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WILEYe BOOK WILEY JOSSEY-BASS PFEIFFER J.K.LASSER CAPSTONE WILEY-LISS WILEY-VCH WILEY-INTERSCIENCE

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HORTICULTURAL REVIEWS Volume 26

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Horticultural Reviews is sponsored by: American Society for Horticultural Science

Editorial Board, Volume 26 Natalia Dudareva Linus U. Opara Dariusz Swietlik

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HORTICULTURAL REVIEWS Volume 26

edited by

Jules Janick Purdue University

NEW YORK

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

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This book is printed on acid-free paper. Copyright © 2001 by John Wiley & Sons, Inc. All rights reserved. Published simultaneously in Canada. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning or otherwise, except as permitted under Sections 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4744. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 605 Third Avenue, New York, NY 10158-0012, (212) 850-6011, fax (212) 850-6008, E-Mail: PERMREQ @ WILEY.COM. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold with the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional person should be sought. Library of Congress Catalog Card Number: 79-642829 ISBN 0-471-38789-4 ISSN 0163-7851 Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1

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Contents

Contributors

ix

Dedication: A. A. De Hertogh

xi

Paul V. Nelson

1. Protea: A Floricultural Crop from the Cape Floristic Kingdom

1

J. H. Coetzee and G. M. Littlejohn I. II. III. IV. V. VI. VII.

Introduction History Reproductive Biology Crop Improvement Physiology Production Conclusion Literature Cited

2. The Molecular Biology of Plant Hormone Reception

2 5 10 13 21 29 38 40

49

Carole L. Bassett I. II. III.

Introduction Current Status of Signal Receptor Research Plant Hormone Receptors

50 53 60 v

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vi

CONTENTS

IV.

Future Prospects Literature Cited

3. Molecular Biology of Cassava

75 78

85

Johanna Puonti-Kaerlas I. II. III. IV. V. VI. VII. VIII. IX. X.

Introduction Botany and Distribution Production and Use Constraints to Cassava Use and Cultivation Traditional Breeding and Biotechnology in Cassava Improvement Molecular Genetics Tissue Culture and Regeneration Genetic Transformation Applications and Potential Molecular Biology Future Prospects Literature Cited

4. Postharvest Physiology and Quality of Coated Fruits and Vegetables

86 87 90 94 94 95 99 112 119 132 134

161

Cassandro Amarante and Nigel H. Banks I. II. III.

IV.

V. VI.

Introduction Permeability of Coating Films and Permeance of Coated Commodities Physico-Chemical Characteristics, and Barrier Properties to Water Vapor and Gases, of Edible Coatings Factors Affecting Water Loss, Gas Exchange, and Modification of Internal Atmosphere of Coated Commodities Postharvest Physiology and Quality of Coated Commodities Summary and Conclusions Literature Cited

162 165

170

181 197 224 227

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CONTENTS

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5. Greenhouse Tomato Fruit Quality

239

M. Dorais, A. P. Papadopoulos, and A. Gosselin I. II. III. IV. V. VI. VII.

Introduction Quality Attributes Genetic Characteristics Affecting Tomato Fruit Quality Environmental Factors Affecting Tomato Fruit Quality Cultural Practices Affecting Fruit Quality Postharvest Conclusion Literature Cited

240 243 262 264 271 291 294 296

Subject Index

321

Cumulative Index

323

Cumulative Contributor Index

345

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Contributors Cassandro Amarante, Centre of Agricultural, Horticultural and Veterinary Sciences, Santa Catarina State University, Caixa Postal 281, CEP 88520-000, Lages, SC, Brazil, [email protected] Nigel H. Banks, Centre for Postharvest and Refrigeration Research, Institute for Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand, [email protected] Carole L. Bassett, USDA-ARS, Appalachian Fruit Research Station, 45 Wiltshire Road, Kearneysville, WV 25430, [email protected] J. H. Coetzee, Agricultural Research Council: Fynbos, Private Bag X1, Elsenburg, 7607 South Africa, [email protected] M. Dorais, Agriculture and Agri-Food Canada, Greenhouse & Processing Crops Research Centre, Harrow, ON, N0R 1G0, Canada, [email protected] A. Gosselin, Horticulture Research Centre, Laval University, Sainte-Foy, QC, G1K 7P4, Canada G. M. Littlejohn, Agricultural Research Council: Fynbos, Private Bag X1, Elsenburg, 7607 South Africa, [email protected] Paul V. Nelson, Department of Horticultural Science, North Carolina State University, Raleigh, NC 27698-7609, [email protected] A. P. Papadopoulos, Agriculture and Agri-Food Canada, Greenhouse & Processing Crops Research Centre, Harrow, ON, N0R 1G0, Canada [email protected] Johanna Puonti-Kaerlas, Institute for Plant Sciences, ETH-Zentrum/LFW E 17, CH-8092 Zürich, Switzerland, [email protected]

ix

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August A. De Hertogh

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Dedication: August A. De Hertogh Who can deny that the profusion of spring flowering bulbs in the landscape and interior environments are truly wondrous? While flowering bulbs are miracles of nature, we have August (Gus) De Hertogh to thank for helping to successfully bring these gifts into our lives. Dr. De Hertogh’s contributions to horticulture are truly outstanding. He is a world authority on flowering bulb plants (geophytes) and his career embraces the science, technology, industry practice, and education in that world. His integration of science, technology, economic application, and public education serves as a model for all horticulturists. When Gus began his work on flower bulbs at Michigan State University in 1965, it was not possible to effectively control the flowering of tulips or other bulbs following overseas shipment from The Netherlands, the world’s largest producer. He not only developed procedures to successfully ship bulbs across the Atlantic Ocean but also developed production protocols for handling bulbs upon arrival, properly cooling bulbs, and then forcing bulbs in the greenhouse from January through May for pot and cut flower markets. He developed production schedules for every major flowering bulb produced in the United States, The Netherlands, South Africa, Israel, Australia, and New Zealand. In short, he managed, through science and technology, to take an age-old industry and advance it into a new age with many economic opportunities. He has been instrumental in expanding the world market of flower bulbs. Dr. De Hertogh pioneered university-industry partnerships when he began collaborative efforts with the Netherlands Flower Bulb Institute and the Holland Bulb Exporters Association. From 1965 to 1970, Dr. De Hertogh and his team conducted basic physiology studies of flowering bulbs. This laid the foundation for the production of the first of five editions of the Holland Bulb Forcers Guide, which is considered the “bible” for bulb producers and forcers. One of the striking attributes of Dr. De Hertogh’s career is his inexhaustible zeal to educate people from all walks of the geophyte world. The breadth of media for his publications speaks to this desire to communicate equally to scientist, producer, and end user. These publications encompass 52 journal articles, 55 symposia/research reports, 58

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DEDICATION: AUGUST A. DE HERTOGH

extension leaflets, 45 books and book chapters, 25 industry service bulletins, 85 industry popular press articles, 4 Web sites, and 2 software packages. Probably one of his greatest literary contributions to science is the comprehensive treatise co-authored with Dr. Marcel Le Nard on bulb crops, entitled, The Physiology of Flowering Bulbs. It is a one-of-akind work that will be an invaluable reference far into the future. Gus has transferred his knowledge and wisdom in more personal ways than just the written word. He has served as leader, partner, and team player in numerous collaborative programs. These programs have included 42 student trainees from The Netherlands, 17 master’s students, 6 Ph.D. students, 17 post doctorates, and numerous technicians and fellow scientists. His personal touch has had an effect on traditional students at all levels, industry clientele, other scientists, and the public. His leadership and dedication to University service was demonstrated by his 10 years as Department Head of Horticultural Science at North Carolina State University (NCSU). He led the department with an enthusiasm for the advancement of individuals as well as the discipline and industry. He was a mentor to the faculty, a father figure, an always capable “John Wayne” type, who did his best to act in the best interest of everyone. Perseverance has always been one of his hallmarks. His ability to complete a chosen task with focus and organization is legendary in horticulture and in the world flower business. He led a worldwide bulb industry while also leading a large horticulture faculty at NCSU. In 1988 the Japanese Society for Promotion of Science engaged him to evaluate their research program in horticulture. Gus has received many awards and honors during his career. These include Fellow of the American Society for Horticultural Science; Medal of Honor, Ministry of Agriculture and Fisheries, The Netherlands; Floriculture Hall of Fame Award and the Alex Laurie Research and Education Award from the Society of American Florists; the Nicolaas Dames Golden Medal, The Netherlands; the Teaching Award of the American Horticulture Society; the Innovator Award by North Carolina State University; the Gold Pin Award of Dutch Bulb Exporters Association; the Herbert Medal, International Bulb Society, Pasadena, California; and the naming of a hyacinth cultivar in his honor. The bulb industry gave him their highest of honors, the Silver Tulip Award. Dr. De Hertogh has truly exemplified the vision of an exceptional horticultural scientist. His dedication and efforts will long be felt in the accomplishments of his colleagues, the careers of the many students he educated, the commercial successes of an entire geophyte industry

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he supported, and the pleasures of a worldwide consuming public who through his efforts find their lives a shade brighter than before. Gus is truly an ambassador for horticulture and, because of this, we dedicate this volume of Horticultural Reviews in his honor. Paul V. Nelson North Carolina State University

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1 Protea: A Floricultural Crop from the Cape Floristic Kingdom* J. H. Coetzee and G. M. Littlejohn Agricultural Research Council: Fynbos Private Bag X1 Elsenburg 7607 South Africa

I. INTRODUCTION II. HISTORY A. Taxonomy and Cultivation B. Research C. World Industry III. REPRODUCTIVE BIOLOGY IV. CROP IMPROVEMENT A. Genetic Variability B. Selection C. Hybridization D. Interspecific Hybridization E. Cultivars V. PHYSIOLOGY A. Flowering B. Propagation 1. Sexual Reproduction 2. Vegetative Propagation 3. Grafting 4. Tissue Culture C. Water and Nutrient Uptake D. Postharvest Physiology

*Research conducted by the authors was supported by The Agricultural Research Council, the South African Protea Producers and Exporters Association, the European Commission (INCO-DC contract number IC18-CT97-0174), and the International Protea Association. Horticultural Reviews, Volume 26, Edited by Jules Janick ISBN 0-471-38789-4 © 2001 John Wiley & Sons, Inc. 1

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VI. PRODUCTION A. Cultivation B. Pathogens Associated with Diseases of Protea 1. Pathogens of Roots 2. Pathogens of Leaves 3. Pathogens of Shoots, Stems, and Inflorescences 4. Pathogens of Woody Stems C. Phytophagous Insects Fauna of Protea 1. Flower Visitors 2. Borers 3. Folivorous Insects 4. Sap Suckers VII. CONCLUSION LITERATURE CITED

I. INTRODUCTION Protea, the most widely known genus of the Proteaceae, is now an important floral crop. Other genera in this family that are widely used in floriculture are Leucospermum (Criley 1998), Banksia (Sedgley 1998), and Leucadendron. Mimetes, Serruria, Aulax, Telopea, Grevillea, Isopogon, and Paranomus are used to a lesser extent. The name Protea, given by Linnaeus in 1753, referring to the Greek mythical god, Proteus, who could change his shape at will, is truly an apt name due to the wide diversity of this genus. The genus Protea is only found in sub-Saharan Africa and currently 114 species are described (Rourke 1980), with 14 subspecies recognized (Rebelo 1995). The tropical Protea species are widely distributed across sub-Saharan Africa and comprise 35 species (Beard 1992). Three of these tropical species are found in the summer rainfall region of South Africa: P. caffra, P. gaguedi, and P. welwitschii. The 89 species of Protea found in Southern Africa may be sub-divided into 20 groups of closely related species, shown in Table 1.1 (Rebelo 1995). The Cape Floristic Kingdom, a small strip of land between the towns of Grahamstown in the east and Clanwilliam in the west (Fig. 1.1) is home to 69 endemic species of Protea (Rourke 1980). It is from these species that the commercially utilized species derive, and include the stately P. cynaroides with a flower diameter of up to 25 cm and P. scolymocephala with a flower diameter of approximately 5 cm. The Cape Floral Kingdom, one of the world’s six plant kingdoms, is also known as the Flora Capensis or the fynbos biome. This plant kingdom, ranking alongside the Holarctic, Palaeotropic, Neotropic, Australasian, and Antarctic Kingdoms that cover vast areas of the globe, is unique. Plants in this region are adapted to hot dry summer conditions and primarily acidic, nutrient poor soils. It comprises only 0.04% of the earth’s surface, but

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Taxonomic groupings within the genus Protea (summarized from Rebelo 1995).

Group Common Names

Species

Rodent Sugarbush

P. amplexicaulis (Salisb.) R.Br., P. humiflora Andrews, P. cordata Thunb., P. decurrens E. Phillips, P. subulifolia (Salisb. ex Knight) Rourke P. caffra Meisn., P. petiolaris (Hiern) Baker & Wright, P. simplex E. Phillips, P. parvula Beard , P. dracomontana Beard, P. nubigena Rourke P. inopina Rourke, P. glabra Thunb., P. rupicola Mund ex Meisn., P. nitida Mill. P. enervis Wild P. angolensis Welw., P. rupestris R.E. Fr., P. madiansis Oliv., P. rubropilosa Beard, P. comptonii Beard, P. curvata N.E. Br., P. laetans L. E. Davidson P. welwitschii Engl., P. gaguedi J.F. Gmel. P. asymmetrica Beard, P. wentzelana Engl. P. cynaroides (L.) L. P. scolopendriifolia (Salisb. ex Knight) Rourke, P. scabriuscula E. Phillips, P. cryophila Bolus, P. pruinosa Rourke P. roupelliae Meisn., P. eximia (Salisb. ex Knight) Fourc., P. compacta R. Br., P. obtusifolia H. Beuk ex Meisn., P. susannae E. Phillips, P. burchellii Stapf, P. longifolia Andrews, P. pudens Rourke P. repens (L.) L., P. aristata E. Phillips, P. lanceolata E. Mey. ex Meisn. P. laurifolia Thunb., P. neriifolia R. Br., P. lepidocarpodendron (L.) L., P. lorifolia (Salisb. ex Knight) Fourc., P. coronata Lam., P. speciosa (L.) L., P. stokoei E. Phillips, P. grandiceps Tratt., P. magnifica Link, P. holosericea (Salisb. ex Knight) Rourke P. lorea R. Br., P. scorzonerifolia (Salisb. ex Knight) Rycroft, P. aspera E. Phillips, P. scabrapiscina R. Br., P. piscina Rourke, P. restionifolia (Salisb. ex Knight) Rycroft, P. denticulata Rourke P. subvestita N.E. Br., P. lacticolor Salisb., P. punctata Meisn., P. mundii Klotzsch, P. aurea (Burm.f.) Rourke, P. venusta Compton P. caespitosa Andrews P. tenax (Salisb.) R. Br., P. foliosa Rourke, P. vogtsiae Rourke, P. intonsa Rourke, P. montana E. Mey. ex Meisn. P. acaulos (L.) Reichard, P. angustata R. Br., P. laevi R. Br., P. convexa E. Phillips, P. revoluta R. Br. P. mucronifolia Salisb., P. odorata Thunb. P. scolymocephala (L.) Reichard, P. acuminata Sims, P. canaliculata Andrews, P. nana (P.J. Bergius) Thunb., P. witzenbergiana E. Phillips, P. pityphylla E. Phillips P. recondita H. Beuk ex Meisn., P. effusa E. Mey. ex Meisn., P. sulphurea E. Phillips, P. namaquana Rourke, P. pendula R. Br.

Grassland Sugarbush

Shaving Brush Sugarbush Red Sugarbush Mountain Sugarbush

Savanna Sugarbush Moorland Sugarbush King Sugarbush Snow Sugarbush Spoonbract Sugarbush

True Sugarbush Bearded Sugarbush

Dwarf-tufted Sugarbush

White Sugarbush

Bishop Sugarbush Eastern Ground Sugarbush Western Ground Sugarbush Shale Sugarbush Rose Sugarbush

Penduline Sugarbush

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Fig. 1.1. Map of Africa, depicting the Cape Floristic Kingdom and the distribution of tropical Protea throughout Africa.

due to its remarkable plant species diversity (>8500 species of flowering plants) and high level of endemism, has been classified as a distinct phytogeographic region (Bond and Goldblatt 1984). While the prominent use of Protea today is as fresh or dried flower, the plant has had many uses in the past. Early European settlers in South

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Africa used the wood of P. nitida for the manufacture of furniture and wagon wheels. The bark of P. nitida was used in the tanning of leather and the leaves as a source of black ink (Rourke 1980). Protea also had their uses in traditional medicine (Van Wyk et al. 1997). The nectar of P. repens, which is produced in copious amounts, was used by early European settlers as a remedy for chest disorders after being boiled to a syrup. The bark of P. caffra is used to treat bleeding stomach ulcers and diarrhoea.

II. HISTORY A. Taxonomy and Cultivation The early taxonomical and cultivation history of Protea has been reviewed by Rourke (1980). A Dutch trade group collected the first Protea in 1597 and in 1605 Clusius described P. neriifolia. Paule Hermann of the Netherlands collected Protea on Table Mountain in 1672, but the descriptions were published in 1737. Sir Hans Sloane of London described P. repens in 1693 and Plukenett did likewise for P. scolymocephala and P. cynaroides in 1700. European collectors of exotic plants were the first cultivators of Proteaceae, from achenes collected by Masson in 1774. P. repens was the first recorded Protea species to flower outside its natural habitat. In 1803, P. cynaroides flowered in the collection of the Earl of Coventry, Croome, Worcestershire. The largest collection of 35 species was grown by George Hubbert in 1805 in the suburbs of London and, by 1810, 23 species of Protea were already grown at Kew Gardens. The Dutch and French showed great enthusiasm for Protea cultivation during this period. The first commercial distributors of Protea achenes were the London firm, Lee and Kennedy. Among their clientele was Josephine, wife of Napoleon. The industrial revolution in Europe and the British Isles, in the early 1800s, led to wide-scale heating of greenhouses and concomitant high humidity, conditions under which Protea would not grow, leading to a loss of interest in their cultivation. It was only in 1981 that P. cynaroides flowered once again in Kew Gardens. In this rich floral kingdom, the South African wild flower industry had a humble origin. Street hawkers began selling flowers, picked in the surrounding mountains, on the streets of Cape Town, a tradition still in existence (Coetzee and Littlejohn 1995). In the 19th century, European church groups established settlements on their mission stations in the rural areas of the Cape, for people originating from the Khoi-San tribes,

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as well as slaves imported from the East, and European settlers. These inhabitants of the mission stations, at Elim and Genadendal, were the first exporters of dried indigenous flowers to Europe in 1886 (Krüger and Schaberg 1984). However, no interest was shown in the cultivation of Protea in the 19th century. In 1910, A. C. Buller cultivated P. cynaroides commercially for the first time on his farm near Stellenbosch. In 1913 the National Botanical Garden of South Africa at Kirstenbosch was established and proteas were among the first plants cultivated. The first seed trader selling proteaceous achenes was Kate Stanford, who issued a catalogue in 1933 (Rourke 1980). Ruth Middelmann greatly promoted sales of proteaceous achenes, exporting achenes to countries such as New Zealand, the United States of America (California), and Australia (Lighton 1960). The Kirstenbosch botanical garden also introduced a system for the selling of achenes of plants from the Flora Capensis soon after its establishment. Frank Batchelor established the first commercial plantation on his farm in Devon Valley near Stellenbosch, the farm later to be known as Protea Heights, where he harvested the first flowers in 1948. In 1953 P. cynaroides was part of a floral basket sent as a gift from the people of the Cape to Queen Elizabeth on the eve of her coronation (Lighton 1960). This is the first documentation of fresh Protea being exported. Buller and Batchelor can be viewed as the fathers of the fresh, cut flower protea industry in South Africa. The commercialization of the dried flower industry began in the mid 1950s, with the Middelmann family exporting large quantities of dried flowers by ship to Europe. Today there are over 400 flower harvesters collecting plant material from the wild and delivering it to large dried flower businesses for drying and processing for export. In the South African dried flower industry, six Protea species are used (Table 1.2), from which a large number of products are created (Coetzee and Middelmann 1997). Twenty different products that originate from P. repens are sold (Wessels et al. 1997), with more than 20 million inflorescences of P. repens harvested in the natural habitat annually to supply the market. The proteaceous material used in the dried flower industry is primarily harvested from the natural habitat and can have negative effects on the ecology of the fynbos, the re-establishment of the species after fire, and the genetic variability within a population (Coetzee and Littlejohn 1995). The fresh cut flower industry utilizes 12 Protea species and a number of interspecific hybrids, listed in Table 1.3 (Coetzee and Middelmann

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Important Protea species used in the dried flower trade.

Species

Trade Name

P. repens P. compacta P. magnifica P. susannae P. neriifolia P. obtusifolia

Repens flower, rosette Compacta flower, rosette Barbigera flower Susannae rosette Neriifolia bud Obtusifolia flower

1997). Approximately 350 growers cultivate Proteaceae commercially. Although some species of Protea are still harvested in the natural habitat and sold as fresh cut flowers, a recent survey indicated that more than 80% of the cut flower Protea are from cultivation (Wessels et al. 1997). In 1997 the Proteaceae hectarage under intensive cultivation in South Africa was in excess of 400 ha, of which 50% were Protea (Middelmann and Archer 1999). A further 1,000 ha of broadcast sown plantations were recorded. During 1998, 3,666 tons of fresh cut flowers were exported from South Africa, of which 30% was represented by genus Protea. The top-selling products exported by South Africa are P. magnifica, Table 1.3. Important Protea species used in the fresh cut flower trade (Middelmann and Coetzee, 1997), with their natural flowering times in the Southern Hemisphere (Rebelo 1995). Flowering time Protea Species P. compacta P. cynaroides P. eximia P. grandiceps P. lacticolor P. magnifica

Trade name

J

F

M

A

M

J

J

A

S

O

N

D

Kingz Duchessz

*

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* *

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* * *

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*

*

*

*

* *

*

*

*

*

*

* * * * * *

* * * * * *

* * * * * *

* *

*

* Barbigeraz or Queeny

P. mundii P. nana P. neriifolia Minky P. pityphylla P. repens Sugarbushy P. scolymocephala Scolyz z

South Africa USA

y

* *

*

*

*

*

*

*

*

*

*

*

*

*

* * *

* * *

* *

*

*

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P. repens, and P. eximia, representing 58% of exports of flowering stems. Large quantities of bouquets, many containing P. eximia or P. compacta, are also exported from South Africa. Export and local sale of Protea is throughout the year, with a peak in export quantities during October. B. Research The domestication of Protea in South Africa began in 1913, with the inauguration of the National Botanical Garden at Kirstenbosch. The establishment of a collection of Proteaceae led to the publication of an article on cultivation, titled “The cultivation of proteas and their allies” (Matthews 1921). It was only in the late 1950s that a scientific manual was published on protea cultivation: Proteas: Know Them and Grow Them (Vogts 1959). Due to the growing interest in South Africa in proteas as a floricultural crop, the South African Department of Agriculture initiated a research program on proteas in the 1960s, under the leadership of Dr. Marie Vogts. The first research phase dealt with the identification, collection, and establishment in cultivation of the protea species in South Africa with floricultural potential. The collection of economically important species was established at Oudebosch near Betty’s Bay. Ten years of research resulted in the identification of horticultural variants within species. The characteristics of these variants were stable when propagated by achenes (Vogts 1971). In 1973, a breeding and selection program was initiated at Tygerhoek, near Riviersonderend, about 150 km from Cape Town. The collection of proteas was moved from Oudebosch to the new site. The first Protea cultivar resulting from this program was Guerna (Plate 1), a P. repens selection (Brits 1985). During the period 1988 to 1992 the germplasm collection of Proteaceae, or what is known as the field genebank (Littlejohn and de Kock 1997) was moved from Tygerhoek to a new site, Elsenburg, an experimental farm near Stellenbosch. In April 1992, the genebank collection was transferred to the Agricultural Research Council (ARC), a non-profit, non-governmental organization. The ARC is responsible for the maintenance of the field genebank and to research the commercialization of Southern African Proteaceae. Research in other countries where Protea is cultivated has been undertaken by various research organizations, with individuals within the organizations playing critical roles. In Hawaii, research on propagation, cultivation, selection, and diseases has been undertaken since the 1960s by the University of Hawaii. In California, the University of California

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has played an instrumental role in importing new plant material and in research on leaf blackening. Proteaceae research in Australia is conducted by a number of different organizations in Western Australia and Queensland, while in New Zealand the Horticulture Research Centre in Levin conducted Proteaceae research (Matthews and Carter 1983). In France, research on cultivation in soilless medium under glass at the Sophia Antipolis INRA station is underway, and in Tenerife, Spain, the University of La Laguna is active in Proteaceae research. The Volcani Institute in Israel has done excellent research on cultivation of Proteaceae in calcareous soils. However, worldwide research on Proteaceae as a horticultural crop is decreasing, although many problems for cultivators of cut flowers still exist. In the 1980s an active International Protea Working Group was inaugurated (Lamont 1984) but, by the late 1990s, the membership had dwindled to five researchers. C. World Industry The Proteaceae of Southern Africa are also cultivated in many other countries, such as Australia, Chile, El Salvador, France, Israel, New Zealand, Spain (Canary Islands), Portugal, the United States of America (California, Hawaii), and Zimbabwe (Leonhardt and Criley 1999). Cultivation in many countries developed simultaneously with the industry in South Africa. In Australia, the Botanical Garden in Adelaide began cultivating Cape flora in 1871 (Lighton 1960). The cut flower industry in Australia gained impetus when immigrants from South Africa, such as the Wood family, sold their farm in South Africa in 1984 and emigrated to Western Australia with large quantities of seed. Today, South African Proteaceae are cultivated in South Australia, Victoria, New South Wales, Queensland, and Western Australia, but no data exists on the extent of cultivation of the genus Protea. Large commercial plantations are especially found in the Busselton/Margaret River area of Western Australia. The largest nursery producing potted plants of various Proteaceae species is situated in Monbulk, Victoria, and is owned and run by the Matthews family. In New Zealand, origins are unclear, but it is widely believed that South African Proteaceae were brought there by soldiers returning from the Anglo-Boer War during the period 1899 to 1906 (Matthews and Carter 1983). In 1922, Duncan and Davies Nursery offered P. repens in their catalogue and Stevens Brothers began selling proteaceous cut flowers in 1945. Achenes imported from South Africa were used to hybridize the well-known Leucadendron cultivar, Safari Sunset. A Protea cultivar

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that originated from New Zealand is the P. repens hybrid, Clark’s Red. Proteaceae cut flowers are an important New Zealand export commodity, and are sold primarily to Japan and the Far East. There are no statistics on the extent of Protea plantations (Soar 1998). The industry in Hawaii developed from a research project on new cut flower crops at the University of Hawaii. While a Visiting Professor in Hawaii, Sam McFadden, University of Florida, imported a wide variety of propagative material in 1964. Included were proteaceous achenes. In 1968, Phillip Parvin joined the Faculty as Research Horticulturist at the Maui Agricultural Research Center, and spent the next 25 years assisting in the development of the protea cut flower industry in Hawaii. Today, approximately 60 ha of Proteaceae are cultivated in Hawaii (Wilson 1998). The cultivation of South African Proteaceae in California was promoted by Howard Asper of Escondido, who imported many species during the 1960s. Today, approximately 450 ha are under woody Southern Hemisphere plants for cut flower production, of which approximately 20% is the genus Protea (Perry 1998). Zimbabwe is a recent entrant to the international trade in Proteaceae. The primary initiators of cultivation on a commercial scale were the Miekle family in the late 1970s. The first Protea cut flower exports were made in 1981. The Australian cultivar Pink Ice was cultivated on a large scale in Zimbabwe, but recent problems with disease and insects have drastically reduced the hectarage. Other Protea cultivars and species are being used and approximately 78 ha are under plantations, with 140 ha of other Proteaceae (Middelmann and Archer 1999). The area under cultivation of Proteaceae in South America is approximately 8 ha, with 0.5 ha in Chile (Lobos 1998) and 7.5 ha in El Salvador (Veltman 1998). Spain and Portugal have approximately 30 ha of cultivated Proteaceae, located mainly on the islands of Madeira (Fernandes and Blandy 1998) and Tenerife (J. A. Rodríguez-Pérez, pers. comm.).

III. REPRODUCTIVE BIOLOGY The genus Protea range in size from small prostrate shrubs, some with underground stems, to large trees. All are evergreen, woody perennials with sclerophyllous leaves suited to withstand periods of hot, dry weather. Regeneration can take place through sprouting from the lignotuber in some species or by release of achenes, from infructescences

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maintained on the plant. The foliage varies from fine needle-like leaves in P. aristata to the petiolate oval or obovate leaves of P. cynaroides. The commercially valuable product in Protea is the terminal inflorescence. It is the size and color of the involucral bracts of the inflorescence, which range from greenish white through all shades of orange, pink, red to brownish-red that give the Protea their aesthetic appeal. The genus Protea is distinguished from all other African genera of the Proteaceae by its flowers. The perianth is bipartite, bilaterally symmetrical with the three adaxial perianth segments fused from the base of the tube to the tips of the limbs, forming a distinct sheath, while the abaxial perianth segment separates completely from the adaxial perianth sheath, falling free as each individual flower opens (Rourke 1980). Each flower is composed of four perianth segments and the individual flowers are aggregated together on the inflorescence, surrounded by a prominent involucre of colored and often tufted bracts. The involucral bracts provide the main floral display. The individual flowers develop spirally from the outer edge of the involucral receptacle. Three anthers are attached to the three fused perianth segments; the fourth anther is attached to the free perianth segment. The central pistil consists of an ovary containing a single ovule, a long style and a small stigmatic region at the tip of the style enclosing the stigmatic groove. The distal portion of the style is specialized to form the pollen presenter, the external morphology of which varies between species (Rourke 1980). The pistil of P. repens can be roughly divided into four major regions: the stigma, a vertebra-shaped upper style, a heart-shaped lower style, and the ovary (Van der Walt and Littlejohn 1996a). The upper pistil is modified to form the pollen presenter, an elongated, ridged structure where pollen is deposited prior to anthesis and a longitudinal obliquely placed terminal groove on the upper adaxial side of the stigma, the stigmatic groove. A layer of interlocking epidermal cells fringes the margin of the stigmatic groove. A stylar canal appears to run the length of the style, surrounded by densely packed transmitting tissue. The stylar canal joins up with the cavity formed between the ovule and the inner ovary wall. The ovary is partially embedded in the woody involucral receptacle of the inflorescence and contains one acutely obovate-shaped ovule. The observed pistil structure of P. repens is very similar to P. cynaroides (Vogts 1971), Macadamia (Sedgley et al. 1985), and Banksia (Clifford and Sedgley 1993). In all cases the style is woody, containing many sclerenchyma cells, but in Macadamia and Banksia the stylar canal does not extend along the entire length of the style.

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Trichomes are found on the outer surface of the ovary. After flowering, the fertilized ovules develop into obconic achenes, densely pubescent with long straight hairs, brown, rust-colored, black, or white (Rourke 1980). The viable achenes tend to be found in distinct groups, or clusters on the receptacle, which may be a mechanism to reduce insect predation (Mustart et al. 1995). It appears that the plant actively controls the clustering, but the mechanism of control is unknown. The achenes formed may be stored in infructescences, the woody flower receptacle enclosed by woody involucral bracts, on the plant (Bond 1984, 1985), with release being triggered when water supply to the infructescence stops, such as during a fire, at plant death, or when insects consume the infructescence stem. Protea adapted to arid conditions, such as P. glabra and P. nitida, release their achenes four to seven months after flowering. The function of the trichomes on the achenes is fourfold: (1) expansion of drying achenes assists in forcing the achenes from the drying infructescence, (2) on an airborne achene they assist with buoyancy in high winds, (3) they assist in anchoring the achene to the ground, and (4) they orientate the achene on the soil surface to ensure optimum water uptake for germination (Rebelo 1995). The flowers of Protea are protandrous, with the anthers dehiscing prior to the flower opening (Van der Walt and Littlejohn 1996b; Vogts 1971). The anthers deposit their pollen on the pollen presenter. During anthesis foraging fauna collects the pollen. Three types of fauna assist in pollination of Protea: birds (predominantly Promerops cafer, the Cape Sugarbird); small mammals such as mice, rats, and voles; and many types of insects (Collins and Rebelo 1992). The shape of the style in mammal pollinated Protea is curved (Plate 2), while bird and insect pollinated species have straighter styles. It is generally accepted that the Protea with large conspicuous inflorescences are bird pollinated, but species differ in dependency on birds as pollinators. Inflorescences of P. nitida, P. cynaroides, and P. repens bagged to exclude bird pollinators, but not insects, set achenes at the same rate as unbagged inflorescences (Coetzee and Giliomee 1985; Wright et al. 1991). In P. neriifolia, P. magnifica, and P. laurifolia the bagged inflorescences set significantly fewer achenes. At anthesis the stigmatic groove has not yet become receptive to pollen. In a study on P. repens and P. eximia, the stigmatic groove was open at its widest between three and six days after anthesis (Van der Walt and Littlejohn 1996b). The number of pollen tubes per style and the achene set recorded from controlled pollination indicated that peak receptivity of the stigma was between two and six days after anthesis.

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Stigmatic secretions in P. eximia increased as the stigmatic groove opened. The genus Protea has an inherently low achene set, between 1% and 30% under natural pollination conditions (Rebelo and Rourke 1986; Esler et al. 1989). Reasons cited for low achene set range from direct plant control of achene set numbers, pollinator limitation, insect and mammal predation, and poor nutrition. The percentage of florets with pollen tubes, the percentage of ovules penetrated by a pollen tube, and the achene set in P. repens and P. eximia are highly correlated, indicating that entry of a viable pollen tube into the stylar canal results in a viable achene. In P. repens the achene set from controlled self-pollination, open pollination, and pollination between different clones of P. repens resulted in the same high achene set percentages of between 40% and 74% (Van der Walt 1995), while the achene set of P. eximia did not exceed 10%. This is contrary to the generally accepted view that all Protea are obligatory cross-pollinators (Horn 1962) and supports the observation that achenes resulting from insect pollination are likely to be from self pollen (Wright 1994a). Pollination does not occur without a pollen vector, such as an insect or bird (Brits 1983).

IV. CROP IMPROVEMENT A. Genetic Variability The growth habit differences between species range from the Eastern and Western ground sugarbushes that have underground stems, to upright bushes typified by P. eximia, and to trees, such as P. nitida (Rebelo 1995). Some species have a lignotuber (a swelling of the stem at or just below ground level, covered in dormant buds that can regenerate after a fire), such as P. cynaroides and P. welwitschii, but most species do not. Protea are described as evergreen, but species differences occur, with some species having leaves that live for one year, e.g., P. nitida, and others with leaves remaining on the bush for up to 6 years, e.g., P. neriifolia. Leaf shape varies from the narrow, elongated leaves of P. longifolia to the ovate leaves of P. cynaroides that have a prominent leaf stalk. Interspecific hybrids exhibit characteristics intermediary to the parental species, allowing for ease of identification of the parents of interspecific hybrids (Vogts 1989). The color of the involucral bracts varies from brown, through shades of deep crimson, red and pink, to white or pale green, both within and between species. Further variation in flower

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appearance occurs due to differences in the color of the trichome tufts, or beard, at the ends of the inner and outer involucral bracts, especially in P. magnifica. Plant species with a predominantly outcrossing breeding system generally show high levels of phenotypic variability. The amount of phenotypic variability within species differs widely between species of Protea. In species with a wide habitat range, such as P. cynaroides, P. neriifolia, and P. magnifica, distinct horticultural forms (Plate 3) can be recognized (Vogts 1989). Studies indicated that the variation observed between seedling populations of P. cynaroides sampled from different localities was consistent when the plants were cultivated at a single locality, and therefore had a genetic basis (Vogts 1971). This was useful in selecting achene propagated populations that could flower at different times of the year and thus supply marketable flowers for 12 months of the year. In species with smaller habitat ranges, such as P. compacta, few observable differences are recognized between populations (Vogts 1989). Currently studies using RAPD-PCR analysis are being done by the Agricultural Research Council in South Africa to compare the extent of variation between species with a wide habitat range and those with a small habitat range. This information will assist in determining the extent to which populations must be sampled from, to try to maximize the variation within species kept in genebanks, botanical gardens, and in cultivation. There is a high level of genetic variation present in P. neriifolia based on analysis of segregation after self-pollination (G. M. Littlejohn, unpubl.). Measurements of various traits on mature seedling plants obtained by self-pollination of a single selected clone of P. neriifolia showed significant variation between seedlings. The type of traits measured included growth habit, plant height, flower color, leaf length and width, inflorescence length and width, inflorescence mass, style length, and the concealment of the inflorescence by the leaves. Genetic improvement is closely linked to the process of domestication of an essentially wild plant, such as the Protea (Brits et al. 1983). Domestication generally follows three phases: (1) the harvesting of wild flowers; (2) the selection of superior populations or clones; and finally, (3) the development of new variations by hybridization, aimed at improving traits of importance in cultivation (Brits 1984). In a woody, perennial plant the breeding process is lengthy. The duration from collected wild plant material to acceptance of a cultivar developed by controlled hybridization can take up to 40 years (Fig. 1.2). This time span allows only for evaluation at one site, and no regional evaluation. Regional evaluation would increase the time span by four to six years (Wessels et al. 1997).

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Fig. 1.2. Sequence of events and time lapse in the development of a Protea cultivar, from selection of wild harvested material through controlled hybridization and the selection of a superior hybrid.

B. Selection The first stage in selection is the selection of species suitable for cultivation. Vogts (1989) provided Protea enthusiasts with a book on the Proteaceae and information on how to cultivate them. Of the species

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described in the book, 150 were identified as suitable for cultivation, with 86 having very good market potential (Brits et al. 1983). Characteristics sought for in suitable species included: attractive and arresting appearance, color, shape and size of flower head, foliage attractive but not dominating; flower head neither hidden nor pendulous; erect growth providing long, straight flower stems; good cultivation potential and ease of achene propagation; stability of characters; desired flowering time; post harvest quality; no obnoxious odor. Selection within a species can take two forms: selection for an improved population or selection of a unique individual from a population that is propagated clonally. Both of these methods have been used in Protea. The identification of horticultural variants within certain Protea species identified populations suitable for use in initiating mass selection for improving populations (Vogts 1989). Brits (1985) documented the selection of an achene propagated cultivar of P. repens, Guerna, which comprised 18 similar clones. Achene propagation or clonal propagation could be used. The success of selection of unique individual plants from within a population is dependent on the level of genetic variation present in the population from which one is selecting (Vogts 1989). Selection criteria for single plant selections are determined by the flower traits together with the producer requirements. These are summarized in Table 1.4. Single plant selections that have become successful cultivars include P. eximia cv. Fiery Duchess, P. magnifica cv. Atlantic Queen, and P. cynaroides cv. Red Rex (see Table 1.5).

Table 1.4. The characteristics desirable in a single plant selection for use as a cut flower cultivar. Production characteristics

Flower characteristics

High yield Growth vigor Longevity Ease of rooting Good regeneration after pruning Tolerance to different soil types Tolerance to cold Tolerance to heat Insect resistance Ability to shift flowering time

Flower head color Flower head shape Attractive foliage that does not conceal flower head Terminal flower with no secondary growth (bypass) Straight stems Stems longer than 40 cm Ease of packing Resistance to leaf blackening Vase life of 10 days minimum Involucral bracts that retain their turgidity and color and do not brown under hot, dry conditions Ease of removal of leaves on lower flower stem

Disease resistance

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Table 1.5. Some cultivars in Protea, selected predominantly from chance hybrids or as single plant species selections, and recorded in the International Protea Register (Sadie 1998). Putative parentage

Cultivar namesz

P. burchelli/P. longifolia P. compacta/P. burchellii P. compacta/P. eximia P. compacta/P. magnifica

Nomad (SA) Brenda (SA) Pink Duke (SA) Andrea (SA), Lady Di (SA), Margot (SA), Pink Velvet (SA) Carnivalz (SA) Red Baron (SA) Pink Ice (Aus) Artic Ice (NZ), Attaturk (Zim), Clarez (SA), Ivory King (Zim), Florindinaz (SA), Madibaz (SA), Red Rexz (SA) Cottontop (SA), King Grand (SA) Valentine (SA) Duchess of Perth (Aus), Fiery Duchess (SA) Baron (Aus), Cardinal (SA), Sylvia (SA) Helzaan (SA) Ivy (SA) Pretty Annez (SA) Atlantic Queen (SA), Chelsea (SA) Sheilaz (SA), Kurrajong Rose (Aus) Princess (SA) Pinitaz (SA), Possum Magic (Aus) Pacific Queen (NZ), Venetia (SA) Candidaz (SA), Ruthz (SA) Susara (SA) Empathy (Zim) Frosted Fire (Aus), Pretty Belindaz (SA) Nataliaz (SA) Barber’s Hybrid (NZ), Anneke (SA) Davidz (Is), Jossef z (Is), Michalz (Is), Shlomoz (Is) Ansiz (SA), Lizlz (SA), Petrouxz (SA), Riaz (SA) Pixiez (Aus) Embers (SA), Guerna (SA), Rubens (SA), Sneyd (SA), Sugar Daddy (SA) Liebencherryz (SA) Sweet Suzyz (SA) Venusz (SA) Clark’s Red (NZ) Kurrajong Petite (Aus)

P. compacta/P. neriifolia P. compacta/P. obtusifolia P. compacta/P. susannae P. cynaroides

P. cynaroides/P. grandiceps P. cynaroides/P. compacta P. eximia P. eximia/P. susannae P. glabra/P. laurifolia P. lacticolor/P. mundii P. laurifolia/P. sulphurea P. magnifica P. magnifica/P. burchellii P. magnifica/P. laurifolia P. magnifica/P. longifolia P. magnifica/P. neriifolia P. magnifica/P. obtusifolia P. magnifica/P. susannae P. mundii/P. subvestita P. neriifolia P. neriifolia/P. repens P. neriifolia/P. longifolia P. obtusifolia P. pityphylla/P. effusa P. pudens/P. longifolia P. repens P. repens/P. longifolia P. repens/P. mundii P. repens/P. aristata P. repens/P. aurea P. repens/P. pudens

Key: z Protected by plant breeder’s rights, or under application; Aus = Australia, Is = Israel, NZ = New Zealand, SA = South Africa, Zim = Zimbabwe

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Early in the development of the fledgling protea industry in South Africa, it was observed that chance occurring interspecific hybrids produced new, unique flower forms, the plants often exhibiting greater vigor than either parental species (Vogts 1989). This led to the active search for interspecific hybrids by growers and the selection of many of these as cultivars, all clonally propagated by means of cuttings (see Table 1.5). This was also the impetus behind the initiation of a controlled breeding program, based primarily on the development of interspecific hybrids. C. Hybridization The controlled pollination method developed for Leucospermum has been extensively used in Protea hybridization (Brits 1983). The method entails covering the inflorescence of the female parent to exclude all possible pollinating fauna after removing any flowers with dehisced anthers. Two days later the unopened flowers are all removed from the center of the inflorescence, leaving a single ring of approximately 40 to 60 flowers that are newly opened. The pollen from the pollen parent is applied by using a style with pollen on the pollen presenter as a “brush” applicator. The inflorescence is re-covered. Mature achenes are harvested between nine and twelve months later. The achene set obtained by using this technique in Protea have been dismally small (Brits 1992), except in the case of intraspecific hybridization in P. cynaroides and P. repens (Table 1.6). Modifications to this technique have been made, using information gleaned from the studies of natural pollination. Firstly, it has been found that viable achenes are often found clustered on the involucral receptacle and this appears to be under direct control of the female plant (Wright 1994a,b; Mustart et al. 1995). Secondly, visual observation of the Table 1.6. Controlled hybridization results in Protea using the Leucospermum hybridization technique (Brits 1983). Female parent

Male parents

P. aristata P. compacta P. cynaroides P. eximia

P. aristata, P. repens P. compacta, P. eximia, P. cv. Sylvia P. cynaroides P. eximia, P. eximia/P. compacta, P. repens P. repens, P. obtusifolia, P. cv. Ivy P. repens

P. pudens P. repens

Average achene set (%) 0 1.2 20.4 0 0 24.6

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involucral receptacle indicates that space could be a limiting factor in achene development, similar to that observed in Banksia (Fuss and Sedgley 1991a,b). Therefore the pollination technique was modified so that at the first visit after bagging the inflorescence only 10 to 20 flowers are pollinated, with the removal of the next spiral of flowers. This is done repeatedly over three to four successive visits to pollinate flowers on the inflorescence, with a final visit to remove the central, remaining flowers. While very time consuming, the increase in success of obtaining mature, viable achenes makes the effort worthwhile (Table 1.7). The full scope of interspecific hybridization can only be utilized if pollen can be successfully stored for use on species or clones flowering at different times of the year. The pollen of four Protea species was successfully stored for 12 months, desiccated, either at –18°C in an ordinary household deep freeze or in liquid nitrogen (Van der Walt and Littlejohn 1996c). D. Interspecific Hybridization Interspecific incompatibility can be exhibited at different stages during the reproduction process or in the interspecific hybrid plant. The simplest form of incompatibility takes place prior to fertilization, where pollen tube growth from a “foreign” species cannot grow down the style

Table 1.7. Some successful cross combinations obtained in Protea by using the modified controlled pollination technique. Seed parent

Pollen parent

P. aurea P. neriifolia P. magnifica/P. laurifolia P. pudens P. pudens P. neriifolia/P. burchellii P. eximia P. compacta/P. neriifolia P. lepidocarpodendron/P. neriifolia P. magnifica/P. laurifolia P. lepidocarpodendron/P. neriifolia P. eximia/P. susannae P. compacta P. compacta P. burchellii P. magnifica/P. laurifolia

P. lacticolor/P. mundii P. holosericea P. holosericea P. acuminata P. nana P. holosericea P. compacta P. repens/P. aristata P. magnifica/P. neriifolia P. neriifolia P. laurifolia/P. magnifica P. compacta P. compacta/P. burchellii P. longifolia/P. burchellii P. compacta/P. burchellii P. magnifica/P. obtusifolia

Achene set (%) 19 9 20 63 27 8 16 1 5 2 8 5 15 15 8 1

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of the seed parent and no fertilization occurs (Van Tuyl 1989). Studies on P. repens and P. eximia indicated that the ten-fold decrease in achene set observed after interspecific pollination compared to intraspecific pollination was due to pollen tube growth being interrupted while growing down the style of the female parent (Van der Walt and Littlejohn 1996a). High correlation was observed between the number of flowers in which pollen tubes observed entered the ovule and the percentage achene set recorded. This indicates that in these two species, post fertilization mechanisms to inhibit interspecific hybridization were not active. Incompatibility can also be detected in poor vigor and growth of interspecific hybrids. In general, interspecific hybrids in genus Protea are vigorous (Brits 1983). A further level of incompatibility is chromosomal incompatibility, leading to loss of sexual reproduction capacity in interspecific hybrids. Pollen grain infertility is a good indicator of meiotic disturbances during the development of the pollen grains (Van Tuyl 1989). In genus Protea the fertility of pollen ranges from 0% in the case of P. cynaroides interspecific hybrids to 89% in a P. laurifolia hybrid (Van der Walt and Littlejohn 1996b). No pattern of relatedness between parental species and pollen fertility was detected. Pollen size varied significantly between and within species. Meiotic analysis of interspecific hybrids of Protea has not yet been done and is complicated by the small size of the chromosomes and the woodiness of the flowers. No differences in the basal chromosome number of 12 have been recorded between species (De Vos 1943). E. Cultivars The aim of a breeding program is to develop cultivars (see Table 1.5) suitable for commercial exploitation for cut flower production. Currently cultivars of genus Protea originate from three sources: selection of individual superior plants from within species, selection of chance hybrids (Plate 4), and selection from achenes obtained from controlled hybridization (Plate 5) (Table 1.8). The parentage of chance hybrids is deduced from knowledge of characteristics of taxonomic importance between the seed parent and possible pollen parents growing in the vicinity of the seed parent. Prior to 1973, commercial plant resources were undescribed and traded collectively under their old specific names, e.g., P. barbigera Meisn. for P. magnifica Link. In 1973 an international cultivar registration program for Proteaceae was launched, South Africa having obtained authority from the International Society for Horticultural Science to act

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Examples of cultivars derived from different sources within genus Protea.

Source

Cultivar name

Putative parentage

Selection of superior plants within species

Fiery Duchess Atlantic Queen Snow Queen Cardinal King Grand Susara Madiba Clare

P. eximia P. magnifica P. magnifica P. eximia/P. susannae P. cynaroides/P. grandiceps P. magnifica/P. susannae P. cynaroides/P. cynaroides P. cynaroides/P. cynaroides

Selection of chance interspecific hybrid Selection derived from controlled pollination

as the International Registrar of all protea cultivars falling within the South African genera (Brits et al. 1983). Some of the well known Protea cultivars incorporated in the international register are listed in Table 1.5.

V. PHYSIOLOGY Protea exhibits some unique physiological traits, such as the role of roots in water and nutrient uptake and carbohydrate metabolism in the cut flowering stems. The understanding of many physiological processes is incomplete, but this provides a fertile area for continued research. A. Flowering Protea species growing in their natural habitat are observed to flower at distinct times of the year (see Table 1.3). The majority of commercially used Protea flower naturally during the autumn to spring months of the Southern Hemisphere. The high demand for flowers in Europe, the dominant market for South African Proteaceae, is mid spring to mid summer, a time when few species flower. This has resulted in studies aimed at elucidating how flowering is initiated and if it can be manipulated. The Protea stem grows in spurts (called flushes) during loosely defined growth periods during the year. This produces clearly defined growth flushes on the stem. Under the climatic conditions of the Western Cape, the predominant growth periods are: Winter (March to August), Spring (September to November), Summer (December to January), and Autumn (February to March) (Malan 1993). The number of flushes, ranging from none to two, produced during each growth period, is influenced by the environmental conditions and the species. The Protea inflorescence is borne terminally on a shoot consisting of two or more

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growth flushes. The flushes arise in succession from a distal axillary bud, with flushes exhibiting strong apical dominance during active growth. Inflorescence initiation in Protea cultivar Carnival, a putative hybrid between P. compacta and P. neriifolia takes place after cessation of growth of the spring or summer flush under conditions in the Western Cape, South Africa. Generally two or more successive flushes are required for an inflorescence to initiate (Greenfield et al. 1993). A spring flush must be subtended by at least one previous flush for flower initiation to take place. Although not investigated in other species or hybrids, the requirement for at least two growth flushes subtending a flower is likely to hold for all other species. In some species, such as P. neriifolia, flowers are produced on secondary growth flushes that initiate below the current flower head, during the same season. This appears to be species specific, and will only occur on flowering stems with a large diameter (G. M. Littlejohn, pers. obs.). A minimum diameter of the flush subtending the inflorescence, a possible requirement for flowering to take place, has not been determined for any of the Protea. There are indications that the sink capacity of the stem plays a role in the ability of a stem to initiate an inflorescence (De Swardt 1989). Pruning studies on Protea cv. Carnival have shown the possibility of manipulating the flowering time, stem length, and production of mature bushes by manipulating the pruning time (Gerber et al. 1993; Hettasch et al. 1997). Pruning the plant during the early spring months results in no flowering in the following spring, probably due to limited leaf area. Inflorescences are initiated on the spring and summer flushes of the following year, resulting in peak flowering during February as opposed to normal peak flowering during April. The bearing cycle of the plant is transformed in this way from an annual cycle to a biennial cycle. This also allows each stem to develop more growth flushes, which results in longer stems and a greater marketable harvest. The precise environmental and intraplant factors triggering inflorescence initiation are still unclear. Dupee and Goodwin (1990a) observed flower initiation on the first spring flush in P. neriifolia cv. Salmon Pink, while seedlings of the Long Leaf variant of P. cynaroides initiated flowers on the summer flush as well as the autumn flush. The flowering time and number of flowers harvested from different Protea species changed, depending on the site at which they were planted (Dupee and Goodwin 1990b, 1992). A delay in flowering, of approximately six months, and a reduction in flower number occurred at the site with the highest altitude, lowest mean winter temperature and largest difference in day length between summer and winter. ‘Guerna’ produces only 18 flowers per bush during the period of December to February at 33°

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Table 1.9. Comparison of flowering times and flower yield of Protea cultivars grown in Hawaii (21°N, 900 m) and South Africa (33°S, 177 m ), with flowering season corrected to the Southern Hemisphere. Months Flowering

Cultivarz

Location y

Guerna

Hawaii South Africa

Brenda

Cardinal

Red Baron

J

F

M

A M

J

J

A

S

O

N

*

*

*

*

*

*

*

*

*

*

*

*

Hawaii

*

*

South Africa

*

*

*

Hawaii

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

Hawaii

*

*

*

*

*

South Africa

*

*

*

*

*

*

86

*

18

*

*

Hawaii

Flowers harvested

210 20

South Africa

South Africa Sylvia

*

D

*

*

*

*

*

31 35

*

*

*

*

*

86 24

*

*

*

*

*

*

*

66 38

z

Refer to Table 1.8 for parents. Hawaii data based on Criley et al. (1996); South African data based on Littlejohn (unpublished).

y

South, compared to 86 stems per bush at 21° North spread over twelve months of the year (Table 1.9). In other cultivars, differences in flower time and flower number per plant per annum occurred when grown in Hawaii or South Africa. While flower numbers can be accounted for by differences in soil fertility, the time of flowering appears dependant on differences in day length. It would appear that in the absence of clear environmental cues, such as changes in day length, many Protea produce a flower on a stem when sufficient carbohydrate source is available in the stem. This latter method is employed by ‘Sylvia’, a backcross of P. susannae on a hybrid between P. eximia and P. susannae (Malan and Le Roux 1995). Although ‘Sylvia’ naturally flowers during the late summer and autumn in South Africa, flowering over the full year can be obtained if pruning is scheduled to occur throughout the year. B. Propagation 1. Sexual Reproduction. The fruits of the Protea species are held on the woody receptacle enclosed by the involucral bracts. The Protea species

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J. COETZEE AND G. LITTLEJOHN

found in the savanna areas outside the Cape Floral Kingdom release their achenes between two and four months after flowering (Rebelo 1995). The Eastern Ground and Western Ground Protea (Table 1.1) generally release their achenes one to two years after flowering. The remaining species store the achenes in the infructescence indefinitely, a process called serotiny. The achenes are subject to large variations in temperature and the infructescence may become waterlogged during heavy rains, but germination will only take place after the achenes fall to the ground (Rebelo 1995). About 80% of viable achenes will germinate within 90 days, if kept sufficiently moist and at temperatures ranging from 5° to 25°C (Van Staden 1966). The duration from fertilization until harvest of achenes of Protea affects the germination rate and amount of achenes germinating (Van Staden 1978; Le Maitre 1990). Dormancy seems to be imposed by a low temperature requirement and by the action of the pericarp, which prevents simultaneous germination of all achenes (Deall and Brown 1981). Scarification, stratification, and incubation in pure oxygen improved the germination of P. compacta (Brown and Van Staden 1973). Treatment of P. compacta with Promalin, a solution containing GA4/GA7 and benzyladendine, increased germination, as did a stratification treatment of 60 days at 5°C, but treatment with GA3 reduced germination (Mitchell et al. 1986). Rodríguez Pérez (1995) observed an improvement in germination after imbibition with GA3 in P. neriifolia and P. eximia, but no significant difference in P. cynaroides. The optimum cues for maximum germination are likely to differ between the Protea species, as has been observed in Leucospermum (Brits 1990c). 2. Vegetative Propagation. Members of the Proteaceae can be propagated by vegetative cuttings. The selection of single plants for use as clonally propagated cultivars depends upon the ability to propagate the plant material vegetatively. Most commercial Protea species are propagated by using approximately 20 cm long terminal, semi-hardwood cuttings (Malan 1993). Sub-terminal cuttings can be successfully used in some cultivars (Harre 1995) and may be the preferred type of cutting (Montarone et al. 1997). Sub-terminal cuttings of ‘Sylvia’ and ‘Cardinal’ delivered more vigorous plantlets with improved branching complexity at an earlier age. Rooting of leaf bud cuttings is also possible in P. obtusifolia (Rodríguez Pérez 1992). In general a 5 sec basal dip in indole butyric acid at 1,000 to 4,000 ppm is followed by setting the cuttings in well aerated medium with intermittent mist and bottom heat at 22° to 25°C (Malan 1993; Harre 1995). Rooting generally occurs within six to 16 weeks. Auxin concentration (Perry 1988), auxin carrier (Gouws et al. 1990), and hormone mixtures (Criley and Parvin 1979; Gouws et al. 1990) all influence rooting success. Specific requirements have to be adapted for each

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cultivar for optimum results (Harre 1995). The frequency of misting (Perry 1988), bottom heat temperature, light intensity, and rooting medium aeration (Harre 1995) also affect rooting. The time of harvesting cuttings is important in Protea, where growth flushes are not always well synchronized (Malan 1993), because the physiological status of the new growth flushes may not be consistent. Scarring of the base of the cutting is effective in promoting rooting of some Protea cultivars (Rodríguez Pérez 1990). Control of diseases while plants are rooting is important to ensure success (Benic 1986) and includes proper sanitation in the mother plants. 3. Grafting. Grafting of Protea has focussed on using alkaline tolerant P. obtusifolia as a rootstock (Brits 1990a,b). The most successful method is the grafting or budding onto cuttings. The cutting can be rooted or unrooted. With unrooted cuttings, rooting and graft union are achieved simultaneously in a mist propagation facility. This latter technique has been successfully applied to Leucadendron (Ackermann et al. 1997). Factors requiring more research in Protea grafting are ease of rooting of the rootstock and selection for low phenolic production in the rootstock and scion, or methods to control blackening of the cut surfaces (Brits 1990b). Low grafting success in Protea was not ascribed to incompatibility between scions and rootstock. An extensive search for rootstocks within Proteaceae resistant or tolerant to root rot caused by Phytophthora cinnamomi highlighted successful scion and rootstock combinations within the different genera and indicated combinations where graft incompatibility occurred (Moffat and Turnbull 1995). P. cynaroides grafted successfully onto a variety of Protea species, but graft union failure occurred after one to two years, with eventual death of the scion. The most successful rootstocks tested were Protea cultivar Pink Ice and P. roupelliae. 4. Tissue Culture. Tissue culture techniques for propagation of Protea (Rugge 1995) have been developed. The major problem in genus Protea is the browning of the tissue due to phenolic compounds (Malan 1993), however, shoot proliferation has been obtained in P. repens, P. obtusifolia, and P. cynaroides. Successful transplanting of rooted shoots to soil has not been achieved. Callus and proteoid roots have been raised from mature cotyledons of Protea (Van Staden et al. 1981). C. Water and Nutrient Uptake Most species of Protea are adapted to nutrient-poor soils derived from Table Mountain Sandstone, with a pH (KCl) between 4 and 6 and a clay content of less than 20%. P. obtusifolia is found only on limestone

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J. COETZEE AND G. LITTLEJOHN

calcareous sands with a pH (KCl) as high as 8 and P. susannae on the fringes of the limestone areas with pH (KCl) in the region of 6 to 7. P. laurifolia can be found on shale soils with a higher silt content. The two rare species, P. mucronifolia and P. odorata, are adapted to growing on shale derived soils (Rebelo 1995). The most striking adaptation of the Proteaceae to the nutrient-poor soils on which they are found is the presence of proteoid roots, first described by Purnell (1960). The root system of Protea consists of a deep tap root, primarily a root for sourcing water, and shallow, lateral roots in the upper five to 10 cm that bear clusters of proteoid roots. Proteoid roots are specialized lateral roots that are diarch, show limited growth, and do not undergo secondary thickening. They bear profuse root hairs that are ephemeral and sometimes branched. Under natural conditions they first appear on roots of seedlings about six months old when the cotyledons are just withering away. The proteoid roots enable the plant to efficiently extract soil phosphorus (Lamont 1982), nitrogen, and potassium (Vorster and Jooste 1986a,b). In Protea growing under seasonally dry conditions, such as their natural habitat, proteoid roots are seasonal structures. Proteoid and other roots are only formed during the wet season (Lamont 1983). Shoot growth is predominantly during the dry, warm season. High nutrient levels in the soil, especially phosphates, inhibit the formation of proteoid roots in many of the Proteaceae (Grose 1989; Silber et al. 1997). Proteaceae are also characterized by highly efficient utilization of P within the plant (Grundon 1972; Grose 1989). The use of tissue and soil samples to determine the seasonal nutritional requirements has not been entirely successful (Parvin 1986). Seasonal and interplant differences in the cycle of growth flushes makes interpretation of leaf samples difficult (Barth et al. 1996). Leaf nutrient composition for ‘Pink Ice’ was studied in detail and the results are summarized in Table 1.10. The range in nutrient concentrations is given for the two periods of the year, i.e., mid summer and late autumn through winter, when the variation between samples and plants was the least. Significant positive and negative correlations were observed between nutrients, e.g., N concentrations were positively correlated with P, K, Na, and Zn and negatively correlated with Ca, Mg, and Fe concentrations. These significant relationships may indicate synergistic and antagonistic interactions between nutrients that need to be considered when interpreting plant nutrient data. Research effort has focussed on the cultivation of Protea in soilless media (Montarone and Allemand 1993). This has led to clarification of the total plant uptake of nutrients for certain species and clones (Mon-

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Table 1.10. Range in mean nutrient concentrations of leaf samples of Protea cultivar Pink Ice during two periods of the year.z Nutrient

December to February

May to August (% dry weight)

N P K Ca Na S

0.82–0.83 0.06–0.07 0.37–0.41 0.46–0.51 — 0.11–0.13

Cu Zn Mn Fe

— 12–15 43–44 —

0.77–0.86 0.05–0.06 0.18–0.21 0.63–0.68 0.14–0.18 0.09–0.10 (mg/kg) 3.5–4.5 — — 51–54

z

Data from Maier et al. (1995).

tarone and Ziegler 1997). It is obvious that differences between species exist in terms of their requirements for different nutrients (Claassens 1986). Water requirements of the different species grown under soilless conditions differ (Montarone and Ziegler 1997), with P. cynaroides requiring twice the amount of water required by P. eximia. Water requirements can be deducted by knowledge of where species grow naturally, i.e., species growing in wet valleys or near water sources have higher water requirements than species preferring dry areas (Manders and Smith 1992). Protea, however, will not grow under waterlogged conditions (Vogts 1989). Investigations on the water requirement of cultivated Protea under irrigation indicated that maintenance of a high soil water capacity was essential to the field survival of rooted cuttings of the Protea cv. Cardinal (Van Zyl et al. 1999). Active consumption of water continued throughout the year and maintenance of high soil water levels increased the shoot lengths and biomass production on cultivar Cardinal in comparison with lower soil water levels. D. Postharvest Physiology In the genus Protea, vase life reduction is associated with the phenomenon of leaf blackening due to oxidation of phenolic compounds in the leaves (McConchie et al. 1991). The vase life of Protea is generally three

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J. COETZEE AND G. LITTLEJOHN

to four weeks, but postharvest leaf blackening reduces the vase life to approximately one week. Discoloration of Protea leaves can be induced by mechanisms such as pre-harvest mechanical damage, insect or fungal attack, or excessive heat; however, postharvest leaf blackening occurs on leaves without any physical damage (Jones et al. 1995). Although pre-harvest conditions such as waterlogging, drought, and harvesting stems from aged plants have been reported to affect the extent of leaf blackening (De Swardt 1979), little is known of the possible mechanisms involved. Symptoms of leaf blackening occur within 2 to 5 days after harvest in P. eximia and P. neriifolia (McConchie et al. 1991). The extent of leaf blackening varies widely between species (McConchie and Lang 1993), clones within species (Paull and Dai 1989), and the time of year. Paull and Dai (1989) found a reduction in leaf blackening if inflorescences were harvested in the afternoon compared to the morning and if inflorescences were harvested when the involucral bracts had just opened rather than at the soft bud stage. Fumigants used for insect disinfestation of inflorescences after harvest can also increase leaf blackening (Coetzee and Wright 1990; Karunaratne et al. 1997). Removal of the inflorescence significantly delays the onset of leaf blackening (Reid et al. 1989; Dai 1993). The inflorescence continues to expand after harvest and exhibits a high rate of respiration (Ferreira 1986) with a large volume of nectar production when open (Cowling and Mitchell 1981). Removal of the inflorescence, girdling of the stem just below the inflorescence (Dai 1993; Reid et al. 1989), adding 2.5% to 5% of sucrose to the vase solution (Dai 1993), or placing the floral stems in bright light (Reid et al. 1989) delays or even prevents leaf blackening. The starch and sucrose concentration in leaves declines in stems held in the dark rather than in the light (McConchie et al. 1991; Bieleski et al. 1992). The physiological basis of leaf blackening is still poorly understood. It appears to be a complex cascade of events that lead to the oxidation of phenolic compounds (Jones et al. 1995). This occurs, either enzymatically via polyphenol oxidase or peroxidase, or non-enzymatically after cleavage of phenolic glycosides by glucosidases. It is still not clear if membrane degradation occurs during leaf blackening (Jones et al. 1995). A reduction in leaf carbohydrate levels is coincident with leaf blackening. Dai and Paull (1995) concluded that leaf blackening in Protea is a result of depletion of carbohydrate by the inflorescence. This was due primarily to the sugar demand for nectar production.

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VI. PRODUCTION A. Cultivation Cultivation techniques, describing the basic cultivation practices in different regions of the world, have been published in books by Matthews (1993), Vogts (1989), and Harre (1995). The Agricultural Research Council of South Africa has compiled a handbook on cultivation of Proteaceae (1998). The cultivation of Protea is limited by the availability of suitable soils and climatic conditions (Vogts 1989). The soils must be well drained and acidic, except in the case of lime tolerant species such as P. obtusifolia. Clay content less than 20% is preferred, but up to 50% clay will be tolerated by some species as long as the drainage is excellent. Hot, humid conditions are not well tolerated by Protea and sufficient air movement is required for healthy growth. High light intensity is required. Protea are generally cultivated without protection and in open soil. In South Africa, two forms of cultivation are practiced: intense cultivation of clonal and seed material in rows, and broadcast seed sowing. The latter is used primarily for P. repens and other species used in the dried flower industry (Coetzee and Littlejohn 1995). Cultivation under glass in soilless media is possible (Montarone and Allemand 1993) and is considered economically viable in the south of France. The general recommendation is to use a between row spacing of 3.5 to 4.0 m and a within row spacing of 0.8 to 1.0 m, giving a plant density of 2,500 to 3,560/ha. In practice, much closer spacing, with plant densities of up to 6,000/ha, is used by many farmers. The most important factors determining plant spacing are the size of the farm implements available to the farmer and the size of the plantation. In plantations small enough to be managed with hand labor only, plants are more closely spaced, but in large plantations wide inter-row spacing is required for the mechanical equipment. Soil preparation prior to planting depends on the soil type and depth. In very shallow soils, ridging is recommended to improve the depth of soil available for plant growth. Ridging is also used to improve the drainage of heavy soil. In very rocky soil, or on very steep slopes, no soil preparation is done. In soils of a good depth, liming and adjustment of the macro and micro nutrient levels by fertilization prior to soil preparation to a depth of 1 m is recommended. Drip irrigation is the preferred method of supplying water to Protea during the dry season. Overhead irrigation is not suitable as it increases

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J. COETZEE AND G. LITTLEJOHN

the possibility of diseases and large droplets can damage the flower heads and leaves. The Protea species and hybrids used in cultivation will tolerate dry summer periods, but sensitivity to lack of water during the winter varies, e.g., P. repens will tolerate dry winter conditions in a summer rainfall area, but P. stokoei will not. Inorganic and organic mulches are widely used. The choice of the type of mulch depends on the soil type, soil temperatures, and cost of the mulch. Low growing cover crops that have a low cutting frequency are recommended between rows to assist in weed control. Fertilization programs differ from locality to locality, depending on the chemical and physical properties of the soil, the biomass removed annually from the plants during harvest and pruning, and the cultivar being grown. The general recommendations are not to apply large amounts of phosphates, nor use fertilizers in which more than 50% of the nitrogen is bound in nitrates. Top-dressing with potassium during the life of the plant will be necessary. Maintenance of the immature bushes requires pruning to develop a complex structure of bearers as soon as possible. Under conditions where the plants grow slowly, annual pruning is sufficient, but in warmer areas where plants grow faster, pruning will be required two to three times a year during the first two years. Protea cultivars are generally able to bear a harvest of flowering stems of sufficient length two to four years after planting, depending on the parentage of the cultivar. Bushes in production will be pruned to leave bearers for the following crop during the harvest of flowering stems, with additional pruning to remove unwanted vegetative stems as required. Pruning to achieve biennial production requires leaving a long bearer when the flowering stems are harvested. This long bearer is then re-cut during the early spring to remove any new shoots, thereby timing the initiation of the new shoots correctly for manipulation of the flowering time. The number of bearers, and therefore shoots per plant, at any stage of the plant’s development is dependent on the cultivar and its interaction with the climatic and soil conditions. Flowering stems are harvested at any stage between soft-bud, or anthesis of the outer ring of florets (Plate 6). The stems are best placed immediately in water, with cooling to 2° to 5°C within 60 minutes after harvest. Thereafter the cool chain should be maintained until the stems are sold to the florist or consumer. In exporting countries the cold chain is of necessity broken during air transport. The stem length categories for export standards from South Africa start at a minimum of 40 cm, with an increase in length of 10 cm for the next category. The stem length of the longest and shortest stem packed in a carton may not differ by more than 5 cm and the stem may not deviate by more than 5 cm from straight.

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The Protea with small flower heads, such as P. nana, may be exported from 25 cm in length and longer. Maximum allowable blemishes, either physical or due to disease, on the involucral bracts and leaves are also defined, but each importing country sets its own phytosanitary restrictions. The cultivation of Protea, both within its natural habitat and in other regions is increasing annually (Middelmann and Archer 1999). Species such as P. cynaroides grow under a wide variety of conditions, but other species, such as P. compacta and P. magnifica, grow poorly when cultivated outside their natural habitat range. The interspecific hybrids registered as cultivars (Table 1.5) are generally easily cultivated under a diversity of conditions. B. Pathogens Associated with Diseases of Protea There are a number of unique pathogens associated with Protea species, as well as some wide host range pathogens that attack these plants. References are also made to fungi that attack proteas when they are cultivated outside their natural habitat (Forsberg 1993; Ziehrl et al. 1995; Swart et al. 1998; Swart 1999). The first protea disease was described by Cooke (1883) and since then more than 30 pathogens have been isolated from Protea, of which nine can be considered as economically important diseases of Protea species (Table 1.11). Diseases are one of the limiting factors in the commercialization of proteas. Diseases can lead to the total destruction of cultivated proteas. Infected foliage and/or stems of protea flowers are esthetically not acceptable and lead to phytosanitary problems during international trade. In the past, disease resistance was not taken into account with cultivar development, as selections were primarily aimed at flower characteristics (Knox-Davies et al. 1986). As a result epidemic disease problems can occur with intensive cultivation of clonal proteas. The most important diseases of Protea species can be grouped into root diseases, leaf spot diseases, diseases of the shoots, stem and inflorescence, and the cankers. With the exception of one bacterial disease, all of these diseases are caused by fungi. There have been no confirmed reports of Protea infected by viruses. 1. Pathogens of Roots. Phytophthora cinnamomi is an important root pathogen of Proteaceae in Australia (Forsberg 1993), New Zealand (Greenhalgh 1981), South Africa (Knox-Davies et al. 1986), and the U.S.A., especially Hawaii (Kliejunas and Ko 1976; Rohrbach 1983). The disease causes root and crown rot, and is commonly referred to as

Leaf spot

Mycosphaerella jonkershoekensis P.S. Van Wyk, Leaf spot Flower head blight Anthracnose/tip die-back Root pathogen Fusarium wilt Root and collar rot Root and collar rot Damping-off Basal stem, crown or collar rot and root rot Shoot wilting and chlorosis of foliage

Phyllachora proteae Wakef.

Botrytis cinerea Pers: Fr.

Collectotrichum gloeosporioides (Penz.) Penz. and Sacc

Armillaria luteobubalina Watling and Kile

Fusarium oxysporum Schltdl.: Fr.

Macrophomina phaseolina (Tassi) Goid.

Phytophthora nicotianae Breda de Haan

Rhizoctonia solani J.G. Kühn

Rosellinia De not. sp.

Verticillium dahliae Kleb.

(Forsberg 1993)

(Forsberg 1993)

(Rohrbach 1983)

(Forsberg 1993)

(Benic 1986)

(Swart et al. 1998)

(Forsberg 1993)

(Benic & Knox-Davies 1983)

(Serfontein & Knox-Davies 1990b)

(Wakefield 1922)

(Van Wyk et al. 1975a,b)

(Van Wyk 1973a)

(Van Wyk 1973a)

(Saccardo 1910)

(Marasas et al. 1975)

(Van Wyk 1973a)

Reference

10/16/2000 9:03 AM

Marasas and Knox-Dav.

Leaf spot

Leaf spot

Coleroa senniana (Sacc.) Müller & Arx Leaf spot

Leaf spot

Batcheloromyces proteae P.S. Van Wyk and Knox-Dav.

Mycosphaerella proteae Sacc.

Root and crown rot

Phytophthora cinnamomi Rands

Leptosphaeria protearum Syd. and P. Syd.

Name of Disease

Economically relevant diseases of Protea species.

Pathogen

Table 1.11.

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Bacterial leaf spot Leaf spot Leaf spot Leaf tip disease Leaf spot

Pseudomonas syringae Moffatt

Cercostigmina protearum (Cooke) U. Braun

Clasterosporium proteae M.B. Ellis

Coniothyrium Corda emend. Sacc. Species

Mycosphaerella bellula Crous and M.J. Wingf.

Leaf spot

Trimmatostroma macowanii (Sacc.) M.B. Ellis

Silver leaf Trunk rot Die back Die-back

Schizophyllum commune Fr.: Fr.

Sclerotinia Fuckel. sp.

Phomopsis (Sacc.) Sacc. sp.

Dothiorella Sacc. sp.

Chondrostereum purpureum (Perd.: Fr.)

Leaf spot Leaf spot

Didymosporium congestum Syd.

Marasas and Knox-Dav.

Leaf spot Leaf spot

Teratosphaeria proteae-arboreae P.S. Van Wyk,

(Orffer and Knox-Davies 1989)

(Benic 1986)

(S. Denman, pers. comm.)

(Forsberg 1993)

(Anon. 1991)

(Diodge 1950)

(Ellis 1976)

(Van Wyk et al. 1975a)

(Sydow and Sydow 1912)

(Crous and Wingfield 1993)

(Van Wyk 1973a)

(Ellis 1976)

(Crous and Braun 1996)

(Paine and Stansfield 1919)

(Benic 1986)

10/16/2000 9:03 AM

Teratosphaeria fibrillosa Syd. and P. Syd.

and Crous var. protearum

Damping-off

Pythium vexans De Bary

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J. COETZEE AND G. LITTLEJOHN

the sudden death syndrome. Infected plants become chlorotic and wilt as a result of extensive root rot (Von Broembsen 1979, 1989; Cho 1981). Most protea deaths occur during hot dry periods and on badly drained soils (Newhook and Podger 1972; Pegg and Alcorn 1972; Van Wyk 1973b). P. cinnamomi can be isolated from seedlings with damping-off symptoms in seedbeds and from cuttings in nursery beds (Benic 1986; Forsberg 1993). Symptoms are generally less severe and develop more slowly on Protea than on other Proteaceae such as Leucospermum and Leucadendron. Protea cynaroides, P. neriifolia, and P. repens appear to be resistant to P. cinnamomi. Other soil-borne pathogens of Protea are listed in Table 1.11. 2. Pathogens of Leaves. Protea species are generally more prone to leaf spot diseases than other Proteaceae (Van Wyk 1973a) and the only bacterium, Pseudomonas syringae, was isolated from the leaves of P. cynaroides in England (Paine and Stansfield 1919) and Australia (Wimalajeewa et al. 1983). Bacterial leaf spot has not been recorded in South Africa (Knox-Davies et al. 1986). Batcheloromyces proteae Marasas is one of the economically important pathogens of Protea leaves. The leaf spots are not destructive but decrease the quality of the leaves for commercial use. The most typical lesions are black, with a red-brown to purple-black discoloration of the leaf tissue (Marasas et al. 1975). The host range includes the following economically important proteas, P. cynaroides, P. grandiceps, P. magnifica, P. neriifolia, P. punctata, and P. repens (Marasas et al. 1975; Smith et al. 1983; Van Wyk et al. 1985; Knox-Davies et al. 1986; Swart 1999). Coleroa senniana was first described by Saccardo (1910) on leaves of P. gaguedi (P. abyssinica) from North Africa. The fungus commonly occurs on leaves of Protea species in Southern Africa (Doidge 1941) and is, except for Mycosphaerella proteae, probably the most widespread pathogen of Protea species. C. senniana produces tiny black specks (pseudothecia of the fungus) on the upper surface of Protea leaves. On P. magnifica the specks are yellow to brown (Van der Byl 1929; Serfontein and Knox-Davies 1990a). Coleroa senniana occurs on leaves of summer and winter rainfall Protea throughout sub-Saharan Africa (Saccardo 1910) and was also isolated on cultivated Protea in California, U.S.A. (Swart 1999). Leptosphaeria protearum causes leaf spots that are necrotic and sunken, with raised, dark brown margins (Van Wyk 1973a). Most economically important proteas are affected by L. protearum, but P. mag-

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nifica is particularly susceptible. Leptosphaeria protearum appears specific to Protea species (Von Broembsen 1989). Mycosphaerella proteae is the most common pathogen on Protea species in South Africa (Van Wyk 1973a) and the host range includes winter and summer rainfall proteas (Saccardo 1891; Sydow and Sydow 1914; Doidge 1921; Van Wyk et al. 1975a,b; Swart 1999). The leaf spots caused by M. proteae on the different hosts are quite variable in appearance but the spots are amphigenous and bright red-purple to red-brown. Mycosphaerella jonkershoekensis has so far only appeared on P. repens and P. magnifica (Van Wyk 1973a; Van Wyk et al. 1975a,b) and causes greyish to light brown leaf spots with raised, dark brown margins. Phyllachora proteae lesions are typically necrotic with a raised margin and move from the leaf tip inwards and finally cover the entire leaf surface (Wakefield 1922; Van Wyk 1973a; Van Wyk et al. 1975a). The host range includes P. acaulis, P. magnifica, P. neriifolia, and P. repens (Van Wyk 1973a; Van Wyk et al. 1975a). Van Wyk (1973a) stated that Phyllachora proteae must be reclassified as a species of Botryosphaeria. P. proteae has been reclassified as Botryosphaeria proteae Wakef. Denman & Crous. (Denman et al. 1999). Vizella interrupta G. Winter, S. Hughes causes brown lesions on Protea leaves, which often coalesce. The ascocarps form black spots on slightly discolored leaf tissue on Protea species. The host range includes P. cynaroides, P. grandiceps, P. magnifica, and P. neriifolia (Van Wyk 1973a; Van Wyk et al. 1975b, 1976; Swart 1999). 3. Pathogens of Shoots, Stems, and Inflorescences. Colletotrichum gloeosporioides, or colletotrichum die-back, is the most important disease of Protea species (Coetzee et al. 1988). The die-back of young shoot tips is the most characteristic symptom. Other symptoms include necrotic stem and leaf lesions, stem rot, sunken stem cankers, seedling damping off, seedling blight, and cutting die-back (Von Broembsen 1989; Forsberg 1993). Colletotrichum lesions on one side of the stem cause the new growth to bend. This is referred to as shepherd’s crook disease of proteas. All economically important Protea species are affected by colletotrichum die-back in South Africa, Australia, and Hawaii (Greenhalgh 1981; Benic and Knox-Davies 1983; Benic 1986; Knox-Davies et al. 1986; Anon. 1991). Botrytis cinerea causes blight of the flowering branches and inflorescence heads. In Protea species, B. cinerea is a strong, active pathogen that can invade actively growing tissues and inflorescences (Rohrbach 1983). Brown spots develop on the leaves and inflorescence buds. The lesions expand and inflorescence buds can be killed, with necrosis

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extending down the inflorescence stalks, causing death of affected parts and new shoots (Serfontein and Knox-Davies 1990b; Forsberg 1993). Infected shoot tips collapse, darken, and die. Bending of affected shoots is typical of botrytis damping-off (Forsberg 1993) and has been recorded on cuttings showing die-back symptoms (Benic 1986). The host range includes P. cynaroides and P. repens in South Africa and Hawaii (Swart 1999). 4. Pathogens of Woody Stems. Botryosphaeria species that cause cankers and die-back of injured tissue are a common problem and cause considerable losses in the production of Protea cut flowers. The most important species associated with Protea are Botryosphaeria dothidea (Moug.: Fr.) Ces and De Not., or Botryosphaeria ribis (Tode: Fr.) Grossenb. and Duggar. The host range includes P. compacta, P. cynaroides, P. eximia, P. grandiceps, and P. repens (Van Wyk 1973a; Knox-Davies et al. 1981; Swart 1999). In South Africa, only two chemicals are registered for the control of diseases on proteas. Looking at the complexity of the proteaceous pathogens, as well as the lack of control strategies of the diseases, it becomes evident that diseases are the most limiting factor in the commercialization of proteas. To prevent the development of diseases, the breeding and selection of resistant or tolerant cultivars will play an important role in the future. C. Phytophagous Insect Fauna of Protea From studies on the insect guilds of P. repens (Coetzee and Latsky 1986), P. cynaroides and P. neriifolia (Coetzee 1989), P. magnifica and P. laurifolia (Wright 1990), and P. nitida (Visser 1992), it is clear that proteas harbor a rich and distinct entomofauna. Insects associated with Protea species play an important ecological role as pollinators (Coetzee and Giliomee 1985), folivores (Wright and Giliomee 1992), and seed predators (Myburg and Rust 1975). Protea insects of significant economic importance can be divided into flower visitors, endophagous or borers, folivorous insects, and sap-suckers. 1. Flower Visitors. The nectar and pollen rich protea flower attracts more than 200 insect species (Gess 1968) with, in many cases, high population levels (Visser 1992). Sugar birds like Promerops cafer (Mostert et al. 1980) and rodents (Cowling and Richardson 1995) pollinate proteas and it is also possible for insects to successfully pollinate Protea species (Coetzee and Giliomee 1985). Collins and Rebelo (1987) suggested that bird pollinated seed would be of genetically higher quality than seeds

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resulting from insect pollination, as birds have a larger foraging range and this could result in greater heterozygosity. Insects pollinating Protea species are generalist flower visitors and it is possible that larger beetles (Coleoptera, Scarabaeidae) may be more important pollinators than smaller insects (Wright 1990), again due to the greater mobility of larger insects. The presence of insects in cut flowers is one of the most serious limiting factors influencing the South African protea industry (Wright and Saunders 1995). Research on the use of a negative pressure fumigation system, based on a forced cooling system, provided excellent insect control using dichlorvos aerosol (Wright and Coetzee 1992; Wright 1992). 2. Borers. Inflorescences and infructescences of Protea species are attacked by the larvae of a range of insects (Coetzee and Giliomee 1987a,b). The endophagous predators of serotinous protea seed are listed in Table 1.12. Insect seed predation of canopy stored Protea seed banks may be a factor that reduces the potential of proteas to form monospecific stands (Wright 1994b). Borers attacking Protea infructescences are also an important guild of pests of cultivated Protea, attacking young shoots and flower buds (Myburg and Rust 1975). On P. cynaroides, the larvae of the protea butterfly, Capys alphauses, has been recorded destroying up to 40% of the flower buds. Endophagous larvae cause phytosanitary problems, when present in cut flowers. Infested infructescences serve as a reservoir where pest numbers can increase and orchard sanitation is a practice that should be applied to reduce borer incidence (Coetzee et al. 1988). 3. Folivorous Insects. As the foliage of protea cut flowers must be esthetically acceptable, the leaves must be free of insect damage. Leaves of proteas are attacked by herbivores, leafminers, and gall forming insects. Leaf Table 1.12. Genus Sphenoptera Genuchus Euderes Capys Orophia Argyroploce Bostra Tinea

Endophagous insects of Protea species. Species

G. hottentottus (Frabricius) E. lineicollis (Wiedemann) C. alphaeus (Cramer) O. ammopleura (Meyrick) B. conspicualis Warren

Family

Order

Buprestidae Scarabaeidae Curculionidae Lycaenidae Oecoporidae Torticidae Pyralidae Tineidae

Coleoptera Coleoptera Coleoptera Lepidoptera Lepidoptera Lepidoptera Lepidoptera Lepidoptera

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feeders can remove 5% to 22% of the leaf surface (Coetzee 1989; Wright and Giliomee 1992). Leaf miners cause scarring of leaves, which renders the final product unmarketable, while gall forming insects are a phytosanitary risk (Wright and Saunders 1995). Young protea leaves are protected by a range of unique anti-herbivore mechanisms such as phenolic compounds (tannins) and a pronounced cyanogenic capacity. Some species cover their young leaves with a thick layer of trichomes. This strategy has led to insects avoiding the more succulent and nutritious young leaves in favor of older, tougher leaves (Coetzee et. al. 1997). However, some of the most important herbivores on Protea species (Bostra conspicualis Warren, Pyralidae, Lepidoptera, and Afroleptops coetzeei) (Oberprieler), Curculionidae, (Coleoptera), have alimentary tract pH levels which suggest adaptation to a tannin rich diet (Wright and Giliomee 1992), which allows them to utilize older leaves in spite of the presence of tannins. Leafminers on Protea species are a guild of micro-lepidoptera that have successfully overcome the defense mechanism of young Protea leaves. The micro-lepidoptera belong to the families Phyllocnistidae, Incurvanidae, and Gracillaniidae. Only Proteaphagus capensis (Scoblein), Incurvariidae, found on P. cynaroides has been identified. The rest are still unknown and very little is known about their life cycle. Gall insects that belong to the Psyllidae (Hemiptera) can form galls on leaves of P. repens and cause phytosanitary problems. 4. Sap Suckers. A selection of sap suckers feed on proteas. These can transfer diseases by means of their mouth parts, but cause little physical damage. Stressed plants can die when infestations are not controlled. Sedentary sap suckers include mealy bug (Pseudococcidae) and scale insect species of the Coccidae and Diaspididae (Coetzee 1989), which causes phytosanitary problems with the export of flowers. Insects cause serious problems where proteas are cultivated in their natural habitat. Where proteas are cultivated outside their natural habitat, no serious insect problems have been experienced. This indicates that insects cannot easily overcome the defense mechanisms of the genus Protea.

VII. CONCLUSION Protea have become an established horticultural crop, with a world sale of approximately 8 million flowering stems. In South Africa 3.01 million stems are exported, 1.14 million sold through the formal market, and 1.01 million sold by the informal sector. Other producing countries

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do not have figures for sales of Protea, but total sales are estimated at 3.00 million. Less than 1.5 million stems are sold through the Dutch auction system annually. The total share of the world flower market filled by Protea is very small, but it is the flower identified with South Africa. P. cynaroides is the national flower of South Africa and is the symbol of its sports teams. In the Cape Floristic Region, Proteaceae is an important component of the agricultural sector and the industry provides many job opportunities. Cultivation in areas outside their natural habitat has increased dramatically, both within South Africa and in other countries with similar climate and soil conditions. This has led to large quantities of proteas on the international market originating from regions other than the endemic environment from which Protea originate. Thus the Cape region and the people who initiated the protea industry run the risk of losing their market share. The stipulations of the Convention on Biological Diversity, which focus on benefit sharing related to commercial exploitation of genetic resources, would appear to have no practical application to the Protea genetic material. Protea species propagation material is widely available from around the globe. The majority of the most widely used cultivars originate in South Africa, but practical and financially viable methods of ensuring that royalties are returned to the legal owners of cultivars are insufficient. Solutions to this problem are being sought. An interesting pattern in the development of the indigenous cut flower industry in South Africa is that changes in the industry have most often been preceded by research activities. The challenge for South Africa is to produce high-quality blooms for the Western European market during the hot dry summer months. The majority of the Protea bloom during the early winter to late spring, while the Western European markets buy Protea during their Northern Hemisphere winter period from September to May. Selection and breeding has resulted in cultivars that flower in the summer, but more types are needed. It is also necessary to develop cultivars of similar appearance, but successive flowering periods, to provide a continuous supply of blooms to the market. An increase in the cultivation of the winter flowering species in the Northern Hemisphere could negatively impact on the Southern Hemisphere countries. Leaf blackening remains a problem in all regions where Protea are grown. Leaf blackening reduces the appeal of Protea to the consumer. It may be possible to reduce leaf blackening by genetic manipulation. If cultivars with reduced potential for leaf blackening can be developed, it would impact positively on the industry. There are pests and diseases of Protea that are common to the different regions in which they are cultivated. South Africa has the challenge

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of cultivating Protea in their natural habitat, with all the co-evolved insects and pathogens present in the natural fynbos. It is necessary to continuously research chemical and biological control measures. Environmentally sound practices must include the breeding of disease resistant cultivars to reduce the dependence on chemical control. Refinement of cultivation practices, such as pruning, fertilization, and irrigation, is required to maintain the economic return of Protea as a crop and to ensure the delivery of quality blooms to a very competitive international market. The challenges of cultivating Protea differ from region to region, but the basic plant physiology controlling the plant’s reaction to environmental stresses remains the same. Funding for basic research has, in the past, been generously supplied by government organizations, but in the economic climate of the late 1990s, government support of research is dwindling. This is especially true in South Africa, where flowers in general are still minor crops. The international flower markets are always searching for new, exciting products. Protea can fulfil this demand. A larger variety of cultivars, with different forms and colors, longer vase life, exceptional quality, and extended availability during the year are needed to maintain and increase the market share. These goals will only be achieved by continued research.

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Littlejohn, G. M., and M. B. de Kock. 1997. Review of the fynbos genebank project. Acta Hort. 453:81–86. Littlejohn, G. M., I. D. van der Walt, G. C. van den Berg, W. de Waal, and G. J. Brits. 1995. “Marketable” product approach to breeding Proteaceae in South Africa. Acta Hort. 387:171–176. Lobos, A. M. 1998. Area Report: Chile. 9th Biennial Int. Protea Ass. Conf and Int. Protea Working Group Workshop Abstr., Int. Protea Assoc., Cape Town, South Africa. Maier, N. A., G. E. Barth, J. S. Cvecil, W. L. Chvyl, and M. N. Bartetzko. 1995. Effect of sampling time and leaf position on leaf nutrient composition of Protea ‘Pink Ice’. Austral J. Expt. Agr. 35:275–283. Malan, D. G. 1993. Propagation of Proteaceae. Acta Hort. 316:27–34. Malan, D. G. 1995. Crop science of Proteaceae in Southern Africa: Progress and challenges. Acta Hort. 387:55–62. Malan, D. G., and R. le Roux. 1995. Preliminary investigation into the effect of time of pruning on shoot growth and flowering time of Protea. Acta Hort. 387:91–98. Manders, P. T., and R. E. Smith. 1992. Effects of watering regime and competitive ability of nursery-grown Cape fynbos and forest plants. S. Afr. J. Bot. 58:188–194. Marasas, W. F. O., P. S. Van Wyk, and P. S. Knox-Davies. 1975. Batcheloromyces, a new genus of annellidic dematiaceous Hyphomycetes on Protea cynaroides in South Africa. S. Afr. J. Bot. 41:41–45. Matthews, J. W. 1921. The cultivation of proteas and their allies. J. Bot. Soc. S. Afr. 7:15–16. Matthews, L. 1993. The protea growers handbook. Kangaroo Press, Manakau, New Zealand. Matthews, L., and Z. Carter. 1983. South African Proteaceae in New Zealand. Matthews, Manakau, New Zealand. McConchie, R., and N. S. Lang. 1993. Postharvest leaf blackening and preharvest carbohydrate status in three Protea species. HortScience 28:313–316. McConchie, R., N. S. Lang, and K. C. Gross. 1991. Carbohydrate depletion and leaf blackening in Protea neriifolia. J. Am. Soc. Hort. Sci. 116:1019–1024. Middelmann, M. C. 1998. Area Report: South Africa. 9th Biennial Int. Protea Ass. Conf and Int. Protea Working Group Workshop Abstr., Int. Protea Assoc., Cape Town, South Africa. Middelmann, M. C., and C. Archer. 1999. Overview of Southern Africa protea industry. Proc. 5th Austral. Wildflower Conf., Melbourne, Australia, p. 70–73. Mitchell, J. J., J. van Staden and N. A. C. Brown. 1986. Germination of Protea compacta achenes: the relationship between incubation temperature and endogenous cytokinin levels. Acta Hort. 185:31–38. Moffatt, J. and L. Turnbull. 1995. Grafting Proteas. Nanju Protea Nursery, Queensland, Australia. Montarone, M., and P. Allemand. 1993. Growing Proteaceae soilless under shelter. Acta Hort. 387:73–84. Montarone, M., D. Savignac, and P. Bearex. 1997. Effect of the position of cuttings along the mother stem on the rhizogenesis and the ramification in soilless cultivation. Acta Hort. 453:29–34. Montarone, M., and M. Ziegler. 1997. Water and mineral absorption for two protea species (P. eximia and P. cynaroides ) according to their development stage. Acta Hort. 453: 135–144. Mostert, D. P., W. R. Siegfried, and G. N. Louw. 1980. Protea nectar and satellite fauna in relation to the food requirements and pollinating role of the Cape Sugarbird. S. Afr. J. Sci. 76:409–412.

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2 The Molecular Biology of Plant Hormone Reception Carole L. Bassett USDA-ARS Appalachian Fruit Research Station 45 Wiltshire Road Kearneysville, WV 25430

I. INTRODUCTION A. Overview of Signal Transduction B. Why Study Signal Transduction? II. CURRENT STATUS OF SIGNAL RECEPTOR RESEARCH A. Animal models 1. Ligand-gated and Voltage-gated Ion Channels 2. Seven-transmembrane Receptors 3. Receptors with Guanylate Cyclase Activity 4. Steroid Receptors 5. Serine/threonine-phosphorylating Receptors 6. Protein Tyrosine Receptor-like Phosphatases 7. Tyrosine-phosphorylating Receptors B. Bacterial Models 1. Histidine/aspartate-phosphorylating Receptors 2. Serine/threonine-phosphorylating Receptor Protein Kinases III. PLANT HORMONE RECEPTORS A. Histidine/aspartate-phosphorylating Receptors: Two-component Reception 1. Ethylene Reception 2. A Cytokinin Two-component Receptor B. Serine/threonine-phosphorylating Receptor Protein Kinases 1. Orphan Receptors 2. A Possible Brassinosteroid Receptor Protein Kinase 3. The Clavata1 Receptor Protein Kinase C. Seven-transmembrane Receptors 1. Is GCR1 a Second Cytokinin Receptor? 2. Is a Seven-transmembrane Protein a Possible Gibberellin Receptor?

Horticultural Reviews, Volume 26, Edited by Jules Janick ISBN 0-471-38789-4 © 2001 John Wiley & Sons, Inc. 49

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D. Current Status of the Perception of Other Plant Hormones 1. Are There Multiple Auxin Receptors? 2. Are There Multiple ABA Receptors? 3. Jasmonic and Salicylic Acid Receptors 4. Ca+2 Ion Channels IV. FUTURE PROSPECTS LITERATURE CITED

I. INTRODUCTION A. Overview of Signal Transduction All living organisms must be capable of responding to external and internal signals to survive in a constantly changing environment. The process by which living cells perceive and respond to these signals is called signal transduction, and the pathways they have evolved to accomplish this task are at the same time both highly conserved and incredibly variable in order to provide flexibility for adaptation. In the simplest sense, a cell must be able to recognize specific signals and transmit them (transduction) to the appropriate target molecules to elicit responses at the biochemical and/or molecular levels. This can be accomplished via several different mechanisms. For example, intercellular signaling between animal cells occurs by the rapid exchange of ions and other small molecules through channels called gap junctions. Gap junctions are composed of a family of integral membrane proteins (connexins) that join the cells through the gap junctions. Aside from roles in nerve transmission and conferring selective permeability to numerous small molecules, gap junctions have also been hypothesized to influence growth, differentiation, and developmental signaling (Goodenough et al. 1996). Another mechanism of signal transmission involves the guanine-nucleotide-binding (G) proteins that transduce signals regulating a variety of cellular processes. There are two types: the heterotrimeric (three different subunits) G-proteins generally associated with seventransmembrane receptors (see Section IIA2), and the small, monomeric GTPases represented by Ras and Rab that bind other proteins that modulate their activity. Small GTPases are associated with several important cell functions, including signal transduction, e.g. Ras (as well as heterotrimeric G-proteins), and vesicular transport, e.g. Rab. Finally, the most extensively characterized mechanism of signal transduction is the phosphorelay system involving the transfer of phosphate molecules from one protein to another, causing the target protein to become either activated or deactivated (Fig. 2.1). Although other types of posttranslational modification of proteins affect their activity, phosphorylation

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Fig. 2.1. Hypothetical phospho-transfer signaling pathway. A hypothetical pathway for signaling via phospho-transfer through a receptor protein kinase (RPK) is illustrated. The signal event is initiated by the binding of a signal molecule (L = Ligand) to the receptor protein kinase. Ligand binding alters the conformation of the RPK and stimulates autophosphorylation of the receptor kinase, thus activating the protein kinase function. The active RPK phosphorylates a cytoplasmic protein kinase (PK), converting it from inactive to active status. In this illustration the hypothetical cytoplasmic protein kinase has two target proteins. One is an enzyme in some biochemical pathway. Phosphorylation of this enzyme inactivates it and shuts down the biochemical pathway with which it is associated. It should be noted that the target enzyme may be another PK that in turn phosphorylates yet another PK, forming a cascade that broadens the response potential of the ligand. The second target is a transcription factor that is activated upon phosphorylation. The active transcription factor binds to certain DNA sequences and activates transcription of specific genes. The forward and reverse arrows indicate reactions that proceed in either direction: forward arrows represent the reaction catalyzed by phosphorylation and reverse arrows indicate reactions catalyzed by removal of the phosphate by protein phosphatases. Small black circles represent phosphate groups.

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appears to be the most global form of response to signaling. Indeed, both the connexins of gap junctions and G-proteins have been shown to be modified by phosphorylation. Phospho-transfer reactions are catalyzed by an enormously complex group of enzymes called protein kinases; the system is usually returned to the pre-responsive state by the removal of phosphates from target proteins via enzymes known as phosphatases. Since a pathway can consist of numerous, sequential protein kinases each with different target proteins, signaling pathways resemble modern computer arrays where a series of “off/on” switches set at specified positions determines a particular end result. However, unlike simple, linear computer arrays, protein kinases may have single (“dedicated”) or multiple targets, including protein kinases in other pathways, thus webs symbolize their interactions more accurately than straight lines. It is obvious that this arrangement provides remarkable flexibility for cellular responses and ensures that different pathways can “communicate” with one another. It is also apparent that unraveling the chain of events in just a single pathway could be quite challenging, particularly if the pathway involves many different protein kinases. B. Why Study Signal Transduction? Signal transduction not only provides the means for an organism to “sense” its environment, but also represents the process through which organisms grow and develop. Adaptation to adverse external conditions includes defensive responses to pathogen and pest attack, to mechanical damage, to temperature extremes, and to all other physical/chemical stimuli having a negative impact on the organism’s survival. Signal transduction regulates cell division and cell expansion and therefore controls the organism’s growth. Likewise, signaling pathways regulate differentiation (for example, in response to hormones), and thereby control the development and maturation of the organism. It is safe to say that signal transduction occupies a central position in cellular metabolism. As a result, it is important for us to identify the components of crucial signaling pathways and understand how they are regulated with regard to the pathway itself and with respect to each other. It is only then that we will be able to logically manipulate the components in ways that can lead to improved cultivars. As applied to plants, for example, the ability to boost a plant’s defenses through manipulation of relevant signaling pathways would result in greater resistance to diseases, pests, damage, or temperature extremes, depending on the specific pathway targeted for manipulation. This is a major goal of current research aimed

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at improving the plant’s ability to cope with biotic and abiotic stresses. Regarding control of cell division and differentiation applied to plants, manipulation of signaling pathways through genetic engineering will lead to, for example, better control of flowering, fruit development/ ripening, vegetative and seed development, and senescence. One obvious application would be to extend the life of cut flowers by interfering with signaling pathways controlling senescence, e.g. ethylene response in the flower. Almost any process involving traits of agricultural or horticultural interest is likely regulated by phosphorelay signal transduction and could theoretically be manipulated to improve the trait once the signaling pathway(s) are identified and characterized.

II. CURRENT STATUS OF SIGNAL RECEPTOR RESEARCH A. Animal Models Much of what we know today about phospho-transfer signal transduction comes from research using animal and bacterial systems. Nearly 40 years ago, Krebs and Fischer (1962) described what is now a classical example of an enzyme, glycogen phosphorylase, whose activity was modulated by phosphorylation/dephosphorylation at a specific serine residue. The phosphorylating (protein kinase) and dephosphorylating (phosphatase) enzymes represent one component of a signal transduction pathway that controls the metabolism of starch. Since then, other components have been identified, e.g. receptors, transducers, and second messengers. There are at present seven major types of receptor/signaling pathways that have been well characterized from various animal systems and two from bacteria (Fig. 2.2). It is anticipated that many others will also be identified and characterized in the near future. 1. Ligand-gated and Voltage-gated Ion Channels. Extracellular ligandgated (ELG) receptor channels combine ion-selective functions with those for ligand binding and signal transduction within a multi-subunit molecular assembly. In general, activation of the receptors results in the opening of cation-influx (depolarizing) channels, while inhibition of the receptors is generally associated with increased chloride ion permeability and hyperpolarization. Examples of ELG receptor-channels include those directly gated by neurotransmitters, e.g. acetylcholine and 5-hydroxytryptamine, which allow the influx of Na+, K+ and/or Ca+2 into the ligand-stimulated cell. ELG channel activity is independent of any intracellular or membrane-diffusible factors, although phosphorylation is

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Fig. 2.2. Summary of model phosphorelay signaling pathways. I: Voltage-gated ion channel. Subunits are designated α1, α2, β, γ and *. The alpha 1 subunit perceives the electrical signal and forms the channel. II: Seven-transmembrane pathway. cAMP/cGMP: 3′,5′ cyclic adenosine/guanosine monophosphate; DAG: diacylglycerol; IP3: myo-inositol 1,4,5 triphosphate; PKC: protein kinase C; PKA: protein kinase A; Cm: calmodulin; CDPK: calcium dependent protein kinase; α, β and γ inside open geometrical shapes: subunits of a heterotrimeric G-protein coupled to receptor; = ligand. III: Receptor with guanylate cyclase activity: = guanylate cyclase domain; = region with homology to protein kinases. IV: Steroid pathway: = ligand; = receptor. V. Pathway involving serine/threonine-phosphorylating receptors: ligand; = type I receptor; = type II receptor. VI: Pathway involving receptor protein tyrosine phosphatase: = domain with phosphatase activity. VIIa and b: Pathways involving protein tyrosine kinases. VIIa: JAKSTAT pathway. JAK: Janus class of protein kinases = ; STAT (signal transducers and activators of transcription) = ; = signal transducing polypeptide; , = ligands. VIIb: Other receptor tyrosine kinases, e.g., growth factor and insulin receptors. = phosphatidylinositol kinase, ras or phospholipase C (gamma); = enzyme target. VIII: Bacterial two-component pathway. = ligand; = enzyme; = response regulator. IX: Bacterial serine/threonine-phosphorylating receptors. = ligand; = target (β-lactamase); C = carboxyterminus; N = aminoterminus. Objects drawn in dotted lines indicate suspected, but not yet demonstrated components. Open objects represent targets for phosphorylation; Phosphates are represented by small black circles(•).

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a major mechanism for regulating the function of these channels (Swope et al. 1992). Structural similarities among subunits of different ELG receptor-channel proteins predict a common transmembrane topology that underlies functional relatedness (Betz 1990). Voltage-gated ion channels are responsible for the generation of electrical signals in cell membranes through changes in permeability to sodium, calcium, or potassium ions (Catterall 1995). These channels exist in one of three functionally distinct states: resting (non-conducting), active, and inactive (depolarizing). Since Ca+2 plays a significant role in overall signaling as a type of second messenger (see Section IIA2), the remainder of this section will focus on the characterization of voltagegated Ca+2 ion channels. These channels are composed of a complex of five different proteins: γ, α1, α2, β, and δ (Fig. 2.2, I). Each α and β subunit is represented by more than one gene, and the expression of different α and β subunits can alter the response of the ion channel to stimulation and/or depolarization. The α1 subunit alone is capable of forming a voltage-gated ion channel; the other subunits apparently modulate its activity. β subunits can be phosphorylated by numerous protein kinases and selectively dephosphorylated by protein phosphatases. Dephosphorylation can influence the number of active Ca+2 channels and the extent of their inactivation after stimulation. The fourth hydrophobic segment (S4) in each α subunit domain is thought to serve as the voltage sensor (receptor). A ryanodine-sensitive, calcium release channel distinct from the plasma membrane-associated channel also exists in cells, but is located in the endoplasmic reticulum and is associated with the storage and release of internal Ca+2. 2. Seven-transmembrane Receptors. As the name implies, receptors in this category (Dohlman et al. 1991) traverse the plasma membrane seven times through sequences of hydrophobic amino acids comprising membrane-spanning regions (Fig. 2.2, II). A classic example of a seventransmembrane receptor is the β2-adrenergic receptor that recognizes epinephrine and norepinephrine as inducing signals (ligands). These receptors are coupled to heterotrimeric G-proteins that transduce signal “information” from the receptor to an effector. The effector can be an ion channel, e.g. calcium, or an enzyme(s) that catalyzes synthesis of second messengers such as cyclic adenosine and guanine monophosphates (cAMP, cGMP), diacylglycerol (DAG), and/or myo-inositol 1,4,5 triphosphate (IP3). In general, pathways utilizing cAMP and cGMP activate protein kinase A (also known as cAMP-dependent protein kinase), whereas DAG and IP3 activate protein kinase C. Calcium appears to act essentially as a second message, activating Ca+2- and calmodulin-dependent protein

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kinases (CDPK and CmDPK, respectively). The seven-transmembrane receptors have no direct phosphorylating capacity by themselves, but must work through a second message system to activate the pathway and transfer “information.” 3. Receptors with Guanylate Cyclase Activity. Peptide hormones, such as the atrial natriuretic factor (ANF), which regulate salt-water homeostasis in vertebrates, bind to two types of receptor. The best characterized of these receptors is known to stimulate the synthesis of cGMP upon ligand binding. The structure of this guanylate cyclase type receptor (Fig. 2.2, III) consists of an extracellular ligand-binding domain and an intracellular guanylate cyclase activity (Rosenzweig and Seidman 1991). There is also a cytoplasmic domain with structural similarity to protein kinases that appears to repress guanylate cyclase activity in the absence of ANF binding. 4. Steroid Receptors. Unlike most receptors, steroid hormone receptors are actually transcription factors (Fig. 2.2, IV) that exist in an inactive state either in the cytoplasm or nucleus (Tsai and O’Malley 1994). The steroid hormone signal crosses the plasma and/or nuclear membrane and binds to the receptor, causing it to become activated. This hormonereceptor complex then binds the appropriate DNA response element(s) and activates transcription of any gene containing this sequence. DNA elements recognized by the receptor complex are composed of palindromic sequences that “read” the same from both directions. Steroid receptors are highly phosphorylated and it is thought that the degree of phosphorylation modulates the activity of the receptor by enhancing its ability to activate cognate genes (Ali et al. 1993) or by increasing its retention in the nucleus (DeFranco et al. 1991). Interestingly, several steroid receptors can be activated by other compounds in the absence of normal hormone activation. For example, dopamine can activate both progesterone and estrogen receptors (Power et al. 1991). 5. Serine/threonine-phosphorylating Receptors. These receptors are actually composed of two receptor types, I and II, and are best exemplified by the transforming growth factor-β (TGF-β) family of receptors (Josso and di Clemente 1997). The primary receptor is probably a dimer of type II proteins that can bind signal in the presence or absence of the type I polypeptides (Fig. 2.2, V). In the former case, the signal can only bind the type II polypeptide when it is associated with type I polypeptides (Yamashita et al. 1996), whereas, in the latter case, binding of the signal to the type II dimer results in the recruitment of type I polypep-

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tides (Luo and Lodish 1996). Both types have transmembrane domains and protein kinase activity; however, only the type I protein kinase activity is inducible. Binding of the signal results in the phosphorylation of the type I kinase by the type II kinase. The type I kinase then transduces the signal through its protein kinase activity to a target protein that is subsequently translocated to the nucleus, where it presumably activates transcription. 6. Protein Tyrosine Receptor-like Phosphatases. The prototypical receptor-like tyrosine phosphatase is the hematopoietic cell surface protein CD45, which plays a crucial role in lymphocyte activation (Walton and Dixon 1993). The structure of this receptor type is similar to that of other single transmembrane receptors. It consists of an extracellular ligandbinding domain having varying numbers of immunoglobulin-like and fibronectin type III-like repeats (Fig. 2.2, VI). A cysteine-rich region precedes the single membrane-spanning domain, followed by the intracellular catalytic region consisting of two phosphatase subdomains. Although the signal transduction pathway(s) has not been fully identified, at least one target of CD45 has been clearly demonstrated. p56lck is a tyrosine kinase that is activated upon dephosphorylation of Tyr-505 by CD45. Although other homologues of CD45 have been isolated from various animal cells/tissues, only the biological role of CD45 has been well characterized, and that only for T-lymphocytes. 7. Tyrosine-phosphorylating Receptors. There are two types of tyrosinephosphorylating receptors: cytokine receptors and receptor tyrosine kinases (Schindler and Darnell 1995). The cytokine receptor family is large and heterogenous and can respond to a variety of cytokines and growth factors, such as growth hormone. These receptors consist of heterotetramers composed of two signal binding polypeptides and two signal transducing polypeptides (Fig. 2.2, VIIa). Binding of the signal to the receptor results in the activation of the Janus class of protein kinases (JAK) that are physically associated with these receptors. Activation of the JAK kinases results in phosphorylation of a specific tyrosine in the signal transducing polypeptide component of the receptor. STAT (signal transducers and activators of transcription) polypeptides can associate with the phosphorylated receptor transducing polypeptide and are subsequently phosphorylated by the JAK kinases. Activated STAT proteins undergo dimerization and translocate to the nucleus, where they effect changes in transcription. The receptor tyrosine kinases (RTKs) constitute the other class of kinases that promote phosphorylation of tyrosine residues (Fig. 2.2,

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VIIb). Unlike cytokine receptors, receptor tyrosine kinases possess intrinsic kinase activity and can directly transfer phosphate groups to other proteins (Fantl et al. 1993). In this way the binding function is covalently linked to the transducing function. Like cytokine receptors, RTKs exist as multimers and have only a single transmembrane spanning region, usually located between the signal binding domain and the tyrosine kinase domain. Many of these receptors bind peptide and protein hormones that regulate development and metabolism, e.g. epidermal growth factor, tumor necrosis factor, and insulin, to name a few. There is evidence that these receptors can also activate JAK kinases. B. Bacterial Models 1. Histidine/aspartate-phosphorylating Receptors. Receptors that phosphorylate his/asp residues are known as two-component systems, because the simplest system has only two protein components: a sensor and response regulator (Parkinson and Kofoid 1992). These receptors mediate important cellular functions such as chemotaxis, nitrogen fixation, and sporulation, among other processes. The sensor component is associated with the cellular membrane through membrane-spanning regions in the input domain, the portion that binds or detects the signal. A second domain (“transmitter domain”) contains the histidine kinase activity oriented towards the cytoplasm. The transmitter domain autophosphorylates a conserved histidine residue, and the phosphate group is subsequently transferred to a conserved aspartate in the “receiver” domain of the response regulator (Fig. 2.2, VIII). The output domain of the response regulator may have enzymatic or transcriptional activity; for example, CheB, which is associated with chemotaxis, has methylesterase activity (Simms et al. 1985), whereas FixJ, which is involved in nitrogen fixation, acts as a transcription factor (Kahn and Ditta 1991). The response regulator may or may not be covalently linked to the sensor. Although this system seems very simple, in reality it is quite complex and has a variety of possible arrangements of sensors and response regulators (Fig. 2.3A). 2. Serine/threonine-phosphorylating Receptor Protein Kinases. This class of receptors has only recently been documented in prokaryotes [see Zhang (1996) for a review of ser/thr/tyr kinases, both cytoplasmic and receptor types]. Genes encoding two receptors have been isolated: pkn2 from Myxococcus xanthus (Udo et al. 1995) and afsK from Streptomyces coelicolor (Matsumoto et al. 1994). Like eukaryotic ser/thr and tyr receptor kinases, pkn2 and afsK have a single membrane-spanning region that

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Fig. 2.3. Comparison of ethylene receptors by structure and amino acid sequence. Ethylene receptors are similar to bacterial two-component signaling pathways. (A) Illustration of the modular nature of bacterial two-component systems, having four major components: an input domain, a transmitter domain, a receiver domain, and an output domain. Immediately below the modules are examples of the structures comprising the ETR- and ERS-type ethylene receptors, indicating that the ERS-types lack the receiver domain, and both types lack a covalently-linked output domain. (B) A distance tree showing relationships among ethylene receptors based on comparison of their amino acid sequences. The tree is drawn such that close sequence similarities result in close positions on the tree. Two major subfamilies are apparent from the analysis. The ETR1-like subfamily comprises receptors similar to the Arabidopsis ETR1 sequence and includes not only a phylogenetic variety of ETR receptors, but also two ERS sequences. The ETR2-like subfamily consists of sequences related to the Arabidopsis ETR2 receptor and includes the Arabidopsis ERS2 sequence. The fact that ERS2 is not as similar to other ERS sequences as to the ETR2 gene suggests that it arose from an ETR2-type ancestral gene and that loss of the receiver domain occurred independently from that of ERS1 during evolution, i.e., ERS2 did not arise from ERS1 (or vice versa) despite similarity in structural domains (IT). 59

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separates the “input” domain from the kinase domain, and all the conserved subdomains diagnostic for eukaryotic ser/thr kinases can be found in the kinase domain of both gene products. Unlike eukaryotic ser/thr/tyr receptor kinases, the kinase domain of the bacterial receptors is located in the amino terminal region of the polypeptide (Fig. 2.2, IX). This arrangement does not affect the relative orientation of these receptors, as the carboxyterminal “input” region is predicted to lie on the outer face of the cytoplasmic membrane, while the kinase activity remains cytoplasmic. The only role of pkn2 so far identified appears to be the phosphorylation of β-lactamase, which prevents its export from the cell and likely inhibits its activity. AfsK phosphorylates the global regulatory protein, AfsR, required for multiple antibiotic production.

III. PLANT HORMONE RECEPTORS A. Histidine/aspartate-phosphorylating Receptors: Two-component Reception 1. Ethylene Reception. The best characterized receptor of plant hormone signaling is the ethylene receptor (Fluhr and Mattoo 1996; Chang and Stewart 1998). In the 1980s a significant effort was made to dissect the ethylene perception pathway via genetic and molecular analysis of Arabidopsis mutants. Among the different mutants obtained was a group showing insensitivity to external (and presumably internal) ethylene, therefore having characteristics predicted for a receptor of ethylene or at least a component acting early in the signaling pathway. A mutant designated etr1-1 was used to obtain the altered gene for comparison to the wild type (Chang et al. 1993). The resulting product showed considerable similarity to the bacterial two-component signaling system in the carboxy-terminal half, but contained unique sequences in the aminoterminal half. Transformation of wild type Arabidopsis with the etr1-1 gene carrying a single missense mutation resulted in ethylene insensitivity in the transgenic plants, strongly suggesting that ETR1 was an ethylene receptor. Proof that ETR1 encoded a receptor of ethylene was obtained when saturable ethylene binding sites were demonstrated in yeast transformed with the ETR1 gene and expressing the ETR1 polypeptide, since yeast are not normally capable of binding ethylene (Schaller and Bleecker 1995). In addition to ETR1, at least four other genes encoding ethylene receptors have been identified and isolated. There appear to be two different component classes of receptor as exemplified by ETR1 and ERS1 genes,

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i.e. ETR1 receptors have the input (I), transmitter (T), and receiver (R) domains seen in some bacterial receptors; ERS1 receptors lack the receiver domain (Table 2.1; Fig. 2.3A). However, comparison of gene and amino acid sequences (Fig. 2.3B), the presence of three or four potential transmembrane regions at the amino terminus, and the number/location of introns suggest two gene families (Hua et al. 1998; Bleecker 1999), examples of which include Arabidopsis ETR1 and ERS1 (ETR1-family) and ETR2, ERS2, and EIN4 (ETR2-family). Further demonstration that two-component systems like those in bacteria also function in plants comes from the isolation of five homologues of bacterial response regulators from Arabidopsis, ARR3-7, and the demonstration that they are capable of being phosphorylated at the appropriate aspartate both in vitro and in vivo by an artificial histidine transmitter (Imamura et al. 1998). Other response regulator homologues have also recently been identified from plants (Brandstatter and Kieber 1998; Sakakibara et al. 1998; Urao et al. 1998). ETR1 has histidine kinase autophosphorylating activity (Gamble et al. 1998), as would be predicted from its similarity to the bacterial twocomponent receptors. In addition it has recently been demonstrated that binding of ethylene to ETR1 requires a copper co-factor (Rodriguez et al. 1999). Of several metal ions tested, only silver mimicked the effect of copper, an observation consistent with previously reported inhibitory effects of silver on ethylene responses. Experiments have demonstrated that ETR1 forms dimers and that this structure, or some multiple thereof, is likely to form the actual ethylene binding site at the amino terminus of the receptor (Schaller et al. 1995). Further dissection of the ethylene signaling pathway using recessive “loss-of-function” mutations in ETR1, ETR2, EIN4, and ERS2 (Hua and Meyerowitz 1998) suggests that, unlike most animal and bacterial signaling systems, the ethylene pathway is negatively regulated (Fig. 2.4). In this model, the receptor is in the active state in the absence of ethylene and interacts in some fashion with the ser/thr cytoplasmic protein kinase, CTR1 (Clark et al. 1998), causing it to become activated. Genetic studies indicate that CTR1 represses ethylene responses, since null mutations show ethylene responses even in the absence of the hormone (Kieber et al. 1993). Therefore, the activated state of CTR1 is believed to shut down ethylene responses either by inactivating positive effectors (like Ein 2) or by activating negative effectors or both. Binding of ethylene deactivates the receptor and prevents the activation of CTR1. This allows positive effectors to act on enzymes and/or genes giving rise to ethylene responses; conversely, negative effectors would be inactive, so their inactivation would also positively affect ethylene response.

62

Typez

ITR

ITR

ITR

IT

IT

ITR

ITR

IT

ITR

ETR1y

ETR2

EIN4

ERS1

ERS2

LeETR1x

LeETR2

LeETR3 (never-ripe)

CKI1w Not reported

Expression increased at fruit climacteric, in flowers during early stages of senescence, and in the flower abscission zone

Transient increase prior to germination of imbibed seed; decrease in senescing leaf petioles

Mostly constitutive expression

Elevated in developing stamens, carpels, and in septum epidermis; otherwise constitutive

Elevated in anther locule, carpel, and developing flower buds; otherwise constitutive

Elevated in tapetum and developing pollen cells; otherwise mostly constitutive

Kakimoto 1996

Wilkinson et al. 1995; Payton et al. 1996 Kakimoto 1996

Lashbrook et al. 1998

Lashbrook et al. 1998

Hua et al. 1998

Hua et al. 1998

Hua et al. 1998

Sakai et al. 1998; Hua et al. 1998

Chang et al. 1993; Hua et al. 1998; Zhou et al. 1996

Reference

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Elevated in flower, particularly carpels, and in leaf; otherwise mostly constitutive

Elevated in anther locule and carpels; otherwise mostly constitutive expression

Expression Characteristics

Comparison of plant two-component systems for hormone perception.

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Gene

Table 2.1.

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R

R

R

R

R

R

R

ARR4/IBC7/ATRR1

ARR5/IBC6/ATRR2

ARR6

ARR7

ATRR3

ATRR4

ZmCip1

Sakakibara et al. 1998

Urao et al. 1998

Urao et al. 1998

Imamura et al. 1998

Imamura et al. 1998

y

I = input domain, T = transmitter domain (his-kinase activity), R = receiver (part of response regulator). ETR/ERS = Arabidopsis genes encoding ethylene receptors. x LeETRn = Tomato genes encoding ethylene receptors. w Putative cytokinin receptor.

Constitutive

Constitutive

Constitutive

Predominant expression in roots and in response to cytokinin; otherwise mostly constitutive

Predominant expression in roots and in response to cytokinin; otherwise mostly constitutive

Imamura et al. 1998; Brandstatter and Kieber 1998; Urao et al. 1998

Imamura et al. 1998; Brandstatter and Kieber 1998; Urao et al. 1998

Imamura et al. 1998

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Predominant expression in roots and in response to cytokinin; otherwise mostly constitutive (ATRR2 is also induced by low temperature)

Predominant expression in roots and in response to cytokinin; otherwise mostly constitutive (ATRR1 is also induced by low temperature)

Predominant expression in roots and in response to cytokinin; otherwise mostly constitutive

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z

R

ARR3

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63

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Fig. 2.4. Hypothetical ethylene response pathway in higher plants. The pathway illustrated is based primarily on information obtained from genetic studies in Arabidopsis. (A) Proposed operation of the pathway in the absence of ethylene. The receptor kinase (ETR1 or ERS1) is shown inserted into the plasma membrane through the three transmembrane domains (black ovals). In the absence of ethylene, both the receptor and the ser/thr protein kinase, CTR1, are active. This is hypothesized because mutations in CTR1 result in the constitutive expression of ethylene responses, indicating that CTR1 negatively regulates the pathway. It is thought that in its active state, CTR1 de-activates an integral membrane-transducer (EIN2). When EIN2 is de-activated, it cannot transmit signals to its target protein. (B) Proposed pathway operation in the presence of ethylene. It is hypothesized that when ethylene binds the receptor, it results in de-activation of the receptor and, as a result, de-activation of CTR1. In the absence of active CTR1, EIN2 is free to transduce its signal. Transmission of the signal may be through several intermediates (illustrated as squares), but it is thought that the ultimate target of the signal is EIN3, a DNA binding protein located in the nucleus. It is proposed that activation of EIN3 results in its interaction with an EREBP (ethylene response element binding protein), leading to the formation of a DNA binding complex that activates transcription of genes known to be up-regulated by ethylene.

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Important questions regarding ethylene signal transduction have yet to be answered. For example, besides CTR1, what other downstream protein kinases are associated with this pathway? Recent research suggests that mitogen-activated protein kinases (MAPKs) might be involved (C. Chang, personal communication), but definitive proof is lacking at this time. Do the downstream protein kinases branch in such a way as to provide cross-talk with other phytohormone signaling pathways? Again, recent evidence indicates that ethylene signal transduction can crosstalk with a glucose sensing pathway (Zhou et al. 1998). The presence of five related receptor genes in a genome implies that 25 different combinations are possible if the receptor is dimeric; can such combinations account for previously observed changes in ethylene sensitivity during development, senescence, and in different tissues? How do previous observations regarding site I and II ethylene metabolic pathways relate to these ethylene receptors? These and other questions will be answered in the near future and will perhaps provide insight into the regulation of other plant hormones. 2. A Cytokinin Two-component Receptor. An interesting approach to identifying cytokinin-responsive mutants was recently described by Kakimoto (1996). Activation T-DNA tagging was used to create dominant mutants exhibiting a constitutive cytokinin response, i.e., typical cytokinin responses occur in the absence of added cytokinin as if the system were always sensing hormone. Since the T-DNA sequence can act as a “tag” for nearby sequences “turned on” by the strong promoter (activator of transcription) in the T-DNA, isolation of the responsive genes is facilitated. In this way a gene, CKI1, encoding a two-component system polypeptide was isolated. Sequence analysis of this gene and comparison to the ethylene receptor, ETR1, revealed extensive similarity in the histidine kinase and response regulator domains. As would be expected for receptors binding different ligands, the amino terminal region sequences were significantly different between the two predicted polypeptides, and the number and arrangement of putative membrane spanning domains was also different. That some part of cytokinin signaling acts through a two-component system was further strengthened when it was demonstrated that the bacterial response regulator homologues, ARR3-7 identified in Arabidopsis and ZmCip1 in maize (Table 2.1), were all responsive to cytokinin treatment (Brandstatter and Keiber 1998; Sakakibara et al. 1998; Taniguchi et al. 1998). Although it is tempting to speculate that CKI1 encodes a cytokinin receptor, further demonstration that loss-of-function mutants result in cytokinin insensitive phenotypes and that the polypeptide product actually binds cytokinin

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will be required to demonstrate unequivocally that CKI1 is a bona fide cytokinin receptor (see also Section IIIC1). B. Serine/threonine-phosphorylating Receptor Protein Kinases 1. Orphan Receptors. At least five different classes of ser/thr receptor protein kinases (RPKs) (Fig. 2.5) have been identified from higher plants (Walker 1994). All share some structural similarities to the animal Type II ser/thr receptors. For example, they have a single membrane-spanning region flanked by an amino terminal extracellular domain and a carboxyterminal cytoplasmic domain. They differ from the bacterial ser/thr receptors in the organization of their domains, i.e. bacterial receptors have a cytoplasmic amino terminal kinase domain and an extracellular carboxyterminal receptor domain, as discussed in a previous section. Although the animal ser/thr RPKs appear to act in concert with a second, closely associated receptor, the Type I receptor, there is little evidence at present to indicate a similar relationship in plants (see Section IIB3). In only two cases have putative ligands been tentatively identified, i.e. brassinosteroid(s) and clavata3. For the nearly 100 or so other plant RPKs so far discovered, no ligand has been identified, thus the description “orphan” receptor. The identification of ligands for these receptors is the single biggest challenge facing plant signal transduction research. 2. A Possible Brassinosteroid Receptor Protein Kinase. Screens for brassinosteroid-insensitive mutants from Arabidopsis led to the isolation of BRI1, a gene encoding a member of the leucine-rich repeat (LRR) class of ser/thr receptor kinases (Li and Chory 1997; Altmann 1998). Mutant seedlings were phenotypically similar to brassinosteroiddeficient mutants; however, unlike the deficient mutants, bri1 seedlings could not be rescued by application of brassinosteroids. Other genetic approaches to isolating seedlings defective in brassinosteroid signal transduction have all yielded only differently mutated alleles of BRI1 (25 so far), and interestingly none of these mutations maps to the LRR region (Li and Chory 1999). The severity of the bri mutant phenotypes suggested that this gene encoded an early component of the brassinosteroid response pathway. In addition, a small island of ca. 70 amino acids exists between LRRs 21 and 22, and two mutations map to this island, suggesting that it is important specifically to brassinosteroid signaling. Is BRI1 a brassinosteroid receptor? Successful binding studies between BRI1 and brassinosteroids have not yet been reported; however, if BRI1 is like the animal Type 1 ser/thr receptor kinases, it would not be

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Fig. 2.5. Classes of ser/thr receptor-like protein kinases in higher plants. Illustration of the five classes of plant receptor-like kinases (RLKs) recognized to date (after Walker 1994). The classes are based on sequence similarities in the ligand interacting domain to proteins of known function or structure. S-Locus: RLKs whose receptor domain is similar to S-locus glycoproteins associated with reproductive self-incompatibility in Brassica (Stein and Nasrallah 1993; Walker and Zhang 1990). Leucine-rich Repeat: RLKs containing 5-26 leucine-rich repeats in the receptor domain (refer to Walker 1994). Growth factor-like: RLKs having sequences similar to animal epidermal growth factor and tumor necrosis factor receptors (Kohorn et al. 1992). Lectin-like: RLKs with sequences similar to plant lectins (Herve et al. 1996). Class V: RLK with no similarity to any other reported protein sequence (Schulze-Muth et al. 1996).

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expected to bind brassinosteroid in the absence of a Type II component. In addition, since all known animal steroids have been shown to bind receptors that are transcription factors, demonstrating that BRI1 is a brassinosteroid receptor component has important implications for the evolution of steroid perception in plants. 3. The Clavata1 Receptor Protein Kinase. Unlike other mutants in Arabidopsis with defects in shoot and/or floral meristem development, clavata1 mutants exhibit a phenotype specific to meristem development (Clark et al. 1997). These mutations result in enlargement of the shoot and floral meristem caused by accumulation of undifferentiated cells. Thus, clavata mutations shift the balance between cell proliferation and cell differentiation in both types of meristems. Isolation and sequencing of the gene encoding Clavata1 revealed that it encodes a typical LRR receptor kinase and represents a novel signal transduction pathway. Recently it was reported that Clavata1 is actually part of two protein complexes, the larger of which requires Clavata3 for formation (Trotochaud et al. 1999). This complex also contains a kinase associated phosphatase and a Rho-like (small GTPase) G-protein. Clavata3 most likely represent a gene whose product acts at the same position as Clavata1 or upstream of it. It is possible that Clavata3 is the ligand that activates Clavata1 or, like the animal ser/thr receptor kinases, Clavata 3 may be required for either ligand binding or for activation of Clavata1. The resolution of these questions awaits the identification of the gene encoding Clavata3. C. Seven-transmembrane Receptors 1. Is GCR1 a Second Cytokinin Receptor? In addition to strong evidence supporting the existence of a cytokinin two-component receptor, there is a recent report identifying a seven-transmembrane receptor from Arabidopsis associated with cytokinin signaling. The gene, designated GCR1, is the first definitive example of this type of receptor in plants (Josefsson and Rask 1997; Plakidou-Dymock et al. 1998). GCR1 is expressed at very low levels in a variety of tissues. Antisense inactivation of GCR1 in Arabidopsis reduced sensitivity to cytokinins in roots and shoots without demonstrably affecting other plant hormone responses. As with the putative cytokinin two-component receptor, binding of the hormone has not yet been unequivocally demonstrated. On the other hand, several soluble and membrane-bound proteins have been shown to bind specifically to cytokinin. The soluble barley

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protein is large (67 kDa) and activates RNA synthesis in the presence of zeatin in isolated nuclei (Kulaeva et al. 1995). In addition, antibodies raised against a maize cytokinin binding protein (CBP) cross-reacted with the barley protein and blocked the previously observed activation of RNA synthesis (Kulaeva et al. 1998). Interestingly, a monoclonal antibody to the maize CBP activated the barley CBP in the absence of zeatin. A recent report identified a membrane-bound CBP (Brault et al. 1999), but it was not clear whether this CBP is identical or related to either the putative two-component cytokinin receptor or GCR1. Some studies have also shown that cytokinin can bind to enzymes and or proteins with other physiological functions (Schnorr et al. 1996; Fujimoto et al. 1998). This complexity in hormone binding is similar to that seen with other phytohormones (Sections IIIC2 and IIID1). 2. Is a Seven-transmembrane Protein a Possible Gibberellin Receptor? Although recent progress in gibberellic acid (GA) signaling has identified several components of the signaling pathway, e.g. protein kinases and G-proteins (Bethke and Jones 1998), little progress has been made on the identification and isolation of a GA receptor since this topic was reviewed by Stoddart (1986) and Srivastava (1987). Early research suggested that GA binding took place at both external and internal sites (Goodwin and Carr 1972; Sinjorno et al. 1993; Smith et al. 1993; Hamabata et al. 1994). More recent studies favor GA binding at the external face of the plasma membrane in oat and barley protoplasts (Hooley et al. 1991; Gilroy and Jones 1994; Lovegrove et al. 1998). Of course, these studies do not completely exclude GA binding a soluble receptor, particularly if multiple binding sites correlate with different GA responses. In attempts to identify other components in the GA signal transduction pathway, Ritchie and Gilroy (1998) obtained evidence that a calcium dependent protein kinase was associated with the aleurone GA response. Furthermore, Jones et al. (1998b) and Hooley (1998) have suggested a role for heterotrimeric G-protein coupling in GA responses using Mas7, a peptide that enhances G-protein action, to stimulate α-amylase production and excretion in the absence of GA. Taken together these results could be explained by the action of a seven-transmembrane/G-protein coupled stimulation of Ca+2 release and/or influx into the target cell and suggest that GA might act through such a receptor. Alternatively, GA may act through a yet to be identified class of receptors that cross-talk with a seven-transmembrane/G-protein pathway. This does not necessarily exclude an additional cytoplasmic receptor site. To date no GA receptor has been unequivocally identified.

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D. Current Status of the Perception of Other Plant Hormones 1. Are There Multiple Auxin Receptors? Binding of auxins to particulate fractions from plants was first investigated over 25 years ago. A number of these studies suggested a plasma or vacuolar membrane location for an auxin receptor (Napier and Venis 1990; Lomax and Hicks 1992). For example, photoaffinity labeling using an auxin analog identified two polypeptides (40 and 42 kDa) from zucchini and a 23 kDa polypeptide from maize plasma membranes (Hicks et al. 1993). Likewise, a group of 14 kDa proteins with auxin-binding activity has been identified from tobacco plasma membrane preparations (Szponarski et al. 1997). These proteins, as well as a larger (57 kDa) one from rice (Kim et al. 1998), may be involved with auxin perception regulating the proton pump of the plasmalemma. However, confirmation that these proteins have sequences that interact with membranes or membrane components awaits further characterization. Recently, genes encoding the major auxin binding polypeptide, ABP1 (auxin binding protein, also known as site I), have been isolated from a variety of plants, including maize (Inohara et al. 1989), tobacco (Watanabe and Shimomura 1998) and Arabidopsis (Palme et al. 1992). The protein predicted from the ABP1 gene sequence possesses a signal peptide, but no membrane-spanning domain. It does have the KDEL (oneletter amino acid code for lysine-aspartate-glutamate-leucine) amino acid motif associated with proteins targeted to the endoplasmic reticulum (ER) and a block of amino acids that may be associated with auxin binding, since antibodies raised against this peptide provoke an auxin response in vitro in the absence of auxin (Venis et al. 1992). Localization of ABP1 at the cellular level has resulted in the identification of several sites. Although earlier studies indicated a plasma membrane association for ABP1 (Shimomura et al. 1986; Barbier-Brygoo et al. 1991), the bulk of ABP1 in maize appears to be associated with the ER; however, some of it is distributed at other sites within the cell, as well as at the plasma membrane and cell wall. Furthermore, only a fraction of the translated ABP1 protein from Arabidopsis was shown to be translocated to the ER lumen in vitro (Palme et al. 1992). Interestingly, binding of auxin to ABP1 could not be demonstrated within the ER (Tian et al. 1995), raising the possibility that the ER lumen may not be the “functional” location for ABP1. Aside from binding auxin, additional evidence that ABP1 is an auxin receptor comes from transgenic studies in tobacco and maize and from electrophysiological experiments using tobacco protoplasts. Overexpression of the Arabidopsis ABP1 gene in tobacco under control of an

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inducible promoter resulted in increased cell expansion in response to auxin (Jones et al. 1998a). Likewise, the same gene constitutively expressed in maize cell lines resulted in increased ability to respond to auxin as evidenced by larger cell size. Another study that supports a role for ABP1 in auxin perception is based on previously observed changes in membrane polarization in protoplasts exposed to auxin. The tobacco ABP1 and a carboxy-terminal synthetic peptide derived from it were shown to induce hyperpolarization of tobacco protoplasts in the absence of auxin (Leblanc et al. 1999). Furthermore, transgenic protoplasts expressing RolB (increases sensitivity to auxin) were observed to be even more sensitive to ABP1 and its peptide than untransformed protoplasts. These experiments demonstrate that ABP1 can replace auxin in activating the auxin response pathway from the outer face of the plasma membrane. These data, taken together with the transgenic studies in tobacco and maize, strongly suggest that ABP1 functions like a receptor for cell expansion and membrane hyperpolarization despite the fact that the protein does not share similarity with any other receptor described to date nor with the mechanism of reception described for any living system. Several groups have reported the identification of soluble ABPs. For example, a soluble ABP (ca. 50 kDa) from tobacco cells has been partially purified and shown to stimulate in vitro transcription in isolated nuclei, although the magnitude of stimulation was variable (Mennes et al. 1987). Prasad and Jones (1991) identified a 65 kDa protein in the nucleus and cytoplasm of several different plant species, suggesting that this ABP might have an effect on transcription. Similarly, Sakai et al. (1986) reported the stimulation of RNA synthesis by two ABPs from mung bean, and Jacobsen et al. (1987) suggested that ABPs from pea could stimulate transcription. ABPs in the former studies enhanced transcription independently of auxin, while those in the latter study were reportedly auxin-dependent. The sizes and auxin binding affinities of these polypeptides strongly suggest that soluble ABPs significantly differ from ABP1. One note of interest reflects similarities between auxin response elements (AuxREs) and glucocorticoid (a steroid) response elements (GREs). These are sequences found in the promoters of genes responding specifically to the hormone. Analysis of AuxREs indicated that, like GREs, these elements had specific hormone response sequences (TGTCTC for AuxREs and TGTTCT for GREs) that were symmetrical sense antisense repeats, i.e., TGTCTCxxxxxxxGAGACA (Ulmasov et al. 1997). Such similarities in DNA response elements suggest that there might be similarities between the animal steroid receptors and auxin receptors. Although it is possible that the soluble ABPs represent a

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‘steroid-like’ signaling mechanism, until the genes encoding these polypeptides are isolated and characterized as to their DNA binding capability, other mechanisms cannot be ruled out. A number of studies have identified proteins with enzymatic activity or associated with specific physiological functions as ABPs. The list includes β-glucosidase (Campos et al. 1992), glutathione S-transferase (Bilang et al. 1993), glutathione-dependent formaldehyde dehydrogenase (Sugaya and Sakai 1996), 1,3, β-glucanase (Macdonald et al. 1991), and manganese superoxide dismutase (Feldwisch et al. 1995). In addition, two germin-like proteins from peach have been shown to have auxin-binding capability, although with lower affinity than that of ABP1 (Ohmiya et al. 1998). It has been speculated that germins that are associated with the cell wall may have a direct role in auxin-mediated cell expansion during germination. The biological significance of diverse auxin-binding proteins in the plant cell is not clear. Certain auxin responses (i.e. cell wall expansion and cell division effects) are separable, implying different auxin interaction mechanisms exist in the cell. There is precedence for ligand binding to multiple receptors in animals (see Section IIA4), and evidence for multiple receptors is accumulating for plants as well (e.g. ethylene and cytokinin receptors). Whether auxin binding proteins represent different classes of receptors or a means of compartmentalizing and/or finetuning hormone responses, remains to be elucidated. 2. Are There Multiple ABA Receptors? As with other plant hormones, an early approach to identifying ABA signaling components was to isolate mutants in Arabidopsis affected in response to ABA. Three complementation groups were identified (Koornneef et al. 1984), and genes encoding two of the mutant versions (Abi1 and Abi2) have been isolated (Leung et al. 1997), as well as a third gene family member, AtP2C-HA (Rodriguez et al. 1998). The genes Abi1 and Abi2 encode functional protein phosphatases and act in a negative regulatory manner with regard to several ABA-controlled responses. Overlapping functions assigned to the three abi mutants suggest either multiple ABA receptors or multiple branches in the ABA signaling pathway (Fig. 2.6). Support for branching in this pathway comes from identification of the cyclic nucleotide, cADPR, as being a central, positive mediator of ABA signaling (Wu et al. 1997) through Ca+2 release, as opposed to negative regulation via alkalinization. This aspect of ABA signaling through a cyclic nucleotide intermediate is consistent with a G-coupled, seven transmembrane receptor; alternatively, given the rapid release of free Ca+2 into the cytoplasm of guard cells stimulated by ABA, a ligand-gated ion channel is another

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Image Not Available

Fig. 2.6. Hypothetical ABA signal transduction pathway. Based on genetic and physiological data, a pathway for ABA signaling has been proposed that utilizes either separate receptors or a single receptor that regulates at least two different pathways. In this illustration, ABA binds an unidentified receptor in the plasma membrane. A second possible receptor is illustrated by dotted lines, indicating that there is no direct evidence of its existence. For the single receptor, binding of ABA results in both the alkalinization of the cytosol and the synthesis of cyclic adenosine 5′-diphosphate ribose (cADPR). cADPR acts like other cyclic nucleotide second messengers and results in the release of calcium ions sequestered in the endoplasmic reticulum or other organelle. Calcium ions trigger the response presumably through calcium-dependent PKs or through calcium/calmodulinactivated PKs. Alkalinization of the cytosol activates the protein phosphatases, ABI1 and ABI2. It is hypothesized that removal of the phosphate(s) by the ABI phosphatases from the target molecule (thought to act as a repressor of ABA responses; open circles) results in inactivation of the target and prevents it from repressing ABA responses. Thus, binding of ABA is proposed to have both positive and negative effects regarding regulation of the signaling pathway (after Grill and Himmelbach 1998).

possible candidate for an ABA receptor (Grill and Himmelbach 1998). Although no ABA receptor has so far been identified, ABA-specific binding to membrane preparations of Arabidopsis (Pedron et al. 1998) and rice (Schultz and Quatrano 1997 ) has recently been described, so it is likely that identification of an ABA receptor is imminent. However, given the overlapping nature of the ABA-insensitive mutants, a cytoplasmic receptor similar to animal steroid receptors also remains a distinct possibility.

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3. Jasmonic and Salicylic Acid Receptors. In plants jasmonates and salicylate act as signals for induction of plant defense responses (Reymond and Farmer 1998). In addition, jasmonates (JA), including jasmonic acid and its derivatives, affect a number of other processes, such as pollen development, fruit ripening, root growth, and senescence. JA is a major component required for plant defense against insect predation, acting in concert with ethylene to promote expression of proteinase inhibitors in wounded tissues. Although a receptor for JA has not yet been identified, a mutant, coi1, in Arabidopsis has been described with reduced sensitivity to JA (Feys et al. 1994). The gene encoding the wild type version (COI1) was recently isolated (Xie et al. 1998). Sequence analysis indicates that the protein contains imperfect LRRs and an F-box, both of which imply that COI1 interacts with other proteins. Another Arabidopsis mutant, jar1, shows decreased sensitivity to JA (Staswick et al. 1992), although its identity has not yet been reported. Further research in this area should help elucidate a role for COI1, whether it represents a novel type of receptor or plays some other role in this signal transduction pathway. Early studies to elucidate the mechanism of action of salicylic acid (SA) in plant defense led to the demonstration that SA could interact with catalase and ascorbate peroxidase, major peroxide-scavenging enzymes (Chen et al. 1993a,b; Durner and Klessig 1995). Although these studies suggested that SA acted through peroxide metabolism, more recent studies have shown the opposite, i.e. that peroxide induces SA accumulation. Thus it was postulated that SA-induced activation of defense responses must act through other proteins or factors. As a result, recent research has led to the identification of an SA-binding protein from tobacco designated SABP2 (Du and Klessig 1997). The binding affinity of this protein for SA and SA analogs is at least two orders of magnitude higher than the interaction between SA and peroxidescavenging enzymes. Although the binding characteristics of SABP2 are consistent with its being an SA receptor, confirmation awaits the elucidation of its identity through genetic and molecular methods. It is interesting that like previously discussed hormones, SA appears to interact with multiple cellular proteins, most of which, however, do not seem to be acting as receptors in the strictest definition. Preliminary genetic studies have led to the isolation of several types of SA response mutants in Arabidopsis, including a class that is SA insensitive (Shah et al. 1997). So far none of the cognate genes identified in these different SA response mutants has characteristics of known receptors. Interestingly, a class of mutants that suppress the SA insensitive phenotype appears to modulate crosstalk between the SA pathway and ethylene/JA

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defense pathway (Shah et al. 1999). It will be of interest to understand the mechanism(s) through which these defense-related signaling pathways communicate and to see whether or not these hormones can interact directly with protein components of the various defense pathways. 4. Ca+2 Ion Channels. Calcium is involved in a number of plant cellular processes, including pollen tube growth, gravitropic responses, root growth, and light responses. Although technically Ca+2 is not a hormone in the classical definition of the word, there is a large body of evidence indicating that it acts in plants as a second (intracellular) message. Entry of calcium into the cytoplasm is through plasma membrane (external) or ER/vacuolar (internal) ion channels. A number of electrophysiological studies of Ca+2 influx and release have been performed. Thuleau et al. (1998) and Trewavas and Malhó (1998) have summarized recent experiments involving Ca+2 signaling in plants. At the moment, almost nothing is known about the composition of these ion channels and whether any function as ligand-gated channels, as has been documented in animals. This area of research should be a major focus in future signaling research, because Ca+2 appears to function as an intermediate in a wide variety of hormone-regulated pathways.

IV. FUTURE PROSPECTS A wealth of information regarding signaling in plants has been steadily accumulating during the past decade. Components of various pathways having some similarity to animal or bacterial signaling have been identified in diverse plants, including both types of G-proteins, and seven transmembrane and two-component receptors. The current status of plant hormone reception is summarized in Fig. 2.7. Zucconi and Bukovac (1988) reviewed the status of phytohormone research in the 1980s and concluded that new approaches were necessary to determine correlations between a given hormone and a specific physiological event. Their critique of then current approaches and methodology has proven valid, as is seen in the case of ethylene signaling where a combination of classical genetics, molecular biology, and biochemistry have not only demonstrated the role of ethylene in the triple response, but have been crucial in identifying the components of the pathway leading to a number of ethylene responses. Several recent reviews of plant hormone signaling are available (Iten et al. 1999; Sopory and Munshi 1998; Redhead and Palme 1996), and the interested reader is also referred to Volume 1, No. 5 of Current Opinion in Plant Biology (1998) and volume 353

Fig. 2.7. Plant signaling pathways. Illustration of the putative plant signal transduction pathways initiated by various hormones. Dotted lines indicate proposed pathways or signaling components. Question marks indicate uncertainty regarding a particular component. cADPR: cyclic 5′-adenosine diphosphate ribose; cAMP/cGMP: 3′, 5′-cyclic adenosine or guanosine monophosphate; IP3: myo-inositol 1,4,5 triphosphate; DAG: diacylglycerol; PKA and PKC: protein kinase A (cAMP-dependent) and protein kinase C, respectively; Cm: calmodulin; CDPK: calcium-dependent protein kinase; CmDPK: calcium/calmodulin-dependent protein kinase; RLK: receptor-like protein kinase; ER: endoplasmic reticulum; ABP: auxin-binding protein.

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no. 1374 of Philosophical Transactions of the Royal Society of London Biological Sciences (1998). As research in the area of plant hormone signal transduction progresses, more similarities between animal and plant signaling may become apparent, e.g. finding type I ser/thr transducers in plants. By the same token, it is anticipated that substantial differences will continue to be seen between signaling components and/or mechanisms in these two groups of organisms. The architecture of signaling pathways suggests that manipulation of these pathways by mutation or genetic engineering is feasible, as interruption at any point in the chain would affect the signaling event. In addition, affecting events at early steps, such as when the signal is perceived by the receptor, is likely to have broader effects on the end result of the signaling event than events at the latter stages of transduction, particularly if pathways diverge. In addition, pathways that are induced by signals may be manipulated to be expressed constitutively. In this manner plant defensive genes that normally are induced by pathogen or pest attack could be engineered to be continually “on” and perhaps provide better protection. Indeed, the manipulation of signal transduction component genes via genetic engineering has already been successfully attempted with regard to disease resistance (Cao et al. 1998; Lund et al. 1998; Hoffman et al. 1999). Other traits of economic significance that could be manipulated by engineering genes encoding signal transduction components include: senescence (e.g. extending the life of cut flowers), fruit ripening (e.g. delayed ripening to improve shipping and handling of stone fruits), development (e.g. controlling flowering in orchards to maximize fruit size), manipulating pigment biosynthesis/degradation to enhance or eliminate specific colors (e.g. redder apples whether exposed to sunlight or not), improved flavors, nutrients, or “phytoceuticals” (e.g. increasing taxol production), improving stress responses (e.g. enhancing cold resistance), and improving morphological traits that directly or indirectly respond to hormones (e.g. production of genetically dwarf fruit trees to lower production costs). Furthermore, identifying target proteins that may be phosphorylated at different positions by different pathways could allow us to disconnect one signaling event from others simply by removing one of the target amino acids or by designing peptides that compete for the protein kinase. Finally, attaching the “antenna” region of one receptor kinase to the protein kinase domain of another could bring some pathways under control of additional signals. The prospects are as exciting as they are challenging in uncoupling or coupling pathways in unique ways to improve plant traits having human/animal health benefits or other economic

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potential. Identifying receptors for the familiar plant hormones and identifying ligands for the ever-increasing list of plant receptor protein kinases represents the first important step in understanding and manipulating signal transduction to benefit horticulture.

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3 Molecular Biology of Cassava Johanna Puonti-Kaerlas Institute for Plant Sciences ETH-Zentrum/LFW E 17 CH-8092 Zürich Switzerland

I. INTRODUCTION II. BOTANY AND DISTRIBUTION III. PRODUCTION AND USE A. World Production B. Role of Cassava in Food Security in Developing Countries IV. CONSTRAINTS TO CASSAVA USE AND CULTIVATION V. TRADITIONAL BREEDING AND BIOTECHNOLOGY IN CASSAVA IMPROVEMENT VI. MOLECULAR GENETICS A. Molecular Markers and Mapping B. Gene Cloning C. Phylogenetic Mapping VII. TISSUE CULTURE AND REGENERATION A. Shoot Tip and Meristem Culture B. Disease Elimination C. Embryo and Anther Culture D. Micropropagation E. Germplasm Conservation F. International Exchange of Material G. De Novo Regeneration 1. Multiple Shoot Induction 2. Somatic Embryogenesis 3. Friable Embryogenic Callus and Embryogenic Suspensions 4. Protoplasts 5. Organogenesis VIII. GENETIC TRANSFORMATION A. Meristems B. Somatic Embryos C. Embryogenic Suspensions D. Organogenesis Horticultural Reviews, Volume 26, Edited by Jules Janick ISBN 0-471-38789-4 © 2001 John Wiley & Sons, Inc. 85

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IX. APPLICATIONS AND POTENTIAL OF MOLECULAR BIOLOGY A. Disease Control B. Virus Control C. Pest Resistance D. Quality and Yield 1. Cyanogenesis 2. Stay-green Index 3. Starch 4. Micronutrients 5. Physiological Post-harvest Deterioration X. FUTURE PROSPECTS LITERATURE CITED

I. INTRODUCTION Cassava (Manihot esculenta Crantz, Euphorbiaceae) is one of the most important food crops and a key component in food security in the tropics. It is grown for its starch-containing roots, which feed over 500 million people worldwide. Traditional breeding of cassava has limitations, and although improved cultivars have been produced by using conventional methods, there is a strong need for additional methods for genetic improvement. Recent reviews have been published on agricultural practices (Onwueme and Charles 1994), breeding (Byrne 1984), and cytogenetics (Bai 1987) of cassava, so this review will concentrate on biotechnology and molecular biology of this species. An attempt to give an overview on the most important topics relating to cassava biotechnology is made, but due to the broad spectrum of the review, some omissions are unavoidable. A more comprehensive view on the field of cassava biotechnology can be obtained from the proceedings of the meetings of the Cassava Biotechnology Network (Roca and Thro 1993; Cassava Biotechnology Network 1995; Thro and Acoroda 1997; Pires de Matos and Vilarinhos 1998). The biotechnological tools that will be discussed in this review can be used to produce and micropropagate disease-free material, to preserve and characterize germplasm, to understand the genomic structure and gene function (a prerequisite for eventual modifications of biochemical pathways), and to produce new, improved cultivars by transgene technology. The construction of molecular markers and maps will enable more efficient breeding. Understanding plant-pathogen interactions and the evolution of and the variability within the various pest and disease populations and the development of tools for pathogen indexing and disease diagnostics are integral for developing resistance strategies. Despite its importance to food security in the tropics, cassava has long been

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neglected in biotechnology programs, but recently significant progress has been made in developing and adapting biotechnological methods to cassava.

II. BOTANY AND DISTRIBUTION Cassava is a 1–5 m high woody perennial shrub that produces starchy tuberous roots (Fig. 3.1) and belongs to the spurge family (Euphorbiaceae), sub-family Crotonoidea and tribe Manihotae. The Euphorbiaceae also contains other commercially important plants such as castor bean (Ricinus communis L.) and rubber (Hevea brazilienesis L.). The genus Manihot comprises 98 species of herbs, shrubs, and trees. All species in this genus studied so far have a chromosome number of 2n = 36 (Rogers 1963; Rogers and Appan 1973; Nassar 1978; Hershey 1983; Bai et al. 1993). Several species contain lactifers and produce latex and cyanogenic glucosides (Bailey 1976), and M. glaziovii is used as a minor source of rubber (Cock 1985). Common names for the crop include yuca (most Spanish-speaking countries), manioc (French-speaking countries), tapioca (Asia), and mandioca (Portuguese-speaking countries). Cassava is native to tropical South America, and it is one of the oldest cultivated crops (Jennings 1976) with possibly two centers of origin. One of these has been suggested to be in lowland tropical Americas (Smith 1968), the other in the Brazilian-Paraguayan area (Vavilov 1992). Since cassava is not known to exist in a wild state, its wild progenitors and the regions of its domestication are disputed (Renvoize 1972; Rogers and Appan 1973; Rogers and Flemming 1973; Nassar 1978; Allem 1994; Olsen and Schaal 1999). The crop may have been cultivated in Colombia and Venezuela as early as 3,000–7,000 years ago (Lathrap 1970), and Ugent et al. (1986) cite evidence for its domestication on the Peruvian coast before 4000 B.C. Cassava reached the Caribbean Islands and Central America probably in the 11th century (Brucher 1989). From Latin America it was introduced to Africa in the 16th century (Jones 1959) and to Asia during the late 17th century (Leone 1977). Today it is cultivated worldwide in more than 80 countries between 30° north and south. Cassava is best suited to warm, humid lowland tropics, but it can be grown in most areas where the mean annual temperature exceeds 20°C and the annual precipitation varies between 500 mm and 8000 mm. After an initial establishment period, cassava is able to survive even prolonged seasonal drought. This makes it suited to semiarid regions and areas with erratic or unpredictable rainfall. Cassava also tolerates other

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Fig. 3.1.

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Cassava roots (A) and a young cassava plant (B).

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adverse environmental conditions, producing dry weight yields that are 1.5 to 3 times more than those of cereals on marginal areas, including acidic infertile soils with high aluminum content (Thro et al., in press). Planting cassava thus allows acceptable harvests even on infertile and eroded soils with pH ranges from 3.0 to 9.5 that are unable to support most other crops without costly external inputs. Cassava roots, which can reach a diameter up to 20 cm and a length of over 1 m, are usually harvested 6–12 months after planting, but in areas where a cool season (e.g. at high altitudes) arrests plant growth and the accumulation of starch in the roots, the growing period can be up to three years. New, early-maturing cultivars in India can be harvested after 6 months. Cassava has an advantage over cereal crops in that its planting and harvesting times are flexible. While the root quality may decline somewhat if harvest is delayed for a considerable period, harvest rate can be adjusted to take advantage of favorable conditions for harvesting and marketing. This also makes cassava an excellent famine reserve because the roots can be stored for several years in the field on growing plants or be partially harvested, when needed. Cassava is traditionally propagated vegetatively from lignified stem cuttings, “stakes,” which means that none of the edible portion, the roots, need be set aside to secure planting material for the next season. In the tropics cassava is the most important root crop and the fourth most important calorie source after rice, maize, and sugarcane, providing a basic staple for over 500 million people. In many developing countries, cassava is the least expensive calorie source. Cassava produces the highest calorie yields per hectare of all staple crops and has a higher efficiency of energy unit per labor input ratio than most tropical crops (Table 3.1) (De Vries et al. 1967; Coursey and Haynes 1970; Onwueme and Charles 1994). Cassava is commonly grown by small-scale and subsistence farmers in the less developed regions in the tropics, but large commercial plantations exist in Brazil, Venezuela, and Thailand.

Table 3.1. Production, labor requirement and calorie yield estimates of some tropical crops (De Vries et al. 1967; Coursey and Haynes 1970; Onwueme and Charles 1994).

Crop Cassava Maize Rice Yams

Daily food energy (kcal/ha)

Yield (kcal/ha)

Monthly yield (kcal/ha)

Labor energy (person days/ha)

250 200 176 266

12000 8000 5000 7000

1100 1800 1000 800

180 80 200 300

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III. PRODUCTION AND USE A. World Production In 1996, cassava was grown on 16.3 million ha, 60% of which were in Africa, 24% in Asia, and 16% in Latin America (Table 3.2).The annual production of cassava in 1997 was over 165 million t (Table 3.3). The production in Africa, Asia, and Latin America/Caribbean is 48%, 32%, and 20% of the total, respectively, and the largest cassava producers are Nigeria, Thailand, Indonesia, Brazil, and the Democratic Republic of Congo (Zaire). The world average yields are about 10 t/ha fresh roots, and the highest yields are obtained on Barbados (27.3 t/ha) and in India (23.5–31 t/ha), whereas the lowest yields only reach 1.8 t/ha in Sudan (Gosh 1994; FAO 1997a, 1998c). Cassava production has risen steadily during the past decades, partly due to increased growing area, but also due to introduction of new high-yielding cultivars. Much of the increase has been due to the expansion of cassava cultivation in Thailand (Table 3.3), which exports most of its production as dried chips and pellets to the European Union. Of the world production, 59% is used for food, 24% for feed, and 17% for other uses (FAO 1997a,b). As food, cassava is used either like potato or in various processed forms as different types of flours or pastes (Hahn

Table 3.2.

Cassava production in 1996, consumption in 1995 (FAO 1997). Area harvested (106 ha)

Yield (t/ha)

Production (106 t)

Annual consumption (kg/person)

Africa Congo Dem. Rep. Mozambique Nigeria

9.9 2.1 1.0 2.9

8.4 8.3 4.2 10.7

83.2 17.5 4.2 31.5

— 346.8 194.7 172.0

Asia India Indonesia Thailand

3.5 0.3 1.3 1.2

13.0 23.5 12.1 13.3

46.3 6.0 15.4 16.0

— 6.0 54.9 3.8

Latin America and Caribbean Brazil Colombia Paraguay

2.7 1.94 0.18 1.75

11.8 12.6 9.8 14.9

31.4 24.6 18.0 2.6

— 52.5 36.2 148.8

World

16.3

9.98

162.9

—

Location

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Cassava production 1970–1999 (FAO 1997, 1998). Production (million t)

Location

1970

1980

1985

1990

1992

1994

1996

1997

Africa Nigeria Congo Dem. Rep. Mozambique

40.5 10.2 10.3 2.9

48.3 11.5 13.1 3.6

58.2 13.5 15.5 3.6

64.1 17.6 17.0 5.0

80.9 29.2 20.2 3.2

82.9 31.0 18.1 3.4

83.2 31.4 16.8 4.7

85.0 31.4 16.8 5.3

Asia India Indonesia Thailand

23.2 5.2 10.6 3.4

45.8 5.8 13.6 16.5

48.5 5.7 14.0 19.3

52.0 5.0 16.3 21.9

51.4 5.8 16.5 20.4

49.3 5.8 15.7 19.1

47.7 4.7 17.0 17.4

48.5 4.7 17.2 18.1

Latin America and Caribbean Brazil Colombia Paraguay

34.7 29.5 1.2 1.6

29.2 23.5 2.2 2.0

29.6 23.1 1.4 2.9

33.7 25.4 4.0 4.0

28.6 22.0 1.7 2.6

31.2 24.5 1.8 2.5

32.1 24.9 2.0 2.6

32.4 25.4 1.8 2.6

World

98.7

124.1

135.7

152.4

161.1

163.5

162.9

165.3

1989; Nweke 1994a; Agbor Egbe et al. 1995; Bokanga 1995; Dufour et al. 1996). In Latin America, 60%–70% of cassava is used for food, while in Africa 90% of the harvest is used for human consumption (Balagopalan et al. 1988; Bokanga 1994a; Onwueme and Charles 1994). In Africa up to 60% of the daily calorie intake derives from cassava, and some of the indigenous groups in the Amazonian area get over 80% of their food energy from cassava alone (Roca 1984; Cock 1985; CIAT 1994; Dufour 1994, 1995; Koch et al. 1994). In Asia cassava is mainly used for food in Kerala (India) and in Indonesia. The highest annual cassava consumption per capita on a country basis is 347 kg in the Democratic Republic of Congo, followed by Congo (255 kg) and Ghana (246 kg). Of the 12 countries where the annual consumption per capita exceeds 100 kg, 11 are in sub-Saharan Africa, the twelfth is Paraguay (Table 3.2) (FAO 1997a). In Africa 70% of the total production is processed to pastes, flour granules, or starches (Nweke 1994b). Some of the main products at home or at the village level are toasted flour (farinha in Brazil, gari in West Africa), flat bread (casabe in the Caribbean) or foo-foo (a paste-like meal made from cooked fermented roots or cassava flour) and dried chips (gaplek) in Indonesia. The use of cassava flour in bread making shows interesting potential (Eggleston et al. 1992; Eggleston and Omoaka 1994). In certain regions of Africa and Asia cassava leaves are also used as a major component of the diet to provide supplementary protein, minerals,

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and vitamins to complement the carbohydrate rich staple (Bokanga 1994b; Dahniya 1994; Lutaladio and Ezumah 1981). In the Democratic Republic of Congo cassava leaves account for 68% of all vegetable output (Tshibala and Lumpungu 1989). As raw material, cassava can be processed into a wide variety of products for feed and industrial uses. Much of the processing can be done locally, providing rural employment and income (Balagopalan et al. 1988; Carrizales 1991; Hershey et al. 1997a,b). Cut and dried flakes, chips, and pellets of cassava are used for animal feed. In addition, cassava leaves are used in Asia for small household-level fish production. In Brazil, cassava leaves are mixed with cassava chips or starch waste for on-farm pig feed. Buitrago (1990) and CIAT (1989) provide comprehensive lists on cassava uses in animal feed. Information on fermentation technologies for cassava waste treatment including possibilities for production of value-added animal feed can be found in Balagopalan and Rey (1993) and Balagopalan (1996). The dry matter content of cassava roots is 34%; of this 74% to 85% is starch (Rickard et al. 1991; Blanshard 1995) with an amylose content of 13% to 28% (Zakhia et al. 1995). Starch yields of up to 11.5 t/ha have been reported (Sriroth et al. 1998). The quality of cassava starch is high and it is well suited for specialty uses. It is highly digestible, acid-free, resistant to freezing and shearing stress and has excellent thickening and textural qualities. It also has low or no protein contamination. In addition to unmodified (native) starch, the main starch-based products are modified starches for industrial purposes, e.g. for paper, textiles and construction, for sweeteners including fructose syrup, glucose, dextrin and monosodium glutamate, and for pharmaceuticals. A treatise on cassava use and processing can be found in Balagopalan et al. (1988). FAO (1997b) analyzed cassava utilization trends and projected cassava production to be 209 million t by 2005, with 58% used for food, 22% for feed, and 20% for other purposes. So far cassava has been considered to be a subsistence crop, especially in Africa, but even there it already contributes considerably to household income. On the average, 40% of cassava in Africa is planted for sale (Nweke and Lynam 1997). Despite its reputation, cassava has potential also for large plantations for industrial purposes. B. Role of Cassava in Food Security in Developing Countries Hunger and malnutrition are among the most severe problems in many areas of the world, particularly in Africa. At present over 800 million people are starving while the world population increases by 85 million

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people every year. Of this growth 80% takes place in developing countries (FAO 1998a,b). The reasons for food insecurity are partly economical, political, social, and educational, but one major cause is the decrease in agricultural productivity per capita for the past 10 years (Brown and Kane 1994). At the same time, the land available for agriculture is declining due to urbanization, soil erosion, and salinization; the speed of the decline is illustrated by the world grain harvest area, which shrank from 740 million ha in 1980 to 680 million ha in 1995 (Brown and Kane 1994; FAO 1998a,b). Thus the future increase in food production must come from higher productivity and more efficient use of the existing resources under sustainable conditions. Especially in Africa, food security has been steadily deteriorating, and the marginalization of this region will continue in the future (Brown and Kane 1994; Dresrüsse 1996; FAO 1996, 1998a,b; Pinstrup-Andersen et al. 1995; Singer 1996). In sub-Saharan Africa cereal production per capita has constantly declined since 1970 (Singer 1996; FAO 1998a,b), and most countries are dependent on net cereal imports, which have increased from 1.2 million t per year in 1961 to 18.2 million t in 1990, and on direct food aid, on which 20% of the population are dependent today (FAO 1998a,b). The food imports and food aid notwithstanding, the daily calorie gap per person has increased by 670 kilocalories between 1961 and 1993, and the number of chronically undernourished people has more than doubled since 1970, from about 69 million to over 200 million (FAO 1998a,b). The population in sub-Saharan Africa continues to increase at the highest population growth rate in the world; an annual rate of 3.1% is enough to double the population in one generation (World Bank 1995; FAO 1998a,b). At present 40% of the people in sub-Saharan Africa suffer from malnutrition, and according to FAO (1998a,b) this number is expected to increase to 50% by the year 2000. It has been estimated that food production has to be doubled during the next 25 years in order to keep up with the population growth. Moreover, in order to reach a well-balanced diet, people will have to diversify their food intake. To eliminate hunger and qualitative malnutrition, the amount of plant-based calorie production has to be increased by sevenfold (FAO 1998a,b). The small-scale and subsistence farmers in Africa have little opportunity to invest in commercially produced inputs, e.g. fertilizers or pesticides needed to increase their yields, and thus have little possibility of meeting the increasing demand for food. Crops that can be produced with limited inputs offer a potential of increasing food security in these countries. Cassava has been grown as a subsistence and famine reserve crop in Africa for centuries, and in the past years its importance as a reliable crop giving acceptable yields with limited

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inputs has also been recognized internationally. The reliability and productivity of cassava has made it the most important locally-produced food in a third of the world’s low-income, food-deficit countries. Cassava is the main staple in sub-Saharan Africa, providing food for over 300 million people, many of them among the poorest in the world. In 15 sub-Saharan countries, 30 million people get up to 60% of their daily energy intake from cassava (Cock 1985; CIAT 1994; Koch et al. 1994). Although most countries that depend on cassava are in Africa (Angola, Benin, Cameroon, Central African Republic, Congo, Democratic Republic of Congo, Equatorial Guinea, Gabon, Ghana, Guinea, Ivory Coast, Liberia, Madagascar, Malawi, Mozambique, Nigeria, Rwanda, Sierra Leone, Tanzania, Togo, Uganda, and Zambia), cassava also plays an important role in Latin America (Cuba, Dominican Republic, Haiti, Paraguay, Nicaragua, and certain regions of Brazil and Colombia) (CIAT 1994, 1996; FAO 1997a, 1998c,d).

IV. CONSTRAINTS TO CASSAVA USE AND CULTIVATION Despite its integral part in food security in developing countries, cassava was long neglected in biotechnology and breeding programs, and was often considered to be a hardy crop with few problems. Up to 80 t/ha roots can be produced under optimal conditions in a 12-month growth period (CIAT 1980), but the grower yields are severely reduced due to poor agricultural practices and infestations by insect pests and diseases (Table 3.2). On the average, various pests and diseases are estimated to cause 20%–50% yield losses worldwide, and locally they can lead to total crop failures. Other important, still unsolved problems are the low protein content of the roots, the poor storability of freshly harvested roots, and the cyanogenic nature of cassava. Participatory studies have shown that the highest priorities among cassava farmers are total yields, production stability, access to markets, and stable prices (Thro et al. 1994, 1997; Henry and Howeler 1995). The following chapters describe some of these problems and discuss the potential and the state of art in the development of molecular biology tools to understand and solve them.

V. TRADITIONAL BREEDING AND BIOTECHNOLOGY IN CASSAVA IMPROVEMENT Cassava plants do not reproduce true to type via seeds. In addition, the low fertility and outcrossing nature linked to strong inbreeding depres-

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sion of the plants prevent breeding the plants to homozygosity. The limited gene availability in the sexually compatible germplasm imposes further restrictions on the use of traditional breeding in cassava improvement. Many of the available resistance traits are polygenic and/or recessive, which makes breeding for such characteristics complicated. Thus introgression of the desired traits to the numerous land races (as cassava is vegetatively propagated, every cultivar is a clone), adapted to specific environments, will be complicated and slow. Despite these problems, traditional breeding has produced a number of improved lines (Ssemakula et al. 1997; for a review, see Byrne 1984). The development of triploid lines has shown promise for yield improvement (Nassar 1992; Hahn et al. 1994; Sreekumari and Abraham 1997). Different aspects and progress in breeding of cassava can be found in Byrne (1984), Acosta (1991), Mahungu et al. (1994), Ofori and Hahn (1994), and Kawano et al. (1998). Biotechnology is a powerful tool to complement traditional breeding, and can extend the genetic pool for useful gene sources over species barriers. Transgene technology also offers the possibility of transferring single traits, without the problems encountered in traditional breeding that arise from the introduction of additional, often undesirable genetic material. The transfer of quantitative traits also lies within the scope of biotechnology. The development of reliable in vitro culture and regeneration methods compatible with transformation methods allows the routine and efficient production of transgenic plants, easily transferable between different laboratories. Characterization of the cassava genome, gene isolation, and the development of molecular maps are prerequisites for the development of marker-assisted breeding programs and mapbased gene cloning, and will also aid the elucidation of metabolic pathways in cassava. Understanding the plant-pathogen interactions and the evolution of the pathogens is essential for devising resistance strategies, as is the development of diagnostic tools for detection of pathogens and methods for pathogen elimination for germplasm preservation and international exchange.

VI. MOLECULAR GENETICS Genetics of cassava are still poorly understood in comparison to the other major staple crops. There is no classical genetic map of cassava, as the use of traditional linkage analysis is constrained by the paucity of morphological markers (Hershey and Ocampo 1989; Byrne 1984). Cassava is a monoecious, predominantly outcrossing and highly

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heterozygous species. Considerable selfing may, however, occur because the protogynous flowering mechanism prevents selfing in the same inflorescence but not in the same plant (Kawano et al. 1978). Recent studies on interspecific hybrids have shown that aposporic apomixis occurs at a low frequency in cassava (Grattapaglia et al. 1996; Ogburia and Adachi 1996; Nassar et al. 1998a,b). The results from cytogenetic studies have led to the proposition that cassava (2n = 36) is either a segmental allotetraploid (Magoon et al. 1969) or an allopolyploid (Umannah and Hartman 1973). The haploid genome contains 6 identical and 3 different pairs of homologous chromosomes and two sets of nucleolar organizing regions (Magoon et al. 1969; Umannah and Hartman 1973). Isozyme studies, as well as RFLP and microsatellite analyses, however, have revealed a predominantly disomic inheritance pattern (Lefevre and Charrier 1993; Sarria et al. 1993a; Fregene et al. 1995, 1997; Chavarriaga-Aguirre et al. 1998), and chromosome pairing behavior (Bai et al. 1993) indicates that cassava now behaves like a diploid, while the data from studies using molecular markers suggest that cassava may not be a strict allopolyploid, because random pairing might take place in certain regions of the genome (Fregene et al. 1998). A. Molecular Markers and Mapping Molecular markers will be indispensable for cassava breeding, as cassava and its pests and diseases have evolved in three separate regions of the world. In addition to quarantine problems during germplasm movements, this limits germplasm introduction between Latin America, the primary center of diversity, and Africa and Asia. Breeding Latin American material for resistance against pests or pathogens not present but presenting potential future hazards is facilitated if molecular markers can be used. Also, the long growing cycle of cassava, 9–18 months for most cultivars, makes improvement schemes time consuming, while the low seed yield per cross and inbreeding depression make introgression of traits difficult. Several different molecular markers have been developed for cassava, and their number is constantly increasing. Isozyme markers (Ocampo et al. 1993; Lefevre and Charrier 1993; Sarria et al. 1993a), minisatellite markers (Bertram 1993), chloroplast, ribosomal and mitochondrial DNA markers (Fregene et al. 1994; Carvalho et al. 1993) as well as cDNAs (Beeching et al. 1993; Haysom et al. 1993) have been used to assess the diversity in different cassava collections. The alpha beta esterase system was found to be the most informative isozyme marker, and both

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isozymes and minisatellites have been used to identify duplicates in the CIAT cassava collection (Ocampo et al. 1993, 1995). RAPD markers developed for cassava were used to characterize elite cassava germplasm held at CIAT (Bonierbale et al. 1993; Gomez et al. 1996). RAPD markers were also used to estimate genetic distances in African cultivars (Marmey et al. 1994; Mignouna and Dixon 1997) and Brazilian cultivars (Carvalho et al. 1993, 1998). RAPD analysis revealed a high degree of diversity in Brazilian germplasm, especially in the local landraces (Colombo et al. 1998). RFLP markers were also developed to study the variability of cassava germplasm (Beeching et al. 1993; Haysom et al. 1993) and to assist the construction of the cassava molecular map (Angel et al. 1993). A low number of randomly distributed duplications of RFLP loci was observed in cassava genome (Fregene et al. 1997). AFLP markers were used to assess the frequency of apomixis (Grattapaglia et al. 1996) and the genetic diversity and phylogenetic relations of cassava (see Section IVC). A unique AFLP fragment in the intergenic spacer region of ribosomal DNA was shown to be present in some African accessions but not in the Latin American ones (Roa et al. 1997). Recently microsatellite markers were developed for cassava and used to characterize a sub-set of the CIAT cassava collection and to identify duplicates in the collection (Agyare-Tabbi et al. 1997; Chavarriaga et al. 1998). Fourteen microsatellites with GA repeats were used to test 48 accessions, and 500 accessions were studied by 32P labeled probes using fluorescence based genotyping. The observed heterozygosity values varied between 0.00 and 0.88, and the number of alleles detected by the microsatellites varied from 1 to 15. Most microsatellites recognized 1–2 alleles per locus, indicating a low degree of microsatellite duplication in cassava. With one exception, which showed male distortion for one locus, most of the microsatellites were shown to segregate in expected ratios (Chavarriaga-Aguirre et al. 1998). To overcome the constrains caused by the lack of a classical genetic map of cassava, a recently constructed genetic linkage map should greatly help in understanding the complexity of the cassava genome (Fregene et al. 1997). A set of RFLP markers was developed (Angel et al. 1993) in order to select those showing the highest polymorphism for the construction of a framework molecular map. RFLP and RAPD markers were developed to identify the intraspecific cross to select the heterozygous parents used for producing an F1 mapping population (Gomez et al. 1996). As the parents used to produce the intraspecific F1 mapping population were heterozygous, the framework map consisted of female and male parent maps, and covered approximately 60% (931.6 cM) of the cassava genome. The female parent map contained 132 RFLP, 30

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RAPD, 3 microsatellite, and 3 isozyme markers, and consisted of 20 linkage groups spanning about 60% of the cassava genome, with an average marker density of 1 marker per 7.9 cM. The second map consisting of 107 RFLP, 50 RAPD, 1 microsatellite and 1 isozyme markers was constructed for the male parent. The maps revealed a higher recombination rate in the gametes of the male parent than in the female parent; also the pattern of marker distribution showed that recombination does not occur uniformly in the cassava genome. The joining of the female and male maps and the addition of more markers to produce a highly saturated map with tightly linked markers will allow its use in marker assisted breeding, map-based gene isolation, and will produce more detailed characterization of the genome structure in cassava. B. Gene Cloning Genomic and cDNA libraries from leaves, roots, harvested roots, and cotyledons of cassava have been constructed (Han et al. 1998; Hughes et al. 1992; Salehuzzaman et al. 1992; Tenjo and Mayer 1993; Bohl et al. 1997, 1998). These have been used to isolate genes and promoters from cassava, and also to characterize cassava germplasm (Beeching et al. 1993). Branching enzyme (Salehuzzaman et al. 1992) and granule-bound starch synthase (Salehuzzaman et al. 1993a,b) genes were cloned as cDNAs from cassava root libraries. The genes involved in the breakdown of cyanogenic glucosides, linamarase (Hughes et al. 1992), and α-hydroxynitrilase (Hughes et al. 1994) as well as the gene encoding the last enzyme in the linamarin synthesis pathway UDPG-glugosyltransferase (Hughes and Hughes 1994) were cloned as cDNAs and genomic clones (Hughes et al. 1997, 1998). Isolation of a genomic clone of cassava PEP carboxylase (Tenjo and Mayer 1993), the cassava Ef1a gene and promoter (Suhandono et al. 1998), AGPase S and B cDNAs (Munyikwa et al. 1998a,b) and a number of yet unidentified root specific (Bohl et al. 1998; Marello et al. 1997) and post-harvest deterioration-related (Huang et al. 1998) sequences have also been reported. Other examples of genes cloned from cassava can be found in Section VII. The development of a bacterial artificial chromosome library (Fregene et al. 1998) should further speed up the isolation and characterization of cassava genes. C. Phylogenetic Mapping RFLP analysis of chloroplast DNA and ribosomal DNA of cassava and its wild relatives first suggested that M. aesculifolia would be most similar to cassava (Bertram 1993), and later studies showed that M. esculenta

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subsp. flabellifolia could possibly be the wild ancestor of cassava (Fregene et al. 1994). RFLP analysis of chloroplast DNA has revealed 9 different chloroplast types, 3 in cassava cultivars and 6 in its wild relatives (Fregene et al. 1994). Roa et al. (1997) and Second et al. (1997) used AFLP analysis to further assess the diversity of cassava and some wild taxa to estimate genetic relationships within the genus Manihot. A mixed group, consisting of M. esculenta subsp. flabellifolia, M. esculenta subsp. peruviana, and M. tristis, was found to be most similar to cassava. Microsatellite analysis has confirmed the close relationship of M. esculenta subsp. flabellifolia to cassava (Cabral et al. 1998a; Roa et al. 1998). Unique AFLP products were detected in samples of three wild Manihot species, which may be useful in future elucidation of the evolution of cassava (Roa et al. 1997). The results from the AFLP analysis thus suggested the existence of intraspecific gene pools and also revealed that the genetic diversity among cassava accessions is less than among its closest wild relatives and supported the hypothesis that cassava was domesticated from a complex of the two M. esculenta subspecies. Second et al. (1997), however, postulated that other species may have contributed to the crop as well, supporting the results of Schaal et al. (1997) from ribosomal DNA analysis. A recent phylogeographic study based on a single copy nuclear gene G3pdh of cassava and its wild relatives, however, indicated that cassava is more likely to have been domesticated from wild M. esculenta in the southern border of the Amazon basin, and not derived from several progenitor species (Olsen and Schaal 1999).

VII. TISSUE CULTURE AND REGENERATION Tissue culture is used routinely for production of disease-free planting material and for mass propagation of selected cassava lines (Roca 1984; Raemakers et al. 1997a). Conservation of germplasm as slow-growth cultures in vitro or as cryopreserved material ensures the maintenance of variability. De novo plant regeneration methods are essential for the development of transformation systems, and embryo rescue and culture techniques facilitate the production of hybrid lines, while anther cultures are required for production of haploids. A. Shoot Tip and Meristem Culture Successful in vitro culture of cassava meristems was first reported in 1974 by Kartha, who regenerated shoots from 5 cassava cultivars and showed that growth and shoot regeneration from the meristems is

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promoted by addition of 0.1 mg/L BA, 0.04 mg/L GA3, and 0.2 mg/L NAA into the culture medium (Kartha 1974). Today, meristem culture is a routine and integral step in a number of tissue culture applications, including micropropagation, cryopreservation, and de novo regeneration. Roca (1984) provides an overview on the use of cassava meristems, and describes detailed protocols used for culturing meristems and shoots.

B. Disease Elimination Cassava is propagated vegetatively, and hence it is susceptible to diseases that are carried from one planting generation to the next through infected planting material. Build-up of systemic pathogens is one of the main reasons for yield decline. International exchange of plant material and also to a large extent the maintenance of plant germplasm require the use of disease-free stocks (Roca et al. 1982; Schilde-Rentschler and Roca 1987). Meristem cultures can be used to maintain and propagate cassava cultivars in vitro and to produce disease-free planting material. Meristem cultures can be used to produce plants free from African cassava mosaic virus, when explants smaller than 0.4 mm are used (Kartha and Gamborg 1975a,b). Meristem culture combined with thermotherapy improves the elimination rate of a number of viruses and bacteria, and it also allows the use of larger meristem explants up to 0.8 mm (Kartha and Gamborg 1975a,b). For disease elimination from field grown material, cassava stem cuttings are grown in the greenhouse at 35°C or 40°C day/35°C night for 3–4 weeks, after which the meristems of the sprouting shoots are isolated and cultured in vitro for 3–6 weeks. The shoots formed by the meristems are tested for the presence of infectious agents, and if disease-free are used for in vitro storage, micropropagation, and for germplasm exchange. The meristems from infected shoots can be reisolated and subjected to thermotherapy in vitro (Roca 1984; Ng et al. 1990; Roca et al. 1991a; Frison 1994). By use of meristem culture and thermotherapy 90%–100% success has been reported in eliminating, among others, cassava bacterial blight (Xanthomonas campestris pv manihotis), frog skin disease (uncharacterized virus) (Schilde-Rentschler and Roca 1987), cassava brown streak virus (Kaiser and Teemba 1979), and African cassava mosaic virus (Adejare and Coutts 1981). Freeing the material of pathogens has been shown to increase the productivity of cassava 50% to 100% in the farmers’ fields (Mabanza et al. 1995). Diagnostic tools for molecular detection of some of the main pathogens are listed later in Section VII.

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C. Embryo and Anther Culture The survival and germination rate of cassava seed is low, which can severely constrain breeding programs (Byrne 1984; Bai 1987; Ogburia and Adachi 1995). Plant germination from seed could be improved from 1%–15% to 41%–80% by isolating and culturing embryonic axes from mature seeds at 30°C on a medium containing 0.25 mg/L IBA under continuous light (Biggs et al. 1986). Newly matured seeds had the highest germination rate, and in aging seeds the germination rate fell rapidly. Both mature and immature embryos of cassava and wild Manihot species could be induced to germinate in vitro on a medium containing halfstrength MS (Murashige and Skoog 1962) salts. Up to 100% plantlet growth was obtained when mature embryos were cultured on a medium supplemented with 3% sucrose, while immature embryos responded best on a medium supplemented with 4% sucrose, 15% coconut water, and 5 mg/L IAA (Ng 1992a; Ng and Ng 1997). When cultured on a medium supplemented with 2% sucrose and 1 mg/L GA3, immature embryos isolated 35 days after pollination showed the highest germination rate (Catano et al. 1993). Recently, the development of an efficient culture system for both immature and mature embryos was reported (Guevara et al. 1998). The embryo culture system has been used successfully to recover interspecific hybrid plants (M. esculenta × M. aesculifolia) and to produce triploid clones of cassava (Asiedu et al. 1992; Roca 1984; Ng 1992a,b; Byrne 1984). A common problem encountered with the wild relatives of cassava is the lack of root formation on stem cuttings. Although improved protocols allowing root formation have been developed (Cabral et al. 1998b), embryo culture has been suggested as a means to circumvent this problem. Use of high temperature (25° to 30°C) was optimal for in vitro germination of M. glaziovii embryos on a woody plant medium (McCown and Lloyd 1981) containing activated charcoal (Mendes et al. 1997). Since cassava cannot be bred to homozygosity, breeding for traits controlled by recessive genes is difficult, especially if they are inherited tetrasomically. Therefore the production of haploid lines via tissue culture would provide valuable information about gene function. Cassava pollen could be cultured to the microcallus stage on a medium containing 5% sucrose (Catano et al. 1993). Pollen cultures could also be used to germinate cassava pollen in vitro (Mbahe et al. 1994). Callus formation from cassava anthers was reported (CIAT 1982; Mukherjee 1995), but to date plant regeneration from anther cultures has not been reported. Immature inflorescences of ‘MCol1505’ (Woodward and Puonti-Kaerlas 1998, in press) were used as source material to induce

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somatic embryogenesis on MS medium supplemented with 2% sucrose and 12 mg/L picloram. Although anthers, uninucleate microspores or floret wall tissues failed to form somatic embryos, up to 78% of macerated male inflorescences and 30% of half male florets formed somatic embryos, which, after maturation, could be regenerated via organogenesis. The regenerated plants are currently being analyzed for their ploidy levels, and if they are haploid, this method can be used for rapid production of pure lines after chromosome doubling, which would accelerate the breeding programs and mutant selection. D. Micropropagation Micropropagation techniques are indispensable for the conservation of cassava diversity and as tools for technology transfer through safe exchange of germplasm. Multiplication and delivery of improved lines and disease-free plants to the farmers are essential to ensure sustained cassava cultivation. Conventional propagation of cassava via stem cuttings is slow, as one plant can produce only 10–20 propagules per year, a multiplication rate only about 10% of that of cereals (Roca 1984). In addition, field propagation involves the risk of re-entry of systemic diseases and accumulation of pathogens. Techniques based on single leafbud cuttings increase the potential multiplication rate to 1:300,000 per year (Roca et al. 1980). Micropropagation in vitro based on meristem culture has even greater potential. Meristems cultured in vitro can be induced to form multiple shoots on cytokinin-containing medium. BA at concentrations between 0.5 and 10 mg/L alone or in combination with low amounts of auxins can be used to break apical dominance of axillary or apical meristems, which allows the proliferation of multiple shoots that can be harvested over a period of several months. Culturing cassava shoot tips on a medium containing 1 mg/L BA led to the formation of rosette cultures, from which up to 20 shoots per original explant could be harvested weekly (Roca 1984). In theory, the potential of the most efficient multiple shoot induction protocols has been estimated to be up to 1.2 ⋅ 1020 new shoots in one year (Smith et al. 1987). The potential of multiplication via de novo regeneration is even higher. Transfer of micropropagated plantlets to the field can be problematic (Roca et al. 1985; Jorge 1997; Ng and Llona 1989). The routine method applicable to many cultivars is described by Roca (1984), and the hardening and acclimatization methods have been further improved (Jorge 1997; Ajonye et al. 1998; Woodward et al. 1998). The use of mycorrhizal inoculation has been shown to improve the survival rate of seedlings upon transfer to the field (Azcon-Aguilar et al. 1997), and in China

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800,000 transplantable plantlets of a high-yielding cultivar ‘Nan-Zhi188’ were produced for distribution to the farmers in two years using an efficient low-cost in vitro propagation system (Guo and Liu 1995). For physiological and gene expression studies in vitro tuberization has been attempted, but so far without success (Salehuzzaman et al. 1994), while in vitro flowering of cassava shoots has been reported (Tang et al. 1983). E. Germplasm Conservation Because cassava does not breed true to type, the cultivars cannot be stored as seeds. Cassava gene banks throughout the world conserve cassava germplasm either as field collections or in vitro. In addition to a number of local gene banks, the largest collections are maintained at Centro Internacional de Agricultura Tropical (CIAT, Cali, Colombia), International Institute of Tropical Agriculture (IITA, Ibadan, Nigeria), Central Tuber Crops Research Institute (CTCRI, Trivandrum, India), L’Institut français de recherche scientifique pour le développement en coopération (ORSTOM, Montpellier, France), and CENARGEN/ EMBRAPA (Brasilia, Brazil). Field collections are maintained as continuous clonal cultivations, which are bulky and occupy large areas. Also, the plants are exposed to environmental hazards, genetic erosion, diseases, and pests. In vitro gene banks are an alternative for field collections. To reduce the amount of labor involved and to minimize the risk of infections through frequent subculturing in maintenance of in vitro banks, slow growth shoot culture techniques have been developed (Roca et al. 1989, 1991b). The development of an in vitro gene bank system involves different steps from sampling and characterization of field material, micropropagation, thermotherapy, virus indexing, and in vitro culture to development of information systems to monitor the status of the collection. Indexing techniques used at CIAT include field symptomatology, graft inoculation, ELISA tests, and dsRNA as well as PCR tests (Roca et al. 1991a,b). As a tool for more efficient conservation of genetic variability, molecular markers, including isozymes, minisatellites, and microsatellites, have been developed for identification of duplicates (Ocampo et al. 1993, 1995; Chavarriaga et al. 1998). In the slow growth system, shoots are maintained under low light conditions on modified MS medium supplemented either with no growth regulators or with 0.01 mg/L NAA, 0.02 mg/L BA, and 0.1 mg/L GA3 (Roca et al. 1989). CIAT has a world mandate for cassava, and maintains the largest in vitro collection of cassava containing almost 6,000 accessions, requiring about 50 m2 space (Escobar et al. 1993, 1997). Recent

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improvements to the culture system include the use of silver nitrate to reduce the shoot growth rate further (Mafla et al. 1998). The suitability of in vitro collections for maintaining genetic stability in cassava was confirmed on the molecular level in plants retrieved from cultures kept under slow growth conditions for ten years (Angel et al. 1996). Even slow growth cultures may, however, be subject to genetic and physiological changes. The most stable form of germplasm conservation is cryopreservation, where the tissues are frozen and stored at –196°C under liquid nitrogen. Cryopreservation of cassava meristems, seeds, and somatic embryos is possible (Bajaj 1977; Kartha et al. 1982; Mycock et al. 1995). Cryopreservation of zygotic embryos and seeds allowed up to 97% survival rates and plant recovery from all surviving seeds and of 34% of zygotic embryos (Marin et al. 1990). Recently an improved protocol for cryopreservation of cassava shoot tips allowing 50%–70% plant recovery was reported (Escobar et al. 1997). Shoot tips were isolated from in vitro-grown shoot cultures, pre-cultured for 3 days on a medium containing 182.2 g/L sorbitol and 0.78% dimethyl sulfoxide (DMSO), treated with cryoprotectant (182.2 g/L sorbitol, 10% DMSO, and 4% sucrose), for 2 h and dehydrated for 1 h on filter paper, after which they were cooled stepwise to –40°C, and finally transferred to liquid nitrogen. Regrowth of shoots from cryopreserved shoot tips after rapid warming was initiated on shoot culture medium after a recovery period of two days first on 25.7% sucrose and 0.25% activated charcoal and then on 1⁄3 strength MS medium with 12.8% sucrose and 1 g/L inositol in the dark. Originally 11 out of 15 tested cultivars could be successfully regenerated after cryopreservation. Later modifications of the protocol allowed some of the initially recalcitrant cultivars to be regenerated at a recovery rate of 50%–70%. Rapid freezing of pre-treated meristems led to 80%–93% viability and 40%–50% plant recovery of ‘MCol22’, while two other cultivars had 9%–77% shoot regeneration frequency (Escobar and Roca 1997). A further refinement of the technique using shoot tips encapsulated in 3% Na-alginate, dipped in 0.1M CaCl2 and pre-treated with 3% sucrose before slow dehydration to 15%–20% internal water content allowed direct freezing of the material by transfer to liquid nitrogen (Escobar et al. 1998). F. International Exchange of Material Germplasm exchange involves a risk of dissemination of pathogens and pests. The transport of cassava stem cuttings as well as of seeds can lead to the introduction of a pest from one country or region to another. Minor pests have developed into major ones when introduced to new

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environments. Pests and pathogens originating from South America, which were inadvertently introduced to Africa, include cassava mealybug, green spider mite, and cassava bacterial blight. Internationally, due to the phytosanitary regulations, most material is exchanged as sterile in vitro shoots, derived from the main gene banks. The appropriate steps involved in germplasm transfer have been described by Roca (1984). Currently CIAT is shipping over 3,000 disease-free plantlets annually, and IITA is distributing germplasm to over 40 African countries. As an alternative to seed stock transfer, the use of in vitro-cultured zygotic embryo axes has been proposed. A new method was developed for excision and culture of the embryos. Studies on establishment rate showed that 85% of mature embryos and 100% of the immature embryos could be induced to produce plants (Guevara et al. 1998). G. De Novo Regeneration 1. Multiple Shoot Induction. Meristems cultured on shoot culture medium containing no or low amounts of growth regulators produce one shoot, but can be induced to form multiple shoots on cytokinin-containing medium. Most of the shoots are derived from pre-existing axillary meristems, but also de novo formation of new meristems and shoots has been described (Konan et al. 1994a, 1995, 1997). Axillary buds were induced to swell to bulb-like structures on media containing 2–10 mg/L BA. Transfer of such structures to media supplemented with either 0.1 mg/L NAA, 1 mg/L BA, 0.1 mg/L GA3, or with 1–2 mg/L BA led to production of up to 14 multiple shoots per explant (Konan et al. 1994a; Frey 1996; Puonti-Kaerlas et al. 1997a). Bhagwat et al. (1996) showed that a pre-treatment in liquid culture medium containing 0.025–0.05 mg/L thidiazuron increased the frequency of multiple shoot formation from nodal explants in several cultivars on solid culture medium containing 0.5 mg/L BA and 0.5 mg/L GA3. Addition of the surfactant Pluronic-F68 to the medium for a two-week period during the multiple shoot induction on 10 mg/L BA also increased the shoot proliferation in cultivars responding poorly to BA treatment alone, and in the best responding cultivars allowed up to 63% of the explants to produce at least 25 shoots that could be harvested (Konan et al. 1997). Using a similar protocol, multiple shoots could also be induced from apical meristems, but the number of shoots was lower than when axillary buds were used (Frey 1996; Puonti-Kaerlas et al. 1997a). 2. Somatic Embryogenesis. Production of somatic embryos was first reported from cotyledons and embryonic axes of cassava zygotic

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embryos in 1982 (Stamp and Henshaw 1982), and somatic embryogenesis is now the most commonly used regeneration method for cassava (Fig. 3.2). The competence for somatic embryogenesis in cassava is restricted to meristematic and embryonic tissues, and somatic embryos can only be induced on a limited number of explants, e.g. cotyledons or embryonic axes from zygotic embryos (Stamp and Henshaw 1982; Konan et al. 1994a,b), immature leaf lobes (Stamp and Henshaw 1987a; Szabados et al. 1987; Mathews et al. 1993; Raemakers 1993; Raemakers et al. 1993a; Li et al. 1995, 1996, 1998a; Puonti-Kaerlas et al. 1997b), meristems and shoot tips (Szabados et al. 1987; Narayanaswami et al. 1995; Frey 1996; Puonti-Kaerlas et al. 1998), anthers (Mukherjee 1995), and immature inflorescences (Woodward and Puonti-Kaerlas 1998, in press) on auxin-containing media. Picloram at 1–12 mg/L, dicamba at 1–66 mg/L, and 2,4-D at 1–16 mg/L have been used to induce primary somatic embryogenesis. The efficacy of the different growth regulators depended on the explant and the cultivar used (Ng 1992a; Mroginski and Scocchi 1993; Sudarmonowati and Henshaw 1993; Taylor et al. 1993; Li et al. 1995, 1998a). In contrast to previous reports (Raemakers et al. 1995a; Sofiari et al. 1997), NAA and also IBA were recently reported to induce primary somatic embryogenesis from immature leaf lobes of ‘NanZhi188’ (Ma et al. 1998). Embryogenic potential in cassava is highly genotype-dependent, and there are cultivars from which, despite repeated efforts, no somatic embryos could be obtained. It is often possible to induce proembryonic structures, or even globular embryos on explants from recalcitrant cultivars, but these do not develop further to torpedo-shaped or maturing embryos (Sudarmonowati and Bachtiar 1995; Raemakers et al. 1997a; Puonti-Kaerlas, unpublished). Pre-treatment of the donor plants with 2,4-D (Matsumoto et al. 1991; Raemakers 1993), and the addition of ABA (Konan et al. 1994b), or supplementary cupric sulphate to the embryo induction medium (Schöpke et al. 1993), were shown to improve the embryo induction frequency in some cultivars. The sucrose concentration of the medium can also affect the embryogenic response. The formation of primary somatic embryos was inhibited by 6% sucrose, which was optimal to embryo development after the induction phase (Konan et al. 1994b). When maintained on auxin-containing medium, some somatic embryos spontaneously start developing into torpedo-shaped embryos and eventually produce mature embryos with greening cotyledons. This process is enhanced if the embryos are transferred to a hormone-free medium or to a medium containing low amounts of BA and auxin (Stamp and Henshaw 1982, 1987a). The frequency of germination of

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Fig. 3.2. In vitro culture and regeneration methods in cassava: Cassava shoot tip (a), multiple shoots (b), primary somatic embryos (c), secondary cyclic somatic embryo cluster (d), germinating somatic embryos (e), organogenesis: shoot primordia developing on cotyledon explant (f), rooted plant (g), embryogenic suspension (h), protoplasts (i), friable embryogenic callus regenerated from protoplasts (j), regenerated plants in the greenhouse (k).

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mature somatic embryos, however, is usually low, and the root development is incomplete. A two-step protocol is therefore normally applied first to allow shoot elongation of the mature somatic embryos on a medium containing 0.1–1.0 mg/L BA and 0–0.01 mg/ 2,4-D and then to root the shoots on a hormone-free medium or a medium containing low amounts of auxin. A new method including a culture step on hormonefree medium containing 0.5% activated charcoal followed by desiccation before transfer to hormone-free medium allowed up to 83% plant regeneration from germinating embryos with normal root development (Mathews et al. 1993). Likewise, additional GA3 was shown to be beneficial for the further development of shoots from somatic embryos (Matsumoto et al. 1991; Szabados et al. 1987). The addition of AgNO3 was reported to improve embryo induction and maturation (Zhu et al. 1998). Highly efficient plant regeneration via germination of somatic embryos, allowing the production of 21 shoots per original explant, was reported by using 4% maltose instead of sucrose in the embryo culture medium, and combining 0.1 mg/L paclobutrazol with 6 mg/L 2,4-D in the embryo induction medium (Li et al. 1998a). Primary somatic embryos can be induced to produce secondary somatic embryos by further subculturing on auxin-containing medium (Stamp and Henshaw 1987b). By constant subculturing of somatic embryos, a cyclic embryogenesis system can be established either in liquid or solid medium, where the embryos rarely pass the torpedo stage, until transferred to germination medium. Paclobutrazol used in combination with 2,4-D has been shown to increase the frequency of secondary somatic embryogenesis, and substituting maltose for sucrose in the embryo induction medium increased the rate of somatic embryogenesis, germination, and plant regeneration, allowing up to 24 plants to be regenerated per original explant (Li et al. 1995,1998a). The efficiency of secondary somatic embryogenesis was shown to be higher in liquid cultures than on solid cultures and the use of NAA or IBA allowed efficient production of somatic embryos with high germination capacity (Raemakers et al. 1993b,c, 1995a; Li et al. 1995; Sofiari et al. 1997; Ma 1998; Ma et al. 1998). To obtain maturing embryos with complete shoot and root poles from NAA-induced embryo cultures, desiccation and further culture on a medium containing 0.1–4 mg/L BA was necessary. NAA-induced embryos matured more efficiently than embryos maintained on 2,4-D (Raemakers et al. 1997b; Sofiari et al. 1997). In most tested cultivars, BA treatment enhanced both germination and root formation of NAA induced embryos, while in 2,4-D induced embryos, with one exception, only the root formation frequency was improved. Controlled desiccation experiments showed that the highest germination rate

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was obtained when embryos were allowed to dry to 60% of their original weight (Raemakers et al. 1995b). Method improvements have increased the efficiency of regeneration via somatic embryogenesis considerably, and the protocols are robust enough to allow transfer between laboratories and extension to several cultivars. By careful monitoring of the cultures and frequent selection and transfer of embryogenic material, regeneration can be achieved even with cultivars that have a very low initial response. By partial germination of embryos and use of cotyledons for each cycle of embryogenesis, cultivars previously considered recalcitrant could be regenerated (Puonti-Kaerlas et al. 1998). 3. Friable Embryogenic Callus and Embryogenic Suspensions. A fraction of the cycling somatic embryos maintained on a MS medium supplemented with 12 mg/L picloram produce a new tissue type that consists mainly of small globular embryo-like structures. When isolated, it produces a highly friable embryogenic callus (FEC) (Taylor et al. 1996). The frequency of FEC production was increased by substituting the MS medium with GD medium (Gresshoff and Doy 1974). The combination of picloram with NAA increased the embryogenic response of 8 out of 10 cultivars and increased the frequency of FEC induction, allowing induction from primary somatic embryos that were developmentally younger than cotyledonary stage (Taylor et al. 1997, 1998a). Once pure FEC is obtained it can be transferred to liquid culture to establish a rapidly proliferating embryogenic suspension in SH (Schenk and Hildebrandt 1972) medium supplemented with 6% sucrose and 10–12 mg/L picloram. By transfer to hormone-free medium, development of maturing embryos is induced. The efficiency of the maturation step was improved by transferring the cultures maintained on 10 mg/L picloram to a medium supplemented either with 1 mg/L picloram or 1 mg/L NAA, which allowed the globular embryos to continue their development to torpedo stage and germinating embryos. The conversion of globular embryos is the limiting step of the regeneration protocol, as only a small fraction of the embryos is able to develop into torpedo stage embryos, and the variation in the regeneration capacity of cultures is high (Raemakers et al. 1997b). Also the frequency of somaclonal variation can be up to 50% of the regenerated plants (Raemakers et al. 1998). The multistep regeneration protocol is laborious. To improve efficiency, a method based on culturing the suspension cells on rafts floating on liquid medium, which allows regeneration in one step, was described (Schöpke et al. 1997b). Culturing the suspension for two weeks without subculture before transfer to maturation medium increased the conver-

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sion rate (Munyikwa et al. 1998a), and the transfer of the embryogenic clusters to 1⁄2 strength MS medium supplemented with low concentrations of picloram or NAA followed by culture on NAA containing medium improved the maturation rate (Raemakers et al. 1997b; Taylor et al. 1997). Another type of embryogenic callus formation was reported by Groll et al. (1997), who induced FEC from thin layer explants of stem internodes and petioles on MS medium supplemented with 3% sucrose, 2.15 mg/L kinetin, and 0.018 mg/L IAA. Radicle elongation of the embryos was observed, but plant regeneration from these cultures has not been reported. 4. Protoplasts. Anthony et al. (1995) described sustained cell division of leaf protoplasts from in vitro cultured shoots. The protoplasts were cultured for up to 50 days in liquid, ammonium-free MS medium, overlaying agarose-solidified B5 medium with short glass rods embedded and protruding from the agarose layer. Microcallus formation was reported using liquid culture medium (McDonnell and Gray 1997). The development of protoplasts isolated from somatic embryos required both 2,4-D and NAA in combination with BA in culture medium (Sudarmonowati 1992). Until the development of friable embryogenic callus culture, however, no plant regeneration from cassava protoplasts was possible. Protoplasts isolated from friable embryogenic callus and from suspensions of cassava genotype TMS60444 and cultured in a medium supplemented with 0.5 mg/L NAA and 1 mg/L zeatin began dividing after 3 days, and after 2 months of culture, friable calli looking identical to the original FEC were obtained. After two rounds of selection of 3 weeks each, regeneration of the protoplast-derived FEC through a series of steps on different media was started. First, the protoplastderived FEC were cultured for 11 weeks on maturation medium on which torpedo shaped embryos were produced. These were isolated from the calli and transferred to fresh medium for maturation. Mature embryos were multiplied by secondary somatic embryogenesis on a medium supplemented with 8 mg/L 2,4-D. About 30% of the mature secondary somatic embryos developed shoots after transfer to a medium supplemented with 1 mg/L BA (Sofiari et al. 1998). The development of protoplast regeneration methods will allow the production of improved cassava lines via the production of somatic hybrids, cybrids, and asymmetrical hybrids. 5. Organogenesis. Early results of shoot regeneration from cassava callus (Tilquin 1979) could never be repeated, and only recently an efficient

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alternative regeneration system via organogenesis was developed in order to circumvent the problems encountered when regenerating plants via germinating somatic embryos (Li et al. 1995, 1996, 1998a). Shoot primordia were induced directly on cotyledon explants from germinating cycling somatic embryos on a medium containing cytokinins. A cycling system where the secondary somatic embryos were induced on cotyledon explants from maturing somatic embryos was established. To induce shoot organogenesis, cycling somatic embryos were transferred to maturation medium containing 0.1 mg/L BA, and the developing young green cotyledons were harvested, cut to pieces, transferred to organogenesis medium containing BA alone or combined with different growth regulators and cultured for 20 days in the darkness. Combining BA with either NAA or IBA resulted in the highest organogenesis frequencies. The number of regenerating shoots that were rooted and transplanted to soil was highest on medium containing BA and IBA followed by NAA. The response on a medium containing 2,4-D combined with BA was very low. The combination of 1.0 mg/L BA and 0.5 mg/L IBA gave the highest shoot induction rate, with an average regeneration rate of 8.7 transplantable shoots per explant. After a passage on elongation medium containing 0.4 mg/L BA, the regenerating shoots were easily rooted on hormone-free medium and transplanted into soil in the greenhouse. Shoot induction frequency of different cassava cultivars varied between 42% and 67% and shoot primordia could be induced on cotyledons from cycling embryos maintained either on 2,4-D or picloram. Cotyledon explants derived from cycling somatic embryos showed the highest competence for organogenesis, while those from primary somatic embryos responded very poorly. The transferability of this protocol has been demonstrated with 10 different cultivars. A report on one Chinese cultivar indicated that BA and thidiazuron are more efficient in inducing organogenesis than kinetin or N-isopentenyladenine (Ma 1998), thus confirming the earlier results. This also showed that while the frequency of primordia induction is higher using thidiazuron than BA, the number of shoots regenerated is lower. Direct organogenesis from immature cassava leaves from ‘Nan-Zhi188’ was achieved by using high concentrations (10 to 80 mg/L) of NAA (Ma et al. 1998) and by culturing immature leaves of ‘Somal1’ for ten days on a medium containing 6 mg/L 2,4-D and then transferring the explants to 5 mg/L zeatin (Mussio et al. 1998). Compared to regeneration via germination of embryos derived from suspensions, shoot regeneration via organogenesis is faster, thus requiring less time in tissue culture. Using organogenesis, transplantable shoots can be regenerated from cotyledon explants within 60–65 days. In addition, the germination/maturation

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steps in the protocol ensure the selection for highly regeneration competent embryos, hence minimizing the risk of producing embryogenic cultures that will be arrested in their development.

VIII. GENETIC TRANSFORMATION Genetic engineering can be used to introduce new traits precisely into existing cultivars. Production of stably transformed plants, however, requires several factors to be fulfilled. First, an efficient in vitro culture system that allows regeneration of plants has to be developed. Plant cells are generally considered to be totipotent, thus being able to regenerate whole plants from single cells in vitro. The ability to react to external stimuli and to regenerate in vitro is, however, often limited to certain tissues and developmental stages, and the requirements for transformation and regeneration competence may not always be compatible. Second, a method for efficient transfer and stable integration of the transgenes into the plant genomic DNA is essential; this also includes means for identifying and selecting for transformed cells. The main constrain is usually not the delivery of foreign DNA to the regenerable cells, but the recovery of the transformed cells. Finally, the introduced genes must be correctly expressed in the primary transgenic plants and stably transmitted to their progeny. In the case of cassava, which is vegetatively propagated, the transgenes can be fixed already at the level of the primary transgenic plants, and stable inheritance is of concern only when the transgenic plants are to be incorporated into breeding programs. Of the several methods for delivering foreign DNA into plant cells (for a set of protocols, see Potrykus and Spangenberg 1995), the most commonly used are Agrobacterium-mediated gene transfer and particle bombardment. As stable transformation frequencies are low, the use of different marker genes is necessary to allow the identification and selection of the stably transformed cells (for review, see Schrott 1995). The most commonly used visual markers are GUS encoded by the uidA gene (Jefferson 1987; Jefferson et al. 1986), the luciferase genes from the firefly Photinus pyralis (Ow et al. 1986) and from the soft coral Renilla reniformis (Mayerhofer et al. 1995), and the green fluorescent protein (Chalfie et al. 1994). Selectable marker genes render plant cells resistant to an antibiotic, a metabolic analogue, or a herbicide, thus allowing the cells containing and expressing the transgene to survive and proliferate, while the wild-type cells are either arrested in their growth or killed. The most commonly used selectable marker genes encode resistance to aminoglycoside antibiotics (nptII) (Bevan et al. 1983; Fraley et al. 1983;

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Herrera-Estrella et al. 1983), hygromycin (hpt) (van den Elzen et al. 1985; Waldron et al. 1985), and phosphinotricin, the active ingredient in many herbicides including Basta (pat and bar) (Murakami et al. 1986; De Block et al. 1987; Thompson et al. 1987; Wohllenben et al. 1988). As public concern regarding the use of antibiotic resistance genes in transgenic plants has become an important factor for decision making, the method development is now moving towards other selection systems, e.g. the use of antibiotic resistance genes the expression of which in micro-organisms can be inhibited by the introduction of intron into the coding regions of the gene (Wang et al. 1997). New non-antibiotic methods based on positive selection, which favors the regeneration and growth of transgenic cells while suppressing the growth and proliferation of non-transgenic ones, have also been developed recently. The use of a glucuronide derivative of the cytokinin benzyladenine (Joersbo and Okkels 1996), xylose (Haldrup et al. 1998a,b), and mannose (Joersbo et al. 1998), were shown to significantly improve the frequencies of transgenic plant regeneration in tobacco, potato, and sugar beet, respectively, when compared to antibiotic selection schemes. Cassava was shown early to be susceptible to Agrobacterium (Calderon-Urrea 1988), but the efficiency of different strains is highly variable and genotype-dependent (Chavarriaga-Aguirre et al. 1993; Sarria et al. 1993b; Li et al. 1996; Puonti-Kaerlas et al. 1997b). Until lately, mainly due to problems in using selectable marker genes, cassava was considered recalcitrant to genetic engineering. A breakthrough was achieved recently when first reports on regeneration of transgenic cassava plants were published (Li et al. 1996; Raemakers et al. 1996; Schöpke et al. 1996). A. Meristems Transient and stable expression of both GUS and luciferase were demonstrated in meristems and meristem-derived somatic embryos and multiple shoot clusters after particle bombardment (Puonti-Kaerlas et al. 1997a) or cocultivation with Agrobacterium (Konan et al. 1995; PuontiKaerlas et al. 1997a). Transgenic sectors were detected in the developing shoots, but no fully transgenic plants have been regenerated. B. Somatic Embryos In cassava both primary and secondary somatic embryos develop from groups of cells, usually located at or near the vascular tissue (Stamp 1987; Raemakers et al. 1995b). The multicellular origin of cassava

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somatic embryos makes them poorly suited for genetic engineering, and their location under the plant epidermis also limits their accessibility to Agrobacterium. Transgenic sectors were detected in somatic embryos after electroporation, but no transgenic plants have been regenerated from these embryos (Luong et al. 1995, 1997). Particle bombardment of embryogenic clusters led to high transient expression of visible marker genes in several laboratories, but only sectorial transgenic embryos could be regenerated from bombarded embryos. Transgenic callus can be easily obtained from explants from cycling somatic embryos, but as a rule the competence for embryogenesis is lost when the cultures are treated with antibiotics to select for transformed cells (Chavarriaga-Aguirre et al. 1993; Schöpke et al. 1993; Puonti-Kaerlas, unpublished). There is only one report on successful regeneration of transgenic cassava plants via secondary somatic embryogenesis with molecular data to show the presence of transgenes in one of the 15 regenerated Basta resistant plant lines (Sarria et al. 2000). In this case, secondary somatic embryos were induced on cotyledon explants from primary somatic embryos of ‘MPeru183’ after cocultivation with the wild-type Agrobacterium strain CIAT 1182 carrying the genes encoding GUS and phosphinotricin resistance. Selection on 8–32 mg/L Basta allowed the regeneration of putative transgenic secondary embryos that could be germinated to plants. ‘MPeru183’ and the Agrobacterium strain CIAT 1182 were selected after a screen for the most efficient combination to ensure high transformation frequency (Sarria et al. 1993b). The reproducibility of this method has not been assessed so far, and the CIAT 1182 is oncogenic. Thus its use for production of transgenic plants on a routine basis is still limited. Disarming of the vector should offer great potential for further improvement of Agrobacterium-mediated transformation methods. The use of immature cassava leaves to regenerate transgenic plants after cocultivation with Agrobacterium was reported (Arias-Garzon and Sayre 1998), but needs verification on the molecular level. C. Embryogenic Suspensions In contrast to the primary or secondary somatic embryos, the new embryogenic units in friable embryogenic callus develop from the surface cells of the globular embryo clusters, and appear to be of single cell origin, which makes them good targets for transformation (Taylor et al. 1996). Particle bombardment of embryogenic suspensions of ‘TMS 60444’ allowed regeneration of transgenic cassava plants via three different approaches. The first is based on antibiotic selection using paro-

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momycin (Schöpke et al. 1996; González et al. 1998) or hygromycin (Zhang and Puonti-Kaerlas, in press), the second on visual selection using firefly luciferase as a screenable marker gene (Raemakers et al. 1996) or on a combination of antibiotic selection and luciferase screening (Munyikwa et al. 1998a), and the third on positive selection (Zhang and Puonti-Kaerlas, in press). When paromomycin selection was used, the bombarded tissues were first grown for two weeks after transformation in liquid medium containing 12 mg/L picloram, after which paromomycin was added to the liquid culture at 15 mg/L. After 4–5 weeks of liquid selection the developing embryogenic units were transferred to solid culture medium under the same selective conditions and cultured for another 4 weeks. Regeneration of plants was only possible when no selection was applied. Therefore, after the transgenic units were multiplied as friable embryogenic callus, shoot regeneration was initiated by sequential transfer and culture on a series of media to induce the differentiation of globular and torpedo stage embryos (1.2 mg/L picloram), development of cotyledons (0.93 mg/L NAA), and maturation (0.5% activated charcoal, no growth regulators) of embryos. Before attempting to root the regenerants, a multiplication step to induce multiple shoot formation from the apical meristem of the germinating embryos was applied (1 mg/L BA). The regeneration capacity differed greatly between the selected lines, and in some cases no plants could be regenerated. Southern data were published to prove the presence of the uidA gene in one regenerated shoot. When hygromycin was used, selection was started 3 days after bombardment. The cultures were grown in a liquid medium with 12 mg/L picloram and 50 mg/L hygromycin for 2–4 weeks, after which they were transferred to a solid medium supplemented with 1 mg/L NAA and 25 mg/L hygromycin for 2–8 weeks to allow FEC formation and embryo emergence. The hygromycin resistant embryos developed into shoots upon transfer to an elongation medium containing 0.4 mg/L BA and no hygromycin. GUS assays, PCR, RT-PCR, as well as Southern and Northern analyses confirmed the transgenic nature and stable expression of the transferred genes into two regenerated plant lines. Paromomycin (Schöpke et al. 1997a; Gonzalez et al. 1998) and hygromycin based (Zhang et al., submitted) selection systems have also been adapted to Agrobacterium-mediated transformation of embryogenic suspension cultures. After a 2-day cocultivation period of the suspension cells with Agrobacterium strain ABI (Konz and Schell 1986) containing a plasmid carrying the nptII and intron-interrupted uidA genes, the cultures were grown for 8–10 days without selection, followed

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by culture in liquid SH medium containing paromomycin for 5–6 weeks and on solidified selective medium for 4 weeks. They were then transferred to regeneration medium without selection for embryo differentiation (4 weeks), cotyledon development (4 weeks), maturation (2 weeks), shoot development (4 weeks), and rooting (4–8 weeks) using the media described for bombarded suspensions. Southern blot analysis demonstrated stable integration of the uidA gene into the cassava genome in two plant lines. After 3–4 days cocultivation with Agrobacterium strain LBA4404 (Hoekema et al. 1984) containing a plasmid carrying introninterrupted hpt (Wang et al. 1997) and uidA genes, the tissue was cultured for 3 days without selection and then for 15 days in liquid medium containing 12 mg/L picloram and 50 mg/L hygromycin. After another 15 days of liquid culture using 25 mg/L hygromycin, the suspensions were transferred to solid medium with 1 mg/L NAA and 25 mg/L hygromycin. Embryos developed on this medium in 4–8 weeks, and shoots were regenerated from the cotyledonary stage embryos on a medium with 0.4 mg/L BA without hygromycin in 4 weeks. Molecular analyses confirmed the transgenic nature of 12 regenerated lines. When firefly luciferase was used to identify and select for transgenic tissues, the embryogenic suspensions were cultured for one day after bombardment on solid medium containing 6% sucrose, and then transferred either to liquid medium containing 6% sucrose or to solid medium on which the sucrose concentration was reduced from 6% to 2% in two 3-day subculture steps. Two weeks after bombardment the cultures were monitored for luciferase activity, and clusters of embryogenic units at and around the luciferase positive spots were isolated and cultured further. The luciferase screen and tissue selection was repeated at 2-week intervals, until 2 months after bombardment the friable embryogenic calli clusters containing at least 1% luciferase positive units were transferred to maturation medium containing 1 mg/L picloram for development of somatic embryos. The maturing luciferase positive embryos were further multiplied via secondary somatic embryogenesis on media containing either 10 mg/L NAA or 8 mg/L 2,4D, and in subsequent steps of cyclic somatic embryogenesis 10 mg/L NAA. After desiccation, the somatic embryos were induced to germinate on a medium containing 1 mg/L BA, and then multiplied as shoot cultures by nodal cuttings. The efficiency of the secondary embryo formation from selected embryos was 83%, but only 1–15% of the embryos could be germinated to transplantable shoots. Southern data confirming the transgenic nature of three plants were presented. A method combining antibiotic selection using phosphinotricin and luciferase showed

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that use of 20 mg/L phosphinotricin allowed the development of both transformed and non-transformed maturing embryos from bombarded friable embryogenic callus, but the luciferase screening could be used to exclude escapes, while inclusion of 20 mg/L phosphinotricin in the maturation medium could not completely block the maturation of nontransgenic embryos (Snepvangers et al. 1997). Later studies demonstrated that strict visual selection using luciferase was more efficient and led to the production of transgenic lines faster than antibiotic selection. Southern analysis confirmed the transgenic nature of 18 plant lines produced by combining antibiotic and visual selection (Munyikwa et al. 1998a). A positive selection system was recently developed for embryogenic suspensions (Zhang and Puonti-Kaerlas, in press; Zhang et al., submitted). After transformation, the cells were grown for 4 weeks in liquid culture using a modified SH medium containing 12 mg/L picloram, 4% mannose, and 1% sucrose, before transfer to solid medium containing 2% sucrose, 2% mannose, and 1 mg/L NAA for 2 weeks. After another 4 weeks of culture on the same medium without mannose, shoots were regenerated in 4 weeks from embryos cultured on a medium containing 2% sucrose and 0.1 mg/L BA. GUS assays and molecular analyses confirmed the transgenic nature of 14 regenerated plant lines. The use of the protocols described above depends on the availability of embryogenic suspensions, the establishment of which is still genotype-dependent and labor-intensive. The low regeneration rates and the regeneration of possibly abnormal plants reduce the transformation efficiencies obtained after transformation of embryogenic suspensions (Taylor et al. 1996; Raemakers et al. 1997b; Snepvangers et al. 1997; Schöpke et al. 1997b). As the use of paromomycin selection reduces the regenerative potential of the transgenic material, relatively complicated, timeconsuming and labor-intensive regeneration schemes must be followed. This has led to somaclonal variation and reduced growth rates in up to 50% of the regenerants, and to a lower survival rate (40% versus 90% in controls) of the plantlets upon transplanting to the greenhouse (Raemakers et al. 1997b; Munyikwa et al. 1998a). Luciferase, though a noninvasive detection method, requires access to costly equipment including a coupled device camera for detection and localization of the bioluminescence. Combining GUS assays with hygromycin or mannose selection, on the other hand, should allow rapid and easy selection of cultures for regenerating, resulting in 100% selection efficiency (Zhang and Puonti-Kaerlas, in press) and possibly reducing the length of time required for tissue culture before plant regeneration.

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D. Organogenesis The shoots regenerated via organogenesis develop from cells at or close to the cut edges of the cotyledon explants, which makes them good targets for Agrobacterium mediated gene transfer. In contrast to the protocols based on somatic embryo production, both callus and shoot development were inhibited by similar amounts of antibiotics, and geneticin, hygromycin, and phosphinotricin could all be used as selective agents (Li et al. 1996, 1998b; Puonti-Kaerlas et al. 1997a,b). In compatibility tests conducted with four different Agrobacterium strains carrying an intron-interrupted uidA gene, cassava cotyledon explants cocultivated with LBA4404(pTOK233) (Hoekema et al. 1984; Hiei et al. 1994) and LBA4404(pBin9GusInt) (Holtorf et al. 1995) showed the highest transient transformation frequencies. Pre-induction of the Agrobacteria with acetosyringone increased the transformation frequency considerably, and a two-hour pre-induction time was found to be optimal. Extending the cocultivation time to four days resulted in the highest transient transformation rates without excessive bacterial contamination. The developmental state of the explants was also found to be a critical factor in the transformation procedure. Cotyledons from newly germinated embryos were very sensitive to Agrobacteria and survived the cocultivation procedure poorly, which resulted in low transformation rates. Cotyledon explants from older embryos survived better, but explants from germinating embryos older than 20 days regenerated less efficiently. The highest regeneration and transformation frequencies could be obtained by using cotyledon explants from somatic embryos of ‘MCol22’ cultured for 15 days on maturation medium. Following cocultivation, callus and small resistant shoot primordia developed on selection medium containing 15 mg/L hygromycin or 20 mg/L geneticin from the cotyledon explants cocultured with LBA4404(pBin9GusInt) or LBA4404(pTOK233), respectively. In GUS assays three out of 27 regenerated geneticin resistant shoot primordia and six out of 30 hygromycin resistant shoots stained blue. After rooting, the putative transgenic shoots were transferred to soil in the greenhouse (Li et al. 1996). Cloned plant material was stained for GUS activity in order to assess the expression of the 35S promoter in different cassava tissues. In contrast to earlier reports based on transient assays after particle bombardment (Arias-Garzón and Sayre 1993), the 35S promoter was shown to be highly expressed in all cassava tissues, including all parts of the roots. The highest expression levels, as determined by the intensity of the blue color, were in the youngest tissues, including apical and axillary meristems and root tips (Puonti-Kaerlas et al. 1997a,b). The sta-

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ble integration of the transgenes into cassava nuclear DNA was demonstrated in five transgenic plants and Northern data to prove the transcriptional activity of the transgenes were presented. The selection system needs further improvement, as many escapes were found. Southern analysis of 18 lines selected on geneticin verified their transgenic nature (Li et al. 1998b; Puonti-Kaerlas, unpublished). Recently, the organogenesis protocol was adapted to use with the Particle Inflow Gun to allow the widest possible range of cultivars to be transformed (Legris et al. 1998a; Zhang and Puonti-Kaerlas, in press; Zhang et al., in press). The bombardment parameters were partially optimized and the selection procedure was modified. Using 7.5 mg/L hygromycin for the first 10 days after bombardment, and then 15 mg/L hygromycin for 2 weeks, followed by culture of the developing shoot primordia on 10 mg/L hygromycin, increased the stringency of the selection. Molecular analyses confirmed the transgenic nature of nine regenerated plant lines. The positive selection system using mannose was shown to be compatible also with the organogenesis-based regeneration mode of cassava (Zhang and Puonti-Kaerlas, in press). Bombarded cotyledon pieces were first cultured for 3–4 weeks on a medium containing 1.0 mg/L BA, 0.5 mg/L IBA, 1% mannose, and 0.5% sucrose, then the developing shoot primordia were transferred for 3 weeks to elongation medium containing 0.4 mg/L BA, 2% sucrose, and 1% mannose. The regenerated shoots were maintained on a medium supplemented with 1% mannose. Molecular analyses confirmed the transgenic nature of two plant lines. In order to improve the selection efficiency of both antibiotic and positive selection, rooting assays were developed. The rooting of axillary shoots of non-transgenic cassava was inhibited by 5.5 mg/L hygromycin and 8 mg/L geneticin (Zhang et al., in press) and 1% mannose (Zhang and Puonti-Kaerlas, in press), while the transgenic plants could root normally. The rooting tests allow an efficient secondary screening for elimination of escapes from primary selection. IX. APPLICATIONS AND POTENTIAL OF MOLECULAR BIOLOGY Biotechnological tools complement those of traditional breeding, and besides facilitating traditional breeding, production, and micropropagation of disease-free material, preservation and characterization of germplasm, they will allow development of new, improved cultivars by transgene technology. Understanding plant-pathogen interactions and pest and pathogen populations, as well as diagnostic methods for diseases are integral to

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developing sustainable resistance strategies. In this section the current knowledge on some of the main problems of cassava is described, with some insights for future possibilities offered by biotechnology. A. Disease Control There are over 30 main pathogens that attack cassava, including cassava bacterial blight, anthracnose caused by the fungus Colletotrichum gloeosporoides, and superelongation disease caused by Elsinoe brasiliensis. In addition, a number of viruses cause severe problems. Cassava bacterial blight caused by Xanthomonas campestris pv manihotis (Xanthomonas axonopodis pv manihotis) is one of the main biotic constraints in cassava cultivation worldwide, and heavy infestations of bacterial blight can destroy the whole crop (Lozano 1979; Mahungu et al. 1994; for reviews, see Boher and Verdier 1994; Verdier et al. 1997). Build up of the pathogen in infested planting material also causes gradual yield declines, eventually leading up to 80% in yield losses. The main local dispersal mechanism of the pathogen is rain splashes, and the use of infected planting material and tools can spread the disease over considerable distances. The disease is transmitted both in vegetative and sexual seed, and thus represents a threat to the international exchange of germplasm (Elango and Lozano 1980). Some resistant cultivars were produced by traditional breeding, but the resistance obtained appeared to be effective only under low infestation pressure (Cooper et al. 1994). Genetic studies showed that the resistance to bacterial blight appears to be polygenic and additively inherited (Sanchez et al. 1998). Molecular markers for resistance were identified using RFLP and RAPD analysis (Jorge et al. 1998; Sanchez et al. 1998), and the first results indicated that the resistance is widely distributed in cassava germplasm. RFLP mapping also identified a region in the cassava genome accounting for 80% of the variance for resistance to one strain of cassava bacterial blight, and 50% of this variance was shown to be linked to two markers separated by 4 cM (Jorge et al. 1998; Thro et al., in press). Valuable information on plant-pathogen interactions is accumulating as well (Verdier et al. 1997), as the pathogen is being characterized and its diversity and evolution elucidated by use of AFLPs, RFLPs, and ribotyping (Verdier et al. 1994; Restrepo et al. 1997, 1998, 1999). The first pathogenesis-related genes of Xanthomonas have been characterized (Verdier et al. 1997). Detection systems for bacterial blight now include polyclonal and monoclonal antibodies (Boher 1996; Boher et al. 1997; Chalida et al. 1988), and PCR techniques based on primers for the patho-

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genecity gene. The primers were able to detect all tested 107 pathogenic strains and to differentiate between the pathogenic strains and 27 other Xanthomonads tested (Verdier et al. 1998). RAPDs, RFLPs, and ELISA tests are currently also used to characterize Phythophthora strains (Alvarez et al. 1998). The infection of cassava leaves by Xanthomonas induces enzymes of the phenylpropanoid pathway, and the expression of phenylalanine ammonia lyase (PAL) increases in stems and leaves after injury. A PCR fragment of the gene that was cloned is currently being characterized. First results indicated that the gene may be a member of a small gene family, that is mainly expressed in leaves (Pereira et al. 1997, Pereira and Erickson 1998). Understanding the plant defense reactions will allow the development of sustainable resistance strategies, and the use of molecular markers will facilitate breeding of new, improved cassava cultivars with multiple resistance to bacterial blight in the future. As an alternative strategy, the rice gene Xa21, which was shown to confer resistance to bacteria in transgenic plants (Wang et al. 1996), was transformed to cassava to assess its usefulness in engineering bacterial blight resistance in cassava (Taylor et al. 1998b). The use of agrochemicals to protect plants against most fungal and bacterial diseases is in principle possible, even if not economically viable or environmentally sustainable, but in the case of viral diseases this option does not exist, as there are no “viricides.” The only means to protect the plants from the viruses are to try to prevent their spread by controlling the vectors, and hence resistance strategies are of highest priority. The potex virus, cassava common mosaic virus (CsCMV), causing up to 20% yield losses (CIAT 1991), is at present the most destructive viral disease of cassava in Latin America. No vector is known for this virus, and it is spread mainly by poor agricultural practices. As a first step toward developing resistant cultivars, the sequence of CsCMV was determined (Calvert et al. 1996), and a modular promoter isolated from this virus was shown to be active in tobacco, cassava, and rice (Verdaguer et al. 1996, 1998). In contrast to the CsCMV, whose genomic organization is similar to that of other potexviruses (Calvert et al. 1996), the cassava vein mosaic virus (CVMV), which is widely spread throughout the northeastern region of Brazil, was shown to be a distinct pararetrovirus (Calvert et al. 1995). Sequence analysis of CVMV showed that its genomic organization was different from that of either the caulimoviruses or badnaviruses. The coat protein gene of CsCMV was transferred to cassava as a potential source of resistance (Schöpke et al. 1998).

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B. Virus Control African cassava mosaic disease (ACMD) causes losses of up to 40%–50% of total yields throughout the African continent, and can locally result in complete crop failure (Lozano and Booth 1974; Thresh et al. 1994a,b; Otim-Nape 1995). It has been ranked as the most important vector-borne disease of all African food crops (Geddes 1990). ACMD is caused by the African cassava mosaic virus (ACMV), which is transmitted by white flies (Bemisia tabaci), and also via infected tools and planting material, but not via true seed (Swanson and Harrison 1994). There have been several epidemics of ACMV in the past, and the occurrence of the disease throughout the continent is high. Caused by a new strain of ACMV, the most recent pandemic, which has resulted in famine-related deaths and huge losses in cassava production, is spreading from north to south through Uganda and western Kenya at the rate of 15–20 km/year (OtimNape 1995; Otim-Nape et al. 1994a,b, 1997a,b; Gibson et al. 1996). Immune cultivars have not been produced by traditional breeding (Hong et al. 1996), but in Uganda the use of new cultivars and disease-free planting material have produced a positive effect (Bock 1994; OtimNape et al. 1997a,b). It has been estimated that the use of disease-free planting material could increase yields two- to threefold, without any external inputs (Lozano 1979; Mabanza et al. 1995). Due to the high infection pressure in the field, however, the benefits of cleaning planting material may be short-lived. In the worst cases, most of the plants would be re-infected within three to six months from planting, unless resistant varieties can be used (Otim-Nape 1993; Thresh et al. 1994a,b,c). The resistance to the vector and the virus seems to be determined by two different genetic mechanisms (Fargette et al. 1996), and yield losses in the currently resistant lines are in the range of 10%–30% (Fargette et al. 1996). RAPD markers were used to detect resistance genes in cassava, and new sources were recently identified in African clones (Mignouna and Dixon 1997). On the other hand, as the resistance to ACMV appears to be recessive and polygenic (Hahn et al. 1980), breeding of new cultivars by using traditional methods can lead to the loss of local land races, and genetic transformation technology may be necessary to transfer the desired traits. ACMV is a typical subgroup III geminivirus with a genome that consists of two covalently closed circular single-stranded DNA (ssDNA) molecules known as DNA A and DNA B (Stanley 1983). The replication mechanism of ACMV is well known (Saunders et al. 1991, 1992; Stanley 1995) and DNA A has been shown to be responsible for the replication of both DNA components (Etessami et al. 1991; Townsend et al.

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1986), virus proliferation rates (Haley et al. 1992; Hong and Stanley 1995), and together with DNA B for vector transmission and virus spread (von Arnim et al. 1993; Haley et al. 1995; Liu et al. 1997; Briddon et al. 1998). Both genomic components are also necessary for the systemic infection of susceptible host plants (Stanley 1983). DNA B is involved in cell to cell and long distance virus spread and production of disease symptoms (von Arnim et al. 1993; Haley et al. 1995). The promoter activity of ACMV is regulated by its own gene products (Haley et al. 1992; Hong and Stanley 1995). Understanding the regulation of ACMV replication and gene expression will allow the development of new resistance strategies using transgene technology. Recent studies on the simultaneous regulation of both sides of the bi-directional promoter of ACMV using dual luciferase assays have, however, shown differences between cassava and model plants, indicating that more work is still required in this field (Frey 2000). The cassava mosaic disease group comprises three serologically distinct geminiviruses, which are now regarded as separate viruses: the African cassava mosaic (ACMV), East African cassava mosaic (EACMV), and Indian cassava mosaic (ICMV) virus (Hong et al. 1993; Swanson and Harrison 1994; Harrison et al. 1997a). The occurrence of a new virus was shown to be involved in the recent epidemic in East Africa. This epidemic is characterized by extremely severe symptoms in the infected plants (Gibson et al. 1996). The novel type of ACMV, the Uganda variant (UgV), is a recombinant between the ACMV and EACMV (Deng et al. 1997; Zhou et al. 1997), and it has been detected in severely affected plants from the epidemic area (Zhou et al. 1997). Despite the hybrid nature of their coat protein, UgV isolates are indistinguishable from ACMV in tests using monoclonal antibodies, including seven that react with ACMV but not EACMV. Most plants containing ACMV alone express mild or moderate mosaic symptoms, whereas very severe mosaic disease develops in most plants containing UgV and ACMV and a few of those containing only UgV (Harrison et al. 1997b). Recently, a new ACMD type has been described in South Africa (Berrie et al. 1998a). As the Ugandan variant has been shown to have arisen by recombination, information of the recombination ability and frequency are crucial to understanding the evolution of this group of viruses (Roberts and Stanley 1994; Frischmuth and Stanley 1998; Zhou et al. 1998). ACMV diagnostic tools include sap inoculation and grafting tests on cassava and indicator species (Adejare and Coutts 1981; Kaiser and Teemba 1979), ELISA tests for ACMV (Kounounguissa et al. 1989), ICMV (Malathi et al. 1989; Swanson and Harrison 1994), double and triple antibody sandwich ELISAs for ACVM, EACMV, and ICMV (Deng et al. 1994;

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Swanson and Harrison 1994; Harrison et al. 1997a), and PCR detection systems (Harrison et al. 1997a). The ELISA and PCR methods are presently being tested in a number of laboratories in different African countries, and the first results indicate a successful transfer of this technology to Africa (Ogbe et al. 1997; Mignouna and Dixon 1997; Woodward et al. 1998). At present, ACMV is a severe constraint to cassava cultivation. There are few means to protect the plants in the field from being infected with the virus, except by trying to control the spread of the vector, whitefly, by unspecific agrochemicals, which, for the African farmers, are economically nonviable in addition to being environmentally unsound (Fishpool and Burbank 1994). So far ACMV has not reached Latin America, but a new biotype of the vector that feeds on cassava has been recently found in the neotropics (CIAT 1990; Franca et al. 1996). This now makes ACMV a serious threat to cassava production in Latin America, as the germplasm in America is highly susceptible to the virus. Virus resistance has been introduced to over 20 plant species by genetic engineering (for review, see Kahl and Winter 1995), and there are indications from heterologous species that resistance against ACMV could also be transferred to cassava. Expressing the Defective Interfering DNA (Stanley et al. 1990; Frischmuth and Stanley 1991), the movement protein of the tomato golden mosaic virus (von Arnim and Stanley 1992), the AC1 gene of ACMV (Hong and Stanley 1996), dianthin (Hong et al. 1996, 1997), and a mutated replicase (Sangare et al. 1999) in transgenic model plants has been shown to increase their resistance to the virus in varying degrees. In addition to these, novel strategies are being designed for engineering sustainable resistance to ACMV (Berrie et al. 1998b; Schärer-Hernandez et al. 1998). Preliminary results using a virusinduced cell death system show that it is possible to reduce ACMV replication by 37–99% in transgenic model plants (Frey 2000). C. Pest Resistance Due to its long growth period, 8–24 months, cassava is susceptible to prolonged and repeated attacks from several insect pests (Bellotti 1979; Bellotti et al. 1994). There are 17 major cassava pests (Bellotti and Schoonhoven 1978), and a recent review describes the state of art in pest management of cassava (Bellotti et al. 1999).The repeated or continual use of pesticides in prevention of pest attacks is neither economically nor environmentally sustainable for cassava farmers (Bellotti et al. 1994). The main arthropod pests throughout the cassava belt in Africa are cassava mealybug (Phenacoccus manihoti) and green mite (Monony-

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chellus tanajoa), both of which originated in South America. The cassava mealybug, which devastated cassava in Africa, is now increasingly being controlled by the use of a parasitoid wasp (Neuenschwander 1994). Studies on the signaling between the infected plants and the parasitoids showed clear differences between different parasitoid species in their specificity to mealybug-infested cassava (Bertschy et al. 1997). Understanding the behavior of the beneficials is vital for the development of new biological control methods. In contrast to the mealybug, cassava green mite, which causes losses up to 80% (Yaninek 1994), remains a severe problem (Yaninek et al. 1998; Bellotti et al. 1999). Locally, a number of other pests cause considerable damage as well. White flies can severely damage the plants, in addition to spreading ACMV. In Africa, variegated grasshopper (Zonocerus variegatus L.) was identified as a major pest in a number of regions, and in Nigeria and Uganda over 50% yield losses and the eradication of complete stands was reported after heavy infestations (Bellotti 1979; Bellotti et al. 1994; Le Rü and Calatayud 1994; Modder 1994). Nematodes, mainly Meloidogyne incognita, M. javanica, and Pratylenchus bracheyurus are emerging as a severe problem, especially in regions of intensive cassava cultivation, where crop rotation and fallow periods are either shortened or abandoned altogether and cassava is increasingly grown in monoculture. The yield losses caused by heavy infestations of nematodes can reach up to 98% (Coyne 1994). An in vitro culture method was developed to allow rapid screening for nematode resistance in cassava cultivars and used to show differences in susceptibility in 10 cassava lines. A modified nematode culture medium could be used to grow cassava root cultures, which can be infected with sterilized egg masses, and the rate of gall formation and the number of females reaching maturity inside the galls could be easily monitored (van Vuuren and Woodward 1998). In Latin America, lepidopteran insects are currently the main cassava pests. These include stem borer (Chilomina clarkei) and hornworm (Erinnyis ello). Hornworm causes varying yield losses along the northern coast of South America and in the Caribbean. The larvae can cause complete defoliation of the plants and also attack tender stem parts and lateral buds, killing young plants. Defoliation and stem damage reduce the starch quality of the roots and also cause yield losses between 10% and 64%, depending on plant age and the number and intensity of the attacks (Bellotti and Arias 1979; Arias and Bellotti 1984). Stem borer not only causes considerable yield loss, but as the larvae live and feed inside cassava stems, protected from external applications of insecticides, a severe attack can lead to reduced quality or even complete loss of planting material. During the past years stem borer has caused considerable

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damage on over 7,000 ha of cassava plantings in Colombia (Bellotti et al. 1999). The soil bacterium Bacillus thuringiensis (Bt) carries a set of cry genes encoding insect specific δ-endotoxins (Bt toxins) that are efficient in combating a variety of insects. Spraying Bacillus thuringiensis was shown to be efficient in biological control of cassava hornworm (Bellotti and Arias 1979), and the spraying of Bt toxins has been recommended as a biological control method for cassava hornworm (Anon. 1978). Expression of the cry genes in transgenic cassava would complement the available methods for pest control in an environmentally and economically sustainable way. Several groups are currently working on this area, and the regeneration of the first putative transgenic plants has been reported (Legris at al. 1998b). D. Quality and Yield 1. Cyanogenesis. Cassava is cyanogenic, i.e. hydrogen cyanide (HCN) is produced in all parts of the plant when the tissues are damaged. HCN is the breakdown product of cyanogenic glucosides, linamarin, and lotaustralin, which are derived from valine and isoleusine, respectively, via 3 hydroxylation steps followed by glucosylation (Koch et al. 1992, 1994). When cassava tissues are disrupted, linamarin and lotaustralin are brought into contact with a β-glucosidase, linamarase, which catalyses their hydrolysis to glucose and cyanohydrins. The breakdown products of cyanohydrins are a ketone and HCN. This reaction is catalyzed by a α-hydroxynitrilase, but can also proceed spontaneously if the pH is higher than 4.0 (for review, see Hughes et al. 1995). On the average, unprocessed fresh cassava roots contain about 150 mg/kg (15–440 mg/kg) cyanide equivalents, while high cyanide cultivars can contain up to 1500 mg/kg (O’Brien et al. 1992). The safety limit for cyanogens for cassava products has been set at 10 mg/kg dry weight (Codex Alimentarius Commission 1989), and a cyanide dose of 50–100 mg can be lethal within minutes (Rosling et al. 1993). To prevent cyanide poisoning, linamarin and lotaustralin have to be removed by laborintensive processing, and shortcuts in processing can have fatal consequences (Akintonwa and Tunwashe 1992; Akintonwa et al. 1994; Mlingi et al. 1992). All known cassava cultivars contain cyanogenic glucosides, and despite considerable efforts no acyanogenic cultivar was found or produced (Jennings 1976; Bokanga 1994a; Dixon et al. 1994). High cyanide cultivars are favored in many areas, as they are considered higher yielding, tolerant to environmental stress, and also possibly safe from theft by mammals (Rosling et al. 1993; Kapinga et al. 1997). The

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occurrence of severe neurological disorders is closely linked to long-time exposure to cyanide (Osuntokun and Monekosso 1969; Tylleskär 1994; Tylleskär et al. 1991, 1992, 1995). Recent studies also seem to indicate a neurotoxic effect of linamarin itself (Banea-Mayambu et al. 1997). In addition to the health risks of cassava-based food, the waste waters from cassava processing plants often contain toxic amounts of cyanide, and consequently can be serious pollutants if not managed properly (Manilal et al. 1983). During cassava starch production 4000–6000 liters of waste water are produced per ton of starch, with the cyanide content being up to 66 mg/L (Balagopalan et al. 1995; Balagopalan and Rajalakshmy 1998). The ground water in the vicinity of starch factories can exceed the threshold values set for safe drinking water by as much as 32 times (Balagopalan and Rajalakshmy 1998). Balagopalan and Sundar (1997) discuss different possibilities of waste water treatment for reduction of toxic wastes from cassava starch factories. There are limited data on the function of the cyanogens in cassava. Defense mechanisms against pests have been suggested, but the data are controversial (Byrne et al. 1983; van Schoonhoven 1974; Jones 1998), and little evidence, apart from that on the variegated grasshopper (Zonocerus variegatus) and burrowing bug (Cryptomenus bergi) (Bellotti and Arias 1993; Bellotti and Riis 1994) support this hypothesis. In fact, many cassava pests seem to have adapted to the high cyanogen levels (Calatayud et al. 1997). Cyanogenic glucosides may have a role as storage forms of nitrogen and energy, as turnover of cyanogenic glucosides via the β-cyanoalanine pathway in roots and leaves has been described (Nambisan 1993; Nambisan and Sundaresan 1994). The main catabolic pathway appears to be different in leaves and roots, leading to ammonia and asparagine, respectively (Elias et al. 1997a,b). The cyanogenic potential of cassava is highly variable between individual plants, and even between individual roots of one plant, and it is also influenced by the environment and plant age (Bokanga 1994a; Bokanga et al. 1994; McMahon et al. 1995; Sriroth et al. 1998). The regulation of cyanogen formation is poorly understood, but correlations were shown to exist between increasingly marginal conditions and stress situations and cyanogen content (Sriroth et al. 1998). On the other hand, no correlation was found between the biosynthetic capacity of cassava and cyanogenic glucoside levels in the different tissues or between those of the leaves and roots (Bokanga and Møller 1993; Chukwumah et al. 1997; McMahon and Sayre 1997). An inhibitor was postulated to be involved in the regulation of the synthesis of cyanogenic glucosides (Bokanga and Møller 1993), and the rate of cyanogen metabolism was shown to affect the cyanogenic levels as well (Chukwumah et al. 1997).

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Early studies suggested that linamarin was produced solely in the leaves and transported to the roots (Selmar 1994), but part of the linamarin synthesis is now known to take place in roots (White et al. 1994; Du et al. 1995; McMahon and Sayre 1997). Of the genes in the biosynthesis pathway, the key enzymes, cytochrome P450 oxidases (Koch et al. 1994), were recently isolated and characterized (B. L. Møller, pers. comm.). The genes involved in the breakdown of cyanogenic glucosides, linamarase (Hughes et al. 1992), and α-hydroxynitrilase (Hughes et al. 1994), as well as the gene encoding the last enzyme in the linamarin synthesis pathway UDPG-glugosyltransferase (Hughes et al. 1993; Hughes and Hughes 1994), were cloned as cDNAs. The α-hydroxynitrilase genes were expressed in E. coli for structural and biochemical studies and for future modification for processing purposes (Hughes et al. 1997, 1998; Keresztessy et al. 1994). Genomic clones of linamarase and α-hydroxynitrilase were also isolated and characterized (Hughes et al. 1997, 1998; Liddle et al. 1997). Both genes appear to belong to multigene families (Hughes et al. 1995, 1997, 1998) and to contain 12 and 2 introns, respectively. The promoter region of the linamarase gene that was isolated is currently being characterized (Liddle et al. 1997) and the use of GUS fusion constructs in transient assays revealed high expression levels in leaves and roots of in vitro seedlings after particle bombardment. Expression studies on the α-hydroxynitrilase showed that the expression of the different members of the gene family were developmentally and tissuespecifically regulated and that variations in sequences and gene regulation existed between different cassava cultivars. The absence of α-hydroxynitrilase in roots and stems was shown to be associated with very low steady-state transcript levels (White et al. 1998). The cyanogenic nature of cassava makes it necessary to process the roots or leaves before consumption, which can be time-consuming and laborious, and if not conducted properly also presents a health hazard to consumers. By manipulating the key enzymes in linamarin synthesis, the cytochrome P-450 oxidases, e.g. by downregulation via antisense technology, the levels of cyanogenic glucosides in cassava roots could be reduced. As the fear about possible poisoning is an obstacle to selling fresh cassava roots directly from the farms in some parts of Africa, production of acyanogenic cassava would also contribute to the household income in these regions. Acyanogenic cassava could considerably reduce the emission of pollutants from starch factories. Alternatively, increased expression of linamarase and α-hydroxynitrilase, the enzymes breaking down linamarin, would offer a way to enhance the rate of HCN release during cassava processing. The residual cyanohydrins in processed cassava are the main source of dietary cyanide (Tylleskär et

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al. 1992), and thus maintaining a high activity of the α-hydroxynitrilase during cassava processing would be of high interest. To enhance the breakdown of cyanohydrins during cassava root processing, production of transgenic cassava overexpressing the α-hydroxynitrilase gene is in progress (Arias-Garzon and Sayre 1998). 2. Stay-green Index. One of the problems of cassava consumers is the low protein content of the roots, which can lead to qualitative protein malnutrition in areas where the diet is based mainly on cassava. Also, protein deficiency has been shown to aggravate symptoms related to cassava toxicity (Rosling 1988). Synthetic storage proteins designed to improve the protein quality and quantity have been introduced to potato (Yang et al. 1989) and to sweetpotato (Prakash and Egnin 1997). Introduction of such a storage protein gene into cassava roots to improve the nutrient balance of cassava and provide a cheap protein source is currently pursued. On the other hand, cassava leaves contain valuable highquality protein and provide a reliable, low cost source of vitamins, minerals, and proteins (Balagopalan et al. 1988; Bokanga 1994b; Dahniya 1994). Excessive leaf harvesting, however, reduces storage root production, and therefore leaves can only be harvested every two months in order to minimize losses in root yields (Bokanga 1994b). Leaf longevity has been shown to be one of the main traits associated with high yields, together with a leaf area index of 3–3.5 (Cock 1979; Hunt et al. 1977). On the other hand, high drought tolerance and productivity were associated with leaf retention during drought (El Sharkawy et al. 1992; Osiru et al. 1994), and the leaf retention capacity during periodic drought was also positively correlated with root quality. Prolonging the life of individual leaves could help to produce cultivars with improved yield and root quality and to permit more frequent harvesting of leaves while maintaining a satisfactory photosynthetic area to ensure storage root production. As the market value of leaves in the areas where they are consumed is often higher than that of the roots (Lutaladio and Ezumah 1981), this could also contribute to household economies. Prolongation of photosynthetically active leaf life has already been achieved in transgenic tobacco carrying the ipt gene encoding cytokinin production under the control of a senescence-regulated promoter from Arabidopsis thaliana (Gan and Amasino 1995). Transgenic cassava plants containing the same construct have been produced and are currently being analyzed (Li et al. 1998b). 3. Starch. On a dry weight basis, 74% to 85% of the cassava root is starch (Rickard et al. 1991; Blanshard 1995), with an amylose content of 13%

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to 28% (Zakhia et al. 1995). Starch content and quality of the roots is dependent on the age of the plants and of the growing conditions (Asaoka et al. 1992; Sriroth et al. 1998). Depending on the season, during an 18-month growing period interrupted by a dry season, in addition to the variation in the contents of cyanogenic glucosides, the starch content in fresh roots was shown to vary between 20.4% and 30.3% FW, and that of the amylose content between 17.3% and 23.9%. The environment and plant age were also shown to affect the quality of flour used for bread (DeFloor et al. 1994). The development of cassava cultivars with different starch composition would increase the value of cassava as an industrial crop. Specialized uses could be found for amylose-free starch, for example, and for cassava roots with increased levels of sugars. For this purpose, the branching enzyme (Salehuzzaman et al. 1992) and two isoforms of the granule-bound starch synthase genes (Salehuzzaman et al. 1993a,b; Munyikwa et al. 1997), which control the amylose/amylopectin ratio in starch, were cloned as cDNAs from cassava root libraries. Expression studies showed that the branching enzyme and the granule-bound starch synthase I genes were highly expressed in roots, but the branching enzyme signal was also highly abundant in the stems, while that of the granule-bound starch synthase II was abundant only in leaves (Salehuzzaman et al. 1994; Munyikwa et al. 1997). The expression pattern of the genes in plants grown in vitro differed from that of the greenhousegrown plants, due to the induction of these genes by sucrose present in the culture medium, and no correlation was found between the starch content of the different tissues and the mRNA levels of branching enzyme and granule-bound starch synthase (Salehuzzaman et al. 1994). Both the ADP glucose pyrophosphorylase B gene, encoding the key enzyme in starch synthesis, and the granule-bound starch synthases were shown to be low copy number genes, while the branching enzyme appeared to be a single copy gene (Salehuzzaman et al. 1992, 1993a,b; Munyikwa et al. 1995). The expression of ADP glucose pyrophosphorylase B gene in antisense in transgenic cassava led to plants with little or no transcript and reduced starch levels in the thickened stems (Munyikwa et al. 1998a,b). 4. Micronutrients. Vitamin A is vital to normal development in humans, and the consequences of vitamin A deficiency range from night blindness to total blindness to reduced resistance to various terminal diseases (Sommer 1988; West Jr. et al. 1989). According to UNICEF, approximately 124 million children in the world suffer from vitamin A deficiency (Sommer 1988; Humphrey et al. 1992). Expressing the cor-

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responding genes from plants producing β-carotene, the precursor for the synthesis of vitamin A, in cassava roots would provide a step toward alleviating this problem. Cassava roots have a basic capacity for βcarotene synthesis, as shown by the identification of cassava cultivars with yellow roots containing carotenoids (Moorthy et al. 1990; Adewusi and Bradbury 1993a,b). The carotenoid content of cassava roots has been shown to be as high as 2 mg/10 g fresh wt (Iglesias et al. 1997), but a large part of this may exist in non-provitamin A-like forms, e.g. as luteolin (Adewusi and Bradbury 1993a,b). Breeding for high carotenoid content was reported to have produced cassava lines with up to sevenfold higher carotene contents in the roots (Nair and Pillai 1996). Thus the expression of the first enzyme in the pathway, phytoene synthase, might be sufficient to produce cassava roots with high β-carotene content in lines favored by farmers, without changing their other characteristics. Should this prove insufficient, it is now also possible to transfer the whole pathway required for β-carotene production, as recently demonstrated with transgenic rice (Burkhardt et al. 1997; Ye et al. 2000). Likewise, iron deficiency anemia, one of the most serious deficiencies in the developing countries and affecting over one billion people, could be combated by increasing iron content and improving its availability in cassava roots using transgene technology (Lucca 1999; Anon. 1999; Gura 1999). 5. Physiological Post-harvest Deterioration. The poor storability of fresh cassava roots is one of the main constraints for urban marketing and industrial use of cassava, and also one of the main reasons for cassava processing. Cassava roots can remain on the growing plant for years, but once harvested they must be processed or packaged quickly to prevent their deterioration, which in most cultivars takes place within 2–7 days of harvest (for reviews, see Wenham 1995; Beeching et al. 1998). In most cultivars the physiological deterioration of the roots commences within 24 h, and is first characterized by vascular discoloration (streaking), followed by general discoloration of the storage parenchyma and secondary deterioration caused by microbial pathogens, which renders the roots unacceptable for human, animal, or industrial use. For subsistence farmers, perishability may not be a problem, but in most cases it leads to unstable prices, increased cost, and crop losses, especially in areas already facing food shortages (Westby et al. 1997). Consequently, fresh cassava, the least expensive food in rural areas, can become costly by the time it reaches the urban areas if the roots are not properly handled. Maintaining high humidity, e.g. by packing the roots in plastic bags, sealing them with wax, or burying them in soil, as well as the use of

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refrigeration, help to prolong the self-life of harvested roots by slowing down PPD. Although still poorly understood, many of the metabolic changes during the initial physiological deterioration resemble those observed during normal plant wound response reaction, and it has been speculated that the cause of the physiological deterioration could be a sustained wound reaction spreading systemically from the wound site into the whole root (Beeching et al. 1994, 1995). If this is the case, the regulation of the wound reaction and wound healing in cassava roots, which have no function in plant propagation and are physiologically inactive in comparison to other tuber and root crops such as yams, potato, or sweet potato, may be deficient. Increased levels of, among others, flavonols and other secondary metabolites are produced during the primary deterioration, and this is accompanied by increased activity of peroxidases, activation of the enzymes of the phenylpropanoid pathway, and also by initiation of de novo protein synthesis including phenyalanine ammonia lyase (PAL), leading to accumulation of phenolics (Tanaka et al. 1984; Rickard 1985; Wheatley and Schwabe 1985; Beeching et al. 1994, 1995). A cDNA library was constructed from roots stored for 48 h after harvesting (Han et al. 1998; Beeching et al. 1997) and cDNA and genomic clones of PAL, the key enzyme in phenylpropanoid pathway (Han et al. 1998; Beeching et al. 1997; Li et al. 1998c), were isolated and sequenced, in addition to other genes involved in plant defense and wound healing (Han et al. 1998). Although regulation of PAL activity during the deterioration process was demonstrated in cassava root slices, the role of PAL is not clear, as inhibitors of the enzyme appeared to have no effect on the deterioration pattern (Pereira and Erickson 1998). Recently cDNA and genomic clones of ACC oxidase, a key enzyme in the ethylene pathway (Li et al. 1998d), and catalase (Reilly et al. 1998) were isolated, and further studies on these will bring light to bear on the role of these enzymes and of oxidative stress in cassava post-harvest deterioration. By use of differential cDNA display 90 transcription-derived DNA fragments were isolated to study the regulation of gene expression during post-harvest deterioration (Huang et al. 1998).

X. FUTURE PROSPECTS The development of new techniques allowing more efficient use and improvement of cassava has proceeded rapidly in the past few years. New processing technologies provide possibilities for more diversifica-

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tion in the cassava industry and for more efficient treatment of effluents. Genetic characterization and map construction of cassava are progressing, and will create the basis for marker-assisted breeding, as well as for map-based gene isolation. Robust micropropagation methods will allow rapid multiplication and dissemination of pathogen-free material to the farmers. The gene banks will guarantee the maintenance and availability of the genetic diversity of cassava and its wild relatives. Isolation of promoters and genes as cDNAs and genomic clones from cassava will contribute to the increased knowledge of metabolic pathways, their regulation, and eventually, their genetic engineering, allowing the production of new cassava cultivars resistant to pests and diseases with improved nutritional quality or altered starch composition. As soon as more information is available about physiological post-harvest deterioration of cassava roots, means to control this problem may also be developed, possibly by manipulating the regulation of the key enzymes involved in the wound response reaction in roots. Future possibilities of adding value to cassava as an income-generating crop include the production of biodegradable plastics. As a first step toward production of biodegradable plastics in plants, accumulation of polyhydroxyalkanoates has been demonstrated in transgenic Arabidopsis plants expressing bacterial PHA genes (Poirier et al. 1995), and an ex-ante study indicated that production of biodegradable plastic in cassava may well be a viable option (Stoeckli 1998). Other possible strategies for valueadded cassava could include the improvement of iodine content and availability, production of cyclodextrins, improved baking quality of cassava flour, nematode resistance, and herbicide resistance. It is now possible to regenerate transgenic cassava plants, which is the prerequisite for genetic improvement of cassava, but so far there is little information on the transferability of the current protocols to other cultivars beyond the model cultivars, or to other laboratories. The genetic improvement of cassava via biotechnology is constrained by the lack of routine, inexpensive, efficient, and genotype-independent transformation methods. Despite the recent breakthroughs, the absence of efficient and reliable technology will be one of the greatest bottlenecks in applying genetic engineering to cassava improvement. Different transformation systems may be needed for different cultivars, and thus both direct gene transfer methods as well as those based on Agrobacterium should be developed further. The high proliferation rate of embryogenic suspensions will make the multiplication of transgenic tissues efficient. On the other hand, organogenesis may be a less genotype-dependent regeneration mode for cassava than germination of somatic embryos, and it may also allow more flexibility in the choice of selectable marker genes.

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The main limitation to the rapid development of new techniques for cassava remains the lack of funding for research. It is to be hoped that the progress achieved with limited resources will help to increase the interest in this important crop. Cassava biotechnology has the potential to alleviate poverty, increase food security, and promote the efficient use and conservation of genetic resources, provided that its results are available to cassava growing countries. Access to this technology should be equitable, and it should not create new dependencies, nor lead to raw material substitution in industrial countries. As cassava remains irreplaceable in marginal environments, the people living in these areas should be the first to benefit from the new technology.

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4 Postharvest Physiology and Quality of Coated Fruits and Vegetables Cassandro Amarante Centre of Agricultural, Horticultural and Veterinary Sciences, Santa Catarina State University, Caixa Postal 281, CEP 88520-000, Lages, SC, Brazil Nigel H. Banks Centre for Postharvest and Refrigeration Research, Institute for Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand.

I. INTRODUCTION II. PERMEABILITY OF COATING FILMS AND PERMEANCE OF COATED COMMODITIES III. PHYSICO-CHEMICAL CHARACTERISTICS, AND BARRIER PROPERTIES TO WATER VAPOR AND GASES, OF EDIBLE COATINGS A. Lipids and Resins B. Polysaccharides C. Proteins D. Composite and Bilayer Coatings IV. FACTORS AFFECTING WATER LOSS, GAS EXCHANGE, AND MODIFICATION OF INTERNAL ATMOSPHERE OF COATED COMMODITIES A. Coating Formulation B. Coating Deposit C. Mode of Application D. Relative Humidity

This review was part of a research project on “Gas exchange, ripening behaviour and postharvest quality of coated pears,” with financial assistance from the Brazilian Scientific and Technological Council (CNPq, Brasília, Brazil) and the New Zealand Apple and Pear Marketing Board.

Horticultural Reviews, Volume 26, Edited by Jules Janick ISBN 0-471-38789-4 © 2001 John Wiley & Sons, Inc. 161

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E. Temperature 1. Effects on Barrier Properties of Coating Films 2. Effects on Commodity Physiology F. Fruit-Coating Interaction V. POSTHARVEST PHYSIOLOGY AND QUALITY OF COATED COMMODITIES A. Physiological Basis for the CA/MA Storage of Fruits and Vegetables 1. Respiration 2. Ethylene Biosynthesis and Action B. Postharvest Physiology of Coated Commodities 1. Coating Effects on Ripening of Fruits and Vegetables 2. Mode of Action of Surface Coatings in Delaying Ripening 3. Optimization of Surface Coatings in Relation to Commodity Internal Atmosphere Composition C. Postharvest Quality of Coated Commodities 1. Skin Finish 2. Disorders 3. Diseases 4. Insects 5. Flavor VI. SUMMARY AND CONCLUSIONS A. General Review B. Prospects for Future Research LITERATURE CITED

I. INTRODUCTION The magnitude of postharvest losses in fresh fruits and vegetables is estimated to be 25–80%, depending upon the commodity and the technological level of postharvest operations (Wills et al. 1981). This reflects a lack of knowledge by postharvest handlers of the biological and environmental factors involved in deterioration or the absence of adequate postharvest technologies required to preserve fresh quality (Kader 1992). These losses represent a large proportion of total costs of the hortbusiness, greatly reducing the profitability of the marketing chain. In recent years, much attention has been paid to exploring the potential of surface coatings to maintain quality of harvested fresh produce and to reduce the volume of disposable non-biodegradable packaging materials (Rose 1992). The close attachment of the coating to the product surface results in a different set of benefits and risks to those encountered with plastic film wraps. Cosmetic enhancement is directly linked to the product rather than being a removable part of its packing. The risk of condensation on the inner surface, and the associated exacerbation of rots, is eliminated. On the other hand, the absence of opportunity for exchange of gases between the product surface and applied film can result in much more dramatic changes in the commodity internal atmos-

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phere and increase in risk of anaerobiosis than wrapping in a plastic film (Ben-Yehoshua et al. 1985; Banks et al. 1993a). Therefore, the decrease in total permeance to O2 and CO2 exchange will be much smaller for a sealed than for a coated commodity, in spite of using films with the same permeability in both techniques (Banks et al. 1993a). Surface coatings have been used extensively on bulky organs to modify internal atmosphere composition and thereby delay ripening (Banks 1984a; Smith et al. 1987; Banks et al. 1993a, 1997b; Baldwin 1994; Amarante et al. 1997a,b), to reduce water loss (Hagenmaier and Baker 1993a, 1994a; Amarante et al. 1997a,b), and to improve the finish of the skin (Mellenthin et al. 1982; Hagenmaier and Baker 1994b, 1995; Johnston and Banks 1998; Amarante 1998). The latter attribute has been considered by the fruit industry to be the main benefit from a marketing point of view, without much consideration about the physiological impacts of coatings on other aspects of fruit quality. Coatings may have very limited effects in delaying ripening at low temperature, when the modification of internal atmosphere is small (Banks 1984c; Smith and Stow 1984; Amarante 1998). At high temperatures, when respiration is high and a substantial decrease of O2 (and increase of CO2) occurs, inhibition of ripening can be excessive and the tissue may ferment (Magness and Diehl 1924; Smock 1935; Trout et al. 1953; Davis and Hofmann 1973; Hagenmaier and Baker 1993a, 1994b; Banks et al. 1997a,b; Amarante 1998). The degree of reduction in water loss and modification of internal atmosphere is also greatly affected by the permeance of the coating film itself (Hagenmaier and Baker 1993a,c, 1994a,b, 1995, 1996) and the character of cover of commodity skin by the surface coating (Banks et al. 1993a, 1997b; Hagenmaier and Baker 1993a, 1994a,b, Amarante 1998). Coated fruits and vegetables may also manifest many physiological disorders during refrigeration or shelf life (Smock 1935; Hitz and Haut 1938; Trout et al. 1953; Farooqui and Hall 1973; Smith and Stow 1984; Van Zyl et al. 1987; Kerbel et al. 1989; Edward and Blennerhassett 1990; Lau and Yastremski 1991; Lau and Meheriuk 1994; McGuire and Hallman 1995; Amarante 1998). As the potential impact of these effects is not always fully appreciated by people in the marketing chain, use of coatings may be far from optimal and can, in such cases, impair rather than enhance product quality. The majority of published papers dealing with surface coatings do not provide full descriptive information about the main components and their concentrations in the formulations applied for reasons of commercial sensitivity. This makes the interpretation of results published by various authors quite difficult, limiting the potential to improve coating formulations for different products and storage conditions.

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Most of the literature about surface coatings has been largely empirical, describing the quality changes occurring from the application of a particular coating treatment, without providing information about the degree of change in permeance to gases and how this is related to the extent of internal atmosphere modification in coated commodities. Several authors have published values of permeance to gases and water vapor of coating films (Kamper and Fennema 1984a,b; Elson et al. 1985; Kester and Fennema 1989a,b,e; Rico-Peña and Torres 1990; Hagenmaier and Shaw 1990, 1991a,b, 1992; Martín-Polo et al. 1992; Wong et al. 1992; Gennadios et al. 1993; Avena-Bustillos and Krochta 1993; Donhowe and Fennema 1993; Gontard et al. 1993, 1996; Koelsch 1994; McHugh and Krochta 1994b; Hagenmaier and Baker 1994a, 1996; Mannheim and Soffer 1996), with some published information for coated commodities (Banks 1984a; Ben-Yehoshua et al. 1985; Paull and Chen 1989; Hagenmaier and Baker 1993a; Avena-Bustillos et al. 1994, 1997; Banks et al. 1997a,b; Amarante et al. 1997a,b; Amarante 1998; Johnston and Banks 1998). Permeance values of commodities treated with surface coatings can be very different from the permeance of the coating films themselves. Coatings mainly exert their effects on skin permeance to gases by blocking a greater or lesser proportion of the pores on the product surface (Banks et al. 1993a, 1997b; Hagenmaier and Baker 1993a; Amarante 1998). As a result, fruits with different skin characteristics can have very different responses to a certain coating by exhibiting distinct types of interaction with the surface coating (Amarante et al. 1997b; Amarante 1998). Published information about barrier properties of coating films has been obtained using several different techniques in a variety of environmental conditions such as temperature, relative humidity (RH), and gas partial pressures (McHugh and Krochta 1994a). In most cases, these conditions are very distinct from the real situation encountered by the surface coating during the storage of a coated product. This, coupled with the variety of terms used to characterize the barrier properties of films (such as transmission rate, permeability, permeability coefficient, permeance and resistance) and the multitude of units used to express each term (Donhowe and Fennema 1994), makes it difficult for the end users of data to compare results from different studies. Conversions made from alternative systems of units is time consuming and must rely on assumptions that may not be true (Banks et al. 1995). This review outlines the main factors to be considered in order to gain a better understanding of the physical and physiological issues involved in preserving postharvest quality of coated fruits and vegetables. The most pertinent literature available is presented and discussed, high-

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lighting the most important benefits and risks of coatings, which may have substantial effects on final product quality. The review focuses on fruits and fruit structures in fruit and vegetable crops and it covers only the treatment of fresh commodities. Information about application of coatings in processed horticultural products can be obtained in recent publications elsewhere (Wong et al. 1994; Baldwin et al. 1995a,c; Nisperos-Carriedo and Baldwin 1996).

II. PERMEABILITY OF COATING FILMS AND PERMEANCE OF COATED COMMODITIES For coating films, when there are no pores, faults, or membrane punctures, the primary mechanism of gas and water vapor transfer through a coating film is by activated diffusion; i.e. the permeant dissolves in the film matrix at the high concentration side, diffuses through the film driven by a concentration gradient, and evaporates from the outer surface (Kester and Fennema 1986). The dissolution and evaporation steps are influenced by solubility of the permeant in the film (Pascat 1985). The diffusion component depends upon size, shape, and polarity of the permeant as well as polymer-chain segmental motion within the film matrix (Kester and Fennema 1986). Factors affecting segmental motion of the polymer chain include inter-chain attractive forces such as hydrogen bounding and van der Waals’ interactions, degree of crosslinking, and amount of crystallinity (Kumins 1965). Thus, permeability of material k to gas j (Pj,k, mol⋅s–1⋅m⋅m–2⋅Pa–1) is defined by the product of the solubility coefficient (Sj,k, mol⋅m–3⋅Pa–1), representing concentration of j in k in equilibrium with a given external partial pressure of j and the diffusion coefficient (Dj,k, m2⋅s–1), representing the mobility of molecules j in k: Pj,k = Sj,k⋅Dj,k

[1]

Permeability of a coating material k to gas j at steady state can also be estimated by Fick’s First Law of Diffusion (Banks et al. 1995):

P j,k

r j′ ⋅ ∆x ∆p j ⋅ A

[2]

where: r j′ = rate of transfer of gas j through a coating film made of k (mol⋅s–1); ∆x = film thickness (m); ∆pj = difference in partial pressures of gas j across the film k (Pa); and A = surface area (m2).

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For coated commodities, it may be difficult to separate the gas exchange into the solubility and diffusivity components, as diffusion through the skin involves parallel transfer through pores and cuticle (Banks et al. 1993a). Also, the surface coating on treated commodities may be heterogeneous or of unknown thickness. For these reasons, permeance is generally used to characterize the gas exchange properties of coating films (Banks et al. 1995). Permeance of a coating film made of k to gas j (P j,k ′ , mol⋅s–1⋅m–2⋅Pa–1) is related to permeability by:

P j,k ′ =

P j,k ∆x

[3]

From Banks et al. (1993a), the total permeance to gas j (P j,′ total) of a commodity coated with a tightly adhering film actually comprises the effective permeance of the commodity skin (P j,′ skin) and the coating barrier k (P j,k ′ ) operating in series:

1 1 1 = + P j,total P j,skin P j,k ′ ′ ′

[4]

From equations 2, 3, and 4, the magnitude of difference between internal and external atmosphere composition of the fruit can be seen to be directly proportional to rate of transfer of gas j and inversely proportional to total permeance of the coated commodity to gas j:

∆p j = p ij − p ej =

r j′ P j,total ⋅A ′

[5]

where: p ij = partial pressure of gas j in the internal atmosphere (Pa) and pej = partial pressure of gas j in the external atmosphere (Pa). In this case, r ′j is given by the product of rj (the specific rate of transfer of gas j between the commodity internal and external atmospheres; mol⋅kg–1⋅s–1) and M (the commodity mass; kg). Eq. 5 is only accurate if internal atmosphere composition is reasonably uniform within the organ (Cameron and Yang 1982). This is not always true in commodities with high-density flesh tissue or following flooding of the intercellular spaces associated with ripening. Problems caused by heterogeneity of internal atmosphere composition can, in some cases, be minimized by sampling internal gas composition non-destructively from under the skin using an external chamber (Amarante 1998).

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Gases diffuse mainly through pores in the skin, while water moves preferentially by a different pathway, probably through a liquid aqueous phase in the cuticle where water solubility is high (Ben-Yehoshua et al. 1985; Banks et al. 1993a). Gases are constrained from using this pathway due to their low solubility in the water and wax components of the cuticle matrix, with SH2O >> SCO2 > SO2 (Foust et al. 1980; Banks et al. 1993a), and low diffusivity in liquid water, which is 104-fold less than in air (Foust et al. 1980). As a result, improving the blockage of pores in the skin with a surface coating results in P′O2 < P′CO2 << P′H2O (Banks 1984a; Ben-Yehoshua et al. 1985; Amarante 1998). Covering the pores of the skin provides some reduction in P′H2O (Amarante 1998), but there is still an extensive diffusion of water through the cuticle and the coating film (Ben-Yehoshua et al. 1985; Banks et al. 1993a). However, covering the pores in the skin has a dramatic effect in reducing P′O2 and P′CO2, especially P′O2 (Amarante 1998), since O2 has a lower solubility in the water and wax components of the coating matrix than CO2 (Trout et al. 1953; Banks 1984a; Banks et al. 1993a). On citrus fruit, waxing with a coumarone indene resin depressed P′H2O, P′CO2 and P′O2 to about 85%, 40%, and 30% of the control values, respectively (Ben-Yehoshua et al. 1985). On banana coated with TAL Pro-long 1.5% w/v P′CO2 and P′O2 were depressed to about 64% and 20% of their controls, respectively (Banks 1984a). This results in coated commodities having more substantial modification of O2 than of CO2 internal concentration particularly at high temperatures (Trout et al. 1953; Ben-Yehoshua 1967; Banks 1984a; Amarante 1998). Permeance to ethylene (C2H4, P′C2H4) in coated commodities has been reported to be reduced to levels between those observed for P′O2 and P′CO2 (Banks 1984a; Ben-Yehoshua et al. 1985). Mannheim and Soffer (1996) observed a relationship between PH2O of coating films and weight loss of coated fruit, but not between PCO2 and PO2 of the coating films and concentrations of these gases in the fruit. This is in agreement with observations made by Banks et al. (1997b). According to these authors, PH2O of the coating film is much more important than pore blockage in reducing fruit water loss. On the other hand, the extent of fruit internal atmosphere modification is more strongly determined by the proportion of pores blocked by the coating than by PO2 and PCO2 of the coating film. However, Hagenmaier and Baker (1993a) observed that for both CO2 and water vapor, the skin permeance of coated fruit is reduced by both the coating’s tendency to seal pores in the fruit peel and the barrier properties of the coating material itself. Amarante (1998) has shown that changes in skin permeance of pear treated with different concentrations of a carnauba-based commercial coating was dependent on the nature of the skin. For cultivars with

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non-lignified cells in the skin (‘Bartlett’, ‘Doyenne du Comice’, and ‘Packham’s Triumph’), more substantial reductions of P′H2O (more than 30%) were achieved with small increases in coating concentration (from 0% to 20% v/v concentration of commercial formulation). Blockage of cracks on the skin of such coated fruit was observed by means of confocal microscopy. According to the authors, coating the fruit with a low concentration of a wax could cover these imperfections, potentially providing a substantial reduction in P′H2O of these areas of the skin (Fig. 4.1). Further increases in coating concentration might then have a small effect by improving the character of cover and increasing its thickness (reducing P′H2O to about 50% of non-coated fruit). However, P′H2O of pear treated with the undiluted commercial formulation of coating was still about 20 times higher than P′CO2 since water is still several orders of magnitude more soluble in the water and the coating film than CO2. Also, the ratio P′CO2/P′O2 increased by increasing the coating concentration, as a result of a more substantial reduction of P′O2 than of P′CO2. ‘Beurre Bosc’, with lignified cells in the skin, had only small changes in P′H2O with waxing. In addition, P′O2 and P′CO2 decreased to a similar extent and more gradually than in the other cultivars with increasing coating concentration, diminishing to close to zero for the undiluted coating. The lignified cells in the epidermis of ‘Bosc’ seemed to have high P′H2O and very low P′CO2, with almost all O2 and CO2 exchange occurring through the lenticels (Fig. 4.2). Increasing coating concentration was not effective in covering the lignified cells in the epidermis but did gradually block the lenticels in the skin, resulting in substantial reduction of P′O2 and P′CO2 but not of P′H2O (Fig. 4.2). According to the authors, approaches to optimization of surface coatings must take into account differences in the nature of the skin, since substantial reduction of skin permeance to water and gases may be achieved with small increases in coating concentration only for fruit without lignified cells in the skin. Since the nature of the commodity skin affects its interaction with a surface coating and, hence, the permeance of the coated commodity (Claypool and King 1941; Amarante 1998), this, in association with the product respiration rate, determines the level of internal atmosphere modification and the resulting effects on quality attributes (Amarante 1998). Therefore, although information about the effects of film type, formulation, temperature, and RH on permeability is quite useful for evaluating edible films, information pertaining to permeance to gases of coated fruit would provide a more realistic approach for selection of surface coating formulations. With direct measurement of commodity permeance to gas exchange under controlled environmental conditions (temperature, RH, and pressure) it would be possible to determine in

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Image Not Available

Fig. 4.1. Hypothetical model for the differences in skin permeance to O2 (↓), CO2 (↑) and water vapor (↑) in a non-coated (A) and a coated (B) pear without lignified cells in the skin. Arrow size is proportional to the differences in permeance values between gases and between coated and non-coated fruit (cr = crack in the cuticle; c = cuticle; e = epidermis; l = lenticel; se = sub-epidermis; w = wax coating layer). Source: Amarante 1998.

loco if the coating film exhibits the properties required to attain an optimized modified internal atmosphere for a given commodity in a specific combination of storage conditions (Hagenmaier and Baker 1993a; Amarante et al. 1997b; Amarante 1998).

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Image Not Available

Fig. 4.2. Hypothetical model for the differences in skin permeance to O2 (↓), CO2 (↑) and water vapor (↑) in a non-coated (A) and a coated (B) pear with lignified cells in the skin. Arrow size is proportional to the differences in permeance values between gases and between coated and non-coated fruit (e = epidermis; l = lenticel; se = sub-epidermis; w = wax coating layer). Source: Amarante 1998.

III. PHYSICO-CHEMICAL CHARACTERISTICS, AND BARRIER PROPERTIES TO WATER VAPOR AND GASES, OF EDIBLE COATINGS Many different coating formulations have been investigated for their potential benefits in preserving the quality of harvested fruits and vegetables. These studies have mainly focused on assessing barrier proper-

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ties of coating films made of components of different chemical natures and composite films made of mixtures of these. The literature concerned with the composition of these coatings and their main physicochemical properties is discussed below. A. Lipids and Resins Lipids and resins are added to coating formulations to reduce gas exchange, but mainly to impart hydrophobicity (to reduce water loss) and gloss (Baldwin 1994; Hernandez 1994; Baldwin et al. 1997). Lipid components of coatings include natural waxes such as carnauba wax, candelilla wax, rice bran wax, and beeswax; petroleum-based waxes such as paraffin and polyethylene wax; petroleum-based oils, mineral oil (mixture of paraffinic and naphthenic hydrocarbons), and vegetable oils (corn, soybean, or palm); and acetoglycerides and oleic acid (used as components of coatings to alter their mechanical and permeability properties). Resins are represented by shellac, wood rosin, and coumarone indene and these are the main coating components used to impart gloss to the commodity (Hagenmaier and Baker 1994b, 1995). However, fruit coated with resins have been reported to develop a whitening of the skin due to condensation that develops when they are brought from cold storage to ambient temperature (Hagenmaier and Baker 1994a). Waxes are less likely to whiten and can also add some sheen to the product (Nisperos-Carriedo and Baldwin 1996). Other compounds added to formulations of these coating materials include plasticizers, emulsifiers, lubricants, binders, de-foaming agents, or formulation aids. Most natural waxes (beeswax, carnauba wax, and candelilla wax) also have emulsifying properties, as they are long-chain alcohols and esters (Baldwin et al. 1997). The common lipid compounds and additives permitted for use as components in commercial and experimental coatings for food systems have been listed recently by Hernandez (1994) and Baldwin et al. (1997). The permeability of lipid components of coatings to water vapor and gases depends on chain length, polarity, and degree of saturation and branching of their main components (Kamper and Fennema 1984a,b; Kester and Fennema 1989a,b,e; Hagenmaier and Shaw 1990; Donhowe and Fennema 1993; Baldwin et al. 1997). Lipid molecules with long chain length, low polarity, high saturation, and high linearity tend to produce films with high degrees of cohesiveness and rigidity as a result of stronger inter-chain attractive forces such as van der Waals’ interactions, and low permeability, as opposed to short chain polar molecules with a high degree of branching and low saturation. Molecules with high

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linearity and long chain length have a more efficient packing of acyl chains. This, in addition to high saturation levels, tends to reduce the hydrocarbon chain mobility, reducing the diffusion of gases and water vapor molecules through, and their solubility in, the coating matrix. Martín-Polo et al. (1992) observed that the proportion of liquid and solid components of a hydrophobic film affects moisture transfer through coatings. For coating films containing different proportions of a solid phase (paraffin wax or n-octacosane) mixed with a liquid phase (paraffin oil or n-hexadecane), a significant increase in PH2O of the coating film was obtained for mixtures containing less than 25% solids. The results suggest that up to 75% liquid lipids may be used in coating formulations to overcome the problem of the rigidity of solid materials like edible waxes and to improve their flexibility and adhesiveness and to improve the character of cover without seriously diminishing their moisture barrier properties. The excellent barrier properties of lipid crystals to moisture and gases is also dependent on the packing of the lipid crystals and their orientation to the direction of permeant flow. Kester and Fennema (1989a,b) reported a dependence of P′H2O and P′O2 of lipids on polymorphic form and the structural morphology of the fatty acid coating film. Fatty acids of the α-polymorphic form in the solid state, with molecular packing in a hexagonal system, had higher P′H2O and P′O2 than those fatty acids of the more stable β and β′ polymorphic forms, packed in a common orthorhombic system. Tristearin (a triacylglycerol) and acetylated monoglyceride have the hexagonal type, while straight chain lipids such as fatty acids (stearic acid), fatty alcohols (stearyl alcohol), and alkanes, have the orthorhombic type. In the orthorhombic form, hydrocarbon chains are aligned with each other and arranged in sheets with strong van der Waals’ interaction between laterally adjacent chains. The hydrocarbon chains of the α-polymorphic form are packed in a hexagonal orientation that is molecularly less dense and possesses greater mobility than the orthorhombic lateral packing, resulting in a higher P′H2O and P′O2. Kester and Fennema (1989c) reported that changing the polymorphic form of lipid films from α to β polymorph substantially decreased P′O2 but not P′H2O. These results may indicate that the hydration capacity of the various polymorphs, which may not be significantly different between the polymorphs, is more important for water permeation. Kester and Fennema (1989a,b) suggested that differences in packing density and molecular mobility between the hexagonal and orthorhombic orientations are of relatively minor importance in comparison to the structural morphology of the coating film. According to these authors, it appears that the properly layered morphology with platelets that com-

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pactly overlay one another is the predominant factor accounting for the low P′H2O and P′O2 of some lipid films. Kester and Fennema (1989d) also observed that tempering a stearyl alcohol coating film improved the gas exchange barrier properties of the film, despite no apparent changes in the polymorphic form of the lipid during the tempering period. Tempering at 48°C for 35 days caused P′O2 and P′H2O to decrease 45% and 33%, respectively. According to the authors, likely mechanistic explanations included the healing of crystal imperfections and the development of a more extensive and betterlinked arrangement of lipid crystalline platelets. Resins have polar groups and therefore have higher PH2O and lower PO2, PCO2, and PC2H4 than waxes (Hagenmaier and Shaw 1992). Coatings formed on polymer films had lower P′H2O when made with wax microemulsions rather than with mixtures of wax with shellac and resin (Hagenmaier and Baker 1994b). Hagenmaier and Shaw (1992) reported that for commercial coatings, PO2 at 50% RH and 30°C ranged from 5.5 × 10–5 pmol⋅m⋅s–1⋅m–2⋅Pa–1 for a shellac-based coating to 2.5 × 10–3 pmol⋅m⋅s–1⋅m–2⋅Pa–1 for a coating made of waxes (natural and synthetic) and fatty acids, with PCO2 two to eight times as high. The PC2H4 was between the values of PO2 and PCO2 for wax-based coatings, but lower than PO2 for shellac and resin-based coatings. The choice of solvent and neutralizing agent is important in determining the coating film permeability (Hagenmaier and Shaw 1991a,b). Hagenmaier and Shaw (1991a) observed that shellac coatings made from water-soluble formulations were more permeable to O2, CO2, and water vapor than formulations made with ethanol, especially at RH above 80%. Using shellac solubilized in water with morpholine instead of shellac in ethanol increased PO2 by 200% and PCO2 by 435%, resulting in an increase of PCO2/PO2 from 3.5 to 6.3 at 30°C and 75% RH. At 75% RH shellac solubilized with NaOH was about 17 times as permeable to water as shellac solubilized with morpholine. The authors attributed the high permeability of films made of shellac solubilized in water to the addition of morpholine or NaOH to neutralize the shellac (a weak acid). Hagenmaier and Shaw (1991b) also reported higher PO2 and PCO2 for polyethylene-based coatings containing ammonia or morpholine instead of non-volatile surfactants. According to these authors, ammonia and morpholine keep the polyethylene dissolved and then evaporate as the coating dries. Thus, these have a minimum effect on the permeability of dried coatings. In contrast, non-volatile surfactants (that remain in the coating after drying) are polar and tend to reduce the coating permeability (Ashley 1985). Differences in formulation of coating films made with the same basic

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components can also affect permeability. Shellac varnish was more permeable to O2 (5.16 × 10–5 pmol⋅m⋅s–1⋅m–2⋅Pa–1) than bleached shellac (3.04 × 10–5 pmol⋅m⋅s–1⋅m–2⋅Pa–1) at 28.5°C and 55% RH (Hagenmaier and Shaw 1991a). High density polyethylene wax had PH2O, PO2, and PCO2 that were 30–50% of the corresponding values for low density polyethylene waxes (Hagenmaier and Shaw 1991b). This was thought to be the result of denser polymers having a more crystalline structure and a lower permeability (Ashley 1985). For wax emulsions, the size of the suspended globules in the emulsion affects the permeability of the film to water vapor and gases (Baldwin et al. 1997). Macroemulsions (turbid and milky in color, with particle size of 2,000 to 100,000 Å) are thermodynamically less stable than microemulsions (clear in color and with particle size of 1,000 to 2,000 Å; Hernandez 1994), with macroemulsions being expected to impart higher permeability and lower gloss to the coated fruit than microemulsions (Baldwin et al. 1997). Therefore, the method of emulsion preparation, by affecting particle size in the emulsion, may have a substantial impact on final quality of the coated commodity. However, most research on preparation of wax emulsions, particularly formulations of edible coatings, is proprietary and little information is available (Hernandez 1994). This makes it very difficult for researchers to compare results published in the literature for coatings having similar formulations but containing different additives and having been prepared by different methods of emulsification. In some published papers, the only information of this kind is that the commodity was coated with a ‘wax-based coating’ without specifying the main components in the formulation. Donhowe and Fennema (1993) reported the values of PO2 and PH2O for candelilla, carnauba, beeswax, and microcrystalline waxes. The x-ray diffraction scans of the four wax films showed that all waxes were partially crystalline with characteristic orthorhombic packing. Candelilla wax had the lowest PH2O of the waxes tested, reflecting the low concentration of polar compounds and the large concentration of alkanes in its composition. Beeswax had the greatest PH2O; this may have been the result of higher concentrations of fatty acids, fatty alcohols, and esters in this wax than in the other three. The PH2O of candelilla wax was somewhat less than that of polypropylene and somewhat greater than that of high-density polyethylene films. Carnauba and candelilla waxes had the lowest PO2. Values for PO2 of beeswax and microcrystalline wax were about six to nine times greater than those of candelilla and carnauba waxes. The greater PO2 of beeswax and microcrystalline wax was thought to be the result of these waxes containing crystals in both the hexagonal and orthorhombic systems. Candelilla contained crystals only

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in the orthorhombic system. Carnauba wax had some proportion of crystals in the hexagonal system but, because of its rigid nature, as reflected by its high terminal melting point and lack of low-melting fraction, the PO2 was comparatively low. Carnauba is presently the most commonly used wax component, while polyethylene wax is permitted for use as a coating component for some fresh produce where, generally, the peel is not normally ingested, such as avocado, citrus, banana, pineapple, melon, mango, pumpkin, and papaya (Hernandez 1994). Consumer trends are leading toward more natural products. As a result, petroleum-based waxes, such as polyethylene and paraffin, and resins (mainly wood rosin and coumarone indene, a petroleum-based product), are becoming increasingly unpopular and restricted in use (Baldwin 1994; Hernandez 1994). Only shellac resin has recently been granted the GRAS (“generally recognized as safe”) status by the American authorities (Baldwin 1994; Hernandez 1994). Some countries have imposed strong restrictions on the use of waxes. Norway has recently prohibited all imports of waxed fruit, while petroleum waxes, morpholine, and carnauba wax are prohibited in the UK and petroleum wax is prohibited in Japan (Baldwin 1994). It seems likely that the naturally derived waxes, such as beeswax, carnauba wax, and candelilla wax, will be considered more acceptable in the next few years. The same reasoning applies to oils, with mineral oil expected to be replaced by vegetable-based oils. This has also led toward increasing research for alternative coating formulations based on composite edible films made of polysaccharides and proteins in combination with lipids (mainly fatty acids), with all components having GRAS status. The goal is to combine the desirable properties of the polymer structural matrix (good barrier to gases, strength, and film forming attributes) with the hydrophobicity and flexibility imparted by the lipid. B. Polysaccharides Polysaccharide-based coatings have been extensively studied for their selective permeabilities to O2 and CO2, resulting in modified internal atmosphere composition and delayed ripening in fruits and vegetables. These coating films are very effective barriers to O2 and CO2 but not to water (Wong et al. 1992). This property is probably related to the dense structure and high polarity of the film (McHugh and Krochta 1994a). The inability of these coatings to provide sufficient gloss or prevent moisture loss can be improved by the incorporation of functional food ingredients such as resins and rosins, plasticizers, oils, waxes, and

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emulsifiers (Hagenmaier and Shaw 1990; Nisperos-Carriedo 1994; Hagenmaier and Baker 1996). This group of coatings is mainly represented by cellulose derivatives, namely methylcellulose (MC), hydroxypropylcellulose (HPC), hydroxypropyl methylcellulose (HPMC), and carboxymethylcellulose (CMC; Kester and Fennema 1986; Nisperos-Carriedo 1994). Changing the level of methoxyl, hydroxypropyl and carboxymethyl substitution affects a number of physical and chemical properties such as water retention properties, sensitivity to electrolytes and other solutes, dissolution temperature, gelation properties, and solubility in non-aqueous systems (Kester and Fennema 1986; Nisperos-Carriedo 1994). The number of substituted hydroxyl groups per monomeric unit, expressed as degree of substitution (DS), can vary from zero to three, with higher DS resulting in increased solubility, compatibility with other ingredients (e.g. salts), and acid stability of the cellulose derivative (Nisperos-Carriedo 1994). The nonionic cellulose esters MC, HPMC, and HPC are available in powder or granular form in varying molecular weights and DS. They are soluble in cold but not in hot water, and they are also soluble in organic solvents (except for MC with low DS). Solutions of these cellulose coatings are stable at pH 2–11 (Nisperos-Carriedo 1994). These compounds are good film formers owing to the linear structure of the polymer backbone. They yield tough and flexible transparent films. Plasticity can be improved by adding polyols such as glycerol, sorbitol, mannitol, sucrose, propylene glycol, and polyethylene glycol (Kester and Fennema 1986; Koelsch 1994). Several formulations have been developed by Nisperos-Carriedo and co-workers, containing different concentrations of MC, HPMC, and HPC, and registered as Nature Seal (NisperosCarriedo 1994). The anionic cellulose ester CMC is soluble in either hot or cold water, but insoluble in organic solvents. However, the gum dissolves in suitable mixtures of water and water-miscible solvents such as ethanol or acetone. CMC solutions are stable at pH 7–9 (Nisperos-Carriedo 1994). Lowings and Cutts (1982) proposed coating fruits and vegetables with a semi-permeable film composed of CMC and sucrose fatty acid esters. These coatings reduced O2 uptake without causing an equivalent increase in CO2 level in internal atmospheres of fruit and vegetables tissues (Banks 1984a). Commercial coating formulations based on CMC and sucrose esters have included TAL Pro-long (later called simply Prolong) and subsequently Semperfresh. These coatings extended the shelf life and preserved important flavor components of some fresh commodities (Banks 1984a; Smith and Stow 1984; Drake et al. 1987; Van Zyl

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et al. 1987; Kerbel et al. 1989; Santerre et al. 1989; Nisperos-Carriedo et al. 1990; Köksal et al. 1994; Lau and Meheriuk 1994; Sümnü and Bayindirli 1994; 1995a,b; Bayindirli et al. 1995). Chitosan (2-amino-2-deoxy-β-D-glucan), another water-soluble polysaccharide coating, is a linear polyamine obtained by alkaline deacetylation of chitin (Wong et al. 1992). Chitosan can form a semi-permeable coating that can modify the internal atmosphere (El Ghaouth et al. 1992b) and reduce weight loss (El Ghaouth et al. 1991b), thereby delaying ripening and preserving postharvest quality of fruits and vegetables. However, Wong et al. (1992) reported that chitosan film formed a very effective barrier to O2 and CO2 but not to water. It can inhibit the growth of several fungi and reduce postharvest decay (El Ghaouth et al. 1991a,b, 1992a,b, 1997; Cheah et al. 1997). Nutri-Save, a chitosan-based commercial coating has been shown to delay ripening and preserve postharvest quality of several pome fruits (Elson et al. 1985; Meheriuk and Lau 1988; Meheriuk 1990; Lau and Yastremski 1991; Lau and Meheriuk 1994). C. Proteins Proteins have been less investigated as film formers than lipids and polysaccharides. The use of protein-based coatings on fruits and vegetables has been restricted due to the high PH2O of such films (Gontard et al. 1993, 1996; Gennadios et al. 1994; Koelsch 1994; McHugh and Krochta 1994a). Protein films are good O2 and CO2 barriers at low RH, but not in high humidity environments because of protein film’s susceptibility to moisture absorption and swelling (Gontard et al. 1996). However, the development of composite or bilayer coatings, combining proteins with hydrophobic materials, has been suggested to present many opportunities for coating fresh commodities (Avena-Bustillos and Krochta 1993; Koelsch 1994; Gennadios et al. 1994; Hagenmaier and Baker 1996). Several chemical and physical treatments show some effectiveness in promoting cross-linking, “hardening” the protein film structure, and improving film barrier and mechanical properties (Gennadios et al. 1994). Film formation is facilitated by the development of hydrophobicity and hydrogen and disulfide bonds in the film matrix. Such films are brittle, as a result of extensive intermolecular associations (Gennadios et al. 1994). Addition of plasticizers is necessary to disrupt some of these associations and induce film flexibility (Koelsch 1994; McHugh and Krochta 1994b). McHugh and Krochta (1994b) observed improved properties of whey protein films plasticized with glycerol and sorbitol. Glyc-

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erol was more effective than sorbitol as a coating plasticizer in that lower concentrations of the glycerol were required to increase the tensile strength, elongation, and elastic modulus of whey protein films; such films also had higher P′O2. According to the authors, the smaller size of glycerol enabled it to influence film mechanical properties (and possibly gas exchange barrier properties) more readily than sorbitol. Avena-Bustillos and Krochta (1993) observed that adjusting the pH of edible films made of sodium caseinate to the protein isoelectric point (pH 4.6) reduced PH2O by 36%. However, Gennadios et al. (1993) observed that film formation was inhibited by poor protein dispersion around the isoelectric pH region of soy protein isolate (pH 4.5) and wheat gluten (pH 7.6) films, resulting in poor water vapor barrier properties. D. Composite and Bilayer Coatings Several composite and bilayer films have been investigated with the goal of combining the desirable properties of different materials to improve permeability characteristics, gloss, strength, flexibility, nutritional value, and general performance of coating formulations. Plasticizers have been incorporated into edible coatings as a processing aid, to facilitate coating application and to increase machinability (Koelsch 1994). The most commonly used plasticizers are polyols (such as sorbitol and glycerol), mono-, di-, or oligo-saccharides, lipids and derivatives (Gontard et al. 1993). The plasticizing effect is due to their ability to reduce internal hydrogen bonding while increasing intermolecular spacing (Lieberman and Gilbert 1973). By decreasing the intermolecular forces along polymer chains (of lipids, proteins, or cellulose entities), plasticizers reduce the brittleness of the film but increase its PH2O (Gontard et al. 1993; Koelsch 1994), PO2 (McHugh and Krochta 1994b), and PCO2 (Lieberman and Gilbert 1973). Plasticizers weaken the intermolecular forces between polymer chains, and therefore increase the diffusion constant. Using more hydrophobic plasticizers, such as lipids, would be less likely to increase P′H2O of polymeric coating films (Kester and Fennema 1986). Lipid compounds added to polysaccharide- and protein-based coating formulations improve the mechanical properties of the film, by acting as plasticizers and emulsifiers. Commonly used lipids for these purposes are oils, lecithin, fatty acids and derivatives, and waxes (Kester and Fennema 1986; Hernadez 1994; Donhowe and Fennema 1994). Emulsions formed by adding lipid materials to hydrophilic coatings can sometimes improve their moisture barrier properties. Hagenmaier

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and Shaw (1990) have shown that the poor water vapor barrier properties of HPMC can be improved by increasing the chain length and the amount of fatty acids added to the coating. The tensile strength of the polysaccharide film was increased while PH2O was substantially reduced (from 5.88 pmol⋅m⋅s–1⋅m–2⋅Pa–1 to 2 × 10–2 pmol⋅m⋅s–1⋅m–2⋅Pa–1) by increasing the stearic acid composition from 0% to 40% (total solids w/w). However, at high RH (94–97% RH) the film tended to swell, thus increasing PH2O. Wong et al. (1992) showed that the uniformity of the distribution of the lipid molecules might be a crucial factor in the determination of the effectiveness of the water barrier of a composite film made of chitosan. The authors observed that a chitosan-laurate film had low PH2O, while films containing other fatty acids (including palmitic acid, a fatty acid of longer chain length) or esters were not effective in this respect. The authors postulated that the unique properties of the chitosan-laurate film could suggest the importance of the morphological arrangements of the lipid within the chitosan matrix. Lauric acid was believed to be the only fatty acid incorporated evenly throughout the film. These results further suggest that the uniformity of the distribution of the lipid molecules might also be a crucial factor in the determination of the effectiveness of the composite surface coatings water barrier. Kamper and Fennema (1984b) studied the improvement of water vapor moisture barrier properties of HPMC film by adding a blend of stearic and palmitic acids to the coating formulation. The film provided considerable protection against moisture transmission at RH up to 90%. Above this humidity, the film became progressively hydrated, leading to a loss of structural integrity and increased P′H2O. Kester and Fennema (1989e) observed that the use of a blend of MC and HPMC (70% MC and 30% HPMC) instead of HPMC alone improved the barrier properties of the polysaccharide-lipid-based coating film, and covering this film with a beeswax layer greatly reduced PH2O. According to the authors, MC is less hydrophilic than HPMC. Hence, it improves lipid solubilization, which may cause a greater percentage of the fatty acids to became entrapped in the bulk of the cellulose ether matrix during film formation, improving the moisture barrier properties. The hydrophobic nature of beeswax and its morphology of tightly packed small crystals greatly improved the moisture barrier of the bilayer film. Kamper and Fennema (1984a,b) tested composite films for P′H2O. Films were prepared by coating lipids (hydrophobic) onto a dried HPMC film (bilayer technique) or by adding the lipids to the film-forming solution containing HPMC (emulsion technique) before the film formation. They were tested at 25°C and with a RH differential of 85%. The P′H2O of the

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HPMC film was 3.05 nmol⋅s–1⋅m–2⋅Pa–1. Lipid layers applied to the surface of the HPMC film, forming a bilayer film, decreased P′H2O, with better results achieved with lipids of decreased fluidity (greater saturation). Bilayer films containing solid lipids, such as beeswax, paraffin, or hydrogenated palm oil yielded P′H2O of 0.06 nmol⋅s–1⋅m–2⋅Pa–1 or less, which was much smaller than that for a 25.4 µm thick low-density polyethylene film (0.11 nmol⋅s–1⋅m–2⋅Pa–1 at 37.8°C and RH differential of 90%). Permeance through emulsion films was also highly dependent on the amount of lipid added to the film forming solution and their degree of saturation and chain length. Small increases of stearic acid concentration in the coating film provided large reductions of P′H2O, with smaller additional beneficial effects being achieved at higher lipid concentration levels. Introduction of the one double bond to the fatty acid hydrocarbon chain increased P′H2O from 0.01 nmol⋅s–1⋅m–2⋅Pa–1 for stearic acid (C18:0, solid at 25°C) to over 1.21 nmol⋅s–1⋅m–2⋅Pa–1 for oleic acid (C18:1, liquid at 25°C). The double bond in the oleic acid hydrocarbon chain was presumed by the authors to have changed the packing of the lipid molecules at the air-water interface of the film-forming solution, producing a more expanded layer with greater molecular mobility. Decreasing the chain length of the saturated fatty acid increased P′H2O of the coating film. The emulsion films of stearic acid (C18:0) and lauric acid (C12:0) had P′H2O values of 0.01 and over 0.06 nmol⋅s–1⋅m–2⋅Pa–1, respectively. Fatty acids with shorter chain length have greater mobility and are consequently more permeable to water vapor. When a blend of stearic and palmitic acids was used in both film forming techniques (bilayer and emulsion techniques), the HPMC film coated with the lipids had P′H2O of 0.28 nmol⋅s–1⋅m–2⋅Pa–1 as compared to P′H2O value of 0.02 nmol⋅s–1⋅m–2⋅Pa–1 for the emulsion film, even though the lipid layer in the emulsion film was about one-tenth as thick as that of the coated film (90 g lipid⋅m–2 film vs. 8 g lipid⋅m–2 film; Kamper and Fennema 1984a). Since in the emulsion technique the fatty acids were allowed to orient at the air-water (film-forming solution) interface before allowing the lipid to solidify on the HPMC matrix, this greatly improved the moisture barrier properties of the coating film. The film was also very flexible and quite resistant to mechanical damage. Hagenmaier and Baker (1996) reported that the addition of protein or polysaccharide components into candelilla wax formulations to improve the gloss also decreased PO2 and increased PH2O of the film; apparently, the mixing of hydrophilic components with waxes may have detrimental effects on permeability of the composite film. Commodities treated with such coatings may be rendered anaerobic and still have high water loss.

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Avena-Bustillos and Krochta (1993) evaluated the water vapor barrier properties of edible films made of sodium or calcium caseinate and from emulsions of these proteins with acetylated monoglyceride, beeswax, and stearic acid. Adjustment of pH to the protein isoelectric point (pH 4.6), calcium ion crosslinking, and combined effects of calcium ascorbate buffer (pH 4.6) reduced PH2O of sodium caseinate films by 36%, 42%, and 43%, respectively. Incorporation of beeswax into sodium caseinate film was more effective in reducing PH2O than stearic acid and acetylated monoglyceride. Calcium caseinate-beeswax emulsion films had PH2O as low as 10% of permeance values reported for pure sodium caseinate films.

IV. FACTORS AFFECTING WATER LOSS, GAS EXCHANGE, AND MODIFICATION OF INTERNAL ATMOSPHERE OF COATED COMMODITIES Apart from the intrinsic physical barrier properties of coatings to gas exchange, the degree of modification of the internal atmosphere of the fruit is greatly dependent on character of cover of the skin by the surface coating, fruit physiological status, and environmental conditions. The latter have an impact on both metabolic activity of the fruit and permeability characteristics of the coating film. The main factors affecting gas exchange, modification of internal atmosphere and postharvest physiology of fruits and vegetables, which are relevant for the optimization of surface coatings, are discussed below. A diagrammatic model (Fig. 4.3) is presented, highlighting the interaction of physical and physiological factors affecting the performance of a surface coating in maintaining quality of coated commodities. A. Coating Formulation The physico-chemical properties of coating films affect their barrier properties to water vapor and gases (Section III) and may also be important in determining the potential of the coating formulation in blocking pores in the skin, both of which can be important in reducing the gas exchange of gases and water vapor of coated commodities (Fig. 4.3; Hagenmaier and Baker 1993a). Claypool and King (1941) observed that the type of wax used seemed to be more important than the film thickness in reducing water loss. Pear, sweet cherry, apricot and tomato lost less water when treated with wax formulations that had a higher proportion of low polarity waxes in

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Image Not Available

Fig. 4.3. Schematic diagram of the effects of surface coatings and environmental conditions on gas exchange and physiology of coated commodities. Arrow thickness is proportional to the magnitude of physical, compositional, or physiological effect. Arrows represent: the effects of coatings on skin permeance, modification of internal atmosphere and ripening (→) ; effects of relative humidity (RH; →) and temperature (T; →) on permeance attributes; effects of T rise on ripening and fermentation (→); and physical and/or physiological factors that may contribute to the manifestation of physiological disorders during cold storage of coated pear (→). Subscript “volatile” represents anaerobic volatiles that accompany fermentation. P′j = permeance to gas j; pij = internal partial pressure of gas j; rCO2 = rate of total respiratory CO2 production (rCO2(tot) from Fig. 4.4 and Eq. 6). Source: Amarante 1998.

their composition (high proportion of paraffin to carnauba wax; Claypool 1939; Claypool and King 1941). Hagenmaier and Baker (1993a) have shown that for waxes and resin coatings the chemical nature is far more important than coating thickness for CO2 exchange, while for water vapor, coating thickness seems as important as coating type. Grapefruit treated with polyethylene wax and shellac coatings (both with a concentration of 14% w/v) had P′CO2 depressed to 26.0% and 6.5% of control values, respectively. For both formulations, fruit treated with thicker coatings tended to have lower P′CO2, though these differences were not as large as those observed between coating formulations. Fruit treated with the thinnest shellac coating (0.9 g⋅m–2) had P′CO2 corresponding to 40% of fruit treated with

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the thickest polyethylene wax (5.3 g⋅m–2). Citrus fruit coated with shellac and resin had higher internal concentrations of CO2 and ethanol and a poorer flavor than fruit coated with polyethylene and carnauba waxes, as a result of over-restriction of gas exchange and fermentative metabolism on the former group (Hagenmaier and Baker 1993a, 1994b). The authors observed that for P′H2O, both coating thickness and coating type seemed to be important. Increasing the concentration of polyethylene wax and shellac from 5% to 25% (w/v) reduced the weight loss from 40% to 50% and from 30% to 40% of that of non-coated grapefruit, respectively, at 21°C and 50% RH. The lowest concentration of polyethylene wax (5% w/v) was as effective as the highest concentration of shellac (25% w/v) in reducing weight loss. Hagenmaier and Baker (1993a) found that the reduction of P′CO2 and P′H2O in coated citrus fruit was caused more by sealing pores than reducing gas movement through the cuticle. This indicates that changes in the formulation of hydrophobic coatings (waxes) to reduce pore blockage may improve coating performance. For example, wax emulsions that have larger globule size (macroemulsions) might not be particularly effective in sealing pores. In this way, the coatings may suppress water loss without the fruit becoming anaerobic. Resin-based coatings may penetrate and block skin pores that wax microemulsions do not, rendering the commodity anaerobic. Mannheim and Soffer (1996) reported that a mixture of carnauba wax and shellac (Primafresh 30) had the lowest PH2O and was the best coating for reducing citrus fruit water loss. This coating also did not excessively modify the fruit internal atmosphere (the fruit did not have very low O2 and high CO2 internal partial pressures), and did not result in excessive ethanol accumulation and development of off-flavors. The fruit treated with this coating also had very high scores for appearance. However, coating films made of carnauba wax emulsion or shellac had very low values for permeability to gases, and fruit treated with them had very low levels of internal O2 and high CO2 and ethanol, and the fruit developed off-flavors. These results contradict other published results which indicate that coatings made of waxes are more permeable to O2 and CO2 than coatings containing shellac. Interpretation of such effects remains difficult whilst commercial secrecy prevents the emulsification method and composition of coating formulations (including both solids and solvents contents) being reported. Hagenmaier and Baker (1993a) observed that increasing the alcohol content of a shellac formulation increased wettability by reducing coating surface tension. However, coating formulations with different alcohol content applied to the fruit with brushes resulted in fruit having

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virtually the same respiration, internal CO2, and weight loss. These authors observed that citrus fruit treated with water-based commercial wax coatings had higher respiration and skin permeance, and lower internal CO2 and weight loss, than those treated with shellac dissolved in 10–25% alcohol, despite both formulations having equivalent surface tension values. These results suggest that the surface tension is apparently less important than the chemical formulation (which determines the coating potential for blocking pores in the skin) for performance of the coating applied to fruit surface by brushing. Orange treated with water-based wax coating had higher weight loss than fruit treated with solvent-based wax, and while multiple coatings of solvent-based wax further reduced weight loss, the same was not observed for water-based wax (Davis and Hofmann 1973). Trout et al. (1953) observed higher P′CO2 for wax emulsion coatings than for corresponding alcoholic solutions. The results indicate that in water-based wax coatings the continuous aqueous soap phase can facilitate the diffusion of water and gases through wax emulsion coatings. Wax- and shellac-based coatings were more effective in reducing water loss and delaying ripening than polysaccharide-based coatings in several coated commodities. Sweet cherry coated with wax formulations had lower P′H2O, lower weight loss and better firmness retention than fruit coated with Semperfresh, 1.0% w/v (Drake et al. 1988). Guavas coated with Nature Seal emulsions (HPC), 2% or 4% w/v, lost more weight during storage than fruit coated with a carnauba wax (5% w/v), while the extent of ripening retardation was not greatly affected by coating type (McGuire and Hallman 1995). Also, guavas treated with palm oil (20% w/v) had lower weight loss than fruit treated with Semperfresh, 0.75% w/v (Mohamed et al. 1994). Nature Seal, 1 and 2% w/v, did not significantly reduce O2 internal concentration to delay ripening of avocado (Bender et al. 1993). Surface coatings made of carnauba wax and/or polyethylene wax mixed with shellac had more positive effects than Semperfresh in reducing weight loss and respiration of pear (Sümnü and Bayindirli 1994) and apple (Sümnü and Bayindirli 1995b) and in reducing the weight loss of mandarin (Bayindirli et al. 1995), resulting in better overall retention of quality. Baldwin et al. (1995b) reported that citrus fruit coated with a shellac-based coating had lower internal O2 and higher internal CO2 than fruit coated with a polysaccharide-based coating when the fruit were stored at 21°C. However, Drake et al. (1987) observed that apple coated with a commercial wax had similar, if not identical, quality attributes in comparison with control apple and that Semperfresh (1.0% w/v) coated apple had the best quality retention. In most instances, apple coated with the Semperfresh + wax showed qual-

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ity attributes similar to the singular use of Semperfresh. The authors concluded that the benefits of using a mixture of Semperfresh + wax could be derived from enhanced quality attributes coupled with a desirable fruit finish, but no reference was made to the main components and concentration in the wax formulation used. Protein-based coatings have shown some potential to reduce water loss and delay ripening of coated commodities. Park et al. (1994) reported delayed changes in color, softening, and weight loss in tomato treated with a corn zein coating (about 25 µm thick, made of zein, glycerin, and citric acid). Shelf life was increased by six days as evidenced by sensory evaluation. Avena-Bustillos et al. (1994) found that calcium caseinate and acetylated monoglyceride emulsion films were more effective than Semperfresh in reducing water loss from zucchini. Using response surface methodology, it was concluded that 0.9% calcium caseinate and 1.1% acetylated monoglyceride film would be the most effective for increasing zucchini shelf life by reducing P′H2O to ∼ 60% of the control. Composite coatings have been tested with the addition of hydrophobic components to protein- and polysaccharide-based coatings to improve their water vapor barrier properties. However, the addition of hydrophobic components has generally not substantially reduced P′H2O and these composite coatings tend to have low P′O2. Hagenmaier and Baker (1996) reported that grapefruit coated with composite formulations made by the addition of protein or polysaccharide components into candelilla wax had lower internal O2 and faster weight loss than when coated with candelilla wax alone. A protein content in the film above 20–30% (w/w total solids) resulted in increased internal CO2 of coated grapefruit and a marked elevation of ethanol content, a strong indication that anaerobic respiration had occurred. Baldwin et al. (1997) observed that the addition of soybean oil or carnauba wax to an HPC coating (Nature Seal) did not improve the water barrier effect of the coating on sweet cherry or cucumber. B. Coating Deposit Improving the amount of coating deposited on the skin by increasing the concentration of Semperfresh substantially delayed fruit ripening and reduced weight loss in pear (Sümnü and Bayindirli 1994), apple (Sümnü and Bayindirli 1995b), and apricot (Sümnü and Bayindirli 1995a) held at ∼ 20°C. Meheriuk and Lau (1988) reported a significant effect of coating concentration of Pro-long and Nutri-Save in delaying ripening and reducing physiological disorders of ‘Bartlett’ and ‘d’Anjou’ pears. Linear

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relationships were observed between coating concentration and firmness and color retention for both cultivars, and for reduction of core breakdown in ‘Bartlett’, and superficial scald in ‘d’Anjou’. Lau and Meheriuk (1994) also observed a better retention of firmness and acidity in apple with increasing concentrations of Pro-long or Nutri-Save. Improving the character of cover by increasing the concentration of wax in the coating formulation achieved more beneficial effects in reducing weight loss of mango (Yuniarti and Suhardi 1992) and citrus fruit (Hagenmaier and Baker 1994b) and in reducing weight loss, delaying ripening, and reducing the incidence of decay and of some physiological disorders in apple and pear (Smock 1935; Farooqi and Hall 1973). In tomato, increasing the concentration of chitosan- (El Ghaouth et al. 1992b) or corn zein-based (Park et al. 1994) coatings resulted in more substantial delay in ripening, as a result of a larger reduction of internal O2. Increasing coating concentration may delay ripening and improve some aspects of quality, but very high concentrations may predispose the commodity to physiological disorders and production of off-flavors (Fig. 4.3). Heavily coated apple may develop off-flavors, skin damage and decay, probably due to excessive restriction of gas exchange, rendering the fruit anaerobic and increasing the wastage after long-term storage, especially after exposure to high temperatures (Magness and Diehl 1924; Farooqi and Hall 1973). Banana (Ben-Yehoshua 1966) and orange (BenYehoshua 1967; Cohen et al. 1990) treated with very thick polyethylene wax developed off-flavors. Beneficial effects in delaying ripening and reducing weight loss have been reported for several commodities treated with low concentrations of coating materials. In orange, small increases in thickness of a polyethylene coating had the largest proportional effects in reducing weight loss (Ben-Yehoshua et al. 1970) and O2 internal concentration (BenYehoshua 1967; Ben-Yehoshua et al. 1970) and increasing CO2 internal concentration (Ben-Yehoshua 1967); smaller additional effects were observed at higher levels of coating thickness. Substantial effects in delaying ripening of banana were achieved with small increases in thickness of a polyethylene coating (Ben-Yehoshua 1966). Hagenmaier and Baker (1994a) observed that orange coated with a wax coating (50% oxidized polyethylene wax and 50% petroleum wax) had most of the beneficial effect in reducing weight loss by treating the fruit with a very thin coating film. They observed more than 50% reduction in weight loss by increasing the amount of coating from 0 to 10 mg/fruit (coating total solids of ∼ 0.5 g⋅m–2), with further smaller beneficial effects when increasing the amount of coating. Another 30% reduction in weight loss was achieved by increasing the amount of coating to 100 mg⋅fruit–1

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(coating total solids of ∼ 50 g⋅m–2). These results are in agreement with those reported by Amarante (1998), who observed more substantial reduction in skin permeance to gases, reduction of weight loss, and delay in ripening of pear having smooth skin by treating the fruit with small concentrations of a carnauba-based coating. If a similar response to low concentrations of wax coating can be achieved with other fruits, this treatment could represent a technology with a high potential to reduce water loss and delay ripening, without the excessive modification internal atmosphere composition that causes anaerobiosis. Coating avocado with a polyethylene wax did not provide a complete cover of the fruit, with several discontinuities of coating film being observed on the skin (Durand et al. 1984). As a result, coating had relatively little effect on internal CO2, O2, and C2H4 levels, with a small impact on delaying fruit softening. However, weight loss was reduced substantially by waxing. In pineapple, treating the fruit with increasing concentrations of a paraffin/polyethylene-based commercial coating formulation from 0 to 50% (v/v) resulted in a decreased level of chilling injury, enhanced fruit shell appearance, and reduced shell degreening rate, though weight loss was not reduced (Rohrbach and Paull 1982). Hagenmaier and Baker (1994b) observed that the effect of coating deposit on water loss and modification of internal atmosphere was dependent on coating formulation. Weight loss of citrus fruit decreased with increasing coating deposits of wax-based coatings (polyethylene and carnauba wax) but not for shellac and resin coatings. Orange and grapefruit coated with shellac and resin had lower internal O2 than fruit coated with waxes, but increasing the coating deposit on the fruit of different coatings had little effect on internal O2. This may be the result of a limited improvement of skin pore blockage (the main path for O2 exchange) by increasing the coating deposit. However, increasing the thickness of shellac and resin coating films substantially increased the internal CO2, while polyethylene wax and carnauba wax did not have this effect. This increase in internal CO2 of fruit coated with thicker films of shellac and resin may be the result of a decrease of P′CO2 and/or stimulation of fermentation. Since wax films have a higher P′CO2 than resin films and the wax coatings did not reduce the internal O2 content to very low levels, the fruit may still maintain aerobic respiration, avoiding substantial accumulation of CO2. C. Mode of Application Drake and Nelson (1990) investigated the differences in hot (60°C) and cold (0°C) drying techniques after waxing on ‘Delicious’ apple and noted

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no major difference in fruit quality. However, they reported that a 60°C wax-drying temperature on ‘Golden Delicious’ apple resulted in a more uniform color after shelf life, but that fruit had higher internal C2H4, higher weight loss, and increased firmness, possibly due to desiccation. For pear, Drake et al. (1991) observed that the half-cooling time for hot (60°C) and cold (0°C) dried waxed fruit in boxes were identical and equal to 17 h. However, waxed, hot-dried pear required an additional 21 h to equilibrate to the cold storage temperature (48 h and 69 h for cold and hot dried waxed fruit, respectively). As a result, after prolonged storage, waxed, cold-dried pear had better retention of color and firmness than waxed, hot-dried pear. The authors affirmed that the wax dried with cold air as fast as with hot air (Postharvest News and Information 1993). Hagenmaier and Baker (1993b) reported that washing citrus fruit with rotary brushes in the pack house could increase shrinkage rates by 50% to 150%. Fruit washed with polypropylene bristles (of medium stiffness) lost 15% more water than fruit washed with polyethylene bristles (of low stiffness). The water loss of orange washed with a sponge (to cause minimum abrasion) was not significantly different from non-washed fruit. They also observed that waxed fruit obtained from packhouses and cleaned with rotary brushes and waxed had the same shrinkage rates as those of non-washed controls. Ben-Yehoshua (1967) and Ben-Yehoshua et al. (1970) reported similar results, for orange coated with polyethylene wax in commercial packhouses. These figures show that in some commercial handling systems, waxing may be only overcoming the loss of skin resistance to water vapor caused by the abrasion during the washing, not providing any additional benefit to reduce water loss in comparison to field-run fruit. Thus, controlling the washing process, by using less abrasive cleaning methods before waxing, may be an important issue to consider in achieving the best benefit of waxing to reduce water loss. Coating orange (Ben-Yehoshua 1967) and avocado (Johnston and Banks 1998) by dipping resulted in a more substantial reduction of weight loss and a larger reduction of O2 internal concentration than spraying on brushes, as done commercially in packhouses. This may have been the result of abrasion on the brushes during the washing removing the natural waxes on the cuticle (Ben-Yehoshua 1967; BenYehoshua et al. 1970; Hagenmaier and Baker 1993b). Alternatively, it may have been that commercial spraying and brushing techniques created a coating that was thinner or less complete than the dipping method (Ben-Yehoshua et al. 1970; Cohen et al. 1990). More variable results were achieved between fruit coated commercially than for fruit coated by dipping (Johnston and Banks 1998). This may be due to more variation in

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the amount of coating received by each fruit and in the volume of coating application and/or speed of fruit flow through the packing line (Ben-Yehoshua et al. 1970). Therefore, the optimization of coating concentration in the laboratory should be followed by a trial under normal packhouse conditions, for adjustments on volume of coating application and speed of fruit flow through the packing line to achieve the best results. Mode of coating application can be critical, depending of the nature of the product skin, such as the presence of hairs or trichomes. In zucchini, dipping was 20% more effective than brushing in reducing water loss (Avena-Bustillos et al. 1994). This vegetable has trichomes, which can be removed by mechanical abrasion if coated by brushing, leaving wounds through which large amounts of water loss may occur. D. Relative Humidity Although most plastic films are not greatly affected by RH, films made from biological materials such as edible surface coatings may undergo substantial changes in their mechanical and barrier properties in high moisture conditions (Gontard et al. 1994, 1996). The permeability of coating films to gases and water vapor can increase with increases in RH as a result of presence of hydrophilic components in coating formulations that interact strongly with migrating water molecules. As the environmental RH is increased, water activity within the film matrix can be elevated substantially due to the strong sigmoidal shape of the sorption isotherms for polar hydrocolloids (Rico-Peña and Torres 1990). The elevation in sorbed moisture has a plasticizing effect that increases the diffusion constant for water vapor. Since permeability is determined by the diffusion constant and the solubility coefficient (Eq. 1), effective PH2O is greatly increased (Fig. 4.3). Similar but smaller changes in PO2 and PCO2 of the film may also be expected (mainly for CO2, which is more soluble in water), as high moisture content increases the solubility of these gases in the watery film and improves their diffusion by the plasticizing effect of moisture sorption (Fig. 4.3). Elson et al. (1985) reported that Nutri-Save films (a chitosan-based coating) were impermeable to O2 and CO2 at RH below 70%. The PO2 through MC-palmitic acid (Rico-Peña and Torres 1990), HPMC-stearic acid (Hagenmaier and Shaw 1990), whey protein (McHugh and Krochta 1994b), collagen (Lieberman and Gilbert 1973), wheat gluten (Gontard et al. 1996), and shellac (Hagenmaier and Shaw 1991a) coating films have likewise been reported to increase exponentially with increases in RH.

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Hagenmaier and Shaw (1991b) observed for different formulations of polyethylene wax that increasing the RH on the coated side of the film increased PH2O. The PH2O increased from two to five times when the RH on the coated side of the film was increased from 32% to 75%. For coating films made of lipids and cellulose ethers, P′H2O increased approximately five-fold as the RH on the low water vapor-pressure side of the film was elevated from 0 to 65%, while the high water vapor-pressure side had a RH of 97% (Kester and Fennema 1989e). Increasing the RH gradient (with 0% RH in the drier side) or keeping the same RH gradient (32% RH), but exposing the film to higher RH values increased P′H2O of the composite film (Kamper and Fennema 1984b). A greater increase in P′H2O occurred when one side of the coating was exposed to RH higher than 90% (Kamper and Fennema 1984b). Hagenmaier and Shaw (1992) reported that more polar coating films, such as shellac and resins, underwent a significant increase in PH2O by changing RH from 75% to 92% RH, while increasing RH from 50% to 85% had almost no effect on PO2. Gontard et al. (1996) observed that wheat gluten edible coatings substantially increased in PO2 and PCO2 with increases in RH. At RH higher than 50%, PO2 and PCO2 increased exponentially with increases in RH. However, at high RHs, the increment in PCO2 was larger than that observed for PO2. From 0% to 60% RH, the selectivity of coating films (expressed by the PCO2/PO2 ratio) was four to six and increased to 28.4 at 94.5% RH. This value corresponds to the ratio of CO2 and O2 solubility in water, since the free water of the film becomes the main medium for the transport of these gases through the coating. The selectivity values decreased when lipidic components were added to wheat gluten, as a result of a much higher depression of PCO2 than PO2. While PO2 (at 25°C and 91% RH) was reduced by 30% (from 0.98 to 0.69 fmol⋅m⋅s–1⋅m–2⋅Pa–1), PCO2 was reduced by 73% (from 24.50 to 6.61 fmol⋅m⋅s–1⋅m–2⋅Pa–1), by adding 30% beeswax to the wheat gluten formulation, with a consequent reduction in selectivity of the coating film from 25 to 9.6. However, PCO2 in any situation was several times higher than PO2. While these studies have demonstrated substantial effects of RH on permeability of coating films, the extent of the impact of environmental RH on permeance to water vapor and gases of fruits and vegetables treated with the coating films is not well defined. For apple (Lau and Meheriuk 1994) and pear (Meheriuk and Lau 1988) coated with NutriSave or Pro-long and held at 20°C, exposure to low (∼ 42%) or high (∼ 85% for apple and ∼ 63% for pear) RH had no consistent effect on ripening and fruit quality. The high RH values tested may have not been high enough to substantially affect the permeance properties of coated fruit. Smith and Stow (1984) did not observe any effect of high or low

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humidity on quality of apple coated with TAL Pro-long and held at 3.3°C. However, the authors did not report the RH values for the different humidity storage conditions. Green bell pepper fruit treated with cellulose- and protein-based edible coatings and stored at 10°C and 80–85% RH for 20 days did not have suppressed respiration rates or color changes, due to very minor modification of internal gas composition (Lerdthanangkul and Krochta 1996). The authors suggested that the absence of any effect was a result of the high permeance of fruit coated with hydrophilic coatings to gas exchange at the high RH of the storage environment, as well as the low respiration rates of pepper, especially at the temperature applied. Only the mineral-oil-based coating significantly reduced moisture loss, thus maintaining fruit firmness and prolonging fruit freshness. Baldwin et al. (1995b) reported a maximal internal CO2 concentration in shellac coated orange of approximately 9% after about one week storage at 21°C and 95% RH, which is considerably lower than the 16–18% CO2 reported by Hagenmaier and Baker (1993a) for shellac and resincoated orange of the same cultivar (‘Valencia’) stored for up to one week at 21°C and 50% RH. This may be the result of higher skin permeance of coated fruit left at higher RH, as observed for coating films (Hagenmaier and Shaw 1992). Overall, it seems that the effect of RH on coating films in intimate contact with the almost water-saturated surface of the commodity is not as large as that observed for the assessment made with coating films themselves. Perhaps, the water transfer from the fruit or vegetable sustains a high water activity in the coating film, with a resultant reduction in the effect of external RH of the air on gas exchange properties of the coating. On this basis, only for highly hydrophilic coatings in a situation of high water vapor deficit between the commodity and the air would substantial relative humidity-induced changes in permeance be anticipated. This issue deserves further investigation. E. Temperature 1. Effects on Barrier Properties of Coating Films. Increasing temperature increases film permeability (Fig. 4.3) due to its impact on diffusivity and solubility of the permeant gas in the coating material (Kester and Fennema 1986). The influence of temperature on gas or water vapor transfer across coating films usually conforms to the Arrhenius relationship (Kester and Fennema 1989a,b). An Arrhenius plot of logarithm of permeability against reciprocal of absolute temperature normally yields a straight line with the slope being proportional to activation energy (Ep)

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for permeation of permanent gases and water vapor. Gases migrating through polymeric films generally exhibit Ep of 12.6 to 62.8 kJ⋅mol–1 (Donhowe and Fennema 1994). Kester and Fennema (1989a) observed that lipid films having a good character of cover on a polar support (filter paper) had positive Ep for O2 at 0% RH. However, they reported unusually high Ep (115.1 kJ⋅mol–1) for acetyl monoglyceride. These authors attributed this to the low melting point range and heterogeneous composition of the acetoglyceride. As temperature is elevated, the proportion of lipid components in the fluid state increases markedly, causing a rapid increase in PO2. Permeability of beeswax to O2 also displayed a relatively strong dependence on temperature (Ep of 62.8 kJ⋅mol–1), likely due to the heterogeneous composition of the wax. Activation energies for stearyl alcohol and tristearin were 29.3 and 31.4 kJ⋅mol–1, respectively. Stearic acid had a negative Ep of –72.0 kJ⋅mol–1, perhaps as a result of increasing temperature causing crystalline expansion sufficient to close or lessen the size of interplatelet channels in the film through which O2 diffused. A bilayer film in which the first layer comprised a blend of MC, HPMC, and stearic and palmitic acids and the second layer was beeswax had an Ep for water vapor (with 100% RH gradient across the film) of 59.4 kJ⋅mol–1 (Kester and Fennema 1989e). Hagenmaier and Shaw (1991a) reported an Ep for water vapor of 50.2 kJ⋅mol–1 for shellac films cast from propanol (with 100% RH gradient across the film). Donhowe and Fennema (1993) observed a high dependence on temperature for PO2 (at 0% RH) and PH2O (with 100% RH gradient across the film) of wax films. Values of Ep for O2 for beeswax and microcrystalline wax (48.0 and 51.0 kJ⋅mol–1, respectively) were higher than for candelilla and carnauba wax (40.0 and 30.0 kJ⋅mol–1, respectively). Likewise, values for water vapor were higher for beeswax and microcrystalline wax (29.0 kJ⋅mol–1 for both waxes) than for candelilla and carnauba wax (17.0 and 21.0 kJ⋅mol–1, respectively). According to the authors, the larger Ep values of beeswax and microcrystalline wax are likely attributable to the larger amount of low-melting components of these waxes. As temperature increased, the liquid fraction would have increased to a greater degree in these waxes than in candelilla and carnauba waxes, and this, in turn, would favor increased transport of water vapor and gases. Permeability of shellac films has been shown to be dependent on temperature, with an Ep for O2 from 53.1 (shellac cast from ethanol) to 64.9 kJ⋅mol–1 (for water-soluble shellac) at 55% RH (Hagenmaier and Shaw 1991a). Hagenmaier and Baker (1996) reported an Ep for PO2 of a microemulsion made of candelilla wax with 20% gelatin of 19.7 kJ⋅mol–1

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at 75% RH. Films made with microemulsions of polyethylene wax had an average Ep for O2 of 19.7 kJ⋅mol–1 at 50% RH (Hagenmaier and Shaw 1991b). These values of RH are higher than those normally used to assess the barrier properties of coating films to O2 (0% RH; McHugh and Krochta 1994a). Increasing RH, mainly for coatings made of hydrophilic components, may increase the permeability to gases (Section IVD). The impact of different RH on Ep for coating films of different levels of hydrophobicity has not been reported and should be more extensively investigated. Films made with microemulsions of waxes may have lower Ep for O2 relative to films made with molten waxes or resins due to the more porous nature of films formed from a globular formulation. Kamper and Fennema (1984b) and Kester and Fennema (1989e) observed a decrease in moisture barrier properties of composite films made from lipids and polysaccharides when temperature was reduced to 4–5°C. According to the authors this may have been the result of increased film hydration at lower temperatures, which would favor increased permeability. Alternatively, low temperatures may fracture the film as a result of lipid rigidity coupled with lipid contraction. This may have detrimental effects on the potential of such coatings to reduce water loss of commodities subjected to hot air drying followed immediately by refrigeration (Hernandez 1994). Kester and Fennema (1989b) reported that when the dependence on temperature of P′H2O was examined using a polar support (filter paper), the regression lines of Arrhenius plots for water vapor transport were negative for most of the films, thus yielding negative Ep. The polar filter paper exhibited a negative Ep when in contact with water vapor, thereby decreasing the overall Ep of the composite film. As temperature is increased, the equilibrium amount of sorbed water at a given RH is reduced: the water solubility coefficient decreases as temperature rises. According to these authors, since sorbed water acts as a plasticizer, a decline in its content at elevated temperatures tended to lessen the temperature-induced rise in the effective diffusion constant: the overall Ep for water vapor was reduced in magnitude. This shows that when a hydrophilic structural polymer is embedded in a lipid film, the temperature dependence of P′H2O is very strongly influenced by the moisture sorption behavior of the polar component. For such composite films, raising temperature should exert almost no reduction in the barrier properties of the films to water vapor. Gontard et al. (1993) investigated the interaction of film water activity and temperature on mechanical and P′H2O of a wheat gluten film. The plasticizing effect of water was highly temperature-dependent. At low levels of water activity, increasing the water content enhanced

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mechanical properties of the film at all temperatures tested (characterized mainly by an increase of puncture strength) as a result of the plasticizing effects of water in the gluten. However, a sharp decrease in puncture strength, elasticity, and an increase in extensibility and P′H2O were observed at 5°C, 30°C, and 50°C for respective water contents of 30%, 15%, and 5%. According to these authors, this was related to disruptive water-polymer hydrogen bonding and glass-to-rubber transition. Plasticization by water affects the glass transition temperature of amorphous or partially crystallized hydrophilic compounds such as gluten, resulting in a drop in the glass transition temperature. Increasing film water content results in a loss of cohesiveness and elasticity of the gluten protein, since under this condition, water-polymer interactions probably develop in detriment to polymer-polymer bonds, resulting in a rupture of the inter-chain bonds. This increases free volume, allowing increased backbone chain segmental mobility and reducing the glass transition temperature, with a resultant decrease of mechanical and water vapor barrier properties of the coating film. 2. Effects on Commodity Physiology. Temperature also affects the rate of physiological processes in harvested crops. Increases in respiration of fruits and vegetables associated with temperature are often described as power (Dadzie et al. 1993; Yearsley et al. 1997a) or exponential (Cameron et al. 1994) functions of temperature, though there is some indication that the relationship flattens off towards high temperatures (Banks et al. 1997a; Yearsley et al. 1997a). Yearsley et al. (1997a) reported a Q10 ([rate of O2 uptake at (T+10°C)] / [rate of O2 uptake at T]; T = temperature in °C) of 2.0–2.5 for apple across temperatures from 0°C to about 25°C. Banks et al. (1997a) observed that raising temperature from 0°C to 25°C had a larger effect in increasing respiration rate than in increasing permeance of apple treated with different concentrations of a carnauba/ shellac-based coating. While respiration increased approximately sevenfold across the range of experimental temperatures, skin permeance to gases only increased by a factor of about two. Therefore, the extent of internal atmosphere modification increased progressively with increases in temperature, with an increasing proportion of fruit fermenting at high coating concentrations (low skin permeance) and high temperature. These results show that for commodities treated with thick coating films, the increase in O2 demanded for respiration may not be accompanied by a comparable increase in P′O2 with increasing temperature. This may result in a drop of internal partial pressure of O2 (p Oi 2) below the internal lower O2 limit, resulting in fermentation and accumulation

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of anaerobic volatiles. Raising temperature would be expected to exert smaller increases in P′O2 than in P′CO2, since CO2 moves more readily through the cuticle than O2, and to have a much smaller effect on P′H2O, which moves much more readily through the cuticle (Fig. 4.3). On this i basis, pCO would not be expected to increase less than p Oi 2 would be 2 depressed, at least whilst the coated commodity is respiring aerobically. For successful commercial application of a particular coating, the character of cover needs to be tailored to achieve a P′O2 required for the extremes of environmental temperature conditions to which the commodity will be exposed. F. Fruit-Coating Interaction Cameron and Reid (1982) reported great differences in natural permeance to gas exchange between commodities. As an example, P′O2 of banana and orange were about 9 to 10 times higher than P′O2 of tomato and apple. Values of P′H2O of banana were 7, 25, and up to 175 times higher than those of tomato, orange, and apple, respectively. Dadzie (1992) found that skin permeance of apple to gas diffusion differed substantially among cultivars, with the cultivar having the highest value being about three times more permeable than the cultivar having the lowest value. However, he reported between two- and sevenfold variation in skin permeance for individual fruit of the same cultivar. Similar variability in P′H2O has been observed between cultivars and between fruit of the same cultivar (Maguire 1998). These differences may be due to anatomical differences such as size of intercellular spaces near the fruit surface; size, number and distribution of functional pores (stomata and lenticels) on the skin; and nature, thickness and imperfection (cracks) of wax deposits of the cuticle. These differences in skin permeance between commodities and the variability observed among cultivars of the same commodity and among fruit of the same cultivar, imply that the variable nature and barrier properties of the commodity skin should not be overlooked in the optimization of surface coatings. This, coupled with the differences in respiration rate and surface area, can cause large variations in product internal atmosphere composition (Amarante 1998). As shown in Section II, surface coatings mainly exert their effects on skin permeance to gases by blocking a greater or lesser proportion of the pores on the fruit surface (Banks et al. 1993a, 1997b; Hagenmaier and Baker 1993a; Amarante 1998). Therefore, it might be expected that commodities with different skin characteristics might have very distinct types of interaction with a surface coating (Figs. 4.1 and 4.2). For coated

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commodities held in the same environmental conditions, this may result in some fruit having a very small modification of internal atmosphere composition while others become anaerobic. The small modification of fruit internal atmosphere may be the result of an ineffective cover of pores on the skin and/or a low respiration rate. Although these differences would be small at low temperatures, a much larger variation between individual fruit could occur at higher temperatures due to an increase in respiration rate. Apples of different cultivars have been shown to vary considerably in their reaction to skin coatings (Trout et al. 1953; Meheriuk and Porritt 1972; Banks 1984c). Trout et al. (1953) observed a great variability of the internal atmosphere composition between coated fruit of different cultivars and among coated fruit of the same cultivar. They attributed this to variability in skin permeance and, to a lesser extent, the variability in respiration rate between fruit. Larger modifications of internal atmosphere composition were expected for fruit having naturally low skin permeance and high respiration rate before coating. Claypool and King (1941) observed a greater variability in coating thickness in fruit having a rough skin, such as pear and nectarine, than in fruit with smooth skin, such as tomato. In the first group, the presence of large lenticels and cracks on the skin made the coverage of these pores very difficult, with coatings providing variable results in skin coverage and being less effective in reducing water loss. Similar results were reported by Amarante (1998), who observed that, for pear cultivars with lignified cells on the skin, treating fruit with increasing wax coating concentration resulted in minor reductions of P′H2O, P′O2 and P′CO2, while cultivars with nonlignified cells in the skin had substantial reductions of P′H2O, P′CO2, and P′O2 with small increases in coating concentration. Apple cultivars with a high proportion of open calyx have shown limited benefits from coatings, since highly variable results were achieved by exacerbating natural variability of internal gas composition between fruit (Trout et al. 1953). Small reductions in pOi 2 have only minor impact on respiration and C2H4 production, but progressively greater suppression of both processes is achieved as pOi 2 is depressed more and more (Dadzie et al. 1996; Yearsley et al. 1996; Amarante 1998), as shown in Section VA. The application of a surface coating thick enough to block the pores on the skin and reduce pOi 2 to low levels does not produce perfectly uniform pOi 2 and can result in a great variability in ripening between coated fruit of the same batch, since small differences in pOi 2 at these low levels of O2 may result in large differences in ripening rates (Banks et al. 1997b; Amarante 1998). This can result in variable response of individual fruit to similar coating treatments (Amarante 1998) as discussed in Section VB2.

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V. POSTHARVEST PHYSIOLOGY AND QUALITY OF COATED COMMODITIES A. Physiological Basis for the CA/MA Storage of Fruits and Vegetables Surface coatings can delay ripening of fruits by modifying their internal atmospheres, achieving similar beneficial effects of CA/MA storage. Generally, the effects of reduced O2 and/or elevated CO2 on reducing respiration rate and processes linked to respiration, as well as C2H4 synthesis and action, have been assumed to be the primary reasons for the beneficial effects of CA/MA on fruits and vegetables (Kader 1989; Kader et al. 1989). 1. Respiration. The majority of literature concerning optimization of CA/MA storage has focused on the effects of low O2 levels, since smaller and less consistent effects of high CO2 have been reported on respiratory metabolism of fruits and vegetables in comparison to that observed for low O2 (Boersig et al. 1988; Dadzie et al. 1996; Yearsley et al. 1996; Amarante 1998). The relationship between respiratory oxidative CO2 production [rCO2(ox), which is equal to respiratory O2 consumption (rO2) when O2 is non-limiting and the respiratory quotient [RQ] is ∼1] and either internal (pOi 2, Fig. 4.4) or external partial pressure of O2 (p eO2) of fruits and vegetables has been modelled using the Michaelis-Menten equation, comprising a gradual decrease of CO2 production at relatively high O2, but becoming steeper as O2 approaches 0 Pa (Fig. 4.4; Solomos 1982, 1985; Andrich et al. 1991; Banks et al. 1993a; Cameron et al. 1995; Dadzie et al. 1996; Peppelenbos et al. 1996; Peppelenbos and van’t Leven 1996; Amarante et al. 1997a,b; Amarante 1998). However, at very low O2 partial pressures, there is a risk of inducing fermentation (Peppelenbos et al. 1996; Peppelenbos and van’t Leven 1996; Yearsley et al. 1996; Amarante 1998), which leads to additional CO2 production (Fig. 4.4). Therefore, modelling the total respiration rate (rCO2(tot)) of a product needs a distinction between CO2 produced by oxidative metabolism (rCO2(ox)) and that produced by fermentative metabolism (rCO2(fer), Fig. 4.4): rCO2(tot) = rCO2(ox) + rCO2( fer)

[6]

Beaudry et al. (1993), Andrich et al. (1994), Yearsley et al. (1996), and Amarante (1998) used exponential functions to describe rCO2(fer), while Banks et al. (1993a) and Peppelenbos et al. (1993) used different functions that modelled O2 as an inhibitor of fermentative CO2 production.

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Fig. 4.4. Effect of internal partial pressure of O2 (pOi 2) on relative respiration rate (relrr, expressed as a proportion of respiration rate maximum when O2 is not limiting) for oxidative [rCO2(ox)], fermentative [rCO2(fer)], and total [rCO2(tot)] CO2 production. Arrows indicate internal “Anaerobic Compensation Point” (ACPi ) and internal “Fermentation Threshold” (FT i).

Under anaerobic conditions, the energy produced during carbon metabolism from the Krebs cycle is reduced, such that glycolysis contributes an increasing fraction as fermentative carbon flux increases (Kader 1995). Pyruvic acid is no longer oxidized but is reduced to lactate and/or decarboxylated to form acetaldehyde, CO2, and, ultimately, ethanol; this results in development of off-flavors and tissue breakdown after long-term stress (Kader 1986). The major function of the fermentative metabolism is to utilize NADH and pyruvate when electron transport and oxidative phosphorylation are inhibited, so that glycolysis can continue. This allows some production of ATP through substrate phosphorylation, which permits the plant tissue to survive, at least temporarily (Kader 1995). Ethanol is usually the major product of the pathway in low O2-stressed fruit (Ke et al. 1991; Ke and Kader 1992). In some plant tissues, ethanol reacts with acetyl coenzyme A to produce ethyl acetate, catalyzed by the enzyme alcohol acyltransferase. Ethyl acetate accumulated in CA-treated pear and strawberry, but not in avocado and lettuce (Ke et al. 1993). Some products, such as carrot, lettuce, and avocado, may also divert pyruvate to produce lactate (Leshuk and Salveit 1991; Ke et al. 1993). Elevated CO2 levels have also been reported to induce fermentation (Ke et al. 1990, 1994), and the combination of low O2 (0.25%) and

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high CO2 (80%) seems to have an additive effect on acetaldehyde and ethanol accumulation in avocado and pear (Ke et al. 1993). Increasing CO2 levels can reduce respiration rate, depending on the crop and partial pressure of CO2. An inhibition of rCO2(ox) by increased CO2 concentrations has been reported for asparagus, broccoli, mungbean sprout, and cut chicory, but not for apple (Peppelenbos et al. 1996; Peppelenbos and van’t Leven 1996). Evidence that elevated CO2 has little or no effect in reducing respiration rate has been reported for banana (Young et al. 1962) and mushroom (Peppelenbos et al. 1993). Joles et al. (1994) reported that partial pressures of CO2 < 17 kPa did not affect O2 uptake for raspberry, and Beaudry (1993) that partial pressures of CO2 > 20 kPa resulted in only a small reduction in O2 uptake of blueberry. Apple exposed to 5 kPa CO2 for four days (Peppelenbos et al. 1996; Peppelenbos and van’t Leven 1996) or to 8 kPa CO2 for up to seven days (Yearsley et al. 1997b) at ∼ 20°C did not undergo a suppression in aerobic respiration. However, by modelling data from the literature (data from Fidler and North 1967) on the respiratory effects of O2 and CO2 of apple stored in CA for 50–200 days, Peppelenbos et al. (1996) identified an effect of CO2 on O2 consumption. This may indicate that long-term stress of high CO2 at low temperature (and also low O2) may exert different effects on respiratory metabolism to short-term stress at ambient temperature. Presumably, this is an issue that should be considered when designing experiments to optimize CA/MA storage. Since the main goal of these techniques is to extend the storage period at low temperatures, the long exposure to the imposed atmospheric conditions may have a stronger influence on metabolic processes than short-term exposure. More work on the effects of low O2/high CO2 for the optimization of CA/MA storage conditions should be done after long-term exposure at low temperature to different concentrations of these gases. Yearsley et al. (1997b) did not observe an effect of external CO2 levels between 0 and 8 kPa on the internal lower O2 limit in apple at 20°C. However, the authors reported an increase in the internal lower O2 limit at 0°C, when the external CO2 level was increased from 0 to 8 kPa, that they attributed to a higher solubility of CO2 at the lower temperature. These results indicate the possibility of a lower tolerance of the produce to high levels of CO2 during low temperature CA storage, affecting the internal lower O2 limit and commodity quality, especially after longterm exposure to these atmospheres. 2. Ethylene Biosynthesis and Action. Tissue exposure to low levels of O2 and/or high levels of CO2 suppresses C2H4 biosynthesis and action of climacteric fruits. Reducing O2 levels decreases C2H4 biosynthesis by

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fresh fruits and vegetables and reduces their sensitivity to C2H4 (Kader 1986). Under anoxic conditions, conversion of 1-aminocyclopropane-1carboxylic acid (ACC) to C2H4 can be completely inhibited because O2 is a co-substrate in the oxidation of ACC to C2H4 by ACC oxidase (ACCO; Yang 1985). Elevated CO2 levels can enhance, reduce, or have no effect on C2H4 biosynthesis in fruit tissue, depending upon the tissue and amount of CO2 present (Kader 1986). The increase in C2H4 production by some commodities during and/or following exposure to high CO2 may be the result of physiological injury. In general, non-stressful CO2enriched atmospheres have been reported to reduce C2H4 biosynthesis in numerous climacteric fruits (Li et al. 1983; Kerbel et al. 1988; Kubo et al. 1990; Chavez-Franco and Kader 1993; Gorny and Kader 1996a,b). The combination of low O2 and elevated CO2 can have a synergistic effect in suppressing C2H4 biosynthesis (Gorny and Kader 1996a). The activity of the enzymes involved in C2H4 biosynthesis, ACC synthase (ACC-S) and ACC-O can also be affected by reduced O2 and/or elevated CO2 concentrations (Yip et al. 1988; Poneleit and Dilley 1993; Gorny and Kader 1996a,b). Gorny and Kader (1996b) reported that autocatalytic (System II) C2H4 biosynthesis in climacteric apple was inhibited by short-term exposure (four days at 20°C) to reduced O2 (0.25%) or elevated CO2 (20%). Reduced O2 and/or elevated CO2 concentrations may inhibit C2H4 biosynthesis by impeding the binding of C2H4 to the receptor (Burg and Burg 1967) blocking System II upregulation of C2H4 biosynthesis (Gorny and Kader 1996a). Gorny and Kader (1996a) also reported that the non-autocatalytic (System I) C2H4 biosynthesis in preclimacteric apple was reduced by low O2 and/or high CO2 (2% O2, 5% CO2, and 2% O2 + 5% CO2) during long-term cold storage (for up to four months at 0°C). As observed for respiration, the relationship between rate of C2H4 production and O2 concentration is reasonably described by a MichaelisMenten type hyperbolic curve, with a much greater reduction on C2H4 production at lower O2 partial pressures. The Km with respect to O2 in the external atmosphere depends on the commodity, having been estimated as 1.7–2.2% O2 in banana (Banks 1985a; Elyatem et al. 1994) and 1.2–1.3% O2 in apple (Banks et al. 1985; Bufler and Streif 1986). For intact apple, Dadzie et al. (1996) reported a Km of ∼ 1.8 kPa for pOi 2. B. Postharvest Physiology of Coated Commodities 1. Coating Effects on Ripening of Fruits and Vegetables. Surface coatings have been reported to reduce respiration rate (Smock 1935; BenYehoshua 1966; Blake 1966; Farooqui and Hall 1973; Banks 1984a,

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1985b; Elson et al. 1985; Drake et al. 1987; Kerbel et al. 1989; El Ghaouth et al. 1991a,b, 1992b; Miszczak 1994; Sümnü and Bayindirli 1994, 1995a; Bayindirli et al. 1995; Díaz-Sobac et al. 1996), and C2H4 evolution (Meheriuk and Porritt 1972; Farooqui and Hall 1973; Banks 1984a, 1985b; Elson et al. 1985; Drake et al. 1987; Kerbel et al. 1989; El Ghaouth et al. 1992b; Miszczak 1994; Bauchot et al. 1995b) of several commodities held at ambient temperatures. Greater suppression of respiration rate (Smock 1935; Ben-Yehoshua 1966; Blake 1966; Farooqui and Hall 1973; Elson et al. 1985; Kerbel et al. 1989; Sümnü and Bayindirli 1994), C2H4 evolution (Farooqui and Hall 1973; Elson et al. 1985; Kerbel et al. 1989) and ripening (Magness and Diehl 1924; Ben-Yehoshua 1966; Farooqi and Hall 1973; Banks 1984c; Smith and Stow 1984; Elson et al. 1985; Meheriuk and Lau 1988; Kerbel et al. 1989; Castrillo and Bermudez 1992; Lau and Meheriuk 1994; Sümnü and Bayindirli 1995b) were observed by increasing the concentration of total solids in the coating formulation, and also with coating formulations having low permeance to gases and with more effective cover of pores in the skin. However, commodities treated with very high concentrations of wax (Magness and Diehl 1924; Smock 1935; Trout et al. 1953; Ben-Yehoshua 1966; Cohen et al. 1990; Edward and Blennerhassett 1990, 1994), polysaccharide(Van Zyl et al. 1987) or protein-based (Park et al. 1994) surface coatings may have excessive restriction of gas exchange through the skin, resulting in anaerobiosis and development of off-flavors when the fruit are exposed to high temperatures. Coatings are reported to have limited effects in delaying ripening of coated commodities during cold storage (Magness and Diehl 1924; Trout et al. 1953; Banks 1984c; Smith and Stow 1984; Elson et al. 1985; Meheriuk and Lau 1988; Kerbel et al. 1989; Santerre et al. 1989; Drake and Nelson 1990; Köksal et al. 1994; Lau and Meheriuk 1994; Miszczak 1994), with more substantial results being achieved during shelf life (Magness and Diehl 1924; Banks 1984a,c, 1985b; Elson et al. 1985; Erbil and Muftugil 1986; Dhalla and Hanson 1988; Meheriuk and Lau 1988; Kerbel et al. 1989; Drake and Nelson 1990; Meheriuk 1990; El Ghaouth et al. 1991a,b, 1992b; Castrillo and Bermudez 1992; Yuniarti and Suhardi 1992; Lau and Meheriuk 1994; Sümnü and Bayindirli 1994, 1995a,b; Bayindirli et al. 1995; Díaz-Sobac et al. 1996). Less significant differences in ripening between coated and non-coated fruit were observed for fruit treated in a more advanced maturity/ripening stage (Banks 1985b; Kerbel et al. 1989; Drake and Nelson 1990; Sümnü and Bayindirli 1995b) and with polysaccharide- instead of lipid- and resin-based coatings (Drake et al. 1987, 1988; Sümnü and Bayindirli 1994). The delay in ripening was largely dependent on commodity (Elson et al. 1985; Van

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Zyl et al. 1987; Köksal et al. 1994) and cultivar of a given commodity (Magness and Diehl 1924; Trout et al. 1953; Meheriuk and Porritt 1972; Elson et al. 1985; Drake and Nelson 1990; Lau and Meheriuk 1994). Coatings have been reported to produce variable results in delaying softening. In some cases, this may reflect differences in water loss between coated and non-coated commodities. Trout et al. (1953) observed that sensory tests often indicated that coated apples were crisper and juicier than non-coated fruit, but that this difference was frequently not reflected in penetrometer values, with non-coated fruit being firmer than coated ones. According to the authors, this was probably due to the greater water loss from non-coated than from coated fruit. In general, wilting was found to toughen the flesh and cause higher penetrometer readings that did not then truly reflect the stage of ripening. Therefore, the likelihood of experimental conditions causing excessive water loss should be taken when interpreting results presented in the literature about the effects of coatings in delaying softening. Methods other than penetrometer readings to assess flesh texture should be explored. Differences in effects on firmness, skin color, soluble solids, and acidity among commodities and between cultivars of the same commodity treated with the same coating formulation presumably relate to variation in initial quality (maturity/ripening stage), respiration and ripening rates, and character of skin cover by the surface coating. Items with high respiration might be expected to have larger modification of internal atmosphere when coated, leading to more substantial reduction in respiration, C2H4 biosynthesis and action and, therefore, potential to achieve a greater relative delay in ripening. Products on which the character of cover results in low skin permeance to gases would be affected similarly. On the other hand, the scope to delay ripening by coating following exposure to higher temperatures would be constrained by the inherently rapid rate of ripening under these conditions, despite the substantial modification of internal atmosphere achieved. Coatings have been reported to exert more substantial effects in slowing change in skin color than in retarding loss of firmness at ambient temperatures (Smith and Stow 1984; Köksal et al. 1994; McGuire and Hallman 1995; Amarante et al. 1997a,b; Amarante 1998). Guava treated with HPC (2% or 4% w/v) or a carnauba wax (5% w/v) fails to develop a full color during shelf life (McGuire and Hallman 1995). Similarly, coated pear treated with a very thick coating layer may remain green while still being able to soften (Smock 1935; Amarante et al. 1997a,b; Amarante 1998). This seems to be the result of differences in responses to internal atmosphere modification between physiological ripening processes in some coated commodities (Amarante 1998).

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Coatings have been reported to improve retention of vitamin C in apple (Sümnü and Bayindirli 1995b), pear (Sümnü and Bayindirli 1994), apricot (Sümnü and Bayindirli 1995a), peach (Erbil and Muftugil 1986), mango (Dhalla and Hanson 1988), lime (Paredes-López et al. 1974), and mandarin (Bayindirli et al. 1995) during shelf life. The decrease in ascorbic acid loss by means of coatings is presumably due to reduced internal O2 retarding ascorbic acid oxidation (Sümnü and Bayindirli 1995a,b). 2. Mode of Action of Surface Coatings in Delaying Ripening. Research on the use of surface coatings to maintain quality in harvested fruits and vegetables through their effects on internal atmosphere began in the 1920s (Magness and Diehl 1924). About the same time, Kidd and West started their pioneering work in UK on controlled atmosphere storage of apple and pear (Kidd et al. 1927). The initial work focused on inhibitory effects of reduced concentrations of O2 and elevated concentrations of CO2 on ripening, representing the start point for all subsequent research on the physiological and biochemical basis of CA/MA storage of fruits and vegetables. The effects of low O2 and high CO2 in suppressing the main metabolic processes related to ripening are well known (Section VA). However, there is some contradiction in the literature about the main mechanism by which coatings achieve their effects. Are these effects exerted by reducing O2, by increasing CO2, or both? Magness and Diehl (1924) first reported that coating apple with paraffin wax or oil reduced skin permeance to gases, resulting in a marked increase in CO2 and a decrease of O2 within the tissue. The authors observed for apple stored at 0°C an abundance of O2 (pOi 2 > 10 kPa, except for heavily oiled fruit, which had ≅ 3 kPa O2), and a large increase of i internal partial pressure of CO2 (p CO > 10 kPa, compared to about 2 kPa 2 for the controls) in coated fruit. As a result of substantial accumulation of internal CO2, the authors suggested that elevated p iCO2, instead of low pOi 2, was responsible for suppressing respiration rate and delaying softening and skin color change at 0°C. Kerbel et al. (1989) and Lau and Yasi tremski (1991) also observed substantially higher p CO in coated apple 2 (4 to 6 kPa as against 1 to 2 kPa for the controls) immediately after removal from storage at 0°C (with fruit treated with higher concentrations of coatings having higher p iCO2), while the pOi 2 was no less than i 15–16 kPa. In pear, Amarante (1998) also reported higher p CO for coated 2 pear stored at 0°C, particularly for fruit treated with higher concentrations of coatings (up to 5 kPa compared to 1 kPa for the controls), while pOi 2 was no less than 11–12 kPa. Substantial reductions in respiration rate are achieved by reducing pOi 2 below 4–5 kPa for apple (Dadzie et al. 1996; Yearsley et al. 1996) and pear (Amarante et al. 1997a,b; Amarante 1998).

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These pOi 2 values are much lower than those pOi 2 values reported above for coated apple and pear left in cold storage and indicate that coating effects on respiration and ripening during low temperature storage occur i mainly by increasing p CO . 2 Gas solubilities in liquids are highly dependent on temperature. The solubility in water of CO2 is more than 25 times higher than that of O2 at 20°C, and this difference increases at lower temperature values since by decreasing temperature there is a greater increase in CO2 than in O2 i solubility (Foust et al. 1980). This implies that for the same pOi 2 and p CO 2 in the intercellular gas phase, there would be much higher concentrations of CO2 than of O2 dissolved in the cell sap of fruit stored at low i temperature. The increased p CO in the intercellular space at low tem2 perature observed for coated fruit combined with the greater solubility of CO2 in the cell sap in such conditions would result in a much higher increase of CO2 concentration in the liquid phase. This supports the notion that the increase of CO2 concentration in the liquid phase could make a substantive contribution to delay in ripening of coated fruit at low temperatures. Since the early work done by Magness and Diehl (1924), some authors i have continued to focus on increase of p CO in coated commodities held 2 at ambient temperatures as the main way by which coatings delay ripening (Smock 1935; Meheriuk and Porritt 1972; Smith and Stow 1984; Drake et al. 1987; Smith et al. 1987; Drake and Nelson 1990; Miszczak 1994). Although an effect of high CO2 seems likely at low temperatures, this does not appear to be the case at high temperatures. Smith and Stow (1984) reported that ‘Cox’s Orange Pippin’ apple treated at harvest with Pro-long (1.25% w/v) had a significant retention of skin color but not of firmness after 4–5 months in cold storage. More substantial retention of firmness and skin color was achieved for fruit treated with higher coating concentrations (from 1% up to 4% w/v) and, relative to their respective controls, held at higher temperatures (from 3.5°C up to 18°C), as a result of greater modification of fruit internal atmosphere. These authors also reported a larger modification in internal CO2 than in internal O2 at higher temperatures, suggesting that coatings delay skin color change and softening mainly by increasing CO2 instead of depressing O2. However, Banks (1984c), working with the same cultivar and similar concentrations of Pro-long (from 1% up to 3% w/v), reported much larger reductions of internal O2 concentrations than those reported by Smith and Stow (1984) for fruit held at similar temperatures. Smith et al. (1987) held apple at 18°C and exposed them to i CA conditions to create pCO similar to those achieved by coating (8.5 kPa 2 CO2 for fruit coated with 3% Pro-long and 7.9 kPa CO2 for CA stored

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fruit). There was a larger inhibition of color change in the coated fruit rather than in CA stored fruit, while firmness was little affected by either treatment in comparison to the controls but the authors did not assess pOi 2, which might have explained the differences in fruit skin color between treatments. Instead, they suggested that coatings might have interfered with color change by a mechanism other than by modifying fruit internal atmosphere. For example, the coating may have penetrated through the skin and directly interfered with the degradation process of chlorophyll in the chloroplasts. However, Bhardwaj et al. (1984) did not observe a migration of a similar coating formulation (TAL Pro-long containing [14C]-labeled sucrose esters of fatty acids in its composition) into the pulp of coated banana, apple, or pear. Therefore, the mode of action of coatings in retarding ripening seems unlikely to be dependent upon migration into the pulpy tissue. Instead, the major effect of coating on ripening seems to result from blockage of pores on the skin, physically impeding gaseous diffusion, modifying the commodity internal atmosphere (Section II). Microscopic evidence of such pore blockage has been reported for banana coated with TAL Pro-long labelled with aurothioglucose; the presence of gold in stomatal apertures on areas of coated skin was demonstrated using energy dispersive X-ray analysis (Banks 1984b). Magness and Diehl (1924) reported that, for coated apple held at temperatures of 18°C and 26.5°C, the reduction in pOi 2 (from about 14 kPa to i 2 kPa) was greater than the increase of pCO (from about 9 to 14 kPa). Sim2 ilar results have been reported for coated apple by others (Magness and Diehl 1924; Trout et al. 1953; Banks 1984c; Kerbel et al. 1989; Banks et i al. 1997b). A larger modification of pOi 2 than of p CO has also been 2 reported for coated pear (Amarante et al. 1997a,b; Amarante 1998), banana (Ben-Yehoshua 1966; Banks 1984a, 1985b), orange (Ben-Yehoshua 1967; Nisperos-Carriedo et al. 1990; Hagenmaier and Baker 1993c, 1994b, 1995), grapefruit (Hagenmaier and Baker 1994a,b, 1996), and tomato (El Ghaouth et al. 1992b) held at ambient temperatures. The i and pOi 2 can be seen in Fig. 4.5 for a coated relationship between p CO 2 commodity held at ambient temperatures. Increasing the amount of coating deposit on the commodity surface results in a larger reduction i of pOi 2 than an increase of p CO as long as the level of O2 is not so low as 2 to induce fermentation (Banks et al. 1997a,b; Amarante 1998). The larger i modification of pOi 2 than of p CO at high temperatures is the result of 2 increased respiration rate coupled with the limited and differential permeance to these gases in coated fruit skins (Section II). However, when pOi 2 is reduced below the internal lower O2 limit (LOLi) and the commodity starts fermenting (Fig. 4.5), the differential effect of coating on

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i Fig. 4.5. Plot of internal partial pressure of CO2 (p CO 2) versus internal partial pressure of i O2 (p O2) to identify the internal lower oxygen limit (LOL i ) of coated fruit (modified from Banks et al. 1997b).

i permeance to the two gases is offset by the increase in RQ, and p CO is 2 dramatically increased (Banks et al. 1997b; Amarante 1998). Amarante (1998) observed for coated pear held at 20°C that the physiological process of respiration, softening, and skin color change were well described by a Michaelis-Menten model when plotted against pOi 2, i while p CO had no explanatory power for these variables. A canonical 2 correlation analysis of the data showed a high correlation (0.92 to 0.97) between the first pair of canonical variables of gas composition (p Oi 2 and i p CO ) and ripening attributes (respiration, softening, and skin color 2 change), with a much higher standardized coefficient for pOi 2 than for i p CO for the gas composition canonical variable. Thus, modification of 2 i pOi 2 rather than of p CO seems to be the principal means by which coat2 ings achieve their effects on delaying ripening at ambient temperatures. The Michaelis-Menten constant values (Km) for plots against pOi 2 were lower for softening than for color change in coated pear kept at ambient temperatures (Amarante et al. 1997a,b; Amarante 1998). This resulted in color change being retarded by any level of depression in pOi 2 created by coating, while firmness was substantially reduced only at much lower pOi 2 during shelf life. The enzymatic system for chlorophyll oxidation

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seems to have a much lower affinity for O2 than that upon which softening is dependent (Amarante 1998). The Km values for plots of aerobic respiration and rate of firmness change against pOi 2 were similar. Perhaps, by reducing the respiratory energetic metabolism, coatings may have suppressed de novo synthesis of cell wall degrading enzymes and delayed softening at ambient temperatures (Amarante 1998). The higher sensitivity of color change than softening to pOi 2 may have important implications for postharvest quality of coated commodities. The commodity may fail to change in color while still being able to soften during shelf life when treated with coatings (Smith and Stow 1984; Amarante 1998) or stored in plastic films (Geeson et al. 1991a,b). Amarante (1998) showed that variable ripening quality would result from variable levels of pOi 2 in coated pear: fruit would have large variability in color at high to moderately low pOi 2, while the variability in firmness would dominate at moderately low to very low pOi 2 (Fig. 4.6). This would add to the naturally high variability in rates of color change and firmness among fruit at a given level of pOi 2 (Amarante 1998).

Image Not Available

Fig. 4.6. Changes in firmness and skin color of coated pear in relation to internal O2 partial pressure (piO2). In the light grey region, reduction of piO2 would result in more substantial inhibition of changes in color than firmness. In the dark grey region, reduction of piO2 would result in more substantial inhibition of firmness than changes in color (Amarante 1998).

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For pear (Meheriuk and Lau 1988; Amarante 1998) and banana (BenYehoshua 1966; Blake 1966), coatings can cause uneven color change characterized by skin blotchiness, with yellow areas interspersed with green tissue. This may be the result of uneven cover of the skin, with the coating failing to block some large pores (Amarante 1998). The skin and flesh tissues of pear and banana can have large resistance to gas exchange, especially when the fruit ripens, as a result of flooding of intercellular spaces. This may create substantial gradients of pOi 2 between different areas under the skin. While the high pOi 2 in tissues near the uncovered skin would allow chlorophyll degradation, pOi 2 would be low enough to inhibit color change of covered areas nearby, resulting in uneven color change of the skin. During cold storage of pear, Amarante (1998) observed that coatings had their main effect on delaying softening rather than color change, the opposite of the pattern observed at ambient temperatures. This difference appeared to be the result of a differential response of softening and skin color change to gas composition at different temperatures. The degreening of the skin was highly dependent on pOi 2 at ambient temperatures; this also seems likely to be true at low temperatures. As only high coating deposits substantially reduced pOi 2 at low temperatures, only these treatments had a small effect in delaying color change during cold storage. Since rate of softening was greatly suppressed only when pOi 2 was reduced below ∼ 2.5 kPa at ambient temperatures, and coatings did not drop pOi 2 to such low levels during cold storage, it seems likely that i the increase in pCO of fruit left in cold storage played a major role in sup2 pressing fruit softening. Surface coatings may also delay ripening by inhibiting C2H4 biosynthesis and action. Coatings have been reported to increase (Drake and Nelson 1990; Lau and Yastremski 1991; Baldwin et al. 1995b; Bauchot et al. 1995b) or reduce (Banks 1984a; Drake et al. 1987) the internal C2H4 level in coated commodities. Immediately after removal from cold storage, apple coated with Nutri-Save (Lau and Yastremski 1991) and Semperfresh (Kerbel et al. 1989; Bauchot et al. 1995b) had higher internal C2H4, with the concentration increasing in proportion to the total solids concentration in the coating formulation (Kerbel et al. 1989; Lau and Yastremski 1991). However, control fruit tended to reach higher concentrations of internal C2H4 than Semperfresh-coated apple during ripening at 20°C (Kerbel et al. 1989). The contrasting published results for internal concentration of C2H4 probably reflect natural variability in C2H4 production rates between commodities or cultivars of the same commodity; differences in fruit ripening stage; differences of coating

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treatments in modifying fruit internal atmosphere (which is dependent on temperature and respiration rate of the commodity). It would also relate to the inherent nature of the skin, chemical composition, and total solids concentration of the coating mixture and the interaction between the two, which would determine the extent of pore blockage by the coating. As shown in Section VA2, tissue exposure to low levels of O2 and/or high levels of CO2 suppresses C2H4 biosynthesis and action of climacteric fruits (Kader 1986; Gorny and Kader 1996a,b). Therefore, coatings can inhibit non-autocatalytic (System I) C2H4 biosynthesis during cold storage to an extent that depends on how much the commodity-coating interaction modifies the internal atmosphere. For those coated commodities that have suppressed non-autocatalytic (System I) C2H4 production during cold storage as a result of substantial modification of internal atmosphere (mainly accumulation of CO2), accumulation of C2H4 may be suppressed. If the modification of internal atmosphere during the storage at low temperature is not large enough to inhibit System I C2H4 biosynthesis, some C2H4 driven ripening may occur during this period, but this may be drastically reduced once the commodity is transferred to ambient temperatures. Under these conditions, the internal atmosphere modification (mainly the reduction of O2) might suppress System I and System II C2H4 production, as well as C2H4 action, in spite of some internal accumulation of the hormone caused by reduced PC2H4 of the coating films (Hagenmaier and Shaw 1992). Ben-Yehoshua et al. (1985) reported a 50% reduction of P′C2H4 for orange coated with a coumarone indene resin and Banks (1984a, 1985b) reported an 80% reduction in P′C2H4 for banana coated with TAL Pro-long (1.5% w/v). Nevertheless, coatings generally retard ripening changes, even though they increase levels of C2H4, indicating that modification of fruit internal composition of O2 and CO2 is the main way by which coatings delay ripening. This suggests that coatings are effective by suppressing aerobic respiration and C2H4 biosynthesis and action (Fig. 4.3) or direct effects on other processes such as color change. The published literature indicates that by increasing temperature there i is a change from a minor effect of high p CO to a more dramatic effect of 2 i pO2 in delaying ripening. Since the reduction of pOi 2 has a stronger effect in reducing respiration, C2H4 biosynthesis and action and delaying ripening, we can expect a more substantial effect of coatings in preserving the commodity quality at higher temperatures. However, this is set against the background of much more rapid ripening at high temperatures, which would constrain the scope of beneficial effects of coating.

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3. Optimization of Surface Coatings in Relation to Commodity Internal Atmosphere Composition. Oxygen has a stronger effect than CO2 on respiration and much effort has been made to characterize the relationship between respiration and O2 level for the optimization of CA/MA storage. One approach to optimize CA/MA storage of fruits and vegetables has been focused on identifying the lower O2 limit (LOL), the O2 level below which accumulation of products of fermentation, development of off-flavors, and degradation of the tissue become likely (Banks et al. 1993b). Several authors have used different approaches to identify the LOL. Blackman (1928) used the term “Extinction Point” (EP), defined as the threshold O2 concentration at which all anaerobic respiration was just extinguished. Thomas and Fidler (1933) defined the EP as the lowest O2 concentration at which ethanol production ceased. Boersig et al. (1988), studying the aerobic-anaerobic transition zone in pear, proposed an alternative concept, the “Anaerobic Compensation Point” (ACP). They defined the (ACP) as the O2 concentration at which CO2 production was minimal (the point at which total respiration was minimal in Fig. 4.4). However, anaerobic respiration starts at a partial pressure of O2 higher than that corresponding to the ACP (represented by the increase of rCO2(fer) in Fig. 4.4). Beaudry (1993) defined the concept of “Respiratory Quotient Breakpoint” (RQB), as the O2 partial pressure at which the steady-state RQ begins to increase as O2 decreases. All of the above-published work dealing with the identification of the LOL has been based on peO2 instead of pOi 2. Studies with peO2 are simpler to undertake and more readily applicable to empirical design of packages. However, fruit tissue responds more directly to the cell sap O2 concentration that can be assumed to be close to the equilibrium with the pOi 2 in the internal atmosphere (Banks et al. 1993b; Dadzie et al. 1996). This is certainly highly relevant when considering the determination of LOL of coated fruits and vegetables, when the main interest is in characterizing modification of the internal atmosphere, which is dictated by the permeance and respiration rate of the coated product. This led Yearsley et al. (1996) to propose the use of LOL based on pOi 2 (LOL i ) instead of peO2 (LOLe), for optimization of CA/MA storage. For estimation of LOL i they proposed the internal ACP (ACP i) and the internal “Fermentation Threshold” (FT i). ACP i was estimated by plotting internal partial presi i sure of CO2 (p CO ) versus pOi 2. In such plots, the increase in p CO when 2 2 i pO2 approaches 0 kPa is related to rates of fermentation. The FT i was described in terms of plots of RQ (FT iRQ) and internal ethanol concentration (FT iEtOH) versus pOi 2 (Fig. 4.7). Beaudry (1993) arbitrarily selected a 20% deviation of RQ from the asymptote of the fitted curve to estimate the LOLi, while the analogous deviation used by Yearsley et al. (1996)

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Fig. 4.7. Plots of respiratory quotient (RQ) and internal ethanol concentration (c iEtOH) versus internal partial pressure of O2 (piO2) to identify the internal “Fermentation Threshold” (FT i) of fruits and vegetables (modified from Yearsley et al. 1996).

was 10%. For coated fruit, Banks et al. (1997b) have proposed plots of i p CO versus pOi 2 to identify the LOLi (Fig. 4.5), equivalent to ACP i 2 described by Yearsley et al. (1996). Increasing the amount of coating i deposited on the fruit surface depresses pOi 2 and increases p CO . As the 2 i pO2 decreases, the respiration rate is suppressed and the negative slope of this relationship decreases and becomes positive at a certain pOi 2 level. i , indicating the With further decreases in pOi 2 there is an increase in p CO 2 i transition to fermentation and that pO2 has dropped below LOL i (Fig. 4.5). The LOLi has been explored by Amarante (1998) for the optimization of surface coatings for pear. This approach, in addition to FTiRQ and FT iEtOH, can be used to identify the safe internal O2 levels in the optimization of surface coatings for fresh fruits and vegetables. The assessment of rCO2(tot) alone does not necessarily reveal if the coating treatment has a beneficial effect in preserving the commodity quality. As observed by Magness and Diehl (1924), treating different apple cultivars with paraffin wax or oil coatings resulted in a reduced respiration rate of fruit held at 0°C, 18°C, or 26.5°C. However, while at 0°C the RQ was ≅1, indicating the predominance of aerobic respiration, at

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18°C the O2 within the tissue was highly depleted, and some fermentation apparently occurred in coated fruit, for the RQ was >1, and at 26.5°C the RQ was >> 1, indicating marked levels of fermentation. In non-coated fruit, the RQ at all temperatures was ≅ 1, indicating the absence of significant fermentation. As coatings reduce commodity P′CO2 and P′O2, they might reduce CO2 evolution or O2 consumption rates. Plots of rCO2(tot) versus pOi 2 can aid in identifying the LOL i of coated commodities, corresponding to the ACP i (Fig. 4.4) described by Boersig et al. (1988). This approach has been used by Amarante (1998) to identify the LOLi for the optimization of surface coatings for pear. Since the relationship between rCO2(ox) versus pOi 2 is described by a Michaelis-Menten relationship, this approach also permits the identification of the affinity for O2 of respiration (represented by the Km) in different commodities. This can be used to characterize the potential benefit of reducing pOi 2 (in CA/MA storage, including surface coatings) in suppressing rCO2(ox) (Amarante 1998). The LOL depends on commodity (Ke and Kader 1992), cultivar (Yearsley et al. 1996; Amarante 1998), physiological age (Boersig et al. 1988; Nanos et al. 1992; Ke et al. 1993; Amarante 1998), temperature (Ke et al. 1990, 1993; Yearsley et al. 1997a), and duration of exposure (Boersig et al. 1988; Ke et al. 1993). Therefore, all these aspects have to be considered for the optimization of surface coating for fruits and vegetables. Amarante (1998), estimating the LOLi of coated pear, reported differences in tolerance to hypoxia between cultivars and increasing sensitivity to hypoxic conditions in more advanced ripening stages. According to the authors, the disorganization of mitochondrial activity, starting with the respiratory climacteric, might lead to loss of tight metabolic control increasing the LOLi. From these results, it is clear that the optimization of surface coatings should take into account differences between cultivars and ripening stage at which fruit are coated. LOLi (as opposed to LOLe) seems to be temperature insensitive for temperatures ≤ 28°C (Yearsley et al. 1997a). Its estimation for different commodities, cultivars, and ripening stages should provide valuable advances for optimization of surface coatings in that composition and amount of coating deposit could be tailored to achieve a level of pOi 2 near and above the LOLi for a certain combination of temperature and RH. This would help to achieve the best results in delaying ripening without the detrimental effects of fermentation. The optimization of surface coatings should also be based upon the assessment of sensory attributes and incidence of physiological disorders after long-term storage in cold storage and also after a shelf life period (Amarante 1998) . Short-term exposure of the commodity to low

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O2 and/or high CO2 only indicates fruit tolerance to MA treatments of the same duration. Longer studies are required to ensure the best retention of product quality over long term commercial storage. C. Postharvest Quality of Coated Commodities 1. Skin Finish. Coatings improved skin finish of apple, with no evident difference among apple waxed with shellac-, carnauba-, or resin-based coatings after long-term cold storage (Drake and Nelson 1990). Glenn et al. (1990) reported that fruit finish in apple coated with a wax was highly dependent of the extent of fruit cracking on the skin. Polishing the fruit before waxing revealed a reticulate pattern of cracks that deeply penetrated the cuticle. More cracking occurred near the calyx end than at the pedicel end of the fruit, and cracking tended to be more severe on surface exposed to the sun. However, waxing the fruit did not always completely fill in the cracks and other irregularities on these regions of the skin, resulting in lower light reflectance and poor finish of coated fruit. Similarly on pear, Amarante (1998) observed that cultivars with high natural gloss had larger increases in skin gloss with increasing coating concentration. ‘Bosc’, with lignified cells in the skin, had the least natural gloss and increasing coating concentration had only a small effect on enhancing surface finish. Hagenmaier and Baker (1994b) reported higher gloss on the skin of orange and grapefruit treated with shellac and resin than with polyethylene and carnauba waxes, while fruit treated with the latter waxes had higher gloss than non-coated fruit. However, fruit treated with shellac and resin underwent a larger decrease in gloss during a two-week shelf life period than fruit coated with polyethylene and carnauba waxes, resulting in no significant difference between coating treatments at the end of the shelf life period. Hagenmaier and Baker (1995) observed that the use of bilayer films on citrus fruit could achieve both benefits of reducing water loss and improving gloss. This approach involved applying two coatings, the first coating being a moisture-barrier wax (petroleum wax) and the second a polyethylene wax or a mixture of shellac and resin. Fruit gloss decreased more rapidly during one week at 20°C with a single glossy coating than with the same coating applied as a second layer over a waxbased first coating. These results reflect differences in water loss of fruit treated with different coatings. Because shellac and resin coated fruit lost more water than polyethylene- and carnauba wax-coated fruit, the wrinkling of their skin resulted in lower gloss after long shelf life period. The application of an initial coating with low permeance to water can reduce shrivel and maintain the shine imparted by the glossy coating layer.

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Hagenmaier and Baker (1996) observed that the gloss of candelilla wax coating films was improved by addition of gelatine and to a lesser extent by adding HPMC. The gloss of candelilla coating film was also affected by thickness and drying temperature; increasing thickness and drying temperature increased gloss. The gloss levels of candelilla and candelilla plus carnauba wax, but not of a candelilla plus beeswax formulation, were increased by adding gelatine or soy isolate (25% protein). Coating banana with a polyethylene-wax emulsion improved the appearance of the skin by imparting gloss and preventing shrinkage and darkening (Ben-Yehoshua 1966). A carnauba-paraffin mixture improved the finish of the skin in sweet cherry and nectarine (Claypool 1939). In mandarin, a carnauba-based wax improved gloss (Farooqi et al. 1988) while, in papaya, only some wax-based coatings improved gloss of the skin (Paull and Chen 1989). Chitosan-coated carrot had a glossier appearance than untreated root (Cheah et al. 1997). Increasing the concentration of total solids in polyethylene and carnauba waxes improved the gloss of coated pear (Amarante 1998) and avocado (Johnston and Banks 1998). Mellenthin et al. (1982) observed that in-line application of a composite coating consisting of water-soluble polysaccharides, natural wax, and emulsifiers (‘Fresh-Cote’) on pear at the waxing location of the packing line reduced peel discoloration of ‘Bartlett’, but not ‘d’Anjou’, caused by brush friction. Fruit were also subjected to a return flow belt for five minutes to simulate the sorting sequence during packing. The coating substantially reduced the susceptibility of both pear cultivars to peel discoloration due to belt friction. Amarante (1998) reported a beneficial effect of a carnauba-based coating in reducing brush-induced friction discoloration of pear. Increasing the concentration of total solids increased exponentially the efficiency of the coating in reducing the damage. However, extending the storage period in cold storage increased the susceptibility to friction discoloration. This required an increase in coating concentration for riper fruit (left longer in cold storage) to achieve greater beneficial effects in reducing skin browning. It was suggested that coatings may have reduced fruit susceptibility to friction discoloration by delaying ripening, reducing weight loss, reducing p iO2 (that may reduce the activity of polyphenol oxidase and browning of the skin), and by forming a physical protective layer on the skin. 2. Disorders. Apple harvested early in the season and coated with wax (Hitz and Haut 1938) or Semperfresh (Kerbel et al. 1989) had increased incidence of scald. In contrast, Bauchot et al. (1995a) and Bauchot and John (1996) reported a nil effect of Semperfresh on scald incidence in

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apple. The coating did not appear to have any effect in modifying the volatility of α-farnesene or representing a physical barrier to trap it in the fruit. In apple, Pro-long had no effect on scald incidence (Miszczak 1994) and Nutri-Save reduced the incidence of scald and core flush (Elson et al. 1985). Farooqi and Hall (1973) reported that increasing the concentration of a carnauba-based coating on apple provided better control of skin disorders (Jonathan spot and soft scald on ‘Jonathan’ and superficial scald and senescent blotch on ‘Granny Smith’) and flesh disorders (internal breakdown in ‘Granny Smith’). Smock (1935) reported 20% less scald in waxed apple. Pro-long and Nutri-Save reduced the incidence of scald in apple, and reduced the incidence of core flush in some cultivars but increased it in others (Lau and Meheriuk 1994). However, increasing the concentration of Nutri-Save, but not Pro-long, exacerbated the incidence of skin purpling in all cultivars. Lau and Yastremski (1991) also observed that apple treated with high concentrations of Nutri-Save developed substantial levels of skin damage. Since skin injury seems to be a symptom of low O2 injury, Lau and Meheriuk (1994) suggested that these differences reflect a higher permeance to gases of fruit coated with Pro-long than with Nutri-Save. Fruit treated with Nutri-Save may have accumulated anaerobic volatiles after longterm storage. Apple treated with Semperfresh did not develop any internal disorders after long-term storage (Santerre et al. 1989). Pro-long increased core flush and low temperature breakdown incidence in ‘Cox’s Orange Pippin’ stored below 3.5°C, both disorders known to be exacerbated by increased internal CO2 levels at close to chilling temperatures (Smith and Stow 1984). The coating did not induce any internal physiological disorder or cause accumulation of alcohol if applied after cold storage. Waxing significantly reduced core flush incidence in ‘McIntosh’, while breakdown was not affected in a number of other apple cultivars (Meheriuk and Porritt 1972). Meheriuk and Lau (1988) reported that the incidences of core breakdown and senescent scald in ‘Bartlett’ pear and superficial scald in ‘d’Anjou’ pear were lower in fruit coated with Pro-long or Nutri-Save. Semperfresh delayed ripening and reduced incidence of senescent breakdown (Van Zyl and Wagner 1986; Van Zyl et al. 1987) and senescent scald (Köksal et al. 1994) in ‘Bartlett’ pear. ‘Bartlett’ treated with wax (Claypool 1939) or mineral oil (Reyneke and Stubbings 1940) had lower incidence of senescent scald. The coatings, however, adversely affected normal pear ripening. Coated fruit tended to ripen unevenly, lost the capacity to ripen fully, and developed a blotchy appearance of green interspersed with yellow when held at ripening temperatures

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(Smock 1935; Claypool 1939; Farooqi and Hall 1973; Elson et al. 1985; Van Zyl et al. 1987; Meheriuk and Lau 1988; Sümnü and Bayindirli 1994). High concentrations of a carnauba-based coating reduced the incidence of senescent breakdown and senescent scald in pear, but caused internal disorders associated with high CO2/low O2 in cultivars having very high respiration rates (‘Bartlett’) or low epidermal permeance to gases (‘Bosc’) when fruit were coated at harvest and stored at 0°C (Amarante 1998). However, if these cultivars were coated after cold storage, the fruit did not develop the disorder. The results show that the development of this disorder requires low temperature as well as the high p iCO2 and/or low p iO2 of fruit treated with a high coating concentration. According to Amarante (1998), although modifications of internal atmosphere would have been less severe at the storage temperature of about 0°C, the effects of coating may have been exacerbated by the enhanced solubility of CO2 at low temperature. Surface coatings can also increase the internal concentration of anaerobic volatiles such as ethanol, acetaldehyde and ethyl acetate (Nisperos-Carriedo et al. 1990; Hagenmaier and Baker 1993c, 1994b; Baldwin et al. 1995b), and their accumulation may contribute to development of internal disorders (Fig. 4.3; Toivonen 1997). However, it is not clear if the accumulation of these volatiles results from increasing anaerobic respiration (as a result of low p iO2 and/or high p iCO2; Hagenmaier and Baker 1994b) or from reduction in skin permeance to these compounds by the surface coating (Fig. 4.3; Baldwin et al. 1995b). The increase in the concentrations of these anaerobic volatiles has been shown to be a natural event during ripening and senescence of fruits (Nanos et al. 1992; Ke et al. 1994), and reducing skin permeance by coating may contribute to their accumulation and exacerbate the incidence of internal disorders. Coatings with lower permeance to gases, such as shellac, may be expected to cause larger depletion of O2 and build-up of CO2. In addition, they may also increase the accumulation of anaerobic volatiles by reducing their escape through the skin (Baldwin et al. 1995b), increasing the risk of fermentation and/or internal disorders (Amarante 1998). Wax and polysaccharide-based coatings are more permeable and, therefore, might present less risk (Hagenmaier and Shaw 1992; Baldwin et al. 1995b). This issue deserves further investigation. Blake (1966) observed blotchiness of banana treated with paraffin coating. Coating banana with a polyethylene-wax emulsion increased the irregularity in duration of skin degreening, a problem that could be overcome by treating the fruit with C2H4 (Ben-Yehoshua 1966). Immersing grapefruit in vegetable oils and vegetable oil-water emulsions prior to storage at 3°C markedly delayed and reduced symptoms

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of chilling injury (Aljuburi and Huff 1984). Chilling injury (rind breakdown, or pitting) of grapefruit was also reduced substantially by treatment with emulsions of polyethylene before storage (Davis and Harding 1960). Wild (1991) reported an increase in the postharvest incidence of peteca rind pitting in lemon by brushing and waxing. Mango coated with a composite coating made of CMC and sorbitan fatty acid ester had lower rates of electrolyte leakage than non-coated fruit (Díaz-Sobac et al. 1996). The coated fruit did not show the incidence of dark spots or discoloration on the skin or in the pulp 24 days after shelf life, although these symptoms occurred in the control fruit after six days of shelf life. After 24 days of shelf life, the coating was removed by washing, and the fruit reached full ripeness three to four days later. Therefore, by delaying the loss of cell integrity, coatings can reduce undesirable reactions leading to tissue browning without impairing the ability of mango fruit to ripen. HPC (2% or 4% w/v) and carnauba wax (5% w/v) coatings negatively affected fruit quality of guavas by increasing the incidence of blackening of the peel after their removal from cold storage to ripening temperature (McGuire and Hallman 1995). McGuire (1997) reported that 17% of guavas treated with a carnauba wax (5% w/v) coating failed to ripen. The fruit remained green and had lower acidity and soluble solids concentrations. Edward and Blennerhassett (1994) reported that a polyethylene wax (‘Citruseal’) reduced chilling injury in ‘Honeydew’ melon stored at 3°C for up to four weeks. According to these authors, the decrease in chilling injury achieved by waxing the fruit was most likely due to the reduction of water loss. Semperfresh significantly increased the severity of brown speckle on the rind in ‘Honeydew’ melon (Edward and Blennerhassett 1990). Sriyook et al. (1994) reported that coating materials were effective in preventing durian fruit dehiscence. The coatings reduced weight loss and modified internal atmosphere composition. According to the authors, since C2H4 seems to be the main factor influencing durian fruit dehiscence, coatings can prevent the problem by reducing C2H4 production and action via its effects in reducing water loss and modifying fruit internal atmosphere. Waxing fresh pineapple with a paraffin-polyethylene coating diluted to 20% of the commercial formulation (v/v) before storage at 8°C for up to four weeks reduced the incidence and severity of internal browning caused by chilling injury to 15 and 31%, respectively, of the non-waxed control (Rohrbach and Paull 1982). Further increases in coating concentration up to 50% (v/v) had a comparatively small beneficial effect

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in decreasing the injury. Waxing did not reduce crown leaf chilling injury symptoms. Semperfresh had no effect on the incidence of internal breakdown in plum. However, Semperfresh increased the wooliness in nectarine, with some cultivars having an unacceptable taint on the skin (Van Zyl et al. 1987). Nature Seal (York 1994) and chitosan (Zhang and Qhantick 1997) significantly reduced pericarp browning in litchi, but the treatment effects were not substantial enough to make these formulations attractive commercially. Undesirable enzymatic browning reactions in mushroom slices were prevented by the application of a polysaccharide edible coating, with the coating’s anti-browning property being further improved by the incorporation of 1% ascorbic acid (food approved antioxidant) and a chelator (0.2% calcium disodium EDTA; NisperosCarriedo et al. 1991). Lidster (1981) reported that sweet cherry treated with emulsified coatings to decrease weight loss had reduced incidence of discolored stems and surface pitting in storage. According to the author, these treatments may reduce the disorders by the inhibition of net volume loss (mainly water loss) from mechanically damaged fruit. Because coating the fruit with a vegetable oil emulsion did not reduce weight loss but reduced the incidence of surface pitting, this may indicate that factors other than weight loss may be involved with the inhibition of surface pitting. Drake et al. (1988) reported that surface pitting and stem discoloration in sweet cherry, two physiological disorders associated with high rates of water loss, were not substantially reduced by wax coatings, especially when the fruit were stored at higher temperatures. Under these conditions, wax coatings did not provide enough water loss control for sweet cherry to reduce the incidence of the disorders. Ben-Yehoshua (1966) reported that coating banana with a polyethylene-wax emulsion not only delayed but also reduced the darkening of the skin of ripe coated fruit. Non-coated fruit left at 10°C developed chilling injury (darkening of the skin), while coated fruit did not. Coatings appeared to have a direct effect in reducing the darkening reaction by reducing the internal O2 concentration and therefore the activity of polyphenol oxidase (Ben-Yehoshua 1966; Lidster 1981; Amarante 1998). 3. Diseases. Claypool (1939) reported that waxing reduced decay of deciduous fruits. Farooqi and Hall (1973) observed a reduction of postharvest decay in coated apple. These authors observed that increasing coating concentration resulted in a better control of decay, presumably as a result of slowed ripening, but fruit treated with excessively high concentrations tended to have a higher incidence, probably by render-

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ing the fruit anaerobic and therefore more prone to pathogenic infection. Waxing cucumber also seemed to induce anaerobiosis and increased incidence of decay (Risse et al. 1987). Waxing with a polyethylene wax (‘Citruseal’) did not reduce breakdown caused by bacteria or fungi (Alternaria spp. and Fusarium spp.; Edward and Blennerhassett 1994) in ‘Honeydew’ melon. Likewise, Semperfresh did not reduce the incidence of fungal breakdown but increased the incidence of Alternaria rots (Edward and Blennerhassett 1990) in this fruit. Coating banana with a polyethylene-wax-based coating delayed and reduced the incidence of decay, particularly of the cut surfaces of fruit (Ben-Yehoshua 1966). However, Blake (1966) observed an increase of decay in paraffin coated fruit in periods when the occurrence of anthracnose was high, making the use of coatings viable only for banana in a good phytosanitary condition. Orange treated with a shellac-based coating had a lower percentage of postharvest decay than non-coated fruit, but there was no beneficial effect if fruit were treated with a MC-based coating (Potjewijd et al. 1995). HPC (2% or 4% w/v) and carnauba wax (5% w/v) coatings did not affect the incidence of postharvest decay of guavas (McGuire and Hallman 1995). Baldwin et al. (1997) reported that cucumber treated with a Nature Seal (1–2% HPC) coating, with or without the addition of carnauba wax microemulsion, had lower incidence of decay than controls. Better decay control was achieved with the composite coating than with Nature Seal alone. In citrus, lower levels of decay were achieved by coating the fruit with a candelilla wax coating and ‘Tag’, a polyethylene wax emulsion, but not with ‘Flavorseal’, a resin-based coating (Lakshminarayana et al. 1974). Chitosan, besides its effects in delaying ripening and senescence (El Ghaouth et al. 1992b) has been shown to be effective against decay by inhibiting fungal activity (El Ghaouth et al. 1991a, 1992a, 1997). Tomato coated with chitosan (1% and 2% w/v) had lower incidence of decay, mainly caused by Botrytis cinerea Pers.:Fr. (El Ghaouth et al. 1992b). Coating cucumber and bell pepper with chitosan (1.0% and 1.5% w/v) reduced fungal infection caused by Botrytis cinerea and by species of Erwinia and Alternaria (El Ghaouth et al. 1992b). Strawberry coated with chitosan was less affected by postharvest decay compared to the controls. There was no significant difference between chitosan- and fungicide-treated berries up to 21 days storage at 13°C. Thereafter, fungicide-treated berries decayed at a higher rate than chitosan-coated berries (El Ghaouth et al. 1991a). The mechanism by which chitosan reduced the decay appear to be related to its fungistatic property rather than to its ability to induce defense enzymes such as chitinase, chitosanase, or beta-1,3-glucanase in the tissue (El Ghaouth et al. 1992a). In pepper,

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chitosan restricted the proliferation of Botrytis cinerea and markedly reduced the maceration of the host cell wall components, pectin and cellulose. This seems to result from the ability of chitosan to cause severe cellular damage to Botrytis cinerea and to interfere with its capability to secrete polygalacturonase (El Ghaouth et al. 1997). Cheah et al. (1997) reported that chitosan (2 and 4% w/v) significantly reduced the growth of Sclerotinia sclerotiorum (Lib.) de Bary in vitro and significantly reduced the incidence of rot (from 88 to 28%) and also the lesion size (from 26 to 12 mm) of carrot inoculated with the pathogen and left for five days at 22°C. Microscope studies revealed that fungal mycelium exposed to chitosan appeared to be deformed and dead, whereas untreated mycelium was normal in appearance. Apple coated with Nutri-Save (a chitosan-based coating) had a lower incidence of decay (Elson et al. 1985). However, in litchi, the application of chitosan coating only partially inhibited decay of fruit during storage, with best control being achieved with fungicide [thiobendazole; 2-(4-thiazolyl) benzimidazole] treatment (Zhang and Qhantick 1997). Fungicides mixed with wax coating material can provide good control of postharvest decay. Fresh market tomato treated with a fungicidal wax containing 2.5% o-phenylphenol (OPP) in a commercial packhouse had lower incidence of decay than fruit treated with plain wax, and almost no chemical residue of OPP could be detected on fruit treated with the fungicidal wax mixture (Hall 1989). In orange, Brown (1984) observed that the control of stem end rot with fungicides incorporated in wax coating required doubling the fungicide concentration used for application in water. However, the total amount of fungicide required to treat a certain quantity of fruit is not doubled because less wax than water is lost from the brushes during application. The author observed less control of green mold [caused by Penicilium digitatum (Pers.:Fr.) Sacc.] developed from infections through minute punctures with wax treatment. This may have been the result of non-availability of the fungicide at the infection site because of encapsulation by the wax (Tugwell 1973), variable wax coverage (Norman et al. 1972), and higher viscosity of wax than water, which may impede depositing of fungicides in certain infection courts (Brown 1984). Incorporation of bio-control organisms into the coatings to restore surface populations of beneficial micro-organisms can provide an opportunity for biological control of postharvest decay pathogens (McGuire and Baldwin 1994; Potjewijd et al. 1995). One bio-control candidate, the yeast Candida oleophila Montrocher, has been shown to prolong the storage life of grapefruit, but its growth on the fruit is dependent upon the coating composition. McGuire and Baldwin (1994) observed that

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films based upon polysaccharides can support very high populations of this species. In a liquid shellac coating, the added yeast were quickly killed and the few survivors did not multiply, with its population remaining low relative to that of fruit coated with cellulose. Apparently, shellac and wax coatings can be toxic to the yeast, due to the addition of alcohols and bases such as KOH, NH4OH, and morpholine that are used to dissolve the primary constituent. Potjewijd et al. (1995) observed that varying the cellulose component of polysaccharide coating formulations affected the survival of two yeast bio-control agents, Candida guillermondii (Castelani) Langeron and Guerra strain US7 and Debaryomyces sp strain 230. Using MC as the main film-former gave a higher recovery of the yeast after an incubation period (at 26°C) for both strains as opposed to using CMC or HPC. The authors suggested that MC might be a better source of nutrients and less toxic to the antagonists than CMC and HPC. Significant control of decay on orange was demonstrated by yeast antagonists incorporated in a MCbased coating for the first two to four weeks of storage (at 16°C and 90% RH), with Candida guillermondii strain US7 providing more promising results than Debaryomices sp strain 230. Use of a strain of Bacillus subtilis (Ehrenberg) Cohn (designated B-3) as a biological control agent has been reviewed by Pusey (1989), with emphasis on the postharvest application to stone fruit for control of brown rot caused by Monilinia fructicola (Wint.) Honey. Brown rot control by B-3 was demonstrated on peach, nectarine, apricot, plum, and sweet cherry. Application of the antagonist was shown to be compatible with commercial fruit waxes (water-based and mineral oil- and paraffin-based waxes) commonly applied to harvested stone fruit. In packing line trials, B-3 applied with a water-based wax was as efficient as the benomyl [benzimidazolecarbamic acid, 1-(butylcarbamoyl)-, methyl ester] in controlling brown rot. Antifungal activity of B-3 was shown to be retained during low-temperature storage of fruit. B-3 had little or no effect against Rhyzopus rot, another important postharvest disease of stone fruit. The addition of dichloran (2,6-dichloro-4-nitroaniline; the fungicide commonly used for Rhizopus control) to B-3 formulation was required for the control of brown rot and Rhizopus rot. In vivo activity of B-3 against fungi was also shown for apple rots caused by M. fructicola, Botrytis cinerea, and Glomerella cingulata (Stoneman) Spauld. & Schrenk, and for grey mold of grapes caused by B. cinerea. Coating can reduce decay by delaying ripening and water loss. Both these processes lead to senescence, making the commodity more prone to pathogenic infection as a result of loss of cellular integrity and tissue natural defense mechanisms. Besides these physiological effects, coatings

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can also form a physical barrier against pathogenic infection, reducing incidence of postharvest diseases. There are many cases where coating may actually increase decay, such as when the spore load on the fruit from the field is high or the sanitary conditions of the packing shed are poor (Blake 1966). Therefore, the sanitation of the fruit before coating is very important. A widely adopted packhouse practice is to wash the fruit with disinfectant solution (e.g. sodium hypochlorite and sodium ophenylphenate) before spraying with the coating. The discontinuance of the coating bath in favor of the line spray method largely eliminated decay resulting from contamination of the waxing solution by decayed fruit (Claypool 1939). There are cases when coating may be so restrictive to gas exchange that it may induce physiological disorders on the skin, possibly by inducing fermentation and accumulation of toxic metabolites. This situation may lead to cellular death of commodity tissue and increase the incidence of decay (Farooqi and Hall 1973; Risse et al. 1987). This issue is vital in assessing the potential of a particular coating formulation in maintaining product quality. 4. Insects. Recently, coatings have been shown to have the potential for postharvest disinfestation. Coating grapefruit infested with larvae of Caribbean fruit fly, Anastrepha suspensa (Loew), provided a significant insect control, with better results being achieved by a combination of coating plus hot-air treatment (Hallman et al. 1994) or coating plus insecticide (Hallman and Foos 1996). Coating plus insecticide provided a better insect control than insecticide or coating alone, and the combined treatments may also reduce the insecticide residue in the fruit (Hallman and Foos 1996). Coating formulations with greater gas barrier properties (those containing resin or a high concentration of MC or HPC, in association with shellac in its formulation) were more effective in controlling fruit fly (Hallman et al. 1994, 1995; Hallman and Foos 1996), but they resulted in fruit with higher content of methanol and ethanol at 20°C, possibly reflecting the occurrence of fermentation in the fruit (Hallman et al. 1994). No larvae emerged from coated grapefruit treated with hot air (48°C for 60 min), whereas 24% survived treatment of noncoated fruit (Hallman et al. 1994). However, the combination of wax and hot air treatment (46°C for 35 min) for disinfestation of guavas increased the percentage of fruit that failed to ripen properly (McGuire 1997). Coatings also provided a significant control of fruit fly larvae in mango, carambola (Hallman et al. 1994), and guavas (Hallman et al. 1995). Coatings providing a good character of cover increased the insect mortality and also delayed larval emergence (Hallman et al. 1995). Coating of cold-stored mango and carambola did not increase fruit fly mortality

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(Hallman et al. 1994). Oxygen uptake by the fruit reduced at lower temperatures, resulting in a small modification of fruit internal atmosphere. Insects require less O2 at these lower temperatures. Therefore, coating may not prove effective for disinfestation during cold storage, but can be very effective in combination with short exposure to heat treatment. The killing of immature fruit fly inside coated fruit seems to be a combined effect of reduced p iO2 and increased p iCO2 (Hallman et al. 1994, 1995; Hallman and Foos 1996). Hallman et al. (1994) have also suggested that internal accumulation of certain volatiles, such as methanol or ethanol, may have contributed to reduced emergence of Caribbean fruit fly from coated grapefruit. In addition, delayed ripening of coated fruit (McGuire and Hallman 1995) might result in a less favorable environment (firmer tissue, less sugar) for larval development, resulting in a delay in larval emergence (Hallman et al. 1995). Fruit coatings can also be used for disinfestation of surface pests. Hallman (1994) reported that in South America coatings are used to desinfest lime and cherimoya of a surface mite, Brevipalpus chilensis (Baker). According to the author, coatings probably kill a surface pest by adhering it to the fruit surface and plugging its respiratory and alimentary openings. This represents a new technology with a high potential for postharvest disinfestation of pests, which may permit a substantial reduction of chemical residues of products currently being used for this purpose. By exposing coated fruit to higher temperatures for a given period of time, it is possible to achieve efficient insect control without causing severe stress that would impair fruit quality. The goal is to select a coating formulation that will permit a substantial modification of fruit internal atmosphere during exposure to short periods at high temperature, and will provide a fruit skin permeance high enough to permit recovery from disinfestation temperatures during subsequent exposure to ambient temperatures. More research will be required to characterize the relationships between coating treatments, internal atmosphere of the fruit at different temperatures and times of exposure, insect mortality, and postharvest quality of the product. This will provide a more mechanistic approach for setting a combination of temperature and exposure time to provide enough modification of internal atmosphere to kill the insect but without impairing fruit final quality. 5. Flavor. The benefit of coatings in maintaining and improving desirable elements of flavor, instead of inducing the development of fermentative flavors, should be thoroughly studied to evaluate the performance of different postharvest coating treatments. Commodities

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treated with coating formulations that provide high reductions in skin permeance to gases might accumulate anaerobic volatiles and develop off-flavors after long-term cold storage (Magness and Diehl 1924; Trout et al. 1953) or exposure to high temperatures (Dhalla and Hanson 1988; Cohen et al. 1990; Hagenmaier and Baker 1993a, 1994a,b; Mannheim and Soffer 1996). However, if excessive accumulation of anaerobic volatiles is avoided, coatings can improve the quality of coated fruit destined for fresh market or for juice processing (Lakshminarayana et al. 1974; Nisperos-Carriedo et al. 1990; Baldwin et al. 1995b). Nisperos-Carriedo et al. (1990) have shown that the use of edible coatings in orange generally increased the levels of the volatile components acetaldehyde, ethyl acetate, ethyl butyrate, methyl butyrate, ethanol, and methanol of fruit left at 21°C for up to 12 days. Use of beeswax emulsion and TAL Pro-long (alone or in combination with other coating components) were the most effective coatings in retaining or increasing volatile components. However, the authors did not carry out a sensory analysis of coated fruit to determine if these changes in volatiles were large enough to significantly affect the flavor, but an informal tasting did not detect noticeable off-flavors of treated fruit. Acetaldehyde, ethyl acetate, and ethyl and methyl butyrate are known to be important in improving flavor of orange juice (Ahmed et al. 1978). Therefore, coatings can retain volatile flavor components in orange during storage that may improve the quality of fruit destined for fresh market or for juice processing.

VI. SUMMARY AND CONCLUSIONS A. General Review A sound knowledge of the physical and physiological mechanisms by which surface coatings exert their effects on storage and shelf life behavior is central to the development of optimized coating treatments for fruits and vegetables. Use of coatings to retard ripening in refrigerated storage by means of internal atmosphere modification is likely to be limited by the small modification of internal atmosphere composition at low temperatures. Some beneficial effects can be achieved by increasing the barrier properties of the applied coating sufficiently to substantially modify the fruit internal atmosphere at low temperatures and thereby to delay ripening. However, this may result in variable modification of internal atmosphere and failure to produce a commodity of sufficient uniform quality to be of commercial benefit. It may also result in unde-

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sirably large modifications of the internal atmosphere following transfer to high temperatures, rendering the product anaerobic. This should constrain attempts for the commercial application of surface coatings to attain MA benefits during cold storage. Rather, coatings should generally be relied upon to achieve some measure of reduced water loss and enhanced gloss at levels that do not excessively modify internal atmosphere composition. This may be achieved by treating commodities with low concentrations of hydrophobic coatings (waxes) that seal cracks in the outer layers of the skin and achieve substantial reduction of P′H2O. This technology has a high potential to reduce water loss, without adversely affecting internal atmosphere composition and causing anaerobiosis in commodities less tolerant of hypoxic conditions. Hydrophilic coatings have little scope for reducing water loss but they can excessively modify internal atmosphere, causing fermentation and development of off-flavors under conditions of high temperature and low RH. Since coatings have more limited effects in delaying ripening in cold storage than during shelf life, an alternative approach might be to optimize the surface coatings for treatment of the commodity upon removal from refrigerated/CA storage in order to delay deterioration during marketing, although this might still cause variable ripening, mainly in terms of color change. Coatings may have a greater potential for use in commodities with colored skin (having high anthocyanin and carotene contents in the epidermal cells), for which the problems with variable degreening caused by coatings would not be such a great concern. Coatings can be used as carriers of other functional ingredients such as bio-control agents. Their use as a means of generating temporary anaerobiosis during short-term high temperature treatments makes good use of the principle of internal atmosphere modification summarized here. Overall, surface coatings offer a valuable additional tool to postharvest technologists for managing quality of harvested fruits and vegetables and for probing the dependence of different physiological processes on internal atmosphere composition. The further development of coating formulation and application technologies, and strategies for their use in different environments, holds exciting potential for the horticultural industry. B. Prospects for Future Research One issue that would be valuable in future publications about surface coatings would be a clear statement of the chemical composition and method of emulsion preparation of the coating formulation. There are so many different coating formulations on the market now that it is of

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very limited value to compare them using trade names within the literature. In several published scientific papers, only the trade name of the coating used is referred. Other papers reported using a “wax coating” and declined to provide details of the wax base. Both these types of omissions eliminate many valuable opportunities to make worthwhile comparisons. It is also suggested that the application rate should be standardized and expressed in terms of surface coating dry matter/commodity skin area or coating thickness, to provide a useful standard base for comparison. Most of the reported assessments of film permeability characteristics are conducted at temperatures between 25°C and 30°C, under conditions of 0% RH for O2 and RH gradient close to 100% for water vapor. Whilst this is worthwhile for comparing various attributes of films, it does not replicate the conditions on the coated commodity. The value of the information gained could be enhanced by using 98%/60% RH gradient in the chambers for gases and water vapor permeability assessment, and at standard temperatures of 0°C (mimicking cold storage temperatures) and 20°C (mimicking shelf life temperature conditions). The level of modification of internal atmosphere results mainly from the interaction of character of cover of the skin by the coating and respiration rate of the commodity. Direct measurement of commodity permeance to gas exchange under controlled environmental conditions should be preferred to characterization of the film itself for selection and optimization of surface coatings. It would be interesting to investigate how coatings interact with the different skin characteristics of distinct commodities. As the permeability of coating films is so variable at different temperatures and levels of RH, it is important to report the environmental conditions experienced by the coated commodities during postharvest assessments. Besides water vapor, O2, and CO2 permeance estimation, the permeance of surface coatings to toxic volatiles (such as acetaldehyde and ethanol) should be investigated. This may help in the selection of edible coatings with low risk of inducing off-flavors and internal disorders. Characterization of temperature effects on rCO2 and of temperature and RH effects on permeance attributes of commodities treated with different levels of coating deposit should help to improve the tailoring of surface coating treatments for different environmental conditions. Characterizing LOLi as a function of ripening stage could help in establishing the optimum amount of coating that can be applied to each type of commodity to ensure maximum retention of postharvest quality without undue risk of fermentation. These pieces of information will be fundamental to produce a more robust mechanistic model for optimizing the

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use of surface coatings for different commodities and environmental storage conditions. Information will be required about the relationship between the extent of internal atmosphere modification and changes in individual physiological ripening processes, such as respiration, softening, and skin color change. Sensory attributes and incidence of physiological disorders after long-term cold storage and also after a shelf life period should be considered in experimental coating programmes. Further research into the factors causing and solutions for minimizing variability between and within lines of fruit would be useful. At present in order to reduce the risk of a few fruit turning anaerobic, the coating level needs to be much less than optimum for the majority of the fruit. It is important that coating optimization experiments conducted within the laboratory are repeated in commercial packhouse conditions to ensure recommendations are relevant to the industry. Collaborative work between engineers designing waxing plants and the chemists formulating the coatings could help industry to optimize coating applications for various commodities.

LITERATURE CITED Ahmed, E. M., R. A. Dennison, and P. E. Shaw. 1978. Effect of selected oil and essence volatile components on flavor quality of pumpout orange juice. J. Agr. Food Chem. 26:368–372. Aljuburi, H. J., and A.. Huff. 1984. Reduction in chilling injury to stored grapefruit (Citrus paradisi Macf.) by vegetable oils. Scientia Hort. 24:53–58. Amarante, C. V. T. 1998. Gas exchange, ripening behaviour and postharvest quality of coated pears. Ph.D. Dissertation, Massey Univ., Palmerston North, New Zealand. Amarante, C., N. H. Banks, and S. Ganesh. 1997a. Permeance to gases, internal atmosphere modification and ripening of coated pears. p. 145–150. Vol. 2. In: E. J. Mitcham (ed.), Proc. Seventh Int. Controlled Atmosphere Research Conference, Univ. California, Davis. Amarante, C., N. H. Banks, and G. Siva. 1997b. Gas exchange and ripening behaviour of coated pears. p. 193–202. In: Proc. Australasian Postharvest Horticulture Conference, Univ. Western Sydney Hawkesbury, NSW, Australia. Andrich, G., R. Fiorentini, A. Tuci, A. Zinnai, and G. Sommovigo. 1991. A tentative model to describe the respiration of stored apples. J. Am. Soc. Hort. Sci. 116:478–481. Andrich, G., A. Zinnai, S. Balzini, S. Silvestri, and R. Fiorentini. 1994. The kinetics effect of PCO2 on the respiration rate of Golden Delicious apples. Acta Hort. 368:374–381. Ashley, R. J. 1985. Permeability and plastics packaging. p. 269–308. In: J. Comyn (ed.), Polymer permeability. Elsevier Applied Science Publ., New York. Avena-Bustillos, R. J., and J. M. Krochta. 1993. Water vapour permeability of caseinatebased edible films as affected by pH, calcium crosslinking and lipid content. J. Food Sci. 58:904–907. Avena-Bustillos, R. J., J. M. Krochta, and M. E. Saltveit. 1997. Water vapor resistance of Red Delicious apples and celery sticks coated with edible caseinate-acetylated monoglyceride films. J. Food Sci. 62:351–354.

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Avena-Bustillos, R. J., J. M. Krochta, M. E. Saltveit, R. J. Rojas-Villegas, and J. A. SaucedaPérez. 1994. Optimization of edible coating formulations on zucchini to reduce water loss. J. Food Eng. 21:197–214. Baldwin, E. A. 1994. Edible coatings for fresh fruits and vegetables: Past, present, and future. p. 25–64. In: J. M. Krochta, E. A. Baldwin, and M. O. Nisperos-Carriedo (eds.), Edible coatings and films to improve food quality. Lancaster, Basel, Technomic Publ. Co. Baldwin, E. A., M. O. Nisperos-Carriedo, and R. A. Baker. 1995a. Edible coatings for lightly processed fruits and vegetables. HortScience 30:35–40. Baldwin, E. A., M. O. Nisperos-Carriedo, and R. A. Baker. 1995c. Use of edible coatings to preserve quality of lightly (and slightly) processed products. Crit. Rev. Food Sci. Nutr. 35:509–524. Baldwin, E. A., M. O. Nisperos-Carriedo, R. D. Hagenmaier, and R. A. Baker. 1997. Use of lipids in coatings for food products. Food Technol. 51:56–62, 64. Baldwin, E. A., M. O. Nisperos-Carriedo, P. E. Shaw, and J. K. Burns. 1995b. Effect of coatings and prolonged storage conditions on fresh orange flavor volatiles, degrees Brix, and ascorbic acid levels. J. Agr. Food Chem. 43:1321–1331. Banks, N. H. 1984a. Some effects of TAL Pro-long coating on ripening bananas. J. Expt. Bot. 35:127–137. Banks, N. H. 1984b. Studies of the banana fruit surface in relation to the effects of TAL Pro-long coating on gaseous exchange. Scientia Hort. 24:279–286. Banks, N. H. 1984c. Internal atmosphere modification in Pro-long coated apples. Acta Hort. 157:105–112. Banks, N. H. 1985a. The oxygen affinity of 1-aminocyclopropane-1-carboxylic acid oxidation in slices of banana fruit tissue. p. 29–36. In: J. A. Roberts and G. A. Tucker (eds.), Ethylene and plant development. Butterworth, London. Banks, N. H. 1985b. Responses of banana fruit to TAL Pro-long coating at different times relative to the initiation of ripening. Scientia Hort. 26:149–157. Banks, N. H., S. M. Elyatem, and M. T. Hammat. 1985. The oxygen affinity of ethylene production by slices of apple fruit tissue. Acta Hort. 157:257–260. Banks, N. H., Q. Cheng, S. E. Nicholson, A. M. Kingsley, and P. B. Jeffery. 1997a. Variation with temperature in effects of surface coatings on gas exchange of apples. Invited presentation to International Congress for Plastics in Agriculture, Tel Aviv, Israel, 9–15 March 1997 (in press). Banks, N. H., D. J. Cleland, A. C. Cameron, R. M. Beaudry, and A. A. Kader. 1995. Proposal for a rationalized system of units for postharvest research in gas exchange. HortScience 30:1129–1131. Banks, N. H., D. J. Cleland, C. W. Yearsley, and A. M. Kingsley. 1993b. Internal atmosphere composition—a key concept in responses of fruits and vegetables to modified atmospheres. p. 137–143. Proc. Australasian Postharvest Conference. University of Queensland, Gatton College, Lawes, Queensland, Australia. Banks, N. H., J. G. M. Cutting, and S. E. Nicholson. 1997b. Approaches to optimising surface coatings for fruits. New Zealand J. Crop Hort. Sci. 25:261–272. Banks, N. H., B. K. Dadzie, and D. J. Cleland. 1993a. Reducing gas exchange of fruits with surface coatings. Postharv. Biol. Technol. 3:269–284. Bauchot, A. D., and P. John. 1996. Scald development and the levels of α-farnesene and conjugated triene hydroperoxides in apple peel after treatment with sucrose esterbased coatings in combination with food-approved antioxidants. Postharv. Biol. Technol. 7:41–49.

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Bauchot, A. D., P. John, Y. Soria, and I. Recasens. 1995a. Sucrose ester-based coatings formulated with food-compatible antioxidants in the prevention of superficial scald in stored apples. J. Am. Soc. Hort. Sci. 120:491–496. Bauchot, A. D., P. John, Y. Soria, and I. Recasens. 1995b. Carbon dioxide, oxygen, and ethylene changes in relation to the development of scald in Granny Smith apples after cold storage. J. Agr. Food Chem. 43:3007–3011. Bayindirli, L., G. Sümnü, and K. Kamadan. 1995. Effects of Semperfresh and Johnfresh fruit coatings on poststorage quality of “Satsuma” mandarins. J. Food Process. Preserv. 19:399–407. Beaudry, R. M. 1993. Effect of carbon dioxide partial pressure on blueberry fruit respiration and respiratory quotient. Postharv. Biol. Technol. 3:249–258. Beaudry, R. M., E. R. Uyguanco, and T. M. Lennington. 1993. Relationship between headspace and tissue ethanol level of blueberry fruit and carrot roots in sealed LDPE packages. p. 87–94. In: Proc. Sixth Int. Controlled Atmosphere Research Conference, Cornell Univ., Ithaca, NY. Bender, R. J., J. K. Brecht, S. A. Sargent, J. C. Navarro, and C. A. Campbell. 1993. Ripening initiation and storage performance of avocados treated with an edible-film coating. Acta Hort. 343:184–186. Ben-Yehoshua, S. 1966. Some effects of plastic skin coating on banana fruit. Trop. Agr. 43:219–232. Ben-Yehoshua, S. 1967. Some physiological effects of various skin coatings on orange fruit. Israel J. Agr. Res. 17:17–27. Ben-Yehoshua, S., S. P. Burg, and R. Young. 1985. Resistance of citrus fruit to mass transport of water vapor and other gases. Plant Physiol. 79:1048–1053. Ben-Yehoshua, S., M. J. Garber, and C. K. Huszar. 1970. Use of a physiological parameter as means for operational control of application of orange skin-coating in packing plants. Trop. Agr. 47:151–155. Bhardwaj, C. L., H. F. Jones, and I. H. Smith. 1984. A study of the migration of externally applied sucrose esters of fatty acids through the skins of banana, apple and pear fruits. J. Sci. Food Agr. 35:322–331. Blackman, F. F. 1928. Analytical studies in plant respiration: I. The respiration of senescent ripening apples. Proc. Royal Soc., London 103:412–445. Blake, J. R. 1966. Some effects of paraffin wax emulsions on bananas. Queensland J. Agr. Anim. Sci. 23:49–56. Boersig, M. R., A. A. Kader, and R. J. Romani. 1988. Aerobic-anaerobic respiratory transition in pear fruit and cultured pear fruit cells. J. Am. Soc. Hort. Sci. 113:869–873. Brown, G. E. 1984. Efficacy of citrus postharvest fungicides applied in water or resin solution water wax. Plant Dis. 68:415–418. Bufler, G., and J. Streif. 1986. Ethylene biosynthesis of ‘Golden Delicious’ apples stored in different mixtures of carbon dioxide and oxygen. Sci. Hort. 30:177–185. Burg, S. P., and E. A. Burg. 1967. Molecular requirements for the biological activity of ethylene. Plant Physiol. 42:144–152. Cameron, A. C., R. M. Beaudry, N. H. Banks, and M. Yelenich. 1994. Modified-atmosphere packaging of blueberry fruit: Modeling respiration and package oxygen concentrations at different temperatures. J. Am. Soc. Hort. Sci. 119:534–539. Cameron, A. C., and M. S. Reid. 1982. Diffusion resistance: importance and measurement in controlled atmosphere storage. p. 171–180. In: D. G. Richardson and M. Meheriuk (eds.), Controlled Atmospheres for Storage and Transport of Perishable Agricultural Commodities. Oregon State Univ. School of Agriculture, Symposium Series 1, Corvallis.

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trolled Atmospheres for Storage and Transport of Perishable Agricultural Commodities. Oregon State Univ. School of Agr., Symposium Series 1. Solomos, T. 1985. Interaction between O2 levels, rates of respiration and gas diffusion in five apple varieties. p. 10–19. In: S. M. Blankenship (ed.), Proc. Fourth National Controlled Atmosphere Research Conference, North Carolina State Univ., Raleigh, NC. Sriyook, S., S. Siriatiwat, and J. Siriphanich. 1994. Durian fruit dehiscence—water status and ethylene. HortScience 29:1195–1198. Sümnü, G., and L. Bayindirli. 1994. Effect of Semperfresh and Johnfresh fruit coatings on poststorage quality of “Ankara” pears. J. Food Process. Preserv. 18:189–199. Sümnü, G., and L. Bayindirli. 1995a. Effects of sucrose polyester coating on fruit quality of apricots (Prunus armenaica (L)). J. Sci. Food Agric. 67:537–540. Sümnü, G., and L. Bayindirli. 1995b. Effects of coatings on fruit quality of Amasya apples. Lebensm. Wiss. Technol. 28:501–505. Thomas, M., and J. C. Fidler. 1933. Studies in zymasis. VI. Zymasis by apples in relation to O2 concentration. Biochem. J. 27:1629–1642. Toivonen, P. M. A. 1997. Non-ethylene, non-respiratory volatiles in harvested fruits and vegetables: their occurrence, biological activity and control. Postharv. Biol. Technol. 12:109–125. Trout, S. A., E. G. Hall, and S. M. Sykes. 1953. Effects of skin coatings on the behaviour of apples in storage: I. Physiological and general investigations. Austral. J. Agr. Res. 4:57–81. Tugwell, G. L. 1973. Postharvest fungicides. J. Austral. Inst. Agr. Sci. 39:167–173. Van Zyl, H. J., H. Tormann, and L. J. von Mollendorff. 1987. Effect of wax treatment on fruit quality of pears, apples, plums and nectarines after cold storage. Decid. Fruit Grower 37:169–172. Van Zyl, H. J., and J. W. Wagner. 1986. Keeping quality and shelf life of Bon Chretien pears as affected by calcium, Alar and Semperfresh. Acta Hort. 194:223–228. Wild, B. L. 1991. Postharvest factors governing the development of peteca rind pitting on ‘Meyer’ lemons. HortScience 26:287–289. Wills, R. H., T. H. Lee, D. Graham, W. B. McGlasson, and E. G. Hall. 1981. Postharvest: An introduction to the physiology and handling of fruit and vegetables. AVI Publ. Co., Westport, CT. Wong, D. W. S., W. M. Camirand, and A. E. Pavlath. 1994. Development of edible coatings for minimally processed fruits and vegetables. p. 65–88. In: J. M. Krochta, E. A. Baldwin, and M. O. Nisperos-Carriedo (eds.), Edible coatings and films to improve food quality. Lancaster, Basel, Technomic Publ. Co. Wong, D. W. S., F. A. Gastineau, K. S. Gregorski, S. J. Tillin, and A. E. Pavlath. 1992. Chitosan-lipid films: microstructure and surface energy. J. Agr. Food Chem. 40:540–544. Yang, S. F. 1985. Biosynthesis and action of ethylene. HortScience 20:41–45. Yearsley, C. W., N. H. Banks, and S. Ganesh. 1997a. Temperature effects on the internal lower oxygen limits of apple fruit. Postharv. Biol. Technol. 11:73–83. Yearsley, C. W., N. H. Banks, and S. Ganesh. 1997b. Effect of carbon dioxide on the internal lower oxygen limits of apple fruit. Postharv. Biol. Technol. 12:1–13. Yearsley, C. W., N. H. Banks, S. Ganesh, and D. J. Cleland. 1996. Determination of lower oxygen limits for apple fruit. Postharv. Biol. Technol. 8:95–109. Yip, W., X. Jiao, and S. F. Yang. 1988. Dependence of the in vivo ethylene production rate on 1-aminocyclopropane-1-carboxylic acid content and oxygen concentrations. Plant Physiol. 88:553–558. York, G. M. 1994. An evaluation of two experimental polysaccharide Nature Seal coatings in delaying the post-harvest browning of the lychee pericarp. Proc. Fla. State Hort. Soc. 107:350–351.

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5 Greenhouse Tomato Fruit Quality M. Dorais and A. P. Papadopoulos Agriculture and Agri-Food Canada, Greenhouse & Processing Crops Research Centre, Harrow, ON, N0R 1G0, Canada A. Gosselin Horticulture Research Centre, Laval University, Sainte-Foy, QC, G1K 7P4, Canada

I. INTRODUCTION II. QUALITY ATTRIBUTES A. Appearance B. Firmness and Texture C. Fruit Dry Matter D. Organoleptic Compounds and Relation to Sensory Properties E. Health Influencing Compounds III. GENETIC CHARACTERISTICS AFFECTING TOMATO FRUIT QUALITY IV. ENVIRONMENTAL FACTORS AFFECTING TOMATO FRUIT QUALITY A. Effect of Light Intensity B. Effect of Temperature C. Effect of VPD D. Effect of CO2 Enrichment V. CULTURAL PRACTICES AFFECTING FRUIT QUALITY A. Hydroponics and Growing Media B. System and Growing Regime C. Effect of Irrigation D. Mineral Nutrition E. Effect of Electrical Conductivity VI. POSTHARVEST VII. CONCLUSION LITERATURE CITED

Horticultural Reviews, Volume 26, Edited by Jules Janick ISBN 0-471-38789-4 © 2001 John Wiley & Sons, Inc. 239

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I. INTRODUCTION Tomato (Lycopersicon esculentum Mill., Solanaceae) is one of the three most important horticultural crops. The plant originates from Central America and was an important crop for the ancient Aztecs and Incas. History, taxonomic status, flower and fruit anatomy and development have been well described by Davies and Hobson (1981), Taylor (1986), Atherton and Harris (1986), and Ho and Hewitt (1986). In terms of human health, tomato is a major component of daily meals in many countries and constitutes an important source of minerals, vitamins, and antioxidant compounds. In 1998, 89.8 million tonnes of tomatoes were produced worldwide (FAO 1998, www.fao.org), of which around 11.6 million tonnes were produced under greenhouse conditions. Virtually all greenhouse production is sold for the fresh market and often transported over long distances. Different types of greenhouses and protection structures can be found, ranging from wooden structures covered with plastic film to glasshouses fully equipped for automatic climatic control. The surface of greenhouses in the world is greater than 306,500 ha (Wittwer and Castilla 1995) to 450,000 ha (Martinez 1999). This is mainly concentrated in China, Japan, Korea (close to 270,000 ha) (Martinez 1999), and the Mediterranean basin (100,000 ha) (Baudoin 1999). In those countries, unheated plastic house structures are mostly made of wooden posts and iron wire covered with plastic film, and have no climate control, or are iron/wood frame walk-in tunnels with partly automated climate control. Glasshouses, widely used in northern latitudes, are relatively scarce (8,000 ha) in the Mediterranean region, and are mostly found in France (2,700 ha), Italy (2,300 ha), and Turkey (2,000 ha) (Castilla and Hernandez 1995). Greenhouses found in northern European and American industries are glass (higher light transmittance) and polyethylene greenhouses providing a maximum climatic control and an environment that optimizes a high year-round productivity and a high product quality. Light, temperature, air, and media humidity, nutrition, and CO2 content of the atmosphere are computer-programmed 24 h a day to achieve maximum crop yield and quality, all as part of dynamic climate and fertigation control systems. The countries with the highest greenhouse (heated and non-heated plastic and glass greenhouses) crop production of tomato are Spain, 12–15,000 ha (Munoz-Carpena 1999, pers. commun.); Japan, 6,488 ha (Ito 1999); Saudi Arabia, 5,527 ha (El-Aidy 1992); Italy, 5,452 ha (Tesi 1992); Greece, 1,765 ha (Olympios 1991); the Netherlands, 1,150 ha (Van Os and Benoit 1999); France, 1000 ha; Belgium, 600 ha (Van Os and Benoit 1999); Canada, 399 ha (Auger 2000); USA, 314

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ha (Auger 2000); Mexico, 290 ha (Auger 2000); the UK, 299 ha; and Germany, 200 ha. In North America, the vegetable greenhouse industry has recently been expanding rapidly (around 20% per year). Demand for high-quality products throughout the year by consumers sustains this wide-spread greenhouse industry. Tomato fruit quality for fresh consumption is determined by appearance (color, size, shape, freedom from physiological disorders, and decay), firmness, texture, dry matter, and organoleptic (flavor) and nutraceutic (health benefit) properties. The organoleptic quality of tomato is mainly attributed to its aroma volatiles, sugar, and acid content, while the nutraceutical quality is defined by its mineral, vitamin, carotenoid, and flavonoid content. Postharvest durability and fruit safety are also very important quality criteria for product distribution and marketing (Grierson and Kader 1986; Kader 1986). Fruit quality as well as postharvest durability are greatly influenced by genetic characteristics of tomato cultivars. Greenhouse cultivars are indeterminate plants, as opposed to determinate field and processing tomato plants, and all hybrids come from traditional breeding techniques (McGrath 1998). Cultivar development ranges from the sweet-tasting small cherry to large beefsteak tomato fruit with a high yield and disease resistance, good shelf-life, and flavor (Van de Vooren et al. 1986). These cultivars can be grouped by color, size, shelf-life, and type of harvest (loose or truss harvest). Most of the greenhouse industry grows red fruit, but there is an increasing market interest for yellow, orange, and pink tomatoes. Pink cultivars (80–210 g) are mostly grown in Japan and Canada. A wide range of size grades exists, from the small cherry (10–20 g) to the beefsteak fruit (Van de Vooren et al. 1986). Beefsteak tomato (180–250 g, 5 locules or more) is the most popular group in North America, representing more than 85% of the area (Anon. 2000), and has an important market in Germany and France, while round fruit (70 g, 2–3 locules) is mostly grown in Europe. Some long life cultivars such as ‘Daniela’ have greater postharvest longevity compared to traditional cultivars and are mostly produced in Spain, Morocco, and Israel for export markets. The cultivation of tomato that can be harvested as a truss (or cluster) has taken a firm hold within the European greenhouse tomato industry (20–40%) and has increased in importance in North America within the last few years, representing more than 10% of the total greenhouse tomato production (Anon. 2000). A number of characteristics such as truss shape, fruit firmness, calyx quality, truss shelf-life, fruit color, and taste are important for the truss tomato market. Truss shape should be flat (fish-bone trusses) to ensure an attractive presentation. Moreover, fruit must stay on the truss for a longer time than the standard types.

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After picking, the truss must be able to withstand a certain degree of handling and maintain a fresh appearance. The fruit requires a green calyx that stays fresh for a long time. Truss tomatoes stay juicier because they continue to absorb moisture from the vine. Also, fruit on the truss must stay firm during the red stage, and must not crack. A deep red fruit color is preferred by consumers. The production of high-quality fruit is also controlled by climate factors and cultural pratices. Climate factors are defined by light intensity, ambient temperature (day/night), vapor pressure deficit (VPD), and CO2 enrichment of the atmosphere concentration. For early yield and high quality of greenhouse tomato production in northern Europe (Ho 1996b) and North America, the most limiting climatic factor is low light. Cultural pratices such as growing methods (growing media, planting density, cluster size, deleafing, root system), the irrigation regime, and the composition and concentration of the nutrient solution, as well as the time of harvest and storage conditions, all influence the quality of the final product. Previous reviews have discussed fruit components and quality, mostly for field and processing tomato (Davies and Hobson 1981; Petro-Turza 1986; Stevens 1986; Stevens and Rick 1986), or on a specific aspect of fruit quality such as health benefit (Steinmetz and Potter 1996; Gerster 1997; Ness and Powles 1997; Clinton 1998; Giovannucci 1999), genotypic variation of field and processing tomato quality and breeding (Stevens 1986; Stevens and Rick 1986), or fruit ripening and postharvest quality (Grierson and Kader 1986; Sams 1999). Even though there is an abundant literature on greenhouse tomato and an increasing interest in high-quality fruit product, no review has been made of the factors that influence greenhouse tomato fruit quality. The present review focuses on greenhouse tomato fruit quality and summarizes research over the past 30 years on the influence of greenhouse climate factors and cultural practices during growth and postharvest conditions on tomato fruit quality, with emphasis on the current state of knowledge of organoleptic and nutraceutic qualities of tomato fruit. Although greenhouse tomato fruit quality is emphasized, we occasionally refer to field or processing tomato studies when no exhaustive greenhouse scientific literature is available. We first outline tomato quality attributes and review recent work on the major organoleptic and nutraceutic components of tomato fruit. Preharvest and postharvest factors that affect greenhouse tomato fruit quality are presented and discussed. We conclude this review by identifying several prospects for future research, including greenhouse plant breeding and crop environment.

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II. QUALITY ATTRIBUTES A. Appearance 1. Color. Color has a strong influence on the buying behavior of the consumer (Francis 1980; D’Souza et al. 1992). Consumers generally relate fruit color to its organoleptic quality. It is therefore essential to offer fruit with uniform color and tints to satisfy consumer demand. Tomato fruit is currently available in red, pink, yellow, and orange colors. These colors depend on fruit carotenoids that are contained in small globules suspended in the fruit pulp. Carotenoid concentration and distribution in the fruit are documented in Davies and Hobson (1981) and Thakur et al. (1996). Carotenoid concentration increases 12-fold during fruit ripening, mainly due to a 460-fold increase in the concentration of lycopene (Fraser et al. 1994). The outer pericarp in tomato fruit is highest in total carotenoids, whereas locular contents are highest in carotene (Thakur et al. 1996). Their concentration in the fruit depends on the cultivar and growing conditions. Gould (1992) has noted that summer- or wintercultivated greenhouse fruit has lower carotene concentrations than summer, field-grown fruit. The influence of climate factors, cultural practices, and postharvest on fruit color are discussed later in Sections IV, V, and VI. 2. Fruit Shape and Size. Fruit shape and size are determined a priori by cultivar but also vary according to ambient climatic conditions and the cultural method used. Fruit number per plant, cluster size, fruit position on the cluster, seed content, and plant density as well as temperature and light intensity determine fruit shape, dry matter accumulation, and size (Bertin et al. 1998; De Koning 1994; Cockshull and Ho 1995). Moreover, growing conditions that favor vegetative growth will lead to fruit malformation and size reduction (De Koning 1994). Misshapen fruit of the second to the fifth cluster obtained under spring growing conditions are a common problem for tomato greenhouse growers in Canada. This problem can cause significant quality losses (up to 45%) and is often due to low light conditions (source limitation and high competition for assimilates) and inappropriate temperature regimes. The potential size of tomato fruit is mainly determined by the number of cells in the pre-anthesis ovary (Ho 1996a). However, the final size of a tomato fruit is determined by both the rate and duration of cell enlargement (Ho 1992). From a physiological standpoint, Thompson et al. (1998) showed that the pericarp exerts tissue pressure on the epidermis in tomato fruit, suggesting that fruit growth rate is determined

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by the physical properties of the epidermal cell walls. Furthermore, results from epidermal and pericarp activities of xyloglucan endotransglycosylase and peroxidase suggested that fruit expansion can be regulated by the combined action of these enzyme activities in the fruit epidermis (Thompson et al. 1998). These authors observed a good temporal correlation between epidermal peroxidase activity and growth termination. This result supports the hypothesis that the tomato epidermis has a special role in regulation of fruit growth (Thompson et al. 1998). Tomato expansion is affected by the availability of photoassimilates and minerals, temperature, and water supply (Ho 1996a). Bussière (1993, 1994, 1995, 1999) has proposed a model of tomato fruit expansion, which should contribute to a better understanding of the variables determining fruit size and its dry matter content. This model can possibly be a tool to control tomato fruit quality. Descriptive and explanatory modelling of biomass production and yield of greenhouse tomato crops has also been reported by different authors (Spitters et al. 1989; Jones et al. 1991; Tchamitchian and Longuenesse 1991; Bertin and Heuvelink 1993; Gauthier 1993; Dayan et al. 1993; De Koning 1994; Heuvelink 1995a; Bertin and Gary 1998; Marcelis et al. 1998; Heuvelink 1999), with special reference to the simulation of leaf area, light interception, production, partitioning, and content of dry matter. Fruit weight of round and beefsteak greenhouse tomato increased with higher truss position on the stem (De Koning 1994; Bertin 1995). This effect has been ascribed to apex enlargement and the subsequent increase in the number of fruit cells (De Koning 1994). However, a recent study by Bertin et al. (1998) on three tomato cultivars (‘Daniela’, ‘Recento’, and ‘Trust’) on the first 15 trusses did not confirm this increase. Different climatic conditions and sourcesink balances during early fruit development are likely to be the main causes for these differences (Bertin et al. 1998). Indeed, the interaction between truss position (ontogeny) and radiation (season) may be ascribed to an increasing cell number due to a faster ontogenic enlargement of the apex at higher radiation (De Koning 1994). Similarly, it has been shown that within each truss proximal fruits reach larger potential weights than distal fruit because of the natural flowering sequence and the higher number of cells in proximal ovaries at anthesis (Bohner and Bangerth 1988; Bertin et al. 1998), while a higher IAA content in proximal fruit may explain their greater sink activity (Bangerth and Ho 1984; Bangerth 1989). However, this response may change with cultivar. Bertin et al. (1998) found that variability in potential weight among trusses was mainly related to the cultivar, whereas differences between proximal and distal fruit were significant for beefsteak tomato only. Under competitive growth conditions (high fruit load compared to low fruit load),

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they observed that the weight of distal fruit was reduced more than that of proximal fruit, the latter having a lower cell number reduction (Bohner and Bangerth 1988). The greater ability of proximal fruit to compete for assimilates under limited supply has also been reported by several authors (Bangerth and Ho 1984). It results in a faster accumulation of dry matter during the first 25 days after anthesis, and was attributed to their higher capacity to receive phloem mobile assimilate and to synthesize starch (Ho et al. 1983). Fruit size is also positively correlated with seed number (Rylski 1979b) but can change among cultivars and under competitive conditions (Bertin et al. 1998). The potential number of seeds could be related to the number of ovules that have been shown to be proportional to the cell number in the fruit (Ho 1996a). Thus, the application of cytokinin and gibberellin can promote cellular division and sink size of tomato fruit (Ho 1996b). However, the link between fruit weight and seed content has been attributed to sink activity induced by the developing seeds at an early stage rather than to a direct effect of the seed-synthesized auxins on cell enlargement in the fruit (Varga and Bruinsma 1976). Given that other plant organs are sinks of lower activity than fruits, a change in the number of fruit per plant will affect mainly fruit weight and shape rather than the harvesting index (economic yield/biological yield × 100) (Ho 1992). In greenhouse tomato plants, 64–67% of photoassimilates (Hurd et al. 1979; Cockshull et al. 1992), expressed on a dry matter basis, are directed to fruit organs, whereas for field tomato this proportion is only 55% (Ho 1996a). 3. Physiological Disorders Fruit Cracking. In North America, depending on the time of the year, greenhouse fruit cracking may cause losses in the order of 35%. Although this physiological disorder causes considerable economic losses in greenhouse and field-grown tomato (Hassan 1978; Abbott et al. 1985; Peet 1992; Welles et al. 1992; McAvoy 1995; Opara et al. 1997), greenhouse fruit is more vulnerable to fruit cracking losses because most of the cultivars used lack crack resistance and because fruit is generally harvested at the “pink” stage (30–60% of the surface shows pink or red color) or later (Peet and Willits 1995). Depending on the extent of this physiological disorder, fruit cracking: (1) reduces fruit appeal (Peet and Willits 1995), (2) reduces fruit shelf-life (Hayman 1987; Bakker 1988; Welles et al. 1992); (3) increases fruit susceptibility to pathogens (Hassan 1978; Peet 1992; Peet and Willits 1995), and (4) reduces fruit marketability (Peet 1992; Welles et al. 1992; McAvoy 1995). The influence

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of cultivars, climate factors, and cultural practices on fruit cracking susceptibility is discussed later in Sections III, IV, and V. There are several types of fruit cracking injury: fruit bursting, radial cracking (star-shaped originating from the peduncle), concentric cracking (circular cracks originating from the peduncle), and cuticle cracking (russeting). Cuticle cracking is among the most commonly observed greenhouse fruit cracking, while radial cracking may also be present. The importance of this disorder varies according to: (1) time of year (more severe during spring, summer, and the early part of the fall) (Koske et al. 1980); (2) cultivar (Koske et al. 1980; Cortés et al. 1983); and (3) ambient conditions (Abbott et al. 1986). Fruit cracking is generally associated with the rapid movement of water and sugars towards the fruit when cuticle elasticity and resistance are weak. This physiological disorder occurs generally six to seven weeks after fruit set (Bakker 1988). The first occurrence of cuticle cracking in cherry tomato fruit, however, was recognized two weeks after anthesis (Ohta et al. 1995). A physical model, based on the theory of shells (Timoshenko and WionowskyKrieger 1959) and the linear theory of elasticity (Considine et al. 1974; Considine and Brown 1981), showed that the site of initiation of cuticle cracking is between locular contents, and the most important stress zone is located near the calyx. Many characteristics are associated with tomato fruit cracking: (1) fruit shape and size (Gill and Nandpuri 1970; Considine and Brown 1981); (2) extensible and thick cuticle (Gill and Nandpuri 1970); (3) deep penetration of cutin inside the cuticle (Hankinson and Rao 1979); (4) thick pericarp (Peet 1992; McAvoy 1995); (5) number of fruits per plant (Peet 1992; McAvoy 1995); (6) fruit position in the plant (Peet and Willits 1995); (7) soluble sugar content (Frazier 1934; Peet 1992); (8) development of vascular tissues in fruit (Cotner et al. 1969); and (9) plant architecture (Peet 1992). Considine and Brown (1981) suggested that as the fruit increases in size, more physical stress is applied against the epidermis, and this leads to an increasing susceptibility to fruit cracking. On the other hand, Ehret et al. (1993) have shown that there was no correlation between fruit size and their relative growth rate, or with fruit susceptibility to cuticle cracking. Small cluster size (one or two fruit per cluster), however, may increase the percentage fruit surface area cracked and the severity of cuticle cracking (Ehret et al. 1993). The number of fruit per plant (Peet 1992) and the position on the plant (Peet and Willits 1995) are also very important factors. A high number of fruit per plant increases the competition between fruit for carbohydrates, thus reducing the supply of sugars and water to each fruit and, as a consequence, their susceptibility to radial and cuticle cracking. A

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fruit:leaf ratio of 1.24:1 to 1.28:1 is generally optimal. In addition, a significant increase in fruit cracking incidence of fruit on upper clusters (linear function) has been observed (Peet and Willits 1995), with the percentage of fruit affected by cracking increasing from 2% (1st cluster) to 38%, 41%, and 45% for clusters 5, 6, and 7, respectively. Many factors can explain this greater susceptibility of fruits of upper clusters to cracking such as a higher irradiance received by fruits of upper clusters and a higher fruit temperature. These factors tend to favor pulp expansion towards the interior of the fruit and, consequently, a weakening of the cuticle. Fruit with a high content in soluble sugars (low solute potential, Ψs) generally have a greater supply of water (Bussières 1995), thereby increasing the pressure applied against the cuticle and fruit susceptibility to cracking. An elevated osmotic concentration in cherry fruit juice is associated with fruit susceptibility to cracking (Ackley and Krueger 1980). It is possible to limit fruit cracking by eliminating the supply of sugars to fruit by the phloem (destruction of the phloem by thermal shock), thus strengthening the hypothesis that soluble sugar concentration in fruits plays an important role in the appearance of cracking (Peet and Willits 1995). An imbalance between the supply (influx) and loss (efflux) of water will also cause fruit cracking. Tomato cultivars with a highly developed system of vascular tissues are therefore more resistant to this physiological disorder (Cotner et al. 1969). Similarly, plant architecture and planting density play an important role in affecting fruit susceptibility to cracking by modifying the degree of leaf shade on fruit (Peet 1992). In periods of rapid plant growth (for example, under high irradiance, PPF), an accelerated cellular enlargement and fruit development require an additional supply of nutrients such as calcium, an important nutrient in the prevention of fruit physiological disorders such as fruit cracking (Simon 1978). Gibberellic acid is a phytohormone involved in the process of fruit ripening and softening of the cuticle (Ben-Arie et al. 1995). The presence of gibberellic acid can alter the calcium dynamics at the level of the pericarp (Bush et al. 1989) by increasing the elasticity of the cuticle (Larson et al. 1983). The application of gibberellin and calcium reduced the tendency of tomato fruits to cracking in some studies (Dickinson and McCollum 1963; Larson et al. 1983; Peet 1992), although inconclusive results were reported by Igbokwe et al. (1987). Gold Specks. Gold specks have been identified as cells containing a granular mass of tiny calcium oxalate crystals (Den Outer and van Veenendaal

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1988; De Kreij et al. 1992). These tiny yellowish spots are regular, less than 0.1 µm across and often observed around the calyx and shoulders (De Kreij et al. 1992). Their presence affects the external appearance of the fruit (Goossens 1988) and reduces their shelf-life (Janse 1988a). Blossom-End Rot. Blossom-end rot (BER) is a greenhouse and field physiological disorder that causes extensive yield losses. Fruit are generally more sensitive to BER 7–10 days (Sonneveld and Van Der Burg 1991) to 21 days (Ho 1999) after anthesis. BER can develop as a visible external depression of black necrotic tissue affecting the distal part of the placenta and the adjacent locular contents as well as the pericarp (Willumsen et al. 1996). Internal BER, called “black seeds”, may also be present on the same fruit. In this case, black necrotic tissue is restricted to the adjacent parenchyma tissue around the young seeds and the distal part of the placenta (Adams and Ho 1992). Interactions between daily irradiance, air temperature, water availability, salinity, nutrient ratios in the rhizosphere, root temperature, air humidity, and xylem tissue development in the fruit control the incidence of BER. For example, Ho et al. (1993) found a linear relation between the incidence of BER in all trusses and the product of average daily irradiance and daily temperature throughout the year. Lack of co-ordination between accelerated cell enlargement, due to high import of assimilates, and inadequate supply of calcium, due to poor development of xylem within the fruit, is generally linked to fruit susceptibility to BER (Ehret and Ho 1986b; Adams and Ho 1992; Belda and Ho 1993; Ho et al. 1993; Ho et al. 1995; Belda et al. 1996; Ho 1996a). The development of BER is positively correlated to the leaf K:Ca ratio, but is weakly correlated to the K:Ca ratio in mature fruits (Bar Tal and Pressman 1996). A high concentration of organic acids in the fruit may reduce the availability of Ca in the tissues, making the fruit more susceptible to BER (Adams and Ho 1993). Willumsen et al. (1996) suggested that incidence of both external and internal BER strongly depends on the ion activity (a, in mol L–1) ratios between K and Ca + Mg (aK/√[aCa + aMg]) and between Mg and Ca (aMg/aCa) in the root zone at both moderate (3 to 6 mS cm–1) and high (6 to 12 mS cm–1) salinity levels. The higher the activity ratios, the higher the risk of BER due to a lower uptake of Ca and a reduced availability of Ca in the fruit tissue caused by increased concentration of organic acids in the fruit juices. By maintaining the ion activity ratios at optimum levels (about 0.1 for the first ratio and 0.2 to 0.4 for the second) these authors reported that it is possible to avoid or reduce the occurrence of BER when salinity of the root zone is increased to improve the fruit taste.

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Recently, Cornillon et al. (1999) have mapped the fruit axial region and determined the variations in dry matter, potassium, calcium, and magnesium distribution in order to better understand the relationship between dry matter and cation content in the fruit and BER. Their results showed that the distribution of these compounds are not uniform and that their behavior varies in space. No relation between the distribution of dry matter and cations in the fruit slice was found. Although there is a gradient of calcium concentration in the interior of the fruit (Adams and Ho 1992), no difference has been observed in the distribution pattern of calcium in the interior of healthy fruit compared to fruit affected by BER (Nonami et al. 1995). Nonami et al. (1995) have concluded that calcium content in fruit is not directly related to BER. They proposed that this physiological disorder results from the expression of some genes under conditions of stress. In order to avoid BER, different strategies can be used: (1) choose resistant cultivars; (2) optimize calcium and phosphate supply; (3) keep a dynamic balance between calcium and potassium, and between nitrate and ammonium that will ensure sufficient calcium uptake; (4) use low EC; (5) optimize the irrigation frequency in order to avoid water stress or waterlogging; (6) avoid high root temperature (≥ 26°C, Adams and Ho 1993) and low oxygen concentration (Tachibana 1988) which will reduce calcium uptake; (7) avoid excessive canopy transpiration by deleafing, shading, roof sprinkling, and greenhouse fogging; (8) keep a proper fruit:leaf ratio that will provide an adequate fruit growth rate; and (9) spray young expanding fruit with 0.5–0.65% calcium chloride solution (Igbokwe et al. 1987; Dorais et al. 2000; Ho 1999). Watery Fruit. Watery tomato is a physiological disorder resulting from an imbalance between plant water absorption and ambient climatic conditions. The massive influx of water into the fruit, due to an excessive root pressure, increases the volume of cells and can sometimes damage them. The organoleptic quality of fruit is then negatively affected and shelf life is much reduced. Maintaining plant leaf area index at a reasonable level during summertime helps reduce root pressure and minimize the incidence of this physiological disorder. Over-irrigation by the end of the day and a strong root system development also favor the appearance of watery tomato due to an elevated root pressure. Puffiness. Puffiness, also known as hollowness or boxiness, refers to the existence of open cavities between the outer walls and the locular contents in one or more locules (Grierson and Kader 1986). Puffer fruit do not travel well because they are soft and are not appreciated by

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consumers because of the lack of gel in the locules. The percentage of fruit affected is related to genotype and growing conditions that cause improper pollination (Grierson and Kader 1986). B. Firmness and Texture Fruit firmness, often associated with the ripeness stage and shipping ability, is controlled by the integrity of cell wall tissues, the elasticity of pericarp tissues, and the enzymatic activity involved in fruit softening during the process of ripening (Wann 1996). Many studies have focused on the repression of genes involved in the process of fruit senescence (Theologis et al. 1992; Gray et al. 1994). Ethylene regulates fruit ripening by co-ordinating the expression of many genes involved in metabolic processes, such as increasing fruit respiration rates (in climacteric tomato fruit), autocatalytic production of ethylene, chlorophyll degradation, synthesis of carotenoids, conversion of starch into sugars, and the activity of several enzymes involved in the degradation of cell walls (Theologis et al. 1992). Studies on polygalacturonase (PG antisense gene), an enzyme involved in cell wall hydrolysis and fruit softening, have not been conclusive. Despite gene repression and inhibition of RNAm PG accumulation and its enzymatic activity, fruit softening still occurs (Sheehy et al. 1988; Smith et al. 1988), suggesting that polygalacturonase is not the only enzyme involved in the process of fruit softening. On the other hand, studies on the physico-chemical and sensory properties of fruits with reduced levels of PG activity (PG antisense gene) showed that this genetic modification improved color and many sensory attributes (Porretta and Poli 1997; Porretta et al. 1998). Pectinesterases, which are present in most tissues and are responsible for most of the demethylation of cell wall pectins, are thought to facilitate cell wall hydrolysis by the polygalacturonases. Nevertheless, the inhibition of the expression of this enzyme (antisense PE) is rendered difficult owing to the presence of isoenzymes (Hall et al. 1993). Recent advances made on β-galactosidase and other cell wall modifying enzymes (Seymour and Gross 1996; Chapple and Carpita 1998) may allow further genetic examination of texture and fruit softening using sense and antisense technology to develop new lines. The inhibition of ethylene synthesis may be an approach to halt fruit ripening. The inhibition of ACC oxydase (which catalyses the reaction of ACC to ethylene) or of ACC synthase (which catalyses the conversion of AdoMet to ACC) by RNA antisenses as well as overexpression of ACC deaminase (enzyme metabolizing ACC to α-ketobutyrate) also inhibit fruit ethylene production by 90 to 99% (Hamilton et al. 1990; Klee et al. 1991; Oeller

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et al. 1991). Using tACC2 antisense RNA, 90 to 120 days may be required before orange coloring appears in fruit (Oeller et al. 1991). Texture is an important constituent of tomato fruit quality and consumer acceptability. Texture is greatly influenced by fruit internal structure (pericarp:locules ratio), cuticle thickness, and flesh firmness. For instance, the granular or mealy sensation of tomato fruit negatively affects its quality. Bourne (1980) has stated that texture is composed of several properties such as mechanical (hardness, chewiness, and viscosity), geometrical (particle size and shape), and chemical (moisture and fat content) characteristics. Factors such as cellular organelles and biochemical constituents, water content or turgor, cell wall composition, and abiotic factors (water and nutrient availability, temperature, and relative humidity) that affect these traits, will affect fruit texture (Sams 1999). These factors vary not only among cultivars but also as a function of growing conditions. Preharvest factors affecting postharvest texture of many species have been recently reviewed by Sams (1999), while the chemical changes associated with textural changes (van Buren 1979; Seymour and Gross 1996; Harker et al. 1997), and methods of measuring texture are well documented (Bourne 1979a, 1979b; Harker et al. 1997; Abbott et al. 1998). C. Fruit Dry Matter Generally, 5 to 7.5% of tomato content is dry matter (Davies and Hobson 1981) with approximately 1% in cuticle and in seeds, and 4 to 6% in soluble solids (Petro-Turza 1986). Soluble solids of tomato fruit increase linearly along the proximal-distal axis (Peiris et al. 1999), and account for 75% of the dry matter of ripened fruits (Hewitt and Garvey 1987). Despite the fact that cultivars with high soluble solids have high sugar concentrations, soluble solids do not relate to a perception of sweetness by consumers. Soluble solids relate to the perception of fruit acidity, bitterness, astringency, and saltiness (Baldwin et al. 1991a). Unfortunately, there is often an inverse relation between yield and total solids (dry weight as a percent of fresh weight) content (Davies and Hobson 1981). Tomato fruit display little variability in soluble solids content between pericarp and locular portions (Stevens et al. 1977b; Peiris et al. 1999). Reducing sugars as glucose and fructose represent 50% of the dry matter content of tomato fruit (Davies and Hobson 1981; Thakur et al. 1996), while proteins, pectin, cellulose, hemicellulose, organic acids, minerals, pigments, vitamins, and lipids represents the other half of fruit dry matter content (Petro-Turza 1986). Minerals account for 8% of the dry matter

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(Davies and Hobson 1981). Among the mineral elements, potassium (3–4%) together with nitrogen (0.6%) and phosphorus (0.4%) account for more than 90% of the total minerals (Ho and Hewitt 1986). Tomato fruit is relatively rich in selenium (0.5 to 10 mg 100g–1), copper (90 mg 100g–1) and zinc (240 µg 100g–1) (Grolier and Rock 1998). Fiber content (lignin and polysaccharides from plant cell walls) is around 0.6 to 0.7% of fresh fruit weight (Davies and Hobson 1981). Citric and malic acids are the main organic acids found in tomato fruit and constitute approximately 10–13% of dry matter content (Davies 1964; Ho and Hewitt 1986; Petro-Turza 1986). Their content varies with genotype, ripening stage, nutritional status of the plant (Mahakun et al. 1979), and the environment (Winsor 1979). Dry matter content in tomato fruit is inversely proportional to fruit size but positively related to total sugars content and to the soluble solids:total solids ratio (Ho 1996a). Fruit dry matter content is determined by the accumulation of photoassimilates and water. Photoassimilate availability depends on light (photosynthesis) and temperature (metabolic activity) whereas water availability varies with plant water status. During tomato fruit growth, about 85% of the water import is via phloem, together with import of assimilate (Ho et al. 1987). Having said this, water import to the fruit is independent of assimilate concentration and is determined by plant water relations. Thus, fruit size is inversely related to the EC of the nutrient solution, while the dry matter content of the fruit is linearly increased by the EC (Ho 1999). As dry matter in the fruit is determined principally by carbon compounds derived from canopy photosynthesis, a higher total fruit dry matter production can be achieved by improving either canopy photosynthesis and/or the partitioning of assimilate within a plant in favor of fruit production (Heuvelink 1995a). In general, dry matter partitioning is regulated by fruit sink activity (fruit sink strength) rather than leaf (source) activity. Ho (1996a) suggested that the cell number in the preanthesis ovary could be a good measure of sink size, which may in turn determine the potential sink strength (sink size and sink activity) for assimilate in a tomato fruit. As reported by Ho (1988, 1999), the sink strength of a tomato fruit should be determined by the product of the number of storage cells and the storage capacity of individual cells. Starch accumulation at the beginning of the cellular expansion phase provides a greater storage capacity of sugars and reflects a greater capacity for photoassimilate import. In developing tomato fruit, sucrose metabolizing enzymes may regulate sucrose unloading and sink strength and thus fruit dry matter accumulation. Recently, it has been shown that acid invertase may not play an important role in the regulation of assimilate import by the tomato

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fruit (N’tchobo et al. 1999; Dorais et al. 1999a). Overexpression of sucrose phosphate synthase (SPS) in field tomato fruit increased sucrose synthase (Susy) activity by 27%, and 70% more sucrose was unloaded in transformant fruit (20 days after anthesis) compared to untransformed control (Nguyen-Quoc et al. 1999; Dorais et al. 1999a). Acid invertase and ADPGlc PPase activities remained at similar levels or were slightly lower than in untransformed control. Unexpectedly, D’Aoust et al. (1999) further showed that the repression of Susy activity in the fruit (antisense cDNA of the tomato fruit specific Susy, TOMSSF) did not affect the unloading capacity when compared to the untransformed plants. These authors suggested that four sucrose turnover cycles (I-sucrose degradation and resynthesis in the cytosol, II-sucrose degradation in the vacuole and resynthesis in the cytosol, III-sucrose degradation in the apoplast and resynthesis in the cytosol, and IV-starch degradation and synthesis in the amyloplast) may control sink activity of tomato fruit (Nguyen-Quoc et al. 1999; Dorais et al. 1999a). Thus, sink utilization (i.e. respiration, cellular structure, growth, and storage) determines the sucrose import rate. Sucrose metabolizing enzymes may affect the unloading rate but they are not the main regulatory factors. For greenhouse tomato plant, we recently observed that fruit dry weight of overexpression SPS plants (maize SPS cDNA with the Rubisco SSU promoter) was 15–18% higher than untransformed plant under enriched CO2 atmosphere and supplemental lighting, while their fruit soluble solids were 13–20% higher (unpublished data). Recent approaches to manipulating sink strength in order to improve harvest index and thereby crop yield and quality have been discussed by Herbers and Sonnewald (1998). In tomato fruit, about 25% of the imported carbon is lost through respiration (Ho et al. 1987). Therefore, it is conceivable that the net dry matter accumulation could be improved not only by increasing the import of assimilate, but also by reducing respiratory loss of carbon (Ho 1999). Ho (1999) suggested that if the CO2 refixation processes can be enhanced by gluconeogenesis, the net loss of carbon from the fruit should be reduced and the accumulation of sugars increased. However, the occurrence of gluconeogenesis and its contribution to sugar accumulation in tomato is not well understood (Ho 1999). D. Organoleptic Compounds and Relation to Sensory Properties Flavor is a complex sensation based on the taste and aroma of several compounds (Stevens et al. 1977a; Shewfelt 1993). Taste is defined by the sum of the sensory attributes arising from the stimulation of the

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organoleptic receptors on the tongue, while aroma is detected when volatiles enter the nasal passages at the back of the throat and are perceived by receptors of the olfactory system (Baldwin et al. 1998). The development of flavor and aroma volatiles in the ripening tomato fruit has been studied extensively (Kazeniac and Hall 1970; Buttery et al. 1971, 1987, 1988, 1989; Dirinck et al. 1976; McGlasson et al. 1987; PetroTurza 1986; McGlasson 1989; Baldwin et al. 1991a,b, 1998; Buttery and Ling 1993a,b; Linforth et al. 1994; Ke and Boersig 1996). Relationships between flavor and aroma compounds, and sensory flavor perception have been reported (Maul et al. 1997; Baldwin et al. 1998; Krumbein and Auerswald 1998; Bucheli et al. 1999a,b). Many studies have shown that tomato flavor is related to the balance between sugars and organic acids (sugars:acids ratio) in the fruit (Simandle et al. 1966; Lower and Thompson 1967; Davies and Winsor 1969; DeBruyn et al. 1971; Stevens 1972; Stevens et al. 1977a, 1979; Jones 1986; Hobson and Bedford 1989; Ke and Boersig 1996; Auerswald et al. 1999b; Bucheli et al. 1999a,b), total sugar or acids content (Jones and Scott 1984; Stevens et al. 1977a,b), and the interaction with volatile compounds content and profile (Buttery et al. 1971; Dirinck et al. 1976; Kavanagh and McGlasson 1983; Buttery et al. 1988; Thakur et al. 1996; Baldwin et al. 1998; Krumbein and Auerswald 1998). Sugar concentration varies largely with species (Yelle et al. 1988, 1991; Husain et al. 1999), cultivar, 1.66 to 3.99% fresh weight (Stevens 1972), and growing conditions, 3.05 to 4.65% fresh weight (Dorais et al. 1999b, 2000). Other types of sugar such as sucrose, < 0.01% of fresh weight (Davies and Hobson 1981; Thakur et al. 1996), raffinose, arabinose, xylose, galactose, and myoinositol may also be present (Thakur et al. 1996) in small concentrations. Types of carbohydrates present vary according to its stage of development and ripening (Winsor et al. 1962; Ho and Hewitt 1986; Ho 1996a; Husain et al. 1999). Sugars and starch each account for about 10% of the dry weight in one-week-old fruit. The proportion of dry matter in the starch component increases to 20% in three-week-old fruit and then declines to 1% by the mature green stage. In contrast, the proportion of sugars increases steadily to about 50% (Ho 1996a). During the early stages of development, fruit content in glucose is higher than fructose (glucose:fructose ratio is around 1:8), but during fruit growth and ripening, the concentration in fructose increases significantly. In some cultivars, the glucose:fructose ratio decreases all the way to 1.0. Three amino acids: glutamic acid, γ-aminobutyric acid, and glutamine represent 65% of amino acids found in tomato fruit. Glutamic acid concentrations increase during ripening and range between 50 and

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300 mg per 100 g of fresh tissue (Davies and Hobson 1981). Aspartic acid may also be abundant and increases during ripening (Davies and Hobson 1981). Changes in amino acid concentration in the fruit during the course of ripening may affect flavor development (Petro-Turza and Teleky-Vamossy 1989; Fuke and Konosu 1991). Tomato fruit can also contain glycoalkaloids such as tomatine, which causes bitterness when harvested immature (Heftmann 1965; Juvik and Stevens 1982; Chen and Miller 2000). These glycoalkaloids are commonly considered to be defensive compounds that protect plants from insect herbivores and pathogens (Friedman and Levin 1998; Chen and Miller 2000). However, their concentration is negligible when fruits are picked ripened (0.3 to 3.9 mg kg–1 for red fruit and 32 to 567 mg kg–1 for green fruit). Recent studies have identified a second glycoalkaloid called dehydrotomatine (Friedman et al. 1994, 1997a). Its content for red fruit ranged from 0.05 to 0.42 mg kg–1 of fresh weight, while the corresponding range for green tomatoes was from 1.7 to 45 mg kg–1 (Friedman and Levin 1998). Their content is strongly influenced by environmental factors as well as by maturity and cultivar (Friedman and Levin 1998). Fruitiness, which best describes overall tomato flavor, depends on growing conditions and cultivar, and was linked to increased levels of reducing sugars and decreased glutamic acid content (Bucheli et al. 1999a,b). Sweetness and sourness are each influenced by the amount of acids and sugars present (Malundo et al. 1995). Sensory analysis indicated that an acceptable fruit is high in tomato-like flavor intensity and sweetness, but intermediate in sourness (Jones 1986; Ke and Boersig 1996; Baldwin et al. 1998). Also, Malundo et al. (1995) reported that at given levels of sweetness there are optimal acid concentrations beyond which there is decreased flavor acceptability. For example, in processed tomato juice samples, a soluble solids:total acidity ratio lower than 10:1 or greater than 18:1 were found to be unacceptable for flavor (Gould 1978). Earlier, Simandle et al. (1966) found a significant correlation between the aroma of fruit and their soluble solids, pH and soluble solids:titratable acids ratio. A highly significant negative correlation has been reported between pH and titratable acidity (Lower and Thompson 1967). De Bruyn et al. (1971) have concluded that a high fruit acid, and, to a lesser extent, a high sugar concentration, generally improve the organoleptic quality of greenhouse tomato. The sweetness of tomato is mainly attributed to fructose, whereas the acidic taste is associated with citric acid (Stevens et al. 1977a). Although malic acid taste is 14% stronger than that of citric acid, its concentration in most ripe tomato genotypes is lower than that of citric acid (Stevens et al. 1977a). Malic acid predominates in immature green fruits,

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with citric acid forming only about 25% of the total acidity (Davies and Hobson 1981). In ripe fruit, however, citric acid accounts for 40 to 90% of the total acidity (Stevens 1972; Davies and Hobson 1981). Sugar and organic acid contents of about 3.0 g 100 ml–1 and 8.0 meq 100 ml–1 of expressed juice, respectively, are associated with a good fruit organoleptic quality (Cronin and Houlihan 1985). Citric and malic acid concentrations between 7.7 and 10.4 meq 100 ml–1 have been reported for different tomato (Davies and Hobson 1981; Ho 1996a). The overall range of pH encountered for different tomato cultivars varies from 3.9 to 4.9 (Davies and Hobson 1981). Other compounds that contribute to tomato flavor have been identified in tomato fruit by gas chromatography-mass spectrometry. The main groups of compounds are aliphatics, alcohols, aldehydes, ketones, esters, and acids. These flavor volatiles are formed by different pathways, including the deamination and decarboxylation of amino acids (3methylbutanal and 3-methylbutanol; Yu et al. 1968) and lipid oxidation of unsaturated fatty acids (hexanal, hexanol, hexenal and hexenol; Galliard et al. 1977; Hatanaka et al. 1986). Some compounds such as 6methyl-5-hepten-2-one, geranylacetone, and β-ionome are derived from oxidative carotenoid breakdown (Buttery et al. 1988). Among the volatile compounds, cis-3-hexenal makes the greatest contribution to the tomato aroma (Buttery et al. 1971, 1989). The volatiles cis-3-hexenol, 2+3methylbutanol, and hexanal have all been shown to have odor units above zero (Guadagni et al. 1963), which indicates that they are present in tomato at levels above their odor threshold (Buttery 1993). Geranylacetone, on the other hand, has a relatively low odor unit, and thus may or may not contribute significantly to tomato flavor directly, but may have an effect through interactions with other flavor components (Baldwin et al. 1998). Early sensory analysis showed that hexanal, trans-2hexenal, 2-methyl-2-hepten-6-one, 2-isobutylthiazole, and valeronitrile were important contributors to the fresh tomato flavor (Dirinck et al. 1977). The trans-2-hexanal and the cis-3-hexanal have been shown to be responsible for the herbaceous green leaf aroma of tomato plants (Heath 1978) as well as cis-3-hexenal, cis-3-hexenol, trans-2-hexenal (Baldwin et al. 1991b; DeRovira 1996), whereas ketones such as acetone, geranylacetone, and β-ionone generally contribute to the fruity aroma of fruits (Heath 1978; DeRovira 1996), and 2-isobutylthiazole (Petro-Turza 1986) and ethanol (Rothe and Schrodter 1996) to the sugary smell. High concentrations of 1-penten-3-one and 6-methyl-5-hepten-2-one was correlated with aroma and overall flavor (Ke and Boersig 1996). Using gas chromatography-olfactometry and aroma extract dilution analysis, Krumbein and Auerswald (1998) reported that cis-3-hexenal (fresh

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green, sweet), hexanal (green, grassy), 1-octen-3-one (mushroomy), methional (cooked potato), 1-penten-3-one (green), and 3-methylbutanal (unpleasant) belonged to the most odor-active aroma volatiles in fresh tomato. Thus of the 400 volatiles compounds identified in the ripening fruit (Buttery and Ling 1993a,b; Buttery et al. 1989; Petro-Turza 1986), the most important volatile compounds contributing to the aroma of tomato fruit are: hexanal, β-ionome, 1-penten-3-one, cis-3-hexenal, cis-3-hexanol, cis-3-hexenol, trans-2-hexenal, 2+3-methylbutanol, 3-methylbutanal, 2-isobutylthiazole, 6-methyl-5-hepten-2-one, trans-2heptenal, 1-nitro-phenylethane, methyl salicylate, geranylacetone, isobutyl cyanide, 2-phenylethanol, trans-2-hexanal, cis-3 hexanal, and trans-2-trans-4-decadienal (Dirinck et al. 1976; Buttery et al. 1987, 1989, 1990; Baldwin et al. 1991a, 1998; Krumbein and Auerswald 1998). The composition of the tomato aroma volatiles is affected by many exogenous and endogenous factors such as growing conditions, management practices, postharvest treatments and storage conditions, cultivars, and metabolic changes taking place during the course of fruit ripening, and is discussed later in Sections III, IV, V, and VI. Recently, Baldwin et al. (1998) established a relationship between taste descriptors and fruit components [soluble solids (SS), titratable acidity (TA), sucrose equivalent (SE) ] and their ratios, and aroma descriptors and measurement of volatile compounds. They found that certain flavor compounds contributed to overall acceptability (SS/TA, SE/TA, TA, cis-3hexenol), perception of tomato-like flavor intensity (SE, SE/TA, geranylacetone, 2+3-methylbutanol, 6-methyl-5-hepten-2-one), sweetness (SE, pH, cis-hexenal, trans-2-hexenal, hexanal, cis-3-hexenol, geranylacetone, 2+3-methylbutanol, trans-2-heptenal, 6-methyl-5-hepten2-one, and 1-nitro-2-phenylethane), sourness (SS, pH, acetaldehyde, acetone, 2-isobutylthiazole, geranylacetone, β-ionone, ethanol, hexanal, cis3-hexenal), astringency (SS), bitterness (SS), and saltiness (SS). They also concluded that SE/TA and SE were slightly more useful in predicting overall acceptability and sweetness, respectively, than SS/TA or SS and both were better than SS/TA in predicting tomato-like flavor. Moreover, they reported that levels of SS showed a better relationship to sourness than to sweetness, and was more strongly correlated to this flavor aspect than TA. For greenhouse cultivars, Auerswald et al. (1999b) found positive correlations between reducing sugar and titratable acid concentrations and the intensity of several sensory attributes of smell (sweetness), flavor (tomato-like, mouldy, fresh cut grass), and aftertaste (intensive) evaluated by a descriptive analysis. From the principal component analysis technique, Krumbein and Auerswald (1998) associated cis-3-hexenal,

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1-penten-3-one and 2-methyl-4-pentenal with the attributes fruity and sweet, while other components contributed to tomato-like perception (trans-2-hexenal, 1-octen-3-one, trans,cis-2,4-heptadienal, trans,trans2,4-hexadienal, and reducing sugars) and sourness (trans-2 heptenal and titratable acid). Dangyang and Boersig (1996) found that high concentrations of 1-penten-3-one and 6-methyl-5-hepten-2-one correlated with aroma and overall flavor. For a characterization of the taste of tomato, several physical and chemical parameters of tomato fruit quality were determined (Verkerke and Janse 1997; Verkerke et al. 1998). In agreement with previous results, Janse and Schols (1995) found that mealiness and sweetness are two prominent attributes of tomato fruit taste, since aroma was highly correlated with sweetness. Sweetness can be determined by measuring the percentage juice and soluble solids content (explained more than 90% of the variation in sweetness), while mealiness can be predicted from the percentage juice and texture parameters of the pericarp (Verkerke 1996; Verkerke and Janse 1997). The distribution of different taste constituents in the interior of the fruit is variable. Thus, fruit pulp (approximately 70% of the fruit) and cuticle (approximately 10% of the fruit) are rich in C5-C13 volatile compounds, whereas the fluid part of fruit locular contents (approximately 20% of the fruit) has a low concentration in cis-3-hexenal (Buttery et al. 1988). Analysis of excised tomato tissues showed that pericarp (including columnella) had twice as much of 16 aroma volatiles when compared to locular gel (442 and 203 µL L–1, respectively) (Maul et al. 1998). No significant contribution of seed (approximately 0.3% of the fruit) to taste has been reported (Buttery et al. 1988). Mahakun et al. (1979) have measured a higher pH in the fruit pericarp (pH 4.49) when compared to locular contents (pH 4.40) and to whole fruit (pH 4.37) in many tomato cultivars. They also reported that total acidity of locular contents (9.07 meq 100 g–1) was higher than values for the whole fruit (6.90 meq 100 g–1) and fruit pericarp (5.80 meq 100 g–1). Citric acid concentration of locular contents, whole fruits, and pericarps was 10.98, 6.30, and 5.42 meq 100 g–1, respectively. Significant differences in malic acid concentrations between various tissue components were not observed (0.56 to 0.61 meq 100 g–1). The soluble solids:total acidity ratio of the pericarp (0.61) was higher than that of the whole fruit (0.59) and of locular contents (0.47). E. Health Influencing Compounds Recently, interest in the nutritional and health aspects of tomato fruit and their products has increased greatly (Stahl and Sies 1996a,b; Stein-

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metz and Potter 1996; Gerster 1997; Ness and Powles 1997; Clinton 1998; Giovannucci 1998, 1999). In epidemiological studies, tomato fruit had one of the highest inverse correlations with cancer risk and cardiovascular disease including stroke (Franceschi et al. 1994; Giovannucci et al. 1995; Ness and Powles 1997; Offord 1998). Many protective compounds have been identified from fruit and vegetables, such as antioxidants (vitamins, flavonoids, carotenoids), isothiocyanates, monoterpenes, organosulphides, isoflavones, phytosterols, selenium, potassium, folate and dietary fiber (Steinmetz and Potter 1996). The tomato provides most of these protective elements (Steinmetz and Potter 1996; Gester 1997; Giovannucci 1999). Carotenoids, in addition to their role in fruit coloring, are an excellent source of vitamin A and antioxidizing agents, and thus play an important role in preventing cancer and heart disease (Krinsky 1991, 1992). Antioxidant vitamins can counteract the oxidizing effects of lipids by scavenging oxygen free radicals that have been found to be major promoters of such diseases. Evidence for the antioxidant properties of dietary agents has been reviewed in Rice-Evans (1998), Rice-Evans et al. (1996), and Jovanovic et al. (1998). Of the more than 600 known carotenoids in the plant kingdom (Olson and Krinsky 1995; Britton 1995), about 40 are normally consumed (Gerster 1997). As a result of selective uptake by the digestive tract, only 14 carotenoids (major ones are lycopene, α- and β-carotene, lutein + zeaxanthin and β-cryptoxanthin) with some of their metabolites have been identified in human plasma and tissues. Of the 14 carotenoids found in human serum (Gerster 1997), nine are derived from tomato (fresh and processed), which is the predominant source of about half of the carotenoids (Khachik et al. 1995). Carotenoids isolated in tomato fruit are lycopene, lycope-5,6diol, α-carotene, β-carotene, γ-carotene, δ-carotene, lutein, xanthophylls, neurosporene, phytoene, phytofuene (Thakur et al. 1996), β-cryptoxanthin, lycoxanthin, neolycopene (Abushita et al. 1997), 5,6-dihydroxy5,6-dihydrolycopene, 1,2-epoxide, and lycopene 5,6-epoxide (Khachik et al. 1992). Carotenoid content varies according to cultivar, crop management, and stage of fruit ripening at harvest (see Sections III, IV, V, and VI). In the plant, the best-characterized natural functions of carotenoids are to serve as light-absorbing pigments during photosynthesis and to protect cells against photosensitization (Demmig-Adams et al. 1996). The regulation of carotenoid and isoprenoid formation in tomato has been reviewed by Gray (1987) and Bramley (1997). Lycopene accounts for 30% (0.5 to 0.8 µm) of the total carotenoids in plasma (Stahl and Sies 1996b). Inverse associations have been found between lycopene intake and cancers of the prostate, pancreas, and

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possibly stomach (Gerster 1997; Clinton 1998; Giovannucci 1998). Data are also suggestive of a benefit for cancers of the lung, colon and rectum, esophagus, oral cavity, breast, and cervix. In a recent review, Giovannucci (1999) examined the epidemiological evidence from 72 studies relating consumption of tomato and related products to the risk of cancer at various body sites. Lycopene appears to be the most efficient quencher in vitro of singlet oxygen and free radicals among the common carotenoids. The quenching constant of lycopene was found to be more than double that of β-carotene and 10 times more than that of α-tocopherol (Di Mascio et al. 1991). The potential benefits of lycopene have also been examined with regard to cardiovascular disease, immune system function (HIV infection), hyperlipidemia, age-related macular degeneration, Parkinson’s disease, inflammatory conditions, and artheosclerosis (Gerster 1997; Bilton 1998; Clinton 1998; Aviram and Fuhrman 1998; Gerber 1998). However, fundamental studies concerning the biology of carotenoids are required to prove their benefits as health supplements. In the tomato fruit, lycopene is responsible for the red color and constitutes 75 to 83% of the total pigment content (Abushita et al. 1997), whereas β-carotene is about 3 to 7% of the total carotenoids content (Gould 1992). Lycopene is one of the major carotenoids in North American and European diets and is mostly found (more than 80–85%) in tomato and tomato products (Hart and Scott 1995; Gerster 1997). In humans, lycopene is destroyed in the skin in response to UV exposure and therefore may represent a first line of defense against UV-induced oxidative damage (Ribaya-Mercado et al. 1995). Phytofluene (colorless) is detected only in the outer flesh, whereas lycopene appears in both pericarp and locular tissue. The content (µg g–1 fresh weight) of translycopene, trans-carotene, and lutein vary among different cultivars and may range from 0.21 to 49.58 mg, 0.93 to 22.32 µg, and 0.48 to 2.04 µg, respectively (Hart and Scott 1995). Offord (1998) reported a concentration of 31 µg glycopene, 7 µg β-carotene, and 1 µg lutein, while Khachick et al. (1992) reported that fresh tomato contains 39.2 µg lycopene, 5.3 µg of 5,6 epoxide lycopene, 2.8 µg β-carotene, 8.4 µg ξcarotene, 1.3 µg lutein, 6 µg phytoene, 5.1 µg phytofluene, and 3.0 µg neurosporene per g fresh weight. Ronen et al. (1999) has recently described regulation of lycopene accumulation in tomato fruit. Studies on pigment content of the inner pulp and in the outer region of the pericarp during growth and ripening fruit (Laval-Martin et al. 1975) showed that pigment distribution and formation are different. β-carotene, lutein, violaxanthin, auroxanthin, neoxanthin, and chlorophylls a and b accumulate in both parts of the growing fruit, then remain constant, except

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for the chlorophylls and neoxanthin, which decrease. Phytofluene and lycopene appear only at the end of ripening and almost exclusively in the external part of the fruit; lycophyll and lycoxanthin can be detected during the entire growth and ripening period (Laval-Martin et al. 1975). Depending on cultivar and growing conditions, concentrations of lycopene reported by different authors range from 43.6 to 181.2 µg per g of fresh weight (Tomes 1963; Thompson et al. 1965; Meredith and Purcell 1966; Davies and Hobson 1981; Hart and Scott 1995). Numerous other potentially beneficial compounds are present in tomato, and, conceivably, complex interactions among multiple components (folate, vitamins C and A, potassium, carotenes, phenylpropanoids, phytosterols, and flavonoids) may contribute to the anticancer properties of tomato (Giovannucci 1999). Flavonoids (diphenylpropanes; C6-C3-C6), which generally occur in foods as O-glycosides with sugars bound at the C3 position (Hertog et al. 1992), have antimutagenic and anticarcinogenic effects in vitro and in vivo (Franscis et al. 1989; Verma et al. 1988). Flavonoids and simple phenolics are also effective scavengers of reactive species (Pannala et al. 1998, 1997). In addition, folic acid is an extremely important micronutrient for prevention of neural tube defects (Offord 1998). Quercetin (flavonol) levels in tomato ranged from 4.6 to 11 µg g–1 of fresh weight. Kaempferol levels of less than 2 µg g–1 of fresh weight have been reported (Hertog et al. 1992; Offord 1998), while other authors (Grolier and Rock 1998) have reported a flavonoid concentration of 5 to 50 µg g–1 of fresh weight. Folate content (as folic acid) of fresh tomato is low, 11 µg per 100 g of fresh weight (Vahteristo et al. 1997) in comparison with other vegetables (broccoli 114 µg per 100 g of fresh weight, brussel sprout 94 µg per 100 g of fresh weight, cauliflower 85 µg per 100 g of fresh weight). Offord (1998) reported that fresh tomato is an important source of vitamin C (22 mg 100g–1), folic acid (39 mg 100g–1 of fresh weight) and vitamin E (0.9 mg 100g–1), providing up to 40%, 20%, and 10%, respectively, of the recommended dietary allowance, and a smaller amount of vitamin A (0.117 mg 100g–1), vitamin B1 (0.06 mg 100g–1), vitamin B2 (0.04 mg 100g–1), vitamin B6 (0.1 mg 100g–1), and niacin (0.6 mg 100g–1). In addition, Grolier and Rock (1998) reported that tomato fruit is relatively rich in phenolic acids (1–2000 mg 100g–1) such as ferulic, chlorogenic, and cafeic acids, and have small quantities of vitamin E (0.49 mg 100g–1). Tomato glycoalkaloids such as tomatine may have beneficial effects in the diet. For example, Friedman et al. (1997b) have observed that feeding commercial tomatine to hamsters induced a significant reduction in plasma low-density lipoprotein cholesterol and that the reduction with high-tomatine green tomato diets was greater than with low-tomatine red

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tomato diets. Development of new medicines containing these glycoalkaloids to attack cancer cells has been reported and will be an interesting future research area (Chen and Miller 2000). Although high levels of glycoalkaloids have toxic effects on humans, low levels in edible organs are important for human safety (Chen and Miller 2000).

III. GENETIC CHARACTERISTICS AFFECTING TOMATO FRUIT QUALITY The choice of cultivar has become a major factor in determining the profitability of a crop and constitutes an important management decision. It must be made according to target markets and production conditions. For example, differences in susceptibility to fruit cracking among various tomato cultivars have clearly been established in the past (Cortés et al. 1983; Abbott et al. 1985; Abbott et al. 1986). In some cultivars, cutin may surround the epidermal cells, even going between and below the first layer of collenchyma. This may explain part of the enhanced crack-resistance shown by ripening fruit of such lines (Voisey et al. 1970; Davies and Hobson 1981). Many genes may be involved in the process of fruit cracking, and each type of cracking may be controlled by specific genes (Cuartero et al. 1981), making it difficult when selecting for resistant cultivars (Peet 1992). As a consequence, very few cultivars resistant to cracking are currently available for greenhouse production (Peet and Willits 1995). Resistant cultivars currently available are of Dutch origin and developed for growing conditions characterized by low light intensities such as those prevailing in northern Europe. Under conditions conducive to more rapid growth, these cultivars are very susceptible to fruit cracking. The susceptibility to gold specks (Royle 1985; Cools and Jansen 1988; Stolk 1988) and BER (Adams and Ho 1992) also varies among tomato cultivars. BER is hardly known in cherry tomato, while it is notoriously severe in plum and beefsteak tomato (Ho 1999). Choosing between extended shelf life, slow ripening or conventional ripening cultivars will also influence fruit organoleptic quality and shelf life. Long shelf life cultivars are generally less savoury than traditional cultivars (Jones 1986). Concerning a wide range of fresh market tomato in the Netherlands, a study has reported that cherry tomato cultivars had the best flavor (Janse 1994). Also, vitamin content of fruit is primarily under the genetic control of the plant. Orange tomato cultivars had a higher content of carotenoids, vitamin A, and volatile compounds, while yellow cultivars contain up to ten times less lycopene than red cultivars (Hart and Scott 1995). Wild cultivars can contain up to twice the lycopene and vitamin C concentration of the commercial cultivars

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(Grolier and Rock 1998). For 17 tomato cultivars, concentrations of total carotenoid, β-carotene, total tocopherol, α-tocopherol, and ascorbic acid ranged from 18.45 to 60.75 µg g–1, 1.13 to 3.74 µg g–1, 1.17 to 3.98 µg g–1, 0.96 to 3.15 µg g–1, and 0.22 to 0.48 µg g–1, respectively (Abushita et al. 1997). Others have reported a range of 0.06 to 0.23 µg g–1 for ascorbic acid over a large number of cultivars (Bajaj et al. 1990; De Serrano et al. 1993), 7 µg g–1 (Offord 1998), and 1.4 to 11.91 µg g–1 (Davies and Hobson 1981) of β-carotene. Recent studies on consumer habits regarding fresh vegetables in North America have shown that taste and aroma are the most important factors in the selection of a product. A study done with 3,000 consumers in Berlin showed that the percentage of a potential sales price increase that consumers were willing to pay for improved quality attributes of fruits and vegetables was 19% for a better flavor, 18% for less chemical residue, 16% for higher vitamin content, and 16% for less nitrate content (Auerswald et al. 1999a). Bittenbender and Kelly (1988) reviewed early studies on the nutraceutical breeding efforts for carotenoids and ascorbic acid content. Despite the efforts of tomato breeders, fresh tomato often do not meet the high standards of flavor required by consumers (Speirs et al. 1998). Many breeders are now concentrating on improving sugar and acid levels. Genetic manipulation of alcohol dehydrogenase (ADH 2; alcohol: NAD+ oxidoreductase implicated in the interconversion of the aldehyde and alcohol forms of flavor volatiles, EC 1.1.1.1, Sieso et al. 1976; Bicsak et al. 1982) levels in ripening tomato fruit, with either the constitutive cauliflower mosaic virus 35S promoter or the fruit-specific tomato polygalacturonase promoter, influenced the balance between some of the aldehydes and the corresponding alcohols associated with flavor (Speirs et al. 1998; Prestage et al. 1999). Hexanol and cis-3-hexenol levels were increased in fruit with increased ADH activity and reduced in fruit with low ADH activity (Speirs et al. 1998). These authors suggest that a balance between aldehydes and alcohols is essential to the development of the ripe-fruit flavor. In a preliminary taste trial, fruit with elevated ADH activity and higher levels of alcohols were identified by these authors as having a more intense ripe fruit flavor. Using a low shear maceration procedure, which is more representative of the conditions found in the mouth during eating than a high-speed homogenization technique (Linforth et al. 1994), Prestage et al. (1999) reported that down-regulation of ADH2 caused a significant decrease in the amount of cis-3-hexenol but no increase in cis-3-hexenal. In contrast, the amount of 3-methylbutanal increased while the amount of 3-methylbutanol did not change significantly (Buttery et al. 1987; Prestage et al. 1999). In order to increase carotenoid levels in tomato, genetic manipulation, using

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Agrobacterium-based transformation with isoprenoid/carotenoid cDNAs or bacterial genes can also be done (Bramley 1998). Genetic manipulation of phytochrome genes (PHY) can improve yield or quality traits in tomato. Alba et al. (1999) have shown that a “MoneyMaker” line containing a PHYB1 mutation increased total yield by 74% and 39% when grown in the greenhouse or the field, respectively. A PHYA mutant had lower yields, but fruit dry weight:fresh weight ratio and soluble solids content were higher. Ripe fruit from the PHYA mutant line had 23% less citric acid and 364% more sucrose than the wild-type. More work has to be done in order to generate new PHY over-expression in cultivars used by the industry and to provide new knowledge about the function of PHY in tomato maturation and its potential for improving tomato crop production and quality. Soluble sugars, titratable acids, and electrical conductivity of the fruit for several cultivars of greenhouse tomato grown in nutrient film technique (NFT) varied from 4.7 to 5.1%, 7.7 to 8.5 meq and 545 to 591 µ5 cm–1, respectively, while firmness varied from 25 to 28 N m–2 (Gormley and Maher 1990). The malic to citric acid ratio is considered to be a cultivar attribute (Davies and Hobson 1981). Cultivars also affect fruit amino acid concentration. Fruit of wild species have generally higher glutamic acid (De Bruyn et al. 1971) and soluble sugar content (Yelle et al. 1991; Husain et al. 1999). Cultivars with a large locular portion and with high concentration of acids have been found to be flavorful (Stevens et al. 1977b). On the other hand, of the 17 aroma volatiles quantified by Baldwin et al. (1998), all but β-ionone showed significant differences among different cultivars. The concentration of cis-3-hexenal and hexanal is lower in yellow cultivars than in red cultivars (Buttery et al. 1987). Moreover, we found that the volatile compound concentration for ‘Campari’, a round greenhouse cultivar, was three times higher than in ‘Blitz’ (beefsteak cultivar). Extended shelf-life cultivars had lower volatile content than a traditional tomato cultivar (Baldwin et al. 1991c). Recently, Gray et al. (1999) showed that cultivar and fruit fatty acyl (α-linoleic acids) content are important in determining the composition of the volatile compounds in tomato fruit. IV. ENVIRONMENTAL FACTORS AFFECTING TOMATO FRUIT QUALITY A. Effect of Light Intensity Most of the solar radiation received by greenhouse tomato plant is reflected (<10%), transmitted (<10%), or absorbed (>80%) (Dorais et al.

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1995). Of the absorbed radiation, a small proportion (5%) is used for biological reactions such as photosynthesis and a large proportion is dissipated by transpiration or convection. The light intensity received by the plant affects not only soluble sugars (Davies and Hobson 1981), ascorbic acid (Giovanelli et al. 1998), and pigments (lycopene) in the fruit, but also influences the quantity of photoassimilates available to the fruit. As did Winsor and Adams (1976), we observed that the soluble sugar concentration of tomato fruit (red stage) followed the pattern of solar radiation (unpublished data). Fruit exposed to light also have a greater capacity for storing starch (higher fruit weight) due to an increased activity of the ADPG pyrophosphorylase enzyme (Ho 1996a). High light intensity is closely associated with a high fruit content in vitamin C (Venter 1977). Thus, plant population and greenhouse cover (plastics or glass) affect fruit vitamin content. On the other hand, too high light intensity or strong direct radiation on fruit reduces quality. Adegoroye and Jolliffe (1987) have reported that various ripening processes, such as ethylene production, lycopene synthesis, conversion of protopectine into soluble pectine and pectic acids involved in fruit softening, were inhibited when green fruit were exposed to 650 W m–2 for 1.5 to 4 h. High light intensity may also play a role in fruit cuticle cracking, not only through effects on fruit temperature but also by increasing sugar content of fruit. However, detailed studies of cracking in relation to light have not been conducted. BER is also linearly related to the energy sum of light and temperature throughout the season (Ho et al. 1993; Ho 1999). Low light intensity reduces pigment synthesis, resulting in uneven fruit coloring, and leads to the formation of swollen and hollow fruit or fruit with a mealy taste. Although the formation of carotenoids in ripening fruit does not require induction by light (Raymundo et al. 1976), shaded fruits have lower carotenoid content (McCollum 1954). Whereas red light influences chlorophyll breakdown, carotenoid synthesis is enhanced by blue light (Jen 1974). Under very low light intensities, the fruit-set is reduced due to a lack of photoassimilates. Studies have shown that a 1% reduction of light level in winter time is translated into a weight loss of 0.6% in young plants and 1% in older plants, while the loss in fruit yield is approximately 1%. Fresh tomato weight is estimated to be produced at 18–26.5 g per MJ of solar radiation incident on the crop (Cockshull et al. 1992; De Koning 1994). Despite the fact that increasing light intensity also increases fruit dry matter and soluble sugars content, it has almost no effect on organic acid concentration (Janse 1984). A high fruit dry matter content is generally associated with high firmness, while a low fruit concentration in soluble sugars is linked to a “watery” taste of tomato.

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B. Effect of Temperature Temperature may influence the distribution of photoassimilates between fruit and vegetative parts as well as their rate of growth (Heuvelink 1995b; De Koning 1992a, 1994, 1996). High temperature favors the distribution of the assimilates to fruit, at the expense of vegetative growth (De Koning 1989). Moreover, there exists a positive correlation between photoassimilate import into fruit and fruit temperature (sink strength increases with temperature) (Walker and Ho 1977; Ho and Hewitt 1986). It is generally reported that increasing ambient temperature by 1°C increases fruit dry matter content by 0.07% (De Koning 1992b) and weekly formation of clusters by 0.05%. Similarly, water movement to the tomato fruit increases with temperature, while temperatures equal to or lower than 15.5°C reduce water absorption by the fruit considerably (Bohning and Lusananda 1952; Koske et al. 1980). A physiological model predicting water import by tomato fruit according to sap viscosity, osmotic potential (Ψs), and temperature has also been developed (Bussières 1995). Fruit relative growth rate (FGR) is strongly related to the ambient temperature and to the water supply (Pearce et al. 1993). FGR increases rapidly at the beginning of the day, peaks at midday, and then decreases by the end of the day (Ehret and Ho 1986a). The increase in growth rate during the morning may well be related to temperature increase when plant water status is not limiting (Pearce et al. 1993). Pearce et al. (1993) reported significant correlations of temperature and growth rate with temperature sensitivities of approximately 5 µm h–1°C–1. At night, FRG is about one-third of that in daytime (Ehret and Ho 1986a). It has, however, been observed that elevated temperatures reduce final fruit size and duration of fruit growth (De Koning 1994). Thompson et al. (1998) reported that this result might be expected if the appearance of the peroxidase activity responsible for growth termination was increased at high temperatures. Temperature also plays an important role in pollen characteristics and fruit set (Dane et al. 1991; Wacquant 1995; Peet and Bartholemew 1996; Lohar and Peat 1998). Low temperatures (<13°C) reduce pollen viability while high temperatures (>30°C) favor an excessive growth of the style. This reduces pollen viability, and as a result increases defects of poor fertilization such as misshapen fruits (Wacquant 1995). Many floral anomalies such as stigma exertion without anthesis, empty flowers, and persistent flowers without fruit-set were also observed in the 35°/30°C (day/night) regime (Lohar and Peat 1998). Peet and Bartholemew (1996) showed that total and percentage normal pollen grains were higher in plants grown at night temperatures of 18° and 22°C than at 24°

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and 26°C, but germination was the highest in pollen produced at 26°C. However, they observed that numbers of flowers and fruit on the first cluster were lower in the 26°C night treatment than in the other night temperature treatments. Temperature has a direct influence on metabolism and, thus, affects cellular structure and other components that determine fruit quality such as color, texture, size, and organoleptic properties. High temperatures accelerate fruit development and reduce the time required for ripening, but also decrease their size, and therefore their quality. They also favor an increase in the number of hollow fruit in the winter and miscolored fruit in the summer, and the number of misshapen and soft fruits. Tomato fruit color develops best when ambient temperature inside the greenhouse is kept between 12° and 21°C. Very low (<10°C) or very high air temperature (>30°C) inhibits normal fruit ripening and the development of lycopene (Tomes 1963; Koskitalo and Ormrod 1972). Conversely, low temperatures favor large irregularly shaped fruit with their carpels often being separated and the placenta exposed. Also, low night temperature slows down fruit ripening. Recently, Tomer et al. (1998) observed that low night temperature (≤12°C) increased the tomato plant susceptibility to pointed-fruit malformation, which is associated with deformed locules (placenta was retarded or absent, absence of seeds and jelly). Night temperature below 10°C for a substantial part of the night during flower development caused the formation of fasciated fruits, a condition referred to as “catfacing” (Rylski 1979a). Lozano et al. (1998) reported that tomato flower and fruit development were severely affected when plants were grown at low temperature (17°C day/7°C night compared to 26°C day/18°C night), displaying homeotic and meristic transformations and alterations in the fusion pattern of the organs, which could be related to expression of MADS-box genes. As a consequence, abnormal fruits of low economic value are produced. On the other hand, under a large day-night temperature differential (14°C DIF compared to 5°C DIF) early in fruit development, Gent and Ma (1998) reported that tomato fruit ripened earlier, fruit size increased, and the incidence of irregular fruit was reduced. In fruit cultured in vitro at 16°C, lycopene concentration (580 µg per g of fresh weight) was 1.7 times higher that of the 26°C treatment and around 10 times that of fieldgrown tomato fruit (Ishida 1998; Ishida et al. 1998a). Ishida (1998) and Ishida et al. (1998a) suggested that cool temperatures (16°C), which involve tomato TAG1 gene activation (Ishida 1998; Ishida et al. 1998b), leads to elevated lycopene production and may, in part, explain this high concentration. Also, addition of 75 mg L–1 2-(4-chlophenylthio) triethyamine (CPTA) to the medium is known to increase lycopene pro-

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duction (Rabinowitch and Rudich 1972; Chang et al. 1977), and results in further increases in lycopene (1.14-fold at 16°C and 1.88-fold at 26°C). Sudden temperature changes or high day/night temperature variation will favor the cracking of fruit (Peet 1992; McAvoy 1995). Changes from night to day temperature need to be made before sunrise in order to avoid water vapor condensation on fruit. Low night temperature favors a negative pressure in fruit, whereas high day temperature increases both gas and hydrostatic pressure of fruit pulp on the epidermis, resulting in a weakening or cracking of the cuticle (Peet 1992; McAvoy 1995). Moreover, high temperature reduces cuticle resistance and firmness (Lang and Düring 1990; McAvoy 1995). Studies have shown that an air temperature of 23°C improves the organoleptic quality of tomato and increases fruit dry matter and K:Ca ratio; it also reduces softer and mealy fruit as compared to 17°C (Janse and Schols 1992). Such fruit also has a less resistant cuticle, despite a higher content in reducing sugars and a diminution of their concentration in titratable acids (Janse and Schols 1992). Studies conducted in The Netherlands have shown that organoleptic quality of fruit is improved when temperatures are increased from 19.2°C to 20.9°C and 23°C. Increasing temperature in the beginning of the growing period (spring) along with an increase in the EC (3.8, 6.3, and 8.1 mS cm–1) greatly improves fruit flavor (Janse and Gielesen 1991). The temperature of the rooting system generally has an influence on the rate of absorption of water and nutrients. For example, increasing the temperature of the rooting system from 14 to 26°C increased the quantity of water absorbed during a day by 30%, the rate of absorption of N, K, and Mg by 21–24%, and the rate of absorption of Ca and P by 45% and 64%, respectively. On the other hand, fruit quality and yield were reduced (Hurd and Graves 1985) when the temperature of the root system was increased from 11 to 27°C at a fixed greenhouse temperature of 13°C. Rooting temperatures of 15–18°C are generally recommended at the beginning of a new crop and a rooting temperature of 25°C is recommended during the growing period (Graves 1986). A rooting temperature too high at the beginning of the growing season results in an increase in the number of boxy fruit. C. Effect of VPD Although vapor pressure deficit (VPD, 0.2 to 1.0 kPa) has almost no effect on growth and development of horticultural crops (Grange and Hand

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1987), the relative humidity in a greenhouse nevertheless affects fruit quality. Fruit produced under a high VPD (low relative humidity) are firmer, juicier, less mealy and have less physiological disorders such as cracking and gold specks than fruit produced under low VPD (Janse and Schols 1992). However, under low relative humidity, 24–59% of fruits can be affected by BER, while the corresponding figure for conditions of high relative humidity is 19% (De Kreij 1992; De Kreij 1996). In fact, calcium intake into tomato fruit was greater under low VPD (Adams and Ho 1995; Paiva et al. 1998b) or when nights were humid rather than dry (Bradfield and Guttridge 1984). Research conducted at Laval University showed that fruit produced under day/night VPD of 0.5/ 0.3 kPa was of higher quality (firmness, soluble sugars) compared to fruit produced under a VPD of 0.3/0.2 kPa (Iraqi 1995). No significant differences, however, were found when VPD treatments of 0.4/0.4 and 0.2/0.2 kPa were compared (Dorais et al. 2000). We also found (unpublished data) that fruit had a more intense coloration when tomato plants were grown under a high VPD. Under conditions of extreme low humidity (1.5–2.2 kPa or 10–14 g kg–1), there is a reduction in the photosynthetic rate due to stomatal closure, in growth, in fruit size, and in total yield. Moreover, there is an increase in the number of fruit affected by BER due to a high foliage transpiration rate, thereby limiting the supply of xylem sap to the fruit. Low VPDs (high humidity conditions) cause a decrease of plant transpiration, a decrease of nutrient uptake, an increase in root pressure, and generally favor fruit cracking (Peet and Willits 1995). High relative humidity and temperature conditions increase the pressure inside the fruit, since the tomato fruit cuticle is impermeable (97%) to gases (Corey and Tan 1990). The return of water to vegetative parts is unlikely under these conditions, and fruit cracking occurs. Under a low VPD, fruit are generally smaller, softer, and misshapen. There is also a reduction in the ovule fertilization rate due to a more difficult release of pollen. Moreover, a low VPD (0.1 to 0.3 kPa or 0.6 to 2 g kg–1) influences fruit color (marbling) and increases gold specks incidence. Yield reductions between 18–21% under a VPD of 0.1 kPa have been observed compared to a VPD of 0.5 kPa. Such yield reductions are probably related to a diminution of leaf size due to a calcium deficiency in the foliage (Armstrong and Kirkby 1979; Holder and Cockshull 1988) and a reduced fruit growth rate (Bakker 1990). For VPDs varying from 0.2 to 0.8 kPa, Bakker (1990) reported low yields, low fruit size, and short fruit shelf life under high humidity (low VPD).

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D. Effect of CO2 Enrichment CO2 enrichment for greenhouse crops increases plant growth, fruit set, the number of fruit, and the average fruit weight (Frydrych 1984; Yelle et al. 1987, 1990; Nederhoff 1994). Several studies have demonstrated a linear effect of CO2 on the photosynthetic rate at concentrations up to 1,000–1,500 ppm. Depending on the growing conditions, a CO2 concentration in the atmosphere of 1,000 ppm as compared to a normal concentration (340 ppm), increases the photosynthesis rate by 50 to 70%. Increasing the CO2 concentration, on the other hand, had almost no effect on the fruit firmness and organoleptic quality (Davies and Winsor 1967; Nilsen et al. 1983; Slack et al. 1988) or on dry matter partitioning to fruit vs. foliage in summertime (Nederhoff 1994). Frydrych (1984) observed that CO2 enrichment in tomato plants increased fruit set and thus dry matter partitioning to the fruit. In controlled-environment rooms, Madsen (1975) and Behboudian and Tod (1995) reported that fruit grown under CO2 enrichment (1,000 ppm) atmosphere had higher concentration of sucrose, glucose, fructose, and total soluble solids than ambient-CO2 fruit. HighCO2 fruit also ripened more slowly and were characterized by lower respiration and ethylene production rates than ambient-CO2-fruit. They noted that concentrations of N, P, and K were lower in the high-CO2 fruit, whereas those of S, Ca, and Mg were the same for both treatments. Inconsistent results can be explained by different growing conditions and by the fact that the increase in sugars imported to the fruit due to CO2 enrichment might be accompanied by a similar increase in water absorption by the fruit. Kretchman and Bauerle (1971) observed a reduction in the number of cracked fruit and non-uniform fruit color, by increases in CO2, but the percentage of BER was unaffected (Nederhoff 1994). Morphological adaptations due to high CO2 in the form of reduced leaf area (Nederhoff 1994) may also affect fruit quality, since fruit are more exposed to solar radiation (Adegoroye and Jolliffe 1987; Janse 1988b). Plants grown under conditions of salt stress have been reported to show a higher growth response to elevated CO2 than non-stressed plants (Schwarz and Gale 1984; Li et al. 1999b). Recently, Li et al. (1999a) observed that high CO2 (1,200 ppm) slightly increased fruit quality (total sugars, soluble solids) of tomato plants grown under high salinity (5.2 and 7.0 mS cm–1). In addition, fruit ripening was about 10 days earlier under CO2 enrichment, regardless of salinity treatments. Thus, these authors suggested for a salt-tolerant tomato cultivar that a combined utilization of high salinity and CO2 supplementation may enable the production of high-quality fruits without incurring all the inevitable loss in yields associated with salt treatment.

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V. CULTURAL PRACTICES AFFECTING FRUIT QUALITY A. Hydroponics and Growing Media Hydroponic culture is a technology for growing plants in nutrient solution or, in a more general sense, in medium separated from the soil in situ (e.g. sand, gravel, vermiculite, rockwool, peat moss, coir, sawdust, clay pellets). It is the most intensive method of crop production in today’s agricultural industry, and generally permits the production of higher quality fruit than in soil (Morard 1995). The use of a nutrient film technique (NFT) system, as compared with the traditional soil system, increases fruit firmness and concentrations of vitamin C, sugars, titratable acids, phosphorus, potassium, calcium, and magnesium in the fruit (Benoit and Ceustermans 1987). Plants grown in NFT yield fruit higher in dry matter content, soluble sugars, and titratable acids compared to solid substrates (Adams and Winsor 1976). Recently, Premuzic et al. (1998) showed that fruit grown on organic substrates (100% vermicompost or 50% vermicompost:50% soil) and irrigated with water contained significantly more calcium and vitamin C and less iron than fruit grown on hydroponic media (sand or peat-perlite) with a complete nutrient solution, but no difference was found for phosphorus and potassium content. They related the higher concentration of vitamin C to reduced foliage development on the organic substrates (more light on the fruit), while higher calcium levels in organically grown tomatoes were associated with reduced levels of other cations such as sodium, magnesium, and potassium. On the other hand, Mzouri et al. (1996) did not find any significant differences after 21 weeks of harvesting plants growing in peat media compared to NFT systems; the average yield, fruit weight, percent of marketable, small or misshapen fruit, as well as the number of fruit affected by BER were similar. Mzouri et al. (1996) have also not observed significant differences between these two growing media for ‘Trust’ and ‘Cencara’ in terms of fruit firmness, color, soluble sugars, and organic acids. Also, Gül and Sevgican (1994) found no significant effect of the substrate on fruit quality when different growing media (perlite, perlite/sand, peat/sand, sand, lava, sawdust/perlite, coarse sawdust/perlite, pine and bark of pine/perlite) were compared. Growing media had no significant effect on fruit composition, firmness, and aroma (Gormley and Egan 1978). However, Cronin and Walsh (1983) have reported a higher fruit content in sugars, ascorbic acid, and in dry matter in peat-based media, while fruit content titratable acids, potassium, and fruit aroma were higher under an NFT growing system. Finally, few differences in fruit physical, chemical,

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and organoleptic properties were observed on tomato grown in peat and NFT (Cronin and Houlihan 1985). Moreover, Maas and Adamson (1971) showed that good-quality tomato can be successfully grown in a soilless medium composed completely of sawdust if adequately enriched with essential mineral nutrients. Because of its high availability (waste product), low cost, light weight, cleanliness, and ease of placement, sawdust has became the preferred medium for greenhouse tomato production in western Canada and New Zealand since the 1970s. The controversy between the different studies probably reflects the level of control of the growing regimes as a function of the substrates used. Each growing substrate has its own demands and responds more or less rapidly (buffer effect) to changes in growing conditions due to daily climatic variations. Moreover, substrates differ in adsorption capacity for specific ions. For example, some volcanic rocks will have a high adsorption capacity for phosphorus, whereas sand or rockwool will have a weak or no adsorption capacity for phosphorus. Thus, the quality of fruit obtained with an optimal growing regime will be generally similar for the different types of substrates. The effect of growing media on fruit quality depends to a large extent on the experience and knowledge acquired by each grower in executing fertigation management. B. System and Growing Regime 1. Plant Density. The effect of plant density on the interception of light (photosynthetic photon flux, PPF) and of the photosynthetic rate of the plant canopy on plant growth and fruit development, and on the distribution of carbohydrates, has been reviewed by Papadopoulos and Pararajasingham (1997). An increase in plant density is positively correlated with fruit yield (kg m–2) but negatively correlated with fruit size due to an insufficient supply of photoassimilates to each fruit. Monthly PPF received by plants varies during the course of the growing period from 359 to 614 MJ m–2 from March to September and from 130 to 250 MJ m–2 from October to February in Quebec, Canada. For an extended production period, light can become limiting. Changes in plant density during the high light growth period by the development of a lateral stem optimizes light interception by the plant canopy and fruit yield (Cockshull and Ho 1995). The choice of a target plant density must be based on plant vigor, on the cultivar, and on the prevailing light levels. For densities of 3.06 compared to 2.04 plants m–2, Cockshull and Ho (1995) observed yield increases of approximately 15% under high densities, although fruit size was reduced. Reducing the density from 2.92

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to 1.87 plants m–2 did not affect fruit composition, color, firmness, flavor, or shelf-life significantly (Gormley and Maher 1990). 2. Pruning. It is generally agreed that the distribution of assimilates between vegetative and generative parts is influenced by fruit load (Hurd et al. 1979; Heuvelink 1997). Thus, tomato fruit production and fruit size can be adjusted to the level of available photoassimilates if cluster pruning is co-ordinated with the growing period (Cockshull and Ho 1995). Since fruit constitute a major portion of photoassimilates, the variation in their number will influence their size rather than the fruit:leaf ratio (Ho 1992). Ehret et al. (1993) has nevertheless observed a higher foliage:fruit ratio when there were 1–2 fruit per cluster as compared to a normal cluster size (3–4 fruit per cluster) and an increase of about 50–60% of the average fruit weight. Cockshull and Ho (1995) also noted that removing 30% of the available fruit from the distal end of the first three clusters increases average fruit weight of the remaining fruit and the yield of top clusters, thus avoiding yield losses. Nevertheless, a high foliage:fruit ratio resulting from fruit pruning significantly increases the number of fruit affected by cracking (Ehret et al. 1993). Indeed, during the months of September to December, Straver (1995) observed that while a reduction in the number of fruit per plant increased their size, it also increased the number of fruit affected by cracking (russeting and radial cracking). Excessive vegetative growth, low fruit load, and rapid fruit growth favor a disequilibrium between xylemic and phloemic sap absorption by the fruit, in favor of the phloemic sap, and lead to calcium deficiency in the fruit and increase the appearance of BER (De Kreij 1992). 3. Deleafing. Severe deleafing of plants reduces the photosynthetic capacity of the canopy and the remobilization of mobile elements. Fruit dry matter, reducing sugars, and soluble solids are reduced, but titratable acids are increased. Leaves growing below an immature cluster should not be removed because they contribute to the formation of photoassimilates and to the remobilization of mobile nutrients (Slack 1986). An increased rate of deleafing, at least to the fifth leaf above the ripening truss, slightly reduced the plant leaf:fruit ratio but did not significantly affect fruit cracking incidence (Ehret et al. 1993). However, a decrease in the leaf:fruit ratio reduces the transpiration rate of leaves and thus favors calcium distribution towards the developing fruit and reduces the susceptibitlity to BER. A severe deleafing, however, exposes fruit to high solar radiation, which often results in the appearance

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of physiological disorders such as fruit cracking and unequal fruit ripening. 4. Volume of Root System. The development of the root system can influence the ion uptake and fruit quality. For example, the absorption of potassium occurs through the entire root system (Clarkson and Hanson 1980) and its accumulation by a tomato fruit is mainly from the phloem sap (Ho and Adams 1995), whereas the absorption of calcium occurs mainly in newly formed zones and moves almost exclusively through the xylem (Hanson 1984). Root restriction can improve fruit quality by increasing the dry matter concentration and reducing the incidence of BER (Bar Tal and Pressman 1996). The increase in dry matter content can be explained mainly by a reduction in fruit water content, while the reduction of BER incidence can be explained mainly by an increase in the fruit:leaf ratio, by a reduction in the plant growth rate, and by a lower K:Ca ratio in tissues. However, a restricted root system also reduced plant growth (>30%), total yield (20%), fruit size, and K concentration in plant organs (Bar Tal and Pressman 1996). The absorption of potassium per unit of fresh root weight remains relatively unchanged, however, and plant growth reduction is not related to a nutrient deficiency but rather to hormone synthesis (ethylene, cytokinins, gibberellins) and root metabolism (Peterson et al. 1991). Recently, it has been observed that root restriction of young tomato plants increased soluble sugar and starch concentration in leaves by two-fold compared to an unrestricted control (Nishizawa and Saito 1998). C. Effect of Irrigation The frequency of irrigation and the quantity of nutrient solution provided to the plants affect yield and fruit quality (Mitchell et al. 1991; Tüzel et al. 1993; Peet and Willits 1995). Irrigation control can influence fruit size. Thus, when tomato plants are irrigated to 60% as compared to a normal irrigation regime, the weight of fruit is 84% of the weight of the control (Adams 1990). Several studies have shown a reduction in carbohydrate hydrolysis and a reduction in the translocation of organic leaf compounds to the fruit following a water stress, resulting in the accumulation of starch and soluble sugars in leaves (Crafts and Crisp 1971; Hsiao 1973; Zrenner and Stitt 1991; Kameli and Lösel 1996; Foyer et al. 1998; Thomas and James 1999). Increasing the rate of irrigation of greenhouse tomato plants can lead to reductions in soluble solids and dry matter of fruit (Tüzel et al. 1993).

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Excess irrigation can also produce physiological disorders such as epinasty, reduction of stem elongation, premature senescence of leaves, high concentration in abscissic acid, and poor root health (Basiouny et al. 1994), which indirectly influence fruit quality. Tomato plants are generally more sensitive to water stress during flowering or fruit set (Helyes 1994). Moreover, increasing the water supply increases fruit yield, but fruit quality is negatively affected (Rudich and Luckhinsky 1986). A high irrigation regime reduces fruit quality due to a high water content (reduction in soluble sugars, organic acids, vitamins, minerals, and volatile compounds) and due to the tendency of fruit to crack (Abbott et al. 1985; Abbott et al. 1986; Peet 1992; McAvoy 1995; Peet and Willits 1995). An excess of water increases root pressure and, as a consequence, fruit turgor pressure (Peet and Willits 1995). It has been shown (Kamimura et al. 1972) that a sudden increase in media water content reduces the elasticity of the tomato cuticle and increases root pressure. Abbott et al. (1986) observed a reduction in the incidence of greenhouse tomato fruit cracking when the daily irrigation frequency was changed from 1 to 4 waterings per day, while total daily irrigation quantity remained the same. It is also possible to reduce fruit susceptibility to cracking by reducing the total daily supply of water (Peet and Willits 1995). To reduce cracking, they suggest that the amount of water provided to plants should be based on the amount of water the plants are using at the time. In fact, results from a radiation-based water management study showed that the percentage of cracked fruit did not increase when irrigation frequency (100 ml plant–1 per irrigation) was increased from 612 to 468 KJ m–2 of solar radiation received (Chrétien et al. 2000). Restriction in the water supply has been shown to improve fruit organoleptic quality. Reductions in fruit water content as well as increases in fruit soluble solids, sucrose, hexoses, citric acid, and potassium have been reported in tomato plants growing under water stress conditions (Adams 1990; Mitchell et al. 1991; Pulupol et al. 1996). The increase in fruit mineral content under conditions of water stress appears to be related to a reduction of their water content rather than to an increase in the accumulation of the solutes following an osmotic adjustment (Mitchell et al. 1991). However, plants growing under high water stress tend to suffer from significant growth and yield reductions (Adams 1990; Mitchell et al. 1991; Shinohara et al. 1995). High water stress due to an inappropriate irrigation schedule significantly increases the leaf abscissic acid (ABA) content, which influences stomatal closure and ethylene production (Burg 1973; Aharoni 1978). Higher levels of ethylene favor an increased activity of cellulase and polygalacturonase, and then air pockets in tomato cells also called silver

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spotlight or pith autolysis (Huberman et al. 1993). Ethylene increases the concentration of carotenoids in fruit (Paz et al. 1982), with the lycopene peak concentration corresponding to the maximum production of ethylene (Ishida et al. 1993). This probably explains the more intense coloring of fruit growing under water stress (Brecht et al. 1994; Pulupol et al. 1996). The rates of CO2 production are higher in fruit growing under a water deficit (Pulupol et al. 1996), and may reduce fruit shelf-life. The accumulation of ammonium and the presence of polyamines (putrescine, spermidine, and spermine) in leaves are indices of water stress following a poor irrigation regime (Kubis and Krzywanski 1991; Feng and Barker 1992). D. Mineral Nutrition In closed systems the supply of nutrients should be equal to the absorption of nutrients by the crop. Over the whole growing period of the tomato crop, the mean values of uptake concentrations of tomato to obtain optimum yield and product quality are 9.6 mM N, 6.1 mM K, 2.2 mM Ca, 1.2 mM S, 1.1 mM P, and 0.9 mM Mg (Voogt and Sonneveld 1997). For a production lasting 238 days, White (1993) estimated the mineral absorption (Kg ha–1) at 790 N, 170 P, 1415 K, 237 S, 606 Ca, 112 Mg, 70 Na, 97 Cl, 14 Fe, 4.5 Mn, 0.8 Zn, 0.5 Cu, and 1.5 B. The rate of absorption of nutrients increases from planting time to the first week of harvesting, then decreases due to the stress of high fruit production and root death. The rate of absorption then gradually increases again and reaches its initial high level. The xylem and phloem supply the fruit with calcium (100 and 0%), magnesium (2 and 98%), potassium (1 and 99%), and sodium (6 and 94%). There exists an antagonism between various cations at the root uptake level (Adams 1999). An excessive concentration of one or several cations in the nutrient solution can reduce or block the uptake of the other. Recently, Carvajal et al. (1999) reported that the calcium concentrations of tissues of all tomato vegetative organs were reduced by magnesium (0.5 to 10 mM) and unaffected by NaCl (1 to 60 mM). They suggested that 0.5 mM Mg in the nutrient solution is adequate for optimum fruit yield, whereas in the presence of 20 mM NaCl or more, a Mg concentration of 1 mM is required. Gunes et al. (1998) suggested that the correct nutrient solution composition for young tomato should be 11.7 mM N (8.2 mM NO3 and 3.5 mM NH4), 0.9 mM H2PO4, 4.9 mM K, 2.1 mM Ca, 0.12 mM Mg, 35.7 µM Fe, 6.2 µM Zn, and 10.9 µM Mn for optimal plant growth. On the other hand, reductions of macronutrient concentrations to 50% or 25% of the control level (control level: 11 mM NO3-N, 0.8 mM H2PO4, 8.0 mM K, 4.0 mM

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Ca, 2.5 mM Mg, 4.1 mM SO4, 1.0 mM Cl, 20 µM Fe, 4 µM Zn, 7 µM Mn, 50 µM B, 0.75 µM Cu, and 0.5 µM Mo) had no adverse effect on growth, fruit yield, and fruit quality such as size, shape, cull, unripe, element composition, firmness, dry matter, soluble sugars, acidity, and electrical conductivity of the juice (Siddiqi et al. 1998). In non-recirculating systems, this strategy would reduce the release of nitrate and phosphate to the environment. 1. Calcium. The function of calcium in plants has been reviewed by Hanson (1984), Kirkby and Pilbeam (1984), and Marschner (1995). Inside the cell, calcium linked to pectic acids of the middle lamellae is responsible for maintaining cell wall and tissue rigidity (Marschner 1995). Calcium pectate is involved in cell wall plasticity and elongation (Yamauchi et al. 1986). Under high light conditions, calcium pectates increase tissue resistance to degradation by polygalacturonase and to fungal or bacterial attacks (Marschner 1995). Calcium is also essential for cellular membrane stability, and cellular compartmentation and integrity (Marschner 1995). The concentration of calcium oxalate in the vacuole is also important for cell osmoregulation. In periods of rapid plant growth (for example, under high irradiance), an accelerated cellular enlargement and fruit development require an additional supply of nutrients such as calcium, an important nutrient in the prevention of tomato fruit cracking (Simon 1978) and fruit with BER (Ho 1999). Due to the immobility of Ca in the phloem, Ca in the leaves will not be remobilized to the fruit and Ca supply to the fruit is restricted to the xylem water, which accounts for less than 15% of total water import by a fruit (Ho et al. 1987). Therefore, Ca distribution to fruit is less than 2% of total calcium content (Ehret and Ho 1986b; Ho 1999). Normally, calcium content in fruits increases with age from 0.1–0.3 mg after 11 days to 3.5–4.0 mg after 66 days of growth and is strongly correlated to fruit dry or fresh weight (El-Gizawy et al. 1986). However, the accumulation rate of calcium in the fruit is maximal 22 to 55 days after anthesis. The concentration of calcium in the distal fruit on the cluster tends to be lower than in the proximal ones (Petersen et al. 1998), indicating that physiological disorders associated with Ca are more likely to develop in fruit at the end of the truss than in the fruit close to the main stem. Studies have shown that the uptake rate of calcium in the peduncle, the calyx, and the fruit is higher during the night than during daytime (Masuda et al. 1996). Ho (1989) suggested that the higher import of calcium into tomato fruit at night may be due to less competition for calcium flux from the major transpiring organs rather than to a higher root

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pressure, while Masuda et al. (1996) reported that root pressure appears to be responsible for this difference. Under high humidity, the import of calcium by the fruit may depend on the balance between the transpiring organs and the development of root pressure (Ho 1989), whereas under high ECs (Ehret and Ho 1986a) reduction of calcium uptake can be explained by a low Ψs, a reduction in water absorption, and an antagonistic effect between potassium and calcium absorption by the roots (Ehret and Ho 1986a). Moreover, high ECs tend to reduce root pressure, which promotes nighttime redistribution of calcium (Ehret and Ho 1986a). To insure an optimal absorption of calcium, the temperature has to be between 18 to 22°C. An adequate supply of Ca to the fruits is essential for their firmness and for their shelf-life. An insufficient supply of Ca will increase the number of fruits affected by BER, but may stimulate ethylene synthesis (Bangerth 1979) and consequently the biosynthesis of carotenoids (Kays 1991; Paiva et al. 1998a). Nevertheless, the presence of high levels of calcium in the fruit negatively affects their organoleptic quality and their shelf-life (De Kreij 1995). High levels of potassium in the root environment interfere with calcium, thus increasing the risk of BER incidence (Voogt 1988; Nukaya et al. 1995a; Bar Tal and Pressman 1996). Calcium levels in the fruit increased with increasing calcium concentrations in the nutrient solution (Bradfield and Guttridge 1984; Paiva et al. 1998a), but magnesium, potassium, total lycopene, and carotene levels decreased (Paiva et al. 1998a). Fruit firmness can be improved by spraying with calcium salts (Cooper and Bangerth 1976) and tomato ripening delayed by increasing fruit calcium content from 0.11 mg g–1 fresh weight to 40 mg g–1 (Wills et al. 1977). 2. Potassium. Fruit constitute important reserves of photoassimilates and minerals, notably potassium (Ho 1996a). Potassium is involved in several metabolic processes such as synthesis of proteins, enzymatic activation (glycolysis, sucrose and starch synthesis, nitrate reduction), membrane transport processes, charge balance, and the generation of turgor pressure (Evans and Sorger 1966; Jackson and Volk 1968; Epstein 1972; Clarkson and Hanson 1980; Leigh and Wyn Jones 1984; Hsiao and Lauchli 1986). By the 8th week from planting, the absorption of K by the tomato plant, relative to Ca and Mg uptake, has increased considerably (Voogt and Sonneveld 1997). When the uptake of potassium is lower than the demand, the foliar potassium is remobilized to the fruit. An inadequate potassium concentration in the nutrient solution reduces plant growth, has a negative effect on fruit set in young reproductive plants (Besford

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and Maw 1975), and decreases dry matter distribution to leaves and roots of fruiting plants (Haeder and Mengel 1972). Potassium is positively related to a nice fruit shape, the reduction of ripening disorders, and the increase of fruit acid concentration (Adams et al. 1978; Mahakun et al. 1979). Potassium plays an important role in the maintenance of electroneutrality of organic acids in the fruit (Davies 1964; Mitchell et al. 1991), and a positive correlation between citric and malic acid content in the fruit and potassium content in the soil has been observed (Davies 1964; Winsor and Baker 1982). Potassium content of fruit was negatively correlated with fruit pH (Winsor and Massey 1958; Mahakun et al. 1979; Picha 1987). Winsor and Baker (1982) did not find any influence of potassium on the fruit sugar and dry matter content, but in greenhouse tomato, Davies and Winsor (1967) have observed a positive response of plants to potassium in terms of fruit acidity, sugars, dry matter, and organoleptic quality. Increasing nutrient solution potassium also increased lycopene concentration (Trudel and Ozbun 1971). The incidence of both external and internal blotchy ripening is decreased with increased potassium supply (Ozbun et al. 1967; Trudel and Ozbun 1971), and is negatively correlated with potassium status of rhizosphere, plant (Hartz et al. 1999), and fruit (Winsor and Massey, 1958). For example, within the range of potassium concentrations in the nutrient solution of 4.6, 7.2, and 9.7 mM, the number of fruit of uneven coloration were reduced from 40 to 21 and 12%, respectively (Gormley and Maher 1990). The K:Ca ratio affects the granular or floury sensation of fruit due to a weak cohesion between cells. A high K:Ca ratio improved fruit firmness and acidity while it reduced the sugar content (Janse and Gielesen 1991), increased the number of fruit affected by BER (Van der Boon 1973), but reduced the incidence of fruit with gold specks (Voogt 1987; Nukaya et al. 1995b). Recently, Bar Tal and Pressman (1996) have noted that increasing the potassium concentration from 2.5 to 10.0 mM reduced the marketable yield (14%) due to a reduction of the calcium uptake and to an increase (4 times) in the number of fruits affected by BER. However, no variation in the potassium content in ripened fruits was observed, whereas the potassium concentration in the roots, stems, and leaves was significantly increased. A low K:Ca ratio in the nutrient solution increased the number of fruit with gold specks (Nukaya et al. 1995a) thereby reducing their shelf life (Janse 1988a). In order to obtain high marketable yields, it has been recommended (Voogt 1988) that a molar ratio of K:Ca in the nutrient solution supplied should be around 6.3:1.7 (K:Ca ratio of 3.7 mM mM–1), whereas others (Nukaya et al. 1995a) have suggested a K:Ca ratio of 2 in mM mM–1 for susceptible cultivars to BER, between 2.0 to 3.0 in mM mM–1 for moderately susceptible cultivars, and

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4.0 in mM mM–1 for resistant cultivars. The calcium concentration in foliar tissues as well as the K:Ca ratio in leaves are not good indicators of the calcium content in fruit or their K:Ca ratio. 3. Phosphorus. Low concentrations in phosphorus adversely affect reproductive growth. Phosphorus (P) is essential to the development of flowers, probably through its role in cytokinin transport (Menary and van Staden 1976) and on fruit development because of its buffer effect. Mahakun et al. (1979) have reported that P content was negatively correlated with H+:total acidity ratio. At the pericarp tissue level, P is positively correlated with pH and negatively correlated with the titratable acids of the tissue. Increasing the phosphorus concentration of the nutrient solution from 0.02 to 3.0 mM, which reduces the incidence of BER (from 25 to 0.1%) (Cerda and Bingham 1978; Cerda et al. 1979; Sonneveld and Straver 1988; Voogt and Sonneveld-Van Buchem 1989), stimulates the absorption and distribution of calcium in the fruit (Cerda and Bingham 1978; Cerda et al. 1979; De Kreij 1996), and favors the incidence of gold specks (Voogt and Sonneveld-Van Buchem 1989; De Kreij et al. 1992). Phosphorus and calcium concentrations in the rhizosphere of 1.7 and 8–9 mM, respectively, are required in order to reduce the incidence of BER (De Kreij 1996). Increasing the P supply also increases the whole tomato plant tolerance to NaCl (Awad et al. 1990). Thus, plants grown under high salinity levels require a supplementary contribution of P. On the other hand, excess P in a growing medium results in an imbalance that affects the availability, uptake, and utilization of other essential elements. For example, P enhances the availability and uptake of Mn, but significantly reduces the uptake and utilization of Zn, and to a limited degree, Fe (Jones 1998). 4. Nitrogen. The nitrogen supply affects the size, the color, and the characteristics of the fruit cuticle (Locascio et al. 1984). A very high nitrogen concentration negatively influenced the color, delayed ripening, caused uneven ripening, and reduced fruit soluble solids content (Locascio et al. 1984). It also increased fruit acid concentration and decreased fruit organoleptic quality (glycosides, esters, alkaloids) (Locascio et al. 1984; Thakur et al. 1996). A high nitrogen concentration also interfered with calcium nutrition and, as a consequence, increased postharvest quality losses and the number of fruit affected by BER. A very low concentration in nitrogen decreased the development of the foliar canopy and, consequently, the quantity of photoassimilates available to fruit. This reduced the size, color, and the yield of tomato fruit. Ascorbic acid accumulation was higher under nitrogen deficiency

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(Giovanelli et al. 1998), decreasing linearly with nitrogen fertilizer application (Montagu and Goh 1990). In fact, high nitrogen concentration in the nutrient solution favors plant leaf area development, thereby decreasing light penetration in the canopy and fruit content in vitamin C (Locascio et al. 1984). The nitrogen source provided to the plants can also influence fruit quality. Ho (1996b) has reported that ammonium (NH4) increases fruit sugars content but decreases calcium concentration. Similarly, Pivot et al. (1997) have reported that an excess of NH4 in the nutrient solution results in a reduction of the calcium content in the fruit and an increase in the number of fruit affected by BER. Ten to 20% of total nitrogen as NH4, compared to 0% of total nitrogen as NH4 favors plant growth but decreases fruit size and total or marketable yield due to a reduction of calcium and magnesium absorption and an increase in the number of fruit affected by BER (Hohjo et al. 1995). Depending on cultivar, ammonium concentrations varying from 0.7 to 2.0 mM can increase BER from 7 to 77% (Nukaya et al. 1995a; 1995b). Generally, nitrogen supply in the form of NH4 should be lower than 10% of total nitrogen in order to minimize the susceptibility of fruit to BER (Adams 1999) and fruit with gold specks (Voogt 1987; Nukaya et al. 1995b). Applications of NH4 compared to NO3 increased glutamic acid levels in the fruit. Nitrogen supply in the form of urea had almost no effect on dry matter, sugars, and ascorbic and organic acids content in the tomato fruit compared to that in the NO3 form (Kowalska and Sady 1996). In order to obtain high-quality fruit, the K:N ratio should be 1.2:1 for young plants (until the first inflorescence) and 2.0–2.5:1 when the 9th cluster is in flower (Ho and Adams 1995; Adams 1999) 5. Sulphate. Sulphate accumulation generally occurs in closed growing systems. High sulphate levels in the nutrient solution does not directly influence fruit quality, but may affect it indirectly through an increase in salinity and a change in ionic activities (Adams 1971; Lopez 1998). Thus, high sulphate concentrations (>40.9 mM) may affect the absorption of P, Ca, and Mg ions and increase the number of fruit affected by BER (Lopez 1998). 6. Sodium. Sodium is not an essential mineral element for tomato, but can replace a certain proportion of potassium without a negative effect on growth (Marschner 1995) and fruit quality (Adams 1989; Adams 1991; Petersen et al. 1998; Dorais et al. 1999b, 2000). However, studies conducted in the Netherlands, UK, and Canada have shown an improvement of the organoleptic quality of fruit after NaCl addition. This effect

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may be related to higher levels of soluble sugars in tomato fruit (Adams 1989; Adams 1991). For example, in the storage tissue of sugar beet, sodium is more effective than K in stimulating sucrose accumulation (Marschner 1995), and seems to be related to stimulation of ATPase activity at the tonoplast of sugar beet cells (Willenbrink 1983). Increasing the EC with NaCl reduces titratable acids, potassium, and nitrogen in fruit but increases their sodium and calcium content (Adams 1991; Adams and Ho 1989; Dorais et al. 1999b), and also reduces the number of fruit affected by BER (Dorais et al. 2000). The reduction in fruit titratable acids can be explained by the interference of sodium in the absorption and transportation of potassium (Läuchli 1986). However, Mizrahi et al. (1986) reported an increase in fruit sodium and organic acids, and a lower fruit pH, in greenhouse tomato grown under 54 mM NaCl compared to the control nutrient solution (0 mM NaCl). Petersen et al. (1998) further explained the effect of salinity source on titratable acid content (as found by Adams 1991) by the achievement of a much higher ion activity ratio ak/√(aCa + aMg) in the root zone when major nutrients rather than NaCl were applied to increase the salinity. They observed that increasing the salinity with NaCl improves the perception that the tomato fruit is sweet more than other salinity sources, although no differences in dry matter, soluble solids, glucose, fructose, or titratable acid were found. Similarly, results of sensorial testing experiments showed that fruit from higher EC-treated plants with 24 to 29 mM NaCl improved the sensory evaluation of sweetness and had a better flavor compared to fruit from non NaCl-treated plants (Dorais et al. 1999b). On the other hand, high Na+ concentration may displace Ca2+ in plant cell membranes (Cramer et al. 1985), and several physiological disorders may occur under such conditions. Atta-Aly et al. (1998) reported that high salinity provided by NaCl or 3:1 NaCl:CaCl2 (7.5 g L–1) may induce ripening in the non-ripening field tomato mutants. Addition of 22 mM Na (5.5 mS cm–1) to the nutrient solution did not reduce fruit yield but increased the percentage of first-class fruits (Adams 1989). A concentration of 67 mM Na (9.8 mS cm–1) had little if any effect on fruit BER incidence. Moreover, for an EC of 12 mS cm–1, the addition of macronutrients considerably reduced fruit yield as compared to the addition of NaCl (Adams 1990). Under these conditions (high levels of macronutrients), the percentage of marketable fruit was only 30% (35% of fruit with BER vs. 2% with NaCl). The inconsistency of these results can very likely be explained by (1) different growing conditions (cultivars, irradiance, p[CO2], temperature, VPD); and (2) different ion activity ratios ak/√(aCa + aMg) and aMg/aCa in the root zone.

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7. Chloride. Chloride has a number of non-specific functions in the plants. It raises the cell osmotic pressure and, as a result of the hydrophilic nature of the ion, increases the hydration of plant tissues (Inal et al. 1998). Chloride ions can replace nitrate ions in their colloidchemical functions and may have a positive effect on the N content of plant organs by helping to prevent excessive NO3 concentrations (Bergman 1992). NO3 accumulation in edible plant tissues is undesirable in terms of human health because it can result in the formation in the blood of NO2, causing methemoglobinemia (Wright and Davidson 1964), and nitrosamines which are believed to be carcinogenic (Craddock 1983). Recently, we found for a spring tomato crop production that increasing EC with NaCl (12–24 mM compared to 0 mM) increased fruit Cl concentration (Dorais et al. 1999b), and reduced the nitrogen concentration in tomato shoots (Chrétien et al. 2000) and fruit (Dorais et al. 1999b). An increase in the Cl concentration (8–13 mM as compared to 3 mM) in the nutrient solution stimulated the absorption of Ca and reduced BER incidence (De Kreij et al. 1992; De Kreij 1995) but increased the number of fruits with gold speck injury (Nukaya et al. 1991, 1992). Thus, a threshold of 7.5 mM Cl in the rooting zone has been established (Voogt 1992). It has been suggested (Ho and Adams 1995) that substituting NO3-N with KCl, NaCl or CaCl2 does not affect fruit quality if the minimal concentration of NO3-N in the nutrient solution is 120 mg L–1 and the K:N ratio is kept between 2 and 4, but increased fruit Cl concentrations (Dorais et al. 1999b). 8. Boron. Boron is an essential micronutrient for plant growth and development. Inadequate boron supply significantly changes the activity of numerous enzymes and consequently affects the metabolism of higher plants. A boron deficiency can considerably reduce fruit yield but its effect of fruit quality is less well known. Yamauchi et al. (1986) showed that boron plays an important role in calcium metabolism of the cell wall. A boron deficiency in tomato plants can decrease the calcium concentration associated with pectic compounds. Similarly, boron has a stabilizing effect on calcium complexes of the middle lamella and is thus essential to the maintenance of the structure of the cell wall (Clarkson and Hanson 1980). Early works showed that boron and calcium sprays individually or in combination were highly significant over the control in reducing tomato cracking (Wilson 1957; Gill and Nandpuri 1970). Weekly cluster calcium and boron spraying (6.6 g L–1 CaCl2 + 3g L–1 borax) on 40-day-old fruit reduced significantly the proportion (20%) and the severity (50%) of fruits with cuticle cracking and increased the

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proportion of fruits of Class 1 (Dorais et al., unpublished data). Boron concentration in those fruits was two times higher than control fruits (water spraying), which is beneficial for human health (Hunt and Stoecker 1996). We found similar effects with commercial foliar spraying (6.6 g L–1 CaCl2 + 0.75 g L–1 borax) without boron toxicity. 9. Bicarbonate. Some studies suggest that bicarbonate (HCO3–) in the rooting environment of tomato plants can significantly increase fruit quality and yield. It has been reported (Bialczyk et al. 1996) that the addition of 5.68 mM KHCO3 per dm3 of the standard nutrient solution increases glucose and fructose in tissue (pericarp + locules) content of unripened green fruit from 34 to 46% and of mature red fruit from 23 to 30%. The glucose:fructose ratio is generally lower in plants treated with bicarbonate. Fruits of treated plants also have a higher organic acid content (27–36%) and a higher fructose:organic acids ratio. The effect of bicarbonate on fruit quality is more profound under high salinity conditions. An EC of 6–7 mS cm–1 together with a bicarbonate supply can increase the accumulation of total solids in the fruit (Gao et al. 1996). E. Effect of Electrical Conductivity 1. Fruit Quality. Several studies have shown that increasing the electrical conductivity (EC) of the nutrient solution in tomato plants by either augmenting the macronutrients (NO3, NH4, K, Ca, Mg, H2PO4, SO4) or by adding NaCl, KCl, or CaCl2 in the nutrient solution increases external and internal tomato fruit quality (Stevens 1979; Mizrahi 1982; Mizrahi et al. 1986; Voogt 1987; Hobson 1988; Matan and Golan 1988; Mizrahi et al. 1988; Sonneveld and Welles 1988; Mitchell et al. 1991; Sonneveld and Van Der Burg 1991; Cornish 1992; Janse 1995; Nukaya et al. 1995b; Cho et al. 1997; Auerswald et al. 1999b; Lin and Glass 1999; Li et al. 1999a). Under such conditions, tomato fruit generally have better organoleptic and nutraceutic properties, a thicker and more resistant cuticle, a lower turgor pressure (Verkerke and Schols 1992) and, as a consequence, a reduced firmness (Verkerke et al. 1991) and a lower susceptibility to fruit cracking (Sonneveld and Van Der Burg 1991). For example, increasing the EC by 1.3 times of the control EC (2.6–4.6 compared to 2.0–3.5 mS cm–1) in a greenhouse tomato spring crop reduced the incidence of fruit with cracking by 68% (Chrétien et al. 2000). However, Peet and Willits (1995) have shown that increases in fruit cracking incidence were the same, irrespective of how water was provided, i.e. as a nutrient solution or water only.

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Table 5.1 summarizes the effect of EC level on greenhouse tomato fruit quality and yield. Fruit firmness and concentrations of dry matter, soluble solids, fructose, glucose, titratable acid, volatile compounds, minerals, carotene, and vitamin C in the fresh fruit increase with increasing salinity (Steven et al. 1979; Mizrahi et al. 1986; Hobson 1988; Sonneveld and Welles 1988; Adams 1989; Holder and Christensen 1989; Gormley and Maher 1990; Sonneveld and Van Der Burg 1991; Solimann and Doss 1992; Petersen et al. 1998; Dorais et al. 1998). In contrast to previous studies (Adams 1991), Petersen et al. (1998) reported that increases in those fruit components under increasing salinity are independent of the source of salinity, if it results in only minor changes in the ion activity ratio ak/√(aCa + aMg) and aMg/aCa in the root zone. Increasing the EC may also lead to an increase in fruit color indices (Sonneveld and Van Der Burg 1991), the proportion of Class 1 fruits (from 76 to 93%; Adams 1991), as well as a reduction of uneven ripening fruit (Sonneveld and Van Der Burg 1991). An EC of 4.6–8 mS cm–1 reduced fruit yield because of a reduction in fruit size, whereas an EC of 12 mS cm–1 reduced both the number of fruit and their size, and the whole fruit sugar and acid content (Gormley and Maher 1990; Adams 1991; Hao et al. 2000). For cluster tomatoes grown in NFT at EC’s of 3.0, 5.0, and 8.0 mS cm–1, Gough and Hobson (1990) found that the most flavorful fruit was grown under an EC of 5 mS cm–1. ECs of 8 mS cm–1 were shown to yield fruit more resistant to mechanical manipulation, while a much faster fruit ripening and a reduction in fruit shelf life has been reported when plants are grown under extreme salinity levels (103 mM NaCl, 11.4 mS cm–1) (Mizrahi 1982). Generally, the number of fruit affected by BER increases with salinity (Adams and Ho 1992; De Kreij 1992; Ho et al. 1993; Willumsen et al. 1996; Ho 1999) and the ion activity ratios in the root zone (Willumsen et al. 1996). The ideal EC of the nutrient solution, relative to preventing BER in the greenhouse tomato, is 2.0 to 2.5 mS cm–1, with a calcium concentration of 7 mM (De Kreij 1992). However, these levels are not optimal for other tomato quality attributes such as fruit flavor, fruit firmness, and fruit cracking. Recently, Auerswald et al. (1999b) observed that the sensory changes caused by increasing nutrient solution EC from 1.0 to 6.0 mS cm–1 with macronutrients improved the quality of the conventional round cultivar ‘Counter’ but not that of the longlife one ‘Vanessa’. For ‘Vanessa’, higher EC led to a much stronger intensity of negative flavor attributes such as “mouldy,” “spoiled sweetish,” “bitter,” and of the aftertaste attributes such as “mouldy” and “burning”, which contribute to the off-flavor. In

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286 Table 5.1. authors.

M. DORAIS, A. P. PAPADOPOULOS, AND A. GOSSELIN Influence of EC levels on greenhouse tomato fruit quality and yield according to various

EC levels (mS cm–1)

Firmness

Color

Size

–

+

–

Cuticle cracking

BER

Gold specks

Uneven ripening

pH

EC

–

+

Dry matter

Soluble solids

11.4 1.0 to 6.0

2.0 to 3.5–5.0

+

3.0 to 5.0

–

+

8.0

–

+

3.0 to 8.0

–

12.0

o/+

–

2.5 to 5.2

+

3.0 to 9.0

3.0 to 8.7

–

+

+ –

–

+

+

o/+

–

o

–

+

+

+

2.0 to 10.0

–

1.0 to 6.0

+

1.8 to 5.1

o

–/o

+

+

–

o

–

+/o

2.0 to 6.0–10.0

–

+

2.0–3.5 to 2.6–4.6

–

1.8 to 5.6

o

o

–

+ = positive effect, – = negative effect, o = no effect

o +

–

+

+

2.1–3.3 to 3.0–4.6

–

+

+/o

+/o

+

o

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287

Influence of EC levels on greenhouse tomato fruit quality and yield according to various

Soluble Volatile sugars Acids compounds

Minerals

+

+

+

+

o

+

+

o

+

+

o

o

–

–

+

+

Carote- Vitanoids mins

Folate

+

+

+

+

+

+

o

o

Fruit number

o

o

Mizrahi et al. 1986 –

Gormley and Maher 1990

o

o

Gough and Hobson 1990

o

–

Gough and Hobson 1990

–

Adams 1991

–

–

–

Adams 1991

+

o/–

–

Sonneveld and Van Der Burg 1991

–

Willumsen et al. 1996

+

Petersen et al. 1998 o

–

–

+

o

o

o

o

o

+

Dorais et al. 1999b

+

+

o

Hao et al. 1999 Lin and Glass 1999

–

+ +

Stanghellini et al. 1998 Auerswald et al. 1999

o

+

Authors Mizrahi 1982

o

+

Yield

–

o

o

+

Shelflife

o

Chrétien et al. 2000

o

Dorais et al. 2000

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contrast, for ‘Counter’, the more intense fruity flavor, juiciness, and firmness were judged by consumers to improve fruit quality. Thus adjusting the EC allows greenhouse growers to modify water availability to the crop and hence improves fruit quality (Massey et al. 1984; Holder and Christensen 1989). However, at some point increases in EC limit marketable yield (Sonneveld 1988). The exact point of decline varies with interactions between cultivars (Janse 1995; Auerswald et al. 1998; Stanghellini et al. 1998), environmental factors such as temperature and light, composition of the nutrient solution, and crop management (Sonneveld and Welles 1988; De Kreij et al. 1997; Stanghellini et al. 1998; Kläring et al. 1999). For example, Stanghellini et al. (1998) reported that depressing tomato plant transpiration under high EC (10 mS cm–1) might significantly reduce incidence of BER. According to different studies, salinities higher than 2.3–5.0 mS cm–1 result in an undesirable yield reduction (Massey et al. 1984; Sonneveld and Van Der Burg 1991; Verkerke and Schols 1992; Dorais et al. 1998), while ECs of 3.5–9.0 mS cm–1 improve tomato fruit quality. 2. Constant and Variable EC. Nutrient solutions used for growing tomato plants in soilless systems often have a daily constant salinity level in the range of 30–75 mM total ion concentration (equivalent to 2–5 mS cm–1, and 0.07–0.18 Mpa osmotic pressure, Van Ieperen 1996). However, positive effects of the use of a low salinity level (0.3 mS cm–1) during the day combined with a high salinity level during the night (10 mS cm–1) on vegetative growth and leaf area of young plants were observed by Bruggink et al. (1987) but was not confirmed by Van Ieperen (1996), probably due to different growing conditions. Studies conducted to improve tomato fruit quality subjected to intermittent salinity stress (two periods of 30 minutes per day) have not been conclusive (Niedziela et al. 1993). Similarly, Adams and Ho (1989) found no advantage of using variable EC (8 mS cm–1 during the day and 3 mS cm–1 at night time, 8/3 mS cm–1) as compared to a constant EC of 5 mS cm–1. A variable EC (8/3 mS cm–1) reduced the number and size of fruits, and affected calcium uptake and distribution to the fruit (Adams and Ho 1989; Ho and Adams 1989). In contrast, Van Ieperen (1996) reported that fluctuating day/night salinity could be advantageous as a tool to reduce BER and to enhance yield. In spring and summer, he observed that a day/night salinity level of 1/9 increased yield by 20%, and reduced BER (from 2.2–3.0% to 0.4–0.6%) compared with similar 24 h average EC (5/5 control treatment). In addition, dry-matter distribution towards the fruits was increased at 1/9 compared with 5/5, while dry-matter percentage of the harvested fruits was slightly lower

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at 1/9 compared with 5/5. Contrary to some authors (Ehret and Ho 1986c; Adams and Ho 1989) but in agreement with others (Gough and Hobson 1990; Adams 1991), differences in yield were not only due to differences in fruit water content but also were caused by a change in the dry weight per fruit. In a summer/autumn tomato crop, Nederhoff (1999) concluded that normal day-time EC with high night-time EC (2/8) can be useful to improve tomato fruit quality (soluble sugar and acid content), without significant loss of production. In general, the effect of daytime EC proved to be more important than of the nighttime EC (Adams and Ho 1990; Van Ieperen 1996; Nederhoff 1999), because the bigger proportion of water and nutrients is taken up at day, and this uptake is hampered by high salinity (Ehret and Ho 1986a). 3. Water and Nutrient Uptake. The flavor increase and yield decline under high salinity may be associated with a reduction in water absorption by roots because of a decrease in the osmotic potential (Ψs) of the nutrient solution combined with an increase in the resistance of the xylem transport system inside the fruit. Ehret and Ho (1986a) have reported reduced water absorption capacity per plant (from 415 to 98 ml) in young tomato plants when the EC was increased from 2 to 17 mS cm–1 and that the absorption rate of Ca was reduced by 87%. Excessive salinity can reduce Ca absorption (Adams and El-Gizawy 1986; Ho and Adams 1989; Minamide and Ho 1993; Ho and Adams 1994), the number of xylem vessels and the ionic exchange capacity (Belda and Ho 1993), and the tomato leaf water potential (Scholberg and Locascio 1999). Under –0.6 and –0.9 Mpa NaCl salinity levels, nitrogen uptake (15N) by 21-day-old tomato plants was reduced compared to –0.3 and –0.03 Mpa (Pessarakli and Tucker 1988). The concentration of the phloem mobile elements (K, P, and N) does not vary significantly as fruit develop (Ehret and Ho 1986a). Nevertheless, increasing the EC of the nutrient solution from 2 to 17 mS cm–1 reduced the fruit phosphorus concentration, increased potassium concentration, and had no effect on nitrogen concentration (Ehret and Ho 1986a). 4. Dry Matter Partitioning. Neither the fruit strength nor the quantity of photoassimilates imported by the fruit was affected by a high EC and by a reduction in water absorption (Ehret and Ho 1986c; Ho 1996a,b). Under an EC of 6 mS cm–1, biomass and dry matter partitioning among fruit (52%), vegetative parts (44%), and roots (4%) were not affected (Ehret and Ho 1986c; Ho 1996a). Nevertheless, an EC of 10 mS cm–1 reduced plant dry weight by 19% as compared to an EC of 2 mS cm–1, but did

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not always influence dry matter partitioning (Ehret and Ho 1986c). An EC of 17 mS cm–1, however, slightly reduced dry matter distribution to the fruits. Yield reductions under high EC conditions were proportional to plant growth reductions (Ehret and Ho 1986c). De Koning (1992b) has reported a 0.17% increase in fruit dry matter content following an increase of EC by one unit. 5. Fruit Growth Rate. Increasing salinity in the nutrient solution can substantially reduce the rate of fruit growth and the final fruit size. At the beginning of a new crop (grown under low light intensity), an EC of 17 mS cm–1 reduced the fruit growth rate by only 25% compared to a standard EC (3 mS cm–1) (Pearce et al. 1993). During the summer season (grown under high light intensity), however, the fruit growth rate was greatly reduced by an elevated EC. Ehret and Ho (1986a) also observed that tomato grown at high salinity (17 mS cm–1 compared to 2 mS cm–1) showed reduced daytime growth rate of fruit, while the growth rate at night was similar for fruit grown at both low and high EC. Thus, the diurnal growth cycle was less evident in fruit grown at high EC. The rate of blossom production and the length of the fruit-growing period did not seem to be influenced by high ECs. 6. Carbohydrate Accumulation. Starch accumulation in the fruit and the activity of the sucrose synthase enzyme were intensified when plants were cultivated under a high salinity (Ho 1996a,b). Ehret and Ho (1986c) have reported that carbohydrate partitioning between starch and soluble sugars can be influenced by the osmotic potential of young fruit. Under saline conditions, the starch concentration in the fruit can account for as much as 40% of dry matter. Recently, Gao et al. (1998) have shown that NaCl salinity (0, 50, or 100 mM NaCl) enhanced the transport of 14C-assimilates from the pulse leaf to adjacent fruits and the diversion of 14C label to the starch fraction of the fruit. It also prolonged the period of starch accumulation in developing fruit. These authors suggested that sucrose unloading to the developing fruit is enhanced under high salinity by increased starch, which will be later hydrolyzed back to soluble hexoses during fruit maturation, with the resulting improvement of fruit quality. Thus, under saline conditions, both a higher concentration of sucrose in the leaves (higher activity of sucrose phosphate synthase, lower acid invertase activity) and a faster rate of starch synthesis in the immature fruit (higher activity of ADP-Glc-Ppase) may constitute part of a mechanism responsible for a higher sugar content in the mature fruit.

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VI. POSTHARVEST Postharvest fruit quality is defined by the fruit shelf life once the product is harvested, by its organoleptic and nutraceutic qualities, and by its appearance at the time of purchase. It is highly dependent on pre-harvest factors (see Sections IV and V) and stage of fruit ripening at the time of picking. It was reported that both total and soluble solids, and acidity, decreased as color increased in typical processing tomato cultivars (Young et al. 1993; Renquist and Reid 1998). The maximum soluble solids were found in the turning-red stages with soluble solid content decreasing in the ripest stage (Renquist and Reid 1998). Similarly, Kavanagh et al. (1986) reported that tomato harvested at breaker stage (<10% red coloration) were superior in flavor to either vine-ripe or green-harvested fruit. Those results may partially be explained by a dilution mechanism whereby water uptake as a fruit develops exceeds the production of sugars and organic acids (Renquist and Reid 1998) or aroma compounds. Generally, the synthesis of volatile compounds contributing to fruit flavor increases with color changes (synthesis of lycopene) and, the production of ethylene and CO2 (Baldwin et al. 1991a; Baldwin et al. 1992). Hayase et al. (1984) have reported an increase in the concentration of tomato fruit volatile compounds (alcohols, cetones) when the fruit turned to the pale red stage. Some volatile compounds in fruit may be regulated by ethylene either directly or indirectly via lycopene (Baldwin et al. 1991a). On the other hand, change in acid and sugar levels in ripening tomato is independent of ethylene or CO2 production (Jeffrey et al. 1984; Baldwin et al. 1991a). Concentration of antioxidant vitamins also changes with fruit ripening. Ascorbic acid and γ-tocopherol levels increased until fruit turned yellow in color and then decreased, while β-carotene increased with ripeness in accordance with the rapid accumulation of red pigment as α- and β-tocopherol (Giovanelli et al. 1998; Abushita et al. 1997). According to Laval-Martin et al. (1975), lycophyll and lycoxanthin increase gradually throughout development and maturation of the fruit. Lycopene and phytofluene, on the other hand, are detected only at the breaker stage of fruit ripening, primarily in the outer pericarp. Studies have showed that the time of fruit harvest may influence the postharvest tomato quality (Brecht et al. 1976; Kader et al. 1977; Watada and Aulenbach 1979). Early harvesting is common practice in order to obtain firmer fruit suitable for transport, and to attain a longer marketable period (Auerswald et al. 1999a). However, trade journals recommended that fruit be harvested at a ripe stage in order to satisfy the

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demand for high-quality product (Janse and Konys 1995; Watzl et al. 1995). Harvested immature green fruit do not ripen as well as mature green fruit (Kader et al. 1978; Maul et al. 1997). Reduced organoleptic or nutraceutic qualities, however, is also related to the harvesting of mature but non-ripe fruits (Watada et al. 1976; Kader et al. 1977; 1978; Watada and Aulenbach 1979). Studies have shown that fruit harvested at earlier stages of maturity and room-ripened are less sweet, more sour, less tomato-like, and had more off-flavor than those harvested at the full red color stage (Kader et al. 1977; Kader et al. 1978; Shewfelt 1990). Vineripe fruit were considered sweeter than fruit harvested at mature-green and breaker stages by sensory panels despite no significant differences in soluble solids content or dry matter (Watada and Aulenbach 1979). The soluble solids:acidity ratio is higher in tomato fruits ripened on the plant compared to room ripened fruits (Bisogni and Armbruster 1976). A higher concentration of C9-C12 compounds and terpene esters have been observed in fruit ripened on the plant compared to those ripened artificially. Fruit artificially ripened, however, had high concentrations of C4-C6 compounds such as 1-butanol, 3-pentanol, 2-methyl-3-hexanol, 3-methyl-butanal, 2,3-butanedione, propyl acetate, and isopentyl butyrate (Shah et al. 1969). Others (Buttery et al. 1987) have reported no differences in the concentrations of C5-C9 volatile compounds between fruits ripened on the plant and fruits artificially ripened, but have measured a cis-3-hexenal concentration ten times lower on fruits ripened at 2°C compared to fruits ripened on the plant or at ambient temperature. The quantity of volatile compounds in fruits ripened after harvest is always lower than the concentration observed on fruits ripened on the plant (Stern et al. 1994). Depending upon harvest time (varying field ripening conditions such as temperature and light) and cultivar, roomripened fruit could have only 41% of the reduced ascorbic acid content of field-ripened fruits, higher locular material content (higher titratable acid), and lower overall quality and flavor (Bisogni and Armbruster 1976). Moreover, shelf-ripened tomato fruits contain more α-tomatine than vine-ripened tomato fruits which are generally α-tomatine free (Chen and Miller 2000). The period of storage also influences fruit quality. Recently, quantitative descriptive analyses of sensory tomato attributes and chemical analysis of red-harvested tomato at 0, 4, and 7 days of storage showed that most attributes (surface and flesh red color, tomato-like smell and flavor, sweetish smell and aftertaste, raw potato smell, sour flavor and aftertaste, mouldy flavor, firmness, juicy, pulpy, grainy and gristly) were changed by 4 days, while the fruit content of the reducing sugars remained relatively constant and the concentration of titratable acid

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decreased by 22% (Auerswald et al. 1999a). During short-term storage, Janse (1995) and Peters et al. (1998) also observed a decrease of titratable acid content. Storage-ripened green fruit usually have much lower levels of carotene when compared to field-ripened fruits (Gould 1992). Cold storage has a negative effect on fruit aroma and consequently on its organoleptic quality (Lammers 1981). Work done during the 1960s in The Netherlands suggested a minimum storage temperature of 16°C for green fruit and 8°C for red fruit, allowing them to be kept for 3 to 4 weeks, while in Canada it was recommended that mature green tomato fruits should be stored at 10°C and ripened at 21°C for 2 to 6 days, and then held at 10°C for a further 8 to 10 days (Davies and Hobson 1981). The buildup of volatile compounds is significantly reduced when fruit are ripened at temperatures lower than 10°C, while temperatures higher than 20°C favor the production of volatile compounds (Stern et al. 1994). The development of volatile compounds involved in fruit taste depends on the final ripening temperature rather than the initial storage temperature (Stern et al. 1994). Nevertheless, fruit stored for 7 days at 5°C and ripened at 20°C had a more acidic taste and a mediocre flavor (Kader et al. 1978). Fruit exposed to low (chilling) temperatures had a higher citric acid content while malic acid, fructose, and sucrose concentrations were reduced (Buescher 1975). Similarly, fruit ripened at 20°C under conditions of low air circulation were characterized by a reduced organoleptic quality when compared to fruit ripened in a well-aerated storage area (Kader et al. 1978). For a long life tomato cultivar, intermittent warming temperature (fruit stored at 9°C were warmed to 20°C for 1 day every week) produced a better cuticle color and flavor than storing fruit at a constant 9°C (Artés et al. 1998a). At the end of cold storage (21 days) and after post-storage ripening (3 days), there was no significant change in the soluble solids content, while intermittent warming slightly reduced titratable acidity (Artés et al. 1998b). Studies of the effects of controlled atmospheres on stored tomato fruit have suggested different mixtures of oxygen, carbon dioxide, nitrogen, and carbon monoxide for retarded ripening and fungal growth (Davies and Hobson 1981). Physical damage incurred during the growing period or the handling process increases the rate of respiration, ethylene production, and fruit water loss. These factors also reduce fruit content in desirable aromas and fruit shelf-life and are an excellent entry point for pathogens. Tomato fruit is particularly vulnerable to microbial infections due to the fact that the calyx scar constitutes an entrance for microorganisms. Fruit disinfecting is a way to increase their shelf-life. Treating fruit with a 13 mM cinnamaldehyde solution (natural compound in this species)

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significantly reduced the number of bacteria and fungi, and delayed the occurrence of these microorganisms on the fruit by seven days (Smid et al. 1996). Recently, Maharaj et al. (1999) observed that postharvest treatment of tomato fruit with ultraviolet radiation (UV–3.7 × 103 J m–2) delayed ripening and senescence. Additionally, the climacteric peaks of respiration and ethylene production were retarded by 7 days. They suggested that this effect might be attributable to the maintenance of a high level of putrescine (Maharaj et al. 1999). Surface sterilization of tomato fruit for sanitation purposes with sodium hypochlorite has been found to lead to physiological and biochemical alterations (Hong and Gross 1998).

VII. CONCLUSION Numerous genetic, climatic, and cultural factors affect the organoleptic and nutraceutic properties of greenhouse tomato fruit. In the face of a global market economy, obtaining high yields of tomato fruit of very high quality and flavor is essential for insuring consumer satisfaction and for the success of the greenhouse industry. One way to reach this objective is to characterize and genetically improve the major compounds responsible for tomato flavor, health benefit, and shelf-life in greenhouse tomato cultivars. Many criteria have been involved in tomato cultivar selection: (1) resistance to diseases and physiological disorders; (2) yield (early as well as total); (3) short-term and long-term plant vigor; (4) growth rate; (5) fruit color; (6) type of fruit; (7) fruit flavor; and (8) shelf-life. The final choice is often a compromise between sales objectives, agronomic criteria, and production constraints. In the past, greenhouse cultivar selection by breeders emphasized yield, fruit type, disease resistance, and fruit color, but not organoleptic and nutraceutic aspects of fruit quality (McGrath 1998). Recently, studies have shown that sugar:titratable acid ratio, soluble solids and sugar content, the percentage juice, and texture parameters of the pericarp are all good organoleptic descriptors of overall tomato fruit acceptability, sweetness, and mealiness, which may predict tomatolike flavor (Baldwin et al. 1998), fruitiness (Bucheli et al. 1999a, 1999b), and pleasantness (Verkerke and Janse 1997; Verkerke et al. 1998). For a practical application, those descriptors are reliable, quick and easy to measure, and could be used by growers to guarantee the organoleptic quality of their fresh product. Certain flavor volatile compounds were identified as contributing to overall acceptability, perception of

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tomato-like flavor intensity, specific aroma, sweetness, sourness, and other taste descriptors (Baldwin et al. 1998; Krumbein and Auerswald 1998). However, measurements of volatiles are expensive and complex and can be avoided for predicting tomato fruit quality (Janse and Schols 1995). Tomato fruit improvement could be achieved by either traditional breeding techniques, modern biotechnology, or a combination of the two approaches. In the near future, with the debate surrounding transgene technology, classical breeding will probably remain the main part of greenhouse tomato breeding programs worldwide (McGrath 1998). In order to speed up the selection process of a particular trait, QTLs (qualitative trait loci) have been identified. Despite the complex lineage between desirable traits already present in modern cultivars, the genetic coding for organoleptic and nutraceutic qualities can be tracked by these “biochemical markers.” For example, genetic manipulation of certain enzymes such as ADH2 (Speirs et al. 1998), ∆9-desaturase (Wang et al. 1996), and lipoxygenase (Kausch and Handa 1997) showed that tomato fruit flavor may be improved. Interactions between aroma compounds and sugars, acids and other compounds both chemically, and in terms of flavor perception, are also a fertile area for additional research (Baldwin et al. 1998; Verkerke et al. 1998). Further work to characterize the action of selective acyl hydrolase enzymes in ripened tomato fruit and to analyse the detail of acyl composition and distribution between individual lipid molecules would provide crucial information for any genetic modification strategies designed to enhance the flavor of tomatoes (Gray et al. 1999). Moreover, tomatoes are an excellent health product due to the balanced mixture of antioxidants including vitamins C and E, lycopene, β-carotene, lutein, and flavonoids such as quercetin (Offord 1998). Actually, only a limited number of genes are available for biotechnology work on protected cropping. Thus, identification of organoleptic and nutraceutic genes would be a fertile area of research for the next years. Another way to improve organoleptic and nutraceutic qualities of greenhouse tomato fruit is to maintain proper environmental parameters in the greenhouse (light, temperature, humidity, CO2 enrichment) and implement new growing methods (higher EC, optimum irrigation, and nutrient solution). However, synergistic and antagonistic effects of climate and cultural factors occur, and more knowledge is required to optimize tomato fruit quality. From a cultural and physiological point of view, research should be conducted with the aim of: (1) optimizing the greenhouse climate; (2) optimizing the absorption of nutrients and their partitioning; (3) co-ordinating the supply of water, nutrients, and

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photoassimilates with the stage of development of plant organs in order to get products of very high quality (Ho and Adams 1995); (4) controlling sucrose hydrolysis and membrane transport of photoassimilate imported by fruit (Ho 1996a,b); and (5) providing scientific information on the effects of environment and cultural factors on tomato fruit organoleptic and nutraceutic components.

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Plate 1 Protea repens cv. Guerna, the first Protea cultivar released in 1978 from the South African Proteaceae breeding project.

Plate 2 Protea holosericea, a mammalpollinated, endangered species found in two isolated populations in the Worcester district within the Cape Floristic Kingdom.

Plate 3 The large-leaf, summer flowering horticultural variant of Protea cynaroides in its natural habitat.

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Plate 4 Protea cv. Sheila, a putative hybrid between P. magnifica and P. burchellii, is an example of an interspecific hybrid produced by natural pollen vectors and selected for its unique involucral bract color, flower head shape, and the plant vigor.

Plate 5 Protea cynaroides cv. Madiba, the result of controlled hybridization, was selected for its late spring flowering time, red involucral bract color, small leaves, thin stems, and strong plant vigor.

Plate 6 The different flowering stages of a Protea repens hybrid, moving from left to right, the hard bud, soft bud, anthesis of first florets and progression of anthesis. The correct cut flower harvesting stage is from soft bud to anthesis of the first florets.

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Subject Index Volume 26 A

P

Asexual embryogenesis, 105–110

Physiology, hormone reception, 49–84 Postharvest physiology, coated fruits and vegetables, 161–238 Protea, 1–48

C Cassava, molecular biology, 85–159

R D Dedication, De Hertogh, A. A., xi–xiii

Root and tuber crops, cassava molecular biology, 85–159

F

S

Floricultural crops, Protea, 1–48 Fruit coating physiology, 161–238

Signal transduction, 49–84 T

G Growth substances, hormone reception, 49–84

Tissue culture, cassava, 99–119 Tomato (greenhouse), quality, 239–319 V

I In vitro, cassava, 99–119 M

Vegetable crops: cassava, 85–159 coating physiology, 161–238 tomato (greenhouse), quality, 239–319

Molecular biology: cassava, 85–159 hormone reception, 49–84

Horticultural Reviews, Volume 26, Edited by Jules Janick ISBN 0-471-38789-4 © 2001 John Wiley & Sons, Inc. 321

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Cumulative Subject Index (Volumes 1–26)

A Abscisic acid: chilling injury, 15:78–79 cold hardiness, 11:65 dormancy, 7:275–277 genetic regulation, 16:9–14, 20–21 mechanical stress, 17:20 rose senescence, 9:66 stress, 4:249–250 Abscission: anatomy and histochemistry, 1:172–203 citrus, 15:145–182, 163–166 flower and petals, 3:104–107 regulation, 7:415–416 rose, 9:63–64 Acclimatization: foliage plants, 6:119–154 herbaceous plants, 6:379–395 micropropagation, 9:278–281, 316–317 Actinidia, 6:4–12 Adzuki bean, genetics, 2:373 Agapanthus, 25:56–57 Agaricus, 6:85–118 Agrobacterium tumefaciens, 3:34 Air pollution, 8:1–42 Alkaloids, steroidal, 25:171–196 Almond: bloom delay, 15:100–101 in vitro culture, 9:313 postharvest technology and utilization, 20:267–311 Alocasia, 8:46, 57, see also Aroids

Alternate bearing: chemical thinning, 1:285–289 fruit crops, 4:128–173 pistachio, 3:387–388 Aluminum: deficiency and toxicity symptoms in fruits and nuts, 2:154 Ericaceae, 10:195–196 Amarcrinum, 25: 57 Amaryllidaceae, growth, development, flowering, 25:1–70 Amaryllis, 25:4–15 Amorphophallus, 8:46, 57, see also Aroids Anatomy and morphology: apple flower and fruit, 10:273–308 apple tree, 12:265–305 asparagus, 12:71 cassava, 13:106–112 citrus, abscission, 15:147–156 embryogenesis, 1:4–21, 35–40 fig, 12:420–424 fruit abscission, 1:172–203 fruit storage, 1:314 ginseng, 9:198–201 grape flower, 13:315–337 grape seedlessness, 11:160–164 heliconia, 14:5–13 kiwifruit, 6:13–50 magnetic resonance imaging, 20:78–86, 225–266 orchid, 5:281–283 navel orange, 8:132–133

Horticultural Reviews, Volume 26, Edited by Jules Janick ISBN 0-471-38789-4 © 2001 John Wiley & Sons, Inc. 323

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324 Anatomy and morphology: (cont.) pecan flower, 8:217–255 petal senescence, 1:212–216 pollution injury, 8:15 waxes, 23:1–68 Androgenesis, woody species, 10:171–173 Angiosperms, embryogenesis, 1:1–78 Anthurium: see also Aroids, ornamental fertilization, 5:334–335 Antitranspirants, 7:334 cold hardiness, 11:65 Apical meristem, cryopreservation, 6:357–372 Apple: alternate bearing, 4:136–137 anatomy and morphology of flower and fruit, 10:273–309 bioregulation, 10:309–401 bitter pit, 11:289–355 bloom delay, 15:102–104 CA storage, 1:303–306 chemical thinning, 1:270–300 fertilization, 1:105 fire blight control, 1:423–474 flavor, 16:197–234 flower induction, 4:174–203 fruiting, 11:229–287 fruit cracking and splitting, 19: 217–262 in vitro, 5:241–243; 9:319–321 light, 2:240–248 maturity indices, 13:407–432 mealiness, 20:200 nitrogen metabolism, 4:204–246 replant disease, 2:3 root distribution, 2:453–456 stock-scion relationships, 3:315–375 summer pruning, 9:351–375 tree morphology and anatomy, 12:265–305 vegetative growth, 11:229–287 watercore, 6:189–251 weight loss, 25:197–234 yield, 1:397–424 Apricot: bloom delay, 15:101–102 CA storage, 1:309 origin and dissemination, 22:225–266

CUMULATIVE SUBJECT INDEX Aroids: edible, 8:43–99; 12:166–170 ornamental, 10:1–33 Arsenic, deficiency and toxicity symptoms in fruits and nuts, 2:154 Artemisia, 19:319–371 Artemisinin, 19:346–359 Artichoke, CA storage, 1:349–350 Asexual embryogenesis, 1:1–78; 2:268–310; 3:214–314; 7:163–168, 171–173, 176–177, 184, 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 Auxin: abscission, citrus, 15:161, 168–176 bloom delay, 15:114–115 citrus abscission, 15:161, 168–176 dormancy, 7:273–274 flowering, 15:290–291, 315 genetic regulation, 16:5–6, 14, 21–22 geotropism, 15:246–267 mechanical stress, 17:18–19 petal senescence, 11:31 Avocado: CA and MA, 22:135–141 flowering, 8:257–289 fruit development, 10:230–238 fruit ripening, 10:238–259 rootstocks, 17:381–429 Azalea, fertilization, 5:335–337 B Babaco, in vitro culture, 7:178 Bacteria: diseases of fig, 12:447–451 ice nucleating, 7:210–212; 11:69–71 pathogens of bean, 3:28–58 tree short life, 2:46–47 wilt of bean, 3:46–47 Bacteriocides, fire blight, 1:450–459 Bacteriophage, fire blight control, 1:449–450

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CUMULATIVE SUBJECT INDEX Banana: CA and MA, 22:141–146 CA storage, 1:311–312 fertilization, 1:105 in vitro culture, 7:178–180 Banksia, 22:1–25 Bean: CA storage, 1:352–353 fluid drilling of seed, 3:21 resistance to bacterial pathogens, 3:28–58 Bedding plants, fertilization, 1:99–100; 5:337–341 Beet: CA storage, 1:353 fluid drilling of seed, 3:18–19 Begonia (Rieger), fertilization, 1:104 Biennial bearing. See Alternate bearing Biochemistry, petal senescence, 11:15–43 Bioreactor technology, 24:1–30 Bioregulation: See also Growth substances apple and pear, 10:309–401 Bird damage, 6:277–278 Bitter pit in apple, 11:289–355 Blackberry harvesting, 16:282–298 Black currant, bloom delay, 15:104 Bloom delay, deciduous fruits, 15:97 Blueberry: developmental physiology, 13:339–405 harvesting, 16:257–282 nutrition, 10:183–227 Boron: deficiency and toxicity symptoms in fruits and nuts, 2:151–152 foliar application, 6:328 nutrition, 5:327–328 pine bark media, 9:119–122 Botanic gardens, 15:1–62 Bramble, harvesting, 16:282–298 Branching, lateral: apple, 10:328–330 pear, 10:328–330 Brassicaceae, in vitro, 5:232–235 Breeding. See Genetics and breeding Broccoli, CA storage, 1:354–355 Brussels sprouts, CA storage, 1:355 Bulb crops: See also Tulip

325 development, 25:1–70 flowering, 25:1–70 genetics and breeding, 18:119–123 growth, 25: 1–70 in vitro, 18:87–169 micropropagation, 18:89–113 root physiology, 14:57–88 virus elimination, 18:113–123 C CA storage. See Controlled-atmosphere storage Cabbage: CA storage, 1:355–359 fertilization, 1:117–118 Cactus: crops, 18:291–320 reproductive biology, 18:321–346 Caladium. See Aroids, ornamental Calciole, nutrition, 10:183–227 Calcifuge, nutrition, 10:183–227 Calcium: bitter pit, 11:289–355 cell wall, 5:203–205 container growing, 9:84–85 deficiency and toxicity symptoms in fruits and nuts, 2:148–149 Ericaceae nutrition, 10:196–197 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 Carbohydrate: fig, 12:436–437 kiwifruit partitioning, 12:318–324 metabolism, 7:69–108 partitioning, 7:69–108 petal senescence, 11:19–20 reserves in deciduous fruit trees, 10:403–430 Carbon dioxide, enrichment, 7:345–398, 544–545 Carnation, fertilization, 1:100; 5:341–345 Carrot: CA storage, 1:362–366 fluid drilling of seed, 3:13–14

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326 Caryophyllaceae, in vitro, 5:237–239 Cassava, 12:158–166; 13:105–129; 26:85–159 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 Cell membrane: calcium, 10:126–140 petal senescence, 11:20–26 Cellular mechanisms, salt tolerance, 16:33–69 Cell wall: calcium, 10:109–122 hydrolases, 5:169–219 ice spread, 13:245–246 tomato, 13:70–71 Chelates, 9:169–171 Cherimoya, CA and MA, 22:146–147 Cherry: bloom delay, 15:105 CA storage, 1:308 origin, 19:263–317 Chestnut: blight, 8:281–336 in vitro culture, 9:311–312 Chicory, CA storage, 1:379 Chilling: injury, 4:260–261; 15:63–95 injury, chlorophyll fluorescence, 23:79–84 pistachio, 3:388–389 Chlorine: deficiency and toxicity symptoms in fruits and nuts, 2:153 nutrition, 5:239 Chlorophyll fluorescence, 23:69–107 Chlorosis, iron deficiency induced, 9:133–186 Chrysanthemum fertilization, 1:100–101; 5:345–352 Citrus: abscission, 15:145–182 alternate bearing, 4:141–144 asexual embryogenesis, 7:163–168 CA storage, 1:312–313 chlorosis, 9:166–168

CUMULATIVE SUBJECT INDEX cold hardiness, 7:201–238 fertilization, 1:105 flowering, 12:349–408 honey bee pollination, 9:247–248 in vitro culture, 7:161–170 juice loss, 20:200–201 navel orange, 8:129–179 nitrogen metabolism, 8:181 practices for young trees, 24:319–372 rootstock, 1:237–269 viroid dwarfing, 24:277–317 Clivia, 25:57 Cloche (tunnel), 7:356–357 Coconut palm: asexual embryogenesis, 7:184 in vitro culture, 7:183–185 Cold hardiness, 2:33–34 apple and pear bioregulation, 10:374–375 citrus, 7:201–238 factors affecting, 11:55–56 herbaceous plants, 6:373–417 injury, 2:26–27 nutrition, 3:144–171 pruning, 8:356–357 Colocasia, 8:45, 55–56, see also Aroids Common blight of bean, 3:45–46 Compositae, in vitro, 5:235–237 Container production, nursery crops, 9:75–101 Controlled-atmosphere (CA) storage: asparagus, 12:76–77, 127–130 chilling injury, 15:74–77 flowers, 3:98; 10:52–55 fruit quality, 8:101–127 fruits, 1:301–336; 4:259–260 pathogens, 3:412–461 seeds, 2:134–135 tropical fruit, 22:123–183 tulip, 5:105 vegetable quality, 8:101–127 vegetables, 1:337–394; 4:259–260 Controlled environment agriculture, 7:534–545, see also Greenhouse and greenhouse crops; hydroponic culture; protected culture Copper: deficiency and toxicity symptoms in fruits and nuts, 2:153 foliar application, 6:329–330

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CUMULATIVE SUBJECT INDEX nutrition, 5:326–327 pine bark media, 9:122–123 Corynebacterium flaccumfaciens, 3:33, 46 Cowpea: genetics, 2:317–348 U.S. production, 12:197–222 Cranberry: botany and horticulture, 21:215–249 fertilization, 1:106 harvesting, 16:298–311 Crinum, 25:58 Cryopreservation: apical meristems, 6:357–372 cold hardiness, 11:65–66 Cryphonectria parasitica. See Endothia parasitica Crytosperma, 8:47, 58, see also Aroids Cucumber, CA storage, 1:367–368 Cucurbita pepo, cultivar groups history, 25:71–170 Currant, harvesting, 16:311–327 Custard apple, CA and MA, 22:164 Cyrtanthus, 25:15–19 Cytokinin: cold hardiness, 11:65 dormancy, 7:272–273 floral promoter, 4:112–113 flowering, 15:294–295, 318 genetic regulation, 16:4–5, 14, 22–23 grape root, 5:150, 153–156 lettuce tipburn, 4:57–58 petal senescence, 11:30–31 rose senescence, 9:66 D Date palm: asexual embryogenesis, 7:185–187 in vitro culture, 7:185–187 Daylength. See Photoperiod Dedication: Bailey, L.H., 1:v–viii Beach, S.A., 1:v–viii Bukovac, M.J., 6:x–xii Campbell, C.W., 19:xiii Cummins, J.N., 15:xii–xv Dennis, F.G., 22:xi–xii De Hertogh, A.A., 26:xi–xii Faust, Miklos, 5:vi–x Hackett, W.P., 12:x–xiii

327 Halevy, A.H., 8:x–xii Hess, C.E., 13:x–xii Kader, A.A., 16:xii–xv Kamemoto, H., 24:x–xiii Looney, N.E., 18:xiii Magness, J.R., 2:vi–viii Moore, J.N., 14:xii–xv Pratt, C., 20:ix–xi Proebsting, Jr., E.L., 9:x–xiv Rick, Jr., C.M., 4:vi–ix Ryugo, K., 25:x–xii Sansavini, S., 17:xii–xiv Sherman, W.B., 21:xi–xiii Smock, R.M., 7:x–xiii Weiser, C.J., 11:x–xiii Whitaker, T.W., 3:vi–x Wittwer, S.H., 10:x–xiii Yang, S.F., 23:xi Deficit irrigation, 21:105–131 Deficiency symptoms, in fruit and nut crops, 2:145–154 Defoliation, apple and pear bioregulation, 10:326–328 ‘Delicious’ apple, 1:397–424 Desiccation tolerance, 18:171–213 Dieffenbachia. See Aroids, ornamental Dioscorea. See Yam Disease: and air pollution, 8:25 aroids, 8:67–69; 10:18; 12:168–169 bacterial, of bean, 3:28–58 cassava, 12:163–164 control by virus, 3:399–403 controlled-atmosphere storage, 3:412–461 cowpea, 12:210–213 fig, 12:447–479 flooding, 13:288–299 hydroponic crops, 7:530–534 lettuce, 2:187–197 mycorrhizal fungi, 3:182–185 ornamental aroids, 10:18 resistance, acquired, 18:247–289 root, 5:29–31 stress, 4:261–262 sweet potato, 12:173–175 tulip, 5:63, 92 turnip moasic virus, 14:199–238 waxes, 23:1–68 yam (Dioscorea), 12:181–183

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328 Disorder: see also Postharvest physiology: bitterpit, 11:289–355 fig, 12:477–479 pear fruit, 11:357–411 watercore, 6:189–251; 11:385–387 Dormancy, 2:27–30 blueberry, 13:362–370 release in fruit trees, 7:239–300 tulip, 5:93 Drip irrigation, 4:1–48 Drought resistance, 4:250–251 cassava, 13:114–115 Durian, CA and MA, 22:147–148 Dwarfing: apple, 3:315–375 apple mutants, 12:297–298 by virus, 3:404–405

CUMULATIVE SUBJECT INDEX apple bioregulation, 10:366–369 avocado, 10:239–241 bloom delay, 15:107–111 CA storage, 1:317–319, 348 chilling injury, 15:80 citrus abscission, 15:158–161, 168–176 cut flower storage, 10:44–46 dormancy, 7:277–279 flowering, 15:295–296, 319 flower longevity, 3:66–75 genetic regulation, 16:6–7, 14–15, 19–20 kiwifruit respiration, 6:47–48 mechanical stress, 17:16–17 petal senescence, 11:16–19, 27–30 rose senescence, 9:65–66 Eucharis, 25:19–22 Eucrosia, 25:58 F

E Easter lily, fertilization, 5:352–355 Embryogenesis. See Asexual embryogenesis Endothia parasitica, 8:291–336 Energy efficiency, in greenhouses, 1:141–171; 9:1–52 Environment: air pollution, 8:20–22 controlled for agriculture, 7:534–545 controlled for energy efficiency, 1:141–171; 9:1–52 embryogenesis, 1:22, 43–44 fruit set, 1:411–412 ginseng, 9:211–226 greenhouse management, 9:32–38 navel orange, 8:138–140 nutrient film technique, 5:13–26 Epipremnum. See Aroids, ornamental Eriobotrya japonica. See Loquat Erwinia: amylovora, 1:423–474 lathyri, 3:34 Essential elements: foliar nutrition, 6:287–355 pine bark media, 9:103–131 plant nutrition, 5:318–330 soil testing, 7:1–68 Ethylene: abscission, citrus, 15:158–161, 168–176

Feed crops, cactus, 18:298–300 Feijoa, CA and MA, 22:148 Fertilization and fertilizer: anthurium, 5:334–335 azalea, 5:335–337 bedding plants, 5:337–341 blueberry, 10:183–227 carnation, 5:341–345 chrysanthemum, 5:345–352 controlled release, 1:79–139; 5:347–348 Easter lily, 5:352–355 Ericaceae, 10:183–227 foliage plants, 5:367–380 foliar, 6:287–355 geranium, 5:355–357 greenhouse crops, 5:317–403 lettuce, 2:175 nitrogen, 2:401–404 orchid, 5:357–358 poinsettia, 5:358–360 rose, 5:361–363 snapdragon, 5:363–364 soil testing, 7:1–68 trickle irrigation, 4:28–31 tulip, 5:364–366 Vaccinium, 10:183–227 zinc nutrition, 23:109–128 Fig: industry, 12:409–490 ripening, 4:258–259

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CUMULATIVE SUBJECT INDEX Filbert, in vitro culture, 9:313–314 Fire blight, 1:423–474 Flooding, fruit crops, 13:257–313 Floral scents, 24:31–53 Floricultural crops: see also individual crops: Amaryllidaceae, 25:1–70 Banksia, 22:1–25 fertilization, 1:98–104 growth regulation, 7:399–481 heliconia, 14:1–55 Leucospermum, 22:27–90 postharvest physiology and senescence, 1:204–236; 3:59–143; 10:35–62; 11:15–43 Protea, 26:1–48 Florigen, 4:94–98 Flower and flowering: Amaryllidaceae, 25:1–70 apple anatomy and morphology, 10:277–283 apple bioregulation, 10:344–348 aroids, ornamental, 10:19–24 avocado, 8:257–289 Banksia, 22:1–25 blueberry development, 13:354–378 cactus, 18:325–335 citrus, 12:349–408 control, 4:159–160; 15:279–334 development (postpollination), 19:1–58 fig, 12:424–429 grape anatomy and morphology, 13:354–378 honey bee pollination, 9:239–243 induction, 4:174–203, 254–256 initiation, 4:152–153 in vitro, 4:106–127 kiwifruit, 6:21–35; 12:316–318 Leucospermum, 22:27–90 orchid, 5:297–300 pear bioregulation, 10:344–348 pecan, 8:217–255 perennial fruit crops, 12:223–264 phase change, 7:109–155 photoperiod, 4:66–105 pistachio, 3:378–387 postharvest physiology, 1:204–236; 3:59–143; 10:35–62; 11:15–43 postpollination development, 19:1–58 protea leaf blackening, 17:173–201 pruning, 8:359–362

329 raspberry, 11:187–188 regulation in floriculture, 7:416–424 rhododendron, 12:1–42 rose, 9:60–66 scents, 24:31–53 senescence, 1:204–236; 3:59–143; 10:35–62; 11:15–43; 18:1–85 sugars, 4:114 thin cell layer morphogenesis, 14:239–256 tulip, 5:57–59 water relations, 18:1–85 Fluid drilling, 3:1–58 Foliage plants: acclimatization, 6:119–154 fertilization, 1:102–103; 5:367–380 Foliar nutrition, 6:287–355 Freeze protection. See Frost protection Frost: apple fruit set, 1:407–408 citrus, 7:201–238 protection, 11:45–109 Fruit: abscission, 1:172–203 abscission, citrus, 15:145–182 apple anatomy and morphology, 10:283–297 apple bioregulation, 10:348–374 apple bitter pit, 11:289–355 apple flavor, 16:197–234 apple maturity indices, 13:407–432 apple ripening and quality, 10:361–374 apple weight loss, 25:197–234 avocado development and ripening, 10:229–271 bloom delay, 15:97–144 blueberry development, 13:378–390 cactus physiology, 18:335–341 CA storage and quality, 8:101–127 chilling injury, 15:63–95 coating physiology, 26:161–238 cracking, 19:217–262 diseases in CA storage, 3:412–461 drop, apple and pear, 10:359–361 fig, 12:424–429 growth measurement, 24:373–431 kiwifruit, 6:35–48; 12:316–318 loquat, 23:233–276 maturity indices, 13:407–432 navel orange, 8:129–179

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330 Fruit: (cont.) nectarine, postharvest, 11:413–452 nondestructive postharvest quality evaluation, 20:1–119 olive processing, 25:235–260 peach, postharvest, 11:413–452 pear, bioregulation, 10:348–374 pear, fruit disorders, 11:357–411 pear maturity indices, 13:407–432 pear ripening and quality, 10:361–374 pistachio, 3:382–391 plum, 23:179–231 quality and pruning, 8:365–367 ripening, 5:190–205 set, 1:397–424; 4:153–154 set in navel oranges, 8:140–142 size and thinning, 1:293–294; 4:161 softening, 5:109–219; 10:107–152 splitting, 19:217–262 strawberry growth and ripening, 17:267–297 texture, 20:121–224 thinning, apple and pear, 10:353–359 tomato parthenocarpy, 6:65–84 tomato ripening, 13:67–103 Fruit crops: alternate bearing, 4:128–173 apple bitter pit, 11:289–355 apple flavor, 16:197–234 apple fruit splitting and cracking, 19:217–262 apple growth, 11:229–287 apple maturity indices, 13:407–432 apricot, origin and dissemination, 22:225–266 avocado flowering, 8:257–289 avocado rootstocks, 17:381–429 berry crop harvesting, 16:255–382 bloom delay, 15:97–144 blueberry developmental physiology, 13:339–405 blueberry harvesting, 16:257–282 blueberry nutrition, 10:183–227 bramble harvesting, 16:282–298 cactus, 18:302–309 carbohydrate reserves, 10:403–430 CA and MA for tropicals, 22:123–183 CA storage, 1:301–336 CA storage diseases, 3:412–461 cherry origin, 19:263–317 chilling injury, 15:145–182 chlorosis, 9:161–165

CUMULATIVE SUBJECT INDEX citrus abscission, 15:145–182 citrus cold hardiness, 7:201–238 citrus, culture of young trees, 24:319–372 citrus dwarfing by viroids, 24:277–317 citrus flowering, 12:349–408 cranberry, 21:215–249 cranberry harvesting, 16:298–311 currant harvesting, 16:311–327 deficit irrigation, 21:105–131 dormancy release, 7:239–300 Ericaceae nutrition, 10:183–227 fertilization, 1:104–106 fig, industry, 12:409–490 fireblight, 11:423–474 flowering, 12:223–264 foliar nutrition, 6:287–355 frost control, 11:45–109 grape flower anatomy and morphology, 13:315–337 grape harvesting, 16:327–348 grape nitrogen metabolism, 14:407–452 grape purning, 16:235–254, 336–340 grape root, 5:127–168 grape seedlessness, 11:164–176 grapevine pruning, 16:235–254, 336–340 honey bee pollination, 9:244–250, 254–256 jojoba, 17:233–266 in vitro culture, 7:157–200; 9:273–349 irrigation, deficit, 21:105–131 kiwifruit, 6:1–64; 12:307–347 longan, 16:143–196 loquat, 23:233–276 lychee, 16:143–196 muscadine grape breeding, 14:357–405 navel orange, 8:129–179 nectarine postharvest, 11:413–452 nondestructive postharvest quality evaluation, 20:1–119 nutritional ranges, 2:143–164 olive salinity tolerance, 21:177–214 orange, navel, 8:129–179 orchard floor management, 9:377–430 peach origin, 17:331–379 peach postharvest, 11:413–452 pear fruit disorders, 11:357–411 pear maturity indices, 13:407–432 pecan flowering, 8:217–255 photosynthesis, 11:111–157 Phytophthora control, 17:299–330 plum origin, 23:179–231

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CUMULATIVE SUBJECT INDEX pruning, 8:339–380 rambutan, 16:143–196 raspberry, 11:185–228 roots, 2:453–457 sapindaceous fruits, 16:143–196 short life and replant problem, 2:1–116 strawberry fruit growth, 17:267–297 strawberry harvesting, 16:348–365 summer pruning, 9:351–375 Vaccinium nutrition, 10:183–227 water status, 7:301–344 Fungi: fig, 12:451–474 mushroom, 6:85–118 mycorrhiza, 3:172–213; 10:211–212 pathogens in postharvest storage, 3:412–461 truffle cultivation, 16:71–107 Fungicide, and apple fruit set, 1:416 G Galanthus, 25:22–25 Garlic, CA storage, 1:375 Genetic variation: alternate bearing, 4:146–150 photoperiodic response, 4:82 pollution injury, 8:16–19 temperature-photoperiod interaction, 17:73–123 Genetics and breeding: aroids (edible), 8:72–75; 12:169 aroids (ornamental), 10:18–25 bean, bacterial resistance, 3:28–58 bloom delay in fruits, 15:98–107 bulbs, flowering, 18:119–123 cassava, 12:164 chestnut blight resistance, 8:313–321 citrus cold hardiness, 7:221–223 cranberry, 21:236–239 embryogenesis, 1:23 fig, 12:432–433 fire blight resistance, 1:435–436 flowering, 15:287–290, 303–305, 306–309, 314–315 flower longevity, 1:208–209 ginseng, 9:197–198 in vitro techniques, 9:318–324; 18:119–123 lettuce, 2:185–187 loquat, 23:252–257 muscadine grapes, 14:357–405

331 mushroom, 6:100–111 navel orange, 8:150–156 nitrogen nutrition, 2:410–411 pineapple, 21:138–164 plant regeneration, 3:278–283 pollution insensitivity, 8:18–19 potato tuberization, 14:121–124 rhododendron, 12:54–59 sweet potato, 12:175 sweet sorghum, 21:87–90 tomato parthenocarpy, 6:69–70 tomato ripening, 13:77–98 tree short life, 2:66–70 Vigna, 2:311–394 waxes, 23:50–53 woody legume tissue and cell culture, 14:311–314 yam (Dioscorea), 12:183 Geophyte. See Bulb, tuber Geranium, fertilization, 5:355–357 Germination, seed, 2:117–141, 173–174; 24:229–275 Germplasm preservation: cryopreservation, 6:357–372 in vitro, 5:261–264; 9:324–325 pineapple, 21:164–168 Germplasm resources: pineapple, 21:133–175 Gibberellin: abscission, citrus, 15:166–167 bloom delay, 15:111–114 citrus, abscission, 15:166–167 cold hardiness, 11:63 dormancy, 7:270–271 floral promoter, 4:114 flowering, 15:219–293, 315–318 genetic regulation, 16:15 grape root, 5:150–151 mechanical stress, 17:19–20 Ginseng, 9:187–236 Girdling, 4:251–252 Glucosinolates, 19:99–215 Gourd, history, 25:71–171 Graft and grafting: incompatibility, 15:183–232 phase change, 7:136–137, 141–142 rose, 9:56–57 Grape: CA storage, 1:308 chlorosis, 9:165–166 flower anatomy and morphology, 13:315–337

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332 Grape: (cont.) harvesting, 16:327–348 muscadine breeding, 14:357–405 nitrogen metabolism, 14:407–452 pollen morphology, 13:331–332 pruning, 16:235–254, 336–340 root, 5:127–168 seedlessness, 11:159–187 sex determination, 13:329–331 Gravitropism, 15:233–278 Greenhouse and greenhouse crops: carbon dioxide, 7:357–360, 544–545 energy efficiency, 1:141–171; 9:1–52 growth substances, 7:399–481 nutrition and fertilization, 5:317–403 pest management, 13:1–66 vegetables, 21:1–39 Growth regulators. See Growth substances Growth substances, 2:60–66; 24:55–138, see also Abscisic acid, Auxin, Cytokinins, Ethylene, Gibberellins abscission, citrus, 15:157–176 apple bioregulation, 10:309–401 apple dwarfing, 3:315–375 apple fruit set, 1:417 apple thinning, 1:270–300 aroids, ornamental, 10:14–18 avocado fruit development, 10:229–243 bloom delay, 15:107–119 CA storage in vegetables, 1:346–348 cell cultures, 3:214–314 chilling injury, 15:77–83 citrus abscission, 15:157–176 cold hardiness, 7:223–225; 11:58–66 dormancy, 7:270–279 embryogenesis, 1:41–43; 2:277–281 floriculture, 7:399–481 flower induction, 4:190–195 flowering, 15:290–296 flower storage, 10:46–51 genetic regulation, 16:1–32 ginseng, 9:226 grape seedlessness, 11:177–180 hormone reception, 26:49–84 in vitro flowering, 4:112–115 mechanical stress, 17:16–21 meristem and shoot-tip culture, 5:221–227

CUMULATIVE SUBJECT INDEX navel oranges, 8:146–147 pear bioregulation, 10:309–401 petal senescence, 3:76–78 phase change, 7:137–138, 142–143 raspberry, 11:196–197 regulation, 11:1–14 rose, 9:53–73 seedlessness in grape, 11:177–180 triazole, 10:63–105 H Haemanthus, 25:25–28 Halo blight of beans, 3:44–45 Hardiness, 4:250–251 Harvest: flower stage, 1:211–212 index, 7:72–74 lettuce, 2:176–181 mechanical of berry crops, 16:255–382 Hazelnut. See Filbert Heat treatment (postharvest), 22:91–121 Heliconia, 14:1–55 Herbaceous plants, subzero stress, 6:373–417 Hippeastrum, 25:29–34 Histochemistry: flower induction, 4:177–179 fruit abscission, 1:172–203 Histology, flower induction, 4:179–184, see also Anatomy and morphology Honey bee, 9:237–272 Horseradish, CA storage, 1:368 Hydrolases, 5:169–219 Hydroponic culture, 5:1–44; 7:483–558 Hymenocallis, 25:59 Hypovirulence, in Endothia parasitica, 8:299–310 I Ismene, 25:59 Ice, formation and spread in tissues, 13:215–255 Ice-nucleating bacteria, 7:210–212; 13:230–235 Industrial crops, cactus, 18:309–312 Insects and mites: aroids, 8:65–66

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CUMULATIVE SUBJECT INDEX avocado pollination, 8:275–277 fig, 12:442–447 hydroponic crops, 7:530–534 integrated pest management, 13:1–66 lettuce, 2:197–198 ornamental aroids, 10:18 tree short life, 2:52 tulip, 5:63, 92 waxes, 23:1–68 Integrated pest management: greenhouse crops, 13:1–66 In vitro: abscission, 15:156–157 apple propagation, 10:325–326 aroids, ornamental, 10:13–14 artemisia, 19:342–345 bioreactor technology, 24:1–30 bulbs, flowering, 18:87–169 cassava propagation, 13:121–123; 26:99–119 cellular salinity tolerance, 16:33–69 cold acclimation, 6:382 cryopreservation, 6:357–372 embryogenesis, 1:1–78; 2:268–310; 7:157–200; 10:153–181 environmental control, 17:123–170 flowering bulbs, 18:87–169 flowering, 4:106–127 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 Iron: deficiency and toxicity symptoms in fruits and nuts, 2:150 deficiency chlorosis, 9:133–186 Ericaceae nutrition, 10:193–195 foliar application, 6:330 nutrition, 5:324–325 pine bark media, 9:123 Irrigation: deficit, deciduous orchards, 21:105–131 drip or trickle, 4:1–48 frost control, 11:76–82 fruit trees, 7:331–332

333 grape root growth, 5:140–141 lettuce industry, 2:175 navel orange, 8:161–162 root growth, 2:464–465 J Jojoba, 17:233–266 Juvenility, 4:111–112 pecan, 8:245–247 tulip, 5:62–63 woody plants, 7:109–155 K Kale, fluid drilling of seed, 3:21 Kiwifruit: botany, 6:1–64 vine growth, 12:307–347 L Lamps, for plant growth, 2:514–531 Lanzon, CA and MA, 22:149 Leaves: apple morphology, 12:283–288 flower induction, 4:188–189 Leek: CA storage, 1:375 fertilization, 1:118 Leguminosae, in vitro, 5:227–229; 14:265–332 Lemon, rootstock, 1:244–246, see also Citrus Lettuce: CA storage, 1:369–371 fertilization, 1:118 fluid drilling of seed, 3:14–17 industry, 2:164–207 seed germination, 24:229–275 tipburn, 4:49–65 Leucojum, 25:34–39 Leucospermum, 22:27–90 Light: fertilization, greenhouse crops, 5:330–331 flowering, 15:282–287, 310–312 fruit set, 1:412–413 lamps, 2:514–531 nitrogen nutrition, 2:406–407

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334 Light: (cont.) orchards, 2:208–267 ornamental aroids, 10:4–6 photoperiod, 4:66–105 photosynthesis, 11:117–121 plant growth, 2:491–537 tolerance, 18:215–246 Longan: See also Sapindaceous fruits CA and MA, 22:150 Loquat: botany and horticulture, 23:233–276 CA and MA, 22:149–150 Lychee: See also Sapindaceous fruits CA and MA, 22:150 Lycoris, 25:39–43 M Magnesium: container growing, 9:84–85 deficiency and toxicity symptoms in fruits and nuts, 2:148 Ericaceae nutrition, 10:196–198 foliar application, 6:331 nutrition, 5:323 pine bark media, 9:117–119 Magnetic resonance imaging, 20:78–86, 225–266 Male sterility, temperature-photoperiod induction, 17:103–106 Mandarin, rootstock, 1:250–252 Manganese: deficiency and toxicity symptoms in fruits and nuts, 2:150–151 Ericaceae nutrition, 10:189–193 foliar application, 6:331 nutrition, 5:235–326 pine bark media, 9:123–124 Mango: alternate bearing, 4:145–146 asexual embryogenesis, 7:171–173 CA and MA, 22:151–157 CA storage, 1:313 in vitro culture, 7:171–173 Mangosteen, CA and MA, 22:157 Mechanical harvest, berry crops, 16:255–382 Mechanical stress regulation, 17:1–42

CUMULATIVE SUBJECT INDEX Media: fertilization, greenhouse crops, 5:333 pine bark, 9:103–131 Medicinal crops: artemisia, 19:319–371 poppy, 19:373–408 Meristem culture, 5:221–277 Metabolism: flower, 1:219–223 nitrogen in citrus, 8:181–215 seed, 2:117–141 Micronutrients: container growing, 9:85–87 pine bark media, 9:119–124 Micropropagation, see also In vitro; propagation: bulbs, flowering, 18:89–113 environmental control, 17:125–172 nuts, 9:273–349 rose, 9:57–58 temperate fruits, 9:273–349 tropical fruits and palms, 7:157–200 Microtus. See Vole Modified atmosphere (MA) for tropical fruits, 22:123–183 Moisture, and seed storage, 2:125–132 Molecular biology: cassava, 26:85–159 hormone reception, 26:49–84 Molybdenum nutrition, 5:328–329 Monocot, in vitro, 5:253–257 Monstera. See Aroids, ornamental Morphology: navel orange, 8:132–133 orchid, 5:283–286 pecan flowering, 8:217–243 Moth bean, genetics, 2:373–374 Mung bean, genetics, 2:348–364 Mushroom: CA storage, 1:371–372 cultivation, 19:59–97 spawn, 6:85–118 Muskmelon, fertilization, 1:118–119 Mycoplasma-like organisms, tree short life, 2:50–51 Mycorrhizae: container growing, 9:93 Ericaceae, 10:211–212

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CUMULATIVE SUBJECT INDEX fungi, 3:172–213 grape root, 5:145–146 N Narcissus, 25:43–48 Navel orange, 8:129–179 Nectarine: bloom delay, 15:105–106 CA storage, 1:309–310 postharvest physiology, 11:413–452 Nematodes: aroids, 8:66 fig, 12:475–477 lettuce, 2:197–198 tree short life, 2:49–50 Nerine, 25:48–56 NFT (nutrient film technique), 5:1–44 Nitrogen: CA storage, 8:116–117 container growing, 9:80–82 deficiency and toxicity symptoms in fruits and nuts, 2:146 Ericaceae nutrition, 10:198–202 fixation in woody legumes, 14:322–323 foliar application, 6:332 in embryogenesis, 2:273–275 metabolism in apple, 4:204–246 metabolism in citrus, 8:181–215 metabolism in grapevine, 14:407–452 nutrition, 2:395, 423; 5:319–320 pine bark media, 9:108–112 trickle irrigation, 4:29–30 vegetable crops, 22:185–223 Nondestructive quality evaluation of fruits and vegetables, 20:1–119 Nursery crops: fertilization, 1:106–112 nutrition, 9:75–101 Nut crops: almond postharvest technology and utilization, 20:267–311 chestnut blight, 8:291–336 fertilization, 1:106 honey bee pollination, 9:250–251 in vitro culture, 9:273–349 nutritional ranges, 2:143–164 pistachio culture, 3:376–396

335 Nutrient: concentration in fruit and nut crops, 2:154–162 film technique, 5:1–44 foliar-applied, 6:287–355 media, for asexual embryogenesis, 2:273–281 media, for organogenesis, 3:214–314 plant and tissue analysis, 7:30–56 solutions, 7:524–530 uptake, in trickle irrigation, 4:30–31 Nutrition (human): aroids, 8:79–84 CA storage, 8:101–127 steroidal alkaloids, 25:171–196 Nutrition (plant): air pollution, 8:22–23, 26 blueberry, 10:183–227 calcifuge, 10:183–227 cold hardiness, 3:144–171 container nursery crops, 9:75–101 cranberry, 21:234–235 ecologically based, 24:156–172 embryogenesis, 1:40–41 Ericaceae, 10:183–227 fire blight, 1:438–441 foliar, 6:287–355 fruit and nut crops, 2:143–164 ginseng, 9:209–211 greenhouse crops, 5:317–403 kiwifruit, 12:325–332 mycorrhizal fungi, 3:185–191 navel orange, 8:162–166 nitrogen in apple, 4:204–246 nitrogen in vegetable crops, 22:185–223 nutrient film techniques, 5:18–21, 31–53 ornamental aroids, 10:7–14 pine bark media, 9:103–131 raspberry, 11:194–195 slow-release fertilizers, 1:79–139 O Oil palm: asexual embryogenesis, 7:187–188 in vitro culture, 7:187–188

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336 Okra: botany and horticulture, 21:41–72 CA storage, 1:372–373 Olive: alternate bearing, 4:140–141 salinity tolerance, 21:177–214 processing technology, 25:235–260 Onion: CA storage, 1:373–375 fluid drilling of seed, 3:17–18 Opium poppy, 19:373–408 Orange: see also Citrus alternate bearing, 4:143–144 sour, rootstock, 1:242–244 sweet, rootstock, 1:252–253 trifoliate, rootstock, 1:247–250 Orchard and orchard systems: floor management, 9:377–430 light, 2:208–267 root growth, 2:469–470 water, 7:301–344 Orchid: fertilization, 5:357–358 pollination regulation of flower development, 19:28–38 physiology, 5:279–315 Organogenesis, 3:214–314, see also In vitro; tissue culture Ornamental plants: Amaryllidaceae Banksia, 22:1–25 chlorosis, 9:168–169 fertilization, 1:98–104, 106–116 flowering bulb roots, 14:57–88 flowering bulbs in vitro, 18:87–169 foliage acclimatization, 6:119–154 heliconia, 14:1–55 Leucospermum, 22:27–90 orchid pollination regulation, 19:28–38 poppy, 19:373–408 protea leaf blackening, 17:173–201 rhododendron, 12:1–42 P Paclobutrazol. See Triazole Papaya: asexual embryogenesis, 7:176–177 CA and MA, 22:157–160 CA storage, 1:314 in vitro culture, 7:175–178

CUMULATIVE SUBJECT INDEX Parsley: CA storage, 1:375 drilling of seed, 3:13–14 Parsnip, fluid drilling of seed, 3:13–14 Parthenocarpy, tomato, 6:65–84 Passion fruit: in vitro culture, 7:180–181 CA and MA, 22:160–161 Pathogen elimination, in vitro, 5:257–261 Peach: bloom delay, 15:105–106 CA storage, 1:309–310 origin, 17:333–379 postharvest physiology, 11:413–452 short life, 2:4 summer pruning, 9:351–375 wooliness, 20:198–199 Peach palm (Pejibaye): in vitro culture, 7:187–188 Pear: bioregulation, 10:309–401 bloom delay, 15:106–107 CA storage, 1:306–308 decline, 2:11 fire blight control, 1:423–474 fruit disorders, 11:357–411 in vitro, 9:321 maturity indices, 13:407–432 root distribution, 2:456 short life, 2:6 Pecan: alternate bearing, 4:139–140 fertilization, 1:106 flowering, 8:217–255 in vitro culture, 9:314–315 Pejibaye, in vitro culture, 7:189 Pepper (Capsicum): CA storage, 1:375–376 fertilization, 1:119 fluid drilling in seed, 3:20 Persimmon: CA storage, 1:314 quality, 4:259 Pest control: aroids (edible), 12:168–169 aroids (ornamental), 10:18 cassava, 12:163–164 cowpea, 12:210–213 ecologically based, 24:172–201 fig, 12:442–477 fire blight, 1:423–474

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CUMULATIVE SUBJECT INDEX ginseng, 9:227–229 greenhouse management, 13:1–66 hydroponics, 7:530–534 sweet potato, 12:173–175 vertebrate, 6:253–285 yam (Dioscorea), 12:181–183 Petal senescence, 11:15–43 pH: container growing, 9:87–88 fertilization greenhouse crops, 5:332–333 pine bark media, 9:114–117 soil testing, 7:8–12, 19–23 Phase change, 7:109–155 Phenology: apple, 11:231–237 raspberry, 11:186–190 Philodendron. See Aroids, ornamental Phosphonates, Phytophthora control, 17:299–330 Phosphorus: container growing, 9:82–84 deficiency and toxicity symptoms in fruits and nuts, 2:146–147 nutrition, 5:320–321 pine bark media, 9:112–113 trickle irrigation, 4:30 Photoautotrophic micropropagation, 17:125–172 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 bitter pit, 11:289–355 blueberry development, 13:339–405 cactus reproductive biology, 18:321–346 calcium, 10:107–152 carbohydrate metabolism, 7:69–108 cassava, 13:105–129 citrus cold hardiness, 7:201–238 conditioning 13:131–181 cut flower, 1:204–236; 3:59–143; 10:35–62

337 desiccation tolerance, 18:171–213 disease resistance, 18:247–289 dormancy, 7:239–300 embryogenesis, 1:21–23; 2:268–310 floral scents, 24:31–53 flower development, 19:1–58 flowering, 4:106–127 fruit ripening, 13:67–103 fruit softening, 10:107–152 ginseng, 9:211–213 glucosinolates, 19:99–215 heliconia, 14:5–13 hormone reception, 26:49–84 juvenility, 7:109–155 lettuce seed germination, 24:229–275 light tolerance, 18:215–246 loquat, 23:242–252 male sterility, 17:103–106 mechanical stress, 17:1–42 nitrogen metabolism in grapevine, 14:407–452 nutritional quality and CA storage, 8:118–120 olive salinity tolerance, 21:177–214 orchid, 5:279–315 petal senescence, 11:15–43 photoperiodism, 17:73–123 pollution injury, 8:12–16 polyamines, 14:333–356 potato tuberization, 14:89–188 pruning, 8:339–380 raspberry, 11:190–199 regulation, 11:1–14 root pruning, 6:158–171 roots of flowering bulbs, 14:57–88 rose, 9:3–53 salinity hormone action, 16:1–32 salinity tolerance, 16:33–69 seed, 2:117–141 seed priming, 16:109–141 subzero stress, 6:373–417 summer pruning, 9:351–375 sweet potato, 23:277–338 thin cell layer morphogenesis, 14:239–264 tomato fruit ripening, 13:67–103 tomato parthenocarpy, 6:71–74 triazoles, 10:63–105; 24:55–138 tulip, 5:45–125 vernalization, 17:73–123 volatiles, 17:43–72

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338 Physiology (cont.) watercore, 6:189–251 water relations cut flowers, 18:1–85 waxes, 23:1–68 Phytohormones. See Growth substances Phytophthora control, 17:299–330 Phytotoxins, 2:53–56 Pigmentation: flower, 1:216–219 rose, 9:64–65 Pinching, by chemicals, 7:453–461 Pineapple: CA and MA, 22:161–162 CA storage, 1:314 genetic resources, 21:138–141 in vitro culture, 7:181–182 Pine bark, potting media, 9:103–131 Pistachio: alternate bearing, 4:137–139 culture, 3:376–393 in vitro culture, 9:315 Plantain: CA and MA, 22:141–146 in vitro culture, 7:178–180 Plant protection, short life, 2:79–84 Plum: CA storage, 1:309 origin, 23:179–231 Poinsettia, fertilization, 1:103–104; 5:358–360 Pollen, desiccation tolerance, 18:195 Pollination: apple, 1:402–404 avocado, 8:272–283 cactus, 18:331–335 embryogenesis, 1:21–22 fig, 12:426–429 floral scents, 24:31–53 flower regulation, 19:1–58 fruit crops, 12:223–264 fruit set, 4:153–154 ginseng, 9:201–202 grape, 13:331–332 heliconia, 14:13–15 honey bee, 9:237–272 kiwifruit, 6:32–35 navel orange, 8:145–146 orchid, 5:300–302 petal senescence, 11:33–35 protection, 7:463–464 rhododendron, 12:1–67

CUMULATIVE SUBJECT INDEX Pollution, 8:1–42 Polyamines, 14:333–356 chilling injury, 15:80 Polygalacturonase, 13:67–103 Postharvest physiology: almond, 20:267–311 apple bitter pit, 11:289–355 apple maturity indices, 13:407–432 apple weight loss, 25:197–234 aroids, 8:84–86 asparagus, 12:69–155 CA for tropical fruit, 22:123–183 CA storage and quality, 8:101–127 chlorophyll fluorescence, 23:69–107 coated fruits & vegetables, 26:161–238 cut flower, 1:204–236; 3:59–143; 10:35–62 foliage plants, 6:119–154 fruit, 1:301–336 fruit softening, 10:107–152 heat treatment, 22:91–121 lettuce, 2:181–185 low-temperature sweetening, 17:203–231 MA for tropical fruit, 22:123–183 navel orange, 8:166–172 nectarine, 11:413–452 nondestructive quality evaluation, 20:1–119 pathogens, 3:412–461 peach, 11:413–452 pear disorders, 11:357–411 pear maturity indices, 13:407–432 petal senescence, 11:15–43 protea leaf blackening, 17:173–201 quality evaluation, 20:1–119 seed, 2:117–141 texture in fresh fruit, 20:121–244 tomato fruit ripening, 13:67–103 vegetables, 1:337–394 watercore, 6:189–251; 11:385–387 Potassium: container growing, 9:84 deficiency and toxicity symptoms in fruits and nuts, 2:147–148 foliar application, 6:331–332 nutrition, 5:321–322 pine bark media, 9:113–114 trickle irrigation, 4:29 Potato: CA storage, 1:376–378

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CUMULATIVE SUBJECT INDEX fertilization, 1:120–121 low temperature sweetening, 17:203–231 tuberization, 14:89–198 Processing, table olives, 25:235–260 Propagation: see also In vitro apple, 10:324–326; 12:288–295 aroids, ornamental, 10:12–13 bioreactor technology, 24:1–30 cassava, 13:120–123 floricultural crops, 7:461–462 ginseng, 9:206–209 orchid, 5:291–297 pear, 10:324–326 rose, 9:54–58 tropical fruit, palms 7:157–200 woody legumes in vitro, 14:265–332 Protaceous flower crop: see also Protea Banksia, 22:1–25 Leucospermum, 22:27–90 Protea, leaf blackening, 17:173–201 Protected crops, carbon dioxide, 7:345–398 Protoplast culture, woody species, 10:173–201 Pruning, 4:161; 8:339–380 apple, 9:351–375 apple training, 1:414 chemical, 7:453–461 cold hardiness, 11:56 fire blight, 1:441–442 grapevines, 16:235–254 light interception, 2:250–251 peach, 9:351–375 phase change, 7:143–144 root, 6:155–188 Prunus: see 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 and vegetables, 20:1–119, 121–224

339 nondestructive, 20:1–119 texture in fresh fruit, 20:121–224 R Rabbit, 6:275–276 Radish, fertilization, 1:121 Rambutan. See Sapindaceous fruits Rambutan, CA and MA, 22:163 Raspberry: harvesting, 16:282–298 productivity, 11:185–228 Rejuvenation: rose, 9:59–60 woody plants, 7:109–155 Replant problem, deciduous fruit trees, 2:1–116 Respiration: asparagus postharvest, 12:72–77 fruit in CA storage, 1:315–316 kiwifruit, 6:47–48 vegetables in CA storage, 1:341–346 Rhizobium, 3:34, 41 Rhododendron, 12:1–67 Rice bean, genetics, 2:375–376 Root: apple, 12:269–272 cactus, 18:297–298 diseases, 5:29–31 environment, nutrient film technique, 5:13–26 Ericaceae, 10:202–209 grape, 5:127–168 kiwifruit, 12:310–313 physiology of bulbs, 14:57–88 pruning, 6:155–188 raspberry, 11:190 rose, 9:57 tree crops, 2:424–490 Root and tuber crops: Amaryllidaceae, 25:1–79 aroids, 8:43–99; 12:166–170 cassava, 12:158–166; 26:85–159 low-temperature sweetening, 17:203–231 minor crops, 12:184–188 potato tuberization, 14:89–188 sweet potato, 12:170–176 sweet potato physiology, 23: 277–338 yam (Dioscorea), 12:177–184

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340 Rootstocks: alternate bearing, 4:148 apple, 1:405–407; 12:295–297 avocado, 17:381–429 citrus, 1:237–269 cold hardiness, 11:57–58 fire blight, 1:432–435 light interception, 2:249–250 navel orange, 8:156–161 root systems, 2:471–474 stress, 4:253–254 tree short life, 2:70–75 Rosaceae, in vitro, 5:239–248 Rose: fertilization, 1:104; 5:361–363 growth substances, 9:3–53 in vitro, 5:244–248 S Salinity: air pollution, 8:25–26 olive, 21:177–214 soils, 4:22–27 tolerance, 16:33–69 Sapindaceous fruits, 16:143–196 Sapodilla, CA and MA, 22:164 Scadoxus, 25:25–28 Scoring, and fruit set, 1:416–417 Seed: abortion, 1:293–294 apple anatomy and morphology, 10:285–286 conditioning, 13:131–181 desiccation tolerance, 18:196–203 environmental influences on size and composition, 13:183–213 flower induction, 4:190–195 fluid drilling, 3:1–58 grape seedlessness, 11:159–184 kiwifruit, 6:48–50 lettuce, 2:166–174 lettuce germination, 24:229–275 priming, 16:109–141 rose propagation, 9:54–55 vegetable, 3:1–58 viability and storage, 2:117–141 Secondary metabolites, woody legumes, 14:314–322

CUMULATIVE SUBJECT INDEX 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 Sodium, deficiency and toxicity symptoms in fruits and nuts, 2:153–154 Soil: grape root growth, 5:141–144 management and root growth, 2:465–469 orchard floor management, 9:377–430 plant relations, trickle irrigation, 4:18–21 stress, 4:151–152 testing, 7:1–68; 9:88–90 zinc, 23:109–178 Soilless culture, 5:1–44 Solanaceae: in vitro, 5:229–232 steroidal alkaloids, 25:171–196 Somatic embryogenesis. See Asexual embryogenesis Sorghum, sweet, 21:73–104 Spathiphyllum. See Aroids, ornamental Squash, history, 25:71–170 Stem, apple morphology, 12:272–283 Sternbergia, 25:59 Steroidal alkaloids, solanaceous, 25:171–196 Storage: see also Postharvest physiology, Controlled-atmosphere (CA) storage cut flower, 3:96–100; 10:35–62 rose plants, 9:58–59 seed, 2:117–141

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CUMULATIVE SUBJECT INDEX Strawberry: fertilization, 1:106 fruit growth and ripening, 17:267–297 harvesting, 16:348–365 in vitro, 5:239–241 Stress: benefits of, 4:247–271 chlorophyll fluorescence, 23:69–107 climatic, 4:150–151 flooding, 13:257–313 mechanical, 17:1–42 petal, 11:32–33 plant, 2:34–37 protectants (triazoles), 24:55–138 protection, 7:463–466 salinity tolerance in olive, 21:177–214 subzero temperature, 6:373–417 waxes, 23:1–68 Sugar: see also Carbohydrate allocation, 7:74–94 flowering, 4:114 Sugar apple, CA and MA, 22:164 Sugar beet, fluid drilling of seed, 3:18–19 Sulfur: deficiency and toxicity symptoms in fruits and nuts, 2:154 nutrition, 5:323–324 Sweet potato: culture, 12:170–176 fertilization, 1:121 physiology, 23:277–338 Sweet sop, CA and MA, 22:164 Symptoms, deficiency and toxicity symptoms in fruits and nuts, 2:145–154 Syngonium. See Aroids, ornamental T Taro. See Aroids, edible Tea, botany and horticulture, 22:267–295 Temperature: apple fruit set, 1:408–411 bloom delay, 15:119–128 CA storage of vegetables, 1:340–341 chilling injury, 15:67–74 cryopreservation, 6:357–372 cut flower storage, 10:40–43

341 fertilization, greenhouse crops, 5:331–332 fire blight forecasting, 1:456–459 flowering, 15:284–287, 312–313 interaction with photoperiod, 4:80–81 low temperature sweetening, 17:203–231 navel orange, 8:142 nutrient film technique, 5:21–24 photoperiod interaction, 17:73–123 photosynthesis, 11:121–124 plant growth, 2:36–37 seed storage, 2:132–133 subzero stress, 6:373–417 Texture in fresh fruit, 20:121–224 Thinning, apple, 1:270–300 Tipburn, in lettuce, 4:49–65 Tissue: see also In vitro culture 1:1–78; 2:268–310; 3:214–314; 4:106–127; 5:221–277; 6:357–372; 7:157–200; 8:75–78; 9:273–349; 10:153–181, 24:1–30 cassava, 26:85–159 dwarfing, 3:347–348 nutrient analysis, 7:52–56; 9:90 Tomato: CA storage, 1:380–386 chilling injury, 20:199–200 fertilization, 1:121–123 fluid drilling of seed, 3:19–20 fruit ripening, 13:67–103 galacturonase, 13:67–103 greenhouse quality, 26:239 parthenocarpy, 6:65–84 Toxicity symptoms in fruit and nut crops, 2:145–154 Transport, cut flowers, 3:100–104 Tree decline, 2:1–116 Triazoles, 10:63–105; 24:55–138 chilling injury, 15:79–80 Trickle irrigation, 4:1–48 Truffle cultivation, 16:71–107 Tuber, potato, 14:89–188 Tuber and root crops. See Root and tuber crops Tulip: See also Bulb fertilization, 5:364–366 in vitro, 18:144–145 physiology, 5:45–125

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342 Tunnel (cloche), 7:356–357 Turfgrass, fertilization, 1:112–117 Turnip, fertilization, 1:123–124 Turnip Mosaic Virus, 14:199–238 U Urd bean, genetics, 2:364–373 Urea, foliar application, 6:332 V Vaccinium, 10:185–187, see also Blueberry; Cranberry Vase solutions, 3:82–95; 10:46–51 Vegetable crops: aroids, 8:43–99; 12:166–170 asparagus postharvest, 12:69–155 cactus, 18:300–302 cassava, 12:158–166; 13:105–129; 26:85–159 CA storage, 1:337–394 CA storage and quality, 8:101–127 CA storage diseases, 3:412–461 chilling injury, 15:63–95 coating physiology, 26:161–238 ecologically based, 24:139–228 fertilization, 1:117–124 fluid drilling of seeds, 3:1–58 gourd history, 25:71–170 greenhouse management, 21:1–39 greenhouse pest management, 13:1–66 honey bee pollination, 9:251–254 hydroponics, 7:483–558 lettuce seed germination, 24:229–275 low-temperature sweetening, 17:203–231 minor root and tubers, 12:184–188 mushroom cultivation, 19:59–97 mushroom spawn, 6:85–118 N nutrition, 22:185–223 nondestructive postharvest quality evaluation, 20:1–119 okra, 21:41–72 potato tuberization, 14:89–188 pumpkin history, 25:71–170 seed conditioing, 13:131–181 seed priming, 16:109–141 squash history, 25:71–170

CUMULATIVE SUBJECT INDEX steroidal alkaloids, Solanaceae, 25:171–196 sweet potato, 12:170–176 sweet potato physiology, 23:277–338 tomato fruit ripening, 13:67–103 tomato (greenhouse) quality: 26:239–319 tomato parthenocarpy, 6:65–84 tropical production, 24:139–228 truffle cultivation, 16:71–107 yam (Dioscorea), 12:177–184 Vegetative tissue, desiccation tolerance, 18:176–195 Vernalization, 4:117; 15:284–287; 17:73–123 Vertebrate pests, 6:253–285 Vigna: see also Cowpea genetics, 2:311–394 U.S. production, 12:197–222 Viroid, dwarfing for citrus, 24:277–317 Virus: benefits in horticulture, 3:394–411 dwarfing for citrus, 24:277–317 elimination, 7:157–200; 9:318; 18:113–123 fig, 12:474–475 tree short life, 2:50–51 turnip mosaic, 14:199–238 Volatiles, 17:43–72; 24:31–53 Vole, 6:254–274 W Walnut, in vitro culture, 9:312 Water relations: cut flower, 3:61–66; 18:1–85 deciduous orchards, 21:105–131 desiccation tolerance, 18:171–213 fertilization, greenhouse crops, 5:332 fruit trees, 7:301–344 kiwifruit, 12:332–339 light in orchards, 2:248–249 photosynthesis, 11:124–131 trickle irrigation, 4:1–48 Watercore, 6:189–251 pear, 11:385–387 Watermelon, fertilization, 1:124 Wax apple, CA and MA, 22:164 Waxes, 23:1–68 Weed control, ginseng, 9:228–229

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CUMULATIVE SUBJECT INDEX Weeds: lettuce research, 2:198 virus, 3:403 Woodchuck, 6:276–277 Woody species, somatic embryogenesis, 10:153–181

343 Y Yam (Dioscorea), 12:177–184 Yield: determinants, 7:70–74, 97–99 limiting factors, 15:413–452

X

Z

Xanthomonas phaseoli, 3:29–32, 41, 45–46 Xanthophyll cycle, 18:226–239 Xanthosoma, 8:45–46, 56–57, see also Aroids Sugar: see also Carbohydrate allocation, 7:74–94 flowering, 4:114

Zantedeschia. See Aroids, ornamental Zephyranthes, 25:60–61 Zinc: deficiency and toxicity symptoms in fruits and nuts, 2:151 foliar application, 6:332, 336 nutrition, 5:326; 23:109–178 pine bark media, 9:124

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Cumulative Contributor Index (Volumes 1–26)

Abbott, J. A., 20:1 Adams III, W. W., 18:215 Aldwinckle, H. S., 1:423; 15:xiii Amarante, C., 28:161 Anderson, I. C., 21:73 Anderson, J. L., 15:97 Anderson, P. C., 13:257 Andrews, P. K., 15:183 Ashworth, E. N., 13:215; 23:1 Asokan, M. P., 8:43 Atkinson, D., 2:424 Aung, L. H., 5:45 Bailey, W. G., 9:187 Baird, L. A. M., 1:172 Banks, N. H., 19:217; 25:197; 26:161 Barden, J. A., 9:351 Barker, A. V., 2:411 Bass, L. N., 2:117 Bassett, C. L., 26:49 Becker, J. S., 18:247 Beer, S. V., 1:423 Behboudian, M. H., 21:105 Bennett, A. B., 13:67 Benschop, M., 5:45 Ben-Ya’acov, A., 17:381 Benzioni, A., 17:233 Bevington, K. B., 24:277 Bewley, J. D., 18:171 Binzel, M. L., 16:33 Blanpied, G. D., 7:xi Bliss, F. A., 16:xiii Borochov, A., 11:15 Bower, J. P., 10:229

Bradley, G. A., 14:xiii Brennan, R., 16:255 Broadbent, P., 24:277 Broschat, T. K., 14:1 Brown, S., 15:xiii Buban, T., 4:174 Bukovac, M. J., 11:1 Burke, M. J., 11:xiii Buwalda, J. G., 12:307 Byers, R. E., 6:253 Caldas, L. S., 2:568 Campbell, L. E., 2:524 Cantliffe, D. J., 16:109; 17:43; 24:229 Carter, G., 20:121 Carter, J. V., 3:144 Cathey, H. M., 2:524 Chambers, R. J., 13:1 Charron, C. S., 17:43 Chen, Z., 25:171 Chin, C. K., 5:221 Clarke, N. D., 21:1 Coetzee, J. H., 26:1 Cohen, M., 3:394 Collier, G. F., 4:49 Collins, G., 25:235 Collins, W. L., 7:483 Colmagro, S., 25:235 Compton, M. E., 14:239 Conover, C. A., 5:317; 6:119 Coppens d’Eeckenbrugge, G., 21:133 Coyne, D. P., 3:28 Crane, J. C., 3:376 Criley, R. A., 14:1; 22:27; 24:x

Horticultural Reviews, Volume 26, Edited by Jules Janick ISBN 0-471-38789-4 © 2001 John Wiley & Sons, Inc. 345

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346 Crowly, W., 15:1 Cutting, J. G., 10:229 Daie, J., 7:69 Dale, A., 11:185; 16:255 Darnell, R. L., 13:339 Davenport, T. L., 8:257; 12:349 Davies, F. S., 8:129; 24:319 Davies, P. J., 15:335 Davis, T. D., 10:63; 24:55 DeEll, J. R., 23:69 DeGrandi-Hoffman, G., 9:237 De Hertogh, A. A., 5:45; 14:57; 18:87; 25:1 Deikman, J., 16:1 DellaPenna, D., 13:67 Demmig-Adams, B., 18:215 Dennis, F. G., Jr., 1:395 Dorais, M., 26:239 Doud, S. L., 2:1 Dudareva, N., 24:31 Duke, S. O., 15:371 Dunavent, M. G., 9:103 Duval, M. -F., 21:133 Düzyaman, E., 21:41 Dyer, W. E., 15:371 Early, J. D., 13:339 Elfving, D. C., 4:1; 11:229 El-Goorani, M. A., 3:412 Esan, E. B., 1:1 Evans, D. A., 3:214 Ewing, E. E., 14:89 Faust, M., 2:vii, 142; 4:174; 6:287; 14:333; 17:331; 19:263; 22:225; 23:179 Fenner, M., 13:183 Fenwick, G. R., 19:99 Ferguson, A. R., 6:1 Ferguson, I. B., 11:289 Ferguson, J. J., 24:277 Ferguson, L., 12:409 Ferree, D. C., 6:155 Ferreira, J. F. S., 19:319 Fery, R. L., 2:311; 12:157 Fischer, R. L., 13:67 Fletcher, R. A., 24:53 Flick, C. E., 3:214 Flore, J. A., 11:111 Forshey, C. G., 11:229

CUMULATIVE CONTRIBUTOR INDEX Fujiwara, K., 17:125 Geisler, D., 6:155 Geneve, R. L., 14:265 George, W. L., Jr., 6:25 Gerrath, J. M., 13:315 Gilley, A., 24:55 Giovannetti, G., 16:71 Giovannoni, J. J., 13:67 Glenn, G. M., 10:107 Goffinet, M. C., 20:ix Goldschmidt, E. E., 4:128 Goldy, R. G., 14:357 Goren, R., 15:145 Gosselin, A., 26:239 Goszczynska, D. M., 10:35 Grace, S. C., 18:215 Graves, C. J., 5:1 Gray, D., 3:1 Grierson, W., 4:247 Griffen, G. J., 8:291 Grodzinski, B., 7:345 Gucci, R., 21:177 Guest, D. I., 17:299 Guiltinan, M. J., 16:1 Hackett, W. P., 7:109 Halevy, A. H., 1:204; 3:59 Hallett, I. C., 20:121 Hammerschmidt, R., 18:247 Hanson, E. J., 16:255 Harker, F. R., 20:121 Heaney, R. K., 19:99 Heath, R. R., 17:43 Helzer, N. L., 13:1 Hendrix, J. W., 3:172 Henny, R. J., 10:1 Hergert, G. B., 16:255 Hess, F. D., 15:371 Heywood, V., 15:1 Hogue, E. J., 9:377 Holt, J. S., 15:371 Huber, D. J., 5:169 Hunter, E. L., 21:73 Hutchinson, J. F., 9:273 Hutton, R. J., 24:277 Indira, P., 23:277 Isenberg, F. M. R., 1:337 Iwakiri, B. T., 3:376

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CUMULATIVE CONTRIBUTOR INDEX Jackson, J. E., 2:208 Janick, J., 1:ix; 8:xi; 17:xiii; 19:319; 21:xi; 23:233 Jarvis, W. R., 21:1 Jenks, M. A., 23:1 Jensen, M. H., 7:483 Jeong, B. R., 17:125 Jewett, T. J., 21:1 Joiner, J. N., 5:317 Jones, H. G., 7:301 Jones, J. B., Jr., 7:1 Jones, R. B., 17:173 Kagan-Zur, V., 16:71 Kang, S. -M., 4:204 Kato, T., 8:181 Kawa, L., 14:57 Kawada, K., 4:247 Kelly, J. F., 10:ix; 22:xi Kester, D. E., 25:xii Khan, A. A., 13:131 Kierman, J., 3:172 Kim, K. -W., 18:87 Kinet, J. -M., 15:279 King, G. A., 11:413 Kingston, C. M., 13:407–432 Kliewer, W. M., 14:407 Knight, R. J., 19:xiii Knox, R. B., 12:1 Kofranek, A. M., 8:xi Korcak, R. F., 9:133; 10:183 Kozai, T., 17:125 Krezdorn, A. H., 1:vii Lakso, A. N., 7:301; 11:111 Lamb, R. C., 15:xiii Lang, G. A., 13:339 Larsen, R. P., 9:xi Larson, R. A., 7:399 Leal, F., 21:133 Ledbetter, C. A., 11:159 Li, P. H., 6:373 Lill, R. E., 11:413 Lin, S., 23:233 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

347 Lorenz, O. A., 1:79 Lu, R., 20:1 Lurie, S., 22:91–121 Lyrene, P., 21:xi Maguire, K. M., 25:197 Manivel, L., 22:267 Maraffa, S. B., 2:268 Marangoni, A. G., 17:203 Marini, R. P., 9:351 Marlow, G. C., 6:189 Maronek, D. M., 3:172 Martin, G. G., 13:339 Mayak, S., 1:204; 3:59 Maynard, D. N., 1:79 McConchie, R., 17:173 McNicol, R. J., 16:255 Merkle, S. A., 14:265 Michailides, T. J., 12:409 Michelson, E., 17:381 Mika, A., 8:339 Miller, A. R., 25:171 Miller, S. S., 10:309 Mills, H. A., 2:411; 9:103 Mills, T. M., 21:105 Mitchell, C. A., 17:1 Mizrahi, Y., 18:291, 321 Molnar, J. M., 9:1 Monk, G. J., 9:1 Monselise, S. P., 4:128 Moore, G. A., 7:157 Mor, Y., 9:53 Morris, J. R., 16:255 Murashige, T., 1:1 Murr, D. P., 23:69 Murray, S. H., 20:121 Myers, P. N., 17:1 Nadeau, J. A., 19:1 Nascimento, W. M., 24:229 Neilsen, G. H., 9:377 Nelson, P. V., 26:xi Nerd, A., 18:291, 321 Niemiera, A. X., 9:75 Nobel, P. S., 18:291 Nyujtò, F., 22:225 O’Donoghue, E. M., 11:413 Ogden, R. J., 9:103

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348 O’Hair, S. K., 8:43; 12:157 Oliveira, C. M., 10:403 Oliver, M. J., 18:171 O’Neill, S. D., 19:1 Opara, L. U., 19:217; 24:373; 25:197 Ormrod, D. P., 8:1 Palser, B. F., 12:1 Papadopoulos, A. P., 21:1; 26:239 Pararajasingham, S., 21:1 Parera, C. A., 16:109 Paris, H. S., 25:71 Pegg, K. G., 17:299 Pellett, H. M., 3:144 Perkins-Veazil, P., 17:267 Pichersky, E., 24:31 Piechulla, B., 24:31 Ploetz, R. C., 13:257 Pokorny, F. A., 9:103 Poole, R. T., 5:317; 6:119 Poovaiah, B. W., 10:107 Portas, C. A. M., 19:99 Porter, M. A., 7:345 Possingham, J. V., 16:235 Prange, R. K., 23:69 Pratt, C., 10:273; 12:265 Preece, J. E., 14:265 Priestley, C. A., 10:403 Proctor, J. T. A., 9:187 Puonti-Kaerlas, J., 26:85 Quamme, H., 18:xiii Raese, J. T., 11:357 Ramming, D. W., 11:159 Ravi, V., 23:277 Reddy, A. S. N., 10:107 Redgwell, R. J., 20:121 Reid, M., 12:xiii; 17:123 Reuveni, M., 16:33 Richards, D., 5:127 Rieger, M., 11:45 Roper, T. R., 21:215 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

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CUMULATIVE CONTRIBUTOR INDEX Rudnicki, R. M., 10:35 Ryder, E. J., 2:164; 3:vii Sachs, R., 12:xiii Sakai, A., 6:357 Salisbury, F. B., 4:66; 15:233 Saltveit, M. E., 23:x San Antonio, J. P., 6:85 Sankhla, N., 10:63; 24:55 Saure, M. C., 7:239 Schaffer, B., 13:257 Schenk, M. K., 22:185 Schneider, G. W., 3:315 Schuster, M. L., 3:28 Scorza, R., 4:106 Scott, J. W., 6:25 Sedgley, M., 12:223; 22:1; 25:235 Seeley, S. S., 15:97 Serrano Marquez, C., 15:183 Sharp, W. R., 2:268; 3:214 Sharpe, R. H., 23:233 Shattuck, V. I., 14:199 Shear, C. B., 2:142 Sheehan, T. J., 5:279 Shipp, J. L., 21:1 Shirra, M., 20:267 Shorey, H. H., 12:409 Simon, J. E., 19:319 Sklensky, D. E., 15:335 Smith, G. S., 12:307 Smock, R. M., 1:301 Sommer, N. F., 3:412 Sondahl, M. R., 2:268 Sopp, P. I., 13:1 Soule, J., 4:247 Sparks, D., 8:217 Splittstoesser, W. E., 6:25; 13:105 Srinivasan, C., 7:157 Stang, E. J., 16:255 Steffens, G. L., 10:63 Stevens, M. A., 4:vii Stroshine, R. L., 20:1 Struik, P. C., 14:89 Studman, C. J., 19:217 Stutte, G. W., 13:339 Styer, D. J., 5:221 Sunderland, K. D., 13:1 Sung, Y., 24:229

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CUMULATIVE CONTRIBUTOR INDEX Surányi, D., 19:263; 22:225; 23:179 Swanson, B., 12:xiii Swietlik, D., 6:287; 23:109 Syvertsen, J. P., 7:301 Tattini, M., 21:177 Tétényi, P., 19:373 Theron, K. I., 25:1 Tibbitts, T. W., 4:49 Timon, B., 17:331 Tindall, H. D., 16:143 Tisserat, B., 1:1 Titus, J. S., 4:204 Trigiano, R. N., 14:265 Tunya, G. O., 13:105

349 Wann, S. R., 10:153 Watkins, C. B., 11:289 Watson, G. W., 15:1 Webster, B. D., 1:172; 13:xi Weichmann, J., 8:101 Wetzstein, H. Y., 8:217 Whiley, A. W., 17:299 Whitaker, T. W., 2:164 White, J. W., 1:141 Williams, E. G., 12:1 Williams, M. W., 1:270 Wismer, W. V., 17:203 Wittwer, S. H., 6:xi Woodson, W. R., 11:15 Wright, R. D., 9:75 Wutscher, H. K., 1:237

Upchurch, B. L., 20:1 Valenzuela, H. R., 24:139 van Doorn, W. G., 17:173; 18:1 van Kooten, O., 23:69 Veilleux, R. E., 14:239 Vorsa, N., 21:215

Yada, R. Y., 17:203 Yadava, U. L., 2:1 Yahia, E. M., 16:197; 22:123 Yan, W., 17:73 Yarborough, D. E., 16:255 Yelenosky, G., 7:201

Wallace, A., 15:413 Wallace, D. H., 17:73 Wallace, G. A., 15:413 Wang, C. Y., 15:63 Wang, S. Y., 14:333

Zanini, E., 16:71 Zieslin, N., 9:53 Zimmerman, R. H., 5:vii; 9:273 Ziv, M., 24:1 Zucconi, F., 11:1