Yang 2003; Ben-Yehuda et al. 2005). Accumulated evidence from studies on the effects of fruit consumption is now abundant and consistent in suggesting a protective effect of fruit intake leading to lower risk of cardiovascular disease, cerebrovascular disease, and stroke. Apples in particular have been associated with lowered risks of cancer and cardiovascular diseases, which are thought to be caused by oxidative processes. Polyphenolics are the major source of antioxidants in apple, and this fruit is a very important source of flavonoids in U.S. and European diets. In the United States, 22% of the phenolics consumed from fruit come from apples, making them the largest source of these compounds. Several studies have linked apple consumption with reduced risk for cancer, especially lung cancer, cardiovascular disease, asthma, pulmonary problems, diabetes, and obesity. These effects are due to the fruit’s antioxidant activity, antiproliferative activity, inhibition of lipid oxidation, and cholesterollowering effects (Boyer and Liu 2004). Antioxidants are localized mainly in the apple peel, but cultivars exhibit a wide variation in the distribution pattern (Eberhardt et al. 2000; Lata 2007). Flavan-3-ols and procyanidins are the most important contributors to the in vitro antioxidant activity of apples, while procyanidin B2 and epicatechin are the most important individual antioxidants in apple. Also, hydroxycinnamic acids may have a significant antioxidant role in the flesh (Tsao et al. 2005). Anthocyanins and copigment flavonols produce most of the pink, red, mauve, and blue color found in higher plants (Lancaster 1992). In apple fruits, the red color is produced by anthocyanins, which accumulate as granules within vacuoles (Bae and Kim 2006) (Plate 9.1), while the

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Plate 9.1. ‘Royal Gala’ apple epidermal skin cells, showing distinctive, anthocyaninfilled vacuoles. Cells were peeled from red sectors of the fruit (a, c) or yellow sectors (b), and viewed at 100 magnification (a) or 400 (b, c). (See insert for color representation.)

background color is determined by the green and yellow chlorophylls and carotenoids in plastids (Lancaster et al. 1994). Copigmentation is a solution phenomenon in which pigments and other noncolored organic components form molecular associations, resulting generally in an enhancement in light absorbance and, in some cases, a shift in the wavelength of the maximum absorbance of the pigment (Boulton 2001). Evidence suggests that copigmentation does not explain differences in shade of red observed in the peel of different apple genotypes. Instead, this variation might be associated with the visual blending of chlorophyll, carotenoids, and anthocyanins (Lancaster et al. 1994). C. Anthocyanin Accumulation Apple peels consist of a thick cuticle, with a layer of wax on the outside, an epidermis, and a hypodermis of thick-walled cells (Walter 1966). Apple fruit color is determined primarily by the ground color of the peel, which is initially green in immature fruit and may fade in varying degrees as the fruit ripens, and second by the superimposed anthocyanin pigmentation, if present (Janick et al. 1996). When red pigmentation is present, there are two peaks of coloration during fruit development. The first occurs during the phase of intense cell division in the fruit, and the second coincides with ripening of red cultivars (Saure 1990). Red pigmentation can adopt different patterns, from small red flecks to bold stripes, and from a weak blush to solid red. The expression of these characters can be affected by environmental, nutritional, and orchard management factors, the stage of maturity of the fruit, and the microenvironment within the canopy (Janick et al. 1996). In general, apple peel coloring most often takes the form of striping, but blushes may also occur. The blush (unbroken area with red pigment accumulation) may be pronounced but in some cases is ephemeral, being extremely light-sensitive. For instance, quite a number of normally

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Plate 9.2. The two different types of fruit peel pigment patterns in ‘Honeycrisp’ apple. Distribution of anthocyanin in apple peels of blushed (a) and striped (b) fruits of ‘Honeycrisp’. (See insert for color representation.)

yellow apples will, if exposed to full sun, accumulate red pigments in part of their peel. There are very few apple cultivars that are truly blushed (David Bedford, Univ. Minnesota, pers. commun.), with blushed being defined as lacking obvious peel stripes (Plate 9.2a). Most fruits are ‘‘solidly striped,’’ an appearance that is hard to distinguish from truly blushed in fruit grown under good light conditions. But unlike truly blushed fruit, solidly striped fruit display a range of obviously striped phenotypes (Plate 9.2b) when grown under poor coloring conditions, such as shading. A good example of a blushed fruit is ‘Connell Red’, which is a blushed sport of ‘Fireside’ (striped). These two cultivars show no differences when molecular marker profiles are compared (Cabe et al. 2005), indicating that observed differences may be caused by very few DNA sequence changes or even a single mutation. In ‘Honeycrisp’, fruit pigmentation can adopt two basic patterns: blushed or striped, where blushed fruits are defined as those that never show stripes, even in less colored areas of the peel. These two types of fruit appear together on the same tree, and even on the same spur, a very unusual phenomenon in apple. In contrast to what is observed in ‘Honeycrisp’, neither ‘Connell Red’ nor ‘Fireside’ seems to deviate from its respective patterns. The striped pattern might be detrimental for fruit marketing, especially in poor coloring conditions, since striped fruits are less red on average than blushed fruits (Telias et al. 2008). Anthocyanin is found in cells of the peel epidermis and subepidermal layers, but not all cells are pigmented (Plate 9.1). Some apples have no pigment in the epidermis, and the color intensity depends on the proportion of cells in each subepidermal layer that contain pigment.

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The peel of both ‘Delicious’ and its bud sport ‘Starking’ contains many stripes, but since the epidermal layer of these cultivars is colorless, the difference in color between a stripe and the adjacent area involves only the hypodermal layers. In ‘Northern Spy’, which has a small proportion of pigmented cells in the epidermis, red stripes have a greater proportion of pigmented epidermal cells than adjacent areas as well as more intense color (Dayton 1959). Striped patterns in ‘Royal Gala’ and ‘Honeycrisp’ apples are caused by higher anthocyanin accumulation in the epidermis and hypodermal layers in red stripes as compared to green stripes (Telias 2009). Light and electron microscopy studies in ‘Fuji’ apple have revealed that peels with fully developed red color have more layers of anthocyanin-containing epidermal cells than those of green cultivars. The density of anthocyanin is higher in cells of the outer layer of the fruit peels than in inner layers (Bae and Kim 2006). D. Color Measurements Given the importance of peel color for describing and identifying apple cultivars, color measurement tools can be very valuable for apple breeders and the apple industry in general. Color measurements in plants are performed using a variety of methods. Color assessment by visual comparison to color charts, such as the one published by the Royal Horticultural Society (Royal Horticulture Society 1995), is the most economical, but also the most tedious and subjective. Colorimeters provide, at a higher cost, an objective measurement, but usually of a localized area. Software packages have been implemented to perform evaluations of the fruit surface (e.g., detection of fruit blemishes; see Du and Sun (2004) and references therein), but none of these methods can perform color measurements of large surface areas automatically, on a large set of images. More recently, new image-processing tools to measure color and other fruit quality variables automatically have been developed and made freely available (Darrigues et al. 2008; Telias et al. 2008). These tools enable the measurement of average fruit color (or other characteristics of interest) from digital images. By measuring color in the entire fruit peel surface, they provide more informative values than, for instance, the measurement of small peel regions that are obtained with standard colorimeters. Color comparisons between blushed and striped apples can be performed using these tools as they provide a measurement of the average color for the peel surface for both kinds of fruit, independently of the pattern of pigment accumulation. Multiple color spaces can be used to define color. The CIE L a b (CIELAB) is a color space specified by the International Commission on

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Illumination. The three coordinates of CIELAB represent the lightness of the color (L ) ranging from 0.0 for black and 100.0 for diffuse white, its position between red/magenta and green (a ) and between yellow and blue (b ). Hue is defined as the attribute of a visual sensation to which an area appears to be similar to one of the perceived colors such as ‘‘red,’’ ‘‘yellow,’’ and so on and can be computed from the a and b values (Fairchild 2005). Hue angle and the a /b ratio can be used as predictors of anthocyanin concentration in red raspberry fruit (Moore 1997). Greer (2005) compared different methods to measure color change in apples throughout development, concluding that hue angle is the best indicator, as compared to chlorophyll fluorescence, chroma, or lightness coefficient. Studies of the diffuse reflectance spectra of whole apple fruit in the 400–800 nm range with a spectrophotometer allow for development of an index linearly related to anthocyanin concentration (Merzlyak et al. 2003).

III. GENETIC CONTROL OF ANTHOCYANIN ACCUMULATION A. Inheritance Two categories of genes affect anthocyanin biosynthesis (Table 9.1). The first category that has been widely studied in apple encodes enzymes required for pigment biosynthesis (structural or biosynthetic genes) (Honda et al. 2002; Kondo et al. 2002; Takos et al. 2006b; Ben-Yehuda et al. 2005). The second category is comprised of transcription factors, which are regulatory genes that influence the intensity and pattern of anthocyanin accumulation and generally control expression of many different biosynthetic genes (Goodrich et al. 1992; Allan et al. 2008). Based on segregation patterns, the red color of apple peel appears to be controlled by two complementary loci (Pratt et al. 1975; White and Lespinasse 1986). More recently, Chen et al. (1996) reported that the red/yellow peel color dimorphism is controlled by a monogenic system with red pigmentation being dominant. The MYB1-1 allele of MYB1 identified by Takos et al. (2006a) (see ‘‘Regulatory Genes’’ section) produces segregation for red peel color, suggesting that it is the peel color locus in the cultivars tested. Red coloration in fruit cores and foliage are both controlled by the Rni locus (Chagne et al. 2007). There is still insufficient information to determine the genetic control of fruit pigmentation patterns. According to Janick et al. (1996), when blushed apples are intercrossed, the offspring will normally be either blushed or nonpigmented but not striped, suggesting an additional

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Table 9.1. Structural and regulatory genes in the anthocyanin biosynthetic pathway in apple. Gene name

References

Structural genes PAL (phenylalanine ammonia lyase) CHS (chalcone synthase)

CHI (chalcone isomerase) F3H (flavanone-3 b-hydroxylase) DFR (dihydroflavonol-4reductase) LDOX (leucoanthocyanidin dioxygenase) UFGT (UDP-glycose: flavonoid-3-Oglycosyltransferase)

Ju et al. 1995b, 1999 Ju et al. 1995a, 1999; Honda et al. 2002; Kondo et al. 2002; Ben-Yehuda et al. 2005; Takos et al. 2006a,b; Espley et al. 2007 Takos et al. 2006a,b; Espley et al. 2007 Ju et al. 1999; Honda et al. 2002; Kondo et al. 2002; Kim et al. 2003; Ben-Yehuda et al. 2005; Takos et al. 2006a,b; Espley et al. 2007 Ju et al. 1997, 1999; Honda et al. 2002; Kondo et al. 2002; Kim et al. 2003; Ben-Yehuda et al. 2005; Takos et al. 2006a,b; Espley et al. 2007 Ju et al. 1999; Honda et al. 2002; Kondo et al. 2002; Kim et al. 2003; Ben-Yehuda et al. 2005; Takos et al. 2006a,b; Espley et al. 2007 Ju et al. 1995, 1999; Honda et al. 2002; Kondo et al. 2002; Kim et al. 2003; Ben-Yehuda et al. 2005; Takos et al. 2006a,b; Espley et al. 2007

Regulatory genes MdMYB10/MdMYB1/ MdMYBA MdbHLH3 MdbHLH33

Takos et al. 2006a; Ban et al. 2007; Espley et al. 2007, 2009 Espley et al. 2007, 2009 Espley et al. 2007, 2009

role for genetic regulation. The random amplified polymorphic DNA (RAPD) genetic marker developed by Cheng et al. (1996) did not correlate with the presence or absence of stripes on red fruit or with the intensity of the anthocyanin pigmentation. B. Biosynthetic Genes Flavonoids are synthesized by the phenylpropanoid metabolic pathway in which the amino acid phenylalanine is used to produce 4-coumaroylCoA (Fig. 9.1). This compound can in turn be combined with malonylCoA to yield the backbone of all flavonoids, a group of compounds called chalcones, which contain two phenyl rings. The conjugate ring-closure of chalcones results in the familiar form of flavonoids, the three-ringed structure of a flavone. The metabolic pathway continues through a series of enzymatic modifications to yield flavanones, dihydroflavonols, and

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Phenylalanine PAL

Hydroxycinnamic acids

Coumaroyl-Co-A Malonyl-Co-A

CHS

Chalcones

Dihydrochalcones

CHI

Flavanones F3H

MdMYB10

Dihydroflavonols

FLS

GT

Flavonols

DFR

Leucoanthocyanidins

LAR

Flavan-3-ols

LDOX

Anthocyanidins

ANR

Condensensed tannins

UFGT

Anthocyanins Fig. 9.1. Schematic representation of the flavonoid biosynthetic pathway in apple regulated by MdMYB10. Flavonoid intermediates (gray boxes) and end products (black boxes) are indicated. Enzymes required for each step are shown in bold uppercase letters (PAL, phenylalanine ammonia lyase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone-3 b-hydroxylase; FLS, flavonol synthase; GT, unidentified enzyme encoding a glycosyl transferase for flavonol glycone synthesis; DFR, dihydroflavonol-4-reductase; LAR, leucoanthocyanidin reductase; LDOX, leucoanthocyanidin dioxygenase; ANR, anthocyanidin reductase; UFGT, UDP-glycose:flavonoid-3-O-glycosyltransferase. Source: Adapted from Takos et al. 2006.

anthocyanins. Along this pathway, many products can be formed, including flavonols, flavan-3-ols, and proanthocyanidins or tannins (Ververidis et al. 2007). Five genes encoding the anthocyanin biosynthesis enzymes, chalcone synthase (CHS), flavonone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX), and UDP glucose: flavonoid 3-O-glucosyltransferase (UFGluT), are coordinately expressed during apple fruit development, and their transcription levels are positively correlated with anthocyanin concentration (Honda et al. 2002; Ben-Yehuda et al. 2005; Takos et al. 2006a; Espley et al. 2007). Expression of these genes in different cultivars may be controlled by

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regulatory genes, and environmental factors might affect anthocyanin synthesis through these regulatory genes (Honda et al. 2002; Ben-Yehuda et al. 2005). However, low levels of expression of the biosynthetic genes do not induce anthocyanin accumulation in ripening fruits of ‘Orin’ 168 days after full bloom but give rise to its accumulation in immature fruits of ‘Jonathan’ 142 days after full bloom, suggesting that there are additional mechanisms controlling red coloration during fruit ripening in addition to the regulation of gene expression (Honda et al. 2002). Ju et al. (1997) found no significant correlation between DFR activity and anthocyanin accumulation in apple fruit. It appears that DFR is necessary, but not a limiting point, for anthocyanin synthesis in apples. Deduced amino acid sequences of the genes encoding F3H, DFR, LDOX, and UFGluT (prepared from the peel tissues of ‘Fuji’ apple) showed high homology to corresponding protein sequences from other plants and indicate that each protein is encoded by a multigene family (Kim et al. 2003). The mRNAs of anthocyanin biosynthetic genes are detected preferentially in the peel tissue, and transcription of the genes was coordinately induced by light (Kim et al. 2003). The transcripts were detected abundantly in the peel of red cultivars but rarely in that of a cultivar bearing nonred fruit, which confirms that these genes have major roles in determination of apple peel color (Kim et al. 2003). Southern hybridization using fragments of anthocyanin structural genes as probes revealed little polymorphism between green (‘Mutsu’) and red cultivars (‘Fuji’ and ‘Jonathan’), demonstrating that both green and red cultivars carry anthocyanin structural genes. This finding further indicates that the expression of the anthocyanin genes in green cultivars is somehow affected, leading the authors to suggest that transcription factors might be altered in the green cultivar (Kim et al. 2003). The enzyme phenylalanine ammonia-lyase (PAL) is critical in the regulation of flavonoid synthesis in apple (Saure 1990), but while PAL catalyzes a reaction to produce precursors of anthocyanin synthesis, changes in anthocyanin accumulation can occur independent of changes in PAL activity under conditions of sufficient precursors (Ju et al. 1995b). Further support is provided by Ju et al. (1999b), reporting that red-striped areas have more anthocyanin and higher UDP galactose : flavonoid-3-O-glucosyltransferase (UFGalT) activity than adjacent areas, but no differences in PAL and CHS activities are observed. The higher abundance of cyanidin 3-galactoside as compared to cyanidin 3-glucoside in apple peel suggests that the functional enzyme is an UFGalT, which is responsible for the transfer of galactose to the 3-O position of flavonoids. However, researchers could not determine whether the apple UFGluT functions as UFGalT by transferring the

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galactosyl moiety to cyanidin (Honda et al. 2002). Kondo et al. (2002) have reported that UFGluT transcripts are not detected when anthocyanins do not appear later in development. However, MdCHS, MdF3H, MdDFR, MdLDOX, and MdUFGluT transcripts are all detected at 20 days after full bloom in both dark-grown fruit without anthocyanin accumulation and light-grown fruit with anthocyanin accumulation. These results, in agreement with those of Ju et al. 1995, suggest that the role of UFGluT may change during fruit development and that anthocyanin formation depends on cyanidin presence rather than on UFGalTactivity. Finally, UDOP-glucose 4 epimerase (UGE), an enzyme that catalyzes the reversible epimerization of UDP-glucose to UDP-galactose (the major sugar donor for cyanidin-glycoside in apple), is more active in apple peels that accumulate anthocyanin, indicating its possible contribution to red coloration (Ban et al. 2007a). C. Regulatory Genes Regulatory genes, controlling the expression of genes of the anthocyanin pathway, have been identified in many plant species. These genes influence the intensity and pattern of anthocyanin biosynthesis and generally control expression of many different structural genes (Holton and Cornish 1995; Mol et al. 1998; Spelt et al. 2002; Espley et al. 2007; Gonzalez et al. 2008). The R/B gene family of transcription factors determines the timing, distribution, and amount of anthocyanin pigmentation in maize. This family encodes proteins with homology with the basic helix-loop-helix (bHLH) motif in the MYC transcription activator (Chandler et al. 1989). Accumulation of anthocyanins in competent tissues of maize plants requires that members of the R/B family interact with either C1 (in the seed) or Pl (in the plant tissue). C1 and Pl share sequence similarity with the DNA binding domains of the MYB oncogenes and encode proteins with 90% or more amino acid identity in domains important for the regulatory function of the C1 protein (Cone et al. 1988). Pigment production in any particular part of the maize plant requires the interaction of a member of the R/B family and a member of the C1/Pl family (Cocciolone and Cone 1993). Studies with petunia have indicated that floral anthocyanin production requires a WD repeatcontaining protein (AN11), a MYB (AN2), and bHLH (AN1) transcription factor. These genes control not only synthesis of anthocyanins but also acidification of vacuoles in petal cells and the size and morphology of cells in the seed coat epidermis (Spelt et al. 2002). In apple, three groups have independently identified an R2R3 MYB transcription factor responsible for anthocyanin accumulation (Takos

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et al. 2006a; Ban et al. 2007b; Espley et al. 2007). MdMYB10 transcript levels strongly correlate with peel anthocyanin levels, and this gene is able to induce anthocyanin accumulation in heterologous and homologous systems (Espley et al. 2007). In addition, MdMYB10 cosegregates with the Rni locus, a major genetic determinant of red foliage and red color in the flesh of apple fruit (Chagne et al. 2007). MdMYB1 transcription also correlates with anthocyanin synthesis and is higher in red-fruit peel sectors (more exposed to light) and in red-peel cultivars (including ‘Cripps’ Red’, ‘Gala’, and ‘Galaxy’) than in green-peel sectors or nonred cultivars (including ‘Golden Delicious’, ‘Granny Smith’, and ‘Grandspur’). Transcription of MdMYB1 increased in dark-grown apples once exposed to light, providing additional evidence of its role as an anthocyanin regulator (Takos et al. 2006a). The expression of several anthocyanin pathway genes was found to be regulated by both MdMYB10 and MdMYB1 (Takos et al. 2006a; Espley et al. 2007) (Fig. 9.1). MdMYBA is also more highly expressed in redder peels and the redder cultivars, such as ‘Jonathan’, as compared to paler cultivars, such as ‘Tsugaru’, and its transcription is induced by UV-B light and low temperature (Ban et al. 2007b). The coding region of MdMYB1 is 100% and 98% identical to MYBA and MYB10, respectively (Ban et al. 2007b). In addition, MdMYB10 and MdMYBA have been mapped to the same region on linkage group 9 (Chagne et al. 2007; Ban et al. 2007b), suggesting that these three genes differ minimally and are probably alleles of each other. The striking phenotype of some apple cultivars producing red-fleshed fruit and red foliage (Plate 9.3) is caused by the occurrence of an autoregulatory mechanism associated with MdMYB10. In these genotypes, five direct tandem repeats of a 23-base pair sequence in the promoter of MdMYB10, 275-bp upstream of the ATG translation start codon, are the target of the MdMYB10 protein itself, and the number of repeat units correlates with an increase in transcription activation caused by the MdMYB10 protein (Espley et al. 2009). In contrast, no sequence differences were detected when comparing promoter and coding regions of MdMYB10 among ‘Fireside’, ‘Connell Red’, and blushed and striped ‘Honeycrisp’, using RNA and DNA extracted from peel tissue (Telias 2009), implying that different pigment patterns in these cultivars are not caused by changes at the primary DNA sequence level. Transcript levels of MdbHLH3 (similar to Arabidopsis TT8) and MdbHLH33 (similar to snapdragon Delila) remain more constant throughout apple fruit development and do not follow the same pattern as the biosynthetic genes or MdMYB10 (Espley et al. 2007). Results from reporter assays suggest that in cultivars carrying the 23-bp sequence

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Plate 9.3. Apple cultivars that carry the R6-MdMYB10 allele accumulate anthocyanin throughout the plant. The R6 mutation causes ectopic anthocyanin accumulation independent of other signals, such as light and tissue specificity. Pictures of 91.136—progeny of Redfield OP—have red foliage (a), petals (b), fruitlets (c), and fruit flesh (d). Source: Courtesy of the P&FR Pipfruit Breeding Program. (See insert for color representation.)

repeats and the MdMYB10 autoregulatory system, the influence of MdbHLH3 on MdMYB10 transcription is reduced (Espley et al. 2009). Repressors of anthocyanin production also have been identified within the MYB class of transcription factors. These include FaMYB1 in strawberry (Aharoni et al. 2001) and AtMYBL2 in Arabidopsis (Dubos et al. 2008; Matsui et al. 2008). FaMYB1 is upregulated jointly with late anthocyanin pathway genes, and it has been suggested that its role is to balance anthocyanin levels produced at later states of strawberry maturation (Aharoni et al. 2001). A gene in apple, MdMYB17, shows high homology to AtMYB4, a repressor of sinapate esters production in Arabidopsis. AtMYB4 expression is downregulated by exposure to UV-B light, indicating that derepression is an important mechanism for acclimation to UV-B in this species (Jin et al. 2000). AtMYB4 has been found to repress its own expression in a negative autoregulatory loop (Zhao et al. 2007b).

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Both MdMYB10 and MdMYB17 transcript levels correlate with anthocyanin concentration in stripes of both ‘Honeycrisp’ and ‘Royal Gala’, with higher mRNA levels in red stripes as compared to green stripes. Transcript levels of structural genes follow the same pattern as those of MdMYB10 and MdMYB17, suggesting that the presence of stripes is correlated with differential transcript accumulation of MdMYB10 and MdMYB17 in the differentially pigmented stripes, which in turn regulates transcription of structural genes. Levels of MdbHLH3 and MdbHLH33 transcripts do not differ in green and red stripes and therefore correlate poorly with anthocyanin concentration (Telias 2009). IV. FACTORS AFFECTING ANTHOCYANIN ACCUMULATION IN APPLE A. Light Elevated sunlight promotes ripening associated pigment changes in apple peel, including more profound breakdown of chlorophyll, induction of carotenoid synthesis, and specific changes in carotenoid patterns (Solovchenko et al. 2006). Anthocyanin production in apple peels has a tight dependence on light, not only the intensity but also the quality of light influences anthocyanin formation, with blue-violet and UV light being most effective and far red being least effective or even inhibitory (Saure 1990). Both phytochrome and specific UV-B photoreceptors appear to be involved in a synergistic activation of anthocyanin synthesis (Arakawa 1988). Studies of the effect of fruit position on the tree on flavonoids and chlorogenic acid content showed that both anthocyanin and quercetin-3-glycoside concentration are tightly linked to light levels. There is a critical far red/red light ratio of approximately 1, below which no anthocyanin and only minimal quercetin 3-glycosides are formed. In addition, photosynthesis is necessary for the full expression of the response (Awad et al. 2001). UV treatments at 20 C were more successful at increasing anthocyanin levels in fruit peels than treatments at 10 C. The effect of temperature during UV irradiation depended on the cultivar tested (Lancaster et al. 2000). Postharvest irradiation is effective in increasing red color only when applied to apples at commercial harvest, and not earlier, indicating that light responses are developmentally controlled (Marais et al. 2001) When fruits are unbagged and exposed to light, MdCHI transcription increases by 240-fold, followed by MdCHS and MdLDOX at 80- and 60fold, respectively (Takos et al. 2006b), in agreement with results obtained

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by Ben Yehudah et al. (2005). MdMYB1 was identified as the lightresponsive regulatory factor controlling transcription of apple flavonoid genes (Takos et al. 2006a). In different studies, transcription of MdMYBA, MdCHS, MdDFR, MdLDOX, and MdUFGluT in apple peels was also found to be regulated by light, particularly UV-B radiation (Ju et al. 1997; Ubi et al. 2006; Ban et al. 2007). Condensed tannin production, however, was not light induced (Takos et al. 2006). Flavonols accumulate in apple peel during acclimation to strong sunlight. They can serve as an efficient UV-B screen, playing an important role in the resistance of the photosynthetic apparatus to the UV-B component of solar radiation. Anthocyanins do not exhibit a detectable synergistic effect in UV-B protection and seemingly serve as protection from damage only by radiation in the blue-green part of the visible spectrum (Solovchenko and Schimtz-Elberger 2003). Reay and Lancaster (2001) studied the potential of detached fruit to accumulate anthocyanins and quercetin glycosides and found that the shaded side of the fruit has a much greater potential than the exposed side. They concluded that the fruit peel’s previous exposure to light is a modifying factor in the potential for accumulation of anthocyanins by ‘Gala’ and ‘Royal Gala’. A study of the effect of light irradiation on the accumulation of phenolic compounds in slices of apple flesh indicated that phenolic acids, anthocyanins, and flavonols increase rapidly by irradiation whereas flavanol, procyanidin, and dihydrochalcone levels remain unchanged (Bakhshi and Arakawa 2007).The position of the fruit on the tree can affect the pattern of anthocyanin deposition in ‘Honeycrisp’ apple. More striped fruit are produced on southwest- facing branches (most sun exposed in the northern hemisphere); these fruits were additionally more strongly striped than those on the least sun exposed northeast branches. These results suggest that in this cultivar, higher light incidence or temperature on the bud or the fruit correlates with an increase in the occurrence and strength of stripes (Telias et al. 2008). B. Temperature Low temperatures promote, and high temperatures in autumn inhibit, anthocyanin synthesis in apple fruit (Marais et al. 2001; Saure 1990). The effect of low temperatures can be explained, at least in part, by a promotion of MdCHS, MdLDOX, and MdUFGluT transcription (Ubi et al. 2006). The amount of pigment in the fruit is more closely correlated to average night temperature than to the average day temperature. The negative effect of higher temperature on color formation in ‘McIntosh’

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apples is more pronounced in attached fruit (no color formation) than in detached fruit (slight color formation), suggesting that factors necessary for the formation of red color in apple may not be the same for detached and attached fruit (Uota 1952). Curry (1997), working with peel tissue discs, found that the optimal temperature for anthocyanin accumulation in pre-climacteric fruit tissue is 25 C. When fruit has entered the climacteric phase of ripening, it shows a greatly reduced capacity for anthocyanin formation, indicating that delaying harvest in order to increase anthocyanin accumulation under advanced ripening conditions might be ineffective. The study also showed that a period of cold temperature stimulates anthocyanin production in attached fruit; two or three nights with temperatures in the range of 2 C to 5 C followed by warm sunny days promote red color development. Bakhshi and Arakawa (2007), however, found that synthesis of phenolic acids, anthocyanins, and flavonols in slices of apple flesh is maximum at 24 C regardless of maturity stage and cultivar. Under otherwise identical conditions, fruits on trees with higher crop loads ripen later and postpone anthocyanin accumulation as compared to those on trees with lighter crop loads. This finding suggests that the increase in anthocyanin in autumn appears to be more closely related to ripening, as indicated by the rise in ethylene, than to a fall in temperature (Faragher 1983). C. Mineral Nutrition Generally, surplus nitrogen fertilization is associated with a reduction in the percentage of well-colored fruits at harvesttime, although the total yield of well-colored fruit may be higher (Beattie 1954). Reay and Lancaster (2001) found that the application of urea increases the chlorophyll and carotenoid concentrations in the fruit peel and reduces anthocyanin concentrations in the blush side of the fruit at maturity. Awad and de Jager (2002b) obtained negative correlations between nitrogen concentration in the fruit and anthocyanin and total flavonoid concentration at maturity. Nitrogen may inhibit flavonoid synthesis by enhancing the channeling of L-phenylalanine toward protein synthesis, or, alternatively, it might negatively influence the enzyme system involved in the biosynthesis of phenolics. Strissel et al. (2005) studied the effect of nitrogen on the activities of anthocyanin biosynthetic enzymes and found that PAL activity seems to be downregulated by high nitrogen levels. In tomato, Bongue-Bartelsmann and Phillips (1995) found that nitrogen deficiency increases anthocyanins and flavonol concentration two- to threefold in leaves and produces an increase in the steady-state

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mRNA levels of CHS and DFR, while chalcone isomerase (CHI) levels decreased. Results in Arabidopsis indicate that anthocyanin accumulation is an early senescence response to nitrogen deficiency and is controlled by the NLA gene (Peng et al. 2008). The effects of other mineral nutrients on apple peel color are not so well documented. Several authors found a positive effect of potassium application on anthocyanin formation in apple, as reviewed by Saure (1990), and a recent study by Funke and Blanke (2006) found an increase in fruit anthocyanins with monophosphate application in ‘Elstar’ apple. D. Carbohydrate Availability In Arabidopsis, PAP1, a MYB transcription factor controlling anthocyanin biosynthesis, upregulates anthocyanin production in response to high sucrose levels (Teng et al. 2005). In addition, PAP1 expression is induced by light, nitrogen, and phosphorus deficiencies and is therefore considered to be a key mediator in environmental regulation of anthocyanin biosynthesis (Rowan et al. 2009). In apples, evidence for sugar involvement in anthocyanin accumulation comes from results indicating lower sugar concentration in fruits that do not develop satisfactory color as well as from experiments with peel discs. Sugars may also be involved in a reduction of anthocyanidin degradation; an increase in galactose prior to harvest could enable anthocyanidin to accumulate by forming the glycoside (anthocyanin). Several studies have found no correlation between sugar content and color development at the time of fruit maturity. Some treatments that may promote anthocyanin formation can even reduce sugar content (Saure 1990). E. Growth Regulators Ethylene is a key factor in the regulation of anthocyanin biosynthesis and color development in apples, with positive correlations being found between ethylene and total anthocyanin but not with other flavonoid compounds (Whale and Singh 2007). According to Faragher and Brohier (1984), ethylene initiates rapid anthocyanin accumulation during apple ripening by increasing the level of PAL in the peel. A transgenic line of ‘Royal Gala’ apple that produces no detectable levels of ethylene was produced to study ethylene response in this cultivar. In response to ethylene application, 17 genes in the phenylpropanoid biosynthetic pathway were upregulated, including PAL (Schaffer et al. 2007). Awad and de Jager (2002a) have reported that the accumulation of anthocyanin is clearly stimulated by the ethylene-releasing agent

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ethephon and delayed by ABG-3168 (a likely inhibitor of ethylene production), whereas maturity-related fruit characteristics are not significantly affected by the different treatments. From this they concluded that anthocyanin formation in apple peel is not simply a ripening-related phenomenon but apparently is regulated by both the developmental signals and ethylene signaling. Whale et al. (2008) reported that the best fruit color and firmness are obtained with an application of the ethylene synthesis inhibitor aminoethoxy vinylglycine (AVC) 5 weeks before harvest, followed by an ethephon application 2 weeks later. The application of ethephon and a seniphos-like substance (a phosphorus-calcium mixture) produces an enhancement of red peel color and an increase in concentration of flavonoid compounds (Li et al. 2002). In these experiments, PAL and UFGalT activities are not closely related to increased ethylene concentration, while the increase in CHI activity possibly may be caused by an increase in ethylene. Ethylene is not involved in the coloration responses due to sunlight (Arakawa et al. 1985), temperature (Blankenship 1987), or bagging (Kubo et al. 1988). Gibberellic acid reduces anthocyanin accumulation, without influencing fruit maturation (Awad and de Jager 2002a). However, application of the gibberellin inhibitors cycocel and prohexadione-calcium does not significantly influence the formation of anthocyanin or fruit maturation. In another study, Mata et al. (2006) found that prohexadione-calcium applications increase red color in ‘Fuji’ but not in ‘Royal Gala’ apples. Jasmonic acid increases anthocyanin concentration in apples (Kondo 2006), and synergistic or additive responses were found between ethylene and methyl jasmonate in apple peel pigment synthesis pathways (Rudell and Mattheis 2007). Abscisic acid, auxins, and cytokinins previously have been implicated in anthocyanin regulation, but a clear role for these growth regulators has not yet been established (Saure 1990). F. Orchard Management Practices Bagging is widely applied in Japan as an effective practice for inducing some color development even in cultivars that do not usually show any red color upon ripening (Yonomori 2009). Bagging, by covering apples with bags made of paper or other materials, is done about 1 month after full bloom. Once fruits are bagged, anthocyanin accumulation is inhibited, consistent with the requirement of light for anthocyanin accumulation. Following removal of the bags some months later, fruit rapidly develop red color, and after several days, the coloration exceeds that of

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control fruits in most cultivars. However, differences at harvest may be negligible, especially if a decreasing temperature promotes color formation in untreated fruits of red cultivars (Saure 1990). Bagging also reduces chlorophyll levels in the fruit peel, and therefore can improve red coloration since anthocyanin accumulation is induced on a practically white background (Yonemori 2009). Arakawa 1988 noted that fruits initially produce more anthocyanin at immature and mature stages after bagging than control fruits when exposed to white and UV-B light. However, anthocyanin synthesis decreases rapidly toward harvest, whereas in the control fruit it increases further for varying periods of time. Bagging is also a useful model for studying anthocyanin synthesis and gene expression in apples. The potential of bagged fruit to synthesize anthocyanin when exposed to light remains constant during 5 months of cold storage (Ju 1998). Another practice that increases anthocyanin concentration is to cover the orchard floor with reflecting films. These films stimulate internal ethylene synthesis and elevate UFGalT activity (Ju et al. 1999a). The use of a white polypropylene ground cover (ExtendayÔ), an aluminized plastic film, and a reflective foil all increase apple peel red color (Funke and Blanke 2005; Glenn and Puterka 2007; Jakopic et al. 2007). Fruit thinning can increase total red color in ‘Honeycrisp’ fruit peels, as demonstrated in experiments by Delong (2006) and Wright et al. 2006. Crop loads can additionally affect the pattern of pigment accumulation in this cultivar, with higher crop loads being associated with a lower percentage of blushed fruit (Telias et al. 2008). Storage conditions used after harvest can have an effect on the flavonoid levels in the fruit. Antioxidant status is maintained more efficiently in controlled atmosphere than in common cold storage if fruit are stored for 90 days (Lata 2008). In Japan, where extremely high-quality fruit is desired, growers have developed additional management practices to obtain optimum fruit color, including leaf pruning and apple turning. Leaf pruning involves the removal of leaves blocking light incidence on the fruit, while fruit turning seeks to expose the least colored side of the fruit by turning the branch and securing it in its new position (Yonemori 2009). The light reaction can be used to label apples by applying films with opaque areas to apple surfaces before color development (Janick 1983). In some apples, imprints can be applied to poorly colored areas of the fruit after harvest by exposing apples to artificial light (P. Hirst, pers. commun.).

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V. MECHANISMS AFFECTING ANTHOCYANIN ACCUMULATION PATTERNS IN APPLE PEELS A. Chimeras A chimera is defined as ‘‘an organism containing a mixture of genetically different tissues’’ (Oxford English Dictionary 2009). The stratified arrangement of cells in the apical meristem is essential for chimeral plants. Angiosperms shoot apices commonly have two tunica layers where anticlinal divisions preponderate, over a corpus, in which cell divisions occur in different planes. Derivatives cells of the outermost layer of the tunica (L-1) give origin to the epidermis, while derivatives of L-2 form the subepidermal tissues. Derivatives of the corpus (L-3) form the pith and parts of the vascular tissues (Pratt 1983). Chimeras arise when mutations occur in cells of the apical meristem, giving origin to regions that are affected in one or more characteristics. In apple production, new materials with a chimeral origin are known as sports and are selected for their improved fruit or tree characteristics. Many apple sports have been identified and selected for changes in fruit color. Some cultivars, such as ‘Cox’s Orange Pippin’, ‘Delicious’, and ‘Elstar’, are prone to produce mutants with increased amounts of anthocyanin in the outer cell layers of the fruit peel, while other widely grown cultivars are stable and seem seldom to mutate (Janick et al. 1996). Red sports of a single cultivar can differ in the area of the fruit affected and in the intensity of the pigmentation in the epidermal and hypodermal layers (Dayton 1959). Often the mutation is limited to a single cell layer in the apical meristem; therefore, the plant is likely to be a periclinal chimera. The mutation is usually not heritable unless the second layer (L-2), which gives rise to gametes, is involved (Pratt 1983). Pratt et al. (1975) suggested that blushed fruited sports of ‘Northern Spy’, a striped apple, have a mutation for color pattern in L-1 and L-2. According to Dayton (1969), many striped strains of ‘McIntosh’ are the result of undesirable mutations from the color pattern of the original ‘McIntosh’. Mutations in L-1 probably account for this altered pigmentation, and results show that this cultivar also may be quite heterogeneous in its internal layers. Dark stripes on the fruits of the pear clone P1571, which have a layer pattern of L-1/green, L-2/green, L-3/red, indicate areas where the L-3 contribution to the flesh color is close enough to the epidermis to receive light and develop anthocyanins (Chevreau et al. 1989). McMeans et al. (1998) were unable to obtain tissue culture plants of ‘Gala’ and ‘Royal Gala’ that produced pure red or pure green fruits from

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leaf explants, suggesting that the source of color patterning in these striped cultivars is not chimeral. Trees in their experiment were obtained from leaf tissue culture and were classified as red or green depending on the pigments present in leaves and stems. However, the distribution of anthocyanin in the tree depends on so many factors, and there are so many exceptions to correlations between red fruit color and anthocyanin pigmentation of other parts of the trees, as to make this of no value as a method for predetermining fruit color (Janick et al. 1996). ‘Honeycrisp’ trees were propagated from buds occurring on branches with only striped (‘‘striped trees’’) or only blushed (‘‘blushed trees’’) fruits in order to study the stability of the blushed and striped traits. Striped trees not only produce a significantly higher percentage of striped fruit, but they also produce fruit with a higher intensity of striping when compared to blushed trees. The percentage of blushed fruit on any given tree or branch changes from year to year, indicating that in the case of ‘Honeycrisp’, the variation in pigment pattern is not chimeral in origin (Telias et al. 2008). B. Cytosine Methylation The addition of methyl groups to cytosines in DNA is a chemical modification that can be inherited and subsequently removed without changing the original DNA sequence. DNA methylation is part of the epigenetic code and is the best-characterized epigenetic mechanism. In plants, cytosines are methylated both symmetrically (at CG dinucleotides or CHG trinucleotides) and asymmetrically (at CHH), where H represents any nucleotide but guanine. In eukaryotes, DNA methylation functions as a gene-silencing mechanism that regulates endogenous genes and protects the genome by inactivating selfish DNA elements (Chan et al. 2005). Environmental factors can affect the methylation state of DNA: Steward et al. (2002) observed, for instance, that exposure to cold stress resulted in genome wide demethylation in maize seedling roots. Methylation in Arabidopsis is the result of three overlapping systems: (1) CG methylation controlled primarily by methyltransferase 1; (2) CHG methylation, maintained by chromomethylase 3 interacting with Histone 3 lysine 9 dimethylation pathway; and (3) CHH methylation, maintained by domains rearranged methylase 1 and 2 and requiring the active targeting of small interfering RNAs (Zhang 2008). Demethylation by DNA glycosylases of the demeter family also can control the resulting methylation landscape (Penterman et al. 2007). Microarray experiments indicate that 20% of the Arabidopsis genome is methylated (Zhang 2008). Methylated regions consist mostly of trans-

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posable elements and other repeats while gene promoters are rarely methylated, probably because methylation in these regions can interfere with transcription. Unexpected results indicated that methylation in transcribed regions is present in one-third of Arabidopsis genes; this type of methylation is enriched in the 3’ half of the transcribed regions and occurs primarily in CG sites (Zhang 2008). In rice, DNA methylation of the transcribed region is enriched for and associated with a larger suppressive effect than DNA methylation of the promoter region (Li et al. 2008). Cocciolone and Cone (1993) reported that differential DNA methylation in the 3’ untranslated region of Pl-Bh, a MYB transcription factor allelic to Pl regulating anthocyanin accumulation in maize, can cause striped patterns in all plant tissues. The level of anthocyanin production within the stripes is not uniform; rather, pigmentation levels vary from cell to cell. The common pattern for all blotchy organs is that not every cell within a pigmented sector is pigmented to the same degree. Additionally, pigmented cells frequently occur in clusters, resembling clonal sectors. Patterns of cell division appear to dictate the spatial arrangement of pigmented cells within any organ. The authors hypothesized that early during development, the Pl-Bh gene would be differentially methylated, and this methylation would be more or less maintained through subsequent cell divisions, producing clonal sectors in plant tissues of predominantly pigmented cells (unmethylated) and sectors of predominantly unpigmented cell (methylated). This hypothesized mechanism can account for the activation of anthocyanin synthesis early in development by Pl-Bh within some cells only, resulting in clonal sectors of pigmentation. It is also capable of explaining the cell-to-cell variability of gene expression, where not all cells attain the fully activated state. This regulatory mechanism is also heritable and can explain the stable transmission of the Pl-Bh phenotype to the progeny from generation to generation (Cocciolone and Cone 1993). Methylation plays a role in the regulation of additional genes controlling pigment production. Sekhon and Chopra (2009) identified a gene called Ufo1, which controls methylation levels in P1, a gene that regulates phlobaphene (brick-red flavonoids) biosynthesis in maize kernel pericarp and cob glumes, and whose activity may also produce variegation in maize pericarp. Ectopic expression of P1-wr correlated with hypomethylation of an enhancer region, 5 kb upstream of the transcription start site. The molecular nature of Ufo1 is still unknown. Cytosine methylation was found to be involved in the reduced expression of endogenous duplications. The duplicated R and Sn genes regulate the maize anthocyanin biosynthetic pathway and encode tissue-

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specific products that are homologous to bHLH transcriptional activators. As a consequence of their coupling in the genome, Sn is partially silenced. Researchers found that differences in pigmentation are inversely correlated with differences in the methylation of the Sn promoter. Accordingly, treatment with 5-azacytidine (AZA), a demethylation agent, restores a strong pigmentation pattern that is transmitted to the progeny and that is correlated with differential expression of the Sn transcript (Ronchi et al. 1995). In apple, a region of the MdMYB10 promoter, encompassing the region between nucleotide positions 1411 and 555 relative to the translation start site, is highly methylated in ‘Royal Gala’ and ‘Honeycrisp’ apple (Telias 2009). Green stripes of ‘Honeycrisp’ show higher methylation levels for MdMYB10 than red stripes, with the largest differences being found within the highly methylated region; methylation levels therefore correlate with transcript levels of MdMYB10. Although differences between red and green striped are consistent, overall methylation levels vary between years. Similar trends are observed in ‘Royal Gala’, except that the differences between red and green stripes are smaller and significant only for a portion of the highly methylated region. Overall, higher methylation levels are observed for ‘Royal Gala’ than ‘Honeycrisp’. Comparisons between red and green regions of the peel of blushed ‘Honeycrisp’ apples indicated no methylation differences, and interestingly, in two out of the three promoter regions studied, red stripes have methylation levels comparable to those in the peels of blushed apples, while green stripes have methylation levels higher than those of red stripes or red and green regions of blushed apples. As suggested for maize (Cocciolone and Cone 1993), one hypothesis is that early in apple fruit development, differences in MdMYB10 methylation are present among individual cells. Throughout fruit growth, these differentially methylated cells would give origin to sectors of peel varying in their ability to accumulate anthocyanins. Methylation of the MdMYB10 promoter may affect transcription through interference with the RNA-polymerase transcription complex or by preventing binding of additional factors required for transcription. It is known that within the most methylated region of the MdMYB10 promoter are five putative E-box motifs (Espley et al. 2009), a bHLHrelated cis-acting element (CACATG) (Atchley and Fitch 1997; Heim et al. 2003). The region also has a motif needed for MdMYB10 transactivation by the MdMYB10 protein itself (Espley et al. 2009). The occurrence of DNA methylation might therefore interfere with these regulatory mechanisms.

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Different methylation levels early in apple fruit development could be mitotically maintained from those in the meristematic cells that gave origin to the fruit, or could result from demethylation or de novo methylation. Results in ‘Honeycrisp’ suggest that there is at least some mitotic maintenance of methylation states, given that trees clonally propagated from buds on branches with exclusively blushed fruits tend to produce a higher percentage of blushed fruit (Telias et al. 2008). Nonetheless, results from the same study indicate that additional factors can influence the pattern in the peel, namely position of the fruit on the tree and crop load. An assessment of the exact location and context of MdMYB10 DNA methylation, as well as other types of epigenetics marks such as histone methylation, can shed light on the mechanism involved in MdMYB10 regulation. C. Transposable Elements The presence of transposable elements can affect gene expression both at the transcriptional (e.g. through the introduction of an alternative transcription start site) and at the posttranscriptional level (Feschotte 2008). Evidence linking transposable elements to pigment variegation in plants was first found by Barbara McClintock during the 1940s in her studies of the Dissociator (Ds) and Activator (Ac) loci in maize (McClintock 1949). She later worked on the Spm family of transposable elements, which comprises fully functional autonomous transposable elements and a variety of moderately to severely disabled relatives in maize. Her studies focused on defective Spm (dSpm) insertions into the A and A2 loci, both of which encode enzymes in the anthocyanin biosynthetic pathway. In Spm-suppressible plants, the transposon-disrupted gene continued to be expressed, but only in the absence of the autonomous Spm element. When Spm was present in the same plant, this type of transposondisrupted gene was not expressed. In Spm-dependent plants, the gene with the transposon insertion was generally expressed only in the presence of a fully functional Spm. Spm codes for a trans-acting protein that binds to the transposon sequence inserted into or near the gene. Binding of these proteins either increases or decreases expression of the transposon-disrupted gene. In some cells, these proteins, probably acting together with other cellular proteins, excise the transposon from the gene and move it to a different location (Fedoroff 1996). Expression and transposition of the Spm transposon of maize is controlled by interacting epigenetic and autoregulatory mechanisms. Methylation of critical transposable element sequences prevents both transcription and transposition in maize, heritably inactivating the Spm

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element. The promoter of the transposable element and a highly GC-rich downstream sequence are the methylation target sequences. The transposable element encodes two proteins necessary for transposition, TnpA and TnpD. In addition to its role in transposition, TnpA is both a positive and a negative regulator of transcription. TnpA represses the promoter of the transposable element when it is not methylated. When the transposable element is inactive and its promoter is methylated, TnpA activates the methylated promoter and facilitates both its transient and heritable demethylation (Fedoroff 1996). Pooma et al. (2002) investigated the effects of Mutator, another type of transposable element, and Spm insertions on the expression of maize a1, encoding for the enzyme dihydroflavonol-4-reductase, independently regulated by the transcription factors C1 and P. The a1-mum2 and a1-m2 alleles carry Mu1 and Spm insertions respectively in the anthocyanin regulatory element (ARE), a conserved motif present in several flavonoid biosynthetic gene promoters where MYB transcription factors bind and activate transcription. a1-m2 belongs to the Spm-dependent class of alleles in which the expression of the a1 gene happens only in the presence of trans-active, nondefective Spm elements. The a1-mum2 allele carries the nonautonomous Mu1 transposable element inserted in the ARE element. It belongs to the Mu-suppressible mutant class, in which the mutant phenotype (no anthocyanin pigmentation) is suppressed, resulting in pigmentation in the absence of the autonomous MuDR element. In the presence of MuDR, excision of Mu1 in the aleurone results in the formation of frequent revertant sectors on a colorless background, because of the inhibitory effect of MuDR on the expression of a1. The presence of MuDR is associated with an extensive hypomethylation of Mu1 and may interfere with the activation of a regulatory element present in Mu1. The unexpected pigmentation patterns provided by the Mu1 and Spm insertions are a consequence of the disruption by the transposons of cis-regulatory elements important for the regulation of a1. Studies in grape indicated that the presence of a retrotransposon named Gret1 in the promoter of MYB transcription factors controlling anthocyanin production in this species leads to the white-fruited phenotype (This et al. 2007; Walker et al. 2007). Subsequent studies by Cadle-Davidson and Owens (2008) further demonstrated that Gret1 copy number is significantly higher in white fruited accessions. Transposon activation and suppression may be responsible for some of the genetic variation that occurs in color or spur habit in pome fruits (Brown 2003). Retrotransposons have been identified in apple, including TRIM retrotransposons (Antonius-Klemola et al. 2006), copia-like

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retrotransposons (Tignon et al. 2001; Zhao et al. 2007a), and the dem1 retroelement (Yao et al. 2001). Transposable elements identified in apple include Ars1 and Ars2, two short repeat sequences present in high number in apple genome. Ars1 is thought to represent miniature inverted-repeat transposable elements (MITEs), while the identity of Ars2, though it is undoubtedly a transposable element, remains undetermined (Hadanou et al. 2003). Finally, Han and Korban (2007) reported the presence of Spring transposons in apple. Despite ample evidence of the presence of multiple kinds of transposable elements, there is no evidence associating transposable elements with fruit peel variegation in apple. Tignon et al. (2001) looked for cDNA fragments that might be differentially expressed in relation with apple fruit color, by comparing mRNA populations extracted from the fruit peel of ‘Jonagold’ and some of its mutants. A partial reverse transcriptase gene of several copia-like retrotransposons was isolated from expressed messenger RNAs from only one of the two color mutants of ‘Jonagold’ studied, suggesting that vegetative multiplication of apple could induce retrotransposon activation and lead to mutations linked to the interruption of genes by retrotransposons. A simple genetic basis for the red/yellow peel color polymorphism in apple was verified using RAPD markers. The A1 marker co-segregates with red color, while a1 and a2 are associated with yellow peel color (Cheng et al. 1996). Subsequent studies performed by Wakasa et al. (2003) demonstrated that the sequence information of the a1 and a2 fragments were virtually identical to A1 except for their respective insertions. Sequence data revealed that the 76-bp a1 insertion is an inverted repeat, while the 163-bp insertion in a2 contains a duplication of a 10-bp target site. The 163-bp insertion of a2 is called Majin and is characterized as a mobile element. There are approximately 6000 copies of Majin per haploid apple genome. Sequence-specific amplified polymorphisms (S-SAP), which allow the identification of dominant markers for the detection of variation in the DNA flanking retrotransposon insertion sites, were used to distinguish several clones of the cultivars ‘Gala’ and ‘Braeburn’. Bud mutations, which have generated new patented varieties of ‘Gala’ and ‘Braeburn’, appear to derive from retrotransposon insertions (Venturi et al. 2005). A sequence highly homologous to TRIM2, a terminal-repeat retrotransposon in miniature (Antonius-Klemola et al. 2006), is present 2.5 kb upstream of the MdMYB10 translation start site in several cultivars differing in the total levels and pattern of anthocyanin accumulation, indicating that the presence of the transposon alone does not explain

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pigment differences between cultivars. This transposon does not seem to insert new transcription start sites, since no transcription of the downstream MdMYB10 gene from the transposon was observed in the cultivars tested (Telias 2009). D. Other Mechanisms Other mechanisms might be responsible for color variability in apple fruit, including paramutation (Chandler et al. 1996), recurrent somatic mutations (Park et al. 2004), and the role of specific genome locations in controlling pigment patterns (Abe et al. 2002). These phenomena have not been implicated in regulation of apple anthocyanins, but the conservation of anthocyanin pathways across plant species leads to the possibility that similar mechanisms control accumulation in this fruit crop.

VI. CONCLUSIONS Apple peel color is an important fruit quality trait that affects consumer demand. Red color in apple is determined by the levels of anthocyanin, a subclass within the group of secondary metabolites flavonoids. Given that the levels of different flavonoids influence the antioxidant activity of the fruit, understanding the regulation of flavonoid biosynthesis becomes a priority for the improvement of the nutritional value of the fruit. Pigment accumulation in apple peels is determined by the genetic composition of the plant and the environmental conditions during fruit development, including light and temperature. A number of agricultural practices, including bagging, use of reflective films, application of growth regulators, and thinning, are used by growers around the world to obtain redder and more attractive fruits. Modern image-processing tools have enabled more precise color measurements that are valuable both for researchers and fruit processors. In the field of genetics, important recent discoveries include the identification of MdMYB10, a key transcriptional regulator of the anthocyanin pathway in apple, as well as the discovery of a novel MdMYB10 self-regulatory mechanism controlling the increased levels of anthocyanin accumulation in apple peel, apple flesh, and vegetative plant parts in ultra-red cultivars. These naturally occurring allelic variants are being deployed in breeding programs to produce marketable red-fleshed apple cultivars that are expected to impact the apple industry in the 21st century. In the field of epigenetics, studies have indicated an association

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between methylation levels in the promoter of MdMYB10 and the pattern in which anthocyanins are distributed in the peel. These exciting discoveries are broadening our understanding of the complex regulation of anthocyanin accumulation in plants. This new knowledge, coupled with a greater understanding of the effects of light, temperature, and other environmental factors, management practices, and the role of tissue organization, will undoubtedly provide new tools for achieving outstanding fruit quality, both in terms of visual appeal, and nutritional value.

LITERATURE CITED Abe, H., M. Nakano, A. Nakatsuka, M. Nakayama, M. Koshioka, and M. Yamagishi. 2002. Genetic analysis of floral anthocyanin pigmentation traits in Asiatic hybrid lily using molecular linkage maps. Theor. Appl. Genet. 105:1175–1182. Aharoni, A., C.H.R. De Vos, M. Wein, Z. Sun, R. Greco, A. Kroon, J.N.M. Mol, and A.P. O’Connell. 2001. The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco. Plant J. 28:319–332. Allan, A.C., R.P. Hellens, and W.A. Laing. 2008. MYB transcription factors that colour our fruit. Trends Plant Sci. 13:99–102. Antonius-Klemola, K., R. Kalendar, and A.H. Schulman. 2006. TRIM retrotransposons occur in apple and are polymorphic between varieties but not sports. Theor. Appl. Genet. 112:999–1008. Arakawa, O. 1988. Photoregulation of anthocyanin synthesis in apple fruit under UV-B and red light. Plant Cell Physiol. 29:1385–1389. Arakawa, O., Y. Hori, and R. Ogata. 1985. Relative effectiveness and interaction of ultraviolet-B, red and blue light in anthocyanin synthesis of apple fruit. Physiol. Plant. 64:323–327. Atchley, W.R., and W.M. Fitch. 1997. A natural classification of the basic helix loop helix class of transcription factors. Proc. Nat. Aca. Sci. (USA) 94:5172–5176. Awad, M.A., and A. de Jager. 2002a. Formation of flavonoids, especially anthocyanin and chlorogenic acid in ‘Jonagold’ apple skin: Influences of growth regulators and fruit maturity. Sci. Hort. 93:257–266. Awad, M.A., and A. de Jager. 2002b. Relationships between fruit nutrients and concentrations of flavonoids and chlorogenic acid in ‘Elstar’ apple skin. Sci. Hort. 92:265–276. Awad, M.A., P.S. Wagenmakers, and A. de Jager. 2001. Effects of light on flavonoid and chlorogenic acid levels in the skin of ‘Jonagold’ apples. Sci. Hort. 88:289–298. Bae, R., and K. Kim. 2006. Anatomical observations of anthocyanin rich cells in apple skins. HortScience 41:733–736. Bakhshi, D., and O. Arakawa. 2007. Induction of phenolic compounds biosynthesis with light irradiation in the flesh of red and yellow apples. J. Appl. Hort. 9:101–104. Ban, Y., C. Honda, H. Bessho, X. Pan, and T. Moriguchi. 2007a. Suppression subtractive hybridization identifies genes induce in response to UV-B irradiation in apple skin: Isolation of a putative UDP-glucose 4-epimerase. J. Expt. Bot. 58:1825–1834. Ban, Y., C. Honda, Y. Hatsuyama, M. Igarashi, and H. Bessho. 2007b. Isolation and functional analysis of a MYB transcription factor gene that is a key regulator for the development of red coloration in apple skin. Plant Cell Physiol. 48:958–970.

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Subject Index

Almond origin and dissemination, 23–81 Anthocyanin accumulation in apple, 357–391 Apple: anthocyanin accumulation, 357–391 deficit irrigation, 166–177 Apricot deficit irrigation, 160–163

Genetics and breeding: anthocyanin in apple, 363–370 vegetable, 324–344 Irrigation, deciduous fruits, 149–189 Juvenililty, rooting, 225–226

Cherry deficit irrigation, 163–164 Container-grown ornamentals, management, 253–297 Dedication: Thompson, M.M., xiii–xv Deficit irrigation, deciduous fruits, 149–189 Disease, hot water treatment, 191–212 Fruit crops: almond origin and dissemination, 23–81 apple deficit irrigation, 166–177 apricot deficit irrigation. 160–163 cherry deficit irrigation, 163–164 deficit irrigation, 149–189 hot water treatment, 191–212 olive oil composition, 83–147 peach deficit irrigation, 151–160 pear deficit irrigation, 177–180 plum deficit irrigation, 165–166 prune, deficit irrigation, 165–166

Nutrition (plant), ornamentals in containers, 253–297 Olive oil composition, 83–147 Ornamental plants: nutrient management in containers, 253–297 water management in containers, 253–297 Peach deficit irrigation, 151–160 Pear deficit irrigation, 177–180 Physiology: adventitious rooting, 213–225 vines, 1–21 Plum deficit irrigation, 165–166 Postharvest physiology, hot water treatment, 191–212 Propagation, adventious rooting in trees, 213–225 Prune deficit irrigation, 165–166

Horticultural Reviews, Volume 38 Edited by Jules Janick Copyright Ó 2011 Wiley-Blackwell. 393

394

SUBJECT INDEX

Root, adventitious, 213–225

Vines, biology and physiology, 1–21

Tree adventitious rooting, 213–225

Water relations: container-grown ornamentals 253–297 deficit irrigation, 149–189 deciduous orchards, 149–189

Vegetable crops: breeding, 324–344 hot water treatment, 191–212 world industry, 299–356

Cumulative Subject Index Volumes 1–38

A Abscisic acid: chilling injury, 15:78–79 cold hardiness, 11:65 dormancy, 7:275–277 genetic regulation, 16:9–14, 20–21 lychee, 28:437–443 mango fruit drop, 31:124–125 mechanical stress, 17:20 rose senescence, 9:66 stress, 4:249–250 Abscission: anatomy & histochemistry, 1:172–203 citrus, 15:145–182, 163–166 flower & petals, 3:104–107 mango fruit drop, 31:113–155 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, see Kiwifruit 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 Allium: development, 32:329–378 phytonutrients, 28:156–159 Alkekenge, history & iconography, 34:36–40

Almond: bloom delay, 15:100–101 breeding, 34:197–238 in vitro culture, 9:313 origin and dissemination, 38:23–81 postharvest technology & utilization, 20:267–311 wild of Kazakhstan, 29:262–265 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 & toxicity symptoms in fruits & 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 & morphology: Allium development, 32:329–378 apple flower & fruit, 10:273–308 apple tree, 12:265–305 asparagus, 12:71 cassava, 13:106–112 citrus, abscission, 15:147–156 daylily, 35:196–203 embryogenesis, 1:4–21, 35–40 fig, 12:420–424; 34:127–137 fruit abscission, 1:172–203 fruit storage, 1:314

Horticultural Reviews, Volume 38 Edited by Jules Janick Copyright Ó 2011 Wiley-Blackwell. 395

396 Anatomy & morphology (Continued ) 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 orchid, 5:281–283 navel orange, 8:132–133 pecan flower, 8:217–255 plant architecture, 32:1–61 pollution injury, 8:15 red bayberry, 20:92–96 waxes, 23:1–68 Androgenesis, woody species, 10:171–173 Angiosperms, embryogenesis, 1:1–78 Anthocyanin accumulation in apple, 38:357–391 Anthurium, fertilization, 5:334–335. See also Aroids, ornamental Antitranspirants, 7:334 cold hardiness, 11:65 Apical meristem, cryopreservation, 6:357–372 Apple: alternate bearing, 4:136–137 anatomy & morphology of flower & fruit, 10:273–309 anthocyanin accumulation, 38:357–391 bioregulation, 10:309–401 bitter pit, 11:289–355 bloom delay, 15:102–104 CA storage, 1:303–306 chemical thinning, 1:270–300 cider, 34:365–415 crop load, 31:233–292 deficit irrigation, 38:166–177 fertilization, 1:105 fire blight control, 1:423–474 flavor, 16:197–234 flower induction, 4:174–203 fruit cracking & splitting, 19:217–262 fruiting, 11:229–287 functional phytonutrients, 27:304 germplasm acquisition & resources, 29:1–61 in vitro, 5:241–243; 9:319–321 light, 2:240–248 maturity indices, 13:407–432

CUMULATIVE SUBJECT INDEX mealiness, 20:200 nitrogen metabolism, 4:204–246 pollination, 34:267–268 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 & anatomy, 12:265–305 vegetative growth, 11:229–287 watercore, 6:189–251 weight loss, 25:197–234 wild of Kazakhstan, 29:63–303, 305–315 yield, 1:397–424 Apricot: bloom delay, 15:101–102 CA storage, 1:309 deficit irrigation, 38:160–163 origin & dissemination, 22:225–266 wild of Kazakhstan, 29:325–326 Arabidopsis, molecular biology of flowering, 27:1–39, 41–77 Architecture, plant, 32:1–61 Aroids: edible, 8:43–99; 12:166–170 ornamental, 10:1–33 Arsenic, deficiency & toxicity symptoms in fruits & 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, 185–187, 187–188, 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 Aubergine, see Eggplant 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 mango fruit drop, 31:118–120

CUMULATIVE SUBJECT INDEX mechanical stress, 17:18–19 petal senescence, 11:31 Avocado: CA & 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 Banana: botany, dispersal, evolution, 36:117–164 CA & MA, 22:141–146 CA storage, 1:311–312 fertilization, 1:105 in vitro culture, 7:178–180 Banksia, 22:1–25 Barberry, wild of Kazakhstan, 29:332–336 Bean: CA storage, 1:352–353 fluid drilling of seed, 3:21 resistance to bacterial pathogens, 3:28–58 rust, 37:1–99 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 Belladonna, history & iconography, 34:14–19 Biennial bearing, see Alternate bearing Bilberry, wild of Kazakhstan, 29:347–348 Biochemistry, petal senescence, 11:15–43 Bioreactor technology, 24:1–30 Bioregulation, apple & pear, 10:309–401. See also Growth substances Bird damage, 6:277–278

397 Bitter gourd, 37:101–141 Bitter pit in apple, 11:289–355 Black currant, bloom delay, 15:104 Black pepper, 33:173–266 Blackberry: harvesting, 16:282–298 wild of Kazakhstan, 29:345 Bloom delay, deciduous fruits, 15:97 Blueberry: developmental physiology, 13:339–405 harvesting, 16:257–282 nutrition, 10:183–227 Boron: deficiency & toxicity symptoms in fruits & 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 Brassica classification, 28:27–28 Brassicaceae, in vitro, 5:232–235 Breeding, see Genetics & breeding Broccoli, CA storage, 1:354–355 Brussels sprouts, CA storage, 1:355 Bulb crops. See also Tulip development, 25:1–70 flowering, 25:1–70 genetics & breeding, 18:119–123 growth, 25: 1–70 industry, 36:1–115 in vitro, 18:87–169; 34:427–445 micropropagation, 18:89–113 root physiology, 14:57–88 virus elimination, 18:113–123 Bunch stem necrosis of grape, 35:355–395 C CA storage, see Controlled-atmosphere storage Cabbage: CA storage, 1:355–359 fertilization, 1:117–118 Cactus: crops, 18:291–320 grafting, 28:106–109 reproductive biology, 18:321–346

398 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 & toxicity symptoms in fruits & 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 Capsicum pepper, history & iconography, 34:62–74. See also Pepper Carbohydrate: fig, 12:436–437 grapevine, 37:143–211 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 postharvest physiology, 30:284–288 Caryophyllaceae, in vitro, 5:237–239 Cassava: crop physiology, 13:105–129 molecular biology, 26:85–159 multiple cropping, 30:355–50 postharvest physiology, 30:288–295 root crop, 12:158–166 Cauliflower, CA storage, 1:359–362 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

CUMULATIVE SUBJECT INDEX 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 & MA, 22:146–147 pollination, 34:266–267 Cherry: bloom delay, 15:105 CA storage, 1:308 deficit irrigation, 38:163–164 origin, 19:263–317 wild of Kazakhstan, 29:326–330 Chestnut: blight, 8:281–336 botany & horticulture, 31:293–349 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 China, protected cultivation, 30:37–82 Chlorine: deficiency & toxicity symptoms in fruits & 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 Cider, 34:365–415 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

CUMULATIVE SUBJECT INDEX functional phytochemicals, fruit, 27:269–315 honey bee pollination, 9:247–248 in vitro culture, 7:161–170 irrigation, 30:37–82 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 Classification: Brassica, 28:27–28 lettuce, 28:25–27 potato, 28:23–26 tomato, 28:21–23 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 & 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 & greenhouse crops; Hydroponic culture; Protected culture

399 Copper: deficiency & toxicity symptoms in fruits & nuts, 2:153 foliar application, 6:329–330 nutrition, 5:326–327 pine bark media, 9:122–123 Corynebacterium flaccumfaciens, 3:33, 46 Cotoneaster, wild of Kazakhstan, 29:316–317 Cowpea: genetics, 2:317–348 U.S. production, 12:197–222 Cranberry: botany & horticulture, 21:215–249 fertilization, 1:106 harvesting, 16:298–311 wild of Kazakhstan, 29:349 Crinum, 25:58 Crucifers phytochemicals, 28:150–156 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 grafting, 28:91–96 Cucumis melo, see Melon Cucurbita pepo, cultivar groups history, 25:71–170 Currant: harvesting, 16:311–327 wild of Kazakhstan, 29:341 Custard apple, CA & 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 mango fruit drop, 31:118–120 petal senescence, 11:30–31 rose senescence, 9:66

400 D Date palm: asexual embryogenesis, 7:185–187 in vitro culture, 7:185–187 Datura, history & iconography, 34:44–51 Daylength, see Photoperiod Daylily, 35:193–220 Dedication: Bailey, L.H., 1:v–viii Beach, S.A., 1:v–viii Bukovac, M.J., 6:x–xii Campbell, C.W., 19:xiii–xiv Cantliffe, D.J., 33:xi–xiii Cummins, J.N., 15:xii–xv De Hertogh, A.A., 26:xi–xiii Dennis, F.G., 22:xi–xii Faust, Miklos, 5:vi–xvi Ferguson, A.R., 35: xiii Goldman, I.L., 37:xiii–xxi Hackett, W.P., 12:x–xiii Halevy, A.H., 8:x–xii Hess, C.E., 13:x–xii Kader, A.A., 16:xii–xv Kamemoto, H., 24:x–xiii Kester, D.E., 30:xiii–xvii Looney, N.E., 18:xii–xv Magness, J.R., 2:vi–viii Maynard, D.N., 36:xiii–xv Mizrahi, Y., 34:xi–xv 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 Sedgely, M., 32:x–xii Sherman, W.B., 21:xi–xiii Smock, R.M., 7:x–xiii Sperling, C.E., 29:ix–x Stevens, M.A., 28:xi–xiii Thompson, M.M., 38:xiii–xv Warrington, I.J., 31:xi–xii Weiser, C.J., 11:x–xiii Whitaker, T.W., 3:vi–x Wittwer, S.H., 10:x–xiii Yang, S.F., 23:xi Deficiency symptoms, fruit & nut crops, 2:145–154

CUMULATIVE SUBJECT INDEX Deficit irrigation: 21:105–131; 32:111–165; 38:149–189 Defoliation, apple & pear bioregulation, 10:326–328 ‘Delicious’ apple, 1:397–424 Desiccation tolerance, 18:171–213 Dieffenbachia, see Aroids, ornamental Dioscorea, see Yam Disease: air pollution, 8:25 aroids, 8:67–69; 10:18; 12:168–169 bacterial, of bean, 3:28–58 bean rust, 37:1–99 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 hot water treatment, 38:191–212 hydroponic crops, 7:530–534 lettuce, 2:187–197 melon, 36:185–190 mycorrhizal fungi, 3:182–185 ornamental aroids, 10:18 resistance, acquired, 18:247–289 root, 5:29–31 rust, bean, 37:1–99 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 also Postharvest physiology bitterpit, 11:289–355 fig, 12:477–479 grape physiological, 35:355–395 pear fruit, 11:357–411 watercore, 6:189–251; 11:385–387 Dogrose, botany, breeding, horticulture, 36:199–255 Dormancy, 2:27–30 blueberry, 13:362–370 fruit trees, 7:239–300 tulip, 5:93 Drip irrigation, 4:1–48 Drought resistance, 4:250–251 cassava, 13:114–115

CUMULATIVE SUBJECT INDEX Durian, CA & MA, 22:147–148 Dwarfing: apple, 3:315–375 apple mutants, 12:297–298 by virus, 3:404–405 E Early bunch stem necrosis of grape, 35:355–395 Easter lily, fertilization, 5:352–355 Eggplant: grafting, 28:103–104 history & iconography, 34:25–35 phytochemicals, 28:162–163 Elderberry, 37:213–280 botany, 37: 215–226 horticulture, 37:226–224 wild of Kazakhstan, 29:349–350 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 bloom delay, 15:107–111 CA storage, 1:317–319, 348

401 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 mango fruit crop, 31:120–122 mechanical stress, 17:16–17 1-methylcyclopropene, 35:263–313 petal senescence, 11:16–19, 27–30 rose senescence, 9:65–66 Eucharis, 25:19–22 Eucrosia, 25:58 F Feed crops, cactus, 18:298–300 Feijoa, CA & MA, 22:148 Fertilization & 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: botany, horticulture, breeding, 34:113–195 industry, 12:409–490 ripening, 4:258–259 Filbert, in vitro culture, 9:313–314

402 Fire blight, 1:423–474 Flooding, fruit crops, 13:257–313 Floral scents, 24:31–53 Floricultural crops. See also individual crops Amaryllidaceae, 25:1–70 Banksia, 22:1–25 China, protected culture, 30:141–148 daylily, 35:193–220 dogrose, 36:199–255 fertilization, 1:98–104 flower bulb industry, 36:1–115 growth regulation, 7:399–481 heliconia, 14:1–55 Leucospermum, 22:27–90 postharvest physiology & senescence, 1:204–236; 3:59–143; 10:35–62; 11:15–43 Protea, 26:1–48 Florigen, 4:94–98 Flower & flowering: Amaryllidaceae, 25:1–70 apple anatomy & 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 bulb industry, 36:199–255 cactus, 18:325–335 citrus, 12:349–408 control, 4:159–160; 15:279–334 daylily, 35:193–220 development (postpollination), 19:1–58 fig, 12:424–429 girdling, 20:1–26 grape anatomy & morphology, 13:354–378 homeotic gene regulation, 27:41–77 honey bee pollination, 9:239–243 in vitro, 4:106–127 induction, 4:174–203, 254–256 initiation, 4:152–153 kiwifruit, 6:21–35; 12:316–318 Leucospermum, 22:27–90 lychee, 28:397–421 orchid, 5:297–300 pear bioregulation, 10:344–348

CUMULATIVE SUBJECT INDEX 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 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 strawberry, 28:325–349 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 industry, 31:47–112 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 & morphology, 10:283–297 apple bioregulation, 10:348–374 apple bitter pit, 11:289–355 apple crop load, 31:233–292 apple flavor, 16:197–234 apple maturity indices, 13:407–432 apple ripening & quality, 10:361–374 apple scald, 27:227–267 apple weight loss, 25:197–234 avocado development & ripening, 10:229–271 banana, 36:117–164

CUMULATIVE SUBJECT INDEX bloom delay, 15:97–144 blueberry development, 13:378–390 CA storage & quality, 8:101–127 cactus physiology, 18:335–341 chilling injury, 15:63–95 coating physiology, 26:161–238 cracking, 19:217–262; 30:163–184 diseases in CA storage, 3:412–461 drop, apple & pear, 10:359–361 elderberry, 37:213–280 fig, 4:258–259; 12:409–490; 34:113–195 fresh cut, 30:185–251 functional phytochemicals, 27:269–315 grape, 35:355–395 growth measurement, 24:373–431 jujube, 32:229–298 kiwifruit, 6:35–48; 12:316–318 loquat, 23:233–276 lychee, 28:433–444 mango fruit drop, 31:113–155 maturity indices, 13:407–432 melon, 36:165–198 navel orange, 8:129–179 nectarine, postharvest, 11:413–452 nondestructive postharvest quality evaluation, 20:1–119 olive physiology, 31:157–231 olive processing, 25:235–260 pawpaw, 31:351–384 peach, postharvest, 11:413–452 pear, bioregulation, 10:348–374 pear, fruit disorders, 11:357–411 pear maturity indices, 13:407–432 pear ripening & quality, 10:361–374 pear scald, 27:227–267 pear volatiles, 28:237–324 pistachio, 3:382–391 phytochemicals, 28:125–185 plum, 23:179–231 pollination, 34:239–275 pomegranate, 35:127–191 quality & pruning, 8:365–367 red bayberry, 30:83–113 ripening, 5:190–205 rose, wild of Kazakhstan, 29:353–360 set, 1:397–424; 4:153–154 set in navel oranges, 8:140–142 size & thinning, 1:293–294; 4:161 softening, 5:109–219; 10:107–152 splitting, 19:217–262

403 strawberry growth & ripening, 17:267–297 texture, 20:121–224 thinning, apple & pear, 10:353–359 tomato cracking, 30:163–184 tomato parthenocarpy, 6:65–84 tomato ripening, 13:67–103 volatiles, pear, 28:237–324 Fruit crops. See also Individual crop almond origin and dissemination, 38:23–81 alternate bearing, 4:128–173 apple bitter pit, 11:289–355 apple crop load, 31:233–292 apple deficit irrigation, 38:166–177 apple flavor, 16:197–234 apple fruit splitting & cracking, 19:217–262 apple germplasm, 29:1–61, 63–303 apple growth, 11:229–287 apple maturity indices, 13:407–432 apple scald, 27:227–267 apple, wild of Kazakhstan, 29:63–303, 305–315 apricot deficit irrigation, 38:160–163 apricot, origin & dissemination, 22:225–266 apricot, wild of Kazakhstan, 29:325–326 architecture, 32:1–61 avocado flowering, 8:257–289 avocado rootstocks, 17:381–429 banana, 36:117–164 barberry, wild of Kazakhstan, 29:332–336 berry crop harvesting, 16:255–382 bilberry, wild of Kazakhstan, 29:347–348 blackberry, wild of Kazakhstan, 29:345 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 CA & MA for tropicals, 22:123–183 CA storage, 1:301–336 CA storage diseases, 3:412–461 carbohydrate reserves, 10:403–430 cherry deficit irrigation, 38:163–164

404 Fruit crops (Continued ) cherry origin, 19:263–317 cherry, wild of Kazakhstan, 29:326–330 chilling injury, 15:145–182 chlorosis, 9:161–165 cider, 34:365–414 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 citrus irrigation, 30:37–82 citrus nutrition diagnostics, 34:277–364 cotoneaster, wild of Kazakhstan, 29:316–317 cranberry, 21:215–249 cranberry harvesting, 16:298–311 cranberry, wild of Kazakhstan, 29:349 currant harvesting, 16:311–327 currant, wild of Kazakhstan, 29:341 deficit irrigation, 21:105–131; 38:149–189 dormancy release, 7:239–300 elderberry, 37:213–280 elderberry, wild of Kazakhstan, 29:349–350 Ericaceae nutrition, 10:183–227 fertilization, 1:104–106 fig, industry, 12:409–490; 34:113–195 fireblight, 11:423–474 flowering, 12:223–264 foliar nutrition, 6:287–355 frost control, 11:45–109 gooseberry, wild of Kazakhstan, 29:341–342 grape flower anatomy & morphology, 13:315–337 grape harvesting, 16:327–348 grape irrigation, 27:189–225 grape nitrogen metabolism, 14:407–452 grape physiological disorder, 35:355–395 grape pruning, 16:235–254, 336–340 grape root, 5:127–168 grape seedlessness, 11:164–176 grape, wild of Kazakhstan, 29:342–343 grapevine carbohydrates, 37:143–211 grapevine pruning, 16:235–254, 336–340

CUMULATIVE SUBJECT INDEX greenhouse, China, 30:149–158 honey bee pollination, 9:244–250, 254–256 hot water treatment, 38:191–212 jojoba, 17:233–266 jujube, 32:229–298 in vitro culture, 7:157–200; 9:273–349 irrigation, deficit, 21:105–131 kiwifruit, 6:1–64; 12:307–347; 33:1–121 lingonberry, 27:79–123 lingonberry, wild of Kazakhstan, 29:348–349 longan, 16:143–196 loquat, 23:233–276 lychee, 16:143–196; 28:393–453 melon, 36:165–198 mango fruit drop, 31:113–155 mountain ash, wild of Kazakhstan, 29:322–324 mulberry, wild of Kazakhstan, 29:350–351 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 oleaster, wild of Kazakhstan, 29:351–353 olive oil composition, 38:83–147 olive physiology, 31:157–231 olive salinity tolerance, 21:177–214 orange, navel, 8:129–179 orchard floor management, 9:377–430 packaging, modified humidity, 37:281–329 pawpaw, 31:351–384 peach deficit irrigation, 38:151–160 peach orchard systems, 32:63–109 peach origin, 17:331–379 peach postharvest, 11:413–452 peach thinning, 28:351–392 pear deficit irrigation, 38:177–180 pear fruit disorders, 11:357–411; 27:227–267 pear maturity indices, 13:407–432 pear scald, 27:227–267 pear volatiles, 28:237–324 pear, wild of Kazakhstan, 29:315–316 pecan flowering, 8:217–255 photosynthesis, 11:111–157

CUMULATIVE SUBJECT INDEX Phytophthora control, 17:299–330 plum deficit irrigation, 38:165–166 plum origin, 23:179–231 plum, wild of Kazakhstan, 29:330–332 pollination, 34:239–275 pomegranate, 35:127–191 prune deficit irrigation, 38:165–166 pruning, 8:339–380 rambutan, 16:143–196 raspberry, 11:185–228 raspberry, wild of Kazakhstan, 29:343–345 red bayberry, 30:83–113 roots, 2:453–457 sapindaceous fruits, 16:143–196 sea buckthorn, wild of Kazakhstan, 29:361 short life & replant problem, 2:1–116 strawberry fruit growth, 17:267–297 strawberry harvesting, 16:348–365 strawberry, wild of Kazakhstan, 29:347 summer pruning, 9:351–375 Vaccinium nutrition, 10:183–227 vacciniums, wild of Kazakhstan, 29:347–349 viburnam, wild of Kazakhstan, 29:361–362 virus elimination, 28:187–236 water status, 7:301–344 water stress, 32:111–165 Functional phytochemicals, fruit, 27:269–315 Fungi: bean rust, 37:1–99 fig, 12:451–474 mushroom, 6:85–118 mycorrhiza, 3:172–213; 10:211–212 pathogens in postharvest storage, 3:412–461 rust, bean, 37:1–99 truffle cultivation, 16:71–107 Fungicide, & apple fruit set, 1:416 G Galanthus, 25:22–25 Gboma eggplant, history & iconography, 34:25 Garlic, 33:123–172 CA storage, 1:375

405 Genetics & breeding: almond, 34:197–238 anthocyanin in apple, 38:363–370 aroids (edible), 8:72–75; 12:169 aroids (ornamental), 10:18–25 bean, bacterial resistance, 3:28–58 bitter gourd, 37:120–131 bloom delay in fruits, 15:98–107 bulbs, flowering, 18:119–123 cassava, 12:164 chestnut blight resistance, 8:313–321 citrus cold hardiness, 7:221–223 cranberry, 21:236–239 daylily, 35:207–214 dogrose, 25:225–244 embryogenesis, 1:23 fig, 12:432–433; 34:165–170 fire blight resistance, 1:435–436 flower bulb crops, 36:16–36 flower longevity, 1:208–209 flowering, 15:287–290, 303–305, 306–309, 314–315; 27:1–39, 41–77 ginseng, 9:197–198 gladiolus, 36:20–23 grafting use, 28:109–115 horseradish, 35:247–255 in vitro techniques, 9:318–324; 18:119–123 iris (bulbous), 36:23–25 kiwifruit, 33:1–121 lettuce, 2:185–187 lily, 36:25–29 lingonberry, 27:108–111 loquat, 23:252–257 macadamia, 35:1–125 melon, 36:165–198 muscadine grapes, 14:357–405 mushroom, 6:100–111 narcissus, 36:29–30 navel orange, 8:150–156 nitrogen nutrition, 2:410–411 pineapple, 21:138–164 plant regeneration, 3:278–283 pollution insensitivity, 8:18–19 pomegranate, 35:172–175 potato tuberization, 14:121–124 rhododendron, 12:54–59 sweet potato, 12:175 sweet sorghum, 21:87–90 tomato parthenocarpy, 6:69–70

406 Genetics & breeding (Continued ) tomato ripening, 13:77–98 tulip, 36:30–33 tree short life, 2:66–70 vegetable, 38:324–344 Vigna, 2:311–394 waxes, 23:50–53 woody legume tissue & cell culture, 14:311–314 yam (Dioscorea), 12:183 Genetic variation: alternate bearing, 4:146–150 banana, 36:117–164 dogrose, 36:225–244 flower bulb crops. 36:16–36 kiwifruit, 33:1–121 melon, 36:165–198 photoperiodic response, 4:82 pollution injury, 8:16–19 temperature-photoperiod interaction, 17:73–123 wild apple, 29:63–303 Geophyte, see Bulb, tuber Geranium, fertilization, 5:355–357 Germination, seed, 2:117–141, 173–174; 24:229–275 Germplasm: acquisition, apple, 29:1–61 characterization, apple, 29:45–56 cryopreservation, 6:357–372 in vitro, 5:261–264; 9:324–325 macadamia, 35:1–125 pineapple, 21:133–175 pomegranate, 35:134–141 resources, apple, 29:1–61 Gibberellin: abscission, citrus, 15:166–167 bloom delay, 15:111–114 citrus, abscission, 15:166–167 cold hardiness, 11:63 dormancy, 7:270–271 floral promoter, 4:114 flowering, 15:219–293, 315–318 genetic regulation, 16:15 grape root, 5:150–151 mango fruit drop, 31:113–155 mechanical stress, 17:19–20 Ginger postharvest physiology, 30:297–299 Ginseng, 9:187–236

CUMULATIVE SUBJECT INDEX Girdling, 1;416–417; 4:251–252; 30:1–26 Glucosinolates, 19:99–215 Gooseberry, wild of Kazakhstan, 29:341–342 Gourd, history, 25:71–171 Graft & grafting: herbaceous, 28:61–124 history, 35:437–493 incompatibility, 15:183–232 phase change, 7:136–137, 141–142 rose, 9:56–57 Grape: CA storage, 1:308 carbohydrates, 37:143–211 chlorosis, 9:165–166 flower anatomy & morphology, 13:315–337 functional phytochemicals, 27:291–298 harvesting, 16:327–348 irrigation, 27:189–225 muscadine breeding, 14:357–405 nitrogen metabolism, 14:407–452 physiological disorder, 35:355–395 pollen morphology, 13:331–332 pruning, 16:235–254, 336–340 root, 5:127–168 seedlessness, 11:159–187 sex determination, 13:329–331 wild of Kazakhstan, 29:342–343 Gravitropism, 15:233–278 Greenhouse & greenhouse crops: carbon dioxide, 7:357–360, 544–545 China protected cultivation, 30:115–162 energy efficiency, 1:141–171; 9:1–52 growth substances, 7:399–481 nutrition & 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 bloom delay, 15:107–119

CUMULATIVE SUBJECT INDEX 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 girdling, 20:1–26 grape seedlessness, 11:177–180 hormone reception, 26:49–84 in vitro flowering, 4:112–115 mango fruit drop, 31:113–155 mechanical stress, 17:16–21 meristem & shoot-tip culture, 5:221–227 1-methylcyclopropene, 35:355–395 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 Hawthorne, wild of Kazakhstan, 29:317–322 Hazelnut, wild of Kazakhstan, 29:365–366. See also Filbert Health phytochemicals: fruit, 27:269–315 horseradish, 35:243–244 pomegranate, 35:175–177 vegetables, 28:125–185 Heat treatment (postharvest), 22:91–121

407 Heliconia, 14:1–55 Henbane, history & iconography, 34:10–14 Herbaceous plants, subzero stress, 6:373–417 Hippeastrum, 25:29–34 Histochemistry: flower induction, 4:177–179 fruit abscission, 1:172–203 Histology, flower induction, 4:179–184. See also Anatomy & morphology History & iconography: alkekenge, 34:36–40 aubergine, see Eggplant belladonna, 34:14–19 capsicum pepper, 34:62–74 datura, 34:44–51 eggplant, 34:25–35. gboma eggplant, 34:25 grafting, 35:437–493 henbane, 34:10–14 husk tomato, 34:40–44 Lycium spp., 34:23 mandrake, 34:4–10 potato, 34:85–89 scarlet eggplant, 34:25 Scopolia spp., 34:20–23 Solanaceae, 34:1–111 Solanum dulcamara, 34:25 Solanum nigrum, 34:23–24 tobacco, 34:51–62 tomato, 34:75–85 Withania spp., 34:19–20 Honey bee, 9:237–272 Honeysuckle, wild of Kazakhstan, 29:350 Horseradish: botany, horticulture, breeding, 35:221–265 CA storage, 1:368 Husk tomato, history & iconography, 34:40–44 Hydrolases, 5:169–219 Hydroponic culture, 5:1–44; 7:483–558 Hymenocallis, 25:59 Hypovirulence, in Endothia parasitica, 8:299–310 I Ice, formation & spread in tissues, 13:215–255

408 Ice-nucleating bacteria, 7:210–212; 13:230–235 Iconography, see History 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; 34:417–445 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; 34:417–445 geophytes, 34:417–445 pear propagation, 10:325–326 phase change, 7:144–145 propagation, 3:214–314; 5:221–277; 7:157–200; 9:57–58, 273–349; 17:125–172 thin cell layer morphogenesis, 14:239–264 woody legume culture, 14:265–332 Industrial crops, cactus, 18:309–312 Insects & 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 particle film control, 31:1–45 tree short life, 2:52 tulip, 5:63, 92 waxes, 23:1–68 Integrated pest management, greenhouse crops, 13:1–66 Invasive plants, 32:379–437 Iron: deficiency & toxicity symptoms in fruits & nuts, 2:150 deficiency chlorosis, 9:133–186

CUMULATIVE SUBJECT INDEX Ericaceae nutrition, 10:193–195 foliar application, 6:330 nutrition, 5:324–325 pine bark media, 9:123 Irrigation: citrus, 30:37–82 deficit, deciduous orchards, 21:105–131; 32:111–165, 38:149–189 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 scheduling, 32:111–165 Ismene, 25:59 J Jojoba, 17:233–266 Jujube, 32:229–298 Juvenility, 4:111–112 pecan, 8:245–247 rooting, 38:225–226 tulip, 5:62–63 woody plants, 7:109–155 K Kale, fluid drilling of seed, 3:21 Kazakhstan, see Wild fruits & nuts Kiwifruit: botany, 6:1–64 genetic resources and breeding, 33:1–121 nutrition and vine growth, 12:307–347 L Lamps, for plant growth, 2:514–531 Lanzon, CA & MA, 22:149 Leaves: apple morphology, 12:283–288 flower induction, 4:188–189 Leek: CA storage, 1:375 fertilization, 1:118 Leguminosae, in vitro, 5:227–229; 14:265–332 Lemon, rootstock, 1:244–246. See also Citrus

CUMULATIVE SUBJECT INDEX Lettuce: CA storage, 1:369–371 classification, 28:25–27 fertilization, 1:118 fluid drilling of seed, 3:14–17 industry, 2:164–207 seed germination, 24:229–275 tipburn, 4:49–65 Leucadendron, 32:167–228 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 wild of Kazakhstan, 29:348–349 Longan, CA & MA, 22:150. See also Sapindaceous fruits Loquat: botany & horticulture, 23:233–276 CA & MA, 22:149–150 Lychee. See also Sapindaceous fruits CA & MA, 22:150 flowering, 28:397–421 fruit abscission, 28:437–443 fruit development, 28:433–436 pollination, 28:422–428 reproductive biology, 28:393–453 Lycium spp., history & iconography, 34:23 Lycoris, 25:39–43 M Macadamia, genetic resources & development, 35:1–125 Magnesium: container growing, 9:84–85 deficiency & toxicity symptoms in fruits & nuts, 2:148 Ericaceae nutrition, 10:196–198 foliar application, 6:331

409 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 Mandarin, rootstock, 1:250–252 Mandrake, history & iconography, 34:4–10 Manganese: deficiency & toxicity symptoms in fruits & 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 & MA, 22:151–157 CA storage, 1:313 fruit drop, 31:113–155 in vitro culture, 7:171–173 Mangosteen, CA & MA, 22:157 Master Gardener program, 33:393–420 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 Taxus, 32:299–327 Melon: diversity, 36:176–198 grafting, 28:96–98 Meristem culture, 5:221–277 Metabolism: flower, 1:219–223 nitrogen in citrus, 8:181–215 seed, 2:117–141 1-Methylcyclopropene, 35:263–313 Micronutrients: container growing, 9:85–87 pine bark media, 9:119–124 Micropropagation. See also In vitro; Propagation bulbs, flowering, 18:89–113 environmental control, 17:125–172

410 Micropropagation (Continued ) nuts, 9:273–349 rose, 9:57–58 temperate fruits, 9:273–349 tropical fruits & palms, 7:157–200 Microtu, see Vole Modified atmosphere (MA) for tropical fruits, 22:123–183 Modified humidity packaging, 37:281–329 Moisture & 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 Momordica charantia, see Bitter gourd 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 red bayberry, 30:92–96 Moth bean, genetics, 2:373–374 Mountain ash, wild of Kazakhstan, 29:322–324 Mulberry, wild of Kazakhstan, 29:350–351 Multiple cropping, 30:355–500 Mung bean, genetics, 2:348–364 Musa, see Banana 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: biochemistry & biology, 36:257–287 container growing, 9:93 Ericaceae, 10:211–212 fungi, 3:172–213 grape root, 5:145–146 Myrica, see Red bayberry N Narcissus, 25:43–48 Navel orange, 8:129–179

CUMULATIVE SUBJECT INDEX 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 & toxicity symptoms in fruits & 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 trickle irrigation, 4:29–30 vegetable crops, 22:185–223 Nomenclature, 28:1–60 Nondestructive quality evaluation of fruits & vegetables, 20:1–119 Nursery crops: fertilization, 1:106–112 nutrition, 9:75–101 Nut crops. See also individual crop almond breeding, 34:197–238 almond postharvest technology & utilization, 20:267–311 almond, wild of Kazakhstan, 29:262–265 chestnut blight, 8:291–336 chestnut, botany & horticulture, 31:293–349 fertilization, 1:106 hazelnut, wild of Kazakhstan, 29:365–366 honey bee pollination, 9:250–251 in vitro culture, 9:273–349 macadamia, 35:1–125 nutritional ranges, 2:143–164 pine, wild of Kazakhstan, 29:368–369 pistachio culture, 3:376–396

CUMULATIVE SUBJECT INDEX pistachio, wild of Kazakhstan, 29:366–368 walnut, wild of Kazakhstan, 29:369–370 Nutrient: citrus diagnotics, 34:277–364 concentration in fruit & 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 & 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 phytochemicals in fruit, 27:269–315 phytochemicals in vegetables, 28:125–185 steroidal alkalois, 25:171–196 Nutrition (plant): air pollution, 8:22–23, 26 blueberry, 10:183–227 calcifuge, 10:183–227 citrus diagnostics, 34:277–364 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 & nut crops, 2:143–164 ginseng, 9:209–211 greenhouse crops, 5:317–403 kiwifruit, 12:325–332 mycorrhizal fungi, 3:185–191 navel orange, 8:162–166 nitrogen in apple, 4:204–246 nitrogen in vegetable crops, 22:185–223 nutrient film techniques, 5:18–21, 31–53 ornamentals in containers, 38:253–297 ornamental aroids, 10:7–14 pine bark media, 9:103–131 raspberry, 11:194–195 slow-release fertilizers, 1:79–139

411 O Oil palm: asexual embryogenesis, 7:187–188 in vitro culture, 7:187–188 Okra: botany & horticulture, 21:41–72 CA storage, 1:372–373 Oleaster, wild of Kazakhstan, 29:351–353 Olive: alternate bearing, 4:140–141 oil composition, 38:83–147 physiology, 31:147–231 pollination, 34:265–266 processing technology, 25:235–260 salinity tolerance, 21:177–214 Onion: CA storage, 1:373–375 fluid drilling of seed, 3:17–18 Opium poppy, 19:373–408 Orange. See also Citrus alternate bearing, 4:143–144 sour, rootstock, 1:242–244 sweet, rootstock, 1:252–253 trifoliate, rootstock, 1:247–250 Orchard & 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 physiology, 5:279–315 pollination regulation of flower development, 19:28–38 Organic horticulture, sustainability, 36:257–287; 37:331–362 Organogenesis, 3:214–314. See also In vitro; Tissue culture Ornamental plants. See also individual plant Amaryllidaceae, 25:1–70 Banksia, 22:1–25 cactus grafting, 28:106–109 chlorosis, 9:168–169 cotoneaster, wild of Kazakhstan, 29:316–317 fertilization, 1:98–104, 106–116 flowering bulb roots, 14:57–88 flowering bulbs in vitro, 18:87–169

412 Ornamental plants (Continued ) foliage acclimatization, 6:119–154 foliage industry, 31:47–112 geophytes, in vitro, 34:417–445 heliconia, 14:1–55 honeysuckle, wild of Kazakhstan, 29:350 Leucadendron, 32:167–228 Leucospermum, 22:27–90 nutrient management in containers, 38:253–297 oleaster, wild of Kazakhstan, 29:351–353 orchid pollination regulation, 19:28–38 poppy, 19:373–408 protea leaf blackening, 17:173–201 rhododendron, 12:1–42 rose, wild of Kazakhstan, 29:353–360 Salix, 34:447–489 viburnam, wild of Kazakhstan, 29:361–362 water management in containers, 38:253–297 Osier, see Salix P Paclobutrazol, see Triazole Papaya: asexual embryogenesis, 7:176–177 CA & MA, 22:157–160 CA storage, 1:314 in vitro culture, 7:175–178 Parasitic weeds, 33:267–349 Parsley: CA storage, 1:375 drilling of seed, 3:13–14 Parsnip, fluid drilling of seed, 3:13–14 Parthenocarpy, tomato, 6:65–84 Particle films, 31:1–45 Passion fruit: in vitro culture, 7:180–181 CA & MA, 22:160–161 Pathogen elimination, in vitro, 5:257–261 Pawpaw, 31:351–384 Peach: bloom delay, 15:105–106 CA storage, 1:309–310 deficit irrigation, 38:151–160 orchard systems, 32:63–109 origin, 17:333–379 postharvest physiology, 11:413–452

CUMULATIVE SUBJECT INDEX short life, 2:4 summer pruning, 9:351–375 thinning, 28:351–392 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 deficit irrigation, 38:177–180 decline, 2:11 fire blight control, 1:423–474 fruit disorders, 11:357–411; 27:227–267 fruit volatiles, 28:237–324 in vitro, 9:321 maturity indices, 13:407–432 root distribution, 2:456 scald, 27:227–267 short life, 2:6 wild of Kazakhstan, 29:315–316 Pecan: alternate bearing, 4:139–140 fertilization, 1:106 flowering, 8:217–255 in vitro culture, 9:314–315 Pejibaye, in vitro culture, 7:189 Pepper (Capsicum): CA storage, 1:375–376 fertilization, 1:119 fluid drilling in seed, 3:20 grafting, 28:104–105 phytochemicals, 28:161–162 Pepper (Piper), 33:173–266 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 parasitic weeds, 33:267–349 particle films, 31:1–45

CUMULATIVE SUBJECT INDEX 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 & toxicity symptoms in fruits & 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 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 also Postharvest physiology abuscular mycorrhizae, 36:257–290 adventitious rooting, 38:213–225 Allium development, 32:329–378 apple crop load, 31:233–292 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 citrus irrigation, 30:55–67 conditioning 13:131–181

413 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 flower bulb crops, 36:36–49 fruit ripening, 13:67–103 fruit softening, 10:107–152 ginseng, 9:211–213 girdling, 30:1–26 glucosinolates, 19:99–215 grafting, 28:78–84 grapevine carbohydrates, 37:143–211 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 lychee reproduction, 28:393–453 male sterility, 17:103–106 mango fruit drop, 31:113–155 mechanical stress, 17:1–42 1-methylcyclopropene, 35:253–313 mycorrhizae, 36:257–289 nitrogen metabolism in grapevine, 14:407–452 nutritional quality & CA storage, 8:118–120 olive, 31:157–231 olive salinity tolerance, 21:177–214 orchid, 5:279–315 particle films, 31:1–45 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 red bayberry, 30:96–99 regulation, 11:1–14 root pruning, 6:158–171 roots of flowering bulbs, 14:57–88 rose, 9:3–53 salinity hormone action, 16:1–32

414 Physiology (Continued ) salinity tolerance, 16:33–69 seed, 2:117–141 seed priming, 16:109–141 strawberry flowering, 28:325–349 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 vines, 38:1–21 volatiles, 17:43–72 water relations cut flowers, 18:1–85 watercore, 6:189–251 waxes, 23:1–68 Phytochemicals, functional: fruits, 27:269–315 vegetables, 28:125–185 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 Pine, wild of Kazakhstan, 29:368–369 Pine bark, potting media, 9:103–131 Pineapple: CA & MA, 22:161–162 CA storage, 1:314 genetic resources, 21:138–141 in vitro culture, 7:181–182 Piper, see Black pepper Pistachio: alternate bearing, 4:137–139 culture, 3:376–393 in vitro culture, 9:315 pollination, 34:264 wild of Kazakhstan, 29:366–368 Plant: architecture, 32:1–63 classification, 28:1–60 protection, short life, 2:79–84 systematics, 28:1–60

CUMULATIVE SUBJECT INDEX Plantain: CA & MA, 22:141–146 in vitro culture, 7:178–180 Plastic cover, sod production, 27:317–351 Plug transplant technology, 35:397–436 Plum: CA storage, 1:309 deficit irrigation, 38:165–166 origin, 23:179–231 wild of Kazakhstan, 29:330–332 Poinsettia, fertilization, 1:103–104; 5:358–360 Pollen, desiccation tolerance, 18:195 Pollination: apple, 1:402–404 artificial, 34:239–276 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 lychee, 28:422–428 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: chilling injury, 15:80 in horticulture, 14:333–356 mango fruit drop, 31:125–127 Polygalacturonase, 13:67–103 Pomegranate, 35:127–191 Poppy, opium, 19:373–408 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

CUMULATIVE SUBJECT INDEX asparagus, 12:69–155 bitter melon, 35:343–344 CA for tropical fruit, 22:123–183 CA for storage & quality, 8:101–127 carrot storage, 30:284–288 cassava storage, 30:288–295 chlorophyll fluorescence, 23:69–107 coated fruits & vegetables, 26:161–238 cucumber, 35:325–330 cucurbits, 35: 315–354 cut flower, 1:204–236; 3:59–143; 10:35–62 fig, 34:146–164 foliage plants, 6:119–154 fresh-cut fruits & vegetables, 30:85–255 fruit, 1:301–336 fruit softening, 10:107–152 ginger storage, 30:297–299 hot water treatment, 389:191–212 Jerusalem artichoke storage, 30:271–276 heat treatment, 22:91–121 lettuce, 2:181–185 low-temperature sweetening, 17:203–231; 30:317–355 luffa, 35:344–345 MA for tropical fruit, 22:123–183 melon, 35:330–337 modified humidity packaging, 37:281–329 navel orange, 8:166–172 nectarine, 11:413–452 nondestructive quality evaluation, 20:1–119 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 potato low temperature sweetening, 30:317–355 potato storage, 30:259–271 protea leaf blackening, 17:173–201 pumpkin & squash, 35:337–341 quality evaluation, 20:1–119 scald, 27:227–267 seed, 2:117–141 sweet potato storage, 30:276–284 taro storage, 30:295–297 texture in fresh fruit, 20:121–244

415 tomato fruit ripening, 13:67–103 tomato posthavest losses, 33:351–391 vegetables, 1:337–394 watercore, 6:189–251; 11:385–387 watermelon, 35:319–325 wax gourd, 35:342 Potassium: container growing, 9:84 deficiency & toxicity symptoms in fruits & 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 classification, 28:23–26 fertilization, 1:120–121 history & iconography, 34:85–89 low temperature sweetening, 17:203–231; 30:317–353 phytochemicals, 28:160–161 postharvest physiology & storage, 30:259–271 tuberization, 14:89–198 Processing, table olives, 25:235–260 Propagation. See also In vitro adventitious rooting in trees, 38:213–225 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 foliage plants, 31:47–112 ginseng, 9:206–209 macadamia, 35:92–95 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 Protea: floricultural crop, 26:1–48 leaf blackening, 17:173–201 Proteaceous flower crop: Banksia, 22:1–25 Leucospermum, 22:27–90 Leukcadendron, 32:167–228 Protea, 17:173–201; 26:1–48

416 Protected crops, carbon dioxide, 7:345–398 Protoplast culture, woody species, 10:173–201 Prune deficit irrigation, 38:165–166 Pruning: alternate bearing, 4:161 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 physiology, 8:339–380 plant architecture, 32:1–63 root, 6:155–188 Prunus. See also 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 & 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, CA & MA, 22:163. See also Sapindaceous fruits Raspberry: harvesting, 16:282–298 productivity, 11:185–228 wild of Kazakhstan, 29:343–345 Red bayberry, 30:83–113 Rejuvenation: rose, 9:59–60 woody plants, 7:109–155 Replant problem, deciduous fruit trees, 2:1–116

CUMULATIVE SUBJECT INDEX 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 Rice bean, genetics, 2:375–376 Root: adventitious, 38:213–225 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 & tuber crops: Amaryllidaceae, 25:1–79 aroids, 8:43–99; 12:166–170 carrot postharvest physiology, 30:284–288 cassava crop physiology, 13:105–129 cassava molecular biology, 26:85–159 cassava multiple cropping, 30:355–50 cassava postharvest physiology, 30:288–295 cassava root crop, 12:158–166 horseradish, 35:221–265 low-temperature sweetening, 17:203–231; 30:317 -355 minor crops, 12:184–188 potato low temperature sweetening, 30:317–355 potato tuberization, 14:89–188 sweet potato, 12:170–176 sweet potato physiology, 23:277–338 sweet potato postharvest physiology, 30:276–284 taro postharvest physiology, 30:295–297 yam (Dioscorea), 12:177–184 Rootstocks: alternate bearing, 4:148 apple, 1:405–407; 12:295–297

CUMULATIVE SUBJECT INDEX avocado, 17:381–429 citrus, 1:237–269 clonal history, 35:475–478 cold hardiness, 11:57–58 fire blight, 1:432–435 light interception, 2:249–250 macadamia, 35:92–95 navel orange, 8:156–161 root systems, 2:471–474 stress, 4:253–254 tree short life, 2:70–75 Rosa, see Dogrose; Rose Rosaceae, in vitro, 5:239–248 Rose: dogrose, 36:199–255 fertilization, 1:104; 5:361–363 growth substances, 9:3–53 in vitro, 5:244–248 wild of Kazakhstan, 29:353–360 S Salinity: air pollution, 8:25–26 citrus irrigation, 30:37–83 olive, 21:177–214 soils, 4:22–27 tolerance, 16:33–69 Salix, botany & horticulture, 34:447–489 Sambucus, see Elderberry Sapindaceous fruits, 16:143–196 Sapodilla, CA & MA, 22:164 Scadoxus, 25:25–28 Scald, apple & pear, 27:227–265 Scarlet eggplant, history & iconography, 34:25 Scopolia spp., history & iconography, 34:20–23. Scoring & fruit set, 1:416–417 Sea buckthorn, wild of Kazakhstan, 29:361 Secondary metabolites, woody legumes, 14:314–322 Seed: abortion, 1:293–294 apple anatomy & morphology, 10:285–286 conditioning, 13:131–181 desiccation tolerance, 18:196–203 environmental influences on size & composition, 13:183–213 flower induction, 4:190–195

417 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 & storage, 2:117–141 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 & toxicity symptoms in fruits & nuts, 2:153–154 Soil: grape root growth, 5:141–144 management & root growth, 2:465–469 orchard floor management, 9:377–430 plant relations, trickle irrigation, 4:18–21 stress, 4:151–152 testing, 7:1–68; 9:88–90 zinc, 23:109–178 Soilless culture, 5:1–44 Solanaceae: history and iconography, 34:1–111. in vitro, 5:229–232 steroidal alkaloids, 25:171–196 Solanum dulcamara, history & iconography, 34:25 Solanum nigrum, history & iconography, 34:23–24 Somatic embryogenesis. See Asexual embryogenesis Sorghum, sweet, 21:73–104 Spathiphyllum, see Aroids, ornamental Squash, history, 25:71–170

418 Stem, apple morphology, 12:272–283 Sternbergia, 25:59 Steroidal alkaloids, solanaceous, 25:171–196 Storage. See also Controlled-atmosphere (CA) storage; Postharvest physiology carrot postharvest physiology, 30:284–288 cassava postharvest physiology, 30:288–295 cut flower, 3:96–100; 10:35–62 ginger postharvest physiology, 30:297–299 Jerusalem artichoke postharvest physiology, 30:259–271 low temperature sweetening, 17:203–231; 30:317–353 potato low temperature sweetening, 30:317–353 potato postharvest physiology, 30:259–271 root & tuber crops, 30:253–316 rose plants, 9:58–59 seed, 2:117–141 sweet potato postharvest physiology, 30:295–297 taro postharvest physiology, 30:295–297 Strawberry: fertilization, 1:106 flowering, 28:325–349 fruit growth & ripening, 17:267–297 functional phytonutrients, 27:303–304 harvesting, 16:348–365 in vitro, 5:239–241 wild of Kazakhstan, 29:347 Stress: benefits of, 4:247–271 chlorophyll fluorescence, 23:69–107 climatic, 4:150–151 flooding, 13:257–313 irrigation scheduling, 32:11–165 mechanical, 17:1–42 olive, 31:205–217 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

CUMULATIVE SUBJECT INDEX Sugar. See also Carbohydrate allocation, 7:74–94 flowering, 4:114 Sugar apple, CA & MA, 22:164 Sugar beet, fluid drilling of seed, 3:18–19 Sulfur: deficiency & toxicity symptoms in fruits & nuts, 2:154 nutrition, 5:323–324 Sustainable horticulture, 36:289–333; 37:331–362 Sweet potato: culture, 12:170–176 fertilization, 1:121 physiology, 23:277–338 postharvest physiology & storage, 30:276–284 Sweet sop, CA & MA, 22:164 Symptoms, deficiency & toxicity symptoms in fruits & nuts, 2:145–154 Syngonium, see Aroids, ornamental Systematics, 28:1–60 T Taro, postharvest physiology & storage, 30:276–284. See also Aroids, edible Taxonomy, 28:1–60 Taxus, 32:299–327 Tea, botany & 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

CUMULATIVE SUBJECT INDEX Texture in fresh fruit, 20:121–224 Thinning: apple, 1:270–300 peach & Prunus, 28:351–392 Tipburn, in lettuce, 4:49–65 Tissue 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. See also In vitro culture bulb organ formation, 34:417–444 cassava, 26:85–159 dwarfing, 3:347–348 geophyte organ formation, 34:417–444 nutrient analysis, 7:52–56; 9:90 Tobacco, history & iconography, 34:51–62 Tomato: CA storage, 1:380–386 chilling injury, 20:199–200 classification, 28:21–23 fertilization, 1:121–123 fluid drilling of seed, 3:19–20 fruit cracking, 30:163–184 fruit ripening, 13:67–103 galacturonase, 13:67–103 grafting, 28:98–103 greenhouse quality, 26:239 history & iconography, 34:75–85 parthenocarpy, 6:65–84 phytochemicals, 28:160 postharvest losses, 33:351–391 Toxicity symptoms in fruit & nut crops, 2:145–154 Transport, cut flowers, 3:100–104 Tree: adventitious rooting, 38:213–225 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 & root crops, see Root & tuber crops Tulip. See also Bulb fertilization, 5:364–366 in vitro, 18:144–145 physiology, 5:45–125 Tunnel (cloche), 7:356–357 Turf grass, fertilization, 1:112–117 Turnip, fertilization, 1:123–124

419 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 wild of Kazakhstan, 29:347–349 Vase solutions, 3:82–95; 10:46–51 Vegetable crops. See also Specific crop Allium development, 32:329–378 Allium phytochemicals, 28:156–159 aroids, 8:43–99; 12:166–170 asparagus postharvest, 12:69–155 bean rust, 37:1–99 bitter gourd, 37:101–141 breeding, 38:324–444 cactus, 18:300–302 carrot postharvest physiology & storage, 30:284–288 cassava crop physiology, 13:105–129 cassava molecular biology, 26:85–159 cassava multiple cropping, 30:355–50 cassava postharvest physiology & storage, 30:288–295 cassava root crop, 12:158–166 CA storage, 1:337–394 CA storage & quality, 8:101–127 CA storage diseases, 3:412–461 caper bush, 27:125–188 chilling injury, 15:63–95 coating physiology, 26:161–238 crucifer phytochemicals, 28:150–156 cucumber grafting, 28:91–96 cucurbit postharvest, 35:315–354 ecologically based, 24:139–228 eggplant grafting, 28:103–104 eggplant phytochemicals, 28:162–163 fertilization, 1:117–124 fluid drilling of seeds, 3:1–58 fresh cut, 30:185–255 ginger postharvest physiology & storage, 30:297–299 gourd history, 25:71–170 grafting, 28:61–124 greenhouses in China, 30:126–141 greenhouse management, 21:1–39

420 Vegetable crops (Continued ) greenhouse pest management, 13:1–66 honey bee pollination, 9:251–254 horseradish, 35:221–265 hot water treatment, 191–212 hydroponics, 7:483–558 Jerusalem artichoke postharvest physiology & storage, 30:271–276 lettuce seed germination, 24:229–275 low-temperature sweetening, 17:203–231 melon, 36:165–198 melon grafting, 28:96–98 minor root & tubers, 12:184–188 mushroom cultivation, 19:59–97 mushroom spawn, 6:85–118 nondestructive postharvest quality evaluation, 20:1–119 nutrition, 22:185–223 okra, 21:41–72 packaging, modified humidity, 37:281–329 pepper phytochemicals, 28:161–162 phytochemicals, 28:125–185 plug industry & technology, 35:387–436 potato low temperature sweetening, 30:317–353 potato phytochemicals, 28:160–161 potato postharvest physiology & storage, 30:271–276 potato tuberization, 14:89–188 pumpkin history, 25:71–170 root & tuber postharvest & storage, 30:295–297 seed conditioning, 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) fruit cracking, 30:163–184 tomato (greenhouse) quality, 26:239–319 tomato parthenocarpy, 6:65–84 tomato phytochemicals, 28:160 tropical production, 24:139–228 truffle cultivation, 16:71–107

CUMULATIVE SUBJECT INDEX watermelon grafting, 28:86–91 world industry, 38:299–356 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 Viburnam, wild of Kazakhstan, 29:361–362 Vigna. See also Cowpea genetics, 2:311–394 U.S. production, 12:197–222 Vines, biology and physiology, 38:1–21 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; 28:187–236 fig, 12:474–475 tree short life, 2:50–51 turnip mosaic, 14:199–238 Volatiles, 17:43–72; 24:31–53; 28:237–324 Vole, 6:254–274 W Walnut: in vitro culture, 9:312 wild of Kazakhstan, 29:369–370 Water relations: citrus, 30:37–83 container-grown ornamentals, 38:253–297 cut flower, 3:61–66; 18:1–85 deciduous orchards, 21:105–131; 32:111–165; 38:149–189 deficit irrigation, 21:105–131, 32:111–165; 38:149–189 desiccation tolerance, 18:171–213 fertilization, greenhouse crops, 1:117–124 grape & grapevine, 27:189–225 kiwifruit, 12:332–339 light in orchards, 2:248–249 packaging, modified humidity, 37:281–329 photosynthesis, 11:124–131 trickle irrigation, 4:1–48

CUMULATIVE SUBJECT INDEX Watercore, 6:189–251 apple, 6:189–251 pear, 11:385–387 Watermelon: fertilization, 1:124 grafting, 28:86–91 Wax apple, CA & MA, 22:164 Waxes, 23:1–68 Weed control, ginseng, 9:228–229 Weeds: invasive, 32:379–437 lettuce research, 2:198 parasitic, 267–349 virus, 3:403 Wild fruit & nuts of Kazakhstan, 29:305–371 almond, 29:262–265 apple, 29:63–303, 305–315 apricot, 29:325–326 barberry, 29:332–336 bilberry, 29:347–348 blackberry, 29:345 cherry, 29:326–330 cotoneaster, 29:316–317 cranberry, 29:349 currant, 29:341 elderberry, 29:349–350 gooseberry, 29:341–342 grape, 29:342–343 hazelnut, 29:365–366 lingonberry, 29:348–349 mountain ash, 29:322–324 mulberry, 29:350–351 oleaster, 29:351–353 pear, 29:315–316 pine, 29:368–369 pistachio, 29:366–368 plum, 29:330–332

421 raspberry, 29:343–345 rose, 29:353–360 sea buckthorn, 29:361 strawberry, 29:347 vacciniums, 29:347–349 viburnam, 29:361–362 walnut, 29:369–370 Willow, see Salix Withania spp., history & iconography, 34:19–20 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 Zantedeschi, see Aroids, ornamental Zephyranthes, 25:60–61 Zinc: deficiency & toxicity symptoms in fruits & nuts, 2:151 foliar application, 6:332, 336 nutrition, 5:326; 23:109–178 pine bark media, 9:124 Zizipus, see Jujube

Cumulative Contributor Index Volumes 1–38

Abbott, J.A., 20:1 Adams III, W.W., 18:215 Afek, U., 30:253 Aharoni, N., 37:281 Albrigo, L.G., 34:277 Aldwinckle, H.S., 1:423; 15:xiii; 29:1 Allen, A.C., 38:357 Alonso, J.M., 34:197 Aly, R., 33:267 Amarante, C., 28:161 Anderson, I.C., 21:73 Anderson, J.L., 15:97 Anderson, P.C., 13:257 Andrews, P.K., 15:183 Ascough, G.D., 34:417 Ashworth, E.N., 13:215; 23:1 Asokan, M.P., 8:43 Atkinson, D., 2:424 Aung, L.H., 5:45 Babadoost, M., 35:221 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., 2:41; 36:xiii Bartz, J.A., 30:185; 33:351 Bar-Ya’akov, I., 35:127 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; 38:149

Behera, S., 37:101 Behera, T.K., 37:101 Ben-Jaacov, J., 32:167 Bennett, A.B., 13:67 Benschop, M., 5:45; 36:1 Ben-Ya’acov, A., 17:381 Ben-Yehoshua, S., 37:281 Benzioni, A., 17:233 Bevington, K.B., 24:277 Bewley, J.D., 18:171 Bharathi, L.K., 37:101 Bieleski, R.L., 35:xiii Binder, B.M., 35:263 Binzel, M.L., 16:33 Blanpied, G.D., 7:xi Blenkinsop, R.W., 30:317 Bliss, F.A., 16:xiii; 28:xi Boardman, K., 27: xi Borochov, A., 11:15 Bounous, G., 31:293 Bower, J.P., 10:229 Bowling, A.J., 38:1 Bradeen, J.M., 38:357 Bradley, G.A., 14:xiii Brandenburg, W., 28:1 Brecht, J.K., 30:185 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 Burger, Y., 36:165

Horticultural Reviews, Volume 38 Edited by Jules Janick Copyright Ó 2011 Wiley-Blackwell. 423

424 Burke, M.J., 11:xiii Buwalda, J.G., 12:307 Byers, R.E., 6:253; 28:351 Byers, P.L., 37:213 Caldas, L.S., 2:568 Campbell, L.E., 2:524 Cantliffe, D.J., 16:109; 17:43; 24:229; 28:325; 35:397 Carter, G., 20:121 Carter, J., 35:193 Carter, J.V., 3:144 Cathey, H.M., 2:524 Chambers, R.J., 13:1 Chandler, C.K., 28:325 Charlebois, D., 37:213 Charles, J., 34:447 Charron, C.S., 17:43 Chen, J., 31:47 Chen, K., 30:83 Chen, Z., 25:171 Chin, C.K., 5:221 Clarke, N.D., 21:1 Coetzee, J.H., 26:1 Cohen, M., 3:394 Cohen, S., 37:281 Cohen, R., 36:165 Collier, G.F., 4:49 Collins, G., 25:235 Collins, W.L., 7:483 Colmagro, S., 25:235 Compton, M.E., 14:239 Connor, D.J., 31:157 Conover, C.A., 5:317; 6:119 Coombs, B., 32:xi Coppens d’Eeckenbrugge, G., 21:133 Corelli-Grappadelli, L., 32:63 Costa, G., 28:351 Costes, E., 32:1 Coyne, D.P., 3:28 Crane, J.C., 3:376 Criley, R.A., 14:1; 22:27; 24:x Crowley, W., 15:1 Cuevas, J., 34:239 Cutting, J.G., 10:229 Daie, J., 7:69 Dale, A., 11:185; 16:255 Darnell, R.L., 13:339; 28:325 Daunay, M.-C., 34:1

CUMULATIVE CONTRIBUTOR INDEX Davenport, T.L., 8:257; 12:349; 31:113 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; 36:1 Deikman, J., 16:1 DellaPenna, D., 13:67 DeLong, J.M., 32:299 Demers, D.-A., 30:163 Demmig-Adams, B., 18:215 Dennis, F.G., Jr., 1:395 Dickson, E.E., 29:1 Dorais, M., 26:239; 30:163 Doud, S.L., 2:1 Dudareva, N., 24:31 Duke, S.O., 15:371 Dunavent, M.G., 9:103 Duval, M.-F., 21:133 D€ uzyaman, E., 21:41 Dyer, W.E., 15:371 Dzhangaliev, A.D., 29:63, 305 Early, J.D., 13:339 Eastman, K., 28:125 Eizenberg, H., 33:267 Ejeta, G., 33:267 Elfving, D.C., 4:1; 11:229 El-Goorani, M.A., 3:412 Erwin, J.E., 34:417 Esan, E.B., 1:1 Esposto, S., 38:83 Evans, D.A., 3:214 Ewing, E.E., 14:89 Fallik, E., 39:191 Famiani, F., 38:83 Faust, M., 2:vii, 142; 4:174; 6:287; 14:333; 17:331; 19:263; 22:225; 23:179 Felkey, K., 30:185 Fenner, M., 13:183 Fenwick, G.R., 19:99 Fereres, E., 31:157 Ferguson, A.R., 6:1; 33:1 Ferguson, I.B., 11:289; 30:83; 31:233 Ferguson, J.J., 24:277 Ferguson, L., 12:409

CUMULATIVE CONTRIBUTOR INDEX Ferree, D.C., 6:155; 31:xi Ferreira, J.F.S., 19:319 Fery, R.L., 2:311; 12:157 Field, S.K., 37:143 Finn, C.E., 37:213 Fischer, R.L., 13:67 Flaishman, M.A., 34:113 Fletcher, R.A., 24:53 Flick, C.E., 3:214 Flore, J.A., 11:111 Forshey, C.G., 11:229 Forsline, P.L., 29:ix, 1 Franks, R. G., 27:41 Fujiwara, K., 17:125 Gadkar, V., 36:257 Galvano, F., 38:83 Gazit, S., 28:393 Geisler, D., 6:155 Geneve, R.L., 14:265 George, K.J., 33:173 George, W.L., Jr., 6:25 Gerrath, J.M., 13:315 Gilley, A., 24:55 Giovannetti, G., 16:71 Giovannoni, J.J., 13:67 Girona, J., 38:149 Glenn, G.M., 10:107; 31:1 Goffinet, M.C., 20:ix Goldschmidt, E.E., 4:128; 30:1; 35:437 Goldy, R.G., 14:357 Goren, R., 15:145; 30:1 Gosselin, A., 26:239 Goszczynska, D.M., 10:35 Grace, S.C., 18:215 Gradziel, T.M., 30:xiii; 34:197; 38:23 Graves, C.J., 5:1 Gray, D., 3:1 Grierson, W., 4:247 Griesbach, R.J., 35:193 Griffen, G.J., 8:291 Grodzinski, B., 7:345 Gucci, R., 21:177 Guest, D.I., 17:299 Guiltinan, M.J., 16:1 Gulia, S.K., 35:193 Hackett, W.P., 7:109 Halevy, A.H., 1:204; 3:59 Hallett, I.C., 20:121

425 Hammerschmidt, R., 18:247 Hanson, E.J., 16:255 Hardie, W.J., 37:143 Hardner, C.M., 35:1 Harker, F.R., 20:121 Hatib, K., 35:127 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; 31:47 Hergert, G.B., 16:255 Hershenhorn, J., 33:267 Hess, F.D., 15:371 Hetterscheid, W.L.A., 28:1 Heywood, V., 15:1 Hjalmarsson, I., 27:79-123 Hogue, E.J., 9:377 Hokanson, S.C. 29:1 Holland, D., 35:127 Holt, J.S., 15:371 Holzapfel, B.P., 37:143 Hoover, E.E., 38:357 Huang, Hongwen, 33:1 Huber, D.J., 5:169 Huberman, M., 30:1 Hunter, E.L., 21:73 Hurst, S., 34:447 Hutchinson, J.F., 9:273 Hutton, R.J., 24:277 Hummer, K., 38:xiii–xv Indira, P., 23:277 Ingle, M. 27:227 Inglese, P., 38:83 Isenberg, F.M.R., 1:337 Iwakiri, B.T., 3:376 Jackson, J.E., 2:208 Jahn, M., 37:v Janick, J., 1:ix; 8:xi; 17:xiii; 19:319; 21:xi; 23:233; 34:1; 35:437 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 Jiang, W., 30:115 Joel, D.M., 33:267 Joiner, J.N., 5:317

426 Jones, H.G., 7:301 Jones, J.B., Jr., 7:1 Jones, R.B., 17:173 Joseph John, K., 37:101 Kagan-Zur, V., 16:71 Kalt, W. 27:269; 28:125 Kamenetsky, R., 32:329; 33:123; 36:1 Kang, S.-M., 4:204 Kapulnik, Y., 36:257 Karp, A., 34:447 Kato, T., 8:181 Katzir, N., 36:165 Kawa, L., 14:57 Kawada, K., 4:247 Kays, S.J., 30:253 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 Kirschbaum, D.S. 28:325 Kliewer, W.M., 14:407 Knight, R.J., 19:xiii Knox, R.B., 12:1 Kodad, O., 34:197 Kofranek, A.M., 8:xi Koltai, H., 36:257 Korcak, R.F., 9:133; 10:183 Kozai, T., 17:125 Krezdorn, A.H., 1:vii Kushad, M.M., 28:125 Kuzovkina, Y.A., 34:447 Labrecque, M. 34:447 Laimer, M., 28:187 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 Laterrot, H., 34:1 Lauri, P.E. 32:1 Layne, D.R., 31:351 Lea-Cox. J.D., 38:253 Leal, F., 21:133 Ledbetter, C.A., 11:159

CUMULATIVE CONTRIBUTOR INDEX Lee, J.-M., 28:61 Le Nard, M., 36:1 Levy, Y., 30:37 Lewinsohn, E., 36:165 Li, P.H., 6:373 Liebenberg, M.M., 37:1 Lill, R.E., 11:413 Lin, S., 23:233 Liu, M., 32:229 Liu, Z., 27:41 Lipton, W.J., 12:69 Littlejohn, G.M., 26:1 Litz, R.E., 7:157 Lockard, R.G., 3:315 Loescher, W.H., 6:198 Lopez, G., 38:149 Lorenz, O.A., 1:79 Lowe, A.J. 35:1 Lu, R., 20:1 Luby, J.J., 29:1; 38:358 Lurie, S., 22:91-121 Lyrene, P., 21:xi Maguire, K.M., 25:197 Mahovic, M.J., 33:351 Majsztrik, J.C., 38:253 Malik, A.U., 31:113 Manivel, L., 22:267 Maraffa, S.B., 2:268 Marangoni, A.G., 17:203; 30:317 Marini, R.P., 9:351; 32:63 Marinoni, D.T., 31:293 Marlow, G.C., 6:189 Maronek, D.M., 3:172 Marsal, J., 38:149 Martin, G.G., 13:339 Masiunas, J., 28:125 Mattoo, A.K., 37:331 Mayak, S., 1:204; 3:59 Maynard, D.N., 1:79; 35:315 McConchie, R., 17:173 McConnell, D.B., 31:47 McIvor, I., 34:447 McNicol, R.J., 16:255 Merkle, S.A., 14:265 Merwin, I.A., 34:365 Meyer, M.H., 33:393 Michailides, T.J., 12:409 Michelson, E., 17:381 Michler, C.H., 38:213

CUMULATIVE CONTRIBUTOR INDEX 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 Mohankumar, C.R., 30:355 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 Mu, D., 30:115 Mudge, K., 35:437 Mulwa, R.M.S., 35:221 Murashige, T., 1:1 Mureinik, I., 34:xi Murr, D.P., 23:69 Murray, S.H., 20:121 Myers, P.N., 17:1 Nadeau, J.A., 19:1 Nair, R.R., 33:173 Naor, A., 32:111 Nascimento, W.M., 24:229 Nayar, N.W., 36:117 Neal, J., 35:1 Neilsen, G.H., 9:377 Nelson, P.V., 26:xi Nerd, A., 18:291, 321 Niemiera, A.X., 9:75; 32:379 Nobel, P.S., 18:291 Norman, D.J., 31:47 Norton, M.A., 35:221 Nybom, H., 36:199 Nyujto`, F., 22:225 Oda, M., 28:61 O’Donoghue, E.M., 11:413 Ogden, R.J., 9:103 O’Hair, S.K., 8:43; 12:157 Okubo, H., 36:1 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

427 Padilla-Zakour, O.I., 34:365 Palser, B.F., 12:1 Papadopoulos, A.P., 21:1; 26:239; 30:163 Pararajasingham, S., 21:1 Parera, C.A., 16:109 Paris, H.S., 25:71; 36:165 Parthasarathy, V.A., 33:173 Peace, C., 35:1 Pegg, K.G., 17:299 Pellett, H.M., 3:144 Perkins-Veazil, P., 17:267 Phillips, G., 32:379 Pichersky, E., 24:31 Pickering, A.H., 35:355 Piechulla, B., 24:31 Pijut, P.M., 389:213 Pisanu, P., 35:1 Ploetz, R.C., 13:257 Pokorny, F.A., 9:103 Pomper, K.W., 31:351 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; 32:299; 35:263 Pratt, C., 10:273; 12:265 Predieri, S., 28:237 Preece, J.E., 14:265 Pretorius, Z.A., 37:1 Priestley, C.A., 10:403 Proctor, J.T.A., 9:187 Puonti-Kaerlas, J., 26:85 Puterka, G.J., 31:1 Qu, D., 30:115 Quamme, H., 18:xiii Rabinowitch, H.D., 32:329 Raese, J.T., 11:357 Ramming, D.W., 11:159 Ransom, J.K., 33:267 Rapparini, F., 28:237 Ravi, V., 23:277; 30:355 Raviv, M., 36:289 Reddy, A.S.N., 10:107 Redgwell, R.J., 20:121 Regnard, J.L., 32:1 Reid, M., 12:xiii; 17:123 Reuveni, M., 16:33

428 Rich, P.J., 33:267 Richards, D., 5:127 Rieger, M., 11:45 Ristvey, A.G., 38:253 Rodov, V., 34:113; 37:281 Romero, M.A., 34:447 Roper, T.R., 21:215 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 Rubiales, D., 33:267 Rudnicki, R.M., 10:35 Ryder, E.J., 2:164; 3:vii; 38:299 Sachs, R., 12:xiii Sakai, A., 6:357 Salisbury, F.B., 4:66; 15:233 Salova, T. H., 29:305 Saltveit, M.E., 23:x; 30:185 San Antonio, J.P., 6:85 Sankhla, N., 10:63; 24:5 Sargent, S.A., 35:315 Sasikumar, B., 33:173 Sauerborn, J., 33:267 Saure, M.C., 7:239 Schaffer, A.A., 36:165 Schaffer, B., 13:257 Schenk, M.K., 22:185 Schneider, G.W., 3:315 Schneider, K.R., 30:185; 33:351 Schotsmans, W.C., 35:263 Schuster, M.L., 3:28 Scofield, A., 35:437 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 Servili, M., 38:83 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 Shehata, A., 35:221 Shipp, J.L., 21:1 Shirra, M., 20:267 Shorey, H.H., 12:409

CUMULATIVE CONTRIBUTOR INDEX Silber, A., 32:167 Silva Dias, J., 38:299 Simon, J.E., 19:319 Simon, P.W., 37:101 Singh, B.P., 35:193 Singh, N.B., 34:447 Singh, S.H., 34:277 Singh, Z., 27:189; 31:113 Skirvin, R., 35:221 Sklensky, D.E., 15:335 Smart, L.B., 34:447 Smith, A.H., Jr., 28:351 Smith, G.S., 12:307 Smith, J.P., 37:143 Smith, M.A.L., 28:125 Smock, R.M., 1:301 Socias i Company, R., 34:197 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 Spooner, D.M., 28:1 Srinivasan, C., 7:157 Srivastava, A.K., 34:277 Stang, E.J., 16:255 Staub, J.E., 37:101 Steffens, G.L., 10:63 Stern, R.A., 28:393 Stevens, M.A., 4:vii Stoffella, P.J., 33:xi Stover, E., 34:113 Stroshine, R.L., 20:1 Struik, P.C., 14:89 Studman, C.J., 19:217 Stutte, G.W., 13:339 Styer, D.J., 5:221 Sunderland, K.D., 13:1 Sung, Y., 24:229 Suranyi, D., 19:263; 22:225; 23:179 Swanson, B., 12:xiii Swietlik, D., 6:287; 23:109 Syvertsen, J.P., 7:301, 30:37 Tadmor, Y., 36:165 Talcott, S.T., 30:185 Tattini, M., 21:177 Teasdale, J.R., 37:331

CUMULATIVE CONTRIBUTOR INDEX Tellias, A., 38:357 Teodorescu, T.L., 34:447 Tet enyi, P., 19:373 Theron, K.I., 25:1 Thomas, A.L., 37:213 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 Trybush, S., 34:447 Tunya, G.O., 13:105 Turekhanova, P.M., 29:305 Uchanski, M., 35:221 Upchurch, B.L., 20:1 Urbani, S., 38:83 Valenzuela, H.R., 24:139 Valois, S., 34:365 van den Berg, W.L.A., 28:1 van Doorn, W.G., 17:173; 18:1 Van Iepersen, W., 30:163 van Kooten, O., 23:69 van Nocker, S., 27:1 van Staden, J., 34:417 Vaughn, K.C., 38:1 Veilleux, R.E., 14:239 Vizzotto, G., 28:351 Volk, T.A., 34:447 Vorsa, N., 21:215 Wallace, A., 15:413 Wallace, D.H., 17:73 Wallace, G.A., 15:413 Walters, S.A., 35:221 Wang, C.Y., 15:63 Wang, L., 30:115

429 Wang, S.Y., 14:333 Wann, S.R., 10:153 Warrington, I.J., 35:355 Watkins, C.B., 11:289 Watson, G.W., 15:1 Webster, B.D., 1:172; 13:xi Weichmann, J., 8:101 Weih, M., 34:447 Werlemark, G., 36:199 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 Woeste, K.E., 38:213 Woodson, W.R., 11:15 Wooley, D.J., 35:355 Wright, R.D., 9:75 W€ unsche, J.N., 31:23 Wutscher, H.K., 1:237 Xu, C., 30:83 Yada, R.Y., 17:203; 30:317 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 Zhang, B., 30:83 Zieslin, N., 9:53 Zimmerman, R.H., 5:vii; 9:273 Ziv, M., 24:1 Zucconi, F., 11:1