Volume 23, Horticultural Reviews

HORTICULTURAL REVIEWS

Volume 23

Horticultural Reviews is sponsored by: American Society for Horticultural Science

Editorial Board, Volume 23 Leo Gene Albrigo Richard L. Fery Paul K. Hasegawa

HORTICULTURAL REVIEWS Volume 23

edited by

Jules Janick Purdue University

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

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Copyright © 1999 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 Cataloging-in-Publication Data: ISBN 0-471-25445-2 ISSN 0163-7851

10987654

Contents List of Contributors

ix

Dedication

xi

1. Plant Epicuticular Waxes: Function, Production,

and Genetics

1

Matthew A. Jenks and Edward N. Ashworth

I. II. III. IV. V. VI.

Introduction Nature of Epicuticular Waxes Role of Epicuticular Waxes in Responses to Biotic and Abiotic Stresses Production of Plant Epicuticular Waxes Genetics of Epicuticular Waxes Summation Literature Cited

2. Applications of Chlorophyll Fluorescence Techniques

in Postharvest Physiology

2 3

10 31 50 53 54

69

Jennifer R. DeEll, Olaf van Kooten, Robert K. Prange, and Dennis P. Murr

I. II. III. IV.

Introduction Chlorophyll Fluorescence Measurements Applications of Chlorophyll Fluorescence Concluding Remarks Literature Cited

3. Zinc Nutrition in Horticultural Crops

70 73 79 99

101 109

Dariusz Swietlik

I. II.

Introduction Zn in Soils

110 114 v

vi

CONTENTS

III. Factors Affecting Zn Availability IV. Function, Absorption, and Transport of Zn in Plants V. Zn Deficiency and Toxicity Symptoms VI. Effects of Zn Applications on Plants VII. Technology of Zn Applications VIII. Zn Fertilizers IX.

Conclusions Literature Cited

4. Origin and Dissemination of Plums

124 127 133 136 157 161 162 164

179

Miklos Faust and Dezso Suranyi

I. II.

III. IV.

Introduction Classification History Conclusions Literature Cited

5. Loquat: Botany and Horticulture Shunquan Lin, Ralph H. Sharpe, and Jules Janick

I. Introduction II. Botany III. Physiology IV. Horticulture V. Future Prospects Literature Cited

6. Crop Physiology of Sweetpotato V. Ravi and P. Indira

I. II.

III. IV. V. VI. VII.

Introduction Shoot System Root System Source and Sink Relationship Dry Matter Production and Harvest Index Shoot Removal and Storage Root Yields Response to Growth Regulators and Chemicals

179 184 202 224 225 233

234 237 242 252 266 269

277 278 280 289 298 300 301 302

vii

CONTENTS

VIII. IX. X.

Response to Stress Propagation Physiology Conclusion Literature Cited

303 309

314 316

Subject Index

339

Cumulative Subject Index

340

Cumulative Contributor Index

362

Contributors Edward N. Ashworth, Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907-1165 Jennifer R. DeEll, Agriculture and Agri-Food Canada, Horticultural Research and Development Centre, 430 Boulevard Gouin, Saint-Jean-sur-Richileiu, QC, J3B 2E6 Canada Miklos Faust, Fruit Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, Beltsville, MD 20705 P. Indira, Central Tuber Crops Research Institute, Sreekariyam, Trivandrum, 695 017 India Jules Janick, Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907-1165 Matthew A. Jenks, Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907-1165 Shunquan Lin, Institute of Subtropical Fruits, Fujian Agriculture University, Fuzhou, 350002 China Dennis P. Murr, Department of Horticultural Science, University of Guelph, ON, N1G 2W1 Canada Robert K. Prange, Agriculture and Agri-Food Canada, Atlantic Food and Horticulture Research Centre, 32 Main Street, Kentville, NS, B4N 1J5 Canada V. Ravi, Central Tuber Crops Research Institute, Sreekariyam, Trivandrum, 695 017 India Ralph H. Sharpe, Horticulture Sciences Department, University of Florida, Gainesville, FL 32611 Dezso Suranyi, Fruit Research Station, Cegled, Hungary Dariusz Swietlik, Texas A & M University-Kingsville, Citrus Center, P.O. Box 1150, Weslaco, TX 78599-1150 Olafvan Kooten, Agrotechnological Research Institute (ATO-DLOl, P.O. Box 17, 6700 AA Wageningen, The Netherlands

ix

Shang Fa Yang

Dedication: Shang Fa Yang This volume is dedicated to Dr. Shang Fa Yang, an outstanding scientist, postharvest physiologist, and teacher who was pivotal in elucidating the biosynthetic pathway of the plant hormone ethylene. Shang Fa was born in 1932 in Taiwan, where he received his B.S. and M.S. degrees in Agricultural Chemistry from the National University in the late 1950s. His M.S. thesis on banana fruit ripening shows his early interest in postharvest physiology. He received a scholarship to do graduate work at Utah State University and received his Ph.D. there in 1962, followed by postdoctoral studies in fat metabolism in higher plants with Dr. Paul K. Stumpf at the University of California, Davis (UCD). He was eager to see the East Coast and with Dr. Stumpf's help obtained a fellowship to the New York University (NYU) Medical School. His studies there on liver enzymes did not pique his interest as did his studies in plant biochemistry, but his stay on the East Coast was very rewarding personally since that is where he met his wife, Eleanor, a student in accounting at NYU. He returned to California the next year to do another postdoctoral with Dr. Andrew A. Benson at Scripps Institute ofOceanography in La Jolla, California, where he met Dr. Jacob B. Biale, a leading figure in postharvest studies, who was there on a sabbatical. Their interactions rekindled Shang Fa's interest in postharvest research. Dr. Yang returned to UCD in 1966 as a postharvest biochemist in the Department of Vegetable Crops. Shang Fa initially shared a lab with Dr. H. K. Pratt, an early researcher in ethylene physiology, in the newly constructed Mann Laboratory. Although space was limited and researchers were crowded, he published the first of his over 200 refereed papers on postharvest physiology and ethylene biosynthesis, the very same year he arrived at UCD. Years later, he would comment that an abundance of ideas and hard work was more important that square feet of bench space. As a new faculty member at UCD, Shang Fa queried the older faculty for their thoughts on what postharvest topics needed to be studied. Dr. H. K. Pratt had been involved in studying ethylene physiology for a number of years and had cobbled together one of the first gas chromatographs on the West Coast that could easily measure the hormonal xi

xii

DEDICATION: SHANG FA YANG.

levels of ethylene, and made a very persuasive case that the biochemistry of ethylene synthesis would be a rewarding field of study. Ethylene, one of the traditional five plant hormones, has great agricultural value, as well as scientific importance. Since 1934, when ethylene was conclusively shown to be produced by ripening fruit, a great deal of effort had been expended to discover its biosynthetic pathway. The modern search for the metabolic pathway began in 1965 when M. Lieberman and L. W. Mapson observed that methionine was converted to ethylene in a model system, and in 1966 their research group confirmed the biological production of ethylene from methionine. Shang Fa's first paper on ethylene in 1966 explored the intricacies of this model system. With the discovery of methionine's involvement in ethylene biosynthesis, Shang Fa and many other scientists in the United States, Europe, and Asia entered the quest to identify the subsequent steps in the pathway. In 1977, Shang Fa and a Ph.D. student, Doug Adams, demonstrated that methionine was converted to S-adenosylmethionine (SAM) and that SAM was a precursor of ethylene. Methionine pools are too low in plant tissue to sustain the observed rates of ethylene synthesis. The recycling of the methylthio group, released from SAM during the synthesis of ACC, to maintain methionine levels was christened the Yang cycle in F. B. Abeles, P. W. Morgan, and M. E. Saltveit's 1992 book on ethylene in plant biology. The pace quickened and a real race ensued to identify the final step. Years ofintensive effort culminated in 1979 when Adams and Yang identified the final precursor of ethylene as 1-aminocyclopropane-1-carboxylic acid (ACC). Surprisingly, ACC had been known for a number of years as a non-proteinacious amino acid of unknown function and was available in crystalline form for around $3/100 g from a number of chemical supply houses. Shang Fa immediately recognized the importance of their discovery and convinced everyone in his lab to set aside their ongoing studies and participate in the expanding inquiry into the regulation and control of the synthesis of ACC and its conversion to ethylene. At lab meetings he kept asking, "How can I help your work?" and "What can I do to make your work more efficient?" He was always thinking of new experiments to try to follow up on the ACC story and was a constant source of inspiration to his colleagues. Although fellow researchers in ethylene biosynthesis quickly realized the importance of this discovery, it took a few years before other plant biochemists and physiologists recognized what this discovery would portend. After a few years, interest in ACC reached such intensity that Sigma almost ran out of ACC, and it was rationed for a time.

DEDICATION: SHANG FA YANG

xiii

Shang Fa was able to succeed where many others had failed because he has an intensity of focus, and an amazing understanding of organic reaction mechanisms and ethylene biochemistry. He had an uncanny ability to keep everyone's research project in mind, even though there may have been 10 or 12 people working in his lab. This, coupled with his affable nature and genuine concern for his students and colleagues, allowed him to assemble a powerful and effective research group that shared his vision and strove to match his intensity. He also has an uncommon faith in humanity, extolling students to always expect the best of people. He is humble and has always been willing to share credit. Although intense, he has the rare gift of always being able to maintain an open mind. While diligently developing a series of experiments to test one hypothesis, he has always been able to step back and consider alternative interpretations of the data. This allowed him to abandon his favorite ideas when they proved untenable, and to incorporate the newest discoveries into his developing research paradigm. Even though his research was very basic, Shang Fa has never lost sight of the practical side of his work or the importance it would have on postharvest biology. He rarely ever missed participating in the weekly seminar held by the UCD Postharvest Group and contributed penetrating questions about the harvesting, handling, and marketing of horticultural crops. He assisted many faculty and colleagues with their practical studies on ethylene. He has continually enlarged his panoply of research tools to encompass all the new technologies of molecular biology and genetic engineering. Shang Fa has figured prominently at many national and international research conferences over the years and has served on the editorial board of leading journals and is a member of many learned societies. He has won many awards and honors, including the Campbell Award of the American Institute of Biological Science in 1969, a Guggenheim Fellowship in 1982, the International Plant Growth Substances Association Research Award in 1985, the Outstanding Researcher Award from the American Society of Horticultural Science in 1992, and the UCD Faculty Research Lecturer in 1992. In 1990 he was elected to the National Academy of Science, USA and received the prestigious Wolf Prize in Agriculture in 1991. In 1992 he was elected to the Academia Sinica, Taipei. Dr. Yang retired from UCD in 1994 and until recently served as Professor in the Department of Biology at the Hong Kong University of Science and Technology and as Distinguished Research Fellow and Director of the Institute of Botany at Academia Sinica, Taipei. Since 1996 he has been Vice President of the Academia Sinica.

xiv

DEDICATION: SHANG FA YANG

Shang Fa and his wife Eleanor have raised two fine sons, Albert and Bryan, and have established an extensive network of friends throughout the worldwide community of scholars and scientists. Shang Fa Yang has touched many lives and contributed to our understanding of the natural world. His legacy is an inspiration to us all. Mikal E. Saltveit Department of Vegetable Crops University of California, Davis

1 Plant Epicuticular Waxes: Function, Production, and Genetics Matthew A. Jenks and Edward N. Ashworth Department of Horticulture and Landscape Architecture, Purdue University West Lafayette, Indiana 47907-1165 1. Introduction II. Nature of Epicuticular Waxes III. Role of Epicuticular Waxes in Responses to Biotic and Abiotic Stresses A. Epicuticular Waxes and Fungal Pathogens 1. The Effect of Wax Structure and Chemistry on Plant Pathogens 2. Leaf Surface Wettability and Plant Fungal-Susceptibility 3. Leaf Surface Permeability and Plant-Fungal Susceptibility B. Epicuticular Waxes and Phytophagous Insects C. Epicuticular Waxes and Drought 1. Effect of Waxes on Plant Water Loss 2. Wax Reflectance of Solar Radiation and Plant Water Loss 3. Wax and the Boundary Layer Above Plant Surfaces D. Epicuticular Waxes and Freezing Temperatures E. Epicuticular Waxes and Solar Radiation F. Epicuticular Waxes and Agricultural Sprays 1. Epicuticular Waxes and Surface Retention 2. Epicuticular Waxes and Surface Penetration 3. Effect of Epicuticular Waxes on the Sorption of Agricultural Chemicals G. Epicuticular Waxes and Air Pollutants IV. Production of Plant Epicuticular Waxes A. Epicuticular Wax Biosynthetic Pathways 1. Elongases 2. Thioesterases 3. Reductases and Decarbonylases 4. Oxidases and Transacylases 5. Potential Novel Functions in Epicuticular Wax Biosynthesis B. Epicuticular Wax Secretion 1. Early Studies of Epicuticular Wax Secretion 2. Cellular Origins for Epicuticular Wax Secretion

Horticultural Reviews, Volume 23, Edited by Jules Janick ISBN 0-471-25445-2 © 1999 John Wiley & Sons. Inc. 1

2

M. JENKS AND E. ASHWORTH

3. Transport of Epicuticular Wax Precursors through the Cytoplasm and Plasmalemma 4. Transport of Epicuticular Wax Precursors through the Cell Wall and the Cuticle Proper 5. Cuticular Involvement in Shaping Wax Morphology 6. Crystallization of Epicuticular Wax on Plant Surfaces V. Genetics of Plant Epicuticular Waxes A. Genetic Involvement in Epicuticular Wax Diversity B. Cloning Epicuticular Wax Genes VI. Summation Literature Cited

I. INTRODUCTION

Essentially all aerial plant surfaces are covered by epicuticular waxes that form an important interface between a plant and its environment. These epicuticular waxes have diverse crystallization patterns, chemical compositions, and relative abundance that change with plant age, development, and environment. The physical and chemical properties of these surface waxes play an important role in plant resistance to a variety of biotic and abiotic stresses, including those caused by fungal pathogens, phytophagous insects, drought, solar radiation, freezing temperatures, mechanical abrasion, and anthropogenic influences such as acid rain and ozone. In addition, epicuticular waxes also influence the uptake and efficiency of plant growth regulators, pesticides, and herbicides. In addition to their ecological importance, plant epicuticular waxes also have significant industrial value. For example, plant epicuticular wax extracts are used in a variety of industrial products such as polishing agents, candles, cosmetics, protective coatings, lubricants, and medicinals. Carnauba wax extracted from the leaves of tree of life (Copernica cerifera Mart.) is a familiar industrial plant wax. Others include bayberry wax from Myrica species, candelilla wax from Euphorbia species, reed wax from Esparto grass (Stipa tenacissima L.), fir wax from Douglas fir (Pseudotsuga menziesii (Mirb.) Franco), and cane wax from sugar cane (Saccharum officinarum L.). Comprehensive information on industrial plant waxes is available in a review by Bennett (1975). Epicuticular waxes often contribute to the esthetic value of many ornamental plants. For example, epicuticular wax crystals on needles of Colorado blue spruce (Picea pungens Engelm. var. glauca) give the plant an attractive glaucous whitish-blue coloration. By comparison, noncrystalline epicuticular waxes that form smooth layers over leaf surfaces of Japanese cleyera (Ternstroemina gymnanthera (Wright & Arn.) T. Sprague) create an attractive glossy appearance.

1. PLANT EPICUTICULAR WAXES: FUNCTION, PRODUCTION, AND GENETICS

3

Clearly, epicuticular waxes have significant ecological, industrial, and aesthetic values that justify continued research into their function, production, and genetics. The purpose of this review is to provide an overview ofthe biology of epicuticular waxes found on important horticultural and agronomic plants. One important objective is to identify topics and research areas where additional study on epicuticular waxes may assist efforts in crop improvement. This review includes an overview of the nature of epicuticular waxes, a discussion of the role these waxes play in plant resistance to stress, current knowledge of wax biosynthetic and secretory mechanisms, a discussion of genes that influence production, and potential avenues for the application of genetic engineering to alter epicuticular waxes. In addition to this review, readers are referred to several useful reviews and books on epicuticular waxes (Cutler et a1. 1982; Walton 1990; Hamilton 1995; WettsteinKnowles 1995; Lemieux 1996; Post-Beittenmiller 19~6; Kerstiens 1996). II. NATURE OF EPICUTICULAR WAXES

Epicuticular waxes on most plant surfaces are deposited as a smooth, transparent layer. However, surface waxes on many plants crystallize into structures that are visible as a whitish-bluish colored coating. Plants hav~ ing this feature are said to be glaucous or to have a wax bloom. When glaucous surfaces are examined at higher magnifications using scanning electron microscopy (SEM), a myriad of unique epicuticularwax crystals specific to various species and their organs have been observed. Such structures include tubular filaments, dendritic structures, plate-like structures, flat plates, umbrella-shaped crystals, cylinders, irregular globs, and many others~ Two representative examples are presented in Fig. 1.1 and 1.2 showing crystalline morphologies of cabbage (Brassica oleracea L.) adaxial leaf surfaces, and sorghum (Sorghum bicolor L.) abaxial sheath surfaces, along with respective genetically similar mutants. Plant epicuticular waxes are composed of complex mixtures of hydrophobic compounds. These typically include long-chained hydrocarbons, ketones, esters, aliphatic alcohols, fatty acids and aliphatic aldehydes. Other lipoidal compounds, such as branched-chain hydrocarbons, terpenoids, and aromatic compounds are also found within epicuticular waxes (Walton 1990). The chemical composition of waxes can differ markedly among plant species (Bianchi and Bianchi 1990) and with ecotypes of the same species (Rashotte et a1. 1997). The structure and chemistry of epicuticular waxes are not static, but instead change during plant development. For example, young expand-

4

M. JENKS AND E. ASHWORTH

Fig. 1.1. Scanning electron micrographs of epicuticular wax crystals on adaxial leaf blade surfaces of cabbage. A. Normal 'Round-up'. The glossy mutants (B-H). B. BrocS. C. PI261S97. D. Broc3. E. 'Glossy Andes'. F. 'Glazed Vates'. G. PI234599. H. 'Green Glaze'. Bar = 10 JAm. Source: Eigenbrode et a!. 1991.

1. PLANT EPICUTICULAR WAXES: FUNCTION. PRODUCTION, AND GENETICS

5

Fig. 1.2. Scanning electron micrographs of epicuticular wax crystallization patterns on abaxial leaf sheath surfaces of sorghum. A. wild-type P954035. The sparse-bloom (h) and bloomless (bm) sorghum epicuticular wax mutants. B. h13·1. C. h2O·1. D. bm4-2. E. bm11-1. F. bm2·4. Bar 10 J..Lm. Source: Jenks et a1. 1992.

=

ing leaves of both monocots and dicots have much less epicuticular wax per unit leaf area than older leaves, and the amount of waxes generally decreases during leaf senescence (Rich 1994; Jenks et al. 1996b). The crystallization patterns of epicuticular waxes can also change dramatically during development. Young, expanding plant organs usually lack wax crystals, but begin producing wax crystals very early in their development. The formation of new crystals usually continues until late in organ development. As tissues senesce, wax crystals degrade due to a combination of weathering by wind, solar radiation, and mechani. cal abrasion, coupled with the cessation of wax production. In addition, the chemical composition of waxes often changes during development.

6

M. JENKS AND E. ASHWORTH

Shorter-chain homologues of wax constituents are often more prevalent in the youngest tissues compared to newly matured leaves of some species (Atkin and Hamilton 1982; Jenks et a1. 1996b). The nature of these developmental shifts in epicuticular wax levels, crystallization patterns, and chemical compositions vary considerably among plant species and tissues. Besides differences in epicuticular waxes associated with the age of the plant organ, different organs of the same plant often exhibit quite distinct surface wax characteristics. For example, leaf surfaces of the dicot arabidopsis (Arabidopsis thaliana (L.) Heynh.) lack wax crystals, whereas stem surfaces have a white glaucous coating created by reflective ornate wax crystals (Fig. 1.3). Moreover, the total amount of epicuticular waxes per area is 25-fold higher on arabidopsis flowering stems than leaves, and leaves possess only trace amounts of secondary alcohols, ketones, and esters, which are major constituents on the stems (Jenks et a1. 1995). Also, the C3l homologues are the major alkanes on arabidopsis leaves, whereas the C29 homologues are the predominant stem wax alkanes. Other species, including beech (Fagus sylvatica L.), maize (Zea mays L.), prickly pear (Opuntia engelmannii Salm-Dyck), cistus (Cistus albidus L.), jojoba (Simmondsia chinensis (Link) C. K. Schneid), and sorghum, also show differences in wax profiles among different organs (Table 1.1). Differences have even been observed on different parts of the same leaf, as in wheat (Triticum aestivum L.) and sorghum (Table 1.1). For example, leaf sheaths of sorghum have a reflective white waxy bloom, whereas leaf blades generally have a green nonglaucous surface. The chemical composition of epicuticular waxes on different parts of the sorghum leaf also varies. Sheath waxes are over 90 percent fatty acids, whereas fatty acids occur in much lower proportions on leaves (Table 1.1). The physiological significance of such differences is unknown, but would be an intriguing topic for future research. The microclimate in which a plant grows can have a dramatic effect on epicuticular wax content. For example, lower temperatures increased the total amount of waxes per leaf area on Brussels sprouts (Brassica oleracea var. gemmifera L.) growing at temperatures between 15° and 25°C (Reed and Tukey 1982) and tobacco (Nicotiana tabacum L.) between 18° and 28°C (Wilkinson and Kasperbauer 1972). In contrast, when carnation (Dianthus caryophyllus L.) were grown between 15° and 25°C, lower temperatures reduced total wax loads (Reed and Tukey 1982). In addition, the amount of wax on leaves of rape (Brassica napus L.) grown under 400/0 and 600/0 full sun at 12°, 15°, and 27°C was higher at lower temperatures except when grown under 1000/0 full sun (Whitecross and Armstrong 1972). The combined results indicate that the effect

1. PLANT EPICUTICULAR WAXES: FUNCTION, PRODUCTION, AND GENETICS

7

Fig. 1.3. Scanning electron micrographs of epicuticular waxes on wild-type arabidopsis A. flowering stems. B. adaxial leaf surfaces. Bar = 51J.m. Source: Jenks et a1. 1995.

of temperature on wax production may vary in different plant species, and that temperatures may interact with light levels in controlling wax synthesis. In general, growing environments with higher light levels tend to result in more waxes being produced per unit leaf area. For example, epicuticular waxes on Brussels sprouts (Baker 1974), rape (Whitecross and Armstrong 1972), barley (Hordeum vulgare L.) (Giese 1975), and

M. JENKS AND E. ASHWORTH

8

Table 1.1. Chemical composition of epicuticular waxes from various organs of several plant species. Values represent the percentage of each chemical class identified in the respective study. These values may vary at different development stages and in different environments. FA =fatty acids. AIde. =aldehydes. 1-Alc. =primary alcohols. Alk. = alkanes. 2-Alc. =secondary alcohols. Ket. =ketones. Est. =esters. Terp. =triterpenoids. P-dkt. = p-diketones. Composition (%) Species and Organ

Cistus albidus z Leaf blade Sepal Petal Stamen Seed coat Opuntia engelmanniiY Cladophyll Bud Simmondsia chinensis x Leaf blade Seed coat Fagus sylvaticaw Leaf blade Seed shell Seed coat Sorghum bicolorv Leaf blade Leaf sheath Stalk Panicle Grain Triticum aestivum u Leaf blade Leaf sheath Zeamayst Leaf blade Husk Kernel Seedling Arabidopsis thaliana s Leaf blade Flower stem

Acids

AIde. 1-Alc. Alk. 2-Alc. Ket.

4.8 4.5 4.6 5.4 30.0

0.5 0.4 1.1 3.9 2.6

9.4 9.1 31.2 30.9 18.0

1.0 2.0

0.7 6.0

59.6 32.0

Est. Terp. P-dkt. 16.3 57.7 22.0 51.1 26.1 21.6 12.2 1.8 34.0 0.0 36.8 56.0

2.0 4.0

35.0 8.0

3.0 8.0

36.0 16.0

6.0 4.0

19.0 56.0

0.0 8.0

8.1 39.5 42.1

10.3 5.9 9.9

34.8 10.0 6.1

17.0 5.4 6.0

17.4 19.5 20.2

0.0 3.3 6.1

25.0 91.4 0.0 0.0 12.0

9.0 1.0 73.0 16.0 46.0

16.0 2.2 27.0 12.0 13.0

10.0 1.0 0.0 68.0 8.0

5.0 1.1 0.0 0.0 8.0

22.0 1.0

10.0 4.0

10.0 1.0 42.0 4.0 64.0 21.0 76.0 5.0 16.0 0.0

14.0 8.0 11.0 0.0

9.0 3.0 0.0 20.0

14.0 0.0 2.0 63.0

17.0 4.0 6.0 1.0

2.6 2.1

1.5 2.8

23.7 6.4

57.9 44.5

0.3 11.2

0.8 25.4

0.1 2.9

23.0 57.0

1.0 3.1

ZHennig et a1.1988. YWilkinson and Mayeux 1990. xGiilz 1983. wGiilz et a1.1989. vRich 1994. uTulloch 1973. tBianchi and Avato 1984. BJenks et a1.1995.

1. PLANT EPICUTICULAR WAXES: FUNCTION, PRODUCTION, AND GENETICS

9

carnation (Reed and Tukey 1982) occurred in greater amounts on leaves grown in high light environments than low light environments. The size, shape, and distribution of these wax crystals were also quite different under different light regimes. Although high light generally boosts wax load, Brussels sprouts grown at 25°C did not have increased wax levels under the high light levels as did comparable plants grown at 15°C (Reed and Tukey 1982). As suggested above, light and temperature may interact in regulating wax production by plants. It is unclear whether the induction of increased wax levels by high light intensity plays an ecologically important role in plant protection against higher levels of mutagenic ultraviolet solar radiation or heat loading via longer wavelength solar radiation. In other studies, bell pepper (Capsicum annuum L.) leaves had more wax per unit area when grown over white plastic mulches that created an environment enriched in white light than leaves grown over red or black plastic mulches that created an environment enhanced in the red wavebands (Kasperbauer and Wilkinson 1995). These authors associated changes in specific wax chemical classes with differences in the far red/red light ratios, and thereby implicated a possible phytochromemediated response. Further, longer photoperiods increased the chain length of alkanes in leaf epicuticular waxes of tobacco (Wilkinson and Kasperbauer 1972). These results suggest that the wavelength distribution of radiation may also influence wax production mechanisms and that phytochrome could playa role in the regulation of wax production. Potentially, phytochrome-mediated wax synthesis could determine the wax chemical profiles on different organs exposed to different light regimes within a plant canopy. Plant water status also appears to influence epicuticular wax production. Growth in low humidity conditions increased the total amount of waxes on Brussels sprouts (Baker 1974) and on plantlets from various species grown in vitro (Ritchie et a1. 1991; Zaid and Hughes 1995). Potentially, reduced moisture may have induced epicuticular wax production as an adaptation to prevent plant desiccation in more arid environments. Likewise, it is not clear whether drought conditions in the soil directly or indirectly affect epicuticular wax production processes. For example, cotton (Gossypium hirsutum L.) plants grown under water-limiting conditions produced more epicuticular wax per unit surface area than plants grown under irrigated conditions (Bondada et a1. 1996). However, whether increased wax per unit leaf area was a function of induced wax biosynthesis by epidermal cells is still unclear. An alternative explanation is that water stress simply impeded the expansion of

10

M. JENKS AND E. ASHWORTH

epidermal cells, and these smaller cells produced the same total wax per cell as the more expanded cells of irrigated plants. Besides climatic influences, growth regulators, inorganic nutrients, and chemical treatments can alter wax production. For example, adding 10 J.lM paclobutrazol or 200/0 polyethylethene glycol as media supplements increased the amount of waxes on micropropagated plantlets and resulted in better ex vitro establishment (Ritchie et al. 1991; Zaid and Hughes 1995). Moreover, inorganic nutrient status also appears to influence epicuticular waxes. For example, Douglas fir treated with increased levels of nitrogen and potassium fertilizers had more ornate wax crystallization patterns, but showed no difference in the amount of wax produced (Chiu et al. 1992).· Although not determined, presumably changes in wax chemical composition were responsible for these altered crystallization patterns.

III. ROLE OF EPICUTICULAR WAXES IN RESPONSES TO BIOTIC AND ABIOTIC STRESSES

The variation and plasticity of the plant epicuticular wax profile discussed above may provide differential plant resistance to environmental stresses in different stages of growth, organ types, and environments. This section discusses current knowledge of the role of epicuticular waxes in plant resistance to biotic and abiotic stresses. A. Epicuticular Waxes and Fungal Pathogens The layer of epicuticular waxes on aerial plant surfaces serves as the outermost barrier through which most fungal pathogens must enter uninjured plants. Even pathogens, such as rusts, that enter through stomata must penetrate the wax and cuticle layers lining the stomatal chamber. Potentially, these surface waxes could impede the entry of fungal pathogens by providing a physical barrier to penetration, via chemical signals that inhibit fungal development, or by increasing the hydrophobicity of plant surfaces, which results in less water retention, thereby removing moisture required for spore germination. Fungal hyphae presumably penetrate surface lipids either by physically forcing (via turgor pressure) their infection hyphae through contiguous wax layers or naturally occurring breaks in these layers (Bonnen and Hammerschmidt 1989; Cruickshank 1995), or by enzymatic degradation and softening of the surface lipids and subtending cuticle (Kolat-

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tukudy 1985). In addition, the chemical composition of epicuticular waxes likely influences fungal development on plant surfaces. Koller (1991) has noted that wax components appear to act as chemical signals in the interaction of plants with microorganisms. 1. The Effect of Wax Structure and Chemistry on Plant Pathogens. Cuticle thickness and the amount of waxes on plant surfaces directly increase plant resistance to fungal pathogens. For example, increased cuticle thickness has been correlated with increased fungal disease resistance in coffee (Goffea arabica L.) (Nutman and Roberts 1960), strawberry (Fragaria x ananassa Duchesne) (Peries 1962), sorghum (Jenks et a1. 1994a), rose (Rosa hybrida L.) (Hammer and Eversen 1994) and several species of vegetables (Louis 1963). Leaves of a sorghum mutant with thinner cuticles exhibited more lesions caused by the fungal pathogens Exserohilum turcicum ((Pass.) K. J. Leonard & E. G. Suggs) and Puccinia purpurea (eke) (Jenks et a1. 1994a). In addition, Jenks, Peters, and Axtell (unpublished) recently observed that sorghum bloomless mutants with reduced epicuticular waxes were more susceptible to the pathogens E. turcicum and P. purpurea in the field than wildtypes. While a relationship between host susceptibility and the thickness of epicuticlar wax and cuticle layers has been observed, it has not been established that increasing the amount and thickness of epicuticular waxes would provide additional levels of host plant resistance. In addition to total wax quantity, the crystallization patterns of epicuticular waxes may also affect the susceptibility of plantsto fungi. For example, berries of Vitis vinifera L. cv. Thompson Seedless that developed in contact with other berries had poorly developed wax crystals in areas where surfaces contacted compared to non-contact surfaces (Marois et a1. 1986). When inoculated with gray mold (Botrytis cinerea Pers. ex Fr.), contact surfaces had 64 percent more infections than noncontact surfaces. How epicuticular wax structure affects fungal growth and development is not clear, but several explanations are tenable. For instance, epicuticular wax crystals may elevate the fungal spores above the leaf surface, thereby limiting the spore's ability to receive physical or chemical signals from the plant, which are needed to direct spore development. Alternatively, wax structures may disorient fungal hyphae growth across plant surfaces. For example, rust fungi are known to use the stomatal ridge as a physical signal to induce hyphal penetration of the stomata (Goodman et at 1986). In such a situation, altered surface topography, due to altered wax crystallization patterns, might influence the number of successful penetrations. Although several studies suggest that wax crystals can significantly

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influence plant fungal-susceptibility, others show no such correlation. Reddy and coworkers (1992) reported no association between the density of wax crystals and susceptibility of Rosa taxa to black spot (Diplocarpon rosae WolD. They evaluated a collection of species and cultivars, which had diverse wax crystal structure, and found no association among resistant and susceptible types, suggesting that factors other than wax crystal structure and density limited blackspot disease susceptibility. It has been suggested that the cutin meshwork within the cuticle proper likely plays a more physical role, whereas the epi- and sub-cuticular waxes playa more chemical role, in inhibiting fungal penetration (Kolattukudy 1985; Kolattukudy et al. 1987; Flaishman et al. 1995). There have been several reports that specific chemical constituents of epicuticular waxes inhibit the development of fungal pathogens. For instance, Yang and Ellingboe (1972) reported that the powdery mildew fungus (Erysiphe graminis DC) produced more malformed appressoria on leaves of barley mutants exhibiting altered wax crystallization patterns than on wildtype leaves. Malformed appressoria also developed on recrystallized waxes from wildtype barley. Since these recrystallized waxes presumably had the same chemical composition as wildtype waxes in situ, but had different crystallization patterns, wax crystallization pattern alone was thought responsible for altered fungal development. However, E. graminis appressorium formation was normal on barley in which epicuticular wax crystals were removed using solvents (Carver and Thomas 1990). Thus, important questions about the Yang and Ellingboe (1972) and Carver and Thomas (1990) studies still remain. It is still unclear what effect wax removal had on the exact proportions of wax chemical constituents at the interface between the plant surface and fungal infection structures. Moreover, it is unclear whether the exact chemical profile of wax constituents was the same on recrystallized surfaces as wildtype barley leaves, or how the chemical profiles of wax constituents on mutants differed from wildtype. Without this information, it is difficult to distinguish whether wax crystal structure or chemical composition was the primary feature mediating E. graminis development. Erysiphe graminis germlings develop normally on the adaxial leaf surface of ryegrass (Lolium spp.), but abnormally on abaxial leaf surfaces (Carver et al. 1990). Adaxial surface waxes have plate-like crystalline patterns, whereas abaxial surface waxes have amorphous sheet morphologies. Interestingly, normal E. graminis development occurred on abaxial surfaces if waxes were removed. Since normal appressoria of these E. graminis were shown to form on inert surfaces, it is likely that some factor present in the abaxial Lolium surface wax is acting as an

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inhibitor (Carver et al. 1996). Chemical analysis of waxes on the abaxial and adaxial leaf surfaces showed that the abaxial leaf surfaces contained significantly higher levels of long-chain aldehydes, alkyl esters, and primary-alcohols than the adaxial surfaces. It has also been suggested that acidic wax constituents on plant surfaces may have antifungal activity. For example, the unsaturated fatty acids, linoleic acid and linolenic acid extracted from waxes of rye (Secale cereale L.) had anti-fungal activity (Honkanen and Virtanen 1960), and saturated and unsaturated fatty acids (C n to Cts) from lime (Citrus aurantifolia (Christm.) Swingle) exhibited fungistatic activity against the pathogen that causes withertip disease (Martin 1964). Similarly, an acidic substance extracted from apple leaf wax using organic solvents was found to be toxic' to apple mildew (Martin et al. 1957). There is also indirect evidence that fatty acids within epicuticular waxes may influence sorghum resistance to two fungal pathogens, E. turcicum and P. purpurea. In sorghum, the ratio of long (>C zo )- to short-chain fatty acids (C t4 to Cts ) was very low inepicuticular wax mutants that were susceptible to these pathogens, but were high in resistant lines (M. Jenks, P. Peters, and ]. Axtell, unpublished). Whether differential susceptibility was due to differences in these compounds is not clear, but using such mutants provides an excellent model system for assessing the role of wax fatty acids in plant fungal-susceptibility. Such studies are particularly suited for sorghum, since it is the only plant known to have fatty acids as the major leaf wax constituents (Jenks et al. 1994b; Rich 1994). While fatty acids appear to be antifungal, other long-chain wax constituents appear to stimulate fungal development. For example, 1,16hexadecanedial and 1,16-hexadecanediol induced appressorium development in the rice blast fungus (Magnaporthe grisea) (Gilbert et al. 1996). By comparison, the CZ4 and longer carbon length primary alcohols from avocado (Persea americana Mill.) induced spore germination and appressorium formation in Colletotrichum gloesoporioides (Penz.) spores (Podila et al. 1993). Interestingly, non-host wax extracts with even greater amounts of these long-chain primary alcohols actually inhibited fungal development. These authors speculated that a balance between appressorium-inducing primary alcohols and the absence of inhibitors may serve as a trigger for germination. Many aromatic epicuticular wax constituents have also been implicated in host plant resistance to fungal pathogens. For instance, isomeric diols, ex and f3 isomers of 4,8,13-duvatriene-1,3-diol found in the chloroform-soluble leaf surface extracts from tobacco (Nicotiana tabacum L.) were fungitoxic to Peronospora tabacina (Adam) (Cruickshank et al.

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1977). N. tabacum showed resistance to P. tabacina, whereas N. debneyi (Domin), a related species lacking these duvatriene diols, was susceptible. Moreover, N. tabacum leaves dipped for one second in acetone lost most fungitoxic activity. However, when these surface compounds were reapplied to these same leaves, rates of P. tabacina sporangial germination were greatly reduced (Reuveni et al. 1987). Since epicuticular waxes often do not recrystallize in the same patterns as seen in situ without special procedures (Jeffree et al. 1975; Jetter and Riederer 1995; M. Jenks, unpublished), wax chemistry and not wax crystallization pattern was likely playing the dominant role in determining tobacco plant susceptibility to these pathogens. It is not known whether pathogenic fungi secrete epicuticular-waxspecific degrading enzymes to facilitate host penetration. However, it is known that fungal cutinase genes and their gene products can be induced by contact with constituents of plant cuticles (Woloshuk and Kolattukudy 1986; Podila et al. 1988) and that cutinase is secreted during fungal penetration of the plant host (Shaykh et al. 1977). Cutin, which exists as a polyester composed of primarily C16 and C18 fatty acids having hydroxyl groups in ro- and midchain positions, is a major constituent of the cuticle that underlies the epicuticular wax layer. The cuticle also contains significant amounts of waxes. Many cutinases have specificity for primary alcohol esters of the cutin polyester, however, chain-length specificity for ester constituents appears to vary widely with fungal genera (Kolattukudy 1984). Further studies are needed to assay fungal secretions for their ability to degrade epicuticular wax constituents. The chemical composition of epicuticular waxes is diverse, and it is difficult to determine what role particular wax constituents play in plant-pathogen interactions, and whether changing wax chemistry would be an effective way to enhance plant resistance. Such investigations would be facilitated by using mutation induction, backcross breeding, or recombinant DNA technology to develop near-isogenic lines with different epicuticular wax chemical profiles. The disease susceptibility of these lines could be subsequently compared. 2. Leaf Surface Wettability and Plant Fungal-Susceptibility. Another means by which epicuticular waxes may influence plant fungalpathogen susceptibility is by altering moisture levels on plant surfaces. In order to germinate, most fungal spores (except conidia like those of powdery mildews) require free water, or relative humidities above 95 percent, for a finite period of time (Blakeman 1973). The water-shedding properties of epicuticular waxes could therefore indirectly impede fun-

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gal development on plant organs by reducing the available moisture on plant surfaces. Raspberry (Rubus spp.) cultivars that had a dense waxy bloom over cane surfaces were more resistant to Botrytis cinerea than those lacking the waxy bloom, and this resistance was attributed to increased run-off of water (Mendgen 1996). Potentially, plant surface waxes may serve as a sort of "raincoat" that sheds irrigation, precipitation, and condensed moisture, thereby improving plant resistance to fungal pathogens. Wettability of a leaf surface is a function of water droplet contact angles, and contact angles are affected by the structure and chemical composition of leaf epicuticular waxes. For instance, large wax structures tend to hold water droplets above the leaf surface, creating large contact angles (Holloway 1969). Likewise, hydrophobic wax constituents, such as alkanes, secondary alcohols, ketones, and esters, create high surface water contact angles and would shed water more efficiently than leaf surfaces covered with less hydrophobic compounds, and thereby indirectly decrease susceptibility to foliar fungal pathogens. The highest contact angles of water droplets on abaxial leaf sheaths of genetically similar lines of wheat were associated with thicker glaucous coatings, higher numbers of tubular wax crystals, and greater proportions of ~-diketone and hydroxy-~-diketones (Netting 1973). Whether differences in surface wettability in turn affected spore germination and pathogen development were not investigated. 3. Leaf Surface Permeability and Plant-Fungal Susceptibility. A variety

of chemical constituents have been observed to diffuse from internal tissues to the plant surface. For example, simple sugars, amino acids, organic acids, growth regulators, vitamins, alkaloids, and phenols have all been found within water droplets on plant surfaces (Blakeman 1973). In fact, competition for nutrients on leaf surfaces may limit spore germination of certain plant pathogens (Blakeman 1973). As a barrier to water movement, epicuticular waxes also have the potential to influence the diffusion of nutrients, growth factors, and antifungal compounds (phytoalexins) to leaf surfaces, but this has not been examined in detail. Mechanical removal of plant epicuticular waxes has been shown to influence fungal development. For example, Cruikshank (1995) demonstrated that formation of the penetration peg and hyphae development of Colletotrichum gloeosporioides were enhanced by removal of surface waxes from tomato (Lycopersicon esculentum Mill) fruit. This effect was thought due to increased diffusion of nutrients and plant substances that stimulated fungal metabolism (Cruikshank 1995). Studies to measure the amount of fungi-active compounds that diffuse through plant

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wax and cuticle layers of various thickness, especially those of genetically similar lines, may provide an effective means of elucidating what role these surface lipids play in determining the amount of plant leachates that actually diffuse onto plant surfaces. Presumably, the diffusion of pathogen-derived compounds into plant organs would also be affected by properties of epicuticular waxes. B. Epicuticular Waxes and Phytophagous Insects As the interface between insect pests and potential hosts, epicuticular waxes play an important role in plant-insect interactions. Chapman and Bernays (1989) proposed that all phytophagous insects make some sensory examination of plant surfaces prior to feeding, and several thorough reviews on the effects of plant epicuticular waxes on insect feeding and behavior are available (Woodhead and Chapman 1986; Juniper 1995; Eigenbrode and Espelie 1995; Eigenbrode 1996). The observations that chemical and physical properties of surface waxes can alter the interactions between insect pests and crop plants has important agricultural implications and has been an active area of research. Feeding-related behaviors of many insects are inhibited on plant surfaces that have thick coatings of epicuticular wax crystals. For example, mature leaves of two Eucalyptus species (E. nutans F.J. Muell. and E. globulus Labill) were susceptible to the Eucalyptus tortoise beetle (Paropsis charybdis Shill, whereas the juvenile leaves of these same Eucalyptus species were resistant (Edwards 1982). These differences were apparently due to developmental differences in epicuticular wax coatings. The juvenile Eucalyptus leaves had a glaucous coating created by dense epicuticular wax crystals, whereas the adult leaves had a nonglaucous surface with reduced amounts of wax crystals. Experimental observations showed that beetles clung less effectively onto the glaucous juvenile leaves compared to the nonglaucous adult leaves. Reduced clinging efficiency meant less time spent by the beetles feeding and ovipositing on juvenile Eucalyptus leaves. Similarly, mustard beetles (Phaedon cochlearlae (Fabricius» adhered better to glossy than glaucous leaves of Brussels sprouts, and thus glaucous lines were less susceptible to mustard beetle damage (Stork 1980). Setae on mustard beetle tarsi appear adapted for clinging to smooth plant surfaces, since wax particles tended to accumulate on the beetle's setae and inhibit insect movement. Likewise, studies by Mulroy (1976) suggested that the glaucous ecotypes of Dudleya britonii (Johans.) were more resistant than glossy ecotypes to the stem boring larvae of the pyralid moth (Rhagea stigmella. (Dyar)). Whether, in fact, properties of surface waxes were the basis for pyralid

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moth resistance in D. britonii has yet to be established. Additional evidence for the role of epicuticular waxes in inhibiting insect feeding behaviors came from studies on cabbage and rape. The flea beetle (Phyllotreta cruciferae (Goeze» fed at higher rates on the glossy mutant leaves than the glaucous cabbage leaves (Bodnaryk 1992; Stoner 1992). In the same manner, mechanical removal of the waxy bloom from cabbage and rape increased feeding by flea beetles, P. cruciferae and P. striolata (Fabricius) (Bodnaryk 1992). Similarly, polishing or rinsing glaucous surfaces with solvents to remove wax deposits increased ovipositing on cabbage leaves by both diamondback moth (Plutella xyJostella L.) (Uematsu and Sakanoshita 1989) and the cabbage root fly (Delia radicum L.) (Prokopy et al. 1983), and more ovipositing on olive (Olea europaea L.) fruits by the olive fruit fly (Dacus oJeae (Gmelin)) (Neuenschwander et al. 1985), than on respective normal glaucous surfaces. In many plant-insect associations, it is a reduction in epicuticular wax crystals that impedes the selection of host plants by phytophagous insects. For example, glossy seedlings of sorghum were more resistant to the sorghum shoofly (Phaonia soccata (Walker» than the normal glaucous lines when grown in the field (Maiti et al. 1984). Likewise, the populations of cabbageworm larvae (Pieris rapae L.) and cabbage aphids (Brevicoryne brassica L.) were reduced on glossy lines compared to glaucous lines of cabbage grown in the field (Stoner 1992). When ovipositional nonpreference was removed as a factor by artificially infesting leaves with both cabbageworm larvae and eggs, the glossy lines were still more resistant than the normal glaucous lines to both insect species. Similarly, glossy cabbage lines exhibited reduced survival of diamondback moth larvae when compared to the wildtype glaucous lines in the field (Eigenbrode et al. 1991a). Recent findings by Eigenbrode et al. (1995,1996) suggest that the field resistance in glossy lines may be due to increased predation of phytophagous larvae by predatory insects. Specifically, three predator species, Chrysopa bicarnea (Banks), Hippodamia convergens (Guerrin-Menneville), and Orius insidiosus (Say), were more mobile and more effective predators of diamondback moth larvae on glossy cabbage wax mutants than on wild-type varieties (Eigenbrode et al. 1996). Glossy and glaucous lines were equally susceptible to diamondback moth in greenhouse studies where entomophagous insects were not present. This observation contributed support to the idea that surface waxes affected these tritrophic interactions. In like manner, glossy lines of pea (Pisum sativum L.) exhibited a 50 percent reduction in pea aphid (Acyrthosiphon pisum (Harris» populations compared to aphid populations on glaucous lines in the field (Eigenbrode, personal communication). Similar aphid numbers on glossy and

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glaucous lines in controlled cage studies contribute to evidence that lack of surface wax crystals may cause increased predation by certain generalist predators. Investigations in sorghum noted that bloomless and sparse bloom mutants were more resistant in the field to the greenbug aphid (Schizaphis graminum (Rondani)) than wildtype. As in previous studies, this difference did not exist in field cages (Starks and Weibel 1981; Weibel and Starks 1986). Thus, a combination of observations indicate that epicuticular waxes may influence insect pest resistance in the field by affecting the mobility and adherence of phytophagous and entomophagous insects on plant surfaces. In addition to crystallization patterns, insects often use the chemical compositions of epicuticular waxes as cues for host-plant selection. For example, many insects prefer to feed on artificial mediums impregnated with epicuticular waxes from their host plant rather than comparable artificial mediums in which the host wax extracts were omitted. In addition, insects presumably avoid feeding on mediums containing plant waxes from a non-host (Woodhead and Padgham 1988; Braker and Chazdon 1993). Thus, a common research objective has been to identify individual wax constituents that, in situ, serve as primary cues for host-plant selection. Long-chain alipathic epicuticular wax constituents have been shown to influence plant-insect interactions. For example, primary alcohols and free fatty acids will stimulate feeding or ovipositing behaviors of aphids (Greenway et al. 1978) and silk worm larvae (Bombyx mori) (Mori 1982) when added to artificial diets. In whole plant studies, leafcutter bees (Megachile sp.) made more cuts on the glaucous leaves of Mexican redbud (Gercis canadensis var. mexicana L.) than on the leaves of a glossy ecotype (Eigenbrode et al. 1998). In addition to lacking wax crystals on the adaxial leaf surface, the glossy ecotype had a 6-fold reduction in the relative content of triacontanol (C30 primary alcohol) in its surface waxes. There are other reports of long-chain alipathic epicuticular waxes affecting insect feeding preferences. For example, third instar locust (Locusta migratoria L.) preferred feeding on older leaves, rather than younger leaves, of sorghum (Atkin and Hamilton 1982). It was proposed that gradual increases in the relative hydrocarbon abundance and hydrocarbon chain-length distribution with increasing age in sorghum leaf waxes explain, at least in part, the increased deterrence exhibited by young plants. By comparison, wax alkanes and esters on sorghum apparently deterred feeding by L. migratoria, with the shorter-length homologues being more effective (Woodhead 1983). Moreover, greater proportions of wax alkanes induced more intense searching behavior on leaves of maize by fall armyworm (Spodoptera jrugiperda (J. E. Smith))

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(Yang et al. 1993), suggesting that alkanes may serve as a deterrent that prevented larvae from settling to feed. Bergman et al. (1991) noted that increased amounts of wax esters were associated with increased resistance of alfalfa (Medicago sativa L.) to the alfalfa aphid (Therioaphis maculatus (Buckton)). Using a rapid near-infrared screening technique, Rutherford and Staden (1996) predicted that either elevated wax alcohols, reduced aldehydes, and/or reduced chain length for wax hydrocarbons on sugarcane contributed to reduced sugarcane stalk bore (Eldana saccharina (Walker)) larvae survival. Interestingly, other evidence has arisen to implicate short-chain alcohols in plant insect-resistance. Greater resistance in tobacco to the tobacco budworm (Heliothis viresens (Fabricius)) was associated with higher levels of docosanol (C 22 primary alcohol) (Johnson and Severson 1984). Thus, the long-chain hydrocarbons clearly play an important role in host-plant selection by phytophagous insects, but the mechanism is unclear. In addition to the hydrocarbon fractions, there are reports that aromatic constituents also affect insect selection of plant hosts. Increased amounts of a- and p-amyrins in the surface waxes of Rhododendron species were correlated with resistance to the azalea lace bug (Stephanitis pyrioides (Scott)) (Balsdon et al. 1995). p-amyrin was also associated with greater resistance in Rubus idaeus (raspberry) to the raspberry aphid, Amphorophora idaei (Van der Goot)) (Robertson et al. 1991). Likewise, amyrins in grasses were shown to be deterrents to Locusta migratoria (Bernays and Chapman 1977). By comparison, free and esterified triterpenols increased aphid resistance in sorghum when present at high levels (Heupel 1985). Prophenylbenzenes, coumarins, and a polyacetylene in leaf epicuticular waxes of carrot (Daucus carota L.) appeared to stimulate ovipositing in the carrot fly (Psila rosae (Fabricius)) (SUidler and Buser 1984). Mixtures of wax constituents have been shown to act synergistically in affecting insect behavior (SHidler and Buser 1984; Spencer 1996). Aromatic components of epicuticular wax vary widely, and can be extremely diverse on plant species, being found in only trace amounts on some plants and as dominant constituents on others, and it is likely that many of these compounds will affect plantinsect interactions. C. Epicuticular Waxes and Drought Epicuticular waxes play an important role in plant-water relations. The chenlical constituents of epicuticular waxes are thought to create a continuous hydrophobic water barrier, which impedes water loss from plant organs. The presence of wax structures can also create a still-air

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boundary layer both above the cuticle surface, and within and above stomatal pores. As with wax hydrophobicity, an enhanced boundary layer would likely reduce the rate of transpiration from plant tissues. In addition, epicuticular waxes on some plants reflect light, decreasing the radiation heat load, and thus reducing the transpiration rate. Therefore, the mechanisms by which surface waxes affect water loss can be varied, involving both chemical and physical methods. 1. Effect of Waxes on Plant Water Loss. Reduced amounts of epicuticular wax on plant surfaces have been shown to be associated with increased rates of transpiration. Brushing waxes off the excised leaves significantly increased the rate of water loss (Hall and Jones 1961). Similarly, leaves of rice (O.cyza sativa L.), dipped for two seconds in chloroform to remove epicuticular waxes, exhibited more than a two-fold increase in cuticular conductance to water vapor compared to control leaves (O'Toole et a1. 1979). Excised wheat leaves from nonglaucous lines had 28 percent less epicuticular wax and 33 percent higher water loss rates than genetically similar glaucous lines (Clarke and Richards 1988; Johnson et a1. 1983). Premach~ndra and co-workers (1992, 1994) reported a negative correlation between the amount of epicuticular wax and cuticular conductance in sorghum leaves. The amount of epicuticular wax on various sorghum cultivars was negatively correlated with excised-leaf water loss rates when wax loads were between 0.1 and 0.03 g m-2 (Jordan et a1. 1984). However, these transpiration rates did not decrease significantly as wax loads increased above 0.067 g m-2 • Wax quantities above 0.067 g m-2 gave no added benefit for sorghum resistance to water loss. Drought-stressed plants generally have greater amounts of epicuticular wax per unit leaf area than non-stressed plants. However, the increased amount of wax is not always associated with greater plant resistance to water loss. For example, crested wheatgrass (Agropyron desertorum Willd.) (Jefferson et a1. 1989), Triticum species (Johnson et a1. 1983), and various sorghum cultivars (Premachandra et a1. 1992; Blum 1975) grown under water-limiting conditions had more wax per unit leaf area than plants grown in irrigated field plots. As might be expected, the drought-stressed sorghum plants had reduced stomatal conductance to water vapor, compared to respective irrigated plants (Blum 1975; Johnson et a1. 1983; Premachandra et a1. 1992, 1994). Somewhat surprisingly, however, the relationship between stomatal conductance and wax content was not observed in crested wheatgrass, even though the amount of wax was significantly higher in the stressed plants (Jefferson et a1. 1989). Similarly, other studies showed no correlation

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between epicuticular wax load and plant water loss. For example, the amounts of leaf wax were not related to either epidermal (Araus et a1. 1991) or stomatal conductance (Johnson et a1. 1983) in Triticum species, to epidermal conductance of several western U.S. conifers (Hadley and Smith 1990), or to excised-leaf water loss rates from oats (Avena sativa L.) (Bengston et a1. 1978). The absence of a correlation between the amounts of epicuticular wax and water loss in these studies may have .occurred because, despite large differences in the amounts of epicuticular wax, each line still had sufficient wax to provide maximum resistance to water loss, as discussed by Jordan et a1. (1984). While such an explanation is plausible, it does not explain why drought-stressed plants often accumulate additional epicuticular wax. Water loss through plant cuticles may be more complicated than simple diffusion through a waxy layer. For instance, water flow through the cuticle layers may be directed through the preferred polar pathways described by Schonherr (1976a). Theoretically, plant cuticles could differ greatly in the number of these polar pathways. As water is a polar molecule, more and larger polar pathways in a cuticle would presumably lead to greater amounts (and rates) of water loss. Studies using isolated cuticular membranes suggested that subcuticular waxes, and not epicuticular waxes, played the major role in reducing cuticular water permeability (Schonherr 1976a, 1976b). Individual chemical constituents and mixtures of constituents that compose the plant epicuticular wax layer likely differentially affect epidermal conductance to water vapor. For example, laboratory studies using plastic membranes coated with either grape epicuticular waxes or selected classes of wax components (at 30-70 Jlg cm-2) found that the hydrocarbon, alcohol, and aldehyde fractions effectively limited water transport through the artificial membranes. By contrast, fatty acids restricted water transport only slightly, whereas wax triterpenoid conjugates had no effect (Grncarevic and Radler 1967). Such results are not surprising since fatty acids and terpenoids are much less hydrophobic than those other wax constituents. Thus, increasing amounts of hydrophobic components within the surface waxes should lower epidermal conductance to water vapor. In addition to producing greater amounts of wax, plants also have been found to alter the chemical compositions of their epicuticular waxes in response to water deficits. In cotton, the leaves, bracts, and bolls produced longer-chain-length epicuticular wax alkanes in drought-stressed plants compared to irrigated plants (Bondada et a1. 1996). Longer-chain alkanes are more hydrophobic and their induction by drought suggests their importance in reducing water loss during periods of water shortage.

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Such studies suggest that particular combinations of wax constituents may provide an adaptive advantage under drought conditions. 2. Wax Reflectance of Solar Radiation and Plant Water Loss. In addition to their assumed role as a hydrophobic barrier, evidence suggests that some epicuticular wax structures may reflect significant amounts of solar radiation and thereby reduce the radiation heat load and water use via transpiration. For example, Clarke and Richards (1988) determined that among seven genetically similar populations of wheat, which had similar levels of wax but differed in surface glaucousness (based on visual ratings), the glaucous lines had, on average, 10 percent lower rates of excised-leaf water loss compared with the nonglaucous lines. Thus, it is suggested that increased reflectance apparently reduced leaf water loss. However, this result is not definitive, since potential differences in wax fine structure and chemistry among these populations may have reduced water loss through other means. Moreover, Johnson et a1. (1983) could show no significant reduction in stomatal conductance or transpiration between glaucous and glossy wheat lines differing by between 8 and 15 percent in reflectance of photosynthetically active radiation. Other evidence for a role of wax reflectance in reducing water loss comes from observations of desert plants. Plant species adapted to arid environments, in general, have a whitish surface and reflect more radiation than do mesophytic plants (Gates et a1. 1965), perhaps as a means of reducing heat load and water used for evaporative cooling. 3. Wax and the Boundary Layer Above Plant Surfaces. Epicuticular wax

crystals that protrude above the plant surface may increase the thickness of the still-air boundary layer. This can reduce transpiration by increasing surface resistance to diffusion of water vapor. For example, the lower excised-leaf water loss rates in glaucous wheat may be due to the greater surface boundary layer created by the protruding wax crystals. Potentially, these wheat lines could provide a model system for dissecting the role of wax "induced" boundary layers in plant water loss. Another excellent model plant system for studying the effect of leaf wax on boundary layers may be the near-isogenic wax mutants of sorghum. The epicuticular wax layer over the wildtype abaxial sheath surfaces of sorghum can reach 2 mm in thickness, while the bJoomless and sparse-bloom mutants have wax layers whose thicknesses are reduced in a continuum down to a glossy surface. Leaf waxes may also directly affect stomatal conductance via effects on boundary layers. Jeffree et a1. (1971) calculated the effect of epicuticular wax tubes inside the stomatal antechamber of Sitka spruce (Picea

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23

sitchensis (Bong.) Carr.) needles, and suggested that increased wax occlusion of stomatal openings increased stomatal resistance to water vapor. Presumably, waxes impeded the diffusion of water vapor within the stomatal antechamber. A similar situation may also occur in sorghum. Scanning electron micrographs show that waxes can occlude the stomatal pore (McWhorter et a1. 1990; Jenks, unpublished). The observation by Chatterton et a1. (1975) that normal sorghum had significantly lower stomatal transpiration rates than comparable bloomless lines may reflect differences in stomatal wax occlusions. Whether, in fact, these differences were due primarily to wax effects on stomatal conductance, or epidermal conductance through the cuticle, still needs to be established. D. Epicuticular Waxes and Freezing Temperatures There are two ways in which epicuticular waxes may facilitate plant survival at sub-zero temperatures. One way is by reducing winter desiccation in evergreen species. The leaves of evergreen species are subjected to desiccation stress during the winter (Sakai 1970; Tranquillini 1979). During cold periods, water within the soil, stems, and branches is often frozen and thus the uptake of water to replenish water lost via transpiration is prevented. Under winter conditions, leaves can lose water by either sublimation of intercellular ice crystals on cold days, or by evaporation of liquid water from within leaves during days when the sun warms the needles above O°C. Since the path of water loss in both cases is primarily through the cuticle, it would seem likely that leaves having well-developed layers of epicuticular wax may have an adaptive advantage in these conditions. Herrick and Friedland (1991) observed that red spruce (Picea ruben Sarg.) needles that had lower cuticular resistance to water loss were more likely injured by winter desiccation than comparable needles with better-developed cuticles. Although the reason for the lowered cuticular resistance was not determined in this study, a well-developed epicuticular wax layer could directly impede water loss, or indirectly reduce cuticular transpiration by reducing the absorption of incident solar radiation and lowering leaf temperature. The presence of epicuticular wax has also been postulated to affect frost damage. Thomas and Barber (1974a) observed that glaucousness of leaf surfaces was more prevalent in Eucalyptus urnigera (Hook. f.) at higher elevations on Mount Wellington, and linked this characteristic to frost hardiness. They observed that glaucous leaves shed water more effectively than comparable non-glaucous leaves, and that dry leaves would supercool to much lower temperatures prior to freezing. Other

24

M.~NKSANDE.ASHWORTH

studies have also noted that leaves with surface moisture freeze at warmer temperatures than comparable leaves having a dry exterior surface (Ashworth 1992). The most likely explanation is that water first freezes on the leaf surface, and subsequently inoculates freezing internally. Therefore, a well-developed layer of epicuticular wax would both shed surface water and impede ice propagation into subtending tissues. However, while this appears to be an attractive hypothesis, it is neither clear how surface moisture affects leaf freezing, nor how external ice crystals trigger freezing within the leaf. There have been no other reports linking leaf glaucousness and frost susceptibility, despite the diversity of wax levels present in some species.

E. Epicuticular Waxes and Solar Radiation It is a common observation that plants growing in environments with high levels of solar radiation, as occurs in desert and alpine regions, often have leaf surface features that reflect light (Billings and Morris 1951; Gates et at 1965). One such feature is the presence of a thick layer of epicuticular wax. Plants growing in high solar radiation environments are often glaucous, and the presence of epicuticular wax crystals on leaf surfaces attenuates light exposure to the subtending tissues. For example, several investigators have demonstrated that plants having thick layers of epicuticular wax can reflect between 20 and 80 percent of the incoming radiation, whereas non-glaucous plants typically reflect less than 10 percent (Thomas and Barber 1974b; Clark and Lister 1975; Reicosky and Hanover 1978; Mulroy 1979; Vogelmann 1993). The reflective character of glaucous leaves was reduced to the level of the non-glaucous leaves by treatments that removed the epicuticular waxes (Thomas and Barber 1974b; Clark and Lister 1975; Reicosky and Hanover 1978; Mulroy 1979). The size, distribution, and orientation of wax crystals, and other surface features, determines the extent to which light is scattered at the tissue surface (Barnes and Cardoso-Vilhena 1996). Generally, radiation is scattered across the spectrum, and increased reflectance of ultraviolet (UV), visible, and infrared radiation has been observed (Thomas and Barber 1974b; Clark and Lister 1975; Reicosky and Hanover 1978; Mulroy 1979; Vogelmann 1993; Barnes and Cardoso-Vilhena 1996; and others). However, in some plant species there is preferential scattering of shorter wavelength radiation. For example, leaves of blue spruce (Picea pungens Engelm. var. hoopsii) reflected a higher proportion of UV and blue light than did needles of either Douglas fir (Pseudotsuga menziesii [Mirb.] Franco.) or Sitka spruce (Clark and Lister 1975). The bluish appearance of these leaves, and the enhanced reflection of UV radiation was shown

1. PLANT EPICUTICULAR WAXES: FUNCTION, PRODUCTION, AND GENETICS

25

to be due to the presence of epicuticular wax deposits (Clark and Lister 1975). Dense deposits of epicuticular wax apparently provide an adaptive advantage to plants growing in high light environments. Much of the research to this point has focused on whether the presence of epicuticular wax deposits would reduce exposure levels of damaging UV radiation. UV irradiance increases at higher elevations, and plants that grow at higher elevations often have thick layers of epicuticular wax (Billings and Morris 1951; Gates et a1. 1965; Thomas and Barber 1974b; Clark and Lister 1975; and others). Measurements of UV penetration into tissues have shown that species vary in their ability to screen out UV-B radiation, and that most of the UV radiation is attenuated in the cuticle and epidermal cell layer (Bornman and Vogelmann 1988; Day et a1. 1992, 1993; Krauss et al. 1997). Most of the attenuating effect of the cuticle and epidermal layer is due to the presence of flavonoids and related phenolic compounds that absorb UV-B (Vogelmann 1993; Barnes and Cardoso-Vilhena 1996; Krauss et a1. 1997). Epicuticular waxes from most species do not absorb significant amounts of radiation in this portion of the spectrum. However, epicuticular waxes on some species may provide protection by scattering and reflecting incoming UV radiation, and thus reducing exposure levels in underlying tissues (Clark and Lister 1975; Mulroy 1979; Vogelmann 1993; Grant et a1. 1995; Barnes and Cardoso-Vilhena 1996). Epicuticular wax deposits can also act as a photoprotectant in the visible portion of the spectrum. Robinson and co-workers (1993, 1994) found that removal of reflective surface waxes from Cotyledon orbiculata L. made plants susceptible to photoinhibitory damage. The white waxy coating on this succulent plant reflects about 60 percent of the incident light, and thus reduces the amount of light that reaches the interior of the leaf. A third way in which epicuticular waxes may provide an adaptive advantage in high radiation environments is by reflecting a portion of the incident solar radiation and reducing the absorbance of visible and infrared radiation. This, in turn, should lead to cooler leaf temperatures, and thus reduced transpiration rates (Reicosky and Hanover 1978; Mulroy 1979; Barnes and Cardoso-Vilhena 1996). Consistent with this hypothesis are reports indicating that plants with well-developed layers of epicuticular wax have lower leaf and canopy temperatures, reduced rates of transpiration, and improved water status relative to comparable controls (Johnson et a1. 1983; Jefferson et a1. 1989). While the presence of epicuticular wax apparently affects the exposure of underlying tissues to solar radiation, it is interesting to note that the

26

M. JENKS AND E. ASHWORTH

light environment also affects the quantity and composition of epicuticular wax (Whitecross and Armstrong 1972; Baker 1974; Giese 1975; Reed and Tukey 1982; Tevini and Steinmiiller 1987; Barnes et a1. 1994). Such indirect evidence also supports the hypothesis that epicuticular waxes may provide an adaptive advantage in high solar radiation environments. F. Epicuticular Waxes and Agricultural Sprays A diverse array of agricultural chemicals are sprayed onto horticultural and agronomic plants for a variety of reasons, including insect, pathogen, and weed control, foliar fertilization, and growth regulation. Epicuticular waxes are a significant barrier to both spray retention and subsequent penetration into plant organs. Nearly all plant surface waxes are hydrophobic and thus tend to repel water-based liquid sprays. For that reason, most agricultural sprays are formulated as either oil-based solutions, or adjuvants like wetting agents, spreaders, or stickers are added to facilitate spray droplet retention, distribution, and penetration into the plant surface. While permeability of the cuticular membrane to agricultural chemicals has been examined in numerous studies, fewer studies have examined the specific role of epicuticular waxes on surface retention, permeability, and sorption of these sprays. 1. Epicuticular Waxes and Surface Retention. It has been demonstrated

that the more polar the surface wax components, the lower the contact angle of water droplets on the plant surface, and that this generally correlates with better spray retention (Holloway 1969). Similarly, in peach (Prunus persica L. Batsch 'Red Haven'), increasing amounts of alkanes, esters, and total surface waxes during leaf development correlated with a decrease in wettability as measured by increased droplet contact angles (Bukovac et a!. 1979). Formulating sprays and their adjuvants to improve their interaction with hydrophobic surface waxes has been used commercially to enhance spray retention. Future approaches might involve crop improvement strategies to alter surface waxes and enhance the retention of pesticides. Alternatively, reducing herbicide retention on the surface of crop plants might also be useful. Epicuticular waxes could be made thicker or more hydrophobic using genetic approaches such that herbicides could be applied at greater rates in field cropping systems without damaging the crop. 2. Epicuticular Waxes and Surface Penetration. As an external barrier, epicuticular waxes often impede the penetration of agricultural chemicals into the interior of leaves and other organs. For example, several

1. PLANT EPICUTICULAR WAXES: FUNCTION, PRODUCTION, AND GENETICS

27

investigators have demonstrated that the physical or chemical removal of cuticular waxes increased the penetration of a range of agricultural chemicals (Sharma and VandenBorn 1970; Bukovac et a1. 1971; Kirkwood et a1. 1972; Norris and Bukovac 1972; Norris 1974; and Schonherr and Riederer 1989). There was a strong negative correlation between the amount of surface waxes per leaf area and foliar penetration of the 14C_ radiolabeled lipophilic herbicide, diuron, in nine different plant species (Gouret et a1. 1993). A negative correlation was also reported between foliar penetration of the 14C-radiolabeled growth regulator, napthaleneacetic acid (NAA), and total surface wax levels per leaf area during leaf expansion (Bukovac et a1. 1979). In other studies, Santier and Chamel (1992) demonstrated that glyphosate herbicide penetration through tomato cuticles was greater in organs that had less cuticular wax. Greater amounts of epicuticular waxes reduced leaf phytotoxicity to surfactants, likely due to reduced penetration of the surfactant through the leaf cuticle (Knoche et a1. 1992). The more wax crystals on the surface of plant leaves, the more tortuous the pathway that agricultural chemicals must traverse before entering epidermal cells (Riederer and Schreiber 1995). In situations where the amount of surface wax controls penetration of spray materials, partial removal, disruption, or increased diffusion through plant surface wax layers would likely increase permeability of agricultural chemicals. Interestingly, the thinnest areas of the plant surface waxy barrier may be found associated with the stomatal pore and substomatal chamber; however, whether this general situation is true for most plants has not been verified. If so, spray adjuvants targeted to the stomatal cuticular boundaries might be effective. While many studies have demonstrated a strong negative correlation between the amount of epicuticular wax and permeability of agricultural chemicals, some studies have reported little or no such correlation. For example, Baker and Hunt (1981) could not demonstrate a clear correlation between foliar penetration of the 14C-radiolabeled growth regulator NAA and the total amounts of wax per leaf surface area. Norris (1974) similarly found no correlation between the amount of cuticular waxes and the permeability of 2,4-D through the cuticles often different plant species. Leaf surfaces of tomato had higher permeabilities than leaf surfaces of pepper, even though the tomato leaves had much higher proportions of wax associated with their cuticles (Chame11986). While it has been assumed that the amount of epicuticular and subcuticular waxes plays a major role in limiting the conductance of agricultural chemicals through plant cuticular layers, instances where no correlation between the amount of cuticular waxes and cuticular permeability suggest that wax thickness alone does not fully explain the physicochemical basis for permeability.

28

M. JENKS AND E. ASHWORTH

Possible explanations for the lack of correlation between wax amounts and spray penetration may be that differences in the specific chemical constituents of both the plant cuticular waxes and agricultural sprays influence the permeability of plant surfaces. For example, increasing amounts of alkanes and esters during leaf development in peach correlated with a decrease in permeability of 14C-radiolabeled NAA (Bukovac et a1. 1979). These differences may be due to reduced wettability of the surface or to reduced penetration through the cuticular wax layers. By comparison, the rate of chemical penetration into leaves was affected by the lipophilicity of spray formulations; that is, cuticular permeability tended to increase in the order of increasing lipophilicity of constituents in the spray (Schreiber and Schonherr 1992b; Schonherr and Baur 1996). 3. Effect of Epicuticular Waxes on the Sorption of Agricultural Chemicals. One fate of agricultural chemicals applied to plants is their sorption (Le., attachment) within or onto the cuticle membrane. Charnel et a1. (1991) showed increased sorption of the plant growth regulator, paclobutrazol, both within and on the surface of isolated cuticular membranes following extraction of the soluble cuticular waxes. The epicuticular waxes themselves have low sorption capacity for most agricultural chemicals; nevertheless, it is unclear why removal of waxes from the cuticle membrane leads to increased sorption of these chemicals to the cuticle matrix (Bukovac et a1. 1990; Charnel et a1. 1991; Scheiber and Schonherr 1992a). Charnel et al. (1991) hypothesized that the presence of epi- and sub-cuticular waxes may, under normal conditions, block sites for chemical sorption within the cuticle. Still, there is no direct evidence supporting this hypothesis. Specific studi~s to elucidate how cuticular waxes might block chemical sorption (and penetration) may lead to novel spray formulations, new spray application procedures, or genetically altered crops with reduced sorption and thus increased rates of agrichemical penetration through the plant cuticle.

G. Epicuticular Waxes and Air Pollutants Since epicuticular waxes are present at the interface between plants and the atmosphere, it is not surprising that both the effects of atmospheric pollutants on epicuticular waxes, and the role of these waxes in tolerance to such pollutants have been investigated. The effects of gaseous pollutants, acid precipitation, and atmospheric deposition on epicuticular waxes has been reviewed by Turunen and Huttunen (1990) and Percy et al. (1994a). A common symptom observed on plants growing in polluted environments is the degradation of surface wax crystals and their accelerated aging. In addition, accompanying changes in chemical

1. PLANT EPICUTICULAR WAXES: FUNCTION, PRODUCTION, AND GENETICS

29

composition, physical characteristics, and rates of biosynthesis have also been documented (Cape 1983; Crossley and Fowler 1986; Turunen and Huttunen 1990; Percy and Baker 1988, 1990; Barnes and Brown 1990; Turunen et a1. 1997). Although the rates at which epicuticular waxes degrade in polluted atmospheres is well correlated with the level of air pollution, it is not clear as to which gaseous pollutant(s) are responsible for symptom development, and the mechanisms involved (Turunen and Huttunen 1990). Exposure to elevated levels of ozone altered epicuticular waxes on conifer needles as measured by altered contact angles (increased wettability) in Norway spruce (Picea abies L.) (Barnes and Brown 1990), and changes in needle wettabilty, wax chemical composition, and reduced rates of wax synthesis in red spruce (Percy et a1. 1992). Such responses are likely dose dependent, as fumigation of Norway spruce with ozone at levels below those used in the previously mentioned investigations, but well above ambient concentrations, did not alter wax structure, surface wettability, or total wax levels (Dixon et a1. 1997). Interestingly, fumigation of in vitro recrystallized epicuticular waxes from Norway spruce, which are composed of nonacosan-10-ol, had no effect on wax crystal structure or chemical composition (Jetter et a1. 1996), suggesting that the degradation of epicuticular waxes on plants growing in ozonepolluted sites is not due to direct oxidative transformation alone, and must involve other mechanisms. Epicuticular waxes are also affected by S02 and N0 2 exposure. Symptoms associated with exposure to elevated levels of these gaseous pollutants either singularly or in combination include altered wax crystal morphology (Karhu and Huttunen 1986), enhanced erosion of epicuticular waxes (Huttunen and Laine 1983; Riding and Percy 1985; Crossley and Fowler 1986; Sauter and Voss 1986; Tuomisto 1988), reduced wax deposition (Riding and Percy 1985), and decreased wettability of needles (Cape 1983). How exposure to the atmospheric pollutants causes such changes is unknown. Direct exposure of recrystallized epicuticular waxes to S02 changed neither the chemical composition nor the morphology of wax crystals, indicating that direct interaction between the pollutant and epicuticular waxes is not the cause for the accelerated erosion observed on plant surfaces (Jetter et a1. 1996). Exposure of these recrystallized waxes to 1% N02 did result in a degradation of wax crystal structure, and a concomitant oxidation of waxes, to cause a change in chemical composition. However, exposure to lower concentration of N0 2 (0.1 %) for prolonged periods had no effect. Since the concentration of N02 used in these in vitro fumigation studies were well above ambient concentrations reported at polluted sites, Jetter and co-workers (1996) concluded that, as with S02' the accelerated erosion of epicuticular waxes was not due

30

M. JENKS AND E. ASHWORTH

to a direct chemical interaction between the gaseous pollutant and epicuticular waxes, and suggest that the degradation of epicuticular waxes must be a secondary effect of tissue interaction with these pollutants. Acidic rain and fog can also affect epicuticular waxes. This has been well documented in conifer species, where a reduction in the hydrophobicity of needle surfaces and an erosion of crystalline wax structures have been reported at both polluted sites, and in response to simulated acid rain treatments (Cape 1983; Huttunen and Laine 1983; Percy and Baker 1988; Barnes and Brown 1990; Turunen and Huttunen 1990, 1991; Percy et al. 1992; Huttunen 1994; among others). Simulated acid rain and acidic fog treatments increased the wettability of leaf surfaces, as measured by decreased water droplet contact angle in several conifer species, including Norway spruce (Barnes and Brown 1990), red spruce (Percy et al. 1992), Sitka spruce (Percy and Baker 1990) and Scots pine (Pinus sylvestris L.) (Cape 1983; Turunen et al. 1997). A similar effect has also been noted in several crop species, including rape, bean (Phaseolus vulgaris L.), pea, and broad bean (Vida laba L.) (Percy and Baker 1987, 1988). Although the reason for the decreased hydrophobicity of leaf surfaces has yet to be resolved, it could occur in response to either changes in surface structural features, altered wax chemical composition, or a combination of both factors. All of these possibilities seem likely, as there is evidence that both the crystalline structure of epicuticular waxes and their chemical composition change in response to simulated acid rain. Epicuticular wax crystals typically weather and degrade to form a more amorphous layer of wax as tissues age, and numerous investigators have reported that this erosion of wax crystalline structures is accelerated in response to acidic precipitation treatments (Cape 1983; Huttunen and Laine 1983; Percy and Baker 1990; Turunen and Huttunen 1990, 1991; Percy et al. 1992; Huttunen 1994; among others). In addition, changes in wax chemical composition in response to simulated acid rain have been reported to occur in several species (Percy and Baker 1987, 1990). How acid rain exposure causes a change in wax crystalline structure, and whether the structural change is linked to changes in wax chemical composition is unclear. Changes in wax structure as a result of chemical interactions between the acidic precipitation and epicuticular wax crystals seems unlikely (Riederer 1989; Percy et al. 1994b; Jetter et al. 1996). In addition, the immersion of recrystallized waxes into sulfuric acid and nitric acid 'mixtures (pH 3) to simulate acid deposition had no effect on wax crystalline structure (Percy et al. 1994b). As an alternative, Percy et al. (1994b) hypothesized that acid rain would affect wax biosynthesis, which would subsequently lead to modifications in wax composition and crystalline structure. They note that enzymes in the wax biosyn-

1. PLANT EPICUTICULAR WAXES: FUNCTION, PRODUCTION, AND GENETICS

31

thetic pathway are sensitive to pH below 5 to 5.5, and that this pH range is ten times less acidic than acid rain, and approaching 500 times less acidic than reported for coastal fog along the eastern coast of North America. These authors also note that exposure to simulated acid precipitation can affect both the rates of wax biosynthesis and the chemical composition (Percy and Baker 1987,1990; Percy et al.1992, 1994b). Although this is an attractive hypothesis, it has not yet been demonstrated that cellular pH levels change to that extent in response to acid rain, or that such changes would lead to altered wax biosynthesis. The well-documented effects of air pollutants and acid rain on epicuticular waxes have been linked to reduced plant growth and forest decline (Turunen and Huttunen 1990). The erosion of epicuticular waxes and the increase in leaf wettabilty would increase the time that water remains on leaf surfaces and facilitate the leaching of mineral nutrients. Erosion of the surface waxes and those associated with stomata may also lead to increased rates of transpiration and predispose tissues to pathogen infection. Therefore, while changes in epicuticular waxes may be one of the first symptoms of air pollution damage, they may also indicate a mechanism by which gaseous pollutants damage plant tissues.

IV. PRODUCTION OF PLANT EPICUTICULAR WAXES

The production of epicuticular waxes over plant surfaces involves a complex integration of biosynthetic and secretory processes. In the following discussion, epicuticular wax production is divided into two general categories including epicuticular wax biosynthesis and epicuticular wax secretion. Epicuticular wax biosynthesis refers primarily to the enzymatic steps and biochemical regulation of wax production. Secretion refers primarily to the physical pathway and transport processes used to move molecules from within epidermal cells to the plant surface, where they are deposited as epicuticular wax. A. Epicuticular Wax Biosynthetic Pathways Depending on plant species, epicuticular waxes are thought to arise from either two or three basic enzymatic pathways, the acyl elongationreduction pathway, the acyl elongation-decarbonylation pathway, and the ~-diketone elongation pathway (Wettstein-Knowles 1995). Fatty acids, aldehydes, primary alcohols, and esters are the primary products of the acyl elongation reduction pathway, whereas fatty acids, aldehydes,

M. JENKS AND E. ASHWORTH

32

and alkanes are products of the acyl elongation-decarbonylation pathway (Fig. 1.4); it is likely that these pathways are found on all plant species (Bianchi and Bianchi 1990; Wettstein-Knowles 1995). In many plants, the acyl elongation-decarbonylation pathway is extended to synthesize secondary alcohols and ketones via enzymatic hydroxylation and oxidation reactions, respectively. The other important group of plant

Activated Fatty Acids

Cuticle Synthesis

bm2 bm6, bm7,g115, waxl - ...-

cer9

I-Alcohols

cer4, giS, bcfl

cer8 bm4, cer-JS9

cer]S, cer6, gl3 *, wb

g12, g14, g116, wa

C21 C23 C25 C27 C29 C31

.:==~~ C32

, I I

I

C38

;'

./'

cer3, cer7S, cerlO, cerl3s, g14*, hlO

C22 C24 C26 C28 C30 C32

..... C37

cerl

Fig. 1.4. Model pathway describing biochemical reactions in plant epicuticular wax production by leaves and putative sites for genetic lesions. Designations of bm represent the bloomless mutants of sorghum, whereas h represents the sparse-bloom mutants of sorghum (Rich 1994), the cer-j59 mutant is an eceriferum mutant of barley (Avato et al. 1982), other cerdesignations represent eceriferum mutants in arabidopsis. The superscript S indicates a stem-specific arabidopsis mutation (Jenks et a1. 1995, M. A. Jenks, unpublished). The waxt and beft are also leaf epicuticular wax mutants in arabidopsis (Jenks et al. 1996a), whereas the designation of gl without asterix represents the glossy mutants of maize (Bianchi et a!. 1985). The gl* designation represents the glossy mutants in various Brassica species (Macey et al. 1970b), whereas wa and wb represent epicuticular wax mutants in pea (Macey et a!. 1970a; Holloway 1977b). The glt, g17, gI8, and gl18 mutations in maize are not shown but were thought to inhibit the production of Ct6 and Ct8 fatty acid wax precursors (Bianchi et a1. 1985).

1. PLANT EPICUTICULAR WAXES: FUNCTION, PRODUCTION, AND GENETICS

33

wax constituents are ~-diketones, hydroxy-~-diketones, and alkan-2-01 esters that arise from a unique biosynthetic pathway called the ~-ketoa­ cyl-elongation pathway that is found in a few plant species, including barley (Mikkelsen and Wettstein-Knowles 1978), carnation, and as minor constituents on cabbage (Post-Beittenmiller 1996). For more information on this ~-ketoacyl-elongation pathway, see the excellent review by Wettstein-Knowles (1995). The following section will focus on the more prevalent acyl elongation-reduction and the acyl elongation-decarbonylation pathways and their associated reactions. Plant epicuticular wax hydrocarbons arise from a pool of Ct6 and Ct8 free fatty acids synthesized within plastids (Post-Beittenmiller 1996). Other studies suggest that cytoplasmic membranes are also sites for synthesis of the Ct6 and Ct8 acid precursors of epicuticular wax (Lessire et a1. 1985). The Ct6 and Ct8 fatty acids arise by activity of the enzyme complex, fatty acid synthetase, which coordinates malonyl-acyl carrier protein's (ACP) sequential donation of seven C2 acyl units to an initial primer, acetyl-Coenzyme A (CoA), to produce the Ct6 fatty acid palmitoyl-ACP. KASII then utilizes the donation of one C2 acyl group from malonyl-ACP to produce the C18 fatty acid, stearoyl-ACP (Ohlrogge and Browse 1995; Wettstein-Knowles 1995; Post-Beittenmiller 1996). Once synthesized, these precursors can be used as substrates in the synthesis of a variety of important plant compounds, including phospholipids, storage lipids, cutin, suberin, and epicuticular waxes. The Ct8 precursors destined for modification into epicuticular waxes undergo further elongation reactions that create chain lengths up to 36 carbons long (Fig. 1.4); the exact length being dependent on plant species, organs, and environments. 1. Elongases. In the past, it was presumed that Ct6 and Ct8 fatty acid precursors of epicuticular waxes were being further elongated by several chain-length-specific acyl-CoA elongases, since certain mutations and chemical inhibitors appeared to suppress production of wax homologues longer than a certain length. For example, the cer-j59 mutant of barley apparently inhibited the putative C24 acyl-CoA elongase, since wax homologues longer than 24 carbons were greatly reduced (Avato et a1. 1982). The bm4 mutation in sorghum (Rich 1994), the cer2 and cer6 mutations in arabidopsis (Jenks et a1. 1995), the wb mutation in pea (Macey and Barber 1970a), and the g13 mutation of cabbage (Macey and Barber 1970b) appeared to inhibit a putative C26 elongase. Elongation of the C28 constituents appeared to be inhibited in the cer19 mutant in arabidopsis (M. A. Jenks, unpublished), the g13 mutant in maize (Bianchi et a1. 1985), and g12 and g15 mutants of cabbage (Macey and Barber

34

M. JENKS AND E. ASHWORTH

1970b). Moreover, the g12, g14 and gliB mutations in maize (Bianchi et a1. 1985) and wa of pea (Macey and Barber 1970a) appeared to suppress a C30 elongase. Although research by Bessoule et a1. (1989) bolstered the concept of multiple elongases by showing that separate C18 -CoA and CzoGoA elongases could be isolated from leek (Allium porrum), the elongases themselves that act on acyl-CoA chains longer than 20 carbons have not been isolated and purified. Thus, whether separate elongases exist for each two-carbon addition to growing acyl chains longer than 20 carbonases has not been proven. Furthermore, thioesterases associated with fatty acid synthase were shown to govern the chain length distribution for synthesis of the 8 to 20 carbon length fatty acids (Voelker et a1. 1997). Thus, a single elongase complex could be responsible for synthesis of all acyl-CoA chains longer than 20 carbons through the mediating activity of thioesterases and other elongase-associated enzymes. In the mutation and inhibitor studies mentioned above, evidence for suppression of single elongation steps could be explained by direct effects on enzymes mediating the activity of a single elongase, rather than inhibition of one of several elongases directly. For these reasons, it is still unclear whether elongation of the very long chain acyl-CoAs in epicuticular wax biosynthesis is performed by one or more elongases. 2. Thioesterases. Previous research suggested that fatty acyl chains were released from elongase complex(es) by either fatty acyl-CoA thioester hydrolysis to fatty acids and CoA, or by fatty acyl-CoA reduction to aldehydes. Acyl-CoA thioesterase activities have been reported in plants (Ohlrogge et a1. 1978; Liu and Post-Beittenmiller 1995). Specifically, Liu and Post-Beittenmiller (1995) isolated an epidermally-expressed acyl carrier protein thioesterase with high specificity for stearoyl-CoA (C 18) substrates. Moreover, Pollard et a1. (1991) cloned an acyl-ACP-thioesterase from bay laurel (Umbellularia california Hook. & Arn.) that terminated elongation to produce medium chain-length acids, and Voelker et a1. (1997) used thioesterase genes to alter medium chain lengths in transgenic rape storage lipids. Although only acyl-ACP thioesterases have been isolated to date, it is possible that additional thioentrases may cleave acyl-CoA and thereby play an important role in determining chain lengths of products released from this pathway. Alterations in epicuticular wax chemical profiles among certain mutant lines may be explained by mutations affecting thioesterases or associated reactions. It has been proposed that low amounts of fatty acids and aldehydes and longer wax chainlengths on cer3, cer7, ceri0, and cer13 mutants in arabidopsis may be due to inhibited release of acyl chains from elongation compartments (Fig. 1.4; Jenks et a1. 1995; Jenks,

1. PLANT EPICUTICULAR WAXES: FUNCTION, PRODUCTION, AND GENETICS

35

unpublished). The h9 mutant of sorghum (Rich 1994) and the g14 mutant of cabbage (Macey and Barber 1970b) had similarly increased chain lengths among epicuticular wax constituents. Potentially, the longer chain length constituents on these mutants could have resulted from suppressed thioesterase activity. 3. Reductases and Decarbonylases. The conversion of fatty acyl-CoA to

aldehydes is thought to be catalyzed by a putative microsomal fatty acyl-CoA reductase that lacks chain-length specificity (Kolattukudy 1971). Recently, two separate acyl-CoA elongases were solubilized from pea (Vioque and Kolattukudy 1997; Kolattukudy 1996), one that apparently generates primary alcohols and another that generates aldehydes from the acyl-CoA precursors. These results suggest that the model in Fig. 1.4 may be modified to include separate aldehyde pools for the 1-alcohol and alkane branches of the pathway. In the next metabolic step, aldehydes can be converted to either primary alcohols or alkanes. A microsomal aldehyde reductase that lacks chain length specificity may produce primary alcohols from aldehydes (Kolattukudy 1971) or alternately via a two-step reduction from the acyl-CoA, Aldehydes may also be converted to alkanes by an aldehyde decarbonylase that, like the reductases, appears to lack chain-length specificity (Cheesbrough and Kolattukudy 1984). Heavy sucrose gradient fractions containing cell wall and cuticle fragments were capable of enzymatic decarbonylation of aldehydes with chain lengths of Ct6 to C32 , while fractions lacking wall and cuticle fragments lacked decarbonylation activity (Cheesbrough and Kolattukudy 1984). These findings suggest that aldehydes produced in intra-cytoplasmic membranes of epidermal cells were likely converted to alkanes by a decarbonylase enzyme located in the cell wall or cuticle region. 4. Oxidases and Transacylases. In many plant species, secondary alco-

hols and ketones constitute a significant portion of epicuticular waxes. For instance, the stems of arabidopsis have a C29 ketone and C29 secondary alcohol as the second and third most abundant constituents, with the C29 alkane being the first (Hannoufa et a1. 1996). Kolattukudy et al. (1973) presented evidence that alkanes are converted into secondary alcohols by a hydroxylase. These alcohols are then converted to the corresponding ketone by an oxidase. However, genes or the enzymes responsible for these oxidative reactions have yet to be isolated. Esters likely arise from esterification of primary alcohols and fatty acylCoA by a acyl-CoA-fatty alcohol transacylase, whose activity was detected in microsomal fractions (Kolattukudy 1967). This transacylase may have

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some specificity toward shorter-chain-length l-alcohol and acid homologues. Nevertheless, although an acyl-CoA:fatty alcohol acyltransferase involved in the synthesis of liquid seed storage wax esters has been isolated from jojoba (Simmondsia chinensis Link) seeds (Shockey et al, 1995), neither genes nor enzymes responsible for esterification reactions in the synthesis of crystalline epicuticular wax esters have yet been isolated. 5. Potential Novel Functions in Epicuticular Wax Biosynthesis. An important question concerning epicuticular wax biosynthetic pathways in plants is whether multiple biosynthetic pathways exist within the same plant. Potentially, more than one pathway might exist in the same tissue. For example, the ~-ketoacyl-elongation pathway apparently exists side-by-side with the reduction and decarbonylation pathways in barley. This is based on chemical analysis of the cer-cqu mutation that apparently affects only the ~-diketone elongation pathway (WettsteinKnowles and Sogaard 1980). Whether multiple acyl elongation pathways exist in single organs has also not been ruled out. Other studies suggest that multiple pathways could exist in different organs of the same plant. For example, many plants have distinct epicuticular wax composition on various tissues (Table 1.1). Also, studies using glossy mutants in maize suggest that two independent epicuticular wax enzymatic systems, designated EDI and EDII (elongation-decarboxylation systems I and II), are predominantly involved in seedling and adult leaf epicuticular wax biosynthesis, respectively (Bianchi et al. 1985). Furthermore, many wax mutations in sorghum were specific to individual tissue types (Jenks et al. 1992; M. A. Jenks, unpublished). Interestingly, even different epidermal cell types may have separate epicuticular wax biosynthetic pathways. The bm3 and bmll mutations in sorghum inhibited wax production from epidermal cork cells but not epidermal long cells, whereas most other wax mutations affected both cork cell and long cells. Wildtype maize has wax crystals over the entire seedling leaf epidermal surface, except that wax crystals are not present over guard cells. It was surprising then to discover that the gIl mutation in maize seedlings inhibited wax production on all epidermal long cells except stomatal accessory cells, which exhibited near-normal wax crystals (Lorenzoni and Salamini 1975). Thus, the GIl gene product may not be as highly expressed in accessory cells as long cells. While such ideas are intriguing, it is still yet to be determined whether multiple wax pathways could exist in single organs, whether wax biosynthetic pathways could be organ- or cell-specific, or whether the findings discussed above are better explained by complicated regulation of a single wax pathway.

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Analysis of other mutants provides evidence that numerous genes contribute to epicuticular wax biosynthesis by specifying complex regulatory functions. For example, the CER-yy gene· in barley may have a regulatory function in epicuticular wax biosynthesis, since the cer-yy mutation converted spike-type to leaf-type epicuticular wax (Lundqvist and Wettstein-Knowles 1982). By comparison, gi15 in maize affected the developmental transition from juvenile to adult leaf epicuticular wax biosynthesis and the recently sequenced GL15 gene has sequence homology to the arabidopsis floral-development regulatory APETALA2 gene family (Moose and Sisco 1995). Arabidopsis cer2 inhibits stem specific C26 elongation but does not affect leaf epicuticular waxes (Jenks et a1. 1995), suggesting either organ-specific expression or differential regulation of acyl-CoA elongation. Likewise, the cer7 and cer13 mutations in arabidopsis may affect wax chain length in a stem-specific manner (Jenks et a1. 1995; M. A. Jenks, unpublished). Genes that dramatically reduce the total amount of epicuticular waxes when mutated are thought to inhibit major regulatory or metabolic functions of early substrate conversion in the wax pathway. For example, cerl and cer16 in arabidopsis (Jenks et a1. 1995), gil, gi7, g18, and gl18 in maize (Bianchi et a1. 1985), and bm2, bm5, bm6, and bm7in sorghum exhibited between three- and ten-fold reductions in total waxes compared to wildtype. The bm2 mutation also reduced the deposition, and altered the ultrastructure of the cuticle (Jenks et a1. 1994a). These mutants likely affect genes that playa role in important early steps in wax biosynthesis. Recently, a new class of epicuticular wax mutants was isolated from aT-DNA mutagenized population of arabidopsis and designated wax, bcf(bicentifoJia), and knb (knobhead) (Jenks et a1. 1996a). These mutants exhibited plieotropic effects on surface wax chemistry, wax crystallization pattern, and leaf cell morphology. Interestingly, tissues on the waxl mutant fused together very early in organ development similar to the fdhl (fiddleheadl) mutant in arabidopsis (Lolle et a1. 1992). The knb mutants are similar to a class of sorghum wrinkled-leaf epicuticular wax mutants identified in an chemically mutagenized population (M. A. Jenks, unpublished). As surface lipid and cell wall constituents are arguably the major secretory products of epidermal cells, these new mutants could have alterations in shared surface lipid/cell wall secretory mechanisms. Conversely, altered cell wall morphology could simply alter secretion of epicuticular wax precursors from the epidermal cytoplasm to the plant surface. Regardless, this class of mutants should provide opportunities for investigating wax biosynthesis and secretion.

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B. Epicuticular Wax Secretion Previous studies have shown that the basic mechanisms controlling cell secretion processes are similar in cells as divergent as yeasts, plants, and mammalian brain neurons (Moore et al. 1991; Barinaga 1993). Thus, secretion of epicuticular waxes likely involves many of these same highly conserved mechanisms. Presumably, surface wax precursors are first synthesized in the cytoplasm of epidermal cells and then secreted in sequence through the cytoplasm, apical plasmalemma, secondary cell wall, primary cell wall, pectic layer, and cuticle (Fig. 1.5). 1. Early Studies of Epicuticular Wax Secretion. In 1679, Malpighi reported using a compound light microscope to identify the outermost plant epidermal layer (see Hallam 1982). The first microscopic description of epicuticular wax deposits may have been Brongniarts's (1834) description of a granular morphology on certain plant surfaces. Von Mohl (1842) used the light microscope to describe two cuticle layers, the primary cuticle and a fibrilla-filled secondary cuticle directly below the

Epicuticular Wax Primary Cuticle Secondary Cuticle

Epidermal Cytoplasm

Fig. 1.5. Diagram showing the arrangement of cuticle and cell wall in the apical portion of the leaf epidermis. The secondary cuticle contains mostly cutin and carbohydrates, the lamellate primary cuticle consist mostly of cutin and subcuticular waxes, whereas the epicuticular wax layer is dominated by aliphatic wax constituents (although aromatic constituents can also occur at high levels in many plant species).

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epicuticular waxes. In following years, numerous theories for production of cuticle components were espoused. Fibrillae in the secondary cuticle led Karsten (1857) to postulate that the cellulose cell walls were chemically modified in situ to form cuticle layers. The first detailed description of epicuticular wax structure by DeBary (1871) led him to postulate that epicuticular waxes were transported to the surface and molded by pores in the cell wall and cuticle. Observing the crystalline appearance of epicuticular wax, Wiesner (1871) hypothesized that these waxes were secreted in a volatile solvent that evaporated after emergence from the cuticle. In later studies, the absence of visible pores or channels traversing the cell wall and cuticle layers led Weber (1942) to suggest that diffusable wax components were secreted in a liquid form that crystallized on the surface. Although recent studies have shed further light on wax secretion mechanisms, many basic questions remain unanswered. 2. Cellular Origins for Epicuticular Wax Secretion. Because wax crystals can be seen using scanning electron microscopy (SEM) over all plant epidermal cell types, it has been assumed that essentially all epidermal cells produce epicuticular waxes. While this assumption likely holds true, some plants have distinct types of epidermal cells that produce specialized epicuticular waxes. McWhorter and Paul (1989) reported on a wax-producing cork-silica cell complex in the epidermal layer of]ohnsongrass (Sorghum halepense L.). They proposed that adjacent cork and silica cells functioned together in the production of filamentous epicuticular waxes. Later studies using sorghum indicated that epidermal cork cells (Fig. 1.6) generated tubular wax filaments (Fig. 1.7) on sorghum leaf surfaces, whether in association with silica cells or not (Jenks et a1. 1994b). Although the role of silica cells is unclear, the specialized wax-producing cork cells on sorghum provide a model cellular system for investigating plant epicuticular wax secretion. Examination of tissues using transmission electron microscopy (TEM) have identified possible epicuticular wax precursors in the cytoplasm of sorghum epidermal cork cells. Large osmiophilic globules were visualized inside both vesicular and plastid membranes of cork cells (Paul and McWhorter 1990). However, similar globules were not found in cork cells during inducible and rapid epicuticular wax secretion that followed exposure to light, even though they were present in cork cells before light exposure (Jenks et a1. 1994b). Moreover, osmium tetroxide preferentially binds unsaturated lipids and epicuticular wax precursors are assumed to exist as primarily saturated lipids. Thus, further studies are needed to determine whether, in fact, these osmiophilic globules are the direct precursors of surface waxes.

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Fig. 1.6. Transmission electron micrograph showing the ultrastructure of an epicuticular wax-producing cork cell in the abaxial sheath epidermis ofsorghum. Notice the thicker cell walls, many small vacuoles, large nucleus, and dense cytoplasm with numerous organelles. The function of the osmiophilic (dark staining) layer in the apical cell wall that contacts the cuticle within the papillae [upper right] is yet unclear. Bar = 5 J..lffi. Source: Jenks at al. 1994b.

There is evidence that cellular membranes playa role in the production of epicuticular waxes. For example, the cytoplasmic density of tubular smooth endoplasmic reticulum membranes increased dramatically in sorghum cork cells during light-induced wax secretion, whereas cork cell wax mutants, unable to produce wax, did not show a similar change in ER membrane density (Jenks et al. 1994b). These studies imply that additional endoplasmic reticulum is produced to support the increased synthesis of waxes following light induction. Perhaps increased endoplasmic reticulum surface area provides increased locations for embedded wax biosynthetic enzymes. Other evidence for endoplasmic reticulum involvement in wax production comes from ultrastructural studies of insect wax-producing cells. The wax gland cells of several insect species were full of smooth endoplasmic reticulum (Marshall et al. 1974; Waku 1978; Percy et al. 1983; Foldi and Pearce 1985). A closer look at the ultrastructure of wax secretion cells of insects and sorghum show significant homologies, indicating that studies on waxes in plants may benefit from comparative studies with insects. Like microscopic analysis, studies involving cell fractionation have also associated cytoplasmic membranes with epicuticular wax production. For example, enzymatic activity associated with elongation of long-

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Fig. 1.7. Low temperature scanning electron micrograph showing hollow filaments of epicuticular wax on abaxial sheath surfaces of sorghum. Groups of filaments are above epidermal cork cells. The entire cuticle surface is densely coated with small plate-like wax crystals. Bar = 20 J.lrn. Source: Jenks et a1. 1994b.

chain epicuticular wax hydrocarbons occurred within at least three cytoplasmic locations. C1a-CoA elongases were located in the endoplasmic reticulum enriched fraction (Cassagne and Lessire 1978; Moreau et al. 1988a, 1988b), C2o-CoA elongases were found in the Goigi apparatus enriched fraction (Moreau et al. 1988a), and ATP independent elongases were in a third uncharacterized sucrose gradient fraction (Moreau et al. 1988a, 1988b). These three elongase activities were not active in either isolated plasma membranes (Moreau et al. 1988a, 1988b) or isolated protoplasts (Mikkelson 1980). Thus, C20 acyl chains may have been synthesized in the endoplasmic reticulum by C1a-CoA elongases, which then may have been transferred to the Goigi apparatus where they were elongated by the C2o-CoA elongases to C22 acyl chains before being passed on to the next elongation reaction. Interestingly, surface wax extracts of most plants generally contain very little of the C20 and C22 fatty acids compared with longer fatty acid constituents. As discussed, synthesis of the C20 and C22 fatty acids may be occurring at locations different from those of the longer fatty acids. Like elongases, acyl-CoA reductase and aldehyde reductase activities were also associated with

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cytoplasmic membranes (Cheesbrough and Kolattukudy 1984). In future studies, antibodies made against purified wax biosynthetic enzymes may be useful, along with TEM-immullocytochemical approaches to localize enzyme activities to specific cellular locations, such as within the endoplasmic reticulum. Although the circumstantial evidence for cytoplasmic membrane involvement in epicuticular wax production is strong, further studies are needed to establish the association of cytoplasmic membranes with wax biosynthetic and secretory enzymes.. 3. Transport of Epicuticular Wax Precursors through the Cytoplasm and Plasmalemma. Studies using TEM have provided evidence that epicuticular wax precursors are transported through the cytoplasm in secretory vesicles. In the case of heterophyllous aquatic plants, Myriophyllum aquaticum (VeIl.) Verde. produced a glaucous coating after emergence above the water surface, whereas M. verticillatum L. produced no visible epicuticular waxes after emergence (Hallam 1982). During this emergence period, the glaucous M. aquaticum produced Golgi derived vesicles that fused with the apical plasmalemma of epidermal cells. Then, these vesicles discharged their osmiophilic contents into the apoplasm. By comparison, the nonglaucous M. verticillatum produced very few vesicles at any time (Hallam 1982). Sorghum cork cell vesicles, possibly carrying epicuticular wax precursors, were also shown to fuse with the apical plasmalemma and then discharge their osmiophilic contents into the apical extra-periplasmic space (Paul and McWhorter 1990; Jenks et al. 1994b). Thus, evidence suggests that epicuticular wax precursor transport in the cytoplasm could utilize vesicles that release their contents to the cell exterior. Vesicular discharge from the cell appears to occur via exocytosis, a highly conserved process exhibited in many diverse organisms (Barinaga 1993). The exocytosis of epicuticular wax precursors, however, has not been demonstrated with certainty, as has been done with other plant secretory products. For example, Ornberg and Reese (1981) presented highresolution electron microscopic images of early vesicle contact and initial pore formation leading to fusion of secretory vesicles with the plasmalemma. Radioactive labeling indicated that these vesicles carried cell wall constituents. Epicuticular wax precursors might likewise be secreted in a similar manner. Methods are needed to tag wax precursors so their transport can be visualized using TEM. It has been demonstrated that clathrin coat proteins are involved in secretory vesicle formation and membrane targeting. Coat proteins were visible on vesicles during budding and transport away from Goigi bodies in a cell free system (Ord et al. 1989). However, similar coat mater-

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ial was not visible on sorghum cork cell vesicles thought to carry epicuticular wax precursors (Paul and McWhorter 1990; Jenks et a1. 1994b). In addition, numerous membrane fusion and associated proteins, like annexin and synexin (Creutz 1992), clearly mediate exocytosis. Whether homologous proteins are involved in epicuticular wax secretion from plant epidermal cells is unknown. Instead of exocytosis, Hallam (1982) suggested that unique enzyme carrier molecules could be used for epicuticular wax precursor transport across the apical plasmalemma. Hallam's postulate was based on a Smith et a1. (1978) description of fatty acid transport by low-density lipoproteins across plasma membranes in protein envelopes with hydrophobic interiors and hydrophylic exteriors. These lipid-protein complexes were visible in electron micrographs as 22 nm diameter osmiophilic globules. As with vesicle coat proteins, globules similar to the lipid-protein carrier molecules have not been visualized on putative epicuticular wax precursor carrying vesicles. In spite of that, these studies raise the possibility that transfer proteins, and not cytoplasmic vesicles, may be the carriers of the plant epicuticular wax precursors in the secretory pathway. Thus far, there have been few studies of epicuticular wax secretion, and our knowledge of this process is extremely limited. Nevertheless, the assumption that secretory mechanisms are highly conserved allows inferences to be made from other systems. Clearly, many important questions still remain in wax cytoplasmic secretion. For example, are vesicles involved in wax secretion? How are wax precursors targeted for transport out of the apical walls of epidermal cells? Do wax vesicles have special apical plasma membrane targeting signals, and does the plasma membrane have wax vesicle receptors? Alternatively, do carrier molecules themselves have a mechanism to target the apical plasmalemma? What other transfer-mediating molecules are involved? Do separate cytoplasmic secretory pathways exist for separate epidermal secretory products, such that separate vesicles carry cell wall and surface lipid components? Presumably, the movement of epicuticular wax precursors through the cytoplasm and plasmalemma involves a host of complicated interactions. 4. Transport ofEpicuticular Wax Precursors through the Cell Wall and

the Cuticle Proper. The outer layers of the epidermis of aerial plant tissues provide a structurally and chemically complex boundary through which epicuticular waxes must be secreted in order to appear on the surface. This part of the secretory pathway is potentially complicated by the requirement that wax precursors must move from the chemically

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reductive environment of the epidermal cytoplasm to the increasingly oxidative environments of the cell wall, cuticle, and plant surface. Epicuticular wax microchannels traversing the cell walls of plants have not been clearly identified, although some investigators have presented evidence for their existence. Cell wall microchannels, visible using freeze-fracture techniques with low temperature SEM, did not appear to extend through the entire cell walls of clover (Trifolium pratense L.), eucalyptus, and cauliflower (Hall 1967a). In addition, cuticular pores of clover imaged using replication techniques with SEM appeared too large and were not clearly associated with epicuticular wax structures (Hall 1967b). Moreover, it is possible that solvents used to remove wax crystals on clover could have created the surface pitting (pores) that were shown. Also, the role of birefringent pectin-filled channels in epidermal cell walls, made visible with the light microscope, are still unclear (Hiilsbruch 1966). Fisher and Bayer (1972) used TEM to describe 2.5 nm electron translucent channels through the cuticle of plantain (Plantago major. L.), but these results were inconclusive, since channels were not visible in all tissue preparations or specimens examined. TEM studies by Jeffree et al. (1976) and Jenks et al. (1994b) supported Weber's (1942) analysis using light microscopy, which indicated that detectable channels did not traverse the cell walls in plants (Fig. 1.8). Such observations suggest that epicuticular wax precursors may diffuse through microscopic spaces within the cell walls, as opposed to mass flow through discrete larger pores or channels, to reach the tissue surface. Paul and McWhorter (1990) described osmiophilic globules within the cell walls of sorghum cork cells as putative epicuticular wax precursors. Since globules in the cell wall were smaller than those in the cytoplasm, they proposed that these globules were being reduced in size as they diffused through the cellulose meshwork of the cell wall. Osmiophilic globules were not, however, visible in the cuticle proper (Paul and McWhorter 1990). Similar results were observed in other species where osmiophilic globules accumulated directly beneath the cuticle, but where not visible within the cuticle (Heide-Jorgenson 1978; Hallam 1982). If these globules represented epicuticular wax precursors, it is yet unclear why they were not visible within the cuticle. Most likely, these globules were either not epicuticular wax precursors as has been suggested, or else these precursors were chemically modified in the cell wall, making them no longer reactive with osmium tetroxide (and thus unable to be visualized using TEM) when in the cuticle. Epicuticular wax precursors must also cross the pectinaceous layer that separates the outer portion of the cell wall and the innermost layers of the cuticle. No studies have yet elucidated how epicuticular wax precursors are trans-

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ported across the cell wall and pectinaceous layer, and this remains an important area for investigation. It is unclear how epicuticular wax precursors are transported through the cuticle. Plant cuticles, like plant cell walls, do not possess microchannels that traverse their entire thickness (see Fig. 1.8; Jenks et a1. 1994a; Anton et a1. 1994). By comparison, insect cuticles have distinct microchannels through which epicuticular waxes are secreted (Locke 1961; Foldi and Pearce 1985). In plant epicuticular wax secretion through the cuticle proper, wax precursors must first traverse the secondary cuticle and then the primary cuticle (Fig. 1.5). The secondary cuticle is composed primarily of a polyester cutin meshwork formed from various Ct6 and Ct8 fatty acid monomers, and is thought to have a lower proportion of waxes than the primary cuticle. Chafe and Wardrop

Fig. 1.8. Transmission electron micrograph showing the ultrastructure of papillae in the apical cell wall of cork cells in the epidermis of sorghum. Small arrows indicate the location from which epicuticular wax filaments emerge. Channels for wax precursor transport are not visible. The function of the osmiophilic layer (larger arrow) in the cell wall that contacts the cuticle within the papillae is yet unclear. Bar = 1 Jim. Source: Jenks et a1. 1994b.

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(1973) suggested that fibrillae within the secondary cuticle layer served as transport channels for epicuticular wax precursors. This hypothesis was supported by Bocher's (1975) light microscope description of cloudy birefringent material, potentially intra-cuticular waxes, at distal ends of microchannels extending into the primary cuticle. Since channels through the primary cuticle were not detected, fibrillae in the secondary cuticle layer were thought to serve only as the beginning of a diffusion pathway. By comparison, the primary cuticle of several plant species possesses alternating opaque and translucent layers (visible with TEM) , composed of cutin biopolyester (Wattendorf and Holloway 1980) and waxes (Heide-Jorgenson 1991), respectively. Interestingly, the alternating layers of chitin microfibrils in insect cuticles are laid down in alternating directions during the day and night (Hadley 1986). In like manner, the layered appearance of plant primary cuticles may be a result of directional night-day deposition of plant cuticle constituents. In spite of many detailed studies, specific ultrastructures within the plant primary cuticle have not been directly implicated in epicuticular wax secretion pathways. Two hypotheses have been set forth to describe mechanisms for the transport of epicuticular wax precursor through cuticle layers. Jeffree et a1. (1976) suggested that epicuticular wax precursors might diffuse through the cuticle layers in association with a volatile carrier. While plants emit a large number of volatile compounds from their surfaces, none of these have been ascribed the function of a wax carrier. An alternative hypothesis is that wax secretion involves lipid transfer proteins. Pyee et a1. (1994) identified a lipid transfer protein as the major protein in the epicuticular waxes of broccoli, and other investigators have detected lipid transfer protein in cell walls and associated with the epidermis (Sterk et a1. 1991; Thoma et a1. 1993). These proteins may act as carriers in transport of epicuticular wax precursors through the cuticle. However, it is difficult to envision how these carrier proteins could traverse the cell wall and cuticle layers and then not accumulate in much larger quantities on the surface than reported (Pyee et a1. 1994). Nevertheless, the surface location of these lipid transfer proteins implies an associated function in epicuticular wax secretion. Finally, the discovery of decarbonylase activity in centrifuge fractions enriched in cell walls and cuticles indicates that chemical modification of aldehydes to alkanes may be occurring outside the cytoplasm in the cell wall or cuticle layers (Cheesbrough and Kolattukudy 1984). Immunolocalization studies using antibodies raised against wax synthesizing enzymes would provide a powerful approach for locating these enzymes within the wax secretory pathway of the epidermis.

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5. Cuticular Involvement in Shaping Wax Morphology. Juniper and Bradley (1958) used carbon-coat replicas and SEM to produce the first high-resolution images of plant epicuticular wax crystals. Using SEM, Hallam (1970a) was unable to detect cuticular pores, and proposed that epicuticular wax precursors diffused evenly through primary cuticle lamellae and crystallized on the surface into unique structures. Jeffree et a1. (1975), using an apparatus that allowed dissolved waxes to crystallize upon extrusion through microscopic pores, determined that the chemical composition of wax constituents played a major role in the formation of epicuticular wax structures, while the size and distribution of surface pores had little effect on crystal morphology. This conclusion was supported by subsequent studies showing that in situ plate-like epicuticular waxes of Johnsongrass could be dissolved with chloroform, and then recrystallized on glass to form nearly identical structures (McWhorter et a1. 1990). Nevertheless, wax chemical composition does not completely dictate epicuticular wax crystallization patterns, since in vitro recrystallized waxes often exhibit different crystallization patterns than observed in situ (Jeffree et a1. 1975; M. A. Jenks, unpublished). Many insects possess unique cuticular modifications that affect the crystallization patterns of their epicuticular waxes. For example, the sinusoidally-corrugated hollow filament epicuticular wax of the insect, Epipyrops anomala, are formed by wax secretion from around the edges of 2 flm diameter lanceolate papillae with longitudinal ridges (Marshall et a1. 1974). Cytoplasmic extensions of 10 nm diameter into the cuticle of E. anomala epidermal cells appeared to transport epicuticular wax precursor to the base of the papillar superficial ridges, at sites where the waxes were then deposited. Wax deposits were then pushed upward and assumed the cross-sectional shape of the papillae as more waxes were deposited below. Another such example occurs in which certain scale insects produce epicuticular wax structures from specialized 18-celled wax glands. Epicuticular wax precursors are secreted through 10 nm pores at the base of secretory holes in a modified cuticle (Foldi 1981). Discs with 25 secretory holes were positioned over each gland. Each hole emitted single 3 nm to 3.5 nm diameter microfilaments that rapidly fused together to form an approximately 30 flm diameter by 1-cm- to 2em-long hollow wax filament. In contrast, the quinquelocular glands of the female scale insect (Pulvinaria regalis Canard.) exuded epicuticular waxes through bifurcate or trifurcate locules producing wax filaments with a C-shaped cross section (Foldi and Pearce 1985). P. regalis also had a tubular duct gland with 1.4 flID ducts with hexagonal shape. Waxes frOIn these duct glands were secreted through the 10 nm pores in a modified cuticle, and molded into the hexagonal shape determined by the

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tubular duct. Thus, insect wax secretion and wax morphology are clearly influenced by structural features at the site of secretion. By comparison, few plants appear to have distinct cuticular morphologies that influence the shape of epicuticular wax structures. For example, the mature leaf surface of New Zealand flax (Phormium tenax J. R. Frost. & G. Forst.) had large numbers of epicuticular wax crystals over regions with thickened secondary cuticles (Jarvis and Wardrop 1974). However, the cuticle appeared to have no influence on individual crystal shape. The only study, to date, that shows a clear effect of secretion site morphology on plant epicuticular wax crystallization patterns was reported by Jenks et al. (1994b). In these studies, epidermal cork cells in sorghum abaxial leaf sheath epidermis produced 1 Ilm diameter tubular waxes from the perimeter of 1 flm diameter papillate structures. These structures were formed by the apical cork cell wall and extended up to 1 flm above the cork cell surface (Fig. 1.6; 1.8). These wax-producing cork-cell papillae appear analogous in function to the 2 flm diameter lanceolate papillae on the wax-producing epidermal cells of the insect, E. anomala, discussed above (Marshall et al. 1974). This is the only example of wax crystallization patterns in plants being influenced directly by cuticular morphology. 6. Crystallization of Epicuticular Wax on Plant Surfaces. The crystallization patterns characteristic of individual, or mixtures of, wax chemical components are thought to play a major role in determining epicuticular wax morphology. For example, plate waxes and lobed plate waxes were associated with alcohol constituents (Hallam and Chambers 1970; Prasad and Giilz 1990), whereas thin tubes, or "loofah-like" tubes, were associated with ~-diketones (Wettstein-Knowles 1974). High concentrations of nonacosonal-10-01 were associated with short, stubby tubes (Jeffree et al. 1976). Jetter and Riederer (1995) recently demonstrated that minor amounts of alkanediols mixed with nonacosan-10-ol acted to stabilize tubular wax structures. Comparably, slight alterations in the relative quantities of hydroxy-~-diketone and ~-diketones determined whether tubular epicuticular wax structures or ribbon-like structures were formed on barley and respective epicuticular wax (eceriferum) mutants (Wettstein-Knowles 1974). Such observations indicate that minor alterations in wax biosynthetic pathways either due to regulation or mutation could result in altered epicuticular wax crystal morphology. Environmental conditions can influence epicuticular wax structure. This may occur due to altered rates of epicuticular wax secretion (Wettstein-Knowles 1974). This mechanism is consistent with von Weir-

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man's empirical law, which states that the more rapid the rate ofnucleation and the greater the number of nuclei formed prior to relief of supersaturation, the smaller the final crystal size (Adamson 1976). Thus, differences in crystallization rates may explain the report by Jeffree et al. (1976) showing that wax structures recrystallized in vitro were often more amorphous than waxes produce in situ. Alternatively, environmental conditions might alter epicuticular wax structure by affecting wax biosynthesis rather than crystallization. Thus, small changes in wax chemical composition could have pronounced effects on crystal structure. The crystallization of plant epicuticular waxes likely occurs according to basic crystallization theory. For example, filaments formed in situ on Douglas fir (Thair and Lister 1975), and Sitka spruce (Johnson and Jeffree 1970) had the same crystal dimensions and orientation as recrystallized wax filaments. Hollow tube waxes with asymmetric end structures were produced both in situ and in vitro. These irregular end structures may be produced by a screw dislocation pattern of crystallization, whereby structural tension induces a shear or slip in the spiral crystal (Adamson 1976). By comparison, carbon-replicas of recrystallized hollow wax filaments from Douglas fir had 26 degree (relative to the long axis of the crystal) pitched striae (Lister and Thair 1981). The spacing between the parallel striae was 10 to 12 nm, and electron diffraction patterns indicated a helical orientation for the recrystallized wax molecules. This basic hollow shape of crystals grown from a solvent may be caused by low solid to liquid surface tension (Adamson 1976). Thus, mechanisms for production of plant epicuticular wax crystalline structures may emulate crystal growth from a solvent with final morphology dependent, in large part, on chemical composition. Likewise, insect epicuticular wax crystallization appears to follow basic crystallization mechanisms. The tubular wax filaments produced on the surface of the woolly alder aphid (Prociphilus tesselatus Fitch), are composed of nearly pure paraffin-like wax molecules, 15-oxotetratriacontyl13-oxodotriacontanoate (Dorset and Ghiradella 1983). Electron diffraction patterns from recrystallized and in situ n-paraffin waxes demonstrated that the hollow filament structures seen on insects are probably formed by a wax monolayer with long chain axes directed along cross-sectional radii. The fluted template from which epicuticular wax precursors arise, and the side-to-side packing of paraffin chains probably contributed to tubular growth patterns. In addition, Van der Waal's forces may provide attraction between side chains of opposite chirality to stabilize the filaments in a helical spiral (Dorset and Ghiradella 1983). Dorset and Ghiradella (1983) surmise that the tubular

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crystals produced on the aphid are entirely consistent with well-known crystallization from circular closure at the secretion site. Clearly, many factors influence the final morphology of epicuticular wax crystals on the surface of plants. Genetic engineering or cultural practices used to create specific alterations in epicuticular wax crystallization patterns may require precise alterations in chemistry or secretion site morphology. In order to produce a specific change, precise modification of wax biosynthetic or secretory pathways may also be required. Obviously, further studies are needed to elucidate the complex physical and chemical factors that determine the final shape of plant epicuticular waxes.

V. GENETICS OF EPICUTICULAR WAXES

Although the environment has significant effects on epicuticular wax structure and chemistry, genes and their products ultimately have dominant control. As reviewed in Section III, the structural and chemical characteristics of epicuticular waxes play a significant role in plant resistance to environmental stress. Therefore, genetic modification of crops to alter their epicuticular wax profiles has tremendous potential for improving crop stress resistance. However, very little is currently known about how genes function in epicuticular wax production. The remainder of this review will discuss current knowledge of genetic involvement in epicuticular wax production, and the potential for isolating wax genes that may be useful for modifying these lipids to improve crop stress resistance. The molecular-genetics of epicuticular waxes has been reviewed by Schnable et a1. (1994), Wettstein-Knowles (1995), Lemieux (1996), and Post-Beittenmiller (1996). A. Genetic Involvement in Epicuticular Wax Diversity

Variation in the epicuticular wax profiles of horticultural and agronomic crops can often be ,explain by differences that exist in the plant genome. For example, the amount of epicuticular wax on various accessions of rice were shown to be under polygenic control (Haque et a1. 1992). However, in many plants, wax production appears to be a qualitative character influenced by a few major genes. For example, variation in pseudostem waxiness of Musa species (bananas and plantains) was under the influence of at least one gene, wx, wherein the recessive allele

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coded for increased waxiness (Ortiz et a1. 1995). In addition, other genes clearly modified the expression of the dominant Wx allele, and an increased dosage effect of wx genes could be seen in comparisons between diploids and tetraploids. Besides naturally-occurring genetic variation, induced genetic variation in epicuticular waxes has been created using chemical, radiation, and insertion mutagenesis. Epicuticular wax mutants have been created in gramineous monocots like sorghum (Peterson et a1. 1982; Jenks et a1. 1992), barley (Lundqvist and Lundqvist 1988), and maize (Lorenzoni and Salamini 1975; Schnable et a1. 1994); and in dicots such as pea (Macey . and Barber 1970a; Holloway et a1. 1977b), and in several Brassica species (Holloway et a1. 1977a; Stoner 1990; Eigenbrode 1991b). Surface wax mutants have also been identified in arabidopsis, which is an excellent model plant for genetic studies of epicuticular wax biosynthesis (Koornneef et al. 1989; Jenks et a1. 1995; Jenks et al. 1996a). Most of these variants likely exhibit differences in a single gene affecting epicuticular waxes. Thus, they provide an excellent resource to elucidate the role of waxes in plant stress resistance, since differential responses to stress can be attributed directly to variations in surface waxes. Mutagenesis of genes affecting surface waxes provides a means for identifying genes involved in wax biosynthesis and secretion. Mutagenesis studies have demonstrated that many loci are involved in epicuticular wax production. In barley, mutagenesis has localized 85 unique loci that influence epicuticular wax production (Lundqvist and Lundqvist 1988; Wettstein-Knowles 1995), and 24 loci were identified in sorghum (Peters 1996). Thus, the production of epicuticular waxes is a complex process involving likely hundreds of individual genes, enzymes, and regulators working together in a concerted action. All induced epicuticular wax mutants identified to date were selected based on visible changes in their surface reflectance. Thus, mutations that did not alter visible surface reflectance would be missed. Screening for alterations in wax total loads or chemical profiles could likely uncover many of these visibly normal mutants. Development of efficient screening procedures is needed. With the possible exception of barley, only one or a few alleles have been identified for each loci, therefore mutagenesis has yet to saturate these genomes for epicuticular wax mutants. Also, epicuticular wax suppressor genes have not been isolated using mutagenesis to revert mutants to wildtype. These suppressor genes would be extremely valuable for dissecting regulatory control in surface wax biosynthesis. Clearly, identification of new genes will help dissect the complex processes of wax production.

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B. Cloning Epicuticular Wax Genes Insertion mutagenesis has an advantage over chemical mutagenesis, because not only are new mutants created, but the genes identified can be "tagged" with a DNA insert. These insertion mutagens interpolate sequences into the gene of interest, and thus provide a method for isolating genes using the insert as a probe (Jenks and Feldmann 1996). Maize and arabidopsis are good systems for isolating epicuticular wax genes by insertion mutagenesis. Maize carries endogenous transposons and arabidopsis is easily transformed with T-DNA. Schnable et a1. (1994) used transposon insertion mutagenesis to create epicuticular wax glossy mutants representing at least 14 Mutator-induced loci. By comparison, T-DNA insertion mutagenesis was used to create arabidopsis epicuticular wax mutants that are allelic to at least four eeeriferum loci (McNevin et al. 1993), and others that represent at least three new T-DNA-induced wax loci, wax, bel, and knb (Jenks et al. 1996a). Recently, several genes involved in epicuticular wax production have been cloned using insertion mutagenesis. For example, the CER2 (Negruk et a1. 1996) and CER3 genes from arabidopsis (Hannoufa et al. 1996) were cloned using T-DNA tagged alleles. CER2 was also cloned using chromosome walking (Xia et a1. 1996), a technique that will become more effective as well-developed chromosome molecular maps become available. Both CER2 and CER3 code for novel proteins. Thus, their function is difficult to predict. Interestingly, the CER2 mRNA appears to be highly expressed in stems but not leaves (Negruk et al. 1996; Xia et a1. 1996). This is consistent with studies by Jenks et a1. (1995) showing that the wax chemical profile of stems, but not leaves, was dramatically altered in the CER2 mutants. CER2 has 63 percent sequence similarity over the entire protein with GL2 from maize. Like the eer2 mutant in arabidopsis (Jenks et al. 1995), g12 of maize has reduced chain length distribution for major wax constituents (Bianchi et a1. 1985). This might suggest that both genes likely playa role in acyl-GoA elongation reactions, with CER2 possibly being a stem-specific regulator. However, Xia et al. (1997) found that the CER2 protein was localized in the nucleus, and thus does not catalyze wax elongation reactions. Jenks et a1. (1995) proposed that CER3 may be involved in the hydrolysis of fatty acyl-GoA into free fatty acids and CoA, but Hannoufa et a1. (1996) found that the CER3 gene lacked homology to members of the fatty acyl-CoA thioesterase gene family. The CER1 gene of arabidopsis was cloned using the heterologous maize transposable element system Enhancer-Inhibitor (En/Spm) (Aarts et a1. 1995). Various transposon systems were also used to isolate the epi-

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cuticular wax genes, GL1 (Hansen et a1. 1997), G12 (Tacke et a1. 1995), GLB (Xu et a1. 1994), and GL15 (Moose and Sisco 1995) from the seedling wax mutants in maize. The deduced amino acid sequence from GL1, although roughly twice as large, was similar to the proteins encoded by the arabidopsis CER1 and Senecio odora (Defl.) EPI23 genes. Moreover, EPI23 was expressed only in the epidermis. Based upon sequence analysis, Hansen et a1. (1997) proposed that the GL1, CER1, and EPI23 belong to a family of membrane-bound receptors. If so, these gene products may be involved in wax secretion. Aarts et a1. (1995) contend that CERl encodes an aldehyde decarbonylase based upon regions of sequence homology to this group of enzymes. The GLB gene in maize has sequence homology to a gene coding the E. coli 3-oxoacyl-ACP reductase (Xu et a1. 1994). Thus, GLB may playa role in reducing ketoacyl intermediates during the acyl-CoA elongation reactions. The GL15 gene, by comparison, has high sequence homology to floral regulatory elements in arabidopsis, suggesting a possible analogous regulatory role in wax biosynthesis (Moose and Sisco 1995). Further mutagenesis studies, using these exogenous and endogenous insertion elements, are needed to tag important epicuticular wax genes and facilitate their cloning. Identifying these genes will not only help to elucidate wax production processes, but also identify candidate genes that might be used in crop improvement programs. VI. SUMMATION

The layer of epicuticular wax that covers the above-ground portions of plant tissues provides a hydrophobic barrier between plants and their environment. As the interface between plants and their external environment, these waxes play an important role in abiotic and biotic stress resistance. Epicuticular waxes have been shown to affect the interactions between plants and other organisms such as phytophagous insects and plant pathogens. Surface waxes also play an important role in plantwater relations, and have been linked to resistance to a variety of environmental stresses, including drought, frost, winter desiccation, air pollution, acid rain, and excess solar radiation. Although all aerial plant surfaces are covered with a layer of epicuticular wax, the chemical composition and appearance of surface waxes can vary within tissues on,a single plant, among ecotypes of the same species, and among species. Tissues may be covered with a thin, transparent layer of epicuticular wax, or a dense network of wax crystals. In addition, the composition and appearance of waxes may vary in response to growing

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conditions and during plant development. This diversity in chemical composition and physical characteristics of epicuticular waxes has often been associated with differences in tolerance to biotic and abiotic stresses. As a result, there have been numerous investigations to determine how waxes affect stress tolerance, and ascertain the contribution of different chemical constituents toward improved plant performance. These investigations have benefited from the identification of mutants that have reduced levels of wax, altered chemical composition, and modified crystallization patterns. Studying such mutants has provided insights into the biosynthesis and secretion of waxes, and facilitated investigations of how specific changes in epicuticular waxes affect physiology and stress tolerance. Our understanding of epicuticular waxes has also benefited from the cloning of genes involved in wax biosynthesis, wax secretion, and their regulation. Such investigations not only provided basic information on the biosynthesis and biology ofepicuticular wax, but provide a foundation for future work in which epicuticular waxes could be modified using recombinant DNA technology in a crop improvement program. This could have relevance for enhanced stress resistance, improved ornamental value, and the development of new industrial waxes.

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Shockey, J. M., R. Rajasekharan, and J. D. Kemp. 1995. Photoaffinity labeling of developing jojoba seed microsomal membranes with a photoreactive analog of acyl-coenzyme A (acyl-CoA). Plant Physiol. 107:155-160. Smith, L. C., H. Pownall, and A. M. Gotto, Jr. 1978. The plasma lipoproteins: structure and metabolism. Annu. Rev. Biochem. 47:751-777. Spencer, J. L. 1996. Waxes enhance Plutella xylostella oviposition in response to sinigrin and cabbage homogenates. Entomol Exp. Appl. 81:165-173. SHidler, E., and H. R. Buser. 1984. Defense chemicals in leaf surface wax synergistically stimulate oviposition by a phytophagous insect. Experientia 40:1157-1159. Starks, K. J., and D. E. Weibel. 1981. Resistance in bloomless and sparse-bloom sorghum to greenbugs. Environ. Entomol. 10:963-965. Sterk, P., H. Booij, G. A. Schellekens, A. Van Kammen, and S. C. DeVries. 1991. Cellspecific expression of the carrot EPZ lipid transfer protein gene. Plant Cell 3:907-921. Stoner, K. A. 1990. Glossy leaf wax and plant resistance to insects in Brassica oleracea under natural infestation. Environ. Entomol. 19:730-739. Stoner, K. A. 1992. Density of imported cabbageworms (Lepidoptera: Pieridae), cabbage aphids (Homoptera: Aphididae), and flea beetles (Coleoptera: Chrysomelidae) on glossy and trichome-bearing lines of Brassica oleracea. J. Econ. Ent. 85:1023-1030. Stork, N. E. 1980. Role of waxblooms in preventing attachment to Brassicas by the mustard beetle, Phaedon cochleariae. Ent. Expt. Appl. 26:100-107. Tacke, E., C. Korfhage, O. Michel, M. Maddaloni, M. Motto, S. Lanzini, F. Salamini, and H. P. Doring. 1995. Transposon tagging of the maize Glossy210cus with the transposable element En/Spm. Plant J. 8:906-917. Tevini, M., and D. Steinmiiller. 1987. Influence of light, UV-B radiation, and herbicides on wax biosynthesis of cucumber seedlings. J. Plant Physiol. 131:111-121. Thair, B. W., and G. R. Lister. 1975. The distribution and fine structure of the epicuticular leaf wax of Pseudotsuga menziezii. Canadian J. Bot. 53:1063-1071. Thoma, S., Y. Kaneko, and C. Somerville. 1993. A non-specific lipid transfer protein from Arabidopsis is a cell wall protein. Plant J. 3:427-436. Thomas, D. A., and H. N. Barber. 1974a. Studies of leaf characteristics of a cline of Eucalyptus urnigera from Mount Wellington, Tasmania. 1. Water repellency and the freezing of leaves. Australian J. Bot. 22:501-512. Thomas, D. A., and H. N. Barber. 1974b. Studies of leaf characteristics of a cline of Eucalyptus urnigera from Mount Wellington, Tasmania. II. Reflection, transmission and absorption ofradiation. Australian J. Bot. 22:701-707. Tranquillini, W. 1979. Physiological ecology of the alpine treeline. Springer-Verlag, New York. Tulloch, A. P. 1973. Composition of leaf surface waxes of Triticum species: variation with age and tissue. Phytochemistry 12:2225-2232. Tuomisto, H. 1986. Use of Picea abies needles as indicators of air pollution: epicuticular wax morphology. Ann. Bot. (Fennici) 25:351-364. Turunen, M., and S. Huttunen. 1990. A review of the responses of epicuticular wax of conifer needles to air pollution. J. Environ. Qual. 19:35-45. Turunen, M., and S. Huttunen. 1991. Effect of simulated acid rain on the epicuticular wax of Scots pine needles under northerly conditions. Canadian J. Bot. 69:412-419. Turunen, M., S. Huttunen, K. E. Percy, C. K. McLaughlin, and J. Lamppu. 1997. Epicuticular wax of subarctic Scots pine needles; response to sulphur and heavy metal deposition. New Phytol. 135:501-515. Uematsu, H., and A. Sakanoshita. 1989. Possible role of cabbage leaf wax bloom in suppressing diamondback moth Plutella xylostella (Lepidoptera: YponomeutidaeJ oviposition. Appl. Ent. Zool. 24:253-257.

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Vique, J., and P. E. Kolattukudy. 1997. Resolution and purification of an aldehyde-generating and an alcohol-generating fatty acyl-CoA reductase from pea leaves (Pisum sativum L.). Arch. Biochem. Biophys. 340:64-72. Voelker, T. A., A. Jones, A. M. Cramer, H. M. Davies, and D. S. Knutzon. 1997. Broad-range and binary-range acyl-acyl-carrier-protein thioesterases suggest an alternative mechanism for medium-chain production in seeds. Plant Physiol. 114:669-677. Vogelmann, T. C. 1993. Plant tissue optics. Annu. Rev. Plant Physiol. Plant Mol. BioI. 44:231-251. VonMohl, H. 1842. Uber die Cuticula der Gewachse. Linnaea 16:401-416. Waku, Y. 1978. Fine structure and metamorphosis of the wax gland cells in a psyllid insect, Anomoneura mori Schwartz (Homoptera). J. Morph. 158:243-274. Walton, T. J. 1990. Waxes, cutin, and suberin. In: J. L. Harwood and J. R. Bowyer (eds.), Methods in plant biochemistry 4:105-158. Academic Press Limited, New York. Wattendorff, J., and P. J. Holloway. 1980. Studies on the ultrastructure and histochemistry of plant cuticles: the cuticular membrane of Agave americana L. in situ. Ann Bot. 46:13-28. Weber, E. 1942. Uber die optik und strukture der Pflanzenwachse. Bericht der Schweizerischen botanischen Gesellschaft 52:111-174. Weibel, D. E., and K. J. Starks. 1986. Greenbug nonpreference for bloomless sorghum. Crop Sci. 26:1151-1153. Wettstein-Knowles, P. von. 1974. Ultrastructure and origin epicuticular wax tubes. J. Ultrastruct. Res. 46:483-498. Wettstein-Knowles, P. von. 1995. Biosynthesis and genetics of waxes. p. 91-129. In: R. J. Hamilton (ed.), Waxes: chemistry, molecular biology and functions. Oily Press Ltd., Dundee, Scotland. Wettstein-Knowles, P. von, and B. Sogaard. 1980. The cer-cqu region in barley: gene cluster or multifunctional gene. Carlsberg Res. Commun, 45:125-141. Whitecross, M. 1., and D. J. Armstrong. 1972. Environmental effects on epicuticular waxes of Brassica napus L. Austral. J. Bot. 20:87-95. Wiesner, J. 1871. Beobachtungen uber die Wachsuberzuge der Epidermis. Botanische Zeitung 29:769-774. Wilkinson, R. E., and M. J. Kasperbauer. 1972. Epicuticular alkane content of tobacco as influenced by photoperiod, temperature and leaf age. Phytochemistry 11:2439-2442. Wilkinson, R. E., and H. S. Mayeux, Jr. 1990. Composition of epicuticular wax on Opuntia engelmannii. Bot. Gaz. 151:342-347. Woloshuk, C. P., and P. E. Kolattukudy. 1986. Mechanism by which contact with plant cuticle triggers cutinase gene expression in the spores of Fusarium solani f. sp. pisi. Proc. Nat. Acad. Sci. (USA) 83:1704-1708. Woodhead, S. 1983. Surface chemistry of Sorghum bicolorand its importance in feeding by Locusta migratoria. Physiol. Entomol. 8:345-352. Woodhead, S., and R. F. Chapman. 1986. Insect behaviour and the chemistry of plant surface waxes. p. 123-135. In: B. Juniper and T. R. E. Southwood (eds.), Insects and the plant surface. Edward Arnold, London. Woodhead, S., and D. E. Padgharn. 1988. The effect of plant surface characteristics on resistance of rice to the brown plant hopper, Nilaparvata Jugens. Entomol. Exp. Appl. 47:15-22. Xia, Y., B. J. Nikolau, and P. S. Schnable. 1996. Cloning and characterization of CE1l2, an Arabidopsis gene that affects cuticular wax accumulation. Plant Cell 8:1291-1304. Xia, Y., B.}. Nikolau, and P. S. Schnable. 1997. Developmental and hormonal regulation of the Arabidopsis CER2 gene that codes for a nuclear-localized protein required for the normal accumulation of cuticular waxes. Plant Physiol. 115:925-937.

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Xu, X., C. R. Dietrich, M. Delledonne, Y. Xia, T.-]. Wen, D. S. Robertson, B. ]. Nikolau, and P. S. Schnable. 1997. Sequence analysis of the cloned gJossy8 gene of maize suggests that it may code for a ~-ketoacyl reductase required for the biosynthesis of cuticular waxes. Plant Physio!. 115:501-510. Xu, X., S. Heinen, T. J. Wen, M. Delledonne, B. J. Nikolau, and P. S. Schnable. 1994. Cloning and sequence analysis of a maize acetyl-CoA carboxylase gene. p. 34. In: Cloning plant genes known only by phenotype. Plant Molecular Genetics Institute, Saint Paul. Yang, G., B. R. Wiseman, D. ]. Isenhour, and K. E. Espelie. 1993. Chemical and ultrastructural analysis of corn cuticular lipids and their effect on feeding by fall armyworm larvae. J. Chem. Ecol. 19:2055-2074. Yang, S. L., and A. H. Ellingboe. 1972. Cuticle layer as a determining factor for the formation of mature appressoria of Erysiphe graminis on wheat and barley. Phytopathology 62:708-714. Zaid, A., and H. Hughes. 1995. In vitro acclimatization of date palm (Phoenix dactyli/era L.) plantlets: a quantitative comparison of epicuticular leaf wax as a function of polyethylene glycol treatment. Plant Cell Rep. 15:111-114.

2

Applications of Chlorophyll Fluorescence Techniques in Postharvest Physiology Jennifer R. DeEll

Agriculture and Agri-Food Canada, Horticultural Research and Development Centre 430 Boulevard Gouin, Saint-Jean-sur-Richelieu, QC J3B 3E6, Canada

Olaf van Kooten Agrotechnological Research Institute (ATO-DLO) P.O. Box 17, 6700 AA Wageningen, The Netherlands

Robert K. Prange Agriculture and Agri-Food Canada, Atlantic Food and Horticulture Research Centre 32 Main St., Kentville, NS B4N lJ5, Canada Dennis P. Murr Department of Horticultural Science, University of Guelph Guelph, ON N1G 2Wl, Canada I. Introduction II. Chlorophyll Fluorescence Measurements III. Applications of Chlorophyll Fluorescence A. Evaluation of Chilling Injury B. Detection of Heat Stress C. Indicator of Atmospheric Stress D. Evaluation of Ripening and Senescence E. Prediction of Shelf-life F. Quality Assessment of Ornamentals G. Prediction of Superficial Scald Development H. Detection of Water Stress Horticultural Reviews, Volume 23, Edited by Jules Janick ISBN 0-471-25445-2 © 1999 John Wiley & Sons, Inc. 69

J. DEELL, O. VAN KOOTEN, R. PRANGE, AND D. MURR

70 IV. Concluding Remarks Literature Cited

I. INTRODUCTION Chlorophyll fluorescence is a nondestructive measurement technique that can be performed relatively fast and with great precision by minimally trained personnel, making it ideal for applied research. Chlorophyll fluorescence reflects the primary processes of photosynthesis that take place in the chloroplasts, such as light absorption, excitation energy transfer, and the photochemical reaction in photosystem II (PSII) (Fig. 2.1). However, since these primary events are integrated into the overall process of photosynthesis, including electron transport, proton transfer across the thylakoid membranes, photophosphorylation, and CO 2 assimilation, the yield of chlorophyll fluorescence is influenced by numerous factors in a very complex manner (Krause and Weis 1988). For example, the following factors will influence fluorescence induction curves: light intensity, temperature, pre-illumination, light-adaptation state, gas composition, humidity, tissue age and the entire "pre-history" of the plant, including possible exposure to environmental stresses (Renger and Schreiber 1986). Control curves can be obtained when all controllable factors are kept at standard values, and then the application of any treatment affecting

Carbon Fixation PS II

~

Photochemistry

Energy transfer Fig. 2.1.

Photosynthetic activity and the role of PSII.

Electron transport

2. APPLICATIONS OF CHLOROPHYLL FLUORESCENCE TECHNIQUES

71

the state of the photosynthetic apparatus should modify the control curve in a characteristic manner (Renger and Schreiber 1986). In principle, even without a proper understanding of the underlying mechanisms, such a method can provide empirical information on the extent of damage caused by such treatments. The response of plants to a diverse range of environmental, chemical, and biological stresses has been assessed by changes in chlorophyll fluorescence (Krause and Weis 1988; Lichtenhaler 1988; Lichtenhaler and Rinderle 1988; Renger and Schreiber 1986; Schreiber and Bilger 1987; Smillie and Hetherington 1983; Snel et a1. 1991). However, application of chlorophyll fluorescence techniques to the field of postharvest physiology has been made only recently, even though the kinetics and fluorescence emission spectra of several fruits are similar to those of green leaves (Gross and Ohad 1983; Smillie 1992). The measurement of stress-induced injuries in plant tissue by chlorophyll fluorescence has several advantages (Smillie et a1. 1987): 1. The measurement is made directly on living plant tissue. 2. The method is nondestructive, so that the same area of leaf or fruit can be measured throughout an experiment. 3. Each variable fluorescence measurement takes a few seconds, thus the method is suitable for screening applications. 4. Cellular injury is detected well in advance of the development of visible symptoms. 5. Measurements can be taken during treatment and can be continued afterwards to follow recovery or deterioration. 6. Portable measuring equipment is available. 7. Fluorescence data can be directly processed by computer or stored on magnetic tape for later processing. Large amounts of data can be acquired in a short time, allowing for a statistical approach with the possibility to follow the response to a stimulus over a prolonged time. The relative ease of acquiring data has allowed the use of chlorophyll fluorescence without careful consideration as to how the process being measured relates to the process under investigation. This is partly due to the vast body of basic research that leaves a newcomer to the field baffled by the many eff~cts studied and the basic lack of agreement about nomenclature or even measurement techniques (van Kooten and Sne11990). It is difficult to decide which chlorophyll fluorescence measurement (Table 2.1) is most appropriate for the intended research. Consequently, it is the instrument at hand that decides the outcome to that question. In this review, we do not intend to give a complete historical overview of the many different measurements researched since Kautsky's first publication on chlorophyll fluorescence (Kautsky and Hirsch 1931). For more information on the kinetics and instrumentation, refer to the reviews by Geacintov and Breton (1987), Havaux and Lannoye (1985),

J. DEELL, O. VAN KOOTEN, R. PRANGE, AND D. MURR

72

Table 2.1.

Recommended chlorophyll fluorescence nomenclature.

Fo

fluorescence intensity minimal fluorescence (dark)

Fi

fluorescence at I level

Fp

fluorescence at P level

Fm

maximal fluorescence (dark)

Fv

variable fluorescence (dark)

Ft

fluorescence at T level

Fs

fluorescence in steady state

Fv/Fm

exciton transfer efficiency (dark) half-time for rise in Fv

F

Tl/2 Fo'

minimal fluorescence (light) Fm' maximal fluorescence (light) variable fluorescence Fv' (light) Fv'/Fm' exciton transfer efficiency (light) qp photochemical quenchingZ non-photochemical ClN quenching quantum yield of cI>pslI photochemistry

actual fluorescence intensity at any time. fluorescence intensity with all PSII reaction centers open while the photosynthetic membrane is in the non-energized state, Le. dark or low light adapted qp = 1 and qN = O. It can also be used for the o level in O-I-D-P-T nomenclature, but it should be clearly described how it is determined. fluorescence intensity at I level (O-I-D-P-T nomenclature). fluorescence intensity at P level (O-I-D-P-T nomenclature). fluorescence intensity with all PSII reaction centers closed (Le. qp = OJ, all non-photochemical quenching processes are at a minimum (Le.
(Fm'-F) / Fm'

ZQther quenching measurements used in the literature, such as "energy" dependent quenching
2. APPLICATIONS OF CHLOROPHYLL FLUORESCENCE TECHNIQUES

73

Krause and Weis (1984), Krause and Weis (1991), Lavorel and Etienne (1977), Lichtenhaler (1992), Papageorgiou (1975), Schreiber (1983), Schreiber and Bilger (1993) and Schreiber et a1. (1986). The focus of this review will be on chlorophyll fluorescence measurements that have been proven to be related to a known physiological process and have been used in the field of postharvest physiology (Table 2.1). In this regard, Table 2.1 contains the recommended chlorophyll fluorescence ternlinology and yet is not a collection of every term used in the chlorophyll fluorescence literature. We will define non-recommended chlorophyll fluorescence terms used by previous researchers when they are first cited in this review.

II. CHLOROPHYLL FLUORESCENCE MEASUREMENTS The primary event of photosynthesis in all green plant tissue is the absorption of light by chlorophyll molecules. This absorption of light causes the energy level of chlorophyll to be raised and thus electrons are displaced into higher energy orbitals. Most of this excitation energy of chlorophyll (-85%) is transferred to the reaction centers of the photosystems and is used to drive the reactions of photosynthesis (Le. oxidation of water, oxygen evolution, NADP+ reduction, membrane proton transport, and ATP synthesis). However, some excitation energy is also lost as heat and as fluorescence (light emission), as the electron moves back to ground state. The fluorescence of green plants, approximately 3-5% of total excitation energy (Walker 1985), is almost exclusively emitted by chlorophyll a. At physiological temperatures, fluorescence is predominantly emitted from PSII, with the main maximum band at 685 nm (Papageorgiou 1975). Thus, the emission peak is of longer wavelength than the excitation energy. The characteristic pattern of fluorescence emitted from the chlorophyll a of dark-adapted plant tissue upon re-illumination is known as the "Kautsky Effect," named after the researcher who first conducted detailed studies on the phenomena (Kautsky and Hirsch 1931). Fluorescence levels of the Kautsky curve are termed 0, I, D, P, S, M, and T (Papageorgiou 1975). a (origin) represents the constant or background fluorescence, from which fluorescence rises to an intermediate or inflection (I), and follows through a dip (D) to a peak (P), within the first 2 s of illumination (Krause and Weis 1984). This is followed by slow decay through the S (semi-steady state or stationary level), M (maximum or intermediate maxima), and T (terminal), which lasts for several minutes.

]. DEELL, O.

74

VAN

KOOTEN, R. PRANGE, AND D. MURR

The height and slope of these peaks are related to the integrity of specific steps in the electron transport system and hence chlorophyll fluorescence can be used to evaluate the photosynthetic activity of plant tissue. Several popular fluorescence measurements represent the levels of fluorescence within the Kautsky curve (Fig. 2.2): Fo (0 level of fluorescence), Fi (I level of fluorescence), Fp (P level of fluorescence), and Ft (T level of fluorescence). In the process of photosynthesis, light energy is captured by PSII and transferred to the reaction center P680 (Stryer 1988). Upon conversion to an excited state, an electron is lost to pheophytin (Ph). Loss of the electron causes P680 to become positively charged, and it then attracts an electron from an adjacent Mn-protein. As the Mn-protein becomes oxidized, it in turn attracts an electron from H20. After the loss of two electrons, H20 becomes split into %02 and 2H+. The electron that was lost to Ph is transferred to a plastoquinone bound to a protein (OA), and finally to a second diffusible plastoquinone (~). With the transfer of two electrons, ~ is reduced to plastoquinol (PQH 2 ). In terms of chlorophyll fluorescence (Krause and Weis 1984; Schreiber and Bilger 1987), the 0 level represents the phase when OA is maximally oxidized. 0 to I represents the reduction of OA as it begins receiving electrons from P680, and I to D is the oxidation of OA as electrons are trans-

Fp

Relative fluorescence

Ft

o

2 Time (s)

Fig. 2.2. Kautsky curve; chlorophyll fluorescence measurements associated with 0, I, D, P, and T levels of fluorescence.

2. APPLICATIONS OF CHLOROPHYLL FLUORESCENCE TECHNIQUES

75

ferred from UA to Qa. D to P represents the reduction of UA that occurs as the plastoquinone (PQ) pool becomes reduced, while P itself is when the PQ pool and UA are "highly" reduced. The decline from P to S-M-T is related to the induction of CO 2 assimilation, with T representing the steady state. The transfer of electrons from UA to Qa lowers the fluorescence yield, as observed by the decline from I to D. However, the overall fluorescence decline can only be partly explained by the reoxidation of ~ (photochemical quenching, qp). In intact systems under most conditions, energy-dependent quenching (~) is also a major component of the fluorescence decline (Krause and Weis 1988). This type of non-photocheznical quenching is related to the light-induced proton gradient across the thylakoid membrane. It is hypothesized that a structural change of unknown nature is induced by low intra-thylakoid pH, which lowers the photochemical efficiency of PSII and transforms the trapped excitation energy to heat. Such a mechanism is thought to function in a regulated manner, serving as protection against damaging effects of excess excitation energy (Weis and Berry 1987). As a result of OA reduction, the fast rise of O-I-D-P is caused predominantly by the removal of qp (Renger and Schreiber 1986). Maximum fluorescence (Fm) is achieved only after complete Ch reduction and exclusion of all other quenching mechanisms. Therefore, Fp is not necessarily equal to Fm. The fluorescence decline through S-M-T reflects the complex interference of different factors that affect the magnitude of both qp and ~. In the second half of the 1980s, the pulse amplitude modulation technique (Schreiber et al. 1986), known previously as the light doubling technique (Bradbury and Baker 1983), revealed three chlorophyll fluorescence parameters that could be linked to well known photosynthetic measurements associated with PSII (Fig. 2.3): q)PSII

qp Fv'/Fm'

quantum yield of photochemistry of PSII redox state of the secondary acceptor UA of PSII exciton transfer efficiency of PSII

(Fm' - F) I Fm' (Fm'- F) I (Fm' - Fo') (Fm' - Fo') I Fm'

where Fm' and Fo' are the maximal and minimal fluorescence, respectively, in any light adapted state and F is the fluorescence intensity at any given time. These three measurements give a quantitative value for the basic functioning of photosynthetic electron transport.

,,-... ~

'2

::s

Q)

-="'0 Q

>=

(l) ()

s::

Fm -----12

5r

:l

-..,J 0')

I

I\....

4

Fv== Fm-Fo

1-------- Fm'

2

(l)

()

tt.l

(l) ~

0

::s G:

~

oL

~

t

sy-------- F0'

S~ ~-AL

O+AL

SP

ML

':""t

+FR

t:l

~

..

t""

.r p

Time

~

z

1

2

qp:l

~ qNqp:O +AL. O

I

qN- O

I

3 l>qp>O l>qN>O

5

~

qp= 1 l>qN>O

~

4

qp=O l>qN > 0

SP

-AL

+FR

o

!'tI "'d

_ Fm' - F

- 1 .-.....F=m.....'_-.. .F. .....o"-' - Fm-Fo

qp- Fm'-Fo' qN-

pSII

=

Measurements of chlorophyll fluorescence by the saturation pulse method. Fluorescence yield is measured with a modulated fluorometer and 5 different states dependent on light conditions are distinguished, with corresponding points in the induction curve characterized by fluorescence yield notations (Fo, Fm, and Fv), quenching coefficients (qp and~) and quantum yield of photochemistry (tPpsn). ML, weak modulated measuring light (- 6 nmol·m-2·s-1 at 660 nm); SP, saturating light pulse (- 10000 lJ.lIl0l·m-2 ·s-1 , 400 nm < 1 < 700 nm, applied for 0.5-2 s); AL, continuous actinic light; FR, far-red light (- 6 JlIIlol·m-2·s-1 , 1> 700 nm). (Modified from van Kooten and Snel1990)

Fig. 2.3.

S!Z

~

> Z

t:l ~

s:c ~

2. APPLICATIONS OF CHLOROPHYLL FLUORESCENCE TECHNIQUES

77

<1>PSII has been shown to correlate negatively with photosynthetic carbon fixation using gas exchange measurements under non-photorespiratory conditions (Genty et a1. 1989; Harbinson et a1. 1990; Heber et a1. 1990). It is interpreted as the efficiency of an absorbed photon to induce an electron to be transported through PSII. If every absorbed photon caused a charge separation to occur in the PSII reaction center complex, then Cl>PSII would equal Fo = 0 and Fv/Fm = 1. Although the photosynthetic machinery is highly efficient in plants, 100 percent efficiency is not possible (2 nd law of thermodynamics). In general, values of Fv/Fm between 0.7 to 0.85 are found for open (i.e., dark adapted) reaction centers in situ (Bjorkmann and Demmig 1987). Exciton transfer efficiency appears to be an underestimation caused by the presence of non-variable chlorophyll fluorescence originating from photosystem I (PSI) (Genty et a1. 1990). If a correction is made, then the real value for the maximum quantum yield is close to or above 0.9, which implies a very high efficiency. Taken together with the absorption coefficient for actinic light alpha (Bjorkmann and Demmig 1987), which is also close to 0.9, then it becomes apparent that the light capturing system is a very efficient funnel for actinic light to the reaction center. Once the light has reached the PSII reaction center, known as P680, photochemistry can occur. An electron can then be transferred to the electron acceptor ~ (an open reaction center) provided it is not already reduced, Le. QA: (a closed reaction center). Electron transfer is the initiation of photosynthetic electron transport, which will culminate in the reduction of NADP+ to NADPH, the reducing equivalents necessary for the chemical fixation of CO2 into carbohydrate chains. The direct link between primary photochemical events and photosynthetic carbon fixation is the reason Cl>PSII' in actinic light, yields the quantum yield of photosynthetic electron transport under non-photorespiratory conditions (Genty and Harbinson 1996). The exciton transfer efficiency can be measured in the dark (Fv/Fm), which implies that all reaction centers are open (i.e. all ~ oxidized or qp =1). Alternately, the exciton transfer efficiency can also be measured in the light (Fv'/Fm'). The capacity to vary the exciton transfer efficiency is a regulatory mechanism of PSII. PSII can reduce the flux of energized electrons through the electron transport chain if the acceptor side cannot cope with the full flux. When Fv/Fm is measured in dark-adapted tissue it is an indicator of the integrity of the reaction center and light harvesting complex of PSII. These two pigment protein complexes are connected through a dipoledipole interaction, which implies that the exciton transfer efficiency is extremely sensitive to variation in the distance between them (1/d 6 )

78

J. DEELL, O. VAN KOOTEN, R. PRANGE, AND D. MURR

(van Grondelle et ale 1994). The bond between these complexes is noncovalent and thus minor disturbances within the thylakoid membrane can have a large effect on this value. Since Fv/Fm can be measured within 1 s, it may be a useful tool for studying membrane altering processes, such as cold adaptation (Sommersalo and Krause 1989), heat stress (Schreiber and Bilger 1987), low O2 stress (Prange et al. 1997), and chilling injury (Tijskens et a1. 1994; van Kooten et a1. 1992). Van Kooten et a1. (1992) demonstrate the complexity in relating Fv/Fm and the integrity of PSII to the complete response of chilling injury. Thus, one should avoid implying that there is a direct connection between chlorophyll fluorescence changes and stress responses. Fv'/Fm' should be measured in parallel with PSII and qp, as these values provide insight into electron transport as a whole and its regulation. PSII measured in the light indicates the efficiency of quantum use for electron transport through PSII. That means if pSlI =0.5, then half of the photons absorbed will produce a photochemical event in PSII. If the spectral absorption cross sections of both PSII and PSI reaction centers are balanced, Le. the number of charge separations per unit area is equal for both types of photosystems, it will result in a balanced flow of electrons between the two photosystems (Harbinson et a1. 1989). The spectral absorption cross section is determined by the stoichiometry of the light harvesting pigment protein complexes between the two photosystem pools. This is regulated through the turnover and insertion rate of the protein complexes and occurs in the time domain of several hours to days (Kyle 1987). It is also possible to exchange light harvesting protein complexes between the two photosystem pools or to change the exciton transfer rate between the light harvesting complex and PSII, within a time domain of minutes (Krause and Weis 1991). When the changes occur faster or when the regulatory mechanisms are hindered, an imbalance occurs in the electron transport between PSII and PSI. Under such conditions, the electron acceptor of PSII becomes either over reduced or totally oxidized, e.g. qp =0 or 1, respectively. An acceleration of electron transport through PSII will result in the case of total oxidation, a situation with which the system is quite capable of coping. However, total oxidation of the electron acceptor of PSII usually leads to an over reduction of~, which is measured as qp tending to zero. When ~ is over reduced, energized electrons are produced at PSII without proper acceptors to neutralize their energy, leading to a breakdown of the PSII protein D1, known as photoinhibition (Kyle 1987; Ohad et a1. 1990). Photoinhibition occurs at a certain rate in all plants exposed to light (Barber and Anderson 1992). However, when the rate becomes too high or the capacity of the plant to renew the D1 protein diminishes,

2. APPLICATIONS OF CHLOROPHYLL FLUORESCENCE TECHNIQUES

79

then the active photosystem pool starts to disappear. Therefore, a sudden decrease of qp can give us an indication that the normal regulatory mechanisms cannot cope with the physiological imbalance induced by the "stress" conditions. Exciton transfer efficiency (Fv'/Fm') will be reduced or the plant will require a large pool of free radical scavengers to cope with the physiological imbalance. Measurements performed in the dark provide us with Fv/Fm, which is a very sensitive indicator of membrane damage or membrane alterations, as shown with chilling injury (Tijskens et al. 1994). Careful auxiliary measurements on membrane structure and composition (Janssen and van Hasselt 1994) improves the interpretation of results. When we want to elucidate the alterations in regulation on a metabolic level, it will be necessary to measure PSII' qp, and Fv'IFm' simultaneously and this cannot be done using dark measurements. When only
III. APPLICATIONS OF CHLOROPHYLL FLUORESCENCE

A. Evaluation of Chilling Injury Chilling injury occurs in several horticultural commodities, especially those oftropical or sub-tropical origin (Wang 1993). This disorder results from the exposure of susceptible tissue to temperatures below 12.5°C, although the critical temperature at which chilling injury develops varies with species and organs (Morris 1982). Visual symptoms of chilling injury can include surface lesions, water-soaked tissue, internal discoloration, breakdown of tissue, failure to ripen, accelerated senescence, and increased decay, all of which will result in unmarketable product and large economic losses. Inhibition of photosynthetic electron transfer on the photo-reducing side of PSII will increase the yield of fluorescence, while inhibition on

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the water-splitting or photo-oxidizing side of PSII will decrease the yield (Papageorgiou 1975). Chilling injury causes inhibition to develop on the water-splitting side of PSII (Smillie and Nott 1979), and thus decreases induced chlorophyll fluorescence. An early postharvest study used chlorophyll fluorescence techniques to follow the development of chilling injury in banana (Musa Group AAA, Subgroup Cavendish) and mango (Mangifera indica L.) (Smillie et al. 1987). Mature green bananas were kept at either O°C (chilling treatment) or 13°C (non-chilling treatment). FR(Fi to Fp level of fluorescence) decreased in fruit held at O°C, but not in fruit held at 13°C. FR/Fo and Fv/Fo gave similar results, both decreasing in line with fruit ripening (as indicated by skin color) at temperatures between 5°C and 15°C. The stress response of chilling injury can be detected and quantified by chlorophyll fluorescence before visual symptoms of injury (peel discoloration) appear in banana. The decrease ofFRin banana during chilling at O°C is linearly correlated with two indices of chilling injury, the post-chilling inhibition of ripening and the post-chilling skin discoloration (R. M. Smillie, unpublished experiments cited in Smillie and Hetherington 1990). As both ripening and chilling injury lead to decreases in variable chlorophyll fluorescence, it is not possible to distinguish one from the other on the basis of Fv/Fo alone (Smillie et al. 1987). Mangoes stored at 5°C, in which ripening was largely suppressed, had relatively high Fv/Fo values (0.94 ± 0.12). In contrast, mangoes stored at O°C had extremely low Fv/Fo values (0.03 ± 0.01) due to severe chilling injury, while mangoes stored at 15°C had low Fv/Fo values (0.20 ± 0.06) associated with advanced ripening. Smillie, Hetherington and Alexander (unpublished experiments cited in Smillie et al. 1987) used chlorophyll fluorescence of fruits to screen for chilling tolerance in cultivars of avocado (Persea americana Mill.), Citrus spp., custard apple (Annona squamosa L.), grape (Vitis vinifera L.), guava (Psidium guajava L.), longan (Dimocarpus longan Lour.), lychee (Litchi chinensis Sonn.), and macadamia nut (Macadamia ternifolia F. J. Muell). Lurie et al. (1994) used chlorophyll fluorescence as a predictor of chilling injury in green pepper (Capsicum annuum L.) before tissue damage became visible. Fruit held at 2°C developed surface pitting after 3 weeks, whereas fruit held at 8°C did not develop chilling injury symptoms. Fm/Fo of green peppers stored at 2°C decreased 90 percent during the first week and remained low thereafter, indicating that the photo-oxidizing side of PSII was inhibited by low temperature. On the other hand, qp (which reflects photosynthetic electron transport) of the peppers was similar at both temperatures and thus cannot be used as an

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indicator of chilling injury in green pepper. Similar response to chilling has been observed in cucumber leaves and isolated chloroplasts, in which electron transport rates are not altered (Peeler and Naylor 1988; Terashima et a1. 1989). Lurie et a1. (1994) also found that ~, which is a measure of the energizing of the thylakoid membranes leading to ATP formation, decreased in peppers stored at 2°C after 2 weeks just prior to the development of surface pitting. Similar results have been found for leaves of Oryza sativa L., in which chilling leads to the disruption of A TP formation earlier than inhibition of the electron transport reactions of photosynthesis (Moll and Steinbach 1986). Thus it appears that the chloroplasts of green pepper respond to low temperature identically to chloroplasts from chilling-sensitive leaves. Van Kooten et a1. (1992) found that cucumber fruit (Cucumis sativus L.) stored for 2 weeks at 10°C and 13°C did not show any change in Fv/Fm (0.77 ± 0.01) during storage, whereas cucumbers stored below these temperatures (4°C and 7°C) exhibited a significant decrease in Fv/Fm, along with discoloration and increased decay incidence. The decrease in Fv/Fm was temperature dependent and was even more pronounced after an additional 6 d at 20°C. The visible symptoms of chilling injury, such as discoloration, are secondary processes that are enhanced by higher temperatures. The primary process of chilling injury is thought to be membrane leakage caused by insufficient scavenging of radicals that form during or after the cold treatment (Hariyadi and Parkin 1991). Membrane leakage is enhanced by lower temperatures and seems to be well correlated with Fv/Fm. It appears that cold storage induces changes in the thylakoid membranes, resulting in a decreased exciton transfer efficiency of PSII, which seems to be temperature dependent and becomes more pronounced in cucumber after 6 d at 20°C. On the other hand, no decrease in Fv/Fm occurs in cucumbers stored at 10°C or higher, which correlates well with the total absence of chilling injury. The decrease in Fv/Fm in chilled cucumber fruit appears to be caused largely by a decrease in Fm. A reduction in Fm can be explained by an inability of the oxygen evolving complex to function, as has been shown to occur in cucumber leaves at O°C (Shen et a1. 1990). Such an inactivation of the donor to PSII would result in an inability to reduce sufficiently the electron carrying redox pool between P680 and cytochrome f. The fact that Fo does not change under most conditions implies that the flux of electrons toward ~ is sufficiently compensated by a drain of electrons toward PSI when the light intensity is low enough, as is presumed to be the case with the measuring light beam. However, storage at 4°C and a subsequent period of 6 d at 20°C results in a further change in the thylakoid membrane, resulting in Fv/Fm reduction that is almost

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solely due to an increase in Fo. Reduction in Fv/Fm that is largely due to higher Fo values could imply a loss of energy transfer efficiency between the light harvesting complex and the reaction center if one reasons according to the model used by Genty et al. (1989). Consequently, one would conclude that fewer PSII complexes remain intact and the bulk of the emitted fluorescence originates directly from the light harvesting complexes. The study by van Kooten et al. (1992) reveals the possibility of using Fv/Fm to detect the effect of chilling on temperature-sensitive fruits and vegetables in the early stages of chilling injury, before the presence of severe visual damage. The exciton transfer efficiency of PSII seems to correlate well with the amount of chilling injury at the membrane level. Thus the possibility exists that chlorophyll fluorescence techniques could be applied as continuous measurements during storage to allow the use of the lowest temperatures possible, while avoiding chilling injury. In later studies, the exciton transfer efficiency of PSII (Fv/Fm) was used as a measure of free radical scavengers in chilling injury of cucumber and bell pepper (Tijskens et al. 1994; van Kooten et al. 1994). A model has been developed based on very fundamental but simplified processes occurring at the membrane and cell plastid level. The model describes the process of chilling injury as dependent on both time and temperature, incorporating a so-called deferred action. This deferred action is described as the degradation of a free radical scavenging system J wherein the free radicals themselves are the direct initiators of chilling injury in an autocatalytic reaction (peroxidative decay of cell membranes) producing more free radicals. For details on the mathematical derivation and validation of the model, and the chemical and physiological background, refer to Tijskens et al. (1994). The proposed model can be applied to study, predict, and prevent the effects of various temperature x time combinations on the behavior of the product. The capacity for free radical scavenging, as a function of temperature and time, is related positively to Fv/Fm (van Kooten et al. 1994). From this model a number of practical applications can be derived, using Fv/Fm as a rapid estimation of active free radical scavenging capacity. There are at least six possible applications. 1. Calculation and determination of the threshold values in free radical concentration or free radical scavenging activity where the net generation of free radicals is zero. This threshold value depends on temperature and time, as scavenging activity decreases with time at low temperatures in chilling sensitive products. Chilling injury will develop if the scavenging activity is less than a threshold value, otherwise no

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chilling injury will develop. A limited amount of chilling injury will develop if the total amount of actual free radical scavenging capacity is large enough that it can cope with the amount of free radicals formed, otherwise extensive chilling injury will develop. 2. Evaluation of post-chilling heat treatment effects to minimize the already induced chilling injury. As with almost all reactions involving free radicals, the chilling injury induction reaction is quite independent of temperature, whereas the scavenging reaction depends very much on temperature. As a consequence, if chilling injury is initiated at low temperatures, it can be slowed somewhat at higher temperatures (12 to 18°C). 3. Determination of prechilling heat conditioning effects to enhance the capability of the produce to resist future chilling. Although the enzyme systems of fruits will be affected by high temperatures (e.g. 28°C), the effect on chilling injury behavior can be explained by the model. Fruits harvested at low temperatures may have an impaired radical scavenging capacity, due to the high temperature sensitivity of the enzymes involved. Saltveit and Cabrera (1987) reported a beneficiary effect of storing tomato at elevated temperatures immediately after harvest, before cooling was applied. The model predicts such behavior if an elevation of the scavenging capacity is assumed in the intermediate high temperature period. Heating in darkness after the chilling period probably induces sources of radical production (e.g. mitochondria), while the scavenging capacity has been impaired. Results with bell peppers (van Kooten et al. 1994) and etiolated chicory (van Kruistum et al. 1994) seem to support this application of the model. 4. Examination of effects of sudden large temperature changes (Le. shock treatment). A sudden exposure to chilling can induce large-scale free radical production in temperature-sensitive plant tissue. After removal from chilling temperatures, a rapid decline in Fv/Fm is observed in bell peppers at 8°C or 16°C (van Kooten et al. 1994). The decrease in Fv/Fm seems to be correlated to the magnitude of the temperature change, while on the other hand the degree of recovery depends on the actual post-storage temperature. 5. Calculation and prediction of the optimal safe storage temperature of each batch of fruits and vegetables separately. Optimal safe storage temperatures depend on the length of storage time and the age of the produce, and hence how much of the scavenging capacity is still intact. 6. Determination, calculation, and application of fluctuating storage conditions or intermittent warming (long cold and short warm) to improve and/or optimize storageability and overall quality with as little as possible actual and potential chilling injury.

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DeEll (1996) showed that Fv, defined as ((Fp-Ft)/Fp)X100 from a nonmodulated fluorometer, has potential to indicate chilling stress in stored apple fruit (Malus x domestica Borkh.). After 3 months of storage in controlled-atmosphere (CA) (2.5 kPa O2 and 4.5 kPa CO 2 ), 'McIntosh' apples stored at O°C had lower Fv values (14.0) than similar apples stored in CA at the optimum temperature of 3°C (Fv =17.3). However, similar results were not found after 6 or 9 months of storage when disorders were present, suggesting that the development of low-temperature disorders and/or fruit aging may interfere with the direct interpretation of fluorescence measurements. It appears that chlorophyll fluorescence is a better indicator of chilling stress when chilling injury has a rapid-onset, such as in banana and mango (Smillie et a1. 1987), green pepper (Lurie et a1. 1994), and cucumber (van Kooten et a1. 1992), than when chilling stress is imposed slowly, such as that which occurs in apples. Abbott et a1. (1993) concluded that chilling injury in eggplant (Solanum melongena L.) fruit could not be detected by chlorophyll fluorescence measurements because of high concentrations of red pigments in the skin. The authors suggested that red pigments absorb the fluorescence and thus may be a limitation in using chlorophyll fluorescence techniques for other commodities with red pigmentation. However, Mir et a1. (1997a) showed that the surface color of apple does not influence chlorophyll fluorescence measurements. B. Detection of Heat Stress Controlled heat treatments (generally >38°C)of fresh fruits and vegetables show promise for decay control (Barkai-Golan and Phillips 1991), disinfestation of insects (Couey 1989), and maintaining postharvest quality (Klein and Lurie 1992). The photosynthetic system in leaves is especially sensitive to heat stress, becoming inactivated at temperatures several degrees below those damaging respiration and other cellular processes (Alexandrov 1964). Of the partial reactions of photosynthesis in leaves, Calvin cycle activity is generally more sensitive to inactivation by heat than either photosynthetic electron transport or photophosphorylation (Bilger et a1. 1986; Weis 1981). Schreiber and Bilger (1987) found the reduction of Calvin cycle activity to precede PSII damage in Arbutus unedo L., expressed by an increase in Fo and a decrease in Fm. On the other hand, Prange et a1. (1990) found high temperature (3D/25°C day/night) caused photosynthetic disruption primarily within PSII of potato plants (Solanum tuberosum L.) but not in the Calvin cycle or PSI. Calvin cycle activity is also more sensitive to heat than the other

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major photosynthetic processes in fruits. Smillie (1992) showed the potential of chlorophyll fluorescence to detect heat stress (48°C for 5 min) in lemon (Citrus limon L.) and in tomato fruit (Lycopersicon esculentum MilL). The initial fluorescence rise to Fm, the decline in fluorescence after Fp, and the quenching of Fm are only partially affected by heat stress, indicating that photoreduction of ~ by PSII, photooxidation linked to PSI, and ATP formation, respectively, are only marginally affected. However, the subsequent relaxation of Fm quenching evident in fruit prior to heating is no longer present after heat treatment. This effect of heat is partially reversible, as 83-85 percent of the relaxation ofFm quenching returns after a further 4 d at 23°C. Smillie (1992) concluded that the nondestructive monitoring of Calvin cycle activity by fluorescence quenching provides a rapid and sensitive means to detect early symptoms of heat stress in chlorophyllous fruits, vegetables, and cut foliage. Smillie (1992) proposed that chlorophyll fluorescence may be a useful indicator of the effectiveness of preconditioning treatments designed to maximize the tolerance of fruits to heat stress. However, Woolf and Laing (1996) found chlorophyll fluorescence to reflect the effect of heat stress in avocado fruit, but not the alleviation of heat damage by pretreatment. The mean Fv/Fm ratio prior to heat treatment in avocado fruit was 0.813 ± 0.001, similar to values for healthy leaves (Adams et a1. 1990; Bjorkman and Demmig 1987). Fv/Fm rapidly decreased to the near-minimal level within 1 h after hot water treatment at 50°C for 1 to 10 min, while only small changes in Fv/Fm occurred during the following 8 d. Fv/Fm 3 to 6 h after treatment was directly related to the duration of hot water treatment. Although pretreatment at 38°C for 1 h almost completely eliminated external browning of avocado fruit, little effect of pretreatment could be detected in Fv/Fm. There was a strong correlation (r =0.93, P < 0.0001) between external browning and Fv/Fm for non-pretreated fruit, but this correlation was not significant when fruit were pretreated. These results suggest that heat treatments of 50°C are higher than even a pretreated avocado photosynthetic system can tolerate, although the other cellular processes and general membrane integrity appeared to successfully acclimate when pretreated. Consequently, while Fv/Fm can provide sensitive information about heat stress to the chloroplast, in relation to the duration of heat treatment applied, it cannot discriminate between damaged and acceptable fruit. Similar conclusions have been made by Joyce and Shorter (1994) with mango fruit. Hot water treatment at 47°C for 1.5 to 2 h caused a significant decrease in Fv/Fm (0.75 to -0.67), which was related to a transient increase in Fo and decrease in Fv. The effects of hot water treatment on

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Fv/Fm were not ameliorated by preconditioning in 37°C air for 7 to 19 h, although preconditioned mango fruit showed less pulp injury on ripening. Jacobi et al. (1995) also found Fv/Fm to decrease in mango fruit treated with hot water at 46°C for 30 min, regardless of whether or not fruit were preconditioned for 4 to 24 h at 39°C in air. However, in the Jacobi study an SF-30 fluorometer (Brancker, Ottawa, Canada) was used, which only measures Fp and not the actual maximal fluorescence (Fm). Although recovery of photosynthesis after a variety of stresses is normal in leaves (Greer and Laing 1988), usually there is little recovery after heat damage at temperatures above 35°C (Havaux 1993a). Chlorophyll fluorescence of both avocado and mango fruit shows little sign ofrecovery following hot water treatment (Joyce and Shorter 1994; Woolf and Laing 1996). The failure to recover probably reflects damage to the electron donating or water-splitting side of PSII, rather than the more easily repairable acceptor side that is damaged by other stresses (Havaux 1993b). In contrast to the above results, Tian et al. (1996) found that Fv/Fm of broccoli (Brassica oleracea L.) decreased immediately after hot water treatment, but then subsequently recovered during storage at 20°C with some treatments (3 or 5 min at 47°C). These results suggest that in broccoli, PSII may recover or be repaired following hot water treatment. In the same study, chlorophyll fluorescence was used to determine the optimum treatment to reduce yellowing of broccoli florets. Hot water treatments that injure broccoli (47°C for 12 or 20 min) caused Fv/Fm to drop immediately to <0.3 and then decrease continuously to -0.1 after 120 h, at which time the florets were decayed. Optimum hot water treatment to prevent yellowing in broccoli (47°C for 7.5 min) caused Fv/Fm to immediately decline to -0.3, but then to remain relatively stable for 120 h. Therefore, chlorophyll fluorescence has the potential to discriminate between the beneficial effects of heat on maintaining green color in broccoli and heat treatments that result in excessive damage. C. Indicator of Atmospheric Stress DeEll et al. (1995) demonstrated that chlorophyll fluorescence techniques can detect low Oz stress in apple fruit prior to the development of off-flavors and skin discoloration, purpling and bronzing, which are associated with low Oz injury. Fv, defined as ((Fp-Ft)/Fp)Xl00 from a non-modulated fluorometer, of apple fruit in low Oz (1-1.5 kPa) decreased from 11.5 to 7.1 after 5 d. Exposure to low Ozfor longer than 5 d did not affect Fv further. DeEll et al. (1995) also found that chlorophyll fluorescence techniques can detect high CO z stress in apple fruit

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prior to the development of off-flavors and small desiccated cavities in the cortex. Fv of fruit in high CO 2 (11-12 kPa) decreased from 11.5 to 3.3 after 5 d, and exposure to high CO 2 for longer than 5 d did not affect Fv further. Later, using a modulated fluorometer, DeEll et al. (199B) found Fv/Fm to be lower and T1/2 (half-time for rise in Fv) to be higher in apples stressed with either high CO 2 (20 kPa) or low O2 (1-2 kPa), compared to control apples in standard CA regimes. For example, Fv/Fm was 0.579 in 'Delicious' and 'Golden Delicious' apples after 1 d of treatment with high CO 2 (control values ~0.604), and T1/2 was 226 ms in 'McIntosh' apples after 1 d of treatment with low O2 (control values -209 ms). Although the effects of high CO 2 or low O2 stress on Fv/Fm and T1/2 can be rapidly observed, the majority of these effects do not change with increased exposure time. 'Golden Delicious' and 'Delicious' apples respond similarly to high CO 2 stress, even though they differ in skin color and relative fluorescence yields. Chlorophyll fluorescence of apples was measured in situ during 15 d at 22°e in O2 levels ranging from 0 to 21 kPa (Mir et al. 1997b). 'Delicious' apples held in 21 kPa O2 showed rapid decreases in Fo and Fm, whereas 'Delicious' held in 0 kPa O2 showed rapid decreases in Fm and Fv/Fm. Some changes in chlorophyll fluorescence were also observed in 'McIntosh' apples held in low 02' The absence of0 2 (0 kPal appeared to reduce the quenching of Fv after 2 and 5 d in 'McIntosh' and 'Delicious', respectively. However, it is difficult to interpret or discuss these results since no statistical differences are reported in the paper by Mir et al. (1997b). Prange et al. (1997) examined the possibility of using chlorophyll fluorescence techniques to control the CA storage conditions for apples. Ethanol production rate increased and off-flavors developed in 'Elstar' apples that were placed in a stressful low O2 atmosphere (0.07 kPa 02' balance N2) at 2°e for 20 d. In addition, Fo increased and Fv/Fm decreased rapidly during such treatment, most likely due to a disassociation of the light harvesting complex and the reaction center of PSII in the thylakoid membrane. This disassociation would decrease the probability of energy transfer and thus energy absorbed in the light harvesting complex would be given off as stray fluorescence from the pigment bed, increasing the Fo value and decreasing the Fv/Fm value. After a shelf-life of 7 d in ambient air at 2°e plus 10 d at 20°C, Fo and Fv/Fm returned to normal. The ability ofFo and Fv/Fm to return to normal suggests that the apples were not permanently damaged by stressful low O2 treatment. Measurements of fruit quality support this suggestion, since there was no visible damage or decay on the apples and

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fruit firmness was the same or better than similar apples storedin ambient air or ultra-low oxygen (1.2 kPa O2 ,2.5 kPa CO 2), While the fruit were in the low O2 atmosphere, there was an exponential relationship between decreasing Fv/Fm and increasing ethanol production rate. This suggests that ethanol accumulation in the tissue and thylakoid membranes can affect chlorophyll fluorescence. Further research is needed to verify the possibility that chlorophyll fluorescence may be affected by ethanol accumulation in the tissue and thylakoid membranes. The addition of 10 kPa CO 2 to the stressful low O2 atmosphere appears to accentuate the increase in Fo, decrease in Fv/Fm, and increase in ethanol in 'Elstar' apples (Prange, unpublished data). Prange and Harrison (1993) used chlorophyll fluorescence to evaluate the effect of CA storage on the postharvest physiology of buttercup winter squash (Cucurbita maxima Duch.). Under low humidity storage (70-800/0 RH) variable fluorescence (calculated as Fp-Fo) was higher in squash stored at 9°C for 3 months in 7 kPa CO 2 than in either 0 kPa or 14 kPa CO 2 , in combination with 1 kPa, 2 kPa or 5 kPa O2, Higher variable fluorescence was associated with the elimination of white mealy breakdown in the squash and less disease development. To investigate the source of CO 2 for the Calvin cycle in fruits, Smillie (1992) used chlorophyll fluorescence as an indicator of photosynthetic activity in tomato and avocado fruit held in either a CO2-free or high CO2 environment. Rates of relaxation for quenching declined more slowly in whole fruit, compared with peel discs or leaves held in CO2-free air, indicating that prolonged continuation of the Calvin cycle in fruit results from the utilization of CO 2 accumulated within the fruit. Further evidence for the utilization of CO 2 from within the fruit is that after cutting the peel from avocado fruit, the rates of relaxation of quenching are high and then quickly decline to zero when fruit discs are placed in a CO2 free atmosphere. This loss of activity is reversible and can be regained when avocados are transferred back into an atmosphere with high CO 2 , Thus it appears that like chloroplasts in leaves, those in avocado peel respond quickly to CO 2 depletion, but in intact fruit these chloroplasts continue to fix CO 2 photosynthetically by using CO 2 present within the fruit. D. Evaluation of Ripening and Senescence During fruit ripening two major changes may affect the level of chlorophyll fluorescence emission (Smillie et a1. 1987). One change may be loss of photosynthetic competence per unit chlorophyll, leading to reduced PSII activity and likely decreased FR (Fi to Fp level of fluores-

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cence) and Fv. Another change may be a decrease in chlorophyll content, which will affect all fluorescence measurements. Using the assumption that Fo is generally unaffected by stress-induced changes in photosynthetic activity per unit chlorophyll, Smillie at a1. (1987) stated that direct effects of chlorophyll loss on fluorescence emission associated with ripening can be monitored by changes in Fo. Changes specifically related to chlorophyll competence can then be followed by the ratio of FR or Fv to Fo. For example, Smillie et a1. (1987) found chlorophyll fluorescence to be a useful indicator of ripening-associated changes in the peel of green fruit. As bananas ripen, both FR and Fo decrease. However, initially FR decreases much faster than Fo, indicating loss of photosynthetic activity per unit chlorophyll as well as chlorophyll loss. It is important to note that accurate Fo values are only obtained when there is no stray light interference, which unfortunately is not possible with most commercially available instruments. Smillie et a1. (1987) used a lab-constructed instrument in which stray light was near zero. Blackbourn et a1. (1990) further examined the functional and structural changes in banana peel tissue during ripening. The fluorescence induction curve of banana is similar to that of leaves, except that there are no smaller peaks following the main Fp peak. There is a progressive loss of Fp and Fm in banana during ripening at 20°C, which corresponds to the loss of chlorophyll associated with PSII and the light harvesting complex of PSII (LHC II) during ripening at 20°C. The decline in Fo during ripening at 20°C (Blackbourn et a1. 1990) is comparable to the gradual decline in Fo during banana ripening at 23°C (Smillie et a1. 1987). The loss of quenching (Fp-Fs) and Fm decline are accelerated during banana ripening at 35°C, compared to those occurring at 20°C (Blackbourn et a1. 1990). Blackbourn et a1. (1990) also compared the fluorescence induction characteristics of plantain (Musa Group, AAB, Subgroup Plantain) to that of banana. Although chlorophyll fluorescence of preclimacteric plantain was similar to that of preclimacteric banana, there was a tran~ sitory increase to a relative peak after Fp, which was not observed in banana. Thus the fluorescence induction of plantain was more similar to leaves than to that of banana (Schreiber 1983). The absolute level of fluorescence in preclimacteric plantain was lower than that of preclimacteric banana, due to a lower chlorophyll content in plantain (Seymour et al. 1987). However, in spite of the lower chlorophyll content, chlorophyll fluorescence parameters of plantain indicate that the photosynthetic apparatus functions normally (Blackbourn et a1. 1990). The loss of fluorescence transients in plantain during ripening at 20°C was relatively similar to the losses observed in banana. However, in contrast

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to bananas ripened at 35°C, there was no detectable increase in either Fo or Fs in plantain ripened at 35°C. Chlorophyll fluorescence is a good indicator of Calvin cycle activity in many fruit species (Smillie 1992). Allowing for differences in chlorophyll content, the photosynthetic activity (based on chlorophyll fluorescence measurements) of the following fruits are comparable to that of the leaves: avocado, blueberry (Vaccinium corymbosUIn Linn.), Citrus grandis L. Osbeck, feijoa (Feijoa sellowiana O. Berg), fig (Ficus carica 1.), guava, kiwifruit (Actinidia deliciosa A. Chev.), lemon, lychee, mandarin (Citrus reticulata Blanco), orange (Citrus sinensis L. Osbeck), persimmon (Diospyros kaki L.), and tomato. Changes in Fm quenching and its subsequent relaxation in tomato fruit at the breaker stage follows the same pattern as that in leaves, with the slow relaxation of quenching indicating that photosynthetic CO 2 fixation is taking place. The decrease in fluorescence after Fp in response to continuous illumination in tomato fruit is more rapid than that of leaves. A more rapid decrease of Fp in fruit has been observed in other comparisons between fruits and leaves, suggesting a more dominant PSI-linked photooxidation of ~ relative to its photoreduction. Fruits and leaves show comparable non-photochemical quenching (qN)' During fluorescence induction there is a rapid increase in ~, followed by marked relaxation after approximately 40 s in lychee, 50 s in lime and blueberry and 80 s in fig. This decrease in ~ agrees with the relaxation of Fm quenching in tomato fruit, indicating strong Calvin cycle activity. Fv/Fo in these and other fruits exceeds 4.0, equivalent to a value of 0.8 for Fv/Fm, indicating that fruits have normal photoreductive systems. Over-mature pears that are softening rapidly have lower than normal Fv/Fo (3.2 compared to normal >4.0). Gross and Ohad (1983) used chlorophyll fluorescence to examine the organization of chlorophyll-containing complexes during the growth and ripening of fruits. Characteristic fluorescence emission peaks are present in the peel and all parts of the green pericarp of mature avocado, cantaloupe (Cucumis melo L. var. cantalupensis) and kiwifruit, and tangerine (Citrus reticuJata Blanco) and tomato fruit after color break. The pattern of fluorescence emission spectra of all fruits, except kiwi, is similar to that of leaves, indicating a normal organization of the chlorophyll-containing complexes of the thylakoid membranes. The characteristic pattern of fluorescence emission spectra for many fruits is characterized by a significantly higher emission at 730-740 nm relative to that of the 686 and 696 nm peaks. On the other hand, the fluorescence emission at 686 and 696 nm is higher than that at 730 nm in kiwifruit, indicating a reduction in the size of PSI antennae chlorophyll. In the innermost yellowish layers of the kiwi pericarp, there is greater loss of

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these antennae chlorophyll and greater disorganization of the PSII complex. Some variable fluorescence is present in all but the innermost layer, in which Fv/Fo is <0.1, compared to 2.1 and 3.6 in the other more photosynthetically active layers. FvlFo values close to 4.0 are equivalent to Fv/Fm values near 0.8, representing healthy plant tissue. As with other studies, Gross and Ohad (1983) also showed that relative fluorescence values decrease as fruits age. DeEll (1996) compared chlorophyll fluorescence measurements with changes in fruit senescence processes in 'Delicious' apples during storage. A rapid decline in Fv/Fm and Tl/2 paralleled fruit senescence, as determined by respiration, ethylene production, and fruit firmness. Fv/Fm and Tl/2 decreased more rapidly in apples stored in air at O°C for 19 weeks than in apples stored in ultra-low Oz (0.70/0 Oz and no COz), which delays fruit senescent processes. DeEll et a1. (1997) found both Fv/Fm and Tl/2 to correlate positively with fruit firmness of 'McIntosh' apples (PpSII values correlated positively with firmness for both apple cultivars, but the relationship was better for 'Elstar' than 'Cox's Orange Pippin'. Furthermore, the correlation found in 'Elstar' occurred only when the apples were at 20°C. When the apples were held at 4°C after removal from ultra-low O2 conditions (1.2 kPa 02' 2 kPa CO 2 , 1.5°C), pSII did not change while firmness decreased, albeit slowly, with time.

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Chlorophyll fluorescence of fruits other than apple has also been found to relate to fruit firmness. Kempler et a1. (1992) found a decline in chlorophyll fluorescence paralleled the decrease in kiwifruit firmness during storage. Fvar, defined as (Fp-Fo)/Fo from a non-modulated fluorometer (Toivonen and Vidaver 1988). decreased more rapidly in kiwifruit stored at 1.0 ± 0.5°C and 970/0 RH than in similar fruit remaining on the vine. Slower Fvar reduction in kiwifruit on the vine indicates that cellular degradation associated with ripening is slower for nonharvested kiwifruit than it is for those that are harvested and placed into storage. Toivonen (unpublished data) found that Fvar of strawberry (Fragaria x ananassa Duch.) sepals was higher in firmer fruit associated with preharvest calcium applications. Using the assumption that calcium affects membrane and cell wall physiology of both fruit and sepal tissue, fluorescence would be a direct measure of the calcium response in the chloroplast membranes of the sepals and hence likely parallels the response to calcium in the fruit tissue. Beaudry et a1. (1997) inferred a positive correlation between fruit firmness and Fv/Fm in freshly harvested peaches (Prunus persica (L.) Batsch), although no significance, r or r 2 values are reported in this paper. Chlorophyll fluorescence was not useful for evaluating apple maturity in terms of optimum harvest time. Van Kooten (1993) found chlorophyll fluorescence to be an unsuitable method for optimum harvest determination of 'Cox's Orange Pippin', 'Elstar', and ']onagold' apples because the variation in fluorescence measurements was too large. This is not surprising, since color indices for skin ground color or flesh cannot be used as the sole determinant of optimum apple maturity (Kingston 1991; Knee et a1. 1989). Chlorophyll content, which can affect chlorophyll fluorescence measurements, is not only influenced by fruit maturity but also by other factors such as light, temperature, and nitrogen in the fruit (Kingston 1991). Chlorophyll fluorescence techniques have been used to assess early postharvest changes in vegetables, such as broccoli (Toivonen 1992). Respiration and vitamin C content are sensitive indicators of changes in broccoli quality, decreasing as the freshness decreases. Fvar, defined as (Fp..Fo)/Fo from a non-modulated fluorometer (Toivonen and Vidaver 1988), decreased with the decline in respiration and vitamin C content in broccoli stored at 1°C. Reduced Fvar indicated a general decrease in chloroplast function, although there was no visual chlorosis or yellowing of the broccoli. Thus, changes in Fvar probably reflected early stages of chloroplast deterioration associated with water loss and not actual senescence. Respiration was highly correlated with Fvar (r = 0.83, P
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Fvar was relatively weak (r = 0.42, P<0.0002). After an initial decline from day 4 to 12, the vitamin C content did not change appreciably in broccoli at 1°C, while respiration and Fvar continued to decrease in a related manner. Hence, chlorophyll fluorescence may be useful as a supplementary indicator of quality loss in green vegetables during storage, especially if it is correlated with respiration or other general indicators of quality. Commercially marketed broccoli can vary in maturity, within certain tolerances (USDA 1943). Therefore, for any technique to be useful in determining the quality loss of broccoli, it must be relatively insensitive to the maturity differences found commercially. Chlorophyll fluorescence appears not to be influenced by broccoli maturity (P. Toivonen and J. DeEll, 1998), and thus would likely be reliable for the assessment of broccoli in the commercial industry. E. Prediction of Shelf-life Chlorophyll fluorescence techniques have been found to be useful in predicting the shelf-life or keeping quality of cucumber, based on initial color (Schouten et a1. 1997; van Kooten et a1. 1997). Color development can be described in a logistic function with a constant reaction rate and a constant value for the final cucumber color after storage. The cucumber color at the start of the storage period is related to growing conditions (plant density and plant nutrients) of the plant (Lin and Ehret 1991). Color development can be described independent of cucumber maturity by applying the concept of a "biological age correction," based on an accurate initial color measurement and chlorophyll fluorescence. The shelf-life of a cucumber can be defined as the time when a color limit (yellowing) is reached and hence the cucumbers are no longer marketable. Predictions of cucumber shelf-life can be made using initial color and chlorophyll fluorescence. Three chlorophyll fluorescence measurements are included in a shelflife prediction model by Schouten et al, (1997) and van Kooten et a!. (1997).
(Harbinson and Woodward 1987; Schreiber et a1. 1988). The rate limiting step for photosynthetic electron transport, which determines ke , precedes P700. Therefore, by measuring how quickly oxidized P700 is reduced by the photosynthetic electron chain, it is possible to calculate

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ke• Measuring the reduction of P700 is done by transiently oxidizing a portion of the P700 pool using a short (1 ms) flash and measuring its relaxation. Using this predictive model, based on an accurate initial color measurement and the three chlorophyll fluorescence measurements, the accounted variability between the predicted and the measured keeping quality for cucumbers of different cultivars and from different growing conditions is 74 percent (Schouten et a1. 1997; van Kooten et a1. 1997). Currently, van Kooten and colleagues are extending the shelf-life prediction model to include different temperatures and relative humidities. They are also trying to predict the occurrence of rubber neck in cucumber, a major physiological disorder. Cucumbers with rubber neck have shriveled and dried stem ends, while the color is still acceptable. Since susceptibility to rubber neck is associated with physiological age (Janse 1995), initial color measurements and chlorophyll fluorescence measurements might be related to this phenomenon. Jolliffe and Lin (1997) were unable to segregate commercial cucumbers based on shelf-life using Fv/Fm and T1/2 fluorescence values. Only Fv/Fm correlated with shelf-life (r =0.22, P<0.05). A multiple regression approach using fluorescence, fruit growth, and color provided some improvement, with the best subset model accounting for 52 percent of the variation in fruit shelf-life. Hence they concluded that this model would be inadequate for the commercial segregation of cucumbers having different shelf-life potentials. Furthermore, some variables of the model would be either unavailable or too time consuming and expensive to obtain commercially. Jolliffe and Lin (1997) used dark-adapted chlorophyll fluorescence measurements and a linear statistical approach, whereas Schouten et a1. (1997) and van Kooten et a!. (1997) used chlorophyll fluorescence measurements in the light and a nonlinear relationship between psII and physiological age. These differences may explain the stronger prediction of cucumber shelf-life by the latter authors. O. van Kooten et a1. (unpublished data) were unable to use chlorophyll fluorescence techniques to predict the shelf-life of leeks obtained from an auction in The Netherlands due to large variations in leek quality and thus in chlorophyll fluorescence measurements. Leeks are not as homogeneous as cucumbers, with considerable variation in diameters and lengths. Differences in shelf-life among cultivars are not known, nor are differences between the storability of thick and thin or long and short leeks. Such unknown information and the fact that leeks are not very morphologically homogeneous makes it virtually impossible to use chlorophyll fluorescence to predict the shelf-life of leeks. Similar large

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variations in product quality and chlorophyll fluorescence measurements make it difficult to predict the shelf-life of 'iceberg' lettuce (J. DeEll, unpublished data). F. Quality Assessment of Ornamentals Van Kooten and Peppelenbos (1993) found chlorophyll fluorescence to be a useful measuring technique to determine the rooting potential of cuttings of chrysanthemum (Chrysanthemum morifolium L.) during storage. Chrysanthemum cuttings are stored at low temperatures (2 to 7°C) for up to 3 weeks, after which root formation is induced by auxins. Although the actual root formation occurs after storage, the ability to form roots is present at the onset of storage and gradually declines during storage. However, the time a cutting can be stored without loss of rooting capability depends on the cultivar, season, storage temperature, and relative humidity. There is a linear relationship between the photosynthetic response (fluorescence) and the quality of flowers evolving from the cuttings several months later. The main varying components of photosynthetic response in chrysanthemum cuttings are qp and cPPSII (van Kooten and Peppelenbos 1993). In comparing the quality (Q) of the cuttings with qp or cPPSII after root formation, the data fit to an exponential sigmoid: qp Q =Qmax/{1 + exp[-k (cPPSII - Oso)ll <1>psII Q =Qmax/{1 + exp[-k (qp - Qso)J} The value of Qrnax (maximum quality) is fixed at 10. The exponential coefficient k is indicative for the steepness of the function and Qso represents the value of qp or <1>PSII at which the quality is half the maximum value (%Qnax)' The exponential sigmoid was chosen because the quality is determined by visual evaluation and is therefore naturally sigmoidal. The fit results in the following values: qp cPPSII

Qso =0.495 ± 0.040 Qso = 0.296 ± 0.006

k = 7.8 ± 3.0 r 2 =0.80 k = 18.2 ± 2.2 r 2 = 0.84

This sigmoidal curve for qp explains 80 percent of the variation in quality, whereas the sigmoidal curve for 4>PSII values explains 84 percent of the variance. Thus, van Kooten and Peppelenbos (1993) concluded that cPPSII is a good indicator of the ability for root formation of chrysanthemum cuttings. On the other hand, they found that the efficiency of energy transfer (Fv/Fm) does not vary much when chrysanthemum cut-

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tings are held in various CA storage treatments that improve or reduce quality. The two fits are equally good, implying that all the variation in psII increased mainly due to an augmentation of Fv/Fm, which recovered to a level above the original. The qp also recovered, but remained below the original value. No adverse effects of transport were visible on Codiaeum variegatum. Van Kooten et al. (1991) concluded that chlorophyll fluorescence can be used to detect whether a potted plant has been exposed to adverse conditions during transport, such as low light. However, the degree to which a decline in PSII correlates to the occurrence of a visually detectable decrease in quality will probably be different among plant species and cultivars.

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Van Kooten et a1. (1991) also used chlorophyll fluorescence to evaluate drought resistance in cut rose flowers. Roses subjected to drought for 12 h hardly responded when measured immediately. However, 4>PSII decreased rapidly after 24 or 36 h of drought but recovered slowly when the roses were placed in water. This decrease of 4>PSII was caused mainly by a decrease in Fv/Fm, which indicates that the diminution in COzfixation, due to a decrease in the water potential of the leaf, was paralleled by a diminution of electron transport caused by a decrease in the flux of photons to the reaction centers. It is expected that, when the regulation mechanism of exciton energy conversion into heat instead of photochemistry fails (Le., when qp diminishes instead of Fv/Fm) , the plant will be unable to fully recover when placed in water after'the storage period. Therefore, a simple 4>PSII measurement should be able to indicate whether the rose has been treated optimally from the moment of harvest until the moment of measurement. Water potential does not appear to be directly related to 4>PSII in cut roses (0. van Kooten, unpublished data). G. Prediction of Superficial Scald Development Superficial scald, a common storage disorder of apples, is characterized by diffuse browning of the skin (Ingle and D'Souza 1989). Chlorophyll fluorescence of 'Delicious' apple strains at harvest was evaluated by DeEll et a1. (1996) as a predictor of superficial scald development during storage. Fv, defined as ((Fp-Ft)/Fp)X100 from a non-modulated fluorometer, at harvest correlated positively (r = 0.28 to 0.50, P<0.05 to P<0.01) with superficial scald development in early-harvested 'Sturdeespur Delicious' apples; fruit with low Fv «10) at harvest was least likely to develop superficial scald, while those with high Fv (>18) were most likely to develop severe superficial scald. However, no such relationship existed for 'Imperial Delicious' apples, or for some 'Sturdeespur Delicious' apples from later harvests. These results suggest that Fv at harvest may be a predictor of superficial scald development, but harvest time and cultivar strain appear to influence its efficacy. Beaudry et a1. (1995) found that Fv/Fm decreased in apples during storage prior to the development of superficial scald but they did not distinguish between the effects of superficial scald development and those of fruit aging on chlorophyll fluorescence measurements. DeEll (1996) also observed that a rapid decline in chlorophyll fluorescence measurements Fv/Fm and T1/2 preceded superficial scald development in apples during storage, while decreases in Fv/Fm and T1/2 also paralleled fruit senescence. Between 4 and 10 weeks of storage, Fv/Fm decreased more rapidly in air-stored (O°C) 'Delicious' apples than in apples stored in

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ultra-low O2 (0.7 kPa O2 and no CO 2 ), The same air-stored apples developed more superficial scald and senesced more rapidly than apples stored in ultra-low 02' which reduces superficial scald development and delays fruit senescent processes. When similar air-stored apples were treated with either diphenylamine to reduce superficial scald incidence or with 1-Methylcyclopropene to delay fruit aging, Fv/Fm decreased at similar rates. Therefore, the effects of fruit senescence will have to be considered if chlorophyll fluorescence is to be used as an indicator of superficial scald development in apple. The ability of chlorophyll fluorescence to detect surface defects on mature apples was examined by Mir et a1. (1997a). Superficial scald, as well as CO 2 injury and bitter pit, caused changes in Fo, Fm and/or Fv/Fm in the areas where surface disorders or damage had developed. Although the authors suggest that chlorophyll fluorescence is a promising tool for sorting apple fruit based on surface defects, no statistical differences or significance are reported in this study. H. Detection of Water Stress Although water stress has been shown to cause substantial changes in the fluorescence induction pattern of leaves (Govindjee et a1. 1981; Havaux and Lannoye 1983; Prange 1986), similarresults have not been found for fruits. DeEll (1996) found chlorophyll fluorescence did not reflect water stress in apples stored in standard CA at 0 or 3°C for 3, 6, and 9 months. The storage humidity affected moisture loss and core browning development but did not influence the Fv values, defined as ((Fp-Ft)/Fp)X100 from a non-modulated fluorometer. It was concluded that chlorophyll fluorescence is not a good indicator for such slowlyimposed stresses, similar to the chilling stress in apples. In support of this theory, reduction in variable fluorescence occurs at much lower water potentials in grapevine leaves when the water stress is slowlyimposed, compared with water stress caused by rapid desiccation (Downton 1983). Considerable osmotic adjustment is observed during slowly-imposed water stress, and the maintenance of turgor pressure associated with osmotic adjustment apparently prevents damage to PSII that occurs during rapidly-imposed stress. Prange and Harrison (1993) used chlorophyll fluorescence to evaluate the effects of storage humidity on buttercup winter squash. Although variable fluorescence (calculated as Fp-Fo) appears to be slightly higher in squash stored for 3 months in CA (combinations of 1, 2 and 5 kPa 02' and 0, 7 and 14 kPa CO2 ) with a low storage humidity (70-800/0 RH) than in squash stored in a high humidity (92-950/0), these data were not sta-

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tistically analyzed for the effect of humidity. Perhaps research using other chlorophyll fluorescence measurements, e.g. cf»psn, qp and/or Fv'/Fm', might be more successful at detecting water stress in fruits and vegetables than the measurements described above in this section. IV. CONCLUDING REMARKS

Chlorophyll fluorescence is a powerful tool in plant stress physiology. The fact that it is nondestructive and can be measured without physical contact to the product surface makes it easily applicable in almost any circumstance fruits and vegetables might encounter in the postharvest phase. Two major phenomena that can be measured, Fv/Fm in the dark and cf»PSII in the light, reveal two totally different characteristics of photosynthetic physiology. Fv/Fm is an extremely sensitive, though temperature independent, measure of membrane constituent integrity, while cf»PSII can be related to an actual current of electrons flowing under the prevailing circumstances. The parameter qp is related to cf»PSII and implies the redox state of the primary electron acceptor of PSII in the current photosynthetic electron flow. Once it is clear what is being measured, it is possible to deduce the physiological implications of chlorophyll fluorescence measurements. Understanding chlorophyll fluorescence allows for the possibility to set up a well-founded hypothesis relating the underlying photosynthetic function to the quality traits under study. In the case of membrane damaging processes such as chilling injury or acute heat stress, Fv/Fm turns out to be a sensitive indicator revealing the initial stages of the damage (Schreiber and Bilger 1987; Tijskens et al. 1994). However, the relationship with the final quality trait under investigation is not straightforward. Measurements of intermediary processes are often necessary to elucidate such possible relationships. In processes such as senescence, Fv/Fm appears to be less useful. Although the tissue is slowly deteriorating, it appears to maintain its photosynthetic capacity up to a point of no return. This is found in many products where gradual senescence is not always accompanied with a gradual decline of Fv/Fm, such as potted plants and cucumber (0. van Kooten, unpublished data). In these cases, Fv/Fm appears to drop rather suddenly when the visual process of senescence has come to a final stage. On the other hand, the regulatory mechanisms of photosynthesis, Le. the rate at which the photosynthetic process moves from one situation of homeostasis to another, can be determined by measuring cf»PSII after a sudden change in the environment and it appears that the acclimation rate

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closely follows the deterioration of the tissue under investigation. These measurements occur after an enhancement of the light intensity (van Kooten et a1. 1997). Many other measurements can be done with chlorophyll fluorescence, some of which can be related to the two types of measurements mentioned before. For example, the induction measurements of Fo-Fi-Fp-Ft are used in (Fp-Ft)/Fp, which is similar to PSII (DeEll et a1. 1995; Maas et a1. 1988), and in (Fp-Fo)/Fp, which is approximately Fv/Fm (Morales et a1. 1991, 1992). For other types of measurements, such as the ratio F735/F68o, the relationship is still totally unknown (Lichtenthaler and Rinderle 1988). Although these measurements may contain valuable information, it is not possible to postulate hypotheses about the relationships between these measurements and the quality traits under investigation. Delayed light emission (DLE) is another possibility to determine the amount of light trapped in PSII when the photosynthetic membrane is cooled down with considerable speed (Abbott et a1. 1997). This technique can give us valuable information about the presence of obstructions in the electron transport chain. When electron transport functions without any obstructions, the amount ofDLE is minimal, which is analogous to Fv/Fm being maximal (qp . . , 1). When electron transport is slightly inhibited this will result in an immediate rise in DLE. Although it has not been proven, it is conceivable, based on our present knowledge of photosynthetic electron transport, that DLE may be similar to 1-qp in chlorophyll fluorescence measurements. Obstruction in the electron transport chain that is not compensated by a diversion of excitons into heat will result in a decrease in Fv/Fm and, consequently, a decrease of qp. Therefore, a rise in 1-qp should be concomitant with a rise in DLE. Since DLE is a measurement prone to complications because it is based on photon counting with the aid of a photomultiplier, the use of DLE may be difficult in practical postharvest applications. Another measurement recently described (Genty and Harbinson 1996) that is similar to the chlorophyll fluorescence measurements uses light absorption at 820 nm to measure the rate constant of photosynthetic electron transport. The value of this rate constant appears to be correlated with the light-saturated CO 2 fixation rate of chloroplasts under nonphotorespiratory conditions. The rate constant is theoretically linked to the conductance of photosynthetic electron transport and appears to be a light-independent physiologically determined value. By combining these measurements, as was done in the keeping quality prediction experiments with cucumbers (van Kooten et a1. 1997), it becomes pos-

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sible to measure both current and resistance of the photosynthetic electron transport chain under rigorously predetermined conditions. In investigating the relationships between the cascade of reactions occurring in senescence or other stress responses, the use of modulated chlorophyll fluorescence is one of the more powerful research tools. It is relatively fast, nondestructive, and measurable without physical contact to the product surface. When care is taken to relate the known physiological processes measured to the complex and often ill-defined quality trait of the product under investigation, it gives us a deeper understanding of the processes involved and finally the possibility of measuring and maintaining quality in these products.

LITERATURE CITED Abbott, J. A., W. R. Forbus Jr., and D. R.Massie. 1993. Temperature damage measurements by fluorescence or delayed light emission from chlorophyll. Proc. Int. Workshop on Nondestructive Technologies for Quality Evaluation of Fruits and Vegetables. p. 44-49. Abbott, J. A., B. L. Upchurch, and R. L. Stroshine. 1997. Technologies for nondestructive quality evaluation of fruits and vegetables. Hort. Rev. 20:1-119. Adams, W. W., B. Demmig.Adams, K. Winter, and U. Schreiber. 1990. The ratio ofvari· able to maximum chlorophyll fluorescence from photosystem II, measured in leaves at ambient temperature and at 17K, as an indicator of the photon yield of photosynthesis. Planta 180:166-174. Alexandrov, V. Y. 1964. Cytophysiological and cytoecological investigations of heat resistance of plant cells towards the action of high and low temperature. Q. Rev. BioI. 39:35-77. Barber, J., and B. Anderson. 1992. Too much of a good thing: light can be bad for photosynthesis; TIBS 17:61-66. Barkai-Golan, R., and D. J. Phillips. 1991. Postharvest heat treatment of fresh fruits and vegetables for decay control. Plant Dis. 75:1085-1089. Beaudry, R. M., J. Song, and W. Deng. 1995. Using chloroplast fluorescence for prediction of scald development in 'Red Delicious' apple fruit. HortScience 30:816 (abstr.). Beaudry, R. M., J. Song, W. Deng, N. Mir, P. Armstrong, and E. Timm.1997. Chlorophyll fluorescence: a nondestructive tool for quality measurements of stored apple fruit. Proc. Int. Conference on Sensors for Nondestructive Testing: Measuring the Quality of Fresh Fruits and Vegetables. p. 56-66. Bilger, W., U. Schreiber, and O. L. Lange. 1986. Chlorophyll fluorescence as an indicator of heat induced limitation of photosynthesis in Arbutus unedo L. p. 391-399. In: J. D. Tenhunen, F. M. Catarino, O. L. Lange, and W. C. Oechel (eds.), Plant response to stress. Springer, Berlin. Bjorkman, 0., and B. Demmig. 1987. Photon yield of O2 evolution and chlorophyll fluorescence characteristics at 77 K among vascular plants of diverse origins. Planta 170:489-504. Blackbourn, H. D., M. J. Jeger, P. John, A. Telfer, and J. Barber. 1990. Inhibition of degreening in the peel of bananas ripened at tropical temperatures. IV. Photosynthetic capac-

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Harbinson, J., and F. 1. Woodward. 1987. The use of light induced absorbance change at 820 nm to monitor the oxidation state of P700 in leaves. Plant Physiol. 103:649660. Hariyadi, P., and K. L. Parkin. 1991. Chilling-induced oxidative stress in cucumber fruits. Postharvest BioI. Technol. 1:33-45. Havaux, M. 1993a. Rapid photosynthetic adaptation to heat stress triggered in potato leaves by moderately elevated temperatures. Plant Cell Environ. 16:461-467. Havaux, M. 1993b. Characterization of the heat damage to the photosynthetic electron transport system in potato leaves. Plant Sci. 94:19-33. Havaux, M., and R. Lannoye. 1983. Chlorophyll fluorescence induction: a sensitive indicator of water stress in maize plants. Irrig. Sci. 4:147-151. Havaux, M., andR. Lannoye. 1985. In vivo chlorophyll fluorescence and delayed light emission as rapid screening techniques for stress tolerance in crop plants. Z. Pflanzenziichtg. 95:1-13. Heber, U., U. Schreiber, K. Siebke, and K-J. Dietz. 1990. Relationship between light-driven electron transport, carbon reduction and carbon oxidation in photosynthesis. p. 17-37. In: 1. Zetlich (ed.), Perspectives in biochemical and genetic regulation of photosynthesis. Alan R. Liss, Inc., New York. Ingle, M., and M. C. D'Souza. 1989. Physiology and control of superficial scald of apples: a review. HortScience 30:1084-1085. Jacobi, K., J. Giles, E. MacRae, and T. Wegrzyn. 1995. Conditioning 'Kensington' mango with hot air alleviates hot water disinfestation injuries. HortScience 30:562-565. Janse, J. 1995. Effect of growing methods on the incidence of rubber necks in cucumber fruits. Acta Hort. 379:281-288. Janssen, L. H., and P. R. van Hasselt. 1994. Temperature effects on chlorophyll fluorescence induction in tomato. J. Plant Physiol. 144:129-135. Jolliffe, P. A., and W. C. Lin. 1997. Predictors of shelf life in long English cucumber. J. Am. Soc. Hart. Sci. 122:686-690. Joyce, D. C., and A. J. Shorter. 1994. High-temperature conditioning reduces hot water treatment injury of 'Kensington Pride' mango fruit. HortScience 29:1047-1051. Kautsky, H., and A. Hirsch. 1931. Neue versuche zur kohlenstoffassimilation. Naturwissenschaften 19:964. Kempler, c., J. T. Kabaluk, and P. M. A. Toivonen. 1992. Effect of environment and harvest date on maturation and ripening of kiwifruit in British Columbia. Can. J. Plant Sci. 72:863-869. Kingston, C. M. 1991. Maturity indices for apple and pear. Hort. Rev. 13:407-432. Klein, J. D., and S. Lurie. 1992. Prestorage heating of apple fruit for enhanced postharvest quality: interaction of time and temperature. HortScience 27:326-328. Knee, M., S. G. S. Hatfield, and S. M. Smith. 1989. Evaluation of various indicators of maturity for harvest of apple fruit intended for long-term storage. J. Hort. Sci. 64:403-411. Krause, G., and E. Weis. 1984. Chlorophyll fluorescence as a tool in plant physiology. II. Interpretation of fluorescence signals. Photosyn. Res. 5:139-157. Krause, G., and E. Weis. 1988. The photosynthetic apparatus and chlorophyll fluorescence: an introduction. p. 3-11. In: H. K. Lichtenhaler (ed.), Applications of chlorophyll fluorescence in photosynthesis research, stress physiology, hydrobiology and remote sensing. Kluwer Academic. Dordrecht. The Netherlands. Krause, G., and E. Weis. 1991. Chlorophyll fluorescence and photosynthesis: the basics. Ann. Rev. Plant Physio!. Plant Mol. BioI. 42:313-349.

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Kyle, D. J. 1987. The biochemical basis for photoinhibition of photosystem II. p. 196-226. In: D. J. Kyle, C. B. Osmond, and C. J. Arntzen (eds.), Photoinhibition, Topics in photosynthesis, vol. 9. Elsevier, Amsterdam, The Netherlands. Lavorel, J., and A.-L. Etienne. 1977. In vivo chlorophyll fluorescence. p. 203-268. In: J. Barber (ed.), Primary processes of photosynthesis. Elsevier/North-Holland Biomedical Press, Amsterdam, The Netherlands. Lichtenhaler, H. K. 1988. In vivo chlorophyll fluorescence as a tool for stress detection in plants. p. 129-142. In: H. K Lichtenhaler (ed.), Applications of chlorophyll fluorescence in photosynthesis research, stress physiology, hydrobiology and remote sensing. Kluwer Academic, Dordrecht, The Netherlands. Lichtenhaler, H. K 1992. The Kautsky effect: 60 years of chlorophyll fluorescence induction kinetics. Photosynthetica 27:45-55. Lichtenhaler, H. K, and U. Rinderle. 1988. The role of chlorophyll fluorescence in the detection of stress conditions in plants. CRC Crit. Rev. Anal. Chern. 19:529-S84. Lin, W. C., and D. L.Ehret. 1991. Nutrient concentration and fruit thinning affects shelflife oflong English cucumber. HortScience 26:1299-1300. Lurie, S., R. Ronen, and S. Meier. 1994. Determining chilling injury induction in green peppers using nondestructive pulse amplitude modulated (PAM) fluorometry. J. Am. Soc. Hort. Sci. 119:59-62. Maas, F. M., E. N. van Loo, and P. R. van Hasselt. 1988. Effect of long-term H2 S fumigation on photosynthesis in spinach. Correlation between CO2 fixation and chlorophyll a fluorescence. Physiol. Plant. 72:77-83. Mir, N., M. Wendorf, R. Perez, and R. M. Beaudry. 1997a. Chlorophyll fluorescence: assessing quality of stored apples. Proc. Seventh Int. Controlled Atmosphere Res. Conference 2:50-56. Mir, N., M. Wendorf, R. Perez, and R. M. Beaudry. 1997b. Variable fluorescence quenching in apple during storage is a function of O2 , Proc. Seventh Int. Controlled Atmosphere Res. Conference 2:162-167. Moll, B., and K. Steinbach. 1986. Chilling sensitivity in Oryza sativa: the role of protein phosphorylation in protection against photoinhibition. Plant Physiol. 80:42Q-423. Morales, F., A. Abadia, and J. Abadia. 1991. Chlorophyll fluorescence and photon yield of oxygen evolution in iron-deficient sugar beet (Beta vulgaris L.) leaves. Plant Physio1. 97:886-893. Morales, F., A. Abadia, J. G6mez-Aparisi, and J. Abadia. 1992. Effects of combined NaCI and CaCl 2 salinity on photosynthetic parameters of barley grown in nutrient solution. Physiol. Plant. 86:419-426. Morris, L. L. 1982. Chilling injury of horticultural crops: an overview. HortScience 17:161-165. Ohad, I., N. Adir, H. Koike, D. J. Kyle, and Y. Inoue. 1990. Mechanism of photoinhibition in vivo. A reversible light-induced conformational change of reaction center II is related to an irreversible modification of the D1 protein. J. BioI. Chern. 265:1972-1979. Papageorgiou, G. 1975. Chlorophyll fluorescence: an intrinsic probe of photosynthesis. p. 319-371. In: W. Govindjee (ed.), Bioenergetics of photosynthesis. Academic Press, New York. Peeler, T. C., and A. W. Naylor. 1988. A comparison ofthe effects of chilling on thylakoid electron transfer in peas (Pisum sativum L.) and cucumber (Cucumis sativus L.). Plant Physiol. 86:147-151. Prange, R. K 1986. Chlorophyll fluorescence in vivo as an indicator of water stress in potato leaves. Am. Potato J. 63:325-333.

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Prange, R. K., and P. A. Harrison. 1993. Effect of controlled atmosphere and humidity on postharvest physiology of buttercup winter squash, Cucurbita maxima Ouch. hybrid 'Sweet Mama'. Proc. Sixth Int. Controlled Atmosphere Res. Conference 2:759-766. Prange, R. K., K. B. McRae, D. J. Midmore, and R. Deng. 1990. Reduction in potato growth at high temperature: role of photosynthesis and dark respiration. Am. Potato J. 67:357-369. Prange, R. K., S. P. Schouten, and O. van Kooten. 1997. Chlorophyll fluorescence detects low oxygen stress in 'Hlstar' apples. Proc. Seventh Int. Controlled Atmosphere Res. Conference 2:57-64. Renger, G., and U. Schreiber. 1986. Practical applications of fluorometric methods to algae and higher plant research. p. 587-619. In: Govindjee, J. Amesz, and O. Fork (eds.), Light emission by plants and bacteria. Academic Press, Orlando, FL. SaltveitJr., M. E., and R. M. Cabrera. 1987. Tomato fruit temperature before chilling influences ripening after chilling. HortScience 22:452-454. Schouten, R. B., E. C. Otma, O. van Kooten, and L. M. M. Tijskens. 1997. Keeping quality of cucumber fruits predicted by biological age. Postharvest BioI. Technol. 12:175-181. Schreiber, U. 1983. Chlorophyll fluorescence yield changes as a tool in plant physiology. I. The measuring system. Photosyn. Res. 4:361-373. Schreiber, U., and W. Bilger. 1987. Rapid assessment of stress effects on plant leaves by chlorophyll fluorescence measurements. p. 27-53. In: J. O. Tenhunen, F. M. Catarino, O. L. Lange, and W. C. Dechel (eds.), Plant response to stress; functional analysis in Mediterranean ecosystems. NATO ASI Series, Vol. G15. Springer-Verlag, Berlin. Schreiber, U., and W. Bilger. 1993. Progress in chlorophyll fluorescence research: major developments during the past years in retrospect. Prog. Bot. 54:151-173. Schreiber, U., C. Klughammer, and C. Neubauer. 1988. Measuring P700 absorbance changes around 830 nm with a new modulation system. Z. Naturforsh. Teil C. 43:686-698. Schreiber, U., U. Schliwa, and W. Bilger. 1986. Continuous recording of photochemical and non-photochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer. Pbotosyn. Res. 10:51-62. Seymour, G. B., P. John, and A. K. Thompson. 1987. Inhibition of degreening in the peel of bananas ripened at tropical temperatures. II. Role of ethylene, oxygen and carbon dioxide. Ann. Appl. BioI. 110:153-161. Shen, J. R., I. Terashima, and S. Katoh. 1990. Cause for dark chilling-induced inactivation of photosynthetic oxygen-evolving system in cucumber leaves. Plant Physiol. 93:1354-1357. Smillie, R. M. 1992. Calvin cycle activity in fruit and the effect of heat stress. Scientia Hort. 51:63-95. Smillie, R. M., and S. E. Hetherington. 1983. Stress tolerance and stress~inducedinjury in crop plants measured by chlorophyll fluorescence in vivo. Chilling, freezing, ice cover, heat and high light. Plant Physiol. 72:1043-1050. Smillie, R. M., and S. E. Hetherington. 1990. Screening for stress tolerance by chlorophyll fluorescence. p. 229-261. In: Y. Hashimoto, P. J. Kramer, H. Nonami, and B. R. Strain (eds.), Measurement techniques in plant science. Academic Press, San Oiego. Smillie, R. M., S. E. Hetherington, R. Nott, G. R. Chaplin, and N. L. Wade. 1987. Applica~ tions of chlorophyll fluorescence to the postharvest physiology and storage of mango and banana fruit and the chilling tolerance of mango cultivars. Asean Food J. 3:55-59. Smillie, R. M., and R. Nott. 1979. Assay of chilling injury in wild and domestic tomatoes based on photosystem activity of the chilled leaves. Plant Physiol. 63:796-801. Sne}, J" O. van Kooten, and L. van Hove. 1991. Assessment of stress in plants by analysis of photosynthetic performance. Trends Anal. Chern. 10:26-30.

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Sommersalo, S., and G. H. Krause. 1989. Photoinhibition at chilling temperature: fluorescence characteristics of unhardened and cold-acclimated spinach leaves. Planta 177:409-416. Song, J., W. Deng, R M. Beaudry, and P. A. Armstrong. 1997. Changes in chlorophyll fluorescence of apple fruit during maturation, ripening, and senescence. HortScience 32:891-896. Stryer, L. 1988. Biochemistry, 3rd ed. W. H. Freeman and Co., New York. Terashima, T., L. K. Huang, and C. B. Osmond. 1989. Effects of leaf chilling on thylakoid functions, measured at room temperature, in Cucumis sativus L. and Oryza sativa L. Plant Cell Physiol. 30:841-850. Tian, M. S., A. B. Woolf, J. H. Bowen, and I. B. Ferguson. 1996. Changes in color and chlorophyll fluorescence of broccoli florets following hot water treatment. J. Am. Soc. Hort. Sci. 121:310-313. Tijskens, L. M. M., E. C. Dtma, and O. van Kooten. 1994. Photosystem II quantum yield as a measure of radical scavengers in chilling injury in cucumber fruits and bell peppers. A static, dynamic and statistical model. Planta 194:478-486. Toivonen, P. M. A. 1992. Chlorophyll fluorescence as a nondestructive indicator of freshness in harvested broccoli. HortScience 27:1014-1015. Toivonen, P. M. A., and J. DeEll. 1998. Differences in chlorophyll fluorescence and chlorophyll content of broccoli associated with maturity and sampling section. Postharvest BioI. Technol. (in press). Toivonen, P., and W. Vidaver. 1988. Variable chlorophyll a fluorescence and CD 2 uptake in water-stressed white spruce seedlings. Plant Physiol. 86:744-748. USDA. 1943. United States standards for grades of bunched Italian sprouting broccoli. USDA-ARS, Fruit and Vegetable Division, Fresh Products Branch. van Grondelle. R, J. P. Dekker, T. Gillbro, and V. Sundstrom. 1994. Energy-transfer and trapping in photosynthesis. Biochim. Biophys. Acta 1187:1-65. van Kooten, D. 1993. Chlorophyll fluorescence as a possible aid to determine the ripeness stage of apples. English summary of: Lavrijsen, P. J. M. and D. van Kooten. 1993. Chlorofylfluorescentie ter bepaling van het pluktijdstip van appels. ATD-DLD, AgrotechnologischDnderzoek Instituut, Wageningen, The Netherlands. van Kooten, 0., M. G. J. Mensink, E. C. Dtma, and W. van Doorn. 1991. Determination of the physiological state of potted plants and cut flowers by modulated chlorophyll fluorescence. Acta Hort. 298:83-91. van Kooten, D., M. G. J. Mensink, E. C. Otma, A. C. R van Schaik, and S. P. Schouten. 1992. Chilling damage of dark stored cucumbers (Cucumis sativus L.) affects the maximum quantum yield of photosystem 2. p. 161-164. In: N. Murata (ed.), Progress in photosynthesis research, vol. IV. Kluwer Academic, Dordrecht, The Netherlands. van Kooten, D., and H. Peppelenbos. 1993. Controlled atmosphere storage of chrysanthemum cuttings. Proc. Sixth lnt. Controlled Atmosphere Res. Conference, 2:610-619. van Kooten, 0., R E. Schouten, and L. M. M. Tijskens. 1997. Predicting shelf-life of cucumbers (Cucumis sativus L.) by measuring color and photosynthesis. Proc. Int. Conference on Sensors for Nondestructive Testing: Measuring the Quality of Fresh Fruits and Vegetables. p. 45-55. van Kooten, D., and J. F. H. Snel. 1990. The use of chlorophyll fluorescence nomenclature in plant stress physiology. Photosyn. Res. 25:147-150. van Kooten, D., L. M. M. Tijskens, and E. C. Dtma. 1994. Photosystem II quantum yield as a measure of radical scavengers in chilling injury in fruits of tropical origin. A model of approach. Proc. Int. Conference on Cooling and Quality of Fresh Vegetables. p.30-41.

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van Kruistum, G., A. R. Biesheuvel, R. C. F. M. van den Broek, P. M. T. M. Geelen, and J. G. M. Jeurissen. 1994. Onderzoek gericht op het voorkomen van lage temperatuurbederf bij witlof. p. 19-26. In: Jaarboek (ed.), 1993/1994 Vollegrondsgroente-teelt, PAGV-publikatienr.73B. Walker, D. 1985. Measurement of oxygen and chlorophyll. p. 95-106. In: J. Coombs, D. Hall, S. Long, and J. Scurlock (eds.), Techniques in bioproductivity and photosynthesis, 2nd ed. Pergamon Press, Oxford, England. Wang, C. Y. 1993. Approaches to reduce chilling injury of fruits and vegetables. Hort. Rev. 15:63-95. Weis, E. 1981. Reversible heat-inactivation of the Calvin cycle: a possible mechanism of the temperature regulation of photosynthesis. Planta 151:33-39. Weis, E., and J. Berry. 1987. Quantum efficiency of photosystem II in relation to 'energydependent' quenching of chlorophyll fluorescence. Biochim. Biophys. Acta 894:198207. Woolf, A. B., and W. A. Laing. 1996. Avocado fruit skin fluorescence following hot water treatments and pretreatments. J. Am. Soc. Hort. Sci. 121:147-151.

3 Zinc Nutrition in Horticultural Crops Dariu8Z Swietlik* Texas A&M University-Kingsville, Citrus Center, P.O. Box 1150, Weslaco, Texas 78599-1150.

USDA-ARS Appalachian Fruit Research Station, 45 Wiltshire Rd., Kearneysville, West Virginia 25430. (current address) I. Introduction

A. Historical Review B. Geographic Distribution of Zn Deficiency and Toxicity C. Scope of the Review II. Zn in Soils A. Content and Chemical Fractions 1. Zn in Soil Solution 2. Surface Adsorbed and Exchangeable Zn 3. Zn Associated with Organic Matter 4. Zn Associated with Hydrous Oxides and Carbonates 5. Zn in Soil Minerals B. Zn Sorption C. Zn Associated with Organic Matter D. Equilibria of Synthetic Zn Chelates E. Zn 2+ Soil Activity and Inorganic Phase Equilibria III. Factors Affecting Zn Availability A. Soil Parent Material and Organic Matter B. Soil pH C. Restricted Root Zones D. Nutrient Interactions E. Temperature F. Soil Moisture G. Light IV. Function, Absorption, and Transport of Zn in Plants A. Function B. Absorption C. Transport *1 thank John E. Fucik, Lloyd B. Fenn and Robert P. Wiedenfeld for their valuable comments.

Horticultural Reviews, Volume 23, Edited by Jules Janick ISBN 0-471-25445-2 © 1999 John Wiley & Sons, Inc. 109

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D. SWIETLIK

V. Zn Deficiency and Toxicity Symptoms A. Deficiency B. Toxicity C. Zn Tissue Concentrations VI. Effect of Zn Applications on Plants A. Vegetative Growth B. Yield C. Crop Quality VII. Technology of Zn Applications VIII. Zn Fertilizers IX. Conclusions Literature Cited

I. INTRODUCTION Zinc is an essential element in the nutrition of plants. The deficiency of this element may lead to serious reductions in yield and/or crop quality. Certain sensitive plant species, e.g., pecan, citrus, rice (Table 3.1), may be particularly affected. Many scientific investigations were conducted in the past to develop the best possible methods of alleviating zinc deficiency. Although rare, zinc toxicity can also be an important economic factor in the culture of horticultural and agronomic crops. A. Historical Review As early as 1863 and 1869, Raulin reported that Zn is essential for the growth of fungi (Thorne 1957). It was not until the 1910s, however, that the essentiality of Zn for the growth of higher plants was proven in a series of experiments conducted by P. Maize and reported in 1914, 1915, and 1919 (Thorne 1957). About fifteen years later, Chandler et al. (1931) demonstrated that "little leaf" or "rosette" on peaches could be corrected with soil Zn applications. Shortly afterwards, this discovery was confirmed by Alben et al. (1932) on pecan and by Johnston (1933) and Parker (1934, 1935) on citrus. B. Geographic Distribution of Zn Deficiency and Toxicity Zinc deficiency is widespread throughout the world (Viets 1966). Deficiency of Zn, next to N, is considered the most widespread nutritional malady of citrus (Chapman 1968). Zinc deficiencies are associated with high pH, calcareous soils in which Zn availability is greatly reduced, and with sandy, highly leached soils because of their low total Zn content (Swietlik 1989). Considerable field variability exists, however, within each soil group, as attested by localized occurrence of Zn deficiency within a given field. Consequently, it is difficult to assign Zn deficiency

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Table 3.1.

Common and scientific names of plants mentioned in the text.

Common name

Scientific name

Alfalfa Apple Apricot Asparagus Avocado Barley Bean Birch Carrot Cherry (tart and sweet) Chickpea Clover Cotton Cucumber Fenugreek Filbert Flax Geranium Grapefruit Grapes Lentil Lettuce Maize Mustard, brown Oat Okra Onion Orange Pea Peach Pear Pecan Peppermint Pistachio Potato Rice Rye Sorghum Sour orange Soybean Spearmint Strawberry Subterranean clover Sudan grass Sugarcane

Medicago sativa L. Malus x domestica Borkh. Prunus armeniaca L. Asparagus officinalis L. Persea americana Mill. Hordeum vulgare L. Phaseolus vulgaris L. Betula sp. Daucus carota L. Prunus cerasus L. and P. avium L. Cicer arietinum L. Trifolium spp. Gossypium hirsutum L. Cucumis sativus L. Trigonella foenum-graceum L. Corylus maxima Mill. Linum usitatissimum L. Pelargonium x hortorum Bailey Citrus paradisis Macf. Vitis vinifera L. Lens culinaris Medik. Lactuca sativa L. Zea maysL. Brassica juncea (L.) Czern. Avena sativa L. Abelmoschus esculentus L. Moench Allium cepa L. Citrus sinensis L. Pisum sativum L. Prunus persica (L.) Batsch Pyrus communis L. Carya illinoensis (Wang.) K. Koch Mentha x piperita 1. Pistacia vera L. Solanum tuberosum L. Oryza sativa L. Secale cereale L. Sorghum bicolor (L.) Moench Citrus aurantium L. Glycine max (L.) Merr. Mentha spicata L. Fragaria x ananassa Duch Trifolium subterraneum L. Sorghum sudanense (Piper) Stapf Saccharum officinarum L. continued

112 Table 3.1.

D. SWIETLIK

Cont.

Common name

Scientific name

Sugar beet Tobacco tree Tomato Tufted hairgrass. Tung Wheat Walnut

Beta vulgaris L. Nicotiana glauca Grah. Lycopersicon escuJentum Mill. Deschampsia caespitosa (L.) Beauv. Aleurites fordii Hemsl. Triticum aestivum L. ]uglans regia L.

to broad regions consisting of specific soil types (Kubota 1980). Obviously, factors other than the soil itself modify Zn availability to plants. Zinc deficiency has been reported to occur in the western United States, on irrigated lands in the Pacific Northwest, California, and in Canada's southern British Columbia (Chapman 1968; Neilsen et al. 1986; Neilsen et al. 1987; Embleton et al. 1988; Neilsen and Neilsen 1994). Zinc deficiency is encountered on citrus grown on calcareous soils of the Rio Grande Valley of Texas (Swietlik 1989) and on Florida's sandy, highly leached soils (Koo 1988). Zinc deficiency symptoms are common on pecan trees grown in Alabama, Arkansas, Florida, Georgia, Louisiana, Mississippi, Oklahoma, South Carolina, and Texas (Sparks 1987). Welch et al. (1991) reports that Zn deficiency in the southeastern United States, east of 100th meridian, is generally less frequent than in the West and is least frequent in the Northeast and Midwest. However, Zn deficiency is a major nutritional malady in tree fruit crops in the Northeast (Stiles and Goff 1965; Stiles 1980, 1987, 1991, 1992, 1993). Growth of young trees is often adversely affected (Stiles 1987). Likewise, yield, size, and color of fruit harvested from mature trees are reduced under low Zn supply conditions (Stiles and Goff 1965; Stiles 1966, 1980, 1987). Also, maize low in Zn was reported in New York, Connecticut, Iowa, and Illinois (Vanden Huevel et al. 1989; Carsky and Reid 1990; Bugbee and Frink 1995; Mallarino and Webb 1995). Zinc deficiency was also reported on Prince Edward Island in Canada (White et al. 1987; Sanderson and Gupta 1990). Zinc deficiency is encountered in most countries that have been studied so far except Belgium and Malta (Sillanpaa 1982 cited by Welch et al. 1991). In Asia, severe Zn deficiencies have been reported on a number of crops in India (Tiwari and Dwivedi 1990, 1994), in calcareous paddy soils of China, rice fields of the Philippines, and in the countries of the Middle East (Welch et al. 1991). Zinc deficiency was also reported in the Caucasian region of Russia (Agaev 1989). Zn deficiency occurs in Brazil and Australia (see papers cited by

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113

Welch et al. 1991). According to Leece (1978a), Zn deficiency is common on maize grown on black earth soils in north-western New South Wales, Australia. Eight to nine million hectares of soils are Zn-deficient in southwestern Australia and yields of cereal crops grown on these soils are greatly reduced unless fertilized with Zn (see Gartell and Glencross 1968 and Donald and Prescott 1975 cited by Brennan 1996). This is the largest contiguous area of Zn deficiency in the world. Zinc toxicity in plants is rare and usually results from soil contaminations caused by mining, metal smelting, and/or disposal of municipal and industrial wastes rich in Zn. Although phytotoxicity of Zn under natural conditions is very rare, it does occur (Welch et al. 1991).

c.

Scope of the Review

There are several comprehensive reviews on zinc in soils and plant nutrition (Thorne 1957; Chapman 1966; Viets 1966; Lindsay 1972; Swietlik 1989; Kabata-Pendias and Pendias 1992). Reviews partially devoted to Zn by Loneragan (1975), Lindsay (1979), Parker et al. (1995a), and Welch (1995) address one or more aspects of Zn in plant nutrition and/or its behavior in soils. In the last two decades, much has been learned about factors controlling the amount of free Zn 2+ in soils; interaction between Zn and other mineral nutrients; the role of Zn in plant physiology, absorption, and transport; and the effect of Zn applications on yield, vegetative growth, and quality of various crops. These advances have prompted this review of the subject. The role of Zn in plant physiology is difficult to study because its level of activity in the rhizosphere required for normal plant growth is very low. Zinc activity (pZn) expressed as -log(Zn 2+) [pZn =-log (Zn2+)], where (Zn 2+) indicates mollar concentration of active Zn 2+, varies between 10 and 11 for most plant species. It comes as no surprise, then, that reproducing varying degrees of Zn deficiency in traditional water culture is extremely difficult because even a trace of Zn contamination is sufficient to greatly affect the nutritional status of plants. The advent of the so-called chelator-buffered nutrient solutions, first proposed by Chaney and co-workers (Chaney 1988; Chaney et al. 1989; Bell et al. 1991a,b), and the development of computerized chemical equilibrium models (Parker et al. 1995b) opened new possibilities for advancing our knowledge of Zn nutrition (Parker et al. 1992; Norvell and Welch 1993; Welch and Norvell 1993; Swietlik and Zhang 1994). It is impossible to include all published information on Zn nutrition in one review, but I have tried to provide a good representation of our current knowledge of the mechanisms involved in controlling soil Zn

114

D. SWIETLIK

availability, the role of Zn in plant physiology, Zn absorption and transport, and the responses of horticultural crops to Zn applications. Technology of Zn fertilizer applications and general descriptions of Zn fertilizers are also included. Although emphasis was placed on horticultural plants, particularly fruit trees, the information on other species was also included whenever it clarified or enhanced the understanding of the subject at hand. II. ZN IN SOILS A. Content and Chemical Fractions Zinc has a complete 3d1 04s 2 outer electronic configuration and unlike the other d block micronutrients, such as Fe, Cu, Mn, and Mo, has only a single oxidation state and hence a single valence of II. Zn shows some similarity to the second main group element, Mg, and can substitute for it in silicate minerals (Chesworth 1991). However, Zn is much more electronegative than Mg, Le., 1.6 vs. 1.2, and forms much stronger covalent bonds. The average concentration of Zn in the earth's crust is approximately 100 ppm in igneous rock, 70 ppm in granitic, and 40 ppm in basaltic rock (Taylor 1964); in sedimentary rocks, Zn concentration is 95 ppm in shale, 20 ppm in limestone, and 16 ppm in sandstone (Turekian and Wedepohl 1961). The total Zn content in soils varies from 3 to 770 ppm, with the worldwide average being 64 ppm (Kabata-Pendias and Pendias 1992). The total Zn is uniformly distributed in soil profiles (Swaine and Mitchell 1960; Follett and Lindsay 1970). However, DTPA-extractable Zn (Table 3.2) declined with depth in 37 profiles of Colorado soils (Follett and Lindsay 1970). A similar trend was also reported for EDTA-extractable Zn (Lindsay 1972) and acetic acid-extractable Zn (Swaine and Mitchell 1960). These results most likely reflect the sorption of soluble Zn on organic and mineral constituents of the soil surface horizon. Table 3.2.

Synthetic chelates mentioned in the text.

Symbol

Chemical name

CDTA DTPA EDTA

trans-1 ,Z-Cyklohexylenedinitrilotetraacetic acid diethylenetriamine-pentaacetic acid ethylenediamine tetraacetic acid N-(Z-Hydroxyethyl)ethylenedinitrilotriacetic acid nitrilotriacetic acid

HEDTA

NTA

3. ZINC NUTRITION IN HORTICULTURAL CROPS

115

There are five major pools ofZn in the soil: (1) Zn in the soil solution, (2) surface adsorbed and exchangeable Zn, (3) Zn associated with organic matter, (4) Zn associated with oxides and carbonates, and (5) Zn in primary minerals and secondary alumino-silicate minerals (Shuman 1991). This classification does not offer clear boundaries between various Zn fractions. Nevertheless, I adopted this classification in this review recognizing that some overlapping between the groups may occur. 1. Zn in Soil Solution. Plants absorb Zn from the soil solution where it is present as a free ion or as a complex with organic or inorganic ligands (Shuman 1991). In New York and Colorado soils, the organic forms constituted from 5% to 90% and from 280/0 to 99% of Zn in the soil solution, respectively (Hodgson et al. 1965, 1966). Zn z+ is the predominant ionic species of Zn in soils, although it hydrolyses as solution pH increases. The ZnOH+ form dominates at pH >7.7 and Zn(OH)~ at pH >9 (Lindsay 1991). Among inorganic complexes, ZnS04 is very important but ZnNOj, Zn(N0 3)z, ZnCI+, ZnCl z, ZnCI 3, ZnCI~-, and ZnHzPO.! are of little importance (Lindsay 1979). ZnHP0 4 contributes significantly to total Zn in solution only in neutral and calcareous soils. Thus, according to Lindsay (1979), the Zn inorganic species that contribute significantly to total Zn in the soil solution are:

Zinc concentrations in solutions obtained by centrifugation from soils of various texture and pH (2.5 to 7.8) showed values ranging from 7137 to 100 Jlg·liter-1 (ppb) (Kabata-Pendias and Pendias 1992). Although, as expected, the general trend was for the values to decrease with increasing pH, a loamy soil with pH 7 to 7.5 had a surprisingly lower soil solution Zn concentration than a more alkaline (pH 7.5 to 7.8) calcareous soil. The mechanism controlling the amount of Zn present in the soil solution is not yet completely understood. Nevertheless, it likely encompasses the dynamic equilibria of various chemical reactions and biological processes and the flow of soluble Zn in soil macropores due to gravitational forces, evapotranspiration, and diffusion (Stevenson 1986; Harter 1991; Lindsay 1991). The following reactions and processes are most likely to affect soil solution Zn concentration: (1) the equilibrium solubility of solid phase minerals containing Zn, e.g., franklinite; (2) specific adsorption on manganese and iron oxides; (3) incorporation into crystalline silicates through isomorphous substitution; (4) cation exchange; (5) formation of complexes with organic ligands; (6) microbial mobilization and sorption; (7) plant uptake; (8) leaching; (9) weathering

116

D. SWIETLIK

of primary minerals; and (10) addition of fertilizers (Jenne 1968; Lindsay 1972, 1991; Harter 1991; Stevenson 1991; Kabata-Pendias and Pendias 1992). Except for leaching, weathering, and addition of fertilizers, all the

other reactions may proceed in two directions, Le., they may remove or add Zn to the soil solution. More recent data suggest that Znz+ activities in the soil solution may actually be controlled by franklinite (ZnFez04) whose equilibrium solubility is similar to that of soil-Zn over the pH values of 6 to 9 (Lindsay 1991; Ma and Lindsay 1993). The mineral will precipitate whenever Zn concentration in the soil solution exceeds the equilibrium solubility of the mineral and it will dissolve whenever the opposite is true, thus providing an effective Zn buffering system. 2. Surface Adsorbed and Exchangeable Zn. In this fraction, Zn is associated with layered alumino-silicates, hydrous oxides of AI, Fe, and Mn, and solid organic matter (Shuman 1991). The exchangeable pool of Zn is considered highly available to plants. Adsorbed Zn is further divided into (1) weakly (nonspecifically) adsorbed, and (2) strongly (specifically) adsorbed. 3. Zinc Associated with Organic Matter. This fraction includes watersoluble and solid organic compounds. The metal is bound via incorporation into organic molecules, chelation, exchange, or by specific and nonspecific adsorption (Shuman 1991). 4. Zn Associated with Hydrous Oxides and Carbonates. Zn is associated

with this fraction via adsorption, surface complex formation, ion exchange, incorporation into the crystal lattice, and co-precipitation (Shuman 1991). Some of these reactions fix Zn rather strongly and are believed to be instrumental in controlling the amount of Zn present in the soil solution (Jenne 1968). 5. Zn in Soil Minerals. Based on the geochemical classification, Zn has predominantly chalcophile chemistry because it largely occurs in the earth's crust in the sulfide mineral sphalerite (ZnB) (Krauskopf 1972). Due to Zn's ability to substitute for Mg and Fe in silicates, however, it also has lithophile characteristics forming silicate minerals in isomorphous substitution sites. Under high pH conditions and a very high Zn z+ concentration in soil solution (>10-4 M), Zn could theoretically precipitate as Zn(OH)z, ZnO (zincite), ZnC0 3 (smithsonite), Zn4 (OHlzBiz0 7 ,HzO (hemimorphite) or Zn zBi04 (willemite) (Krauskopf 1972). However, the above minerals are

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far too soluble to be responsible for very low concentrations of Zn 2+ found in most soil solutions and hence are unlikely to be present in soils (Lindsay 1991). Zinc can also precipitate in soils as ZnS under reduced conditions (Chesworth 1991), but under normal oxidizing conditions the concentration of S2- is far too low for the compound to be stable (Lindsay 1972). Zinc is not sufficiently electronegative to be easily reduced and thus may not persist in the soil in a metallic form. B. Zn Sorption Sorption (adsorption and desorption) refers to reactions at the solid-solution soil interface. Together with precipitation-dissolution reactions, the process has a major influence on the solubility relationships of the soils' mineral elements, including Zn. Zinc may also be removed from the soil solution by substituting for Mg in octahedral positions of silicate clay minerals (Elgabaly 1950; Kabata-Pendias and Pendias 1992). Because the majority of well-known Zn minerals are far too soluble to account for the low Zn solubility in soils (Lindsay 1972), some authors believe that Zn sorption may be the most important mechanism controlling its solubility in soils (Krauskopf 1972; Elvashidi and 0' Connor 1982; Harter 1991). Generally, the following molecular interactions are involved in sorption processes: (1) van der Waars forces (physisorption), (2) ion-dipol forces, (3) hydrogen bonding, (4) electrostatic forces, (5) ion and ligand exchanges, (6) valency forces (chemisorption), and (7) magnetic bonding (Kabata-Pendias and Pendias 1992). Some of the forces, e.g., van der Waal's and valency forces (covalent bonding), do not require the interacting molecules to carry a charge, contrary, for example, to electrostatic or ionic bonding forces. Sorption of solutes on various soil components is classified as specific or nonspecific (Harter 1991). Ion (cation and anion) exchange on clay minerals in soils is an example of nonspecific sorption. The process is very rapid (2-3 minutes) and easily reversible. It involves Coulomb's electrostatic forces of attraction between oppositely charged soil clay colloids and ions. Surface charges on soil clay colloids are induced by ionic isomorphous substitution. For example, the substitution of Mg2+ for AP+ leaves one unsatisfied negative charge from the oxygen anions in the silicate crystal, thus making it capable of adsorbing positively charged cations (Brady 1984). The affinity of cations for this kind of adsorption is closely related to their ionic potential, Le., the ratio between ionic charge and the ion's radius. In that respect, Zn 2+has higher

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affinity for exchange sites than Mn 2 + and Cu 2 +, equal to Fe 2 +, C0 2+, and NP+ and lower than M0 4+, Fe3 +, Cr 3 +, or Cr6 +. For the specific adsorption to occur, the ion's orbital electron configuration must match that of the specific adsorption site (Harter 1991). For this reason, this kind of adsorption is highly specific, making it possible to retain tiny amounts of a specifically adsorbable cation in the soil even in the presence of large quantities of other competing cations. Specifically adsorbed ions are not easily removed from the adsorption sites. Thus, such a process may not be confirmed by subsequent desorption reactions (Elvashidi and O'Connor 1982). Also, its confirmation may be difficult by charge balance calculations, because the adsorption reactions are not accompanied by easily measurable changes in the concentrations of counterbalancing ions in the soil solution (Harter 1991). Soil components involved in sorption of Zn are (1) hydrous oxides of iron and manganese, (2) organic matter, (3) clays, and (4) carbonates (Elgabaly 1950; Jurinak and Bauer 1956; Cavallaro and McBride 1984; Udo et a1. 1970; Reddy and Perkins 1974; Kalbasi et a1. 1978; Elvashidi and 0' Connor 1982; Harter 1991; Kabata-Pendias and Pendias 1992). The conditions under which carbonates are involved in Zn sorption may need further clarification, as Elvashidi and O'Connor (1982) found no correlation between Zn sorption and calcium carbonate content of the soil. Different clay minerals have different capacities to retain Zn, but ranking the clay minerals in that respect by various authors is contradictory (Farrah et a1. 1980; Kabata-Pendias 1980). The fact that Zn is adsorbed in soils when present at activities lower than those required for precipitation of its inorganic minerals in neutral and alkaline, but not acidic soils, is interpreted to indicate that the element is retained on specific sites in neutral and alkaline soils and on nonspecific sites in acid soils (Wakatsuki and Kawaguchi 1975). Exchangeable Zn decreases and the ratio of specific to nonspecific adsorption increases with increasing soil pH (Elsokkary and Lag 1978; Kalbasi et a1. 1978; Iyengar et a1. 1981; Sanders et a1. 1986; Sims 1986). This explains why the water-soluble Zn, and thus available Zn, decreases dramatically with increasing soil pH (EI-Kherbawy and Sanders 1984).

As far as the retention of Zn by Fe and Mn oxides is concerned, the distinction between adsorption and precipitation may not be easily defined (Harter 1991). Co-precipitation of Zn with Fe and Mn oxides has been noted by Sposito (1983). Iron and particularly Mn oxides have strong affinity for metal ions (Chao and Theobald 1976), which explains why they are highly contaminated with trace metal elements (Taylor and McKenzie 1966). The retained metals form inner-sphere complexes, displacing Mn from the mineral (Harter 1991). A similar difficulty exists in

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differentiating between adsorption and precipitation of Zn on carbonate surfaces (Harter 1991) because the metal may be adsorbed or occluded on calcium/magnesium carbonates (Shuman 1991). As mentioned before, the formation of Zn carbonate itself is les~ likely because it is far too soluble to persist in soils (Lindsay 1979). Neither ionic strength nor Zn complexation with SO:-, NO a, or CIaffected zinc sorption by soils, although the presence of EDTA in the soil suspension decreased Zn sorption (Elvashidi and O'Connor 1982). In the same study Ni2+ and Cu 2+ decreased Zn2 + sorption by soils, but only when added in excess of the soils' sorbing capacity. As stated before, zinc hydrolyses in aqueous solutions (Smith and Martell 1989) to ZnOH+ and Zn(OH)g at pH higher than 6 and 8, respectively (Harter 1983). The attachment of OH- increases the affinity of specific adsorption sites for Zn (Harter 1991), which may further help to explain the lower Zn solubility and mobility with increasing soil pH. C. Zn Associated with Organic Matter Only the most fundamental information concerning the formation of Zn organic complexes will be presented here. For a more comprehensive treatment of the subject, the reader is referred to the reviews by Stevenson and Ardakani (1972) and Stevenson (1986, 1991). The importance of the organic Zn fraction in soils stems from its positive contribution to the pool of plant available Zn, particularly in alkaline soils (Lindsay 1972; Stevenson 1991; Kabata-Pendias and Pendias 1992). In such soils, organic complexes account for higher Zn solubility than otherwise would have been observed (Stevenson 1991; KabataPendias and Pendias 1992). Studies show a positive relationship between organic matter content and chemically extractable Zn fractions (Lindsay 1972). There are examples, however, that show organic matter actually decreasing Zn availability to plants by shifting Zn into Mn and Fe oxide fractions in the soil (Shuman 1988), or by creating solid or possibly nonabsorbable organic complexes (Kreij and Basar 1995). The preponderance of experimental data, however, show organic matter amendments to increase soil Zn bioavailability (Singhania et a1. 1983; MandaI and MandaI 1987a,b; MandaI et a1. 1988; Shuman 1991; Tsadilas et a1. 1995). As the pH of the soil increases, so does the pool of organic Zn (Shuman 1986; Sims 1986; Kabata-Pendias and Pendias 1992). Humic substances, such as humic acid (HA) and fulvic acid (FA), are part ofthe soil organic matter that is involved in Zn complexation. Organic compounds bind metals because of the presence of COGH, phenolic-, enolic-, and alcoholic-OH, and ketone (C=O) functional groups (Stevenson 1991).

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As pH increases, not only the amount but also the capacity of humic substances to form complexes with micronutrients increases (Stevenson 1991). Additionally, these complexes are more soluble at higher pH. Generally, FA metal complexes are more soluble than HA complexes due to their higher content of acidic functional groups and lower molecular weight (Stevenson 1991). Zn-organic complexes are more stable at higher pH values (Stevenson and Ardakani 1972). The stability constants for Zn 2 + complexes of FA were determined to be 3.7, 3.6, and 4.83 at pH 5,6, and 8, respectively (Schnitzer and Hansen 1970; Mantoura et a1. 1978; Ryan et a1.1983). For a given pH and ionic strength, humic substances have higher affinity for trivalent cations and Cu, followed by Zn, and Mn (Stevenson 1991). A number of biochemical substances naturally occurring in soils, such as organic acids, hydroxamate siderophores, polyphenols and phenolic acids, and polymeric phenols (lichen acids), form complexes with metal ions, thus increasing their mobility and availability to plants (Graustein et a1. 1977; Powell et a1. 1980, 1982; Stevenson 1986, 1991). Other compounds that may also playa role in chelating micronutrients include polysaccharides, mugineic acid, gluconic, glucuronic, and galacturonic acids, and amino acids (Takagi et al. 1984; Stevenson 1991). As in the case of humic substances, these biochemical substances form the strongest complexes with Fe3+and AP+, followed by Cu 2+, Nj2+, C0 2+, Zn 2+, Fe 2+, and Mn 2+. All of them are produced by roots and/or soil microorganisms and are usually more abundant in the rhizosphere than in bulk soil (Stevenson 1986). D. Equilibria of Synthetic Zn Chelates Zinc complexation with synthetic chelates increases the metal's solubility and mobility in soils, thus generally enhancing Zn availability to plants. By forming nonabsorbable complexes, however, Zn activity may be depressed to such an extent that the metal's bioavailability decreases dramatically (Halvorson and Lindsay 1972; Swietlik and Zhang 1994). Thus, after being transported to the plants' roots, Zn must be freed from these complexes for the plants to benefit from the chelation-improved metal mobility. The extent to which Zn is chelated in the soil depends on the equilibria of chelating compounds with those other cations that compete for the chelate's reactive sites. The following reaction describes the formation of a metal chelate: [2]

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where M and L are a metal and chelate (ligand), respectively, and a and b designate charges. The state of equilibrium between the two opposing processes is described by the equilibrium or formation constant, K: [3]

where (MLa-b), (Ma+), and (Lb-) are the activities of metal chelate, metal ion, and free chelate, respectively. Instead ofK, the log of formation constants are often provided to avoid unduly long numbers. Ion activities are expressed in molar concentrations. For thermodynamic calculations, however, they are not assigned any units, hence formation constants are dimensionless. At very low concentrations, free metal activities and molar concentrations of reactants and products of a reaction are numerically the same, but as ionic strength of an electrolytic solution increases, electrostatic forces depress metal activity below that of free metal concentration. Activities can be converted to concentrations using activity coefficients (Szarawara 1985). Norvell (1991) cOInpared the relative effectiveness of various chelates in complexing Zn 2 + in the model soil solution containing Fe3+, AP+, Ca2+, and Mg2+ as the dominant competing cations at pH ranging from 4 to 9. The comparisons were made by calculating the distribution of chelatedZn among nine different chelating ligands that were assumed to be present in the soil solution in equal concentrations. In alkaline soils, DTPA was suggested to be the most effective chelating ligand, followed by HEDTA, CDTA, and EDTA (see Table 3.2 for chelate descriptions). In the acid to neutral range, HEDTA was suggested to be the best chelator. The above conclusions are supported by other studies (Lindsay and Norvell 1969; Halvorson and Lindsay 1972; Sommers and Lindsay 1979). Norvell's (1991) calculations and the earlier published data (Norvell and Lindsay 1969) revealed that Fe and Ca strongly compete with Zn for chelation in acidic and alkaline soils, respectively. Generally, the extent of free Zn 2+ chelation increases with increasing pH. In highly acidic soils, the soil solution fraction of Zn will mostly consist of free Zn 2 +, In heavy metal contaminated soils, NP+ will strongly compete with Zn for chelation in acid soils and Cd 2+ and Pb 2+ will do the same in alkaline soils (Sommers and Lindsay 1979). The extent of metal chelation in soils may be reduced by adsorption and microbial and light degradation of chelating agents (Norvell 1991). Norvell and Lindsay (1969) reported that 5-25 percent of EDTA added to soil suspensions was lost during the first few days due to adsorption by solid soil phase. The greatest losses were observed following FeEDTA addition to acid soils and the lowest after CuEDTA and ZnEDTA addi-

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tions to alkaline soils. Also, high and low adsorption of FeEDTA and ZnEDTA, respectively, was reported by Lahav and Hochberg (1975). The mechanism involved in the adsorption of metal chelates and chelating ligands by soils is poorly understood, but it is believed that negatively charged chelating ligands are adsorbed on positive charges of Fe and Al oxides and other colloids (Norvell 1991). Norvell (1991) reviewed the subject of microbial degradation of synthetic chelates in soils. He cited studies of other authors that showed 10-46 percent loss of EDTA in 15 weeks from 11 surface soils and only 3-4 percent loss from 3 subsoils. Addition of organic matter accelerated the degradation, but poor aeration inhibited it. The EDTA chelates of Cu, Cd, Zn, Mn, Ca, and Fe degraded at similar rates, but that of NiEDTA was slower. There is very little information on biodegradation of other chelating species, but DTPA, HEDTA, and CDTA are believed to be rather resistant to this process. Norvell and Lindsay (1972) postulated that partial biodegradation of DTPA reduces its complexing capacity. Aminopolyacetate chelates (DTPA, EDTA, HEDTA, etc.) are subject to photodegradation, especially when complexed with Fe 3+(Norvell 1991). Generally, photodecomposition is considered insignificant when chelates are incorporated into the soil, but it may be significant when they are left on the soil's surface or added to nutrient solutions exposed to sunlight.

E. ZnZ+ Soil Activity and Inorganic Phase Equilibria In neutral and alkaline soils, the concentration or activity of free Zn 2+ in soil solutions is too low to be directly measured. Norvell and Lindsay (1982), Ma and Lindsay (1990, 1993) overcame this difficulty by measuring cation activities indirectly using chelation with EDTA or DTPA. The chelation method allows calculation of the activity of the metal in question, e.g., Zn 2 +, using the following equation: (Zn 2+) :F [ZnL]/[NL] x (KNL/Kznd x (N)

(4]

where (N) is the activity of cation that can be easily measured in the soil solution, e.g., Ca; [NL] is the concentration of the cation's complex with a chelating agent L, e.g., DTPA; and K NL K ZnL are the formation constants for the reactions NL :F N8+ + Lb- and Zn 2+ + L :F ZnL, respectively (Ma and Lindsay 1990). The chelating agent is added to the soil as ZnL and NL at various initial molar ratios. As ZnL and NL react with the soil, Zn and N can be gained or lost from the chelate. After the reaction is completed, a new ZnL/NL ratio is determined by means of chemical analyses. The unique [ZnL]/[NL] ratio that is used to solve eq. 4 is the one that neither gained nor lost Zn and N from the chelate.

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Using the above approach, Ma and Lindsay (1990) found that free Zn2+ concentrations in 10 arid calcareous soils with a pH of 6.75 to 8.22 varied from 10-7.9 to 10-10.9 M (0.0008 ppm to 0.0008 ppb Zn) and were inversely related to soil pH. The relationship between the soil pH and log (Zn2 +) was: log (Zn 2+)= 5.7 -2pH

[5]

This solubility expression is very similar to that established in the earlier theoretical development by Lindsay and Norvell (1969). A 100-fold reduction in Zn activity for each unit of pH increase explains why Zn deficiencies are frequently encountered on alkaline soils (Swietlik 1989). In another study, Zn 2 + activities in a number of heavy-metal-contaminated and noncontaminated Colorado soils varied from 10-8·11 to 10-2•26 M (0.0005 ppm to 357 ppm Zn) and from 10-9 .97 to 10-6·49 M (7 ppb to 0.02 ppm), respectively (Ma and Lindsay 1993). The soils were contaminated through mining of Ag and other heavy metals. The solubility ofsoil Zn found for the noncontaminated soils in this (Ma and Lindsay 1993) and the previous study (Ma and Lindsay 1990) matches the solubility of Zn from franklinite (ZnFe 20 4 ) + soil-Fe and franklinite + meghamite (Lindsay 1991). Thus, franklinite may indeed be the mineral phase that controls Zn solubility in alkaline soils. In the mine-contaminated soils that contained much more soluble Zn, the metal's solubility could have been controlled by much more soluble minerals such as ZnSi04 or ZnC0 3 • The Aspen soil, which contained the highest level of soluble Zn, was indeed associated with ZnC0 3 deposits. Several studies were conducted in nutrient solutions to determine the critical Zn2+ activity needed for normal plant growth. Zinc activities in the nutrient solutions were controlled by the addition of strong Zn chelators: DTPA, EDTA, CDTA, or HEDTA. This approach assumed nonabsorption of a Zn-chelate complex by plant roots (Halvorson and Lindsay 1977). The critical Zn 2 + activities, expressed as pZn =-log(Zn2 +), were 10.8 for soybean (Chaney et al. 1989), 10.6 for corn and tomato (Halvorson and Lindsay 1977; Parker et al. 1992), 10.52 for barley (Norvell and Welch 1993), and 9.8 for sour orange (Swietlik and Zhang 1994). Note that the higher the pZn value the lower the Zn 2+ activity, e.g., pZn of 10.8 corresponds to Zn2+ activity of 10-10.8 M. In the most recently published paper, critical Zn 2+ (pZn 2 +) activities were estimated to be 10.18 for maize, 10.82 for wheat, 11.00 for alfalfa and soybean, and 10.52 for tomato (Parker 1997). Using eq. 5, I estimated Zn activity in most south Texas soils (pH = 7.5 to 8.4) to vary between pZn =9.3 and 11.1. These estimates explain why citrus trees grown in these areas frequently develop Zn deficiency symptoms (Swietlik 1989).

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III. FACTORS AFFECTING ZN AVAILABILITY

A. Soil Parent Material and Organic Matter Since granitic and basaltic igneous rocks contain more Zn than sedimentary rocks such as limestone and sandstone (Turekian and Wedepohl1961; Taylor 1964), the soils derived from the former materials are expected to contain more total Zn. From the plant nutritional standpoint, however, the amounts of Zn extractable with chemicals such as DTPA, EDTA, NaOH, and KNO a are more important than total Zn content. This is because Zn extracted with the above compounds is plant available (LeClaire et a1. 1984; Tsadilas et a1. 1995). The fact that the available fraction of Zn decreases with depth (Lindsay 1972) explains why severe cases of Zn deficiency have been observed in areas where the surface soil has been removed during leveling (Parker 1935; Swietlik 1989; Moraghan and Mascagni 1991). In some areas, removal of topsoil uncovers highly calcareous layers of soil that contribute to low Zn availability via elevated pH (Wear 1956) and/or strong Zn adsorption on calcium carbonate (Jurinak and Bauer 1956; Navrot et a1. 1967; Navrot and Ravikovitch 1969). The higher level of Zn in topsoil layers coincides with higher levels of organic matter that increase Zn availability to plants. As discussed before, organic compounds increase solubility and thus mobility of Zn, especially in neutral and alkaline soils (Bar-Tal et a1. 1988). It is therefore not surprising to see manure applications increasing leaf Zn concentrations in avocado trees (Labanauskas et a1. 1958). However, zinc complexation with insoluble organic molecules (humic acids) or those that are nonabsorbable by plant roots, may actually reduce Zn availability (Lindsay 1972; Kreij and Basar 1995). Moraghan and Mascagni (1991) discussed the possibility that root exudates mobilize soil zinc in the vicinity of plant roots via chelation. They also postulated that a similar effect may be induced by elevated microbial activity in the rhizosphere. The microbial activity also includes mycorrhizal associations with plant roots that were shown to improve Zn absorption by plants (Killham and Firestone 1983; Thompson 1994). Decomposing organic matter contributes to widespread occurrence of Zn deficiencies in lowland rice in Asia (Yoshida et a1. 1973 cited by Moraghan and Mascagni 1991). This process supplies high concentrations of HCOi that immobilizes Zn in plant roots and reduces the metal's transport to the tops of plants (Forno et a1. 1975). Sandy, acidic soils are inherently low in total zinc, because quartz is generally low in this element (Lindsay 1972). Leaching, under high rainfall, further contributes to zinc depletion. These conditions prevail in most Florida citrus orchards where Zn deficiency is a problem.

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B. Soil pH Increasing soil pH reduces plant-available Zn due to increased adsorption by inorganic and insoluble organic soil constituents (Kabata-Pendias and Pendias 1992), precipitation of franklinite (Lindsay 1991), and the increased bonding energy of ZnOH+ to clay surfaces compared to Zn 2 + (Bar-Tal et al. 1988; Harter 1991). Contrary to that, acidification greatly improves soil Zn availability. Fenn et al. (1990) reported that acidification of a calcareous soil by applying sulfuric acid in a 15-cmdeep trench on both sides of a pecan tree increased solubility of soil Zn and significantly increased leaf Zn concentration from the 4th to 9th year after treatment. The effect of pH on Zn transformations in soils are discussed in more detail in Section lIB. C. Restricted Root Zones Lindsay (1972) reported that soils with restricted root zones are conducive to the development of Zn deficiency. Similar observations were reported by Swietlik (1989) for citrus trees grown in Texas. Trees whose root system is restricted by compacted soils, hardpans, and/or poor drainage are more prone to develop Zn deficiency. The restriction of the root zone of newly planted citrus trees may also contribute to their higher sensitivity to Zn deficiency. D. Nutrient Interactions The antagonism between P and Zn has been the most widely studied mineral interaction involving Zn. Phosphorus fertilization reduces Zn tissue concentration in a number of plant species (Reuther and Crawford 1946; Bingham and Martin 1956; Bingham and Garber 1960; Labanauskas et a1. 1960; Soltanpour 1969; Rudgers et a1. 1970), but the relationship is not always significant (Reuther et a1. 1949). Phosphorus applications may enhance Zn adsorption in certain soils (Saeed and Fox 1979; Ghanem and Mikkelsen 1988) and reduce free Zn2+ in the soil solution (Norvell et a1. 1987). These effects, however, are contrary to the results of other studies that show P fertilization to increase soil extractable Zn (Bingham and Garber 1960; Moraghan and Mascagni 1991). Zinc phosphate, Zn3 (P0 4)z, is more soluble than the native soil zinc and thus the addition of this compound to the soil should increase the soluble pool of Zn in the soil (Lindsay 1972). Some authors suggest that P antagonism against Zn is a physiological phenomenon whose explanation can be based on P-induced reductions in Zn uptake, transport, and/or utilization in plants (Soltanpur 1969; Terman et a1. 1972; Leece 1978b; Cakmak and Marschner 1987). Cakmak

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and Marschner (1987) concluded that high tissue P levels decrease the physiological availability of Zn. This may explain why high P supply may induce Zn deficiency symptoms without reducing total tissue Zn concentrations. They suggested that water-soluble rather than total plant tissue Zn concentration is a much better indicator of physiologically active Zn. Another means of assessing physiologically active Zn is to measure the activity of carbonic anhydrase enzyme. A reduction in the enzyme activity has been correlated with increased severity of Zn deficiency in several plant species (Bar-Akiva and Lavon 1969; Randall and Bouma 1973; Edwards and Mohamed 1973; Ohki 1976). Loneragan et al. (1982) reported that under low Zn supply to okra, P reaches toxic levels in leaves and induces symptoms that resemble Zn deficiency. These symptoms, however, could be eliminated by increasing Zn supply to the plants. Many other studies also associated elevated P concentration or toxicity in plants with Zn deficiency (Christensen and Jackson 1981; Cakmak and Marschner 1986; Webb and Loneragan 1988; Parker et al. 1992, 1997; Swietlik and Zhang 1994). However, this effect could have been an artifact of the hydroponic systems used in these studies, since no elevated leaf P levels were observed in field-grown Zn deficient citrus trees (Swietlik and LaDuke 1991). Heavy N fertilization was reported to intensify Zn deficiency of citrus in California and Florida (Reuther and Smith 1950; Smith et al. 1954), but Labanauskas et al. (1960) found no such relationship, presumably because of a high zinc level in the soil of the experimental site. Lindsay (1972) reported that acidifying N fertilizers may elevate the absorption of zinc via reduction of soil pH, which increases Zn availability. Studies by Graham et al. (1987) indicated that Zn may playa protective role against excessive B accumulation and toxicity in barley. Similar results were reported by Swietlik (1995) for sour orange seedlings.

E. Temperature Moraghan and Mascagni (1991) reported that Zn deficiency in maize, bean, potato, flax, subterranean clover, and tomato was more pronounced in colder than warmer parts of the growing season. Similar observations were also reported for citrus (Parker 1935; Labanauskas et al. 1963; Swietlik 1989, 1996). Lindsay (1972) offered two explanations that could account for the negative effect of low temperatures on Zn nutrition. First, the root system of annual plants does not develop sufficiently fast in cool soils to extract enough Zn from the soil, and second, due to limited microbial activity in cool soils, insufficient amounts of Zn are released from organic matter. However, other factors such as reduced absorption (Moraghan and Mascagni 1991) and translocation of Zn in plants

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(Edwards and Kamprath 1974; Schwartz et al. 1987) under low temperatures must not be discounted. One may also speculate that physiological activity of Zn may be adversely affected under low temperatures. F. Soil Moisture Low soil moisture should be expected to reduce Zn absorption either by restricting root growth and/or diffusion of Zn to the root surface (Lindsay 1972; Moraghan and Mascagni 1991). However, Nambiar (1976) reported continued absorption of 65Zn from the soil at water potentials lower than -1.5MPa. Zn deficiencies are frequently encountered in flooded rice. Sajwan and Lindsay (1986) proposed that this was due to the antagonism between Fe 2 + and Mn 2+ vs. Zn 2 + under high reducing conditions. They also proposed the precipitation offranklinite as a means of removing soluble Zn from the system. Citing works of other authors, Moraghan and Mascagni (1991) suggested that organic matter additions to poorly aerated soils would aggravate Zn deficiency by increasing the level of Fe 2+, which would further suppress Zn 2+ uptake. G. Light Low light levels were reported to be involved in Zn deficiencies in maize during the early stages of plant development (Edwards and Kamprath 1974). Contrary to that, however, Zhang and Wu (1989) reported that the detrimental effects of Zn deficiency in tomato plants are most likely to develop under high rather than low irradiance. Their interpretation of these results was that plants exposed to high light levels have a higher requirement for Zn. This finding seems to corroborate the observation that Zn deficiency symptoms in citrus are more pronounced on the southern rather than the northern side of the tree (Swietlik 1989). IV. FUNCTION, ABSORPTION, AND TRANSPORT OF ZN IN PLANTS A. Function

Zn activates a number of plant cell enzymes (Romheld and Marschner 1991). However, only a few of these-alcohol dehydrogenase (ADH), superoxide dismutase (Cu-Zn-SOD), carbonic anhydrase (CA), and RNA polYlnerase-contain the metal. Zn can affect carbohydrate metabolism because a number of Zn-dependent enzymes are involved in biochemical reactions involving sugars

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(Romheld and Marschner 1991). For example, carbonic anhydrase, whose activity closely correlates with the plant's Zn nutritional status (BarAkiva and Lavon 1969; Edwards and Mohamed 1973; Randall and Bouma 1973) is thought to facilitate CO 2 diffusion for photosynthetic fixation. Surprisingly, however, only severe cases of Zn deficiency had a negative effect on photosynthesis (Pn) (Randall and Bouma 1973; Romheld and Marschner 1991). Randall and Bouma (1973) reported that the effect of Zn deficiency was observed at CO 2 concentrations> 300-350 Ill/liter, but not at lower concentrations. They concluded that Zn deficiency had a direct effect on the Pn process itself rather than on CA-mediated CO 2 diffusion. A more recent study reported the Hill reaction (photosynthetic electron transfer) to be negatively impacted by Zn deficiency (Sharma et al. 1982). Despite this finding, however, the concentrations of carbohydrates in leaves of Zn-deficient plants were higher than in the control. This suggests that the growth inhibition observed under Zn deficiency is more pronounced than reduction in ,carbohydrate synthesis (Marschner 1986). Cakmak et al. (1989) reported inhibition of protein and auxin (IAA) synthesis and an increased level of amino acids (including tryptophan) and amides in Zn-deficient bean plants. The vital role that Zn plays in auxin synthesis has been reported by other authors (Tsui 1948; Salami and Kenefick 1970). Zn deficiency may reduce auxin synthesis either by inhibition of tryptophan conversion to IAA or via inhibition of protein synthesis (Cakmak et al. 1989). Zinc plays an important role in maintaining cell membrane integrity. Under Zn-deficient conditions, NADPH-dependent superoxide radical (02") generation is enhanced, leading to increased peroxidation of membrane lipids (Cakmak and Marschner 1988a). Concomitantly, the activities of Cu-Zn-SOD and catalase decrease, thus diminishing the plant's natural defense mechanism against superoxide radicals (Elstner 1982). The oxidative damage of phospholipids explains the Zn-deficiencyinduced deterioration of membrane integrity that manifests itself by elevated membrane permeability to organic and inorganic compounds (Cakmak and Marschner 1988b,c). Zinc also contributes to membrane integrity by binding to sulfhydryl (SH) groups of membrane proteins (Romheld and Marschner 1991). These proteins are involved in ion transport processes and prevent, for example, excessive absorption and transport ofP to the shoots (Welch et al. 1982; Marschner and Cakmak 1986). By binding to SH-groups, Zn protects them from oxidation by free radicals (Welch and Norvell 1993). Graham and Webb (1991) cited a number of instances of increased incidence of root infectious diseases under Zn-deficient conditions. This phenomenon may reflect elevated microbial activity in the rhizosphere caused by Zn-deficiency-induced root exudation.

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B. Absorption To be absorbed by the roots, a solute must first enter the roofs free space, Le., a network of cell walls consisting of cellulose, hemicellulose, pectins, and glycoproteins (Marschner 1986). Carboxylic groups (R-COQ-), largely contributed by polygalacturonic acid, act as ion exchangers for cations, particularly the di- and trivalent ones that are preferentially adsorbed by these groups. Zinc concentration in the apoplasm was estimated to be 0.5 mol·m-3 (32.5 J.lg·liter1 ) (Santa Maria and Cogliatti 1988), which is considerably higher than in the rhizosphere. This serves as a proof of considerable binding of Zn. Interfibrillar and intermicellar spaces between the wall constituents are large enough to accommodate Zn and other ions (Marschner 1986). Consequently, not only the rhizodermal but also root cortex cells, outside the Casparian strip, are accessible to plant nutrients. Thus, it comes as no surprise that soluble Zn forms, which can easily diffuse into the free space, are absorbed more readily than those that are sparsely soluble (Stewart et a!. 1955). Lower rates of uptake by leaves and roots of chelated vs. inorganic forms ofZn, e.g., ZnEDTA vs. ZnS0 4 (Stewart et a1. 1955; Barber and Lee 1974), may indicate restricted permeation of chelated zinc into the free space. Moreover, as uncharged chelated forms cannot be adsorbed on cell walls, Zn concentrations at the outer surfaces of plasmalemma would be lower in plants supplied with chelated vs. inorganic forms, further contributing to lower rates of Zn absorption in the chelated form (Barber and Lee 1974). One must also recognize the fact that bulky Zn chelator complexes are poorly or not at all absorbed by plant roots, as shown by a number of studies conducted in water cultures (Halvorson and Lindsay 1977; Chaney 1988; Parker et a1. 1992; Swietlik and Zhang 1994; Parker et a1. 1995a). Under field conditions, however, the addition of a Zn-chelate will elevate the amount of free Zn2+ in the soil solution because of the shift in eq. 2 (Section lID) to the left. Carroll and Loneragan (1969) and Schmid et a1. (1965) reported the daily rate of uptake of Zn by roots to be 2 and 4,000 ngtg of fresh root, respectively, whereas Welch and Norvell (1993) estimated the daily rate to be 5,000 to 80,000 ng of Zn/g dry root. Assuming that roots contain 20 percent dry matter, the latter rates were equivalent to 1,000 to 16,000 ng of Znl g fresh root. Carroll and Loneragan (1968) reported that a soluble Zn concentration of 0.25 J.lM in the rhizosphere produced maximum plant growth with plant toxicity encountered at Zn levels from 3 to 6 J.lM. Early studies indicated the saturation concentration for Zn uptake to be about 50 flM, but recent studies produced more realistic values in the range of 1.5 to 2.2 J.lM (see references cited by Kochian 1991).

130

D. SWIETLIK

Zinc activity or free Zn 2+ concentration may be a better indicator of Zn availability to plants than total soluble Zn, as found in studies with Zn-chelator buffered nutrient solutions (Parker et al. 1992, Swietlik and Zhang 1994; Swietlik 1995). Naturally occurring organic compounds in the soil solution may also render some of the soluble Zn unavailable to plants (see Section lILA). Zinc is compartmentalized in a plant cell between the apoplasm (8-14%), cytoplasm (80/0), and the third compartment that includes vacuolar Zn and presumably that in organic complexes in the vacuole and cytoplasm (76%) (Santa Maria and Cogliatti 1988). The first and the second compartments exchange Zn rather rapidly, with half times of 0.08 and 0.55 hour, respectively. The third one is the slowest, with a halftime of 134 h. There is controversy as to whether Zn absorption by plants is an active or passive process. Partly, this controversy stems from the difficulty of distinguishing between adsorption and absorption of Zn by roots. In a 2-h uptake period, Schmid et al. (1965) found that 60 percent of Zn held by the roots consisted of adsorbed Zn. The absence of a rigorous definition for the active uptake process further contributes to the active vs. passive absorption controversy. In the following discussion, active uptake is defined as transport against the electrochemical potential gradient (Ussing 1949 cited by Kochian 1991). According to this definition, a mere dependence of the uptake process on cellular metabolism does not prove the existence of active uptake. A description of ion absorption phenomenon in higher plants is given below to better introduce the reader to the mechanism involved in Zn transport across cell membranes. Due to space constraints, the description had to remain brief and general. For more detailed treatment of the topic, the reader is referred to the reviews by Poole (1978), Spanswick (1981, 1989), Serrano (1988), and Kochian (1991). The carrier hypothesis is the most widely accepted model of ion absorption by plant cells (Marschner 1986). It is based on the concept that membrane transport proteins, such as ATPases, mediate ion transport across plasmalemma and tonoplast (Kochian 1991; Fig. 3.1). The model encompasses three transport systems. The first one is the primary active transport system, which includes H+-ATPase (proton pump), that is responsible for transporting H+ out of the cytoplasm (Spanswick 1981), K+-ATPase transporting K+ into the cytoplasm (Kochian et al. 1989), and Caz+-ATPase pumping Ca2+out of the cytoplasm (see references cited by Kochian 1991). The secondary active system, or proton cotransport system, utilizes the proton pump-generated energy stored in the electrochemical potential gradient for protons across the membrane. This gradient induces proton

3. ZINC NUTRITION IN HORTICULTURAL CROPS

131

MICRONUTRIENT CATIONS

c:==J

ION CHANNEL

o

ION PUMP

o

CELL WALL (pH == 5.0)

K+

COTRANSPORT

CYTOPLASM (pH == 7.0)

(LOW K;) 1"---+

(ATPase)

H +---t'...-----'I---....

- - - H +---1'..--/.1---....

1---

CI

H+---f'oo...-

~..- -

NOi---I/---...J---.... - - - H + - - - f ' . . - - / l - - -.... SUGAR!

AMINO ACID

' - - - - H +- - - 1 ' -.......... ~--

PLASMA MEMBRANE

Fig. 3.1. Solute transport through the plasma membrane of plant cells: 0 = primary iontransport ATPases with. denoting H+-translocating ATPase (proton pump); 0 = secondary cotransport systems; c:J = ion channels. Question marks indicate more speculative transport systems. Redrawn with permission from Kochian (1991).

132

D. SWIETLIK

movement across the membrane that is coupled with transport of an accompanying ion against its electrochemical potential gradient. When the accompanying ion and proton move in the same direction, this transport is referred to as symport. When the opposite is true, it is referred to as antiport or exchange. The examples of the secondary active system include CI--H+ and N03-H+ symports in barley and corn roots, respectively (Jacoby and Rudich 1980; McClure et al. 1990) and a Na+-H+ antiport in barley roots (see references cited by Kochian 1991). The third system includes passive cation transport down the electrical potential through ion channels (transport proteins). In this system, the proton pump contributes the electrical driving force by transporting net charge across the membrane and maintaining its negative potential at -120 to -180 mV (Kochian 1991). It is believed that this potential is large enough to drive passive uptake of Zn and other micronutrients such as Fe3+/Fe2+, Cu2+, Mn2+, and NP+ (Kochian 1991). It must be recognized that the voltage potential is generated by the proton pump, whose operation involves metabolic processes. This explains why Zn uptake, although passive, has been linked to metabolic processes (Bowen 1969; Giordano et al. 1974). Zinc absorption was strongly inhibited by Cu 2+in sugar cane and barley (Schmid et al. 1965; Bowen 1969). This prompted Kochian (1991) to speculate that the same ion channel may mediate the absorption of both elements. A similar mechanism may also be involved in the reported antagonism between Fe 2+and Zn2+in flooded rice (Sajwan and Lindsay 1986).

c.

Transport

Bukovac and Wittwer (1957) characterized Zn mobility as intermediate, but Kabata-Pendias and Pendias (1992) observed that some authors consider Zn highly mobile, particularly under the condition of luxury consumption. There is very limited knowledge on the short-distance transport ofZn, i.e., within and between plant cells. Scholtz et al. (1987) suggested that nicotianamine may be a symplastic carrier for all of the divalent heavy-metal cations. Also, a certain group of chemicals called phytochelatins, which are induced in plants upon exposure to heavy metals (Kochian 1991), may playa role in the short-distance transport of Zn (Walker and Welch 1987). The long-distance transport of Zn is thought to take place mainly in the xylem (Marschner 1986), but high levels of the element were also found in the phloem sap of Nicotiana glauca Grah. (Hocking 1980) and blighted and healthy orange and grapefruit trees (Taylor et al. 1988). Zn in xylem exudate exists as the free cation or as complexes with citric and malic acids (White et al. 1981a,b). The concentration of Zn in the xylem

3. ZINC NUTRITION IN HORTICULTURAL CROPS

133

sap reported in the literature ranges from 4 to 22 flM (Hocking 1980; White et a1. 1981a; Clark et a1. 1986). Kabata-Pendias and Pendias (1992) reported Zn mobilization from old to new growth, particularly under the condition of Zn luxury consumption. Pearson and Rangel (1994) reported Zn movement from roots and stems to the developing grain in wheat. They also found remobilization of Zn from the flag leaf. These results suggest Zn mobility in the phloem. Contrary to the above data, no evidence was found of Zn moving from Zn-sprayed leaves in citrus and avocado trees using Zn65 isotope (Stewart et a1. 1955; Crowley et a1. 1996) or standard research techniques (Smith 1966a; Labanauskas et a1. 1969; Embleton et a1. 1988; Swietlik and Zhang 1994; Swietlik 1996). In some field studies, however, small increases in Zn concentration were found in leaves formed after Zn foliar sprays were completed (Labanauskas et a1. 1961; Labanauskas and Puffer 1964; Swietlik and LaDuke 1991). It is possible that these increases reflected the root-absorbed Zn, Zn absorbed by the bark and subsequently translocated to new foliage (Stewart et a1. 1955), or Zn contamination of samples collected from the sprayed trees.

V. ZN DEFICIENCY AND TOXICITY SYMPTOMS

A. Deficiency Chandler et a1. (1931, 1932) successfully demonstrated that "little leaf" or "rosette" on peaches could be corrected with soil Zn applications (Plate 3A). The etiology of little leaf was believed to be the same as that of citrus "mottle-leaf," which is characterized by yellowing of the areas between leaf veins, while the leaf tissue adjacent to the midrib and main veins remains green (Plate 3B). Shortly after Chandler's discovery, mottle-leaf of citrus was also corrected with soil and foliar Zn applications (Johnston 1933; Parker 1934, 1935). At about the same time, Alben et a1. (1932) reported that rosetting on pecans was an expression of Zn deficiency (Plate 3C). The most characteristic symptoms of Zn deficiency in dicots include rosetting or little leaf caused by strong inhibition of internode elongation and reduction in leaf size, as exemplified by apple and pistachio shoots on Plate 3D and E (Stiles 1966). Depending on the severity of the deficiency, the plants may be stunted and exhibit shoot dieback, defoliation, and partial or total leaf chlorosis (Chandler et a1. 1931; Haas 1936; Chapman and Vanselow 1937; Wallihan et a1. 1958; Chapman 1966, 1968; Stiles 1966; Embleton et a1. 1973; Marschner 1986; Sparks 1987, 1994; Swietlik 1989). In pecan, avocado, and citrus, rosetting or little leaf, shoot dieback,

134

D. SWIETLIK

and/or defoliation are indicative of severe Zn deficiency (Wallihan et al. 1958; Swietlik 1989; Sparks 1994). When Zn deficiency is severe, citrus yield, fruit size, and juice content are reduced, rind thickness is increased, and fruit shape is abnormal (Parker 1937b; Swietlik 1996). While severe expressions of Zn deficiency are rather rare, leaf mottling affecting several shoots within the tree canopy is common in many citrus-growing areas of the world. Leaf mottling is more pronounced on orange than on grapefruit and usually occurs on the south rather than the north side of the tree. When deficiency is most severe, pecan trees produce rosetted shoots without staminate or pistillate inflorescences (Hu and Sparks 1990). With less severe deficiency, catkin length and weight and number of fruit per shoot are decreased. Also, fruit death and drying are observed. Under severe deficiency, apple trees produce small, pointed, misshapen, and poorly colored fruit that ripen early and lack flavor (Stiles 1966; Stiles and Shaw Reid 1991). Zinc deficiency may be accentuated by high P, hence the condition is called liP-induced Zn deficiency" (Loneragan et al. 1979). It may develop without any reduction in Zn concentration of plant tops. Because it is accompanied by excessive P accumulation, particularly in older leaves, most researchers agree that the observed symptoms of interveinal necrosis and/or chlorosis are the symptoms of P toxicity rather than Zn deficiency (Loneragan et al. 1979, 1982; Christensen and Jackson 1981; Cakmak and Marschner 1986; Webb and Loneragan 1988). Several authors reported that adequate Zn supply is necessary for normal root growth (Potapova 1974; Christensen and Jackson 1981; Loneragan et al. 1982). Swietlik and Zhang (1994) found severe stunting of roots in Zn-deficient sour orange seedlings grown in nutrient solution (Plate 3F). Severely deficient seedlings produced only a few, extremely short, white roots and all of them originated from the lowest portion of the root system. Moderately deficient seedlings produced first-order lateral white roots but they were very short (1 mm) and abnormally thick (2 mm). Second-order laterals were absent. As deficiency further diminished, white roots became more abundant and longer (3-6 mm) and thinner «1 mm). However, the second-order laterals were still absent and all white roots emerged from the lower 1/3 of the root system. The seedlings that received adequate Zn produced first-order laterals up to 25 mm long and second-order laterals up to 10 mm long. The white roots were about 0.5 mm thick, which is typical for nutrient-solution-grown sour orange seedlings. These roots originated from all parts of the root system. In monocots such as maize, Zn deficiency symptoms consist of interveinal chlorosis that broadens into chlorotic or white bands between the midrib and the edge of the leaf (Lindsay 1972) and may include red, spot-

3. ZINC NUTRITION IN HORTICULTURAL CROPS

135

like discolorations (Marschner 1986). Silking and tasseling are delayed and, under severe deficiency, plants are stunted and have short internodes (Chapman 1966; Bergman 1977). On rice plants, the midribs of young leaves become chlorotic, with brown spots or streaks developing at a later stage of growth (Sedberry et aI. 1988). On older leaves, yellow or rusty-brown lesions appear, hence zinc deficiency is often described as "bronzing." Chapman (1966) summarized specific symptomatology of Zn deficiency for a total of twenty-two annual and perennial mono- and dicotyledonous crops. Additional descriptions of Zn deficiency symptoms on a number of deciduous tree fruits and nuts are given by Shear and Faust (1980). Crops differ in their sensitivity to Zn deficiency. Bean, maize, cotton, onion, sorghum, and sweet corn are considered sensitive; barley, lettuce, potato, soybean, Sudan grass, sugar beet, and tomato are medium sensitive; and alfalfa, asparagus, carrot, clover, oat, pea, peppermint, rye, spearmint, and wheat show low sensitivity to Zn deficiency (Chapman 1966; Martens and Westermann 1991). Tiwari and Dwivedi (1990) ranked eight winter crops by their decreasing sensitivity to Zn deficiencyas follows: lentil> chickpea> pea> wheat> flax > mustard> barley> oat. Most of the fruit trees are considered sensitive to Zn deficiency (Chapman 1966). Widespread Zn deficiencies were reported on citrus, peach, avocado, and pecan trees (Wallihan et a1. 1958; Chapman 1966, 1968; Sparks 1987). Not only plant species but also different cultivars within a species differ in their efficiency in acquiring Zn from the soil. Yang et al. (1994) described such differential absorption capacity in rice, and the use of various rootstocks for fruit trees of the same species may alter Zn concentration in scion leaves (Embleton et aI. 1973; Wutscher 1989). B. Toxicity An excess of Zn not only produces Fe deficiency in plants (Chapman et a1. 1940; Chapman 1966; Shear and Faust 1980; Lee et a1. 1996) but also may cause leaf damage and defoliation (Embleton et a!. 1973). Zn toxicity is likely to occur on soils contaminated by zinc from mining operations (Ma and Lindsay 1993), derived from rocks naturally rich in Zn (Ma and Lindsay 1993), overfertilized with Zn (Chapman 1966; Embleton et al. 1973), or on soils where sewage sludges with a high Zn content were disposed of (Marschner 1986). It is generally assumed that leaf Zn levels in excess of 300-600 mg·kg-1 dry weight is considered toxic to plants (Chapman 1966; Embleton et a1. 1973; Marschner 1986; Lee et aI. 1996).

136

D. SWIETLIK

There are genotypical differences in zinc tolerance between ecotypes of noncultivated plants. For example, a zinc-tolerant clone of tufted hairgrass (Deschampsia caespitosa) was shown to actively pump zinc into the vacuoles of root cells where no sensitive metabolic activities take place (Brookes et al. 1981). A Zn-sensitive clone had no such ability. A later study suggested that Zn-tolerance in Deschampsia also relied on detoxifying the excess of vacuolar Zn as zinc phytate (Van Steveninck et al. 1987). Zinc-tolerant genotypes of Betula resisted zinc toxicity by controlling uptake of the metal into their tissues at higher external concentrations (Denny and Wilkins 1987). Genotypical differences in Zn tolerance are not limited to natural vegetation but extend to cultivated plant species as well, e.g., various soybean genotypes (White et al. 1979). C. Zn Tissue Concentrations Wherever possible, an effort was made to classify Zn into deficient, low, normal, high, and excessive ranges of Zn in various plant tissues obtained under different growing conditions (Table 3.3). The deficient and low ranges indicate the potential for an increase in yield and/or crop quality, and the deficient ranges are also associated with visual malnutrition symptoms. The normal range indicates an adequate supply of the element. The high range is not associated with toxicity symptoms but indicates a luxury consumption and possible beneficial effects in terms of yield and/or crop quality when measures that may reduce Zn concentrations are adopted. The excess range indicates a substantial increase in yield and/or crop quality when corrected. In this range, toxicity symptoms and decreased vigor are likely to be observed. It is of interest to note that, in most crops, the critical deficiency levels are below 15-20 mg Zn·kg-1 dry weight of leaves. This is unique, as critical concentrations of other plant micronutrients vary rather widely among species. VI. EFFECfS OF ZN APPLICATIONS ON PLANTS

A. Vegetative Growth Early experiments with Zn applications to citrus in California involved severely Zn-deficient trees (Parker 1934,1935,1936) that showed shoot dieback and as much as 68-100 percent of foliage with Zn deficiency patterns (Parker 1937a,b). Under these conditions, even a single foliar spray with zinc sulfate greatly increased tree vigor. Zinc applications

Table 3.3.

Zn concentrations in tissues of various crops.

Concentration (mg/kg dry wt) Crop

Culture

Tissue

Deficient

Low

Normal

High

Excess

Reference

Apple

Field Field

<14

NID

N/D NID

15-200 35-50

NID NID

NID N/D

Shear and Faust 1980 Stiles and Shaw Reid 1991

Apricot Avocado

Field Field

<12 <20

NID NID

12-11 30-150

NID NID

NID

>300

Shear and Faust 1980 Jones and Embleton 1976

Cherry (sweet & tart)

Field

Leaves, July Leaves, 60-70 days after petal fall Leaves, July Leaf, 5-7 months old, non-fruiting terminal mid-Aug. to mid-Oct. Leaves, July

NID

NID

15-70

NID

N/D

Shear and Faust 1980

Leaves, 60-70 days after petal fall Water Recently matured leaf, 3rd from the top Greenhouse Leaf Field Leaves, Sept. Synthetic medium Field Leaf petioles, opposite flower cluster, full bloom

NID

NID

35-50

NID

NID

Stiles and Shaw Reid 1991

<14

NID

~14

NID

N/D

Ohki 1976

NID NID NID

NID N/D NID

NID

27-85

54.5

NID

NID NID NID

NID

>660

Dang et ale 1990 Shear and Faust 1980 Lee et ale 1996

<15

NID

25-50

NID

NID

Cook and Wheeler 1976

Field

<15

NID

>15

N/D

NID

Viets et ale 1953

NID NID NID

White et ale 1987 Carsky and Reid 1990

NID

NID N/D N/D NID

110-200

75 >2001

Dang et ale 1990 Chapman 1960

25-49

50-200

>200

Smith 1966b

Cotton Fenugreek Filbert Geranium Grapes 'Thompson Seedless' Maize

Field

Field Field

~

t.I)

':l

Onion Orange

Greenhouse Field

Orange

Field

Leaf at silking, 2nd node below ear Leaf at silking stage Whole plants, 3--6 wks old Earleaf at tassel Bulb Leaves,4-10-month-old fruiting terminals Leaves from non-fruiting 4-7-month-old spring cycle shoots

NID NID NID

NID NID NID NID

<18

18-24

~.3

4-15

15-24

~11.0

~16 ~14

25-100

....

Table 3.3.

(cont.)

tJ,)

Concentration (mglkg dry wt)

0:>

Crop

Culture

Tissue

Deficient

Low

Normal

High

Excess

Reference

Orange

Field

<16

16-24

25-100

110-200

>300?

Embleton et al. 1973

Peach

Field Field

<12

NID NID

12-50 35-50

NID NID

NID N/D

Shear and Faust 1980 Stiles and Shaw Reid 1991

Pear

Field Field

<16

NID

NID NID

20-60 35-50

N/D NID

N/D NID

Shear and Faust 1980 Stiles and Shaw Reid 1991

Pecan

Field

<6.1

NID

~14

N/D

N/D

Hu and Sparks 1990

Pecan

Field

Leaves from non-fruiting 5-7-month-old spring cycle shoots Leaves, July Leaves, 60-70 days after petal fall Leaves, Sept. Leaves, 60-70 days after petal fall Leaflets, July, critical values on shoot basis Leaflets, critical values on orchard basis Non-bearing shoots, July-September. Leaves without petiole, uppermost mature branches Third mature leaf from the top at tillering Top leaf 2nd leaf 3rd leaf 4th leaf 5th leaf Leaves, a combined sample of leaves up to 3-month-old Leaves, Aug. Leaves, July

<50

NID

50-200

N/D

N/D

Sparks 1993

N/D

NID

~10

N/D

N/D

Uriu and Pearson 1986

NID

NID

NID

N/D

97-224 Sanderson and Gupta 1990

NID

NID

~6.5

NID

NID

Tiwari and Dwivedi 1994

<10 <10 <9 <13 <24 <16

N/D NID N/D

23-??

N/D N/D NID NID NID N/D

>64 >68 >80 >100 >195

Ohki 1984

NID

NID NID NID NID N/D

<35 <20

NID N/D

50-300 20-200

N/D NID

Pistachio Potato

Field

Rice

Field

Sorghum

Water culture

Sour orange seedlings

Water culture

Tung Walnut

Field Field

NID =not determined.

NID

N/D 16-23

NID

Swietlik. and Zhang 1994

N/D

Shear and Faust 1980 Shear and Faust 1980

N/D

3. ZINC NUTRITION IN HORTICULTURAL CROPS

139

also had a positive effect on vegetative growth of 'Pineapple' orange trees on acidic sandy soils in Florida despite the fact that only occasional Zn deficiency symptoms were noted (Koo and Reese 1971; Koo 1988). However, it took five years before the effect of annual Zn applications became measurable. Swietlik and LaDuke (1985) reported small growth increases from combined Zn, Mn, and Fe foliar sprays applied three times at monthly intervals to 'Ruby Red' grapefruit trees in Texas. The experimental trees were severely pruned after sustaining serious freeze injuries in the year preceding the treatment and contained 19 ppm Zn, 23 ppm Mn, and 59 ppm Fe in their foliage on a dry weight basis, suggesting that the trees were optimally supplied with Fe and were low in Mn and Zn. In fact, mild Zn-deficiency symptoms were observed but Mn and Fe patterns were absent. Although all these facts suggest that the response was induced by Zn, the experiment's design did not allow drawing of a definite conclusion. In a number of other studies on citrus, no growth responses were observed following Zn applications. Griffiths and Enzor (1953) observed no growth responses of 'Valencia' orange trees to one or two annual Zn foliar sprays applied over a four-year period in Florida. The trees showed some deficiency symptoms, but Zn nutritional status was not reported. In another study conducted in Florida over a seven-year period, omission of two annual foliar sprays of Zn did not affect vegetative growth or produce visual Zn deficiency symptoms on 'Pineapple' orange trees despite the fact that in five years Zn concentrations in leaves dropped to a level ~ 15 ppm (Wutscher and Obreza 1987). Swietlik and LaDuke (1991) were unable to observe growth responses in one experiment with 'Valencia' orange and two experiments with 'Ruby Red' grapefruit trees in which one to three annual Zn foliar sprays were applied over a four-year period in Texas. Over the experimental period, leafZn concentrations in control trees varied between 13-26 ppm d.w. in 'Valencia'; 17-46 and 12-31 ppm dry wt. in the first and second experiment with 'Ruby Red' grapefruit, respectively. Zinc-deficiency patterns were mild and affected only 1 to 2 percent of 'Valencia' foliage. The symptoms were absent in the first grapefruit experiment and were transient and obscured by symptoms of B excess in the second experiment. No growth responses to 2-4 annual foliar sprays in winter, spring, and summer with ZnS0 4 and to a single soil ZnEDTA or ZnDTP A application in winter were observed in a study conducted in Texas with 'Rio Red' grapefruit on a calcareous soil with pH 8.03 to 8.31 (Swietlik 1996). The trees were showing severe Zn deficiencies during winter months prior to treatment initiation and contained 6.8 ppm Zn in leaf samples

140

D. SWIETLIK

collected in August (Swietlik 1996). However, the deficiency symptoms were transient and their severity greatly diminished in the spring and summer months, a phenomenon frequently observed on citrus trees with increasing temperatures in spring and summer (Swietlik 1989). The fact that the trees were successfully recovering from freeze injuries sustained one year prior to initiation of the experiment probably also contributed to the reduced severity of Zn-deficiency patterns. In the same study, however, soil and foliar spray treatments with EDTA and ZnS04 , respectively, significantly increased fruit yield, which suggests higher sensitivity of generative than vegetative development to Zn deficiency. In an experiment conducted in water culture under controlled conditions in a growth chamber, Swietlik and Zhang (1994) demonstrated that growth of various tissues of sour orange seedlings varied in their sensitivity to Zn deficiency, Le., root dry weight < leaf number and white roots dry weight < stem dry weight < leaf dry weight < shoot elongation and leaf area (Fig. 3.2). The critical activities of Zn around the roots, expressed as pZn = -log(Zn 2 +), where (Zn 2 +) denotes molar Zn activity, were;:: 10.2 ± 0.2 for root dry weight, 10.1 ± 0.2 for leaf number and white root growth, 10.0 ± 0.2 for stem dry weight, 9.8 ± 0.2 for shoot growth and leaf area. Increases in growth were observed in 11.5 ,----=R:""'""o""""":ot:""'""d7""ry-wt-:--'"---:"l-e-af=-n-o-.- - - - - - - - - - - - ,

11

~

10.5

l:

0-

I

1 st1em d~e: dry 1 Shoot growth leaf area WI

~"" ~

, , _

!:i

§

White root growth

.. " 10 .....

...... ........

- - -..............

......

---

...................

9.5 9

1

...

-

- ..... _---

\..u..L................................uI..u...u..u...u..~L.U.I.lu..u..l..u...u. ............1.LU.J...u..u..u..u...u...u.J..I-U..................................u..u.I...................................

5

21

37

53

69

85

101

Total Zn concentration (uM) Fig. 3.2. Activities of free Zn2+ in the nutrient solutions at the beginning (broken line) and end (solid line) of a 2-week nutrient solution change cycle. The Zn activities coincident with the observed maximum responses of sour orange seedlings for a given growth variable are indicated by arrows. Source: Swietlik and Zhang (1994).

141

3. ZINC NUTRITION IN HORTICULTURAL CROPS

response to Zn applications even in the absence of visible Zn-deficiency symptoms, which is contrary to the results of field studies cited above. Zn foliar sprays were less effective than Zn applications to the roots in increasing vegetative growth and particularly root growth of Zndeficient sour orange seedlings (Swietlik and Zhang 1994). This resulted from the fact that foliar-absorbed Zn was not translocated from the top to the roots and thus could not correct Zn deficiency in the roots. Sparks (1993,1994) reported that vegetative growth of pecan followed the modified Mitscherlich's plant growth model with the threshold value for leaf Zn of 49 ppm for maximum response (Fig. 3.3). The data presented in Fig. 3.3 is a mathematical expression of relative vegetative growth that combines leaf and trunk growth vs. Zn leaf concentrations obtained in several field experiments conducted on high pH soils in Texas and Arizona (Smith and Storey 1979; Malstrom et a1. 1984; Kilby 1985) and acid soils in Georgia (Sparks 1993). The slope of the curve for vegetative growth (Fig. 3.3) was less steep than that for leaf deficiency symptoms (Fig. 3.4) and yield (Fig. 3.5), which indicates that vegetative 100

-t ie

(!)

~

111I

90

80 70 60

... OILBY (1985) • MALSTROM et al (1984) + SMITH & STOREY (1979) 1m SPARKS (unpublished)

:;;

SQ) 50 0)

~ 40 ~

".=

30

~ a: 20

10 0 <:>

~

t>t.f::)

COf::)

'O~ ,~f::)

,,();~ "bt.~

"ro~

,,'O~

~f::) ();~

~<:>

Zn (ppm in dry leaf)

Fig. 3.3. The relationship between vegetative growth and leaf Zn concentration of pecan: Y =98.234 7[1_0.1 e-o.G7925(X-49.038)}, r 2 = 0.504. The threshold value of Zn concentration for 98% maximum response is indicated by the broken line. Redrawn with permission from Sparks (1993).

D. SWIETLIK

142

100 90

;g

!L~ c:

Q)

'0

~

80

70 60

~:::J 50 o

~ 40 ~ ~

F

30

20 10 O-l--+---+--+--+-..J--.t--+---t---"1I---+---t---t---;----t-----1 <)

,<)

~

lO<)

b?

<:><)

ro<)

,,<)

CO<)

0,)<)

,<)<) ,'<)

,~

,lO<)

,'t)..<)

Zn (ppm in dry leaf) Fig. 3.4. The relationship between the percent of pecan trees without Zn-deficiency symptoms and leaf Zn concentrations: Y = 100.2125[1_0.1e-O.l059(X-46.4513)], r 2 = 0.79. The threshold value of Zn concentration for 98% maximum response is indicated by the broken line. Redrawn with permission from Sparks (1993).

growth may be less responsive to Zn deficiency. This concurs with the growth and yield response data for citrus reported by Swietlik (1996) and the results of the study on pecans conducted by Worley et al. (1972) in which yield, but not growth, responses of pecan trees to soil ZnS04 applications were observed. Sparks (1993) indicated that leaf Zn concentrations in pecan as high as 250 ppm had no negative effect on vegetative growth in Zn-foliar treated trees and that leaf Zn concentration of at least 200 ppm was not deleterious to the growth of trees that were soil-treated with zinc. Since it is not certain to what extend analysis of Zn-sprayed leaves reflect absorbed vs. adsorbed Zn, the concentration of 250 ppm may not truly indicate the pecan trees' tolerance of such a high concentration of this element. There is little experimental data on the effect of Zn applications on the growth of other fruit trees. The fact that severe Zn deficiencies result in shortening shoot internodes indicates that correction of the deficiency

143

3. ZINC NUTRITION IN HORTICULTURAL CROPS

100 90 80

~ 70

A BROOKS (1964)

"t:I Q)

::; 60 :J z 50

Q)

> ~

g HUNTER (1965) • MALSTROM at al (1984) + SMITH & STOREY (1979) 181 WORLEY at al (1981)

40

Q)

a: 30 20 10 0 ~

~

~

~

~

~

~

~

~

~

~

Zn (ppm in dry leaf) Fig. 3.5. The relationship between relative nut yield and leaf Zn concentration of pecan: Y =97.059[1_0.l e-o· 1147 (X-50.3876)], r 2 =0.852. The threshold value of Zn concentration for 98% maximum response is indicated by the broken line. Redrawn with permission from Sparks (1993).

with soil and/or foliar Zn application may be expected to increase the amount of shoot growth. Stiles (1987) reported the negative effect of Zn deficiency on the growth of young pome and stone fruit trees in New York. Under severe deficiency conditions, shoot growth is also stunted on mature trees (Stiles 1966; Stiles and Shaw Reid 1991). Neilsen and Hogue (1983) reported increases in the amount of shoot growth of'McIntosh' apple seedlings foliar sprayed with chelated and mineral forms of Zn. The growth increases, however, were not reflected in higher leaf Zn concentrations of the treated plants. The authors concluded that this reflected the dilution effect caused by a higher relative rate of dry-matter production than Zn accumulation. Also, application of Zn to plant roots but not foliar sprays resulted in the highest dry-matter production, which concurs with the data for citrus (Swietlik and Zhang 1994). In a study conducted on 'Antonovka' apple seedlings, ZnS04 foliar sprays increased root growth (Potapova 1974).

144

D. SWIETLIK

Zinc soil applications at the rate of 15 kg/ha in the form of ZnS04 increased vegetative growth of maize containing 17-22 ppm Zn in leaf dry weight in studies conducted in Connecticut (Bugbee and Frink 1995). Depending on the year of study, the soil of the experimental site contained from 0.7 to 2.2 ppm DTPA-extractable Zn, Le., levels t~at were generally higher than the critical concentration of 0.8 ppm described by Lindsay and Norvell (1978). This explains why the growth increases were small and occurred in only two out of four years of the experiment. In another field study with maize conducted on fine sandy loam and loam soils with a pH of 6.0, no significant growth responses were observed when Zn as ZnS0 4 was applied to soil at rates of 11 and 22 kg Zn/ha or when it was foliar sprayed (White et a1. 1987). Leaf tissue Zn concentrations at silking stage were 11 ppm in the untreated plants, suggesting that the critical leaf Zn level on maize grown in the region (Prince Edward Island in Canada) is much less than the 15 ppm suggested by other authors (Table 3.3). The lack of growth responses in maize were also reported by Vanden Heuvel et a!. (1989) in pot experiments conducted under greenhouse conditions in which Zn was applied to the soil as ZnEDTA at the rate of 2.3 kg/ha. A number of Illinois soils were used in these studies but most of them produced plants with Zn concentrations well above 15 ppm. Moreover, no symptoms of Zn deficiency were noted. On one of the soils, the plant tissue Zn level was only 8.5 ppm. Applying Zn in this instance still did not affect growth, although Zn plant concentrations increased as a result of the treatment. Zinc applied as ZnS04 to a calcareous sandy loam (pH 7.8 to 8.3) at rates of 3.3 to 6.6 kg/ha increased the growth of a number of crops such as wheat, barley, oats, lentil, chickpea, pea, flax, and mustard (Tiwari and Dwivedi 1990). Except lentil and chickpea, the level of Zn in straw of the Zn-nontreated plants was less than 15 ppm on a dry weight basis. Also, the soil DTPA-extractable Zn was quite low and ranged between 0.44 and 0.49 ppm. Seventeen field experiments were conducted with rice in which ZnS04 was applied at rates of 1.25,2.5, and 5 kg Zn/ha to soils ranging from loamy sands to silty clay 10ams with pH from 7.22 to 8.15 and DTPA-extractable Zn of 0.25 to 1.5 ppm (Tiwari and Dwivedi 1994). Straw yields increased in response to Zn applications on soils containing less than 1 ppm DTPA-extractable Zn. Forty-five-day-old control plants grown on these soils contained 10.7 to 36.8 ppm Zn in the third leaf. The lower the tissue and soil DTPA-extractable Zn concentrations, the higher the relative growth response. The most pronounced growth

3. ZINC NUTRITION IN HORTICULTURAL CROPS

145

responses occurred when the soil DTPA-extractable Zn was < 0.6 ppm and leaf Zn concentrations ranged from 10.7 to 20.8 ppm. The growth of rice plants measured at midtillering, first joint, and panicle differentiation responded positively to Zn applied at the rate of 1.9 kg/ha to a silt loam soil (Sedberry et al. 1988). Plant tissue Zn concentrations were low in the above-ground parts and ranged from 9 to 14 ppm on a dry weight basis. Also, the soil 0.1N HCI-extractable Zn concentration of 1.6 to 1.8 ppm was considered low. Another example of rice plants responding positively to Zn soil application comes from a greenhouse study in which a clayey calcareous soil containing low DTPAextractable Zn (0.47 ppm) was used (Sajwan and Lindsay 1988). A rather dramatic 64 percent increase in growth was probably caused by a rather low Zn level (14.4 ppm) in tissue samples of the control plants. Excessive levels of soil zinc may be harmful to the growth of cultivated plants. Zinc toxicity can become a problem where industrial or municipal waste products, e.g., ashes from power plants or sewage sludges containing high levels of this element, are disposed of on agricultural lands and result in excessive accumulation of Zn in the plant tissues. However, amending a growing medium, high in organic matter, with a composted sewage sludge containing 186 ppm Zn was not deleterious to the growth of greenhouse-grown cucumbers (Fallahi-Ardakani et al. 1988). Organometallic complexes that apparently formed in this medium were quite effective in binding zinc and other heavy metals and preventing their excessive accumulation in the plant tissues. The application of sewage sludge incinerator ash containing 7213 ppln Zn at rates of 0.95 to 61 g/kg soil increased growth of lettuce and corn in greenhouse studies in which a sandy loam with pH 5.7 and organic matter content of 51 mg/kg soil was used as a growing medium (Bierman and Rosen 1994). The growth increases, however, were most likely caused by the increases in plant P levels, because tissue Zn concentrations in the control were optimal. In lettuce, tissue Zn concentrations increased from 31 to 42 ppm and no change in corn tissue concentrations were noted as a result of ash application. That Zn excess may reduce biomass production is illustrated by studies conducted with geranium grown in a sphagnum peatmoss-perlite mixture under greenhouse conditions (Lee et al. 1996). Elevating the plant tissue Zn concentrations to 660 ppm significantly reduced plant growth. Similarly, application ofZn as ZnS0 4 at rates of~100 mg/kg soil reduced the growth of onion and fenugreek (Dang et al. 1990). Tissue Zn concentrations associated with 10 percent growth reduction were 75 ppm and 54.5 ppm for onion andfenugreek, respectively. The soil used

146

D. SWIETLIK

in the experiment contained only 0.28 percent organic matter and had a pH of8.3. The rate of Zn applied, soil properties such as pH, organic matter, and clay content, and genetic differences between various plant species are the most important factors that determine whether high levels of Zn do or do not negatively affect the growth of cultivated plants. B. Yield

When citrus trees were severely deficient in Zn, as in early trials in California, foliar sprays with ZnS04 improved yields (Parker 1934, 1935, 1936, 1937a, 1937b). In one experiment, the yield of severely Zn-deficient grapefruit trees increased 5.5 fold in response to a single zinc sulfate foliar spray applied during bloom in March (Parker 1937b). The yield increases resulted from increased fruit set and fruit size. When approximately 30-40 percent of foliage showed symptoms, yields increased by only 20 percent and fruit number by 14 percent with fruit size showing only a mild increase. On the trees with 5-10 percent of the foliage showing symptoms, a foliar spray with zinc sulfate elevated the yield by only 10 percent. In a study conducted in Texas, the yield of 'Rio Red' grapefruit trees with 60 percent of the foliage affected by Zn patterns in winter months responded to 2 to 4 foliar sprays with ZnS04 applied in winter (Dec. and Jan.), winter (Dec. and Jan.) and spring (March and June), or a single soil Zn application as ZnEDTA around the trunk at the rate of 30 g Zn/tree (Swietlik 1996). The yield increases resulted from increased fruit set and not fruit size. No significant yield increases were noted when foliar sprays were applied only in spring or when soil applications of ZnEDTA at 10 g Zn/tree or ZnDTPA at 10 and 30 g Zn/tree rates were made. The deficiency symptoms were transient and were greatly diminished in spring and summer months, which explains the lower effectiveness of spring foliar sprays. There was a highly significant relationship between the percent of foliage with Zn-deficiency symptoms one month prior to anthesis vs. fruit yield (Fig. 3.6). The data indicate that yield reductions in grapefruit may be expected when more than 15-20 percent of foliage is affected by zinc deficiency. These results corroborate the findings of Parker (1937b), who concluded that mild symptoms of mottle-leaf have minimal effect on fruit yield. In Swietlik's study, the relationship between yield and leaf Zn concentrations was quite variable, particularly in trees that received Zn foliar sprays (Figs. 3.7 and 3.8). Thus, the severity of zinc deficiency symptoms was the superior criterion determining the yield responses of the trees to corrective Zn treatments.

3. ZINC NUTRITION IN HORTICULTURAL CROPS

Y = 87.7 + 0.025X

140

J.

120 (j) 100 Q)

t:

~

"'0

Q)

>=

147

80

~

X

~

Y =:: 118.2 - 3.8X + 0.05 X2

*+

-0.0003 X3 R2

t+.

60 40

J

20

~

l

=0.70

X

.

+

X

A.

0 0

10

20

30

40

50

60

70

80

.. 90

100

% Leaves with Chlorosis Fig. 3.6. The relationship between the percent of leaves with Zn·deficiency chlorosis in Jan. 1992 and total yield in 1992/93 season for 'Rio Red' grapefruit trees foliar·sprayed annually with ZnS04 in December and January (*), December, January, March and June (-), March and June (+); soil·treated in January 1991 with ZnEDTA at 10 g Zn/tree (A.), or 30 g Zn/tree (X), ZnDTPA at 10 g Zn/tree (X), or 30 g Zn/tree (+), or untreated with Zn (.). Source: Swietlik (1996).

Foliar sprays with ZnS04 failed to increase yields of 'Valencia' and 'Washington' navel oranges with moderate Zn deficiency and symptomless 'Eureka' lemon trees, although the symptoms on both orange cultivars were successfully eliminated or reduced (Labanauskas et a1. 1963; Embleton et a1. 1965,1988; Labanauskas and Puffer 1964; Labanauskas et a1. 1969). However, lemon yields increased in response to soil ZnS04 applications of 294 g Zn/tree when used jointly with MnS04 sprays (Embleton et a1. 1965). Manganese sprays alone had no effect. Leaf Zn concentrations in control trees were: 14 ppm and 12 to 15 ppm for the two experiments with 'Valencia' orange (Labanauskas et a1. 1963; Labanauskas and Puffer 1964); 12 to 23 ppm for 'Washington' navel (Embleton et a1. 1988); and 11 to 20 ppm for 'Eureka' lemon (Embleton et a1. 1965). Although the leaf Zn concentrations in lemon trees were deficient « 15 ppm) on most of the sampling dates (Embleton et a1. 1973), the trees showed no deficiency symptoms. Also, Swietlik (1996) observed that the relationship between leaf Zn concentrations and the

D. SWIETLIK

148 140 Y

=0.0357 (X)2.8867

R2 =0.54

120 100 Q) Q)

80

~

60

t:

"C

(j)

5=

40 20 0 7

9

11

13

15

Leaf Zn (ppm) Fig. 3.7. The relationship between leaf Zn concentrations in February 1992 and yield in 1992/93 season for 'Rio Red' grapefruit trees soil-treated with ZnEDTA or ZnDTPA. Source: D. Swietlik.

percent of grapefruit canopy foliage with Zn deficiency was quite variable (Fig. 3.9). This suggests that the concentration of a yet undetermined "physiologically active" form of zinc should be considered rather thaI,l total Zn level when assessing the nutritional status of citrus leaves. Two annual foliar sprays with zinc during a four-year experimental period had no effect on yield of 'Valencia' orange trees grown on an acidic sandy soil typical of the Lakewood series in Florida and showing moderate Zn deficiency symptoms (Griffiths and Enzor 1953). Similarly, two annual foliar sprays with zinc did not affect yield of 'Pineapple' oranges in another study in Florida, despite the fact that leaf Zn levels dropped to deficient ranges in some years (Wutscher and Obreza 1987). However, no leaf deficiency symptoms were noted. Omission of Zn soil applications to 'Pineapple' orange trees grown on a typical acidic ridge soil in Florida failed to reduce fruit yield in a 20year-long study (Koo 1988) and leafZn concentrations were maintained at approximately 25 ppm. The trees showed only sporadic Zn-deficiency symptoms. In Texas, Leyden (1983) and Leyden and LaDuke (1984) observed no

3. ZINC NUTRITION IN HORTICULTURAL CROPS

149

120,.--------------------------, Y = 5.971 (X)O.5186 R2 = 0.31 100 80

m e~

60

'0 Q)

>= 40 20 0

I

3

. •

18

33

48

63

78

93

108

123

138

153

Leaf Zn (ppm) Fig. 3.8. The relationship between leaf Zn concentrations in February 1992 and yield in 1992/93 season for 4Rio Red' grapefruit trees foliar-treated with ZnS04' Source: D. Swietlik.

effect on yield of one or two annual foliar sprays of zinc as ZnS04 or Zn chelate applied in Mayor May and July on 4Ruby Red' and 'Star Ruby' grapefruit and 'Marrs' orange trees. The deficiency symptoms were moderate on control trees whose leaves contained 16 to 23 ppm Zn in 'Ruby Red', and 11 to 18 ppm in 'Marrs'. The'Star Ruby' trees were judged Zn deficient but the actual leaf Zn concentrations were not reported. Zinc foliar sprays on 'Ruby Red' grapefruit and 'Valencia' orange trees, which had approximately 1 to 2 percent leaves mottled, did not induce significant responses in yield during a 4-year-Iong study in Texas (Swietlik and LaDuke 1991). From one to three foliar sprays of ZnS04 were applied depending on the year of the study. In nonsprayed trees, leaf Zn concentrations ranged from 13 to 26 ppm in 'Valencia' orange, and 17 to 46 and 12 to 31 ppm in 'Ruby Red' in two separate experiments. On the grapefruit trees Zn-deficiency symptoms were either absent or were mild and transient. Zinc foliar sprays were not beneficial in terms of yield when applied to 'Blood Red' sweet orange containing 16 ppm Zn in the foliage (Mann and Takkar 1987). The sprays, however, elevated leaf Zn to 25 ppm.

D. SWIETLIK

150

Y

100

= 1411.6 * (0.706)x

R2 = 0.55

80

;? ~

en

~ (U J!? .2

60

'0 ..... 40 0

:c

(,)

20

0 8

10

12

14

16

Leaf Zn (ppm) Fig. 3.9. The relationship between the percent of leaves with Zn chlorosis in January 1992 and leaf Zn concentrations in February 1992 in 'Rio Red' grapefruit trees soil-treated with Zn-EDTA or Zn-DTPA. Source: Swietlik (1996).

Yield of 'Valencia' orange trees were elevated with a single annual foliar spray containing MnS04 or MnS04 + ZnS04 but not when ZnS04 was used alone, indicating that Mn rather than Zn was responsible for the yield increases (Garcia et al. 1983). Deficiency symptoms of both elements were observed on the trees and the treatments alleviated these symptoms within 60 to 70 days. The common characteristic of the citrus trees used in the experiments in which no yield responses were observed was their mild to moderate expression of Zn deficiency or, in the case of some studies, their total absence. This underscores the fact that the severity of Zn-deficiency symptoms is the most important criterion determining the trees' response to corrective Zn treatments. Thus, judging a tree's Zn nutritional status based only on the results of leaf analysis and leaf standards may not be sufficient to predict tree responses. This fact, however, should not lessen the importance of leaf analysis as the most reliable diagnostic tool, especially in situations involving multi-elemental disorders. Foliar Zn applications did not affect the yield of avocado (Kadman and Lahav 1978), possibly because of poor absorption and mobility of foliar applied Zn (Crowley et al. 1996). Soil applications of Zn (2.9-44.8

3. ZINC NUTRITION IN HORTICULTURAL CROPS

151

kg/hal, B (0.2-4.5 kg/ha), and P (11.2-123.3 kg/hal produced yield interactions of 'Earliglow' strawberries on silt loam at pH 6.5 (May and Pritts 1993). Increasing B fertilization from 0.2 to 4.5 kg/ha had a negative effect on yield at low P application rates. This effect was more pronounced at the high (44.8 kg/hal than the low (2.9 kg/hal Zn application rate. When the same increases in B fertilization occurred at high Prates, however, fruit yields increased. These yield increases were much more pronounced at the low than high Zn application rates. At pH 5.5, strawberry yields were affected by P and B but not Zn treatments. No leaf Zn concentrations were given for plants receiving the lowest Zn application rate, but at the rate of 23.9 kg Zn/ha, leaf lamina contained from approximately 20 to 30 ppm Zn depending upon the month of sampling. Rosette, the primary expression of Zn deficiency in pecan trees, was the most important factor limiting the production of pecan in the southwestern United States in the 1920s and 30s (Sparks 1987). Sparks (1987) provided a very interesting analysis of the effect of Zn treatment on the rate of growth and nut production in a number of southern states. According to this author, after the cause of rosette was discovered and publicized in the 1930s, increases in pecan production accelerated particularly in Georgia, Alabama, Florida, and South Carolina. These increases were associated with higher yields per tree. In the above states, pecan trees grew in mechanically cultivated orchards, Le., under a soil management system conducive to the development of Zn deficiency. Moreover, in these states with predominantly acidic soils, trees responded well to soil Zn treatment, which at the time was the primary method of correcting Zn deficiency. This method was ineffective on alkaline calcareous soils of Arkansas, Louisiana, Mississippi, Oklahoma, and Texas, and thus yield increases were small or absent. Hu and Sparks (1990) reported that the effect of Zn deficiency on reproductive growth of pecan depends on the severity of the deficiency. At the greatest deficiency level, neither staminate nor pistillate inflorescences were produced. At less severe deficiencies, catkin length and weight of staminate flowers decreased with decreasing leaf Zn concentrations. Also, the reduced number of nuts per shoot, fruit death, and drying were associated with increasing Zn deficiency. Smith and Storey (1979) reported increases in yields of pecan trees foliar-sprayed with ZnS0 4 or Zn(NOa)z three times a year in April, May, and June. The highest nut yields were noted when zinc nitrate was used at 10.8 g Znll00 liters of water with 0.50/0 Uran, Le., a liquid nitrogen fertilizer containing urea:NH4N0 3 at 1:1 ratio. The presence of Uran also improved yield responses when foliar sprays of ZnS04 were used. Interestingly, however, with Zn concentrations in the spray solution as

152

D. SWIETLIK

high as 43.1 or 86.3 g/100 liters, leaf Zn concentrations were above ·200-300 ppm and yield increases were less spectacular, pointing to a possible deleterious effect of high Zn on nut yield. Foliar Zn concentrations in September in the nonsprayed control were 23 ppm in the first, 42 ppm in the second, and 23 ppm in the third year of study, while in the treatment producing the highest yields, Zn leaf levels were elevated to 57, 60, and 81 ppm, respectively. Worley et a1. (1972) were unable to show any beneficial effect of soil treatments with Zn as ZnS0 4, ZnO, or Zn chelate (ZnHEDTA and ZnEDTA) on yields during an eight-year-long study on 'Stuart' pecans grown in a slightly acidic soil (pH =5.7-6.7). Zinc was broadcast annually at the rate of 0.82 kg Zn/tree as ZnS04 and ZnO and at the rate of 0.082 kg Zn/tree as ZnHEDTA and ZnEDTA. All the materials were incorporated into the soil with a disk. Leaf Zn concentrations, however, increased significantly as a result of soil Zn treatments. Control trees showed only mild Zn-deficiency symptoms and, depending upon the year of the study, contained between 40 and 67 ppm Zn in the foliage. In another experiment by Worley et a1. (1972), 'Stuart' pecan trees responded with yield increases to annual soil applications of ZnS04 at the rate of 0.82 kg Zn/tree in 2 out of 8 years. However, leaf Zn concentrations increased only after 6 years of zinc applications. This contrasted very sharply with the first experiment in which excellent Zn uptake from soil applications was observed. The differential response could not be explained by differences in soil type, soil pH, method of Zn application, soil management, or soil P. The only known differentiating factor was the presence of crown gall in the second experiment, which might have contributed to inefficient Zn uptake. Leaf Zn concentrations varied between years from about 25 ppm to 60 ppm in the nontreated trees, thus indicating deficient to optimal Zn levels. Sparks (1993) reported that pecan yields followed the modified Mitscherlich's plant growth model, with the threshold value for leaf Zn being 50 ppm for maximum response (Fig. 3.5). The data presented in Fig. 3.5 were obtained in several field experiments conducted on basic soils in Texas (Smith and Storey 1979; Malstrom et a1. 1984) and acid soils in Georgia (Brooks 1964; Lane et a1. 1965). The slope of the curve for yield coincided with the slope for the curve depicting the relationship between leaf Zn and the occurrence of Zn deficiency symptoms (Fig. 3.4). No symptoms were observed when the leaf Zn concentration reached the critical value of 48 ppm. This illustrates that, as in citrus trees (Fig. 3.6), the occurrence of leaf Zn patterns is a prerequisite for yield reductions although, contrary to citrus, pecan trees seemed to respond with yield reductions to even mild Zn deficiencies.

3. ZINC NUTRITION IN HORTICULTURAL CROPS

153

Stiles and Goff (1965) and Stiles (1966) reported a strong positive relationship between deficient Zn leaf levels within the range of 5.9 to 14.4 ppm dry wt and yield of 'McIntosh' apple trees. Severely Zn deficient 'Red Delicious' and 'McIntosh' apple trees responded positively to foliar and soil Zn applications in terms of fruit yield (Stiles 1966). Similar results were obtained in other experiments with soil and foliar Zn applications to these two apple cultivars, but Zn leaf levels were not provided (Stiles and Goff 1965; Stiles 1980). Eliyeva (1975) reported increased yields of apple trees in response to Zn sprays, but the nutritional status of the trees was not reported. Stang et a1. (1978) failed to show the benefits of Zn dormant sprays on fruit set in apple with high leaf Zn levels (117 ppm). Lack of response in terms of the number of flower buds, fruit set, and yield was also observed by Yogaratnam and Greenham (1982) on 'Cox's Orange Pippin' apple trees foliar-sprayed with Zn, despite the fact that the leaves contained only 12 to 13 ppm Zn. No deficiency symptoms, however, were noted on the nontreated trees. Interestingly, however, 'Discovery' apple trees that contained twice as much Zn in the foliage as 'Cox's Orange Pippin' responded to the same treatment with increased numbers of flower buds. However, no yield increases were noted since the increased number of flowers was offset by reduced fruit set. Fruit yield of the parthenocarpic 'La Reine' cucumber was unaffected when grown in a greenhouse in containers filled with equal parts of peatmoss and vermiculite and blended with 00/0, 250/0, or 50% by volume composted ferric-chloride-precipitated, lime-stabilized, digested sewage sludge composted with wood chips (Fallahi-Ardakani et a1. 1988). The compost contained 186 ppm Zn. The fact that leaf Zn concentration in the control plants was rather high (109 ppm) explains why Zn additions with the compost had no positive effect on yield. Under the conditions of this experiment, toxicity of Zn from the compost applications could be expected, but the compost additions had no effect on leaf and fruit Zn even when the growing medium was acidified with sulfur to pH 5.1. Apparently, the highly organic medium buffered the plant-available Zn, preventing its excessive accumulation in the tissues. Only when the medium pH was lowered to 3.4 was there an increase in Zn concentration of fruit tissues that coincided with a tendency for yields to decrease. Such a low pH, however, is unlikely in commercial potting media used for production of greenhouse cucumbers. White et a!. (1987) observed no yield response in maize when ZnS04 was banded and mixed with soil at 11 and 22 kg Zn/ha or when plants were foliar sprayed with the same material at 0.34 kg Zn/ha. Two experiments were conducted on Prince Edward Island, Canada, on fine sandy

154

D. SWIETLIK

loam and loam with a pH of -6. The nontreated plants contained 11 to 14 ppm Zn in the leaves, i.e., a level considered deficient. Both soil and foliar Zn applications increased leaf and whole plant Zn concentrations. Vanden Heuvel et a1. (1989) evaluated over a 3-year period responses of maize to Zn applications at a total of 82 site-years in Illinois. Zn was applied at 1.15 and 2.3 kg/ha before planting as ZnEDTA. In only 3 out of 82 year-locations were grain responses noted. In a few locations, soil Zn applications increased Zn concentrations in whole plant samples at the six-leaf stage or in earleaf samples at silking, but the increases were rather small and statistically significant only at the 10 percent probability level. With one exception, the tissue Zn concentrations in nontreated plants were well above the critical 15 ppm level. It seems that the lack of yield responses in this study could be explained by optimal tissue Zn concentrations and the inability of soil Zn applications to affect the plants' Zn status. Increases in grain yields of maize in response to Zn soil applications at rates of 9 to 15 kg Zn/ha as ZnS0 4 were observed in studies conducted in the northeastern United States (Carsky and Reid 1990; Bugbee and Frink 1995). Zinc fertilizers were broadcast and incorporated into the soil with disking. The tissue Zn concentrations in nontreated plants were 17 to 22 ppm in one study (Bugbee and Frink 1995) and less than 15 ppm in the other (Carsky and Reid 1990). The relative yield increases were much larger in the latter study. Whole plant tissue concentrations showed much closer relationship with yield than did earleaf Zn concentrations. A study conducted with maize in central Iowa indicated that over a 26-year experimental period, annual soil applications of zinc at 11.4 kg Zn/ha as ZnS04 increased, on an average, the grain yield of maize by only 2 bushels per acre (Mallarino and Webb 1995). Yield increases occurred in only 5 out of the 26 experimental years. The study was conducted on a fine loam soil that was calcareous and had a pH from 6.8 to 8.0. The DTPA-extractable Zn was 1.2 ppm and was considered only marginally adequate. Analyses of earleaves at silking at the beginning of the study showed 15 ppm Zn, which is borderline between the deficiency and sufficiency ranges. The continuous applications of high rates of P fertilizers in this study did not induce higher Zn deficiency than that observed under low P application rates. Wijesundara et a1. (1991) observed no effect on grain yield of heavy ZnS04 soil applications to maize during a 23-year period. Depending on the treatment, the cumulative application rates were 270 and 1142 kg Zn/ha, which exceeded the maximum loading rates according to the U.S. Environmental Protection Agency gUidelines. The authors concluded that the absence of yield reductions reflected the adsorption and occlu-

3. ZINC NUTRITION IN HORTICULTURAL CROPS

155

sian of Zn in the three soils used in the study whose pH's ranged from 6.4 to 6.8. Zinc levels in earIeaf and grain ranged from 20 to 38 ppm and 17 to 23 ppm, respectively, indicating normal levels of this metal. Agaev (1989) reported significant increases (22-29%) in yield of potato plants in response to soil and foliar Zn applications in the Caucasus region of the former Soviet Union. Yield increases were caused by both elevated numbers of tubers and their size. In studies conducted in Canada on acid soils (pH 5 to 5.8), however, no responses in potato yields were noted after soil applications of 25 kg Zn/ha as ZnS04 (Sanderson and Gupta 1990). The lack of response may have been caused by adequate levels of Zn in leaves, which ranged in the nontreated plants from 15.4 to 46.8 ppm. The soil treatments elevated leaf Zn concentrations to 18.2 to 69.9 ppm. Foliar ZnS0 4 treatments induced even higher leaf Zn concentrations that ranged from 97 to 224 ppm. These high Zn concentrations proved to be toxic to the plants and were manifested by reduced total and marketable yield, tuber numbers, and starch content. Blaylock (1995) reported that ZnS0 4 applied to the soil at 5.6 or 11.2 kg Zn/ha had no consistent effect on yield of dry bean. Mean plant Zn concentrations, however, increased as a result of the treatments. Rice grain yields responded positively to soil Zn treatments in a number of studies conducted on soils with pH ranging from 5.8 to 8.15 and the texture of sandy loam to silty clay loam (Sedberry et a1. 1988; Sarkunan et al. 1989; Tiwari and Dwivedi 1994). These results indicate that rice plants are particularly responsive to Zn application. In one of the studies, critical leaf Zn for maximum yields was estimated at 26.5 ppm (Tiwari and Dwivedi 1994), which is significantly higher than the usual 15 ppm described for other crops. That yields of different crops respond differently to added Zn is also illustrated by the studies of Tiwari and Dwivedi (1990). Pulses such as lentil, chickpea, and pea were more sensitive to Zn deficiency and had higher demands for Zn to achieve maximum yield compared to cereal crops (wheat, barley, oats), flax, and mustard. All the plant species were treated with broadcast ZnS0 4 at rates of 3.3 and 6.6 kg Zn/ha at sowing. The soil was calcareous sandy loam with a pH of 8.3 to 7.8 and low DTPA-extractable Zn of 0.44 to 0.49 ppm. C. Crop Quality

Crop quality is often assumed to be inferior in Zn-deficient plants. However, information on the effect of corrective Zn treatments on crop quality is rather sketchy and often inconsistent. The inconsistency most

156

D. SWIETLIK

likely is caused by differences in severity of Zn deficiency between experiments and whether the symptoms of Zn deficiency are transient or persistent over the growing season. Early trials in California on grapefruit trees with as much as 100 percent of tree foliage mottled showed that a single foliar spray with ZnS04 greatly increased fruit size, eliminated abnormal fruit shape, reduced rind thickness and eliminated resinlike formations in the albedo, and increased juice content (Parker 1937b). A similar treatment applied to moderately Zn-deficient trees having 30-40 percent of mottled leaves produced only some increase in fruit size and the percent of fruit packout. Fruit quality on trees showing only 5-10 percent of mottled-leaf foliage did not respond to Zn foliar sprays. Zinc foliar sprays to moderately Zn-deficient 'Valencia' orange trees increased the amount of ascorbic acid in juice but also decreased the percent of juice in fruit (Labanauskas et al. 1963). External and internal fruit quality of orange and grapefruit trees with mild Zn deficiency was not influenced by foliar or soil Zn treatments (Embleton et al. 1988; Koo 1988; Swietlik and LaDuke 1991). In another study on grapefruit with severe Zn-deficiency symptoms, foliar and soil treatments with Zn had no effect on fruit size, percent soluble solids (SS), percent total acid (TA), SS:TA ratio, percent juice, percent peel, and percent blush on the fruit surface (Swietlik 1996). However, leaf mottling was severe only in the October-March period and greatly diminished in the spring and summer months, which may explain the lack of response to Zn treatments. Hu and Sparks (1990) reported fruit death and drying with increasing Zn deficiency in pecan. They concluded that Zn deficiency suppressed fruit development and resulted in delayed and staggered shuck dehiscence. In Zn-deficient avocado, the fruit is more round and less pear shaped (Wallihan et al. 1958). ZnEDTA and ZnHEDTA applied to the soil as (1) dry material, (2) pressure-injected into the soil under the trees, or (3) applied with irrigation water completely eliminated Zn-deficiency symptoms. A positive effect of Zn sprays on apple fruit Ca that resulted in less bitter pit was indicated by studies conducted by Schmitz and Engle (1973). The positive effect of Zn on Ca transport in apple trees was also suggested by Stiles (1987) and Fallahi and Simons (1996), who showed that fruit Zn correlated positively with fruit Ca concentrations. This relationship, however, was not confirmed by studies conducted by Martin et al. (1976) and Yogaratnam and Johnson (1982). Fruit Zn concentrations were negatively correlated with fruit color at

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harvest of 'Redchief Delicious' and 'Redspur Delicious' apples (Fallahi and Simons 1996). Similarly, leaf Zn concentrations correlated negatively with fruit color but only for 'Redspur Delicious'. The above results contradict the earlier findings of Stiles (1966), who showed that Zn applications increased the percent of fruit having fancy color. Stiles (1966) and Stiles and Shaw Reid (1991) reported that Zn-deficient apple trees produce small, pointed, and poorly flavored fruit. However, the data of Huguet et a1. (1993) indicate that leaf Zn is positively associated with high acidity in apples. Agaev (1989) reported a positive effect of soil and foliar Zn applications on starch content in potato tubers and tuber size. These data contrast with those reported by Sanderson and Gupta (1990), who reported the lack of or deleterious effect of soil and foliar Zn applications, respectively, on quality of potatoes. Blaylock (1995) reported a positive effect of soil Zn applications on plant tissue Zn concentrations and early maturity of dry bean pods. Acceleration of pod maturity was considered highly desirable, because it decreased the chance of crop losses to early fall frosts. VII. TECHNOLOGY OF ZN APPLICATIONS There are four major techniques of applying Zn to plants, i.e., (1) soil application, (2) foliar or dormant spray applications, (3) trunk injections, and (4) seed applications. The effectiveness of various techniques of applying Zn to agronomic crops has been reviewed by Martens and Westermann (1991). Soil Zn applications to apple, avocado, citrus, and pecan trees have been reported to successfully improve their Zn nutritional status (Leonard et a1. 1956; Leonard et a1. 1958; Wallihan et a1. 1958; Smith and Rasmussen 1959; Stewart and Leonard 1963; Embleton et a1. 1965; Stiles 1966; Smith 1967; Worley et a1. 1972; Smith et a1. 1980; Anderson 1984; Zekri and Koo 1992; Neilsen and Neilsen 1994; Stiles et a1. 1995; Crowley et a1. 1996; Swietlik 1996). To be effective, however, such treatments required large amounts of Zn materials (Leonard et a1. 1956; Leonard et a1. 1958; Embleton et a1. 1965; Stiles and Goff 1965; Stiles 1966, 1992; Smith et a1. 1980; Anderson 1984; Stiles et a1. 1995; Crowley et a1. 1996), special application techniques such as applying Zn materials in concentrated bands (Embleton et a1. 1965; Smith et a1. 1980; Anderson 1984; Neilsen and Neilsen 1994; Crowley et a1. 1996), piles (Leonard et a1. 1956; Leonard et a1. 1958), in peat plugs containing Zn and inserted into the soil (Neilsen and Neilsen 1994), application beneath trickle irrigation

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emitters (Neilsen and Neilsen 1994; Crowley et al. 1996), or injecting Zn materials into the irrigation water (Zekri and Koo 1992; Stiles et al. 1995). Due to the large amounts used, soil Zn applications carry the danger of inducing Zn toxicity (Parker 1934; Orphanus 1982; Neilsen and Neilsen 1994). Limited movement of Zn in the soil is the reason for the poor and/or inconsistent effect of soil treatments. The use of ZnEDTA proved to be more effective than ZnS04 in the studies conducted by Stiles et al. (1995) in an apple orchard and Worley et al. (1972) in a pecan orchard. In the latter case, the treatment effect was inconsistent between two locations of a similar soil type and the authors offered no definite explanation for this differential response. The use of ZnEDTA on Florida's acidic soils proved ineffective in citrus because the compound easily reverted to FeEDTA (Stewartand Leonard 1963). Addition of Na 2S04 to raise pH, however, greatly improved the effectiveness of the treatment. Applying the ZnEDTA + Na 2C0 3 mixture in concentrated spots under the trees maintained Zn in chelated form and increased leaf Zn concentrations. In alkaline soils, applications ofZnEDTA to avocado (170 or 454 g/tree) and grapefruit trees (300 g ZnEDTA/tree) proved to be effective (Wallihan et al. 1958; Crowley et al. 1996; Swietlik 1996), but the cost of such an application is rather high. Leonard et al. (1956, 1958) and Stewart and Leonard (1963) reported much improved Zn uptake by citrus trees when a 1:1 mixture of ZnS04 and CaCl 2 was applied at the rate of 4.6 kg per tree in 10 to 60 concentrated piles beneath the trees' canopy. One possible explanation for the increased Zn uptake is that very high salt concentrations "pruned" the roots beneath the fertilizer piles, causing roots to proliferate at the interface of Zn-saturated soil volumes. A similar phenomenon was reported by Fenn et al. (1990). These authors found that H2 S04 and ZnS04 applied to a calcareous soil in a 15em-wide and 15-cm-deep trench on both sides of pecan trees (3 m from the trunk) lowered soil pH to a depth of 60 em and increased solubility of Zn in the acid band. Tree roots proliferated extensively at the interface of the acidified band, resulting in better Zn uptake, which was reflected in increased Zn leaf concentrations from the 4th to 9th year after treatment. Smith et al. (1980) reported that soil applications of ZnS04 to pecan trees grown on a calcareous soil can increase leaf Zn concentrations, particularly when applied jointly with S to acidify the soil. The applications were made to the soil in the outer one-third of the canopy and incorporated to a depth of 15 em. Zinc sulphate in excess of 20 kg/tree was required to increase leaf Zn concentrations to a desired level of 60 ppm. The authors concluded that foliar Zn applications are the best method of maintaining pecan leaf Zn concentration at the optimal level. Smith and Rassmussen (1959) and Smith (1967) reported success with

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soil Zn applications to citrus trees grown on slightly acidic to neutral soils when ZnS04 or ZnO were broadcast over the soil surface around each tree over an area of 9 to 54 m Z, or when the materials were applied in a narrow, 20-cm-wide, band at the perimeter of a 3-m x 3-m square. Subsoil trenching with any of the materials and the use of ZnS were ineffective in increasing Zn leaf concentrations. In both studies, the rates of Zn used were rather high and ranged from 0.18 to 1.1 kg Zn/tree. The higher the rate of Zn, the higher the leaf Zn concentration. All the fertilizers were shallowly incorporated into the soil. In early studies, various materials such as ZnS04, ZnO, ZnS, and ZnCOa were found approximately equally effective when used as foliar sprays to citrus at equivalent Zn concentrations (Parker 1937a). Dusting the leaves with metallic Zn, however, was generally less effective. More recent studies by Alva and Tucker (1992) indicated, contrary to the literature, rather poor effectiveness of a variety of Zn compounds applied as foliar sprays to navel orange trees. The authors explained this discrepancy by the fact that the sprays were applied with an airblast sprayer calibrated at 1580 liter/ha and not a hand gun whose use results in much higher application volumes. Studies by Storey and his group working on pecan trees in the early 1970s indicated that Zn(N03 lz and a mixed fertilizer containing 5.50/0 Zn and 22% N derived from Zn(N0 3 lz, CO(NHzlz, and NH4N0 3 are more effective in raising leaf Zn levels than ZnS04 and other forms of Zn (Smith et a1. 1972; Smith and Storey 1979; Storey et a1. 1979). More recently, similar results were reported by Alva and Tucker (1992) and Anderson and Leonard (1982) for citrus. Emina et a1. (1980) reported that the mixture of Zn(N0 3h, CO(NH 2h, and NH4N0 3 was effective as Zn foliar fertilizer on a number of container-grown ornamental shrubs. Storey's findings led to the development of the commercial fertilizer NZN®, which is widely used for correcting Zn deficiencies via foliar sprays. Later work by Grauke (1982) and Grauke et a1. (1982) also showed that the Zn absorbed from Zn(N0 3)2' NH4N0 3 , and CO(NH2)2 mixture (the ingredients of NZN®) is more mobile than from ZnS04 • They found that 10 percent of the absorbed Zn from this mixture moved from the point of absorption compared to only 2.8 percent when ZnS04 was used. Neilsen and Neilsen (1994) concluded that there is little difference in effectiveness of various forms of Zn when applied as foliar sprays to apple provided they are equally soluble. However, the nitrate form of Zn was not tested in their studies. Solubility was also an important factor assuring high leaf Zn absorption by citrus leaves (Stewart et a1. 1955). In New York State, however, foliar applications with ZnEDTA are recommended as the most economical approach to alleviating Zn deficiencies on pome and stone fruit trees (Stiles 1992).

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In California, early recommendations for Zn foliar sprays to citrus called for the use of high concentrations of ZnS04 in conjunction with lime, which acted as a precipitating agent. The use of such a mixture resulted in heavy salt deposits on leaf surfaces and a build-up of citrus mites and scale insects (see papers cited by Embleton et al. 1965). Labanauskas et al. (1969) developed low-residue zinc foliar sprays using diluted ZnS0 4 (432 ppm Zn) without lime, which elevated leaf Zn level up to 75 ppm, corrected Zn-deficiency symptoms, caused no leaf injury, and left no visible residue on sprayed leaves. The major setback of Zn foliar sprays to fruit trees is the fact that they have to be repeated every year due to limited Zn translocation from sprayed to nonsprayed new leaves (Stewart et al. 1955; Labanauskas et al. 1961,1963,1964; Smith 1966a; Labanauskas et al. 1969; Embleton et al. 1988; Swietlik and LaDuke 1991; Neilsen and Neilsen 1994; Crowley et al. 1996). The obvious advantage of foliar sprays is their ability to rapidly alleviate Zn-deficiency symptoms. In Florida, application of Zn foliar sprays for citrus are recommended during the post-bloom period in the form of ZnS04 or ZnO (Koo 1984). The standard concentration is 1200 ppm metallic Zn. In California, the standard concentration is 430 ppm Zn in the form of ZnS0 4 applied when the spring flush of growth is two-thirds or almost fully expanded (Platt 1981; Meith 1982). In Texas, Zn foliar sprays are recommended to be applied during winter before bloom at the concentration of 430 ppm metallic Zn in the form of ZnS04 (Swietlik 1996). When deficiency symptoms persist, the sprays should be repeated at the end of bloom and again 2 to 3 months later. In the post-bloom period, the standard concentration is 216 ppm metallic Zn when ZnS0 4 is used. Higher concentrations induce leaf speckling on grapefruit. The use of a suitable surfactant in the tank mix is recommended. When proprietary zinc products are used, the concentrations employed should be those recommended by the manufacturers. Postharvest and dormant Zn foliar sprays are recommended for deciduous fruit trees in many production areas (Swietlik and Faust 1984; Neilsen and Neilsen 1994). However, these sprays have little long-term effect due to limited translocation of absorbed Zn. Fungicides such as Dithane M-45 and Zineb contain Zn and have been found to be a good source of the metal for foliar applications in deciduous fruit trees (Beyers and Terblanche 1971) and citrus (Smith 1966a). In citrus, however, no evidence was found of Zn translocation from the older, sprayed leaves to subsequent growth. Trunk injection of Zn in the form of Zn crystals or a solution of ZnS04 were found not to be commercially feasible in citrus (see Swietlik 1989). Also, trunk injections of Zn(N0 3)2 to avocado trees were generally unsat-

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isfactory (Crowley et a1. 1996). Banin et a1. (1980) reported trunkimplanted Zn-bentonite paste to be a good source of slow-release Zn for pecan trees. This method of application, however, does not appear to be. widely practiced. The seed applications are used for crops grown from seeds and consist of coating the seeds with Zn materials such as ZnS04 or ZnO (Martens and Westerman 1991). VIII. ZN FERTILIZERS Zinc sources for production of single or complex fertilizers containing this element can be divided into three groups: (1) inorganic sources; (2) synthetic and natural chelates; and (3) natural organic complexes. Inorganic sources, along with their Zn concentrations and solubility in water, are listed in Table 3.4. They are marketed as fine powders, granules, or aqueous solutions. In the latter form, the concentrations of Zn Table 3.4.

Inorganic and organic sources of Zn.

Source ZnS04' H20 ZnS04·7H20 ZnCl 2 Zn(NO a)2 . 6H20 ZnS04 - NHa complex Zn(NOaJz + urea + ammonium nitrate (NZN®) Zn oxysulfate ZnS04 . 4Zn(OHh ZnCOa ZnO

Solubility in water

Inorganic compounds Soluble Soluble Soluble Soluble Soluble (liquid) Soluble (liquid) Variable Slightly soluble Insoluble Insoluble

ZnEDTA ZnHEDTA ZnNTA ZnCitrate Zn-glucoheptonate Zn-amino acids

Synthetic and natural chelates Soluble Soluble Soluble Soluble Soluble Soluble

Zn-lignosulfonates Zn-phenolic acids Zn-polyflavonoids

Natural organic complexes Soluble Soluble Soluble

Zn (%) 36

22 47 21 Variable 5

25-60 55 52

60-78

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will differ from those listed for dry materials. Sulphates, nitrates, and chlorides are highly soluble in water and can be used for soil as well as foliar applications. Zinc oxysulfates are obtained by treating ZnO with H2 S04 , Their solubility varies depending on the amount of H2 S04 added. Zinc forms complexes with NH 3 in ammoniated ZnS0 4 solutions. When applied to the soil, the complexes break down, forming Zn2 + ions. As discussed in Section VII, a liquid formulation of Zn(N0 3 h with CO(NH 2h and NH4 N03 has been found to be particularly effective in supplying Zn with foliar sprays and is available as a commercial product called NZN®. Most of the synthetic and natural chelates and natural organic complexes of Zn are marketed in a liquid form at various dilutions and for that reason their Zn concentrations are not provided. Synthetic chelates such as EDTA, HEDTA, and NTA are used for manufacturing single Zn fertilizers or for mixing with other fertilizer materials. Zinc fertilizers are also produced from natural chelates of Zn such as amino acid, citric acid, and glucoheptanates. Some natural organic compounds form complexes with Zn, but the bonds between the metal and these compounds and the stability constants for Zn in these complexes are usually not well defined. Examples of fertilizers in this group consist of Zn complexes with humates, lignosulfonates, phenolic acids, and seaweed extracts. Zn fertilizers can be divided into two large groups: (1) those containing high concentrations of Zn with or without one or more other microelements and (2) those containing macroelements such as N, N-P, or N-P-K with relatively low concentrations of Zn and/or other microelements. In the latter group, microelements may be added by: (1) incorporating them during manufacturing, (2) bulk blending of single granular fertilizers, (3) coating granular N-P-K fertilizers with microelements, or (4) mixing fluid N, N-P, or N-P-K fertilizers with different soluble microelement sources. The advantages and disadvantages of different methods of formulating complex fertilizers and their use under specific conditions are discussed by Mortvedt (1991). IX. CONCLUSIONS

Much information was generated during the last several decades on soil chemistry, sorption, soil activity, and inorganic phase equilibria of Zn. These advances enhanced our understanding of the factors involved in controlling availability of soil Zn to plants. The same can be said about the equilibria reactions of synthetic Zn chelates in soils. This new

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knowledge allows us to predict relative effectiveness of various Zn chelates as sources of the metal for plants under different soil conditions.. Despite all these advances, however, the mechanisms controlling the amount of free Zn 2+ present in the soil solution is not yet completely understood. It likely encompasses the dynamic equilibria of adsorption and desorption, dissolution and precipitation, biological processes, and the flow of soluble Zn in soil due to gravitational forces, evapotranspiration, and diffusion. Relatively little is known about the role of soil organic matter and soil microorganisms in Zn transformations in soils and how these processes affect Zn availability to plants. Definitely, more research needs to be done to gain a better understanding of Zn reactions in soils. Zinc contamination of traditional nutrient solution systems has long hampered research on the role of Zn in plants' physiological processes. The level of free Zn 2+ that is required for normal plant growth is so small that it cannot be measured directly with standard methods. Although purification of chemicals, e.g., with dithizone, helps to remove contaminants, such a system remains extremely sensitive to even the tiniest bit of post-purification Zn contamination. Recent developments in the use of chelator-buffered nutrient solutions have greatly diminished purification and contamination problems. The use of this technique carries a promise of further advances in our understanding of the role of Zn in physiological processes of plants. Still, much needs to be learned about the conditions under which horticultural plants are most likely to respond to corrective Zn treatments. For exa.mple, the critical period for Zn supply in relation to optimal fruit set, fruit growth, and high fruit external and internal quality is unclear. The results of recent studies on grapefruit trees suggest that high Zn supply prior to anthesis is very important for optimal fruit set (Swietlik 1996). However, more research is needed on this and other plant species and under different climatic and edaphic conditions to more accurately predict plant responses to corrective Zn treatments. The results of many trials suggest that zinc deficiency must be quite severe to make Zn applications economically justifiable. This may, at least partially, be explained by considerable variability encountered in field studies. It is common to observe localized occurrences of Zn deficiency within a given field. In well-controlled nutrient solution studies, however; growth responses were realized on plants showing no Zndeficiency symptoms but otherwise judged to be low in Zn (Swietlik and Zhang 1994). Thus, future research must strive to utilize improved methodologies to quantify the impact of various levels of deficiency on plant growth, crop yield, and crop quality.

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Some experimental results suggest that judging the Zn status of plants based only on total tissue Zn concentrations may correlate poorly with Zn-deficiency symptoms. The results of studies by Embleton et al. (1965) on lemons and Swietlik (1996) on grapefruit may serve as examples of such an anomaly. This suggests that the concentration of a yet undetermined "physiologically active" form of Zn should be considered, rather than total Zn level, when assessing the nutritional status of citrus trees and possibly other plant species as well. Sorption and precipitation of Zn is the primary reason for poor movement of the metal in soils and thus its limited availability to plants. This poor movement of Zn has particularly pronounced impact on Zn nutrition of woody plants, because a large portion of their root system occupies deep soil layers. That is why foliar sprays with Zn are generally more effective than soil treatments for correcting Zn deficiencies in fruit trees. Consequently, they are widely recommended by many researchers. However, we may need to re-examine this approach because of poor mobility of foliar-absorbed Zn in fruit trees. Even though Zn foliar sprays are effective in controlling Zn deficiency in leaves, they are not effective in alleviating Zn deficiency in roots or in subsequent flushes of growth (Swietlik and Zhang 1994). This is particularly important in evergreen trees, e.g., citrus, which typically produce several flushes of growth per year. It is therefore possible that the lack of tree responses to foliar Zn sprays in a number of experiments on fruit trees may in fact reflect the inability of foliar sprays to alleviate Zn deficiency in a whole tree. Thus, future research should focus on developing more effective techniques of supplying Zn through the soil. If successful, this would not only alleviate Zn deficiency in all plant parts but also, contrary to foliar sprays, would produce longer-lasting effects. Recent results on apple, avocado, and citrus trees indicate that soil treatments with Zn may successfully alleviate Zn stress (Zekri and Koo 1992; Stiles et al. 1995; Crowley et al. 1996; Swietlik 1996).

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Anderson, C. A.1984. Micronutrient uptake by citrus from soil-applied zinc compounds. Proc. Soil Crop Sci. Soc. Fla. 43:36-39. Anderson, C. A., and C. D. Leonard. 1982. Comparison of several zinc foliar treatments for correction of Zn-deficiency in citrus trees. Proc. Soil Crop Sci. Soc. Fla. 41:169-171. Banin, A., J. Navrot, and Y. Ron. 1980. Tree implanted zinc-bentonite paste as a source of slow-release zinc for 'Dalmas' pecan. HortScience 15:182-184. Bar-Akiva, A., and R. Lavon. 1969. Carbonic anhydrase activity as an indicator of zinc deficiency in citrus leaves. J. Hort. Sci. 44:359-362. Barber, D. A., and R. B. Lee. 1974. The effect of micro-organisms on the absorption of manganese by plants. New Phytol. 73:97-106. Bar-Tal, A., B. Bar-Yosef, and Y. Chen. 1988. Effects of fulvic acid and pH on zinc sorption on montmorillonite. Soil Sci. 146:367-373. Bell, P. F., R. F. Chaney, and J. S. Angle. 1991a. Determination of the free copper2+ activity required by maize using chelator-buffered nutrient solutions. Soil Sci. Soc. Am. J. 55:1366-1374. Bell, P. F., R. L. Chaney, and J. S. Angle. 1991b. Free metal activity and total metal concentrations as indices of micronutrient availability to barley (Hordeum vulgare (L.) 'Klages']. Plant & Soil 130:51-62. Bergmann, W. 1977. Atlas objawow niedoboru lub nadmiaru skladnikow pokarmowych u roslin uprawnych. (Symptoms of mineral nutrients deficiencies and excesses in cultivated plants). Panstwowe Wydawnistwo Rolnicze i Lesne, Warszawa (Warsaw). Beyers, E., and J. H. Terblanche. 1971. Identification and control of trace element deficiencies. I. Zinc deficiency. Decid. Fruit Grower 21:132-137. Bierman, P. M., and C. J. Rosen. 1994. Sewage sludge incinerator ash effects on soil chemical properties and growth of lettuce and corn. Commun. Soil Sci. Plant Anal. 25:2409-2437. Bingham, F. T., and M. J. Garber. 1960. Solubility and availability of micronutrients in relation to phosphorus fertilization. Soil Sci. Soc. Am. Proc. 24(3}:209-213. Bingham, F. T., and J. P. Martin. 1956. Effects of soil phosphorus on growth and minor element nutrition of citrus. Soil Sci. Soc. Am. Proc. 20:382-385. Blaylock, A. D. 1995. Navy bean yield and maturity response to nitrogen and zinc. J. Plant Nutr. 18(1):163-178. Bowen, J. E. 1969. Absorption of copper, zinc, and manganese by sugar cane tissue. Plant Physiol. 44:255-261. Brady, N. C. 1984. The nature and properties of soils. Macmillan, New York. Brennan, R. F. 1996. Availability of previous and current applications of zinc fertilizer using single superphosphate for the grain production of wheat on soils of South Western Australia. J. Plant Nutr. 19:1099-1115. Brookes, A., J. C. Collins, and D. A. Thurman. 1981. The mechanism of zinc tolerance in grasses. J. Plant Nutr. 3:695-705. Brooks, O. L. 1964. Yield and growth response of Stuart pecan trees to zinc sulfate and nitrogen. Univ. of Georgia Agr. Expt. Sta. Cir. N.S. 40. Bugbee, G. J., and C. R. Frink. 1995. Phosphorus and zinc fertilization of com grown in a Connecticut soil. Commun. Soil Sci. Plant Anal. 26:269-276. Bukovac, M. J., and S. H. Wittwer. 1957. Absorption and mobility of foliar applied nutrients. Plant Physiol. 32:428-435. Cakmak, I., and H. Marschner. 1986. Mechanism of phosphorous-induced zinc deficiency in cotton. I. Zinc deficiency-enhanced uptake rate of phosphorous. Physiol. Plant. 68:483-490.

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Tsadilas, C. D., T. Matsi, N. Barbayiannis, and D. Dimoyiannis. 1995. Influence of sewage sludge application on soil properties and on the distribution and availability of heavy metal fractions. Commun. Soil Sci. Plant Anal. 26:2603-2619. Tsui, C. 1948. The role of zinc in auxin synthesis in the tomato plant. Am. J. Bot. 35:172-179. Turekian, K. K., and K. H. Wedepohl. 1961. Distribution of the elements in some major units ofthe earth's crust. Geol. Soc. Am. Bul. 72:175-192. Udo, E. J., H. L. Bohn, and T. C. Tucker. 1970. Zinc absorption by calcareous soils. Soil Sci. Soc. Am. Proc. 34:405-407. Uriu, K., and J. Pearson. 1986. Zinc deficiency in pistachio-diagnosis and correction. California Pistachio Industry Annual Report 1986/87, p. 71-72. Vanden Heuvel, R. M., J. E. Sawyer. M. A. Schmitt, R. G. Hoeft, and G. S. Brinkman. 1989. Corn responses to zinc on Illinois soils. J. Fertilizer Issues 6:68-76. Van Steveninck, R. F. M., M. E. Van Steveninck, D. R. Fernando, W. J. Horst, and H. Marschner. 1987. Deposition of zinc phytate in globular bodies in roots of Deschampsia caespitosa ecotypes; a detoxification mechanism? J. Plant Physio!. 131:247-257. Viets, F. G., Jr. 1966. Zinc deficiency in the soil-plant system. p. 90-127. In: A. Parasad (ed.), Zinc metabolism. Charles C. Thomas Pub!., Springfield, IL. Viets, F. G., Jr., L. C. Boawn, C. L. Crawford, and C. E. Nelson. 1953. Zinc deficiency in corn in central Washington. Agron. J. 45:559-565. Wakatsuki, T. H., and K. Kawaguchi. 1975. Specific and non-specific adsorption of inorganic ions. II. Specific adsorption of cations on kaolinite and kaolinite soil clays. Soil Sci. Plant Nutr. (Tokyo) 21:351-360. Walker, C. D., and R. M. Welch. 1987. Low molecular weight complexes of zinc and other trace metals in lettuce leaf. J. Agr. Food Chern. 35:721-727. Wallihan, E. F., T. W. Embleton, and W. Printy. 1958. Zinc deficiency in the avocado. Calif. Agr. 12(6):4-5. Wear,J. 1.1956. Effectofsoil pH and calcium on uptakeofzinc by plants. SoilSei. 81:311-315. Webb, M. J., and J. F. Loneragan. 1988. Effect of zinc deficiency on growth, phosphorous concentration, and phosphorous toxicity of wheat plants. Soil Sci. Soc. Am. J. 52: 1676-1680. Welch, R. M. 1995. Micronutrient nutrition of plants. Crit. Rev. Plant Sci. 14:49-82. Welch, R. M., W. H. Alloway, W. A. House, and J. Kubota. 1991. Geographic distribution oftrace element problems. p. 31-57. In: J. J. Mortvedt, P. M. Giordano, and W. L. Lindsay (eds.), Micronutrients in agriculture. 2nd ed. Soil Sci. Soc. Am., Madison, WI. Welch, R. M., and W. A. Norvell. 1993. Growth and nutrient uptake by barley (Hordeum vulgare L. cv. Herta): studies using an N-(2-Hydroxyethyl) ethylenedinitrilotriacetic acid-buffered nutrient solution technique. II. Role of zinc in the uptake and root leakage of mineral nutrients. Plant Physiol. 101:627-631. Welch, R. M., M. J. Webb, and J. F. Loneragan. 1982. Zinc in membrane function and its role in phosphorous toxicity. p. 710-715. In: A. Scaife (ed.), Proc. 9th Int. Plant Nutri. Colloq., Warwick, England. Commonwealth Agr. Bureau. England. White. M. C., F. D. Baker, R. L. Chaney. and A. M. Decker. 1981b. Metal complexation in xylem fluid. II. Theoretical equilibrium model and computer program. Plant Physio!. 67:301-310. White, M. C., A. M. Decker. and R. L. Chaney. 1979. Differential cultivar tolerance in soybean to phytotoxic levels of soil Zn. I. Range of cultivar response. Agron. J. 71:121-126. White, M. C.• A. M. Decker, and R. L. Chaney. 1981a. Metal complexation in xylem fluid. I. Chemical composition of tomato and soybean stem exudate. Plant Physiol. 67:292-300.

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Wutscher, H. K., and T. A. Obreza. 1987. The effect of withholding Fe, Zn, and Mn sprays on leaf nutrient levels, growth rate and yield of young 'Pineapple' orange trees. Proc. Fla. State Hort. Soc. 100:71-74. Yang, X., V. Romheld, and H. Marschner. 1994. Uptake of iron, zinc, manganese, and copper by seedlings of hybrid and traditional rice cultivars. J. Plant Nutr. 17:319-331. Yogaratnam, N., and D. W. P. Greenham. 1982. The applications of foliar sprays containing nitrogen, magnesium, zinc, and boron to apple trees. 1. Effect on fruit set and cropping. J. Hort. Sci. 57:151-158. Yogaratnam, N., and D. S. Johnson. 1982. The application of foliar sprays containing nitrogen, magnesium, zinc and boron to apple trees. II. Effects on the mineral composition and quality of fruit. J. Hort. Sci. 57:159-164. Zekri, M., and R. C. J. Koo. 1992. Application of micronutrients to citrus trees through microirrigation systems. J. Plant Nutr. 15:2517-2529. Zhang, G., and Z. Wu. 1989. Relationship between light intensity and requirement for zinc in tomato plants. J. Plant Nutr. 12:633-646.

Plate 3 Expressions of Zn deficiency symptoms on: A. peach, top-deficient leaves, bottom-normal leaves; B. grapefruit, 'leaf mottling'; C. pecan, left-'little leaf' or 'rosette', right-normal leaf; Photographs provided by G. H. Neilsen (A); D. Swietlik (B); J. B. Storey (C)

Plate 3 (continued) Expressions of Zn deficiency symptoms on: D. apple, 'little leaf' or 'rosette'; E. pistachio, 'little leaf' or 'rosette'; F. sour orange, left-inhibited root growth on deficient plant, middleplant supplied with Zn via roots, right-foliar sprayed with Zn, no Zn applied via roots. Note that foliar sprays only partially restored the normal root growth. Photographs provided by E. Fallahi and W. M. Colt (D); 1. Ferguson (E); and D. Swietlik (F).

4 Origin and Dissemination of Plums Miklos Faust Fruit Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, Beltsville, Maryland 20705 Dezso Surdnyi Fruit Research Station, Cegled, Hungary I. Introduction II. Classification A. Botanical 1. Basic Species 2. Garden Plum and Damson 3. Asian Species 4. American Species B. Horticultural III. History A. Archeobotany 1. Europe 2. America B. Antiquity C. Japan D. Europe E. America 1. Native Species and Cultivars 2. Imported Cultivars IV. Conclusions Literature Cited

I. INTRODUCTION Plums are a diverse group of plants. The fruits may be small or large, round or elongated, green, black, purple, blue, red, or yellow; the trees may be shrub-like with slender branches, or have a strongly constructed Horticultural Reviews, Volume 23, Edited by Jules Janick ISBN 0-471-25445-2 © 1999 John Wiley & Sons, Inc. 179

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M. FAUST AND D. SURANYI

framework; and the foliage may be delicate or the leaves may be coarse and heavy and occasionally red, retaining an attractive appearance all summer. Hedrick (1911) commented that the range of fruit size and shape, flavor, aroma, texture, and color in plums is greater than in any of our orchard species. The silhouettes of fruit of a few typical plums are illustrated in Fig. 4.1. The diversity of plums is also expressed in their names. There are plums, prunes, bullaces, damsons, date plums, green-gages, mirabelles, cherry plums, egg plums, and sloes. The origin of plums also varies. Some species originated in Asia, others in Europe and America. Plums are eaten fresh or dried. Accordingly, a distinction is made between plums and prunes. Stubenrauch and Wickson (1927) defined prune as a plum that dries whole without fermenting. The fruits used for making prunes are also used fresh and are then called plums. Plums are used dried, or are made into jelly, jam, juice, liquor, brandy, cognac, and cordials. They are also used in baking and for confection. Plums may have been the first species among all the fruits to attract human interest. Three of the most important species of plums. P. domestica, P. salicina and P. simonii are not known in the wild and presumably were selected and cultivated very early by humans. It is more remarkable that the earliest cultivation of P. domestica began somewhere between Eastern Europe and the Caucasian mountains, whereas P. salicina and P. simonii were brought into cultivation in Asia. In China, since ancient times, millions have extolled plum blossoms. According to the Chinese, "plum" blossoms defiantly brave snow and frost and spread their fragrance when they bloom in cold winter and greet the new spring before all plants begin to wake up. Lu You (11251210) wrote more than 600 poems in praise of "plum" blossoms (Wang 1994). Lin Hejing (420-589) devoted all his efforts to creating paintings of "plum" blossoms. From the Tang Dynasty (618-907) to the Qing Dynasty (1644-1911), there were about 250 famous artists who specialized in "plum" paintings (Wang 1994). Even though the Chinese say "plum," the paintings depict blossoms of Prunus mume, a Prunus species that is closely related to apricots. The early bloom time also indicates that the flowers painted belong to the species P. mume. In Japan, mume is often called "Japanese apricot" or "Japanese plum" (Yoshida 1994) and in China "Mei plum" (Wang and Ma 1986). Close examination of the flowers indicates that the flowers of P. salicina, the Chinese plum, are sufficiently different from those found in the paintings (Fig. 4.2). Although it is beautiful, the Chinese plum art does not illustrate plums per se, but a species closely related to apricots.

4. ORIGIN AND DISSEMINATION OF PLUMS

181

3

30mm

10

11

12

Fig. 4.1. Silhouettes of various types of plums. 1. Sloe (P. spinosa), 2. Bullace (P. insiti· tia), 3. Damson (P. insititia), 4. 'Italian prune' (P. domestica), 5. P. cerasifera, 6. 'Arkansas' (P. munsoniana), 7. 'Santa Rosa' (hybrid), 8. 'Burbank' (hybrid), 9. 'Friar' (hybrid), 10. 'De Soto' (P. americana), 11. 'Green Gage' or 'Reine-Claude' (P. domestica), 12. 'Forest Garden' (P. hortulana mineri), 13. Mirabelle (P. domestica), 14. 'Cheney' (P. nigra), 15. 'Pottawattamie' (P. americana).

M. FAUST AND D. SURANYI

182

B

A

~&~)

~

, (?

c

D

Fig. 4.2. Comparison of flowers. A and B. Colored Chinese woodcuts from 1782. Flowering "plum" trees by an unknown artist in Hsu Hai (V. dynasty) style (Hejzlar 1972). C. Flowers of Prunus mume from the Chinese botanical dictionary. D. Flowers of P. salicina drawn after specimens in the herbarium of the Royal Gardens, Kew, as P. triflora, Roxbg. (Bailey 1927). Note that the flowers of the "plum" painting resemble those of P. mume, (a species closely related to apricots) and not of P. salicin a (the plum of China).

4. ORIGIN AND DISSEMINATION OF PLUMS

183

In Transylvania, Romania, plum seeds were used as the motif in wood carvings on gift items. In these motifs, occasionally two or three seeds were delineated, five oval shapes of seeds were arranged as a rose, or four oval-shaped seeds were carved in a diagonal pattern (Fig. 4.3) (Kos 1980). World production of plums has remained constant during the last decade, with the exception of Asia, where production has doubled since the early 1980s. World production is 7 million tonnes (Table 4.1), a third of which is produced in Europe and an equal amount in Asia. The largest concentration of dried prune production is in California, which dries nearly 90 percent of its prune output. Plum production is divided into two categories. Production of hexaploid prunes (6x), including P. domestica and P. insititia cultivars, in 1987-89 was, in thousand tonnes (numbers in parentheses indicate production of dried prunes): United States 640 (610); former Soviet Union 977; Yugoslavia 780 (37); Germany 431; Hungary 195; France 90 (90); Chile 30 (30); Argentina 30 (30). Production

Fig. 4.3. Various motifs used in carving in Transylvania that were derived from the plum seed.

M. FAUST AND D. SURANYI

184

Table 4.1. Worldwide production of plums in thousand tonnes; data from FAO (1990, 1992, 1994). Continent/years

1979-1981

1988

1989

1990

1991

1992

1994

Africa North/Central America Asia Europe Oceania Former USSR

78

117

154

154

131

144

149

719 863 2862 26 873

755 1341 3208 22 1000

1011 1427 2818 24 1166

843 1582 2196 24 1000

987 1537 2008 23 800

980 1619 2433 24 800

1035 2634 2688 17 738

World

5518

6584

6750

5960

5655

6181

7261

of diploid (2x) plums, including Asian and native American cultivars and their hybrids, was in 1987-89, in thousand tonnes: China 789; United States 226; Spain 77; Italy 58; Argentina 48; Mexico 84; Japan 68; Korea 36; Pakistan 48; Egypt 38; South Africa 17; and Australia 18. (Okie and Weinberger 1996). Outstanding reviews of plum cultivars and history include Hedrick (1911), Wight (1915), Cullinan (1937), Weinberger (1975), Roach (1985), Ramming and Cociu (1990), Korber-Grohne (1996), and Okie and Weinberger (1996). Plum rootstocks have been reviewed by Okie (1987).

II. CLASSIFICATION A. Botanical The early history of genus Prunus is inherently connected with that of plums. Pre-Linnean botanists John Ray (1627-1705), Joseph Pitton de Tournefort (1656-1708), Johann Jacob Dillenius (1687-1747), and Herman Boerhaave (1668-1738) considered Prunus to include only plums. Carolus Linneaus (1707-1778) adapted the name Prunus, used by his predecessors for the plum alone, for a genus in which he placed plums, cherries, apricots, and seven other species. Michael Adanson (17271806) and Antoine Laurent Jussieu (1748-1836) of France returned to the pre-Linnean classification, but Joseph Gaertner (1732-1791) of Germany followed the grouping of Linneaus. The controversy continued. Augustin Pyramus De Candolle (1778-1841), Johan Jacob Roemer (1763-1819) of Switzerland, and Joseph Decaisne (1809-1882) of France held that the plum alone belongs to Prunus. George Bentham (1800-1884) and J. D.

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Hooker (1785-1865), authors of Handbook ofthe British Flora,' Asa Gray (1810-1888), a Harvard botanist, and his coworkers; and Adolf Engler (1844-1930) and Karl Anton Prand (1849-1893), authors of Natilrlichen Pflanzenfamilien, published in Berlin, extended the genus Prunus to all of the stone fruits. Even after the number of plants belonging to the genus Prunus appeared to be settled, Nathaniel Lord Britton and Addiston Brown, Director and President of the New York Botanical Garden, respectively, in their Illustrated Flora of the Northern United States, in 1898, listed only plums and cherries under the genus Prunus. The diversity of views of what species belong to Prunus is an indication that differences separating the species are not very distinct. The difficulty is not eased by restricting discussions to plums alone. Most plum species are diploid 2n =16; the sloe is tetraploid 2n =32, but in the Caucasian mountains in Georgia, P. spinosa has been found with 2n = 16, 32, 48, 64, or 96 and P. cerasifera with 2n = 32 or 48 (Zohary 1992). Prunus hybrids have intermediate chromosome number, 2n =40, and P. domestica and P. insititia are hexaploid 2n =48. Weinberger (1975) lists 18 species that are horticulturally important. Only 12 species are important for the purposes of this historical discussion. For the complete list of cultivated plum species, readers should consult Bailey (1927) or Rehder (1954). 1. Basic Species. The two basic species are Prunus spinosa and P. cocomilia. P. spinosa L., known as sloe or blackt~orn, is a low, spreading, thorny bush. Leaves are small, oblong, elliptic-ovate, and very numerous. Flowers are white, small, borne singly or in pairs and often occur on thorns. Fruit is small, not larger than a large pea, blue, and usually persists until the winter. This species is widespread in Europe, North Africa, and Northern Turkey. Botanically recognized forms are ssp. fruticans (macrocarpa), a larger bush with less thorn and larger fruit and ssp. dasyphylla (tomentosa) , a pubescent bush. Garden forms are: f. plena with double flowers and f. purpurea with pink flowers and purple foliage. Bajashvili (1990) encountered arborescent forms of sloe in the Caucasian mountains with larger leaves and larger, less astringent fruit. Some of these forms appeared to be polyploid, 2n =48, 64. Some forms were closer to myrobalan (see P. cerasifera below), when characterized by various combinations of fruit color, leaf size, and bud size. Pollen of these forms appeared to be closer to the pollen of P. domestica. Russian botanists (see Zerov 1954) described P. spinosa ssp. macrocarpa as P. stepposa, elevating it to the species level. They also described another variant as P. moldavica Kotov. P. spinosa is clearly a polymorphic species.

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P. cocomilia Ten. (Syn. P. pseudarmeniaca Held. and Sart.) is known as mock apricot. It is a bush or small tree with thorny branches. Leaves are oval or broadly ovate, more or less pubescent. Flowers are white or greenish white, in pairs, appearing with the leaves. Fruit is small yellow, oblong-ovoid, rather good for eating. The species has been described as being from Italy, but it is native in the southern part of the Balkan peninsula on the sides of high mountains and near the Mediterranean coast of Turkey (Brovitz 1972). Var. puberula Schneider has been described, but in the opinion of Borvitz (1972) it may be a hybrid of P. spinosa x P. cocomilia. P. cerasifera Ehrh. (Syn. P. domestica var. myrobalan L.; P. myrobalana Loisel.) is known as cherry plum. The French cherry plum, commonly called myrobalan, is widely used as plum rootstock. Cherry plum is a slender, often thorny, shrub-like small tree. Flowers are small, white, comparable with most types of P. domestica. Fruit is small, less than 2.5 em in diameter, globular, cherry like, yellow or red with soft, sweet, juicy flesh, depressed at stem end. The species is native from the Balkans to the Caucasian mountains and southwest Asia. Fresh and dried fruit of P. cerasifera have been used for centuries in the Tien Shan and Pamir mountains and seedlings are widely used as rootstocks for domestica plums. The fruit color of cherry plum is highly variable. D. Suninyi (unpublished) found yellow, red, purple, and black colored fruits of myrobalan when he explored plum species in Asia Minor. Some botanists differentiate between the cultivated and wild forms of P. cerasifera. They contend that the cultivated form is P. cerasifera macrocarpa Erem. & Garkov., whereas the wild form is P. divaricata Ledeb. P. divaricata was collected in the Trans-Caucasian region by Ledebour, who named it in 1820, and is native from Macedonia to northern Persia. The tree is branching from the base with branches quite prostrate with yellow fruit. Bailey (1927) considered the botanical position of P. divaricata Ledeb. as var. divaricata Bailey [Syn. P. divaricata Ledeb., ssp. divaricata (Ledeb.) Schneid.], but decided that it was not different from the main species or at best is only a form of it. Therefore, it does not deserve species status. There are other recognized forms of P. cerasifera based mostly on eco-geographical adaptation: ssp. ursina (P. ursina Kotschy) grows in southeast Turkey and Syria; ssp. CQspicQ (P. caspica Kov. & Ekm.) occurs in the Caspian coast ofCaucasia; var. iraniCQ (Koval.) Erem & Garkov, the Iranian cherry plum; and var. nairicQ Koval., the form frequently found in Armenia. These subspecies were considered separate species, as indicated by the name in parentheses. However, after collecting a large number of specimens, Kovalev (1939) came to the conclusion that the Syrian, Caspian, and Armenian forms

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are not sufficiently different from the main species to consider them distinct species and should be considered only as a botanical variety of P. cerasifera. As mentioned above, there are tetraploid and hexaploid races of P. cerasifera but they are morphologically very similar to the diploid species (Watkins 1981; Beridze and Kvatchadze 1981). Eryomin (1991) remarked that the Iranian and Armenian forms of cherry plum appear to be cultivated types with larger fruit than the other wild types. Cherry plums have many hybrids, among which those that are well known are the Marianna plum, (P. cerasifera x P. munsoniana 'Wild Goose') and 'Methley' (P. cerasifera x P. saUcina). Kovalev (1941) also described P. ferganica, a hybrid of P. divaricata and Amygdalus ulmifolia. There are probably many other hybrids because P. cerasifera can be crossed with several Prunus species. In their deliberations, various authors throughout their botanical sections often noticed and discussed "larger fruited types" of a given species. The larger fruited forms are often designated macrocarpa. In our experience, when wild fruited species are crossed, tree and branch structure and leaf and bud morphology of the seed parent are transmitted to the progeny but fruit size increases. Often we could not distinguish between seed plant and its F1 seedlings until the seedlings fruited. Although our experience is limited regarding plums, we wonder whether the macrocarpa forms are true forms of the species or hybrids with a larger fruited pollen donor. There are ornamental forms of P. cerasifera. Best known among them are the var. planteriensis Hort. with full double red or white flowers; var. pendula Hort., a weeping form; and var. aculifolia or angustifolia Hort., a form with narrow willow-like leaves. Var. pissardii (P. pissardii Carr, P. cerasifera var. atropurpurea Dipp), a form with purple leaves and dark red fruit, was introduced to France by Pissard, the French head gardener to the Shah of Persia in 1880. Today, there are at least 50 cultivars of red leaf plums, excluding known synonyms (Jacobson 1992). Yoshida (1987) increased the importance of this species by suggesting that P. cerasifera is the progenitor of all plum species, because of its wide native range and cross compatibility with many other plum species. 2. Garden Plum and Damson. These include P. domestica and P. insi-

titia and all their variations. P. domeslica L. ( Syn. P. communis Huds., P. mconomica Borkh., P. domestica ssp. oeconomica Schneid., P. sativa Rouy et Cam., P. italica Borkh.) is known as the common garden plum. It is a strong-growing small tree. Leaves are large, thick, dark green. Flowers are white, large, usually in clusters. Fruit varies in form but is firm in texture and usually not depressed at stem end. This is the most

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important plum species in cultivation today. Linneaus, in 1753, divided P. domestica into 14 sub-species using the list of Bauhin published in his Pinax Theatri Botanici in 1623. Both of them included species other than domestica in their description of garden plum. P. domestica is unknown in an originally wild state and the typical form of this species is the prune. Crane and Lawrence (1952) suggested that P. domestica, a hexaploid, originated as a hybrid between P. cerasifera Ehrh., a diploid, and P. spinosa, a tetraploid, via either chromosome doubling of the hybrid tetraploid or a product of unreduced gametes from both parents. Crane (1949) indicated that native areas of P. spinosa and P. cerasifera overlap (Fig. 4.4) and a hybrid could occur. Crane and Lawrence (1952) reasoned that P. cerasifera has a yellow base color and red anthocyanin in its skin, while P. spinosa's has green ground color and blue skin color. The supposed hybrid, P. domestica, has both green and yellow under-color and red and blue skin colors that apparently combine the characteristics of both parents. Rybin (1936) found spontaneous, highly sterile interspecific hybrids between P. cerasifera and P. spinosa in the Caucasus, in the Maikop area. Rybin crossed the two species and

Fig. 4.4. Overlapping areas of P. cerasifera and P. spinosa after Terpo (1974) 1 = P. spinosa; 2 = P. cerasifera. The eastern extent of the range of P. cerasifera is undetermined.

hence the loop is not closed.

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obtained seedlings that he regarded as resynthesized P. domestica. Endlich and Murawski (1962) also crossed P. cerasifera and P. spinosa. The F1 generation was nearly sterile, but the F z generation had about 50 percent of its seedlings with 2n = 48 chromosomes. The hybrid nature of P. domestica was further emphasized by Zhukowsky (1965). Several authors (Johansson and Olden 1962; Webb 1968; Weinberger 1975; Watkins 1976, 1981; and Zeven and De Wet 1982) accepted the explanation that the garden plum is a hybrid of P. cerasifera and P. spinosa. Subsequent cytological studies indicated that the origin of P. domestica is more complex since P. spinosa itself may be a tetraploid containing two different genoms (Salesses 1973; Reynders and Salesses 1990). Eryomin (1991) suggested that P. domestica is a hybrid of P. spinosa and P. cerasifera macrocarpa, the cultivated form of myrobalan, and placed its origin in the area where cultivated forms of cherry plum existed, namely Iran, Trans-Caucasian countries, and Asia. Zohary (1992) argued that the evidence supporting an aHo-polyploid origin of P. domestica and the participation of 4x, P. spinosa in the formation of this species is far from being satisfactory. He proposed that the available cytogenetic evidence seems to indicate that 2x, 4x, or 6x, P. cerasifera was the sole wild stock from which the cultivated 6x, garden plum could evolve. Reynders and Salesses (1991) tried to use Restriction Fragment Length Polymorphism (RLFP) to study the origin of garden plum, but they were unable to obtain restriction patterns sufficiently satisfactory to establish a definitive map. Brovitz (1972) described native or naturalized stands of P. domestica on hillsides and slopes up to 1900 m elevation in Turkey. Lin and Shi (1990) reported stands of P. domestica in wild forests along the IIi River in Northwestern Xinjiang Province of China with neither of the presumed parental species nearby. Since the Ili valley is the major crossing road of the silk route through the Tien Shan mountain range, the possibility exists that seeds of P. domestica were carried there rather than the species being native there. P. domestica is considered a relatively young species. None of the Neolithic seed remains are helpful in establishing the origin of P. domestiea. Werneck (1959), using archeological evidence, concluded that P. domestiea is indigenous to middle Europe. Gavrilovic and Paunovic (1962) pointed to a population of P. domestica widespread in HungarySerbia-Bosnia and the neighboring countries of Romania and Bulgaria, under the names of 'Pozegaca' (Serbia), 'Besztercei' (Hungary), 'Kiistendili' (Bulgaria), often referred to as the Pozegaca/Besztercei group, and placed the origin of this species in the Balkans. Terpo (1974) considered the overlapping areas of P. cerasifera and P. spinosa where a

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M. FADST AND D. SURA.NYI

hybrid could occur and placed the origin considerably further east into northern Turkey or the southern Caucasian area (Fig. 4.4). Eryomin (1991) also considered Trans-Caucasia or Iran as the place of origin for P. domestica. Because of the variable fruit, a number of subgroups have been proposed as subspecies of P. domestica. Such subgroups are the prunes, the Reine-Claude or green-gages, and the yellow egg plums. Bailey (1927) thought that P. insititia was also a variety· of this species. However, there are reasons to believe that P. insititia is distinctly separate and it is regarded here as a separate species. Karpati (1967) studied the flower structure of plums. He especially considered petal shape and color. Based on this study, he rearranged the plum types belonging to the two species, P. insititia and P. domestica, as follows: (1) P. insititia Jusl. includes P. italica (Borkh.) Karp. (damsons); convar. pomarium Boutigny; convar. c1audiana Poiret (Reine-Claude); convar. ovoidea Martens (egg plums); and convar. mamillaris Schub!. et Mart. (date plums). (2) P. domestica 1. includes P. syriaca (Borkh.) Karp. (Mirabelle group); convar. prisca (Werneck) Karp.; convar. cerea (L.) Karp. This arrangement is the opposite of the arrangement of plum groups that other botanists previously believed in. It puts the Reine-Claude group, the egg plums, and the date plums into P. insititia and the Mirabelle group into P. domestica. He has not only rearranged these well-defined plum groups but has also disputed the parental species that produced the hybrid nature of some of them. For example, he claimed that P. syriaca (Borkh.) Karp. is a P. cerasifera x P. domestica hybrid instead of P. syriaca Borkh., a P. domestica x insititia Koch hybrid. His study and reclassification reaffirms our opinion expressed before that plums are a group of fruit with widely hybridized plants and it is difficult to find the original species upon which to base the botanical classification. Petal shape and flower color are variable characteristics and in plants such as petunia, cultivated and perhaps hybridized since ancient times by the South American Indians, a great variability exists that is independent from other characters. Therefore, it is questionable how much advancement Karpati's classification contributes toward sorting out the basic plum species. Prunes. In western America, the word prune is used for plums that can be dried whole. In Europe the term is used to designate a distinct pomological group of plums in which fruit is usually reddish or blue, elongated, high in sugar content, and firm. The fruit is excellent for drying. Prunes are part of the Pozegaca/Besztercei group. There are many cultivars with prune in their name. William Prince (1828) speaks of 'Italian

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Prune', a cultivar originated in the early 1800s in Lombardy, Italy, and William Robert Prince (1831), in his Pomological Manual, describes the 'German Prune' and'Agen', the 'French Prune'. The origin of the 'German Prune' is uncertain. First described in 1771, 'German Prune' is generally believed to have been carried to Europe from Asia during the Crusades. Smith (1978) lists Musquee de Beszterce, and Quetsche Musquee de Hongrie as synonyms for 'German Prune' and remarks that it belongs to the group that in Germany was called small Zwetschen or Hauszwetschen. She remarks that 'German Prune' reached Germany in the 1600s and the name Quetsche or Zwetsche was not widely used until 1700. The origin of 'Agen' also goes back to the times of the Crusades when the Benedictine monks brought the 'Date Plum' from Turkey or Persia. It was planted in their garden in the vicinity of Bordeaux, France, and afterwards became the 'Agen' (Hedrick 1911). It was recorded in 1796 and first called 'd'Ente'. The 'French Prune' belongs to a group of red prunes indistinguishable from 'Agen'. Since the discovery of 'Agen' many of its hybrids have been introduced at various locations (Odier 1993). The locality of origin of 'German Prune' and 'Agen' underscores the opinion of Terpo (1974) and Eryomin (1991) that the origin of P. domestica is located east of the Balkan peninsula. The Romans planted large orchards of prunes in Pannonia (northern Balkans and southern HungaryL which may have caused the confusion about the origin of this group and could explain the Hungarian synonyms of the 'German Prune' described above ('Musquee de Beszterce' and 'Quetsche Musquee de Hongrie').

Reine-Claude Gages. These comprise a number of round, very high quality, golden or green plums classified by botanists as either subspecies or distinctly different species. In 1753, Linnaeus called them P. domestica cereola; in 1803, Borkhausen named them P. italica; in 1904, Poiret separated them under the name P. claudiana. The origin of the ReineClaude group, like the origin of the other P. domestica subspecies, is also unknown. Koch (1876) found plums in the Trans-Caucasian regions that were very similar to Reine-Claude, and assumed that Reine-Claude originated in that region. In 1545, a French botanist, Pierre Below (1517-1564), brought Reine-Claude gages to the Chateau de Blois from Italy (Blanchet 1996). Duhamel de Monceau illustrated it in 1768 (Fig. 4.5). Hogg (1884) thought that Reine-Claude was brought from Greece to Italy and was cultivated under the name ofVerdocia. He probably based his opinion on Parkinson's (1629) cultivar ofVerdoch, which is thought to be the present-day 'Green Gage'. Gallesio (1817-31) illustrated 'Ver-

M. FAUST AND D. SURANYI

192

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dacchia' (Fig. 8.13B) as a prune and not as a 'Green Gage' (or 'ReineClaude'). Hedrick (1911) summarized a number of reasons why Verdocia was not Reine-Claude, which leaves the origin of Reine-Claude uncertain. Yellow Egg Plums. This group has large, distinct, handsome fruit. Parkinson (1629) mentioned 'Imperial', which later became 'Red Magnum Bonum', as a cultivar belonging to the yellow egg plums. In 1676, John Rea described 'Yellow Egg', and Knoop of Holland used the name of

4. ORIGIN AND DISSEMINATION OF PLUMS

193

'Prune d'OeufBlanche' in 1771, indicating a French origin. Duhamel, in Traite des Arbes Fruitiers (1768), described Yellow Egg as the 'Dame Aubert'. Kraft in Pomona Austriaca (1792), used the name 'Die Grosse Weisse Glanzende' or 'Die Albertus Damenpflaume'. These references show that the Yellow Egg group had an early origin. Hedrick (1911) classified a few additional groups, the Perdigon plums, the Imperatrice plums and the Lombard plums. None of these groups received the attention of botanists required to separate them as subspecies or botanical varieties; therefore, their botanical importance is questionable today. P. insititia L. [syn. P. insititia subsylvestris Boutigny, the wild form, P. insititia var. Juliana (subsylvestris) Boutigny, P. domestica var. insititia Bailey, P. domestica subsp. insititia Uusl.) Schneid., P. domestica var. nigra A. and G., P. insititia syriaca (Borkh.) Koechne, P. italica (Borkh.) Ashers and Graebn., P. insititia Borkh., P. c1audiana Poir] includes damson, bullace, mirabelle, and 81. Julien. P. insititia cultivars are readily distinguishable from the garden plum. The trees are dwarfer and more compact, leaves are much smaller and more ovate, fruits are smaller, less than 2.5 em in diameter, purple or yellow without intermediate colors and the stones are smaller but much more swollen than those of the garden plum. During the years since the description of P. insititia, several subdivisions have been proposed. However, according to Brovitz (1972), who studied this species extensively in Turkey, it is difficult to distinguish truly wild and cultivated populations. Some of the subdivisions, such as P. pomarium Boutigny, P. insititia glaberrima Wirtg., var. alpina-orientalis Werneck, and var. leopoldiensis Simk., have lesser importance. Others are easily distinguishable and are important. Among the important subdivisions one must consider P. insititia syriaca (Borkh.) Koechne, the mirabelles, which are used for fruit in France, and P. insititia var. Juliana, the St. Julien plums, which are used extensively as rootstocks. Hedrick (1911) discussed the name bullace. According to him, it is difficult to distinguish between damsons and bullaces. The name bullace refers to the round shape of the fruit but it is not certain who used it first. Apparently there are variations of this name, of which bullis, bulloes, and bullum were the most common. Murray (1888) claims that the word began to be used in 1688 by R. Holme, who used the word to describe a spinous or thorny shrub whose fruit may be eaten as bullas. In 1862, W. Coleman defined bullace by writing that "The Bullace is a plum ... a variety of the common sloe, from which it chiefly differs in the superior size of all its parts especially the fruit" (Murray 1888). Today it is difficult to identify any cultivars clearly belonging to this group. There are differences of opinion about the systematic position of the

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M. FAUST AND D. SURANYI

mirabelle plums, P. syriaca Borkh. (Syn. P. insititia var. syriaca; P. domestica x P. insititia Koch). The tree is small. Leaves are elliptic and pubescent. Flowers are greenish-white. Fruit is round, yellowish or golden, more or less freestone. The assumption is that the word mirabeJ1e is derived from mirable, meaning wonderful, and it was used first by the French. Mirabelle plums are important in France and are regarded as diminutive of Reine-Claude. Hedrick (1911) stated that they are not P. cerasifera hybrids, yet Terpo (1974) still lists them as P. cerasifera x P. domestica. Thus the opinion is divided on the hybrid origin of this type. It is believed that they came from Asia Minor or Armenia and entered into Roman colonies through the Mediterranean area (Wadier 1991) and were introduced to the Lorraine region of France by Rene (1409-1480), provincial monarch of Lorraine. There are early maps indicating that mirabelles

Fig. 4.6. Mirabelle orchards around the village of Soncourt, France, 1749. (After Wadier 1991). The sizeable orchard shows the extent of plum growing at that time. It is notable that the orchards are around the village and not in a medieval walled-in garden, as was customary in the Italian and French gardens a century earlier.

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were planted in orchard settings as early as 1749 around the village of Soncourt, France (Fig. 4.6). St. Julien plums are classified by some as P. insititia var. juliana (subsylvestris) Boutigny. This is an upright group with pubescent leaves beneath, and with medium size, dark blue fruit with long peduncules. In 1754, Miller described a plum as "St. Julien" (Hedrick 1911). In 1804, Poiret reclassified it (Lamark and Poiret 1804) as P. domestica L. subsp. insititia Schneider var. juliana (L) Poiret. It finally became a botanical variety of the species P. insititia. Both Miller in 1754 and Carriere in 1892 speak highly of St. Julien plums as rootstocks (Hedrick 1911). The rootstock characteristics of St. Julien plums were discussed throughout the old horticultural literature. Whether the St. Julien name ever was applied to a specific cultivar is unknown. Kuppers (1976), after studying St. Julien plums for 40 years, came to the conclusion that St. Julien was not a cultivar but a plum type ranging in appearance from small fruited plums to wild bullace to a large-leafed wild plum (P. spinosa), creating an extraordinary picture of hybridization within the section of Prunophora. According to him, there was a group of plums in the Loire Valley in the Nievre region that were processed for prunes in the Middle Ages. As nurseries increased in the 1850s, they planted seeds of"St. Julien" plums for rootstocks. Even at the time of their first use, seedlings of St. Julien were known for their great non-uniformity, susceptibility to diseases, and deficient compatibility with peach. Carriere in 1892 started vegetative propagation of St. Julien and selected better types. As time progressed, only the vegetatively propagated types remained (Kiippers 1976). Among the old recognized forms are 'Petite St. Julien', 'Gros St. Julien', and 'St. Julien de Toulouse.' Several clonal selections have been made, including a series designated by letters (St. Julien A, B, C, J, K) made in East MaIling, England (Tukey 1964) and newer ones made in France. Thus, St. Julien became a well-defined series of rootstock cultivars after about 500 years of existence. 3. Asiatic Species. The Asiatic species include P. saUcina and P. simonii. Prunus salicin a Lind!. (syn. P. triflora Roxb.; P. japonica Hort.; P. Hattan Tamari; P. ichangana Schneid.; P. botan Hort.; P. masu Hort.), known as the Japanese plum, is a strong-growing small tree. Leaves are oblongovate, pointed, shiny, and dull beneath. Flowers are few from each bud (commonly about 3), and white. The fruit is variable, large and firm; skin is yellow or light red. Some cultivars, such as 'Satsuma', have red flesh, and others, such as 'Kelsey', have greenish flesh. As with P. domestica, the wild form of this species is unknown. According to Yoshida (1987),

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P. saUcina may have originated in the Yangtze River Basin and was spread across eastern China. The history of 'Zhui Li' cultivar goes back more than two millennia. Species described under various names, such as P. ussuriensis Kov. and Kost., P. gymnodonta Koehne, P. thibetica Franch., and P. consociiflora Schneid, are all P. saUcina with minor variations and would not be considered separate species today. An early illustration of plum in a book by Hsu Kwan-Chi (1562-1633) probably depicts P. saJicina (Fig. 4.7). Prunus simonii Carr. (syn. Persica simonii Decne.) is known as Simon or apricot plum. First described in 1872 (Kovalev 1941), it has no wild form, but was in cultivation in China, Japan, and Central Asia in the 1st century. The tree is conspicuous because of its narrow, erect habit. Flowers are white, 2 or 3 together, and precede the leaves in the spring. Fruit

Fig. 4.7. Illustration of plum in Hsu Kwan-Chi's (1562-1633) book, Encyclopedia of Agriculture.

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is 2.5 to 5 cm in diameter, firm in texture, smooth, maroon red, adhering to the small pit. Botanical position is doubtful since it has some characters of apricots. Chow (1934) describes it as an occasionally cultivated plant of north China. Because of its resemblance to apricot, it is often considered an apricot-plum hybrid. Okie and Weinberger (199B) think that it is more likely an upright form of P. saUcina. It is also used in the California hybridization programs as parent of the so called California Japanese plum cultivars because of its firm flesh and strong flavor. 4. American Species. Five American species are discussed here: P. americana, P. nigra, P. angustifoUa, P. hortulana, and P. munsoniana. The other species can be found in botanical books by Rehder (1954) or Bailey (1927), or in a description by Hedrick (1895). Other American plum species are less important in the origin of commercial cultivars and are not discussed here in detail. These include P. subcordata Benth., Pacific plum, native of California and Oregon; P. mexicana Wats., big-tree plum, native from southwestern Kentucky to western Tennessee to Oklahoma and Mexico; P. rivularis Scheele, creek plum, native in Texas; P. orthosepaJa Koechne, native from Kansas to Texas; P. alleghaniensis Porter, Allegheny plum, native from Pennsylvania to Connecticut; P. umbellata Ell., black sloe, native near the coast from South Carolina to Florida; P. maritima Marsh., beach plum or shore plum, native from New Brunswick to Virginia; P. gracilis Engelm. & Gray, Oklahoma plum, native to western Arkansas, Oklahoma and northern Texas; P. Janata (Sudw.) Mack. & Bush, common from Illinois to Texas [Gray considers it a variety of P. americana and named it accordingly (P. americana var. mollis Torr. and Gray)]; and P. reverchonii Sarg., hog plum, native in Oklahoma and Texas. Rehder (1954) considers it closely related to P. rivularis and thinks that it may not be different from P. rivuJaris. P. americana Marsh. (syn. P.latifolia Moench.; P. hiemalis Michx.; P. ignota Nels.), known as common wild plum, is a small, thorny, spreading tree. Leaves are oblong-ovate, not glossy, pubescent on the veins. Flowers are large, white, and appear in clusters before the leaves. It is the most widespread among the American plum species. Its native range is from Massachusetts to Georgia to near the Gulf of Mexico and to Utah and New Mexico to the west. In the East, fruit quality is poor, but in the West, edible forms are abundant. It is well adapted to the cold North and several cultivars were developed from this species there. P. nigra Ail. (syn. P. borealis Poir.; P. mollis Torr.; P. americana var. nigra Waugh.), known as Canada plum, is a more showy tree than P. americana. Flowers are large, white changing to pink. Fruit is oblong, orange red; the stone is large and much compressed. Its native range

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M. FAUST AND D. SURANYI

includes New Brunswick (Canada), New England, New York, Michigan, Wisconsin, and northern Ohio. P. angustifolia Marsh. (syn. P. chicasa Michx.; P. stenophyllus Raf.), known as Chickasaw plum, is a small, bushy tree, usually suckering from its roots and forming thickets. Leaves are oblong-Ianceolate, 5 cm long, sparingly pubescent. Flowers are white, preceding the leaves. Fruit is small, cherry-like, red or yellow, yellow dotted, clinging to the small rough stone. Its native range is from Delaware to Florida and Texas. It is abundant on sandy soils. This species gave rise to only a few cultivars, but it was important for southern adaptation. P. hortulana Bailey (syn. P. hortulana var. waylandii Bailey) is known as hortulana plum. The relatively tall (5-10 m) tree is distinct, nonsprouting. Leaves are elliptic-ovate, 7.5 to 10 cm long, shiny, lightly pubescent. Flowers are white, preceding the leaves; fruit is oblong, 2.5 cm in diameter, red to yellow, white dotted, with little or no bloom. Its native range includes Central Kentucky, Tennessee, to Iowa and Oklahoma. The species was first distinguished in 1892 to designate cultivars of plum intermediate between P. americana and P. angustifolia. P. hortulana represents a range of hybrids between P. americana and P. angustifolia (Bailey 1927). P. munsoniana Wight and Hedr. is known as Wild Goose plum. This species represents a range of forms separated out of the old Hortulana class, of larger and freer growth than the variants of P. angustifolia, hardier, with more pointed leaves and larger flowers. Trees are 6.5 to 8 m tall and forming thickets. Leaves are 7.5 to 10 cm long, lanceolate, slightly pubescent on veins beneath. Flowers are white, and appear either with the leaves or before them. Fruit is oval, bright red or yellowish, marked with white dots. Native range includes Kentucky and Tennessee to Mississippi, Texas, Minnesota, and Kansas. According to Bailey (1927), the botanical status of this group is uncertain. Nevertheless, this group gave rise to a number of cultivars.

B. Horticultural The diversity of plums intrigued early horticulturists and they tried to group them according to various criteria. Konrad Gesner in his Horti Germaniae in 1560 identified nine groups of plums: (1) large fruited plums with purple skin, occasionally yellow, acidic; (2) small, late, yellow plums HZipparten"; (3) early, small, multicolored plums ripening with oats, "Haberkriechen"; (4) Neapolitan plums, long, sweet, pale colored, "Curtius Lindau"; (5) Hungarian plums of two kinds, one larger and

4. ORIGIN AND DISSEMINATION OF PLUMS

199

longer, the other sweeter than Curtius Lindau; (6) outstanding Hungarian plums, "Damascena", firm flesh, acidic; (7) yellow Hungarian plums of the size of an egg, with yellow color, and pleasant taste; (8) a diverse group of plums not classified under the previous groups; (9) plums in the forest and in hedges, and sloes. Jaques Dalechamps (1586/87) in his Historia generalis plantarum Lugduni distinguished five kinds of plums: (1) "Damascena", with dark skin, pleasant flesh, and small stone; (2) Perdrigona, syn. Iberica, with firm flesh, sweet and pleasant taste; (3) yellow plums; (4) Asinina, large, purple, elongated plums; (5) Pruni dactyla (date plums), large, purple, egg-shaped plums. In John Gerard's The Herball, enlarged by Thomas (1633), five types of plums are described: damson, myrobalan, almond plum, damascene plum, and the sloe. Four of these are illustrated (Fig. 4.8.). Caspari Bauhin in his Pinax Theatri Botanici in 1623 set up 15 different groups that constituted the basis of Linneaus's classification. Even though Linneaus's classification was botanical, the groups he mentioned under the species constituted a horticultural listing of existing groups. This is clearly indicated on page 475 of Linneaus's Species Plantarum I, which includes the description of the 9th and 10th species of the genus Prunus (P. domestica and P. spinosa) (Fig. 4.9). Taft (1894) commented that relatively little attention had been paid to the diversity of the native American plum until L. H. Bailey studied the plums in detail and made a new classification. Taft classified American plums essentially into four groups: (1) The Americana Group (P. americana). This species is found from New England to the Rocky Mountains and extends to Manitoba and Texas. The group is characterized by a firm, meaty, usually compressed, dull colored, late fruit, with thick and usually very tough skin, and large flattened stone that is often quite free. All the cultivars that belong here have a light purple bloom. (2) Wild Goose Group (P. hortulana). This embraces cuItivars with a wide-spreading growth, a firm, juicy, bright-colored, thin-skinned fruit, a clinging, turgid, comparatively small, rough stone, and with peach-like ovate-Ianceolate, long-pointed leaves. The species is located in the Mississippi valley from Illinois southward. (3) The Miner Group (P. hortulana var. mined). It differs from the basic species because of its dull, comparatively thick leaves and a late, very firm fruit. Cultivars of this group are hardy, and Taft (1894) recommended them for Michigan. (4) Chickasaw Group (P. angustifoJia). The trees of this group have a slender, spreading, and irregular growth. The fruit is small, generally red, and more or less spotted. The flesh is soft, juicy, and adheres very tightly

M. FAUST AND D. SURANYI

200 ~_

••...... _"----~~-:--:-----------------

L 1 B. j.

Of the Hillory of Plants.

1497

----~.-:...-_-------_...:..-_-------------~ Prllft", c.M.r,6"lif1IlI. % 'rMIlS DIJmtjlkli.

The Mlrobalane P1um tree.

The D.:unlon tree.

J

; Prtll1NS AmygdAlinA.

The Almond Plum tree.

Kkkkkk 3

Fig. 4.8.

Plum illustrations in Gerard's HerbaJl (1633).

PTNPIIS nffltjlril. The Sloe cree.

4. ORIGIN AND DISSEMINATION OF PLUMS

201

Page 475 from Species Plantarum I. 1753. (English translation of tbe Latin text. P. domestica, simple flowers. leaves long. oval. P. inermi.f, plum without thorns. leaves are e1ongated-oval. Damaacena, plums are larger sweet. or small blackish-blue. Hungarlca, plums are large. thick. sauerish. Juliana, plums are longish. blue. Pemicona, plums are black with firm flesn. Cerea, plums are wax-yellow. whitish-yellow or colorless. Acinarla, plums are large. red and round. Maliformis, plums are large. yellow. sweet. and apple shaped. Augustana, plums are smaller. ripe in August. Praecox, plums are small early. Cereola, plums are small greenish-yellow. Amygdaliana. plums are almond sbaped' Galatens/s, plums are white. long and sauer. Brlgnola, plums are reddish-yellow with outstanding flavor. Myroba/on, plums are round blackish-purple and sweet. P. spinosa, PrunU8 with thorns and elongated leaves. J Original Latin telt. 9. PRUNUS pedunculis simplicibus. foUis lanceolato-ovatis convolutis Prunus inermis. foUis lanceolata-ovatis. Hart. Cliff 186. Hort. Up8. 124. Mat. Med. 232. Roy.Llugdb.

domestlea

268.

Prunus Bauch. pin. 443.

P Pruna majora dulcia & parva atro-caerula. Bauch. pin. 44.1.

Damascena

., Prona magnacrassa subacida. Bauch. pin. 443. d Prona oblonga caerolea. Bauch. pin. 443. e Pruna nigra: camedura. Bauch. pin. 44.1. z Prona coloris cerae ex candido inluteum pallescente. Bauch. pin. 44.1

juliana pernicona cerea

11 Prona magnarobra rotunda. Bauch. pin. 443. f1 Prona rotundaflava dulcia mali amplitudine. Bauch. pin. 44.1.

aeinarla malifonnis

hungarica

Prona angosto maturescentiaminora & austeriora. Bauch. pin. 44.1.

augU8tana

Prona parva paecocia. Bauch. pin. 44.1. A Pruna parva exviridi flavescentia. Bauch. pi". 443. Jl Pmna amygdalina. Bauch. pi". 44.1. n PruneoU alba oblongiusculi acidL Bauch. pin. 443. o Prona exfJavo rofescentiamixti saporis gratissima. BOlich. pin. 443.

cereola amygdalina galatensis brignola

1. IC

~

Pronus fluClO rotundo nogro purpureodulci. Bauch. pin. 443. Habitat Europae australiori81ocis elevatis.

10 PRUNUS spinosa, folUs lanceolatis Hart. Cliff /86.

praecox

myrobalan spinosa

Fl. Succ. 397. Mat. Med 231. RqyLugdb. 268. Hall. He/v. 355. Prunus sylvestris Ballch. pin. 444. Tabem. ie. 992.

Habitat in Europae collibus apricis.

Fig. 4.9. Description of Prunus domestica by Linneaus in Species Planta"'rum 1. 1753. Original Latin text reproduced as it appears on page 475.

202

M. FAUST AND D. SURANYI

to the small, broad stone. Even in the cultivated state the trees are quite thorny. III. HISTORY A. Archeobotany 1. Europe. Archeobotany of Prunus species was reviewed by Bertsch and Bertsch (1947) and Korber-Grohne (1996). Additional information was provided by Baas (1974), Ermenyi (1975/1977), and Hartyanyi (1978). The oldest remains are stones of P. insititia, damsons and St. Juliens, going back to Neolithic times. In Ukraine, along the river Dnipro, an ancient culture, the Trypilians, existed 3,000 to 8,000 years ago. People belonging to this culture already cultivated plums 6,000 years ago. Seeds found at Novaja Russesti, Ukraine, indicate that the primitive plum grown at that time had a relatively large seed (Ermenyi 1975/1977). The impression of one plum seed, found at Varvarovka XV, from the late Trypilian culture 5,000 years ago, was interpreted by Janusevics (1976) to represent a cherry plum x wild apricot hybrid. Additional archeological evidence indicated that cherry plums and sloe were also present in the area, which means that hybrids could have occurred at this early Neolithic time. Pieces of plum stones uncovered at this site signify that such hybrids did indeed occur (Ermenyi 1975/1977). Stones unearthed in Bedburg, Germany, and in Novaja Russesti, Ukraine, from around 6,000 years ago indicate a relatively large geographic area of Europe where plums were used. According to Knorzer (1974), the damson seed found at Bedburg must have come from orcharding because there were no wild damsons in Germany at that time. At the beginning of the Bronze Age, in Switzerland there were colonies that built their dwellings on poles. Damson seeds have been found in the garbage pits of these colonies at Robbenhausen-PfOffikersee, Switzerland, and Wangen-Bodensee and Sipplingen-Bodensee, Germany. Plum production was also established in England during the very early Neolithic period. Roach (1985) lists several English sites where stones of plums were found. The earliest remains were of the native species, namely the sloe. Stones of sloe were unearthed at the Glastonbury lake village, from the Middle Bronze, Bronze, and Iron Ages. Dimbleby (1985) noted that in a Bronze Age shaft at Wilsford, England, near Stonehenge, Prunus pollen (most probably sloe) was found. Table 4.2 lists representative finds from prehistoric times.

4. ORIGIN AND DISSEMINATION OF PLUMS

203

Table 4.2. Archeological finds of plums from prehistoric times (list is not allinclusive).

Location

Time period

Type of plum

Reference

Ehrenstein, near VIm Germany Dnyetrov-Prut (Novaja Russestii) Kluczbork, Poland Nowe Cerekwiew, Poland Bedburg-Garsdorf, Germany Robenhausen, Switzerland Sipplingen, Germany

4060-3956 B.C.

Damson

Hopf1968

Neolithic

Ermenyi 1975/77

Neolithic Neolithic Late Neolithic

Cherry plum? hybrids Prunus sp. Prunus sp. Damson

Opravil1963 Opravil 1963 Knorzer 1974

Late Neolithic

Damson

Heer 1866

Late Neolithic

Damson, St. Julien

Wyeregg at Untersee, Austria

Late Neolithic

Damson, St. Julien

Ravensburg Salamis, Cyprus

Late Neolithic Late Neolithic

Damson Damson

Wangen

Bronze Age

Damson

Seengen, Riesi, Switzerland Lengyeli, Hungary

Late Bronze Age

Damson

Bertsch and Bertsch 1947; Baas 1971 Bertsch and Bertsch 1947; Baas 1971 Renfrew 1973 Renfrew 1973; Hjelmqvist 1963/64,1973 Bertsch and Bertsch 1947 Neuweiler 1935

Late Bronze Age

P. domestica

Galstonbury, England Schwabish Hall

Bronze/Iron Age Early Iron Age

Sloe Damson

Am Lac de Neuhatel

1050-860 B.C.

Damson

Hartyanyi et a1. 1968 Roach 1985 Bertsch and Bertsch 1947 Jaquat 1988

[Neolithic period from 4000 to 2500 B.C.; Bronze Age from 2500 B.C., includes the Egyptian (from the III to the XXth Dynasty), Sumer, Assyrian, and Minoan civilizations until the time of the Trojan War, about 1200 B.C. The first use of iron was credited to the Hittites in Asia Minor about 1400 B.C. and the Iron Age lasted to about 500 B.C.]

From the Roman period, seeds of damson, garden plum, and interInediates were found. In Saalburg, Germany, in a well of the fort, 41 whole and 22 broken seeds of damsons were unearthed from the period of 83-260. At Rottweil, Germany, during the excavation of the Roman city, 13 seeds of intermediate forms and 3 seeds of P. domestica were found. Slightly more north in Germany, in Aalen and Butzbach, from

M. FAUST AND D. SURANYI

204

Roman wells and at the military camp at Neuss, 42 seeds of damsons and 17 seeds of P. domestica were found. At another Roman military camp at Linz, Austria, from the period 14 to 80, more than 300 seeds were recovered. According to Werneck (1961), the seeds at Linz were Celtic in origin. The Romans had brought them to Linz, where they adapted to local conditions. However, Ermenyi (1975/77) thought that all types of plums were known in Austria and were produced there before the Roman occupation. At Linz, several plum seeds from the period between 380 and 425 were recovered from an area that indicated that the fruit had been used in religious sacrifices. The seed from this period can be classified into 3 groups: (1) 10 to 16 mm long, round or oval, with variable shape; (2) an intermediate group resembling the prunes, 12 to 16 mm in length with pointed ends; (3) true prunes, 18 to 22.5 mm in length with pointed ends and a raised dorsal suture. Plums were recovered from Roman sites at Silchester, Manchester, and Caerwant in Monmouthshire, England. Charcoal remains of bullace were found from the Roman period at Silchester and from the Anglo-Saxon period at Hungate in Yorkshire. Damson and domestic plum stones have been excavated from late Iron Age settlements at Maiden Castle in Dorset and at the Roman site in Silchester. Even though the domestic plum and damson were not native to England, the archeological evidence indicates that they were grown there in the Roman period and perhaps even earlier (Roach 1985). Representative locations from the Roman period where plum seeds were excavated are listed in Table 4.3.

Table 4.3. Archeological finds of plum seeds from Roman times (list is not all-inclusive). Location

Time period

Saalburg, Germany 83 Neuss, Germany 1st century Aalen, Germany 1st century Xanten am Rhein, 1st century Germany Aachen, Garmany 1st century 1st-2nd century Kaln, Germany Rottwail, Garmany 186 Kangan, Germany 141-160 Tac-Gorsium, Hungary 2nd century 2nd-3rd century Ellingen, Germany Balatonbereny, 4th century Hungary

Type of plum

Reference

St. Julien, Damson P. domestica Damson St. Julien, Sloe

Baas 1951 Knorzer 1970 Baas 1974 Knorzer 1981

P. domestica St. Julien, Sloe St. Julien P. domestica, Sloe P. domestica St. Julien P. domestica

Knorzer 1967 Knarzer 1987 Baas 1974 Maier 1988 Hartyanyi et a1. 1968 Frank and Stika 1988 Sagi and Fiizes 1967

4. ORIGIN AND DISSEMINATION OF PLUMS

205

Plum production in Europe increased during the Middle Ages and this is reflected by the increasing number of archeologimil finds. In the Czech Republic, at Uhersky Brodu, 6 domestica and 17 damson seeds were found from the 12th century (Opravil 1976). In Olomouc, Ostrava, Opava, and Plzen (Moravia), many seeds of damsons, mirabelles, and domesticas from the 16th century were found. In Poland, at Szczecin, 58 damson and P. domestica seeds were found. At Gdansk, 1,700 seeds were collected from the 10th-11th century. At Opole, 168 seeds were unearthed from the 12th century. Seeds of various types were found in the Wawel at Krakow from the 12th-13th century, at Posnan from the 12th century, and at Wroc1aw from the 12th-13th century. From the excavation of a medieval fort near Buderick, Germany, very rich plant material was found from the 11th-12th century. Among the material there were seeds of damsons and round- and egg-shaped fruits belonging to the Green-Gage group. In Hungary, seeds were found at Budapest during the excavation of a house (#10) at Disz square, and from a fort at Kereki-Feherko. At Pecs, a plum seed adhered to a jar was discovered, indicating that plums were also in this area. Plum seeds recovered during the Middle Ages are listed in Table 4.4. 2. America. Even though plum was a native fruit in America, it apparently had limited use by the indigenous peoples. A few plum seeds were recovered at the Smiling Dan settlement along the lower Illinois Valley (Asch and Asch 1985). This was a Middle Woodland settlement that lasted from 250 B.C. to 400 A.D. (Sant and Stafford 1985). The inhabitants of Smiling Dan used grapes and hazelnuts extensively, but judging from the few seeds, plum was not an important component of their diet. This is notable, because in the vicinity of the settlement (3 km) there is a creek called "Plum Creek," indicating that plums were present in relatively recent times when the creek was named, and the possibility exists that plums were present at the time of the Indian settlement. Similar to the Smiling Dan inhabitants, Indians of the Bluff-Dweller culture in the Ozark mountains in Arkansas used plums to a negligible extent. Even though the Bluff-Dwellers used grapes and various nuts, plums were not important for them. The Bluff-Dweller culture existed before the earliest Pueblo culture, which existed from the 1st to the 5th century. Beautiful strings of beads, made from Ozark gromwell, a plant of the BOTaginaceae, were unearthed in the settlement, but the only plum seed found was from a chickasaw plum and it was perforated to be used as a bead (Gilmore 1931).

Table 4.4.

N

o0)

Archeological finds of plums from the Middle Ages (list is not all-inclusive).

Location

Time period

Type of plum

Reference

Mikulcice, Czech Rep. Behren-Lubchin Szczecin, Poland Gdansk, Poland Maus Meer, Germany Wroclaw, Poland Lund, Sweden Prague, Czech Rep. Burghausen, Germany Kelheim, Germany Kravin, Czech Rep. Uhersky Brod, Czech Rep. Opava, Czech Rep. Koln, Germany Lubeck Opatovice, Czech Rep. Budapest, Disz square, houses #8 and 10 Ho1l6ko, Hungary Kereki-Feherko, Hu.TJ.gary Olomouc, Czech Rep. Ostrava Bad Windsheim Neuss, Germany Briiggen, Germany Ivancice, Czech Rep. Pees, Hungary Gyongyospata, Hungary

8th-9th century 990-1210 10th-11th century 10th-11th century 11th-12th century 12th-13th century 1200-1300 10th-15th century 1250-1350 1200-1450 13th-14th century 13th-14th century 13th-14th century 13th-14th century 13th-16th century 14th century 14th century

Damson, P. domestica St. Julien, large plums Damson Oval plums Damson, sloe, round plums, egg plums P. domestica types Damson St. Julien, sloe, P. domestica St. Julien, sloe, mahaleb Large plums, sloe, mahaleb Damson, St. Julien, mirabelle, cherry plum St. Julien, mirabelle, sloe St. Julien, mirabelle, Reine-Claude, egg plum St. Julien, sloe, P. domestica Damson, St. Julien Damson P. domestica, cherry plum, sloe

14th century 14th century 14th-15th century 14th-15th century 1400-1500 15th-16th century 1500 16th-17th century 16th-17th century 18th-19th century

P. domestica Damson Damson, cherry plum, P. domestica Damson Damson, egg plum Damson, sloe, egg plum Damson, sloe, St. Julien St. Julien, sloe P. domestica P. domestica

Hartyanyi et al. 1968 Wessely-Meincke 1965 Opravil 1964 Opravil 1964 Knorzer 1971 Opravil 1964 Hjelmqvist 1963/64 Opravil 1986a Gregor 1995a Gregor 1985 Opravil 1988 Opravil 1966 Opravil1966b Knorzer 1987 Kroll 1980 Tempir 1962 Hartyanyi et al. 1968 Hartyanyi and Novaki 1973174 Hartyanyi et al. 1968 Hartyanyi et al. 1968 Burian & Opravil1970 Opravil 1964 Gregor 1995b Knorzer and Muller 1968 Knorzer 1979 Opravil 1985 Hartyanyi et al. 1968 Hartyanyi et al. 1968

4. ORIGIN AND DISSEMINATION OF PLUMS

207

B. Antiquity Written evidence concerning plums surfaces in the Greek and Roman literature. Suranyi (1985) interpreted Herodotus's (484-430 B.C.) and Xenophon's (431-355 B.C.) references to plums to be about cherry plums. According to these Roman writers, there were many cherry plum trees in the mountains populated by Scythians and Armenians. The European plums were mentioned first in Archilochus's Pollux (7th cent. B.C.). In Rhodos, the word brabiila was used for designating damsons. Rhodos was also the location of extensive damson production (Suranyi 1985). Publius Valerius Cato (201 B.C.) wrote about propagation of plums and Vergil (Publius Vergilius Maro, 70-19 B.C.), in his Georgica (IV.145), remarked that sloe was grafted with plum. He also wrote about a "thorny plum" and recommended that it be planted alternately with "obelisks of box" (Georgica IV). Horace (Quintus Horatius Flaccus, 65-8 B.C.) mentioned plums in his letters (1.16:8). Ovid (Publius Ovidius Naso, 43 B.c.-17 A.D.) gave a more detailed description of black and yellow plums. Vergil also wrote (2:53) about yellow plums, which probably belonged to the green-gage group. Plinius (23-79) and, following him, Lucius Junius Moderatus Columella (1st century) wrote about several kinds of plums: cereolum (cherry plum), damasci (Damascus plum), and onychium (mirabelle). Plinius also wrote about the damascene plums introduced earlier into Italy from Syria. In Herculaneum, destroyed by the eruption of Mount Vesuvius in 79, Reine-Claude (Regina Claudia)-like spherical plums were painted on the north side of the Sannitic House, and in Pompeii, on the wall of the tabino on the left of the entrance ofTrebio Valente's house, mirabelle-like violet plums are painted in two different views: one showing the suture, the other the perpendicular rim cavity (Pompeiana 1950). Thus, plum culture was unquestionably established at the time of the Romans. Yet, according to Hedrick (1911), the plum culture was probably not widespread, because there were no seeds found in Pompeii that could be traced to the time when the city was destroyed. Hedrick estimated that plums should have been marketed in Pompeii at the time of destruction (August 1479) and their absence must mean that plum production was limited in southern Italy. Apparently the Romans planted more plums in the territories than on the mainland ofItaly. Emperor Gaius Aurelius Valerius Dioc1etianus (245-313), who was of Dalmatian origin, established large plum orchards on the banks of the Drava and Sava rivers (Croatia) utilizing the cultivar tDe Bosnia', from which tPozegaca' was developed (Ramming and Cociu 1990). That plums were in the Roman territories is also indicated if the archeological sites where plum seeds were found from Roman times are mapped (Korber-Grohne 1996). Sites with plum seed were more numerous in the area of Germany occupied by the

208

M. FAUST ANDD. SURANYI

Romans compared with the unoccupied area. There was a IItavern" at Balaca, north of Lake Balaton in Western Hungary, where a wallpainting from the 1st century illustrates a woman's head surrounded by reddishpurple plums, indicating that the Romans grew plums in Hungary also. The plum in the illustration is probably P. domestica (Fig. 4.10).

Fig. 4.10. Detail from a painting found at Balaca, Hungary, from the 1st century. A. The painting depicts a mask as a veiled woman's head surrounded by plums. The plums are reddish-purple. B. Close-up of the plums.

4. ORIGIN AND DISSEMINATION OF PLUMS

209

C. Japan

Plums were taken from China to Japan quite early. Yoshida (1987) remarks that plum stones have been found from the Yayoi Era, about 300 B.C. and cultivated plums were mentioned in Japanese books from 500. Matsumoto (1977) put the time of introduction somewhat later. According to him, the use of the word ume (Japanese for plums) first appeared in 751, in a collection of Japanese poetry written in the Chinese style. By the early 800s, ume was used frequently as a subject in poetry. From this, Matsumoto (1977) concluded that the introduction of plum to Japan must have been in the mid 700s. The introduction of plums to Japan is traced back to at least two sources. Wani, a naturalized Japanese, was supposed to have brought the plums from the Korean peninsula. Alternatively, plums were introduced to Japan by a Chinese monk of high standing who brought plum trees as a gift to the Emperor (Matsumoto 1977). When considering the situation in Japan, we find that the same problem exists about the use of the word "plum" as in China. Ume is derived from P. mume, a species closer to apricots than to plums. Yoshida (1994) remarked that the mume is often called "Japanese apricot" or "Japanese plum," but its common name is "ume" in Japan. Therefore, because the information above is pertinent to the species P. mume, we are not certain when the introduction of plum (P. saUcina) took place in Japan. Ubai (smoked and dried "plum") had long been used as medicine by the Chinese and was well known to the Japanese before the introduction of plum to Japan. Ubai was prepared by burning tree roots and straw to change the plums into a dry, black form. These smoked plums could be stored longer and were more palatable because some of the sourness was lost in the drying process. The Japanese also pickled plums and apricots for medicine, calling them umeboshi. The oldest record of umeboshi is dated 984 and subsequent dictionaries listed the word, so that it is almost certain that umeboshi was used in the 11th and 12th centuries (Matsumoto 1977). Salted plums were popularly known as a medicine that could be used to prevent running short of breath. Hideyoshi Toyomi, famous for his military leadership in Japan, used salted plums to motivate his soldiers. He would shout, "There is a Japanese plum grove over the mountain!" This encouraged soldiers and hastened their marching (Matsumoto 1977). It must be emphasized again that this information is likely to be about P. mume and not about the true plum species. The Japanese plum, P. salicina, was imported into the United States in 1870 by Mr. Hough of Vacaville, California. The cultivar 'Kelsey' was selected from this import, which was followed by another import in 1884. Dreyer (1985) presents evidence that Luther Burbank imported 210 seedling trees of 12 cultivars from Japan in 1885. From these, Burbank

210

M. FAUST AND D. SURANYI

selected 'Satsuma' and 'Burbank'. In his plum-breeding activities, Luther Burbank used P. salicin a, and this species is in the parentage of almost all of the so-called California Japanese plum cultivars. The interrelationships between the various species in California Japanese plums are described by Li et al. (1997). D. Europe In the early 9th century, Charlemagne included plums in his list of fruit to be grown (Roach 1985). The plant list of al-Biruni from 1050 gives us an idea of which plants were included in the Spanish Islamic gardens. In the middle of the 11th century, essentially the whole range of temperate-zone fruits, including plums, were planted in these gardens (Harvey 1975). The Islamic garden had a principal canal with constantly flowing water that was dissected at right angles by smaller channels. Along the canals, cypress and fruit trees were planted, among which plum was used essentially for shade (Hobhouse 1992). Persian miniature paintings that date from about 1396 illustrated many stories and give us excellent information about the Islamic gardens. Fruit trees were often painted as part of the landscape of these miniatures, and plum was one of the trees pictured (Hobhouse 1992). Plums were important in practically all parts of Europe. In his book Physica (1151-1158), Hildegard described red plums and garden plums. His reference to garden plums was interpreted by Korber-Grohne (1996) to mean damsons. The Westminster Abbey Customary required the monk who was the gardener to supply the monastery with plums, among several other fruits. Although plums were cultivated in some monastery gardens, the majority of people still relied on wild plums, bullaces, and sloes. The inclusion of plums in William Longland's Piers Plowman in 1362, and in Chaucer's translation of the Roman de 1a Rose in 1372 indicates their importance (Roach 1985). In the 16th century, several horticulturists were concerned about plums. In the Grete Herball of 1526, an English translation of Le Grand Herbier, a French work from the press of Peter Treueris, two kinds of plums are mentioned: "blacke and reed." In 1575, Mascall regarded damson as the best type. He advised gathering the fruit when ripe and drying them in the sun or in a hot bread oven to keep them for a long period ofUme. Hyeronymus Bock (1546) wrote about a large black plum, a small green plum, and a yellowish-brown plum. Fuchs (1543) was concerned about the shallow root system of plums, and Konrad Gesner (1565) wrote about the early-ripening plums being small.

4. ORIGIN AND DISSEMINATION OF PLUMS

211

Early in the 14th century, Pietro de' Crescenzi produced a book describing the establishment of medium-size gardens, including orchards, for kings and princesses. He recommended that these gardens be surrounded by hedges. In warm places, a hedge of pomegranates could be used and in cold places, a hedge of nuts or plums. His book was completed in 1305 and Charles V (1337-1380) commissioned its translation into French in 1373. It is likely that this book greatly influenced the establishment and design of the royal garden at Saint-Pol. Charles V, the king of France from 1364 to 1380, established an 8-ha garden at the Hotel de Saint Pol in Paris. The garden deteriorated after the death of the king and was later destroyed. Fortunately, there is a complete inventory of the trees planted in the garden and plums were included (Hobhouse 1992). Pietro Crescenzi's book, Liber ruralium commodorum, long read in manuscript form but actually printed in Italian in 1471, also influenced the development of the Italian Renaissance gardens. In 1460, Bartolomeo Pagello of Vicenza, Italy, fulfilled his desire to build a garden where he could grow many apples, pears, pomegranates, damascene plums, and vines (Hobhouse 1992). An inventory, taken at Villa Lante, north of Rome, after the death of Cardinal Gambra in 1587, describes the fruit orchards and reports that the walls were covered with plum, medlar, pomegranate, and quince (Hobhouse 1992). The superior plum 'Reine-Claude' was named to honor Queen Claude, the wife of Francis I, French king from 1494 to 1547, the period during which the plum was introduced to France (Hedrick 1911). 'Green Gage', the common name for the 'Reine-Claude', derives from the fact that the Gage family of England procured a number of plums from the monastery of Chartreuse at Paris. When the plums arrived, all had labels except one, which was 'Reine-Claude'. When the unlabeled tree started to produce fruit, the gardener simply called it green gage. 'Reine-Claude' apparently was taken to England soon after its establishment in France. 'Green Gage' seeds were recovered from the wreck of the Mary Rose, the flagship of Henry VIII, which sank in 1545 and was raised in 1982. The wreck revealed a basket containing the remains of more than 100 plums. 'Green Gage', 'Catalonia', mirabelles, and myrobalan were identified in the basket (Roach 1985). In Hungary in 1552, the existence of the Hungarian plum 'Besztercei' was mentioned in a Latin language document, "Una libra pruni Besztercei . .. ," and in 1558 the production of dried prunes made from the fruit of this cultivar was described (Tbth and Surflllyi 1980). The quality of Hungarian plums also interested Maximilian II (1527-1576), emperor of the Holy Roman Empire, who sent a letter to Antal Verancsics, arch-

212

M. FAUST AND D. SURANYI

bishop of Esztergom, Hungary, dated March 18, 1573, asking for grafting material from plums, especially of four cultivars: 'Large Duranci', 'Katalan', 'White- and Black Horseye' (Rapaics 1940). Aldrovandi (1522-1605), in his Iconographia Plantarum, illustrates 9 plum cultivars: (1) Pruna Damascena rotunda minora; (2) Pruna Damascena viridia maiora et oblunga rubia, amethystina et viridia (Fig. 4.11); (3) Pruna Gregola vulgo dicta purpurei coloris; (4) Pruna Lutea,' (5) Pruna lutea Augustana; (6) Pruna maxima; (7) Pruna maximilianis congenera; (8) Pruna purpurea. Prune seu Prugne Bon Succino ItaJis; (9) Prunorum oblungorum luteorum due differentie (Baldini 1990). La Quintine, the gardener of Louis XIV, established the royal fruit and vegetable garden between 1677 and 1683 (Tukey 1964). The garden covered 8 hectares and had 29 separate walled enclosures. Enclosure num-

Fig. 4.11.

Pruna damascena viridia maiora from Aldrovandi's Iconographia Plantarum.

4. ORIGIN AND DISSEMINATION OF PLUMS

213

ber 20 was planted with dwarf espaliered plums. We estimate that as many as 200 plum trees were planted in the orchard. In England in 1597, Gerard remarked that he had 60 of the best and rarest cultivars of plums in his garden and every year he received new ones not previously known. He commented that the dried damson prunes were more astringent than those produced in Spain, which were sweeter. Prunes produced in Hungary were long and sweet and those of Moldavia were the best (Roach 1985). He also commented that potatoes are good with plums: "doe boyle them with prunes, and so eate them." Parkinson in 1629 described 61 cultivars of plums and emphasized the role of John Tradescant in bringing new ones to England. In 1664, Janos Lippai, gardener of the archbishop of Pozsony (Bratislava), in his book, Posoni (Pozsonyi) kert, devoted an entire chapter to plums. He described nutritional needs, propagation methods, cultivars, and diseases. Batty Langley (1729), a London nurseryman, listed 'Reine-Claude' and 'GreenGage' separately (Fig. 4.12 illustrates Reine-Claude). In 1731, Philip Miller described 'Green Gage' as one of the best plums of England. Switzer (1724) considered the available plum cultivars excellent and recommended CSt Catherine', 'Reine-Claude', 'Maitre Claud', 'Drap-d'Or', 'Jeanne Hative', 'Mirabelle', 'La-Royal', 'Blue Perrigon', 'Orleance', 'Red Fotheringham', 'Black' 'Damascene', 'Marocco', and 'St Julian'. Fifty years later, John Abercrombie (1779) recommended plum cultivars, some of which were the same as recommended by Switzer (1724) earlier. His list included: 'Black Damask', 'Orleans', 'Queen-Claude', 'Green Gage', 'Perdigon', 'White Magnum Bonum', 'Fotheringham', CSt Catherine', 'Mirabelle', 'Muscle', CSt Julian', 'Damascene', and the cherry plum. In 1800, Jervaise Coe, a gardener at Bury St. Edmunds, Suffolk, introduced a new plum named 'Coe's Golden Drop'. He believed the 'Golden Drop' grew from a stone of 'Green Gage' pollinated by 'White Magnum Bonum'. It turned out to be an excellent late-season dessert plum that could be stored for the winter (Roach 1985). Many of the important cultivars of plums were introduced to England at the beginning of the 17th century and no new cultivars were described until the 19th century. The Royal Horticultural Society encouraged breeding to produce new cultivars. Thomas Andrew Knight tried his hand at plum breeding with limited success. In 1823, he introduced a cultivar that had only medium quality. His work, however, encouraged others to develop new plum cultivars. Thomas Rivers introduced 'Early Rivers' in 1820 and 'Precoce de Tours' in 1834. In 1843 he produced a seedling that was introduced in 1875 under the name of 'Czar', named after the Czar of Russia, who had visited England that year. Rivers intro-

M. FAUST AND D. SURANYI

214

~11"~.Jxxm

c.t'I1txPer.rdrigoJ! .Yt~Iy.JII:_ ~_ ,1/I,lwll. .:.,.. 'd:.ij;'~~~-

L''/i.l.

Fig. 4.12.

Illustration of plums from Langley's Pomona, 1729.

4. ORIGIN AND DISSEMINATION OF PLUMS

215

duced several other cultivars, which included the 'Golden Gage' and 'Late Transparent Gage', 'Monarch', 'President', and 'Wyedale'. The most important English plum cultivar, 'Victoria', was introduced in 1840. 'Victoria' was found as a seedling in a garden at Ald~rton in Sussex. The seedling became known as 'Sharp's Emperor' and 'was sold to a nurseryman, named Denyer, who in turn again sold it as 'Denyer's Victoria' and its name was finally simplified as 'Victoria'. As a self-fertile, heavy-cropping, dual-purpose cultivar, it was widely planted on farms in Kent and most other areas of plum production. Particulars of the improvement in English plums are detailed by Roach (1985). During the 19th century, plums were improved at many diverse locations. Gallesio in 1839 published and illustrated high-quality cultivars (Fig. 4.13) in his Pomona Italiana (Baldini and Tosi 1994). 'Oullins Gage' was a chance seedling found in France at Coligny and introduced by M. Massot, a nurseryman near Lyon, sometime before 1856. 'Count Altham', a cultivar raised in Bohemia, was introduced between 1850 and 1860. E. America 1. Native Species and Cultivars. In 1621, Edward Winslow mentioned

that in Massachusetts plums were used by the Pilgrims, which, according to Sturtevant's notes (Hedrick 1919), was likely a reference to the beach plum (P. maritima). The native American plum (P. americana) was planted by the New England Indians from the early times and the western Indians collected large quantities for drying (Pickering 1879). Plum improvement in America started in the early 19th century with the selections of native American plums, and a great number of cultivars were introduced during the last two decades of the 19th century. The first American plum named 'Miner' (P. hortulana) was found on a Chickasaw Indian reservation in Alabama in 1813. It was soon followed by the most important cultivar among the native American plums. In 1820, a seed of munsonina plum was found in the craw of a goose and planted by M. E. McCance, Nashville, Tennessee. The resulting tree, named 'Wild Goose', produced exceptional fruit and achieved the largest early distribution among American plums. A hybrid of P. cerasifera, the Marianna plum, was introduced in 1884 by Charles Eley, Smith Point, Texas. The originator considered Marianna to be a seedling of Wild Goose, one of the Munsoniana plums, probably fertilized by P. cerasifera. Marianna is a good rootstock for plums. It can be propagated by cuttings but unfortunately produces root sprouts (Yoshikawa et al. 1989).

M. FAUST AND D. SURANYI

216

•

A

B

c

D

Fig. 4.13. Plum illustrations in Pomona ItaJiana by Giorgio Gallesio, 1839. A. 'Basaricatta', fruit is yellow, flesh is yellow. B. 'Verdacchia', fruit is green, flesh is yellow. C. 'Claudia', fruit is greenish-yellow, flesh is green. D. 'Damaschina d'Estate', fruit is yellow with red overlay.

4. ORIGIN AND DISSEMINATION OF PLUMS

217

There were a few individuals who were instrumental in the improvement of American plums. In 1857, H. A. Terry (1826-1909) established a nursery in Crescent City, Iowa. He was greatly interested in American plums and during his lifetime introduced over 50 cultivars of P. americana, P. hortulana, and P. munsoniana types. C. G. Patten, another private breeder and a contemporary of Terry, attempted to improve native plums at Charles City, Iowa, in 1867, but his only introduction was 'Patten', a P. americana species that was classified by Hedrick (1911) as a plum of minor importance. J. W. Kerr, who operated a nursery beginning in 1870 at Denton, Maryland, was also interested in American plums. He introduced three cultivars-'Sophie', 'Choptank' and 'Maryland'-and popularized American plums (Cullinan 1937). Hedrick (1911) described many important cultivars of American plums (Table 4.5.).

Cullinan (1937) gives a complete description of State Experimental Stations involved in the early improvement of plum cultivars. The improvement of native American species continued at least until the 1960s. Okie and Weinberger (1996) list the introductions involving American species hybrids. Improvement of native American species took on importance in the North, where the hardiness of the native Table 4.5. Time of major activity when native American plum species were improved and new cultivars introduced. Year after the name indicates the time of introduction. Hortulana cultivars 'Forest Garden' 1862; 'Golden Beauty' 1874; 'Wayland' 1875; 'Forest Rose' 1878; 'Moreman' 1881; and 'Maquoketa' 1889. Americana cultivars 'Rollingstone' 1852; 'DeSoto' 1853-54; 'Garden King' 1853; 'Gaylord' 1854; 'Orem' 1878; 'Hawkeye' 1882; 'Blackhawk' 1899; 'New Ulm' 1890; 'Gale' 1890; 'Cherokee' 1892; 'Deep Creek' 1892; 'Craig'1900; and 'Golden Queen' 1905. Munsoniana cultivars 'Robinson' 1835; 'Pottawattamie' 1875; 'Pool Pride' 1885; 'Downing' 1885; 'Sophie' 1899; 'Freeman' 1893; and 'Flemming' 1901. Nigra and Angustifolia cultivars 'Caddo Chief' 1887. Mixed species Hybridized by N. E. Hansen, American plums with Japanese plums and with P. Bessey, simoni, and cerasifera. 'Cheresoto', 'Cistena', 'Ztopa', 'Hanska', 'Inkpa', 'Kaga', 'Opata', 'Sapa', 'Wakapa', 'Wohanka', and 'Yuteka', among others introduced between 1808-1810.

218

M. FAUST AND D. SURANYI

species was needed. Centers in the United States that improved native species were located at Brookings, South Dakota; Mandan, North Dakota; and Excelsior, Minnesota; and in Canada at Brooks, Alberta; Brandon and Morden, Manitoba; Rosthern and Saskatoon, Saskatchewan; and Ottawa, Ontario. The introduction of native American species hybrids continued until about 1960. After this time there were only occasional introductions and the improvement of native American species essentially ceased. The native American plums also made their mark on large-scale production. Many species of plums were native to Texas and several of the early horticulturists were enthusiastic about the opportunities for commercial plum growing there. In the 1880s, Gilbert Oderdonk considered using the native chickasaw plum (P. angustifolia) for production for local markets. T. V. Munson introduced the 'Munson' cultivar, an excellent plum, but because of its yellow color, soft texture, and susceptibility to brown rot, it never gained importance. In 1877, A. L. Bruce began crossing Japanese plums with the native chickasaws. By 1900 he had released 14 cultivars. The most remarkable of all was named 'Bruce'. 'Bruce' was pollen sterile, but interplanting it with native plums and providing pollinators assured its fruitfulness. Commercial plum growing in Texas was based on 'Bruce' (Brison 1976). In 1904, plum production in Georgia numbered 900,000 trees, based mostly on 'Wildgoose', but it slowly changed to the new Japanese hybrids (Starnes 1904). 2. Imported Cultivars. In 1629, Francis Higginson recorded that the governor of Massachusetts planted a vineyard and fruit trees, including plums. According to John Josselyn (1865), writing of a voyage to New England in 1663, damsons were the only plums cultivated at that time. Importation of plums to America was also made by the French in Nova Scotia, Prince Edward Island, and the Island of Montreal in the S1. Lawrence River (Hedrick 1911). In his history of North Carolina, written in 1714, Lawson noted that damsons, 'Damazeen', and a large round plum were grown in that state. John Bartram was the first person in Pennsylvania to grow plums, which he located in the Bartram Botanical Garden near Philadelphia in 1728. He had the 'Great Yellow Sweet Plumb', 'Crimson Plumb', 'Chicasaw Plumb', 'Beach or Sea-Side-Plumb', and 'Dwarf Plumb' listed in his garden. In a separate part of the list of the "Catalogue of American Trees and Herbaceous Plants" grown in John Bartram's garden, Prunus americana, P. chicasaw, and P. maritima were mentioned (Reveal 1996). Plums were also introduced to Florida in 1763 at the colony at New Smyrna. In 1730, Robert Prince established his famous fruit tree nursery on Long Island, New York, and

4. ORIGIN AND DISSEMINATION OF PLUMS

219

started to propagate fruit trees. His catalogs mention plums for the first time in 1767, and yet his 1774 advertisement does not mention them. Charleston, South Carolina, had nurseries that distributed plums to the southern states in 1786. The Prince nursery started to offer named cultivars of plums in 1794. In 1797, Samuel Deane, in his book, The New England Farmer or Geological Dictionary, speaks about the horse plum with a pleasant taste, the pear plum that had a distinct pear shape, the wheat plum that had a furrow in the middle, and the green-gage plum that was generally preferred among all the plums. In 1773, Thomas Jefferson made the first notation about plums in his garden book. He noted that he sent Patrick Morton several "slips" of fruit, including a 'Green Gage' plumb. He made additional notes from 1778 to 1814. Among the plums he had were 'Mogul', 'Egg plum', 'Magnum Bonum', 'White Imperial', 'Horse', 'Cherokee', 'Chickasaw', 'Damson', and 'Green Gage' (Baron 1987). The improvement of imported plums began soon after their introduction. William Prince (1828), the third proprietor of the Prince nursery, recorded in his A Short Treatise on Horticulture that his father planted 25 quarts of seeds of 'Green-Gage' in 1790 and the seedling trees produced fruit of every color. Cultivars, well known at that time, that came from this progeny were the 'White Gage', 'Red Gage', 'Prince's Gage', and perhaps the 'Washington'. By 1828, the Prince nursery offered 140 cultivars of plums. Even though the successive owners of the Prince nursery promoted plums, the greatest impetus of plum growing in America, according to Hedrick (1911), was created by William Robert Prince (1795-1869), the fourth proprietor of the Prince nursery, whose writings were characterized by a clear, vigorous style and by accurate statements as he disseminated information about plums. Other nurseries also promoted plums. The Mount Hope Nursery in Rochester, New York, commissioned the best botanical artist at the time to produce lithographs of fruits, including plums, that their salesmen could use when making presentations to customers (Fig. 4.14). In 1858, Joseph Zettel of Switzerland acquired a farm on Door Peninsula, Wisconsin. He established the first commercial orchard there. His high yields and good-quality fruit aroused the interest of Emmett S. Goff and Arthur L. Hatch, who planted an orchard of cherries, apples, and plums in 1892 (Klingbeil 1976). The largest orchard in South Dakota was planted by Mrs. Hurley in 1870. The 52-ha orchard consisted primarily of apples, but also included plums. In 1891, Professor T. A. Williams discovered and collected seed from several superior wild plums in the Badlands of South Dakota. Professor N. E. Hansen was appointed head of horticulture at South Dakota

M. FAUST AND D. SURANYI

220

A

B

c

D

Fig. 4.14. Plums painted by America's premier botanical illustrator, Joseph Prestele. A. Chicasaw plum, lithograph, completed in 1850 for the planned but never completed work of Asa Gray, The Forest Trees ofNorth America. Color of fruit is red. B. Nebraska Seedling Plum (1) and Thompsons Golden Gem Plum (2). Lithograph. Issued by Mount Hope Nursery, Rochester, New York. Fruit color is yellow with red overlay on the sunny side. C. Mc Laughlin Plum. Lithograph. Issued by Mount Hope Nursery Rochester, New York, in 1871. Fruit is yellow, overlayed with red. D. Lawrence'~ Favorite. Unsigned lithograph. Attributed to Gottlieb Prestele, son of Joseph Prestele. Fruit color is dull yellowish-green covered with green bloom.

4. ORIGIN AND DISSEMINATION OF PLUMS

221

State University in 1895 and began his plum improvement program, using native species, which lasted for overtwo decades (Peterson 1976). Indiana had about 700,000 plum trees in 1900 (Tukey 1976). In Iowa, there were sizable plum orchards near Sioux City between 1880 and 1935 (Nichols 1976). In Colorado, the first plantings of plums were made by W. Lee in 1862 east of Golden along Clear Creek. A flood in 1864 washed out all the trees. In 1870, a new shipment of 15,000 trees from Iowa arrived and were planted. By 1877 good crops were being produced, but some plum cultivars and chestnut trees did not survive (Ure and Binkley 1976). In the early days of California, native plums (P. subcordata) were frequently cultivated and, before the introduction of standard European cultivars, attempts were made to improve the fruit of this species by selection. The early Mission plantings (1769-1823) included European cultivars, a few of which were able to survive the abandonment of the Missions in 1834. One cultivar found at Mission Santa Clara was grown and marketed as "Mission prune" as late as 1870. The introduction of improved plum cultivars dates back to 1851, when the first grafted fruit trees were brought to California from Oregon by Seth Levelling. The first importation of prune scions from France by the United States Patent Office in 1854 did not reach California. Two years later, Pierre Pellier, a sailor, brought scions from the famous prune district of Agen in France with him to San Francisco. The scions were grafted in Pellier's brother's nursery in the Santa Clara Valley upon a site that is today the city of San Jose. The prune industry was established in the Santa Clara Valley. In 1863, the first California-grown and cured prunes were exhibited at the State Fair in Sacramento and commercial-scale plantings started in 1870. There was disappointment over the fact that the prunes of 'Agen' were smaller than the prunes marketed by the French. Because of the small size ofPellier's introduction, they christened that cultivar "petite prune d'Agen," and the question was raised whether they had the real prune of'd'Agen'. California growers tried several other larger fruited cultivars, including 'Pond', which was unsatisfactory because of its low sugar content. The dispute finally was settled in 1878 when W. B. West of Stockton visited France and determined that the California cultivar was the real prune of 'd'Agen' (Wickson 1914). For about a decade, plantings were increased, and in 1881 several growers produced 5 to 6 tons of cured fruit. The introduction of the French cherry-plum, myrobalan (P. cerasifera), as a rootstock greatly increased the productivity of the orchards. As time progressed, the prune industry increased. In 1870, there were 260 ha of plums produced for prunes. By 1900 the number had increased to 36,000 ha and by 1926 to 77,400 ha, the all-time high

222

M. FAUST AND D. SURANYI

in the prune industry. The production area slowly decreased to 32,000 ha by 1960 and fluctuated around 30,000 ha in the 1970s. At the head of the list of cultivars was the 'Prune d'Agen', the originally introduced French prune, which proved itself to be well adapted to various conditions and was widely planted in the area. However, there was an interest in large fruited cultivars before 1940. The non-French cultivars comprised 16.7 % of the production in 1940; their proportion decreased to 4.4% by 1978. Mechanical harvesting favored 'French Prunes' because the fruit of this cultivar could be harvested by machines without injury and the tree required no thinning. The large fruited cultivars required hand thinning, which slowly increased the cost of production to an uneconomic level. By 1981, the prune industry became essentially a onecultivar industry based on the 'French Prune' (Chaney 1981). Japanese plums were introduced to California in 1870 by Mr. Hough of Vacaville. John Kelsey of Berkeley, California, produced the first ripe fruit ofJapanese plums in 1877 and 1878. The first wide distribution of Japanese plums fruited by John Kelsey was made by W. P. Hammon & Co. in 1884, which named the fruit after Mr. Kelsey. About the same time, Luther Burbank (1849-1926) started to operate in Santa Rosa, California. Burbank purchased a small farm there and established his breeding activities in 1875. Before Burbank started to improve Japanese plums, only two cultivars, 'Kelsey' and 'Chabot', were available. Burbank produced hundreds of thousands of plum seedlings using practically all plum species under cultivation and selected a dozen or more excellent types. Howard (1945) lists over 100 plum introductions by Burbank, some of which he imported from Japan, but the majority of which were produced by his own hybridization or from open pollination. In 1885, Burbank imported a cultivar with the help of Isaac Bunting, an export-import merchant in Yokohama. It was one of the plums Burbank read about in a travel book by a sailor. It was known as the 'Blood Plum of Satsuma', because the entire inside of the fruit was red. The name later was changed to 'Satsuma' (Howard 1945). Burbank introduced 'Santa Rosa' in 1907, the most important cultivar of this century. 'Santa Rosa' is a complex hybrid purportedly containing P. salicin a, P. simonii, and P. americana, with the salicina character predominating. One of its likely ancestors was 'Satsuma' because 'Santa Rosa' acquired its reddish flesh from some of its parents and 'Satsuma' was the only plum carrying this character at that time. In 1914, Burbank regarded 'Santa Rosa', 'Formosa', 'Beauty' and 'Wickson' as his greatest introductions. Several other breeders continued the work of Burbank. Okie and Weinberger (1996) list the breeders involved in the improvement. In contrast to

223

4. ORIGIN AND DISSEMINATION OF PLUMS

prune production, where cultivars did not change, plum cultivars changed as new cultivars became available (Table 4.6). With the exception of the month of August, at least one or more cultivars ripen weekly, which assures an extended marketing season for plums. Production of Japanese hybrids maintained a planting level of 10,000 ha between 1945 and 1970. Beginning in 1970, the Japanese plum production increased to 14,000 ha and remained at that level through the mid-1980s. During this period, the percentage of'Santa Rosa' decreased from 36 to 70/0 and large black plums, such as 'Friar' and 'Blackamber', gained consumer acceptance. Johnson and LaRue (1989) compiled the changes in plum cultivars between 1945 and 1988 and Okie and Weinberger (1996) listed those in use in 1994. Their lists of cultivars are presented in Table 4.6. The new cultivars lengthened the harvest season, extending it to 16 weeks from the fourth week of May to the third week of September.

Table 4.6.

Major plum cultivars in California from 1945 to 1994.

1945

%

1955

%

1965

%

Santa Rosa Beauty President Duarte Wickson Tragedy Giant Kelsey Burbank Formosa

36 14 10 1.0 5 4 4 3

Santa Rosa Beauty Duarte President L.Santa Rosa Wickson Late Duarte Kelsey EI Dorado Burbank

35 14 12 9 4 3

Santa Rosa Laroda EI Dorado L.Santa Rosa Nubiana Beauty President Burmosa Duarte Queen Ann

31 10 10 10 6 6 4 4

1975

%

1988

%

1994

%

Santa Rosa Casselman Laroda L.Santa Rosa Red Beaut EI Dorado Simka Nubiana Royal Diamond Friar

20 14 10 9 6 6

Friar Red Beaut Santa Rosa Blackamber Casselman Angeleno Queen Ann Simka Black Beaut EI Dorado

18 12 10 8 6 5 4 5 5

Friar Blackamber Angeleno Santa Rosa Red Beaut Simka Laroda Casselman Black Beaut Kelsey

20 12 10 7 7 5

2 2

5

5 3 3

3

3 3 2

3

3

4

4

4 2

224

M. FAUST AND D. SURANYI

IV. CONCLUSIONS

From the developmental point of view, we conclude that two major species provided the basis of the present plum industry. These two species emerged on separate continents-Po domestica in Europe and P. salicin a in Asia. Neither species has wild progenitors and both entered into human use remarkably highly developed. The time period during which these species emerged is relatively recent. Archeological data indicates the widespread use of plums during the Neolithic period, but these were the small damsons or cherry plums, adequate only in rudimentary conditions. The garden plum and the Japanese plum emerged about 300 B.C. as important species. Why and how these plums were selected ten thousand miles apart at about the same time continues to baffle the imagination. Fruits are important to mankind as a source of delicious food and have become admired for their beauty. They have become part of our cultural heritage. Among the stone fruits, peaches and cherries were used in names of localities and artists used them as the subject of their art. There are decorations of codices, frescoes, paintings, and floors produced since the beginning of the Roman empire involving not only the blossoms but also the fruit of these species (Faust and Timon 1995; Faust and Suranyi 1997). Many other fruits such as apples, pears, quinces, and pomegranates are used in cornucopia as an expression of abundance. Plums have been the subject of artistic expression in much fewer cases. Prunes are excellent for health because they help promote digestive regularity. The mention of prunes even today is met with a response of muffled hilarity in the United States. Yet, at the same time, the virtues of plums are recognized in the use of the term to signify the best of everything or a bonus, such as in the saying that someone has a "plum" job. The kinds of plums that are eaten is also a complicated manner. Even though some of the native American plums are yellow and many of the plums of the 19th century were yellow (Ravenswaay 1984), yellow plums are not marketed in America. Today, plums in America are mostly blue, black, or dark red, with a few bright reds becoming popular. In Europe, the yellow or green Reine-Claude gages or Mirabelles are accepted and sometimes even preferred over the blue or red prunes. In Asia Minor, the red or yellow colors of cherry plums are completely acceptable. One must raise the question then as to what was enticing to the American public about the dark colors. Was it a conscious selection by the people or was the dark color merely the preference of Luther Bur-

4. ORIGIN AND DISSEMINATION OF PLUMS

225

bank, who was very successful at breeding plums and whose legacy has influenced the American plum culture. Black plums, especially the cultivars 'Friar' and 'Blackamber', became successful because they produce large fruit, are very productive, and do not show bruises, as the yellow plums or red plums that have yellow shoulders do. Although the garden plum was used for drying in Asia Minor and Central Europe, the rate of drying never came close to the level achieved in apricots. A major change occurred when prunes entered California and processing by sun drying began. Today, a very high percentage of the world production of dried prunes (760/0) is produced on the west coast of the United States, especially in California.

LITERATURE CITED Abercrombie, J. 1779. The British fruit gardener. London. Asch, D. L., and N. B. Asch. 1985. Archeobotany. p. 327-388. In: B. D. Stafford and M. B. Sant (eds.), Smiling Dan. Structure and function at a Middle Woodland settlement in the Lower Illinois Valley. Kampsville Archeological Center, Kampsville, IL. Baas, J. 1951. Die Obsarten aus der Zeit des Romerkastells Saalburg im Taunus bei Bad Homburg v.d. H. Bericht des Saalburgsmuseums 10:14-28. Baas, J. 1971. Pflanzenreste aus romerzeitlichen Siedlungen von Mainz-Weisenau und Mainz-Innenstadtund ihr Zusammenhang mit Pflanzenfunden aus vor- und fruhgeschichtlichen Stationen Mitteleuropas. Saalburg-Jahrbuch 28:61-87. Baas, J. 1974. Kultur- and Wildpflanzenreste aus einem romischen Brunnen von RotweilAltstadt. Fundberichte aus Baden-Wurttemberg 1:373-416. Bailey, L. H. 1927. The standard cyclopedia of horticulture. Macmillan, London. Bajashvili, E. I. 1990. Studies of some species of Prunus Mill. Genus. Acta Hort. 238:31-34. Baldini, E. 1990. Fruits and fruit trees in Aldrovandi's Iconographia Plantarum. Adv. Hort. Sci. 4:61-73. Baldini, E., and A. Tosi. 1994. Scienza e arte nella Pomna Italiana di Giorgio Gallesio. Acad. Georgofili, Firenze. Baron, R. C. 1987. The garden and farm books of Thomas Jefferson. Fulcrum Inc., Golden. Colorado. Bauhin, Caspar (= Bavhini. Caspari). 1623. Pinax Theatri Botanici, Basel. Bentham, G., and J. D. Hooker. 1924. 7th ed. revised by A. B. Rendle. Handbook of the British Flora. Reeve &Co.• London. Beridze, R. K., and M. V. Kvatchadze. 1981. Origin and evolution of cultivated plums in Georgia. Kulturpflanze 29:147-150. Bertsch, K., and F. Bertsch. 1949. Geschichte unserer Kulturpflanzen. Vissenschaft, Stuttgart. Blanchet. P. 1996. La prune Rheine-Claude, historie et genetique. L'Arborie. Fruit. 497:23-26.

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Knorzer, K. H., and G. Miiller. 1968. Mittelalterliche F8kalien-Fassgrube mit Pflanzenresten aus Neuss. Beihefte zum Bonner Jarbuch 28, Rheinische Ausgrabungen 1, Beitriige zur Archaologie des Mittelalters. p. 131-169. Koch, K. 1876. Die Deutchen Obstgeholze. Stuttgart. Korber-Grohne, U. 1996. Pflaumen, Kirschpfaumen Schelen. Thiss, Stuttgart. Kos, K. 1980. Eszkoz, munka, nephogyomany. Kriterion, Bucharest. Kovalev, N. V. 1939. Die okologische Differenzierung der Kirschpflaume, Prunus cerasifera. Dokl. Akad. Nauk. SSSR New Ser. 23:285-288. Kovalev, N. V. 1941. Prunus L. p. 515-517. In: V. L. Komarov, B. K. Siskin, and S. V. X. Juzepczuk (eds.). Flora SSSR. Vol. 10. Izd. Akad. Nauk., Moskva. Kraft. J. 1792. Pomona Austriaca. Abhandlungen von dem Obstbaumen. Wienna. Kroll, H. 1980. MiUelalterlich- fruhneuzeitliches Steinobs aus Liibeck. Lubecker Schriften zur Archaologie und Kulturgeschichte 3:167-173. Kiippers, H. 1976. What is the St. Julien plum rootstock. Dtsch. Baumsch. 28:194-199. DNAL Transl. 32612. 1982. 26 leaves. Lamarck, J., and J. Poiret. 1804. Encyclopedie methodique botanique. Vol. 5. Paris. Langley, Batty. 1729. Pomona or the fruit garden illustrated. London. Lawson, J. 1714. The history of Carolina containing exact description and natural history of that country. London. Li, Shaohua, Xingguo Xiao, and Patrice Blanchet. 1997. II potentiale genetico del susino cinese. Frutticoltura 49:25-28. Lin. P., and L. Shi. 1990. The discovery and distribution ofIli wild P. domestica (P. communis Fritsch) in Xinjiang. p. 282-286. In: Proc. Int. Symp. Hart. Germplasm, Cultivated and Wild. Chin. Soc. Hart. ScL, Beijing. Linneaus, C. (Linne). 1753. Species plantarum. Ray Soc. Pub!. 140. Facsimile ed. 1957. Lippai, J. 1664. Posoni kert. Facsimile ed. Akademiai kiadb, Budapest. 1966. Maier, S. 1988. Botanische Unersuchung romerzeitlicher Pflanzenreste aus dem Brunnen der romischen Zivilsiedlung Kangen (Landkreis Esslingen). p. 291-324. In: Kiinster (ed.), Der priihistorische Mensch und seine Umwelt. Cited by Korber-Grohne 1996. Mason, S. C. 1913. The pubescent fruited species of Prunus of the southwestern States. J. Agr. Res. 1:147-178.

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Matsumoto, Kosai.1977. The mysterious Japanese plum. Woodbridge Press, Santa Barbara. Miller, P. 1731. The gardener dictionary. London. Murray, J. H. H. 1888. A new English dictionary on historical principles. Clarendon Press, Oxford. Neuweiler, E. 1935. Nachtrage urgeschichtlicher Pflanzen,-Vierteljahrschrift der naturforschenden Gesellschaft in Zurich 80:98-122. Nichols, H. E. 1976. Iowa. p. 54-58. In: D. V. Fisher and W. H. Upshall (eds.), History of fruit growing and handling in United States of America and Canada 1860-1972. Am. Pomol. Soc. University Park, PA. Odier, G. 1993. Faunt-il encore ameliorer la prune d'Agen. L'Arboricult. Fruit. 460:32-36. Okie, W. R. 1987. Plum rootstocks. p. 321-360. In: R. C. Rom, and R. F. Carlson (eds.), Rootstocks for fruit crops. Wiley, New York. Okie, W. R., and J. H. Weinberger. 1996. Plums. p. 559-607. In: J. Janick and J. N. Moore (eds.), Fruit breeding, Vol. 1: Tree and tropical fruits. Wiley, New York. Opravil, E. 1963. Rostlinne nalezi z archeologickeho vyzkumu steredoveke Opavy provadeneho v roce 1961. Zvlastoniotisk z Casopis Slezskeho musea. Serie B. Historia, 12:18-29. Opravil, E. 1964. Rostliny ze Stredovekych nalezu v Ostrove. Zvlastni oUsk z Casopis Slezskeho musea. Serie B. Historia 13:9-12. Opravil, E. 1976. Archeobotanicke nalezy z m~stskeho jadra Uherskeho Brodu. Studie Archeologickeho Ustavu Tceskoslovenske Akademie ved V Brne 3:3-59. Opravil, E. 1985. Rostlinne zbytky z areali Byv alneho Bratvskeho sboru v Ivanicich. Venkov v Ivanicich. p. 61-74. Okresni Museum, Brno. Opravil, E. 1986a. Rostlinne makrozbytky z historickeho jadra Prahy. Archeologica Pragensis 7:237-271. Opravil, E. 1986b. Archeobotanicke nalezy z arealu jaktarske brany v Opave. Ces. Slez. Muz. Opava (A) 35:227-253. Opravil, E. 1988. Mittelalterliche pflancenreste aus stadtischen und dorflichen Brunnen und Gruben. p. 389-394. In: Kunster (ed.), Der prahistorische Mensch und seine Umwelt. Quoted by U. Korber-Grohne. Parkinson, J. 1629. Paradisi in Sole. Paradisus Terrestris. London. Peterson, R. M. 1976. South Dakota. p. 123-126. In: D. V. Fisher and W. H. Upshall (eds.), History of fruit growing and handling in United States of America and Canada 1860-1972. Am. Pomol. Soc. Publ., University Park, PA. Pickering, C. 1879. Chronological history of plants: Man's record of his own existence illustrated through their names uses and companionship. Boston. Plinius (Pliny the Elder). 79. Natural history. Transl. by H. Rackham and W. H. S. Jones. London. Vol. VII. 2nd. ed. 1980. Pompeiana, Raccolta di stury per il secondo centennario degliscavi di Pompei. 1950. Gaetano Macchiaroli Editore, Napoli. Prince, W. 1828. A short treatise on horticulture. New York. Prince, W. R. 1831. The pomological manual, or a treatise on fruits. 2nd ed. New York. Ramming, D. W., and V. Cociu. 1990. Plums. p. 235-287. In: J. N. Moore and J. R. Ballington (eds.), Genetic resources of temperate fruit and nut crops. Acta Hort. 290. Int. Soc. Hort. Sci., Wageningen. Rapaics, R. 1940. A magyar gyiimolcs. Budapest. Ravenswaay van, C. 1984. Drawn from nature. The botanical art of Joseph Prestele and his sons. Smithsonian Press, Washington. Ray J. 1688. Historia plantarum, London. Rea, J. 1676. Flora: Seu, De Florum Cultura; or a complete florilage. Marriott, London.

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5 Loquat: Botany and Horticulture Shunquan Lin * Institute of Subtropical Fruits, Fujian Agriculture University, Fuzhou 350002 China Ralph H. Sharpe Horticulture Sciences Department, University of Florida, Gainesville, Florida, 32611-0690 Jules Janick Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana, 47907-1165 I. Introduction A. Origin and History B. World Production II. Botany A. Taxonomy B. Morphology and Anatomy C. Embryology III. Physiology A. Growth and Development B. Chemical Composition C. Plant Growth Regulation 1. Growth Control 2. Pollen Germination 3. Fruit Set 4. Fruit Thinning 5. Induction of Seedlessness D. Sorbitol Physiology E. Temperature Response F. Medicinal Value *1 thank Prof. N. Nito of Saga University, Japan, for his encouragement and constructive comments.

Horticultural Reviews, Volume 23, Edited by Jules Janick ISBN 0-471-25445-2 © 1999 John Wiley & Sons, Inc.

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IV. Horticulture A. Crop Improvement 1. Ploidy Manipulation 2. Hybridization and Selection 3. Biotechnology B. Propagation 1. Seed 2. Vegetative C. Field Culture 1. Orchard Establishment 2. Training and Pruning 3. Flower and Fruit Thinning 4. Water and Soil Management and Fertilizers 5. Tree Protection 6. Harvesting and Handling D. Protected Culture E. Storage and Processing V. Future Prospects A. Crop Improvement B. Culture and Utilization Literature Cited

I. INTRODUCTION

Loquat (Eriobotrya japonica Lindl., Rosaceae, Maloideae) is a subtropical evergreen fruit tree that blooms in fall and early winter. The tree is cold-hardy to -10°C (12°F), but fruits are frozen by winter minimum temperatures of about -3°C (27°F). In the Gulf region of the southern United States, and in many other countries, the tree fruits irregularly but is grown for its handsome foliage (McConnell 1988). Loquat is now commercially produced in many countries (Table 5.1). Fruits can be consumed fresh or processed and can be used for jam, juice, wine, syrup, or as candied fruits (Liu 1982); seeds are rich in starch (200/0) and have been used to make wine. Leaves and fruits of loquats traditionally have been considered to have high medicinal value (Duke and Ayensu 1985; Wee and Hsuan 1992) and there is evidence of pharmaceutically active compounds (Yang 1984; Shimizu et al. 1986; Morton 1987; Noreen et al. 1988; Chen et al. 1991; DeTommasi 1992b). Loquat is highly nectariforous, with a heavy fragrance and high honey potential (Yu 1979). Its wood is pink, hard, close-grained, and medium heavy (Morton 1987). Since Popenoe (1920) wrote a chapter on the basic knowledge of loquat, there have been publications in several languages on various aspects of loquat, including chemical compositions (Shaw 1980) and cultivars in countries other than China (Morton 1987). There are popu-

5. LOQUAT: BOTANY AND HORTICULTURE

235

Table 5.1. Loquat production statistics in selected countries. Source: Fujisaki 1994; Monastra and Insero 1991. CountryZ Location

Area (1,000 hal

China Fujian Zhejiang Taiwan Jiangsu Anhui Jiangxi Sichuan Other Japan Italy Brazil Spain

25.9

Production (1,OOOt) 102.0

11.9 9.1 2.5 0.6 0.5 0.4 0.4 0.5 2.8 NAY 0.3 est 3.9

35 35 13 5 4 3 3 4 13 7

NA NA

zOther countries with some commercial production include India, Turkey, and Israel. The United States has only home garden production. YNA =not available.

lar books on loquat in Chinese (Chen 1988; Wang 1989) and in Japanese (Ichinose 1995). The objective of this chapter is to review the botany and horticulture of loquat and to summarize recent research, with emphasis on Chinese and Japanese investigations. A. Origin and History Records on loquat in China span over 2,000 years (Sima 100 B.C.); there many loquat species occur in the wild state (Zeng 1937; Chen 1954; Zhang 1987; Zhang et al. 1990). The loquat cultivated in Japan was introduced from China in ancient times and loquat cultivation in Japan was described as early as 1180 (Ichinose 1995). Because Japan had been considered the original region ofloquat by Thunberg (1784), the species was named as Mespilis japanica. Since some primitive types of E. japanica occur in several prefectures in Japan, some Japanese authors consider the origin to be both China and Japan (Fujisaki 1994; Ichinose 1995). Most authors around the world now believe loquat originated in China (Popenoe 1920; Shaw 1980; Morton 1987; Zhang 1987, 1990; Campbell and Malo 1986), but the definite region of origin is unknown. Morton (1987) described loquat as indigenous to southeastern China. In fact, various species of Eriabatrya are found in southwestern China. In the 1960s,

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S. LIN, R. SHARPE, AND J. JANICK

a large group of previously unknown Eriobotrya plants were found in the Dadu River Valley, located on the southern slopes of the mountain Gongga in western Sichuan and named E. prinoides Rehd. & Wils var. daduneensis H. Z. Zhang (Zhang et al. 1990). The Dadu River Valley is now considered the center of origin for the genus Eriobotrya in China, and a great number of indigenous communities of E. japonica, E. prinoides, and E. prinoides var. daduneensis are distributed in the 'middle and lower reaches of the river valley (Zhang et a1. 1990). People beyond eastern Asia first learned of the loquat from Kaempfer, who observed it in Japan and described it in Amoenites Exotica in 1712, while the Swedish botanist, Thunberg, in Flora Japonica (1784), provided a more ample description of loquat under the name Mespilus japonica. In 1784, the loquat was introduced from Guangdong into the National Garden at Paris, and in 1787 was introduced into the Royal Botanical Gardens at Kew, England (Condit 1915; Liu 1982). From this beginning, loquat was distributed around the Mediterranean to various countries, including Algeria, Cyprus (Cyprus Agricultural Research Inst. 1987), Egypt, Greece, Israel, Italy, Spain, Tunisia, and Turkey (Demir 1983; Morton 1987). Sometime between 1867 and 1870, loquat was introduced to Florida from Europe and to California from Japan. Chinese immigrants are assumed to have carried the loquat to Hawaii (Morton 1987). By 1915, it had become quite well established in Florida and southern California and several new cultivars had been named. In that year, Condit published 33 pages of information on the culture of loquat in California Experiment Station Bulletin 240. Cultivation spread to India and southeastern Asia, the East Indies, Australia (Goubran and EI-Zeftawi 1983), New Zealand (Burney 1980), Madagascar, and South Africa. Loquats are now distributed in many Asian countries, for example, Laos, Nepal, Pakistan, South Korea, and Vietnam; in Armenia, Azerbaijan, and Georgia (Safarov 1988); and in the Americas, including Argentina, Brazil, Chile, the mountains of Ecuador, Guatemala, Mexico, and Venezuela (Endt 1979). Generally, loquats are found between latitudes 20 and 35 0 North or South, but can be cultivated up to latitude 45 0 under maritime climates. B. World Production

The major producing countries are China and Japan (Table 5.1). Loquats are grown in 19 provinces of China, ranging from the Yangtze River to Hainan Island (south of Hong Kong). Loquat is frequently sold at local markets in China during the fruiting season, from May to June, at a

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237

moderate price that is higher than that for citrus and banana, lower than for longan and litchi, and usually similar to that for apple and pear. In high producing areas such as Fujian and Zhejiang, fruits are shipped to Hong Kong and Shanghai, and are increasingly popular for canned products. In some areas, loquat is confined to home gardens. Loquat was cultivated on 1,700 ha in 1949 but production has increased dramatically in recent years due to the introduction of high-yielding and goodquality cultivars that ripen early enough for the fresh market. Loquats are concentrated in Japan's warm districts, including Kyushu and Shikoku, and Chiba, Hyougo, Wakayama prefectures of Honshu. Loquat is often the most expensive fruit on the market, reflecting the high cost of production, and is now marketed over a very long season. Marketing begins in January with small amounts of the fruit and ends in July, but in exceptional years, a few fruits are marketed in November and December. More than 500/0 of the fruit is marketed in June. Marketing in April is increasing because of the increase in early cultivars growing under protected facilities (Fujisaki 1994). Japan was a leading producer of loquat from the beginning of this century to World War II. The crop area amounted to 4,162 ha in 1934, but declined during and after World War II, and was replaced by citrus. Protected culture has increased since the 1970s (Ichinose 1995). Loquats are grown in northern areas of India. In Italy, loquat production is located in the central and southern coasts, and loquats are cultivated commercially in a small area near Palermo (Monastra and Insero 1991). Loquats are grown on a small scale in southeastern Spain (Galan Sauco 1986; Farra Massip 1993).

II. BOTANY A. Taxonomy Thunberg first described loquat in 1784 and placed it in the genus Mespilus. In 1822, the English botanist, John Lindley, revised the genus Mespilus, and established loquat in a new genus, Eriobotrya (from Greek, erio-, wool, and botrys, a cluster, referring to the woolly, clustered panicles) (Condit 1915; Huxley 1992). The specific epithet japonica was based on Thunberg's belief that the origin of loquat was Japan. In Chinese, loquat has two common names, Juju and biba (southern Chinese) or pipa (northern Chinese). The Japanese name of loquat, biwa, is undoubtedly derived from the southern Chinese name, biba. Loquats cultivated in Japan were called Tang Biwa after the Tang Dynasty,

238

S. LIN, R. SHARPE, AND J. JANICK

618-907. Common names of loquat in various languages in the world are

often derived from the China name or loquat's former scientific name, mispiJus, or medlar. The English name, loquat, is derived from the Chinese luju , while the present names in French are bibassier, derived from biba, or neflier du Japon, literally medlar of Japan. The names in Spanish, German, and Italian are all derived from mespilus, e.g. nispero japones, japanische mispel, and nespola giapponese, respectively (Morton 1987). In Portugal, loquat is called ameixa do Japao, or plum of Japan, and in Florida it was once called Japan plum or Japanese medlar. The number of loquat species is under dispute and the opinions of authors in different countries vary. In Japan, 20 species in the genus are estimated, but only 11 species have been well described (Ichinose 1995). The New Royal Horticultural Society Dictionary of Gardening lists some 10 species of evergreen shrub or trees (Huxley 1992). In several Chinese references, more than 30 species are listed, of which 14 species have originated in China and are fully described (Yu 1979; Chen 1988; Zhang et a1. 1990). However, three species that have been thought to be distributed in China by non-Chinese authors were not included in Chinese publications. This situation can be attributed to the confusion involving genera of Rosacese. Eriobotrya is often confused with Mespillus, and sometimes with Crataegus and Photinia. For example, E. grandiflorapy has been placed in the" genus Mespillus, E. henryi in Crataegus, and E. prionphylla in Photinia. The 16 loquat species and three botanical varieties, which are clearly established, are listed in Table 5.2. Only E. japonica is cultivated for its fruits, but E. deflexa and E. prinoides had been used as rootstocks in China. Variegated forms of loquat have been sold as ornamentals in Europe and the United States (Morton 1987; McConnell 1988). Peroxidase isozymes from shoot and root were clearly different among several species of loquat and can be used for Eriobotrya classification (Zhang et a1. 1990). The loquat cultivars 'Akko l' and 'Akko 13' were distinguished by isozyme patterns for shikimate dehydrogenase, peroxidase, and phosphoglucose isomerase. A third cultivar (whose characters are similar to 'Akko 1'3 ') grown at Zikim, Israel, was distinguished from both cultivars on the basis of its phosphoglucose isomerase banding pattern, and designated 'Zikim' (Degani and Blumenfeld 1986). A number of widely planted cultivars had been classed as either "Chinese" or "Japanese" by some authors, based on distinguishing features that separate the two groups. For example, the Chinese group have nearly round fruits with orange flesh and small, numerous seeds, while the Japanese group have borne long-oval fruit with whitish (yellowwhite) flesh and a few large seeds; however, these differences are no

5. LOQUAT: BOTANY AND HORTICULTURE Table 5.2.

239

Loquat species and varieties.

Eriobotrya species

Representative area

References

E. japonica Lindley E. bengalensis (Roxb) Hook. f forma angustifoJia E. cavaleriei Rehd. E. deflexa Nakai var. buisanesis Kane & Sasaki var. koshunesis Kane & Sasaki E. elliptica Lind!. E. fragrans Champ E. grandiflora Rehd. & Wils E. henryi Nakai E. hookeriana Decne E. malipoensis Kuan E. obovate W. W. Smith E. prinoides Rehd. & Wils var. daduneensis H. Z. Zhang E. salwineses Hand-Mazz E. seguinii Card ex Guillaumin E. serrata Vidal E. tenyuehensis W. W. Smith

Yangtze River valley

Yu 1979

Yunnan Sichuan and Fujian Guangdong and Taiwan Taiwan Taiwan Xizhang(Tibet) Guangxi Sichuan Yunnan Xizhang and Sichuan Yunnan Yunnan Southeastern Yunnan Western Sichuan Northeastern Yunnan Southeastern Yunnan Southern Yunnan Western Yunnan

Yu 1979 Yu 1979 Yu 1979 Huxley 1992 Huxley 1992 Yu 1979 Yu 1979 Huxley 1992 Yu 1979 Huxley 1992 Yu 1979 Yu 1979 Yu 1979 Zhang et al. 1990 Yu 1979 Yu 1979 Yu 1979 Yu 1979

longer typical for each country's cultivars. Among 40 cultivars cataloged by T. Ikeda as more or less important in Japan, most were intro~ duced from China, especially 'Magi' and 'Tanaka', which account for 84 percent of the total area in Japan (Fujisaki 1994). Whitish flesh cultivars make up 30 percent of the number of total cultivars in China (Ding et al. 1995a), and some whitish flesh cultivars, such as 'Zhaozhong' and 'Baiyu', are the leading cultivars in Jiangsu province. There are several low-seeded cuItivars, such as 'Duhe' (single seed) and 'Taicheng No.4' (lor 2 seeds). Using data based on 100 characters, 50 cultivars collected from China and Japan clustered into three major groups (Liu et al. 1993). The first group was characterized by small, pale-color fruits, and consisted mainly of cultivars from Wuxian county, Jiangsu province; and from most areas of Zhejiang province. The second group consisted of cultivars with darker-colored, medium-sized fruits, mainly from Anhui province, some from Fujian province, and a few from Zhejiang province. The third group, with large, dark fruits, were cultivars from Fujian province. Japanese cultivars fell into the first two groups. Loquats have formed different ecological types in various zones during the long course of their cultivation and climatization. Ecotypes in

240

S. LIN, R. SHARPE, AND J. JANICK

China can be divided into two cultivar groups: the north subtropical cultivar group (NSCG) and the south subtropical cultivar group (SSCG) (Ding et a1. 1995a). NSCG distributes in the mid- and north subtropical area, roughly in the provinces in the basin of the Yangtze River, located in the range of 27° to 33°, where its average annual temperature is 15°C to 18°C, with an absolute low temperature of _5° to -12°C, and 800 to 1,500 mm of annual rainfall. Snows and frost can occur. NSCG cultivars are characterized by strong cold-resistance; most of their fruits are late ripening and small but with high quality. Representative cultivars are 'Dahongpao' and 'Luoyangqing' in Zhejiang, 'Baiyu' and 'Zhaozhong' in ]iangsu, and 'Guangrong' in Anhui. In China, these cultivars have been successfully introduced to the south subtropical zones and margins of tropical zones. SSCG is located in the south subtropical zone and margins of the tropical zone, approximately in the area about 19° to 27°N, with only a few days of frost and snow or temperature lower than O°C, and with more than 1,500 mm of annual rainfall. The SSCG cultivars have poor cold-resistance but are high yielding and early, while fruits are large but flavorless. Representative cultivars are ']iefangzhong' and 'Changhong No.3' in Fujian. Flowers and fruits are injured by cold when they are introduced to the north subtropical zones. Introduction of ']iefangzhong' has been attempted in Zhejiang and ]iangsu several times since the 1970s, but it has not been accepted (Ding et a1. 1995a). It appears that both the first and the second groups classified by Liu belong to the north subtropical cultivar group of Ding. As the first group has the distinguishing feature of whitish flesh, cultivars in China can be divided into three groups, namely, whitish group, north subtropical group, and south subtropical group. Most cultivars cultivated in Japan belong to the north subtropical group. Several cultivars, such as 'Shiro Mogi', could be placed in the whitish-flesh group. B. Morphology and Anatomy

The main characteristics of the genus Eriobotrya are as follows (Huxley 1992): leaves alternate, simple, coriaceous, coarsely dentate; petiole short; flowers small, white, in broad pyramidal, usually densely lanatepubescent, terminal panicles; bracts deltoid-ovate, persistent; calyx 5lobed, acute, persistent; petals 5, ovate to suborbicular, clawed; stamens 20-40; styles 2-5, basally connate; ovary inferior, each locule 2-ovulate. Fruit an obovoid to globular pome (Fig. 5.1), with persistent calyx lobes at apex; seeds (1-9) are large. Yu (1979) described the main characteristics of E. japonica as follows: evergreen tree, occasionally up to 10 m; shoot density varies with culti-

5. LOQUAT: BOTANY AND HORTICULTURE

Fig. 5.1.

241

Loquat cluster (x1/4). Source: F. W. Popenoe 1928.

var. Leaves on upper surface usually lustrous, lower surface often with pubescence; blades are narrow or broad, 12-30 cm long and 3-9 cm wide. Inflorescence 10-19 cm long, the main panicle axis bears 5-10 branched secondary axes, with 70-100 flowers, occasionally more than 100; hermaphrodite, flower size 12-20 mm. Fruit shape in longitudinal section as round, obovate, or elliptical; fruit size 2-5 cm; average weight usually about 30-40 g, some cultivars such as ']iefangzhong' average 70 g, the largest one 172 g, peel and flesh white or yellow; fruit apex concave, flat or convex, with calyx cavity closed or open; ease of fruit peeling depending on cultivars; thickness of flesh 0.5-0.8 cm, proportion of flesh usually 60-80%: Brix value 6.7-17°, some cultivars such as 'Huangyang No.5' higher than 20°; number of seeds 1-8, often 3-4, each seed weight 1.1-3.6 g. The loquat has relatively large seeds, as the subfamily Amygdaloideae, but has multiple seeds as do the subfamiles Rosoideae and Maloideae. Scanning electron microscopy of loquat revealed that the fruit skin was composed of only one layer of cells. The stomatal openings and base of trichome were surrounded by small, circular, cuticle ridges. Stomatal differentiation was completed before enlargement of young fruits, while trichomes developed up to the initial stages of fruit enlargement (Yin et al. 1994). Trichome density and the capacity of leaf hairs to protect underlying tissues against ultraviolet-B radiation damage were assessed during leaf

S. LIN, R. SHARPE, AND J. JANICK

242

development (Karabourniotis et al. 1995). Trichomes density and the relative quantities of ultraviolet radiation-absorbing phenolic constituents declined considerably with leaf age. C. Embryology Embryogenesis of several loquat cultivars were observed in paraffin and semi-thin section with light microscopy, ultrathin section with transmission electron microscopy, and from enzymatic isolation of embryo sacs (Lin 1985, 1992; He et al. 1995). Embryo sac of loquat is the Polygonum type (Lin 1992; He et al. 1995). The first divisions of the endosperm are not accompanied by cell-wall formation, so endosperm remains free nuclear in the early stages. As the embryo develops to the globular stage, wall formation commences in the micropylar end of the embryo sac, and then the endosperm passes through an early and late dissociate nuclear stage, completely cellular-stage, followed by disintegration until elimination. When the young fruit is oblong and the peel is yellow (peel covered with yellowish trichomes), the embryo is in the globular stage and the endosperm is late dissociate nucleus cellularstage; when the young fruit is rhomboid and the peel color is green (trichome abscission), the embryo is in the heart stage and the endosperm is completely cellular (Lin 1985, 1992). Semi-thin sections were used to investigate the structure of female organs before and after fertilization, differentiation, and the distribution of transfer cells in the early developmental stage of the endosperm. There are papillose cells on the wet stigma and conducting tissue in the style, which contains transfer cells and annular tracheids (Lin 1992). Transfer cells are also found in locules (He et al. 1995). Some cells in the inner integument and nucellus had outstanding wall ingrowths; exo-layer cells of the endosperm were transfer cells; embryo sac and center cell all had some wall ingrowths or haustorial structures (He et al. 1995). '

.

.

III. PHYSIOLOGY A. Growth and Development

Vegetative growth is in the form of a series of flushes that occur once each season. Summer shoots are the most abundant; spring shoots, summer shoots, and sometimes autumn shoots will be flower branches; winter flushes depend on tree age and nutrition (Chen 1958). In China,

5. LOQUAT: BOTANY AND HORTICULTURE

243

flower bud differentiation occurs from July (warmer climate) to September (cooler climate). Flower differentiation in loquat is basically the same as in other Rosaceae, but the sequences of flowering in autumn and winter is of particular interest (Li 1982). In Zhejiang, China, the main axis of inflorescence panicles differentiate in the beginning of August, secondary axes in the middle or the end of August, sepals and petals in the beginning of September, stamens and pistils in the middle or end of September, and sperm nuclei and egg nuclei in October. The time span from flower bud differentiation to anthesis in November is three months. The summer lateral shoot begins to differentiate flower buds in September, one month later than the spring main shoot, but anthesis also takes place in November, the differential duration just spanning two months. Therefore, flower clusters of summer lateral shoots may be short and small, and should be thinned (Li 1982). Flowering in loquat may extend over 1.5 to 2.5 months, and fruits normally ripen about 150 to 200 days from flowering (Chen 1958). In Israel, the loquat flowers over a three-month period, which permits collection of fruit at all stages of development at a single date (Blumenfeld 1980). Net photosynthetic rate (Pn) of loquat was low during winter, usually less than 1.5 mg CO 2 dm-2 h-1 (Ruan and Wu 1991). The highest Pn was measured in loquat during flowering; the presence of flowers increased the Pn in adjacent leaves but not basal ones. The optimum temperature for photosynthesis during winter was lower than 20°C and was depressed more after exposure to ooe or -2°C. The light saturation point and light compensation point were 18 klx and 360 mmol m-2 s-t, respectively. Photosynthetic induction of loquat leaves (45 min on a cloudy day) occurred more rapidly than that previously reported for 'Satsuma' mandarin. The activity and abundance of flower-visiting insects of loquat were studied in Punjab, India. Apis dorsata (Fabr.) was the main flower visitor. Other species of insects found occasionally included syrphids, houseflies, Myrmeleontidae, Bombinae, and Pieris rapae (L.). Fruit set was 150/0 greater in unbagged than in bagged flowers (Mann and Sagar 1987).

The growth pattern of loquat fruit in Israel (Blumenfeld 1980) is neither sigmoidal, as in most small-seeded pome fruits, or double sigmoidal, as in stone fruits that have a large seed, but is exponential with a rapid growth toward the end of fruit development, in spring, until ripening. The maturation phase is characterized by decreasing acidity, color development, softening of the pulp tissue, sugar accumulation, and a rapid increase in the fresh weight of the pulp tissue. The fruit produces ethylene at the beginning of the maturation phase (Hirai 1980, 1982).

244

S. LIN, R. SHARPE, AND J. JANICK

However, the loquat is a nonclimacteric fruit and shows no respiration climacteric rise and no peak of ethylene production either on the tree or after harvest (Blumenfeld 1980). The fruit does not abscise after ripening but shrinks on the tree. Fruit weight was influenced by the number of days to ripening, heat summation from flowering to ripening, seed number and seed weight, but not number of leaves on bearing shoots. Seed weight was the most influential factor affecting fruit weight (Uchino et al. 1994a). Amitava and Chattopadhyay (1993) reported that fruit acidity increased up to 50 days after fruit set and then declined as maturity approached, resulting in a marked increase in total soluble solids (TSS) and sugar:acid ratios. Leaf N, P, K, and Mg concentrations were lowest at flower initiation and highest at beginning of ripening (Ding et al. 1995b). LeafCu, Fe, and Mn concentration were highest at flower initiation and decreased at the beginning of fruiting. Leaf Na concentration was lowest at flowering and fruiting, and increased markedly before and after harvest ripening. Macroelement concentrations in fruits were in the order N > K > Ca > Mg > P; microelement concentrations were in the order Fe > > Mn > Cu. Burl6 et al. (1988) proposed a method to predict total nutrient content in fruits at various stages based on fruit weight. In Taiwan, soluble carbohydrate in the leaves and soluble solids in the juice decreased as the N content of the leaves of non-fruiting shoots increased. LeafCa content of both shoot types was higher at the flower bud stage than at the young fruit stage. Fruit of loquat grown in some areas were larger, and had higher acid contents and lower ratios of solids to acids than fruits from other areas (Fan 1987a,b). In India, deficiency symptoms of 'Golden Yellow' grown in the greenhouse appeared after one year and increased in severity in succeeding growth flushes; characteristic symptoms of deficiency were described for C, P, K, Ca, Mg, and S (Singh and Lal 1990). Critical limits for C, P, K, Ca, and Mg have been suggested for loquat orchards in Italy (Crescimanno and Barone 1980).

Zn

B. Chemical Composition Chemical composition of loquat fruit is presented in Table 5.3. In various cultivars, sucrose, sorbitol, glucose, and fructose varied almost 6fold, but total sugars varied less than 2-fold (Kursanow 1932; Ito and Sakasegawa, 1952; Hirai 1980; Uchino et al. 1994b). Sucrose accumulated faster than any other sugars at the beginning of fruit maturation and became the predominant sugar in ripe fruit (Hirai 1980), while sorbitol, predominant during fruit development, was reduced to a minor com-

5. LOQUAT: BOTANY AND HORTICULTURE

Table 5.3.

245

Chemical composition of loquat (Church et a1. 1935). Cultivar

Variable Moisture (%) Total solids (%) Insoluble solids (%) Soluble solids (%) Total acid (%) Total malic acid (%) Protein (%) Ether extract (%) Reducing sugars (%) Sucrose (%) Total sugars (%) Alcohol ppt (%) Pectic acid (%) Total ash (%) Soluble ash (%) Insoluble ash (%)

Champagne

Advance

Thales

85.0 15.0 2.3 12.7 0.4 0.3 0.4 0.2 6.7 3.4 10.1 0.6 0.3 0.5 0.4 0.1

84.7 14.3 2.7 11.6 0.5 0.3 0.4 0.2 5.7 3.4 9.1 0.7 0.4 0.5 0.4 0.1

86.1 13.9 2.4 11.5 0.7 0.5 0.4 0.2 6.1 2.4 8.5 0.9 0.4 0.5 0.4 0.1

ponent in ripe fruit. Glucose and fructose contents increased as color intensity increased (Hirai 1980). Malic and citric acid levels increased with fruit maturation, and then decreased, with citric acid decreasing at a faster rate. Traces of tartaric acid that disappeared with maturation were found in green fruit (Kursanow 1932; Church and Sorber 1935; Rajput and Singh 1964). Loquat flesh contained 0.42 g crude protein/l00g fresh wt, 146 mg essential and 387 mg total amino acids (Hall et al. 1980). Ten essential amino acids were measured, with leucine the most abundant and cysteine-cystine the least abundant. Of the eight nonessential amino acids measured, glutamic and aspartic acids were the most abundant, with an unusually high level of proline (9.7 g per 100 g recovered amino acids). The profiles of lipids, long-chain hydrocarbons, desmethyl sterols, and fatty acids were determined by gas-liquid chromatography (GLC). Long-chain hydrocarbons varied from Cn to C31 • Major sterols included ~-sitosterol, campestol, isofucosterol, and cholesterol, in order of their prevalence. Fatty acids consisted of palmitic, oleic, linoleic, linolenic, and stearic (Nordby and Hall 1980). Loquat seeds yielded 0.1 % lipids and consisted of 3.1 % hydrocarbons, 5.30/0 wax esters, 78.6% triglycerides, and 13% (polar fraction) fatty acids, coloring matter, and other

246

S. LIN, R. SHARPE, AND J. JANICK

compounds. After saponification of the fat, the fatty acids C12 -C 24 and fatty alcohols C12 -C 26 were identified by GLC (Raie et al. 1983). The carotenoids of loquat fruit are mainly responsible for flesh and skin color, which varied from yellowish white, yellow to deep orange (Sadana 1949; Gross et al. 1973; Lin and Li 1985; Godoy and Amaya 1995). Total carotenoid values, especially carotene, varies widely in fresh fruit peel and pulp. Total carotenoid values in the peel are several times higher than in the pulp (Gross et al. 1973; Lin and Li 1985). The content of carotene in yellow-orange fruit was 5-10 times higher than in the yellow-white fruits, while the contents of zeaxanthin, lutein, and violaxanthin in yellow-orange fruit were much lower (Lin and Li 1985). The carotenoid compositions in Brazil cultivars were identified by Godoy and Amaya (1995) as follows: ~-carotene (7.8 mg/g), ~-carotene (0.1 f..lg/g) , neurosprence (1.1 f..lg/g), ~-cryptoxanthin (4.8 f..lg/g), 5,6-monoepoxy-cryptoxanthin (0.6 f..lg/g), violaxanthin (1.6 f..lg/g), neoxanthin (0.8 f..lg/g),and auroxanthin (0.9 f..lg/g). Betacarotene and ~-crytoxanthinwere the principal pigments, being responsible for 44 and 27 % , respectively, of the total carotenoid content (17.6 f..lg/g) and were also the principal contributors to the vitamin A value of 175 RE/l00 g. Loquat fruits also contain a number of small carotenoids such as phytofluene, mutatochrome, carbonyl, and cryptoflavin (Gross et al. 1973). Eighteen volatile compounds were identified in a methylene chloride extract of a distilled fraction from loquat fruit. The major components, phenylethyl alcohol, 3-hydroxy-2-butanone, phenylacetadehyde, and hexen-l-ols, and the minor components, ethyl acetate, methyl cinnamate, and ~-ionone, contribute to the fruity-floral flavor of the fruit (Shaw and Wilson 1982). Loquat is a cyanogenic plant and contains three cyanide metabolizing enzymes: ~-cyanoalanine synthase, rhodanses, and formamide hydrolase (Miller and Conn 1980). Loquat tannin was a proanthocyanidin oligomer (Matsuo and Ito 1981). A few specific organic components, 4-methyleneD,L-proline and trans-4-hydroxymethyl-D-proline, have been identified in seeds (Gray 1972; Gray and Fowden 1972). C. Plant Growth Regulation

In China, loquat fruit growth occurs in three stages and levels of phytohormones have been analyzed during each stage (Ding and Zhang 1988). In stage I, the stage of slow fruit growth, from December to the middle of February, indoleacetic acid (IAA), abscisic acid (ABA) and cytokinin are maximal. In stage II, the cell division stage from the end of February to the end of March, ABA decrease gradually to a minimum, while eth-

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ylene, which appears at the end of stage I, increases gradually to a maximum and then gradually decreases. IAA and cytokinin reach a second peak at the end of stage II. In stage III, the stage of rapid enlargement of fruits in the middle of April to fruit maturation, IAA and cytokinin are at a minimum, ABA increases again, and ethylene appears at a second peak (Ye 1988). Endogenous gibberellins in the immature seed and pericap of loquat were first confirmed by Japanese scientists (Yuda 1987; Koshioka et a1. 1988). Gibberellins, including GAg, GA 15 , GA 19 , GA 20 , GA 29 , GA 35 , GA44 , GA 50 , and GA6l , were identified by capillary gas chromatographyl selected ion monitoring in immature loquat seeds. Five unknown GAlike compounds with apparent parent ions of m/z 418, 504, or 506 (as methyl ester trimethylsilyl ether derivatives) were also found in the biologically active fractions. The m/z 418 and 504 compounds may have been C-ll ~-hydroxylated GAg, and dehydro-GA 35 , respectively. The bioassay and GC/MS results suggest that the major gibberellins were GA 50 and five unknown GA-like compounds. In the immature seeds, at least two GA metabolic pathways may thus exist, one being the nonhydroxylation pathway of GA l5 ~ GA Z4 ~ GAg, and the other C-13 hydroxylation pathway of GA 44 ~ GA 19 ~ GA 20 ~ GA 29 . A late C-~-hydroxylation pathway is also possible (Koshioka et a1. 1988). Besides GAg, GAts, GA 35 , and GA so mentioned above, GA 24 , GAzs , GA48 , and GABO were identified by Yuda et a1. (1992). Two of them were determined to be new gibberellins: GA 80 (11 ~-hydroxy-GA7)' and GA 48 (11 ~-hydroxy-GA9)' Based on these results, an early I1-hydroxylation biosynthetic pathway is suggested in the loquat seeds (Yuda et a1. 1992). Synthesis of the methyl ester was used to confirm the structure of the new 11 ~-hydroxy, GA 84 , which isolated together with another gibberellin, GA BO ' from seeds of immature loquat fruits (Kraft-Klaunzer and Mander 1992). 1. Growth Control. Paclobutrazol (PP333) was applied as foliar sprays to three-year-old ']iefangzhong' trees or direct to their soil (Pan et a1. 1995). Four treatments were compared: soil application at 1.0 or 1.5 g m-z of canopy, foliar application at 500 mg L-1 (three times at monthly intervals), and a control. Soil application was inferior to foliar application with regard to advancement of flowering. In the second year, the effect of soil treatment was significant; of the three PP 333 treatments, percentage flowering was highest for the 1 g m-z application rate. In the third year, fruit yields/tree were 23.5 kg (3360/0), 9.9 kg (84%) and 10.7 kg (900/0), respectively, compared with 5.4 kg for the control. Results obtained in Italy showed that PP 333 reduced fruit yields but increased

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fruit size, with no significant effect on juice pH and total soluble solids (TSS) (Pilone and Scaglione 1996). 2. Pollen Germination. The effects of naphthaleneacetic acid (NAA), GA 3 , and IAA in combination with B, Ca, and Mn were analyzed on 8-10-year-old trees of several cultivars for three years. GA 3 and NAA at 1-100 ppm, boric acid and MnS04 at 1-100 ppm, and IAA at 0.1-1.0 ppm all had good effects on pollen germination (Ding et a1. 1991). Eti et al. (1990a) reported that the germination rate of pollen was related to percent fruit set. Pollen germination was highest in 10% sucrose (Singh 1963; Singh et al. 1979). 3. Fruit Set. Fruit set was increased by 54 to 120% in various cultivars treated with plant growth regulators (Ding et al. 1991). Best results with regard to fruit set and fruit quality were obtained with GA at 60 ppm (Singh and Shukla 1978). Mature loquat trees sprayed when fruits were at the pea stage and again one week later with NAA, 2,4,5-T, or GA 3 , each at 10, 20, or 40 ppm, ripened about 10 days earlier than with GA 3 at 10 ppm, gave the best fruit retention (88.5 % ) and greatest fruit volume (20.6 cm3), weight (19.5 g), and pulp content (15.9 g/fruit), and lowest seed total weight (3.6 g/fruit). NAAat 40 ppm gave the highest TSS (13.5) and reducing sugars (8.6 mg/l00 g), and lowest acidity (0.90/0) (Chaudhary et al. 1990, 1993). 4. Fruit Thinning. NAA and NAAm (naphthaleneacetamide) applications (25, 50, or 100 ppm) effectively thinned loquat fruits. Optimum level of thinning was obtained with 25 ppm. The effects of thinning on fruit growth varied with cultivar. Fruits on thinned branches developed more rapidly than non-thinned controls in all cultivars. Thinning had no effect on fruit shape (Eti et al. 1990b; Kilavuz and Eti 1993). 5. Induction of Seedlessness. Several groups of scientists have applied GA to induce seedless fruits (Kumar 1976; Blumenfeld 1980; Kihara 1981; Muranishi 1982; EI-Zeftawi and Goubran 1983a; Goubran and ElZeftawi 1986; Fan 1989; Takagi et a1. 1994). Goubran and EI-Zeftawi (1986) reported that GA at 250 ppm applied after the emergence of floral buds or NAA at 20 ppm applied during full bloom produced seedless fruits. Seedless fruits were smaller, elongated, and matured four to five weeks earlier than seeded fruits. The reduced size was related to early maturity, as much of the fruit weight increase normally occurs just prior to maturation. It is suggested that seedless loquats need further treatment to increase fruit size. GA 3 applied at 250-500 ppm after emer-

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gence of the floral buds in mid-October resulted in the production of seedless fruits with a high flesh content. Seedless fruits were smaller, more elongated and matured about five weeks earlier than seeded fruits. TSS was increased in seedless fruits receiving a second growth regulator [GA3 + benzyladenine (BA)] (Fan 1989). Spraying loquat clusters with an aqueous solution of 500 ppm GA3 or 500 ppm GA3 + 20 ppm kinetin greatly stimulated frost-induced seedless fruits to attain the same size as seeded control ones. The GA-treated seedless fruits were more slender but had a thicker pulp than seeded untreated fruits. Application of GA 3 + kinetin was more effective for enlargement than a single application of GA 3 • If sprayed immediately after a frost, the enlargement response of the seedless fruits was significant. Although treated seedless fruits turned yellow earlier, the total soluble solid content in the juice at harvest was slightly lower than that of seeded fruits. No difference in titrable acidity was found between the treated and control fruits (Takagi et al. 1994). D. Sorbitol Physiology Loquat, like other rosaceous fruits, utilizes sorbitol as the main metabolite of photosynthesis (Hirai 1979,1980,1983; Nii 1993; Nii et al. 1994; Lin et al. 1995). Sorbitol and sorbitol-6-phosphate dehydrogenase (a sorbitol-utilizing enzyme) activity were surveyed in the leaves of mature loquat trees (Hirai 1983). The enzyme activity increased in late autumn and sorbitol content increased in early winter, both reaching maximums in winter and decreasing in spring. In seedlings, the increase in enzyme activity was induced by low temperature. Photoperiod did not affect enzyme activity. During the development of spring leaves, enzyme activity increased during the period in which a leaf had nearly reached its maximum size, the period during which young leaves depend on imported sorbitol from mature leaves. Enzyme activity was considered to be controlled mainly by the amount of enzyme in leaf tissue (Hirai 1983). Although sorbitol content increased during fruit development (from 7 mg/per young fruit in March to about 50 mg/per mature fruit), its percentage relative to total sugar (sucrose, glucose, and fructose, which increased from 0 mg/per young fruit in March to about 500-1000 mg/per mature fruit) decreased during development and maturation (Hirai 1979). Sorbitol in globular, torpedo-shaped, and cotyledonary embryos were 5.7,11.5, and 17.9 mg/g, respectively. Sorbitol increased gradually, while none of the other sugars increased, but D-galactose decreased from 29.7 to 10.7 mg/g (Lin et al. 1995b). In fruits, sorbitol accumulation

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S. LIN, R. SHARPE, AND J. JANICK

paused in the middle of May and resumed after the color-change of the fruit. The regulatory mechanism of the accumulation is unknown, but interconversion between sugar alcohol in the fruit may be a factor in the carbohydrate accumulation. Sorbitol may play an important role in morphogenesis in vitro. Protoplast cultures, cultured in the medium supplemented with a 3 percent or higher level of sorbitol, differentiated shoots (Lin and Chen 1996a). The relationship between sorbitol metabolism and morphogenesis requires further investigation. Some related enzymes such as sorbitol-6-phosphate dehydrogenase have been surveyed. The activity of sorbitol-6-phosphate dehydrogenase both in leaf and in fruit increased before the increase of the sorbitol content (Hirai 1979, 1981, 1983), suggesting that sorbitol metabolism is regulated by sorbitol-6-phosphate dehydrogenase (Hirai 1983). Callus cultured in the medium added with sorbitol increased activity of D-sorbitol dehydrogenase and were richer in organelles (Lin and Chen 1997). Some cells with distinct structural features may be related to the transport of sorbitol. Loquat, like other rosaceous fruit trees, show thickening olthe sieve elements in vascular bundles of several organs. The ingrowth thickening, referred to as nacreous wall formation, was completely different from the apparatus of the transfer cell in other species (Nii 1993; Nii et al. 1994). In reproductive organs, not only outstanding wall ingrowths but transfer cells were found (Lin 1992; He et al. 1995). There may be a relationship between the degree of ingrowth of the nacreous cell and the transported soluble carbohydrate (Nii et al. 1994), and between distribution of the transfer cell and its role in the transport of sorbitol and other nutrients (He et al. 1995). E. Temperature Response Flowers and fruits of loquat show increasing injury from flower state to early fruit. Ovules in the early fruits are killed by brief exposure to -4°C (Yang 1963). Pollen could be stored at-23°C for 26 months (Singh 1963). Loquats were undamaged in southern Florida during January 1977, when temperatures fell to -6.7°C for 12-14 h on three consecutive nights (Dawes 1980), and in the San Giuliano region of France when freezing temperatures occurred every night from January 2 to 12, 1985, with an absolute minimum of -6°C (Vogel 1986). In Mokpo, Republic of Korea, cold injury to fruits was 100/0 after exposure to -2°C, 40-490/0 at -3°C, and 95-1000/0 at -4°C. At -3°C, smaller fruits (diameter < 9.5 mm) were more susceptible to cold injury than larger fruits. The conductivity rate of immature fruit exposed to cold temperatures increased as fruit diameter decreased. The freezing temperature of immature 'Mumok' ('Mogi ')

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fruits, with diameter of 7.5,8.0,9.0,10.2, and 11.2 mm, were -1.4, -1.5, -1.7 and -1.8°C, respectively. High contents of reducing sugars and unsaturated fatty acids were associated with decreased freezing injury (Park and Park 1995). Campbell and Malo (1986) reported -12°C as the critical temperature for wood hardiness and -3°C the killing temperature for young fruit. The latter is the limiting factor for regular cropping in Florida and much of California. In tropical regions, adaptation is obtained at altitudes from 900 to 2100 m. F. Medicinal Value

Loquat has been considered to have health benefits in traditional medicine and there is now evidence of therapeutic effects. The ether-soluble fraction of the ethanolic extract of the leaves showed anti-inflammatory activity when applied topically to rats (Shimizu et al. 1986). Ursolic acid, maslinic acid, methl maslinate, and euscaphic acid were isolated from this fraction. Maslinic acid was shown to be at least partly responsible for the anti-inflammatory activity of the extract. Loquat leaves have been used for the treatment of skin diseases and diabetes mellitus. The alcoholic extract of the leaves produced a significant hypoglycemic effect in normal, but not in alloxan-treat rabbits, but the effect was short-lived, lasting only for 3 h (Noreen et al. 1988). Comparison with the effect of tolbutamide indicated that the hypoglycemic effect of the extract was probably mediated through the release of insulin from pancreatic beta cells. Later, a new polyhydroxylated triterpene was isolated, as well as three known triterpenes. The new compound was identified as 3p,6a,19a-trihydroxyurs-12-en-28-oic acid (Liang et al. 1990). Seven glycosides, five of which are new natural products, were isolated from the methanol extraCt of leaves collected in Italy (De Tommasi 1992a). The three new sesquiterpene glycosides have nerolidol or isohumbertiol as aglycones, and two of these have branched oligosaccharidic chains made up of one P-L-glucopyranosyl and three a-L-rhamnopyranosyl units that link transferuloyl ester moieties. Analysis of the oligosaccharide structures was achieved by 2 D spectral analysis. The two new ionone-derived glycosides isolated from the extract were characterized by chemical and spectral methods. An alcoholic extract has been shown to exhibit anti-inflammatory and hypoglycemic effects. The CHCl 3 extract of leaves from an Italian source contained four new triterpene esters, namely, 23-trans-p-coumaroyltormrntic acid, 23-cisp-coumaroyltormrntic acid, 3-0-trans-caffeoyltormentic acid, and 3-0trans-p-coumaroyltormrntic acid, in addition to three common ursolic

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acid derivatives. Spectral data were used to elucidate their structures. An investigation of the antiviral properties of these compounds revealed that only 3-0-trans-caffeoyltormentic acid significantly reduced rhinovirus and was ineffective towards human immunodeficiency virus type I (HIV-I) and Sindbis virus replication (De Tommasi 1992b). Loquat fruits contained low levels of B vitamins, including thiamine, riboflavin, and niacin (Shaw and Wilson 1981). For at least 40 years, Chinese food stores in the United States have sold a product imported from Hong Kong and recommended for chronic bronchitis, coughs, and lung congestion. Contents are listed as loquat leaves with other herbs (Duke and Ayensu 1985; Wee and Hsuan 1992). IV. HORTICULTURE

A. Crop Improvement 1. Ploidy Manipulation. The number of chromosomes in loquat cultivars cultivated in China are all 2n = 2x = 34 (Lu and Lin 1995). Tetraploidy

was reported in India, and was also attained from colchicine treatment (Kihara 1981), and triploidy was derived from 2x x 4x (Huang 1984, 1989) and by endosperm culture (Lin 1985; Chen and Lin 1991). Hybrids between E. japonica and E. deflexa were obtained and clearly resembled E. deflexa. Hybrids among cultivars are cross compatible. Most of the cultivars are self-fertile, but several cultivars in the United States are selfinfertile (Morton 1987). A sterile plant that sets small fruits with no or few seed was found in Fujian (Lu 1984; Lu and Lin 1995). 2. Hybridization and Selection. Progenies between clones of loquat with round fruit and oblong fruit segregated ranging from round to oblong. Orange flesh appears incompletely dominant to white flesh fruit, indicating that the characteristics may be controlled by several pairs of genes (Zheng et a1. 1993a). Most major cultivars are derived from chance seedlings (Huang et a1. 1990), but breeding programs based on hybridization have been initiated, and several cultivars have been released in China, such as 'Zaozhong No.6', 'Zhongjing', '82-6-26', while 'Nakasakiwase', 'Obusa' (Fujisaki 1994), 'Suzukaze', and 'Yogyoku' (Y. Sato, pers. commun.) have been released in Japan. ']iefangzhong', bearing large fruits, has been a popular parent in China and is the parent of three cultivars (Huang et a1. 1993; Ding et a1. 1995a). Cross-pollination is most successful with flowers of the second flush. Early and late flushes have

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abnormal stamens, very little viable pollen, and result in poor setting and undersized fruits (Morton 1987). There are many cultivars or selections in various provinces of China (Table 5.4). For example, there are 83 cultivars (or selections) in Zhejiang, 78 in Fujian, 57 in Jiangsu, 31 in Anhui, 18 in Hubei, 18 in Guangdong, 14 in Guangxi, 10 in Hunang, 10 in Jiangxi, and 9 in Sichuan (Zhang et al. 1990). The largest collection of germplasm, more than 250 cultivars, is located in Fuzhou, China. Most of the cultivars cultivated in Taiwan and the United States derived from materials introduced from Japan. Although more than 10 cultivars are grown in Japan, three cultivars account for 950/0 of the total area: 'Mogi' (620/0), 'Tanaka' (22%), and 'Nakasakiwase' (11%), followed by 'Obusa' (2°,10). 'Tanaka' was introduced prior to 1900 to the United States and Israel, and has now been introduced to Algeria (Lupescu et al. 1980), Brazil (Godoy and Amaya 1995), India (Testoni and Grassi 1995), Italy (Monastra and Insero 1991), Spain (L6pez-Galvez et al. 1990), and Turkey (Polat and Kaska 1992a-e), as well as China. The major cultivars of loquat in the world are presented' in the Table 5.4. 3. Biotechnology

Endosperm Culture. Endosperm culture has been pursued in order to obtain triploids that might be seedless. The cellular-shaped endosperm inoculated into B5 medium supplemented with 2,4-D and LH medium for two weeks to one month produced callus but induction rate was low. Somatic embryos at various developmental stages appeared on the surface of some callus when the medium was supplemented with 0.1 mg/L 2,4-D. By ultra-thin sectioning, it was found that embryonic cells with much thicker walls, denser protoplasm, and smaller vacuoles from surrounding cells divided and differentiated into embryoids (Chen et al. 1983). Somatic embryos developed similar to zygotic embryos, but cotyledons grew slowly. Normal shoots differentiated on MS supplemented with zeatin (0.25 mg/L) and NAA (0.1 mg/L). Roots were produced when new shoots were transferred into MS medium supplemented with IBA (0.4-0.5 mg/L). (Lin 1985, 1987). The chromosome number of these plants was close to triploid (2n = 45-50) (Chen et al. 1991), but all triploids (100 plants) died before fruiting. Embryo Culture. Mature embryos are easily cultured (Zhuan 1980). Two months after pollination, the embryos were 6-8 mm long and all organs

N

0'1

>l:::o

Table 5.4. Major cultivars in the main producing areas of the world. Country

Area

Australia

Name of cultivar

Origin

Large fruit, acceptable taste, high flesh:seed ratio

Bessel Brown

Brazil

Enormity

Large fruit, acceptable taste

Victory

White to cream-colored flesh, juicy, sweet

Nectar de Cristal

Obtained by open pollination of Togoshi (Japan), 1970s

High yield, fruit uniformity

Parmogi

Obtained by open pollination of the Mogi (Japan), 1970s

High yield, pleasant taste

Mendes da Fonseca China

Outstanding characteristics

Large fruit

Precoce de Itaquera

Selected from Japanese seedling

Very productive

82-6-26 Baiyu

From Jiefangzhong x Baozhu, 1982 Selected from seedling of Baisha, 1980

Cold-resistance, large fruits, good eating quality

Jiangsu Fujian

Changhong No.3

Selected from a natural hybrid seedling of Changhong, 1990

Elongate-obovate fruits, weighing 50 g, ripening in mid-April; high and stable yield

Zhejiang

An old seedling cultivar

Strong growth vigor, stable yield

Jiangxi

Dahongpao Dube (one seed)

Introduced from unknown cultivar, 1958

High yield, single seed, medium eating quality

Anhui

Guangrong

Selected from seedling of Dahongpao Selected from Baisha

Vigorous growth, stable yield, quite large fruit, good keeping quality

Selected from Dazbong seedling, 1950

Large fruits. average 70 g with some fruits as large as 172 g; high yield

Zhejiang

Hubei

HuaboaNo.2

Fujian

Jiefangzhong

Yellowish-white flesh, fruit uniformity, good keeping quality

Cold-resistance, stable yield, good eating quality

Zhejiang

Loyangqing

Selected from Dahongpao, 1980s

Hunang Fujian

Yuanjiang Zaozhong No.6

An old seedling cultivar Jiefangzhong x Moriowase, 1992

Jiangsu

Zhaozhong

Selected from seedling of Baisha (white peel)

Strong disease resistance, high and stable yield, good keeping quality Strong flavor, good eating quality Ripening in the beginning of April, average 53 g, attractive, good quality Yellowish-white flesh, juicy, Brix 12°, good eating quality

Egypt

GoldenZiad Moamora Golden Yellow

Selected from seedling of Premier Selected from seedling of Premier

High yield, early season seedling High yield

India

Pale Yellow Safeda Thames Pride

Israel

Akko 13

Fruit large, flesh white Flesh cream-colored, early to midseason Bears heavily, early in season, juicy, canned commercially Japanese origin

Tsrifin Italy

Nespola di Ferdinando Nespola di Francesco

~

<:J1 <:J1

Nespolo di Palarmo

Early in season (March), juicy, agreeable flavor, good keeping quality, needs cross-pollination Bears regularly and abundantly, excellent quality, stores well

Released from breeding program, 1980s Released from breeding program, 1980s Superior in flavor

N

01

0'>

Table 5.4.

Major cultivars in the main producing areas of the world. Outstanding characteristics

Country

Area

Name of cultivar

Japan

Kyushu

Mogi

From a chance seedling of C1Uneseloquatintroduced from China in 1840

Prone to cold damage, harvest in May, fruit 50-60 g, excellent quality

Honshu

Tanaka Shikoku

Kyushu

Nakasaki-wase

?

Obusa

From a seed brought to Tokyo from Nagasaki, 1888 From Mogi x Hondawase, 1976 From Tanaka x Kushioki, 1967

Harvest in May, fruit weighing 60-70 g, good keeping quality Prone to cold damage, very early ripening, excellent quality Fruit 70 g, good keeping quality, resistant to insects and diseases

A clone of Tanaka obtained in California, 1888-1890

Juicy, sweet flesh, apricot-like flavor, good keeping, ships well Sweet, juicy; pleasant in flavor

A seedling selected, 1897 Selected and introduced, 1908 A seedling of Advance, 1905 Parentage unknown, introduced

A good pollinator Juicy, excellent flavor, good for preserving Early in season, very juicy Good keeping quality and flavor

Turkey

Gold Nugget

Origin

Hafif Cukurgobek United States

California California California Florida

Advance Champagne Eulalia Fletcher

1957

California Florida California Florida

MacBeth Oliver Premier Wolfe

Chance seedling, 1966 ? Tanaka hybrid Originated in California, 1899 A seedling of Advance, released 1965

Flavor pleasing, low acidity, small seed Best cultivar for southern Florida Flesh whitish, juicy, agreeable flavor Pale-yellow flesh, excellent flavor, stable yield, resistant to bruising

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were differentiated. After three days of culture on MS medium in the dark, the embryo enlarged slowly, and then cotyledons opened. Cultures were transferred to light when plumules grew longer than cotyledons. Cotyledons and plumules rapidly turned dark green, after which plumules developed into a main shoot and two laterals and some radides gradually developed and formed a root system. Cultured mature embryos can easily be subcultured from microcuttings after excising the shoot tip. This technique can be used to multiply genotypes obtained from hybridization (Lin et al. 1989; Lin 1991). Several research groups have succeeded in producing embryogenic callus from immature zygotic embryo of E. japonica (Ho 1983; Ho et al. 1986; Teng and Chen 1986; Lin et al. 1989; Lin 1991) and E. prinodes (Lin and Lin 1993). The key step to establishing embryogenic callus is to balance BA and 2,4-D concentrations (Ho et al. 1986; Teng and Chen 1986; Lin et al. 1989; Lin 1991).

Protoplast Culture. Embryogenic callus derived from torpedo-shaped embryos was the optimum material for protoplast isolation (Lin et al. 1989; Lin 1991, 1995). A combination of 20/0 pectinase and 1 % cellulase, combined with 120/0 mannitol as an osmotic agent, yielded 107 protoplasts/g of callus with 95% survival (Lin and Chen 1996a). Sorbitol was less efficient than mannitol as an osmotic agent. The· first mitosis was observed four days after protoplasts were cultured in MS liquid medium supplemented with sucrose in several concentrations, and colonies were subsequently formed (Lin and Chen 1994). Protoplast of E. prinodes were also successfully isolated and cultured to form colonies (Lin et al. 1994). Colonies formed little callus in agarose medium. Shoots differentiated when callus was transferred into the medium supplemented with 3% or 5% sorbitol and developed when the shoot was cultured in MS salts (half strength macroelements) supplemented with 2 mg/L zeatin. At 4 mg/L IBA, 800/0 of shoots rooted with 5.7 roots per shoot and 90% rooting was achieved by another transfer to I mg/L IBA after 10-15 days (S. Q. Lin and F. X. Chen 1996a). In vitro-produced plants of two cultivars, 'Jiefangzhong' and 'Baili', derived from protoplast cultures, were successfully transplanted to soil (Lin 1995; Lin and Chen 1996a). Genetic Transformation. Cotyledons were inoculated with Agrobacterium, A. tumefaciens, and A. rhizogenes, and octopine and nopaline were identified from the subcultured callus, indicating that Ti plasmid had been transferred into callus (Li et al. 1991). There has been no report on target gene transformation.

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B. Propagation 1. Seed. Propagation by seed has been the traditional practice in many

producing countries, and is still occasionally used in China and Japan. Although seedling plants are long lived, this method cannot be recommended because of genetic segregation. However, seedlings are often used as rootstock. Loquat seeds remain viable for 6 months if stored in partly sealed glass jars under high humidity at room temperature; the best temperature for storage is 5°C. Seeds are washed and planted in flats or pots soon after removal from the fruit and seedlings are transplanted to nursery rows when 15-17.5 tall. Seedlings are ready to be topworked when the stem is 1.25 em thick at the base (Morton 1987). 2. Vegetative

. Graft. The rootstock generally is E. japonica itself, although E. deflexa and other species, even Photinia serrulata, have been used for rootstocks in China. Loquat seedlings are preferred over apple, pear, quince, or pyracantha rootstocks under most conditions in Turkey (Polat and Kaska 1992e). Quince and pyracantha may cause extreme dwarfing. Dwarfing on quince rootstocks has encouraged expansion of loquat cultivation in Israel since 1960. The growing of dwarf trees greatly reduces the labor of pruning and flower- and fruit-thinning, bagging, and later, harvesting. Quince rootstock, which tolerates heavier and wetter soils, is widely used in Egypt (Morton 1987). Chip, patch, and T (shield) buddings performed at 15-day intervals from 15 January to 15 May were evaluated in Turkey (Polat and Kaska 1991, 1992d). March was the most suitable month for budding, with 950/0 bud take. Patch budding was more successful than T and chip budding, but the strongest scion shoots were obtained with chip budding. The Chinese have used cleft grafting on large loquat trees for centuries and this method is still used in Japan for cultivar change. An improved method, called a young stock cleft graft, was developed in Fujian, China (Huang 1978) and more than one million plants per year were produced by this method in Putian county alone. Using i-em-diameter stock, cleft grafts are made and held tightly in place with parafilm strips. Because there were several leaves under the graft position, the scion grew quickly and the shoot reached 60-70 em by autumn; survival rates were higher than 800/0. Veneer grafting is commonly practiced in Japan (8ato 1996) and in Jiangsu, China, and has proved to be a superior method in Pakistan.

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Scion are usually grafted to two-year-old seedlings in spring prior to active sap flow. Scions begin budding out about one month after grafting. Whip and tongue and bridge grafting have been practiced in some producing areas of China.

Cutting. In Japan, January cuttings dipped in IBA solution were rooted under mist installation, but survival rate usually was lower than 500/0. In Egypt, 650/0 of cuttings of 'EI-Soukari' (25 cm long with 4 or 5 leaves) rooted when dipped for 10 s in 4,000 ppm IBA solutions, and placed in a 1 sand:peatmoss medium under mist. (EI-Shazly et al. 1994). In China, 70% rooting was obtained when cuttings were placed in a 1 soil:l sand mix (W. Lin 1996, pers. commun.). Air Layering. In China, many farmers successfully propagate selected clones of loquat by air layering (Hu et al. 1988). Micropropagation. Loquat has few buds and so rapid multiplication by conventional vegetative propagation is a problem. Chinese scientists have developed effective and successful micropropagation using shoottip culture (Yang et al. 1983; Yang 1984; Chen et al. 1991, 1995; Chen and Lin 1995; Lin and Chen 1996b). Most of propagules derived from shoot-tip cultures have performed better than grafted plants and a steady flow of plants has been supplied to farmers. Successful micropropagation was achieved from axillary shoots derived from shoot-tip cultures (Yang et al. 1983). When the terminal bud was removed, lateral buds could be induced to develop by cytokinin. The optimum level of BA was 1.5-2 mg/L, producing multiplication rates of 4.25 per month. Rooting is achieved by transferred shoots to 1/2 MS basal medium after immersing the shoot in 100 mg/L IBA. The average survival rate of transplanted plants was 96%(Yang 1984). There are reports of shoot formation from callus derived from shoottip culture (Ho et al. 1986; Higashi and Kuwahata 1989). Embryogenic callus was obtained from 1-2 mm shoot-tip dissected from late fall or winter buds on MS agar medium supplemented with 1 mg/L thiamine, 0.5-2.0 mg/L 2,4-D, and 0.05-1.0 mg/L BA. The differentiation of somatic embryo occurred on the surface of embryogenic callus that germinated on MS medium (Ho 1983). Embryos with secondary embryogenic callus were formed when embryogenic callus was transferred into induction medium (Ho et a1. 1986).

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C. Field Culture 1. Orchard Establishment. Loquat is a long-lived tree and orchards over

30 years old remain productive. Thus, location and site selection are important in planning orchards. In China and India, loquats are grown at elevations up to 2000 m. In Japan, loquats are grown on hillsides to obtain the benefit of good air flow (Kozaki et a1. 1995). In more tropical regions, the tree thrives and fruits well at elevations between 900 and 1200 m, but bears little or not at all at lower levels (Campbell and Malo 1986; Morton 1987). Winter temperature should be higher than -3°C, and summer temperature not over 35°C. The tree requires 1000-1200 mm of rainfall annually and a suitable level of humidity. Soil should be deep and well drained, with an adequate content of organic matter. Sand loams or clay loams with a pH of 5.0 to 8.0 are considered appropriate, with pH 6.0 being optimum. Nursery plants must be transplanted before the growth of spring buds, depending on climate. In China, leaves on the base of nursery plants are removed and the root system is often dipped in mud. Before planting, well-fermented manure is added to planting holes. Loquats are planted at a density of about 500-600 trees/ha, but some cultivars with vigorous and spreading character are established at about 450 trees/ha (about 5 m between rows and 4 m between trees). In Japan, standard plant distance is 5 to 7 m (Sato 1996). In Brazil, a spacing of 7 x 7 m is recommended on flat land, 8 x 5 (or 6) m on slopes. In Putian county, Fujian province of China, loquat is spaced 6 m between rows and 3 m between trees with longan, a tree with a long juvenile period, interplanted 6 x 6 m. Loquat produced the same yield as the normal orchard from the third to the tenth year and then was removed to encourage longan (K. Fan 1995, pers. commun.). 2. Training and Pruning. Loquat trees grow upright and too tall when proper training is neglected, often resulting in damage by strong winds and lower labor efficiency. In China, loquat trees had been traditionally trained as a modified central leader, but are recently trained into an open-center system, where branches are pulled down by string to allow light penetration into the crown to promote fruit set (Fujisaki 1994). Loquat may also be trained into a vase-shape. Pruning is carried out in autumn or winter when flower buds become visible. Overgrown branches of the tree crown are removed with shears or handsaws, and sprouts are removed or cut back. Pruning is indispensable to reduce the number of bearing shoots and to secure sufficient flower buds.

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Renovation pruning improved yields and profits of 'Luoyangqing' and was best carried out in summer, one week after harvest. Pruning should not be too heavy and trees are best trained to a modified leader system (Liu et a1. 1994). 3. Flower and Fruit Thinning. Thinning of flowers and/or thinning of fruit is a basic cultural practice for loquat. Flower bud thinning is aimed at limiting the number of flowers within an inflorescence, encouraging the growth of fruitlets, and shaping the inflorescence for easier bagging. After flower thinning, one inflorescence contains a maximum of ten fruits. Fruit thinning by hand is necessary to reduce the number of fruits to one to four per inflorescence in Japan (Fujisaki 1994; Sato 1996) and four to six in some production areas in China. Flower thinning is not widely practiced in China because small farmers feel anxious about reducing production, but is widely performed in Putian, Fujian. The whole inflorescence is often removed, often as many as 30% of the total. When the flowers on the remaining inflorescence are all blooming, further thinning of the clusters is practiced. This method is considered simple and effective by local growers. In Japan, the usual procedure of flower thinning is to remove the lower two to three peduncles and some upper peduncles on the inflorescence, leaving the middle three to four peduncles on the inflorescence. As flower buds appear over a long period, successive thinning (up to three times) is required (Fujisaki 1994). The fruits to remain are selected by size, the larger the better. The thinning should be done as soon as possible after the danger of cold damage is over. The remaining large and healthy fruits are covered by paper bags in Japan. Bagging is indispensable to obtain fruit of excellent appearance, and particularly to protect pubescence on the peel from being rubbed off. Bagging is carried out simultaneously with fruit thinning. Selection of the paper material of the bag is important; translucent bags accelerate fruit maturity, but tend to increase the incidence of physiological disorder in the fruit (Fujisaki 1994). Old newspapers are used for bagging in Putian county, China. 4. Water and Soil Management and Fertilizers. Loquat, which can tolerate drought, is hardier than orange but not as hardy as the fig (Sawyer et a1. 1985). In general, loquat does not require irrigation, but when the fruits are maturing, sprinkler irrigation is carried out to reduce sunburn. Loquat is usually grown under sod culture in Asia. Orchards are mowed two to three times per year and mowed grass clippings are spread

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under the trees as mulch. Growers improve the soil by providing manure and other organic substrates (Sinkai et al. 1982). Film mulch was found to increase hardiness of loquat in Zhejiang, China. The soil temperature increased by 2°C in a 12-year-old loquat orchard mulched with brownblack or transparent polyethylene film from November to June. Soil moisture, nutrient status, and soil bulk density were improved by mulching and available soil N, P, and K, as well as yield also increased (Xia 1986). The types of fertilizer used and application rates are related to plant age and soil nutrient content. For young juvenile trees, fertilizer is applied every two months. In the orchards with low fertility, 195 kg N, 165 kg P, and 210 kg K/ha were applied each year; while in orchards with higher fertility, 150 kg N, 94 kg P, and 112 kg K were applied. In Japan, standard applications are 170 kg N, 115 kg P, and 125 kg K/ha for 10-year-old trees of 'Mogi' (Sato 1996), and 240 kg N, 190 kg P, and 190 kg Kwhen a yield of 10 t/ha is expected (Fujisaki 1994). Fertilizer schedules in China are shown in Table 5.5. A pit disorder (black fruit disease) was associated with low soil Ca and was corrected with soil applications of 100 kg/ha Ca (Huang and Lin 1996). 5. Tree Protection. In Japan, many insect pests and diseases damage loquat and, although only a few are serious enough to require prevention measures (Table 5.6), these can be difficult to control. Control is mainly by chemical pesticides, but every effort is devoted to keeping loquat trees vigorous by management practices (Fujisaki 1994; Sato 1996). In China, the loquat suffers from few diseases (Table 5.6), compared with fruits such as citrus and apple (Chen et al. 1991). The most severe disease is caused by Rosellinia necatrix. Drenching the soil with a solution of Bavistin 50% WP, Benlate 500/0 WP, or Basamid 85% WP and then covering with transparent polyethylene were potential measures for effective control (Duan et al. 1990). Fumigation tests in Florida with methyl bromide at normal atmospheric pressure indicated that 16 g/m 3 Table 5.5.

Fertilizer application timetable for loquat.

Stage of growth

Percentage of total

Function

After harvest Prior to flowering After fruit set Growth of fruit

50

Resuming vigor Increasing cold resistant Lowering fruit drop Increasing fruit growth

15 25 10 (foliar spray)

263

5. LOQUAT: BOTANY AND HORTICULTURE Table 5.6.

Pests and pathogens in major producing areas (+ ::: minor, +++ = severe).

Pests and diseases INSECT Aprona japonica Thomson Anastrepha suspensa Loew Grapholitha moJesta Busck NippoJachnus piri Matsumura PhaJera flavescens Brem. et Grey Rhynchites heros Roelofs PATHOGEN Bacillus amyJovorus (Burr.) Trev. Cercospora eriobotryae Sawada CoJeopucciniella simplex Hara Entomosporium eriobotryae Prill Erwinia amylovora (Burr.) Winsl. Glomerella cingulata Spauld. & Schrenk Hemiberlesia lataniae Sign Pestalotia funerea Desm. Phyllostica eriobotryae Thumen Pseudomonas eriobotryae Takimoto Rosellinia necatrix (Hart.) Berl.

China

Japan

United States

+++ +++ +++

+++ +++ +++ +++

+++

+++ +

+++

+ +

+++

+++ +++ + +++

+++ +++ +++ +++

+++ + + +++ +++ +++

for 2.5 h at 22.8°C or 32g/m3 for 2.5 h at 18.3-22°C gave adequate protection to loquat and other horticultural crops grown in the greenhouse for latania scale [Hemiberlesia lataniae. (Signoret)] (Witherell 1984). Loquat must be protected from cold, wind, and sunburn. Young fruitlet sets from autumn flowering are sensitive to cold temperature and suffer from damage that results in irregular production. Maintaining trees with vigorous and heavy flower bud thinning results in longer flowering periods, and this may avoid cold damage to some extent. In some districts, cold damage is successfully avoided by covering an inflorescence with about 20 g of wool (Fujisaki 1994). In windy areas, newly planted nursery plants must be supported by poles to protect trees from wind damage. Loquat trees, which have shallow root systems, must be protected by windbreaks (Sato 1996). Loquat fruits are usually protected from sunburn by covering the fruit with bags. 6. Harvest and Handling. Loquats reach maturity in about 150 days from full flower-opening in China and Japan (Chen 1958; Ichinose 1995). As each growing district grows only a few cultivars, the typical period of harvesting is only seven to ten days (Fujisaki 1994). Determination of ripeness is not easy, but is important because unripe fruits

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are excessively acid. Change of skin color to the original ripe color (orange-yellow or yellow-white) is a useful indicator for optimum harvest time. However, in case of yellow-white cultivars, determination of the ripe color is difficult (Sato 1996). There is a relationship between harvest date and skin color and fruit quality (Uchino et a1. 1994b). Soluble solids content increased and the titratable acidity (TA) decreased with maturation. Thus, TA in fruits can be decreased with harvest delay. Malic acid content in the flesh decreased as the color increased, for each harvest date. Citric acid content was higher in immature fruits than mature fruits, whereas succinic and fumaric acid contents rose with maturation. Flesh firmness gradually decreased with maturity. Since loquat fruits are easily injured, fruits should be handed carefully (Sato 1996). The fruits are difficult to harvest because of the thick, tough stalk on each fruit that does not separate readily from the cluster, and the fruits must be picked with stalk attached to avoid tearing the skin. Clusters are cut from the branch with a sharp knife or with clippers. Whole clusters are not considered attractive on the market, therefore the individual fruits are clipped from the cluster, and the fruits are graded for size and color to provide uniform packs. An exception is that whole clusters may be displayed in Spain. In India, usually two grades of fruit are considered, although. three grades can be made, with the poorest fruits (undersized or misshapen) sold for manufacture of jams, jellies, and other by-products (Randhawa and Singh 1970). In Japan, fruits are separated into three to four grades according to quality and four to five grades by size, and packed in a 300-g or a 500-g bag, i-kg or 2-kg carton box. Almost all of these procedures are performed manually. As harvesting and packing are highly labor-intensive operations, this limits the area of loquat production for each grower (Fujisaki 1994). In China, the fruit cluster is cut, packed in wood boxes or bamboo baskets, and shipped to market, where the fruits may be classified into two or three grades. In Putian Fujian, loquat fruits are carefully picked, classified into three grades and packed into various kinds of boxes, then shipped to Hong Kong. D. Protected Culture

Growing loquat in plastic greenhouses first originated to protect the trees and fruits from falling volcanic ashes in Tarumizu, a major loquat growing area in Japan, and later to protect the tree from cold injury. This system proved to be profitable because of higher prices from earlier marketing, stable production, and the spreading of labor requirements. 'Mogi' and 'Nagasaki-wase' are the cultivars grown in protected culti-

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vation (Fujisaki 1994). An ethylene vinyl acetate plastic film cover, supported on a framework commonly used for training grapes in Spain, was placed over an orchard of 'Argelino' and 'Tanaka' loquat on quince restocks from budbreak to ripening. The cover advanced the harvesting date by six days and increased market value. Yields, fruit diameter, and weight were unaffected by the plastic cover (L6pez-GaIvez et al. 1990). Most plastic houses are built of iron pipes and covered with polyvinyl chloride sheets. The plastic is covered from leatbreak to flowering and removed after harvest, but, in some cases, is removed in July after the rainy season is over. Because high humidity during flowering is conducive to the outbreak of gray mold (Botrylis cinerea Person) and lowers fruit setting, it is advisable to apply plastic cover after full bloom where gray mold is a problem. Higher temperatures accelerate the growth of young fruitlets but increase fruit maturation, which results in small fruit. Therefore, minimum night temperature should not be higher than 15°C and maximum temperature not higher than 25°C. Higher temperatures just before fruit maturation may cause physiological disorders in the fruits. (Fujisaka 1994). Losses of soil nutrients decreased during the time that the plastic is covered. Thus, fertilizers should be decreased, according to tree vigor. As higher soil water content promotes fruit growth, irrigation is applied during the fruit growing period, and reduced towards fruit maturation to enhance quality. E. Storage and Processing Data available on fruit storage and processing are quite limited. Loquat fruits are mostly consumed fresh and sold at high prices, especially in Japan. Fruit generally will keep for ten days at ordinary temperatures, and for four weeks to 60 days in cool storage. Sugar loss was minimal with slight decrease in acid, resulting in an overall improvement in taste for the mature fruit during storage (Ogata 1950; Shaw 1980). After removal from storage, the shelf-life may be only three to five days (Ogata 1950; Mukerjee 1958; Guelfat-Reich 1970; Shaw 1980; EI-Zeftawi and Goubran 1983b). Treatment with the fungicide benomyl makes it possible to maintain loquats for one month at 16°C with a minimum of decay (Morton 1987). Cold storage of loquats in polyethylene promotes internal browning and fungal development and alters flavor (Guelfat-Reich 1970; Morton 1987). The 'Tanaka' cultivar had an unacceptable flavor after storage, but not 'Akko 13'. Quality aspects after storage depend on cultivar. In a storage experiment in Italy, good results were obtained with

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'Argelino' and 'Tanaka', which were notable for resistance to mechanical damage. 'Marchetto' and 'Palermo' were superior in organoleptic traits (Testoni and Grassi 1995). Controlled atmosphere (CA, low 02) did not influence the quality of fruits stored at 25 ± 5°C. Low temperature (3 ± 1°C) prolonged storage life, especially when combined with CA; losses of soluble sugar, TSS, titratable acidity, and ascorbic acid slowed, enabling fruits to be stored for longer than 40 days. Furthermore, respiration rate, ethylene production, and fruit rots were kept low by low temperature and low O2 (Q. Lin et a1. 1994). Loquat has been used for canning, jam, juice, syrup, candied fruits, and jellies. Dried fruit has good flavor (C. Campbell, per. commun.). A significant amount of canned loquats was produced in Japan in the 1970s and in Taiwan, China, in the 1980s. Yearly production of canned loquats in Japan was 2254 t in 1970 but gradually declined and is now near zero. In Fujian and Zhejiang, China, canning industries are increasing, but enterprises are usually small. Small amounts of loquats are used for jam. Adam (1950) studied the final pH of loquats canned at 100°C and produced over a period of several years. A pH range of 4.0-5.4 was too high to prevent microbial growth during storage of the product (pH higher than 4.0 was considered unsafe). He recommended taking precautions to reduce the pH of the final canned product to increase storage stability.

V. FUTURE PROSPECTS A. Crop Improvement Research priorities have been listed by several authors (Condit 1915; Chandler 1958; Huang 1989; Chen et a1. 1991). These include cultivar improvement such as development of seedless or low-seeded cultivars and whitish flesh, increased quality and size of fruit, and increased cold-resistance. Dwarf types would be of interest for both growers and homeowners. Seedless or less-seeded cultivars would be a desirable feature. Loquat has ten ovules, and is potentially able to bear ten seeds, but generally no more than eight develop and most frequently only three to four. Both frost-induced seedless fruit (Condit 1915) and GA-induced seedless fruit have been reported (Fan 1989; Takagi et a1. 1994), indicating that it is theoretically possible to produce seedless or less-seeded cultivars. However, the physiology and genetics of seedlessness remain to be deter-

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mined. Seediness might be reduced by using low-seeded cultivars as parents in future crossing programs. Although autopolypoloids have undesirable characteristics (Kihara 1981; Huang 1989), triploids should be further investigated for seedlessness. The extent of induced parthenocarpy in loquat is encouraging (Fan 1989; Takagi et a1. 1994) and BA and other plant-growth regulators could be used as auxiliary measures for the production of seedless cultivars. Whitish flesh, which combines fine and tender texture, high sugar content, and good flavor, has been emphasized by Japanese breeders. Breeders have released a whitish flesh cultivar, 'Shiro Mogi', which originated from an open-pollinated 'Mogi' seed irradiated with 20 KR gamma ray. Fruit size is a quantitative trait. The large fruited cultivar, 'Jiefangzhong' crossed with 'Baozhu' (small, medium size fruit of good quality) produced a selection, 82-6-26, characterized by large fruit, high quality, and cold-resistance. 'Zaozhong No.6' and 'Zhongjing 2', released in Fujian, are seedlings of 'Jiefangzhong' and possess large fruit and good quality (Huang et a1. 1993; Ding et a1. 1995a). There are a number of reports that loquat trees are surprisingly hardy (Dawes 1980; Vogel 1993; Park and Park 1995), although reproductive organs, especially ovules, are cold sensitive. In general, cold injury is a limiting factor for commercial production of loquat. The survey of loquat resources in recent years in China are encouraging. The north part of Jiangsu province is a deciduous tree area, but farmers plant loquat trees around their houses and propagate the trees by seed. Many cold-resistant resources have apparently been developed. For example, some seedlings with cold resistance and large fruit, such as 'Bahong' and.'Shichen', have been selected in Zhenjiang City, Jiangsu. The yield of the original 'Bahong' tree reached 100 kg when the lowest temperature was -11.6°C in the winter of 1976, and in the winter of 1991, when the lowest air and ground temperature was -11.2°C and -20°C, respectively, yields were as high as in normal years. 'Bahong' obviously possesses very high cold resistance (Ding et a1. 1995a). Cold-resistance breeding could focus on selection of seedlings in the marginal area of the north. In marginal areas, seedless fruits are sometimes formed when cold kills the very young embryos but is not severe enough to damage the flesh. The timing of fruit setting in fall and the timing of the cold are factors in fruiting and damage. If minimum night temperatures at bloom time are too high, fruit may not set. When the earliest flowers set in Florida, there can be ripe fruit in early January before the most damaging cold. Higher elevation could possibly affect the temperatures and time of fruit set. A study of the 'Bahong' in Jiangsu province is needed

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S. LIN, R. SHARPE, AND J. JANICK

to determine if there is genetic resistance or avoidance factors in fruit· ing. The presence of resistance, if present, could extend the culture of loquat.

B. Culture and Utilization Loquat areas of production are generally expanding. In China, loquat areas have increased 15·fold and yield increased 38·fold during the past 45 years. In Japan, there has been no increase in loquat cultivation area from the 1940s to the present for many reasons, but loquat production potential is high because the prices are the highest among all kinds of fruits year·round, and in the leading producing prefecture, Nakasaki, production has increased gradually. Loquats have become increasingly popular in Brazil. The range of cultivated loquat is also changing. The leading produc· ing areas of loquat in China are Zhejiang, Fujian, and Taiwan, ranging from 30 0 to 22 0 N latitudes. Two of the leading producing prefectures in Japan are Nakasaki and Chiba, 33 0 and 35 0 N latitudes, respectively. Commercial cultivated areas of loquat in Palermo, Italy, are located at 44°N. It is clear that loquat could be cultivated between 25 0 to 35 0 Nand S latitudes, depending on altitude and climate. If some cultivars with cold resistance are introduced, the areas of production of loquat could be further expanded. In India, a large area of adaptation appears to have a stable production, though specific figures are unavailable. Australia can produce loquat in both western and eastern areas. Adapted cultivars have large firm fruit, and if an export market could be developed, there would be increased commercial potential. Production of loquat in home gardens is high in Australia. Home production also occurs in the Mediterranean area, but with large urban markets there is an opportunity for commercial pro· duction (Mansour and Leaver 1995). There was much early interest in loquat being grown in Florida (Krome 1936; Popenoe 1960), but the fruit is no longer grown commer· cially because of the fruit fly, Anastrepha suspensa. In the Gainesville area, where the fruit fly is not a problem, homeowners welcome the occa· sional crop (spared by frosts) but nurseries offer only seedlings that are inferior to 'Oliver' and 'Advance'. Interestingly, loquat is about the only fruit that does well in the shade of large trees such as hickory and oak, which can sometimes protect flowers and young fruit from freezing. As in Florida, there was also a similar early interest in loquats in California (Condit 1915; Chandler 1958). Both states have a limited region of adaptation and have attracted rapid population growth to their mild

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winter areas. Cultivars are still offered by California nurseries (Whealy 1989). Among them are 'Advance', 'Ben Lehr', 'Big Jim', 'Champagne', Gold Nugget', 'Mogi', 'Mrs. Cooksey' and 'Strawberry'. In the United States, the introduction of loquat would have serious competition in the marketplace because of the presence of many kinds of citrus, apples, and pears from controlled atmosphere storage, peaches and nectarines from Chile, and strawberries from local production in Mexico and California in the early spring. Nevertheless, a limited commercial market for loquat exists among Asian populations in California. Extensive studies have been carried out on harvesting, handling, and packaging of loquat, but the data on fruit storage and processing are limited (Zheng et al. 1993b; Lin and Chen 1994; Testoni and Grassi 1995). Some cultivars, such as 'Argelino' and 'Tanaka', are resistant to mechanical damage, and 'Palermo' is superior in organoleptic traits. Loquat fruits can be stored for longer than 40 days under CA without extensive diminution of quality. This suggests that loquat is a candidate for the export market provided fruit shelf-life can be extended with controlled atmosphere storage. In Fujian and Zhejiang, China, the canning of loquat is increasing, and small amounts of loquat fruits have been used to make jam, wine, syrup, and candied fruits, but these are small-scale operations and large enterprises with better facilities must be established.

LITERATURE CITED Adam, W. B. 1950. pH in fruit and vegetable canning. Food 19:4-7. Amitava, G., and P. K. Chattopadhyay. 1993. Studies on the developmental physiology of loquat fruit. Hort. J. 6:29-33. Blumenfeld, A. 1980. Fruit growth of loquat. J. Am. Soc. Hort. Sci. 105:747-750. Burl6, F., A. Vidal, 1. G6mez, and J. Mataix. 1988. Changes in the mineral fraction in leaves and fruits of loquat (cv. Algeri). Anales Edafologfa Agrobiogfa 47:1607-1618. Burney, B. 1980. Exotics: A guide to some that may be grown in New Zealand. Part I. New Zealand J. Agr. 140:58-59, 62. Campbell, C., and S. E. Malo. 1986. IFAS Fact Sheets 5. The loquat. Univ. Florida, Gainesville. Chandler, W. H. 1958. Pome fruits. Evergreen orchards. Lea and Febiger, Philadelphia. p. 347-350. Chaudhary, A. S., M. Singh, and C. N. Singh. 1990. Effect of plant growth regulators on maturity of loquat. Progress. Hort. 22:184-190. Chaudhary, A. S., M. Singh, and C. N. Singh. 1993. Effect ofNAA, 2,4,5-T and GA on loquat cultivar 'Safeda Batia'. India J. Agr. Res. 28:127-132. Chen, Q. F. (ed.). 1988. Loquat (in Chinese). Fujian ScienTech. Press, Fuzhou, China. Chen, W. X. 1954. The loquats in Putian, Fujian. Acta Agr. 5:199-214.

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Chen, W. X. 1958. Observation of biological characteristics in loquat. J. Fujian Agr. ColI. 1958(7):29-49. Chen, Z. G., and S. Q. Lin. 1991. Some research on micropropagation of horticultural crops. p. 28-32. In: R. Chen (ed.), Advances in biotechnology in China (in Chinese). China Scientech Press, Beijing. Chen, Z. G., and S. Q. Lin. 1995. On research of micropropagation of horticultural crops in China. p. 143-146. In: Chinese Scientech. Committee (eds.), Advances in biotechnology in China (in Chinese). China Scientech Press, Beijing. Chen, Z. G., S. Q. Lin, and Q. L. Lin. 1983. A briefreport on the induction of plantlets from endosperm of loquat. J. Fujian Agr. Univ. 12:243-246. Chen, Z. G., S. Q. Lin, and Q. L. Lin. 1991. Loquat. p. 62-75. In: Y. P. S. Bajaj (ed.), Biotechnology in agriculture and forestry. Vol. 16, Trees III. Springer-Verlag, Berlin/Heidelberg, Germany. Chen, Z. G., B. K. Ma, S. Q. Lin, and J. J. Lei (eds.). 1995. In vitro biology in horticultural plants (in Chinese). China Agr. Press, Beijing. Church, C. G., and D. G. Sorber. 1935. The chemical composition of the loquat. Fruit Prod. J. Am. Vinegar Ind. 14:335-340. Condit, 1. J. 1915. The loquat. California Agr. Expt. Sta. Bul. 250. Crescimanno, F. G., and F. Barone. 1980. Variations in the N, P, K, Ca and Mg contents of loquat during one annual cycle. Tecnica Agriola 32:215-222. Cyprus Agricultural Research Institute (eds.). 1987. Ministry of Agriculture and Natural Resources. Nicosia, Cyprus. Dawes, H. 1980. Reevaluating cold hardiness of certain tropical fruit trees. California Rare Fruit Growers Yearb. 11:46-49. Degani, C., and A. Blumenfeld. 1986. The use of isozyme analysis for differentiation between loquat cultivars. HortScience 21:1457-1458. Demir, S. 1983. Promising loquat cultivars for the Antalya region. Bahce 12:5-16. De Tommasi, N. 1992a. Plant metabolizes. New sesquiterpene and ionone glycosides from loquat. J. Nat. Prod. 55:1025-1032. De Tommasi, N. 1992b. Constituents of Eriobotyra japonica: A study of their antiviral properties. J. Nat. Prod. 55:1067-1073. Ding, C. K., Q. F. Chen, and T. L. Sun. 1995a. Seasonal variant in the contents of nutrient elements in the leaves and the fruits of Eriobotrya japonica Lindl. Acta Hort. 396:235-242. Ding, C. K., Q. F. Chen, T. L. Sun, and Q. Z. Xia. 1995b. Germplasm resources and breeding of Eriobotrya japonica Lindl. in China. Acta Hort. 403:121-126. Ding, C. K., Q. F. Chen, Q. Z. Xia, and T. L. Sun. 1991. The effects of mineral elements and growth regulators on the pollen germination and fruit set of loquat trees. China Fruits 1991(4):18-20,40. Ding, C. K., and H. Z. Zhang. 1988. Effects of hormones on growth and development of loquat fruits. Acta Hort. Siniea. 15:148-153. Duan, C. H., H. W. Tsai, and C. C. Tu. 1990. Dissemination of white root rot disease of loquat and its control. J. Agr. Res. China 39:47-54. Duke, J., and E. S. Ayensu. 1985. Medicinal plants of China. Vol. 2. Reference Publ., Algonac, MI. p. 544. EI-Shazly, S. M., M. B. EL-Safrout, and H. A. Kassem. 1994. Root formation on the stem cuttings of Eureka lemon and 'EL-Soukari' loquat as affected by root-promoting chemicals and mist. Alexandria J. Agr. Res. 39: 559-569. EI-Zeftawi, B. M., and F. H. Goubran. 1983a. Chemical induction of seedless loquat. Austral. Hort. Res. News!. 55:126.

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6 Crop Physiology of Sweetpotato V. Ravi and P. Indira * Central Tuber Crops Research Institute Sreekariyam Trivandrum India 695 017

I. Introduction II. Shoot System A. Stem Branching B. Leaf Characteristics C. Leaf Area D. Photosynthesis E. Respiration F. Translocation III. Root System A. Non-Storage Roots B. Storage Roots 1. Initiation 2. Endogenous Growth Regulators 3. Starch Synthesis 4. Effect of Atmospheric/Soil Factors 5. Storage Root Bulking Pattern IV. Source and Sink Relationship V. Dry Matter Production and Harvest Index VI. Shoot Removal and Storage Root Yields VII. Response to Growth Regulators and Chemicals VIII. Response to Stress A. Water Deficit B. Flood C. Shade D. Salt

*We thank the Director of the Central Tuber Crops Research Institute for providing facilities, Mr. R. Saravanan, CTCRI, for assistance, and referees for their critical but constructive reviews. Horticultural Reviews, Volume 23, Edited by Jules Janick ISBN 0-471-25445-2 @ 1999 John Wiley & Sons, Inc. 277

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IX. Propagation Physiology A. Vine Cuttings B. Root Sprouts C. Cut Root Pieces D. Micropropagation E. True Seed X. Conclusion Literature Cited

I. INTRODUCTION

Sweetpotato (also sweet potato) (Ipomoea batatas (L.) Lam., Convolvulaceae) is a herbaceous dicot widely grown throughout the tropics and warm temperate regions of the world between latitudes 40 0 N and S of the equator and between sea level and 2,300 m altitude (Shukla 1976; Hahn 1977a; Bourke 1982; Jana 1982). The world sweetpotato production has been estimated to be 124,339,000 t, of which 92% is in Asia (FAD 1994; Table 6.1). China produces 84% of the world sweetpotato production. In China, most of the crop is used for animal (pig) feed and some is used in the commercial production of noodles and other consumer food products (Zaag et aL 1991). Sweetpotato, a perennial is commonly cultivated as an annual crop. Depending on the growing conditions and cultivars, crop growth period varies between 12 and 35 weeks (Chen and Xu 1982; Hahn and Hozyo 1984), whereas a long duration of 25-50 weeks also has been reported for some cultivars (Huett 1976; Huett and D'Neill1976). However, most of the cultivars attain maximum storage root yield in 12-22 weeks after planting (WAP) (8teinbaur and Kushman 1971; Huett 1976; Gupta and Ray 1979; Indira and Lakshmi 1984; Nair and Nair 1985; Nair et al. 1986; Sen et al. 1990). Photoperiod is a major factor here. As one approaches the equator with its -12 h photoperiod, the total light energy per day is less and cultivars grow for a longer duration than in the temperate areas to produce yield. Being a shorter duration crop than the other tropical root and tuber crops (O'Hair 1990), sweetpotato can be readily considered a quick source of food. The succulent, starchy storage roots of sweetpotato serve as staple food, animal feed (Ruiz et al. 1980; Lu et al. 1989; Posas 1989; Woolfe 1992), and to a limited extent as a raw material for industrial purposes as a starch source and for alcohol production (Winarno 1982; Yen 1982; Collins 1984). Storage roots contain about 50-79% starch and 4-15% sugar on a dry weight basis (Ashokan and Nair 1983; Liu et al. 1985; Lila and Bala 1987; Lila et al. 1990; Goswami 1991; Li et aI. 1994) or 7-28%

6. CROP PHYSIOLOGY OF SWEETPOTATO

Table 6.1.

279

Sweetpotato area harvested, yield, and production. Area harvested (x 103 hal

Yield (t/ha)

Production (x 10 3 t)

World

9380

13.26

124339

CONTINENTS Asia Africa South America North Central America Oceania Europe

7587 1384 116 166 121 5

15.07 5.02 10.72 6.87 4.95 12.03

114347 6944 1248 1140 600 60

CHIEF COUNTRIES China Uganda Indonesia Japan India Rwanda Philippines Kenya Brazil United States Madagascar Burundi KoreaDRRP Papua New Guinea

6511 478 197 51 138 160 148 66 61 33 95 90 36 107

16.15 4.50 9.42 24.64 8.33 6.25 4.73 9.85 10.33 18.15 5.90 5.63 14.21 4.53

105180 2151 1854 1264 1150 1000 700 650 630 593 560 507 504 484

Location

starch on a fresh weight basis (Huang 1982; Li and Liao 1983; Indira and Lakshmi 1984; Li et al. 1984; Liao et al. 1985; Lee et al. 1985; Hong et al. 1986; Lila et al. 1990; Li et al. 1994). Fresh storage roots can also be a source of vitamin C, provitamin A, vitamin B (thiamine), and iron. They are normally low in protein and lipids (Huang 1982; Yang 1982; Bureau and Bushway 1986). Storage roots of yellow-orange fleshed cultivars contain high amounts of carotenoids (Badillo-Feliciano et al. 1976; Huett 1976; Junek and Sistrunk 1978; Love et al. 1978; Collins and Pope 1979; Ikehashi 1985; Nair et al. 1986; Kukimura et al. 1988; Bhattacharya et al. 1990a; Tanahata et al. 1993). Tender shoot tips and leaves are nutritive and are used as a vegetable and as animal feed. In the tropics and subtropics, sweetpotato is cultivated under rainfed or irrigated conditions in soils of low fertility. In temperate regions, the crop grows in areas with a frost-free period of 16-20 weeks. Sweetpotato

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is generally cultivated as a sole crop, but being a short-duration crop, it often fits well into farming systems such as relay cropping, intercropping, mixed cropping, and rotations with other crops (Wan 1982; Moreno 1982; Sannamarappa and Shivashankar 1988; Caradang and Curayag 1989; Shinohara et al. 1989; Ghosh 1991). Sweetpotato cultivars vary widely in their storage root yield potential. An average fresh storage root yield of about 10-25 t/ha in 16-20 weeks has been obtained in many countries (Bhagsari and Harmon 1982; Li and Kao 1985a; Secreto and Villamayor Jr. 1985; Sen et al. 1988; Bhagsari 1990; Rao and Sultana 1990). The world average storage root yield of sweetpotato has been estimated to be 13.38 t/ha (FAD 1994). However, experimental storage root yields ranging between 30-73 t/ha have been obtained (Hossain et a1. 1987; Siddique et a1. 1988; Hall and Harmon 1989; Bhagsari and Ashley 1990; Varma et a1. 1994). Fresh vine yield varies between 11-45.7 t/ha when harvested as whole shoots (Singh and MandaI 1976; Li and Kao 1985a; Sen et a1. 1990; Mukhopadhyay et al. 1992). Wide variability in storage root yield among sweetpotato cultivars and individual plants of the same cultivar has been attributed to cultivar, propagation material, environment, and soil factors (Lowe and Wilson 1975). Genetic and environmental factors influence leaf area, leaf production and abscission, leaf photosynthesis, storage root formation and development, total dry matter production, and dry matter partitioning. This review attempts to discuss the interaction of physiological traits with environmental and edaphic factors that determine sweetpotato yield, including root and vine.

II. SHOOT SYSTEM A. Stem Branching

Sweetpotato has a decumbent stem and cultivars can be arbitrarily categorized as either erect bushy, intermediate, or spreading, based on the length of their vines (Yen 1974; Kays 1985). Branching is cultivar dependent (Yen 1974) and branches vary in number and length. Normally, sweetpotato plants produce three types of branches, primary, secondary, and tertiary, at different periods of growth (Kays 1985; Somda and Kays 1990a; Sasaki et a1. 1993). The total number of branches varies among cultivars between 3-20, whereas stem length varies between 0.5-2.2 m (Amarchandra et al. 1985; Amarchandra and Tiwari 1987; Li and Yen 1988; Jeong and Oh 1991; Rao et al. 1992; Rajeshkumar et a1. 1993). How-

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ever, the branching system in sweetpotato plants is heavily influenced by spacing, photoperiod, and soil moisture and nutrients. Increasing plant density decreases stem length and total number of branches per plant, presumably due to increases in competition for nutrients and irradiance (Amarchandra and Tiwari 1987; Li and Yen 1988; Ogbuehi et al. 1988; Somda and Kays 1990a; Sasaki 1991). Plant density also influences the initiation time of the three types of branches as well as their number. The increase in stem length and total number of branches per plant at decreasing plant density is primarily due to the formation of secondary branches (Somda and Kays 1990a). Stem length and total number of branches per plant increase with increases in irrigation (Li and Yen 1988; Indira and Kabeerathumma 1990; Nair and Nair 1995) and soil application ofN (Li and Yen 1988; Nayar and Vimala 1990; Nair and Nair 1995). An increase in K up to 75 kg/ha, however, does not significantly increase the total number of branches (Nair and Nair 1995). A long photoperiod (18 h) decreases branch number while increasing the branch length when compared to plants exposed to a 12.5 h photoperiod. A short photoperiod of 8 h increases branch number while decreasing the branch length (McDavid and Alamu 1980a). Light intensity does not have any clear effect on the number of branches (Wilson 1967). B. Leaf Characteristics Sweetpotato cultivars predominantly have prostrate stems with leaves expanded into a horizontal, shallow canopy close to the soil surface, enabling the plant to intercept maximum solar radiation. Leaf shape varies widely among cultivars. The leaves may be round, reniform, cordate, triangular, and lobed moderately or deeply (Yen 1974). Plants have an indeterminate growth habit and continuously produce new leaves. As the number of leaves produced increases throughout the growing period, the percentage of leaves attached to the plant significantly decreases due to shedding (Somda et al. 1991). The total number ofleaves retained per plant at any point in time varies widely among cultivars. The number of leaves depends on the number of branches or growing points, stem and internode length, rate and duration of leaf production, and leaf longevity or leaf shedding. Total number of leaves per plant among cultivars varies between 60-300 (Amarchandra et al. 1985; Somda et al. 1991; Rajeshkumar et al. 1993). The number of leaves per plant increases with decreasing plant density (Somda and Kays 1990b), increasing irrigation (Indira and Kabeerathumma 1990; Holwerda and Ekanayake 1991; Nair and Nair 1995), and N application (Nair and Nair 1995). An increase in K, however, does not appreciably increase the number of

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leaves per plant. Defoliation of vine cuttings prior to planting drastically decreases the number of leaves per plant (Jayakrishnakumar et a1. 1991). Under typical production conditions, leaf shedding is a consistent phenomenon among sweetpotato cultivars. As the sweetpotato plant expands its canopy horizontally, the petioles of newer leaves become progressively longer, placing newer leaves above older leaves in the canopy. Consequently, older leaves become situated in a progressively less favorable position in the canopy's light-reception hierarchy. Thus, leaves within the plant as well as between adjacent plants cause mutual shading of older leaves in the lower strata of the canopy. Ageing and mutual shading cause shedding of older leaves (Somda and Kays 1990b; Somda et a1. 1991; McLaurin and Kays 1993). Leaf shedding is less frequent during the early period of growth but increases progressively during the later period (Somda et a1. 1991). Under normal production conditions, leaf shedding varies among cultivars between 48-63°,10 of the total leaves formed by the end of the growing period (Somda et a1. 1991; McLaurin and Kays 1993). In one study, it was found that prior to shedding, leaves lost 63% of their dry matter and the remaining remobilized within the plant (Somda et a1. 1991). Leaf shedding, therefore, represents a significant loss of dry matter by the plant. The estimated loss of leaf dry matter among cultivars varies between 1.2-2.8 t/ha (Somda et a1. 1991; McLaurin and Kays 1993). A significant portion of P, K, and Mg is also remobilized prior to leaf shedding (Somda et a1. 1991). Positive correlation between leaf shedding and vine dry weight indicate that vigorous vine growth and subsequent shading of older leaves induce high leaf shedding. However, absence of a strong correlation between number of leaves shed and storage root yield suggest that leaf shedding has no negative impact on storage root yield (McLaurin and Kays 1993). The specific leaf weight (SLW) or leafweight/area ratio of sweetpotato cultivars varies between 2--4.4 mg/cm 2 (Bhagsari 1981; Bhagsari and Harmon 1982; Indira and Kabeerathumma 1990; Nair and Nair 1995). The SLW declines at the end of the growth period mainly due to translocation of dry matter from the leaves to the storage roots (Somda and Kays 1990b; Somda et a1. 1991; Nair and Nair 1995). SLW increases with increasing plant density (Sasaki 1991), and CO 2 concentration in the atmosphere (Bhattacharya et a1. 1992; Biswas et a1. 1986). A long photoperiod (24 h) increases leaf dry weight and number as compared to a short photoperiod (12 h) (Bonsi et a1. 1992). Stomatal density of sweetpotato leaves varies between 47-155/mm 2 on the adaxial side and between 151-318/mm2 on the abaxial side (Bhagsari 1981; Bhagsari and Harmon 1982; Bhagsari 1990; Kubota et a1. 1992a). High-yielding cultivars have a greater number of stomata on the

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abaxial surface and a lower number of stomata on the adaxial surface than low-yielding cultivars (Kubota et al. 1993). The concentration of chlorophyll a, ~-carotene, and xanthophyll in leaves varies widely among cultivars (Katayama and Shida 1961). Chlorophyll a content varies between 5.3-7.8 mg/g dry leaf tissue, while the chlorophyll b content varies between 2.4-3.8 mg/g dry leaf tissue (Bhagsari 1981). The total chlorophyll (a + b) content varies between 7.6-10.6 mg/g dry leaf tissue (Bhagsari and Harmon 1982). The leaf chlorophyll content remains relatively constant throughout the growth period, while the chlorophyll a:b ratio increases with plant age (Songhai et a1. 1994). Bhagsari (1981) reported significant positive correlation between leaf chlorophyll and N content. However, leaf chlorophyll content does not show significant correlation with the net photosynthetic rate (Bhagsari and Harmon 1982). Deficiency of Mn, Zn, and Cu causes interveinal chlorosis that leads to complete bleaching of the young and middle leaves (Pillai et al. 1986). Walker and Woodson (1987) reported petiole N0 3-N concentration to be a reliable indicator of current N status of the plants, while the total N concentration of blades appears to be a more reliable predictor of storage root yield. C. Leaf Area Leaf area per plant or the leaf area index (LAI) is the ratio of leaf area to land area. LAI varies widely among sweetpotato cultivars and at different growth periods depending on the number of leaves retained on the stem and their size. Length and breadth, leaf dry weight, and planimeter measurements determine LAI (Ramanujam and Indira 1978; Rao et al, 1979). Shorter photoperiod (McDavid and Alamu 1980a), increasing N application (PatH et al. 1990), and decreasing plant density (Somda and Kays 1990b) significantly increase individual leaf area and leaf area per plant. Changes in LAI during growth occur in three phases. LAI steadily increases from the 2nd WAP in the first phase, reaching a plateau between the 8th-16th WAP in the second phase, and declines during the third phase at the end of the growth period partly due to leaf shedding, mutual shading, and reduced light intensity in the lowermost leaves. The maximum LAI among cultivars during the second phase varies between 2-11 (Yu 1981; Agata 1982; Bourke 1985; Indira and Ramanujam 1985; Li and Kao 1985a; Tiwari et a1. 1985; Mukhopadhyay et a1.1992; Bhagsari and Ashley 1990; Nair and Nair 1995). LAI increases with increase in air temperature (Agata 1982; Mukhopadhyay et a!. 1991), photoperiod (Mukhopadhyay et al. 1991), N application (Bourke 1985; Nair and Nair

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1995). soil moisture (Enyi 1977; Indira and Ramanujam 1985; Chowdhury and Ravi 1990). and due to staking (Bhagsari 1990). A higher dose of K has no effect on LAI (Bourke 1985; Nair and Nair 1995). Brown (1992) estimated that LAI of 3-4 is required to intercept 95°t'o of PAR in sweetpotato. Most of the cultivars. in fact. maintain LAI between 3-4 between 8-16 WAP. At this LAI. maximum weekly crop growth rate (CGR) or dry matter production of cultivars varies between 106-133 g/m 2 (Tsuno and Fugise 1963; Enyi 1977; Agata 1982; Tiwari et al. 1985). D. Photosynthesis The physiology of photosynthesis of sweetpotato is little understood. Photosynthesis of sweetpotato leaves is similar to that of C3 plants (Evans 1976; Bhagsari and Harmon 1982; Hahn and Hozyo 1984; Kays 1985). Net photosynthetic (PN ) rate of individual leaves varies between 12-39 mgCO z·dm-2 ·h-1 among cultivars (Tsuno and Fujise 1965; Spence 1971; Hahn 1977a; Wilson 1977; Kato et al. 1979; Bhagsari 1981; Bhagsari and Harmon 1982; Vines et al. 1983; Bhagsari 1990; Bhagsari and Ashley 1990). The PN rate is highest during early growth period and declines at the end of growth period because the sink attains maximum size at this time (Hozyo et al. 1979. 1980; Bhagsari and Harmon 1982). Lack of consistent PN rate of cultivars in different seasons and at different periods of growth in the same season is primarily due to the interaction of PN with environmental factors and plant growth period (Bhagsari and Harmon 1982). The PN capacity of leaf blade relates to the thickening property of storage roots (Hozyo et al. 1979; Hozyo 1982). In reciprocal grafts between 1. batatas cultivar and I. trifida, a related species which does not form storage roots. PN rate of leaf blade was greater in grafts with larger storage roots than in grafts of smaller storage roots (Hozyo and Park 1971; Hozyo and Kato 1976). The PN rate of individual leaves drastically declines with leaf age (Kato et al. 1979; Bhagsari 1988). The PN rate of individual leaves negatively correlates with individual leaf size and the PN rate per unit leaf area decreases in leaves greater than 50 cm 2 (Bhagsari and Brown 1986). Maximum PN rate occurs at air temperature >25-34°C (Hozyo 1982; Bhagsari and Harmon 1982; Bhagsari 1988. 1990; Bhagsari and Ashley 1990; Kubota et al. 1992a). The PN rate of individual leaves steadily increases with an increase in CO 2 concentration up to 900 ppm in the atmosphere surrounding the leaf (Hozyo and Kato 1976). The PN rate at 900 ppm CO 2 is 1.5-fold greater than the PN rate observed at ambient atmospheric CO 2 concentration. However. in vitro activity of ribulose biphosphate carboxylase (RuBISCO) saturates at a very low CO 2 con-

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centration of 20 Jlmol/mol surrounding the leaf where the stomatal resistance was removed by peeling the abaxial side epidermis (Kubota et al. 1994). The PN rate of sweetpotato leaves saturates at irradiance (I) of 750-900 flmol·m-2·s-1 (Vines et al. 1983; Kubota et al. 1994). The response curve of PN/I depends upon the leaf internal CO 2 concentrations (Gi ) and an increase in inter-cellular CO 2concentrations causes the PNrate to saturate at a high level ofl (Kubota et al. 1992b). However, radiation greater than that required to saturate single leaves may be required to saturate the whole canopy because of lower shaded leaves (Hahn and Hozyo 1984). Photosynthesis of sweetpotato leaves is also influenced by stomatal resistance (SR) because SR regulates Ci (Kubota et al. 1994). High relative humidity (RH) increases PNrate and plants grown under higher RH (850/0) have greater stomatal conductance and PN rates than those grown under low RH (Mortley et al. 1994). The PNrate of sweetpotato leaves significantly decreases during midday (Hahn 1977a; Agata and Takeda 1982; Bhagsari and Harmon 1982; Xu and Shen 1985). The midday depression in PNneither relates to the accumulation of photosynthates nor to changes in CO 2 concentration of the air but closely correlates with an increase in SR of leaves (Xu and Shen 1985) and decrease in irradiance (Agata and Takeda 1982; Bhagsari and Harmon 1982). However, under constant environmental conditions, P Nrate of sweetpotato leaves remains high and relatively stable during the morning and mid-day but decreases toward the late afternoon (Tsuno and Fujise 1965). Hozyo et al. (1979) found the PNrate to be positively correlated with the N content of the leaf blade. However, Bhagsari and Harmon (1982) found the PNrate to have no significant correlation with the N content of sweetpotato leaves. Photosynthesis is markedly higher in sweetpotato leaves containing >40/0 K on a dry weight basis (Tsuno and Fujise 1965). This may be because K increases the rate of translocation of photosynthates from leaves, which in turn accelerates photosynthetic activity. Under P-deprivation, photosynthesis and photorespiration (RJ of isolated sweetpotato leaf cells decreased and the ratio of RL and PN rates increased (He et al. 1992). The optimum P concentration for photosynthesis of isolated leaf cells of sweetpotato is lower with 21 % O2 than with 2% O2 and conditions favoring RL could decrease the demand for P in photosynthesis due to release of P during the hydrolysis of phosphoglycollate to glycollate (He et al. 1993). Inoculation with vesicular arbuscular mycorrhizae in the root system (Potty and Indira 1990) and high concentration of CO 2 in the atmosphere surrounding the leaf (Biswas et al. 1986) increase PN rate of sweetpotato plants. The PN rate shows a negative correlation with starch content of leaves (Hozyo et al. 1979; Nakatani et al. 1988b) due to feedback inhibition of photosynthesis.

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The PN rate of individual leaves in the sweetpotato canopy is variable and therefore the P N rate does not precisely reflect the performance of all leaves together in the canopy. Lack of correlation between P N rate and canopy photosynthetic rate (CPN ) (Bhagsari and Ashley 1990) is presumably due to the indeterminate growth habit that results in the presence of a portion of leaves with high PN rate while a portion of leaves have reduced PN rate. The PN of individual leaves also shows no significant correlation with the total dry matter production and storage root yield (Bhagsari and Harmon 1982; Bhagsari 1990; Bhagsari and Ashley 1990). Maximum CPN rate of sweetpotato cultivars varies between 3.7-6.5 gCO z·m-z·h-1 (Agata and Takeda 1982; Bhardwaj and Bhagsari 1988; Bhagsari 1990). At the end of the growth period, the CP N rate declines to 500/0, presumably due to an increase in the proportion of older leaves in the canopy and the decrease in the PN rate of individual leaves due to the maximum growth of storage roots (Bhardwaj and Bhagsari 1988; Bhagsari 1990). In one study, sweetpotato cultivars differed in CPN rates at each measurement during a 23-week growth period due to interaction of the PN rate with the environment (Bhagsari 1990). This makes the ranking of cultivars for CPN difficult. An increase in plant density does not have a significant effect on CPN (Bhardwaj and Bhagsari 1988). E. Respiration Dark respiration (Ro) of different parts of the sweetpotato plant, including leaves, stem, storage and fibrous roots, has not been extensively investigated. Leaves of the sweetpotato plant have the highest Ro rate when compared to the stem, storage and non-storage roots (Tsuno and Fugise 1964, 1965; Agata 1982; Agata and Takeda 1982). Throughout the growth period, leaf blades show the largest proportion (500/0) of total respiration and there are no cultivar differences (Tsuno and Fujise 1964). The Ro rate of leaves, stems, and storage roots steadily increases during the early period of growth, levels off during the middle of growth period, and then declines at the end of the growth period (Tsuno and Fugise 1965). The non-storage roots show a relatively constant Ro rate throughout the growth period. The higher Ro of leaves, petiole, and stems than is found in storage roots may presumably decrease storage root yield when excessive vine growth occurs.

F. Translocation The carbon fixed by the sweetpotato leaf is translocated as sucrose out of the leaf into the stem. For sweetpotato storage root growth, high shoot

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growth should be combined with efficient translocation of photosynthate. Under low-yielding conditions, translocation limitation is more important than sink limitation (Janssens 1984). Kato et al. (1972) studied the translocation of 14C photosynthate in rooted sweetpotato leaves (phytomodels) with storage roots. They found that 50% of the total 14C disappeared within 24 h of exposure to 14CO Z ' presumably due to respiration, while the rest translocated to storage roots. Kata et al. (1979), using 14COz, reported that both apical and basal leaves on the sweetpotato stem display bidirectional transport, while the lower leaves transport a major portion of their photosynthata in a basipatel direction. However, Kays at al. (1987), by exposing the leaves in a whole plant to 11COz, found that essentially all of the photosynthate from the leaves on the main stem is basipetally translocated toward the roots. That being the case, carbon stored as starch within the apical leaves on the main stem may be recycled for growth of the main stem apex. While acropetal translocation is negligible within the main stem, lateral branches at the base of the plant, which bear numbers of fully developed leaves capable of photosynthate export, exhibit acropetal translocation of some photosynthate derived from the main stem (Kays et al. 1987). It is not clear if the photosynthata from the main stem moves directly into the lateral branches or first moves to the root system and then is translocated into the lateral branches. The significance of acropetal translocation in lateral branches is not known. Sweetpotato leaves export a greater amount of photosynthate when measured during early hours of the forenoon and late hours of the afternoon than during the mid-day. This means basipetal translocation occurs during the dark period. Correspondingly, the export pool ofphotosynthate in leaves is greater in the early forenoon and late afternoon than during the mid-day. In contrast to the export pool, the storage pool of photosynthate within the leaf remains low in the early forenoon and late afternoon but increases during the mid-day (Kays at al. 1987). The speed of basipetal translocation of photosynthate is an important factor for storage root growth and it varies during the day and between various sites along the main stem (Kays et al. 1987). This may be due to influxes of photosynthate from other leaves along the stem and/or changes in the resistance to flow once the photosynthate enters the phloem tissues of the main stem. . Photosynthate translocated toward the root system is partly used for expansion of fibrous, non-storage roots and the rest is deposited in storage roots. Basipetal translocation toward underground parts increases when the storage roots are initiated (Kato and Hozyo 1972,1974). High sink (storage root) potential (Kato and Hozyo 1972, 1974, 1978) and fac-

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tors that increase storage root growth such as moderate soil moisture, RH (Ehara and Sekioka 1962), low soil temperature (Sekioka 1963a,b, 1971) and low light intensities (Sekioka 1962) enhance the rate and speed of basipetal translocation. This is because an increase in sink strength would subsequently decrease concentration of photosynthate in the phloem within the sink, which in turn would increase the concentration gradient between the leaves and the storage roots. Velocities of acropetal translocation are not much affected due to changes in the sink potential (Kato and Hozyo 1978). Movement of photosynthate to storage roots can be influenced by the extent of vascular connections between the stem and the storage root. It has been suggested that the secondary phloem and the phloem vas· cular bundles in the storage root stalk influence the translocation of photosynthate from the shoot to storage roots (Wilson and Lowe 1973). In one study, phloem tissues occupied up to one·third of the cross· sectional area of storage root stalk in a high-yielding cultivar (Wilson 1982). As the number of storage roots increase, the total amount of phloem in the storage root stalk increases (DeCalderon 1981; DeCalderon et al. 1983). However, correlations of phloem crosssectional area with storage root dry weight in a limited number of cultivars do not support this claim (DeCalderon et al. 1983). Regulation of the flow of photosynthate to storage roots at different points in the hierarchy in a single plant is not well understood. Variations in the ability of storage roots to deplete the flow of photosynthate in the phloem and the chronological order of storage root inception can influence the preferential movement of photosynthate to one storage root over another (Kays et al. 1982). Hence, a large storage root with more cells can deplete more photosynthate in the phloem and thus cause greater concentration gradient in the phloem, which in turn enhances subsequent flow of photosynthate to that storage root. Storage roots with identical potential but at different points in the hierarchy may differ in the amount of photosynthate translocated to them (Kays et al. 1982). Inside the storage roots, part of the translocated photosynthate accumulates in a soluble form around the vascular cambium ring (which is near the phloem region) and the remainder moves out toward the external surface and inward toward the center of the storage root. Because the vascular cambium is a metabolically active site and the zone of maximum meristematic activity in the storage root, the accumulated photosynthate is likely to be expended in respiration or moved to other regions of the storage root. Once located in a region other than vascular cambium, photosynthates polymerize and do not readily move to other areas (Chua and Kays 1982).

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III. ROOT SYSTEM The root system of sweetpotato plants comprises non-storage and storage roots (Togari 1950; Wilson 1970; Wilson and Lowe 1973). A. Non-Storage Roots Thin, adventitious roots arise from the internodal regions of vine cuttings or cut sprouts used for propagation (Togari 1950). These internodal roots are typically tetrarch with a central core of xylem with no central pith and four protoxylem points with alternate phloem tissues within the stele with a broad secondary cortex and a limited amount of secondary phloem. These roots develop horizontally or obliquely in the soil. Such roots develop largely into fibrous roots (Togari 1950; Wilson and Lowe 1973). The fibrous roots are less than 5 mm in thickness and are branched and rebranched with lateral roots forming a dense network throughout the root zone and constitute the water- and nutrient-absorbing system of the plant. The fibrous roots and storage roots occupy much of the soil volume. Jones (1961) found that 51 and 920/0 of total roots were within the top 45 and 57 cm, respectively. These roots have heavily lignified stele and very low levels of vascular cambium activity. In one study, fibrous roots accounted for 30/0 of the total plant dry weight (Somda et al. 1991). Light, dry and compact soil (Akita et al. 1962), high levels of N supply (Wilson 1973a,b), low O2 within the root zone (Togari 1950; Chua and Kays 1981), and long photoperiod (Bonsi et al. 1992) are known to favor the development of non-storage, fibrous roots. The optimum pH for better root growth varies between 4.5-7.0, while at pH below 3.5 no root growth occurs (Ilaava et al. 1995). The root cation exchange capacity (CEC) varies between 21-50 me/l00 g dry roots. Because of a strong positive correlation between root CEC and yield of storage roots and lack of variation in root CEC in a particular cultivar at different seasons, the root CEC can be used as a reliable index to reflect the storage root yield (Nair et al. 1981). However, many of the agroclimatic factors, including soil physical characters and fertility, plant spacing, soil moisture, and soil and air temperature, are likely to influence the root system of sweetpotato plants. B. Storage Roots 1. Initiation. Storage roots are capable of storing starch grains through localized lateral bulking in a specific subapical region of thick adventitious roots originating from the nodal region of the underground portion

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of vine cuttings used for propagation (Wilson and Lowe 1973). Such thick roots develop horizontally or obliquely in the soil. The initiation of storage roots can be recognized on the basis of primary stelar structure of thick adventitious roots (Artschwager 1924; Togari 1950). The primary structure of thick roots varies (Togari 1950; Wilson and Lowe 1973). The thick roots are pentarch or hexarch or septarch at the base and tetrarch nearer to the apical meristem and contain a central pith with or without central metaxylem cells. The protoxylem elements often show an incomplete centripetal development, resulting in few of the protoxylem elements being connected to one or more centrally located metaxylem cell while the other(s} remain separated by parenchymatous cells (Wilson and Lowe 1973; Indira and Kurian 1977; Ravi and Indira 1996). The protoxylem elements may also show complete centripetal development, resulting in their connection to one or more centrally located metaxylem cell. Sometimes the primary xylem elements are connected laterally to form a continuous cylinder surrounding the central parenchymatous pith. Such pentarch or hexarch roots are potential storage roots (Wilson and Lowe 1973; Wilson 1982). Some roots that are tetrarch do not differentiate into storage roots. Togari (1950) indicated that storage root initiation preceded the centripetal development of the primary xylem. Togari (1950) considered parenchymatous pith as a transient stage of root development and that failure of cells therein to become meristematic results in their lignification. This means that storage roots initiate in thick roots prior to the centripetal development of xylem by meristematic activity within pith cells. However, storage root initiation does not always precede the completed centripetal development of all the xylem elements, so that one or two xylem elements remain connected to the central metaxylem cells (Wilson and Lowe 1973). The initiation of storage root growth involves secondary growth by genesis of a vascular cambium as well as several anomalous circular cambia in the subapical region of thick roots (Togari 1950; Esau 1965; Wilson and Lowe 1973; Wilson 1982; DuPooly and DuPooly 1989; Nakatani and Komeichi 1991a; Ko et ai. 1993; Ravi and Indira 1996). At the onset of secondary thickening, vascular cambium initials are first laid down within the parenchymatous zone lying between the xylem and phloem and are connected to form a continuous and irregular cylinder through division of the single-layered pericycle (Wilson and Lowe 1973). Subsequent vascular cambial activity leads to centripetal production of thin-walled storage parenchyma, secondary vascular tissues, and a regular cylinder of vascular cambium. Differentiation of vascular cambium is accompanied by the origin of anomalous circular cambia in the central pith around the central metaxylem cell as well as around each

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of the discrete protoxylem elements. These meristems are referred to as anomalous primary cambia (Esau 1965; Wilson and Lowe 1973). In roots having a pith of thin-walled parenchymatous cells and no central metaxylem cells. initiation of anomalous primary cambia is usually associated with meristematic activity within the pith cells (Wilson and Lowe 1973). Anomalous circular secondary cambia also originate around secondary xylem elements derived from the vascular cambium (Togari 1950; Esau 1965; Kokubun 1973; Wilson and Lowe 1973). Phellogen activity on the periphery of the storage roots gives rise to the periderm. The time of initiation of storage roots varies widely among cultivars and may occur between 1-13 WAP (Enyi 1977; Indira and Kurian 1977; Ramanujam and Indira 1979; Wilson 1982; Roberts-Nkrumah et a1. 1986b; Oswald et a1. 1994; Songhai et a1. 1994; Ravi and Indira 1996). by which time the typical storage root number of a cultivar is determined. Growth of the storage roots occurs by the activity of vascular cambium as well as anomalous primary and secondary cambia. Cambial strips unassociated with vascular tissues also develop within the secondary parenchyma and contribute to storage root growth (Wilson and Lowe 1973). Activity of all cambia results in the formation of thin-walled. starch-storing parenchyma cells. The contribution of different cambia in production of storage parenchyma varies among cultivars and appears to be a cultivar characteristic. A high-yielding cultivar will show extensive anomalous circular cambial activity compared to a low-yielding cultivar (Wilson and Lowe 1973). Early in storage root ontogeny. specialized tissues at the distal end of the storage root attribute for the longitudinal growth of the storage root. Later. these tissues assume the structure and the functions of normal secondary thickened roots (Wilson and Lowe 1973). As the storage root develops. earlier deposited carbohydrates are concentrated toward the distal end and growth along the longitudinal axis of the storage root starts at the distal end and progress toward the proximal end (Chua and Kays 1982). Different rates of longitudinal and lateral growth determine the shape of storage roots and increase in storage root length is completed earlier than the width (Lowe and Wilson 1974; Bomda et a1. 1991). Cultivars with high activity of vascular cambium develop narrow. uniform storage roots. whereas cultivars with both vascular cambium as well as anomalous cambial activity develop globular storage roots (Wilson 1982). In some storage roots. cambial activity does not lead to lateral root bulking but results in uniform thickening of the entire root (Togari 1950; Wilson 1970; Wilson and Lowe 1973). In such roots. activity of the vascular cambium is accompanied by some meristematic activity and

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expansion of cells within the stelar parenchyma (Wilson and Lowe 1973). Often, one or more of the protoxylem element remains connected to the central metaxylem cell by a strand of lignified tissue. Failure of further development of these storage roots results from restricted activity of vascular cambium to produce a heavily lignified stele with prominent xylem rays and extensive cortical parenchyma. The parenchyma of primary stele sometimes develops some meristematic activity that contributes to the width of such roots. At the completion of storage root initiation, the proximal end of the storage root (the storage root stalk) differentiates and the stalk length appears to be cultivar dependent. The storage root stalk shows either complete or incomplete lignification of secondary xylem elements, medullary rays, and secondary xylem parenchyma, a considerable amount of secondary phloem, as well as a ring of vascular bundles (phloem bundles) within secondary phloem parenchyma and cortical tissues. These phloem vascular bundles, characteristic of the storage root stalk, contain xylem internally and phloem externally (Wilson and Lowe 1973). The distal region of storage root axis shows normal secondary thickening with a completely lignified tetrarch stele. In the beginning, however, this region shows the structure of a storage root (Wilson and Lowe 1973). 2. Endogenous Growth Regulators. Storage root growth (bulking) involves an increase in size and weight. Increase in storage root size occurs by increase in cell number and cell size, while the storage root weight increases through accumulation ofphotosynthates (Wilson 1967, 1969,1974,1977). Increase in storage root cell number and cell size are under the control of endogenous growth regulators. Several reports suggest a relationship between formation (initiation) of storage roots and cytokinins (Spence and Humphries 1972; Hozyo 1973; McDavid and Alamu 1980b; Oritani et a1. 1983; Matsuo et a1. 1983, 1988; Koda et a1. 1985; Sugiyama and Hashizume 1989; Nakatani and Komeichi 1991a,b; Nakatani and Matsuda 1992). Trans-zeatin riboside (trans-ZR) and 9glucosyl-N-6 (A2-isopentenyl adenosine (i6Ado) are the major cytokinins involved in storage root formation of sweetpotato (Sue et al. 1982; Matsuo et al. 1983). The concentration of i6Ado is much lower than that of trans-ZR throughout storage root growth, while the maximum amount of i6Ado precedes the maximum amount of trans-ZR. The concentration of 6-3-methyl-2-butemyl aminopurine glucoside (iPG) is higher than that of trans-ZR and the pattern of changes in the former is more complex (Matsuo et al. 1988). Longitudinal distribution of cytokinins in developing storage roots shows that the concentration of trans-ZR is higher in parts of the proximal side than in lower parts of storage roots.

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Trans-ZR content of roots increases rapidly when the thick roots begin to appear and declines later in storage root growth. However, trans-ZR content of fibrous roots does not change during the growth period. Trans-ZR content of fibrous roots also does not differ between 1. batatas cultivar and 1. trifida. Trans-ZR content of thick storage roots is 6-7-fold greater than in fibrous roots in the beginning. Trans-ZR content is also higher in storage roots of a cultivar with higher numbers of thick storage roots. However, endogenous trans-ZR does not relate to root thickening after formation of storage roots (Nakatani and Komeichi 1991a). Highest trans-ZR content occurs around the vascular cambium rather than the peripheral secondary phloem as well as peripheral and central xylem (site of anomalous cambial activity) (Nakatani and Komeichi 1991b; Nakatani and Matsuda 1992). Therefore, it appears that cytokinins, and especially trans-ZR, participate in the activation of vascular cambium in sweet potato roots. In potato (Solanum tuberosum L.), cytokinins play an important role in tuberization (Koda and Okazawa 1983) but they are not primary stimuli for tuber initiation and specific stimuli have been identified (Koda et al. 1988). However, in sweetpotato, whether cytokinins are the primary or secondary stimuli for initiation of thick roots is unknown. Because indol-3yl-acetic acid (IAA) induced an increase in the number of xylem elements in ferns and higher plants, the level of IAA was thought to be critical for the pentarch or hexarch condition of the root, a prerequisite for storage root initiation (Wilson 1982). Auxin and cytokinin are known to control the secondary growth of radish and carrot (Dacus carota L.) roots (Torrey 1976). Low IAA levels and high IAA oxidase activity are known to be associated with the lignification in storage root, while increase in cell division and expansion and storage root growth are associated with high IAA levels as well as low IAA oxidase activities (Akita et al. 1962). An increase in the activity of cell wall bound invertase in sweetpotato roots treated with auxin and high levels of IAA oxidase activity in non-storage roots indicates that auxin might playa key role in storage root initiation (Acock 1984). Auxin biosynthetic genes transferred from the plasmid DNA of Agrobacterium tumefaciens to the nuclear DNA of tumor cells formed in the sweetpotato petiole explants induced root morphogenesis (Barringer et al. 1996). Auxin content increases with advancing storage root growth, while the storage roots contain a higher amount of auxins than the fibrous roots (Jimenez and Garner 1983). The IAA content is constant throughout growth period in fibrous and thick roots of 1. trifida (Nakatani and Komeichi 1991a). In 1. batatas cultivar, the IAA content of the storage root remains low initially, increases when the diameter of the storage root

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increases rapidly and later IAA content decreases below the initial level when the rate of increase in storage root diameter becomes slow (Nakatani and Komeichi 1992a). The higher level of endogenous abscisic acid (ABA) in storage roots of 1. batatas cultivar than in non-storage roots of 1. trifida indicates the involvement of ABA in storage root thickening (Oritani et al. 1983). The endogenous level of ABA also shows a positive correlation with the thickening potential of storage roots (Nakatani et al. 1987, 1988b, 1989). ABA level remains much lower than that of trans-ZR throughout the storage root growth (Matsuo et a1. 1988). The ABA content of storage roots remains considerably greater in a cultivar with maximum root diameter than in a cultivar with maximum number of thick storage roots. In 1. batatas cultivar, the ABA content decreases in storage roots at a later period of growth, while the ABA content of fibrous roots remains steady throughout the growth period (Nakatani and Komeichi 1991a). Thick storage roots in 1. batatas cultivar with greater activity of anomalous cambia show high ABA content, whereas thick non-storage roots in 1. trifida that are totally lacking anomalous cambial activity show a lower ABA content (Nakatani and Komeichi 1991a). ABA content is higher in the vascular cambium zone than peripheral phloem and peripheral as well as central xylem (Nakatani and Komeichi 1991b). These results indicate that ABA may be related to the activity of vascular and anomalous cambia and promotes thickening of storage roots by itself or through interaction with cytokinin. Extracts from shoot systems of sweetpotato show the activity of jasmonic acid (JA) or JA-related compounds (JAs). The JA activity is very high in storage roots (Nakatani and Koda 1992, 1993). ]asmonic acid is the substance that induces tuber formation in potato (Koda et a1. 1988). Thick non-storage roots of 1. trifida show low JA or JAs. When applied exogenously, JA increases the root diameter of 1. trifida due to increase in cortex width rather than by the active division of cambia (Nakatani and Koda 1993). In 1. batatas cultivar, JA increases the frequency of storage root formation as well as the diameter of storage roots (Nakatani 1994). The thickening of roots of 1. trifida by grafting with the top organs of 1. batatas cultivar also indicates that some substance(s) that stimulates root thickening is translocated from the 1. batatas shoot to roots of 1. trifida (Hozyo and Park 1971). However, the interaction effect among cytokinin, IAA, ABA, and JA needs further investigation. 3. Starch Synthesis. Because starch is the major storage material in storage roots of sweetpotato, storage root growth is influenced by the extent of starch synthesis and accumulation. Starch content of storage roots also

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varies among sweetpotato cultivars. It is therefore important to understand the enzymic reactions controlling starch accumulation and the regulatory mechanism determining cultivar differences in starch content in storage roots of sweetpotato. Regulation of starch synthesis in sweetpotato storage roots is little understood. As in other crops, starch is synthesized by starch synthase in storage roots of sweetpotato (Murata and Akazawa 1968). There are two forms of starch synthase, one tightly bound to the starch granule (starch granule-bound ADPG (UDPG) starch synthase) and the other a soluble form of the enzyme present in the amyloplasts (soluble ADPG (UDPG) starch synthase). In a developing sweetpotato storage root, starch granule-bound starch synthase activity is high during the early period and it sharply declines during later periods (Lila and Bala 1996). Uridine diphosphate glucose (UDPG) is the predominant nucleotide during the early period, while adenosine diphosphate glucose (ADPG) content is low and the latter increases during active growth (Murata 1970). The starch granule-bound form of starch synthase prefers ADPG to UDPG as a substrate. However, the soluble form of starch synthase shows a similar affinity to both ADPG and UDPG as substrates. Soluble starch synthase activity is much higher than the granule-bound enzyme activity throughout the storage root growth period. Because amylopectin makes up 70 to 800/0 of most starches and soluble starch synthase is responsible for the synthesis of amylose, it is likely that the soluble starch synthase activity is greater than the starch granule-bound starch synthase activity (Lila and Bala 1994, 1996). High activity of bound starch synthase during the early growth period and its sharp decrease at a later time indicates that amylose synthesis takes place early in the storage root growth (Lila and Bala 1996). Although starch synthase is involved in starch synthesis, the enzyme does not account for differences in storage root dry matter and starch content among sweetpotato cultivars (Nakatani and Komeichi 1992b; Lila and Bala 1995). However, ADPG pyrophosphorylase (the enzyme that catalyzes the synthesis of ADPG) activity shows a significant positive correlation with the dry matter and starch content of storage roots (Nakatani and Komeichi 1992b; Tsubone et al. 1997). High ADPG pyrophosphorylase activity occurs in cultivars with high starch content, whereas ADPG pyrophosphorylase activity is low in cultivars with lowest starch content. The rate of increase in starch content declines in parallel with a decrease in ADPG pyrophosphorylase activity (Nakatani and Komeichi 1992b). Therefore, the reaction catalyzed by ADPG pyrophosphorylase appears to be more important than starch synthase in determining starch content of sweetpotato storage roots.

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In growing sweetpotato storage roots, starch phosphorylase (X~1~4-glu­ can: orthophosphate (X-D glucosyl transferase) and ~~amylase are the two enzymes that share a common substrate amylose. Both enzymes are localized in the starch-accumulating amyloplasts and ~-amylase is a non-competitive inhibitor of starch phosphorylase (Chang and Su 1986; Chang et a1. 1987; Pan et a1. 1988). Starch phosphorylase is generally regarded as a starch-degrading enzyme, although the possibility of its role in starch synthesis in some plants has been suggested (Schneider et a1. 1981; Sivak et a1. 1981; Slabnik and Frydman 1970). In sweetpotato, starch phosphorylase concentration is in proportion to the starch content of storage roots, indicating its possible involvement in starch synthesis (Chang et a1. 1987). The inhibitory action of ~-amylase on starch phosphorylase (Pan et a1. 1988) and the preliminary indication that ~­ amylase may share the same sub-cellular loci with starch phosphorylase (Chang and Su 1986) shows that ~-amylase can be a modulator of starch accumulation in storage roots. It appears that starch phosphorylase may be significant in starch accumulation, while the regulatory role that ~­ amylase may play in the starch accumulation still needs clarification. 4. Effect of Atmospheric/Soil Factors. Air and soil temperature, physical characters of the soil, and soil fertility influence sweetpotato storage root formation and growth. Night air temperature seems to be the most critical factor for storage root growth, presumably due to greater translocation of sugar from the shoot to roots during this time. Night temperature between 15-25°C promotes storage root formation and growth. Sweetpotato cultivars yield at their maximum in seasons having night air temperatures between 14-22°C (Singh and MandaI 1976; Nawale and Salvi 1983; Janssens 1984; Ngeve et a1. 1992). Night air temperature higher than 25°C suppresses storage root formation while promoting shoot growth (Kim 1961; Chatterjee and Mandai 1976; Ueki and Sasaki 1987; Nakatani 1989; DuPooly and DuPooly 1989). Night air temperature lower than 15°C suppresses storage root formation, growth, and yield (Janssens 1984; Ngeve et a1. 1992). At air temperature >30°C, an increase in IAA oxidase activity causes reduction in storage root formation and growth, while an increase in gibberellic acid (GA) promotes shoot growth (Chan 1988; DuPooly and DuPooly 1989). Soil temperatures between 20-30°C favor storage root formation and growth, while a soil temperature of 15°C promotes fibrous root formation. Soil temperatures >30°C promote shoot growth at the expense of storage root growth (Hasegawa and Yahiro 1957; Spence and Humphries 1972). Long photoperiod favors storage root growth. In one study, storage root yield

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among cultivars was 20-fold greater under 24 h photoperiod than under 12 h (Bonsi et a1. 1992). Dry and compact soil (Watanabe et al. 1968a,b; Sajjapongse and Roan 1982; Yanfu. et al. 1989) hampers storage root growth. Loose soils or soils of low bulk density «1.3) enhance more vegetative growth, while heavy soils or soils of high bulk density (>1.5) reduce both shoot and storage root growth (Sajjapongse and Roan 1982). In both low and high bulk density soils, thickening of storage roots is reduced by low O2 concentration, the effect being greatest in the low bulk density soil (Watanabe et a1. 1968a,b). Potassium favors storage root growth (Fujise and Tsuno 1967; Scott 1950; Tsuno 1971). The number of secondary xylem vessels associated with the vascular cambium zone and the width of vascular cambium increase in roots with increased K application (Speight et a1. 1967). Conversely, a high N adversely affects the storage root growth (Samuels 1967) because of vigorous shoot growth that would compete for photosynthate. Inadequate K, Mg, or Ca inhibit storage root formation (Spence and Ahmad 1967). Deficiency ofZn, Mn, and Cu causes localized browning of storage root flesh, whereas deficiency of B completely suppresses the storage root formation and growth (Pillai et a1. 1986). 5. Storage Root Bulking Pattern. Sweetpotato storage root yield is determined by the duration and rate of storage root growth, which varies widely among cultivars. Sweetpotato storage root growth fluctuates over a long bulking period due to changes in the agroclimatic conditions. Hence, unlike cereal grains, the sweetpotato storage root can undergo periods of arrested growth during unfavorable conditions and then continues growth once conditions improve. High-yielding cultivars have a high bulking rate over a long period, whereas cultivars with intermediate and low storage root yield have a high bulking rate for a short duration or low bulking rate for a longer duration. In late bulking cultivars, a high bulking rate for a short duration may also result in an increase in storage root yield (Wilson 1982). Early-maturing short-duration cultivars exhibit fast initiation and bulking of storage roots, whereby yields reach a maximum within a growing period of 12-16 weeks (Bitai and Lian 1978). Cultivars are classified into short-duration or early-maturing (12-17 weeks), medium-duration (17-21 weeks), and long-duration or late-maturing (>21 weeks) types (Yanfu et a1. 1989). The bulking rate of storage roots of early-maturing cultivars declines or even pauses at 12 WAP, whereas for the late-maturing cultivars, bulking rate increases at the middle and later growth period. Short-duration cultivars exhibit a maximum bulking rate during a 12-17-week period and the daily rate

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varies between 1.8-7.3 g/plant on a fresh weight basis or 0.7-1.7 g/plant on a dry weight basis (Ramanujam and Indira 1979; Indira and Ramanujam 1985; Mishra et al. 1987; Venkatachalam et al. 1990; Mannan et al. 1992; Mukhopadhyay et al. 1991; Goswami et al. 1995). High- and lowyielding cultivars differ in their bulking rate and the period at which they exhibit the maximum bulking rate (Ramanujam and Indira 1979). Cooler night air temperature (11.3-26.4°C) (Mukhopadhyay et al. 1991), application of K (Mukhopadhyay et al. 1992, 1993), and 2 or 3 subsequent irrigations during the 5th-13th week of the growth period (Goswami et al. 1995) significantly increase the bulking rate of storage roots. The storage root bulking rate shows a positive correlation with rainfall and relative humidity (Chowdhury 1994). IV. SOURCE AND SINK RELATIONSHIP

The yield of a crop depends on the production of assimilates by a "source" and the extent to which they can be accumulated in a "sink" represented by the organs that are harvested (Hahn 1977b). In sweetpotato, the storage roots that accumulate assimilates are the predominant sink. The shoots, mainly leaves, which produce assimilates are the source, although shoot growth is itself an important sink in the early period of crop growth. The photosynthetic rate and the leaf area can be regarded as the "source potential," while the number of storage roots and the mean storage root weight can be regarded as the "sink capacity." The source potential as well as sink potential varies widely among sweetpotato cultivars (Hahn 1977b, 1982). The storage root yield is controlled not only by source potential but also by sink capacity. However, considering the wide variation in source potential and sink capacity among sweetpotato cultivars, it is uncertain whether the source or the sink is limiting the storage root yield. Earlier studies were conducted to gain an understanding of the source and sink relations by changing the sizes of both source and sink. Source size has been varied by removing leaves, while sink size has been varied either by exposing the storage roots to light (Tsuno and Fugise 1965) or to different temperatures (Spence and Humphries 1972) or by removing the storage roots and by treating them with growth regulators (Spence and Humphries 1972). Such treatments, however, were likely to have an adverse effect on other physiological processes. To minimize interference with these processes, reciprocal grafts have been used (Hahn 1977b; Li and Kao 1985b; Bouwkamp and Hassan 1988; Nakatani et al.1987, 1988b; Li and Kao 1990; Zhong 1991; Ko et al. 1992, 1993).

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Some studies indicate that in grafts between 1. hatatas and 1. trifida, plants that had strong sink as stock accumulated dry matter much more abundantly than plants with weak sink (Hozyo and Park 1971; Kato and Hozyo 1974,1978; Hozyo and Kato 1976). Therefore, it was inferred that storage root yield of sweetpotato is determined primarily by sink capacity rather than source potential (Wilson 1967; Hozyo 1970; Hozyo 1977; Zhong 1991; Ko et al. 1992). However, both source potential and sink capacity can be factors limiting storage root yield (Hahn 1977b). The relative contribution of source potential and sink capacity to storage root yield differs during the crop growth period among cultivars (Li and Kao 1985b; Hassan 1986; Bouwkamp and Hassan 1988). The source potential is more limiting than sink during the early growth period but they are equally important in determining storage root yield at a later growth period after the formation of storage roots (Hatten and Garner 1979; Li and Kao 1985b; Nakatani et al. 1988a; Li and Kao 1990). Several studies reveal a positive correlation between shoot weight and storage root weight, indicating that storage root growth is closely associated with shoot growth (Li 1965; Ghuman and Lal 1983a,b; Ashokan et a1. 1984; Varughese et a1. 1987; Ravindran and Bala 1987; Vimala et al. 1988; Syriac and Kunju 1989; Goswami 1991; Sen et a!. 1990; Nair and Nair 1992; Mukhopadhyay et a1. 1992, 1993). However, other studies indicate a negative correlation between shoot weight and storage root weight (Haynes 1970; Kamalam et al. 1977; Gollifer 1980; Ibrahim 1987; Amarchandra and Tiwari 1987; Mukhopadhyay et a1. 1990, 1991; Mortley et al. 1991; Rajeshkumar et a1. 1993; Goswami 1994). This means that storage root growth depends on the shoot growth to a certain extent. Excess shoot growth consumes a greater amount of photosynthates and does not favor storage root growth. The number of branches shows a negative correlation with storage root yield (Thankamma and Easwariamma 1990). LAI has a positive correlation with storage root yield (Tiwari et a1. 1985; Chowdhury 1994). Sinkrelated parameters such as storage root number per plant show positive significant correlation with storage root yield (Kamalam et a1. 1977; Janssens 1984; Amarchandra et al. 1985; Ibrahim 1987; Amarchandra and Tiwari 1987; Bouwkamp and Hassan 1988; Biswas et a1. 1988; Antony and Inasi 1990; Rao and Sultana 1990; Thankamma and Easwariamma 1990; Zhang and Lian 1994). Sink characters such as storage root girth and length, fresh weight per storage root (Amarchandra and Tiwari 1987; Ibrahim 1987; ]anssens1984; Zhang and Lian 1994), and bulking rate (Enyi 1977; Venkatachalam et a1. 1990; Mukhopadhyay et al. 1993; Chowdhury 1994) show significant positive correlation with storage root yield, as would be expected.

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Carbohydrate accumulates in the leaves of shoots grafted onto plants with low sink capacity (Hozyo and Park 1971; Ko et al. 1993). The PN rate drastically declines when root enlargement is restrained (Tsuno and Fujise 1965). When grafted, the high sink capacity of highyielding cultivars increases the source potential of low-yielding cultivars, which in turn increases root yield (Hahn 1977a; Zhong 1991). This is because higher sink capacity stimulates the translocation of photosynthates and thereby reduces the carbohydrate content of the leaves and increases source potential (photosynthetic rate). The balance between source potential and sink capacity changes during the day, at different periods of growth, and due to changes in environmental conditions. This makes it difficult to generalize the relative importance of either source or sink towards storage root development. Therefore, the relative contribution of source and sink toward storage root growth appears not to be constant throughout the growth period and appears to be specific to initiation and the bulking period of storage roots. More vine growth in cultivars represents more shoot activity that competes with storage root growth for assimilates. An active source coupled to a higher sink capacity is desirable, provided that the source component should not be as active as a competitive sink. V. DRY MATTER PRODUCTION AND HARVEST INDEX

Total dry matter (TDM) production and efficiency of dry matter (DM) allocation to storage roots is an important factor determining storage root yield. The increase in TDM as well as storage root dry matter (SRDM) follows a sigmoid pattern in sweetpotato (Huett and O'Neill 1976; Enyi 1977; Bourke 1985; Li and Kao 1985a; Li and Yen 1988; Oswald et al. 1994). A few reports indicate a linear increase in TDM (Li and Yen 1988; Nair and Nair 1995) and SRDM (Nair and Nair 1995). The increase in SRDM is at its maximum during 7-23 weeks. In general, sweetpotato exhibits three growth phases based on dry matter partitioning. During the first phase, shoot growth dominates, with an increasing proportion of DM diverted to shoot growth. This is followed by a second phase of constant partitioning of DM between shoot and storage root growth. During the third phase, a major portion of DM is partitioned to storage roots. High soil moisture prolongs TDM production, reduces the proportion of DM allocation into storage roots, and diverts to shoot growth (Enyi 1977). An increase in Nand K fertilizers considerably increase TDM and SRDM (Bourke 1985; Li and Yen 1988). An increase in plant population decreases SRDM and shoot DM per plant but significantly increases both SRDM and shoot DM yield

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per hectare (Li and Yen 1988). Increase in shoot DM follows a hyperbolic pattern (Li and Kao 1985a; Li and Yen 1988; Oswald et al. 1994). The ratio between the SRDM and the TDM (HI) indicates dry matter partitioning efficiency to storage roots. Accordingly, 80% HI has been estimated to equate storage root yield on the order of 46 t/ha for a 16 weeks crop or 69 t/ha for a 24 weeks crop (De Vries et al. 1967; Wilson 1982). Sweetpotato cultivars differ in TDM production and cultivars with higher TDM divert more DM to storage roots than those with lower TDM (Li and Yen 1988; Huett and O'Neill 1976). High-yielding cultivars divert more DM to storage roots than low-yielding cultivars (Enyi 1977). Huett and O'Neill (1976) compared their results with that of Lowe and Wilson (1974) and found that the cultivar with HI 0.3 yielded 1.3- or 2.6fold greater than cultivars with HI 0.8 or HI 0.6, respectively. Similarly, a cultivar with HI of 0.5 yielded 1.4- or 2.9-fold' greater than the cultivars with HI 0.8 or HI 0.6, respectively. Huett and O'Neill (1976) therefore opined that HI is not necessarily related to storage root yield potential. The HI among sweetpotato cultivars varies between 11-850/0 when harvested during 12-24 weeks (Enyi 1977; Bhagsari and Harmon 1982; Nawale and Salvi 1983; Bourke 1985; Bhagsari and Ashley 1990; Sen et al. 1990; Rao and Sultana 1990; Li et al. 1991; Mukhopadhyay et al. 1991; Somda et al. 1991; Nair and Nair 1992; McLaurin and Kays 1993; Goswami et al. 1995). Excess or inadequate soil moisture reduces HI (Enyi 1977; Mukhopadhyay et al. 1991; Goswami et al. 1995). Application of N fertilizer either has no influence (Bourke 1985) or reduces HI (Nair and Nair 1992), while K increases it. HI has a strong positive correlation with storage root yield (Bhagsari and Harmon 1982; Li and Kao 1985a; Li et al. 1991; Rao and Sultana 1990; Bhagsari 1990; Bhagsari and Ashley 1990). There is a strong, positive correlation between SRDM and TDM and between HI and SRDM. The correlation between HI and TDM is positive but insignificant (Li and Kao 1985a; Li et al. 1991). The HI and storage root dry weight of cultivars respond differently to a change in the environment. Because variation in HI among individual plants within cultivars is small as compared to the SRDM and HI might be influenced to a lesser extent than SRDM by changes in environmental conditions (Li et al. 1991), HI can be used as a reliable selection parameter. VI. SHOOT REMOVAL AND STORAGE ROOT YIELDS The shoot tips of sweetpotato plants are removed for use as a leafy vegetable in Asia and Africa (Magoon 1967; Villareal et al. 1979) and for

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propagation (Edmond and Ammerman 1971; Eronico et al. 1981; Bhuyan and Chowdhury 1984; Chiappe et al. 1984; Balasurya 1991). Sweetpotato vines are also used as a good source of animal feed (Koh et al. 1960; Chen et al. 1977, 1979; pfoulkes et al. 1978; Mena et al. 1979; Ruiz et al. 1980). The amount and frequency of shoot removal and the growth period at which the shoots are removed has a definite effect on the storage root yield. Detopping or artificial defoliation of shoots decreases storage root yield (Gonzales et al. 1983; Dahniya et al. 1985; Villanueva Jr. 1985; Villamayor Jr and Perez 1988b; Nwinyi 1992; Uddin et al. 1994; David et al. 1995). In one study, shoot tip removal at 2, 4, 6, 8, and 10 WAP resulted in 11.6, 15.9, 37, 56, and 63.3% reduction, respectively, in storage root yield (Nwinyi 1992), which indicates that shoot tip removal during the later growth period causes greater reduction in storage root yield. The reduction in storage root yield is more severe when whole shoots are removed than when only shoot tips (15-25 em) are removed (Dahniya et al. 1985; Villamayor ]r and Perez 1988b; Chowdhury and Ravi 1990). In one study, removal of shoot tips or whole shoots caused 45 and 60.5% reduction, respectively, in storage root yield (Dahniya et al. 1985). More frequent shoot removal reduces the number and size of storage roots (Dahniya et al. 1985). In one study, shoot tips removal at 2-, 3-, or 4-week intervals caused 75, 69.5, and 49% reduction, respectively, in storage root yield (Dahniya et al. 1985). Detopping during the dry season decreases storage root yield more than detopping in the wet season (Bartolini 1982). However, Bartolini (1982) and Gamao et al. (1984) found an increase in storage root yield in plants detopped during wet season. Increasing fertilizer application does not overcome the reduction in storage root yield caused by detopping (Gonzales et al. 1977; Bartolini 1982). VII. RESPONSE TO GROWfH REGULATORS AND CHEMICALS Experimental results show that foliar application of growth regulators such as 2-chloroethyltrimethyl ammonium chloride (cce or cycocel or chlormequat) (Tompkins and Bowers 1970; EI-Fouly et al. 1971; Nambiar et al. 1976; Biswas et al. 1980; Khanna et al. 1980; Vaheb and Mohankumaran 1980; Aiazzi et al. 1985; Mishra et al. 1987; Varma and Nedunzhiyan 1996), (2-chloroethyl) phosphonic acid (Ethrel or ethephon) (Shanmugam and Srinivasan 1974; Muthukrishnan et al. 1974; Biswas et al. 1980; Vaheb and Mohankumaran 1980; Mustaffa et al. 1980; Khanna et al. 1980; Rai et al. 1980), and kinetin (Biswas et al.

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1980), and some synthetic compounds such as tri-iodobenzoic acid (TIBA) (Marlowe Jr and Scheuerman 1969), and paclobutrazol (EI-Gamal 1994) increases storage root yield of sweetpotato. However, application of growth regulators and chemicals have proven to be of no commercial value in sweetpotato production. VIII. RESPONSE TO STRESS

Sweetpotato production may be increased by increasing yield per unit area or increasing area under cultivation. Yield increases per unit area can be achieved by a breeding and subsequent selection program. However, most of the land available in the tropics is limited in its productive capacity by either unfavorable soil properties or climatic conditions. Therefore, for increasing area under cultivation, attention must be given to developing cultivars that are resistant to various stress conditions. A. Water Deficit Sweetpotato yields best when irrigated at 25% available soil moisture and there is no increase in storage yield by maintaining soil moisture >500/0 (Hernandez and Barry 1966; Hammett et al. 1982). Under typical production conditions, the crop requires 500 mm water for a 16-20-week growth period (King 1985; Kay 1987; Onyekwere and Nwinyi 1989; Chukwu 1995). However, storage root yields are affected by amount, timing, and distribution of water. Storage root yield decreases under water deficit stress (WDS), particularly when the available soil moisture decreases below 200/0 (Hernandez and Hernandez 1967; Chowdhury and Ravi 1987, 1988; Indira and Kabeerathumma 1988; Nair et al. 1996). Irrigation at less than 50% of the cumulative pan evaporation rate also has been reported to decrease storage root yield (Indira and Kabeerathumma 1990; Chowdhury 1996). The storage root initiation period is the most sensitive to WDS due to its effect on storage root number (Indira and Kabeerathumma 1988; Nair et al. 1996; Ravi and Indira 1996). WDS during the storage root initiation period induces lignification of storage roots and hampers storage root growth. Lignification and reduction in storage root yield is greater in cultivars with weak sink capacity than those with higher sink capacity (Ravi and Indira 1996). The reduction in storage root yield under WDS is also related to physiological and biochemical changes in the leaves. Under WDS·conditions, water potential (WPd or relative water content (RWC) of sweetpotato leaves decreases (Sung 1985a,b; Indira and Kabeerathumma· 1988;

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Chowdhury and Naskar 1993; Ravi and Indira 1995). Leaves permanently wilt whenWPL decreases to -1.3 MPa, and at WP L between -1.6 to -2.0 MPa, the leaves senesce (Sung 1985b; Ravi and Indira 1995). The decrease in WPL increases SR to CO 2 exchange (Ghuman and La11983a; Sung 1985a; Indira and Kabeerathumma 1988,1990), causing reduction in the PN rate (Sung 1985a; Ravi and Indira 1996). Cultivars differ in their tolerance to WDS conditions (Chowdhury and Ravi 1987, 1988; Indira 1989; Ravi and Indira 1996). Tolerant cultivars have greater SR than the susceptible ones (Ghuman and Lal 1983a; Indira 1989; Kubota et al. 1993). High SR in tolerant cultivars may be advantageous for conserving leaf water content at the cost of reduction in photosynthesis under WDS. This helps tolerant cultivars to have a lower desiccation rate in the leaf tissue than the susceptible ones (Indira 1989; Garner et al. 1992; Newell et al. 1994; Naskar and Chowdhury 1995). Under WDS conditions, an increase in CO 2 concentration surrounding the leaf improves the WP L and the storage root yield (Bhattacharya et al. 1990b). The total chlorophyll content of leaves decreases in sweetpotato plants subjected to WDS (Sung 1985a; Indira and Kabeerathumma 1988,1990; Chowdhury and Ravi 1987, 1988). Cultivars tolerant to WDS have lower chlorophyll content than the susceptible ones (Indira 1989). Under WDS conditions, nitrate reductase (NR; the first enzyme of the nitrate assimilatory pathway that reduces NOi to NOi in the cytosol) activity decreases in sweetpotato leaves (Sung 1981; Chowdhury and Ravi 1987, 1988; Indira and Kabeerathumma 1990). In corn (Zea mays L.) and bar. ley (Hordeum vulgare L.) plants, NR activity decreased due to a decrease in protein synthesis but not due to the NOi amount in stressed tissue (Arriaga et al. 1972; Huffaker et al. 1970). Whether the decrease in NR activity in stressed sweetpotato leaves is due to a decrease in the enzyme activity itself because of a decrease in protein synthesis or due to decrease in NOi uptake or both, is not known. In cotton (Gossypium hirsutum L.) plants, during WDS N deficiency promoted ABA accumulation, which in turn induced stomatal closure and a decrease in stomatal conductance at a higher WPL than normal (Radin and Ackerson 1981; Radin et al. 1982). However, in sweetpotato, under WDS, the interaction among low N, WPL, ABA, and stomatal closure is not known. Because the inflow of inorganic N into plants is largely controlled by NR activity, reduction in NR activity under WDS may limit growth, development, and protein synthesis. Sweetpotato cultivars tolerant to WDS have greater NR activity than the susceptible ones (Naskar and Chowdhury 1995). Drought-tolerant sweetpotato cultivars accumulate a greater amount of proline in the leaf and fibrous root tissues than the plants under WDS

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free conditions (Chowdhury and Ravi 1987; Indira and Kabeerathumma 1988; Ravi and Indira 1997). In both tolerant and susceptible cultivars, leaves accumulate a greater amount of proline than the non"storage, fibrous roots (Ravi and Indira 1996). However, some susceptible culti" val'S that do not yield but survive under WDS also accumulate a good amount of proline in their leaf tissues (Ravi and Indira 1997). Because most of the proline accumulation occurs after growth has ceased, proline does not seem to influence sweetpotato plant growth during WDS. However, greater accumulation of proline in the leaves during WDS has been shown in other crops to help the plant to survive and retain the leaves through osmotic adjustment (Ford and Wilson 1981; Hanson and Hitz 1982), strengthening of protein stability (Paleg et al. 1984; Nash et al. 1982), binding of excess photosynthetic energy when stomata are closed (Hanson and Hitz 1982), and forming a readily available pool of carbohydrate (Barnett and Naylor 1966; Stewart et al. 1966) and N (Blum and Ebercon 1976; Itai and Paleg 1982) during recovery from WDS. Sivaramakrishnan et al. (1988) proposed that in (Sorghum bicolor L.) proline may be used by tolerant cultivars to meet the immediate needs of energy and N during recovery from drought. Similar efficiency in poststress recovery, coupled with high sink capacity, may contribute toward better storage root yield in drought-tolerant sweetpotato cultivars. Although susceptible cultivars accumulate a good amount of proline under WDS, their poor sink potential may explain their low yield. Whether sweetpotato plants accumulate other organic solutes under WDS that may contribute toward drought tolerance is not known. B. Flood In the tropics, waterlogging in the field during heavy rain storms or excess soil moisture in heavy soils with poor drainage. impedes the growth of plants by restricting the availability of O2 in the root zone. Because induction and growth of storage roots depend on the presence of sufficient O2 in the soil, anaerobiosis during waterlogging or surplus soil moisture reduces storage root production (Togari 1950; Batty 1975; Watanabe et al. 1968a,b; Chua and Kays 1981; Silva and Irizarry 1981; Ghuman and Lal 1983b; Martin 1983; Li and Kao 1985c; King 1985; Bourke 1985). Excessive vegetative growth under high soil moisture con" ditions results in low storage root production (Hernandez and Hernandez 1967; Watanabe 1979; Goswami et al. 1995). Cultivars differ in their abilities to withstand flooding or surplus soil moisture (Martin 1983, 1984b; Ghuman and La11983b; Li and Kao 1985c;Martin and Carmer 1985; King 1985). In rooted sweetpotato leaves, flooding induces fewer

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storage roots and increases the fibrous root dry weight (Spence and Humphries 1972; Martin 1984b). Flooding induces reduction in the number, size, and diameter of storage roots (Li and Kao 1985c) and increases in the shoot fresh weight, presumably due to the decrease of sink capacity, which in turn inhibits the translocation of photosynthate to storage roots (Martin and Carmer 1985; Li and Kao 1985c). High temperatures during flooding also enhance leaf senescence. Plants subjected to flooding during the early period ofgrowth resume their normal growth better than plants subjected to flooding later in the development cycle (Li et a1. 1989). Therefore, the reduction in storage yield is greater in plants exposed to flooding during the later growth period than plants that are subjected to flooding during the early growth period. Flooding resulted in less reduction of storage root yield in grafted plants with high sink capacity than those with poor sink capacity (Li et a1. 1989). Therefore, it appears that cultivars with high sink potential tolerate flooding better than those with weak sink potential. Transient flooding (flooding two days in a week), however, increases shoot growth and storage root yield (Ghuman and LaI1983b). The physiological and biochemical changes associated with flooding and the mechanism of flood tolerance require investigation. C. Shade

Sweetpotato requires high levels of solar radiation for optimum growth and storage root yield (Hahn 1977a). However, in many tropical countries sweetpotato is grown under sub-optimal light conditions due to reduction of solar radiation by taller-growing adjacent crops in multiple cropping systems. For instance, several reports indicate a reduction in storage root yield when sweetpotato is intercropped with maize (Wan 1982; Moreno 1982; Watson et a1. 1991; Oswald et a1. 1996) and coconut (Zara et a1. 1982). This is mainly due to shade imposed by the taller crop rather than the competition for nutrients. Sweetpotato cultivars vary in their response to shade stress (Zara et a1. 1982; Martin 1984b, 1985; Roberts-Nkrumah et a1. 1986a,b; Demagante et a1. 1989; Oswald et a1. 1994, 1995a). Shade reduces total dry matter (TDM) production primarily due to reduction in storage roots' and secondarily due to reduction in vine length and amount of foliage (Martin 1986). Compared to plants that grow under 1000/0 sunlight and mild shade [20-250/0 light reduction (LR)] , moderate (40-550/0 LR) and deep shade (60-73% LR) significantly reduce TDM and storage root yield (Martin 1984a, 1986; RobertsNkrumah et a1. 1986a,b;Mwanga and Zamora 1988; Itong and Villa-

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mayor Jr 1991; Oswald et al. 1994, 1995a,b}. However, the amount of reduction in TDM and storage root yield is greater under deep shade than moderate shade. Reduction in storage root yield varies widely among cultivars. Mild shade may occasionally increase storage root yield (!tong and Villamayor 1991; Martin 1986). Deep shade retarded storage root initiation whereas mild and moderate shade did not (Wilson 1967; Martin 1985; Roberts-Nkrumah et al. 1986b; Mwanga and Zamora 1988; Demagante et al. 1989; Oswald et aL 1994, 1995b). Therefore, for deep shade conditions, cultivars with an early initiation of storage roots have an advantage over cultivars with a late initiation (Roberts-Nkrumah et al. 1986b). However, under deep shade, Oswald et al. (1994) found no increase in storage root yield due to genetically early initiation of storage roots. Moderate shade reduces the number and size of storage roots mainly because of suppression of storage root growth but not initiation of storage roots. However, deep shade significantly reduces the number and size of storage roots because of complete suppression of both initiation and growth of storage roots (Martin 1985; Roberts-Nkrumah et aL 1986b; Dimagante et al. 1989; Oswald et al. 1995a). Thus, in terms of TDM production and storage root yield, sweetpotato cultivars seem to tolerate moderate shade but not deep shade. Generally, shoot growth is less affected than storage root growth. Compared to 1000/0 sunlight, mild shade increases shoot growth (Itong and Villamayor Jr 1991; Oswald et al. 1995a) whereas moderate and deep shade reduce shoot growth (Oswald et al. 1995a). Reduction in the DM partitioning to storage root growth is presumably attributed to reduction in PN rate and partitioning of photosynthates to shoot growth (Oswald et al. 1994, 1996). Under shade conditions, cultivars with greater storage root production (sink capacity) and lesser shoot growth show lesser reduction in storage root yield, whereas cultivars with greater shoot growth relative to their storage root production show greater reduction in storage root yield (Oswald et al. 1994). Because cultivars with greater storage root production and lesser shoot growth develop a rather small shoot system, yield increases in low irradiance can only be achieved by an improved photosynthetic rate per unit leaf area or a longer leaf area duration. On the other hand, in cultivars with greater shoot growth relative to their storage root production, yield in shade conditions can be increased by a manipulation of both yield components, the assimilate production and the assimilate partitioning (Oswald et al. 1994). Leaf area (LAl) is not a limiting factor for storage root yield in sweetpotato cuItivars under shade conditions. Specific leaf area (SLA, leaf area per gram dry leaf tissue), leaf area ratio (LAR, leaf area/total plant weight) and the leaf size increase in plants grown under increasing levels of shade

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(Roberts-Nkrumah et al. 1986a). Increase in SLA is greater under deep shade than under moderate shade. The increase in leaf size and a decline in leaf number result in similar leaf area in all levels of shade (RobertsNkrumah et al. 1986a). However, shade reduces the leaf thickness or the number of chloroplasts per unit leaf area, which in turn limit dry matter production because of lower assimilatory potential. Thus, moderate and deep shade alters the mode of dry matter partitioning and diverts photosynthates towards shoot growth, thereby weak.ening sink strength (storage root number and growth). Shade also reduces the level of cambial activity, and restricts the development of storage parenchyma and hence sink capacity. This in turn reduces the assimilatory potential, which ultimately reduces storage root DM and yield. It appears that the early growth period of sweetpotato cultivars is more sensitive to shade stress rather than the later growth period (Roberts-Nkrumah et al. 1986a,b). However, Oswald et al. (1995b) reported a greater reduction in storage root yield when shade was imposed at the later growth period than at its beginning because of reduction in storage root growth rather than initiation. D. Salt In tropical regions, soil salinity due to CI- and S04-- salts of Na+, Ca++, and Mg++ is a critical limiting factor for high productivity and expansion in the cultivation of sweetpotatoes (Edmond 1971; Horton 1989). However, the response of sweetpotato cultivars to salt stress is little understood. Cultivars that tolerated an electrical conductivity (EC) of 4.0 dS/m in irrigation water or an EC of soil saturated extract at 6-11.0 dS/m produced 500/0 of the yield of normal plants (Bernstein 1964; Ayers and Westcot 1976; Maas1986). Martin and Carmer (1985) found that watering plants weekly with a 342.24 mM solution of NaCI arrested shoot and root growth. Naskar et al. (1990, 1991), however, found total inhibition of adventitious root initiation from the vine cuttings immersed in saline medium containing 171.12 mM NaCI or 83.07 mM MgS04 • In vines kept immersed in saline medium containingNaCI, MgS04 , and CaCI together at different concentrations, the root production increased initially up to 51.34 mM NaCI with 12.46 mM MgS04 and 19.86 mM CaCl z and declined gradually at higher concentrations. In some cultivars, irrigation once a week with saline water containing 102.68 mM NaC!, 24.92 mM MgS0 4 and 39.72 mM CaCl z yielded storage roots greater than 500 g/plant (Naskar et al. 1991). Application of NaCI during four consecutive years to the same field reduced storage root yield (Worley and Harmon 1974).

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The physiological and biochemical changes associated with salt stress and the mechanism of salt tolerance are unknown. Although weak correlation coefficients were found among stress responses in sweetpotato (Martin an Carmer 1985), the response of cultivars to different stress conditions appears to be different. The mechanism of tolerance to different stress conditions also appears to be different and cultivars vary in their tolerance under different stress conditions. Cultivars with high sink potential appear to be tolerant under different stress conditions than those with weak sink potential (Oswald et al. 1994; Ravi and Indira 1966). Identification of cultivars tolerant under different stress conditions or identification of a single cuItivar tolerant to different stress conditions would greatly help in extending the area of sweetpotato cultivation. IX. PROPAGATION PHYSIOLOGY

Sweetpotato is normally propagated vegetatively by vine cuttings. However, where vines are unavailable for planting, root sprouts and storage root pieces may be used for propagation. Micropropagation techniques and propagation from true seed have been suggested but neither of these methods is practical. A. Vine Cuttings Vine cuttings are used as planting material in tropical regions. Cuttings are normally taken straight from one field being harvested to another being planted. Adequate soil moisture, aeration, light, and heat are necessary for better establishment of vine cuttings. The advantage of vine cuttings is that they are free from soil-borne diseases but not virus leaf diseases (Onwueme 1978; Phills and Hill 1984). The portion of the vine from which cuttings are made, the age of the source plants, the physiological state of the cuttings, and the number of days the cuttings are stored influence the growth and subsequent storage root yield of the new crop. Because sweetpotato storage roots form quite early in the growth period, planting cuttings with greater potential for initiation of storage roots is essential. Cuttings from the shoot apex are better planting material than basal or middle vine cuttings (Shanmugavelu et al. 1972; Eronico et al. 1981; Tindall 1983; Bhuyan and Chowdhury 1984; Chiappe et al. 1984; Villanueva Jr 1985; Choudhury et al. 1986; Villamayor Jr and

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Perez 1988a; Balasurya 1991; Schultheis and Cantliffe 1994). Compared to cuttings from middle and basal portions, apical shoot cuttings grew more vigorously and produced greater storage root yield. However, differences in leaf area development are small (Degras 1969; Eronico et al. 1981; Bhuyan and Chowdhury 1984). Plants grown from apical and middle cuttings accumulate maximum dry matter in the storage roots (Degras 1969). The age of the source plants from which cuttings are taken is a critical factor. Storage root yields are significantly reduced when cuttings from older plants are used (Martin 1984a; Villamayor Jr and Perez 1988a). In one study, when cuttings were taken from 11week-old actively growing plants, storage root yield of plants from basal cuttings was 19% lower than the yield of plants from apical cuttings. Storage root yield of mixed plants from 500/0 apical cuttings plus 50% from basal cuttings was comparable to that of plants from apical shoot cuttings alone. When cuttings were taken from a 16-week-old ready-toharvest stand, apical vine cuttings outperformed both the basal and mixed vine cuttings in terms of storage root yield. The yield of plants from basal cuttings was 560/0 lower than the yield of plants from apical shoot cuttings, whereas the yield of mixed plants was 130/0 lower than the yield of plants from the apical vine cuttings (Villamayor Jr and Perez 1988a). Thus, for better storage root yields apical cuttings should be used. When there is a scarcity of apical vine cuttings, the middle or basal cuttings from any age of the source plant can be used. The presence of leaves on vine cuttings greatly increases adventitious root production, presumably due to the presence of active endogenous root-promoting substances (Fadl et al. 1977,1978). Storage root yield is significantly higher in plants from vine cuttings with foliage than in plants from cuttings without foliage (Ravindran and Mohankumar 1982, 1989). Significant reduction in storage root yield occurs in plants from cuttings stored for four days without foliage (Villamayor Jr 1986). Therefore, stripping of vine cuttings should be avoided if vine cuttings are to be stored. The length of vine cuttings used for planting depends on the vine cuttings' ability to sprout in relation to the number of internodes, cultivar, and season. For better storage, root yield, 20-40 em long vine cuttings should be used (Shanmugavelu et al. 1972; Godfrey-Sam Aggrey 1974; Tanaka and Sekioka 1976; Ravindran and Mohankumar 1982; Chen and Allison 1982; Sanchez et al. 1982; Bautista and Vega 1991; Hall 1986). Plants from cuttings stored for 2-5 days under damp but well-aerated conditions yield better than those from fresh cuttings (Hammett 1983; Villanueva Jr 1985; Martin and Jones 1986; Ravindran and Mohankumar 1989; Nwinyi 1991; Villamayor Jr 1991).

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B. Root Sprouts Sprouts produced from storage roots also may be used for propagation (Onwueme 1978; Martin and Jones 1986; Hall 1992, 1993). In temperate regions, immediately after harvest, storage roots are exposed to higher temperature (about 32°C) and 85% RH for about one week (curing) and then stored at about 16°C and 85% RH (Steinbauer and Kushman 1971). During the next planting season, sound storage roots are taken from the stored lot, exposed to about 32°C and 850/0 RH (presprouting). A brief extension of the curing and presprouting period induce early sprouting and increase sprout production (Hall 1987, 1990, 1992, 1993). The presprouted storage roots are then buried in moist sand or soil beds and stimulated to sprout by application of moisture. The bedded storage roots sprout within 2 weeks, and about 4-6 weeks after bedding, the first batch of sprouts of desirable length (20 cm) is ready for harvest. Subsequent harvests can be made at weekly intervals. Sprouts from the earlier harvests may be stored with their bases dipped into moist sandy loam soil in trays. When sufficient sprouts are collected, they are transplanted to the field. Cutting the storage roots transversely into 3 or 4 sections increases sprout production (Demprey 1961; Welch and Little 1966; Folquer and Mesias 1967; Whatley 1969; Bouwkamp and Scott 1972; Keys 1987; Hall 1990). Experimental results show that ethylene chlorhydrin (Michael and Smith 1952; Hall and Greig 1956; Darhouse 1958), thiourea or acetylene (Michael and Smith 1952), 2,4-D, NAA, amethoxyphenyl acetic acid, ~-naphthoxyacetic acid, 3,4-dichlorophenyl a-methoxy acetic acid (Darhouse 1958), dimethyl sulfoxide (DMSO), 3IBA plus DMSO (Whatley et al. 1968), ethephon (Tompkins and Horton 1973; Tompkins et al. 1973; Tompkins and Horton 1974; Hall 1990) and GA 3 (Tompkins and Bowers 1970; Tompkins and Horton 1974; Hall 1994) increase the number of sprouts from treated storage roots with earlier sprouting in the ethephon or GA 3 treatments. However, sprouts produced through chemical treatments are not suitable for transplantation. C. Cut Root Pieces Several workers studied the potential of using cut storage root pieces directly as planting material (Kodama and Kobayashi 1954; Kays and Stutte 1979; Bouwkamp and Scott 1972; Kim et al. 1983; Mohankumar and Potty 1993). In plants from cut root pieces, Bouwkamp and Scott (1972) recorded storage root yield at par with those from root sprouts. Cutting the storage root into pieces with a thickness of 2.5 cm decreases the proximal dominance and enhances sprout production (Kays and

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Stutte 1979). Larger root pieces produce progressively larger sprouts, but these sprouts are substantially smaller than sprouts produced by intact roots. Cut roots produce more adventitious roots than intact ones. Cut roots produce more vigorous shoot growth but less-uniform storage roots than vine cuttings. Smaller root pieces (6.4 cm 2) produce slightly more sprouts per cm2 of surface area than the larger root pieces. The set size may be 20-50 g (Ikemoto 1971) or 40-50 cm3 (Kim et al. 1983). Curing the cut root pieces for 24 h at 30-35°C and 100% RH or treating them with IBA and BA stimulates formation of adventitious roots and shoots. Mohankumar and Potty (1993) found no significant difference in the yield of marketable storage roots or vine when damaged, non-marketable roots as well as good-quality marketable roots were used as planting material. However, this technique is not yet useful in commercial production. D. Micropropagation There has been recent interest in the micropropagation of sweetpotato through tissue culture. Within 3 to 4 weeks, somatic embryos and plantlets with roots and shoots could be successfully regenerated from embryogenic callus derived from the shoot apical meristem (Elliot 1969; Over de Linden and Elliot 1972; Alconero et al. 1975; Scaramuzzi and DeGaetano 1983; Jarret et al. 1984; Liu and Cantliffe 1984; Chee and Cantliffe 1988; Komaki et al. 1989; Chee et al. 1990; MandaI and Chandal 1991; AVRDC 1991; Acedo 1991; Schultheis and Cantliffe 1992; Mukherjee et al. 1993), stern and root explants (Liu and Cantliffe 1984; Mukherjee et al. 1993), leaf explants (Sehgal 1975; Belarmino et al. 1992), anther (Kobayashi and Shikata 1975; Sehgal 1978; Tsay and Tseng 1979; Tsay et al. 1982; Mukherjee et al. 1991), nodal explants (Unnikrishnan et al. 1991; MandaI and Chandal1990; Paul et al. 1991), petiole explants (Prakash et al. 1996), petiole protoplasts (Murata et al. 1987), stem and petiole protoplasts (Murata et al. 1987; Sihachakr and Ducreux 1987; Belarmino et al. 1994), and mesophyll cell suspension (Murata et al. 1994). Such plantlets could successfully establish in the field. Cantliffe et al. (1988) studied the production of synthetic sweetpotato seed, via somatic embryogenesis, as a means of micropropagation. The success of the synthetic seed system depends on efficient somatic embryo germination. Use of somatic embryogenesis in a seeding system offers several potential advantages, e.g. the production of large quantities of propagules in limited space, maintenance of genetic uniformity, rapid propagule multiplication, and direct planting of somatic embryos in the field, thus eliminating the cost of transplanting (Fujii et al. 1987)

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and reducing the incidence of disease. Even though somatic embryos are morphologically identical to zygotic embryos, the former lack the protective seed coat and nutritional reserves that are typically found in zygotic seeds because cotyledons are not fully developed and no endosperm is present (Chee and Cantliffe 1988). Cantliffe et al. (1988) suggested the suspension of somatic embryos in a viscous gel supplied with growth additives. The addition of salts and carbohydrates within hydroxyethyl cellulose gel increases plantlet production from somatic embryos (Schultheis and Cantliffe 1992). The inclusion of hormones and beneficial microbes may improve embryo growth, or the inclusion of pesticides may prevent the growth of microbes that would inhibit the development of embryos into plantlets. Plants produced from somatic embryos are morphologically identical to normal plants. However, compared with plants from vine cuttings, plants from somatic embryos show consistent reduction in vegetative growth and storage root yields. Storage roots are greater in size in plants from vine cuttings than from somatic embryos. Plants from somatic embryos require more time for roots to bulk than plants from vine cuttings (Templeton-Somers and Collins 1986; Schultheis and Cantliffe 1994). The reduction in storage root yields of plants from somatic embryos is presumably attributed to the reduction in sporamin content in the roots (Schultheis and Cantliffe 1994). Thus, sweetpotato propagation through somatic embryogenesis does not appear practical, given the present level of technology. E. True Seed Use of true seeds for sweetpotato production may simplify the planting process compared with the routine method of planting vine cuttings, but genetic segregation would make true seed propagation an unlikely choice. Many problems affect seed production in sweetpotato (Martin and Jones 1986). Sweetpotato cultivars are sensitive to photoperiod and differ in their flowering habit. Some flower readily at any season, others flower only under shorter photoperiods, and some do not flower under any normal conditions. Cultivars that do not flower readily can be stimulated to flower by various techniques (Wang 1975). However, problems of incompatibility and sterility impede controlled pollination in sweetpotato. Serious physiological problems, occurring as post-pollen germination barriers to fertility, often impede seed production in sweetpotato even when a cross is compatible (Martin 1982). Finally, insect and disease problems affect seed production. Compared with vine cutting planting, storage root yield is significantly reduced in true seed

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planting (Hirosaki and Ono 1970; Yamakawa and Sakamoto 1980a,b, 1981, 1987; Schultheis and CantHffe 1994). Storage root yield varies widely among the true seed population and cultivars tend to have higher storage root weight ratio than the true seed population (Iwama et al. 1990). True seed does not appear to be promising for sweetpotato propagation. X. CONCLUSION It is evident from the literature that a considerable amount of work has

been carried out over the past three decades and that it has helped to expand our knowledge of physiological factors favoring or limiting storage root yield in sweetpotato. Sweetpotato growth occurs in three phases. During the first phase, the greater portion of the photosynthates or dry matter is diverted to shoot growth, including branch and leaf area development. The second phase involves partitioning of photosynthates toward both shoot and storage root growth, and the third phase involves allocation of more photosynthates to storage root growth. Cultivars widely vary in their efficiency of dry matter allocation to shoot and storage root, a factor that determines storage root yield potential. The increase in total dry matter and storage root dry matter follows a sigmoid pattern. The branch and leaf area (LAI) development is at its maximum during the second phase. LA! of 3-4 has been estimated to maximize solar radiation interception and dry matter production. Plant spacing, N application, photoperiod, soil moisture, and nutrients influence branching and leaf production and thus modify the canopy architecture that affects light interception, photosynthate production, and storage root yield. Sweetpotato cultivars widely vary in their PN rate. The PN rate is highest during the early growth period and declines at the end of the growth period because storage roots attain maximum growth at this time. Cultivars do not show a consistent PN rate in different seasons and at different periods of growth in the same season due to the interaction of PN with environmental factors. The PN rate of individual leaves does not show significant correlation with the total dry matter and storage root yield. However, the CPN rate shows significant correlation with storage root yields. Thus ePN appears to be more important than individual leaf PN rate. The respiration rate of leaves, which is higher than it is for other plant parts, may explain the reduction in storage root yield when excess vegetative growth occurs. Sweetpotato cultivars vary in their storage root initiation period. Cytokinins, indolacetic acid,abscisic acid, and jasmonic acid control the formation and growth of storage roots. ADPG pyrophosphorylase activ-

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ity appears to be more important than starch synthase activity in determining the starch content of storage roots. Low night temperature (15-25°C) and soil temperatures of 25-30°C promote formation and growth of storage roots. Temperatures higher than this promote shoot growth at the expense of storage root growth. Duration and rate of storage root growth, which determine storage root yield, vary widely among cultivars. Short-duration cuItivars exhibit fast initiation and bulking of storage roots, thereby reaching maximum yield in a shorter period of time. The relative contribution of source and sink to storage root yield differs at different periods of crop growth and varies widely among cultivars. Source potential is more limiting than sink during the early period of growth, but they are equally important in determining storage root yield after the formation of storage roots. An active source coupled to a higher sink capacity is desirable, provided that the source component should not be so active that it becomes a competitive sink. Increase in Nand K increases total dry matter and storage root dry matter. Detopping of shoots decreases storage root yield: the reduction is more severe when the whole shoots are removed than when only shoot tips are removed. More frequent harvests of shoots should be avoided because of reduction in the number and size of storage roots. Sweetpotato cultivars yield better when irrigated at 250/0 available soil moisture and the yield does not increase by maintaining available soil moisture >50%. Storage root yield decreases when available soil moisture decreases below 25%. It is important to provide adequate soil moisture during initiation of storage roots because water deficit stress during this period causes greater reduction in storage root yield than when the stress occurs at a later period of growth. Under water deficit conditions, stomatal resistance and proline content increase in the leaves, which helps the plants to preserve water content and enzyme proteins. These characteristics, coupled with high sink capacity, help the tolerant cultivars to yield under drought conditions. Flood stress impedes the storage root growth of plants by restricting availability of O2 around the root zone and leaching the soil nutrients. Luxuriant vegetative growth under high soil moisture also contributes to low storage root yield. Plants exposed to flood stress during the early growth period resume their normal growth better than plants exposed to flooding at a later period of growth. Therefore, reduction in storage root yield is greater in plants exposed to flooding during the later period of growth. Cultivars with high sink capacity tolerate flood stress better than cultivars with weak sink capacity. Deep shade (>55%) reduces total dry matter production due to suppression of both initiation and growth of storage

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Yamakawa, D., and S. Sakamoto. 1981. Growth analysis ofthe early stage under true seed planting culture on sweetpotato. Kyushu Agr. Res. 43:44. Yamakawa, 0., and S. Sakamoto. 1987. Response to selection of natural flowering population for adaptability to true seed planting in sweetpotato. Japan J. Breed. 37:66-74. Yanfu, Y., T. Jialan, Z. Yunchu, and Q. Ruilian. 1989. Breeding for early-maturing sweetpotato varieties. p. 67-82. In: K. T. Mackay, M. K. Palomer, and R. T. Sanieo (eds.), Sweetpotato research and development for small farmers. SEAMEO-SEARCA, College Laguna, Philippines. Yang, T. H. 1982. Sweetpotato as a supplemental staple food. p. 31-34. In: R. L. Villareal and T. D. Griggs (eds.), Sweet potato. Proc. 1st Int. Symp. AVRDC, Taiwan, China. Yen, D. E. 1974. The sweetpotato and Oceania. Bishop Museum Press, Honolulu, Hawaii. Yen, D. E. 1982. Sweetpotato in historical perspective. p. 17-30. In: R. L. Villareal and T. D Griggs (eds.), Sweet potato. Proc. 1st Int. Symp. AVRDC., Taiwan, China. Yoshida, T., Y. Hozyo, and T. Murata. 1970. Studies on the development oftuberous roots in sweet potato (Ipomoea batatas. Lam. var. edulis. Mak.). The effect of deep placement of mineral nutrients on the tuber yield of sweet potato. Proc. Crop Sci. Soc. Japan 39:105-110. Yu, Z. Q. 1981. A study ofthe physiological indices and the scientific cultivation of high yielding sweet potato. Sci. Agr. Sin. 6:50-55. Zaag, P. V., D. Qiwei, and X. Liangshang. 1991. Sweet potato in the food systems of Asia with emphasis on China. p. 45-57. In: Sweetpotato cultures of Asia and South Pacific. Proc. 2nd Annu. UPWARD Int. Conf., Las Banos, Philippines. Zara, D. L., S. E. Cuevas, and J. T. Carlos, Jr. 1982. Performance of sweetpotato varieties grown under coconuts. p. 233-242. In: R. L. Villareal and T. D. Griggs (eds.), Sweet potato. Proc. Int. Symp., AVRDC, Taiwan, China. Zhang, L. Y., and X. P. Lian. 1994. Studies on the yield structure ofsweet potatoes. ]iangsu J. Agr. Sci. 10/17:13-17. Zhong, R. S. 1991. Studies on the source-sink relationship in sweetpotato. Jiangsu J. Agr. Sci. 7:44-48.

Subject Index

p

A

Anatomy and morphology, waxes, 1-68

c Chilling injury, chlorophyll fluorescence, 79-84 Chlorophyll fluorescence, 69-107

Physiology: loquat, 242-252 sweet potato, 277-338 waxes, 1-68 Plums, origin, 179-231 Postharvest physiology, chlorophyll fluorescence, 69-107

D

R

Dedication, Yang, S.F., xi Disease, waxes, 1-68

Root and tuber crop, sweet potato physiology, 277-338

F

s

Fertilization and fertilizers, zinc nutrition, 109-128 Fruit: loquat, 233-276 plum, 179-231 Fruit crops: loquat, 233-276 plum origin, 179-231 G

Genetics and breeding: loquat, 252-257 waxes, 50-53 I

Insects and mites, waxes, 1-68

Senescence, chlorophyll senescence, 88-93

Soil, zinc, 109-178 Stress: chlorophyll fluorescence, 69-107 waxes, 1-68 Sweet potato physiology, 277-338

v Vegetable crops, sweet potato physiology, 277-338

w Waxes, 1-68

L

z

Loquat, 233-276

Zinc, nutrition, 109-178 339

Cumulative Subject Index (Volumes 1-23) 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 Actinida, 6:4-12 Adzuki bean, genetics, 2:373 Agaricus, 6:85-118 Agrobacterium tumefaciens, 3:34 Air pollution, 8:1-42 Almond: bloom delay, 15:100-101 in vitro culture, 9:313 postharvest technology and utilization, 20:267-311 Alocasia, 8:46, 57. See also Aroids Alternate bearing: chemical thinning, 1:285-289 fruit crops, 4:128-173 340

pistachio, 3:387-388 Aluminum: deficiency and toxicity symptoms in fruits and nuts, 2:154 Ericaceae, 10:195-196 Amorphophallus, 8:46, 57. See also Aroids Anatomy and morphology: apple flower and fruit, 10:273-308 apple tree, 12:265-305 asparagus, 12:71 cassava, 13:106-112 citrus, abscission, 15:147-156 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 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

341

CUMULATIVE SUBJECT INDEX

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 bitter pit, 11:289-355 bioregulation, 10:309-401 bloom delay, 15:102-104 CA storage, 1:303-306 chemical thinning, 1:270-300 fertilization, 1:105 fire blight control, 1:423-474 flavor, 16:197-234 flower induction, 4:174-203 fruit cracking and splitting, 19:217-262 fruiting, 11:229-287 in vitro, 5:241-243; 9:319-321 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 yield, 1:397-424 Apricot: bloom delay, 15:101-102 CA storage, 1:309 origin and dissemination, 22:225-266 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 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, 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

CUMULATIVE SUBJECT INDEX

342

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,

Broccoli, CA storage, 1:354-355 Brussels sprouts, CA storage, 1:355 Bulb crops. See also Tulip genetics and breeding, 18:119-123 in vitro, 18:87-169 micropropagation, 18:89-113 root physiology, 14:57-88 virus elimination, 18:113-123

c

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

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

Cabbage: CA storage, 1:355-359 fertilization, 1:117-118 Cactus: crops, 18:291-320 reproductive biology, 18:321-346 Caladium, see Aroids, ornamental Calcifuge, nutrition, 10:183-227 Calciole, nutrition, 10:183-227 Calcium: bitter pit, 11:289-355 cell wall, 5:203-205 container growing, 9:84-85 deficiency and toxicity symptoms in fruits and nuts, 2:148-149 Ericaceae nutrition, 10:196-197 foliar application, 6:328-329 fruit softening, 10:107-152 nutrition, 5:322-323 pine bark media, 9:116-117 tipburn, disorder, 4:50-57 Calmodulin, 10:132-134,137-138 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

CUMULATIVE SUBJECT INDEX

fluid drilling of seed, 3:13-14 Caryophyllaceae, in vitro, 5:237-239 Cassava, 12:158-166; 13:105-129 CA storage, see Controlledatmosphere storage 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

343

Citrus: abscission, 15:145-182 alternate bearing, 4:141-144 asexual embryogenesis, 7:163-168

CA storage, 1:312-313 chlorosis, 9:166-168 cold hardiness, 7:201-238 fertilization, 1:105 flowering, 12:349-408 honey bee pollination, 9:247-248 in vitro culture, 7:161-170 juice loss, 20:200-201 navel orange, 8:129-179 nitrogen metabolism, 8:181 rootstock, 1:237-269 Cloche (tunnel), 7:356-357 Coconut palm: asexual embryogenesis, 7:184 in vitro culture, 7:183-185 Cold hardiness, 2:33-34 apple and pear bioregulation, 10:374-375

citrus, 7:201-238 factors affecting, 11:55-56 herbaceous plants, 6:373-417 injury, 2:26-27 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

344 Controlled environment agriculture, 7:534-545. See also Greenhouse and greenhouse crops; Hydroponic culture; Protected culture Copper: deficiency and toxicity symptoms in fruits and nuts, 2:153 foliar application, 6:329-330 nutrition, 5:326-327 pine bark media, 9:122-123 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 Cryphonectria parasitica, see Endothia parasitica Cryopreservation: apical meristems, 6:357-372 cold hardiness, 11:65-66 Crytosperma, 8:47, 58. See also Aroids Cucumber, CA storage, 1:367-368 Currant, harvesting, 16:311-327 Custard apple, CA and MA, 22:164 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

CUMULATIVE SUBJECT INDEX

in vitro culture, 7:185-187 Daylength, see Photoperiod Dedication: Bailey, L.H., 1:v-viii Beach, S.A., l:v-viii Bukovac, M.J., 6:x-xii Campbell, C.W., 19:xiii Cummins, J.N., 15:xii-xv Dennis, F.G., 22:xi-xii Faust, Miklos, 5:vi-x Hackett, W.P., 12:x-xiii Halevy, A.H., 8:x-xii Hess, C.E., 13:x-xii Kader, A.A., 16:xii-xv 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 Sansavini, S., 17:xii-xiv Sherman, W.B., 21:xi-xiii Smock, RM., 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 Deficiency symptoms, in fruit and nut crops, 2:145-154 Deficit irrigation, 21:105-131 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

CUMULATIVE SUBJECT INDEX

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 mosaic virus, 14:199-238 waxes, 23:1-68 yam (DiosGorea), 12:181-183 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 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

345

ginseng, 9:211-226 greenhouse management, 9:32-38 navel orange, 8:138-140 nutrient film technique, 5:13-26 Epipremnum, see Aroids, ornamental Eriobotrya japonica, see Loquat Erwinia: amylovora,1:423-474 lathyri, 3:34 Essential elements: foliar nutrition, 6:287-355 pine bark media, 9:103-131 plant nutrition 5:318-330 soil testing, 7:1-68 Ethylene: abscission, citrus, 15:158-161, 168-176 apple bioregulation, 10:366-369 avocado, 10:239-241 bloom delay, 15:107-111 CA storage, 1:317-319,348 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 F

Feed crops, cactus, 18:298-300 Feijoa, CA and MA, 22:148 Fertilization and fertilizer: anthurium, 5:334-335 azalea, 5:335-337 bedding plants, 5:337-341 blueberry, 10:183-227 carnation, 5:341-345 chrysanthemum, 5:345-352

346

Fertilization and fertilizer (cont'd) 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 Filbert, in vitro culture, 9:313-314 Fire blight, 1:423-474 Flooding: fruit crops, 13:257-313 Floricultural crops. See also individual crops 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 Florigen,4:94-98 Flower and flowering: alternate bearing, 4:149 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

CUMULATIVE SUBJECT INDEX

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 raspberry, 11:187-188 regulation in floriculture, 7:416-424 rhododendron, 12:1-42 rose, 9:60-66 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

CUMULATIVE SUBJECT INDEX

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

I

10:361-374

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 cracking, 19:217-262 diseases in CA storage, 3:412-461 drop, apple and pear, 10:359-361 fig, 12:424-429 kiwifruit, 6:35-48; 12:316-318 loquat, 23:233-276 maturity indices, 13:407-432 navel orange, 8:129-179 nectarine, postharvest, 11 :413-452 nondestructive postharvest quality evaluation, 20:1-119 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

347

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 CA and MA for tropicals, 22:123-183

cactus, 18:302-309 carbohydrate reserves, 10:403-430 CA storage, 1:301-336 CA storage diseases, 3:412-461 cherry origin, 19:263-317 chilling injury, 15:145-182 chlorosis, 9:161-165 citrus abscission, 15:145-182 citrus cold hardiness, 7:201-238

CUMULATIVE SUBJECT INDEX

348

Fruit crops (cont'd) 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, 1:104-1 06 fig, industry, 12:409-490 fireblight,11:423-474 flowering, 12:223-264 foliar nutrition, 6:287-355 frost control, 11:45-109 grape flower anatomy and morphology, 13:315-337 grape harvesting, 16:327-348 grape nitrogen metabolism, 14:407-452 grape pruning, 16:235-254, 336-340 grape root, 5:127-168 grape seedlessness, 11:164-176 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 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 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

CUMULATIVE SUBJECT INDEX

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 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 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

349 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 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 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. See also Abscisic acid, Auxin, Cytokinins, Ethylene, Gibberellins abscission, citrus, 15:157-176 apple bioregulation, 10:309-401

CUMULATIVE SUBJECT INDEX

350

Growth substances (cont'd) 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,

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

Herbicide-resistant crops, 15:371-412

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 in vitro flowering, 4:112-115 mechanical stress, 17:16-21 meristem and shoot-tip culture, 5:221-227

navel oranges, 8:146-147 pear bioregulation, 10:309-401 petal senescence, 3:76-78 phase change, 7:137-138, 142-143 raspberry, 11:196-197 regulation, 11:1-14 rose, 9:53-73 seedlessness in grape, 11:177-180 triazole, 10:63-105 H

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

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

Hypovirulence, in Endothia parasitica,8:299-310

I

Ice, formation and spread in tissues, 13:215-255

Ice-nucleating bacteria, 7:210-212; 13:230-235

Industrial crops, cactus, 18:309-312 Insects and mites: aroids, 8:65-66 avocado pollination, 8:275-277 fig, 12:442-447 hydroponic crops, 7:530-534 integrated pest management, 13:1-66

lettuce, 2:197-198 ornamental aroids, 10:18 tree short life, 2:52 tulip, 5:63,92 waxes, 23:1-68 Integrated pest management: greenhouse crops, 13:1-66 In vitro: abscission, 15:156-157

351

CUMULATIVE SUBJECT INDEX

apple propagation, 10:325-326 artemisia, 19:342-345 aroids, ornamental, 10:13-14 bulbs, flowering, 18:87-169 cassava propagation, 13:121-123 cellular salinity tolerance, 16:33-69 cold acclimation, 6:382 cryopreservation,6:357-372 embryogenesis, 1:1-78; 2:268-310; 7:157-200; 10:153-181 environmental control, 17:123-170 flowering, 4:106-127 flowering bulbs, 18:87-169 pear propagation, 10:325-326 phase change, 7:144-145 propagation, 3:214-314; 5:221-277; 7:157-200; 9:57-58,273-349; 17:125-172 thin cell layer morphogenesis, 14:239-264 woody legume culture, 14:265-332 Iron: deficiency and toxicity symptoms in fruits and nuts, 2:150 deficiency chlorosis, 9:133-186 Ericaceae nutrition, 10:193-195 foliar application, 6:330 nutrition, 5:324-325 pine bark media, 9:123 Irrigation: deficit, deciduous orchards, 21:105-131 drip or trickle, 4:1-48 frost control, 11:76-82 fruit trees, 7:331-332 grape 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 tipburn, 4:49-65 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, 2:4'06-407 orchards, 2:208-267 ornamental aroids, 10:4-6 photoperiod,4:66-105 photosynthesis, 11:117-121 plant growth, 2:491-537 tolerance, 18:215-246 Longan. See also Sapindaceous fruits CA and MA, 22:150

352

Loquat: botany and horticulture, 23:233-276 CA and MA, 22:149-150 Lychee. See also Sapindaceous fruits CA and MA, 22:150 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, temperaturephotoperiod induction, 17:103-106 Mandarin, rootstock, 1:250-252 Manganese: deficiency and toxicity symptoms in fruits and nuts, 2:150-151 Ericaceae nutrition, 10:189-193 foliar application, 6:331 nutrition, 5:235-326 pine bark media, 9:123-124 Mango: alternate bearing, 4:145-146 asexual embryogenesis, 7:171-173 CA and MA, 22:151-157 CA storage, 1:313 in vitro culture, 7:171-173 Mangosteen, CA and MA, 22:157 Mechanical harvest, berry crops, 16:255-382 Mechanical stress regulation, 17:1-42 Media: fertilization, greenhouse crops, 5:333 pine bark, 9:103-131 Medicinal crops

CUMULATIVE SUBJECT INDEX

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 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 fungi,3:172-213 grape root, 5:145-146

353

CUMULATIVE SUBJECT INDEX

N

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 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

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 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 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

354

Okra: botany and horticulture, 21:41-72 CA storage. 1:372-373 Olive: alternate bearing. 4:140-141 salinity tolerance, 21:177-214 Onion: CA storage, 1:373-375 fluid drilling of seed. 3:17-18 Opium poppy. 19:373-408 Orange. See also Citrus alternate bearing. 4:143-144 sour. rootstock. 1:242-244 sweet. rootstock. 1:252-253 trifoliate. rootstock. 1:247-250 Orchard 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 physiology, 5:279-315 pollination regulation of flower development. 19:28-38 Organogenesis. 3:214-314. see also In vitro; tissue culture Ornamental plants: 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

CUMULATIVE SUBJECT INDEX

CA and MA. 22:157-160 CA storage, 1:314

in vitro culture, 7:175-178 Parsley: CA storage. 1:375 drilling of seed. 3:13-14 Parsnip. fluid drilling of seed, 3:13-14 Parthenocarpy. tomato. 6:65-84 Passion fruit: CA and MA. 22:160-161 in vitro culture. 7:180-181 Pathogen elimination, in vitro. 5:257-261 Peach: bloom delay. 15:105-106 CA storage. 1:309-310 origin, 17:333-379 postharvest physiology. 11:413-452 short life. 2:4 summer pruning. 9:351-375 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

CUMULATIVE SUBJECT INDEX

quality, 4:259 Pest control: aroids (edible), 12:168-169 aroids (ornamental), 10:18 cassava, 12:163-164 cowpea, 12:210-213 fig, 12:442-477 fire blight, 1:423-474 ginseng, 9:227-229 greenhouse management, 13:1-66 hydroponics, 7:530-534 sweet potato, 12:173-175 vertebrate, 6:253-285 yam (Dioscorea), 12:181-183 Petal senescence, 11:15-43 pH:

container growing, 9:87-88 fertilization greenhouse crops, 5:332-333 pine bark media, 9:114-117 soil testing, 7:8-12, 19-23 Phase change, 7:109-155 Phenology: 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

355

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 desiccation tolerance, 18:171-213 disease resistance, 18:247-289 dormancy, 7:239-300 embryogenesis, 1:21-23; 2:268-310 flower development, 19:1-58 flowering, 4:106-127 fruit ripening, 13:67-103 fruit softening, 10:107-152 ginseng, 9:211-213 glucosinolates,19:99-215 heliconia, 14:5-13 juvenility, 7:109-155 light tolerance, 18:215-246 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

356

Physiology (cont'd) 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 triazole, 10:63-105 tulip, 5:45-125 vernalization, 17:73-123 volatiles, 17:43-72 watercore, 6:189-251 water relations cut flowers, 18:1-85 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

CUMULATIVE SUBJECT INDEX

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 flower regulation, 19:1-58 fruit crops, 12:223-264 fruit set, 4:153-154 ginseng, 9:201-202 grape, 13:331-332 heliconia, 14:13-15 honey bee, 9:237-272 kiwifruit, 6:32-35 navel orange, 8:145-146 orchid, 5:300-302 petal senescence, 11:33-35 protection, 7:463-464 rhododendron, 12:1-67 Pollution, 8:1-42 Polyamines, 14:333-356 chilling injury, 15:80 Polygalacturonase, 13:67-103 Postharvest physiology: almond,20:267-311 apple bitter pit, 11:289-355 apple maturity indices, 13:407-432 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 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

CUMULATIVE SUBJECT INDEX

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 fertilization, 1:120-121 low temperature sweetening, 17:203-231 tuberization, 14:89-198 Propagation. See also In vitro apple, 10:324-326; 12:288-295 aroids, ornamental, 10:12-13 cassava, 13:120-123 floricultural crops, 7:461--462 ginseng, 9:206-209. orchid, 5:291-297 pear, 10:324-326 rose, 9:54-58 tropical fruit, palms 7:157-200 woody legumes in vitro, 14:265-332

357

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 soJanacearum, 3:33 syringae, 3:33, 40; 7:210-212 Q

Quality evaluation: fruits and vegetables, 20:1-119, 121-224 nondestructive, 20:1-119 texture in fresh fruit, 20:121-224

R Rabbit, 6:275-276 Radish, fertilization, 1:121 Rambutan, see Sapindaceous fruits Rambutan, CA and MA, 22:163 Raspberry: harvesting, 16:282-298 productivity, 11:185-228

CUMULATIVE SUBJECT INDEX

358

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: aroids, 8:43-99; 12:166-170 cassava, 12:158-166 low-temperature sweetening, 17:203-231 minor crops, 12:184-188 potato tuberization, 14:89-188 sweet potato, 12:170-176 sweet potato physiology, 23:277-338 yam (Dioscorea), 12:177-184 Rootstocks: alternate bearing, 4:148 apple, 1:405--407; 12:295-297 avocado, 17:381-429 citrus, 1:237-269 cold hardiness, 11:57-58 fire blight, 1:432-435

light interception, 2:249-250 navel orange, 8:156-161 root systems, 2:471-474 stress, 4:253-254 tree short life, 2:70-75 Rosaceae, in vitro, 5:239-248 Rose: fertilization, 1:104; 5:361-363 growth substances, 9:3-53 in vitro, 5:244-248

s Salinity: air pollution, 8:25-26 olive, 21:177-214 soils, 4:22-27 tolerance, 16:33-69 Sapindaceous fruits, 16:143-196 Sapodilla, CA and MA, 22:164 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 priming, 16:109-141 rose propagation, 9:54-55 vegetable, 3:1-58 viability and storage, 2:117-141 Secondary metabolites, woody legumes, 14:314-322 Senescence: chlorophyll senescence, 23:88-93 cut flower, 1:204-236; 3:59-143; 10:35-62; 18:1-85 petal, 11:15-43 pollination-induced, 19:4-25

CUMULATIVE SUBJECT INDEX

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,

359

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 protection, 7:463-466 salinity tolerance in olive,

2:1-116

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,

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,

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 Somatic embryogenesis, see Asexual embryogenesis Sorghum, sweet, 21:73-104 SpathiphylJum, see Aroids, ornamental Stem, apple morphology,

3:18-19

Sulfur: deficiency and toxicity symptoms in fruits and nuts, 2:154 nutrition, 5:323-324 Sweet potato: clllture, 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

12:272-283

Storage. See also Postharvest physiology; Controlledatmosphere (CA) storage cut flower, 3:96-100; 10:35-62 rose plants, 9:58-59 seed, 2:117-141 Strawberry: fertilization, 1:106 fruit growth and ripening, 17:267-297

harvesting, 16:348-365 in vitro, 5:239-241

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 cut flower storage, 10:40-43

CUMULATIVE SUBJECT INDEX

360

Temperature (cont'd) cryopreservation, 6:357-372 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 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 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 Triazole, 10:63-105 chilling injury, 15:79-80 Trickle irrigation, 4:1-48 Truffle cultivation, 16:71-107 Tuber, potato, 14:89-188

Tuber and root crops, see Root and tuber crops Tulip. See also Bulb fertilization, 5:364-366 in vitro, 18:144-145 physiology, 5:45-125 Tunnel (cloche), 7:356-357 Turfgrass, fertilization, 1:112-117 Turnip, fertilization, 1:123-124 Turnip Mosaic Virus, 14:199-238

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

v Vaccinium, 10:185-187. See also Blueberry; Cranberry 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 CA storage, 1:337-394 CA storage and quality, 8:101-127 CA storage diseases, 3:412-461 chilling injury, 15:63-95 fertilization, 1:117-124 fluid drilling of seeds, 3:1-58 greenhouse management, 21:1-39 greenhouse pest management, 13:1-66 honey bee pollination, 9:251-254 hydroponics, 7:483-558 low-temperature sweetening, 17:203-231 minor root and tubers, 12:184-188 mushroom cultivation, 19:59-97 mushroom spawn, 6:85-118 N nutrition, 22:185-223 nondestructive postharvest quality evaluation, 20:1-119 okra, 21:41-72

361

CUMULATIVE SUBJECT INDEX

potato tuberization, 14:89-188 seed conditioning, 13:131-181 seed priming, 16:109-141 sweet potato, 12:170-176 sweet potato physiology, 23:277-338 tomato fruit ripening, 13:67-103 tomato parthenocarpy, 6:65-84 truffle cultivation, 16:71-107 yam (DioscoreaJ, 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 Virus: benefits in horticulture, 3:394-411 elimination, 7:157-200; 9:318; 18:113-123 fig, 12:474-475 tree short life, 2:50-51 turnip mosaic, 14:199-238 Volatiles, 17:43-72 Vole, 6:254-274

w Walnut, in vitro culture, 9:312 Water relations: cut flower, 3:61-66; 18:1-85 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 Weeds: lettuce research, 2:198 virus, 3:403 Woodchuck,6:276-277 Woody species, somatic embryogenesis, 10:153-181

x Xanthomonas phaseoli, 3:29-32,41, 45-46 Xanthophyll cycle, 18:226-239 Xanthosoma, 8:45-46, 56-57. See also Aroids Y

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

z Zantedeschia, see Aroids, ornamental 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

Cumulative Contributor Index (Volumes 1-23) Abbott, J.A., 20:1 Adams III, W.W., 18:215 Aldwinckle, H.S., 1:423; 15:xiii 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 Barden, J.A., 9:351 Barker, A.V., 2:411 Bass, L.N., 2:117 Becker, J.S., 18:247 Beer, S.V., 1:423 Behboudian, M.H., 21:105 Bennett, A.B., 13:67 Benschop, M., 5:45 Ben-Ya'acov, A., 17:381 Benzioni, A., 17:233 Bewley, J.D., 18:171 Binze!, 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 Broschat, T.K., 14:1 Brown, S. 15:xiii Buban, T., 4:174 Bukovac, M.J., 11:1 362

Burke, M.J., 11:xiii Buwalda, J.G., 12:307 Byers, RE., 6:253 Caldas, L.S., 2:568 Campbell, L.E., 2:524 Cantliffe, D.J., 16:109, 17:43 Carter, G., 20:121 Carter, J.V., 3:144 Cathey, H.M., 2:524 Chambers, RJ., 13:1 Charron, C.S., 17:43 Chin, C.K., 5:221 Clarke, N.D., 21:1 Cohen, M., 3:394 Collier, G.F., 4:49 Collins, W.L., 7:483 Compton, M.E., 14:239 Conover, C.A., 5:317; 6:119 Coppens d'Eeckenbrugge, G., 21:133 Coyne, D.P., 3:28 Crane, J.C., 3:376 CrUey, RA., 14:1; 22:27 Crowly, W., 15:1 Cutting, J.G., 10:229 Daie, J., 7:69 Dale, A., 11:185; 16:255 Darnell, RL., 13:339 Davenport, T.L., 8:257; 12:349 Davies, F.S., 8:129 Davies, P.J., 15:335 Davis, T.D., 10:63 DeEll, J.R, 23:69 DeGrandi-Hoffman, G., 9:237 De Hertogh, A.A., 5:45; 14:57; 18:87 Deikman, J., 16:1 DellaPenna, D., 13:67

CUMULATIVE CONTRIBUTOR INDEX

Demmig-Adams, B., 18:215 Dennis, F.G., Jr., 1:395 Doud, S.L., 2:1 Duke, S.O., 15:371 Dunavent, M.G., 9:103 Duval, M.F., 21:133 Diizyaman, E., 21:41 Dyer, W.E., 15:371 Early, J.D., 13:339 Elfving, D.C., 4:1; 11:229 EI-Goorani, M.A., 3:412 Esan, E.B., 1:1 Evans, D.A., 3:214 Ewing, E.E., 14:89 Faust, M., 2:vii, 142; 4:174; 6:287; 14:333; 17:331; 19:263; 22:225; 23:179 Fenner, M., 13:183 Fenwick, G.R, 19:99 Ferguson, A.R, 6:1 Ferguson, LB., 11:289 Ferguson, L., 12:409 Ferree, D.C., 6:155 Ferreira, J.F.8., 19:319 Fery, RL., 2:311; 12:157 Fischer, RL., 13:67 Flick, C.E., 3:214 Flore, J.A., 11:111 Forshey, C.G., 11:229 Fujiwara, K., 17:125 Geisler, D., 6:155 Geneve, RL., 14:265 George, W.L., Jr., 6:25 Gerrath, J.M., 13:315 Giovannetti, G., 16:71 Giovannoni, J.J., 13:67 Glenn, G.M., 10:107 Goffinet, M.G., 20:ix Goldschmidt, E.E., 4:128 Goldy, RG., 14:357 Goren, R, 15:145 Goszczynska, D.M., 10:35 Grace, S.C., 18:215 Graves, C.J., 5:1 Gray, D., 3:1

363

Grierson, W., 4:247 Griffen, G.J., 8:291 Grodzinski, B., 7:345 Gucci, R, 21:177 Guest, DJ., 17:299 Guiltinan, M.J., 16:1 Hackett, W.P., 7:109 Hallett, I.C., 20:121 Halevy, A.H., 1:204; 3:59 Hammerschmidt, R., 18:247 Hanson, E.J., 16:255 Harker, F.R, 20:121 Heaney, RK., 19:99 Heath, RR, 17:43 Helzer, N.L., 13:1 Hendrix, J.W., 3:172 Henny, RJ., 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 Indira, P., 23:277 Isenberg, F.M.R, 1;337 Iwakiri, B.T., 3:376 Jackson, ].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, RB., 17:173 Kagan-Zur, V., 16:71 Kang, S.-M., 4:204 Kato, T., 8:181 Kawa, L., 14:57 Kawada, K., 4:247

364

Kelly, J.F., 10:ix; 22:xi Khan, A.A., 13:131 Kierman, J., 3:172 Kim, KW., 18:87 Kinet, J.M., 15:279 King, G.A., 11:413 Kingston, C.M., 13:407-432 Kliewer, W.M., 14:407 Knight, RJ., 19:xiii Knox, RB., 12:1 Kofranek, A.M., 8:xi Korcak, RF., 9:133; 10:183 Kozai, T., 17:125 Krezdorn, A.H., l:vii Lakso, A.N., 7:301; 11:111 Lamb, RC., 15:xiii Lang, G.A., 13:339 Larsen, RP., 9:xi Larson, RA., 7:399 Leal, F., 21:133 Ledbetter, C.A., 11:159 Li, P.H., 6:373 Lill, RE., 11:413 Lin, S., 23:233 Lipton, W.J., 12:69 Litz, RE., 7:157 Lockard, RG., 3:315 Loescher, W.H., 6:198 Lorenz, O.A., 1:79 Lu, R, 20:1 Lurie, S., 22:91-121 Lyrene, P., 21:xi Manivel, L., 22:267 Maraffa, S.B., 2:268 Marangoni, A.G., 17:203 Marini, RP., 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, RJ., 16:255 Merkle, S.A., 14:265 Michailides, T.J., 12:409 Michelson, E., 17:381

CUMULATIVE CONTRIBUTOR INDEX

Mika, A., 8:339 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 Neilsen, G.H., 9:377 Nerd, A., 18:291,321 Niemiera, A.X., 9:75 Nobel, P.S., 18:291 Nyujtb, F., 22:225 O'Donoghue, E.M., 11:413 Ogden, RJ., 9:103 O'Hair, S.K, 8:43; 12:157 Oliveira, C.M., 10:403 Oliver, M.J., 18:171 O'Neill, S.D., 19:1 Opara, L.U., 19:217 Ormrod, D.P., 8:1 Palser, B.F., 12:1 Papadopoulos, A.P., 21:1 Pararajasingham, S., 21:1 Parera, C.A., 16:109 Pegg, KG., 17:299 Pellett, H.M., 3:144 Perkins-Veazil, P., 17:267 Ploetz, RC., 13:257 Pokorny, F.A., 9:103 Poole, RT., 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, RK, 23:69 Pratt, C., 10:273; 12:265

CUMULATIVE CONTRIBUTOR INDEX

Preece, J.E., 14:265 Priestley, G.A., 10:403 Proctor, J.T.A., 9:187 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, RJ., 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 Rudnicki, RM., 10:35 Ryder, KJ., 2:164; 3:vii Sachs, R, 12:xiii Sakai, A., 6:357 Salisbury, F.B., 4:66; 15:233 San Antonio, J.P., 6:85 Sankhla, N., 10:63 Saure, M.C., 7:239 Schaffer, B., 13:257 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 Seeley, S.S., 15:97 Serrano Marquez, C., 15:183 Sharpe, RH., 23:233 Sharp, W.R, 2:268; 3:214 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.K, 19:319 Sklensky, n.K, 15:335

365

Smith, G.S., 12:307 Smock, RM., 1:301 Sommer, N.F., 3:412 Sondahl, M.R, 2:268 Sopp, P.L, 13:1 Soule, J., 4:247 Sparks, D., 8:217 Splittstoesser, W.K, 6:25; 13:105 Srinivasan, C., 7:157 Stang, E.J., 16:255 Steffens, G.L., 10:63 Stevens, M.A., 4:vii Stroshine, RL., 20:1 Stroik, P.C., 14:89 Studman, C.J., 19:217 . Stutte, G.W., 13:339 Styer, D.J., 5;221 Sunderland, K.D., 13:1 Suranyi, 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 Tetenyi, P., 19:373 Tibbitts, T.W., 4:49 Timon, B., 17:331 Tindall, H.D., 16:143 Tisserat, B., 1:1 Titus, J.S., 4:204 Trigiano, RN., 14:265 Tunya, G.O., 13:105 Upchurch, B.L., 20:1 van Doorn, W.G., 17:173; 18:1 van Kooten, 0., 23:69 Veilleux, RE., 14:239 Vorsa, N., 21:215 Wallace, A., 15:413 Wallace, D.H., 17:73 Wallace, G.A., 15:413 Wang, C.Y., 15:63 Wang, S.Y., 14:333 Wann, S.R., 10:153 Watkins, C.B., 11:289 Watson, G.W., 15:1 Webster, B.D., 1:172; 13:xi Weichmann, J., 8:101

366

Wetzstein, H.Y., 8:217 Whiley, A.W., 17:299 Whitaker, T.W., 2:164 White, ].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, RD., 9:75 Wutscher, H.K., 1:237

CUMULATIVE CONTRIBUTOR INDEX

Yada, RY., 17:203 Yadava, V.L., 2:1 Yahia, E.M., 16:197; 22:123 Yan, W., 17:73 Yarborough, D.E., 16:255 Yelenosky, G., 7:201 Zanini, E., 16:71 Zieslin, N., 9:53 Zimmerman, RH., 5:vii; 9:273 Zucconi, F., 11:1