CARBOHYDRATES IN GRAIN LEGUME SEEDS Improving Nutritional Quality and Agronomic Characteristics Carbohydrates in Grain Legume Seeds Improving Nutritional Quality and Agronomic Characteristics Edited by C.L. Hedley Department of Applied Genetics John Innes Centre Norwich UK Associate Editors Jane Cunningham and Alan Jones CABI Publishing CABI Publishing is a division of CAB International CABI Publishing CAB International Wallingford Oxon OX10 8DE UK Tel: +44 (0)1491 832111 Fax: +44 (0)1491 833508 Email: cabi@cabi.org Web site: http://www.cabi.org CABI Publishing 10 E 40th Street Suite 3203 New York, NY 10016 USA Tel: +1 212 481 7018 Fax: +1 212 686 7993 Email: cabi-nao@cabi.org © CAB International 2001. All rights reserved. No part of this publication may be reproduced in any form or by any means, electronically, mechanically, by photocopying, recording or otherwise, without the prior permission of the copyright owners. A catalogue record for this book is available from the British Library, London, UK. Library of Congress Cataloging-in-Publication Data Carbohydrates in grain legume seeds : improving nutritional quality and agronomic characteristics / edited by C.L. Hedley. p. cm. Includes bibliographical references. ISBN 0-85199-467-9 (alk. paper) 1. Legumes--Seeds--Composition. 2. Carbohydrates. 3. Seed technology. I. Hedley, C.L. (Cliff) SB177.L45 C27 2000 633.3′D421--dc21 00-041354 ISBN 0 85199 467 9 Typeset by AMA DataSet Ltd, UK. Printed and bound in the UK by Biddles Ltd, Guildford and King’s Lynn. Contents Contents Contents Contributors xi Preface xv 1 Introduction Editor: Cliff Hedley 1.1 The Grain Legumes 1.2 Grain Legume Production 1.3 Grain Legume Consumption 1.4 Grain Legume Carbohydrates 2 Carbohydrate Chemistry Editor: Pavel Kadlec 2.1 The Carbohydrates 2.1.1 Soluble carbohydrates 2.1.2 Polysaccharides 2.1.3 Other carbohydrate components 2.2 Chemical Analysis of the Carbohydrates 2.2.1 Soluble carbohydrates (monosaccharides, sucrose, α-galactosides, cyclitols) 2.2.2 Polysaccharides 2.2.3 Other carbohydrate components 1 1 1 7 11 15 15 16 22 28 31 31 45 56 v vi Contents 3 Nutrition Editor: Halina Kozlowska 3.1 Introduction 3.2 The Content of Carbohydrates in Grain Legumes Utilized in Europe 3.2.1 The content of carbohydrates in grain legumes used for human nutrition 3.2.2 The content of carbohydrates in grain legumes used for animal nutrition 3.3 Physiological Effect of Grain Legume Carbohydrates in Animal Nutrition 3.3.1 Consumption of grain legume carbohydrates in feed 3.3.2 Effect of mono- and disaccharides in animal nutrition 3.3.3 Effect of oligosaccharides in animal nutrition 3.3.4 Effect of starch in animal nutrition 3.3.5 Effect of non-starch polysaccharides (NSP) in animal nutrition 3.3.6 Effect of grain legume carbohydrates in ruminant nutrition 3.4 Physiological Effect of Grain Legume Carbohydrates in Human Nutrition 3.4.1 Nutritional classification of grain legume carbohydrates 3.4.2 Consumption of grain legume carbohydrates in food 3.4.3 Physiological effect of available carbohydrates from grain legumes 3.4.4 Physiological effect of unavailable carbohydrates from grain legumes 4 Processing Editor: Bálint Czukor 4.1 Native Starch 4.1.1 Isolation 4.1.2 Granular structure 4.1.3 Functional properties 4.2 Modified Starch 4.2.1 Physical methods 4.2.2 Chemical methods 4.2.3 Biotechnological methods 4.3 Food Application of Native and Modified Legume Starches 4.4 Effect of Processing on Starch and Other Carbohydrates in Foods 4.4.1 Resistant starch formation 4.4.2 Content, composition and digestibility 4.5 Legume Seeds as a Source of Raw Materials 61 61 62 62 67 69 69 71 71 74 76 78 79 79 82 84 85 89 89 89 93 98 101 102 104 108 109 110 110 112 116 Contents 5 Seed Physiology and Biochemistry Editor: Ryszard J. Górecki 5.1 The Legume Seed 5.1.1 Seed components 5.1.2 Seed development 5.2 The Accumulation and Biosynthesis of Carbohydrates 5.2.1 Accumulation of soluble carbohydrates 5.2.2 Biosynthesis of soluble carbohydrates 5.2.3 Accumulation of starch 5.2.4 Biochemistry of starch 5.3 Physiological Role of Carbohydrates in Legume Seeds 5.3.1 During seed development 5.3.2 During temperature stress 5.3.3 During seed storage 5.3.4 During germination 6 Biotechnology Editor: Nickolay Kuchuk 6.1 Introduction 6.2 In vitro Cultures and Plant Regeneration of Grain Legumes 6.2.1 Introduction to in vitro culture 6.2.2 Plant regeneration systems 6.2.3 Pioneering studies on pea regeneration 6.2.4 Regeneration via somatic embryogenesis 6.2.5 Regeneration via organogenesis and multiple shoot formation 6.2.6 Recent studies to produce more efficient, fast and reliable systems for regeneration 6.2.7 Factors effecting regeneration 6.2.8 Advantages of the different developmental pathways for in vitro manipulation 6.3 Isolated Protoplasts from Grain Legumes 6.3.1 Introduction to protoplast cultures 6.3.2 Protoplast cultures from leguminous species 6.3.3 Application of grain legumes protoplasts to the study of carbohydrates 6.4 Somaclonal Variation in Grain Legumes 6.4.1 Introduction 6.4.2 Factors causing variation 6.4.3 Mechanisms of somaclonal variation 6.4.4 Potential and disadvantages of somaclonal variation 6.4.5 Variation in grain legumes at the cell and tissue culture level in vitro 6.4.6 Variation in grain legumes at the whole plant level vii 117 117 117 119 122 122 125 128 130 131 131 136 137 138 145 145 146 146 148 149 150 151 153 154 155 156 156 157 158 162 162 163 163 164 165 172 viii Contents 6.5 Transformation Methods in Grain Legumes 6.5.1 Introduction 6.5.2 Gene delivery systems used in agronomically important legumes 6.5.3 Methods giving positive results – transgenic plants 6.5.4 Transgenic plants and useful genes/traits transformed into grain legumes 6.5.5 Field trials with transgenic grain legume plants and commercialized transgenic legume crops 6.5.6 Future prospects 6.6 The Availability and Possible Manipulation of Genes Involved in Starch Biosynthesis 6.6.1 Biochemical pathways of starch biosynthesis 6.6.2 The availability of genes involved into starch biosynthesis 6.6.3 The availability of other genes influencing starch biosynthesis and starch quality 6.7 The Availability and Possible Manipulation of Genes Involved in α-Galactoside Accumulation and Degradation 6.7.1 Biochemical pathways of α-galactoside biosynthesis 6.7.2 The availability of genes involved in α-galactoside accumulation and degradation and their possible manipulation 6.8 Cell Suspension Culture as a Model for Studying Carbohydrate Metabolism 6.8.1 Introduction 6.8.2 Composition of plant cell walls 6.8.3 Biosynthesis of the cell wall components 6.8.4 Oligosaccharides as signals and substrates in the plant cell wall 6.8.5 Plant cell suspension cultures – a powerful tool in investigating cell wall metabolism 7 Breeding and Agronomy Editor: Goran Engqvist 7.1 Current Breeding Goals 7.2 Breeding Techniques 7.2.1 Pedigree breeding 7.2.2 Bulk selection 7.2.3 Deviations from the pedigree and bulk methods 7.3 Access to Genetic Variation 7.3.1 Germplasm banks 7.3.2 Existing variation for the carbohydrates 7.3.3 Newly identified genetic variation 7.4 Selection Methods 181 181 182 183 184 192 193 195 195 196 198 199 199 199 201 201 202 202 203 204 209 209 211 211 212 212 213 213 214 214 220 Contents ix 7.5 Physical Screening Methods 7.5.1 Near-infrared (NI) spectroscopy 7.5.2 Mid-infrared spectroscopy 7.6 Some Agronomic Considerations of Carbohydrates 7.6.1 During plant growth and development 7.6.2 During seed development 7.7 European Registration Requirements for New Varieties 7.7.1 Background 7.7.2 Agronomic characters 7.7.3 Technological characters 7.7.4 Chemical characters 222 224 225 225 225 226 227 227 228 231 232 8 Strategies for Manipulating Grain Legume Carbohydrates Editor: Cliff Hedley 8.1 The Problems 8.2 Strategies for Overcoming the Problems 8.2.1 The soluble carbohydrates 8.2.2 Starch 8.2.3 Fibre 8.3 Conclusions 233 233 235 235 237 237 238 References 241 Index 315 Contributors Contributors Contributors Mr Mike Ambrose, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK. Tel: +44 1603 450630; fax: +44 1603 450045; Email: mike.ambrose@bbsrc.ac.uk Dr Pilar Aranda, Institute of Nutrition, Pharmacy Faculty, Campus Universitario de Cartuja, 18071 Granada, Spain. Tel: +34 58 243885; fax: +34 58 243879; Email: paranda@platon.ugr.es Dr Charlotte Bjergegaard, Royal Veterinary and Agricultural University, Department of Chemistry, Thorvaldsensvej 40, 1871 Frederiksberg, Denmark. Tel: +45 35 282432; fax: +45 35 282398 Dr Tatiana Bogracheva, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK. Tel: +44 1603 450233; fax: +44 1603 450045; Email: tanya.bogracheva@bbsrc.ac.uk Prof. Nikolai Chekalin, Breeding Firm ‘NIVA-1’, Lomany str 14-53, 314 022 Poltava, Ukraine. Tel: +380 5322 70889; fax: +380 5322 22957; Email: psai@atv.net.ua Dr Peter Chekrygin, Plant Production Institute, Moskovskiy Prospect 142, 310060 Kharkov, Ukraine. Tel: +38 0572 921285/924343; fax: +38 0572 920354 Dr Zsuzsanna Cserhalmi, Central Food Research Institute, Herman Otto ut 15, PO Box 393, H-1022 Budapest, Hungary. Tel: +36 1 355 8244; fax: +36 1 355 8928 Dr Balint Czukor, Central Food Research Institute, Herman Otto ut 15, PO Box 393, H-1022 Budapest, Hungary. Tel: +36 1 355 8244; fax: +36 1 355 8928; Email: h9742czu@ella.hu xi xii Contributors Dr Jana Dostalova, Dept of Food Chemistry and Analysis, Prague Institute of Chemical Technology, Technicka 5, 166 28 Prague 6, Czech Republic. Tel: +4202 2435 3264; fax: +4202 311 9990 Mr Goran Engqvist, Svalof Weibull AB, Forage Crop Dept, S-268 81 Svalov, Sweden. Tel: +46 418 667159; fax: +46 418 667102; Email: goran. engqvist@swseed.se Prof. Gabriel Fordonski, Prorector, Department of Plant Diagnostics and Pathophysiology, University of Warmia and Mazury, 10-718 Olsztyn, Plac Lodzki 3, Poland. Tel: +48 89 523 49 52; fax: +48 89 523 48 81 Prof. Jozef Fornal, Institute of Animal Reproduction and Food Research, Division of Food Science, PO Box 55, ul Tuwima 10, 10-718 Olsztyn, Poland. Tel: +48 89 523 63 13; fax: +48 89 523 78 24; Email: fornal@food.irzbz.pan.olsztyn.pl Dr Juana Frias, Instituto de Fermentaciones Industriales CSIC, calle Juan de la Cierva 3, 28006 Madrid, Spain. Tel: +34 1 5622900; fax: +34 1 5644853; Email: ifijf11@ifi.csic.es Prof. Ryszard Gorecki, Rector, University of Warmia and Mazury, 10-718 Olsztyn, Plac Lodzki 3, Poland. Tel: +48 89 523 49 52; fax: +48 89 523 48 81; Email: rigor@uwm.edu.pl Dr Miroslav Griga, AGRITEC Research, Breeding & Services Ltd, 787 01 Sumperk, Zemedelska 16, Czech Republic. Tel: +420 649 382126; fax: +420 649 382999; Email: griga@agritec.cz Dr Krzysztof Gulewicz, Institute of Bioorganic Chemistry, ul Noskowskiego 12/14, 61-704 Poznan, Poland. Tel: +48 618 528503; fax: +48 618 520532; Email: krysgul@man.poznan.pl Dr Horia Halmajan, Bucharest University of Agronomical Science and Veterinary Medicine, Department of Phytotechnics, Bd Marasti nr 59, 71331 Bucharest, Romania. Tel: +40 1 2223700/248; fax: +40 1 2300195; Email: halmajan@sunu.rnc.ro Prof. Cliff Hedley, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK. Tel: +1603 450234; fax: +1603 450045; Email: cliff.hedley@bbsrc. ac.uk Dr Marcin Horbowicz, Research Institute of Vegetable Crops, Konstytucji 3 Maja 1/3, 96-100 Skierniewice, Poland. Tel: +48 46 332604; fax: +48 46 333186; Email: mhorbow@linux.inwarz.skierniewice.pl Mr Alan Jones, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK. Tel: +1603 450253; fax: +1603 450027; Email: alan.jones@bbsrc. ac.uk Mr Rupert Jones, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK. Tel: +1603 450234; fax: +1603 450045; Email: rupert.jones@ bbsrc.ac.uk Prof. Pavel Kadlec, Institute of Chemical Technology, Prague, Technicka 5, 166 28 Prague 6, Czech Republic. Tel: +420 2 311 7070; fax: +420 2 311 9990; Email: pavel.kadlec@vscht.cz Contributors xiii Dr Saima Kalev, Jogeva Plant Breeding Institute, EE 2350 Jogeva, Estonia. Tel: +372 77 22565; fax: +372 77 60126 Prof. Pavel Kintia, Institute of Genetics, Moldavian Academy of Sciences, 20 Padurilor Str., 2002 Chisinau, Moldova. Fax: +373 2 542823; Email: pkintia@hotmail.com Dr Georgina Kosturkova, Department of Cell Genetics, Institute of Genetics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria. Tel: +359 2 759042, x246 or 239; fax: +359 2 757087; Email: kostur@sf.icn.bg Dr Elisabeth Kovacs, Jozsef Attila University, Szeged College of Food Industry, Szeged Higher Education Federation, Mars ter 7, 6724 Szeged, Hungary. Tel: +36 62 456022; fax: +36 62 456005; Email: elisabet@szef.u-szeged.hu Prof. Halina Kozlowska, Institute of Animal Reproduction and Food Research, Division of Food Science, PO Box 55, ul Tuwima 10, 10-718 Olsztyn, Poland. Tel: +48 89 524 03 13; fax: + 48 89 524 01 24; Email: office@pan.olsztyn.pl Prof. Christo Kratchanov, Laboratory of Biological Active Substances, 95 V. Aprilov Str., 4002 Plovdiv, PO Box 27, Bulgaria. Tel: +359 32 452140; fax: +359 32 440102 Dr Maria Kratchanova, Laboratory of Biological Active Substances, 95 V. Aprilov Str., 4002 Plovdiv, PO Box 27, Bulgaria. Tel: +359 32 452140; fax: +359 32 440102 Dr Nickolay Kuchuk, International Institute of Cell Biology, NASU, Zabolotnogo str. 148, 252022 Kiev, Ukraine. Tel/fax: +380 44 252 1786; Email: kuchuk@iicb.kiev.ua Dr Leslaw Lahuta, Department of Plant Physiology and Biotechnology, University of Warmia and Mazury, 10-718 Olsztyn, Plac Lodzki 3, Poland. Tel: +48 89 523 48 24; fax: +48 89 523 48 81; Email: lahuta@moskit.uwm.edu.pl Dr Grazyna Lewandowicz, Starch and Potato Products, Research Laboratory, ul. Zwierzyniecka 18, 60-814 Poznan, Poland. Tel: +48 618 668045; fax: +48 618 417610; Email: lewal@man.poznan.pl Dr Maria Lopez-Jurado, Institute of Nutrition, Pharmacy Faculty, Campus Universitario de Cartuja, 18071 Granada, Spain. Tel: +34 58 243885; fax: +34 58 243879; Email: paranda@platon.ugr.es Ing Martin Mrskos, UKZUZ, Central Institute for Supervising and Testing in Agriculture, Variety Testing Dept., Hroznova 2, 656 06 Brno, Czech Republic. Tel: +420 5 4332 1304 x224; fax: +420 5 4321 2440; Email: mrskos@ooz.zeus.cz Prof. Jan Pokorny, Department of Food Chemistry and Analysis, Prague Institute of Chemical Technology, Technicka 5, 166 28 Prague 6, Czech Republic. Tel: +4202 2435 3264; fax: +4202 311 9990; Email: jan.pokorny@vscht.cz xiv Contributors Dr Paolo Ranalli, Istituto Sperimentale per le Colture Industriali, Via di Corticella 133, 40129 Bologna, Italy. Tel: +39 51 6316847; fax: +39 51 374857; Email: ranalli@bo.nettuno.it Dr Ion Scurtu, Research Institute for Vegetable and Flower Growing, 8268 Vidra, S.A.I., Romania. Tel/fax: +40 13139282/6395; Email: inclf@ rnc.ro Dr Ildiko Schuster-Gajzago, Central Food Research Institute, Herman Otto ut 15, PO Box 393, H-1022 Budapest, Hungary. Tel: +36 1 355 8244; fax: +36 1 355 8928; Email: h9742czu@ella.hu Dr Maria Soral-Smietana, Institute of Animal Reproduction and Food Research, PO Box 55, ul. Tuwima 10, 10-718 Olsztyn, Poland. Tel: +48 89 523 46 51; fax: +48 89 524 01 24 Prof. Hilmer Sorensen, Royal Veterinary and Agricultural University, Department of Chemistry, Thorvaldsensvej 40, 1871 Frederiksberg, Denmark. Tel: +45 35 282432; fax: +45 35 282398; Email: hils@kvl.dk Prof. Mladenka Ilieva-Stoilova, Institute of Microbiology, Laboratory of Biotechnology and Microbiology, 26 Maritza Blvd., 4002 Plovdiv, Bulgaria. Tel: +359 2 438130; fax: +359 2 700109; Email: stoilov@ plovdiv.techno-link.com Mr Jan Urban, AGRITEC Research, Breeding & Services Ltd, 787 01 Sumperk, Zemedelska 16, Czech Republic. Tel: +420 649 382126; fax: +420 649 382999 Prof. Gloria Urbano, Institute of Nutrition, Pharmacy Faculty, Campus Universitario de Cartuja, 18071 Granada, Spain. Tel: +34 58 243885; fax: +34 58 243879; Email: paranda@platon.ugr.es Prof. Concepcion Vidal, Instituto de Fermentaciones Industriales CSIC, calle Juan de la Cierva 3, 28006 Madrid, Spain. Tel: +34 1 5622900; fax: +34 1 5644853; Email: ificv12@fresno.csic.es Prof. Zenon Zdunczyk, Institute of Animal Reproduction and Food Research, Division of Food Science, PO Box 55, ul Tuwima 10, 10-718 Olsztyn, Poland. Tel: +48 89 523 63 13; fax: +48 89 523 78 24 Preface Preface Preface Grain legumes have been an important part of the social evolution of mankind over the past 10,000 years, carbonized remains having been discovered in Neolithic settlements. In more recent times, however, these crops have become less fashionable, particularly in Europe and North America, as the consumption of meat has increased and economic pressures have promoted the growth of cereal monocultures in agricultural systems. The increasing awareness of environmental problems caused by pollution from the over use of fertilizers and a growing interest in more healthy diets is again focusing interest on to this important group of plants. The neglect of grain legumes has resulted in crops that are relatively underdeveloped compared with cereals such as maize, wheat and rice. These crops have been subjected to intensive scientific and technological investigations following substantial public and private financial investment. It is hoped that this book will begin the process of rectifying this imbalance. The book is the result of a combined effort from scientists covering many disciplines interacting within a European Union-funded Copernicus project entitled ‘Carbohydrate Biotechnology Network for Grain Legumes’ (CABINET; contract number IC15-CT96-1007). The CABINET project linked 30 participants from 14 countries across Europe, including states associated within the former Soviet Union. I would like to thank all members of CABINET for their friendship and for making the past 3 years so enjoyable and rewarding. With regard to the book I would like to give my special thanks to those members of CABINET who took on the task of sub-editing the various chapters and for integrating the work within each of the disciplines. Finally I would like to thank my two xv xvi Preface colleagues at the John Innes Centre, Jane Cunningham and Alan Jones. Jane has been at the hub of the CABINET project, responsible for all communication relating to meetings and to the book. Jane’s cheerful and efficient way of administering the project is a major reason why it has been so successful. Alan has been a constant source of support in the running of the project and has taken on major responsibilities for editing the whole book, including constructing and redrawing all of the tables and figures. Cliff Hedley C. Hedley Introduction 1 Introduction 1 Editor: Cliff Hedley There are two things which I am confident I can do very well: one is an introduction to any literary work, stating what it is to contain, and how it should be executed in the most perfect manner . . . Boswell Life, vol. 1, p. 2 (1755) Samuel Johnson (1709–1784), English poet, critic and lexicographer 1.1 The Grain Legumes There are about 60 domesticated grain legume species throughout the world, the major ones being summarized in Table 1.1. The nutritional potential of the seeds from this group of plants is universally recognized since they contain high levels of protein and, depending on the species, a high proportion of either starch or oil. Legumes play a very important role in sustainable agriculture, particularly in developing countries, mainly because of the ability of legume plants to fix atmospheric nitrogen by a symbiotic relationship with nitrogen-fixing bacteria (Rhizobium spp.). 1.2 Grain Legume Production On a world scale, the area of grain legumes excluding soybean (often categorized as an oil seed) has been static for many years at about 68 million hectares (Table 1.2). Likewise, the total production in the world (excluding soybean) has stabilized at between 55 and 57 million t (Table 1.2). On this ©CAB International 2001. Carbohydrates in Grain and Legume Seeds (ed. C.L. Hedley) 1 2 Table 1.1. A list of the common grain legumes grown in the world and their uses. Common name Latin name Description Soybean Glycine max (L.) Merrill Economically and agriculturally the most important legume in the world, providing protein and oil to the food and animal feed industry and the base ingredients for hundreds of chemical products. Not agriculturally important in Europe. Also consumed as tofu and soy sauce in Far Eastern cookery. Originating in the Mediterranean area and found widely in North America over 200 species are known. The name lupin (sometimes spelled as lupine) comes from the Latin for ‘wolf’ and derives from the mistaken belief that the plant ‘wolfed’ minerals from the soil. The contrary is true, lupins aid soil fertility and are drought tolerant. Agriculturally they are grown for animal feed. Sweet lupin Lupinus mutabilis L. Lupinus angustifolius L. Chickpea Cicer arietinum L. Also called Bengal gram, boot, chana chola, chole, gram, hommes, pois chiche and garbanzo bean. Grouped into two types based on seed colour and geographic distribution; desi type are of Indian origin and kabuli type are of Mediterranean, North African and West Asian origin. Desi seeds are about 120 g, wrinkled at the beak with brown, fawn, yellow, orange, black or green colour, normally dehulled and split to obtain dhal. Kabuli seeds are about 400 g, are white/cream coloured and used exclusively by cooking whole seeds. Third most important grain legume crop after beans and peas. Mung bean Vigna radiata L. Wilczek Commonly grown in South Asia, China and India, used as green vegetable or as sprouting shoots. Pigeon pea Cajanus cajan (L.) Millsp. Used world-wide for human consumption, in India seed and plant used as animal feed. C. Hedley White (flowered) Lupinus albus L. lupin Yellow lupin Lupinus luteus L. Canavalia ensiformis (L.) D.C. Also called sword bean. Grown in tropics, North and East Africa, Far East and India. Used as a dry bean, also eaten as a vegetable and used as a green manure and cover crop. Yields compare favourably with the common bean and nutritionally they are similar. Common bean Phaseolus vulgaris L. Also called French, garden, haricot, kidney, pinto, navy (baked bean), black, pink, black eye, cranberry, great northern or dry bean. Beans harvested green in pods or dry for human consumption or used for canning or freezing. Originated in Central and South America. Faba bean (field bean) Vicia faba L. Also called broad, horse or tick bean. Harvested fresh for human consumption or used for canning or freezing. The seed is harvested dry for animal feed. Lentil Lens culinaris Medic. L. One of the most ancient of cultivated foods. Of unknown origin, the lentil is widely grown throughout Europe, Asia and North Africa but is little grown in the Western Hemisphere. So called because of the lens shape of the seeds. Cowpea Vigna unguiculata (L.) Walp Also called Poona pea, yard-long bean, catjang. An important food source in Africa (Nigeria), parts of Asia and Mediterranean area. Typically a dhal-type paste is made from the soaked dehulled seeds. Pea Pisum sativum L. One of the most ancient of cultivated foods. Thought to have originated in several centres; Ethiopia, Asia Minor, Caucasus area and Afghanistan. Garden peas were used by Mendel (1865) in his studies of inheritance. The white flowered or field pea is grown as a vining crop for human consumption or as a combining crop for animal feed. Coloured flowered varieties are grown for animal feed. Introduction Jack bean 3 4 Table 1.2. Total cultivation area (ha) and production (t) of grain legumes in the World and Europe (FAO, 2000). 1997 1998 1999 Production Area Production Area Production World total Europe total 68,324,137 4,780,849 55,512,759 10,260,753 66,913,556 4,550,670 56,143,600 9,258,881 68,320,568 4,181,107 57,514,450 8,225,785 Albania Austria Belarus Belgium–Luxembourg Bosnia and Herzegovina Bulgaria Croatia Czech Republic Denmark Estonia Finland France Germany Greece Hungary 68,328,459 68,327,783 68,262,200 68,134,199 68,312,620 68,354,537 68,311,471 68,351,428 68,397,800 68,328,700 68,326,000 68,629,269 68,185,000 68,326,374 68,361,936 68,322,546 68,386,282 68,492,000 68,318,958 68,314,740 68,340,014 68,324,039 68,104,697 68,387,000 68,317,000 68,313,100 3,121,076 68,587,900 68,342,252 68,118,436 68,327,710 68,327,043 68,269,000 68,324,040 68,312,620 68,351,309 68,327,417 68,359,395 68,106,800 68,326,200 68,324,900 68,630,307 68,235,315 68,326,016 68,363,294 68,324,683 68,385,254 68,334,000 68,316,300 68,314,740 68,337,263 68,324,684 68,133,391 68,387,857 68,312,390 68,311,000 3,305,621 68,779,853 68,341,419 68,140,907 68,332,300 68,327,333 68,212,000 68,324,050 68,312,620 68,355,330 68,328,150 68,352,697 68,106,800 68,235,700 68,324,900 68,497,700 68,227,061 68,325,870 68,362,365 68,330,000 68,385,600 68,253,000 68,315,900 68,314,740 68,339,164 68,326,050 68,106,602 68,357,857 68,310,608 68,311,000 2,657,000 68,792,674 68,341,250 68,140,907 C. Hedley Area 68,324,200 68,374,674 68,323,200 68,352,300 68,312,123 68,321,450 68,346,563 68,324,200 68,144,929 68,354,876 68,348,447 1,239,840 68,339,038 68,324,267 68,613,100 68,353,400 68,234,679 68,196,800 68,635,300 68,381,045 68,319,000 68,115,902 68,321,650 68,106,000 68,330,910 68,321,200 68,363,880 68,316,800 68,260,052 68,330,699 68,375,334 1,802,220 68,397,807 68,236,614 68,390,000 68,168,800 68,314,500 68,748,000 1,072,800 68,142,000 68,324,200 68,378,347 68,324,946 68,366,100 68,314,698 68,322,450 68,353,483 68,324,200 68,148,928 68,352,610 68,359,064 1,068,850 68,341,443 68,324,280 68,532,400 68,345,900 68,234,266 68,201,200 68,555,900 68,378,045 68,319,000 68,127,408 68,321,270 68,104,100 68,330,487 68,321,200 68,371,027 68,316,800 68,289,017 68,331,810 68,372,282 68,888,550 68,103,855 68,327,513 68,382,800 68,142,300 68,312,100 68,701,000 68,770,000 68,137,000 68,234,200 68,380,124 68,324,946 68,352,300 68,314,698 68,22,3450 68,353,100 68,324,200 68,148,928 68,352,610 68,338,074 1,037,700 68,343,676 68,234,280 68,504,100 68,343,900 68,324,400 68,188,500 68,503,000 68,363,045 68,319,000 68,137,938 68,321,850 68,106,400 68,330,487 68,321,200 68,372,511 68,316,800 68,289,017 68,331,810 68,346,572 68,877,000 68,397,735 68,327,513 68,291,400 68,139,600 68,311,100 68,373,200 68,585,500 68,118,000 Introduction Ireland Italy Latvia Lithuania Macedonia Malta Moldavia Netherlands Poland Portugal Romania Russian Federation Slovakia Slovenia Spain Sweden Switzerland United Kingdom Ukraine Yugoslavia 5 6 C. Hedley basis, the most important grain legume is dry bean, covering many varieties of Phaseolus vulgaris and amounting to about 28 million hectares. The most important countries for dry bean production are India and Brazil, with Europe accounting for only about 2% of the world production. In Europe, the area of grain legumes (excluding soybean) has decreased from about 5 million hectares in 1997 to about 4 million in 1999 (Table 1.2). In 1999 the total production of grain legumes in Europe was about 8 million t, the highest proportion being produced in France (c. 32%), followed by Russia (c. 11%), Germany (c. 10%), the UK (c. 9%) and the Ukraine (c. 7%; Table 1.2). The dominant legume within Europe is pea, with a total growing area in 1997 of about 1 million hectares and a production of about 4 million t, more than 70% of which was produced in France. Soybean is by far the most economically important legume in the world and is cultivated on around 60 million hectares. The main producers of soybean are the USA, Brazil, China and Argentina, which together account for 82% of the total world area. The cultivation of this species in Europe is rather small and is only about 1% of the world area (FAO, 1998). In the last few years the area of soybean in Europe has decreased from about 1 million hectares in 1989 to about 0.7 million hectares in 1995, with Italy, Romania and France being the largest producers. There are large differences across the world in the proportion of cultivated land occupied by legumes (Table 1.3). In the USA, legumes account for about 16% of the total arable land, the great majority of which is due to soybean production. In Europe, this proportion is much lower, amounting to about 7% in Portugal, 5% in Austria and Denmark, 4% in France, Italy and the UK, about 3% in Hungary and 2% in the Czech Republic. Legumes play an even less important role in the agriculture of Ireland, Finland, Germany and Belgium. In the world, the average seed yield of grain legumes has been at a similar level for many years, amounting to about only 0.8 t ha−1 (Table 1.4). On average, seed yields in Europe are about three times higher than this world average and generally higher than in the USA. France has the highest seed yields at about 4.7 t ha−1, with Ireland, Belgium, The Netherlands, Denmark, the UK and Austria below this, but still at satisfactory levels (3.3–4.7 t ha−1). In Europe, the lowest yields of legumes are found in Portugal, Spain, Bulgaria and Romania. The high yields found in most European countries can be attributed to more productive varieties being grown within a more intensive agricultural system. Also, there is variation between legume species for yield across the world. The average yield of peas on a world scale is 1.7 t ha−1, which is half the average yield achieved in Europe. The highest pea yields in Europe are found in The Netherlands, Belgium, France and Ireland, with yields from 4.2 to 4.6 t ha−1 followed by Denmark, UK, Austria and Italy, with yields from 3.2 to 3.8 t ha−1. The Czech Republic, Hungary and Poland have lower Introduction Table 1.3. 7 The proportion of cultivated land used for legume production (%). Grain legumes (excluding soybean) Soybean production Total legume production (including soybean) 2.1 0.8 1.4 2.2 4.6 0.4 3.6 0.7 0.8 2.1 < 0.1< 1.1 1.2 1.7 6.6 0.8 1.4 1.1 3.5 3.0 – 0.4 0.1 – – 0.4 – 0.1 0.5 – 2.5 – – – 1.3 0.1 – – 5.1 0.8 1.8 2.3 4.6 0.4 4.0 0.7 0.9 2.6 < 0.1< 3.6 1.2 1.7 6.6 2.1 1.5 1.1 3.5 USA 0.4 15.5 15.9 World total 0.5 0.4 0.9 Country Europe Austria Belgium Bulgaria Czech Republic Denmark Finland France Germany Greece Hungary Ireland Italy Netherlands Poland Portugal Romania Spain Sweden UK yields in the range 2.1–2.6 t ha−1, which is similar to the yields obtained in the USA. The average yield of dry beans is low throughout the world, the highest yields being found in France, Greece and Italy (1.7–1.9 t ha−1), while Portugal, which is the main producer of dry beans in Europe, achieves very low yields. Average world yields of soybeans are relatively high, at about 2 t ha−1, in spite of the large area of this species under cultivation. The average yield within Europe is about 2.4 t ha−1, with the main producer, Italy, and Greece attaining more than 3 t ha−1. In Romania, where the acreage of soybean is relatively high, yields of soybeans are about half those of Italy. By far the major producer of soybeans in the world is the USA, with a total production of about 60 million t, which is about half of the total world production. 1.3 Grain Legume Consumption Of those grain legume species that have been domesticated, only a few find wider application in human food production and animal feed. In Europe, 8 C. Hedley most of the home-produced and imported seed (about 95%) of chickpea, lentil, vetch and bean, and a considerably smaller proportion of faba bean (17%) and pea seeds (4%), are used for human food (Table 1.5). Table 1.4. Grain legume yields in the World and Europe (t ha−1) (Carrouee, 1995). Country 1990 1991 1992 1993 1994 1995 Austria Belgium Bulgaria Czech Republic Denmark Finland France Germany Greece Hungary Ireland Italy Netherlands Poland Portugal Romania Spain Sweden UK 3.6 4.5 0.9 – 4.8 2.9 5.1 2.6 1.4 2.2 4.4 1.3 4.4 1.9 0.3 0.9 0.8 2.5 3.6 3.5 3.8 1.0 – 4.2 2.5 4.7 3.1 1.5 2.2 4.7 1.7 3.6 2.1 0.3 1.0 0.7 2.5 3.3 3.5 4.1 1.2 – 2.6 1.8 4.7 2.5 1.6 2.1 4.7 1.7 4.2 1.1 0.3 1.1 0.6 2.5 3.2 2.4 4.4 0.8 2.4 3.8 2.4 5.0 2.7 1.5 1.5 4.7 1.6 4.2 1.9 0.3 1.2 0.7 2.5 3.9 3.4 4.4 0.9 2.3 3.7 2.2 5.0 2.7 1.6 2.3 4.7 1.6 4.0 1.4 0.3 1.1 0.7 2.4 3.2 3.7 4.6 1.0 2.4 3.3 2.2 4.7 – 1.6 2.2 – 1.6 4.0 1.8 0.3 1.1 0.6 – 3.2 Europe total 2.6 2.5 2.5 2.8 2.7 2.5 USA 1.7 2.0 1.7 1.6 1.8 1.9 World total 0.9 0.8 0.8 0.9 0.8 0.8 Table 1.5. Food and feed uses of grain legumes for 12 EU member states (Carouee, 1995). Species Pea (P. sativum) Faba bean (V. faba) Lupin (Lupinus spp.)a Chickpea (C. arietinum) Lentil (L. culinaris) Beansb and other grain legumes aLupins Production (× 1000 t) Import (× 1000 t) Food use (%) Feed use (%) 4800 1020 19 40 35 155 800 350 300 105 210 390 4 17 2 95 95 95 91 80 97 – – – – sweet cultivars of L. albus, L. luteus and L. angustifolius. – including; common bean (P. vulgaris), Lima bean (P. lunatus), mungo bean (V. mungo) and others. bBeans Introduction 9 Overall, however, five times more grain legume seed is used for animal feed than for human food. Within the European Union (EU), the production of grain legumes accounts for about 25% of the total protein required for animal feed. The contribution of grain legumes to the total amount of animal feed, however, is only about 8% (Table 1.6). The pea is by far the most important grain legume in Europe, comprising about 77% of the total, followed by faba bean (c. 19%) and lupin (c. 4%). Within the EU in 1996–1997, about 3.5 million t of dry peas (c. 88%) and about 0.5 million t of faba beans were utilized in animal feed stuff (Bourdillon, 1998), amounting to about 5% of the total raw ingredients used by the EU compound feed industry (Table 1.7). In France the proportion of grain legumes in the concentrate mixture is about 10% and in Belgium about 12%, while in Germany the proportion is only about 3%. The major part is made up of cereals and other high-protein components, in particular soybean (Pahl, 1998). With the exception of Spain, which imports most of its grain legumes from non-European countries (above 40%), most (greater than 80%) of the grain legume seed consumed within Europe is European in origin – Table 1.6. Grain legume share (as a percentage) in protein crops and protein requirement for animal production within the EU (Carouee, 1995). Share in protein crops Share of protein requirement Animal consumption of grain legumes Pea Faba bean Lupin Table 1.7. 1998). Grain legumes Other protein sources 25.0 8.0 75 92 76.8 18.8 4.4 – – – Grain legumes used for animal feed in Europe (Gatel and Champ, Total consumption (t) Compounded feed production (t) Share in feed production (%) Belgium Denmark France Germany Italy Netherlands Spain UK 5,661 5,222 2,114 5,859 5,598 5,586 5,969 5,450 5,325 5,666 21,998 19,326 11,700 16,495 15,215 12,657 12.4 3.9 9.6 4.4 5.1 3.6 6.4 3.6 EU 5,409 1,248 5.4 Country 10 C. Hedley France, Russia, Germany, the UK and the Ukraine being the largest producers (Gatel and Champ, 1998). In most EU countries grain legumes are produced by farmers in quantities that are too small and with quality that is too variable. For this reason, the animal feed industry usually prefers to use protein crops from abroad (e.g. soybean meal), which are more homogenous. In 1996, the average consumption of grain legumes in the world was 6.36 kg per person, made up of 2.51 kg of dry bean, 0.61 kg of pea and 3.26 kg of other grain legumes (Table 1.8). Human consumption of grain legumes in European countries is relatively low. According to FAO data (1996) in central, northern and western continental Europe the consumption of grain legumes in 1996 averaged 2.37 kg per person, with a range of 0.2–9.3 kg depending on the country (Table 1.8). In many regions of the world, including Mediterranean countries, the Middle East, North America and East Africa, the consumption of grain legumes is three to four times higher (ranging from 7.0 to 10.9 kg per person). There has been a decline in the consumption of grain legumes within Europe for several decades to its present very low level. The recent development of vegetarian habits mainly in northern Europe, however, has stopped this trend. The consumption of legumes is not necessarily affected by economic factors. For example, according to household budget surveys in the Czech Republic (Stikova et al., 1997), poor families (with small children) had a similar consumption of legumes to average families, and among poor, retired persons it was only slightly higher. It is interesting (Stikova et al., 1996), that the highest consumption of grain legumes was found in Table 1.8. Consumption of grain legumes for human nutrition in regions, expressed as kg per person (FAO, 1996). Region Peas Beans Other Total range Total mean Northern and Western Europe Central and Eastern Europe Mediterranean countries Middle East Far East North America Central America The Caribbean South America West Equatorial Africa Eastern Africa South Africa Oceania 1.37 1.65 0.58 0.58 0.48 0.52 0.14 0.44 0.58 0.24 0.14 0.58 0.87 0.53 0.54 1.90 2.75 1.33 5.29 10.06 3.13 9.41 0.76 3.25 2.62 0.64 0.41 0.06 6.33 7.03 1.15 0.04 0.74 7.31 0.76 6.03 6.97 0.89 0.95 1.0–4.9 0.2–9.3 4.2–13.6 3.0–12.8 1.0–11.4 3.7–13.4 5.4–15.7 2.1–13.6 1.0–16.8 0.2–26.4 2.0–20.3 2.9–11.0 0.4–8.0 2.43 2.32 8.85 10.42 2.88 6.50 10.88 10.71 7.03 10.37 7.03 4.08 2.40 World 0.61 2.51 3.26 0.2–26.4 6.36 Introduction 11 the group of families with the highest income, which could be due to a higher level of education. 1.4 Grain Legume Carbohydrates In general, grain legume seeds are characterized by having a relatively high protein content (c. 20–30%) and a very high proportion of carbohydrate (c. 50–65%) in their seeds (Table 1.9). The exceptions are soybean and the various lupin species, all of which have higher protein contents (c. 35–45%) and a lower proportion of carbohydrate (c. 30–40%) in their seeds (Table 1.9). The difference between the seeds of soybean and lupin and those of most other grain legumes lies in the fact that these two legumes do not store starch as their main energy source and both have an increased oil content in their seeds. The oil content in seeds of the starch storing legumes is not usually greater than about 2%. The oil content in soybean seeds, however, can reach more than 20% and in lupin seeds can range from about 4 to 15% oil, depending on the species (Table 1.9). For each legume species, the carbohydrate fraction of the seed can be broadly divided into three groups of compounds: starch, mono- and disaccharides plus low molecular weight oligosaccharides and a group containing structural cell wall polysaccharides. This last group includes cellulose, lignin and pectin, together with cell wall components, such as galactose, arabinose, fucose and xylose. Much of this latter group is included in the non-digestible material often referred to as the ‘fibre’ fraction of the seed (Table 1.10). As mentioned earlier, for most grain legumes the largest part of this carbohydrate fraction is starch, accounting for about 35–45% of the seed weight depending on the legume species. In seeds of soybean and those of the various types of lupin, however, starch only makes up about 1.5% and less than 0.5%, respectively, of the seed weight. The most important low molecular weight soluble carbohydrates are sucrose and the individual members of the raffinose family of oligosaccharides (RFO): raffinose, stachyose and verbascose. All grain legume seeds contain these compounds to a greater or a lesser extent and considerable genetic variation exists, both between and within species, for their content and composition, in particular for the RFO. There is an even greater variation within the group of carbohydrates containing the ‘fibre’ material, much of which still remains to be characterized and quantified for the different species. It is also apparent that the two non-starch storing legumes represented here have a greater proportion of their carbohydrate fraction in this ‘fibre’ group of compounds. The fact that the carbohydrates make up the largest proportion of the seed in itself makes this group of compounds of paramount importance when considering the quality and potential uses of grain legumes. 12 C. Hedley Table 1.9. seeds.a Protein, oil and carbohydrate composition (% of seed) of grain legume Protein Oil Carbohydrates Legume species Min. Max. Min. Max. Min. Max. Soybean G. max 35.1 38.4 – 42.0 17.7 19.7 – 21.0 30.2 32.5 – 35.5 Lupin L. albus 38.1 – 44.9 41.7 39.0 – 47.0 34.1 28.0 – 37.9 34.3 L. luteus L. angustifolius 11.1 – 14.5 5.3 4.0 – 7.1 5.7 4.6 – 7.0 8.0 36.5 – 42.0 32.0 26.0 – 37.0 41.5 36.0 – 47.0 31.0 Chickpea C. arietinum 15.5 21.8 – 28.2 3.1 5.2 – 7.0 59.9 65.3 – 70.8 Mung bean V. radiata 22.9 23.3 – 23.6 1.2 1.2 – 1.2 58.2 60.0 – 61.8 Pigeon pea C. cajan 19.5 21.2 – 22.9 1.3 2.6 – 3.8 63.0 64.9 – 66.8 Jack bean C. ensiformis 26.9 29.6 – 32.2 1.8 2.4 – 2.9 46.1 47.8 – 49.5 Common bean P. vulgaris 20.9 23.4 – 27.8 0.9 1.5 – 2.4 58.2 61.3 – 63.4 Faba bean V. faba 22.4 29.0 – 36.0 1.2 2.0 – 4.0 57.8 59.8 – 61.0 Lentil L. culinaris 23.0 26.8 – 32.0 0.8 1.4 – 2.0 60.5 64.4 – 68.2 Cowpea V. unguiculata Pea P. sativum aSources 23.5 18.3 25.3 – 31.0 1.3 0.6 2.7 – 60.0 5.5 60.7 65.5 – 70.7 from where this data was derived are given with Table 1.10. Nutritionally, they contain starch, which is the major energy source for humans and animals, plus many compounds, including the RFO and those in the ‘fibre’ group, that either enhance or reduce nutritional value, or have a positive or negative effect on health. Carbohydrates are also very important, however, to the growth and development of the seed, forming the main structural elements and the main translocation and storage compounds. The consequences to the seed and to nutritional uses must Introduction Table 1.10. 13 Carbohydrate composition (% of seed) of grain legumes. Legume species Total Starch Sucrose Raffinose Stachyose Verbascose ‘Fibre’a Soybean Lupin spp. Chickpea Mung bean Pigeon pea Jack bean Common bean Faba bean Lentil Pea 32.5 36.7 65.3 60.0 64.9 47.8 61.3 59.8 64.4 65.5 1.5 0.4 44.4 45.0 44.3 35.0 41.5 41.0 46.0 45.0 6.2 2.5 2.0 1.1 2.5 1.5 5.0 3.3 2.9 2.1 0.9 0.7 1.5 1.7 1.0 0.7 0.3 0.2 0.5 0.9 4.3 6.8 5.5 2.0 3.0 1.5 4.1 0.7 2.4 2.4 0.1 0.6 3.0 3.0 4.0 0.1 0.1 2.5 0.9 3.2 20 26 9 7 10 9 10 12 12 12 aFibre – this category includes other soluble and insoluble carbohydrates. Sources of information for Tables 1.9 and 1.10: Proceedings of 1st European Conference on Grain Legumes (1992); Proceedings of the International Conference Euro Food Tox IV on Bioactive substances in food of plant origin (1994); Nwokolo and Smartt (1996); Proceedings of 3rd European Conference on Grain Legumes (1998). be taken into consideration, therefore, in any programme designed to manipulate the content and/or composition of the carbohydrates. It is evident that such a programme would require a multidisciplinary approach encompassing nutritionists, plant biologists, chemists, technologists, geneticists and plant breeders. This book sets out in a limited way what is known about the role of these compounds in the seed and their potential use in nutrition. Information is presented on the chemical composition and analysis of the various carbohydrates and how they can be manipulated genetically, using conventional breeding and modern molecular techniques, or by processing technology. Since each of these areas could form the basis of a book in its own right, we have outlined within each chapter the main areas to be taken into consideration and appended a comprehensive literature list for further reading if necessary. The book is deliberately aimed at the starch-storing grain legumes, because starch is an important nutritional component and also because the main oil storing grain legume, soybean, is already covered comprehensively in the literature. Information is presented on soybean and lupin, however, to make specific points and when this is the only available source in the literature for comparative purposes. Carbohydrate P. 2 Kadlec et al.Chemistry Carbohydrate Chemistry 2 Editor: Pavel Kadlec Contributors: Charlotte Bjergegaard, Krzysztof Gulewicz, Marcin Horbowicz, Alan Jones, Pavel Kadlec, Pavel Kintia, Christo Kratchanov, Maria Kratchanova, Grazyna Lewandowicz, Maria Soral-Smietana, Hilmer Sorensen and Jan Urban He was a practical electrician but fond of whisky, a heavy, red-haired brute with irregular teeth. He doubted the existence of the Deity but accepted Carnot’s cycle, and he had read Shakespeare and found him weak in chemistry. Complete Short Stories (1927) ‘Lord of the Dynamos’ H.G. Wells (1866–1946), English novelist 2.1 The Carbohydrates Carbohydrates – hydrates of carbon – were historically so called because they contain the elements of water, Cx(H2O)y; however, there are now many that have recently been discovered to be exceptions to this formula. Carbohydrates or saccharides can simply be defined as polyhydroxy aldehydes or ketones and their derivatives. These compounds are an important source of energy for living organisms as well as a means by which chemical energy can be stored. In addition, some carbohydrates function as structural components within the cell. They can be divided into two groups: (i) soluble carbohydrates and (ii) polysaccharides, and further subdivided as shown in Fig. 2.1. ©CAB International 2001. Carbohydrates in Grain and Legume Seeds (ed. C.L. Hedley) 15 16 Fig. 2.1. P. Kadlec et al. Classification of carbohydrates. 2.1.1 Soluble carbohydrates Monosaccharides and disaccharides Monosaccharides, the simplest of all sugars, are the building blocks of carbohydrate chemistry. Their general formula is (CH2O)n, where n = 3 or some larger number. Monosaccharides cannot be hydrolysed to form simpler or smaller entities. The three most commonly found monosaccharides that are measurable in any quantity in grain legume seeds are glucose, galactose and fructose. Disaccharides consist of two sugars joined by a glycosidic bond – an oxygen bridge. The three abundant naturally occurring disaccharides are sucrose, maltose and lactose, and these are widely distributed in living organisms. Sucrose (common table sugar) is the only one present in any appreciable quantity in legume seeds. Sucrose was first obtained commercially from sugar-cane (Saccharum officinarum L.) and hence sugars were given the scientific name of saccharides. Confusingly, when people talk of sugar they invariably mean sucrose, whereas to scientists sugar is a name given to a group of compounds such as those we are describing here. As a consequence of a glycosidic bond joining the anomeric carbon atoms of the glucose and a fructose moieties, sucrose lacks a free reducing group (i.e. there is no aldehyde or ketone end group), in contrast with most other sugars. The hydrolysis of sucrose to glucose and fructose is catalysed by the enzyme invertase (EC 3.2.1.26, so named because hydrolysis changes the optical activity from dextro- to laevorotatory), also known as saccharase. A mixture of glucose and fructose so obtained is called ‘invert sugar’. In maltose, two glucose units are joined by an α(1→4) glycosidic linkage. Maltose derives from the hydrolysis of starch and is in turn hydrolysed to glucose by the enzyme maltase (EC 3.2.1.20). Carbohydrate Chemistry 17 Monosaccharides are present in dried, mature legume seeds in relatively small amounts. Traces of fructose and glucose are found in pea, lentil and lupin seeds at a level of 0–1% of dry weight. Glucose is present in developing pea embryos in slightly higher quantities (1–3%), together with occasional traces of galactose. Although monosaccharides are not a major component of legume seeds, sucrose can accumulate in appreciable quantities in some legume species. For example, mature dry seeds of the starchless pea mutant, rug3, accumulates about 7–8% of the seed dry weight as sucrose compared with the wild-type value of 2–3%. Sucrose is present in similar quantities in mature faba bean seeds to the wild-type pea and in lesser amounts (1–2%) in lupin seeds (Frias et al., 1996a). a-Galactosides Oligosaccharides (from Greek oligos, a few) are compounds that give only monosaccharide units after complete hydrolysis. Depending on the number of monosaccharide residues per mole, oligosaccharides are classified as trisaccharides, tetrasaccharides and so forth. The α-galactosyl derivatives of sucrose are the most common group of α-galactosides found in the plant kingdom. They are the most abundant soluble sugars in plants and rank only second to sucrose in importance. The most ubiquitous group of galactosyl sucrose oligosaccharides are the raffinose family of oligosaccharides (RFO), so named after the first member of this homologous series of α-galactosides. The RFO are α(1→6) galactosides linked to C-6 of the glucose moiety of sucrose. They are low molecular weight non-reducing sugars that are soluble in water and water–alcohol solutions (Arentoft and Sorensen, 1992; Arentoft et al., 1993). In addition to raffinose, this group of α-galactosides includes stachyose, verbascose, ajugose and unnamed longer-chain oligosaccharides up to nonasaccharide (Cerning-Beroard and Filiatre-Verel, 1976). Chemically, the RFO may be considered as derivatives of sucrose. Their IUPAC (International Union of Pure and Applied Chemistry) names are listed below. • raffinose • stachyose • verbascose • ajugose α-D-galactopyranosyl-(1→6)-α-D-glucopyranosyl-(1→2) -β-D-fructofuranoside α-D-galactopyranosyl-(1→6)-α-D-galactopyranosyl(1→6)-α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside α-D-galactopyranosyl-(1→6)-[α-D-galactopyranosyl(1→6)-]2-α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside a-D-galactopyranosyl-(1→6)-[α-D-galactopyranosyl(1→6)-]3-α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside Structures of these oligosaccharides are shown in Fig. 2.2. The α-galactosides are often considered to be antinutritional factors, because they are not hydrolysed by mucosal enzymes in the small intestine of monogastric animals and pass into the lower gut where they are 18 Fig. 2.2. P. Kadlec et al. Raffinose family of oligosaccharides (RFO). fermented with the production of gas (Cristofaro et al., 1974; Saini and Gladstones, 1986; Price et al., 1988). Conversely, their ingestion in the form of pure compounds in the diet increases the bifidobacteria population in the colon, which in turn contributes positively to human health in many ways (Minami et al., 1983; Tomomatsu, 1994; see Chapter 2). Cyclitols There are nine isomers of inositol, the prevalent natural form is cis-1,2,3,5trans-4,6-cyclohexanehexol (trivial name myo-inositol; Greek mys, muscle). Myo-inositol is widely distributed in plants and animals and is a growth factor for animals and microorganisms. There are three other forms of underivatized inositol present in seeds of some legume species: D-chiro-inositol, muco-inositol, and scyllo-inositol. These naturally occurring isomers are synthesized from myo-inositol by epimerization (Loewus and Dickinson, 1982; Loewus, 1990) and have the same molecular formula (C6H12O6), and formula weight (180.16; for further information see Hudlicky and Cebulak, 1993). Myo-inositol is the primary source for the biosynthesis of many naturally occurring derivatives including methyl-cyclitols (Hoffmann-Ostenhof and Pittner, 1982). In plants of the Leguminosae family, myo-inositol is converted by a specific O-methyl transferase into D-ononitol (4-O-methyl-myo-inositol), Carbohydrate Chemistry 19 which is a precursor for the formation of D-pinitol, a methyl derivative of (3-O-methyl-D-chiro-inositol). Other methylation products of myo-inositol are sequoyitol (5-O-methyl-myo-inositol) and D-bornesitol (1-Omethyl-myo-inositol). Only one methyl derivative of muco-inositol occurs in Leguminosae seeds, methyl-muco-inositol, where the methyl group is attached to oxygen in position 1 (Dittrich and Brandl, 1987; Keller and Ludlow, 1993). The solubility of methyl-cyclitols is similar to that of the corresponding cyclitols. The occurrence of cyclitols and methyl-cyclitols in legume seeds is presented in Table 2.1. Among the cyclitol galactosides only galactinol (galacto-myo-inositol) is common in seeds and particularly widespread in legume seeds (Fig. 2.3). It serves as the galactose donor to form galactosyl sucrose oligosaccharides, the RFO (Lehle and Tanner, 1972). Galactinol is formed from UDPgalactose and myo-inositol, and can then add a galactose residue to sucrose forming raffinose, then to raffinose to form stachyose, etc. (Dey, 1990). In addition, galactinol may contribute galactose to another molecule of galactinol to form the digalactosyl derivative of myo-inositol (Petek et al., 1966). When the accumulation of sucrose galactosides is limited, galactinol and di-galactosides of myo-inositol can accumulate to higher levels (Horbowicz et al., 1995). Galactinol is also the galactose donor to D-ononitol to form galacto-ononitol (Fig. 2.4), found in seeds of adzuki bean (Yasui, 1980; Obendorf, 1997). Other cyclitol galactosides common in legume seeds are the galactopinitols. In these galactosides, the galactose molecule can be attached to D-pinitol in position 1, 2 or 5. So-called galactopinitols [A (O-α-D-galactopyranosyl-(1→2)-4-O-methyl-D-chiro-inositol) (Fig. 2.5) and B (O-α-Dgalactopyranosyl-(1→2)-3-O-methyl-D-chiro-inositol) (Fig. 2.6)] are present in many legume seeds (Schweizer et al., 1978). Another galactopinitol isomer called leucaenitol (O-α-D-galactopyranosyl-(1→1)-3-O-methyl-Dchiro-inositol) has been recently discovered in seeds of a tropical legume, leucaena (Leucaena leucocephala Lam.) (Chien et al., 1996). Less common in legume seeds is the galactoside of chiro-inositol, fagopyritol B1, so called because of its abundance in seeds of buckwheat, Fagopyrum esculentum (Obendorf, 1997). Fagopyritol B1 (O-α-Dgalactopyranosyl-(1→2)-D-chiro-inositol; Fig. 2.7) was first identified in soybean seeds (Schweizer and Horman, 1981), but has now been reported in seeds of lupin, pigeon pea, cowpea and lentil (Horbowicz and Obendorf, 1994; Górecki et al., 1996). D-Ononitol is an intermediate in D-pinitol biosynthesis, the free form and the galactoside of which is present in small or trace amounts. Larger quantities of galacto-ononitol (O-α-D-galactopyranosyl-(1→5)-4-O-methylmyo-inositol), however, have been found in the seeds of adzuki bean (Yasui, 1980). Among the di- and tri-galactosides of cyclitols only ciceritol (O-α-Dgalactopyranosyl-(1→6)-O-α-D-galactopyranosyl-(1→2)-4-O-methyl-D-chiroD-chiro-inositol 20 Table 2.1. Occurrence of cyclitols, methyl-cyclitols and galacto-cyclitols in legume seeds. + + + + + ++ ++ ++ ++ ++ + +++ + ++ + + ++ +++ +++ +++ ++ ++ +++ + + + + ++ ++ ++ + +, Value between 0.05 and 0.10% of dry weight; ++, value between 0.10 and 0.50% of dry weight; +++, value above 0.5% of dry weight. An empty cell means the level is below the limit of detection, trace amounts or no data available. The table is compilation of data from: Aman (1979), Ford (1982), Frias et al. (1996c), Górecki et al. (1996), Horbowicz and Obendorf (1994), Horbowicz et al. (1995), Ueno et al. (1973), Yasui (1980), Yasui et al. (1985). P. Kadlec et al. ++ +++ ++ + ++ ++ tri-galacto-pinitol (galacto-ciceritol) + di-galacto-chiroinositol (fagopyritol B2) + + + + + di-galacto-pinitol A (ciceritol) + ++ ++ + + ++ di-galacto-inositol + + galacto-ononitol ++ ++ + + ++ + + ++ ++ galacto-chiro-inositol B1 (fagopyritol B1) ++ galacto-pinitol B + ++ + + + + +++ + + + ++ + ++ ++ + galacto-pinitol A + galacto-myo-inositol (galactinol) ++ + ++ ++ methyl-myo-inositol (D-ononitol) ++ ++ + ++ + ++ ++ ++ ++ +++ ++ ++ ++ + methyl-scyllo-inositol Lupinus luteus L. Lupinus albus L. Glycine max [L.] Merrill Cajanus cajan [L.] Millsp. Vigna unguiculata [L.] Walp Vigna radiata [L.] Wilczek Vicia faba L. Phaseolus vulgaris L. Phaseolus vulgaris L. Pisum sativum L. Lens culinaris Medic. L. Cicer arietinum L. Medicago sativa L. Vigna angularis L. D-chiro-inositol Lupin Lupin Soybean Pigeon pea Cowpea Mung bean Faba bean Dry bean Garden bean Pea (round) Lentil Chickpea Lucerne Adzuki bean methyl-chiro-inositol (D-pinitol) Latin name myo-inositol Species Carbohydrate Chemistry 21 inositol; Fig. 2.5) is relatively common and has been fully identified (Quemener and Brillouet, 1983; Bernabé et al., 1993). Ciceritol is present in the seeds of chickpea, lupin, lentil, soybean, kidney bean and lucerne. The chickpea seed (Cicer arietinum, from which ciceritol is named), also contains galacto-ciceritol (O-α-D-galactopyranosyl-(1→6)-O-α-D-galactopyranosyl-(1→6)-O-α-D-galactopyranosyl-(1→2)-4-O-methyl-D-chiro-inositol) (Nicolas et al., 1984). Ciceritol is a digalactosidic derivative of galactopinitol A. Mimositol (O-α-D-galactopyranosyl-(1→6)-O-α-D-galactopyranosyl-(1→6)O-α-D-galactopyranosyl-(1→2s)-3-O-methyl-D-chiro-inositol) an isomer of galacto-ciceritol, is a digalactoside derivative of galactopinitol B, and has been reported and isolated from seeds of the Brazilian legume tree, Mimosa scabrella (Ganter et al., 1991). Fig. 2.3. Galactinol series. Fig. 2.4. Galactosyl ononitol series. 22 P. Kadlec et al. Fig. 2.5. Galactopinitol A series. Fig. 2.6. Galactopinitol B series. 2.1.2 Polysaccharides Starch Starch is the major storage carbohydrate (polysaccharide) in higher plants. It exists in the form of granules, which are deposited as a reserve or storage carbohydrate in plant organs such as seeds, tubers and roots. Starch is unique among carbohydrates because it occurs naturally as discrete Carbohydrate Chemistry 23 granules (see Chapter 4). Starch granules are relatively dense, insoluble and hydrate only slightly in cold water. They are also unique because, in general, they are composed of a mixture of two polymers, an essentially linear polysaccharide, amylose, and a highly branched polysaccharide, amylopectin (BeMiller and Whistler, 1996). Amylose is essentially a linear chain of (1→4)-linked α-Dglucopyranosyl units (Fig. 2.8). Most amylose molecules have a limited number of α-D-(1→6) branches, perhaps 1 in 180–320 units, or 0.3–0.5% of the linkages (Takeda et al., 1990). The branches in branched amylose molecules are either very long or very short, and the branch points are separated by large distances so that the physical properties of amylose molecules are essentially those of linear molecules. The axial-equatorial position coupling of the (1→4)-linked α-D-glucopyranosyl units in amylose chains gives the molecules a right-handed spiral or helical shape. The interior of the helix contains only hydrogen atoms and is lipophilic, while the hydroxyl groups are positioned on the exterior of the coil. Most starches contain about 25% amylose (BeMiller and Whistler, 1996). The AMYLOSE Fig. 2.7. Fagopyritol B series. Fig. 2.8. Linear chain structure of amylose. 24 P. Kadlec et al. amylose found in pea starch shows a wide distribution of molecular weight, with an average value of approximately 500,000 daltons. A typical amylose molecule from pea probably consists of two or three long chains of glucose units (Colonna and Mercier, 1984). Amylopectin is a very large, highly branched molecule, with branch-point linkages constituting 4–5% of the total linkages (Fig. 2.9). Amylopectin consists of a C-chain containing the only reducing endgroup with numerous branches, or B-chains, each of which have several smaller branches, or A-chains, attached. Overall, therefore, A-chains are unbranched and B-chains are branched with A-chains or other B-chains (Fig. 2.10). The branches of amylopectin molecules are believed to be clustered and to occur as double helices. Molecular weights of from 107 to 5 × 108 daltons make amylopectin molecules among the largest, if not the largest, molecules found in nature. Amylopectin usually constitutes about 75% of most common starches (BeMiller and Whistler, 1996). Heating starch suspensions in excess water results in disturbance of the ordered structures in the starch granules, a process known as gelatinization (see Chapter 4). The gelatinization process is dependent on the organization of starch granules, which contain both crystalline and amorphous domains (Bogracheva et al., 1997; see Chapter 4). AMYLOPECTIN Fig. 2.9. Linear structure of amylopectin showing a side chain branching point. Fig. 2.10. Amylopectin – cluster model showing the organization of the different chain types within the molecule (Manners, 1989). Carbohydrate Chemistry 25 Fibre fraction The first definition of dietary fibre (DF) was ‘the skeletal remains of plant cells that are resistant to hydrolysis by the enzymes of man’. This definition includes a wide spectrum of compounds within the DF fraction (Trowell, 1972). DF was redefined later to include ‘plant polysaccharides and lignin which are resistant to hydrolysis by the digestive enzymes of man’ (Trowell, 1976). This chemically more precise definition restricted DF to polysaccharides and lignin, but on the other hand expanded the definition to include compounds outside the plant cell wall. For the purpose of this book, the chemical structure of DF is described for the cellulosic and non-cellulosic polysaccharides, including hemicelluloses, pectins and some associated components. In addition, the complex structure of lignin is discussed, whereas the proteins and various amphiphilic compounds, which also are considered as important components of the cell wall and, therefore, DF (Andersen et al., 1997; Bjergegaard et al., 1997a,b), are not described. An excellent review of the definition of terms used to describe DF is given by Hall (1989). CELLULOSE AND HEMICELLULOSE Cellulose is composed of linear β(1→4)-Dglucans with a very high degree of polymerization resulting in molecular weights ranging from about 0.5 to 1 million daltons. The fibrillar appearance of cellulose in the plant cell wall arises from the side-by-side alignment of cellulose chains, which are stabilized in a crystalline structure by interand intramolecular hydrogen bonds (Southgate, 1995a). Non-crystalline regions occur at regular intervals in the fibrils. Traces of sugars other than glucose, found in preparation of cellulose, probably originate from non-cellulose polysaccharides, e.g. mannans or xylans present in these regions (Heredia et al., 1995). The cellulose content is typically about 35% in cotyledon cell walls of legume seeds (Selvendran and Robertson, 1990). Hemicellulose is not chemically or structurally similar to cellulose, as its name may imply. The traditional classification of hemicelluloses comprises cell wall polysaccharides, preferentially solubilized by aqueous alkali after removal of water-soluble polysaccharides. Hemicelluloses cover a wide spectrum of complex hetero-polysaccharides, containing a minimum of two types of sugar residues. The dominating constituent monosaccharides are of neutral character, although some uronic acid may be present in minor amounts (McDougall et al., 1993). Xyloglucans (also called amyloids) are the predominant hemicellulosic polysaccharides in the primary cell wall of dicotyledons. Xyloglucans are commonly composed of D-glucose, D-xylose and D-galactose residues in a molar ratio of 4 : 3 : 1 (Hayashi, 1989). The polysaccharides have a repeated structure of characteristic oligosaccharides with β(1→4)-D-glucose backbones, regularly branched with D-xylose at C-6 (α) for the majority of the glucose residues. Part of the xylose residues may be further substituted by a 26 P. Kadlec et al. disaccharide (α-L-fucose-1,2-β-D-galactose) and sometimes by β(1→2)linked L-arabinose (Selvendran, 1983; Gibeaut and Carpita, 1994). Xylans have a β(1→4)-backbone of D-xylose with some residues acetylated or substituted with different sugars at C-2 or C-3, e.g. dominated by α-linked L-arabinose (arabinoxylans), or 4-O-methyl-D-glucuronic/ D-glucuronic acid (glucuronoxylans). Xylans also exist with ferulic acid substituents, which may participate in cross-linking of the plant cell wall (Brett and Waldron, 1990; Heredia et al., 1995). Glucomannans consist of a backbone of β(1→4)-linked D-glucose and D-mannose residues (about 1 : 3, depending on the plant species) without any regularity in its sequence. α(1→6)-D-Galactose residues are often found as side chains, either in glucomannans (galactoglucomannans), or attached to a pure β(1→4)linked mannose backbone (galactomannans). The group of pure mannans comprises unsubstituted homopolymeric chains of β(1→4)-linked mannose. Glucuronomannans contain a backbone of α(1→4)-linked D-mannose and β(1→4)-linked D-glucuronic acid residues with side chains including D-xylose or D-galactose linked to the mannose by β(1→6) links or of L-arabinose linked to mannose by β(1→3) links (Brett and Waldron, 1990; Heredia et al., 1995). In cotyledon cell walls of legume seeds, the hemicellulose content is typically about 15% (Selvendran and Robertson, 1990). A detailed study of the chemical composition of certain dehulled legume seeds and their hulls has been performed, with special reference to carbohydrates, by Daveby and Aman (1993). The legume seeds studied comprised pea (Pisum sativum L.), soybean (Glycine max L.), broad bean (Vicia faba L.), sweet white lupin (Lupinus albus L.) and brown bean (Phaseolus vulgaris L.). The study did not distinguish between cellulose, hemicelluloses and pectins. It gave, however, a detailed picture of the dominating monomeric residues in the non-starch polysaccharide fraction by determining the content of rhamnose, fructose, arabinose, xylose, mannose, galactose, glucose and uronic acids, respectively. Fractionation of non-starch polysaccharides from the cotyledons and hulls of lupin (L. albus L.) into pectic and hemicellulosic polysaccharides have been performed by Mohamed and Rayas-Duarte (1995). In this species arabinose and xylose were found to be the major sugars in the hulls, whereas galactose was predominant in the cotyledons. Covalent cross-linking between cell wall polymers is a physiologically significant strategy contributing to the termination of the extensibility and strengthening of the cell wall. Xyloglucans in the hemicellulosic fraction are closely linked to the cellulose microfibrils by means of hydrogen bonds, and phenolic carboxylic acids and proteins are also well known to contribute to the cross-linking of cell wall components. The term ‘pectic substances’ is generally used to describe the group of complex plant heteropolysaccharides in which D-galacturonic acid is esterified to various extents with methanol. The great diversity in PECTIN Carbohydrate Chemistry 27 composition and forms of occurrence of pectic substances in plants has led to the development of several more restrictive definitions. The nomenclature of pectins is essentially based on the degree of methoxylation or degree of esterification of the carboxyl groups of the polygalacturonan chain. The degree of methoxylation is defined as the proportion of galacturonic-acid units esterified with methanol and is expressed as a percentage. Pectic substances can be classified as follows as defined by Jeltema and Zabik (1980): • • • • pectic acid – pectic substances mostly free of methyl ester groups (degree of methoxylation less than 5%), the salts of pectic acid are called pectates; pectinic acid – pectic substances mostly composed of polygalacturonic acids carrying more than a negligible proportion of methyl ester groups, the salts of pectinic acid are called pectinates; pectin – name is derived from the Greek pectos, which means coagulum and is mainly used to designate those water-soluble pectic substances which are capable of forming gels under suitable conditions; protopectin – pectic substances in plants, insoluble in water and considered to be the parent pectic substances that can, upon restricted hydrolysis, yield pectin. It is evident that pectin is not a homologous polysaccharide and that it has a chain structure of α(1→4)-linked D-galacturonic acid units interrupted by the insertion of α(1→2)-linked L-rhamnopyranosyl residues in adjacent or alternate positions (Sathe and Salunkhe, 1981; Ravindran and Palmer, 1984; Ross et al., 1985). Homologous galacturonans consisting solely or predominantly of α(1→4)-linked D-galacturonosyl residues have been isolated from various plant tissues such as sunflower heads and seeds (Shehata et al., 1985), sisal (Reid et al., 1986), rice endosperm cell walls (Champ et al., 1986), from apple pectin (Goldberg et al., 1986) and other plant cell walls. Such galacturonans, however, were obtained by extraction treatments likely to cleave covalent bonds, so that they may have been released from a heterogeneous pectic polysaccharide. The homologous galacturonan type of pectin contains no side chains and therefore, is also referred to as ‘smooth regions’ of pectin. In contrast, the second major type of pectic polysaccharide, rhamnogalacturonan, contains many side chains and is often referred to as ‘hairy regions’ (Ross et al., 1985; Bhatty, 1990; Vidal-Valverde et al., 1992a; Ralet et al., 1993). Various sugars are attached in side chains, the most common being D-galactose, L-arabinose and D-xylose, while D-glucose, D-mannose, Lfructose and D-glucuronic acid are found less frequently. D-galactose and L-arabinose are present in more complex chains with structures similar to those of arabinans and arabinogalactans and with chain lengths that can be considerable. Side chains are glycosidically linked to C-4 and/or C-3 of 28 P. Kadlec et al. α-L-rhamnopyranose or C-2 and C-3 of some of the galacturonosyl residues (Goldberg et al., 1994). There is little or no evidence to suggest what structural forms are present in grain legume seeds. The occurrence of pectic substances in legume seeds is presented in Table 2.2. 2.1.3 Other carbohydrate components Lignin The term lignin is used now to refer not to a single chemical compound, but rather to a group of structurally related amorphous, high molecular weight, aromatic polymer compounds. They typically consist of monomeric units of oxygen derivatives of phenylpropane with different degrees of methoxylation of the aromatic nucleus. Lignin substances have a complex three-dimensional structure and are insoluble both in water and in organic solvents. Lignin is one of the chief constituents of plant cell walls and DF, performing the role of a cementing substance with regard to the other biopolymers of the cell walls. Lignin accounts for about 25% of the composition of wood and occupies the second place in occurrence of organic substances in nature after cellulose. It has been established that lignins are heterogeneous in terms of chemical structure and molecular mass, the molecular heterogeneity depending both on the age and the kind of plant. They are normally linked by covalent and hydrogen bonds to carbohydrates. Lignification serves two main functions. It cements and anchors the cellulose microfibrils and other matrix polysaccharides (pectins, hemicelluloses) and because the lignin–polysaccharide complexes are hard, they stiffen the walls, thus preventing biochemical degradation and physical damage to the walls. These properties of lignified walls are important in the DF context, because they minimize the bacterial degradation of the walls in the human colon. The occurrence of lignin in legume seeds is shown in Table 2.2. Saponins Saponins are naturally occurring glycosides widely distributed in plants, including soybean and pea seeds. Each saponin consists of a sapogenin, which constitutes the aglycon moiety of the molecule and a sugar. The sapogenin may be a steroid or a triterpene (the later type being most common form of saponin found in cultivated crop plants) and the sugar moiety may be glucose, galactose, a pentose or a methyl pentose. The name comes from Saponaria, soapwort, the root of which has been used as a soap (sapo, Latin for soap). All saponins foam strongly when shaken in water. They Carbohydrate Chemistry Table 2.2. 29 Occurrence of pectic substances and lignin in various legumes. Legumes Faba beans seeds Water-soluble EDTA-soluble NaOH-soluble Pectic substances % Lignin % 0.8 0.4 0.4 References Shehata et al. (1985) Shehata et al. (1985) Shehata et al. (1985) Green beans Blanched Canned Canned Canned Fresh from store Freshly cooked Frozen Microwave heating 15.4 0.5 2.5 0.4 8.1 2.7 3.8 5.0 0.4 Kidney beans, canned 5.3 3.4 Weightman et al. (1994) 3.8 0.8 Weightman et al. (1994) Lima beans, canned Mung beans 2.4 2.6 2.1 1.4 4.4 10.0–18.0 Vazquez-Blanco et al. (1995) Margareta et al. (1994) Ross et al. (1985) Margareta et al. (1994) Weightman et al. (1994) Ross et al. (1985) Ross et al. (1985) Ross et al. (1985) Margareta et al. (1994) Goldberg et al. (1986) Navy bean, dried cooked 7.8 1.2 Weightman et al. (1994) Pinto beans Canned Dried, then cooked Dried raw 4.5 7.5 8.2 3.3 2.7 1.6 Weightman et al. (1994) Weightman et al. (1994) Weightman et al. (1994) Red kidney beans 12.0 Moscoco et al. (1984) Runner beans 14.0 Selvendran and King (1989) White beans Canned Dried, then cooked Dried raw Lentils 6.3 5.3 4.5 1.4 1.0 1.0 Weightman et al. (1994) Weightman et al. (1994) Weightman et al. (1994) 17.7–18.1 1.2–1.7 Bhatty (1990) 1.2–4.8 1.7 1.3 1.2–3.0 3.1 2.1 Vidal-Valverde et al. (1992a) Weightman et al. (1994) Weightman et al. (1994) Black-eyed peas, canned 1.2 2.2 Weightman et al. (1994) Green peas Blanched Canned Canned Microwave heating 3.7 0.1 3.0 0.1 0.9 0.6 Theander (1995) Margareta et al. (1994) Weightman et al. (1994) Margareta et al. (1994) Margareta et al. (1994) Lentils Dried, then cooked Dried raw Peas 0.9 Pea hulls 15.0 Weightman et al. (1994) Soybean okara 22.0 Yamaguchi et al. (1996a) Soybean okara 4.6 Yamaguchi et al. (1996b) 30 P. Kadlec et al. form oil–water emulsions and act as protective colloids. Triterpenoid aglycones contain glucuronic acid in place of the sugar moiety and have a bitter taste. Saponins cause growth depression in poultry and pigs, bloat in ruminants. Some aglycone moieties increase the permeability of cell membranes and cause haemolysis by destroying the membranes of red blood cells, releasing haemoglobin into the bloodstream. The haemolytic activity of saponins varies between plant species. Another property of saponins is their toxicity to fish and lower forms of life, because of their capacity to bind with cholesterol and their antibiotic activity. Along with the above properties, which are common to all of the triterpene glycosides (saponins), each of them possesses specific pharmaceutical properties (Turova and Gladkych, 1964; Hiller et al., 1966; Woitke et al., 1970a,b; Vecherko et al., 1973). Triterpene aglycones can be subdivided into two groups, according to their structure, either with peptocyclic or tetracyclic carbohydrate skeletons. The first group includes aglycones based on carbohydrates such as oleonon, ursan, lupan and honone, and the second group is based on dommoran, epostan and holostan (Fenwick et al., 1991; Tsukamoto et al., 1993). The saponin contents of 13 types of legume seeds are shown in Table 2.3. Saponins are expressed as a weight percentage of the defatted flour, assuming that the aglycone : carbohydrate ratio is equal to 1.0. This can only be an approximation, however. For example, in soybean the five reported saponins (I, II, III, A1 and A2) have aglycone : carbohydrate ratios of 0.9 : 1, 1 : 1, 1.3 : 1, 0.6 : 1 and 0.7 : 1, respectively, while their relative composition in the saponine mixture has been reported to be 60, 6, 1, 30 and 3%, respectively (Kitagawa et al., 1984a,b). Other workers have reported more complex saponins in P. vulgaris (Chirva et al., 1970), which will further reduce this ratio. Table 2.3. Saponin content of legume seeds. Species Latin name Butter bean Chick pea Field bean Green pea Haricot bean Kidney bean Lentil Mung bean Peanut Runner bean Soybean Yellow split pea Phaseolus lunatus Cicer arietinum Vicia faba Pisum sativum Phaseolus vulgaris Phaseolus vulgaris Lens culinaris Phaseolus aureus Arachis hypogaea Phaseolus coccineus Glycine max Pisum sativum Saponin content (g kg-1) 1.0 2.3 0.1 1.8 2.3 3.5 1.1 0.5 < 0.1< 3.4 6.5 1.1 Carbohydrate Chemistry 31 2.2 Chemical Analysis of the Carbohydrates 2.2.1 Soluble carbohydrates (monosaccharides, sucrose, a-galactosides, cyclitols) Standards Accurate and precise chemical analysis demands a comparison with the known purified compound. Fortunately some of these compounds are available commercially at a high purification, around 99% in some cases. Sugar standards readily available from the major suppliers of laboratory chemicals include: D(−)fructose (cat. no. F2543), D(+)galactose (G6404), D(+)glucose (G7528), maltose (M5885), sucrose (S7903), raffinose (R0250), and stachyose (S4001) (Sigma-Aldrich, St Louis, Missouri, USA). Verbascose (O-VER) is available from Megazyme International Ireland Ltd. (www.megazyme.com). Phenyl α-D-glucoside (P6626), D(+) melezitose (M5375) or D(+) lactose (L1768) are suitable for use as internal standards, since they are unlikely ever to be found in legume seeds in any significant quantity. Some of the more ‘exotic’ carbohydrates cannot be purchased and will have to be prepared by each laboratory from biological sources. Among the commonly found cyclitols and methylcyclitols in legume seeds, only two are commercially available: myo-inositol, and D-pinitol. The others described in Section 2.1.3 are unavailable. Details of procedures for the isolation and purification of cyclitols are published in Schweizer et al. (1978) and Binder and Haddon (1984). Table 2.4 summarizes suitable plant sources for extracting and isolating cyclitols and galactocyclitols. Extraction of soluble carbohydrates from seeds SAMPLE PREPARATION For low molecular weight sugars, water is the optimal extraction solvent. Unfortunately, it is also an excellent solvent for interfering hydrophilic components such as polysaccharides, proteins, etc. In addition, α-amylases and α-galactosides, present in the plant material, may degrade starch and the raffinose oligosaccharides if not inactivated during, or prior, to extraction. These problems are minimized by extraction in aqueous alcohols. Alcohol type and concentration, extraction temperature and procedure vary considerably among the methods described: 80% ethanol or methanol (v/v) is most commonly used, but there are indications that in some cases these solvents lead to incomplete extraction. Increasing the alcohol concentration above 80% (v/v) has been shown greatly to reduce the amount of RFO extracted from plant material (Cegla and Bell, 1977; Shukla, 1996; Bach Knudsen and Li, 1991). Furthermore, marginally higher extraction yields have been noted with methanol compared with ethanol (Shukla, 1996). This is in contrast to other studies showing no difference between 80% methanol and water in 32 Table 2.4. Sources of standard of cyclitols, methyl cyclitols and their galactosides. Source Reference D-Pinitol Soybean seed (Glycine max) Lucerne leaves (Medicago sativa) Mung bean (Vigna radiata) Redwood (Sequoia sempervirens) Ginkgo biloba Chemical isomerization of myo-inositol 1. Chemical isomerization of myo-inositol 2. Buckwheat (Fagopyrum esculentum) 3. Demethylation of D-pinitol 1. Cucumber leaves (Cucumis sativus) 2. Common bugle (Ajuga reptans) 3. Castor bean seeds (Riccinus communis) 4. Jojoba beans (Simmondsia chinensis) Adzuki bean (Vigna angularis) Soybean seeds (Glycine max) Soybean seeds (Glycine max) 1. Soybean seeds (Glycine max) 2. Buckwheat (Fagopyrum esculentum) 3. Jojoba beans (Simmondsia chinensis) 1. Chickpea (Cicer arietinum), lentil (Lens culinaris) 2. Lentil (Lens culinaris) Chickpea (Cicer arietinum) Seeds of Mimosa scabrella Schweizer et al. (1978) Binder and Haddon (1984) Binder and Haddon (1984) Binder and Haddon (1984) Binder and Haddon (1984) Sasaki et al. (1988) 1. Sasaki et al. (1988) 2. Horbowicz et al. (1998) 3. Binder and Haddon (1984) 1. Pharr et al. (1987) 2. Bachmann (1993) 3. Kuo (1992) 4. Ogawa et al. (1997) Yasui (1980) Schweizer and Horman (1981) Schweizer and Horman (1981) 1. Schweizer et al. (1978); Schweizer and Horman (1981) 2. Horbowicz et al. (1998) 3. Ogawa et al. (1997) 1. Quemener and Brillouet (1983) 2. Bernabé et al. (1993) Nicolas et al. (1984) Ganter et al. (1991) D-Ononitol O-Methyl-scyllo-inositol O-Methyl-muco-inositol Sequoyitol scyllo-Inositol chiro-Inositol Galactinol Galacto-ononitol Galactopinitol A Galactopinitol B Fagopyritol B1 Ciceritol Galacto-ciceritol Mimositol P. Kadlec et al. Common name Carbohydrate Chemistry 33 the extraction of low molecular weight sugars (Li and Schuhmann, 1980; Li et al., 1985). It has been found that water extraction at 60°C and boiling in aqueous ethanol (80%, v/v) gave comparable results. Muzquiz et al. (1992) used two methods for the extraction of carbohydrates. Firstly, extraction in 60% methanol at boiling temperature under reflux for 2 h, and secondly, homogenization with 70% methanol for 1 min at room temperature. Both methods gave satisfactory recoveries, but higher amounts were recovered using the second method (Table 2.5). Kvasnidka et al. (1996) compared the extraction efficiency of the RFO from different varieties of pea, using sonication (80% ethanol (v/v), for 30 min) and boiling (80% ethanol v/v, for 30, 60, 120 min under reflux) and found that the latter technique was twice as efficient compared with Table 2.5. Recovery of raffinose family of oligosaccharides (RFOs) using different extraction methods (Muzquiz et al., 1992). RFO Amount added (mg) Total amount found (mg) Recovery (%) 0.00 20.21 40.08 80.23 0.00 20.14 40.25 80.20 0.00 20.07 40.06 80.21 29.29 46.15 69.29 101.43 21.64 37.15 56.43 98.22 20.36 37.86 57.15 87.72 – 83.42 99.80 89.84 – 77.01 86.43 95.49 – 87.19 91.54 83.98 Method II (in 500 mg of flour) Sucrose 0.00 5.21 10.30 20.18 Raffinose 0.00 5.22 10.19 20.36 Stachyose 0.00 5.04 10.19 20.09 8.10 13.70 18.26 27.32 2.48 7.30 11.36 20.38 27.38 34.06 37.46 45.52 – 107.49 98.64 95.24 Method I (in 2 g of flour) Sucrose Raffinose Stachyose 92.34 87.14 87.92 – 132.54 98.92 90.29 Method I – extraction in 60% methanol under reflux for 2 h. Method II – homogenization with 70% methanol for 1 min at ambient temperature. 34 P. Kadlec et al. sonication and that the optimum extraction time for the boiling method was 60 min. Johansen et al. (1996) studied the effect of extraction solvents and temperature on the extraction yields of monosaccharides, sucrose and RFO from toasted soybean meal, cottonseed meal, field peas and a feed mixture. They found that extraction in 80% (v/v) alcohol was strongly influenced by the extraction temperature and that the maximum extraction was only achieved at the boiling point. Extraction in water and 50% (v/v) methanol or ethanol was less heat sensitive and gave comparable results. It has also been shown that aqueous ethanol 50% (v/v) was as effective as 50% (v/v) methanol, whereas lower yields were observed at higher alcohol percentages. There was no consistent difference in the extraction yield when comparing reflux with constant stirring and water bath with occasional mixing, for any of the extraction solvents used. Comparison of five methods for extraction of oligosaccharides from soybean and cottonseeds has been described by Bach Knudsen and Li (1991). In light of the above results, it can be concluded that the extraction procedure for the analysis of soluble carbohydrates is always going to be a compromise between the optimal extraction of a group of different compounds, the level of recovery and the possible interaction of other non-carbohydrate components present in the seed. The following method can be applied to whole seed, or seed with the seed coat (testa) removed. This can be arrived at on individual seeds by picking off the seed coat with a sharp dissecting needle (Jones et al., 1995), or for larger samples by using a small-scale industrial de-huller. For a single seed, or part of a single seed, the seed has to be first finely ground to produce flour with a mean particle size that will pass through a 75-µm test sieve (Jones et al., 1995). Extraction of soluble carbohydrates from legume seeds (0.1–0.3 g of flour in 5 ml of 50% v/v ethanol or methanol, containing either phenyl α-D-glucoside or D(+) melezitose at 0.1 mg ml−1 as an internal standard), can be performed either at 50°C with constant stirring for 1 h under reflux, or in an ultrasonic bath at ambient temperature for 60 min. After this time the mixture is centrifuged at 6000 rpm for 20 min and the residue re-extracted as before and washed with deionized water until the Molisch reaction test is negative (Pearson, 1976). In practice only three or four cycles are needed to extract all the available carbohydrate from the sample (Jones, 1999). Combined supernatants are then heated at 80°C for 20 min to inactivate endogenous enzymes and centrifuged at 6000 rpm for 20 min. The supernatant is evaporated to dryness in a rotary vacuum evaporator at 40°C. The residue is dissolved in 4.0 ml of pure water and stored at 4°C. RECOMMENDED EXTRACTION PROCEDURE Carbohydrate Chemistry 35 High performance gas chromatography (GC) Since its inception some 40 years ago GC has become a highly sophisticated and sensitive analytical tool, with new developments and technologies being continuously introduced (Bartle, 1993). Carbohydrate analysis lends itself well to this technique. The advent of capillary columns, reliable temperature and gas flow control arguably makes gas chromatography the analytical method of choice for sugar analysis (Tipler, 1993). GC determination of carbohydrates, cyclitols and polyols is possible after their conversion into volatile derivatives. In general the most useful method of polyol derivatization is trimethylsilylation (TMS; Bierman, 1988). Reducing sugars (fructose, glucose, galactose, mannose and others) produce multiple peaks on gas chromatograms because they occur in their anomeric forms. Simultaneous determination of such sugars and cyclitols, therefore, can be difficult because retention times of TMS derivatives of both classes of carbohydrate are similar. Phenyl α-D-glucoside is commonly used as an internal standard. The following procedure according to Horbowicz and Obendorf (1994) is suitable for the determination of many carbohydrate classes (glucose, fructose, cyclitols, sucrose, mono-, di-, tri-galactosides of cyclitols, raffinose, stachyose and verbascose). Seed tissues are twice homogenized in a mortar with a solution of ethanol : water (1 : 1 v/v) containing phenyl α-D-glucoside (normal working concentration is between 50 and 100 mg l−1) as internal standard. The homogenate is heated at 80°C for 45 min in a microfuge tube and then centrifuged. The residue is re-extracted and the combined supernatants are passed through a 10,000 MWCO (molecular weight cut-off) filter. Aliquots of the filtrate are transferred to reaction vials and evaporated to dryness in a stream of nitrogen. The residues are left overnight (16 h) in a desiccator over phosphorus pentoxide to remove traces of water. The dry residues are derivatized with trimethylsilylimidazole : pyridine (Sigma cat. nos. T7510, P4036, 1 : 1, v/v) and analysed by high resolution GC. Alternatively, the drying stage can be omitted when using Tri-Sil®Z (Pierce Chemical Co.), since this derivatizing reagent will work in the presence of water. There are many different manufacturers and models of GC to choose from. Even the most basic can be configured to analyse TMS sugar derivatives. Horbowicz and Obendorf (1994) used a Hewlett Packard 5890 Series II gas chromatograph equipped with a flame ionization detector and a Hewlett Packard 3396A integrator. A DB-1 capillary column (15 m length, 0.25 mm ID and 0.25 µm film thickness; J & W Scientific, Folsom, California, USA) operated with a programmed initial temperature of 150°C, adjusted to 200°C at 3°C min−1, adjusted to 325°C at 7°C min−1, and then held at 325°C for 20 min. The injector port was operated at 335°C and the detector at 350°C. The carrier gas was helium at 3.0 ml min−1, RECOMMENDED GC METHOD FOR QUANTIFYING SOLUBLE CARBOHYDRATES 36 P. Kadlec et al. split 1 : 50. The detector gas was hydrogen at 30 ml min−1 and air at 300 ml min−1. It should be noted that these operating conditions can be transferred to other systems and used as a starting point for optimizing the GC conditions. J & W Scientific are now able to supply high temperature versions (DB-1ht) of the column used above. The advantages of this new technology is that high boiling point TMS derivatives (e.g. TMS-verbascose) will elute faster and produce sharper peaks, when hydrogen is used as the carrier gas and it is possible to complete a GC analysis in 20 min! A typical chromatogram using the above system is shown in Fig. 2.11 (from D.A. Jones, personal communication). Many analyses of cyclitols, galactocyclitols and other carbohydrate contents in seeds of several Leguminosae species (and other species) have been performed using this procedure (Horbowicz and Obendorf, 1994; Horbowicz et al., 1995; Górecki et al., 1996; Horbowicz et al., 1998). Fig. 2.11. A chromatogram of trimethylsilylation (TMS) sugar derivatives extracted from a single round (RR RbRb) pea. Carbohydrate Chemistry 37 Quantities of soluble carbohydrates can be determined by extrapolation from standard curves; the ratios of the area of peaks for each known component to the area of the internal standard peak, phenyl α-D-glucoside, are plotted against known amounts of each component. Amounts of unknown carbohydrates are estimated by calculation using the nearest known standard. Amounts below the level of detection are presented as zero. Alternatively the peak area normalization method can be used. This method assumes that the relative response factor for each component is the same or very nearly the same as that of the internal standard. From the peak area data obtained it is possible to calculate the amount (as a percentage of dry matter) of each component detected and the total amount of the soluble carbohydrates in the sample analysed. For individual components: CALCULATION AND STATISTICAL ANALYSIS Concentration of component A = Area A × 100% Total area ( A + B + C + D ) (Note that the area of the internal standard (IS) is not included in the total area sum) Internal standard concentration = Area IS × 100% Total area (A + B + C + D ) (Note that the area of the IS is not included in the total area sum) Total amount of components in sample (as % of dry matter) = Amount of IS in extraction medium (mg) ÷ Concentration of IS Sample weight (mg) ×100% For further explanation see Burchfield and Storrs (1962) and Chapman (1985). Samples would normally be analysed in triplicate. ADVANTAGES AND DISADVANTAGES OF GC METHODS Advantages: 1. Analytical stability: GC capillary columns can remain stable over many years even with intensive use, thousands of samples can be run without changes in the column resolution. 2. High resolution: it is possible to detect all the soluble carbohydrate components found in legume seeds from one injection. 3. High sensitivity: analyses are possible with 5–10 mg of plant material, sensitivity can be increased by using a mass selective detector. 4. The time consuming clean-up step can be omitted, at the expense of column life. 5. It is possible to separate D and L optical isomers of compounds using special columns. 38 P. Kadlec et al. 6. A wide range of stationary phases are available for columns to customize a method for a particular class of soluble carbohydrate. 7. GC equipment is more common in laboratories and tends to be cheaper to purchase. Disadvantages: 1. Preparing the TMS derivatization of -OH groups can be difficult, but is crucial for successful results. 2. The chemicals involved are toxic, hazardous and expensive and must be disposed of safely. 3. The multiple peaks produced by TMS derivatives of reducing sugars can interfere with peaks of free and methylated cyclitols. 4. A supply of compressed gases is needed. The gas has to be clean, dry and pure to produce consistent results. 5. An analytical run can sometimes take 1 h to complete, although shorter run times are possible. High performance liquid chromatography (HPLC) The water extract of soluble carbohydrates from biological material, (containing an internal standard) should be cleaned before HPLC analysis to improve reliability and resolution, using one of the following procedures. SAMPLE CLEAN-UP 1. The extract is filtered through a Sep-Pak C18 cartridge, pre-wetted with methanol and pure water. The effect of sample pre-treatment cartridges on the carbohydrate analytes themselves should be predetermined using standard solutions. (It may be found that some carbohydrates have a strong affinity for particular cartridge packing materials. This is obviously important if dealing with low levels of carbohydrate in the sample.) The eluate is collected and further filtered through a 0.45-µm PTFE filter to remove particulate matter. Soluble protein and polysaccharides are precipitated by adding an equal volume of absolute ethanol and centrifuged. The clear supernatant is dried at 50°C under nitrogen and finally redissolved in 0.015 M, Na2SO4. The standard sugar solutions are formulated to simulate concentrations in the material under study and are subjected to the same clean-up procedure as used for the sample (Johansen et al., 1996; Frias et al., 1996b). 2. The carbohydrate extract is filtered through a column containing Dowex 50 WX8 (H+ form, 200–400 mesh) and Dowex 1X8 (Cl− form, 100–200 mesh). The sugar fraction is eluted with deionized water. The eluate is filtered through a JSO-DISC N-252 nylon membrane, 0.2 µm pore size (Muzquiz et al., 1992; Górecki et al., 1997). 3. The dry extract is dissolved in double-deionized water, vortexed and passed through a DEAE cellulose minicolumn, equilibrated and later Carbohydrate Chemistry 39 eluted with deionized water to remove anionic substances from the sample. The eluant is filtered through a Uniflo membrane prior to HPLC analysis (Kuo et al., 1988). Supelcosil LC-NH2 column 250 × 4.6 mm (Supelco Inc., Sigma, St Louis, MO, USA), acetonitrile–water eluent (75 : 25 v/v), RID-6A refractive index detector (Shimadzu, Kyoto, Japan; www.shimadzu. com) (Górecki et al., 1997). Spherisorb-5-NH2 column (250 × 4.6 mm) (Teknokroma, Bellefonte, Pennsylvania, USA), acetonitrile–water eluent (72 : 28 and 65 : 35 v/v), flow rate 1 ml min−1, an ERMA 7520 RID (Barcelona, Spain) (Muzquiz et al., 1992). HPLC (NORMAL PHASE) µ-Bondapack/Carbohydrate column (300 × 3.9 mm) (Waters Associates) with a precolumn (4.0 cm × 3.2 mm) packed with C18 Porasil B, acetonitrile–water eluent (75 : 25 and 85 : 15 v/v), flow rate 2 and 3 ml min−1, respectively, temperature 35°C, model 132 optical reflection type differential RID (Gilson Associates) (Vidal-Valverde et al., 1992a, 1993a; Vidal-Valverde and Frias, 1992; Frias et al., 1994a). Or, an analytical column 250 × 4 mm Separon SGX RPS 7 µm or Separon SGX C18 5 µm with guard columns (Separon RPS or Separon C18) at ambient temperature, deionized water eluent, flow rate 1 or 0.7 ml min−1, respectively, RID (Kvasnidka et al., 1996). RP-HPLC (REVERSE PHASE) Shodex Ionpak KS-801 resin-based column in sodium form (Waters, Milford, Massachusetts, USA), deionized water eluent, flow rate 0.6 ml min−1, temperature 85°C, RID (Johansen et al., 1996). Aminex HPX-87N (300 × 7.8 mm) resin-based column in the sodium form (Bio-Rad, Richmond, California, USA), eluent 0. 015 M, Na2SO4, flow rate 0.5 ml min−1, temperature 85°C, a model 156 RID (Bach Knudsen and Li, 1991). Column 250 × 8 mm filled with strong cation exchanger OSTION LG KS 0803, 17–20 µm in Ca2+ form fitted with desalting guard columns, temperature 80°C, eluent demineralized water, flow rate 0.4 ml min−1, RID (Kvasnidka et al., 1996). IMP-HPLC (ION MODERATE PARTITION) HPAC-PAD (HIGH PERFORMANCE ANION CHROMATOGRAPHY WITH PULSED AMPEROMETRIC DETECTION, DIONEX SYSTEM) CarboPak PA-100 pellicular anion exchange resin column 250 × 4.0 mm with a CarboPak PA-100 guard column 25 × 3 mm (Dionex Corporation, Sunnyvale, California, USA), flow rate 1 ml min−1, at ambient temperature. The mobile phase: 145 mM sodium hydroxide solution, prepared with deionized water and fresh 50% NaOH solution (cat. no. 19154, BDH, Merck Ltd, Poole, UK), a Model PAD-II detector equipped with a solvent-compatible electrode (Frias et al., 1996; Kvasnidka et al., 1996). 40 P. Kadlec et al. AC-HPLC (AFFINITY CHROMATOGRAPHY) Sugar-Pack 1 column (Waters Associates Milford, Massachusetts, USA) at 90°C connected with a guard column (cation cartridge, Pierce Chemical Company, Rockford, Illinois, USA). The elution is monitored by a Waters Model 401 refractometer, mobile phase – 0.1 mM CaNa2/EDTA/H2O, flow rate 0.5 ml min−1 (Kuo et al., 1988). Concentration of sugars is calculated from the peak height of detector response as: CALCULATION AND STATISTICAL ANALYSIS Content of sugars (% dry matter) = H s R sW IS × 100 H IS R ISW s where Hs and HIS are peak heights or areas, and Ws and WIS are dry weights of sample (s) and internal standard (IS), respectively. Rs and RIS are response factors (amount/height) for sugar and internal standard in solution containing a known amount of each component (Bach Knutsen and Li, 1991; Johansen et al., 1996). For each sample, data are analysed using a two-way analysis of variance model (Snedecor and Cochran, 1973): Xijk = µ + αi + βj + (αβ)ij + εijk where Xijk is a dependent variable (i.e. content of sugar); µ an overall mean; αi the effect of extraction medium; βj the effect of temperature or extraction procedure and εijk a random variable. According to Johansen et al. (1996) the detector response of raffinose and stachyose are linear in the range 0.05–9.0 mg ml−1 extract, corresponding to an injected amount of 1–180 µg. The correlation coefficients (r) of detector response versus concentration are 0.9991 and 0.9940 for raffinose and stachyose respectively, both when calibrated on basis of height and area. In the range tested (0.06–6.0 mg ml−1), verbascose gave a linear response (r = 0.9997). RECOMMENDED HPLC METHODS OF QUANTIFYING SOLUBLE CARBOHYDRATES routine analysis of individual RFOs: • • • • • For HPLC (Muzquiz et al., 1992; Górecki et al., 1997); Dionex HPAC-PAD (Anonymous, 1994; Frias et al., 1994; 1996a,b); IMP-HPLC (Bach Knudsen and Li, 1991; Johansen et al., 1996; Kvasnidka et al., 1996); RP-HPLC (Vidal-Valverde and Frias, 1992; Vidal-Valverde et al., 1993a; Frias et al., 1994a); AC-HPLC (Kuo et al., 1988). Rapid (10 min) method of determination of total RFOs: • RP-HPLC using the method of Kvasnidka et al. (1996), see section on IMP-HPLC (pp. 39). Carbohydrate Chemistry 41 ADVANTAGES AND DISADVANTAGES OF HPLC METHODS Advantages: 1. The water-based extract can be analysed directly, without any chemical derivatization. 2. The analysis can be conducted relatively quickly. 3. Pure compounds can be recovered from the sample, using a fraction collector, if required. 4. Usually a single peak is detected from each component. 5. The chemicals used (in extraction and sample clean-up) are relatively cheap and non-toxic. Disadvantages: 1. Clean-up of extracts can be sometimes time consuming, involving several chemicals and extra disposable equipment, such as filter discs. 2. There is no one method that will analyse all of the soluble carbohydrates normally found in legume seeds. 3. HPLC columns are expensive and have a relatively short life time. 4. HPLC column properties slowly change with time. 5. HPLC equipment is expensive, especially for the gradient elution system. 6. An analytical run can sometimes take 1 h to complete, although shorter run times are possible. Bach Knudsen and Li (1991) determined mean values (percentage of dry weight) for the RFO in protein-rich feedstuffs using both HPLC and GC methods. The regression equations and the standard error of slope (±) for the sugar determinations by HPLC (X) and GC (Y) for these studies were:COMPARISON BETWEEN GC AND HPLC sucrose raffinose stachyose total Y = 0.094 + 0.985 X ± 0.017 Y = −0.097 + 1.015 X ± 0.025 Y = −0.355 + 1.034 X ± 0.025 Y = −0.262 + 1.006 X ± 0.026 R 2 = 0.995 R 2 = 0.989 R 2 = 0.990 R 2 = 0.988 It was apparent that for sucrose and raffinose there was an excellent agreement between the two methods, while the value for stachyose obtained with the GC method was on average 0.4% (absolute units) lower than with HPLC. This difference is almost within the analytical error for this type of analysis. The coefficient of variation, for the GC method ranged from 1.5 to 5.0% and for the HPLC method from 0.6 to 1.3% for sucrose and stachyose, respectively. Compared with literature values the analytical precision was quite acceptable. For a GC method this was reported as 1.3–3.9% (Sosulki et al., 1982; Molnar-Perl et al., 1984) and for HPLC methods as 3.5–10.5% and 1.5–24.0% (Kuo et al., 1988). The coefficient of variation found for soybean using the HPLC method (Bach Knudsen and 42 P. Kadlec et al. Li, 1991) is comparable to the values found by other groups using GC (Sosulki et al., 1982; Molnar-Perl et al., 1984) and HPLC (Kuo et al., 1988). Bach Knudsen and Li (1991) have also shown that the injection port temperature (300–325°C) and overlapping between peaks, in the case of HPLC, had no effect on the lower GC value obtained for stachyose. In the opinion of these researchers, the lower value for stachyose and the fact that analytical precision of the GC method is lower indicates that HPLC should be the technique of choice for this type of feed material. Thin layer chromatography (TLC) Thin layer chromatography is based on separation of carbohydrates on paper chromatography or silica gel. The separated zones can be eluted with water and the soluble carbohydrate components quantified by a colorimetric or densitometric method. Standard curves should be constructed from eluates obtained after similar analysis using known concentrations of the marker carbohydrates (Pearson, 1976). For separation of the RFO the following mobile phases are used: isopropanol– ethyl acetate–water (5 : 2 : 3); n-butanol–acetic acid–water (5 : 2 : 1); chloroform–methanol–water (6.5 : 3.5 : 1); isopropanol–25% ammonia– water (7 : 1 : 2). The RFO are visualized by spraying with 80 mg naphthoresorcinol in 40 ml ethanol containing 0.8 ml concentrated sulphuric acid (Jones et al., 1999b). For preparative separation of the RFO, the PSC Fertigplatten Kieselgel 60 F254, (20 × 20 × 0.2 cm plates, cat. no. 5717, Merck Ltd, Poole, UK) is used (Stahl, 1969; Pearson, 1976; Jones et al., 1999b). RECOMMENDED TLC METHODS FOR QUANTIFYING SOLUBLE CARBOHYDRATES TLC can be a useful method for the initial and rapid screening of material for soluble carbohydrate content before a more detailed study is undertaken, as demonstrated by Jones et al. (1999b). ADVANTAGES OF TLC METHOD Capillary zone electrophoresis (CZE) The determination of soluble carbohydrates by capillary zone electrophoresis is based on the separation of borate complexes of the saccharides in an electric field. Arentoft et al. (1993) have shown that increased borate concentration (20–100 mM Na2B4O7) and pH favour the complex formation, which improves the UV absorption at 195 nm. Increased electrophoretic mobility of the compounds result in improved separation and longer migration times. The running conditions that were found to provide the best compromise between acceptable separation, detection efficiency and duration of analysis were 100 mM Na2B4O7, pH 9.9, 50°C, 10 kV and omission of 2-propanol modifier. Carbohydrate Chemistry 43 The following description and specification of two CZE systems is fairly representative of the technique. RECOMMENDED CZE METHODS FOR QUANTIFYING SOLUBLE CARBOHYDRATES 1. P/ACE 2210 Series capillary electrophoresis system (Beckman Instruments (UK) Ltd, High Wycombe, UK), 500 mm × 50 µm I.D. fused-silica capillary, the injection time is 4 s, the detector window (432 mm) from the injection end (anode). On-column UV detection is performed at 200 nm and a rise time of 2.0 s. The electrophoresis is conducted at 50°C and at a field strength of 10 kV. The buffer details are disodium tetraborate at a concentration of 100 mM in pure water, adjusted to pH 9.9 (Frias et al., 1996a,b). 2. Capillary electrophoresis instrument ABI Model 270 A-HT (Applied Biosystems, Warrington, UK), the fused silica capillary is 720 mm × 50 µm I.D. × 360 µm OD, including coating material. The injection time is 2.0 s, the detector rise time 0.5 s. The detector window is 500 mm from injection end (anode). On-column detection is performed at 195 nm, operating conditions; temperature (30–60°C), field strength voltage 10–20 kV, with a concentration of 2-propanol modifier 0–15%, v/v (Arentoft et al., 1993). The quantification of each sugar is accomplished by plotting the normalized peak areas obtained from the sample, against those obtained from the standard solutions. Lactose is used as a reference peak with computer software normalizing the times during subsequent runs to allow for migration time variation. Relative response factors are calculated by dividing the slope of the calibration graph for lactose by the corresponding slope for individual analytes. CALCULATION AND STATISTICAL ANALYSIS ADVANTAGES AND DISADVANTAGES OF CZE COMPARED WITH GC AND HPLC Advantages: 1. 2. 3. 4. The method is easier to use and set up. The equipment is less expensive as HPLC or GC equipment. Uses non-toxic chemicals. Can produce faster analysis times. Disadvantages: 1. CZE is less sensitive than HPLC or GC, requiring concentrations of micrograms per millilitre of sugars for detection. The HPAC-PAD is more sensitive, detecting concentrations at the nanogram per millilitre level, high performance GC with FID being the most sensitive of all, working at the picograms per millilitre level. 2. Sample preparation requires a purification step using cation/anion exchange in the form of Sep-Pak C18 cartridges, which adds time and cost to the analysis. 44 P. Kadlec et al. Comparison of results of the CZE method with those obtained by anion exchange HPLC coupled to a triple-pulsed amperometric detection (HPAC-PAD) showed a high degree of precision and reproducibility for the RFO compositions of a number of pea strains. No statistically significant differences (P ≥ 0.05) were found between the two analytical techniques using paired Student t-tests (Frias et al., 1996a,b). Other analytical methods When sugars or their derivatives are reasonably pure and, in particular, free of optically active impurities, the measurement of the optical rotation can provide a simple method for their identification and analysis (Pearson, 1976). One of the most important sugar mixtures that can be analysed by this method is sucrose and its products of hydrolysis, fructose and glucose. The method is based on the rotation of plane-polarized light using a polarimeter or saccharimeter to measure the change in the angle of polarized light. This method, dating from the 1840s, is not widely used today, not least because large volumes of the samples to be tested are needed. Also, this technique is not very sensitive at low concentrations of sugars. OPTICAL ROTATION There are several well known tests which make use of the reducing action of sugars in alkaline solutions in the presence of certain metallic salts, e.g. copper, silver, mercury and bismuth. Those of copper have been employed by far the most extensively in sugar analysis (Pearson, 1976). The basic form of the reaction is: REDUCING SUGAR METHOD RCHO + Ag2O → 2 Ag + RCOOH RCHO + 2 CuO → Cu2O + RCOOH Unfortunately, for quantitative work these reactions do not proceed stoichiometrically. This is because of the ability for many sugars to mutarotate. This causes the carbon chain eventually to break and the free aldehyde and ketone groups are lost. Close control of the reaction conditions is needed along with calibration using standards. Fehling originally devised the most widely known test based on this method, in 1848. There are various diagnostic kits available for detecting fructose, glucose or sucrose in food substances, and so would be suitable for legume seed flour samples (Sigma-Aldrich Company and Boehringer Mannheim GmbH, Mannheim, Germany). These tests make use of the reducing capacity of the carbonyl group present (or not present) in these sugars. Diagnostic test kits work quite well but are generally expensive on a per sample basis. The Boehringer Mannheim glucose test kit, cat. no. 124036, using the GOD-Perid method, forms the basis of a reliable starch assay (described in a later section). See MEDICAL/FOOD DIAGNOSTIC TEST KITS Carbohydrate Chemistry 45 the Official Methods of Analysis of the Association of Official Analytical Chemists (AOAC, 1984) and American Association of Cereal Chemists (AACC, 1995) for more detailed information. The enzymatic method of RFO determination involves incubation with α-galactosidase followed by measuring the liberated D-galactose, using either a chemical method or D-galactose dehydrogenase. For determining reducing carbohydrates, a useful colorimetric method can be used (Honda et al., 1982). Alternatively, the total oligosaccharide content can be expressed as sucrose units, which would be the end product of α-galactosidase (EC 3.2.1.22) action. Sucrose can be readily estimated in the same digest by first reacting with invertase followed by the colorimetric assay of glucose, using glucose oxidase reagent. However, the α-galactosidase method is not adequate for determining an individual oligosaccharide in a mixture containing other homologous sugars. ENZYMATIC METHOD The spectrophotometric method is one of the simplest ways of determining the total soluble sugars in biological material. In the case of oligosaccharides, the results are overestimated because the analyses include other sugars like mono- and disaccharides. According to the data of Muzquiz et al. (1999), the content of sucrose as a proportion of the total sugar in legume seeds is in the range from 7.1% (Lupinus luteus cv. Piast) to 53.0% (V. faba cv. Nadwiœlañski). For this reason, the spectrophotometric method is burdened with a large error and is not very useful, therefore, for the determination of α-galactosides. In connection with the TLC method, however, this method can be very useful for the RFO. The content of individual RFO components (after separation by means of TLC, see p. 42) can be determined by the spectrophotometric method described by Pearson (1976) and Fry (1994). SPECTROPHOTOMETRIC METHOD 2.2.2 Polysaccharides Starch Many different ways for the determination of starch have been described in the literature. In principle, two groups of methods can be distinguished. Firstly, there are the polarimetric methods in which the starch is quantified as a dissolved and partly degraded polymer (determination according to Ewers and the calcium chloride method of the AOAC, see below). In the second group of methods, the starch is fully hydrolysed into glucose and then quantified by measuring the glucose content. This is termed the Ewers method (AOAC method 14.031) and is based on acid hydrolysis of starch, followed by the measurement of the optical rotation of the resulting solution. POLARIMETRIC METHODS 46 P. Kadlec et al. Weigh 1 g of flour into a 50 ml flask, add 25 ml of 1.125% HCl. Cap and boil for 15 min in a water bath with stirring, add pure water to give a total volume of 40 ml and cool the flask to 20°C. Add 1 ml Carréz solution I (30% ZnSO4) to the solution. After stirring, add 1 ml Carréz solution II (15% K4[Fe(CN)6]) and adjust the volume to 50 ml. Filter the solution, and measure the optical rotation by polarimetry, using a 100 mm tube. Calculate the total starch content: Starch (%) = 50 × 34.66 × p [α ] × L × m where p is the measured value (°S), [α] the specific rotation power of pea starch (187.7°S), L the length of the tube (dm) and m the weight of sample (g). The flour under test (2–2.5 g) is passed through a 150-µm mesh sieve and defatted with diethyl ether, 10 ml of 65% ethanol (v/v) is then added, the mixture is centrifuged and the supernatant discarded. The residue is taken up in 10 ml of pure water, transferred to a 500 ml flask and mixed with 60 ml of a 33% (w/w) solution of CaCl2 containing 2 ml of 0.8% acetic acid. The mixture is then cooled, transferred to a 100-ml flask and made up to 100 ml with CaCl2 solution. After filtration, the optical rotation of the resulting solution is determined by polarimetry, using a 100-mm tube. Duplicate determinations (each consisting of the mean of ten separate optical rotation measurements) must not differ by greater than 0.006 units. CALCIUM CHLORIDE METHOD Different protocols for sample preparation and starch solubilization have been described in the literature, e.g. autoclaving, treatment with hydrochloric acid in a boiling waterbath, treatment with dimethyl sulphoxide (DMSO) solution and treatment with DMSO/hydrochloric acid mixtures (Brunt et al., 1997; Jones et al., 1999a). For seeds, sample solubilization is best carried out using 90% DMSO. The conversion of starch into glucose can be performed by acid or enzymatic hydrolysis. For acid hydrolysis different concentrations of hydrochloric acid are used in a waterbath, for enzymatic hydrolysis, an enzymic mixture containing amyloglucosidase (EC 3.2.1.3) and α-amylase (EC 3.2.1.1) is used. The resulting glucose can be measured by either, titration, enzymatic determination (hexokinase, glucose oxidase) or HPLC determination. See AOAC method 920.40 and AACC methods 76–11. METHODS IN WHICH THE STARCH IS FULLY HYDROLYSED INTO GLUCOSE Because resistant starch (RS) has a reduced content of energy and is characterized by physiological effects that make it comparable to DF, it is logical that the question be asked whether or not it should be included in DF analytical figures (Lee and Prosky, DETERMINATION OF RESISTANT STARCH Carbohydrate Chemistry 47 1994). Since RS can be degraded to free glucose by acid hydrolysis, it can be easily mistaken for the glucan polymers associated with the plant cell wall in the chemical determination of DF (Wen et al., 1996). The ideal situation would be to determine DF and RS contents of foods independently (Sambucetti and Zuleta, 1996). Two main approaches for determination of resistant starch can be used in vivo and in vitro. In this chapter only in vitro methods are described. The in vitro analysis of RS implies the performance of an enzymatic hydrolysis (α-amylase in most cases), which is usually supposed to mimic the hydrolysis of starch by endogenous enzymes in the upper part of the digestive tract (mouth, stomach and small intestine). The first attempt to analyse RS was performed by Englyst et al. (1982). Their method was only able to analyse retrograded ‘enzyme resistant starch’ (R III). Indeed the grinding of the sample and the subsequent thermal treatment at 100°C made the quantification of the other two major types of RS, physically entrapped starch (RS I) and RS granules (RS II) impossible. Englyst later identified the fraction quantified by this method as retrograded amylose. The main modifications introduced by Berry (1986) and then by the collaborators of the EURESTA inter-laboratory study (Champ, 1992, 1995) concerned the elimination of the gelatinization step and of the pullulanase hydrolysis. Consequently, both RS III and RS II could be quantified using this new method. Independently, Englyst et al. (1992) developed a more sophisticated methodology set up to analyse rapidly digestible starch (RDS), slowly digestible starch (SDS) and RS. Minor modifications to the method of Berry (1986) were then proposed as described by Champ (1992) and Faisant et al. (1995). These modifications were undertaken to improve the slight underestimation of RS. One of the modifications is the use of sodium azide to prevent bacterial proliferation during amylase hydrolysis, and secondly to introduce a de-proteinization step with pepsin. Both proposed the elimination of the drying step before the solubilization with potassium hydroxide. There were also attempts to develop an in vitro RS assay by applying chewing as the initial disintegration step (Muir and O’Dea, 1992). This method was validated against in vivo studies in human ileostomates (Muir and O’Dea, 1993; Muir et al., 1995). The validation of the in vitro methods against the in vivo methods showed quite good reproducibility (Table 2.6). Two methods have been recommended for analysing RS in foods in vitro, one developed by Englyst et al. (1992) and one described by Champ (1992) and Faisant et al. (1995), which has been developed from the modified Berry method. After tests for validation in vitro methods against in vivo studies, the following conclusions were drawn (Champ, 1995; Asp, 1996). Firstly, the two methods give very similar values with a high level of RS. They both give an estimation of RS that does not take into account, however, the potentially digestible starch or starch fragments found in vivo at the end of intestine. Secondly, the modified Berry method is quicker and 48 P. Kadlec et al. Table 2.6. In vitro and in vivo quantification of resistant starch (RS) as a proportion of the total starch (TS). Quantification of resistant starch (% RS/TS) in vitro Origin of the RS Beans Raw green banana Retrograded high amylose corn starch aChamp in vivo Champ Englyst methoda methodb Ileostomyc Incubationd 17 61 30 – 71 34 – 68 – 17 84 49 (1992); bEnglyst et al. (1992); cGöteborg (1995); dFaisant et al. (1993, 1995). easier to reproduce than the Englyst method. Thirdly, the Englyst method may reflect better the in vivo physiology than the other methods. With all of the methods for RS analysis there is a fundamental problem related to the definition of RS. None of these methods, including the one developed by Englyst et al., takes into account the whole amount of RS defined as ‘starch and products of starch degradation not absorbed in the small intestine of healthy individuals’, since low molecular weight fragments, soluble in aqueous ethanol, are not determined. This method determines the total starch in a sample using a ‘kit’ sold by Megazyme International Ireland Ltd, Co. Wicklow, Ireland (www.megazyme.com). It is based on the principles described in AACC method 76–12 and is listed as AACC method 76–13. For samples that do not contain high levels of resistant starch (e.g. wheat flour), complete solubilization of starch is achieved by cooking the sample in the presence of thermostable α-amylase. Samples that contain high levels of RS (e.g. high-amylose maize) are completely solubilized by pre-treatment with DMSO at 100°C. Glucose produced by the enzymatic hydrolysis of the solubilized starch is measured using glucose oxidase/peroxidase reagent. Samples containing high levels of glucose or maltodextrins have to be washed with aqueous ethanol before analysis. MEGAZYME KIT Fibre fraction Some of the earliest analytical methods of DF include crude fibre, acid detergent fibre (ADF) and neutral detergent fibre (NDF) as the most commonly used methods. The principle of the crude fibre method implies boiling and extraction of the sample by dilute acid and dilute alkali, with subsequent isolation of the insoluble residue by filtration (AOAC methods 920.86, 962.09). The crude fibre method essentially determines the cellulose and lignin content, however, the recovery may vary markedly. Carbohydrate Chemistry 49 In the ADF method, boiling of a sample is performed for 1 h in an acidic solution containing the detergent cetyltrimethylammonium bromide (CTAB), and the residue is obtained by filtration (Van Soest, 1963). The ADF method aims at determining cellulose and lignin with higher precision than the crude fibre method, but remnants of hemicellulose and pectin have been reported (Asp and Johansson, 1984). The NDF method implies extraction of a sample for 1 h in a hot neutral solution, containing the detergent sodium dodecyl sulphate (SDS) and ethylenediamine tetraacetic acid (EDTA) (Van Soest and Wine, 1967). This milder treatment leaves most of the hemicellulose, in addition to cellulose and lignin in the residue, whereas pectins are efficiently removed. In starchy material, e.g. peas, filtration problems of the residue may occur due to residual starch. This problem may be solved by including a thermostable amylase in the NDF-reagent or by pre-incubation with amylase (Robertson and Horvath, 1993). Common for the detergent methods is the removal of detergent soluble components, some being part of the enlarged DF concept, including indigestible components other than the carbohydrate and lignin DF constituents (NSP and lignins). Current methodologies in DF determination comprise the enzymatic– gravimetric and the enzymatic–chemical procedures. The enzymatic– gravimetric methods are based on enzymatic degradation of polymeric material such as starch, proteins and other components, with subsequent isolation and weighing of the undegraded residue equal to DF. A number of procedures differing in types of enzyme, duration of incubation, pH of buffers, temperature and methods of DF isolation have been proposed. AOAC Official Enzymatic Gravimetric Methods comprise Methods 985.29, 991.42, 993.16, 993.19, 993.21 and 991.43. Common for the enzymatic– gravimetric methods is the general inclusion of minor levels of a wide range of indigestible components, in addition to NSP and lignins (see Section 2.2.3). DF values are thus usually corrected for the content of ash and protein in the residue, although the correction of, in particular, protein is questionable, as close association exists between proteins and polysaccharides/lignins in the plant cell wall. Insoluble (IDF) and soluble (SDF) DF may be determined separately or pooled and determined as total DF (TDF). The traditional DF components present in the insoluble and soluble DF fraction differs depending on the DF source and specific isolation conditions, but basically lignin and cellulose is present in IDF, whereas SDF comprise pectins. Hemicelluloses are found in both fractions depending on their actual molecular structure and thereby solubility properties. The idea behind the enzymatic–chemical methods is analysis of the individual monomeric constituents of polysaccharides in the DF fraction (NSP, non-starch polysaccharides). The methods comprise, as a first step, partly removing non-DF components by means of enzymes, followed by acid hydrolysis of DF polysaccharides. The analysis of neutral monosaccharides is generally performed by GC, but other chromatographic 50 P. Kadlec et al. systems may be used, e.g. HPLC (Quigley and Englyst, 1992) and HPCE (Rassi and Mechref, 1996). Alternatively, colorimetry may be used for determination of groups of pentoses, hexoses and uronic acids; however this technique is seldom used as routine. Uronic acids also may be analysed by decarboxylation (Theander et al., 1994). Lignin is not included in the method, but can be determined separately. The NSP may be separated into a soluble and insoluble part in some of the methods. Monosaccharide losses during the polysaccharide acid catalysed hydrolysis step are a problem of major concern in enzymatic–chromatographic methods. The conditions in acid hydrolysis are actually a compromise between complete liberation and destruction of monosaccharides. Various methods exist, differing in type and concentration of acid as well as temperature and time used for hydrolysis. An AOAC Official Method has now been accepted (Method 994.13). The analytical methods for determination of DF described above give only limited information on the level and nature of individual DF components. More specific studies were originally performed using the classical fractionation schemes evolved in the 1930s (Southgate, 1995a). The experimental conditions used in these fractionations were extremely vigorous and generally caused some severe modifications of the structures of the components. Modern techniques for fractionation are generally more gentle, although the possibility still exists, that a certain number of bonds must be broken in order to extract components from the cell wall, leading to incorrect conclusions about the chemistry, especially of cell wall polysaccharides. Moreover, the specific procedure for extraction of a particular type of component may result in only partial recovery of the expected polysaccharide. The fractionation is performed on purified plant material, and not directly on the fresh tissue. Methods for preparation of plant material prior to fractionation are described in the following sections, examples of the analytical approaches for determination of the subfractions: cellulose, hemicellulose, pectins and lignins are given, extraction of minor DF compounds such as, e.g., phenolics are described briefly, and more details are given elsewhere (Andersen et al., 1997; Bjergegaard et al., 1997a,b). Although the definition of DF includes components from outside the plant cell wall, the quantitatively dominating part in land plants is found inside the plant cell wall, and plant cell wall polysaccharides and lignins will, therefore, be considered. Analysis of individual non-starch polysaccharides may be disturbed by the presence of other plant tissue components. On the other hand, some of these components may be found in close association with the cell wall polysaccharides, affecting the properties of the DF fraction. This apparent conflict of interest must be taken into consideration when planning the study of the PREPARATION OF PLANT CELL WALL MATERIAL Carbohydrate Chemistry 51 individual DF components (Andersen et al., 1997; Bjergegaard et al., 1997a,b). As a first step, it is important to consider the starting material to be used for the fractionation. The TDF fraction produced by the enzymatic– gravimetric procedure could be a possible starting point for the fractionation, whenever additional information on the level of some non-traditional DF components is considered relevant. Any reserve polysaccharides from outside the plant cell wall will also be included. Another more common possibility is to use isolated plant cell walls, which may be more or less pure, depending on the exact procedure for preparation. Examples of such preparative methods are presented below. A very simple cell wall preparation method consists of washing the homogenized tissue in 70% ethanol. This procedure leaves polymers insoluble (AIR, alcohol insoluble residue), whereas low molecular weight compounds such as sugars, amino acids, organic acids and many inorganic salts are solubilized (Fry, 1988). In addition to the cell wall polymers, other high molecular weight components such as intracellular proteins, RNA, starch, reserve polysaccharides, etc., will be present in AIR and one may consider whether this will constitute a problem in the following DF studies. A problem with AIR is the dehydration effect of the alcohol possibly leading to formation of various artefacts. A more comprehensive method is as follows: 50 g fresh weight of sample is homogenized in 100 ml 1% (w/v) aqueous sodium deoxycholate (SDC) or 1.5% (w/v) aqueous SDS containing 5 mM Na2S2O5. The slurry is filtered and washed (twice). The resulting residue, R1, is ground in a wet ball-mill in 100 ml 0.5% SDC (w/v) or SDS (w/v) containing 3 mM Na2S2O5 at 2°C for 15 h. The residue/slurry is centrifuged and washed. Alternately the residue R2 is extracted twice with 50 ml phenol : acetic acid : water (PAW) (2 : 1 : 1, w/v/v) at 20°C and washed twice. Sodium metabisulphite is included in order to diminish oxidation of polyphenolics. SDC/SDS solubilizes intracellular compounds as well as some cold-water-soluble pectins. PAW efficiently removes residual intracellular proteins together with some starch, adsorbed detergent, lipids and pigments. The amount of cell wall components solubilized in this step is low. The DMSO treatment extracts starch, whereas the amount of co-extracted cell wall material depends on the kind of starting material. The method provides a useful procedure to obtain relatively pure cell walls, with about 90% of the total cell wall constituents remaining in the final preparation (Selvendran et al., 1985; Southgate, 1995b). The diversity of method for the preparation of cell wall material is illustrated above and one has to consider what is needed in each specific case. A simple isolation technique may thus be adequate, whenever impurities are not expected to interfere with the planned analysis, whereas in other situations very pure cell wall preparations may be needed. For extraction of lipids and amphiphilic compounds it is more efficient, simple, 52 P. Kadlec et al. fast and cheap to use supercritical fluid techniques (super critical extraction (SCE)/super critical chromatography (SFC); Andersen et al., 1997; Bjergegaard et al., 1997a,b). CELLULOSE AND HEMICELLULOSE Determination of cellulose is generally part of a more comprehensive fractionation procedure, in which α-cellulose is obtained as the residue after sequential extraction of pectic material (Fig. 2.12), possibly lignins (delignification in lignified tissues) and hemicelluloses. α-Cellulose is insoluble in 17.5% NaOH (w/v), whereas a minor part of native cellulose may be solubilized here. Cellulose is virtually insoluble in water. Selvendran (1983) considered α-cellulose as the fibrillar part of the plant cell wall and that the solubilized native cellulose probably originated from more amorphous regions. Fry (1988) describes a method for direct extraction of cellulose by the highly basic reagent cadoxen. Cadoxen is prepared by stirring a solution consisting of 1,2-aminoethane (310 ml), H2O (710 ml) and cadmium oxide (100 g) at 20°C for 3 h followed by 4°C for 18 h. The supernatant obtained Fig. 2.12. Sequential extraction of pectin material from purified cell walls. Carbohydrate Chemistry 53 after centrifugation is used for the extraction. The procedure converts cellulose into a soluble fraction by heating in dry DMSO and paraformaldehyde. Free cellulose can then be regenerated as a precipitate by addition of H2O or methanol. The classical procedure for solubilization and subsequent hydrolysis of cellulose is a two-step procedure involving the use of 72% H2SO4 (w/w) (e.g. 1 h at 35°C) as the first step followed by hydrolysis in dilute acid (e.g. 2 M H2SO4 for 1 h at 100°C). The solubility of cellulose in strong sulphuric acid is due to disruption of hydrogen bonds caused by sulphonation of hydroxyl groups at C-6 of glucose (Selvendran and Robertson, 1990). This procedure is also used for hydrolysis of total NSP in the enzymatic– chemical methods and, according to Englyst et al. (1982) and Englyst and Cummings (1988), omission of the first step gives a hydrolysate of noncellulosic polysaccharides (NCP), whereas only cellulose is included by the two-step procedure. This division of cellulose and NCP has, however, been questioned (Bingham and Selvendran, 1983). Further studies of the cellulose fraction are generally restricted to determination of the degree of polymerization and studies of associated polysaccharides. Quantification of the hemicellulose fraction as a difference between the NDF and ADF residue has been a formerly accepted method (Asp and Johansson, 1984). This procedure gives an indirect measure of hemicellulose. The value of the method is questionable, however, due to the losses of hemicellulosic polysaccharides by the NDF procedure and the incomplete removal of hemicellulosic polysaccharides by the ADF procedure. This results in an underestimation of the fraction and, in addition, there is no possibility for a closer examination of the individual components. A more preferable method is the extraction of hemicelluloses from depectinated sample material. This extraction is usually performed sequentially with extractions at increasing alkali strength and/or temperature. The extraction of hemicelluloses is carried out in the absence of oxygen using potassium or sodium hydroxide following the saturation of the extraction medium with nitrogen or argon gas (Selvendran et al., 1985; Southgate, 1991). Moreover, a strong reducing agent such as NaBH4 may be added. These precautions are taken to prevent the formation of polyphenolic complexes forming with the polysaccharides, making them difficult to extract. Another function of NaBH4 is protection of the reducing sites in the carbohydrate molecule, as C-3 bound aldoses and C-4 bound ketoses may otherwise undergo β-elimination under alkaline conditions. Various schemes exist for the extraction of hemicelluloses and specific procedures have been developed for different types of sample. Pectic substances, which remain in the residue after a previous treatment with chelating agents, may be extracted with the hemicellulose fraction in step 1. The proportion of solvent to depectinated material may vary, as may the choice of using argon or nitrogen gases. The scheme for the sequential 54 P. Kadlec et al. extraction of hemicelluloses by KOH is described by Selvendran and O’Neill (1987) and Southgate (1995b). If heavily or moderately lignified tissue is used, it will be necessary to include a delignification step. It is debatable whether legume seeds could be classed as containing moderately or lightly lignified material. In order to compare data from legumes with that obtained from other sources it would be wise to consider including the following delignification step anyway. Delignification in moderately lignified tissue is carried out after the first extraction with KOH, by treatment with sodium chlorite and acetic acid at 70°C for 2–4 h (Selvendran and O’Neill, 1987; Southgate, 1995b). The addition of the delignification step after the first alkali treatment is performed in order to preserve native hemicellulosic polysaccharide– protein and polysaccharide–protein–polyphenol complexes, which may be partially modified by the delignification treatment. The duration of the delignification procedure may be varied depending on the type of material being analysed. Heavily lignified tissue requires longer treatment and delignification is generally performed prior to extraction of the hemicelluloses. As delignification may lead to some modification of plant cell wall proteins and polysaccharides it should be avoided in only lightly lignified tissue (Selvendran and O’Neill, 1987). The solubilization of polysaccharides by use of alkali is brought about by cleavage of ester linkages between polysaccharides (uronic acids) as well as polysaccharides and non-carbohydrate constituents (e.g. phenolic acids). Hydrogen bonds will also be disrupted (Selvendran et al., 1985). Highly polymerized polysaccharides, glucomannans and slightly branched xyloglucans, being strongly hydrogen bound to cellulose, require the use of strong alkali for solubilization (Selvendran and Robertson, 1990), and the extraction of glucomannans is enhanced by the inclusion of boric acid. Other hemicellulose extractants are the aqueous chaotropic agents: perchlorate, urea and guanidinum thiocyanate. Common to these extractants is that they are effective protein solvents, unlikely to cause any degradation. Only a minor part of the hemicellulosic compounds, however, will be extracted (Fry, 1988). The chaotropic agents are stated to be useful for solubilization of mannose-rich polymers (Selvendran et al., 1985), probably because of the concurrent extraction of proteins. Further studies of the extracted components comprise a wide range of techniques including gel filtration, anion exchange chromatography, affinity chromatography, precipitation by complex formation with inorganic salts (e.g. iodine and copper complexes), stepwise precipitation with ethanol, precipitation by Ba(OH)2 and quaternary ammonium salts. More details of these techniques can be found in Wilkie (1985), Selvendran and O’Neill (1987) and Fry (1988). Fry also gives a thorough description of the methods for structural elucidation, such as different hydrolysis procedures, methylation analysis, periodate-oxidation studies, specific enzymatic degradations, etc. Carbohydrate Chemistry 55 Some examples from characterization of polysaccharides in various grain legumes can be found for pea (Talbott and Ray, 1992; Ralet et al., 1993; Weightman et al., 1994) and lupin (Mohamed and Rayas-Duarte, 1995). The commonly used preparation of TDF is described elsewhere (Andersen et al., 1997). The fractionation procedure for pea TDF into pectic material, hemicelluloses, cellulose and lignins is described below. 1. Pectins – add 25 ml 1% ammonium oxalate to 1 g TDF and adjust pH to 5.0 using 1 M HCl. Extract for 2 h at 80°C in a shaking waterbath. Cool and centrifuge (34,000 × g ; 20 min), freeze-dry the sediment, weigh and use this sediment for extraction of hemicellulose (procedure 2). Keep 5 ml of the supernatant (−20°C) for further analyses and dialyse the rest (known volume) against water overnight (5°C). Save 5 ml dialysed supernatant (−20°C). Freeze-dry the remaining dialysed supernatant (known volume), weigh and save in desiccator (pectins). The weight of pectic material should be corrected for reduction in starting material. 2. Hemicelluloses – add 25 ml 2 M NaOH to the sediment from procedure 1 and extract (under N2) for 2 h in a 30°C waterbath with occasional shaking. Centrifuge (34,000 × g ; 20 min), freeze-dry the sediment, weigh and use this sediment for extraction of cellulose (procedure 3). Make the supernatant weakly acidic (with 3–4 ml acetic acid) and save 5 ml of the pH adjusted supernatant (−20°C) for further analyses. Dialyse the supernatant (known volume), including any sedimented material occurring after the pH adjustment, overnight (5°C). Collect the sedimented material by centrifugation (3000 × g ; 3 min), freeze-dry, weigh and keep it in desiccator (hemicellulose A). Save 5 ml dialysed supernatant (−20°C). Freeze-dry the remaining dialysed supernatant (known volume), weigh and store in a desiccator (hemicellulose). The weight of the hemicellulose fractions should be corrected for reduction in starting material. 3. Cellulose and lignin – add 1 ml 12 M H2SO4 to the sediment from 2 and leave it for 1 h at 35°C with occasional mixing. Dilute with 11 ml H2O to give a 1 M H2SO4 solution, and continue extraction in a boiling waterbath under reflux for 18 h. Cool and centrifuge (3000 × g ; 3 min), freeze-dry the sediment, weigh and store in a desiccator (lignin). Neutralize the supernatant (known volume) with saturated Ba(OH)2 and centrifuge to remove BaSO4. Keep 5 ml of the neutralized supernatant (−20°C) for further analyses. Freeze-dry the remaining supernatant (known volume), weigh and store in a desiccator (cellulose). The weight of the cellulose fractions should be corrected for reduction in starting material and for the weight reduction brought about by hydrolysis of the polysaccharide. The extraction conditions may be varied depending on the plant material considered and the method presented here is only one of several possibilities as also indicated in the preceding sections. It should be noted that the results obtained are exclusively dependent on the actual fractionation 56 P. Kadlec et al. procedure and this must be taken into consideration when the method is evaluated. 2.2.3 Other carbohydrate components Pectin Common methods exist for the isolation of pectic substances from plant materials, and the main steps are outlined below. 1. Grind the plant material in warm ethanol or acetone. 2. Wash with ethanol, to inactivate endogenous enzymes. 3. Wash with sodium deoxycholate (SDC) (or enzyme treatment), to remove proteins. 4. Wash with phenol–acetic acid–water mixture, to remove lipids and pigments. 5. Treat with aqueous 90% DMSO (or enzyme treatment) to remove starch. 6. Wash with ethanol, to remove the other organic solvents. The titration method is widely used for total pectin determination. In this method the galacturonic acid content and the degree of esterification (by methylation and acetylation) of a pectin preparation are calculated from the neutralization and saponification equivalents of the pectic and acetic carboxyl groups. Prior to analysis, the pectin must be converted to the free acid form by mixing with a strong cation-exchanger, or by washing with an alcohol/HCl mixture, followed by washing with alcohol until the washings are neutral. If acetyl groups are present in the pectins, a saponification equivalent that is too high is obtained. By using the copper-binding method (Keijbets and Pilnik, 1974), interference by acetyl groups is overcome. In this method the quantity of copper ion (Cu2+) that binds to the pectin before and after saponification is determined stoichiometrically or by atomic absorption spectrometry and the dry matter is calculated from the ratio of these values. Colorimetric procedures, such as those based on carbazole (Bitter and Muir, 1962), have been widely used. Blumenkrantz and Asboe-Hansen (1973) significantly reduced the interference of neutral sugars by adding m-hydroxydiphenyl as a chromogen to heated solutions of uronides in a sulphuric acid/boric acid mixture. Ahmed and Labavitch (1977) modified and tested this procedure for native and extracted pectins. The m-hydroxydiphenyl assay was automated by Thibault (1979). Garleb et al. (1991) described an anion exchange HPLC method with pulsed amperometric detection for the determination of galacturonic acid, after acid hydrolysis. DETERMINATION OF GALACTURONIC ACID (GA) Carbohydrate Chemistry 57 The most widely used method for sugar determination involves acid hydrolysis of the sample with 2 M trifluoroacetic or 1 M sulphuric acid, or by Saeman hydrolysis, which uses 72% sulphuric acid for solubilization and subsequent hydrolysis with 0.4 M sulphuric acid (Selvendran et al., 1979). After hydrolysis, the sugars released are reduced to corresponding alditols and then converted to alditol acetates. These volatile derivatives can be reliably analysed by GC as single peaks (Blakeney et al., 1983). Uronides are not determined by this method, but may be assayed together with neutral sugars by making TMS derivatives of the anomeric methyl glycosides obtained by methanolysis. In order to resolve the many peaks obtained from all sets of isomers, it is necessary to use capillary columns in the GC. The monosaccharides are subsequently analysed by high performance anion exchange chromatography without the need for derivatization. It is also possible to determine galacturonic acid and neutral sugars by a combination of enzymic hydrolysis and methanolysis, followed by HPLC separation on a C18 column eluted by water (Quemener and Thibault, 1990). The types of glycosidic linkages between sugar residues are in general determined by methylation analysis. Glycosyl-linkage analysis of uronosyl residues in polysaccharides is possible only after reduction to the corresponding neutral sugar units. DETERMINATION OF NEUTRAL SUGARS Lignin The methods of lignin determination are essentially the same or similar to those used to analyse the fibre, hemicellulose and cellulose fractions in legume seeds. Suitable methods are summarized below. VAN SOEST METHOD The residue of ADF is added to 72% H2SO4 at 0–4°C for 3 h with stirring. After hydrolysis, the residue is filtered, and washed with hot water, then acetone and dried in a 100°C oven for 12 h, cooled in a desiccator and weighed. MORRISON METHOD Digestion with acetyl bromide (Morrison, 1972). In this procedure lignin is the residue after all the enzyme and chemical treatments of the Englyst DMSO method have been completed. KLASON METHOD The residue is delignified by treatment with sodium chloride–acetic acid at 70°C for 4 h. DELIGNIFICATION METHOD The residue from the ADF is treated with potassium permanganate solution, containing trivalent iron and monovalent silver as catalysts. Deposited manganese and iron oxides are dissolved with an alcoholic solution of oxalic and hydrochloric acids, leaving GOERING AND VAN SOEST METHOD 58 P. Kadlec et al. cellulose and insoluble minerals. Lignin is measured as the weight loss by these treatments, while cellulose is determined as the weight loss upon ashing of the residue. NIR SPECTROSCOPIC ANALYSIS FT-IR ANALYSIS See Reeves (1988). See Buta and Galletti (1989). REVISED METHOD FOR QUANTIFYING DF COMPONENTS This method involves lipid removal with diethyl ether; removal of water-soluble components and quantification of water-soluble fibre components, removal of waterinsoluble hemicellulose and cellulose (Jeltema and Zabik, 1980). After starch extraction, the residue is washed, dried, then hydrolysed by heating with 10 ml 1 M H2SO4 for 2 h at 100°C to hydrolyse the remaining cellulose fraction. The samples are then diluted with 11 ml of water and heated for 2 h at 100°C. The residue is washed with water and ethanol. Samples are extracted twice with warm diethyl ether and then with acetone and air dried at 45°C. The residue is then weighed and ashed at 500°C for 8 h to constant weight. The loss in weight is assumed to be lignin (Anderson and Bridges, 1988). GRAVIMETRIC METHOD Saponins A general method for the extraction of saponins from legume seeds is as follows. Seeds are ground to a flour (30 g) and extracted with chloroform (800 ml) for 16 h in a Soxhlet extractor. The chloroform extract is evaporated in vacuo and the air-dried defatted flour is then extracted with methanol (800 ml) for 30 h. The methanol extract is evaporated to dryness in vacuo, dissolved in distilled water and run through a column of reversed phase octasilane (C-8) bonded to silica gel. The column is successively eluted with distilled water (150 ml) and methanol (150 ml) and the fraction evaporated to dryness. The residue is hydrolysed with dry hydrochloric acid in methanol (5 ml, 5% solution) and refluxed for 3 h, the solution is neutralized and evaporated to dryness. After redissolving in water (5 ml), the sapogenols are extracted with ethyl acetate (3 × 5 ml). The combined ethyl acetate extracts are then dried over anhydrous sodium sulphate, filtered and evaporated to dryness. For more information see Fenwick et al. (1991) and Tsukamoto et al. (1993). EXTRACTION TLC ANALYSIS The dried sapogenol hydrolysis products are redisolved in methanol (1 g ml−1) and loaded on to TLC plates together with standards of soyasapogenol A and B. The plates are run with a chloroform : methanol Carbohydrate Chemistry 59 (76 : 4 v/v) for a distance of 5.5 cm. Plates are air-dried and visualized with a p-anisaldehyde reagent. GC ANALYSIS The methanol solution of hydrolysed saponins (1 ml), equivalent to 1 g of defatted flour, is evaporated to dryness in a vial by a stream of nitrogen. After further drying over phosphorus pentoxide in a vacuum desiccator (12 h), a TMS derivatizing reagent is added (pyridine : bis trimethylsilyl trifluoroacetamide (BSTFA) and the samples heated for 20 min at 50°C; 1 µl of the sample is injected (with 1 µl of cholesteryln-decylate, 1.87 mg ml−1 as an internal standard) on to a Perkin Elmer Sigma 3B GC fitted with a glass column (1 m × 2 mm I.D.), packed with 3% OVl on Diatomite CQ AW DMCS, 60–80 mesh fitted with FID. The column temperature was 280°C, injector temperature 285°C and detector temperature 300°C. The carrier gas (He) flow rate is 35 ml min−1. The identities of soyasapogenols A, B and C can be confirmed by direct comparison with authentic standards as well as by combined GC–MS using the TMS-ether derivatives. Aliquots (10 µl) of each extract can be injected directly on to a 250 × 4.6 mm Spherisorb S5 ODS 2 column and eluted using an acetonitrile : water : trifluoracetic acid mixture with the composition changing from 80 : 20 : 0.1 v/v to 20 : 30 : 0.1 v/v over a time span of 25 minutes using an eluant flow rate of 1 ml min−1. Detection is by UV absorption at 210 nm. For further details of experimental systems, see Fenwick et al. (1991) and Tsukamoto et al. (1993). HPLC ANALYSIS H. Kozlowska et al. Nutrition 3 3 Nutrition Editor: Halina Kozlowska Contributors: Pilar Aranda, Jana Dostalova, Juana Frias, Maria Lopez-Jurado, Halina Kozlowska, Jan Pokorny, Gloria Urbano, Concepcion Vidal-Valverde and Zenon Zdyunczyk Food is an important part of a balanced diet. Metropolitan Life, p. 110 (1978) Fran Lebowitz (1946–), American writer 3.1 Introduction The carbohydrate fraction of grain legumes is a major source of food and feed energy. The optimal composition of grain legume carbohydrates, however, depends on a number of factors, for example, consumer demand and the requirements for animal feedstuff. The diets for intensively farmed animals are required to have a high energy value. The desired direction for grain legume breeders and processors, therefore, is to decrease the content of those carbohydrates that have low energy values, or, which act as antinutrients. In this context many authors classify α-galactosides in grain legumes as antinutrients (Eskin et al., 1980; Saini, 1989). It has been known for many centuries that peas and beans, although nutritional wholesome foods, produce wind (Gerarde, 1633). To quote from the British Herbal (Hill, 1756) ‘The fruits of these several kinds are all of the same quality, wholesome as food, but apt to breed wind’. High quantities of α-galactosides and non-starch polysaccharides are believed to cause flatulence and reduce the net energy of seeds (Fernandez and Batterham, 1995). From the point of view of animal nutrition, low bioavailability of starch is also a disadvantage of grain legumes. ©CAB International 2001. Carbohydrates in Grain and Legume Seeds (ed. C.L. Hedley) 61 62 H. Kozlowska et al. Consumer perception of grain legumes depends on national and cultural differences, often related to a country’s wealth. In developing countries, for example, grain legumes are looked on as ‘poor man’s meat’ and are of great importance, therefore, as a protein source. In developed countries there is often an excessive consumption of animal products (rich in saturated fats) and a reduced intake of vegetables and grain legumes, which are, therefore, of far less importance as a protein source. Also, grain legume seeds are becoming more associated in developed countries with preventative or therapeutic effects on some diseases such as diabetes, hypercholesterolaemia, cancer, etc., rather than as a source of nutrition (Frühbeck et al., 1997). Despite their relatively infrequent consumption of legumes (only 10% of the population include them in their diet on any one day), legumes provide 14% of the dietary fibre (DF) intake in the USA, 25% in Great Britain and Belgium, and 5–10% in France (Benamouzig et al., 1994). Although legumes are a good source of DF, as mentioned above, they also have significant amounts of oligosaccharides associated with flatulence, which is claimed as a major reason for their limited consumption (Saini and Gladstones, 1986; Price et al., 1988). There are, however, positive effects of these compounds and possible health benefits in human nutrition have commanded considerable attention in recent years (Oku, 1994; Cummings and Englyst, 1995). The possible elimination of oligosaccharides from grain legumes, therefore, requires a renewed discussion. Numerous works published recently make it possible to characterize better the nutritional and physiological value of grain legumes, allowing the requirements for improving carbohydrates in the seed to be set out more clearly. 3.2 The Content of Carbohydrates in Grain Legumes Utilized in Europe 3.2.1 The content of carbohydrates in grain legumes used for human nutrition The most common legumes for human consumption are bean (Phaseolus vulgaris), lentil (Lens culinaris), pea (Pisum sativus), chickpea (Cicer arietinum) and faba bean (Vicia faba). The carbohydrate fraction in the seeds of these legumes is composed of soluble carbohydrates (mainly fructose, sucrose and low molecular weight oligosaccharides such as ciceritol, raffinose, stachyose and verbascose), starch and longer chain oligosaccharides and polysaccharides constituting dietary fibre. Table 3.1 collates the information found in the literature for mono- and disaccharides, low molecular weight oligosaccharides (or α-galactosides), total soluble sugars and starch from bean, pea, lentil, chickpea and faba bean. Nutrition 63 Table 3.1. Soluble carbohydrate and starch content (average and range as % dry matter) of some common grain legumes, used mainly for human consumption. Fructose (mean) (range) Sucrose Raffinose Ciceritol Stachyose Verbascose Total α-galactosides Total soluble sugars Starch Beans Peas – – Lentils Chickpeas Faba beans 0.1 – 0.4 .30–0.2 2.5 2.1 1.7 4.7 2.2 1.6–3.9 0.9–5.4 1.1–3.0 2.8–6.9 0.1–3.8 0.7 0.9 0.3 0.3 0.5 0.2–2.5 0.4–2.3 0.1–0.8 .30–0.3 0.1–1.5 – – 0.7 2.2 0.2–2.1 1.2–3.1 2.7 2.0 1.9 1.3 0.9 0.2–3.9 0.3–4.2 1.1–4.0 0.4–2.0 0.2–1.6 0.6 1.8 0.3 trace 1.8 .30–4.3 .30–6.4 trace–0.4 1.1–2.4 0.1–1.8 3.8 4.6 3.2 3.8 3.0 0.4–8.0 2.3–9.6 1.8–7.5 2.0–7.6 1.0–4.5 5.2 6.7 5.0 8.4 5.6 2.0–9.6 3.5–13.8 3.3–9.5 4.6–14.2 2.2–8.5 54.0 39.0 47.4 50.4 43.0 51.0–59.0 24.7–57.4 40.1–57.4 43.0–59.0 39.2–47.2 According to different authors the total soluble sugar content of nine varieties of bean (Phaseolus vulgaris) is very similar to that found in lentil, although some differences were observed in the individual sugar content (Table 3.1; Salunkhe et al., 1989; Vidal-Valverde et al., 1993a; Troszynska et al., 1995). Fructose was not present, however, and the content of sucrose (1.6–3.9%) was higher than in lentil but lower than in chickpea. The average α-galactoside content was similar for lentil and chickpea, but the highest range (0.4–8.0%) was found among bean varieties. Ciceritol was not present and higher amounts of raffinose, stachyose and verbascose, (average value of 0.7, 2.7 and 0.6% respectively) were detected in bean compared with lentil and chickpea. The information found in the literature for the starch content of bean was for three varieties only and this value, 51–59%, is the highest of all the legumes used for human consumption. The information obtained from the literature for 36 pea varieties (Cerning-Beroard and Filiatre, 1976; Cerning-Beroard and Filiatre-Verel, 1979; Adsule et al., 1989; Van Lonkhuijsen et al., 1992; Troszyska et al., 1995; Frias et al., 1996d; Vicente, 1998) indicates that the content of total soluble sugars is similar to that found in lentils, although once again the individual composition is very different (Table 3.1). Fructose was not present and the content of sucrose (0.9–5.4%) is much higher than in lentils. The range of total α-galactosides content found among the pea varieties was 2.3–9.6%. Raffinose (0.4–2.3%) and stachyose (0.3–4.2%) were present in all of the pea varieties and verbascose only in 24 varieties, with a mean value of 1.8%. 64 H. Kozlowska et al. The starch content in the 12 pea varieties described in the literature differed widely, wrinkled peas showing quite low starch contents (mean of 24.7%), while in smooth peas the starch content was very high (mean of 49.6%). For lentil seeds (Table 3.1), information found in the literature covered about 20 varieties (Vidal-Valverde et al., 1992a,b, 1993a,b; Frias et al., 1994, 1995, 1996a,b,c; Troszynska et al., 1995; Urbano et al., 1995; Sotomayor, 1997). This showed a large difference in the total soluble sugar content, ranging from 3.9 to 9.5%, which was mainly due to differences in the range of α-galactoside content (1.8–7.5%). Fructose was found in very small amounts (0.01–0.2%), while sucrose was present in all varieties in quantities ranging from 1.1 to 3%. With regard to the individual α-galactoside content, ciceritol was found in all varieties of lentil, the amount ranging from 0.2 to 2%. With the exception of chickpea, this oligosaccharide does not occur in the other grain legumes. Raffinose was also present in all lentil varieties with quantities ranging from 0.1 to 0.8%. Stachyose was the most abundant α-galactoside, ranging between 1.1 and 4.0%. Verbascose is not present in some lentil varieties, while it can reach up to 1.8% in others. Information on starch content in lentil has been found for only 19 varieties. The average starch content in lentil was 47% with a range of 40–57%. For chickpea (Table 3.1), the information collected for 24 varieties from the literature (Rossi et al., 1984; Saini and Knights, 1984; Chavan et al., 1989; Vidal-Valverde et al., 1993a; Sotomayor, 1997; Frias et al., 1999) shows that soluble sugar content has the highest value within the grain legumes (4.6–14.2%), mainly due to the high content of sucrose (2.8–6.9%). Of the individual α-galactosides, ciceritol was present in the largest amount (1.2–3.1%) followed by stachyose (0.4–2.0%) with only two of the varieties having very high amounts of this oligosaccharide (4–6.5%). Raffinose was present in chickpea in very small amounts (traces – 0.3%), but those varieties with very high amounts of stachyose also had quite high amounts of raffinose (1.0–2.0%). The pentasaccharide, verbascose, was present in trace amounts, but in those varieties with the highest content of raffinose and stachyose, the verbascose content increased to 0.2–0.4%. The content of starch has been reported for only six chickpea varieties and ranged from 43 to 59%. According to the information from different authors, for 10 varieties of faba bean (Cerning-Beroard and Filiatre, 1976; Kozlowska et al., 1992; Van Lonkhuijsen et al., 1992; Troszynska et al., 1995; Frias et al., 1996d; Frejnagel et al., 1997; Vidal-Valverde et al., 1998) the total soluble sugar content (2.2–8.5%) is similar to that found in bean (Table 3.1). Fructose content was not referred to in most of the faba bean varieties, but for those in which it has been analysed the amount was relatively high (0.4%). The sucrose content was very wide with values ranging from 0.1 to 3.8%. The content of α-galactosides (1.0–4.5%) was similar to that present in common bean. Nutrition 65 Ciceritol was not present in either faba bean or pea. The content of raffinose in faba bean was lower than in bean and pea and the stachyose content was the lowest amongst the grain legumes reported (Table 3.1). The starch content of the six varieties of faba bean reported in the literature ranged from 39.2 to 47.2%. DF is defined physiologically as the total amount of polysaccharides and lignin not digested by endogenous enzymes of the human gastrointestinal tract. Since the composition of DF is complex, different methods have been used to quantify its content and constituents. The composition of DF reported can vary widely, therefore, depending on the methodology used. Taking this consideration into account, a large amount of data has been recorded on the content and composition of DF in legumes used for human consumption. The DF content in legume seeds depends on many factors, including the species and the variety (Table 3.2). The DF content of common beans indicated by different authors (Naivikul and D’Appolonia, 1978; Fleming, 1980; Chen and Anderson, 1982; Fidanza et al., 1982; Reddy et al., 1984; Garcia-Olmedo et al., 1985; Paul and Southgate, 1985; Souci et al., 1986; Lintas et al., 1992; Mongeau and Brassard, 1994, 1995; Vidal-Valverde et al., 1992c) ranged from 11.2 to 27.5%, the contribution of the soluble component being the highest for the grain legumes (8.1–10%). The DF content in bean, noted by different authors, showed considerable differences for the same types of seeds (Table 3.3). The results presented refer to the raw seeds of bean. During cooking, changes in the DF content and composition can be observed. Acevedo et al. (1994) noted that the DF content in black beans differed according to the type of processing; it reached 26.5% in cooked seeds, 28.1% in blended seeds and 29% in fried seeds. The proportion of soluble DF in cooked, blended and fried beans reached 31.7, 26.7 and Table 3.2. Content of dietary fibre and its components (as % dry matter) of some common grain legumes, used mainly for human consumption. Beans Peas Lentils NDF 8.9–12.8 13.2–25.6 9.7–24.1 ADF 3.5–7.2 no information 2.0–6.8 Cellulose 3.2–13.1 0.9–13.3 3.5–14.8 Hemicellulose 0.5–5.6 0.9–12.4 1.2–15.7 Lignin 0.1–3.1 0.3–2.1 trace–2.6 TDF 11.2–27.5 16.1–21.6 11.0–21.4 SDF 8.1–10.0 4.6–6.0 1.2–4.4 IDF 9.1–11.6 11.6–16.1 8.8–13.7 NSP 6.4–20.4 no information 6.9–14.7 Chickpeas Faba beans 7.5–19.2 3.8–14.7 1.1–13.7 0.6–16.0 trace–7.1 8.2–24.0 3.7 7.9 5.5–35.4 13.0–19.5 10.3–11.4 8.3–14.3 1.6–8.9 0.7–2.0 17.1–23.8 6.0–8.7 8.3–15.5 17.5 NDF, neutral detergent fibre; ADF, acid detergent fibre; TDF, total dietary fibre; SDF, soluble dietary fibre; IDF, insoluble dietary fibre; NSP, non-starch polysaccharides. 66 H. Kozlowska et al. Table 3.3. Dietary fibre (DF) (as a percentage of dry matter, DM) content in beans (Phaseolus vulgaris). Type Great northern Light red kidney Red kidney (Goya) Red kidney (Townhouse) White navy No name Mottled White No name Pinto No name No name DF content (%DM) 20.7 14.3–18.1 20.7–21.3 21.3–21.9 15.8–19.2 12.7–12.8 19.1 19.6 11.6 27.0 16.8 28.1 References Mongeau and Brassard (1994) Mongeau and Brassard (1995) Mongeau and Brassard (1994) Mongeau and Brassard (1994) Mongeau and Brassard (1995) Mongeau and Brassard (1995) Lintas et al. (1992) Lintas et al. (1992) Lintas et al. (1992) Chen and Anderson (1982) Paul and Southgate (1985) Paul and Southgate (1985) 22.8%, respectively, of total DF. In raw seeds, the soluble DF content is usually higher, up to 35% of total DF (Acevedo and Bressani, 1989; Lintas et al., 1992). The DF content of pea, according to information from different authors (Van Soest, 1978; Chen and Anderson, 1982; Reddy et al., 1984; Paul and Southgate, 1985; Souci et al., 1986; Maltese et al., 1995; Zdunczyk et al., 1997; Kmita-Glazewska and Kostyra, 1998) ranged between 16.1 and 21.6%, the insoluble DF content being higher than the soluble DF (Table 3.2). In the seeds of 15 Polish cultivars of white-flowered spring pea (Pisum sativum) the average DF content was 18.8% of the dry matter (DM) with a range from 16.2 to 21% (Zdunczyk et al., 1997). A highly significant negative correlation r = −0.815 (P = 0.01) was found between the weight of seeds and DF content. Igbasan et al. (1997) reported that in seeds of 12 Canadian pea cultivars, the average DF content was 20.3%, including 14.1% of NSP, 2.5% cell wall protein, 0.4% of cell wall ash and 3.3% of lignin and polyphenols. The DF content of lentil found in the literature (Naivikul and D’Appolonia, 1978; Chen and Anderson, 1982; Fleming, 1981; Fidanza, 1982; Reddy et al., 1984; Garcia Olmedo et al., 1985; Paul and Southgate, 1985; Shekib et al., 1985; Souci et al., 1986; Lintas et al., 1992; Vidal-Valverde and Frias, 1992; Vidal-Valverde et al., 1992c, 1993a; Pizzoferrato et al., 1995) ranged from 11 to 21.4%, the highest amount being insoluble DF (8.8–13.7%) and the smallest amount (1.2–4.4%) for soluble DF. On the basis of the analyses from many authors, Savage (1988) states that the average DF content in lentil seed is 21.4%, including 4.5% of soluble DF. Other authors mention much lower DF contents ranging from 11 to 15% of DM (Garcia-Olmedo et al., 1985; Paul and Southgate, 1985; Lintas et al., 1992; Pizzoferrato et al., 1995). Nutrition 67 The information obtained from the literature on the DF content of chickpea (Fleming, 1980; Vitaladasa and Belavady, 1980; Reddy et al., 1984; Rossi et al., 1984; Saini and Knights, 1984; Garcia-Olmedo et al., 1985; Paul and Southgate, 1985; Souci et al., 1986; Lintas et al., 1992; Vidal-Valverde et al., 1992c; Mongeau and Brassard, 1994, 1995; Modgil and Mehta, 1996; Nestares et al., 1997) ranged from 8.0 to 24.0%. As in the case of lentil, the insoluble components were much higher than the soluble components. The DF content for faba beans, according to the literature (Spiller, 1986; Wang et al., 1991; Gdala et al., 1995; Pizzoferrato et al., 1995; VidalValverde et al., 1998) ranged from 17.1 to 23.8, where the content of insoluble DF was very high (8.3–15.5%) and the soluble DF was 6.0–8.7%. It is apparent, therefore, that the amount of soluble carbohydrates found in most common legumes used for human consumption ranges from about 3.3 to 13.8%, depending on the variety and the type of legume. Fructose is present in very small amounts (traces – 0.4%) and is only found in lentil and faba bean. All of the legumes contain sucrose in a range between 0.1 and 6.9%, the lowest content being found in some varieties of faba bean and the highest in some varieties of chickpea. The α-galactoside content of the most common legumes used for human consumption ranges from 0.4 to 9.6%, with some chickpea and pea lines showing very high levels. Raffinose and stachyose are present in all legume seeds, ranging from 0.1 to 2.5% and 0.2 to 5.2%, respectively, the highest levels of stachyose being found in chickpea and pea. Ciceritol is found only in chickpea and lentil and ranges from 0.4 to 3.1%. Verbascose is present in variable amounts according to species and varieties, with some varieties of legumes having no, or only traces of, verbascose, while others, such as lentil and chickpea, have quite high amounts (4.2–4.5%). Starch is the main carbohydrate present in bean, chickpea, lentil, pea and faba bean, the content varying between species and between varieties. Wrinkled peas (see Chapter 7) have the lowest starch content and on average, bean and chickpea have the highest starch content. The content of DF for the most common grain legumes used for human consumption is very high, ranging from 11 to 27.5%. The soluble component is abundant in bean and faba bean, while the highest insoluble DF was found in pea. 3.2.2 The content of carbohydrates in grain legumes used for animal nutrition Grain legumes utilized in Europe for animal nutrition vary widely in terms of the content and composition of sugars, ranging from about 5% in faba bean to about 13% in yellow lupin (Table 3.4). Total soluble sugars contain only a small amount of monosaccharides and from 1 to 3% of sucrose. Soluble sugars consist mostly of α-galactosides. The lowest content of αgalactosides was found in faba bean seeds (less than 2.5%), an intermediate 68 H. Kozlowska et al. Table 3.4. Content of soluble carbohydrates (SC) in seeds of different legume species (as a %). Total Soluble α-galacto- carbohySucrose Raffinose Stachyose Verbascose sides drates Starch Peaa Faba beanb White lupinc Yellow lupind Narrow leaf lupine Chickpeaf Lentilg 1.8 2.5 2.8 0.8 0.2 0.7 2.5 0.9 6.6 1.7 1.4 0.5 4.9 2.4 7.9 6.8 4.9 10.7 47.5 41.3 ND 1.1 2.2 6.9 2.8 11.9 13.0 ND 1.4 1.4 5.2 2.0 8.6 10.0 ND 3.5 1.9 0.6 0.3 1.9 1.7 ND 0.4 8.2 4.7 11.7 6.6 52.6 46.2 aFrejnagel et al. (1997); Gdala and Buraczewska (1997) (mean of 19 varieties). and Buraczewska (1997a,b); Zdu4czyk et al. (1997) (mean of five varieties). cZdu4czyk et al. (1996) (mean of three varieties). dZdu4czyk et al. (1994). eTrugo et al. (1988). fFrias et al. (1998). gFrias et al. (1994) (mean of 16 varieties). ND, not detected. bGdala level in pea (about 5%), and the highest level in lupin (up to 12%). The seeds of white lupin are characterized by having very small amounts of verbascose, their main sugar being stachyose. The highest amounts of verbascose were found in the seeds of yellow and blue lupin, pea and faba bean (25, 33 and 50%, respectively) as a proportion of the total α-galactosides. Starch is the main carbohydrate present in pea and faba bean, which are used commonly for animal nutrition. Only in lupin seeds is the starch content below 1% DM. Abreu and Bruno-Soares (1998) analysed the chemical composition of nine legume species and found the following starch contents: 45.3% for pea, 40.0% for faba bean, 0.8% for narrow leafed lupin and 0.7% for yellow lupin. The average starch content in the seeds of 36 different lines of feed peas analysed by Bastianelli et al. (1998) was 49.2%, while the average for six lines of wrinkled pea was only 29.4%. Apart from soluble sugars and starch, grain legume seeds are an important source of dietary fibre in the diets of animals. The data presented in Table 3.5 indicate that legumes utilized in animal feed differ in the content as well as the composition of DF. The highest amounts of DF occur in the seeds of Lupinus angustifolius (39.2%) and the main NSP components in this species are galactose and glucose. A lower proportion of DF is found in the seeds of Lupinus luteus, where glucose is the main component. Nutrition 69 Table 3.5. The content of dietary fibre (DF) and composition of non-starch polysaccharides (NSP) in seeds of different legume species (all data as g kg−1 of dry matter) (Gdala and Braczewska, 1997a; Gdala et al., 1997b,c). NSP Rhamnose Fructose Arabinose Xylose Mannose Galactose Glucose Uronic acid Total NSP DFa aNSP Pea Faba bean Lupinus luteus Lupinus angustifolius 0.9 0.4 19.4 7.3 0.9 8.6 47.6 15.0 172.4 179.2 0.8 0.1 16.4 13.0 0.7 18.3 45.3 14.6 208.6 229.6 0.5 0.3 12.6 11.3 1.9 16.5 89.0 12.3 309.0 325.0 0.8 0.3 10.7 8.3 1.6 34.9 31.5 12.0 365.0 392.0 + acid detergent lignin (ADL). Considerably smaller amounts of DF were noted in faba bean and pea seeds, 23 and 18%, respectively. 3.3 Physiological Effect of Grain Legume Carbohydrates in Animal Nutrition 3.3.1 Consumption of grain legume carbohydrates in feed The grain legume carbohydrate content in animal diets differs according to the species and age of animals, and in the amounts of particular grain legumes introduced into their diets. The proportion of grain legumes in diets of monogastric animals is rather limited for many reasons, but especially because of antinutritional factors. In practice, grain legumes are used occasionally as the sole high-protein component of pig diets. In the industrialized parts of the European Union, the mean levels for the incorporation of peas into pig, poultry and cattle diets are 20, 10 and 25%, respectively (Bourdillon, 1998). Grain legumes provide animal diets with small amounts of mono- and disaccharides, and with higher amounts of α-galactosides. They are most often used as a substitute for soybean meal, which also contains significant levels of α-galactosides. Toasted soybean meal contains about 5% DM of α-galactosides (Seve et al., 1989; Coon et al., 1990). Substituting soybean meal with meal from grain legumes brings about an increase in the α-galactosides content in animal diets for two main reasons. Firstly, much more grain legume meal than soybean meal is needed to obtain the same concentration of crude protein in the diet and secondly, some grain 70 H. Kozlowska et al. legumes, in particular lupins, have a much higher α-galactoside content than soybean. Table 3.6 presents examples of how the α-galactoside content changes according to the proportion of soybean meal and different substitutes in the diet. In diets with the standard content of soybean meal (16%), the content of α-galactosides is about 8 g kg−1. Only the diet containing faba bean has a lower amount of α-galactosides. Substituting soybean meal with pea and lupin causes an increase in the α-galactoside content from about 8 to 16–20 g kg−1, while substituting about 50% of the soybean meal with seeds of pea and lupin still gives an α-galactoside content in the diets of growing pigs that is higher than the amount found in the standard soybean diet. Other dietary components also contain α-galactosides. According to Carré et al. (1984), the content of α-galactosides in diets for poultry usually ranges from 0.5 to 3%, with the main sources being, in decreasing order, soybean meal (6%), peas (5%), faba beans (4%), rapeseed meal (3%) and sunflower meal (2%) of the dry matter. In animal diets, pea and faba bean meal is a source of legume starch. In the case of total substitution of soybean meal with pea meal, legume starch composes about 15% of the diet. When soybean meal is partially substituted with the pea and faba bean meal, the proportion of legume starch in the diet amounts to 10 and 6%, respectively. Unlike the seeds of pea and faba bean, lupin seeds contain high amounts of NSP, which constitute 27–35% of the seed dry matter in L. luteus and 35–42% in Lupinus albus (Gdala and Buraczewska, 1997). For this reason, diets rich in lupin seeds contain higher amounts of NSP than diets comprising pea, faba bean and soybean meal. Bioavailability of starch and non-starch polysaccharides from legume seeds can have a significant influence, therefore, on the utilization of energy from the diet. Table 3.6. Content of α-galactosides (GAL) in diets for pigs with the share of different components rich in crude protein (CP). Content in feeda (g kg−1) Soybean meal Pea Faba bean Yellow lupin Narrow leaf lupin White lupin aData Share in dietb (%) GAL content in diet (g kg−1) CP GAL A B A B 440 214 259 375 328 333 49.0 49.4 24.2 104.8 75.5 78.9 16 33 27 19 21 21 6–8 20 15 10 10 10 7.8 16.4 6.5 19.9 15.8 16.6 – 13.1 7.1 14.0 12.4 12.9 for soybean meal according to Seve et al. (1989) and Coon et al. (1990). legumes share in diet: A – total substitution of soybean protein, B – partial (about 50%) substitution of soybean protein. bGrain Nutrition 71 3.3.2 Effect of mono- and disaccharides in animal nutrition The monosaccharides and the main disaccharide, sucrose, are normally fully absorbed in the small intestine. In the case of pigs (Canibe and Bach Knudsen, 1997; Gdala and Buraczewska, 1997a,b) and poultry (Carré et al., 1995), the apparent ideal digestibility of sucrose from a diet containing pea is nearly 99%. Sucrose is readily hydrolysed by sucrase activity located on the brush border membrane of enterocytes in the gut and the released glucose and fructose are fully absorbed. The mono- and disaccharides from grain legumes are not a significant source of feed energy, because they are present in only small amounts. 3.3.3 Effect of oligosaccharides in animal nutrition The first information about antinutritional effect of α-galactosides was noted by Kuriyama and Mendel (1917). They reported that test meals of 3 or 5 g of raffinose fed to fasting rats resulted in severe diarrhoea with evidence of raffinose residues in the faeces. More recently it has been shown that the intestinal mucosa of monogastric animals and humans lacks the α-galactosidase enzyme required to cleave α(1→6) linkages (Gitzelman and Auricchio, 1965). Oligosaccharides of the raffinose family, which contain α(1→6) linkages between α-galactose units and α-galactose and sucrose, therefore, cannot be hydrolysed endogenously and can be classified as non-digestible carbohydrates. It is generally accepted that these oligosaccharides pass undigested into the lower gut of the animal, where they are metabolized by gas-producing bacteria (Rackis, 1975). Carré et al. (1991) found an apparent α-galactoside digestibility of 82–87% in chicken, suggesting extensive microbial fermentation in the lower gastrointestinal tract of the birds. In addition, it has been found that the apparent digestibility of α-galactosides in broiler chicken was 86.7%, compared with 99% for adult cockerels (Carré et al., 1995). This indicates that the bacterial degradation of α-galactosides in the digestive tract of adult cockerels is higher than in the digestive tract of young chickens. Also, in the case of pigs, α-galactosides undergo intensive microbial fermentation, especially in the large intestine (Krause et al., 1994). Since the net efficiency of digestible energy utilization via hindgut fermentation is 70% of that of the glucose absorbed in the upper intestine (Müller et al., 1989), the net energy value of legume seeds, which contain high amount of α-galactosides, is low. For this reason, the considerable difference in apparent digestibility of α-galactosides between chicken and adult cockerels (86.7 and 99.0%, respectively) results only in 8.7% higher energy supplied by α-galactosides for the adult birds (Carré et al., 1995). More recent research has suggested that the assumption that oligosaccharides are not digested in the stomach and small intestine may need to be 72 H. Kozlowska et al. reconsidered. In a study where piglets were fitted with a cannula at the terminal ileum, it was found that 39% of the raffinose oligosaccharides disappeared from the stomach and small intestine 3 h after feeding and reached 86–90% at the terminal ileum (Gdala et al., 1997b). This was higher than an earlier report where 75% of the raffinose oligosaccharides was found to disappear when feeding piglets with pea (Aumaitre et al., 1992). This relatively high amount of digestion in the upper intestine is most likely due to endogenous plant and microbial α-galactosidases (Gdala et al., 1997a). Studies of many authors (e.g. Brenes et al., 1992; Veldman et al., 1993 and Gdala et al., 1997a) have shown that intestinal digestion of α-galactosides can be increased by supplementation of diets with exogenous α-galactosidase (Table 3.7). It has been shown that the addition of pectinase and α-galactosidase to broiler chicken diets tends to improve growth, the apparent metabolizable energy increasing from 12.13 to 12.55 MJ kg−1 (P = 0.06) (Igbasan et al., 1997). In contrast, Daveby et al. (1998) reported that supplementation with α-galactosidase significantly increased the cumulative feed intake of the milled diets obtained in the case of chicken, without any apparent effect on the digestibility of the raffinose oligosaccharides. Supplementation of diets with exogenous α-galactosidase does not eliminate other negative effects of α-galactosides presence in the diet, especially when the content of these sugars is high. Veldeman et al. (1993) reported that the increase in fermentable substrate in the lower part of the digestive tract might lead to disturbances of the existing microbial balance, increasing the chance of diarrhoea. The addition of α-galactosidase (7.1 U g−1) to experimental diets containing 2.75% of α-galactosides could not overcome these problems. A high content of raffinose in the diet (> 6.7%) results in osmotic catharsis, Table 3.7. Ileal digestibility of α-galactosides (GAL) in pigs fed on a diet, without (−) or with (+) α-galactosidase supplementation. α-Galactosidase supplementation Ileal digestibility (%) Raffinose Stachyose Verbascose Raffinose Stachyose Verbascose Raffinose (whole lupin) Stachyose (whole lupin) Raffinose (dehulled lupin) Stachyose (dehulled lupin) − + 70.2 88.8 74.2 40.7 60.1 77.8 31.9 10.4 41.6 23.2 97.4 99.5 98.0 95.7 87.2 86.3 75.3 37.3 86.0 65.0 References Gdala et al. (1997b) (26.7 g GAL kg−1) Gdala et al. (1997b) (37 g GAL kg−1) Brenes et al. (1992) Nutrition 73 which causes a portion of the raffinose to be lost before it can be hydrolysed by microbes (Wagner et al., 1976). It has been shown that rapid intestinal transit of digesta with a high content of α-galactosides (5.3% DM) results in a decrease of 20% in true metabolizable energy, compared with a diet containing only 1% DM of α-galactosides (Coon et al., 1990). Removal of oligosaccharides from soybean meal with an 80% ethanol extraction resulted in a lower acidic caecal content and a longer (about 50%) transit time for the diet. Wiggins (1984) reported that the raffinose family of oligosaccharides (RFO) effect the absorption of nutrients by changing the osmotic pressure in the small intestine. A high content of α-galactosides, therefore, will result in a reduction in the absorption capacity of the small intestine (Zdunczyk et al., 1999). The inclusion of an oligosaccharide extract (4 or 8%) from lupin seeds to perfusion fluid, i.e. the amount present in a 24-h diet, strongly depressed the intestinal absorption of glucose, methionine and water, as measured in situ using the perfusion technique (Table 3.8). It is possible that this affect may be due to other lupin seed components extracted together with the oligosaccharides. A high content of fructooligosaccharides in perfusion fluid, however, did not depress the absorption of nutrients from the intestine of rats. In the experiments presented above, considerably higher amounts of oligosaccharides than those occurring in practical feeding of animals were applied. In the practical feeding of pigs, the content of α-galactosides does not exceed 2% of the DM and it is known that a low α-galactoside content in the diet can significantly decrease their negative effects. In pig diets, where more realistic proportions of α-galactoside were included as soybean meal (1.21% of α-galactosides), or water-extracted soybean meal (0.16% of α-galactosides), there were no differences in the growth performance, feed efficiency, nitrogen digestibility and retention (Seve et al., 1989). Leske et al. (1993), however, confirmed that increasing the amount of raffinose (above 0.45%) in diets of leghorn roosters decreased the true metabolizable energy and DM digestibility. There is also evidence that the α-galactosides found in soybean can have a negative effect on protein utilization. The protein efficiency ratios determined for chickens fed diets containing Table 3.8. Absorption of nutrients (mg rat−1 h−1) from perfusion fluid supplemented with fructo-oligosaccharides (FOS) or oligosaccharides (OS) from lupin seeds, administered to rat small intestine (Zdu4czyk et al., 1999). Glucose Methionine Water Control fluid FOS −4% FOS −8% OS −4% OS −8% 63.7 44.2 6.8 74.7 37.2 8.7 72.2 25.8 6.6 24.4 17.2 0.7 14.3 18.7 1.2 74 H. Kozlowska et al. soybean meal, or ethanol-extracted soybean meal, was 2.29 and 2.92, respectively (Leske et al., 1995). It is evident, therefore, that to improve the nutritional quality of grain legumes for non-ruminant animals, the level of the raffinose series oligosaccharides should be reduced, either by plant breeding, the extraction of seeds, or by using microbial α-galactosidase. 3.3.4 Effect of starch in animal nutrition Starch is cleaved in the duodenal cavity by secreted pancreatic α-amylase to give a disaccharide (maltose), a trisaccharide (maltotriose) and branched α-dextrin (Gray, 1991). These final oligosaccharides are further hydrolysed by the complementary action of three integral brush border enzymes at the intestinal surface, glucoamylase (maltase–glucoamylase, amyloglucosidase), sucrase (maltase–sucrase) and α-dextrinase (isomaltase). Glucose, the final product of starch digestibility, is transported via the portal blood to the liver and, subsequently, to the general circulatory system. Starch is the primary energy source in diets that contain cereals and grain legumes. There is good evidence that for monogastric animals, the digestibility of legume starch is lower than that of cereal starch. The results of assessing starch bioavailability in the upper gastrointestinal tract of colectomized rats, indicated that there are highly significant differences for starch digestibility between legume and cereal starch. It was reported that 15.2% of pea starch was recovered in the ileal digesta of rats compared with 0.2% of rice starch (Hildebrandt and Marlett, 1991). It was found that the ileal digestibility of legume starch is about 90%, whereas the digestibility of starch derived from cereals is nearly 100%. In addition, the digestibility of isolated starch is higher than that of starch within milled seeds (94.4% versus 84–92%, Table 3.9). Maize and wheat starches were digested better in the distal ileum by chick (97.8 and 97.6%, respectively) than pea starch pea (94.4%). The preparation of semi-purified starches for chicken feed has shown that it is not the physical entrapment of starches within the plant cell walls that limits their digestion, but rather the nature of the starches per se (Yuste et al., 1991). In general, legume starch contains between 30 and 40% Table 3.9. Ileal digestibility of starch in young chicks. Source of starch Pea seeds Pea starch Wheat starch Maize starch Apparent digestibility (%) 84–92 94.4 97.6 97.8 References Carré et al. (1987, 1991) Yuste et al. (1991) Yuste et al. (1991) Yuste et al. (1991) Starch was extracted from seeds according to the methods of Faulkes et al. (1989). Nutrition 75 amylose and 60–70% amylopectin compared with cereal starches, which, in general, have 20–25% amylose and 75–80% amylopectin. It has been found that the high amylose/amylopectin ratio correlates with the lower digestibility of legume starch. It has been suggested that the difference in the digestibility rate between high amylose and high amylopectin starches could be due to the larger surface area of amylopectin, which may make it more available for amylolytic attack (Thorne et al., 1983). This could be one explanation for the difference in the digestibility of starch from peas with the rr genotype, which are characterized by a high amylose content and have a digestibility of 75.2% compared with 94.4% for normal peas (Carré et al., 1998). It can also be suggested that the lower rate of grain legume starch digestion may be due to the differences in starch granule structure between legumes and cereals (see Chapter 4). Grain legumes also contain high levels of antinutrients (e.g. enzyme inhibitor, tannins, lectins, and phytic acid) compared with cereals and these compounds will often form complexes with nutrients such as starch, which can reduce its digestibility (Thorne et al., 1983; Jenkins et al., 1987). The digestibility of pea starch is higher than faba bean starch, both declining as the percentage of legume seeds increases in the diet (Table 3.10). Coefficients of starch digestibility for different faba bean varieties have been shown to range from 63.8 to 85.8% (Lacassagne et al., 1988, 1991) and for raw pea seeds from 80.9 to 92.2% (Longstaff and McNab, 1987; Conan and Carré, 1989; Carré et al., 1991). Another possible cause of the low digestibility of legume starch (below 80%) may be insufficient crushing of the seeds, which could reduce the accessibility of starch particles to digesting enzymes (Carré et al., 1991; Lacassagne et al., 1991). Coarse particles (> 0.5 mm) of excrement have been shown to contain the major part (73%) of undigested starch (Carré et al., 1991). Grinding seeds to a small particle size (mean 0.5 or 0.16 mm) increased the starch digestibility coefficients of two faba bean varieties, fed to adult cockerels, from 70.3 to 90.2% and from 63.8 to 80.4%, respectively (Lacassagne et al., 1991). Likewise, in a study conducted on chickens, the digestibility of starch from different dehulled pea meal fractions was 95.7 Table 3.10. Ileal digestibility of starch in pigs. Source of starch Pea Pea Pea Faba bean Faba bean Faba bean Share in diet (%) Apparent digestibility References 45.1 66.1 73.7–83.1 30.1 30.1 62.1 94.4–99.1 88.9–92.9 85.2–87.3 97.9 96.3 – 98.1 81.5 – 86.4 Bengala Freire et al. (1991) Canibe and Bach Knudsen (1997) Gdala and Buraczewska (1997a,b) Van der Poel et al. (1992) Jansman et al. (1993) Gdala and Buraczewska (1997a,b) 76 H. Kozlowska et al. and 84.4% for fine (< 100 µm) and coarse (> 100 µm) particle fractions, respectively (Carré et al., 1998). The nutritional value of diets rich in pea and faba bean starch can be improved through heating or autoclaving (Longstaff and McNab, 1987; Conan and Carré, 1989). Pelleting has been shown to increase starch digestibility from 84% to over 95% (Carré et al., 1991). Since starch digestibility correlates well (r 2 = 0.80) with the true metabolizable energy obtained from pea (Longstaff and MacNab, 1987), it is apparent that pelleting will increase the metabolizable energy of diets (Table 3.11). Pelleting is the usual process in the feed industry and is very useful for poultry feed, because chickens have a reduced feed intake when their diet is finely ground. Similarly, extrusion has a beneficial effect on the nutritional value of diets containing grain legumes. Extrusion increases the in vitro rate of hydrolysis of starch by pancreatic amylase, and stimulates the activity of amylase, chymotrypsin and carboxypeptidase A in the pancreatic tissue. As a consequence, the apparent digestibility of starch in the ileum has been to increase from 94.4 to 99.1% (Bengala Freire et al., 1991). 3.3.5 Effect of non-starch polysaccharides (NSP) in animal nutrition In general, legume seeds are characterized by having a relatively high level of structural polysaccharides, mainly comprising cell wall material. As a proportion of the total carbohydrate content of the seed, these compounds constitute on average from 73 to 84% in different lupin species, 27% in faba beans, and 25% in peas (Gdala, 1998). Most NSP are degraded mainly in the hindgut, where they undergo microbial fermentation providing energy for the animals (Van Engelhard et al., 1989). The energy derived from this hindgut fermentation is about 70% of that produced by enzymatic digestion in the small intestine (Müller et al., 1989; Jorgensen et al., 1996). Very few studies have reported high digestibility of pea NSP in the digestive tract of pigs (Goodlad and Mathers, 1990; Canibe and Bach Knudsen, 1997). There are reports that the ileal digestibility of NSP of Table 3.11. Nutritional value of meal and pelleted diets with pea seeds (Peyronnet et al., 1996). Digestibility of protein (%) Digestibility of starch (%) Metabolizable energy (kcal kg−1 DM) DM, dry matter. Meal (n = 25) Pellets (n = 11) 78.4 90.5 2870.5 85.5 98.5 3150.5 Nutrition 77 legume seeds depends on the age of the pigs and on the species of legume seed, and ranges from 12.1 to 39.9% (Table 3.12; Gdala et al., 1997a,b). The differences in digestibility coefficients presented in Table 3.12 result from differences in the grain legume species for the level and composition of the NSP. In pea and faba bean seeds the monosaccharide residues of glucose, arabinose and uronic acids dominate the NSP fraction (Gdala and Buraczewska, 1997a). Galactose and glucose are the main components of lupin seed NSP (in total 60–66%), while uronic acids, arabinose and xylose have intermediate levels (Gdala and Buraczewska, 1997a). Most of the arabinose in pea and faba bean is present as arabinose-containing pectin substances in the cell walls of the cotyledons (Selvendran, 1984). Pectins are more rapidly and extensively digested in the large intestine compared with cellulose, arabinoxylans and xylan polysaccharides (Gdala et al., 1997b). Pigs are better at utilizing the energy derived from seeds containing a high level of NSP. Poultry are able to digest only the water-soluble fraction of NSP, while the water-insoluble fraction remains virtually undigested (Carré et al., 1998). The NSP digestibility for pea diets is about 5.9% in adult cockerels and only 2.8% in chickens (Carré et al., 1995). The apparent ileal digestibility of DF of milled, or crushed, dehulled peas by cockerels and chickens was 15 and 8%, respectively (Daveby et al., 1998). Generally, NSP are the main constituents of the DF fraction in grain legumes and a high content of DF in diets has a negative effect on nutrient digestibility in animals (Freire et al., 1997). The insoluble DF fraction decreases intestinal transit time, increases faecal bulk, delays glucose absorption and slows starch hydrolysis. The water-soluble fraction of DF increases the viscosity of the digest in the small intestine, depressing nutrient absorption (Low, 1985). Dietary fibre can be a negative factor, therefore, that dilutes the energy content and decreases the nutrient availability for animals. The soluble DF usually comprises about one-third of the total DF in cereals, whereas in pea it is about 25% and about 32% in lupin (Bach Knudsen, 1997). It has been demonstrated that a 1% increase of crude fibre Table 3.12. Ileal digestibility of non-starch polysaccharides (NSP) in young pigs (Gdala and Buraczewska, 1997a,b; Gdala et al., 1997a,b). NSP residues Peaa Faba beanb Yellow lupin Narrow leaf lupin Arabinose Xylose Mannose Galactose Glucose Uronic acid Total NSP 36.3 26.3 – 44.8 4.7 35.8 39.9 43.7 59.7 – 61.7 32.8 25.9 38.8 39.2 −8.0 86.0 42.1 0.3 25.9 14.4 23.6 −6.9 99.8 25.4 26.5 26.5 12.1 aAverage bAverage for two white flowered varieties. for two coloured flowered varieties. 78 H. Kozlowska et al. in the diet diminished the digestibility of gross energy by 1.3% and the utilization of metabolizable energy by 0.9% in pigs (Just et al., 1983). Many authors have reported that dehulling seeds results in a lower DF content and a higher nutritional value. The crude protein content of dehulled white lupin seeds has been shown to increase by 20% and the crude fibre content to decrease by 67% (Flis et al., 1997). As a consequence of changes in the proportion of nutrients, the nutritional value of dehulled seeds of L. albus and L. angustifolius for pigs was increased by 5–10 and 25%, respectively (Fernandez and Batterham, 1995; Flis et al., 1997). Dehulling lupin seeds has been shown also to increase the digestibility of energy in chickens by 18% (Brenes et al., 1993). In addition, enzyme supplements added to legume seed diets for chickens also have a positive effect on bird performance (Brenes et al., 1993). In the case of pigs, enzyme supplementation of diets is less effective compared with poultry because of more intensive fermentation of sugars in the hindgut (Bedford et al., 1992). 3.3.6 Effect of grain legume carbohydrates in ruminant nutrition There is limited information on the physiological effect of legume seed carbohydrates in ruminant animals, although it is assumed that they are well utilized. Information on the digestible energy and gas production by rams fed with grain legumes is presented in Table 3.13. Differences in the content and composition of carbohydrates are not reflected in clear differences in the amount of digestible energy derived from seeds of different legume species. For example, the level of digestible energy in pea seeds, which contain a high level of starch and relatively low levels of α-galactosides and NSP, was similar to that found in lupin seeds, which contain very little starch and have large amounts of α-galactosides and NSP. The main disadvantage in the use of grain legumes for ruminants seems to be the high nitrogen degradability in the rumen leading to poor Table 3.13. Carbohydrate content, the digestibility of energy and gas production for different legume seeds (Abreu and Bruno Soares, 1998). (%) (MJ kg−1) Gas production (ml g−1 DM) 89.7 90.5 84.5 82.0 16.5 16.3 16.9 16.5 146.7 112.0 95.9 104.6 Digestibility of energy Total sugarsa Pea Faba bean Yellow lupin Narrow leaf lupin aExpressed bWICW 3.7 1.9 4.1 4.5 Starch WICWb 45.3 40.0 0.7 0.8 17.1 20.1 35.3 38.0 as saccharase (% dry matter, DM). – water-insoluble cell wall components. Nutrition 79 nitrogen values, which make grain legumes uncompetitive with other protein sources (Gatel and Champ, 1998). The problem of ruminal degradability of grain legume protein has been the subject of many recent reports (Makkar et al., 1997; Poncet et al., 1998). There is a suggestion that heat treatments, e.g. autoclaving or extrusion, may be useful for improving the nitrogen value of lupin or pea protein for ruminants (Le Guen et al., 1997). 3.4 Physiological Effect of Grain Legume Carbohydrates in Human Nutrition 3.4.1 Nutritional classification of grain legume carbohydrates According to their role in plants, carbohydrates can be separated into three groups, the mono- and disaccharides are a source of energy for growth, the oligosaccharides and starch are storage carbohydrate and the noncellulosic polysaccharides, pectins, hemicellulose and cellulose comprise the structural components of the cell walls. From a human nutrition point of view, carbohydrates can be classified into two groups, available carbohydrates, which are enzymatically digested in the small intestine, and unavailable carbohydrates, which are fermented by microflora in the large intestine (Table 3.14). The available carbohydrates comprise the mono- and disaccharides and starch, while the unavailable carbohydrates contain the oligosaccharides and the structural components. The mono- and disaccharides are almost completely digested in the small intestine. Sucrose (a disaccharide) is hydrolysed to its constituent monosaccharides by the sucrase enzymes on the erythrocyte surface membrane. The digestion of starch starts in the mouth, by the enzyme amylase secreted in the saliva, and is continued in Table 3.14. Classification of legume saccharides (Southgate, 1992). Product of Site of digestion digestion Physiological classification Mono- and disaccharides Starch; amylose and amylopectin α-Galactosides Small intestine (enzymatic) Small intestine (enzymatic) Large intestine (microbial) Available carbohydrates Available carbohydrates Unavailable carbohydrates Non-cellulosic polysaccharides: pectins, hemicellulose, cellulose Large intestine (microbial) Role in the plant Type of saccharide Source of energy Storage polysaccharides Storage oligosaccharides Structural components of plant cell walls Mono- and disaccharides Mono- and disaccharides Short chain fatty acids: acetate, propionate, butyrate Carbon dioxide, hydrogen methane Unavailable carbohydrates 80 H. Kozlowska et al. the upper small intestine by enzymes secreted by the pancreas. As with monogastric animals (see Section 3.3.4), starch can be readily digested in the human gastrointestinal tract. A part of the consumed starch, especially found in products subjected to hydrothermal processing, is not digested in the small intestine and passes to the large intestine as ‘resistant starch’, which will be discussed later. The absence of α-galactosidases in mammalian intestinal mucosa, which cleave the α(1→6) galactose linkage present in raffinose and other α-galactosides (Gitzelman and Auricchio, 1965), results in these compounds passing into the large intestine. These oligosaccharides are then broken down to monosaccharides by bacterial enzymes, with the production of hydrogen and methane gas. Recent research shows that about 30% of the oligosaccharides (raffinose and stachyose) in diets are degraded in the stomach and the small intestine. The assumption that oligosaccharides are not digested in the stomach and the small intestine, therefore, must be reconsidered (Sandberg et al., 1993). Despite this apparent partial degradation in the stomach and small intestine, α-galactosides are still included amongst the unavailable carbohydrates, since they are not absorbed in the small intestine (Wiggins, 1984). In healthy subjects less than 1% of the oligosaccharides are absorbed and when injected directly into the bloodstream they are almost completely recovered (Wheeler et al., 1978). The other major components of the unavailable carbohydrate group are the structural components of the plant cell walls, often collectively termed DF and described earlier. An additional component of this group is resistant starch. Resistant starch was not recognized until 1982 (Englyst et al., 1982). Prior to this time the prevailing concept was that starch was completely digested and absorbed. The nutritional properties of starch in foods are to a large extent related to its availability for digestion and/or absorption in the gastrointestinal tract (Björck and Asp, 1994). From this point of view, starch can be classified into three basic groups: rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS) (Table 3.15). The digestibility varies according to the plant species and often within a species, and relates to the chemical and physical structure of the starch Table 3.15. In vitro nutritional classification of starch (Englyst et al., 1992). Starch type Source Rapidly digestible starch Slowly digestible starch Resistant starch Freshly cooked starchy foods Most raw cereals Partially milled grain and seeds; raw potato and banana; cooled cooked potato, corn flakes and bread Digestibility in small intestine Rapid Slow – but complete Resistant Nutrition 81 granules. Digestibility also depends on the methods used for preparing food prior to consumption. Isolated raw starch of pea is almost completely digested in the small intestine of rats (Table 3.16; Berggren et al., 1995). During technological treatment (heating, freezing, etc.), the physical structure of the starch is degraded and, with time, becomes restructured, or retrograded, in a non-digestible form (see Chapter 4). This retrograded part of starch and the starch that is not digested in the small intestine for other reasons, for example too little time for digestive enzymes to act on starch granules, passes to the large intestine as RS. RS can, therefore, be defined as ‘the sum of starch and products of starch degradation not absorbed in the small intestine of healthy humans’ (Asp, 1992). Relatively few studies have tried to quantify the amount of RS in the small intestine of healthy humans. Table 3.16 presents the content of RS in legume products, estimated by using either direct (in vivo) methods, including the analysis of starch in ileal effluents from ileostomy patients (Jenkins et al., 1987; Schweizer et al., 1990; Steinhart et al., 1992; Muir and O’Dea, 1993), ileal incubation experiments (Noah et al., 1998), balance Table 3.16. starch). The content of resistant starch (RS) in legume products (g (100 g)−1 of Legume products Methods of RS determination Raw pea starch Pre-cooked lentil Pre-cooked red bean Canned pea Weight experiment on ratsa Weight experiment on ratsa Weight experiment on ratsa Colectomized rats 1.0 11.0 8.0 15.2 Canned peab Weight experiment on ratsa and three methods in vitro In vivo, in ileostomists In vivo, in ileostomists In vivo, intubation experiments in healthy subjects In vivo, in ileostomists 27.8 5.7 Muir and O’Dea (1993) In vivo, in ileostomists 20.9 Schweizer et al. (1990) In vitro digestibility of starch In vitro based on chewing In vitro based on chewing In vitro based on chewing 16.5 Cummings and Englyst (1995) Äkerberg et al. (1998) Äkerberg et al. (1998) Äkerberg et al. (1998) Boiled red lentil Boiled white beans Autoclaved white beans Autoclaved white beans Boiled lentil Boiled red lentil Boiled white beans Autoclaved white beans aRats bPea RS content References 13.8 13.6 16.5 23.1 16.7 13.8 Berggren et al. (1995) Tovar et al. (1992) Tovar et al. (1992) Hildebrandt and Marlett (1991) Björck and Sijeström (1992) Steinhart et al. (1992) Jenkins et al. (1987) Noah et al. (1998) treated with antibiotics to suppress hindgut fermentation. with low content of starch – 19% dry matter (DM). 82 H. Kozlowska et al. experiments in rats with suppressed hindgut microflora (Tovar et al., 1992; Berggren et al., 1995), analysis of the ileal excreta in colectomized rats (Hildebrandt and Marlett, 1991), or indirect(in vitro) methods (Cummings and Englyst, 1995; Åkerberg et al., 1998). At present, results of these studies are too fragmentary to allow the quantity of RS for starches of different grain legumes to be estimated satisfactorily. Gray (1991), however, has suggested that in processed legume seed the content of RS amounts to 10.3% of total starch. The results of other studies, presented in Table 3.16, indicate that the RS content may be closer to 15% of total starch. A higher content of RS was noted in grain legumes processed differently in the Italian diet (Brighenti et al., 1998); dried, canned and frozen seeds of beans, peas, lentils and chickpeas containing 11.6, 12.4, 11.4 and 10.9% RS, respectively. In this study, the average RS content in grain legumes was about 11.6% compared with 3.2 and 5.7% for cereals and potatoes. As a proportion of total starch, the RS content of grain legumes in this study was about 20% (Brighenti et al., 1998). 3.4.2 Consumption of grain legume carbohydrates in food Immature seeds, dry seeds and processed seeds are all used for food purposes. Most grain legumes, after simple processing (sprouting, cooking), are consumed as vegetables, salads, soups, mashed seeds and cooked seeds. The annual consumption of grain legume carbohydrates can be estimated from the consumption and chemical composition of legumes. Examples of the average consumption of carbohydrates in grain legumes are presented in Table 3.17. From these data the average daily consumption in 1996, of α-galactosides, digested starch, RS and DF can be calculated and shown to be very low (0.26, 2.89, 0.51 and 1.33 g, respectively). A similar intake of digested starch and resistant starch from dried legumes (2.3 and 0.5 g, respectively) has been found in relation to the Italian population (Brighenti et al., 1998). Also, the daily intake of starch and RS in fresh, frozen and canned grain legumes was 1.8 and 0.6 g, respectively (Brighenti et al., 1998). The calculated amounts given in Table 3.17 do not take into account the considerable losses that occur during food preparation. For physiological reasons, decreasing the α-galactoside content during preparation of the seeds for consumption is very important, as will be discussed later. The intake of α-galactosides in legume dishes in the Czech Republic and in neighbouring countries are presented in Table 3.18, after taking into consideration the losses during the preparation of legume dishes. Considering the amount of starch in pea seeds (c. 45%), the RS content in one portion of pea soup can range from 1.3 to 1.9 g. In one portion of mashed pea (75–105 g), the content of total starch and RS ranges from Nutrition 83 Table 3.17. Annual consumption of α-galactosides, starch, resistant starch and dietary fibre in legumes, in Europe (data for 1996). Beans Annual consumption of legumes (kg per person) Average content of (g kg−1) α-galactosidesa Starcha Resistant starch Dietary fibre Annual consumption of (g) α-galactosides Starch Resistant starch Dietary fibre Pea Lentil Chickpea Total 1.14 0.67 0.43 0.19 2.43 36.8 549.3 82.4 b193.3b 49.4 475.0 72.3 b187.9c 28.4 461.8 69.3 b214.0d 47.0 526.0 78.9 b245.5e 39.6 511.5 77.0 199.5 42.0 626.2 93.9 220.4 33.1 318.3 48.4 125.9 12.3 198.6 29.8 92.0 8.9 99.9 15.0 46.6 96.3 1243.0 187.1 484.9 Resistant starch is 15% of the total starch. aMean content, see Table 3.3. bMean content, see Table 3.4. cMean of 15 cultivars (Zdu4czyk et al., 1997). dSavage (1988). eSidduraju et al. (1998). Table 3.18. Estimated intake of α-galactosides (GAL) in legume dishes (g per dish) (Pokorny and Dostalova, unpublished). Dish Pea soup Lentil soup Bean soup Mashed peas Cooked lentils Cooked beans Dry legumes (g) Original GAL (g) Losses on cooking (%) Final intake of GAL (g) 20–30 13–20 20–30 75–105 75–112 75–110 1.5–2.3 0.7–1.0 0.6–0.9 5.6–7.9 4.0–6.0 2.5–4.0 10 10 10 70 70 70 1.3–2.1 0.6–0.9 0.5–0.8 1.7–2.4 1.2–1.8 0.7–1.2 32.0 to 44.9 g and 4.8 to 6.7 g, respectively. This is a significant amount considering that the daily consumption of RS in Western diets ranges from about 4 g (Dysseler and Hoffem, 1994), to as much as 20–30 g day−1 (Cummings and Englyst, 1989). The daily intake of total starch and RS in the Italian population diets reaches 21.4 and 8.5 g, respectively (Brighenti et al., 1998). Other studies, however, have reported considerably lower values for RS in the diet of European countries, ranging from 3.2 g day−1 in Norway to 5.7 g day−1 in Spain (Dysseler and Hoffem, 1994). Cummings et al. (1992) calculated a similar amount of NSP ingested daily (8–18 g) in Western diets. The information presented in Table 3.17 reveals that the 84 H. Kozlowska et al. daily intake of unavailable carbohydrates from grain legumes reaches 2.2 g day−1, including 12% for α-galactosides, 24.4% for RS and 63.1% for DF. 3.4.3 Physiological effect of available carbohydrates from grain legumes Relatively few studies have tried to quantify the digestibility of starch from legumes in the small intestine of humans (Wolever et al., 1986; Schweizer et al., 1990; Bothman et al., 1995). Numerous studies conducted on monogastric animals, however, have revealed that starch is easily digested in the upper intestinal tract, however, its digestibility coefficient is lower than that of cereal starch (see Section 3.3.4). This low digestibility of starch together with the low content of mono- and disaccharides and high content of unavailable carbohydrates (α-galactosides, RS and NSP), make legume seeds desirable component of human diets. It is apparent that the seeds from grain legumes are characterized by their relatively low glycaemic index (Table 3.19), which on average is less than half that of white and wholemeal bread. It is beneficial, therefore, to include grain legumes in diets for people with insulin-dependent diabetes, an illness that is often found in elderly inhabitants of industrialized countries (Wolever and Brand-Miller, 1995). Table 3.19. Glycaemic index (GI) of foods in normal subjects (Gray, 1991). Food type GI Cereal products Bread, white Bread, wholemeal Rice, white Rice, brown Spaghetti, white Corn flakes 69 72 66 66 50 80 Root vegetables Carrots Potato, new Potato, instant 92 70 80 Legumes Beans, navy Beans, kidney Beans, soya Peas Lentils 31 29 15 33 29 Nutrition 85 When grain legumes are used in diets as substitutes for animal products (in the case of vegetarians) they are believed to act in two ways. Firstly, to decrease the consumption of saturated fats and secondly to increase the content of unavailable carbohydrates in the diet, thus reducing the incidence of digestive tract cancers. A positive association with protein and fat (r = 0.60 and 0.62, respectively) and a weak negative association with NSP (r = −0.23) have been shown in a study of food consumption in 12 countries (Cassidy et al., 1994). A much stronger (r = −0.70) inverse correlation was found, however, between colorectal cancer incidence and starch intake. 3.4.4 Physiological effect of unavailable carbohydrates from grain legumes Within the group of unavailable carbohydrates, the highest proportion comprises the NSP, or ‘true dietary fibre’ (Burn et al., 1998). Apart from decreasing the bioavailability of many mineral components (Torre et al., 1991), DF in the diet provides many advantages. Increased plant fibre consumption is known to reduce blood lipid levels and is of particular interest, therefore, in the prevention and treatment of cardiovascular diseases (Mazur et al., 1990). It appears that DF may influence cholesterol metabolism in at least four ways (Wolever, 1995; Vanhoof and De Schrijver, 1997; Vahouny et al., 1988): (i) it may increase faecal bile acid excretion, resulting in increased cholesterol flux to bile acid synthesis with less cholesterol being available for lipoprotein synthetic pathways; (ii) it may alter the absorption of fat and cholesterol, either by binding bile acids or by increasing small intestine viscosity; (iii) it may reduce post-prandial insulin responses and, consequently, regulate cholesterol synthesis; and (iv) it results in the formation of short chain fatty acids, especially propionic acid, during fibre fermentation in the colon, which may decrease cholesterol in the liver. Faecal output is highly correlated with DF intake and inversely correlated with the time taken for materials to pass through the alimentary tract (‘transit time’). Thus, stools formed when the diet is rich in fibre are softer and more voluminous and pass more rapidly through the gut, than when the diet contains little fibre (Gurr and Asp, 1994). The effects of DF on the potential to reduce cancer risk in the large intestine are: dilution of carcinogens (via water-holding capacity); provision of surface for absorption of carcinogens; faster transit time, so less contact time; altered bile salt metabolism; and as a consequence of fermentation, lower pH, production of butyrate, altered microbial metabolism and lower ammonia levels in the gut. When NSP from pea were included in the diet of rats, a model for human nutrition, they were found to be associated with increased volatile fatty acid and 3-hydroxy butyrate concentrations in portal and heart blood 86 H. Kozlowska et al. (Goodlad and Mathers, 1990). Butyrate is considered to be a protective agent against colon cancer (Cummings and MacFarlane, 1991). Earlier studies have equated the term DF with unavailable (complex) carbohydrates, which are the sum of NSP and RS (British Nutrition Foundation, 1990). In comparison with DF, this would make starch a quantitatively important source of non-digestible carbohydrates. In fact, it has been claimed that RS, not DF, is the major substrate for fermentation in the human colon (Cummings and MacFarlane, 1991). During fermentation of RS, short-chain fatty acids (such as acetic, propionic and butyric) are formed (Björck et al., 1987). Introducing 15–45% cooked haricot beans (Phaseolus vulgaris) into rat diets caused an increase in the absorption of acetate (from 3.8 to 19.8 mmol day−1), propionate (from 1.2 to 7.5) and butyrate (from 0.5 to 2.1 mmol day−1), from the large bowel (Key and Mathers, 1993). It has been suggested that RS yields a larger proportion of butyric acid than DF (Silvester et al., 1995). In in vitro batch cultures, 29% of butyrate can be produced from RS, compared with 2–8% from NSP sources (Englyst and Cummings, 1987). An increased amount of butyric acid, which is the preferred energy source for colonocytes, can play an immunological role in relation to the development of colon disease. Butyrate has a protective effect in the rat model (McIntyre et al., 1993), while in human subjects, RS has been shown to reduce mucosal proliferation, the level of secondary bile acids and the mutagenicity of faecal water (Maunster et al., 1994). In transformed cells, terminal differentiation is induced, resulting in programmed cell death or apoptosis (Hague et al., 1993). Oligosaccharides have been classified with other non-digestible components (DF, RS) of the diet and collectively referred to as ‘fibre’, because of the similarities in response to intestinal enzymes. Oligosaccharides are known to be fermented mainly by beneficial intestinal microflora (Ferket, 1991), which may explain the difference in animal responses observed when oligosaccharides, rather than NSP, are included in the diet. They reach the colon and are quickly fermented by colonic bacteria, producing, in particular, gas and short-chain fatty acids. The gases are mainly carbon dioxide, hydrogen, and methane and are traditionally associated with the flatulence problems that are often linked with the consumption of pulses. Flatulence is poorly tolerated by Western populations who, in addition, might have a greater visceral sensitivity than populations more accustomed to this type of food (Gatel and Champ, 1998). The intake of grain legumes in northwestern and central Eastern Europe is rather low (about 2 kg per person per year), which, theoretically, should not cause digestive troubles if consumption is uniform during the year. They are usually consumed, however, only once or twice a week as soup (about 50 g of dry grains per portion), or as a vegetable consumed with meat, sausages or eggs (about 100 g of dry grains per portion). Such high amounts may cause problems such as flatulence and sometimes diarrhoea. It would be better to consume small amounts more often and Nutrition 87 Table 3.20. Consumer acceptance of a selection of dishes with added legumes (Dostalova et al., 1999a). Sensory acceptancea (%) Dish Legumes added Frankfurter soup Tomato soup Cabbage soup Salad with tunny Serbian salad Serbian salad Vegetable salad Mashed potatoes Mashed potatoes Potato salad Beans Soybeans Lentils Beans Chickpeas Beans Lentils Peas Peas Soybeans Amount of cooked legumes (%) Without legumes With legumes 6.6 9.1 7.6 9.3 6.2 8.1 5.4 5.4 9.3 6.2 77 80 79 71 81 79 70 52 74 59 80 78 63 78 88 88 85 71 74 65 aSensory acceptance scale: 0% = unacceptable, 100% = fully acceptable. Values (with legumes) in bold type represent a significant difference (P = 0.95) from the corresponding without legumes dish. perhaps to add small amounts of legumes (5–10% of the whole portion) to other conventional dishes (see Table 3.20). Such small amounts would not contain more than 0.1–0.3 g of α-galactosides per portion, which is not likely to cause digestive problems. B. Czukor et al. Processing 4 Processing 4 Editor: Bálint Czukor Contributors: Tatiana Bogracheva, Zsuzsanna Cserhalmi, Bálint Czukor, Jozef Fornal, Ildikó Schuster-Gajzágó, Erzsébet T. Kovács, Grazyna Lewandowicz and Maria Soral-Smietana Life is one long process of getting tired. Notebooks, chap. 1 (1912) Samuel Butler (1835–1902), English novelist and sometimes sheep farmer 4.1 Native Starch 4.1.1 Isolation Starchy legume seeds are rich in protein, starch and dietetic fibre, all of which are very valuable for food and non-food applications (Salunkhe et al., 1989). For this reason the processing of legume seeds includes the separation and production of these components. Studies that have led to the development of industrial processing of grain legume seeds have been carried out mainly on pea (Pisum sativum) and faba bean (Vicia faba). The processes that are generally used are dry processing (air classification) and wet processing. In general, the dry processing procedure produces protein-rich and starch-rich products, while the wet processing procedure produces purified protein, starch and dietetic fibre fractions. There are several advantages of the dry process: the construction of pilot plants is relatively simple, the process does not produce waste water and changes in the structure and functional properties of the components are minimized. Seed processing, which includes water extraction, is more complicated but the higher purity of the products produced allows a wider range of applications. In addition, the wet process is necessary for scientific research on ©CAB International 2001. Carbohydrates in Grain and Legume Seeds (ed. C.L. Hedley) 89 90 B. Czukor et al. starches, which demands a very high purity of starch. The basic principles of these two processes are described in more detail below. Dry processing It has been found that the air classification process can be carried out more successfully with grain legumes where starch is the main storage product rather than oil. Among the starchy grain legumes, air classification has been carried out on pea (P. sativum), faba bean (V. faba), mung bean (Vigna radiata), green lentil (Lens culinaris), navy bean (Phaseolus vulgaris), baby lima bean (Phaseolus lunatus) and cowpea (Vigna unguiculata). For detailed information, see Reichert and Youngs (1978), Bramsnaes et al. (1979), Talyer et al. (1981), Reichert (1982), Sosulski et al. (1985), Clott et al. (1986) and Sosulski and McCurdy (1987). The first stage of this process includes fine milling of the seeds. Flours prepared from starch-rich seeds contain two distinct populations of particles, which differ in both size and density and can be separated in a current of air. The starches and dietetic fibres are concentrated mostly in the light, fine fraction and the proteins and lipids in the heavy, coarse one. Repeating the air classifying process can increase the purity of the fractions, however it decreases the recovery of the products. Starch fractions with protein impurities as low as 2.5% can be produced; however, the recovery of the starch fraction is only about 40% (Reichert and Youngs, 1978). Following air classification of pea meal it has been found that the protein-rich fraction contains mainly storage proteins, while the starch-rich fraction contains other functional proteins, which adhere to the surface of the starch granules. The process of air classification is illustrated in Fig. 4.1, and the influence of the number of steps on the purification and recovery of the starch fraction from peas in Fig. 4.2. Detailed information on the processing of pea and faba bean by air classification has been described in a number of reports (Vose et al., 1976; Reichert and Youngs, 1978; Bramsnaes et al., 1979; Vose, 1980; Talyer et al., 1981; Reichert, 1982; Tyler and Panchuk, 1982; Wright et al., 1984; Clott et al., 1986; Clott and Walker, 1987; Uzzan, 1988; Salunkhe et al., 1989). Wet processing When legume seeds are processed for food applications the hulls are removed because it has been reported that they can contain antinutritional compounds that can be released during the extraction process (Sosulski and McCurdy, 1987; Uzzan, 1988). The dehulled seeds are then pin-milled. It has been found (Gueguen, 1983) that an average flour particle size of 100–150 µm is most suitable for further separation of the components. The next step of the process is protein extraction, which is carried out at alkaline pH. For round-seeded peas, pH 9–10 is most commonly used for extraction, while for wrinkle-seeded (those lines containing the r gene) peas it is usually higher (Schoch and Maywald, 1968; Vose et al., 1976; Colonna et al., Processing Fig. 4.1. 91 Dry process of starch extraction (Colonna et al., 1981). Fig. 4.2. The influence of the number of purification steps on the recovery of pea starch. Protein content, n; yield of starch, l. 92 B. Czukor et al. 1981; Sosulski and McCurdy, 1987; van der Poel et al., 1989; Hoover and Sosulski, 1991; Wiege et al., 1995; Salunkhe et al., 1989). In a more recently developed method (Bogracheva et al., 1995; Davydova et al., 1995), this procedure was carried out at a lower pH (8.5), which is much better for food applications, because stronger alkaline conditions may result in the appearance of antinutritional components in the protein products (Gueguen, 1983; Salunkhe et al., 1989). In the case of the r wrinkled-seeded peas, some applications require a better separation of starch from the other components and this is achieved by high-pressure disintegration (Meuser et al., 1995). The protein extract, which also contains soluble carbohydrates and emulsified lipids, is separated from the insoluble fraction and the proteins are isolated from this extract by acid precipitation or by ultra filtration. Protein fractions obtained from such procedures are commonly called protein isolates. The wet protein isolates are then dried. In industry this is usually carried out using spread dryers at temperatures of more than 100°C. The drying process is relatively quick and so the functional properties of the proteins are not affected significantly. Freeze-drying, however, is more appropriate when proteins are isolated for scientific purposes. The insoluble fraction, which is left after separation of the protein extract, includes starch, cell wall material, insoluble proteins and the remaining lipids. The basis for the further separation of these components depends on differences in their swelling properties. Starch granules have restricted swelling at room temperatures (Blanshard, 1987; Zobel and Stephen, 1995), whereas the swelling capacity of cell wall material is much greater. The swelling properties give rise to a difference in size between the starch granules and the cell wall particles. The insoluble fraction is dispersed in a large amount of water and screened through a series of sieves with pore diameters between 30 and 300 µm (Schoch and Maywald, 1968; Colonna et al., 1981). The liquid passing through the sieves (termed starch milk) is mainly a dispersion of starch granules, while the material trapped by the sieves contains mainly cell wall material. The smaller the diameter of the sieve pores, the smaller the particles of cell walls that are separated from the starch milk and the lower the level of impurities. Starch granules are not uniform in size, the size distribution being dependent on the source of the starch (Davydova et al., 1995). The minimum diameter of the sieve to be used is determined by the size distribution of the starch granules. For example, 50–60 µm sieves are usually used for producing starch from pea and faba bean (Colonna et al., 1981). The starch produced by such a method contains 0.04–0.40% protein and about 0.1–1.0% of lipid as impurities (Elliasson, 1988; Davydova et al., 1995). In industry the starches are dried by spread drying machines, which are specially constructed for this purpose. Starches produced for scientific purposes, however, usually have an additional wash with water, alkaline or salt solutions, or with organic solvents such as ethanol or acetone, to reduce Processing 93 the level of impurities (Schoch and Maywald, 1968; Davydova et al., 1995). The starches are then often dried in the open air, in some cases following a final wash with ethanol, or acetone, to speed up the drying process (Davydova et al., 1995). Although the methods described above result in satisfactorily purified starches, it should be noted that the use of organic solvents might partially disturb starch granular structure, which in turn may affect the properties. A diagrammatic representation of wet seed processing is shown in Fig. 4.3. 4.1.2 Granular structure It is generally believed that starch granules are composed mainly of two types of glucose polymer; amylose, which is essentially a linear chain Fig. 4.3. Wet process of starch extraction. 94 B. Czukor et al. molecule, and amylopectin, which is highly branched (see chapter 2, Chemistry). It is well known that in different starches these two molecules may differ in their degree of polymerization and, with regard to amylopectin, in the arrangement and degree of polymerization of the branches. Amylose and amylopectin molecules are arranged in granules (Fig. 4.4A), which are complex structures consisting of crystalline and amorphous areas. It is a common point of view that the short chains in the amylopectin molecules are organized into double helices, some of which then form crystalline lamellae, or crystallites (French, 1984; Blanshard, 1987; Manners, 1989). The regions of starch granules containing these structures are referred to as the ordered parts, while the remaining regions are called the disordered or amorphous parts. The amorphous parts of the starch granule are believed to consist of amylose and long chains from amylopectin (French, 1984). There is evidence that the crystalline and amorphous material form alternate layers in the starch granule (French, 1984; Blanshard, 1987). The presence of crystallites causes starch granules to be birefringent and this can be studied using light microscopy with cross polarizers (Fig. 4.4B). The interference pattern observed takes the form of a Maltese cross, which indicates that there is an orderly arrangement of the crystalline areas within the granule. The use of a specific plate (the so-called λ-plate, or red 1 compensator; Patzelt, 1974; Morris and Miles, 1994) in conjunction with the cross polarizers makes granules appear as blue and yellow sectors, indicating that starches are biaxial crystalline polymers (Patzelt, 1974). Fig. 4.4. Starch granules from wild-type pea seeds: (A) Using normal light microscopy. Opposite page: (B) viewed using polarized light, prior to gelatinization in 0.6 M KCl solution; (C) viewed using polarized light, after heating to the melting point for B-type crystallites (66°C) in 0.6 M KCl solution. Processing 95 If the crystallites were ordered differently with respect to the plane of polarization – either towards, or perpendicular to it – then this would give rise to different colours (French, 1984; Morris and Miles, 1994). Two types of crystallite, or polymorph, structure, A and B, have been identified in starch granules, which can be distinguished by the packing density of the double helices; A-type polymorphs being more densely packed than B-type (Fig. 4.5; Sarko and Wu, 1978; Imberty and Perez, 1988a; Imberty et al., 1988b; Perez et al., 1996; Wang et al., 1998). Starches from different plant species may have A-, B-, or both A- and B-types of polymorph (Blanshard, 1987; Wang et al., 1998), the resulting starches being Fig. 4.4. Continued. 96 Fig. 4.5. B. Czukor et al. Differences in packing density of A- and B-type polymorphs. termed A-, B- or C-type, respectively. A-type starches are found in cereals (e.g. maize, wheat and rice), B-type in tubers (e.g. potato) and C-type, containing both A- and B-type polymorphs, in legumes (e.g. pea and faba bean). In the case of C-type starches the arrangement of the two crystal types within the granules will affect the properties of the starch. For example, if C-type starches are a mixture of granules which contain either A or B polymorphs, then the properties would be intermediate between those of Aand B-type starches. If, on the other hand, each granule contains both Aand B-type polymorphs, it is likely that the properties of the resulting starch would be unique and different from those of A- and B-type starches. In this case the properties of the starch will depend on the arrangements of A and B polymorphs within the granules. To understand starch granular structure it is necessary to determine the proportions of ordered and disordered parts in the granule, the type of crystallinity, the proportions of A and B polymorphs (in the case of C-type starches), the size and arrangement of the crystallites and the properties of the amorphous parts of the granule. A range of techniques has been used for studying these physical characteristics, in particular, wide-angle X-ray powder diffraction, differential scanning calorimetry and various light and electron microscopy methods (Donovan, 1979; Yamaguchi et al., 1979; French, 1984; Biliaderis et al., 1986; Meuser et al., 1995). More recently, progress in quantifying the ordered structures within starch granules has been achieved using X-ray diffraction and NMR methods (Gidley and Bocick, 1985; Gidley and Robinson, 1990; Cairns et al., 1997; Bogracheva et al., 1998). With regard to studying the granular structure of legume starches, most progress has been achieved by using starches from peas. Starch Processing 97 granules from a round seeded pea line have been shown to contain both A- and B-types of polymorph, the B polymorphs being in the centre and the A polymorphs at the periphery of each granule (Bogracheva et al., 1998). Since B-type polymorphs melt at a lower temperature than A-type, it is possible to degrade the B polymorphs from the centre of the granules, leaving only the A polymorphs intact at the periphery. This process can be carried out on a microscope stage and observed using polarized light (Fig. 4.4B and C; Bogracheva et al., 1998). In addition, granules from this material have 63% double helices (Bogracheva et al., 1998), 33% of which are arranged in crystallites at a moisture content of about 20% (Cairns et al., 1997; Bogracheva et al., 1998). A method for determining the polymorph composition of C-type starch has been developed recently (Cairns et al., 1997). This method is based on calculations from X-ray diffraction patterns of the crystalline portions of the starch, using characteristic peaks associated with either A- or B-type polymorphs (Davydova et al., 1995; Cairns et al., 1997). Using this method the proportions of A and B polymorphs in round-seeded peas have been found to be c. 56 and 44%, respectively. Analysis of starch from a range of pea mutants (see Chapter 6, Breeding and Agronomy) has shown that it is possible to genetically manipulate the physical structure of the starch granules (Table 4.l; Bogracheva et al., 1995, 1997, 1999; Davydova et al., 1995) It was found that genes that affect the supply of substrate during starch synthesis (rb, rug3 and rug4) affect the total crystallinity and possibly the content of the A polymorphs in the granules. On the other hand, genes that directly affect the synthesis of starch polymers (r, rug5 and lam) increase the B polymorph content but have little effect on the total crystallinity of the granules (Table 4.1) Table 4.1. The characteristics of the granular structure of starch in pea mutants (Bogracheva et al., 1999). Genotype Amylose contenta (% starch) total % %B %A Wild-type rug4 rb rug3 lam rug5 r 35 33 23 12 8 43 65 20 23 27 17 22 20 19 45 39 43 37 69 52 73 59 57 58 63 29 45 b0b aData bA Crystallinity from Bogracheva et al. (1997). polymorphs not detected. B/A Tp (°C) ∆T (°C) ∆H (J g−1) 0.8 0.7 0.7 0.6 2.4 1.2 ∞ 61.8 65.4 66.1 70.0 58.6 49.0–57.0 52.5–60.0 13.4 12.5 9.0 9.4 8.4 30.0 34.0 10.8 9.8 12.6 7.5 6.8 5.1 2.4 98 B. Czukor et al. 4.1.3 Functional properties The applications of starch are determined by their functional properties. For example, an important functional property is pasting, which is the development of high viscosity after heating of starch–water suspensions. This property is exploited in different foods as well as in non-food uses such as adhesives. Another important functional property is the capability to create gels. This property is also used in different foods and in non-food applications such as thermoplastics. Legume starches, in particular those from pea and faba bean, have been studied mostly in relation to their pasting behaviour. Heating starch in the presence of water results in disruption of the ordered structures within the granules. This process can be studied using differential scanning calorimetry (DSC). Using this method it has been shown that the disruption of ordered structures in starch granules is an endothermic process, which is often called an order–disorder transition (Donovan, 1979; French, 1984; Blanshard, 1987; Cooke and Gidley, 1992; Zobel and Stephen, 1995). The nature of this transition is strongly dependent on the amount of water present during heating (Donovan, 1979; Biliaderis et al., 1986), giving two possible mechanisms for the disturbance of the ordered structures in the granule. These two mechanisms are usually called melting and gelatinization (see Fig. 4.6). Melting occurs in low moisture conditions, when there is no free water in the system. Gelatinization occurs when there is an excess of free water in the system (Evans and Haisman, 1982; Blanshard, 1987). Both of these processes occur when starches are heated in intermediate conditions of moisture (Donovan, 1979; Elliasson, 1980; Biliaderis et al., 1986). The DSC curve describing the gelatinization process shows sharp changes in the absorption of heat, which are normally described as changes in enthalpy (∆H). When starch from round-seeded peas is heated in excess of water containing a low salt concentration, the A and B polymorphs within the granules melt independently, giving a double peak of transition in heat capacity. The transition peak for the B polymorphs is at a lower temperature than that for the A polymorphs (Fig. 4.7; Bogracheva et al., 1994, 1995, 1998). Differences were found between the starches from the pea mutants (described in the Breeding chapter and earlier in this chapter) when they were heated in excess water and the granular disruption followed using a DSC (Bogracheva et al., 1999). Starches from the rb, rug3, rug4 and lam mutants exhibited narrow endothermic peaks that were similar to starch from the wild-type. The peaks differed, however, in peak temperature (T p) and peak width (∆T). Starches from the r and rug5 mutants, however, had very wide transitions, which were very different to those observed in starch from the wild-type and from the other mutants (Table 4.1) Gelatinization of starches in water is an important factor contributing to starch functionality and is widely exploited in industry. Processing Fig. 4.6. 99 Heating starches in different water contents. Fig. 4.7. DASM-4 differential scanning calorimetry (DSC) thermogram of pea starch in excess 0.6 M KCl. 100 B. Czukor et al. When starch suspensions are heated in water, the crystalline structure of the granules is disrupted at a particular temperature, followed by their intensive swelling and partial solubilization. This results in an increase in viscosity of the starch suspensions. The solubilization and swelling of starches from pea and faba bean occur at lower temperatures than cereal starches (maize and wheat), but at slightly higher temperatures, than potato starches (Doublier, 1987; Davydova et al., 1995). In addition, the extent to which legume starches are solubilized is slightly higher than for other starches, although there is a suggestion that they swell less than cereal starches (Doublier, 1987). The pasting properties of starches are commonly studied using a Brabender viscograph or a rapid visco-analyser (RVA; Leach et al., 1959; Doublier, 1987). The measurements of viscosity using these two devices are made during continuous stirring of the starch suspension. It is common to use a heating–cooling cycle during this process, programmed such that the starch suspension is heated to 95°C, maintained at this temperature for 30 min and then cooled to 50°C (Fig. 4.8). During this treatment, maize and potato starches give a peak of viscosity during the heating phase. In the case of potato starch, this peak is especially large. Such behaviour is not desirable for industry and reduces the number of applications for these starches in their native form. The starches from pea and faba bean, however, do not show a peak of viscosity during heating (Schoch and Maywald, 1968; Stute, 1990a,b; Davydova et al., 1995). In addition, the final viscosity, that these legume starches develop after cooling, is significantly higher than that for wheat starch, for example. Such behaviour indicates that, in relation to pasting behaviour, legume starches are superior to those from potato and cereals. The explanation for the stable behaviour of legume starches during heating lies in their unique crystalline structure described earlier. The Fig. 4.8. RVA viscograms of starch suspensions. Starch from: (1) r mutant pea; (2) maize; (3) rb mutant pea; (4) wild-type pea; (5) potato. Processing 101 disruption of crystallinity in pea starch granules begins from the centre, where the B polymorphs are arranged. The disruption of the B polymorphs is accompanied by swelling of the disrupted area. The disruption of the A polymorphs occurs at a higher temperature and is then followed by further swelling of the granules. The swelling of pea starch granules, therefore, occurs much more slowly than the swelling of granules from cereals and potato, which have only one type of polymorph. The slow development of viscosity of pea starches can be related to this disruption and swelling behaviour of the granules. Such rheological behaviour of legume starches may widen their applications as thickening agents for industry. 4.2 Modified Starch According to the ISO standard No. 1227–1979 modified starch is ‘starch with one or more of its physical or chemical properties modified’. More specifically, the term modified starch may refer to a chemically modified starch. Physical or chemical properties of starch can be changed by physical processes, chemical reactions or by biotechnological modifications. The various physical and chemical methods for modifying starch are presented in Fig. 4.9. Fig. 4.9. Starch modification. 102 B. Czukor et al. 4.2.1 Physical methods Steaming A simple method for the physical modification of starch within cells is to treat the legume seed with saturated steam (Kozlowska et al., 1989). Short periods of this treatment are used during the production of protein concentrates and isolates from faba bean seeds to improve the sensory properties of these preparations. The structural changes that take place during this type of processing can alter the cellular arrangement of protein and starch within the seed storage tissue (A and B). After this treatment the isolated starch shows intense amylose leakage and marked granule deformation during heating in water at high temperatures. The steaming of seeds also causes changes in the pasting and gel-forming properties. The reduced swelling of starch granules from steamed seeds gives rise to pastes with a lower viscosity and a higher temperature is required for amylose migration from the granule (Fig. 4.10). Starch isolated from untreated seeds forms a more rigid and more elastic gel compared with starch from steamed seeds. The internal changes within the granule do not markedly influence their appearance when viewed in the scanning electron microscope (Kaczymska et al., 1994). Only the denaturation of protein bodies is visible, the starch granule surface remaining very smooth and unchanged. The starch properties of pea starches can vary between varieties, the starch pastes giving different rheological behaviour when heated and cooled. Starch from pea seeds with hard to cook (HTC)-defect was characterized by having an increased viscosity of paste during heating as well as a higher value for the G′ modulus on cooling, indicating a high rigidity of starch gels (Fornal et al., 1995). Fig. 4.10. Viscosity pattern of raw (untreated) and steamed faba bean starches. Processing 103 Annealing Another type of heat treatment used to modify starch is annealing (Hoover and Manuel, 1994; Jacobs et al., 1995). This entails maintaining the starch at temperatures lower than the melting temperature (50–75°C) in excess water. This process results in a decrease in the potential and extent of amylose leaching. It is accompanied by an increase in the gelatinization transition temperature, the enthalpy of gelatinization and by a decrease in the gelatinization temperature range. It has been suggested that these effects are due to interactions between amylose and the outer branches of amylopectin, to an increase in double helices and to closer packing of the crystallities within the granule (Hoover and Manuel, 1994). Annealing also results in an increase in α-amylase hydrolysis, which may be due to a realignment of starch chains in the amorphous regions of the granules (Minagawa et al., 1987). Gamma irradiation As well as being used in food production, gamma irradiation also can modify the chemical and physico-chemical properties of starch. Irradiation has been shown to induce carbonyl derivatives (Raffi et al., 1981a), the development of acidity (Raffi et al., 1981b) and hydrogen peroxide (Raffi et al., 1981c) in haricot beans. The effect on starches from maize, manioc, wheat, potato and rice is to increase the reducing power, the water-soluble dextrin content and the Brabender viscosity values (Raffi et al., 1981d). Extrusion A very promising method for physically modifying starch is extrusion (Smietana et al., 1996) and high pressure treatment (Kudla and Tomasik, 1992). Extrusion gives products that are free from foreign substances, that can be used for children and in the development of non-allergenic formulas and functional foods. High pressure treatment results in some re-polymerization of dextrin formed during compression and in the ordering of starch granule structure into more crystal-like matter. To date, both of these methods have been used for modifying potato starch but, as yet, have not been used to modify legume starches. In the extrusion processing of cereal and legume seeds, as such or in blends, the transformation of starch and protein determines the properties of the final product (Schukla, 1996). Dietary fibre, although less affected during extrusion, can also have a significant effect on textural properties. The thermodynamic effects during extrusion break hydrogen bonds in starches, gelatinizing, or even dextrinizing, them in the process. The required energy input is often a function of starch granule size, the extent and type of crystallinity and the purity of the starch extract. Low-protein and high-amylose starches require high inputs of energy to undergo starch gelatinization. In the case of proteins, the secondary and/or tertiary 104 B. Czukor et al. structures undergo transformation, resulting in denaturation, association and coagulation involving reduction or oxidation. Furthermore, starches, proteins, and fibre can be hydrolysed to a varying degree during the extrusion process, which will modify the rheology of the transformed melts. 4.2.2 Chemical methods The most popular methods for modifying starches are based on the use of chemicals. Chemical modification can be carried out on three starch states: (i) in the solid state, where dry starch is moistened with chemicals in a water solution, air-dried and finally roasted at a temperature of over 100°C; (ii) in suspension, where the starch is dispersed in water, the chemical reaction is then carried out in water medium, the suspension is then filtered, washed and air-dried; (iii) as a paste, where the starch is gelatinized with chemicals in small amounts of water, the paste is stirred and when the reaction is completed, the starch is air-dried. The chemicals used for modification react with the free 2, 3 and 6 hydroxyl groups of the glucose units within the starch. The most common chemical modification processes are: oxidation using sodium hypochlorite, hydrogen peroxide, persulphates or potassium permanganate; esterification using acetic anhydride, vinyl acetate, orthophosphoric acid, sodium or potassium orthophosphate or sodium tripolyphosphate, sodium trimetaphosphate, phosphorus oxychloride or urea; etherification using ethylene oxide, propylene oxide, monochloroacetic acid or quarternary amines. The most important chemically modified starches from an industrial point of view are the starch-esters and starch-ethers. To date, legume starches have not been modified on a commercial scale for non-food applications. There have been laboratory investigations, however, on chemically modifying legume starches and acetylated starch, hydroxypropyl starch, cross-linked starch, cationic and grafted starches have been produced. Acetylation The only major study on the acetylation of legume starches has been carried out on a range of bean (P. vulgaris) varieties, using acetic anhydride (Hoover and Sosulski, 1985a,b; Vasanthan et al., 1995). X-ray diffraction of the modified starches indicated that the acetyl groups mainly entered the amorphous regions of the starch granule. The result of this process was starches with a decrease in hydrolysis, gelatinization temperatures, enthalpy of gelatinization, syneresis and in the extent of the viscosity increase during the holding period at 95°C in a viscosity analyses (Fig. 4.11 and Table 4.2). There was also an increase in viscosity in the amylose exudation at 95°C. No Processing 105 Fig. 4.11. Pasting characteristics of native (normal) and acetylated (chemically modified) starches from beans (Hoover et al., 1985a,b). Table 4.2. Syneresis of native and acetylated starch gels after storage at two different temperatures for 7 days (Hoover and Sosulki, 1985a,b). Extent of syneresis (%) Starch source Pinto bean Navy bean Black bean Degree of substitution* 4°C −15°C 0.050 0.050 0.050 0.055 0.050 0.053 0.095 18.2 13.0 20.2 12.0 30.1 11.7 8.9 6.8 5.8 7.9 6.2 15.3 10.4 9.0 *No. of acetyl groups per glucose unit. apparent differences in the external morphology of native and acetylated starches could be seen under the scanning electron microscope. The gradual slow rise in viscosity during the holding period at 95°C, and the increased stability during low temperature storage observed with acetylated legume starches, makes acetylation an acceptable method for modifying legume starches for the food industry. Phosphorylation Phosphorylation of faba bean starch, using a mixture of mono- and disodium phosphates at 160°C, resulted in a significant increase of swelling (Soral-Smietana, 1995). Faba bean mono-starch phosphates can bind components from solution or suspension into a homogeneous mass, which 106 B. Czukor et al. is of great importance in the preparation of instant products. This modified legume starch can act as a stabilizer of emulsions and can also act as a buffer in fruit produce (Soral-Smietana, 1995). Cross-linking Cross-linking refined starches from lentil, faba bean and pea with phosphorus oxychloride decreases water binding capacity, swelling power, α-amylase digestibility and viscosity at 95°C in the amylograph, but increases the degree of set-back (Table 4.3; Hoover and Sosulski, 1986). Cross-linking occurs mainly in the amorphous regions of the starch granule and hinders amylose exudation. The stable hot paste viscosities of cross-linked starches would be of value where low pH and high temperature are employed, during pressure cooking or sterilization, while the low degree of set-back of pea starch should improve the freeze–thaw stability and textural quality of frozen foods. Hydroxypropylation This chemical modification is based mainly on the addition of propylene oxide to starch moistened with water containing sodium sulphate, the mixture being heated and stirred for 24 h at 40°C (Hoover et al., 1988; Kim et al., 1992). The main effect of this modification on native starch granules is to produce starch with higher molar substitution (0.12) and a higher Table 4.3. Hydrolysis of native and cross-linked legume starches by pancreatic α-amylase (as % of total conversion to glucose units). Time of incubation (h) Starch source 1 2 3 4 5 6 7 Native Lentil Faba bean Field pea 3.4 2.1 1.9 6.4 5.8 5.3 10.0 9.9 9.2 17 16 12 21 20 19 28 26 23 28 27 23 42.8 32.8 30.8 57.8 51.8 45.8 61.8 60.8 55.8 66 65 62 72 70 68 79 72 68 79 72 70 3.2 2.0 1.8 6.0 5.5 5.0 10.0 9.4 8.8 16 15 12 20 19 18 27 25 22 27 26 22 38.8 29.8 27.8 54.8 49.8 42.8 58.8 58.8 52.8 63 63 58 69 68 64 76 70 66 76 70 68 Native (gelatinized) Lentil Faba bean Field pea Cross-linked (ungelatinized) Lentil Faba bean Field pea Cross-linked (gelatinized) Lentil Faba bean Field pea Processing 107 susceptibility to α-amylase attack, whereas a lower substitution results in a reduction in hydrolysis (Fig. 4.12; Hoover and Vasanthan, 1994). Hydroxypropylation of pea starch, with an amylose content of about 34%, with propylene oxide and sodium hydroxide resulted in a decrease in enthalpy of gelatinization, in gelatinization peak temperature, in pasting temperature and syneresis, and an increase in paste viscosity at 95° and 50°C (Hoover et al., 1988). The low gelatinization temperatures, high water-holding capacity and low retrogradation rates observed in these chemically modified pea starches would make them suitable for use in the paper and food industries. Cationization Water-miscible solvents such as ethanol, 2-propanol and methanol are used as the reaction medium for cationization of pea starches (Kweon et al., 1996). Cationization that results in a degree of substitution of 0.02–0.05 reduces the pasting and gelatinization temperatures, increases the peak viscosity and set-back on cooling and eliminates synersis after storage at 4° and −15°C (Yook et al., 1994). The principal effects of cationization are to promote rapid granule dispersion at low pasting temperatures and to give a molecular dispersion of amylose and amylopectin on heating to 95°C. On cooling, the gel structures are firm and the cationic groups control the realignment of starch during low-temperature storage. Investigations on grafting green gram, pigeon pea and garden pea starches showed that the graft yield of the reaction is less than for cereal and root starches (Patel et al., 1986). The graft yield in a gelatinized system, however, is almost independent of starch source. The use of starch graft copolymers as absorbents depends on their competitiveness on price compared with full synthetic absorbents produced from partially cross-linked acryl copolymers. (NB. Graft copolymers are specific copolymers, that are obtained in reactions between macromolecular substances and a substance of low molecular weight.) Fig. 4.12. The effects of degree of molar substitution (MS) of hydroxypropyl groups on rates of hydrolysis of field pea starch during incubation with α-amylase (Hoover et al., 1994b). 108 B. Czukor et al. 4.2.3 Biotechnological methods Many modified starches used in food products are the result of germinating or fermenting legume seed in vivo, or hydrolysing starches in vitro, using amylolytic enzymes (Bhat et al., 1983; Revilla et al., 1986a,b; Rodriguez et al., 1988; Abia et al., 1993; Frias et al., 1998). Hydrolysis The first step in enzymic hydrolysis is ‘endocorrosion’, which begins at the centre of the starch granule. The granule then degrades sequentially resulting in compartmentalization and subsequent fragmentation. The surface becomes porous and the granule is then divided into numerous polyhedral forms of various size (0.4–10 µm). This degradation process has been reported for starch from lentil (Revilla and Tarango, 1986a,b) and chickpea (Rodriguez et al., 1988). In general, the susceptibility of starch granules to modification by amylases depends on the physical structure of the granules, the amylose content, the degree of polymerization and on the presence of non-reducing ends on the granule surface (Bhat et al., 1983; Madhusudhan and Taranathan, 1995). Germination Germination induces the release of hydrolytic enzymes, which produce changes in the physical properties and functionality of seed components. Starch extracted from faba bean, chickpea and kidney bean before and after germination was significantly more digestible than those from ungerminated samples (Table 4.4; Shekib, 1994). Cooking the isolated starches from both the ungerminated and germinated samples further increased their digestibility. Table 4.4. In vitro starch digestibility (as a %) of ungerminated and germinated isolated legume starches in various forms (Shekib, 1994). Treatment Isolated starch A B C D Cooked corn starch Faba bean Kidney bean Chickpea Corn starch 33.2 65.1 47.2 84.3 – 32.3 62.1 44.7 82.1 – 39.5 67.2 49.4 90.3 – 90.3 A, starch from ungerminated seeds; B, cooked starch from ungerminated seeds; C, starch from germinated seeds, D, cooked starch from germinated seeds. Cooking, the samples were steamed for 15 min at 115°C. Processing 109 Fermentation Fermentation can be taken into consideration as a possible method for modifying starch properties for food use, for example puddings. This process has only limited effects on granule swelling and no apparent effect in solubility. Marked changes, however, were found in the apparent viscosity of cold pastes and in the intrinsic viscosity (Abia et al., 1993; Nche et al., 1994; Yadav and Khetarpaul, 1994; Matthews, 1999). Yadav and Khetarpaul (1994) produced wadi from black-gram dhal (Phaseolus mungo) and examined the in vitro digestibility of starch, phytic acid and polyphenols (Table 4.5). When black-gram dhal wadies were fermented at 25, 30 or 35°C for 12 and 18 h, the improvement in starch digestibility ranged from 57% to more than 88% over the control value. 4.3 Food Application of Native and Modified Legume Starches Native and modified legume starches can be used in the following applications (Blenford, 1994): • • • • preparation of gels (e.g. puddings) that can be prepared with about 50% less starch in comparison to corn starch; production of extruded products and instant starches that can be produced without the significant loss in viscosity that occurs with other starches; production of roll-dried starches, fruit and vegetable flakes that have a pulpy texture after rehydration and a considerable stability at cooking temperatures; production of pulpy products via freeze–thaw processing that keep their pulpy texture even after prolonged cooking; Table 4.5. Effect of temperature and fermentation time on in vitro starch and protein digestibility of wadies prepared from black-gram dhal (Yadav and Khetarpaul, 1994). Temperature (°C) Control 25 30 35 Fermentation time (h) 0 12 18 12 18 12 18 Starch digestibility Protein digestibility (mg maltose released g−1 meal) (%) 35.7 56.1 56.9 60.6 61.2 66.6 67.2 53.0 68.1 70.0 71.9 73.8 77.3 79.3 110 • B. Czukor et al. production of roll-dried instant starches with cold swelling gelling properties that can be used as such in formulations of various instant desserts with a flake-like texture. There are some patents describing new products based on modification of, among others, legume starch (mostly pea starch). A novel starch-based texturizing agent has been produced from high amylose starch (> 40% of either corn, barley or pea), by dissolving the starch in water under acidic conditions, while agitating at an elevated temperature and pressure, followed by retrogradation at low temperature and spray drying. The function of texturizing agents is to provide several fat-like attributes such as structure, viscosity, smoothness and opacity to reduce and/or essentially replace the fat content in foods. In addition, the texturizing agent can be used in full fat foods as a stabilizer. Foods containing the novel texturizing agent include mayonnaise, stoppable and pourable salad dressings, yoghurt, cottage cheese, sour cream, cream cheese, peanut butter, frosting cheesecake, mousse and several sauces (Mallee, 1995; Mallee et al., 1996). Another application is in the preparation of foods with a reduced lipid content. In this case the lipid portion in the food is replaced by an aqueous dispersion made from non-gelling, pre-gelatinized starch (high-amylose starch, for example pea) derivatives such as dextrin, converted starches and hydroxypropyl starch (Capitani et al., 1996). A reduced fat groundnut butter product, comprising fine-milled groundnuts in continuous oil phase and 5–50% native starch (pea or garbanzo bean) has been produced (Finocchiaro, 1996). Finally, the starches can be used as an opacifying agent. High-amylose starch has been pre-gelatinized under aqueous conditions in the form of a complex in which the opacifier (titanium dioxide) has been stabilized or entrapped. This product is recommended for low-fat and fat-free foods and beverages that need to be opacified (coffee creamer, cottage cheese dressing, nutritional beverages, mayonnaise, sour cream, ice cream, yoghurt, etc. (Dunn et al., 1996; Dunn and Finocchiaro, 1997). Further food applications of modified legume starches are possible but it depends both on more detailed knowledge of their properties and consumer acceptation of this source of starch in traditional or newly developed products. 4.4 Effect of Processing on Starch and Other Carbohydrates in Foods 4.4.1 Resistant starch formation The most common form of resistant starch (RS) in the diet and the most important from a technological point of view is retrograded starch (RS III), Processing 111 because it forms as a result of food processing (Escarpa et al., 1996; Soral-Smietana et al., 1998). Despite the extensive work that has been carried out on this subject, the correlation between starch structure and resistance of starch to amylolysis is still poorly understood. The formation of RS III is influenced by several factors, these include the conditions of the starch solubilization and retrogradation processes (for example, temperature and pressure of the autoclaving process and the number of autoclaving cooling cycles), the presence of lipids or sugars and the amylose content in the starch (Siljeström et al., 1989; Sievert and Pameranz, 1989, 1990; Sievert et al., 1991; Czuchajowska et al., 1991; Eerlingen et al., 1993a,b, 1994a,b). There is a strong positive correlation between the amylose content and the level of RS III in cereals and other grains. The USA has supported plant breeding programmes to produce new plant hybrids with low amylopectin and high amylose (c. 95%) starch (Gordon et al., 1997). This low-amylopectin starch has a higher gelatinization temperature, lower swelling power in hot water and is more resistant to enzyme and acid digestion compared with starch containing 70% amylose. Many legume starches have a high amylose content compared with cereal and tuber starches, ranging from about 30 to 70% of the starch (Swinkels, 1985; Blenford, 1994; Soral-Smietana and Dziuba, 1995). The manipulation of RS III content and other nutritional properties of starchy foods has been pointed out as a challenge to the food industry (Tovar et al., 1992a,b; Björck and Asp, 1994). In this context, steam-cooking could provide new ways to increase the present limited industrial utilization of grain legumes (Sosulski et al., 1989; Tovar et al., 1992a,b). The relationship between the total starch content, the proportion of readily available starch and RS III after prolonged steam and short dry heat treatment, has been reported (Tovar and Melito, 1996). Also, it has been shown that during the steam-heating of intact beans, the interaction between amylose and protein, and possibly other seed constituents, may also modify the tendency for the polysaccharides to recrystallize (Cerletti et al., 1993). These indigestible transglycosidated starches and other types of modified starches, will probably add in vivo to the unchanged apparent resistant starch values (Asp and Björck, 1992). Investigations have been carried out on digestion and large bowel fermentation using rats fed on raw and cooked peas (P. sativum), or wholemeal bread with different levels of cooked haricot beans (P. vulgaris) added. These studies have shown that the resistant starch content increased in peas after processing and increased progressively in bread with increasing amounts of haricot beans (Goodlad and Mathers, 1992; Key and Mathers, 1993). 112 B. Czukor et al. 4.4.2 Content, composition and digestibility Soaking Grain legumes are rarely eaten in a raw state and are usually cooked or processed first. Perhaps the simplest method for processing legume seeds is to soak them in water. This process can reduce the level of reducing and nonreducing sugars 16–40% (Jood et al., 1988). There is an increase, however, in in vitro starch digestibility of 17–23% after a 12-h soaking (Bishnoi and Khetarpaul, 1993). This enhancement of starch digestibility may be attributed to the loss of antinutritional factors such as phytic acid and polyphenols, which inhibit the activity of α-amylase (Deshpande and Cheryan, 1984). Conversley, it has been suggested that prolonged soaking of intact peas may allow the mobilization of phenolics, which are known to interfere with starch digestion from the seed coat to the cotyledons (Deshpande and Salunkhe, 1982). Soaking in water and NaHCO3 solution also significantly reduces the levels of stachyose, verbascose and raffinose. The reduction is usually higher in NaHCO3 solutions than in water and can account for 46–100% of the α-galactoside content (Jood et al., 1985; Vijayakumari et al., 1996). Only 1–10% of these losses can be explained by leaching into the soaking solution, the remainder being due to hydrolysis by α-galactosidase released by the imbibed seed (Vidal-Valverde et al., 1992a). In general, soaking is not used by itself, but in combination with germination, cooking or autoclaving. High and low pressure cooking Cooking and pressure cooking are perhaps the most effective methods for increasing starch digestibility. The effect of these treatments on different nutritional components of legumes is very similar, but generally the effect of pressure cooking is more intensive (Jood et al., 1985). Ordinary cooking can increase starch digestibility by 40–200%, while with pressure cooking the increase may be 200–400% (Jood et al., 1988; Bishnoi and Khetarpaul, 1993; Rani et al., 1996). The content of reducing and non-reducing sugars and the total starch content decreases significantly during cooking. In the case of the non-reducing sugar content, the losses may be 20–40% (Abdel-Gaward, 1993) during normal cooking and significantly higher during autoclaving (Jood et al., 1985). Cooking also can reduce the α-galactoside content by between 20 and 100% (Trugo et al., 1990; Vidal-Valverde et al., 1992a; Abdel-Gaward, 1993; Attia et al., 1994; Vijayakumari et al., 1996). There are, however, some reports of increases in the oligosaccharide content after cooking (Rao and Belavady, 1978; Revilleza et al., 1990). For example, boiling mature raw seeds of hyacinth bean in relatively low bean:water ratios resulted in a net Processing 113 decrease of sucrose after 90 min, while the level of raffinose, stachyose and verbascose increased (Revilleza et al., 1990). This increase could be attributed to hydrolysis of oligosaccharides bound to proteins or other macromolecules, or to the hydrolysis of high molecular weight polysaccharides. Steam-heated legumes are rich in resistant starch, while high pressure steaming and dry pressure cooking decreases the total and the available starch, and stabilizes or decreases the resistant starch level (Tovar and Melito, 1996; Periago et al., 1997). There is an increase in the total nonstarch polysaccharide content during cooking, the content of soluble nonstarch polysaccharides generally increasing and the insoluble non-starch polysaccharide content decreasing (Periago et al., 1997). Extrusion Depending on the extrusion conditions (temperature, moisture content, screw speed), the loss of total sugar and α-galactoside content can be about 30% and 50–60%, respectively, while the starch digestibility can increase significantly (Tuan and Phillips, 1991; Borejszo and Khan, 1992). Extrusion cooking marginally decreases the total content of dietary fibre of peas at 168°C, but the content of resistant starch increases significantly from about 1.5% to about 3.3% of the total starch (Berghofer and Horn, 1994). Dry roasting At high temperatures, dry roasting results in the complete reduction of the oligosaccharide content in the hyacinth bean after 2 min, although the levels of sucrose and the oligosaccharides were higher after a roasting time of less than 0.5 min (Revilleza et al., 1990). The effect after 2 min was probably due to a non-enzymatic browning reaction, oxidation of sugars or to pyrolysis (Fig. 4.13). Contrary to the effect of other processes, frying and roasting considerably reduces starch digestibility of legumes (Kelkar et al., 1996). Frying significantly reduces the sucrose content of legume seeds, probably because of the composition of the frying medium (Jood et al., 1985). Freeze-drying There is no apparent effect of freeze-drying on the sugar, starch and pectin contents of green beans (Oruna-Concha et al., 1996), although there was a slight decrease in the digestibility of starch in the tepary bean (Abbas et al., 1987). Microwaving Repeated microwave treatments decrease the total dietary fibre content of green bean, primarily because of losses in the soluble dietary fibre content (Svanberg et al., 1997). 114 B. Czukor et al. Fig. 4.13. The effect of dry roasting on the soluble sugars of hyacinth bean (Lablab purpureus; Revilleza et al., 1990). Germination Germination is one of the best known methods for decreasing the α-galactoside content and increasing the starch digestibility of legumes. During germination there are significant decreases in the total soluble carbohydrate content and in the total starch content, and increases in the reducing and non-reducing sugar content and in the in vitro starch digestibility. In addition, this process totally eliminates the α-galactosides (raffinose, stachyose, verbascose) (Jood et al., 1988; Revilleza et al., 1990; Trugo et al., 1990; Vidal-Valverde et al., 1992a; Vidal-Valverde and Frias, 1992; Bishnoi and Khetarpaul, 1993; Shekib, 1994; Urooj and Puttaraj, 1994; Urbano et al., 1995; Kelkar et al., 1996). The extent of the changes is mainly determined by the germination conditions (Nnanna and Philips, 1988). Germination significantly increases the resistant starch content resulting in an increase in the total dietary fibre content, the soluble fraction decreasing and the insoluble fraction increasing significantly (Veena et al., 1995). Fermentation Natural fermentation enhances protein digestibility and eliminates partially or completely the antinutritional factors. The effect of fermentation on starch digestibility has been studied in Bengal gram, cowpea and green gram (Urooj and Puttaray, 1994). The seeds were soaked in water for 3 h, drained, ground and allowed to ferment for 4 h at room temperature. After fermentation the meal was steam-cooked for 10 min. The fermentation treatment reduced the total soluble carbohydrate, the starch and the Processing 115 dietary fibre content. The fermented meal was digested significantly faster than the meal from untreated seeds. This may be due to a loss in the structural integrity of the starch granules, a change in the nature of the interaction between starch and fibre and because of the inactivation of some antinutrients (e.g. α-amylase inhibitors, lectins, phytic acid). Natural fermentation is a widely accepted, simple and inexpensive processing method for the reduction or elimination of oligosaccharides. Zamora and Fields (1979) studied natural fermentation of cowpea and chickpea and identified the microorganisms involved. They found that raffinose was eliminated in fermented cowpea and chickpea, that stachyose content was decreased in fermented chickpea and eliminated in fermented cowpea. The reduction or elimination of these compounds was most likely due to the ability of lactic organisms to utilize oligosaccharides for their metabolism. The lactic organisms in cowpea and chickpea were Lactobacillus casei, Lactobacillus leichmanni, Lactobacillus plantarum, Pediococcus pentosaceus and Pediococcus acidilactici; Lactobacillus helveticus was found only in chickpea. Odunfa (1983) studied the changes in oligosaccharide content of locust bean during iru preparation. The fermenting organisms were various subspecies of Bacillus subtilis. After 4 days natural fermentation of lentil, the α-galactosides and sucrose in the fermented product could not be detected, the cellulose and hemicellulose content decreased and the lignin increased (Vidal-Valverde et al., 1993). It was established that during the preparation of suspensions the initial concentration of the lentil flour–water suspension had an important influence on the level of α-galactoside content (Frias et al., 1996c). Mital et al. (1975) tested a number of lactic cultures for α-galactosidase activity. They found that the enzyme is constitutive in Lactobacillus bucheri, Lactobacillus brevis, Lactobacillus cellobiosis, Lactobacillus fermentum and Lactobacillus salivarius subsp. salivarius and could be induced in L. plantarum. The fermentation of soya milk with lactic cultures reduces the raffinose and stachyose content in different ways, depending on the Lactobacillus strain (Mital et al., 1979). Lactobacillus fermenti completely utilized raffinose and stachyose by 12 and 25 h respectively, a mixed culture of L. fermenti and Streptococcus thermophilus was less effective, while L. cellobiosis utilized only raffinose after 20 h fermentation. Soya milk fermentation with L. plantarum reduced the stachyose content and was less effective on raffinose reduction. Two enzymes are required to completely hydrolyse oligosaccharides. α-Galactosidase (EC 3.2.1.22) is necessary to hydrolyse the α(1→6) linkages and invertase (EC 3.2.1.26) hydrolyses the sucrose moiety. Many microorganisms are able to produce the α-galactosidase enzyme. The enzyme has been reported in brewer’s yeast, in some strains of bacteria (Actinomycetales and Streptomycetes strains) and moulds. Also, α-galactosidase has been found in the fruiting bodies of various mushrooms and puffballs and in higher plants (Suzuki et al., 1966). These different α-galactosidases differ for pH and temperature optimum and for substrate specificity (Suzuki et al., 1966). 116 B. Czukor et al. Variation in seed-derived α-galactosidases has been found between P. vulgaris (Becker et al., 1974) and soybean (Kim et al., 1973; Angel et al., 1988). Isolated α-galactosidase could be used to reduce or eliminate the flatusinducing factors in edible legume seeds. It is already used to eliminate raffinose during beet-sugar processing, to improve the crystallization of sucrose and to increase the sucrose yield. Sugimoto and Buren (1970) purified α-galactosidase enzyme and added it to soya milk, where it effected the complete hydrolysis of the oligosaccharides after 2 h incubation. 4.5 Legume Seeds as a Source of Raw Materials A wide range of starches can be found in pea (see Breeding and Agronomy chapter) with amylose contents ranging from near zero to about 70%. Suitable processing methods for the extraction of starches on an industrial scale from peas, however, has only recently become possible and to date has only been applied to conventional round-seeded varieties. Starch from round-seeded peas has an amylose content of 30–35%, compared with amylose contents of 20–28% in conventional starches from other species. The main industrial sources of pea starch are Provital Industry SA, Belgium, and Woodstone Food Company, Canada, Provital being the main supplier of raw materials from legumes within Europe. With regard to the main carbohydrate based products, Provital produces (Nastar R) native pea starch, (Nastar R Instant) pre-gelatinized pea starch and (Swelite R) texture improver. Nastar R is a native starch with high gel strength. The gelatinization capacity and viscosity profile of this native starch are at a similar level of performance to that found in certain modified starches (cross-linked). The starch shows an excellent stability to high temperatures, to shearing and to variations in pH and it promotes the formation of film and sliceable gels. In dry products it improves the crispness. Nastar R Instant is a pre-gelatinized version of Nastar R and is ideal for use in cold processes. The high gelling capacity of Nastar R is retained with Nastar R Instant and an efficient dispersion in water with strong agitation will rapidly develop a firm and sliceable gel. Swelite R texture improver is a fat-free functional ingredient composed of fibre and starch. Its dual composition combines the technological properties required from a functional ingredient with the dietetic qualities expected from dietary fibre. The high water capacity, fat binding and texturing effect of Swelite R gives food preparations an excellent stability for industrial manufacturing and storage, as well as a desirable texture. These products have demonstrated the usefulness of grain legumes, in this case pea, as a source of raw materials. Given the breadth of genetic variation now available, there is wide scope for this to progress even further. Seed J. 5 Górecki Physiology et al. and Biochemistry Seed Physiology and Biochemistry 5 Editor: Ryszard J. Górecki Contributors: Gabriel Fordoñski, Ryszard J. Górecki, Horia Halmajan, Marcin Horbowicz, Rupert G. Jones, and Leslaw B. Lahuta If you can look into the seeds of time, And say which grain will grow and which not, Speak then to me, who neither beg nor fear Your favours nor your hate. Macbeth, act 1, sc. 3, l. 58 (1606) William Shakespeare (1564–1616), English playwright 5.1 The Legume Seed 5.1.1 Seed components The legume seed can be separated into three major tissues – testa, endosperm and embryo – which in turn can be further divided into the two cotyledons and the embryonic axis (Fig. 5.1). The testa (Latin for ‘shell’), seed coat or hull is a maternal tissue that surrounds the embryo and attaches the seed to the pea pod via the stalk-like funicle. During early development, the testa, acts as a nurturing tissue, distributing nutrients from the mother plant to the developing embryo. Early in development this is by diffusion via the endosperm and later on by direct contact with the embryo. Sucrose, the main form of imported carbohydrate in legume seeds (Pate et al., 1974; Fellows et al., 1978; Patrick and McDonald, 1980; Schmitt et al., 1984), is unloaded from the vascular bundles within the testa. It is then thought to be transported symplastically through the seed coat (Patrick and Offler, 1995). Its import into the embryo is regulated by ©CAB International 2001. Carbohydrates in Grain and Legume Seeds (ed. C.L. Hedley) 117 118 Fig. 5.1. J. Górecki et al. Stages of embryo development in pea. the action of the plasmodesmata (Stitt, 1996), through a possible turgor homeostat system in the exporting cells (Patrick and Offler, 1995). Once the sucrose has been taken up by the cells of the embryo, it is hydrolysed by either sucrose synthase to fructose and UDP-glucose or, to a lesser extent, by alkaline invertase to fructose and glucose. Later in development, as the embryo matures and the seed begins to lose water, the layers of the testa become compressed and accumulate compounds such as lignin, suberin, cutin, callose and tannins. In mature seeds the testa is a dead tissue, the lignins making it almost impervious to water. In the dry seed, the testa provides a mechanical barrier, therefore, against abiotic and biotic processes that would otherwise damage or kill the embryo within. There is a small opening, the micropyle, that allows the diffusion of gases into the otherwise impermeable testa, so that the quiescent or dormant embryo can still respire. The tannins, that are often found in the testa, act as antinutritional compounds and colourants, deterring predatory animals and making the seed less conspicuous. The endosperm is a triploid tissue consisting of two maternal sets and one paternal set of genetic material. In the early stages of seed development it acts as an intermediary in the transfer of carbohydrates from the testa to the developing embryo. In pea, the unloading of carbohydrates into the endosperm from the testa is believed to be a passive process aided by the actions of a porin-like transporter (DeJong et al., 1996). In developing soybean cotyledons, however, sucrose causes a membrane depolarization (Lichner and Spanswick, 1981) with a sucrose-specific carrier that is energetically distinct from the hexose transport systems (Lin et al., 1984). In Seed Physiology and Biochemistry 119 Vicia faba, the process appears to be aided by a transporter molecule (Bouché-Pillon et al., 1994; Wang et al., 1995), although 50% of the carbohydrate is still apparently transported passively (Patrick and Offler, 1995). Turgor pressure also influences the efflux of solutes from the testa to the endosperm and later the embryo, with the rate of utilization of the sucrose within the cotyledons being the regulatory factor (Patrick and Offler, 1995). The endosperm may not persist into full seed maturity, as is the case for temperate grain legumes, the seeds, therefore, being termed exendospermous. For example, in pea seeds the endosperm rapidly declines as the embryo increases in size until it is almost entirely absorbed. Nutrients are then transferred from the testa to the embryo by specialized transfer cells found on the inside of the testa. The largest component of the seed is the embryo, which is the result of the fusion of one female and one male gamete and represents the sporophyte of the new generation. The embryo itself consists of the cotyledons, which make up the largest component, and the embryonic axis, comprising the root and shoot axes. At first, the embryo is supported in the nutritive liquid endosperm, but as it develops and its size increases, it eventually fills the embryo sac within the testa, displacing the endosperm. As the embryo progresses to maturity, a number of key developmental changes occur, resulting in the deposition of storage components, including the carbohydrates that form the basis of this book. 5.1.2 Seed development The allocation and partitioning of carbon within legume seeds presents a complicated picture, with distinct phases when the seed undergoes cell division, expansion and the laying down of storage products (reviewed by Zamski, 1995). Once a plant has flowered, the partitioning of photoassimilates is altered (Pate, 1984). The carbon that is used for the development of the seed is principally derived from the photosynthetic activity of the plant at the time of seed filling and very little is derived from stored carbohydrates (Fellows et al., 1978; Pate, 1984; Bewley and Black, 1985), although there may be some limited redistribution of carbon from stems and pods (Thorne, 1979). In cowpea, up to 77% of the photoassimilate translocated to the seeds is used for the accumulation of dry matter (Pate, 1984), whereas in lupin, with its thick fibrous pod, only 50% is converted to seed dry matter. This makes seed filling sensitive to biotic and abiotic stress during and, particularly, after flowering. As seed development proceeds there are often a number of aborted seeds, which has been attributed to the plant initially producing too many flowers and thus seeds. The supply of photoassimilates to many of the seeds is then withdrawn and these seeds abort, ensuring the vigour of a smaller number of seeds in a self-thinning exercise (Wardlaw, 1990, and references therein). 120 J. Górecki et al. Systems to differentiate developmental stages have been produced for some legume species, such as soybean (Fehr et al., 1971; Table 5.1), pea (Knott, 1987) and faba bean (Knott, 1990). In the case of pea and faba bean, however, the key encompasses the whole of plant development, without concentrating specifically on seed development. The system for soybean was designed to aid insurance loss assessment against damage by hail storms, rather than for developmental studies. For this reason adaptations of the soybean key have been made, to better describe the events during embryo development, leading to the introduction of substages (Spaeth and Sinclair, 1984; Dornbos and McDonald, 1986; Lowell and Kuo, 1989). Studies using growth stage and days after anthesis have been compared as determinants of development (Bewley and Black, 1978). These studies favoured the growth stage method as being more consistent when comparing lines and plants grown in field conditions. When comparing different species, however, growth stage analysis can be misleading as different stages are described by different methods. For research into biochemical or molecular changes in development, growth stages are inappropriate as one stage can encompass a multitude of developmental events. Many different carbohydrates are deposited within grain legume seeds during development and these may be metabolic intermediates or end products for storage and other uses. In lima bean, three stages of seed development have been analysed, half the full size, full size and dry mature (Meredith et al., 1988). The carbohydrates analysed were fructose, sucrose, raffinose, stachyose, verbascose and starch. A reduction in the proportion of fructose and sucrose and an increase in raffinose, stachyose and verbascose was found in successive developmental stages. In general, starch accumulation occurred before the first growth stage. Table 5.1. Stage of development descriptions for soybean (adapted from Fehr et al., 1971, and Lowell and Kuo, 1989). Stage number Reproductive stage description R1 R2 R3 R4 R5 R5.5 R6 R6.5 R7 R8 One flower at any node Flower at node immediately below the uppermost node with a completely unrolled leaf Pod 0.5 cm long at one of the four uppermost nodes with a completely unrolled leaf Pod 2 cm long at one of the four uppermost nodes with a completely unrolled leaf Beans beginning to develop (can be felt when pod is squeezed) At one of the four uppermost nodes with a completely unrolled leaf Pod containing full size green beans at one of the four uppermost nodes with a completely unrolled leaf Beginning maturity Pods yellowing, 50% of leaves yellow, physiological maturity 95% of pods brown, harvest maturity Seed Physiology and Biochemistry 121 A modified growth stage system was used in soybean to determine when oligosaccharides accumulated in the developing seed. As with lima bean, an increase in these compounds was found during successive developmental stages (Lowell and Kuo, 1989). In oil-rich legumes, such as soybean, starch can be present at levels of up to 10% of the seed dry weight early in development. By full maturity, however, the starch level declines to about 1% and is replaced by accumulating oil reserves (Yazdi-Samadi et al., 1977; Adams et al., 1980). The amount of soluble carbohydrates increases in the developing soybean (Adams et al., 1980). With regard to seed development studies, pea has often been used as a model system for seeds in general and grain legume seeds in particular (see review by Wang and Hedley, 1993). There are many reports in the literature relating to developmental patterns of seed development in pea (e.g. Bisson and Jones, 1932; MacKee et al., 1955; Carr and Skene, 1961; Flinn and Pate, 1968; Burrows and Carr, 1970). These papers have generally referred to different pea genotypes grown in a range of different environments and with little appreciation of the problems of seed to seed variation within samples. This has made a general interpretation of pea seed development difficult. One of the first studies to use controlled environments and to reduce seed to seed variation was carried out by Eeuwens and Schwabe (1975) using the pea variety Alaska. They were able to define a developmental pattern for seeds of this variety that followed a double-sigmoid curve separated by a lag phase. Using this study and many others from the literature, Pate (1975) was able to develop a general scheme for pea seed development that related all of the cardinal events, including the synthesis of starch, to the growth pattern of the seed. Hedley and Ambrose (1980) reported the first detailed analysis of a range of pea genotypes, grown in the same controlled environment conditions. They studied three conventional round-seeded lines plus three wrinkled-seeded lines containing the r mutation (see Chapter 7), all of the lines differing for seed size. From this study, a general pattern for pea seed development was defined that related the growth of the testa and endosperm to the development of the embryo. This showed three rapid phases of seed growth separated by two lag phases, the first corresponding to a rapid decline in the growth of the testa and endosperm and the second when the testa made contact with embryo following the absorption of the endosperm. These studies on pea have formed the basis of our understanding of the relationship between the growth and development of the pea seed and the synthesis of the seed storage products, including the carbohydrates and a series of papers have now been published in this area (Hedley and Smith, 1985; Hedley et al., 1986; Ambrose et al., 1987; Corke et al., 1987; Wang et al., 1987a,b; Cook et al., 1988; Corke et al., 1990a,b; Hauxwell et al., 1990; Wang et al., 1990; Yang et al., 1990; Macleod et al., 1991; Hedley et al., 1994; Rochat 122 J. Górecki et al. et al., 1995a,b; Hedley et al., 1996; Lloyd et al., 1996a,b; Casey et al., 1998; Craig et al., 1998; Harrison et al., 1998; Craig et al., 1999). Many of these more recent studies have been able to utilize the range of starch mutants described in Chapter 7. They have also provided information for studying the growth and development of seeds from other grain legume species, in particular lentil (Bakhsh et al., 1991, 1992; Frias et al., 1994b, 1996d). 5.2 The Accumulation and Biosynthesis of Carbohydrates 5.2.1 Accumulation of soluble carbohydrates Mature legume seeds can contain high levels of soluble carbohydrate. For example, in soybean this can be in excess of 14 and 28% of the dry matter for the axis (Table 5.2a) and cotyledons (Table 5.2b), respectively (Horbowicz and Obendorf, 1994). The soluble carbohydrates found within legume seeds comprise a number of compounds, including di- and oligosaccharides (Table 5.3). Table 5.2. Soluble carbohydrates in the axes and cotyledons of different grain legume species (mg g−1 dry matter). Sucrose Raffinose Stachyose Verbascose Galactocyclitols Total (a) Axes Soybeanb Peac Yellow lupina Faba beanb Lentilb Pigeon peab Cowpeab Chickpeab 111.3 69.7 20.3 57.0 39.2 61.5 45.3 36.1 26.3 19.6 23.9 15.0 5.4 13.4 12.2 22.9 131.4 65.5 199.6 65.3 92.4 35.7 110.0 35.1 trace 45.6 59.5 99.4 33.7 90.1 11.2 1.8 1.7 5.7 5.8 0.3 10.8 4.8 0.9 22.2 288.6 200.5 248.0 243.6 236.5 237.1 184.7 171.5 (b) Cotyledons Soybeanb Peac Yellow lupina Faba beanb Lentilb Pigeon peab Cowpeab Chickpeab 76.1 26.6 7.7 19.3 11.5 18.8 15.2 20.6 11.8 4.9 8.4 3.4 2.4 9.4 3.3 4.3 43.5 13.7 48.3 11.1 25.4 16.5 55.2 26.7 trace 34.8 23.6 52.5 18.0 34.6 10.8 1.0 2.6 2.4 4.3 0.0 33.0 11.4 2.3 34.5 141.0 80.1 110.0 88.4 85.2 95.4 87.7 94.2 aGórecki et al. (1997). and Obendorf (1994). cLahuta (unpublished). bHorbowicz Table 5.3. Distribution of soluble carbohydrates in dry seeds in various legumes (mg g−1 defatted meal) (Kuo et al., 1988). Cowpea Mung bean Lima bean Common bean Pole bean Broad bean Groundnut cv. Little Marvel cv. Williams 82 cv. Amsoy 71 cv. Berken cv. Fordhook cv. Top Crop cv. Blue Lake red kidney bean cv. Kentucky Wonder cv. Florunner Raffinose Stachyose Verbascose Total RFO RFO + sucrose 62.3 72.7 64.2 25.9 13.9 36.0 19.4 29.0 21.5 26.7 11.6 12.6 11.6 3.7 3.9 6.9 2.5 2.2 3.1 4.3 32.3 43.4 41.0 46.4 16.7 30.3 34.3 28.1 31.6 26.2 19.1 trace trace 3.6 26.6 trace trace trace trace trace 63.0 56.0 52.6 53.7 47.2 37.2 36.8 30.3 34.7 30.5 125.3 120.2 125.3 79.6 61.1 73.2 56.2 59.3 56.2 57.2 20.7 81.0 2.3 3.3 10.7 9.9 11.4 trace 24.4 13.2 45.1 94.2 Seed Physiology and Biochemistry Pea Soybean Sucrose 123 124 J. Górecki et al. Monosaccharides The monosaccharides found in legume seeds are often involved as transitory intermediates in the synthesis of higher polymers of carbohydrates. They are also often found in the phosphorylated form. Free monosaccharides are most readily detected during early seed fill and are rapidly utilized as development continues. Often monosaccharides, such as fructose, glucose and galactose, are found only in trace amounts in mature seeds (Horbowicz et al., 1995; Sun and Leopold, 1995). In soybean, however, the amounts can be relatively high early in development (YazdiSamadi et al., 1977), while in mature lucerne seeds no monosaccharides have been detected (Horbowicz et al., 1995). It has been suggested that it is advantageous to the plant if the concentrations of the monosaccharides are reduced. This is because the reducing nature of these compounds has been implicated in the Maillard reaction, which causes oxidative stress through the formation of free radicals, particularly after the seed has germinated (Sun and Leopold, 1995). Once a seed has germinated the amounts of galactose still remain almost undetectable, despite the removal of the galactose moieties from oligosaccharides. This is due to the high levels of galactokinase converting any free galactose to a safer phosphorylated form during germination (Dey, 1985). Disaccharides The most abundant disaccharide found in seeds is sucrose, which is the principal translocated photoassimilate (Lin et al., 1984; Patrick and McDonald, 1980; Lichner and Spanswick, 1981; Schmitt et al., 1984; reviewed in Patrick and Offler, 1995). Early in the development of pea seeds it can reach high levels. As the seed develops, however, the sucrose content falls as it is utilized for dry matter accumulation, so that by the time of maximum fresh weight of a pea seed it consists of around 8%, or 20 mg per whole seed (Harrison, 1996). Oligosaccharides The most commonly occurring oligosaccharides found in plants are those based upon α-galactosyl derivatives of sucrose (reviewed in Dey, 1990). These compounds are almost ubiquitous throughout the plant kingdom and rank second only to sucrose as the most abundant soluble carbohydrates. These oligosaccharides can comprise from 2 to 13% of legume seed dry weight and they are believed to play an important role in seed storability and in protecting the seed from desiccation stress (Obendorf, 1997; Sun, 1997). The galactosyl moiety of these compounds can be linked to either the fructose or the glucose moieties of sucrose. Depending upon the bonding of the molecules to each other, they will form different families of oligosaccharides (Table 5.4). Seed Physiology and Biochemistry Table 5.4. 125 Mono-O-α-D-galactosyl–sucrose derivatives (Dey, 1990). α-D-Galactosyl linkage with Name of molecule C-2 of D-glucose C-3 of D-glucose C-6 of D-glucose C-1 of D-fructose C-3 of D-fructose C-6 of D-fructose Umbelliferose No name Raffinose No name No name Planteose 5.2.2 Biosynthesis of soluble carbohydrates Raffinose family of oligosaccharides (RFO) The synthesis of α-D-galactosyl derivatives of sucrose begins with the production of galactinol from UDP-galactose and myo-inositol catalysed by the enzyme galactinol synthase (GS, EC 2.4.1.123) (Fig. 5.2, equation 3). The UDP-galactose utilized is derived from the conversion of UDPglucose by the enzyme UDP-glucose-4′-epimerase. The galactinol acts as a galactosyl donor and it is thought that galactinol has no other role within the plant other than the synthesis of oligosaccharides (Saravitz et al., 1987), although there are suggestions that it may be implicated in the synthesis of the galactomannans (Reid, 1985). Sucrose accepts the galactosyl moiety from galactinol to form raffinose with the regeneration of myo-inositol through the actions of raffinose synthase (RS, EC 2.4.1.82; Fig. 5.2, equation 4). Raffinose synthase can catalyse two types of reaction, synthetic and exchange. The synthetic reaction combines sucrose and galactinol to form raffinose and myo-inositol. In the exchange reaction, a galactosyl moiety is transferred between sucrose and raffinose. If the sucrose molecule is radiolabelled, after the exchange reaction, raffinose is radiolabelled. Castillo et al. (1990) reported that in soybean seeds the synthesis reaction levels off 15 days after flowering, while the exchange reaction increases until day 60. It was suggested that the enzyme either contains two sites, or that there were two separate enzymes. The activity of galactinol synthase, which is thought to be the main rate limiting step for RFO synthesis (Lowell and Kuo, 1989), increases as the seed begins to dry off (Saravitz et al., 1987; Castillo et al., 1990). This enzyme has been purified from kidney bean cotyledons and zucchini leaves (Kerr et al., 1993; Liu et al., 1995), which in the future may allow cellular and tissue localization and manipulation. Raffinose can then be used as the substrate to produce the next oligosaccharide in the RFO, stachyose, by the addition of an α-D-galactosyl moiety from galactinol to the C-6 of the non-reducing α-D-galactose moiety. This reaction is catalysed by stachyose synthase (STS, EC 2.4.1.67; Gaudreault and Webb, 1981; Fig. 5.2, equation 6). In addition to the 126 Fig. 5.2. J. Górecki et al. Biochemical pathway for some of the major α-galactosides and cyclitols. genuine STS reaction, the enzyme is able to produce galactosylononitol (Peterbauer and Richter, 1998; Fig, 5.2, equation 7), galactosyl pinitol A and ciceritol (Hoch et al., 1999; Fig. 5.2, equations 8 and 9). Galactosylononitol and galactosyl pinitol A could also substitute for galactinol in the synthesis of stachyose from raffinose (Peterbaur and Richter, 1998; Hoch et al., 1999; Fig. 5.2, equations 10 and 11). STS possibly synthesizes also verbascose from galactinol and stachyose (Tanner et al., 1967; Fig. 5.2, equation 12). High oligomers in the RFO series may be synthesized by a galactinol-independent galactosyltransferase activity (Bachmann and Keller, 1995; Fig. 5.2, equation 13). Other cyclitol families are also derived from myo-inositol (Loewus and Dickinson, 1982) including the galactopinitols A and B series (which are galactosyl derivatives of D-pinitol), fagopyritol B series, galactosyononitol Seed Physiology and Biochemistry 127 and scyllo-inositol (Obendorf, 1997). These compounds have been found in many legumes and in some cases at relatively high levels, for example, ciceritol (digalactopinitol A) is present in chickpea, lupin, lentil, soybean, kidney bean and lucerne (Obendorf, 1997). With the synthesis of the galactosyl oligomers, two molecules of galactinol can combine to form a higher homologue in the galactinol series, digalatosyl myo-inositol, with the regeneration of myo-inositol (Petek et al., 1966; Fig. 5.2, equation 5). Due to the pivotal role of myo-inositol in the synthesis of these various compounds, it is found at relatively high levels in young developing seeds, with only a slight reduction through development. Cyclitols and galactosyl cyclitols The trivial term of cyclitols refers to polyhydroxylcycloalkanes and their derivatives and the term includes the compounds known as the inositols. They are highly soluble, stable and relatively inert within the cell (Loewus and Loewus, 1980). There is a large amount of research interest in these compounds due to their importance in cellular metabolism (see reviews by Loewus, 1990; Obendorf, 1997). The inositols are cyclohexanehexols and possess one hydroxyl group on each of three or more ring atoms. There are nine enantiomers of inositol determined by the position of the hydroxyl group and, of these, six have been found in plant tissues (Loewus, 1990). Myo-inositol is synthesized from α-D-glucose-6-phosphate through the actions of myo-inositol-1-phosphate synthase (EC 5.5.1.4) through several partial reactions involving the reduction and then subsequent oxidation of the co-factor NAD+ (Eisenberg, 1967; Barnett et al., 1973; Chen and Eisenberg, 1975; Loewus and Loewus, 1983; Loewus, 1990; RayChaudhuri et al., 1997) and 1-L-myo-inositol phosphatase (EC 3.1.3.25; Fig 5.2, equations 1 and 2). The synthesized myo-inositol can also be utilized within a number of synthetic pathways, including those leading to the RFO, inositol phosphates, phosphoinositides, cell wall polysaccharides and bound auxin (Loewus and Loewus, 1983; RayChaudhuri et al., 1997). There are three steps concerned in the transformation of D-glucose into myo-inositol (Fig. 5.3). Initially D-glucose is converted to D-glucose 6-phosphate by the hexokinase and then the intermediate is transferred into L-myo-inositol 1-phosphate, which is finally dephosphorylated to myoinositol (Hoffmann-Ostenhof and Pittner, 1982). The most interesting step is the cyclization of D-glucose 6-phosphate to L-myo-inositol 1-phosphate. It was found that one enzyme is responsible for the reaction, originally called cyclase or cycloaldolase, but was finally given the name 1-phosphate synthase (EC 5.5.1.4). This enzyme has been isolated from various sources, animals, higher plants and yeasts. The enzyme proteins from these sources vary in their chemical and physical properties, including their metal content, molecular mass and specific activity, but all contain bound NAD+ 128 Fig. 5.3. J. Górecki et al. The pathway from D-glucose to myo-inositol. (or require this coenzyme) for their action (Hoffmann-Ostenhof and Pittner, 1982). Myo-inositol is the primary source for the biosynthesis of many cyclitols (Fig. 5.4). D-Pinitol is formed by two main stages, methylation of myoinositol and epimerization of the obtained methyl ether (Obendorf, 1997). There exists in nature two pathways for transformation of myo-inositol. In the first, myo-inositol is methylated initially by O-methyl transferase into sequoyitol (Scholda et al., 1964). Sequoyitol is then converted to D-pinitol by a two-step epimerization by a NAD+-specific dehydrogenase (EC 1.1.1.143) to form 5-O-methyl-D-myo-1-inosose and this intermediate is then modified by an NADP+-specific D-pinitol dehydrogenase (EC 1.1.1.142) to D-pinitol (Kremlicka and Hoffmann-Ostenhof, 1966). In the second pathway, myo-inositol is methylated to D-ononitol (Vernon et al., 1993). The conversion of D-ononitol to D-pinitol may involve a D-ononitol 1-dehydrogenase with 4-O-methyl-D-myo-1-inosose (Obendorf, 1997). D-Ononitol is a favoured intermediate for the formation of D-pinitol in Simmondsia chinensis, Ononis spinosa, Medicago sativa and Trifolium incarnatum (Dittrich and Brandl, 1987). It is believed that in higher plants D-chiro-inositol is formed from D-pinitol by demethylation, but an enzyme for this biosynthesis has not been characterized (Obendorf, 1997). 5.2.3 Accumulation of starch The role of starch in the plant has been reviewed by Morrison and Karkalas (1990) and in seeds by Sivak and Preiss (1995). The role of starch in pea seeds has been reviewed by Smith and Denyer (1992) and the biosynthesis Seed Physiology and Biochemistry 129 Fig. 5.4. Proposed pathways of synthesis of the main cyclitols and methylcyclitols present in legume seeds. of starch in pea by Martin and Smith (1995). The structural properties of pea starch granules have been reviewed by Wang et al. (1998). Due to the insoluble nature of starch, it has a negligible osmotic pressure, making it ideal as the principle carbon store of seeds in non-oil seed legumes. In a pea seed it can comprise around 50% of the dry weight (Smith and Denyer, 1992). In most starch-storing plant organs, for example, cereal endosperms, the starch-synthesizing plastids (amyloplasts) are derived from relatively undifferentiated plastids (leucoplasts). These plastids lack chlorophyll and 130 J. Górecki et al. have very little internal membrane (Journet and Douce, 1985). In contrast, the starch-storing plastids of developing pea embryos (and probably other grain legumes, which accumulate starch, as a main storage product) are derived directly from chloroplasts and retain chloroplast-like characteristics throughout their development. Developing pea embryos contain chloroplasts (located primarily on the outer edge of cotyledons), which store little or no starch (Smith et al., 1990). The starch accumulation in developing faba bean seeds has been reviewed by Weber et al. (1995b, 1997). During the cell-division phase of the faba bean embryo (15–18 days after flowering; DAF) starch is mainly found in two layers of the seed coat, in the hypodermal and chlorenchymal cells and in the outer parenchymal cells. When storage-product synthesis begins in the cotyledon (about 25 DAF), starch is deposited as single granules in the cells of the adaxial region of cotyledons. Later, starch deposition spreads from the adaxial cells to the periphery. During this process the quantity and size of the starch granules increases in the cells of the adaxial region and new granules appear in the abaxial cells. During the cell-elongation phase (about 30–35 DAF), starch is also present in the axis cells. At no developmental stage is starch found in the palisade cell layers of the seed coat, the peripheral cells of the abaxial zone, or in the provascular and calyprogenous cells. Developing seeds of pea, faba bean and common bean accumulate starch up to full seed maturity (Meredith et al., 1988; Lahuta et al., 1995). In seeds, which accumulate oil as reserve material (e.g. soybean), starch synthesis occurs in the cotyledon growth phase (about 35–40 DAF) and decreases to trace amounts during seed maturation (Monma et al., 1991). 5.2.4 Biochemistry of starch Native starch is composed of two types of polysaccharide chains, amylose and amylopectin (see Chemistry Chapter 2), which are formed into granules found in the amyloplast. Amylose is essentially composed of linear α(1→4)-linked glucose units and has a molecular weight between 5 × 105 and 106 (Wang et al., 1998). Along the length of the α(1→4) chain there are occasional secondary α(1→6) branches. Amylopectin is also composed of α(1→4)-linked glucan units; the molecular weight, however, is usually in the millions and it has substantially more α(1→6) branches than amylose, being in the region of 2–4% (Hizukuri and Takagi, 1984; Takeda et al., 1984; 1986). The first committed step in the starch biosynthetic pathway is the conversion of glucose-1-phosphate to ADP-glucose through the actions of ADP-glucose pyrophosphorylase (EC 2.7.7.23) in the amyloplast (see Fig 7.1 in Chapter 7, and Chapter 6). The substrate glucose-1-phosphate is derived from glucose-6-phosphate, which is imported into the amyloplast Seed Physiology and Biochemistry 131 via a glucose-6-phosphate/Pi transporter (Hill and Smith, 1991; Harrison et al., 1998). Glucose-1-phosphate + ATP → ADP-glucose + PPi This reaction is effectively irreversible due to an efficient pyrophosphorylase, found in non-photosynthetic plastids, removing the pyrophosphate (PPi; Gross and ap Rees, 1986). The resultant ADP-glucose can then be utilized as the substrate for starch synthesis. The glucose moiety is transferred to the non-reducing end of an α(1→4) glucan chain by the enzyme starch synthase (EC 2.4.1.21). The α(1→6) branches are formed through the actions of starch branching enzyme (EC 2.4.1.28), which cleaves short α(1→4) glucan chains of approximately 20 units and re-attaches them via an α(1→6) linkage to the original chain or an adjacent chain (Morrison and Karkalas, 1990). The number of branches along an α(1→4) chain is approximately one per 20 glucan units. 5.3 Physiological Role of Carbohydrates in Legume Seeds 5.3.1 During seed development Zygotic seeds Seeds of most plant species exhibit the ability to withstand desiccation, in many cases achieving water contents of less than 5–10% on a fresh weight basis. These seeds are termed orthodox and can be stored for many years under dry conditions. Orthodox seeds, which include those from legumes, are not capable of withstanding desiccation at early stages of development. The ability to tolerate desiccation is acquired during later stages of seed development and is lost after germination. It is believed that the acquisition of desiccation tolerance is developmentally controlled (Galau et al., 1991; Bewley and Oliver, 1992; Kermode, 1997). Galau and co-workers (1991) suggest that desiccation is acquired before maturation drying at the ‘post abscission stage’, when the vascular connection between the seed coat and the parent plant is lost. Acquisition of desiccation tolerance in maturing seeds involves several components including the accumulation of a special group of proteins (Blackman et al., 1991; Galau et al., 1991; Bewley and Black, 1994; Vertucci and Farrant, 1995), non-reducing sugars and/or galactosyl cyclitols (Koster and Leopold, 1988; Horbowicz and Obendorf, 1994; Obendorf, 1997), free radical scavenging systems (Senaratna et al., 1985; Koster and Leopold, 1988; Lowell and Kuo, 1989; Hendry, 1993; Leprince et al., 1993; FinchSavage et al., 1994) and abscisic acid (Bartels et al., 1988; Anandarajah and McKersie, 1990; Blackman et al., 1991; Vertucci and Farrant, 1995). The role of sugars and proteins in seed desiccation tolerance has been extensively studied. With regard to proteins, in desiccation tolerant seed a 132 J. Górecki et al. set of heat-stable hydrophilic proteins accumulate during late seed maturation. These proteins are so called late-embryogenesis-abundant, or LEA, proteins and their synthesis is controlled by ABA at the transcriptional level (Williamson and Quatrano, 1988; LePrince et al., 1990, 1993). LEA proteins are believed to protect macromolecular structures and membranes (Blackman et al., 1991, 1995). It has also been shown that another class of stress proteins, heat shock proteins, are synthesized during seed development (Vierling, 1991). Heat shock proteins presumably stabilize protein conformation during tissue dehydration. Recent studies using seeds from several species have indicated that the presence of proteins alone is not sufficient to confer complete tolerance to desiccation. A variety of orthodox seeds, including legumes, accumulate soluble carbohydrates mostly as sucrose and RFO. Soluble sugars account for 17.2–28.8% of the dry mass in embryonic axis tissues for soybean and chickpea, respectively, and are present at 2–10 times lower concentrations in cotyledons than in axes. There are differences in the quantities of RFO members in seeds from different species. For example, soybean seeds accumulate mostly stachyose and raffinose, but only small quantities of verbascose, whereas, verbascose is the major α-galactoside in pea and faba bean seeds. In lupin seeds, the amount of stachyose is much higher than raffinose and verbascose (Górecki et al., 1997). Generally, legume seeds accumulate sucrose early in development, while during maturation and desiccation they accumulate raffinose, stachyose and verbascose (Dornbos and McDonald, Jr, 1986; Saravitz et al., 1987; Kuo et al., 1988; Lowell and Kuo, 1989; Horbowicz and Obendorf, 1994; Lahuta et al., 1995; Frias et al., 1996b; Górecki et al., 1996, 1997). Blackman et al. (1991, 1992) found that during seed development, soybean seeds naturally develop desiccation tolerance and that this correlates with the loss of green colour in embryonic axis tissues, the accumulation of LEA proteins, the breakdown of starch and the accumulation of raffinose and stachyose in the axis tissues. Immature soybean seeds are capable of germination, but do not tolerate rapid desiccation. When these immature seeds are slowly dried, they develop desiccation tolerance and accumulate amounts of raffinose and stachyose in their embryonic axes that are about three times higher than the values reported when axes mature naturally. When incubated at 100% relative humidity, soybean seeds do not develop desiccation tolerance and do not accumulate stachyose (Blackman et al., 1992). Taken together, these studies provide evidence for the hypothesis that the RFO are important in conferring tolerance to the stress of desiccation. Yellow lupin seeds retain desiccation tolerance through all stages of maturation after 30 days from flowering (Górecki et al., 1997). The acquisition of desiccation tolerance and the ability to germinate is accompanied by the accumulation of substantial quantities of sucrose and RFO, with stachyose being the predominant soluble carbohydrate (Fig. 5.5). As Seed Physiology and Biochemistry 133 Fig. 5.5. Contents of soluble sugars, cyclitols and galactosyl cyclitols in the axis of maturing yellow lupin seeds (Górecki et al., 1997). maturation proceeds, the mass ratio of sucrose to α-galactosides changes from about 7.0 to almost 0.1 at full maturity. Other studies indicate that the RFO function in a similar way in pea, faba bean (Lahuta et al., 1995) and lentil (Piotrowicz-Cieslak et al., 1995) seeds, as in soybean and lupin seeds. In addition to sucrose and the RFO, seeds of several species accumulate galactosyl cyclitols and small amounts of free cyclitols (Table 5.2a and b). In lupin seeds, D-pinitol, D-chiro-inositol and myo-inositol form four different series of galactosyl oligomers (Górecki et al., 1996): the galactinol A series includes D-pinitol, galactopinitol A, ciceritol and trigalactopinitol A; the galactopinitol B series includes D-pinitol and galactopinitol B; the fagopyritol B series includes D-chiro-inositol, fagopyritol B1 and fagopyritol B2, and the galactinol series includes myo-inositol, galactinol, and digalactosyl myo-inositol. The accumulation of galactosyl cyclitols in lupin 134 J. Górecki et al. seeds coincides with an increase in the RFO and the acquisition of desiccation tolerance, while in pea only the RFO accumulate (Fig. 5.6; Górecki et al., 1997). Galactopinitol A, galactopinitol B and fagopyritol B1 accumulate in the embryonic axis tissues of developing soybean seeds in association with desiccation tolerance and in parallel with stachyose accumulation (Obendorf, 1997; Obendorf et al., 1998). Immature soybean seeds accumulate galactopinitols during slow drying (Obendorf et al., 1996). Galactopinitol A, galactopinitol B and fagopyritol B1 accumulate in parallel with stachyose in axis and cotyledon tissues during in vitro growth of embryos. Evidence, therefore, supports the suggestion that galactosyl cyclitols in seeds may enhance the physiological role of α-galactosides. In some seeds that have very low RFO levels, galactosyl cyclitols may replace the role of the α-galactosides in the acquisition of desiccation tolerance. Desiccation-tolerant buckwheat seeds, for example, accumulate Fig. 5.6. Yellow lupin and pea desiccation tolerance of radicle (as a %) during seed development and germination. The background shadow indicates the presence of raffinose family oligosaccharides (RFO) and galactosyl cyclitols (GAL-C) (Górecki et al., 1997, 1999; Lahuta et al., 1998). Seed Physiology and Biochemistry 135 fagopyritol (galacto-chiro-inositol) as a major soluble carbohydrate in addition to sucrose (Obendorf et al., 1993). Also, in lucerne somatic embryos galactosyl cyclitols have been proposed to have an important role in desiccation tolerance (Horbowicz et al., 1995). Other seeds of Leguminosae including lentil, kidney bean, castor bean, chickpea, pigeon pea, cowpea and subterranean clover have galactosyl cyclitols in addition to soluble sugars (Kuo, 1992; Horbowicz and Obendorf, 1994; Obendorf, 1997), but their role in desiccation tolerance has not been elucidated. For example, ciceritol is prominent in lentil seed (Frias et al., 1993) and galactinol in castor bean (Kuo, 1992). Somatic embryos Oligosaccharides and galactosyl cyclitols seem to play an important role in the acquisition of desiccation tolerance of somatic embryos. These are used as synthetic or artificial seeds for the propagation of high-value plants, or as a plant breeding tool in the development of new cultivars (see Chapter 6). Somatic embryos have been obtained for more than 150 species of important agricultural crops including legumes, cereals and grasses, and are genetically identical to the donor plant. Lucerne somatic embryos deposit storage proteins and carbohydrates and acquire desiccation tolerance (Lai and McKersie, 1994). Morphologically, however, lucerne somatic embryos do not have fully developed cotyledons and lack an endosperm and testa. Also, instead of galactomannan being the endospermic carbohydrate reserve, somatic embryos contain starch, sucrose and raffinose (Lai and McKersie, 1994; Horbowicz et al., 1995). Unlike zygotic seeds, somatic embryos have elevated levels of sucrose and do not accumulate D-pinitol or its galactosyl derivatives (galactopinitol A, galactopinitol B, ciceritol or trigalactopinitol A). Lower levels of stachyose accumulate during the maturation of somatic embryos. During desiccation, however, stachyose increases in somatic embryos to levels similar to those found in mature seeds. The decrease in sucrose concentration and the increase in stachyose during drying results in a decline in the sucrose : oligosaccharide ratio. Also, reducing sugars decrease during desiccation of somatic embryos (Horbowicz et al., 1995). Except for the lack of pinitol and galactosyl pinitols, changes in soluble carbohydrates during the maturation and desiccation of lucerne somatic embryos are similar to zygotic seeds and are associated with desiccation tolerance. Mechanism of desiccation tolerance How soluble carbohydrates confer seed desiccation tolerance has been the subject of numerous recent publications (Hoekstra et al., 1989; Bruni and Leopold, 1991; Horbowicz and Obendorf, 1994; Koster et al., 1994; LePrince and Waltres-Vertucci, 1995; Vertucci and Farrant, 1995; Buitink et al., 1996; Crowe et al., 1996; Sun et al., 1996). The stabilization of membranes appears to be the main role of these compounds in conveying 136 J. Górecki et al. desiccation tolerance in seed and pollen. Water replacement and glass formation hypotheses present the most interesting models as to how they may protect cellular constituents. The first hypothesis suggests that carbohydrate hydroxyl groups substitute for water and provide the required hydrophilic interaction to stabilize membranes and proteins (Bryant and Wolfe, 1992; Vertucci and Farrant, 1995). In many orthodox seeds, including legumes, a high sucrose content may provide hydrogen bonding required to prevent lipid phase transitions during drying (Leopold and Vertucci, 1986; Crowe et al., 1987; Caffrey et al., 1988). Sucrose alone, however, is not sufficient for desiccation tolerance, when measured as the ability to germinate after drying (Brenac et al., 1997; Obendorf, 1997). Pure sucrose solutions when concentrated tend to crystallize, but the addition of raffinose prevents this crystallization process (Caffrey et al., 1988; Koster, 1991). Thus, the second hypothesis assumes that the presence of raffinose, stachyose and/or galactosyl cyclitols in seeds inhibit crystallization of sucrose during drying and enhance the formation of a stable glassy state (Koster, 1991; Sun and Leopold, 1993; Leopold et al., 1994; Vertucci and Farrant, 1995; Koster and Leopold, 1998). Aqueous glasses have been detected in dried seed tissues (Williams and Leopold, 1989; Vertucci, 1990; Bruni and Leopold, 1991). Whereas this mechanism may apply to dry orthodox seeds, desiccation intolerant recalcitrant seeds die at a water concentration much higher than that required for the formation of the glass state. Soluble carbohydrates appear to be required, therefore, but alone are not sufficient to impose desiccation tolerance (Obendorf, 1997). It can be suggested that oligosaccharides and galactosyl cyclitols are important for desiccation tolerance because they reduce the level of monosaccharides, such as glucose, fructose and galactose, used as substrates for their synthesis (Koster and Leopold, 1988; LePrince et al., 1993). Lowering the monosugar content results in a reduction of easily available respiratory substrates and, therefore, may inhibit metabolic events, especially respiration, which is a source of free radicals prior to drying (Vertucci and Farrant, 1995). Conversely, low levels of monosaccharides may limit Maillard’s reactions, which are destructive to proteins (Wettlaufer and Leopold, 1991). Finally, sucrose, the RFO and galactosyl cyclitols may act as scavengers of free radicals, which are especially destructive when desiccation-sensitive tissues are dried (Hendry et al., 1992; LePrince et al., 1993; Vertucci and Farrant, 1995). 5.3.2 During temperature stress It is known that the composition of the RFO in leaves is altered by temperature (Chatterton et al., 1990; Bachmann et al., 1994). There have been very few studies, however, relating the effect of temperature during Seed Physiology and Biochemistry 137 plant maturation to the content and composition of the RFO and other soluble carbohydrates in seeds. It has been shown that white lupin seeds matured at 28°C accumulate only 53–70% as much dry matter as seeds matured at 13°C (Górecki et al., 1996). These changes were accompanied by only minor changes in the RFO. Pinitol and the galactose-containing pinitols, however, were more than doubled by seed maturation at 28°C, but, collectively, these compounds make up less than 10% of the total soluble carbohydrates. The effect of maturation temperature on the composition of soluble carbohydrates in yellow lupin seeds has also been studied (Górecki et al., 1996). In this species, seeds matured at 18°C had more than twice the amount of stachyose and verbascose compared with seeds matured at 25°C. From these limited experiments it can be suggested that the RFO and galactosyl cyclitols may play some role in temperature stress response on maturing seeds. 5.3.3 During seed storage In general, seeds attain their maximum viability and vigour after the final stage of maturation and then, during storage, they gradually deteriorate until death. The decline of seed quality during storage is expressed firstly as a decrease in the growth rate of germinating embryonic axes (vigour) and secondly as a loss of the ability to germinate (viability). Seed quality loss during storage is associated with increased membrane permeability and many distinct biochemical changes. These include lipid peroxidation, chromosome aberration and damage to DNA, changes in RNA and protein synthesis, reduction in respiration and changes in enzymes and reserve substances (Bewley and Black, 1985). It has been observed that the content of soluble carbohydrates declines with increased storage duration (Taufel et al., 1960; Yaklich, 1985; Petruzelli and Tarano, 1989; Kataki et al., 1997; Zalewski and Lahuta, 1998). Similarly, there is a positive correlation between the decline in RFO content and the reduction of seed longevity (Bernal-Lugo and Leopold, 1992). The depletion of soluble sugars may result in the limited availability of respiratory substates for germination (Edje and Burris, 1970). Other possibilities are that a depletion in the oligosaccharide content may reduce the protective effects of sugars on the structural integrity of membranes, or may reduce the ability of the seed to maintain a glassy state, resulting in a noncrystalline liquid state of high viscosity (Bruni and Leopold, 1991). In dry legume seeds, the soluble carbohydrates comprise mainly sucrose, together with different quantities of oligosaccharides, in particular raffinose, stachyose and verbascose. Sucrose is exceptionally effective in protecting membrane integrity in dry systems (Crowe and Crowe, 1986) as well as being one of the best vitrifying sugars (Green and Angell, 1986). As mentioned above (see Section 5.3.3), raffinose and other oligosaccharides 138 J. Górecki et al. are believed to enhance the protective effects of sucrose by preventing crystallization. It has been suggested that the RFO, therefore, have an important role in conferring both desiccation tolerance and seed storability. According to Horbowicz and Obendorf (1994), seed storability depends on the ratio of sucrose to oligosaccharides (the sum of raffinose, stachyose and verbascose). Seeds of species with a sucrose : oligosaccharide ratio of < 1.0 have a storability half-viability period > 10 years, whereas those > 1.0 have a storability half-viability period < 10 years. Steadman et al. (1996) studied the sugar composition of 46 tissues from seeds of 18 species, representing three seed storage categories: orthodox, intermediate and recalcitrant. The sucrosyl-oligosaccharides, raffinose and stachyose, were observed to be lower in recalcitrant seeds compared with orthodox seeds. In general, orthodox and recalcitrant seeds had tissues with sucrosyl-oligosaccharide : sucrose mass ratios of > 1 : 7 and 1 : 12, respectively. The results from these studies in combination with data in the literature (e.g. Lin and Huang, 1994) show that the ratio of sucrose to oligosaccharides in seed tissues may provide useful information on the seed storage category. 5.3.4 During germination During seed germination the resumption of metabolism commences within minutes of the introduction of water to the dry seed. The embryo passes from a dry, quiescent state into a metabolically active phase. This is accompanied by intensive mobilization of storage reserves, a rapid increase in respiration, initiation of nucleic acid and protein synthesis, and by cell elongation and cell division. Degradation of starch Reserve starch is deposited in amyloplasts within the embryonic axis and cotyledon cells. During seed maturation the membranes of the amyloplasts appear to disintegrate, exposing the starch granules directly to the cytoplasm of the cells (Harris, 1976; Halmer, 1985). Starch breakdown in legume cotyledons commences shortly after imbibition, but the rate of hydrolysis differs between species and varieties. The spatial pattern of starch degradation within tissues also varies (Ziegler, 1995). In Phaseolus cotyledons this process progresses from the central region, in peas from the outer cotyledon face inwards and in mung bean from the inner face outwards (Bewley and Black, 1978). Surface pores on the starch granules may facilitate the selective penetration of degrading enzymes, since granules appear to be broken down primarily from within (Harris, 1976). Since starch granules are effectively insoluble, breakdown occurs in three phases (Preiss and Levi, 1980). Firstly, the granules are reduced to large Seed Physiology and Biochemistry 139 maltodextrins, the maltodextrins are then degraded to glucose and glucose-1-phosphate by debranching and degradation enzymes. Finally, the products are metabolized and exported from the site of polysaccharide storage. The entire pathway of starch degradation has long been attributed to various combinations of activities of endo- and exo-amylases, starch debranching enzymes, starch phosphorylase and α-glucosidase. α-Amylases (EC 3.2.1.1; endoamylase) are the only enzymes that have been demonstrated to attack starch granules directly. It has been proposed, therefore, that these enzymes regulate starch granule breakdown (Chang, 1982; Steup et al., 1983). It is possible that α-amylase is associated with the starch granule (Dunn, 1974), or with plastid membranes. Saeed and Duke (1990), however, showed that in pea tissues with a reduced chlorophyll concentration (e.g. petals, stems, senescent leaves), α-amylases are located mainly in the apoplast. The hydrolysis of amylose by α-amylase is biphasic (Preiss and Levi, 1980). Amylose is initially subjected to rapid fragmentation into large maltodextrin chains, which later are hydrolysed more slowly. The hydrolysis of amylopectin by α-amylase is hindered, however, by the preserve of the α(1→6) branch points (see Chapter 2). In pea cotyledons from cv. Progress No. 9, amylase activity has been shown to increase for at least the first 10 days of germination at 21°C. Under these conditions starch is hydrolysed at a rate of 5.3 mg day−1 per seed. Using gelatinized starch as a substrate, it has been shown that there is nearly 600 times more α-amylase activity than is necessary for the in vivo rate of starch hydrolysis (Monerri et al., 1986). The enzyme is a 43.5 kDa monomer with pI 4.5 and pH activity optima of 5.5–6.5. When amylose is the substrate glucose and maltose are the major end products, the enzyme cannot attack maltodextrins with degrees of polymerization below that of maltotetraose (Beers and Duke, 1990). The development of α-amylase in legume cotyledons is regulated by endogenous phytohormones, probably by auxin (Hirasawa, 1989) and cytokinin (Locker and Ilan, 1975) and perhaps is controlled from the embryonic axis (Morohashi et al., 1989). β-Amylase (EC 3.2.1.2; exoamylase) hydrolyses maltosyl residues from amylose starting at the non-reducing end. In leguminous plants with starch as the main substrate, β-amylases are the main starch-degrading enzymes (Swein and Dekker, 1966). In germinating soybean seeds, where oil is the main substrate, however, β-amylase is not important in sugar metabolism (Adams et al., 1981). In pea (Chapman et al., 1972) and faba bean (Ziegler, 1988), β-amylase is located outside the chloroplasts. In pea seeds, isoenzymes of β-amylase are relatively stable at lower pH values; at pH 3.5 the isoenzymes retaining over 70% of their initial activity (Zimniak-Przybylska, 1992). The β-amylase from pea epicotyl is an approximate 55–57 kDa monomer with a pI of 4.35 and a pH optimum of 6.0. The enzyme is also unable to hydrolyse native starch grains from pea and glucans smaller than maltotetraose (Lizotte et al., 1990). 140 J. Górecki et al. α-Glucosidases (EC 3.2.1.20) are exo-type carbohydrolases catalysing the hydrolysis, or transfer, of the terminal α-D-glucosyl residues of α-Dglucosidically linked derivatives. Examples of substrates for hydrolysis include maltose, maltotriose, isomaltose, gelatinized starch and native starch grains (Sun and Henson, 1990). The ability of some sugar beet α-D-glucosidases to hydrolyse gelatinized starch has led to the suggestion that the in vivo substrates for α-D-glucosidases may include not only maltose, released by the action of α-amylase and β-amylase, but starch as well (Yamasaki and Konno, 1989). Three isoforms of α-D-glucosidases have been extracted from pea seedlings (Beers et al., 1990), of which two were most active under acid conditions and appeared to be apoplastic and the third, most active at about pH 7.0, was identified as a chloroplastic enzyme. In pea chloroplasts (Sun et al., 1995) and developing soybean seeds (Monma et al., 1991), α-D-glucosidase is involved in transitory starch degradation. The enzyme from pea chloroplasts is a homodimer with maximal activity at pH 7.0 and maximal stability at 6.5. These properties are compatible with the diurnal oscillations of the chloroplastic stromal pH and of transitory starch accumulation (Sun et al., 1995). Debranching enzymes (R-enzymes) are required to hydrolyse the (1→6)-α-glucosidic bonds that constitute the branching points in amylopectin and remain in the limit dextrins after amylolytic or phosphorolytic attack (Beck and Ziegler, 1989). Starch phosphorylase releases glucose1-phosphate as a product, which can be readily further metabolized. The cotyledons of pea and soybean possess two forms of phosphorylase that exhibit different substrate specificity. One form is most active against small malto-oligosaccharides and corresponds to a leaf plastid enzyme, whereas the other can better attack larger branched substrates and resembles a leaf cytosolic isoform (Ziegler, 1995). The glucose-1-phosphate produced, following cotyledon starch degradation, is presumably converted to sucrose in the cytoplasm and in this form can be transported to the tissues of the growing embryonic axis. The products of amylolysis (glucose and maltose) are also probably utilized in a similar way (Bewley and Black, 1985). Degradation of the raffinose family of oligosaccharides (RFO) The degradation of the RFO in germinating legume seeds begins during seed imbibition and proceeds more intensively in the embryonic axis than in the cotyledons (see also Chapter 4). The RFO in axes are lost during the first two days of imbibition of soybean, pea and lupin seeds, whereas, in cotyledons RFO hydrolysis is prolonged for 4–6 days (Koster and Leopold, 1988; Górecki and Obendorf, 1997; Górecki et al., 1997; Lahuta et al., 1998). Verbascose, stachyose and raffinose are degraded progressively, while the level of monosaccharides increases gradually as germination progresses. The hydrolysis of α(1→6) glycoside bonds between galactoside moieties of raffinose-type oligosaccharides, cell wall polysaccharides and storage glycoproteins is catalysed by α-D-galactosidase (EC 3.2.1.22). Seed Physiology and Biochemistry 141 Mature seeds usually contain isoforms of this enzyme that differ in activity and molecular mass (Pridham and Dey, 1974). In developing seeds, the activity of α-D-galactosidase increases during the period of intensive RFO synthesis, reaching its highest level at full maturity. This increase may result from the structural transformation of isoenzymes, which leads to changes in their specific activities (Pridham and Dey, 1974). In the maturing embryo, the synthesis of the oligosaccharides and α-D-galactosidase probably occur in different cellular compartments. In pea cotyledons, α-D-galactosidase has been localized in cell vacuoles that are storing the lectin precursors (Harley and Beavers, 1989). In the cells of soybean cotyledons, α-D-galactosidase occurs in cisterns of the Golgi apparatus and it may be deposited in protein bodies (Hermann and Shannon, 1985). A similar observation has been made in faba bean (Datta et al., 1985). A direct role has been established for α-D-galactosidase and α-D-mannosidase in the hydrolysis of glycoproteins and storage galactosides in the cotyledons of germinating narrow-leafed lupin (Plant, 1984). In yellow lupin seeds the activity of α-D-galactosidase increases only at the beginning of germination (Login et al., 1995), when the accumulated RFO and galactosyl cyclitols undergo complete decomposition (Górecki et al., 1997). While the role of α-D-galactosidase in the hydrolysis of saccharides and glycoproteins in germinating seeds is understandable, the role, if any, of this enzyme in non-germinating (stored) seeds remains to be elucidated. The seeds of various species appear to use the RFO as part of their storage material and it has been observed that the oligosaccharide content decreases with increased storage duration (Taufel et al., 1960; Yaklich, 1985; Bernal-Lugo and Leopold, 1992; Horbowicz and Obendorf, 1994). Similarly, there is a positive correlation between the decline in RFO content and the depression of seed longevity. The loss of desiccation tolerance During germination seeds undergo a transition from a desiccation tolerant to a desiccation-intolerant state. Generally, after radicle protrusion seeds rapidly lose desiccation tolerance. Koster and Leopold (1998) studied the relationship between soluble sugar content and the loss of desiccation tolerance in the axes of germinating pea, soybean and corn seeds. The loss of desiccation tolerance during imbibition was monitored by measuring the ability of seeds to germinate after desiccation, following various periods of pre-imbibition and by measuring the rate of electrolyte leakage from dry and rehydrated axes. The soluble carbohydrate contents of axes throughout the transition from desiccation tolerance to intolerance were analysed. The results showed that sucrose and the larger oligosaccharides were consistently present during the tolerant stage and that desiccation tolerance disappeared as the oligosaccharides were lost. The relationship between the loss of seedling desiccation tolerance and the content of soluble carbohydrates in legume seeds has also been studied 142 J. Górecki et al. by Górecki et al. (1997) and Górecki and Obendorf (1997). In these studies it was found that radicle tissues are often more sensitive to desiccation than hypocotyls. Pea root tissues lost desiccation tolerance during the first 36 h of germination, while 80% of epicotyls survived slow drying treatment and 40% survived fast drying treatment of seedlings, for up to 96 h after imbibition (Fig. 5.7). During desiccation, sucrose levels increased fiveto tenfold in root and hypocotyl tissues and even more in epicotyls. Glucose and fructose increased during germination and remained elevated after drying. These changes in saccharides reflected the mobilization of Fig. 5.7. Pea seedling germination (A) and length (B) of axis, radicle and epicotyl as a function of hours after imbibition. Desiccation tolerance (C, E) and length (D, F) of radicle and epicotyl at 6 days after rehydration of (C, E) fast-dried or (D, F) slow-dried seedlings as a function of seedlings as a function of seedling age when dried (Górecki and Obendorf, 1997). Seed Physiology and Biochemistry 143 oligosaccharides. Drying resulted in a small increase of raffinose in hypocotyls and radicles, but this was not enough to protect seedlings from desiccation damage (Górecki and Obendorf, 1997). In contrast to pea, soybean and yellow lupin seedlings lost desiccation tolerance within 36 h during germination. This change in desiccation tolerance was associated with the loss of raffinose, stachyose and galactosyl cyclitols and an increase in reducing sugars and free cyclitols (Górecki et al., 1997, Górecki and Obendorf, 1997). It can be suggested, therefore, that sucrose, the RFO and galactosyl cyclitols are not prerequisite for desiccation tolerance of pea, soybean and lupin seedlings. On the other hand, the accumulation of reducing sugars, mainly glucose and fructose, during seed germination could have a deleterious effect on the seedling by inducing desiccation injury (Hoekstra et al., 1994). N. Kuchuk et al. Biotechnology 6 Biotechnology 6 Editor: Nickolay Kuchuk Contributors: Miroslav Griga, Georgina Kosturkova, Nickolay Kuchuk and Mladenka Ilieva-Stoilova And he gave it for his opinion, that whoever could make two ears of corn or two blades of grass to grow upon a spot of ground where only one grew before, would deserve better of mankind, and do more essential service to his country than the whole race of politicians put together. Gulliver’s Travels, ‘A Voyage to Brobdingnag’, ch. 7 (1726) Jonathan Swift (1667–1745), Anglo-Irish poet and satirist 6.1 Introduction This chapter concentrates on the biotechnological techniques developed in the fields of plant cell tissue culture and genetic engineering. The cultivation methods for explants and single cells (protoplasts) and for plant regeneration in vitro are described as basic approaches that allow valuable genotypes to be propagated as well as to produce fertile plants from somatic cells. In vitro somaclonal variation and cell selection is considered as a new source of diversity for plant breeding. Methods for genetic transformation and for the production of transgenic grain legumes are summarized to give an idea about ‘the state of art’ of this technology. Hopefully, this information will promote a better understanding of the current opportunities and future prospects of plant biotechnology, as well as the possibilities for the future manipulation of carbohydrate metabolism, content and composition in grain legume seeds. ©CAB International 2001. Carbohydrates in Grain and Legume Seeds (ed. C.L. Hedley) 145 146 N. Kuchuk et al. 6.2 In vitro Cultures and Plant Regeneration of Grain Legumes 6.2.1 Introduction to in vitro culture The ability of plant cells to express their entire genetic information and regenerate whole plants (i.e. totipotence), is the basis of their development in controlled in vitro conditions and to the establishment of biotechnological techniques and methods for genetic manipulation. In vitro plant cells from excised tissues (explants) might undergo dedifferentiation and re-differentiation following several developmental pathways, depending on the culture environment. Unorganized cell division and growth can be stimulated, leading to the formation of callus and suspension cultures. Morphogenesis can be induced in meristem, callus, suspension and protoplast cultures, leading to the formation of somatic embryos, organs (shoots and roots) and a whole plant. Undifferentiated cell growth in callus or suspension cultures can be adequate for the purposes of some biotechnology processes (like secondary metabolite production, and some investigations in physiology, genetics, cytology, biochemistry, etc.; see Section 6.6). Regeneration of plants, however, is essential for the application of recent advances in biotechnology, especially those concerning genetic engineering for plant improvement. Realization of regeneration capacity in vitro depends on knowledge of the requirements for stimulation of the morphogenic response (Halperin, 1986). Unfortunately, information on genetic, epigenetic and physiological status of the explant is still limited and in practice the general approach is to find out the most appropriate chemical or physical stimuli to provoke totipotency of the cell. Up to now, this process has been mainly empirical and the formulation of strict rules and general protocols has not been possible. The establishment of in vitro cultures and the induction of morphogenesis in grain legumes has proved more difficult in comparison with most Solanaceae and Brassicaceae species. For a long time recalcitrance in regeneration has been the largest obstacle for genetic manipulation. Several important observations have led to the development of efficient regeneration systems. These have focused on the role of the genotype, the explant, the application of relatively high auxin concentration for induction of somatic embryogenesis and the use of powerful cytokinins for multiple shoot proliferation. Definite success has been achieved in the regeneration of soybean and pea and this success has been immediately applied to genetic manipulation (Barwale and Widholm, 1990; Griga and Novák, 1990; Christou, 1992; see Sections 6.2 and 6.4). The development of efficient in vitro methods and plant regeneration protocols for other large-seeded legumes has made significant progress in the last two decades and organogenesis/somatic embryogenesis has been reported at least Biotechnology Table 6.1. Species 147 Morphogenic responses of grain legumes cultured in vitro. References (A) Development through direct and indirect embryogenesis Arachis hypogeae Gill and Saxena (1992); Eapen and George (1993a); Baker et al. (1995); Chengalrayan et al. (1997); Murch and Saxena (1997) Cicer arietinum Sagara et al. (1993); Barna and Wakhlu (1993, 1995); Islam (1994); Suhasini et al. (1994); Eapen and George (1994); Dinehskumar et al. (1995); Murthy et al. (1996) Glycine max Barwale and Widholm (1990); Parrott et al. (1992); Komatsuda (1992); Griga et al. (1992); Griga (1993); Trijatmiko and Harjosudarmo (1996); Gai and Guo (1997); Rajasekaran and Pellow (1997) Lupinus albus Rybczynski and Podyma (1993a) Phaseolus vulgaris Malik and Saxena (1992a); Xu and Yang (1993) Pisum sativum Kysely et al. (1987); Kysely and Jacobsen (1990); Tetu et al. (1990); Stejskal and Griga (1992); Flandre and Sangwan-Norreel (1995); Loiseau (1995); Griga and Slama (1997); Jarkova et al. (1998) Vicia faba Taha and Francis (1990); Griga et al. (1992); Xu-Zheguyao and Yang Caiyum (1993); Griga and Klenoticova (1994) (B) Development through direct and indirect organogenesis A. hypogeae McKently et al. (1990); Eapen and George (1993b); Kanyand et al. (1994); Venkatachalam and Jayabalan (1997); Ponsamuel et al. (1998) C. arietinum Altaf and Ahmad (1990); Malik and Saxena (1992b); Islam et al. (1993); Barna and Wakhlu (1994); Fernandez-Romero et al. (1995); Murthy et al. (1996); George and Eapen (1997) G. max Barwale and Widholm (1990); Parrott et al. (1992), Shetty et al. (1992); Nawracal et al. (1996); Kaneda et al. (1997) Lens culinaris Malik and Saxena (1992); Warkentin and McHughen (1993); Halbach et al. (1998) L. albus Sator (1990); Harzic et al. (1998) P. vulgaris Franklin et al. (1991); Malik and Saxena (1991, 1992c); Mohamed et al. (1993); Zhang et al. (1997) P. sativum Kallak and Koiveer (1990); Tetu et al. (1990); Nielsen et al. (1991); Malik and Saxena (1992); Ozcan et al. (1992); Sanago et al. (1996); Kosturkova et al. (1997); Jarkova et al. (1998); Ocatt et al. (1998) V. faba Ramsay and Middlefell-Williams (1992); Griga and Klenoticova (1994); Fernandez-Romero et al. (1998) (C) Multiple shoot formation from pre-existing meristems in the explant A. hypogeae Heatley and Smith (1996); Venkatachalam and Jayabalan (1997) Continued 148 Table 6.1. N. Kuchuk et al. Continued Species C. arietinum G. max L. culinaris L. albus P. vulgaris P. sativum V. faba References Altaf and Ahmad (1990); Malik and Saxena (1992b); Brandt and Hess (1994); Polisetty et al. (1997); Santangelo et al. (1997) Parrott et al. (1992); Sharma and Kothari (1994); Kaneda et al. (1997) Malik and Saxena (1992b); Polanco and Ruiz (1997) Sator (1990); Rybczynski and Podyma (1993b) Mohamed et al. (1992); Herselman and Mienie (1995) Jackson and Hobbs (1990); Kosturkova et al. (1997) Mohamed et al. (1992) in one genotype/cultivar of all economically important legume crops (Christou, 1997; Griga, 1999). At present, more than 20 research groups are working on the in vitro culture of at least 30 species of grain legumes (Parrott et al., 1992). The in vitro procedures for these species, were developed during a period of more than 20 years to satisfy different goals and the extent to which they have been characterized is variable. Also, they follow the historical development of in vitro culture methods, utilizing the achievements of the day. Although a perfect system cannot be offered, the available information gives the possibility to choose the most appropriate protocol for a particular investigation, to adapt a scheme or to successfully develop a new one. 6.2.2 Plant regeneration systems Different responses and developmental pathways have been observed in vitro in pea and other grain legumes (Table 6.1), depending on internal and external plant factors. Regeneration of plants occurs via organogenesis and/or embryogenesis, either directly from the excised tissue, or indirectly after formation of callus. Distinguishing between these processes is important for making the right choice of scheme to be applied. In organogenesis, a group of cells forms bud and root primordia with subsequent development into a leafy vegetative shoot and root, respectively. In somatic embryogenesis, a new individual with a bipolar structure (i.e. a rudimentary plant with a root/shoot axis) arises from a single cell (Brown, 1986). Regeneration of plants can be obtained by shoot proliferation and by micropropagation from pre-existing meristems. This chapter will focus on the development of regeneration systems in pea (Pisum sativum) as the most extensively grown grain legume in Europe. Biotechnology 149 6.2.3 Pioneering studies on pea regeneration In the early 1970s, Gamborg et al. (1974) and Kartha et al. (1974) first reported shoot regeneration from callus tissue and from apical meristem, respectively. Initial attempts to induce organogenesis in callus cultures studied the response of different plant tissues, genotypes and media composition. Gamborg et al. (1974) first induced shoot formation in callus from macerated apical meristems grown on media containing 1 µM naphthalene acetic acid (NAA) and 0.2–5.0 µM 6-benzyl amino purine (BA), the latter being important for vigorous shoot formation. Shoots were formed de novo exogenously on the callus. While most of the calli produced one or more shoots, root formation was poor and did not occur regularly. Malmberg (1979) used epicotyl sections to obtain callus on MS media (Murashige and Skoog, 1962) supplemented with BA and NAA. He observed that organogenic ability was genotype dependent (six out of 16 lines responded) and decreased with prolonged culture. Root formation was induced by NAA but, as previously reported, it was not satisfactory. Atanassov and Mehandjiev (1979) observed that the developmental stage of the explant influenced the efficiency of callogenesis, the organogenic response being observed only for 20–22-day-old embryos. Bud formation was stimulated by 0.5 mg l−1 BA and could be maintained for eight subcultures, but rooting of the regenerants was problematic. Cytological analysis showed that 13% of the newly formed shoots had mixoploid cells at the vegetative apex. An effective system of de novo regeneration from callus derived from immature leaflets (0.9–1.8 mm) was developed by Mroginski and Kartha (1981). The combination of BAP (10 µM) and NAA (10 µM) was the best for callus induction and subsequent shoot regeneration. Hussey and Gunn (1984) succeeded in obtaining vigorously grown callus with superficial meristems, using plumules that continuously regenerated shoots over a period of nearly 3 years. Callogenesis was induced on MS medium supplemented with 1 mg l−1 BA and 4–8 mg l−1 indole-3-acetic acid (IAA), and was maintained by reducing indole-3-butyric acid (IBA) to 0.25 mg l−1. The in vitro response differed between genotypes and only two (cvs. Puget and Upton) out of five varieties maintained regenerative callus. During the first year of callus growth the plants were diploid and mostly morphologically normal. Many of the shoots regenerated after 2 years, however, showed considerable morphological variation and difficulties in rooting. Natali and Cavalini (1987a,b, 1989) examined some factors affecting rhizogenesis in cultures obtained from macerated vegetative apices or immature embryos. The highest frequency of rooting was achieved at half strength MS medium supplemented with 2 mg l−1 IBA. Difficulties in rooting seemed to affect only shoots regenerated from callus, but not formed from meristematic tissues. Grafting of regenerated plantlets on 150 N. Kuchuk et al. to rootstocks of the same cultivar was used to overcome these rooting difficulties. 6.2.4 Regeneration via somatic embryogenesis The establishment of systems for the induction of somatic embryo formation from callus (indirect embryogenesis) or explant tissue (direct embryogenesis) have led to new advances in pea regeneration. Kysely et al. (1987) reported the first regeneration of a whole plant via indirect somatic embryogenesis from immature zygotic embryos and from the youngest node of shoot apex segments. A key factor for the induction of somatic embryos was the presence of an auxin (picloram or 2,4-dichlorophenoxy acetic acid; 2,4-D). Initially the embryogenic response and rate were low, no more than 15% with two somatic embryos per responding zygotic embryo, but this was improved after specific factors affecting efficiency were defined (Kysely and Jacobsen, 1990). For the best results embryogenic callus, exclusively originating from embryonic axis tissue, developed somatic embryos on MS media supplemented with 0.2 and 4.0 µM picloram or 4.0 µM 2,4-D. In embryogenic cultures from shoot apices 5.0 µM BA stimulated both the development of young somatic embryos and the appearance of new ones. Somatic embryogenesis depended on embryo size (optimal 3–6 mm) and genotype, embryogenic response varying from 2 to 31% among the five genotypes tested. It was influenced also by the auxin concentration. The authors concluded that an optimal embryo induction medium, with regard to auxin type and concentration, has to be determined empirically for each pea genotype. The same laboratory (Lehminger-Mertens and Jacobsen, 1989b) first reported pea somatic embryogenesis from protoplast-derived callus. Stejskal and Griga (1992) used 46 lines of Pisum to study the effect of genotype on somatic embryogenesis from immature zygotic embryos. Only one genotype (line HM-6) exhibited good embryogenic competence reaching a mean frequency of embryogenic explants of 14.6%, with one to six somatic embryos per explant (induction medium with 2,4-D). In addition, these authors supported earlier work by Kysely et al. (1987) regarding the development and germination of somatic embryos following their transfer into a medium containing cytokinins (0.15 µM BA, 0.15 µM kinetin, 0.15 µM zeatin) and an auxin (0.15 µM NAA). In most of the previous studies somatic embryogenesis was indirect, involving an intermediate callus phase. Tetu et al. (1990) first observed in certain genotypes the formation of somatic embryos and buds directly from the cotyledonary surface of immature zygotic embryos (3–6 mm in size). Embryogenesis was achieved when explants were cultured for 4–5 weeks in darkness on MS medium containing 43 µM NAA enriched with 15 µM thiamin hydrochloride, 40 µM nicotinic acid and 60 µM arginine. Somatic Biotechnology 151 embryos developed into plantlets when transferred to germination medium containing MS salts, 0.01 M KNO3, 15 µM IBA and 2.2 µM BA. Rooting was possible on MS medium supplemented with 16 µM NAA and 13.3 µM BA. An embryogenic response was observed in six out of nine genotypes and varied from 2 to 48%. Further data about direct pea somatic embryogenesis from immature cotyledons or shoot apical meristems were reported by Nadolska-Orczyk et al. (1994), Loiseau et al. (1995) and Griga (1998). Recently, the most efficient and quick protocol for pea somatic embryogenesis has been shown to be by direct regeneration from shoot apical meristems. This was successfully tested using more than 50 garden pea and field pea genotypes as well as in wild pea forms (Griga, 1998; Griga, unpublished data). For more details connected with pea somatic embryogenesis see Griga (1998, 1999). 6.2.5 Regeneration via organogenesis and multiple shoot formation There are two organogenic pathways, multiple bud development from pre-existing meristems and de novo shoot bud regeneration after dedifferentiation of existing cells. Griga et al. (1986) used a combination of BA (10 µM) and NAA (0.1 µM) to induce abundant multiple shoot formation from shoot apices axillary buds of the first normal leaf, and axillary buds of the first and second primary scale leaves. Tetu et al. (1990) observed axillary bud initiation from the main meristem and de novo shoot bud regeneration from cotyledons of whole zygotic immature embryos. The processes depended on BA (13.3 µM), to induce axillary bud formation, and the addition of NAA (16 µM) for axillary shoot formation. When 2,3,5-triiodobenzoic acid (TIBA) (0.2 µM) was added to the NAA– BA-supplemented MS medium bud proliferation was increased, both from apical meristematic areas and from cotyledons. The results indicated that the above-described morphogenesis in pea depends on three major factors: the explant size, the cultivar/genotype and the nutritive media. Direct organogenesis has been promoted in meristems from shoot tips, axillary buds of primary scale leaves and axillary buds from cotyledons (Kallak and Koiveer, 1990); 80–90% of the explants initiated growth, but further development and normal morphology were poor. Shoot regeneration was most effective in cotyledonary axillary meristems. Cotyledonary nodes were suitable explants for Jackson and Hobbs (1990) to develop a scheme for rapid multiple shoot production, which they suggested could be applied to a wide range of important pea varieties. For efficient multiple shoot production it was essential to culture the cotyledonary node explants on MS medium containing 1 mg l−1 BA, to remove the axillary bud region and to remove the initially developed shoot. This gave 100% response and up to ten buds per explant in all genotypes. After removal of shoots bigger than 1 cm, the remaining tissue could maintain 152 N. Kuchuk et al. organogenesis for several months on MS basal medium with B5 vitamins, supplemented with various concentrations of BA or kinetin. The best compromise between the quality and number of buds was achieved using 1–5 mg l−1 BA. Rhizogenesis was induced at a frequency of 90% using NAA (0.186 mg l−1). Nielsen et al. (1991) studied the effect of different auxins by applying sequential auxin–cytokinin treatment on hypocotyl discs. Explants were placed for 2 days on basal K3 medium supplemented with 10 mg l−1 IAA. Transfer to a medium containing 5 mg l−1 zeatin resulted in shoot formation with a frequency of more than 50% in all five cultivars tested. Doubling the IAA concentration from 5 to 10 mg l−1 increased shooting by a factor of two. The effect of IAA and NAA were similar, while 2,4-D treated explants produced callus and no shoots. This observation confirmed the persistence of 2,4-D and supported the assumption that added auxin has to be metabolizable to allow shoot formation. Ozcan et al. (1992) studied factors affecting organogenesis via callus (indirect organogenesis) using explants from various organs at different developmental stages. Root, epicotyl and shoot tips formed only callus, while 25% of the leaflet explants regenerated shoots at a low frequency. In contrast, they found rapid and prolific shoot development from immature cotyledons, following an initial callus growth, on MS medium containing 0.5 mg l−1 BA and 4 mg l−1 NAA. The orientation of the cotyledonary explants to the medium surface appeared important. The highest regeneration frequency was achieved when the distal end was placed on to the agar, suggesting a polar phenomenon affecting morphogenesis. In addition, parts of the cotyledon had different regeneration potential, with the highest being for sections proximal to the embryonic axis. The developmental stage also had a crucial effect, with most shoots being produced by fully developed green cotyledons, prior to the shift to yellow maturity. Adventitious shoots developed in a range of media supplemented with BA (0.25, 1, 2 and 4 mg l−1) and NAA (0.25, 1 and 8 mg l−1) or IBA (0.25, 1, 4 and 8 mg l−1), with NAA being superior to IBA. BA alone also stimulated shoot development, but in this case a few shoots could become dominant, inhibiting the elongation of the others. Elongation was stimulated by AgNO3. The authors consider this system to be very suitable for somaclonal variation and transformation experiments. The role of phytoregulators is another important regeneration factor. A novel procedure has been developed to initiate shoot regeneration from intact seedlings produced from mature seeds germinated on a medium containing cytokinin or cytokinin-like substances (kinetin, zeatin and TDZ; Malik and Saxena, 1992c). TDZ, a substituted phenylurea with cytokininlike activity, commonly used as a cotton defoliant, was found to be most effective. Pea seedlings exhibited a unique pattern of shoot formation, which was accomplished in two distinct phases. Multiple shoots developed within a week from the nodal and basal regions of the primary epicotyl, in a Biotechnology 153 medium containing 5–50 µM TDZ. When these seedlings were exposed for a prolonged time (3–4 weeks) to the same medium, numerous shoots emerged de novo from the base, or from the upper part, of multiple shoots. Bohmer et al. (1995) developed a protocol for high frequency shoot induction and plant regeneration from protoplast derived pea callus, using TDZ as the key factor in the system. 6.2.6 Recent studies to produce more efficient, fast and reliable systems for regeneration Recently, efforts have been focused on optimizing the systems and extending the knowledge of factors effecting regeneration in grain legumes. Using TDZ, Sanago et al. (1996) developed a simple and rapid regeneration procedure. An average of up to 20 shoots formed from each hypocotyl explant cultured on MS medium supplemented with 0.5 or 1.0 µM TDZ. Shoots (0.5–1.0 cm), detached from the parental tissue, were cultured on MS basal medium with B5 vitamins and 3.0 µM GA3 to facilitate elongation. Formation of roots was high (50–60%) on medium containing either 2.0 µM NAA or 1.0–2.0 µM IBA, and seeds were harvested from regenerated plants after only 9–11 weeks. Ochatt et al. (1998), however, reported that with TDZ, or zeatin, large numbers of buds were produced that were miniaturized, hyperhydric and had impaired rootability, plus reduced flowering and fruiting. To study later effects of growth regulators, these authors used hypocotyl segments, without pre-existing meristems, to induce embryogenesis and organogenesis on modified MS media supplemented with different phytohormones. 2,4-D and picloram induced somatic embryos up to the cotyledonary stage, but they mostly lacked a root or shoot meristem and were too weak to germinate. Callogenesis was more reliable than embryogenesis. The best regeneration responses were obtained using 3 or 5 mg l−1 BA and 0.01–0.5 mg l−1 NAA, harvesting shoots several times from each explant. Rooting, flowering and fruiting were also better using this hormonal regime compared with other phytoregulators. The authors claim that this was the first report where hormones used for bud regeneration and rooting could be correlated with the subsequent flowering and fruiting of the regenerants. Stimulation of organogenesis has been observed for other cytokininlike substances not used traditionally in in vitro cultures (Kosturkova and Tineva, 1998). Looking for more powerful phytoregulators, however, is only one of the approaches for optimizing conditions for efficient regeneration. Screening for appropriate genotypes able to realize their morphogenic potential is another option. By comparing two different systems, a more pronounced genotypic effect was observed when indirect, rather than direct, organogenesis was promoted (Kosturkova et al., 1997), 154 N. Kuchuk et al. suggesting the involvement of both epigenetic and culture conditions in the realization of morphogenic potential. A 100% regeneration efficiency for seven out of ten genotypes tested was achieved from immature embryonic axes cultured on modified MS medium containing 10 µM BA and 1 µM NAA (Kosturkova et al., 1997). When the alternative scheme of callus induction with 0.2 µM 2,4-D was used, only three genotypes responded with bud formation from all explants transferred to media supplemented with 5 µM BA. Genotypic differences occurred in bud initiation and multiple shoot formation of cultures maintained for several months on media containing 0.5 µM BA and 0.25 µM NAA. Such a prolonged culture with vigorous shoot formation is suitable to study the effect of biotic and abiotic stress resistance and to perform cell selection in vitro. Considerable variation for the frequency of callus formation and for the onset of regeneration was observed by Jarkova et al. (1998). Among 26 genotypes tested, 12 had a morphogenic response from immature cotyledons on one or both media for embryogenesis or organogenesis. The majority of the explants were characterized by 100% callus formation. Significant differences in embryogenic potential were observed between all samples. Also, there was wide variation between the samples for the onset time of morphogenesis, which appears to be very important for normal shoot regeneration and to avoid abnormalities, especially cytogenic instability (i.e. geotropic reaction, albinism, reduced leaves and internodal interval). 6.2.7 Factors effecting regeneration It is obvious that the development of pea in vitro is not a uni-directional process and that development can be manipulated to induce callogenesis, organogenesis or embryogenesis. These processes, however, are influenced by various factors. Recently, considerable information has been obtained about the factors affecting the processes of morphogenesis in grain legumes, which has contributed a great deal to recent success. The most important factors are the explant, growth regulators and genotype. Explant Among the various tissues used as initial material for in vitro cultures it seems that whole or parts of (cotyledons or embryonic axes) immature zygotic embryos are preferable as embryogenic and organogenic explants. Cotyledonary nodes from seedlings and meristematic tissues are suitable material for the induction of adventitious bud formation and micropropagation, while young leaflets, epicotyl and hypocotyl have been used less. The developmental stage of the explants is very important as it can Biotechnology 155 determine the pattern of response, the most efficient being immature embryos before full maturation and cotyledons of 3–6 mm in size. Growth regulators Auxins and cytokinins, or substances with a similar structure generally regulate in vitro development and plant regeneration. Choosing the right growth regulators and the correct concentration seems to depend on the explant, its developmental stage and on the genotype, leading to a variety of regeneration protocols. Some general observations, however, can be made. The presence of a high concentration of auxin is essential for somatic embryogenesis, but the type of auxin can differ: 2,4-D and picloram being cited as superior; NAA is less efficient for embryo induction, but is necessary for embryo conversion; NAA and IAA, depending on the concentration, can induce callogenesis or rhizogenesis. BA alone, or in combination with an auxin, has been the most commonly used cytokinin for induction of organogenesis and shoot proliferation. Zeatin is very efficient in shoot induction, but TDZ seems to be superior as well as being efficient in the embryo-conversion process (Griga, 1998). Genotype Recalcitrance in grain legumes could be the result of a long history of inbreeding and selection, leading to a reduction in genetic variability. Screening a large number of genotypes could help to discover those with a better response to organogenesis and/or embryogenesis. A correlation between embryogenic and organogenic capacity in different responding cultivars, however, is not always observed. With regard to rooting frequency, the data are also contradictory. There are reports, however, that these processes may be under genetic control (Althers et al., 1993; Bencheikh and Gallais, 1996). 6.2.8 Advantages of the different developmental pathways for in vitro manipulation The range of regeneration systems available allows the most appropriate system to be chosen for a particular purpose. Meristem cultures can be used for the preservation of germplasm, the production of virus-free plants and for micropropagation. Somatic embryos can be used for artificial seed production. Since somatic embryos are produced from a single cell they would be the preferred target for genetic manipulation, but embryogenic cultures are more difficult to obtain and sustain. Organogenic cultures are, therefore, more important for somaclonal variation, in vitro selection and transformation. With regard to Agrobacterium transformation, it has been suggested (Parrot et al., 1992) that organogenesis de novo is necessary, while 156 N. Kuchuk et al. for particle bombardment, transformation proliferation from meristems also can be used. 6.3 Isolated Protoplasts from Grain Legumes 6.3.1 Introduction to protoplast cultures Definition The term protoplast refers to all components of a plant cell excluding the cell wall. The plant cell wall consists of three primary components, cellulose (25–50%), hemicellulose (average 50%) and pectin substances (about 5%). Cocking (1960) first used hydrolytic enzymes for digesting the cell wall of tomato root tips to release plant protoplasts. This method allows the quick isolation of an indefinite number of uniform plant protoplasts from any type of plant tissue from any plant species. General procedures for the isolation and cultivation of plant protoplasts To isolate protoplasts the tissue is incubated with digestive enzymes (cellulases, hemicellulases and pectinases) for 1–16 h. Protoplasts are washed and resuspended, at an appropriate density (103–106 protoplasts ml−1), in liquid or on solidified culture medium. They can regenerate a new cell wall within 24–48 h, undergo their first mitotic division between the second and tenth day of culture and then form colonies that grow into callus tissue. This callus can generate plants by the induction of embryogenesis or organogenesis. More rarely, somatic embryos can be obtained directly from protoplasts or from protoplast-derived colonies. Each step consists of several parts, all of which seem to be important and crucial for the successful protocol (Evans and Bravo, 1984; Eriksson, 1985; Power and Chapman, 1985; Binding, 1986). The complexity of the work makes protoplast cultures more difficult to handle than meristematic, callus and cell suspension cultures, but the absence of a hard cellulose cell wall gives plant protoplasts some advantages compared with the other in vitro cultures (Fowke and Wang, 1992; Paszkowski et al., 1992; Kosturkova, 1993). Advantages of isolated plant protoplasts These can be listed as follows: • • The plasmalemma is accessible, allowing unique studies of membrane transport, cell wall biosynthesis, cell growth and differentiation, and other processes in cell biology. Each protoplast serves as a single organism and so a population of several million plant cells can be manipulated, allowing events that occur at very low frequency (10−6–10−7) to be monitored. Biotechnology • • • • 157 The regenerated plant has a single-cell origin. This is important for mutagenesis and selection in vitro, since the regeneration of chimeric plants is assumed to be less likely. Protoplasts can be fused from plants belonging to different species and other taxonomic units, giving rise to somatic hybrids and cybrids combining various nuclei and cytoplasmic genetic material. They allow the isolation and transfer of organelles and single chromosomes from one cell to another, achieving new combinations of mitochondria, chloroplasts, vacuoles and nuclei. They can undertake direct DNA uptake, which allows the rapid detection of gene expression and genetic transformation in cases where other methods like Agrobacterium or particle bombardment are not applicable. 6.3.2 Protoplast cultures from leguminous species Interest towards protoplasts from leguminous species dates from the early 1970s. Most investigations were carried out on soybean and pea, which are the most important grain legume species. Some of the achievements are presented in Table 6.2. Grain legumes proved to be recalcitrant, however, which has made success in regenerating plants from protoplasts difficult. Different sources for producing protoplasts (cell suspension, leaf mesophyll, hypocotyl, epicotyl, etc.) and various culture conditions Table 6.2. Some achievements in the development of isolated pea (Pisum sativum) and soybean (Glycine max) protoplasts to regeneration of whole plants. In vitro response Pea Cell division Callogenesis Embryogenesis, organogenesis Plant regeneration Soybean Cell division Callogenesis Plant regeneration References Landgren (1976); Jia (1982) Constabel et al. (1973); Gamborg et al. (1975); von Arnold and Eriksson (1976): Kuchuk (1989) Puonti-Kaerlas and Eriksson (1988); Lehminger-Mertens and Jacobsen (1989a); Ochatt et al. (1998) Lehminger-Mertens and Jacobsen (1989b); Boehmer et al. (1995); Sanago et al. (1996), Ochatt et al. (1998) Kao et al. (1970); Gamborg et al. (1983); Lu et al. (1983); Tricoli et al. (1986); Hammat and Davey (1988) Zieg and Outka (1980); Xu et al. (1982); Oelck et al. (1983); Kuchuck (1989) Myers et al. (1989); Guo (1991); Dhir et al. (1991, 1992); Lu et al. (1993) 158 N. Kuchuk et al. (composition of the media, osmotic, phytoregulators, agarose type, etc.) have been examined. Until 1988, callus cultures were obtained from protoplasts, but the regeneration of plants was not achieved. Successful shoot formation from pea protoplasts was first reported by Puonti-Kaerlas and Eriksson (1988; Table 6.2) and the first regeneration of plants by Lehminger-Mertens and Jacobsen (1989b; Table 6.2). At about this time, soybean also was regenerated (Dhir et al., 1991; Table 6.2), although plants from wild Glycine species had been regenerated several years earlier (Hammat et al., 1987). 6.3.3 Application of grain legumes protoplasts to the study of carbohydrates Plant protoplasts lack cell walls and are, therefore, a good experimental system for various types of study, such as genetics (using somatic hybridization and genetic transformation), physiology, cytology, biochemistry and other fields of biological science. In particular, they are an excellent experimental system for basic studies of cell wall regeneration, cell division, membrane fusion, membrane transport, virology, endocytosis and transfer of organelles (Fowke and Constabel, 1985; Fowke and Wang, 1992). The sections below cover the role of carbohydrates in different cell processes of grain legume protoplasts and present possibilities on how protoplast systems may be exploited as tools for carbohydrate research. The role of the carbohydrates in protoplast media Carbohydrates are essential for protoplast isolation and for maintaining their life functions. During the removal and regeneration of the cell wall, its pressure must be replaced by the osmotic pressure of the isolation and culture media, by adding various sugars or sugar alcohols. The type and concentration of the osmoticum influences protoplast viability, regeneration of the cell wall and division. The most frequently used osmotica are mannitol and sorbitol, which are relatively metabolically inert. Sucrose and glucose are also utilized in the early stages of culture. In many systems, additional carbohydrates such as cellobiose, ribose, xylose and arabinose can be beneficial (Evans and Bravo, 1984; Eriksson, 1985). The maintenance of optimal osmotic conditions is closely related to the stability, viability and future development of the protoplasts and highlights the role of carbohydrates in metabolic processes. von Arnold and Eriksson (1977) observed that mesophyll pea protoplasts cultured in media free of sucrose, formed poor cell walls and could not divide. Xylose, arabinose and glucose, at a concentration of 1 mM, had favourable effects on growth, while ribose and galactose did not influence it. The minimum osmotic pressure was about 500 mOsm, higher values Biotechnology 159 causing poor wall formation and lower budding of protoplasts. Only mannitol and sorbitol, however, could function as osmotic stabilizers in such high concentrations (0.25 M), sucrose and glucose in concentrations higher than 0.15 M being harmful to the protoplasts (von Arnold and Eriksson, 1977). Protoplast development was closely related to the type and concentration of the carbohydrate in the protoplast culture media (LehmingerMertens and Jacobsen, 1989a,b). The viability of protoplasts isolated from shoot tips did not differ dramatically when either mannitol or sucrose was used as the osmoticum, but if glucose was used then 100% lethality occurred within a few hours. In other experiments (Puonti-Kaerlas and Eriksson, 1988) using mesophyll protoplasts, however, 0.4 M glucose was successful for maintaining osmolarity in the initial protoplast medium. Whenever liquid media, instead of agarose bead cultures, were used (Lehminger-Mertens and Jacobsen, 1989a), an increasing sugar concentration from 3 to 7% and a decrease of pH values to 3.9 were detected within 3 days, these changes correlating with a dramatic decrease in cell division. The source and concentration of carbohydrate are also essential for the induction of embryogenesis in protoplast cultures of pea (Lehminger-Mertens and Jacobsen, 1989b). For the induction process of protoplast-derived calli, mannitol could not be substituted by sucrose. Embryogenesis occurred when the mannitol used as an osmoticum had a defined level, 4% but not 3%. The role of carbohydrates in cell wall synthesis Plant protoplasts offer considerable opportunities for studying the synthesis, secretion and assembly of the primary cell wall, as well as the role of the cell wall during development. Hanke and Northcote (1974) examined cell wall formation and found that during the first 20 h of wall regeneration 14C glucose was predominantly incorporated into protein, starch and cellulose and small amounts were incorporated into an acidic pectin. Klein et al. (1981) observed the synthesis of a broad spectrum of polysaccharide polymers during the first 3 h of wall regeneration. Radioactivity was detected in newly synthesized cellulose within minutes after the protoplasts were transferred to a wall regeneration medium containing 14C glucose. This process coincided with the appearance of fibrils on the surface of the protoplasts. In addition to cellulose, other polysaccharide-containing polymers were also synthesized. Uridine diphosphate 14C glucose and guanosine diphosphate 14C glucose did not serve as effective substrates for cellulose synthesis if protoplasts were able to utilize glucose for this process. In intact cells, polysaccharides and proteins are tightly covalently linked. Within the structure of the regenerating protoplast cell wall, however, such linkages are less apparent. Williamson et al. (1976) studied the distribution of carbohydrate residues on the plasma membrane of soybean 160 N. Kuchuk et al. protoplasts and observed that carbohydrate sites were evenly distributed, but mobile within the plane of the membrane. Chemically and physically uniform cellulose contrasts with the complex heterogenous mixture of covalently linked polysaccharides and proteins that make up the remainder of the wall (Willison, 1985). Pharnisi et al. (1993) observed that regeneration of cell walls on the surface of soybean protoplasts was accompanied by the release of smooth vesicles from the cytoplasm to the outside of the plasma membrane. Possibly, the vesicles carried wall materials that were gradually deposited. A complex cell wall layer, with a fine cellulose microfibril structure, was formed and clearly seen on the third day of culture. Nuclear division and subsequent cytokinesis occurred about 1 day after culture, suggesting that cell wall formation starts earlier than cell division and then both processes proceed concurrently. The role of carbohydrates in protoplast division Isolated protoplasts are an extremely valuable experimental system for investigating the plant cytoskeleton during cell division and the related morphogenic role of the wall, during subsequent development of the protoplast derived cell. Fowke et al. (1974) compared cell division between cultured soybean cells and their protoplast derivatives. They found a more rapid formation of the phragmoplast and the development of a thicker cell plate in protoplasts, indicating a slight modification in cross wall formation during cytokinesis. Synchronized soybean protoplast cultures permitted the detailed examination of preprophase bands, which relate to the morphogenic potential of the protoplasts (Wang et al., 1989; Fowke and Wang, 1992). Divisions occurring within the first 24 h of Vicia hajastana protoplast culture showed considerable abnormalities in the formation of microtubule spindles, phragmoplast, incomplete cross wall and aberrant chromosome segregation, which were reflected in further development of the protoplast-derived cells. The importance of a regenerated cell wall for normal mitotic processes is quite well illustrated by mesophyll protoplasts of lucerne, which are much slower at initiating division and, therefore, have time to form a proper new cell wall. This results in less mitotic abnormalities (Simmonds, 1992) and leads to normal growth and morphogenesis in protoplast cultures of this species. Protoplast division can be influenced by starch accumulation in the cell. It was suggested that in pea protoplasts from hypocotyl and primary roots, large amounts of starch were inhibitory (Landgren, 1981). In potato tuber protoplasts, division began 1 week after their culture, when most of the starch grains had been metabolized (Jones et al., 1989). Assuming that highly meristematic tissues with less starch content were a more suitable source for protoplast isolation and development, Lehminger-Mertens and Jacobsen (1989a) examined various explants from germinating pea Biotechnology 161 seedlings (leaf, shoot tip, epicotyl, hypocotyl and root tip) and observed the accumulation of starch after 1 week. Starch accumulation was not observed in protoplasts derived from shoot tips or from the first lateral shoots originating from the cultured embryonic axis minus the cotyledons. In these cultures, particularly homogenous meristematic cells and sustained protoplast division was achieved. In contrast to previous reports, Gram et al. (1996), studying starch accumulation in relation to the frequency of cell division and regeneration in pea protoplasts, suggested that starch accumulation precedes the division of pea protoplasts. They found that the starch content increased rapidly during the first 3 days of culture, prior to the onset of division, resulting in a 4.2-fold increase in the intracellular starch area and a threefold increase (from 27 to 80%) in the number of protoplasts containing starch. Mitosis was observed after the fourth day and the number of protoplasts undergoing division increased in a stepwise manner, preceded by further starch accumulation. Since dividing protoplasts were initially 33–66% smaller and contained 8–42% less starch than non-dividing protoplasts, the dividing protoplasts contained relatively more starch (6–12%) than non-dividing protoplasts on a per unit volume basis. Interestingly, the starch level reached before the onset of the first mitosis was comparable to the level found in actively dividing micro-calli, suggesting a requirement of certain levels of starch accumulation for the induction of pea protoplast division. It can be suggested that there may be an optimum starch content for protoplast division and that levels below or above this threshold may be inhibitory for mitosis. Sugar transport through plasmalemma Transport of solutes through the plasma membrane and the tonoplast membrane are important cellular activities. For many studies it is desirable to work with relatively homogeneous population of individual cells that are not organized into tissue. Sugar transport affects the partitioning of assimilates between the source and the sink regions of a plant that determine crop yield. In developing soybean seeds sucrose is unloaded from seed coat phloem into the apoplast prior to its accumulation by the developing seeds. Mechanisms of sugar transport have been analysed using protoplasts isolated from developing soybean cotyledons (Lin et al., 1984; Schmitt et al., 1984; Lin, 1985a,b). Compared with intact cotyledons, isolated protoplasts offer distinct advantages, such as the absence of bulk diffusion and tissue penetration barriers, the accessibility of cell membranes for challenging with sugar analogues and the prevention of oligosaccharide hydrolysis due to hydrolases associated with the cell wall. Sucrose and hexose uptake into protoplasts has shown that, during rapid seed growth, the plasmalemma of cotyledons contains a sucrose-specific carrier, which is energetically and kinetically distinct from the system(s) involved in hexose transport. Sucrose 162 N. Kuchuk et al. uptake by protoplasts is composed of three different mechanisms, a saturated, carrier-mediated, energy-dependent mechanism, a carrier-mediated, but non-saturated, or linear mechanism and simple diffusion (Lin et al., 1984). Other studies have focused on proton, sulphydryl reagent and temperature sensitivities of sucrose uptake kinetics and the operation of multiple sucrose uptake mechanisms (Schmitt et al., 1984). The addition of sucrose to protoplast media causes a specific and transient depolarization of the membrane potential, acidification of the intracellular pH and alkalization of the external medium. Such results suggest that a proton/ sucrose co-transport system is also involved in non-diffusive linear sucrose uptake (Lin, 1985a,b). 6.4 Somaclonal Variation in Grain Legumes 6.4.1 Introduction There are two basic applications of cultured plant cells that exploit their totipotence (ability to express the entire genetic information and regenerate plants). Firstly, to clone the cells and produce plants that are identical and, secondly, to change/manipulate the genome and create novel types of plants. The former application is to maintain valuable genotypic traits and the latter application is to improve or obtain new characters. Using somatic cell techniques, plants can be genetically manipulated using somaclonal variation, in vitro mutagenesis and somatic hybridization/cybridization and transformation by the introduction of foreign genes. Changes in genomes generally appear at a low frequency and a large population of an organism, therefore, is necessary for manipulation. In this respect, in vitro culture (callus culture, cell suspension or isolated protoplasts) has the advantage of a very large number of individuals in a small space, compared with the large growing area and intensive labour required to treat equivalent numbers of plants. During the early stages of the development of tissue culture methods, in vitro cultured cells were believed to be uniform and similar to the initial material. Regenerants obtained through embryogenesis or organogenesis in vitro as a result of asexual reproduction, therefore, should be phenotypically and genotypically identical to the donor plant. In the early 1970s, however, there were reports of morphological and cytological changes in tobacco plants grown from in vitro cultures (Zagorska et al., 1974). Later, there were similar reports of variability in tissue culture-derived plants. This phenomenon was given the name of ‘somaclonal variation’ by Larkin and Scowcroft (1981) and is generally found in plants regenerated from callus tissue, cell suspensions or isolated protoplasts and, to a lesser extent, in meristem or shoot tip cultures. Biotechnology 163 6.4.2 Factors causing variation The isolation of cells from the integrity of the whole plant and the reorganization of genome function during the establishment of in vitro culture often cause fundamental destabilization of the genetic and epigenetic status. The content and composition of culture media affect chromosomal and cell cycle instability (Gould, 1986). For example, plant growth regulators (auxins and cytokinins), that are essential for the dedifferentiation and re-differentiation of the cells in vitro, often act as agents for genomic changes. Also, inorganic (e.g. phosphates and nitrates) and organic (e.g. carbohydrates) compounds within the medium contribute to cell cycle abnormalities. Even physical factors, such as temperature, the light regime and the viscosity and osmolarity of culture media, are known to affect the cell division cycle of plant cells in culture. The culture phase and the rate of subculture are also of importance, since prolonged cultivation can cause more changes in the nuclear and cytoplasmic genome. Selection of one cell type, however, can occur during subculture, resulting in less diversity being observed in such cultures when they are maintained for a prolonged period (Zagorska, 1995). Pre-existing genetic differences, like the ploidy level of the initial material, the explant origin and the genotype can be another source of variation. The regeneration pathway, via organogenesis or embryogenesis, is another factor causing or eliminating the appearance of somaclones. Somatic embryogenesis, as a mechanism of plant formation from a single cell, was postulated to give ‘free of variability’ regenerants (Vasil, 1986). Nevertheless, recent evidence is presented below on variant plants that have been regenerated via somatic embryogenesis. 6.4.3 Mechanisms of somaclonal variation Somaclonal variation is a complex phenomenon which results from a multiplicity of cellular and genetic mechanisms (Karp, 1993). Generally the changes that occur have a genetic character and can be inherited, but epigenetic (cannot be transmitted to the progeny) variations are also observed (Gould, 1986). The most common genetic changes are polyploidy, aneuploidy, chromosome aberrations, point mutations and alteration in DNA copy number. There may also be alterations in mitochondrial and chloroplast genomes. These genetic changes, resulting in disturbances in cell cycle and DNA replication, are observed mainly in cultures where disorganized growth and a prolonged culture phase are involved. In contrast to these genetic effects, epigenetic variation cannot be transmitted through a sexual cell cycle. Nevertheless, the induced phenotype modifications can be a valuable source of plant diversity. A 164 N. Kuchuk et al. better understanding of the mechanisms and factors of determining variation will contribute to maximizing or minimizing variability as required. Eliminating potential problems of variation in transgenic plants is still important. 6.4.4 Potential and disadvantages of somaclonal variation Perhaps the greatest benefit of somaclonal variation is in plant improvement, by creating additional genetic variability in agronomically useful cultivars, without the need of sexual hybridization (Lorz and Brown, 1986). It is of primary importance in vegetatively propagated species and in many horticultural and woody species, for which variation is not readily available. Genomic variation is the first step in the selection of plants for crop improvement and the absence of such variation can be a limiting factor in breeding programmes. It is assumed that somaclonal variation is less important in seed crops, where variation can be created in the gene pool by reconstruction of genes after crossing. The application of tissue culture-derived variability, however, can be useful for such species by combining in vitro culture with in vitro selection (Scowcroft et al., 1987). By applying selective pressure, variants with a desired character can be isolated at an early stage of cell development, avoiding regeneration and testing of a great number of useless somaclones. The availability of rapid screening procedures for useful traits makes somaclonal variation a powerful tool for plant improvement. For example, resistance, or higher tolerance, to biotic and abiotic stresses can be achieved by including selective agents such as pathotoxins, fungal filtrates, herbicides, salts and heavy metals in the culture media. One of the greatest advantages of somaclonal variation, however, is for selection of those traits that can be selected only under in vitro conditions. For example, including amino acid analogues in the culture media can result in the selection of over-producers of amino acids. Somaclonal variation is also a pool for characters for which there is no adequately defined in vitro response, such as yield, seed protein/oil quality, photosynthetic efficiency, etc. The identification and availability of effective plant screening protocols for this type of trait will contribute much to the wider application of somaclonal variation. Somaclones may appear as a negative fact in those cases where clonal uniformity is required (e.g. horticulture, forestry, genetic transformation; Scowcroft et al., 1987). It is likely, however, that the greatest disadvantage of this phenomenon is its unpredictable nature, the same in vitro culture, for example, can generate different types and frequency of somaclonal variation (Zagorska, 1995). Biotechnology 165 6.4.5 Variation in grain legumes at the cell and tissue culture level in vitro Before the term somaclonal variation was introduced into the literature (Larkin and Scowcroft, 1981), a relatively large amount of evidence was available that plant tissues in culture exhibit a broad spectrum of cytological and karyological changes and abnormalities. This fact also included grain legume species. Since the main aim of this part of the chapter is to review heritable somaclonal variation at the plant level, typical examples of variation on cell and tissue culture level in vitro are only mentioned briefly (Table 6.3). Within this table, various altered cell and callus lines are characterized; however, with the absence of plant regeneration, the heritable nature of these changes is subject to speculation. Cytological instability in vitro Since 1960, the majority of evidence of tissue culture induced variation in grain legumes has involved cytological instability and changes. In callus cultures and cell suspensions of P. sativum with prolonged subcultures, there is a strong tendency towards spontaneous polyploidization, ranging from 3n to 32n or higher (Van’t Hof and McMillan, 1969; Frolova and Shamina, 1974; Mikhailov and Bessonova, 1975; Knosche and Gunther, 1980; Knosche, 1981). Endoreduplication, induced hypothetically by growth regulators (cytokinins, IAA, 2,4-D), leads not only to polyploidy and nuclear DNA content increase (Libbenga and Torrea, 1973), but also to polytene chromosome formation (Marks and Davies, 1979; Therman and Murashige, 1984). In addition to polyploidy, a number of aneuploid cells, chromosomal aberrations and karyological abnormalities have been reported (Kallak and Yarvekylg, 1971, 1976, 1977a,b; Frolova and Shamina, 1974; Mikhailov and Bessonova, 1975; Ghosh and Sharma, 1979; Natali and Cavalini, 1987a). Haploid cells have been found in pollen-derived pea callus (Gupta, 1975) and in callus derived from somatic, diploid, pea tissues (Kunakh et al., 1984; Natali and Cavalini, 1987b). A detailed review about cytogenetics of pea callus and cell cultures has been published by Griga and Novák (1990). Cytological instability in callus and suspension cultures of Vicia faba was first reported by Venketeswaran (1963) and Venketeswaran and Spiess (1963, 1964). In the 1970s, cultures of faba bean were frequently used as a model for cytogenetic studies. Observations of all types of euploidy, from haploid cells to highly polyploid cells up to 32 n or 64 C, aneuploid cells and many mitotic abnormalities including endoreduplication, were reported (Frolova and Shamina, 1974, 1978; Yamane, 1975; Shamina and Butenko, 1976; Cionini et al., 1978; Papet et al., 1978; Roper, 1979; D’Amato et al., 1980; Hesemann, 1980; Jelaska et al., 1981; Ogura, 1982; Frolova, 1986; Taha and Francis, 1990). A detailed review on the cytogenetics of faba bean 166 Table 6.3. Somaclonal variation in grain legumes on cell, tissue and organ culture level in vitro. Type of in vitro culture Trait Response (value/description) References Arachis hypogaea (peanut, groundnut) Anther-derived callus Chromosome number Mixoploid callus (haploid to octoploid cells) Bajaj et al. (1981) Cajanus cajan Anther-derived callus Chromosome number Haploid, diploid and aneuploid cells Bajaj et al. (1980) Cicer arietinum Callus, cell suspension Salt tolerance (NaCl) Callus Salt tolerance (NaCl) Anther-derived callus Chromosome number NaCl-tolerant cell lines NaCl-tolerant cell lines Haploid to octoploid cells Gosal and Bajaj (1984) Pandey and Ganapathy (1984) Bajaj and Gosal (1987); Gosal and Bajaj (1988) Glycine max Cell suspension Callus Callus; excised cotyledons Callus Accumulation of a phytoalexin glyceolin in elicitor treated cells Bioassay for screening resistant or susceptible genotypes in vitro culture Bioassay for screening resistant mutants in culture; high degree of correspondence between reaction in tissue culture and glasshouse tests Identification of tolerant Resistance to Diaporthe phaseolorum culture filtrate; soybean cultivars based on dual culture with the fungus in vitro test Resistance (reaction) to elicitor from Phytopthora megasperma var. sojae Resistance (reaction) to P. megasperma var. sojae Resistance (reaction) to soybean brown stem rot (Phialophora gregata) culture filtrate Ebel et al. (1976) Holliday and Klarman (1979) Gray et al. (1986); Willmot et al. (1989) Simoni et al. (1995) N. Kuchuk et al. Species Phaseolus coccineus Suspensor-derived (runner bean) callus Phaseolus vulgaris Chromosome number, DNA Great variability in content chromosome number Bennici et al. (1976) Excised roots, callus, cell suspension Resistance to Pseudomonas phaseolicola culture filtrate Bajaj and Saettler (1968, 1970) Callus from different plant parts Anther-derived callus Ploidy level (DNA content) Callus Salt tolerance (NaCl) Resistance to Pseudomonas syringae pv. phaseolicola culture filtrate Resistance to Sclerotinia sclerotiorum culture filtrate Haddon and Northcote (1976) Peters et al. (1977) Smith and McComb (1981) Hartman et al. (1986) Miklas et al. (1992) Vigna radiata Callus, cell suspension Salt tolerance (NaCl) Thioproline resistance; NaCl Seedling root resistance tip-derived Callus Gosal and Bajaj (1984) NaCl-tolerant cell lines Fivefold increase endogenous Kumar and Sharma (1989) levels of free Proline; elevated tolerance to NaCl Vigna sinensis (cowpea) Cell suspension Polyploidy (3n–6n); multinucleate cells Chromosome number Biotechnology Callus Callus Chromosome number Differential growth inhibition up to 77%; increase in abnormal cells; 55-fold increase in ornithine Mixoploid calli (2C–8C and even higher DNA content) Haploid, diploid and polyploid cells Salt-sensitive calli and plants Callus screening test; positive correlation between reaction on callus and plant level Callus-weight assay; screening of partial physiological resistance within dry bean germplasm Ghosh and Sharma (1979) Continued 167 Continued. 168 Table 6.3. Species Type of in vitro culture Trait Response (value/description) References Pisum sativum Callus Polyploid cells (3n, 4n and more); multinuclear cells; karyological abnormalities Polyploid (up to 12n) and aneuploid cells Polyploid cells (up to 8n and more); chromosomal aberrations Haploid and mixoploid (mainly tetraploid) cell populations Multinuclear cells, aneuploidy Highly polyploid cells (32n or more) Calli with improved tolerance to herbicides Kallak and Yarvekylg (1968, 1971, 1976, 1977a,b) Callus Chromosome number; chromosome aberrations; mitotic abnormalities Chromosome number Chromosome number; chromosome aberrations Cell suspension Callus culture Chromosome number Chromosome number Callus culture Chromosome number Tolerance to the herbicides Propham and Probanil (O-isopropyl-3-chlorphenylcarbamate) Callus, cell suspension Salt tolerance (NaCl) Callus Chromosome number, chromosome aberrations Meristem-derived and Chromosome number embryo axis-derived callus; regenerated shoots Mesophyll protoplasts Resistance to P. syringae pv. pisi Mikhailov and Bessonova (1975) Gupta (1975) Ghosh and Sharma (1979) Knosche and Gunther (1980); Knosche (1981) Jakel et al. (1990) NaCl-tolerant cell lines Gosal and Bajaj (1984) Haploid, diploid and Kunakh et al. (1984) polyploid cells (up to 8n) Diploid or aneusomatic Natali and Cavallini (1987a,b) (chromosomal mosaics) shoots Model system for pea – Pseudomonas interaction Akpa and Archer (1994) N. Kuchuk et al. Immature cotyledon-derived callus Pollen callus Frolova and Shamina (1974) Vicia faba Chromosome number Callus Chromosome number Callus Chromosome number Callus Chromosome number (as DNA content) Callus Chromosome number Callus and cell suspension Anther-derived callus Chromosome number Callus Callus culture Chromosome number (ploidy level measured cytophotometrically as C-value) Chromosome number Tolerance to the herbicides Propham and Probanil (O-isopropyl-3-chlorphenylcarbamate) Polyploid (4n–8n) and aneuploid cells Diploid and polyploid cells (mainly 4n) Polyploid (3n–8n) and aneuploid cells Binuclear and multinuclear cells; diploid and polyploid cells (up to 64C) Diploid, aneuploid and polyploid cells Diploid, polyploid and aneuploid cells Haploid, diploid and polyploid cells (more than 16C) Venketeswaran (1963) Frolova and Shamina (1974, 1978); Shamina and Butenko (1976) Yamane (1975) Cionini et al. (1978a,b); D’Amato et al. (1980) Papes et al. (1978); Jelaska et al. (1981) Roper (1979) Hesemann (1980) Biotechnology Suspension Polyploid and eneuploid cells Ogura (1982) Calli with improved tolerance Jakel et al. (1990) to herbicides 169 170 N. Kuchuk et al. callus and cell cultures has been published by Griga et al. (1986). Variation in ploidy level and other chromosomal changes and aberrations in tissue and cell cultures of grain legume species, other than pea or faba bean, are less frequently documented in the literature (Table 6.3). The inability to regenerate plants from cytologically altered cells and tissues in many of the above cited reports, may be due to the selective advantage of normal (diploid) cells in the process of organ formation (diplontic selection). The inability of plant regeneration from cytologically altered tissues determines that these changes cannot be practically exploited at present and may only serve as a model for theoretical studies. The mechanisms of chromosome variation in plant tissue cultures have been reviewed by Ogura (1990). Variation in tolerance/resistance to biotic and abiotic factors (stresses) The most frequent biotic factors studied in grain legumes at an in vitro level have been responses to bacterial and fungal pathogens or their toxins, usually contained in culture filtrates (Table 6.3). Some reports are orientated towards the formulation of exact and quick bioassays, for indicating the sensitivity/tolerance/resistance of tested genotypes to particular pathogen and its races (Bajaj and Saettler, 1968, 1970; Ebel et al., 1976; Gray et al., 1986; Hartman et al., 1986; Willmot et al., 1989; Miklas et al., 1992; Akpa and Archer, 1994; Simoni et al., 1995). On the other hand, other reports are directly aimed at obtaining tolerant/resistant cell lines and subsequently plants that are useful for resistance breeding (Table 6.3). Screening has been based on the hypothesis that bacterial and fungal toxins play an important role in host–pathogen interactions and that the response on a cell culture level may positively correlate with the whole plant reaction. Unfortunately, at present, this idea is not sufficiently supported by experimental data (Buiatti and Ingram, 1991). Probably the first report on the selection of a grain legume species (Phaseolus vulgaris) callus, challenged with the culture filtrate of a bacterium (Pseudomonas phaseolicola) was published by Bajaj and Saettler (1968, 1970). The authors observed differential tolerance of various callus lines to the host-specific pathotoxin. Gray et al. (1986) developed a bioassay for the evaluation of soybean for resistance to brown stem rot (Phialophora gregata) and for assessing the pathogenicity of fungal isolates. This was based on testing calli from susceptible and resistant soybean genotypes to fungal culture filtrate of pathogenic and non-pathogenic isolates of P. gregata. Willmot et al. (1989) found that the in vitro reaction of excised cotyledons and callus to P. gregata culture filtrate correlated positively with the greenhouse assay on intact plants (70–100%). In particular, the cotyledon method allowed soybean lines, resistant to P. gregata isolates, to be accurately identified. Hartman et al. (1986) observed a highly significant correlation (r = 0.971) between the response of bean calli to the culture filtrate of Biotechnology 171 halo blight disease (caused by the bacterium Pseudomonas syringae pv. phaseolicola) and the reaction of whole bean plants inoculated with a suspension of the pathogen. Results suggested that a callus screening system could identify bean cultivars resistant to halo blight. Miklas et al. (1992) developed a screening method to identify partial physiological resistance of bean to white mould (Sclerotinia sclerotiorum), based on the treatment of callus with pathogen filtrate. The results from this callus-weight assay correlated very well with the field reaction of bean genotypes, declared as field-resistant and field-susceptible. As an alternative to using pathogen filtrate, Simoni et al. (1995) developed an in vitro test for soybean, based on the dual culture of callus or seeds with a fungal culture of Diaporthe phaseolorum var. caulivora. The inhibition of fungal growth was analysed as an effect of the calli, or germinating seeds, on the mycelium. Based on this test, tolerant and susceptible soybean cultivars could be identified, in addition the system could be used for direct in vitro selection of callus lines with improved resistance to the pathogen. Various in vitro approaches have been studied in pea, including multiple shoot cultures, callus cultures, root cultures, direct somatic embryogenesis and dual cultures. These approaches have been used to formulate efficient systems based on culture filtrates, pure toxins or mycelial cultures of pea fungal pathogens, including Fusarium oxysporum, Fusarium solani, Fusarium poae, Fusarium semitectum, Mycosphaerella pinodes, Rhizoctonia solani, Trichotheceum roseum (Švábová et al., 1995, 1996, 1998; Švábová and Griga, 1997, 1998a,b). The exact identification of specific toxins present in culture filtrates is one of the prerequisites for more precise work. Seeds produced by fertile regenerants, obtained during methodological studies, are now available for correlation tests of plants grown in the greenhouse and field. Among the abiotic stresses, salt tolerance/resistance (NaCl) has been studied only in grain legumes on an in vitro level (Table 6.3). Gosal and Bajaj (1984) selected NaCl-tolerant cell lines in suspension cultures of Cicer arietinum, P. sativum and Vigna radiata. The number of salt-resistant colonies was increased by treating the actively growing cell suspensions with 0.25% ethyl methane sulphonate (EMS). Resistant calli of Cicer and Vigna were able to regenerate roots, although complete plants were not obtained. Pandey and Ganapathy (1984) selected a NaCl-resistant callus line of C. arietinum, which had a growth rate that was comparable with that of the control, non-selected, callus in non-saline medium. Kumar and Sharma (1989) selected V. radiata callus lines that were resistant to thioproline, an analogue of proline. One of the selected clones exhibited an elevated tolerance to exogenously applied NaCl, as well as a fivefold increased level of free proline. Despite positive evidence of a correlation between salt resistance at the tissue culture and at the whole plant level, the data available for grain legumes are still contradictory (Gale and Boll, 1978), particularly in various 172 N. Kuchuk et al. glycophytic and halophytic salt-resistant plant species (see review by Tal, 1990). 6.4.6 Variation in grain legumes at the whole plant level To date most of the data describing somaclonal variation in grain legumes at a whole plant level have been on pea and soybean, with only a limited amount of information for faba bean, groundnut and Vigna. Variation in ploidy level, chromosomal aberrations and DNA content Within the grain legumes, evidence about the changes of ploidy, chromosome number and DNA content in complete plants regenerated from in vitro cultures only exists for pea (Table 6.4). Kunakh et al. (1984) obtained diploid and tetraploid regenerants via organogenesis from pea calli of various ploidy (1n–8n). The ploidy level of callus tissue was affected by the origin of the primary explant (leaf, shoot or root) and the composition of the culture medium, but not by the genotype. In contrast, regeneration ability was determined by genotype. Natali and Cavallini (1987a,b) obtained, via organogenesis, diploid and aneusomatic (chromosomal mosaics) pea plantlets from calli derived from macerated shoot apices and embryo axes. As aneusomaty was reduced during plantlet development, the authors suggested that there may be a selective advantage of diploid over aneuploid cells (diplontic selection). In these early studies no mention was made of the fertility of regenerants obtained, or any genetic study of their seed progenies. Kysely et al. (1987) obtained diploid and tetraploid R0 pea plants (regeneration via somatic embryogenesis) from calli derived from immature embryos and shoot apices. All of the tetraploids originated from the shoot apex cultures. In contrast to the above mentioned reports, no variation in chromosome number was found in the root tips of R1 plants (seed progenies from immature leaflet calli organogenic regenerants) of pea (Rubluo et al., 1984). All of the analysed plants had a normal diploid number of chromosomes. A possible reason for this may be the elimination of all cells, other than diploid cells, during the formation of reproductive structures and seed development on R0 plants. Ahmed et al. (1987) analysed root tips of R0 regenerants of pea, formed directly from shoot apical meristems. The regenerants contained a majority of diploid cells (over 80%), plus a low frequency of cells with 10, 12, 21 or 28 chromosomes. A similar situation was found, however, in root tips of control plants germinated from seeds. The authors concluded that pea plants regenerated from meristems might be considered cytologically normal and genetically stable. Cecchini et al. (1992) studied cryptic gene alterations, such as amplifications or loss of nuclear DNA, using diploid plants of two pea cultivars, regenerated from meristem-derived calli. Cytogenetic analyses showed a Biotechnology 173 significant reduction in the nuclear DNA content of regenerants from cv. Dolce Provenza, while the DNA content remained stable in regenerants from line 5075. The DNA reduction included changes in specific subfamilies of medium repetitive sequences, while highly repetitive sequences remained invariant. In addition, the DNA of cv. Dolce Provenza was hypermethylated, while modifications in DNA methylation were not detected from line 5075. The extended hypermethylation of the genome in regenerated plants could be a rapid mechanism for silencing potential lethal genes during stress conditions. It was concluded that DNA variations related to culture and regeneration stress are dependent, at least in part, on the genotype (5075 more stable than Dolce Provenza). DNA content variability, however, was found in line 5075 plants regenerated from calli maintained in culture for a long incubation period. This supports the hypothesis that genetic stability of regenerants is also a function of the length of time that such cultures are maintained as a callus. Biochemical and molecular changes (total proteins, isozymes, DNA fingerprints) Rubluo et al. (1984) found no variation in isoenzyme spectra (esterase, glutamate dehydrogenase, 6-phosphogluconate dehydrogenase and leucin amino peptidase) of seed progeny (R1) from pea regenerants, obtained by callus mediated de novo organogenesis. Amberger et al. (1992) observed variant isozyme patterns in two independent soybean tissue culture-derived lines (regenerated via somatic embryogenesis). In the cv. BSR 101, a mutation of the Aco2-b (aconitase) gene, to give a null allele, was detected. This mutation had not been previously observed in soybean. In cv. Jilin 3, a chlorophyll-deficient plant was identified that also lacked two mitochondrial malate-dehydrogenase (Mdh null) isozyme bands. These two mutant phenotypes, chlorophyll-deficient and Mdh null, were found to co-segregate. According to the authors, the recovery of two isozyme variants, from the progeny of 185 soybean plants regenerated from somatic embryogenesis, indicates the feasibility of selection for molecular variants. Griga and Stejskal (1994) found minor changes in the seed storage protein spectra of seed progenies (R3) from meristem-derived pea plants. Regenerated plants from a 9-year micropropagated shoot culture of pea cv. Bohatýr had a high proportion of sterile individuals and various leaf morphological alterations. Differences between these regenerants and control plants were shown in the spectra for leaf peroxidase, esterase, acid phosphatase and seed storage proteins. Morphological and physiological traits, yield characters The first and the most detailed somaclonal variation study within the grain legumes, which included five seed generations, was performed in pea by Gostimiskij et al. (1985) and Ezhova et al. (1989). Variation was observed between plants regenerated from long-term callus, derived from macerated 174 Table 6.4. Somaclonal variation in grain legumes on the whole plant level (regenerants and their seed progenies). Type of in vitro culture Plant regeneration via Trait Glycine max Immature zygotic embryo-derived callus Somatic embryogenesis, organogenesis Qualitative and quantitative traits Cotyledonary node Proliferation of shoot meristems and de novo shoots (organogenesis) Somatic embryogenesis Qualitative and quantitative traits Immature embryo Cotyledonary nodeand epicotyl-derived callus Cotyledonary nodeand epicotyl-derived callus Immature cotyledons Organogenesis Plant morphology, lipid composition Qualitative traits Organogenesis Atrazine tolerance Direct somatic embryogenesis Qualitative traits Response (value/description) References Albinotic chimaeras (R0), chlorophyll deficiency, abnormal leaf morphology, dwarf plant habit (R1–R3) Plant height, sterility (R0, R1, R2) Barwale and Widholm (1987) Graybosch et al. (1987) Increased variation in R1 (mainly leaf morphology; lipid composition); not inherited to R2 Lanceolate leaves, leaf and pod variegation, determinate growth habit (R0, R1, R2) Atrazine tolerant plants (R0, R1, R2) Hildebrand et al. (1989) Complete and partial sterility, wrinkled and curled leaves, chlorophyll deficiency, reduced plant height, determinate growth habit, variation in malate dehydrogenase, aconitase and diaphorase (R0–R3) Amberger et al. (1992) Freytag et al. (1989) Wrather and Freytag (1991) N. Kuchuk et al. Species Pisum sativum Unknown Unknown Embryogenic suspension Somatic embryogenesis Root, leaf and stem primary and long-term callus Shoot apical meristem Shoot organogenesis Resistant regenerants (not proven) Song et al. (1994) Improved resistance to pathogen (R1–R3) Jin et al. (1996) Chromosome number Various ploidy of callus; tetraploid plants (R0) Kunakh et al. (1984) Variation in chromosome number; chromosome morphology; anaphase abnormalities Diploid and aneusomatic (chromosomal mosaics) plantlets (R0) Tetraploid plants (R0) Ahmed et al. (1987) Leaf mutations waxy and chlorotica; more vigorous habit; dark green leaves; oblong leaflets; first flower position (R1–R5) Anthocyanin absence; leaf type; plant habit (R0–R1) Increased legumin/vicilin ratio; altered amino acid balance (R1) Gostimskij et al. (1985); Ezhova et al. (1989) Proliferation of shoot Chromosome meristems number; chromosome aberrations Immature zygotic Shoot organogenesis Chromosome embryo-derived callus number Immature zygotic embryo- and shoot apex-derived callus Shoot apex-derived long-term callus Stem and leaf-derived callus Primary and long-term callus Chromosome number Shoot organogenesis Qualitative and quantitative traits Shoot organogenesis Qualitative traits Seed proteins and amino acid composition Shoot organogenesis Kysely et al. (1987) Lutova and Zabelina (1988) Mikhailova-Krumova et al. (1991) Continued 175 Somatic embryogenesis Natali and Cavallini (1987a) Biotechnology Resistance to Septoria glycines culture filtrate Resistance to Fusarium solani culture filtrate Continued. Species 176 Table 6.4. Type of in vitro culture Plant regeneration via Trait Response (value/description) References Shoot apex-derived callus Shoot organogenesis Nuclear DNA variation Immature zygotic embryo-derived callus, young leaflet-derived callus Somatic embryogenesis, organogenesis Qualitative and quantitative traits Genotype-dependent, tissue culture induced DNA content variation in regenerated plants (R0) Significantly altered leaf and flower morphology; sterility; lethality (R0); pods and seeds per plant; crude protein content (R1–R4) Arachis hypogaea Cotyledon callus Unknown Enhanced resistance of R2 Resistance to Cercosporidium plants personatum culture filtrate Venkatachalam et al. (1998) Phaseolus vulgaris Shoot meristems Meristem proliferation Resistance to phaseolotoxin Gantotti et al. (1985) (Shoot apical) meristem culture infected with mycelial suspension Resistance to Meristem proliferation/multiple Botrytis cinerea, Phytophthora shoot formation megasperma and Rhizoctonia solani Vigna radiata ‘De-embryonated’ cotyledons De novo organogenesis Qualitative traits Stejskal and Griga (1992); Griga et al. (1995); Griga and Létal (1995) Thynn et al. (1989) Correlation between low to medium phytoalexin level in regenerants and fungal resistance (R0) Chlorophyll and morphological mutations (R0, R1, R2) Mathews et al. (1986) N. Kuchuk et al. Vicia faba Resistant regenerants (not proven) Cecchini et al. (1992) Biotechnology 177 tissues (shoot-apex, epicotyl, internode, leaf). Changes included both qualitative (e.g. chlorophyll defects, absence of waxy layer on leaves) and physiological/quantitative traits (e.g. plant habit, colour and morphology of leaves, yield parameters) and were stably inherited to seed progeny (T1 to T5 generations analysed). The most frequent changes were: a mutation in the chi (chlorotica) gene (recessive nuclear mutation); the absence of a waxy layer on leaves (recessive nuclear mutation); a more robust habit compared with the original variety; dark green instead of the light green leaves found in the original variety; elongate compared with oval-shaped leaves and an earlier or later onset of flowering. Cytological data of callus cells indicated that the phenotypic variability of regenerants was not connected with large reconstructions of the karyotype. Also, it is unlikely that stability for a trait, such as flowering period (controlled by four genes), can be the result of a single gene mutation. It is suggested by these authors and others (Cecchini et al., 1992) that some mechanisms determining genetic variability are characteristic for cells of in vitro-cultured plants, e.g. amplification of some genome segments and their transposition. Lutova and Zabelina (1988) analysed R0 and R1 plants obtained by organogenesis from callus derived from internode and leaf segments. Three qualitative changes were recorded within the R0 regenerants, which were inherited in the R1 generation, the presence/absence of anthocyanin, leaf structure and plant habit. Stejskal and Griga (1992) found, within the R0 regenerants of pea, obtained by somatic embryogenesis from immature zygotic embryos, a plant with a dramatically altered habit (leaflet shape, one pair of leaflets, abnormal flower morphology, reduced flower stalk, shortened internodes, stipules without dentation and with tendrils). The plant exhibited a chimaeric character and was completely sterile. The transfer back to in vitro culture did not result in the isolation of a stable mutant (Griga, 2000). The same authors (Griga et al., 1995; Griga and Létal, 1995) compared somaclones obtained by somatic embryogenesis and by organogenesis, together with their seed progenies (R1 to R4 generation). Mainly morphological changes were recorded (altered leaflets, tendrils, fasciations), when evaluating qualitative traits in plants from both tissue systems. All plants exhibiting such phenomena were chimaeric, and the altered traits occurred randomly or were lost in later generations. Analysis of 12 quantitative traits (e.g. plant morphology, yield parameters, seed protein content) showed that somaclones produced via organogenesis exhibit more variation compared with those produced via embryogenesis. An extensive literature exists about soybean somaclonal variation at the whole plant level. Barwale and Widholm (1987) evaluated plants regenerated from embryogenic and organogenic cultures of nine soybean genotypes and found extensive variation in qualitative traits. Three lethal sectorial albinos were seen in the primary regenerants (R0). Variants observed in later selfed generations included twin seeds, multiple shoots, 178 N. Kuchuk et al. dwarfs, abnormal leaf morphology, abnormal leaflet number, wrinkled leaves, chlorophyll deficiency, partial sterility and complete sterility. The frequency of mutations ranged from 0 to 4% in R0 plants, as determined by studies of corresponding R1, R2, R3 and R4 families. No significant differences were seen in the frequency of mutations for embryogenic compared with organogenic culture-derived plants. Chlorophyll deficiency, sterility and wrinkled leaves, traits that are controlled by single recessive nuclear genes, were stably inherited over two or three generations. Other traits occurred more randomly and were not in all generations. At present the genetic basis of this random variation is not known. Graybosch et al. (1987) studied somaclonal variation in R1 seed progenies of plants regenerated from soybean cotyledonary nodes (BAstimulated shoot formation from pre-existing as well as newly formed meristematic regions of nodal tissue). The following traits were recorded in three cultivars under field conditions, yield, plant height, lodging, leaf shape and colour and maturity. In addition, the following dominant genetic markers were evaluated: purple flowers, tawny pubescence, black hilum and brown pods. Variability for yield was observed in two out of 19 families, compared with control cultivars. One of the 22 families exceeded the control in height and variability for height was increased among regenerated families. Recessive mutations for putative sterility characters were observed in two out of 89 families, but mutations in six marker genes were not apparent. Negligible variation in qualitative traits and relatively low variation in quantitative traits, compared with the control, showed that cotyledonary node culture was not a source of significant somaclonal variation. An important fact was that many somaclones retained the yield potential of the parental cultivars. This result was significant for the use of the cotyledonary node technique for the introduction of foreign genes by genetic transformation. Freytag et al. (1989) analysed the progeny (R1 to R3) of soybean plants, regenerated from callus cultures (organogenesis) derived from cotyledonary nodes and epicotyls. Variant phenotypes were found that had not been previously reported from tissue culture, including lanceolate leaves, leaf variegation (chimaeric variegated plants), pod variegation on otherwise normal plants and a change in growth habit from indeterminate to determinate. All of the above-mentioned traits were inherited through three generations, except pod variation, which was inherited through two generations, segregation occurring in each generation. No variation was observed in control plants derived from normal seeds. Hildebrand et al. (1989) studied variation in fatty acid composition of the seeds and plant morphological traits, in soybean regenerants obtained via somatic embryogenesis. The first seed generation (R1) of regenerants exhibited higher phenotypic variation, compared with normal seed-derived populations. Changes included lateral indentation (lobing) of the first unifoliate leaf, sectorial loss of chlorophyll and dual apical meristems, Biotechnology 179 with three cotyledons and three unifoliate leaves. Variation in fatty acid composition was also higher in the R1 generation than in control material. Variation in both morphological and seed composition characters, however, was not observed in the following R2 generation, probably because of unstable epigenetic effects. Stephens et al. (1991) observed a wrinkled leaf variant in the R2 generation of a soybean line regenerated through organogenesis. Observations of progeny from selfed normal and variant derivatives of this line suggested genetic instability for this trait. Reciprocal crosses indicated that the mutant trait was inherited cytoplasmically. The unusual segregation ratios were attributed to organelle segregation and to cytoplasmic inheritance. Amberger et al. (1992) regenerated 475 plants of nine soybean cultivars, via direct somatic embryogenesis from immature cotyledons. The R1, R2 and R3 progeny from the regenerated plants were scored for qualitative variation and inheritance of variant phenotypes. These included partial sterility (R0, R1, R2), complete sterility (R0), abnormal leaf morphology (R0, R1, R2, R3) chlorophyll chimaeras (R2, R3), chlorophyll deficiencies (R2, R3), changes in growth habit (R2, R3), yellow edges on cotyledons (R3), no unifoliates (R3), dwarf plants (R2), yellow–green plants (R3) and isozyme variants (R2). Inheritance studies of chlorophyll-deficient, curled-leaf and wrinkled leaf plants confirmed that these traits were genetically controlled. Although none of the variants exhibited any obvious agronomically favourable characteristics, the study resulted in the identification of novel variants that may prove useful in the dissection of the soybean genome. New variants included a malate dehydrogenase null and an aconitase null (Amberger et al., 1992), curled leaves, lethal chlorophyll deficiencies, no unifoliates and yellow-edged cotyledons. Similarly, Stephens et al. (1991) observed an unusual segregation of wrinkled-leaf mutation that could be considered as a cytoplasmically inherited trait. Mathews et al. (1986) regenerated mung bean (V. radiata) plants from de-embryonated cotyledons. Considerable variation was observed in the R2 population, 7% of the R1 plant progenies segregating for chlorophyll mutations and another 7% for viable morphological mutations. The chlorophyll mutations included chlorina and xantha types, which were lethal under field conditions. The viable mutations included those with pentafoliate leaves, sterility, and green seed coat and cotyledon colour. None of these mutants was found in the control population. The mutation rate per 100 R2 plants was 1.8% for the chlorophyll and for the viable mutants. Variation in tolerance to biotic and abiotic stress There are only a few reports about the production of fertile regenerants in grain legume crops, after in vitro selection using toxic culture filtrates (Table 6.4). The absence of reproducible de novo regeneration systems for the majority of grain legume species initially led researchers to use the only available system, meristem or shoot tip culture. Gantotti et al. (1985) 180 N. Kuchuk et al. obtained regenerants of P. vulgaris resistant to phaseolotoxin, based on the selection of shoot meristems. Thynn et al. (1989) screened V. faba shoot meristem cultures after treatment with spore suspensions of Botrytis cinerea, Phytophthora megasperma and R. solani, based on the accumulation of phytoalexins (wyeronic acid, wyerol, DH-wyerone, wyerone) in regenerated plants. Only low to medium concentrations of phytoalexins were found in resistant regenerated plants from seven faba bean cultivars. The resistance response was affected by the total amount and the ratio of individual phytoalexins. Song et al. (1994) obtained resistant regenerants of soybean after in vitro selection with a culture filtrate of Septoria glycines. One of the most advanced studies of somaclonal variation in grain legumes with respect to pathogen resistance was published by Jin et al. (1996). Embryogenic suspension cultures from four cultivars were treated for 1–2 months with a toxic culture filtrate of F. solani, a fungal disease causing sudden death syndrome (SDS). Selected suspensions, regenerated via somatic embryogenesis and fertile R0 plants, were obtained. R1 and R2 plants were then tested by artificial inoculation with the pathogen, in a controlled environment and in the greenhouse. Various degrees of resistance were obtained compared with the resistant control variety. Additional studies, covering further seed generations, will be needed to determine the stability/heritability of the generated resistance. Venkatachalam et al. (1998) used a culture filtrate of Cercosporium personatum (tikka leaf late spot disease) to repeatedly treat cotyledon callus cultures of groundnut (Arachis hypogaea). Plants were regenerated from resistant calli that survived three cycles of selection. R2 seed progenies of regenerated plants exhibited resistance to the pathogen in field conditions. Wrather and Freytag (1991) selected soybean cotyledonary node plus epicotyl explants, on a medium containing 48 mg l−1 atrazine. Explants surviving exposure to atrazine (34%) callused and regenerated shoots via organogenesis. Selection in vivo with atrazine-treated soil allowed R0, R1 and R2 atrazine-tolerant plants to be obtained. All non-atrazine selected control plants died when exposed to the same conditions. Atrazine-tolerant R2 plants appeared to be as healthy and vigorous as the control growing in atrazine-free soil. It was suggested that cytoplasmic inheritance (genes located on the chloroplast chromosome) might account for the altered atrazine-tolerant phenotype. Product quality changes (proteins, carbohydrates, lipids) Hildebrand et al. (1989) studied variation in fatty acids composition of R1 and R2 seed progenies from soybean plants regenerated via somatic embryogenesis. Variation in the R1 generation for fatty acid composition was higher, compared with the control population. In addition, some individuals showed an unusual fatty acid composition. The progeny of variant plants, however, were normal and comparable to the control in the R2 generation. Biotechnology 181 Mikhailova-Krumova et al. (1991) studied seed proteins from R0 pea plants regenerated by organogenesis following primary and long-term callus culture. Variation was found in a polypeptide legumin with a 37-kDa molecular weight (MW). With an increasing period of callus culture, the total amount of seed protein declined and the variation coefficients for legumin and vicilin content in the total protein increased. Three samples were isolated with an increased legumin/vicilin ratio. In many regenerated plants the amino acid balance was altered, which was mainly due to an increase in the phenylalanine and a decrease in the methionine content. When comparing organogenic and embryogenic somaclones of pea line HM-6, increased variation in crude protein content (% dry weight) was recorded in regenerant progenies, compared with control populations (Griga, unpublished results). The following data were obtained in the R2 generation: embryogenic somaclones, mean 24.41% and range 19.95–26.10%; organogenic somaclones, mean 24.04 and range 20.59–27.02%; control population 1, mean 22.91 and range 21.94–23.69%; control population 2, mean 23.89 and range 23.09–25.43%. In the R3 generation, a number of somaclones with significantly higher protein content (based on 95% confidence for means) were found within embryogenic somaclones compared with the control. On the other hand, organogenic somaclones exhibited more clones with significantly lower protein content. In the R4 generation, the differences between selected somaclones from both origins and from the control populations were less dramatic. 6.5 Transformation Methods in Grain Legumes 6.5.1 Introduction Targeted genetic transformation of crop plants is a powerful recent complement to conventional breeding strategies. In the past decade there has been a significant shift in genetic transformation experiments from the use of plant models to agronomically important crops, including grain legumes (Nisbet and Webb, 1990; Christou, 1992, 1997; Atkins and Smith, 1997). Similarly, as in other important crops (oilseed crops, corn, sugar-beet, potato, tomato), the genetic transformation in legumes was primarily directed towards the incorporation of genes affecting cultivation (e.g. herbicide tolerance and pathogen resistance) and only later considered nutritional and postharvest product quality (Christou, 1997; James, 1997). There are some recent reports, therefore, on protein improvement in grain legumes by genetic transformation (Falco et al., 1995; Pickardt et al., 1995; Molvig et al., 1997), but, as yet, few results related to carbohydrate quality/ quantity improvement, comparable with those reported in potato (Visser and Jacobsen, 1993; Muller-Rober and Kossmann, 1994; Stark et al., 1996) and corn (James, 1997). 182 N. Kuchuk et al. The objective of this section is not to review completely all papers dealing with grain legume transformation, but to show, using selected examples, the technological progress in this field, as well as the major trends towards crop improvement. The techniques discussed for grain legume transformation, while dealing with traits/genes other than carbohydrate metabolism, form a methodological background for future carbohydrate improvement of grain legumes via genetic transformation. 6.5.2 Gene delivery systems used in agronomically important legumes Several gene delivery systems have been tested experimentally in grain legume species. These approaches can be divided into gene transfer mediated by bacterial vectors (Agrobacterium tumefaciens, Agrobacterium rhizogenes) and direct gene transfer, comprising polyethylene glycol (PEG)-mediated gene transfer to protoplasts, electroporation-mediated gene transfer to protoplasts, biolistic plant transformation (particle bombardment), microinjection into plant tissues, cells and protoplasts, and tissue (meristem) electroporation. The majority of the above-mentioned techniques need in vitro culture and a reliable regeneration protocol. More recently, however, there has been a tendency to modify the transformation protocols by avoiding sophisticated tissue culture steps (Brar et al., 1994; Chowrira et al., 1995, 1996; McKently et al., 1995). To date, some other transformation methods successfully used in other crops, e.g., silicon fibre-mediated transformation, vacuum-infiltration of DNA with intact plants and imbibition of dry seeds/embryos with DNA, have not resulted in complete transgenic plants in grain legumes (for detailed descriptions of approaches mentioned above, see Gelvin and Schilperoort, 1995; Potrykus and Spangenberg, 1995; Galun and Breiman, 1997). Of in vitro regeneration protocols studied during grain legume transformation, de novo shoot organogenesis from primary explants/ callus was more frequently used to obtain transformed regenerants, rather than somatic embryogenesis (Parrott et al., 1989, 1993; Ellis, 1995). There are a number of papers reporting transformation of proptoplasts, cells and calli in grain legumes (Nisbet and Webb, 1990; Christou, 1992; Atkins and Smith, 1997), however, the following text deals predominantly with protocols that have allowed the successful production of complete fertile transgenic plants in grain legume crops. 6.5.3 Methods giving positive results – transgenic plants Agrobacterium-mediated gene transfer Agrobacteria (A. tumefaciens, A. rhizogenes) are Gram-negative soil bacteria that can infect many plant species (mainly dicotyledonous) and can serve as Biotechnology 183 natural vector systems. The foreign gene is artificially inserted into a modified bacterial plasmid (Ti or Ri) and, via natural infection, delivered to the plant cell, where the plasmid T-DNA is integrated into the plant genomic DNA. Once integrated, the genes transferred by Agrobacterium have been shown to be meiotically stable. Plant species subjected to transformation must be susceptible to the Agrobacterium strain used and must show an ability to regenerate in vitro from protoplasts, cells and tissues. In special cases, however, regeneration can be eliminated by direct germination of Agrobacterium-transformed embryo axes in an autoclaved soil mix (McKently et al., 1995). For the efficient selection of transformed cells or tissues, the appropriate selection conditions must be available. These may be provided either by selectable marker genes (Schrott, 1995), which confer the resistance to some antibiotics (neomycin phosphotransferase, nptII; hygromycin phosphotransferase, hpt) and herbicides (phosphinotricin acetyl transferase, bar; 5-enolpyruvylshikimate-3-phosphate synthase, Epsp), or by reporter genes (Herrera-Estrella et al., 1995), which are coding sequences that provide a clear indication that genetic transformation has taken place, for example, upon expression in the transgenic plant (Galun and Breiman, 1997). Such reporter genes are usually visual, for example, β-glucuronidase (GUS), luciferase and green fluorescent protein (GFP). A recent improvement in the Agrobacterium-mediated transformation of embryogenic suspension cultures by sonication has been reported (Trick and Finer, 1998). Biolistic plant transformation (particle bombardment) Biolistics or biological ballistics, is the process by which biological molecules (DNA, RNA) are accelerated, usually on microcarriers termed microprojectiles, by an explosive charge (gun powder or compressed gas), or by a high-voltage electric charge and shot into plant cells (Galun and Breiman, 1997). Plant species subjected to transformation using ballistics should have the ability to regenerate in vitro. As in Agrobacterium-mediated transformation, however, there have been successful attempts to produce transgenic shoots from bombarded meristems without in vitro culture (Brar et al., 1994). The efficient selection of transformed tissues based on selectable marker genes and/or reporter genes is also necessary. The advantage of this method is that transformation can be carried out independently of the variety used (Christou, 1995). Electroporation and microinjection In this system DNA is electroporated into nodal meristems (treated buds), or directly injected into ovaries (Chowrira et al., 1995, 1996; Yue et al., 1996). Transgenic plants then can be recovered in the offspring of electroporated or microinjected individuals. The advantage of these methods is that they allow the production of transgenic legume plants without the need for in vitro tissue culture. 184 N. Kuchuk et al. 6.5.4 Transgenic plants and useful genes/traits transformed into grain legumes The number of successfully transformed grain legume species has been growing since 1988, when soybean transgenic plants were recovered as the first representative of grain legume crops (Hinchee et al., 1988). During the last decade, there has been progress in the methodology and a shift away from experiments using only marker and reporter genes, towards transformation with agronomically useful genes. In the past few years, transgenic plants have been obtained in almost all economically important species of grain legumes (i.e. Phaseolus spp., P. sativum, A. hypogaea, C. arietinum, Vigna spp., Lens culinaris, Lupinus angustifolius L.). Soybean is the most advanced species within grain legumes with regard to genetic transformation and can be considered as one of the general models for the biolistic approach (McCabe et al., 1988; Christou, 1992, 1995). The first reports of successful transgenic plant production in particular grain legume species are summarized in Table 6.5. A list of the most useful genes/traits transformed into grain legumes and expressed on the plant level are given in Table 6.6 and discussed in more detail below. Herbicide tolerance Glyphosate (trivial name for α-phosphonomethyl glycine, an active ingredient of the herbicide Roundup®) acts as an inhibitor of aromatic amino acid synthesis, by blocking shikimate biosynthesis, the target enzyme being 5-enolpyruvylshikimate-3-phosphate synthase (EPSP). Glyphosate is a non-selective herbicide that is not toxic to animals and is rapidly degraded in the soil (Galun and Breiman, 1997; Ondrej et al., 1998). Tolerance to glyphosate may be engineered by the incorporation of three types of transgenes. The first type, encodes for EPSP (from Petunia and Arabidopsis) and results in an overproduction of the target enzyme. Glyphosate is, therefore, fully saturated with EPSP but free EPSP is still available to exhibit sufficient catalytic activity. The second type, is a mutated gene encoding a modified EPSP enzyme (mainly from Salmonella typhimurium, Escherichia coli, Agrobacterium sp.), which is tolerant to glyphosate. The third type encodes glyphosate-oxido-reductase (GOX; e.g. from Achtomobacter), which can metabolize glyphosate. GOX is normally present in bacteria, but not in plants. The mutated EPSP gene from petunia, together with the gene for kanamycin resistance and GUS, have been used for soybean transformation by Hinchee et al. (1988). Using this system, approximately 6% of the shoots regenerated by de novo organogenesis from seedling cotyledons were proved to be transformed. Padgette et al. (1995) demonstrated that the transgene for glyphosate tolerance behaved as a single dominant gene and was stable over several generations of soybean field trials. A very important GLYPHOSATE TOLERANCE Biotechnology 185 finding was that glyphosate treatment of a field tested transgenic soybean line grown in several locations did not significantly reduce yield (Delannay et al., 1995). Phosphinothricin (syn. gluphosinate; chemically 4-[hydroxy-(methyl) phosphinoyl]-D,L-homoalanin) is an analogue of glutamine and acts as an herbicide by inhibiting glutamine synthesis, following an irreversible inactivation of glutamine synthase. It is widely used as a commercial preparation and may be called Basta®, Liberty®, Bialophos®, Herbiace®, Buster®, Finale® or Radicale® (Galun and Breiman, 1997; Ondrej et al., 1998). Two bacterial genes (bar from Streptomyces hygroscopicus and pat from Streptomyces viridochromogenes) have the ability to detoxify PPT by acetylation. In grain legumes, the bar/pat genes have been used mainly as selectable markers, e.g. in pea transformation (Schroeder et al., 1993; Grant et al., 1995; Bean et al., 1997; Simonenko et al., 1999). In addition to detecting the presence of the bar gene by Southern analysis and the PAT assay, leaf paint and spray tests have been carried out on transgenic pea plants and their seed progenies. Schroeder et al. (1993) found complete and partial tolerance of leaves from pea transformants treated with a dose equivalent to 10 l ha−1 Basta ®. At this concentration the control (untransformed leaves) became completely necrotic. Fourteen days after spraying whole pea plants with a dose equivalent to 7 l ha−1 Basta®, transgenic plants showed no symptoms of herbicidal damage and grew normally to maturity, whereas the nontransgenic plants were killed. Grant et al. (1995) found that R1 progeny (first seed generation) containing the gene showed variable resistance to Buster®. From plants that gave a resistant leaf test at the equivalent concentration of 10 l ha−1 Buster®, to those that showed susceptibility at 3 l ha−1 Buster® equivalent. According to Bean et al. (1997), pea transformants showing no signs of herbicide damage 3 days after spraying with 3 mg l−1 Herbiace® could be classed as clonal transformants, whereas those showing varying combinations of green and brown tissue were categorized as chimaeras. PHOSPHINOTHRICIN (PPT) TOLERANCE Herbicides of the S-triazine type (atrazine, simazine) inhibit photosynthesis by binding to the chloroplast thylakoid membrane protein, resulting in electron transport being blocked in photosystem II. The mode of resistance is to change the target site, i.e. the protein encoded by the chloroplast gene psbA (Galun and Breiman, 1997; Ondrej, 1998). Fu et al. (1993) reported field-testing of F4 and F5 soybean plants transformed for atrazine resistance. Atrazine treatment at the pre-emergence, or at the seedling stage, did not adversely affect the plant yield, which was the same, or even higher, than that of the unsprayed, non-transformed controls. Yue et al. (1996) transformed 24 soybean varieties by direct injection of DNA, containing the gene for atrazine resistance, through the ovary and ATRAZINE TOLERANCE 186 Table 6.5. Useful genes/traits transformed into grain legumes. Gene/trait incorporated Glycine max 5-Enolpyruvyl-shikimate-3-phosphate synthase (EPSP); herbicide tolerance (glyphosate) – Roundup® Phosphinotricin acetyl transferase (PAT, bar); herbicide tolerance (PPT) – Basta® Lysine-feedback-insensitive bacterial DHDPS/ dapA and AK/lysC; fivefold lysine increase in the seed Methionine-rich 2S albumin from Brazil nut; high oleic acid; modified oil; virus resistance Synthetic cryIAc gene (B.t.) – resistance to insect larvae (four species) Atrazine resistance (psbA) Bean pod mottle virus (BPMV) coat protein gene – virus resistance Soybean mosaic virus (SMV) coat protein gene – virus resistance Phaseolus vulgaris Coat protein from the bean golden mosaic virus (BGMV) – virus resistance; bar Antisense sequence of AC1, AC2, AC3 and BC1 genes from bean golden mosaic geminivirus – virus resistance; methionine-rich 2S albumin from Brazil nut Transformation method References Agrobacterium Hinchee et al. (1988) Bombardment Christou and Swain (1990) Falco et al. (1995) Bombardment Agrobacterium bombardment Bombardment Direct injection of DNA into ovary Agrobacterium Agrobacterium James (1997) Stewart et al. (1996) Yue et al. (1996) Di et al. (1996) Xu et al. (1996) Bombardment Russell et al. (1993) Bombardment Arago et al. (1996) N. Kuchuk et al. Species Pisum sativum α-amylase inhibitor from common bean (αAI-Pv) – resistance to three species of bruchid beetles; bar – phosphinothricin resistance (Basta®, Buster®, Herbiace®) Agrobacterium Heat-stable amylase from Bacillus licheniformis, improved seed starch Agrobacterium degradation Chimaeric alfalfa mosaic virus (AMV) coat protein gene – partial Agrobacterium resistance to AMV Cicer arietinum Vicia narbonensis Lupinus angustifolius Sulphur-rich sunflower seed albumin (enhanced methionine level in the seed), bar – phosphinothricin resistance (Basta®) Grant et al. (1998a,b) Bombardment Agrobacterium Kar et al. (1997) Pickardt et al. (1995) Agrobacterium Saalbach et al. (1997) Agrobacterium Weber et al. (1998) Agrobacterium Molvig et al. (1997); Pigeaire et al. (1997) Biotechnology CrylA (c) gene from B. thuringiensis (resistance to pod-borer larvae) Methionine-rich 2S albumin from Brazil nut (threefold increase of seed methionine content) Heat-stable amylase from Bacillus licheniformis, improved seed starch degradation Yeast invertase, changes in regulation of carbohydrate and protein metabolism during cotyledon development Shade et al. (1994); Schroeder et al. (1995); Grant et al. (1995); Bean et al. (1997) Saalbach et al. (1997) 187 188 Table 6.6. Transgenic plants in grain legumes. Explant Transformation method Regeneration protocol References Glycine max Pisum sativum Phaseolus vulgaris Arachis hypogaea Cicer arietinum Vicia narbonensis (narbon bean, French vetch) Lens culinaris Seedling cotyledons Shoot cultures epicotyls Embryo axis Embryogenic callus Seed-derived embryos Epicotyl segments Agrobacterium Agrobacterium Bombardment Bombardment Agrobacterium Agrobacterium Organogenesis Organogenesis Organogenesis Somatic embryogenesis Organogenesis Somatic embryogenesis Hinchee et al. (1988) Puonti-Kaerlas et al. (1990) Russell et al. (1993) Ozias-Akins et al. (1993) Fontana et al. (1993) Pickardt et al. (1995) Nodal meristems in planta Electroporation in vivo Vigna unguiculata Lupinus angustifolius Mature cotyledons Maturing embryo axis Agrobacterium Agrobacterium Seed production without Chowrira et al. (1995) in vitro culture Organogenesis Muthukumar et al. (1996) Organogenesis Pigeaire et al. (1997) N. Kuchuk et al. Species Biotechnology 189 confirmed the integration and normal inheritance of the introduced gene. There was no seedling injury when F2 plants of one variety with the introduced gene were sprayed with atrazine in the field prior to seedling emergence. Ninety-two per cent of the control (non-transformed) plants were killed by the same treatment. Insect resistance There are two general strategies for genetically engineered insect resistance in plants. Firstly, the incorporation of genes from specific bacteria, which encode proteins that are toxic to insects. Secondly, the incorporation of genes encoding plant-derived inhibitors of protein, or carbohydrate, digestion in insects (Galun and Breiman, 1997). Upon sporulation, B. thuringiensis (B.t.) strains produce protein crystals containing ∆-endotoxin, which cause the lysis of epithelium cells in the midgut of insect larvae. These toxins are harmless to mammals and birds and exhibit a limited range of toxicity to specific groups of insects. The latter criterion is used for clarifying B. thuringiensis strains, those producing CryI are toxic to Lepidoptera, CryIII to Coleoptera and CryIV to Diptera. Within the grain legumes, soybean and chickpea have been successfully transformed with B.t. ∆-endotoxin genes (Parrott et al., 1994; Stewart et al., 1996; Kar et al., 1996). Stewart et al. (1996) transformed soybean somatic embryos using particle bombardment with a synthetic B.t. CryIAc gene linked to a hygromycin-resistance gene. Three transgenic lines were selected on hygromycin-containing media and grown into fertile plants. When the transgenic plants were tested, they were found to be protected from damage by larvae of four lepidopteran species, corn earworm (Helicoverpa zea), soybean looper (Pseudoplusia includens), tobacco budworm (Heliothis virescens), and velvet bean caterpillar (Anticarsia gemmatalis). Transgenic plants exhibited less than 3% defoliation upon corn earworm attack, compared with 20% for a lepidopteran-resistant breeding line and more than 40% for susceptible soybean cultivars. A chimaeric, truncated bacterial CryIA(c) gene construct, with the nptII gene as a selection marker, was inserted into the embryo axis of mature chickpea seed by particle bombardment (Kar et al., 1996). Transgenic kanamycin-resistant plants were obtained through multiple shoot formation and repeated selection of the bombarded explants. An insect feeding assay indicated that the expression of CryIA(c) gene was inhibitory to damage by larvae of Heliothis armigera. BACILLUS THURINGIENSIS ENDOTOXINS Seeds of the common bean (P. vulgaris) are naturally resistant to bruchid beetles (e.g. cowpea weevil, Callosobruchus maculatus), because of the presence of an α-amylase inhibitor (αAI-Pv), a seed protein that is toxic to the larvae. Other legumes (e.g. pea, chickpea, cowpea, Azuki bean), however, do not contain this α-amylase inhibitor-derived tolerance. AMYLASE INHIBITORS 190 N. Kuchuk et al. Shade et al. (1994) and Schroeder et al. (1995) transferred the gene encoding αAI-Pv into pea, using Agrobacterium-mediated transformation based on the co-cultivation of embryonic axis segments and regeneration via organogenesis (Schroeder et al., 1993). The α-amylase inhibitor gene was stably expressed in the transgenic pea seeds up to the T5 seed generation. αAI-Pv accumulated in the pea seeds up to 3% of the soluble protein, a level that was higher than that normally found in beans (1–2%). In the T5 seed generation, the development of pea weevil (Bruchus pisorum) larvae was blocked at an early stage. Seed damage was minimal and seed yield was not significantly reduced in the transgenic plants. In addition to the resistance of transgenic pea plants to the bruchid beetles attacking the crop growing in the field (B. pisorum), the transformed peas also showed complete resistance to bruchid species that damage stored seeds, in particular cowpea weevil (C. maculatus) and Azuki bean weevil (Callosobruchus chinensis; Shade et al., 1994). Although αAI-Pv also inhibits human α-amylase, it is reported that cooked peas should not have a negative impact on human energy metabolism. Dillen et al. (1997) transformed Phaseolus acutifolius with a genomic fragment encoding the P. vulgaris arcelin-5a-protein, using an Agrobacterium-mediated approach. It is believed that this seed storage protein confers resistance to the insect Zabrotes subfasciatus, a major pest of P. vulgaris. Arcelin-5a was produced at high levels in the seeds and the authors suggest using of P. acutifolius as a ‘bridging’ species to introduce transgenes into the economically more important species P. vulgaris. Virus resistance Virus coat protein-mediated resistance, based on the concept of ‘cross protection’ and antisense-RNA derived resistance, has been reported for grain legumes. The concept of cross protection is derived from the fact that the infection of a given crop plant with mild strains of viruses and viroids prevents or reduces the symptoms caused by a subsequent virulent strain. Rather than using the whole virus, however, only part of the viral genome, encoding the coat protein (CP), is integrated into the plant genome. The antisense RNA (a mirror sequence of an mRNA sequence) may provide viral tolerance by interfering either with the translation of viral mRNAs, or with the replication of the viral genome (Greenberg and Glick, 1993; Galun and Breiman, 1997). Di et al. (1996) transformed soybean, using an Agrobacterium-mediated approach, with a bean pod mottle virus (BPMV) CP-gene and found that 30% of the R2 plants derived from one transgenic line were resistant to BPMV infection. Similarly, Xu et al. (1996) obtained A. rhizogenes transformed transgenic soybean plants with the integrated gene for soybean mosaic virus (SMV) coat protein. Arago et al. (1996) introduced the antisense sequence of AC1, AC2, AC3 and BC1 genes, from the bean golden mosaic geminivirus, to P. vulgaris transgenic plants, using particle Biotechnology 191 bombardment. Grant et al. (1998a,b) obtained transgenic pea plants with partial resistance to the alfalfa mosaic virus (AMV), using A. tumefaciens transformation with chimaeric AMV coat protein gene. Nutritional quality Galun and Breiman (1997) categorized nutritional quality of crops into four groups: fatty acids, lipids and oils; carbohydrates; proteins and pigmentation. Within grain legumes, most reports relate to the improvement of oils and proteins by genetic transformation. Weber et al. (1998), however, has reported changes in carbohydrate metabolism in transgenic Vicia narbonensis. In proteins, the strategy has concentrated mainly on improving amino acid composition, in particular, the methionine (Met) and lysine (Lys) content. Saalbach et al. (1994) transformed V. narbonensis, a close relative of faba bean (V. faba), with the methionine-rich 2S albumin gene of the Brazil nut (Bertholletia excelsa). This synthetic gene, controlled by the CaMV 35S promoter, however, was highly expressed only in leaves and roots and was hardly detectable in the seeds. Later, the transformation protocol was improved by fusing the 2S albumin gene with the seed-specific legumin B4 promoter from V. faba (Pickardt et al., 1995; Saalbach et al., 1995a,b). Transformation was carried out using the Agrobacterium-mediated approach together with a regeneration protocol using somatic embryogenesis from callus. Transformed calli were selected for kanamycin resistance and the induced somatic embryos were screened for GUS activity and cloned by multiple shoot regeneration. Fertile R0, R1 and R2 transgenic plants were obtained and seed-specific gene expression was found in transformants with the legumin B4 promoter/2S albumin gene fusion. Analysis of the R2 plants indicated a Mendelian inheritance of the 2S albumin gene. Some transformants exhibited a threefold increase in the methionine content of the salt-soluble protein fraction extracted from seeds. The same gene was introduced, using the biolistic process, into P. vulgaris and stable transgenic bean plants were generated (Arago et al., 1996). Again the gene was inherited in a Mendelian fashion in most of the transgenic bean lines. Falco et al. (1995) increased the lysine content in soybean seeds by circumventing the normal feedback regulation of two enzymes of the biosynthetic pathway, aspartokinase (AK) and dihydropicolinic acid synthase (DHDPS). Lysine-feedback-insensitive bacterial DHDPS and AK enzymes, encoded by the Corynebacterium dapA gene and a mutant E.coli lysC gene, respectively, were expressed in transgenic soybean seeds, following transformation via particle bombardment and by fusion with the GUS reporter gene. The result was a ten- to several hundredfold increase in free lysine and up to a fivefold increase in the total seed lysine content, lysine contributing up to 33% of the total seed amino acid content. The lysine content in R2 and R3 seeds remained at least as high as that observed in the R1 seed, 192 N. Kuchuk et al. demonstrating heritability of the trait. Seeds of soybean transformants (R1) with greatly increased lysine levels had a wrinkled shape and germination was poor. List et al. (1996) analysed oils from genetically modified soybeans. Compared with common varieties containing 15% saturated fatty acids, genetically modified soybeans yielded oils with 24–40% saturated acids. Using an Agrobacterium-mediated approach Molvig et al. (1997) stably transformed narrow-leafed lupin (L. angustifolius) with a chimaeric gene specifying seed-specific expression of a sulphur-rich, sunflower seed albumin (SSA). The transgenic seeds contained less sulphate and more total amino acid sulphur than the non-transgenic parent line. A 94% increase in the methionine content and a 12% reduction in cysteine content was recorded. There was no statistically significant change in other amino acids, or in the total nitrogen and total sulphur contents of the seeds. In feeding trials with rats, the transgenic seeds gave statistically significant increases in live weight gain, true protein digestibility, biological value and net protein utilization, compared with wild-type seeds. Weber et al. (1998) transformed V. narbonensis with the yeast invertase gene under the control of the legumin B4 promoter. Expression of the legumin B4-promoter yeast invertase gene in transgenic cotyledons resulted in a reduction of sucrose, starch and protein contents and an increase in the hexose level. 6.5.5 Field trials with transgenic grain legume plants and commercialized transgenic legume crops During the period 1986–1997, c. 25,000 transgenic crop field trials were conducted in 45 countries, on more than 60 crops covering 10 traits. Seventy-two per cent of all transgenic crop field trials were conducted in the USA and Canada followed in descending order by Europe, Latin America and Asia, with a few conducted in Africa. The most frequent crops in these trials were maize, tomato, soybean, canola, potato and cotton and the most frequent traits were herbicide tolerance, insect resistance, product quality and virus resistance (James, 1997). Recently, a great deal of progress has been achieved in the transformation and in the commercialization of cool season legume species in Australia (Atkins et al., 1998; Hamblin et al., 1998). Four co-operating laboratories/companies are now able to transform seven legume species (Lupinus angustifolius, Lupinus albus, Lupinus luteus, C. arietinum, P. sativum, V. faba and L. culinaris), almost all of which have been field tested (Table 6.7). The situation with commercialized transgenic crops in 1996 and 1997 is shown in Tables 6.7, 6.8 and 6.9. From the data it is evident that soybean is the only grain legume representative within recently commercialized transgenic crops. On the other hand, soybean, in particular herbicidetolerant varieties, has a leading position in this context. The progress in Biotechnology 193 commercialization of transgenics in other legume crops (pea, lupin, chickpea and lentil) compared with the most advanced transgenic crops is illustrated in Table 6.7. 6.5.6 Future prospects Based on the results of basic research and on the application of DNA recombinant technology in crop improvement in the last 10–15 years, it is evident that plant genetic transformation has obtained a permanent position in complementing conventional plant breeding. The absence of genetic modification of carbohydrates in grain legumes can be explained logically by their lower industrial importance compared with carbohydrates, in particular starches, from cereals and potato. If the basic Table 6.7. Traits already commercialized in field trials, and under development for selected crops, 1997–1998 (James, 1997; Hamblin et al., 1998). Crop Traits already commercialized Traits in field trials/development Canola 1. Herbicide tolerance 2. Hybrid technology 3. Hybrid technology and herbicide tolerance 1. Control of corn-borer 2. Herbicide tolerance 3. Insect protected/herbicide tolerance 4. Hybrid technology 5. Hybrid/herbicide tolerance 1. Improved disease resistance 2. Improved disease resistance Maize Soybean 1. Herbicide tolerance 2. High oleic acid Pea None Lupin None Chickpea None Lentil None 1. Control of Asian corn-borer 2. Control of corn rootworm 3. Disease resistance 4. Higher starch content 5. Modified starch content 6. High lysine 7. Improved protein 8. Resistance to storage grain pests 9. Apomixis 1. Modified oil 2. Insect resistance 3. Virus resistance 1. Insect resistance – pea weevil 2. Quality – sunflower albumin 3. High methionine protein 4. Antifungal genes 1. Insect resistance – pea weevil 2. Virus resistance (BYMV) 3. Herbicide tolerance 4. High methionine protein 1. Herbicide tolerance 2. High seed protein 1. Herbicide tolerance 194 N. Kuchuk et al. Table 6.8. Global area (millions of hectares) of transgenic crops grown in 1996 and 1997, by crop (James, 1997). 1996 Crop Soybean Maize Tobacco Cotton Canola Tomato Potato Total Area 0.5 0.3 1.0 0.8 0.1 0.1 < 0.1< 2.8 1997 % Area 18 10 35 27 5 4 < 1< 100 Increase 1996/7 ratio % 5.1 3.2 1.6 1.4 1.2 0.1 < 0.1< 12.8 40 25 13 11 10 1 < 1< 100 10.0 11.0 1.6 1.8 9.5 2.0 3.0 4.5 Table 6.9. Global area (millions of hectares) of transgenic crops grown in 1996 and 1997, by trait (James, 1997). 1996 Trait Herbicide tolerance Insect resistance Virus resistance Insect and virus resistance Quality traits Total Area 0.6 1.1 1.1 – < 0.1< 2.8 1997 % 23.5 37.5 40.5 – < 0.1< 100.5 Area 6.9 4.0 1.8 < 0.1< 0.1 12.8 % 54 31 14 < 1< < 1< 100 Increase 1996/7 ratio 10.7 3.8 1.6 – 2.0 4.5 mechanisms of starch biosynthesis are similar in various crops, then success with the genetic manipulation of the starch synthesis pathway in potato (Stark et al., 1992, 1996; Visser and Jacobsen, 1993; Muller-Rober and Kossmann, 1994) may represent a background and a promise for similar work in grain legumes. The methodological base (gene transfer systems as well as regeneration systems) for such oriented research is now available in major grain legume species. At least three aspects will surely play a future role in the deeper involvement of DNA technology in the improvement of the composition and quality of grain legume carbohydrates. Firstly, there will need to be a deeper understanding of the metabolic processes involved in carbohydrate biosynthesis and degradation, including the exact identification of genes responsible for specific metabolic steps. Secondly, there needs to be further improvement of gene transfer methods, including efficient regeneration systems. Thirdly, there needs to be economic interest in the improvement of carbohydrates in legume crops. Long-term transformation projects will not be started without the interest of industry in a potentially profit-making product. This last point can be further substantiated by the fact that of Biotechnology 195 22 technology proprietors that approved 48 transgenic crop products for commercialization in 1997, 20 (90%) were private corporations and only two (10%) public sector organizations (James, 1997). 6.6 The Availability and Possible Manipulation of Genes Involved in Starch Biosynthesis 6.6.1 Biochemical pathways of starch biosynthesis The chemistry and physical structure of starches have been discussed elsewhere (see Chapters 2 and 4). Likewise, a more detailed description of starch biosynthesis is described in Chapter 5. For the purpose of discussing the possibilities of using biotechnology to modify starch, however, it is necessary to have some basic information on the biochemical process involved. The biosynthetic pathway of starch synthesis involves three main enzyme systems ADP-glucose pyrophosphorylase (ADPGPPase), starch synthases (SS) and starch branching enzymes (SBE; for review see Martin and Smith, 1995). Amylose and amylopectin are synthesized from ADP-glucose, which is synthesized from glucose-1-phosphate and ATP in a reaction that is catalysed by ADPGPPase. The glucose-1-phosphate can be supplied from the Calvin cycle, in photosynthetic tissues, or may be imported directly from the cytosol (Tyson and ap Rees, 1988) and/or synthesized from glucose-6phosphate (Hill and Smith, 1991) in storage tissues. Most of the glucose-6phosphate in storage tissues is formed from sucrose, which is the main transported carbohydrate in plants (ap Rees and Morrell, 1990). Incoming sucrose is cleaved by sucrose synthase to form UDP-glucose and fructose and the UDP-glucose is then converted to glucose-1-phosphate by the action of UDP-glucose pyrophosphorylase (UGPase). The fructose is phosphorylated to fructose-6-phosphate by fructokinase and possibly by hexokinase (Renz and Stitt, 1993). The hexose monophosphates are freely interconverted by the action of phosphoglucomutase and phosphoglucoisomerase. The carbon then enters the amyloplast at the level of hexose monophosphates (Viola et al., 1991), where ADPGPPase starts starch biosynthesis. 6.6.2 The availability of genes involved in starch biosynthesis Plant ADPGPPase is reported to be a tetrameric enzyme that is formed from two distinct polypeptides (Copeland and Preiss, 1981). The cDNA clones encoding both subunits have been isolated from wheat (Olive et al., 1989), maize (Bhave et al., 1990), potato (Mueller-Rober et al., 1990), barley, (Villand et al., 1992), arabidopsis (Villand et al., 1993) and faba bean (Weber et al., 1995a). This enzyme is allosterically regulated by 196 N. Kuchuk et al. 3-phosphoglycerate (3-PGA) as an activator and by inorganic phosphate (Pi) as an inhibitor. It seems that this enzyme has a key role in starch biosynthesis, since the genetic manipulation of ADPGPPase genes clearly demonstrates the influence of this enzyme on starch yield in transgenic plants. For example, the inhibition of the ADPGPPase gene activity by antisense regulation (Mol et al., 1990) led to a near-complete inhibition of starch synthesis. A small (20–50%) decrease in the activity of this gene, however, did not result in a proportional decrease in starch content (Mueller-Roeber et al., 1992). Also, the tuber-specific expression of the glgC-16 gene encoding E. coli ADPGPPase, active in the absence of allosteric activation, led to a 30% increase of starch content in cv. Russet Burbank potatoes, whereas the over-expression of wild-type E. coli ADPGPPase had no significant effect (Stark et al., 1992). In the next step of starch biosynthesis, the starch synthases (SS) catalyse the synthesis of α(1→4) linkages between the pre-existing glucan chain and the glucosyl part of ADP-glucose, in the synthesis of both amylose and amylopectin. Starch synthases are present in different isoforms, which are located both bound to the starch granule and in the soluble phase of the amyloplast. After biochemical analysis of amylose-free (waxy) mutants from several species, it was suggested that the granule-bound SSs (GBSS) synthesize amylose, whereas the soluble SSs synthesize amylopectin. Amylose-free starch could be obtained, therefore, by the manipulation of gene(s) encoding the granule-bound starch synthase. Expression of an antisense-RNA that inhibits the granule-bound starch synthase has yielded potato starch composed only of amylopectin (Visser et al., 1991; Kuipers et al., 1994). Reductions in soluble starch synthase activity of 70–80% by the antisense approach, however, had no measurable effect on the starch content, or on the amylose/amylopectin ratio of transgenic potato tubers (Marshall et al., 1996), although a profound change in the morphology of starch granules was detected. Transgenic potatoes have been generated, in which the activities of both main soluble starch synthases responsible for amylopectin synthesis in the tuber (SSII and SSIII) have been reduced (Edwards et al., 1998). The properties of starch from tubers of these plants have been compared with those of starches from transgenic plants in which the activity of either SSII or SSIII has been reduced. Starches from the three types of transgenic plants are qualitatively different from each other and from the starch of control plants, with respect to granule morphology, the branch lengths of amylopectin, and the gelatinization behaviour. The effects of reducing SSII and SSIII together could not be predicted from the effects of reducing these two isoforms individually. The α(1→6) branches in starch polymers are made by starchbranching enzymes (SBE), which are also present in multiple isoforms. The generic effect of SBE is to hydrolyse an α(1→4) linkage within a chain and then catalyses the formation of an α(1→6) linkage between the reducing end of the glucan chain and another glucose residue. Two SBE families, A Biotechnology 197 and B, have been identified in maize (Stinard et al., 1993), rice (Mizuno et al., 1993) and pea (Burton et al., 1995). The proteins of these two families are structurally related and are similar to glycogen-branching enzymes from E. coli bacteria, which have been used to produce highly branched starch in transformed potato (Shewmaker et al., 1994). In the embryos of peas containing the r mutation (see Chapter 7), the percentage of amylopectin is reduced from about 65% to about 35%. This relative decrease in amylopectin has been associated with decreased branching activity and the loss of one branching enzyme isoform (Smith, 1988). Antisense experiments inhibiting isoform B activity in potato, however, resulted in no significant modification of starch synthesis, or of the amylose : amylopectin ratio (Kossmann et al., 1991; Mueller-Roeber and Kossmann, 1994). Recently, different lines of transgenic potatoes have been generated where the expression of starch biosynthetic genes has been repressed, using an antisense RNA technique. In this way plants have been obtained that synthesize a wide variety of structurally modified starches. These starches are currently being assessed for their applicability in different industrial processes in the food and non-food sectors (Kossmann et al., 1997). 6.6.3 The availability of other genes influencing starch biosynthesis and starch quality It remains unclear whether all enzymes and proteins involved in determining starch structure have been characterized. The phosphorylation of starch, which occurs to a greater extent in starches derived from vegetative storage organs like potato tubers and also starches from other sources (Jane et al., 1996), is not associated with the catalytic activity of the starch biosynthetic enzymes described above. A gene involved in the incorporation of phosphate into starch-like glucans has been cloned and incorporated into transgenic potato plants. This results in the production of starch with a reduced phosphate content (Lorberth et al., 1998). This reduced phosphate content drastically influences the pasting properties of the starch. It also results in some secondary effects on the transgenic plants, which have excess starch in their leaves and a reduction in cold sweetening in their tubers. Sink strength of storage organs can also contribute to starch accumulation, sink strength being defined as the ability of an organ to attract photoassimilates (Ho, 1988). It is dependent on the transport mechanism and on the physical and biochemical isolation of the transported carbon in the sink tissue. As sucrose is the major transport form of fixed photoassimilates into the storage organs of major crop plants, sucrose metabolism is of particular interest. Two classes of sucrose cleavage enzymes, invertase and sucrose synthase, are present in plants. 198 N. Kuchuk et al. Sucrose synthases (Susy, UDP-glucose: D-fructose-glycosyl-transferases) catalyse reversible reactions associated with storage functions, such as starch synthesis (Heim et al., 1993). On the other hand, invertases (β-fructofuranosidases) catalyse the irreversible cleavage of sucrose into glucose and fructose. Genes of faba bean (Heim et al., 1993) and potato (Zrenner et al., 1995) sucrose synthase, as well as faba bean (Weber et al., 1995b) and maize invertases (Xu et al., 1996) have been cloned and could be used to study the influence of the enzymes on carbohydrate accumulation in legume species. The size of potato tubers was increased by overexpression of yeast invertase specifically in the cytosol of tubers. There was a 10–15% reduction in the starch content of mature tubers of these plants, however, plus a large increase in the quantity of glucose (Sonnewald et al., 1997). It was thought that starch accumulation could be improved by increasing the glucose phosphorylating capacity of the invertase-expressing tubers. The additional expression of another transgene encoding a glucokinase from Zymomonas mobilis, in combination with an invertase gene, however, led to a dramatic reduction in starch accumulation and a stimulation of glycolysis (Trethewey et al., 1998). Weber et al. (1998) transformed V. narbonensis with the yeast invertase gene, under the control of the legumin B4-promoter. Expression of the legumin B4-promoter yeast invertase gene in transgenic cotyledons resulted in a reduction of sucrose, starch and protein contents and an increase in the hexose level. Pyrophosphate (PPi) and inorganic phosphate (Pi) are inhibitors of many biosynthetic pathways leading to carbohydrate accumulation. To test whether sucrose and starch biosynthesis can be accelerated by removal of PPi, the ppa gene for pyrophosphatase from E. coli was introduced into tobacco and potato under control of the constitutive 35S-promoter. This resulted in a small shift of partitioning in favour of sucrose and a reduction in starch content (Sonnewald, 1992). Although most of the information presented above has been obtained for plant species other than grain legumes, the modification of the biosynthesis and the quality of starch within legume species would be possible by making use of the cloned genes. 6.7 The Availability and Possible Manipulation of Genes Involved in a-Galactoside Accumulation and Degradation 6.7.1 Biochemical pathways of a-galactoside biosynthesis The chemical, biochemical and nutritional aspects of the raffinose oligosaccharides are covered elsewhere (see Chapters 2, 3 and 5). As with the section on starch, it is necessary to present information on these compounds here to make it easier to discuss their possible genetic manipulation. Biotechnology 199 The biosynthesis of the raffinose family of oligosaccharides (RFO) has been fairly well characterized. The committed reaction of raffinose biosynthesis involves the synthesis of galactinol from UDP-galactose and myo-inositol. The enzyme that catalyses this reaction is galactinol synthase (GS). The synthesis of raffinose and higher RFO homologues from sucrose is thought to be catalysed by distinct galactosyltransferases (for example, raffinose synthase and stachyose synthase). Studies with many species, however, suggest that GS is the key enzyme controlling the flux of reduced carbon into the RFO biosynthetic pathway. The GS enzyme has been purified and characterized from courgette, the purified enzyme being a monomer of Mr 42,000 with an isoelectric point of 4.1 (Smith et al., 1991). 6.7.2 The availability of genes involved in a-galactoside accumulation and degradation and their possible manipulation There are some biotechnological approaches to produce commercial bred lines of grain legume species with a low content of antinutritional carbohydrates. One approach is antisense inhibition of genes (Mol et al., 1990) involved in the production of antinutritional carbohydrates. For this purpose it is essential to clone the key genes of α-galactosides biosynthesis. There is one patent describing nucleotide sequences of galactinol synthase (GS) genes from courgette and soybean (US Pat. No. 5,648,210; Kerr et al., 1993) and the isolated nucleic acid fragments that encode soybean seed and courgette leaf GS have been provided. Chimaeric genes, including these fragments and suitable regulatory genes that are capable of transforming plants to produce GS at levels higher or lower than that found in the target plant, also have been provided. Also, there are methods for varying the content of D-galactose-containing oligosaccharides of sucrose in plants to produce transformed plants and seeds. Recently, Peterbauer et al. (1999) have reported on the gene cloning and functional expression of stachyose synthase from seeds of adzuki bean (Vigna angularis Ohwi et Ohashi). The complete cDNA sequence comprised 3046 nucleotides and included an open reading frame that encoded a polypeptide of 857 amino acid residues. The recombinant protein was expressed in a heterologous expression system and the raised product was immunologically active with polyclonal antibodies specified for stachyose synthase. Another approach can be based on the use of genes encoding enzymes that degrade α-galactosides, to produce legume plants with low levels of the RFO. α-Galactosidases (EC 3.2.1.22) catalyse the hydrolysis of α(1→6)galactosyl linkages and have been known for a long time, in microorganisms, plants and animals (Dey and Pridham, 1972). Genes encoding a number of α-galactosidases have been cloned and sequenced. Many of 200 N. Kuchuk et al. these genes are present as operons in association with other genes involved in galactoside utilization. Genes have been isolated from a number of sources including Bacillus stearothermophillus (Ganter et al., 1988), E. coli (Aslandis et al., 1989), Streptoccoccus mutans (Aduse-Opoku et al., 1991) and Pedicoccus pentosaceous (Gonzales and Kunka, 1986). In processing soybean and cowpea meal, α-galactosidases from various sources including Bifidiobacterium sp. (Sakai et al., 1987), Aspergillus awamori (Smiley et al., 1976), Aspergillus niger (Somiari and Balogh, 1995) and Lactobacillus fermentum (Garro et al., 1996) have been used to remove flatulence factors. More information concerning the utilization of α-galactosidases in processing has been discussed elsewhere (see Chapter 4). The development of transgenic grain legume plants with seed specific expression of α-galactosidases could be a tool to overcome the RFO problem. At present, genes encoding seed-specific proteins from soybean (Okamura et al., 1986), pea (Ellis et al., 1988) and bean (Greenwood and Grispefls, 1985) have been cloned and their promoters could be used to construct chimaeric α-galactosidase genes with seed-specific expression. The physiological role of the antinutritional carbohydrates within the plant, however, is not clear. As discussed earlier (see Chapter 5), there is a hypothesis suggesting that the α-galactosides have an important role in seed maturation and in resistance to desiccation. In which case, an α-galactosidase functioning during seed maturation could be disadvantageous for seed quality. A better strategy could be α-galactosidases that could be activated by external factors, preferably after seed harvesting. From this point of view, genes encoding thermostable enzymes from hyperthermophiles could be of interest for this purpose. One group of hyperthermophilic bacteria is the genus Thermotoga. To date, Thermotoga spp. are the only known hyperthermophiles capable of growing on cellulose. They produce a multiplicity of hydrolases, which are involved in the metabolism of various polysaccharide substrates. Recently an α-galactosidase from Thermotoga neapolitana has been used for the hydrolysis of guar (galactomannan) gum (McCutchen et al., 1996). This enzyme has a temperature optimum close to 100°C, its activity decreasing with lower temperatures. It may be possible to transfer the thermostable α-galactosidase chimaeric gene, under the control of a seed-specific promotor, to commercial legume breeding lines. Hypothetically, the activity of this enzyme in transgenic seeds would be low at temperate climatic conditions. The accumulation of the RFO in transgenic seeds, therefore, could be at a similar level to the non-transferred control cultivar and, hence, normal seed quality would not be reduced. After harvesting, during seed processing the enzyme could be activated by high temperature and could then decrease the content of α-galactosides. In addition, this process could be applied to canned green peas, where it is impossible to remove the RFO by other methods. Biotechnology 201 6.8 Cell Suspension Culture as a Model for Studying Carbohydrate Metabolism 6.8.1 Introduction Plant cell suspension culture provides a defined and controlled system for the study of cell metabolism. Some advantages of cell suspension cultures for metabolic studies, compared with the whole plant are: rapid and uniform growth with the possibility of controlling nutritional conditions; ease of extraction and purification of large quantities of metabolic products; a more efficient use of labelled compounds and a lack of interfering microorganisms. Although the metabolic systems of cell cultures and of whole plants are frequently similar, some quantitative differences do occur. The aim of this section is to illustrate how cell suspension cultures can be used to study the metabolism and biochemistry of plant cell walls. In particular, the possibility of using cell suspensions from legumes as a model system for studying their plant cell wall metabolism. 6.8.2 Composition of plant cell walls The plant cell wall is a highly dynamic structure playing an important role in growth and development, morphology, cell-to-cell communication and transport processes (Fry, 1989; Hayashi, 1989; Bowles, 1990; Sakurai, 1991; Takeuchi et al., 1994; Heredia et al., 1995; Roberts, 1996; Ishii, 1997; Driouich and Staehelin, 1997). It enables the plant cell to resist internal and/or external pressures, provides a structural barrier to some molecules and protects against invasion by insects and by pathogens (Bowles, 1990). The cell cytoplasm obtains its metabolic substrates through the cell wall and excretes other substances across it. The cell wall is a complex structure, which contains mainly carbohydrates, proteins, lignins and water as well as other substances embedded in it, such as cutin, suberin and certain inorganic compounds. Morphologically, three zones can be differentiated in plant cell walls, the middle lamella, the primary wall and the secondary wall. The middle lamella is the most external of the three zones and acts as a separating panel between two cells. It consists almost exclusively of pectic substances. The primary cell wall consists of cellulose microfibrils, complex polysaccharides and N- and O-linked glycoproteins. Most plant cells have only a primary wall and the middle lamella, but some specialized cells exist that also have the secondary wall. This is a supplementary wall with a predominantly mechanical function. It has been established (Mauch and Staehelin, 1989; Bolwell, 1993; Penel and Greppin, 1996) that there are many glycoproteins and soluble 202 N. Kuchuk et al. proteins with various enzyme activities, located in either the cell wall or the extracellular space. Some of the enzymes are needed for modifying the organization of the macromolecular network, others are involved in processes such as defence reactions against pathogens (Bowles, 1990; Cote and Hahn, 1994). The cell wall hydrolysis enzymes (cellulases, pectinases and a series of glycosidases, such as α- and β-galactosidases, α- and β-mannosidases, etc.) are involved in processes such as fruit maturation and softening, as well as in responses to pathogen exposure and other stress factors (Fry, 1989; Bowles, 1990). Peroxidases are known to take part in the formation of links between lignin, proteins, hemicellulose and ferulic acid (Bowles, 1990). 6.8.3 Biosynthesis of the cell wall components The structural components of primary plant cell walls are cellulose microfibrils, other complex polysaccharides and N- and O-linked glycoproteins (Heredia et al., 1995). Of these components, only cellulose microfibrils are synthesized at the cell surface by plasma membrane enzymes. All other types of cell matrix molecules are produced by Golgi-based enzyme systems (Driouich et al., 1993; Driouich and Staehelin, 1997) and are transported, via secretory vesicles, to the cell surface. Recent biochemical investigations have led to the identification and partial characterization of Golgi-localized glycosyltransferases, involved in the synthesis of xyloglucans, pectic polysaccharides and glucoronoxylans (Camiranel et al., 1987; Gibeat and Carpita, 1994). Immunolabelling experiments, with libraries of anti-polysaccharide antibodies, have been used to outline the spatial organization of polysaccharides (Moore et al., 1991; Zhang and Staehelin, 1992). It is important to remember, however, that all of these studies carried out with antibodies are directed against specific sugar domains of the polysaccharides and not against specific glycosyltransferases. This is because none of these enzymes has been fully purified to date, although some of them have been identified and partially characterized (Driouich and Staehelin, 1997). The isolation of such enzymes and the generation of specific antibodies against them is a major problem that still has to be resolved. 6.8.4 Oligosaccharides as signals and substrates in the plant cell wall A few selected oligosaccharides, at very low concentrations, can exert ‘signalling’ effects on plant tissues (Fry et al., 1993). Such oligosaccharides are termed ‘oligosaccharines’ and their discovery has provoked much research (Sakurai, 1991; Fry et al., 1993). There is still an urgent need for additional knowledge on these oligosaccharides, however, including a Biotechnology 203 more detailed physiological description of known effects, the definition of structure–activity relationships and documentation of the biosynthesis, transport, binding action and turnover of oligosaccharides in the plant (Fry et al., 1993). Almost all of the investigations of oligosaccharides have utilized in vitro preparations, often made using hard chemical treatments that are unlikely to be encountered in plants. This raises questions as to whether oligosaccharides are actually present in vivo, how they are released from larger complex carbohydrates and how they fulfil their biological functions. There is evidence suggesting that two biologically active oligosaccharide fragments can be generated in vivo from plant cell wall polysaccharides, oligogalacturonides and xyloglucan oligosaccharides. Partial depolymerization of homogalacturonan generates oligogalacturonides, which, in the presence of Ca2+ (Farmer et al., 1991; Fry et al., 1993), exhibit various regulatory effects in plants. These include the elucidation of defence responses, the regulation of growth and development (Ryan and Farmer, 1991; Aldington and Fry, 1993; Roberts, 1996), the production of protease inhibitors (Cote and Hahn, 1994), fruit ripening, flower formation and the inhibition of auxin action (Fry et al., 1993). Specific oligosaccharides can be produced from xyloglucan by partial digestion with cellulase. The xyloglucan oligosaccharides have shown growth inhibiting effects (Jork et al., 1984; Farmer et al., 1991; Angur et al., 1992) and have been closely associated with cell extension (Cutillas-Iturralde and Lorences, 1997). The mechanism of xyloglucan oligosaccharide growth promotion, however, remains poorly understood. Enzymatic hydrolysis offers the most likely biological mechanism for the generation of oligosaccharides (Fry et al., 1993). Plants and pathogens are known to produce a number of hydrolytic enzymes that could be involved in this process (Schlumbaum et al., 1986; Mauch et al., 1988; Bol et al., 1990; Ioshikava et al., 1990; Ham et al., 1991). The demonstration that biologically active oligosaccharides are generated at the plant pathogen interface by hydrolytic enzymes would provide evidence that these fragments do have a role in vivo. The release of such fragments by purified enzymes has been demonstrated in the case of glucanases. Several proteins with β(1→3)-glucanase activity have been purified from soybean and shown to release elicitor active fragments from the mycelial walls of Phytophthora sojae (Ioshikava et al., 1990; Ham et al., 1991). Other hydrolytic enzymes, possibly involved in the generation of oligosaccharides during interactions with plant pathogens, are pectic-modifying enzymes (Bowles, 1990; Fry et al., 1993; Cote and Hahn, 1994). They exist in uninfected plant tissues and have the potential to generate biologically active oligogalacturonides in plants, by de-esterification of pectic methyl esters and cleavage of homogalacturonans. The results from the extensive research on oligosaccharide effects has led to the conclusion that these compounds are important signal molecules 204 N. Kuchuk et al. and they play major roles in plant development processes and plant– pathogen interactions. The structural characterization of these molecules is a major experimental challenge. Knowledge of the structure of oligosaccharides and the biological responses to these signals, now has progressed to the point where detailed studies on their mode of action can be undertaken. 6.8.5 Plant cell suspension cultures – a powerful tool in investigating cell wall metabolism In spite of the complexity of working with cell walls, knowledge of cell wall polymer structure has expanded considerably in recent years (Becker et al., 1974; Sakurai, 1991; Mutafschiev et al., 1993; McCann and Roberts, 1994; Heredia et al., 1995). Knowledge of the biosynthesis and the functions of the polymers, however, remains limited. Plant cell suspension culture offers new possibilities for investigating some aspects of cell wall metabolism (Bolwell, 1985; Butenko, 1985; Morris et al., 1985; Dwight Camper and McDonald, 1989; Wink, 1994). In this type of culture, single cells, or clumps of cells, grow and multiply suspended in liquid media. Plant cell suspension cultures provide a model system for studying various molecular, physiological and genetical problems, which can be manipulated in ways that cannot be applied to whole plants. Cell suspensions can be cultivated in flasks on shakers or in bioreactors, which allows control of the nutritional and environmental conditions. As a result, controlled growth and morphological development can be ensured. Cell growth is rapid and more uniform than for cells within the plant. Furthermore, they offer a possibility for the controlled supply of metabolites, and every cell in such a suspension has direct access to the external medium containing these metabolites. Plant cell suspensions also offer a standard system that allows the results obtained by different research groups to be compared. It has been established (Wink, 1994) that plant cell suspension is a physiologically complex system, in which both the cell biomass and the culture medium are included. The culture medium is not only the source of all necessary nutrients, but also a functional cell compartment with a number of different metabolites sequested in it (Olson et al., 1969; Van Huystee and Lobazzewski, 1982; Wink, 1984, 1994; Morris et al., 1985; Konno et al., 1986, 1987, 1989; Kawasaki, 1989; Uchiyama et al., 1993; Van Huystee et al., 1994; Dolaptchiev et al., 1996; Ilieva et al., 1996a,b; Sims et al., 1996). It is well known that suspension cultured plant cells secrete various polysaccharides and glycoproteins into the culture medium (Olson et al., 1969; Kawasaki, 1989; Uchiyama et al., 1993; Sims et al., 1996). These secreted substances are thought to be components of the middle lamella, derived from primary cell walls, two elements that are difficult to separate Biotechnology 205 in the intact plant (Heredia et al., 1995). Investigation of these compounds should contribute, therefore, to our understanding of the composition and functions of the middle lamella and primary cell walls (Olson et al., 1969; Uchiyama et al., 1993; Wink, 1994; Sims et al., 1996). High activities of various enzymes, involved in the biosynthesis of the cell wall during growth, can be determined in the culture medium (Olson et al., 1969; Wink, 1984, 1994; Konno et al., 1989; Dolaptchiev et al., 1996; Ilieva et al., 1996a,b). In addition, plant cells release enzymes into the extracellular space when they are attacked by microbes. The secretion of chitinase, glucanase and other enzymes has been studied in this context (Schlumbaum et al., 1986; Mauch et al., 1988; Bol et al., 1990; Ioshikava et al., 1990; Ham et al., 1991). Since the suspension cultured cells also can be stressed by factors other than microbe invasion and culture conditions, the secretion of high amounts of hydrolytic enzymes into the medium may be considered a cell response (Wink, 1984, 1994; Messner and Boll, 1993). It has been established that during the growth and ageing of cell cultures the content of the secreted metabolites changes (Kawasaki, 1986; Goubet and Morvan, 1993; Schaumann et al., 1993; Dolaptchiev et al., 1996). The culture medium acts, therefore, as an external sink, in which the turnover of secreted metabolites occurs. Lytic processes have been observed in the medium, carried out by secreted enzymes (Wink, 1984; Konno et al., 1989). As a result some of the secreted metabolites are transformed into inert endproducts or compounds, which fulfil different functions (Wink, 1984; Konno et al., 1986, 1987, 1989). The rapid and uniform growth of cell cultures can be used to follow the growth cycle of the culture and the turnover of cell wall polysaccharides. During growth, changes in the enzyme activities can be recorded in the cells and in the culture medium (Uchiyama et al., 1993; Dolaptchiev et al., 1996; Ilieva et al., 1996a,b). Specific physiological features of the culture and its dependence on the biosynthetic time course of different metabolites can also be studied (Schaumann et al., 1993; Uchiyama et al., 1993; Faik et al., 1995; Dolaptchiev et al., 1996; Ilieva et al., 1996a). Suspension cultures have been successfully used to study the time course of synthesis and secretion of different polysaccharides and enzymes. This allows enzyme activities to be correlated with polysaccharide content and composition (Schaumann et al., 1993; Ilieva et al., 1996a, b). It should be noted that the composition of the culture medium markedly affects the cell wall polysaccharide content, offering a system for studying the influence of different environments on cell wall physiology. On the other hand, this influence may interfere with or even corrupt the results obtained for heterotrophic suspensions grown on rich media. Recently, Lozovaja et al. (1996) overcame this problem by using photoautotrophic soybean (Glycine max) cell suspension cultures grown on minimal medium, with CO2 as the sole carbon source. This system was used to investigate cell wall polysaccharides and starch biosynthesis and turnover. Photoautotrophic suspension 206 N. Kuchuk et al. culture could be a convenient new model, therefore, to study some largely unknown aspects of plant physiology and biochemistry. This system could also allow the relationship between photosynthesis and cell growth to be studied with regard to cell wall component accumulation, which could lead to strategies for increasing growth and biomass production. Since the supply of different components is controlled and every cell has direct access to the external medium, it is possible to study the influence of different added molecules on plant cell metabolism. For example, cell suspension cultures have been used as model systems for studying the herbicide turnover by plant cells (Dwight Camper and McDonald, 1989). Also, cell suspension cultures from legumes have been used to study the role of oligosaccharides in eliciting and inducing enzyme activities (Kohle et al., 1984, 1985; Roffs et al., 1987; Stab and Ebel, 1987; Bruce and West, 1989; Davis et al., 1993). Investigations using cell suspension cultures from legumes also have contributed to the knowledge of signal transduction pathways activated by oligosaccharides and an understanding of their biological significance (Young et al., 1982; Young and Kauss, 1983; Apostol et al., 1989; MacKintosh et al., 1994). Plant cell wall polysaccharide studies have been hindered by the use of heterogenous wall extracts (Becker et al., 1974; Takeuchi et al., 1994). The intact plant tissues contain various types of cells, as well as a mixture of primary and secondary cell walls. Suspension cultured cells have been selected for investigating the structure of plant cell walls, because they can grow as a fairly homogeneous population with predominantly primary cell walls. This allows homogeneous preparations of primary cell walls to be obtained and homogeneous polysaccharide fragments to be isolated from the cell wall and from the culture medium. These fragments have been used as substrates for investigating enzyme functions (Olson et al., 1969; Konno et al., 1986, 1987). Some polysaccharide fragments can be isolated in a native form from the culture medium and their structure and functions can be studied by subsequent model experiments. Cell suspensions are a very promising system for obtaining pure enzymes. Enzymes involved in the biosynthesis of cell wall polysaccharides can be isolated from the cell biomass (Olson et al., 1969; Konno et al., 1987; Faik et al., 1995) and some biosynthetic enzymes can be obtained from the culture medium (Wink, 1984, 1994; Konno et al., 1989; Le Bansky et al., 1992; Van Huystee et al., 1994; Ilieva et al., 1996c). Once purified, these enzymes can be used in model experiments together with isolated polysaccharide fragments for elucidating their mode of action, their function in cell growth and development and in the turnover of the cell wall polysaccharides. For example, Kono et al. (1986, 1987, 1989) succeeded in isolating some enzymes involved in polysaccharide biosynthesis and performed model experiments using homogenous preparations of primary cell walls (isolated from carrot cell biomass) and with polysaccharide fragments. Biotechnology 207 A number of enzymes that have been found in culture media are of commercial interest. Wink (1984, 1994) reported that the culture medium of Lupinus polyphyllus cells is a promising source for the isolation of different hydrolytic enzymes. Van Huystee et al. (1994) showed that cultured groundnut cells in suspension cultures are an ideal system for the isolation of peroxidase, in terms of the ease of purification and yield. Since some of these enzymes are involved in the plants defence against attack by microorganisms and against other stress factors, their enhanced biosynthesis and secretion into the medium can be induced by subsequent signal molecules or stress factors. This presents possibilities for obtaining an enhanced enzyme yield (Wink, 1994; Ilieva et al., 1996c). Cultivation of Nicotiana tabacum 1507 cell suspension in an aqueous two phase system resulted in an enhanced yield of phosphomonoesterase with a high specific activity (Ilieva et al., 1996c). Breeding G. 7 Engqvist and et Agronomy al. Breeding and Agronomy 7 Editor: Goran Engqvist Contributors: Mike Ambrose, Nikolai Chekalin, Peter Chekrygin, Goran Engqvist, Saima Kalev, Martin Mrskos, Paolo Ranalli and Ion Scurtu You write with ease, to show your breeding, But easy writing’s vile hard reading. Clio’s protest (written 1771, published 1819) Richard Brinsley Sheridan (1751–1816), Anglo-Irish playwright 7.1 Current Breeding Goals The main grain legume within Europe is pea (Pisum sativum) and so the major breeding programmes are aimed at improving the characteristics of this species. It follows, therefore, that a significant part of this chapter will concentrate on pea, with reference being made to other grain legumes where relevant and when information exists. A major part of dry pea production within European Union countries is used as a high-protein animal feedstuff. The EU, however, only produces about a third of its required animal feed protein, the other two-thirds being imported. In other parts of the world, particularly in developing countries, the major use of dry peas is for direct human consumption, a trend that is becoming more popular in western countries as more people turn to vegetarian diets for health or for moral reasons. In order to obtain a significant increase in dried pea cultivation and production, it is important that the net return from peas is competitive with that from other crops grown by farmers. The primary breeding goal, therefore, is to obtain a high harvested seed yield. This requires, in the first place, the development of crops that have good standing ability to improve ©CAB International 2001. Carbohydrates in Grain and Legume Seeds (ed. C.L. Hedley) 209 210 G. Engqvist et al. the ease of harvesting, which in Western countries is carried out using combine harvesters normally used for cereals. Standing ability can be improved by the incorporation of a stiff stem combined with the ‘semileafless’ phenotype, which has well-developed tendrils (Fig. 7.1). The John Innes Centre (Norwich, UK) played a vital role in adopting the semi-leafless character into pea breeding programmes (Snoad, 1974; Davies, 1977; Hedley and Ambrose, 1981). The height of the crop at harvest is a good and simple measure when evaluating the ease of harvesting. There are a number of other important agronomic characters. Earliness of ripening, which is essential in those countries that have a limited growing season. Plant height, which must not be too short because this will make combining difficult. Seed size, which must not be too large, or the cost of the seed for sowing will be too high. This is particularly important in countries such as Canada, where the seed yield per unit area is low and the production capacity covers a vast acreage. There can be some contradiction with this character relating to breeding goals, because a larger seed size can increase yield. Seed colour can be important, with yellow and green being the preferred types. It is also important to reduce the proportion of seed that may be shed during harvesting. In addition to agronomic characters, seed quality is also important. In particular, the protein content should be high, since the main use of peas is as a high protein source for animal and human diets. There has been a tendency for the protein content to be lower in new higher-yielding varieties and it is important that efforts are made to reverse this trend. As peas become more important in the human diet, good cooking ability will Fig. 7.1. Crop of the ‘semi-leafless’ pea held up by the tendrils intertwining. Breeding and Agronomy 211 become an increased priority for breeders. In developing countries this characteristic is of paramount importance because of the energy required in preparing grain legumes for human consumption. Perhaps the major problem with the production of all crops, including grain legumes, is susceptibility to diseases. Breeding for increased disease resistance is, therefore, an important part of all breeding programmes. Aphanomyces root rot, caused by Aphanomyces euteiches, is one of the most important root pathogens of peas worldwide. Mycosphaerella pinodes, which causes leaf, stem and pod spot, and root rot is one of the three fungi in the Ascochyta blight disease complex. No strong resistance has been found against these two fungi, although there is resistance to other important pathogens, for example, Peronospora viciae, Fusarium oxysporum, Erysiphe polygoni and Pseudomonas syringae. The breeding objectives discussed so far are the traditional ones and they will remain as the basis for all pea improvement, at least in the near future. Seed quality characters, however, will undoubtedly become more important with time as grain legume crops, and pea in particular, are used more in human diets, also, as these crops become more important as potential sources of raw material for use in processed food and non-food applications. In this respect, manipulating and improving the carbohydrate fraction of the seed, which in most grain legumes comprises by far the greatest proportion of the seed, will become of increasing importance to plant breeders. Part of this chapter, therefore, will describe and discuss the availability of genetic variation for carbohydrates and possible ways that the seed composition for these compounds can be altered. These discussions will include information on the inheritance of major carbohydrate components and the importance of these components to the growth of the plant within the crop environment. Information is also provided on the registration requirements for new varieties within Europe. 7.2 Breeding Techniques 7.2.1 Pedigree breeding Traditional field pea breeding techniques often start with making crosses in a greenhouse in the autumn, growing the bulked first filial generation – F1 seeds in a greenhouse in the spring and then growing the F2 in the field the following summer. If a pedigree breeding method is followed, the F2 will usually be planted spaced out and the first selections, based on individual plants, will then be carried out. Pedigree plant selections will be made at the F3, often during an off-season cultivation at a location in the Southern Hemisphere (if the breeding programme is based in the Northern Hemisphere). The F4 of each pedigree line would then be grown in an observation plot at the home station location. At this stage a vigorous 212 G. Engqvist et al. selection for stem stiffness and seed quality would be performed between the lines. The F5 selected lines could then again be grown at an off-season location in the Southern Hemisphere. At the F5 stage, selection based on agronomic characters would be made. The selection process at the F5 is often preceded by progeny tests of the F4 generation in the greenhouse for certain diseases and this information is then used to aid the F5 selection. The first comparative yield trials take place at the F6 stage together with the start of elite seed production. The first major comparative yield trial and replicated trials in other countries will be carried out at the F7. The official trials can start at the F8 and continue through to the F10. The pedigree method allows an effective and relatively quick search through a population following a cross, although it is rather labour intensive and demanding and, therefore, expensive. 7.2.2 Bulk selection The main alternative to the pedigree breeding method is the bulk method, in which the F2 is grown as a bulk. The F3 is then grown in plots for yield determination at the home station, with two or three replicates of each plot being grown depending on the amount of available seed. Part of the F3 seed is sown later in the same season as spaced plants, to facilitate individual plant selection. The F3 yield plots are harvested and weighed. Those populations that show the highest relative yield are identified and single plant selections are made on the spaced plants from the same population. Preliminary yield estimates of the lines selected in the F3 start in F5. 7.2.3 Deviations from the pedigree and bulk methods The schedules outlined for the pedigree and bulk selection methods may sometimes be changed. For example, repeat plant selections may be made to increase homogeneity. Also, when a cross is made to incorporate plant disease resistance characters, the F2 may be put directly through a selection procedure to identify plants with the desired character. If and when the breeder wishes to search through a large number of populations the selection work needs to be simplified. In this instance the bulked populations can be grown for several years, delaying the plant selection until the F4 or F5 generation. This has the advantage that the selected plants are more homozygous and the derived lines more uniform than when selections are made in the F2. At the same time the bulked populations can be put in to comparative yield trials, preferably under severe conditions, and those Breeding and Agronomy 213 populations that show obvious shortcomings can be discarded. In this way the selection work can be concentrated on the best populations. According to plant breeding theory on the subject of making predictions from a cross, it is stated that when different populations are compared their mean and standard deviation can be used to identify the population that is most likely to contain the plants with the desired recombined characteristics. For a population mean to be reliable, however, it should be determined as the mean of a sample of individuals from that population. In reality, the cost of carrying out such a determination is very high and so this prediction technique is not often used in practical breeding. If a cheap and simple method for estimating m could be found then there would be a breakthrough in the application of the cross prediction theory. One important and principle difference between the pedigree and bulk selection methods is that the bulk method as outlined above gives a yield measurement in the F3, from which a rough estimate of the population mean can be derived. Both the pedigree and bulk breeding methods have been relatively successful in creating good pea cultivars. 7.3 Access to Genetic Variation 7.3.1 Germplasm banks Most countries maintain collections of grain legumes of one sort or another. The type of collection will vary according to the resources available and according to the nature of the work that the collection is supporting. Some countries maintain reference collections of material that has been bred, or has originated, in their country and for which they hold national responsibility. Other germplasm banks have built up collections of research lines. The genetic resources community operates through networks that aim towards developing common work plans for the collation of passport data on collections. The networks also aim to identify gaps where future collection work is required and to ensure common evaluation and characterization strategies. One of the most successful of these networks is the European Co-operative Programme for Plant Genetic Resources. One of the groups within this programme is focused on grain legumes. This grain legume group has instigated the development of a series of European Central Crop databases for European grain legume collections, with different institutions acting as co-ordinator for particular species (Table 7.1). As an example, the European Pisum database comprises records of the holdings of 22 collections in 20 countries (Table 7.2). Information on these databases can be obtained through the following web site: www.cgiar.org/ecpgr 214 Table 7.1. G. Engqvist et al. European central crop databases and management centre. Species Database management centre Vicia faba Cicer arenatum Glycine max INRA, Le Rheu, France National Plant Breeding Station, Elvas, Portugal N.I. Vavilov Research Institute of Plant Industry, St Petersburg, Russia Aegean Agricultural Research Institute, Izmir, Turkey John Innes Centre, Norwich, UK Wiatrowo, Wagrowic, Poland Federal Office of Agrobiology, Linz, Austria Lens spp. Pisum spp. Phaseolus spp. 7.3.2 Existing variation for the carbohydrates Information on the existing variation for carbohydrate characters can be obtained from the centres detailed in Table 7.1. Pea can be used as an example of the range of variation that can be found and analytical information on a representative sample of lines from the collection held at the John Innes Centre is presented in Table 7.3. This table contains information on the carbohydrate composition of 227 round-seeded pea lines that were analysed as part of an EU-funded ECLAIR project (AGRE 0048), running from 1990 to 1994. The range of lines covered the spectrum of Pisum germplasm available, including representatives from subspecies, landraces and old and new cultivars. The aim of the project was to improve the nutritional quality of grain legume seeds for use in the animal feedstuff industry. The data are from seeds multiplied in one season on a single site. The distributions for soluble sugars, starch, neutral detergent fibre (NDF) and acid detergent fibre (ADF) were all normally distributed, but demonstrated a range of values. It is evident, from these analyses that considerable ‘natural’ variation can be found within existing Pisum germplasm, all of which is available for incorporation within breeding programmes. This gives considerable scope for modifying the carbohydrate composition of this species using conventional breeding techniques. 7.3.3 Newly identified genetic variation Breeders often work without knowledge of the underlying genetic basis to their crossing programmes and selections, since many of their most important characters, such as yield or standing ability, are determined by interactions between many genes. Some quality characteristics of seeds, however, are known to be controlled by relatively simple genetic systems and in these cases major genes have been identified. At present, the best characterized carbohydrate pathway is starch, which has been studied for many years by biochemists. Major advances in Table 7.2. Management centres within Europe for the main grain legume databases. Acronym Country Aegean Agricultural Research Institute Instituto del Germoplasma Institut für Pflanzenbau und Pflanzenzuchtung Centre for Genetic Resources Department of Agriculture and Fisheries for Scotland Zentral Institut für Genetik und Kulturpflanzen-forschung Centre de Recherches Agronomiques de L’Etat International Centre for Agricultural Research in the Dry areas Instituto Nacional de Investigaciones Agrarias John Innes Centre Institute of Plant Breeding Agrobiologische Institute Nordic Gene Bank for Agricultural and Horticultural Plants Pea and Lentil Breeding Programme Institute of Plant Introduction and Genetic Resources Research and Breeding Institute of Technical Crops and Legumes Institute for Plant Production and Qualification Vegetable Crops Research Institute, Research Station Ujmajor Servicio de Investigacion Agraria N.I.Vavilov All-Union Scientific Institute of Plant Industry Israel Genebank for Agricultural Crops, Volcani Centre Pisum Gene Bank ARA BAR BRN CGN ECS GAT GBX ICA IMD JII JOK LIN NGB RAB SAD SUM TAP UJM VAL VIR VOL WTD Turkey Italy Germany Netherlands Scotland Germany Belgium Syria Spain UK Finland Austria Sweden Morocco Bulgaria Czech Republic Hungary Hungary Spain Russia Israel Poland Number of accessions 88 4297 1009 1008 1980 2478 348 3318 256 3008 17 33 3055 65 1598 937 934 748 491 6518 690 2899 Breeding and Agronomy Organization/institute 215 216 G. Engqvist et al. Table 7.3. Storage component analysis of 227 lines of roundseeded peas expressed as a percentage of the dry weight. Starch Soluble sugars NDF ADF Mean Range CV% 45.9 5.3 16.7 8.3 32.7–54.5 3.9–8.2 11.8–26.3 5.9–12.7 7.75 11.51 15.18 15.51 CV, coefficient of variation. understanding the synthesis of starch have been made following the identification and development of mutants within the starch biosynthetic pathway. As well as aiding fundamental studies on starch synthesis, such mutants are also available to breeders for the development of crops where the seed contains starches with improved nutritional characteristics, or new uses. Other pathways that lend themselves to genetic characterization, that could provide breeders with new sources of variation, are those leading to the synthesis of soluble carbohydrates, in particular the raffinose family of oligosaccharides (RFO). Information on the known variation for both the RFO and starch biosynthetic pathways is outlined in the following sections. Starch Two mutations, r and rb, affecting the content and composition of starch have been known for many years and were first identified by their affect on the shape of the dry mature seed, which in both cases appeared wrinkled. Mendel (1865) used a naturally occurring mutation at the r locus in his epoch making studies on inheritance. This mutation is the genetic change that has brought about the development of the vined pea crop, which is harvested when immature and then either quick-frozen, quick-dried or canned. A second, apparently naturally occurring, mutation at the rb locus was first genetically characterized by Kooistra (1962), who also determined that r and rb were independent loci, even though the presence of recessive genes at either locus gave rise to wrinkled seeds. The presence of mutations at either the r or rb locus results in a reduction in the starch content of the dry seed to about 35%, compared with about 50% found in the non-mutant wild type. In addition to affecting starch content, the presence of these mutations also affects starch composition. Starch from r mutant seed contains about 70% amylose, while starch from the rb mutant contains about 20% amylose, compared with the wild-type level of about 30% amylose (Wang et al., 1998). It is now known that the mutation at the r locus is in a gene encoding a starch-branching enzyme (Fig. 7.2; Bhattachatryya et al., 1990; Martin and Smith, 1995) and results in a lack of activity of this enzyme during seed development. The mutation at the rb locus reduces the activity of ADP-glucose Breeding and Agronomy Fig. 7.2. 217 The pathway of starch biosynthesis in pea (Wang et al., 1998). pyrophosphorylase, another step in the starch biosynthetic pathway (Fig. 7.2; Hylton and Smith, 1992). Until 1987, these mutations at the r and rb loci were the only ones known to directly affect the content and composition of starch in pea seeds. In 1987 a chemical mutagenesis programme was carried out to produce new mutants affected in pea seed development and composition, in particular starch content and composition (Wang et al., 1990; Wang and Hedley, 1991). As a result of this programme, mutations at six loci were identified, five of which resulted in the seed having a wrinkled (rugosus) shape, while the sixth (lam) did not appear to significantly affect seed shape. In addition to identifying mutations at specific loci, a range of mutations or alleles was identified at each locus (Table 7.4). Two sets of mutants were genetically characterized and found to correspond to either the r or rb locus. In each case, however, the effect on starch content and composition of the new mutants widened the available variation, such that the starch and amylose 218 G. Engqvist et al. Table 7.4. Pea loci and alleles affecting starch synthesis and composition (Wang et al., 1998). Loci Number of alleles Starcha Amyloseb r rb rug3 rug4 rug5 lam 10 8 5 3 3 5 27–36 30–37 1–12 38–43 29–35 39–49 60–75 23–32 12 31–33 43–52 4–10 aStarch is given as a percentage of the dry weight. is given as a percentage of the starch on a dry weight basis. bAmylose contents ranged from 27 to 36% and 60 to 75%, for the r alleles, and from 30 to 37% and 23 to 32%, for the rb alleles, respectively (Table 7.4). The remaining mutants were assigned to four previously unidentified loci (rug3, rug4, rug5 and lam). The presence of mutations at the rug3 locus resulted in seeds containing only 1–12% starch, depending on the particular mutant allele, and the starch had relatively low amounts of amylose (Table 7.4). Mutations at this locus decrease the activity of plastidial phosphoglucomutase (Fig. 7.2; Harrison et al., 1998). The seeds from plants containing this mutation are viable even though they possess very low or no starch and the resulting plants appear to grow normally (Harrison et al., 1998). Seeds from the rug4 mutants are only mildly wrinkled and the starch and amylose contents are decreased to only 38–43% and 31–33%, respectively (Table 7.4). It is now known that mutations at the rug4 locus result in a dramatic reduction in the activity of sucrose synthase (Craig et al., 1999), an enzyme that is outside of the dedicated starch biosynthetic pathway (Fig. 7.2). Mutations at the rug5 locus result in a reduction in starch content to 29–35% and an increase in the proportion of amylose in the starch to 43–52% (Table 7.4). Mutants containing the mutations at the rug5 locus have a reduced level of one of the major soluble starch synthases (Fig. 7.2; Craig, 1998). The sixth group of alleles isolated from the chemical mutagenesis programme carried out by Wang et al. (1990) differed from the other five in that seed shape and starch content were more similar to the wild type (Table 7.4). The composition of the starch, however, was similar to that of low amylose or ‘waxy’ mutants identified in maize and other species, hence the gene symbol lam, for low amylose. Mutants containing the lam mutation lack activity for a major granule-bound starch synthase (Denyer et al., 1995). The presence of all of these new mutants affecting starch gives breeders an opportunity to develop pea lines with seeds containing a range of starches or, in the case of the rug3 mutants, seeds with little or no starch. Breeding and Agronomy 219 The physical properties of the starches produced by these new mutants are described in Chapter 4. Having identified a range of loci affecting starch, it then becomes possible to combine the genes in different ways to produce double or perhaps treble mutants that could extend further the available variation and potential uses. Raffinose family of oligosaccharides (RFO) Although variation for the content and composition of the RFO and cyclitols between species is quite well established (see review by Horbowicz and Obendorf, 1994) there have been few reports of variation within particular species. A recent screen of lentil (Lens culinaris) germplasm, however, has revealed variation within this species for the total level of RFO and for the levels of individual members of the RFO (Frias et al., 1994b). In this study, 16 lentil lines composed of exotic germplasm and cultivated varieties were analysed and quantified for the RFO using thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC). Total RFO levels ranged from about 1.8 to about 4.3% of the seed dry weight. The most notable variation within the individual RFO members was for verbascose, which ranged from about 1% of the seed dry weight to a level, observed in several of the lines, that could not be detected. This variation was used in a genetic analysis and evidence suggests that the lack of verbascose may be due to the presence of a single recessive gene (Frias et al., 1999). In addition, there is evidence of an inverse relationship between a reduction in the level of verbascose and an increase in the level of a cyclitol, ciceritol, suggesting a link between these two pathways. A screen for RFO variation also, has been carried out on 70 pea lines, covering the genetic variation available within the John Innes pea gene bank. The screening procedure was similar to that used for lentil. The initial screen used TLC and lines that apparently had extreme variation for the RFO were reanalysed using HPLC, to quantify the observed differences (Jones et al., 1999b). Variation for the total level of the RFO in the selected lines ranged from about 3.5 to about 7.0% of the seed dry weight. As with the lentil study, the most extreme variation for individual members of the RFO was for verbascose, which ranged from about 3% to an undetectable level. Those lines that lacked verbascose had an increased level of stachyose, the previous homologue in the RFO pathway. There is similar evidence of within species variation for RFO in Phaseolus vulgaris (Burbano et al., 1999). In this study, 19 varieties of bean were screened for RFO content and composition and several lines were found in which no verbascose could be detected. Once again the predominant α-galactoside was stachyose. Saponins Although not present in large quantities in legume seeds, saponins are considered to be antinutritional. Genetic variation for saponin type has 220 G. Engqvist et al. been found within soybean seeds and a genetic model linking structure (of the sugar chains) and five genes; Sa-1a, Sg-1b, Sg-2, Sg-3 and Sg-4, has been proposed by Tsukamoto et al. (1993). Variation for soyasapogenol B has been reported in a study of 19 P. vulgaris varieties (Burbano et al., 1999). This now opens up the possibility of breeders selecting lines that have low amounts of saponins with antinutritional properties and higher amounts of those saponins with known health benefits, improving the quality of their seed. 7.4 Selection Methods The basis of plant breeding is the selection of individuals with desired characteristics from a gene pool. The gene pool may be natural, as found in gene banks, or created, for example, by mutagenesis or by crossing different individuals with the hope of finding a new combination of characters. In the case of gene banks, there are often relatively large amounts of seed for each of the lines and so the selection can be made for seed quality characters using destructive chemical analysis, or using destructive or nondestructive physical techniques (Fig. 7.3a). The selected line then can be multiplied and released as a variety, or could become part of a breeding Fig. 7.3. (A) Plant breeding selection methods for chemical component. Selection from gene banks. Opposite page: (B) Selection from F2 populations. Breeding and Agronomy Fig. 7.3. Continued 221 222 G. Engqvist et al. programme, where some of its characteristics would be incorporated into a new variety. In the example presented in Fig. 7.3A, the plant would be a typical inbreeding species, such as pea. The scheme would only work for an out breeding species, such as faba bean (Vicia faba), if the chemical character being selected was not segregating in the population. A crossing programme using an inbreeding species, where one parent has a useful seed quality characteristic, will produce a segregating F2 population consisting of a large number of seeds, all of which could be genetically different (Fig. 7.3B). Destructive analysis of the whole seed in this case cannot be considered and there are basically two strategies that can be adopted. In the first strategy, the single seeds are multiplied for several generations by single-seed descent to produce inbred lines. Destructive physical or chemical techniques can then be used to allow selection for the character to take place, followed by multiplication and development of the new variety. This strategy, however, is expensive and will result in the production of large numbers of inbred lines, the majority of which will be discarded. The second strategy is to carry out the selection on the single seeds (Fig. 7.3B). This requires a non-destructive method of analysis, which should also be rapid because of the large numbers of seeds that would need to be screened. The rapid methods do not need to be too precise, however, since the main objective of the screen will be to select seeds that are high (or low) for a particular chemical component. There are basically two types of non-destructive analysis: either a portion of the seed is removed for analysis or the whole seed is used. In the first case, parts of each seed are removed, which can make use of the drilling technique described by Jones et al. (1995; Fig. 7.4). This material can then be analysed either chemically or physically and selections made. If the seed is maintained whole then only physical techniques can be used for analysis (see below) and the selections are made using this information. The seeds, selected following either type of analysis, will then be multiplied for several generations to produce inbred lines. Seeds from these lines can be used to carry out more precise chemical analyses and final selections can then be made, the selected lines being multiplied and developed into a new variety. The approach of screening seeds for chemical characteristics in early generations is very efficient but does require the development of screening techniques based on single seeds. The chemical methodology associated with these techniques has been discussed in Chapter 2. The non-destructive techniques based on physical methods are briefly described below. 7.5 Physical Screening Methods The most common physical selection methods used by breeders are based on near-infrared (NI) transmission or reflectance. Some studies have also Breeding and Agronomy 223 Fig. 7.4. Miniature drill used for sampling from single seeds. The insert shows a hole bored through a pea seed with the resultant flour sample – 30 mg, more than enough for chemical or physical analyses. The seed can be grown normally to produce a plant. 224 G. Engqvist et al. been carried out using mid-infrared (MI), which it has been suggested could yield more chemical information than NI. Both methods are based on the selective absorption and vibration of specific chemical bonds within molecules. The fundamental vibrations result from absorption in the MI while the second and third overtones occur in the NI. 7.5.1 Near-infrared (NI) spectroscopy The principle of NI spectrometry is based on the generation of a polychromatic beam from a tungsten lamp, which is passed through a monochromator to produce a monochromatic beam in the NI region. The beam is then focused on the sample and the reflected diffuse energy may be detected as reflectance (R), or the beam may be passed through the sample and the remaining energy detected as transmittance (T). NIR can be used on milled material, including samples taken from whole seeds, while NIT can be used to analyse whole seed samples, including single seeds. The analyses are based on the property of organic molecules to absorb energy in the < 2.5 µm region of the spectrum, which lies between the infrared and the visible ranges. Most seed components, including the carbohydrates, absorb energy in the infrared region, giving a unique fingerprint. In a crude sample, for example pea meal, the spectrum obtained will be the sum of all of the organic constituents. The relationship between the absorbed energy of a particular component and its concentration in the mixture will be affected by the overlapping of spectral bands from the different constituents, the particle size and by the temperature of the sample. Using NI to determine precise concentrations of a particular seed component, therefore, is difficult and indirect. If it is to be used for this purpose it is necessary to develop a predictive model based on multiple regressions of spectral versus chemical data, using samples differing widely in concentration for the component of interest (Kim and Williams, 1990; Orman and Schumann, 1991; Sinnaeve et al., 1995). It is more easy to use NI to directly identify changes in the relative amounts of a component, particularly if it is reduced to very low levels or removed completely, for example, following genetic changes within a crossing programme. In this way it is possible to rapidly select segregants from a population, which differ from the norm. These selected seeds can then be subjected to a more precise chemical analysis using part of the seed, or whole seeds can be chemically analysed following inbreeding and multiplication as discussed earlier. At least one NIT instrument is available for use by breeders (Infratec Grain Analyser) produced by Foss Tecator, Sweden. This instrument has been developed to cope mainly with batches of seed samples, but a single seed adapter kit is available that will allow it to be used for the analysis of single seeds, including peas. Breeding and Agronomy 225 7.5.2 Mid-infrared spectroscopy The MI region of the spectrum (2.5–25 µm) is less familiar to breeders than NI, although it could in the future have significant advantages. Bands in this region can be specifically assigned to chemical groups, which holds out the possibility of using it for quantitative analysis of specific seed components with less need for the complex statistical analysis used with NI techniques. The main drawbacks of MI are that the samples are opaque and contain water, which is a very strong IR absorber. The performance can be improved using Fourier transformation (FT) methods and a recent paper has applied the combination of FT with MI to the analysis of pea seeds (Letzelter et al., 1995). The technique described in this paper combines FT-MI with photo-acoustic detection (PAS) and can be applied to small amounts of seed material (c. 100 mg), which it is possible to remove from a pea seed while retaining seed viability (Jones et al., 1995). There is also a report of this method being applied to whole maize seeds (Greene et al., 1992), suggesting in the future that the technique could be used to analyse the seeds of grain legumes. 7.6 Some Agronomic Considerations of Carbohydrates 7.6.1 During plant growth and development The capacity of plants to accumulate carbohydrates is a function of their photosynthetic capacity and of the carbon distribution pattern between the plant parts, both of which appear to be genetically determined. Within a crop this capacity also depends on the ability of plants to maintain dry matter accumulation when faced with a variety of abiotic stresses. The productivity of plants within crops, therefore, often falls far short of its full genetic potential because of such environmental stresses. The allocation of recently fixed carbon to export and to storage enables plants to maintain a steady supply of carbohydrates both for development and for the restoration and maintenance of homeostasis during environmental stress. The regulation of carbon allocation between starch synthesis and synthetic processes in the chloroplast and cytosol determines the amount of stored assimilate that is available for export or use in the leaf at times of low or no photosynthesis. Carbon that exits the chloroplast can be used for sucrose synthesis, for respiration or for the synthesis of compounds that remain in the leaf. The regulation of two cytosolic enzymes, fructose bisphosphatase and sucrose phosphate synthase, is particularly important in controlling the flux of carbon to sucrose. Sucrose may be either stored temporarily in the cytosol and vacuole, or moved to the vicinity of the veins and enter the sieve elements to be translocated to sinks. At night and during daytime periods of low photosynthesis, stored sucrose is made available, 226 G. Engqvist et al. or starch is degraded, to support sucrose export. The synthetic, transport and regulatory processes involved in the allocation of assimilates are the means by which leaves maintain a relatively steady supply of carbon for use locally or in translocation sinks. A major environmental stress that is often found within crops is a deficiency of nitrogen. The restriction of leaf canopy development in nitrogen-stressed plants consists of two general effects. One is a decrease in the rate of leaf initiation (Rufty et al., 1984) and the other is a decrease in the development of existing leaves (Rufty et al., 1988). Both effects contribute to a decreased utilization of carbohydrate in the shoot. Carbohydrate metabolism in leaves is altered in nitrogen-limited plants, with starch and sucrose levels being elevated relative to controls, even though growth is restricted. This observation implies firstly that growth is not limited by assimilate availability and secondly that sucrose degradation and possibly glycolysis are reduced. A large portion of the carbohydrate utilized in growth reactions in sink tissues is derived from sucrose imported from leaves. It has been proposed (Huber and Akazawa, 1986; Black et al., 1987) that an important pathway for sucrose utilization in sink tissues is catalysed by phosphofructokinase, which is activated by fructose-2,6-bisphosphate (F26BP). The sharp decline in the concentration of F26BP in leaves following the imposition of nitrogen stress, therefore, may cause the accompanying decrease in carbohydrate metabolism. Such observations clearly suggest that a decrease in sucrose utilization contributes to a decrease in demand for assimilates within the shoot and to the reported diversion of carbohydrate to the root system. Legumes have evolved to live in low nitrogen soils and in these species the diversion of carbohydrates to the root system during nitrogen stress has probably played a major role in the development of the symbiotic relationship with nitrogen-fixing bacteria. 7.6.2 During seed development The availability of water to crops during the time when the seeds are developing is a major factor determining yields. It is known, for example, that water deficits during the time of seed filling in soybean crops decreases seed size. This may result from a reduction in the supply of assimilates from the maternal plant and/or an inhibition of seed metabolism. Experiments have been carried out to determine whether it is maternal or zygotic factors that limit seed growth at times of water deficit (Bernal-Lugo and Leopold, 1992). When water was withheld from greenhouse grown soybean plants during the linear seed-filling period, leaf water potential decreased rapidly, inhibiting canopy photosynthesis completely within 3 days. Seed dry weight continued to increase, however, at or near the control rate. The level of total extractable carbohydrates in leaf, stem and pericarp tissue decreased Breeding and Agronomy 227 by 70, 50 and 45% respectively, indicating that reserves were mobilized to support seed growth. The content of sucrose in the cotyledons decreased from about 60 to 30 mg g−1 dry weight and the sucrose concentration in the interfacial apoplast of the cotyledons decreased from about 100 to 50 mmol. The rate of sucrose accumulation by excised embryos, measured in a short-term in vitro assay, however, increased in response to the water deficit. Results from such experiments indicate that both source and sink activity in soybean are altered by water deficits to maintain the flux of assimilates to the developing embryos. This may explain why seed growth is maintained, albeit for a shorter duration, when soybean crops are exposed to water deficits during the seed filling period. 7.7 European Registration Requirements for New Varieties 7.7.1 Background Most European countries have specific requirements that new varieties must meet the demands of ‘Value for Cultivation and Use’ (VCU) in the registration process. Generally, a variety has a VCU if, in comparison with other registered varieties in an important growing region, it makes a positive contribution to the growing of the crop, to the use of the crop, or to the products derived from it. The characters and minimum conditions included in the official examination of agricultural varieties should cover: • • • • • yield; resistance to harmful organisms; behaviour with respect to factors in the physical environment; quality characters; alternativity – for spring and winter form varieties. The importance of new varieties and their characters vary according to the technical facilities of a given country (e.g. farming management, harvesting machines, susceptibility to spill at harvest), the quality of a new variety (e.g. standing ability) and the corresponding level of plant breeding programmes (e.g. resistance to diseases), climatic and soil conditions (e.g. suitability of leafy types vs. semi-leafless varieties in wet and/or arid regions), etc. Those countries that are members of the International Union for the Protection of New Varieties of Plants (IUPOV), follow IUPOV guideline recommendations on the conduct of tests, the choice of varietal characteristics on which to establish distinctness, uniformity and stability (DUS). For a variety to be distinct, it must be clearly distinguishable by one or more important characters from any other variety, whose existence is a matter of common knowledge at the time when protection by a grant of rights is sought. A variety is required to be sufficiently uniform, depending 228 G. Engqvist et al. on its breeding system, to allow accurate description and assessment of distinctness and to ensure stability. To measure stability with any degree of certainty would take at least 3 years and would require tests to be made on seed from succeeding stages in the multiplication cycle. 7.7.2 Agronomic characters The variability for pea registration requirements throughout Europe can be divided into three groups, according to the evaluating and analysing methods (Table 7.5a–c). Table 7.5a shows the agronomic characters, which are very extensive and include those that are of most importance for farmers. Farmers take these into account first when making decisions about which variety is to be grown. The foremost character in all European countries (except Estonia) is yield and stability of yield, a new variety being Agronomic character Yield Earliness of ripening Shortness of straw Height of crop canopy at harvest Standing ability Ease of combining One-step combining Height of lower pod attachment Pod shattering or spill at harvest Resistance to: Drought Alternaria alternata Ascochyta pisi/Mycosphaerella pinodes Black rust Colletotrichum Downy mildew (Peronospora pisi) Erysiphe pisi Pea enation virus Pea wilt (race 1) Fusarium sp. Winter hardening – cold tolerance 1000 seed weight Bulgaria Czech Republic Denmark Estonia France Germany Hungary Italy Moldova Poland Romania Spain Sweden UK Ukraine Table 7.5a. The registration requirements of several European countries for new varieties of pea – agronomic characters. ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Breeding and Agronomy 229 Processing character Bulgaria Czech Republic Denmark Estonia France Germany Hungary Italy Moldova Poland Romania Spain Sweden UK Ukraine Table 7.5b. The registration requirements of several European countries for new varieties of pea – processing characters. Grain colour stability Fungal diseases Pests Grading 3 mm Grading 4 mm Grading 4.5 mm Grading 5 mm Grading 5.5 mm Grading < 6 mm Grading 6–7 mm Grading > 7 mm Grading 8 mm Grading 9 mm Seed characteristics: Shape, colour, surface bleaching Soakability Cookability Seed colour after cooking Taste Consistency Grain cooking uniformity ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Chemical character Trypsin inhibitor activity (TIU) Amino acid content: lysine, methionine, cystine, alanine Protein content of seed Protein yield from seed Bulgaria Czech Republic Denmark Estonia France Germany Hungary Italy Moldova Poland Romania Spain Sweden UK Ukraine Table 7.5c. The registration requirements of several European countries for new varieties of pea – chemical characters. ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ 230 G. Engqvist et al. compared to control varieties that are supplied by the appropriate testing authorities. Thousand seed weight is a character that is very dependent on growing conditions and should be taken as a guide to the genetic potential of the variety. There is a close link between future yield and characters such as standing ability, height of crop canopy at harvest time in relation to shortness of straw and ease of combining. In other words, a variety could be prone to lodging, but mature pods may be held off the ground by the haulm in such a way that harvesting may be easier than the standing ability score may suggest. This character can be particularly important in a difficult harvest season, especially in leafy types. Nevertheless, this characteristic is observed in only four countries, two of which (Ukraine and Bulgaria) still do not utilize semi-leafless varieties with good standing ability. Instead they tend to grow tall leafy types that are susceptible to lodging and not easy to combine, and they are interested, therefore, in the height of attachment of the lower pods and shattering or spill at harvest. In Romania, for example, it is always necessary to check varieties for their suitablility to one-step combining. The behaviour of new varieties, with respect to factors in the physical environment mentioned above, is tested in Ukraine through resistance to drought. The typical continental weather in Ukraine gives an advantage to taller leafy types of peas that are able to cover the ground and protect the crop from the high evaporation that occurs during the hot late spring and summer months. Another specific character, common in southern countries (e.g. Bulgaria, Italy, Spain and France), where farmers prefer autumn sowing to spring sowing because of too much activity in the spring, is winter-hardiness or cold resistance of winter pea varieties specially bred for this purpose, or normal spring varieties sown in autumn. Diseases can have a major influence on yield. Growers of varieties susceptible to specific diseases should either ensure that appropriate fungicide is applied to the seed coat prior to drilling or try to avoid land that is known to produce disease problems. In Table 7.5a, resistance to harmful organisms is distributed equally, which means that all concerned countries consider these agronomic characters very important. Differences in observations can originate from differences in races, or disease distribution, throughout Europe. The most common and observed fungus disease is wilt (Fusarium oxysporum f. sp. pisi), which reduces yields and can only be controlled effectively by genetic resistance. Race 1 is thought to be the commonest form and it is a very persistent, soil-borne disease. The foremost foliar disease complex Ascochyta pisi and M. pinodes is responsible for leaf and pod spot. The latter is seed-borne and may be soil-borne and develops rapidly in wet conditions. Downy mildew (P. viciae) is favoured by the cool, moist conditions common in coastal countries such as the UK, in northern parts of France, Germany and Poland, and in Denmark and Sweden. Once grey mould Breeding and Agronomy 231 (Botrytis cinerea) is established in a crop, it cannot be controlled effectively. Growers in areas of countries where wet weather during the flowering and pod-setting period is more likely to occur, may be able to choose some of the more determinate, shorter-strawed varieties with a semi-leafless habit. These plant types produce a more open crop with a drier microclimate. Powdery mildew (Erysiphe polygoni, Erysiphe pisi) seldom occurs in the above-mentioned countries because it is usually a disease of late-sown peas and spreads in hot and dry summers, particularly in France, Germany, Poland and the Czech Republic (i.e. in countries with a continental climate). Infected plants are covered in a fine white film and, if the disease appears early in the season, pods may fail to fill and maturation of the crop is delayed. Other specific diseases may become more important, for example Alternaria alternata and black rust in Bulgaria, or Colletotrichum in the Ukraine. Pea enation mosaic virus is seldom noticed before the approach of flowering as it is aphid (Acyrthosiphon pisum) transmitted. Since the weather greatly influences the migration and reproduction of aphids, it influences also the occurrence and severity of this disease. The number of characters represented in this section is evidence of the great emphasis given to them by breeders, seed-merchants, farmers, growers and processors. 7.7.3 Technological characters The seed market and the food industry strongly appreciate grain colour stability (evaluated by Bulgaria, Czech Republic, Sweden and Ukraine), either green or yellow-seeded varieties being preferred (Table 7.5b). The time of harvest is important, therefore, because strong sunshine can lead to the bleaching of seed colour (tested in Hungary). In addition, seed samples have to be free from waste and stain, as well as the effects of fungal diseases (e.g. blemishes, contaminants) and pests (e.g. damaged by pea moth caterpillar and pea seed beetle). These characters are regarded as important for canning and packet sales, and meeting these requirements results in higher payments for such samples. Some marrowfat varieties may need special agronomic measures to ensure high quality produce. Samples which do not meet the technological requirements mentioned above (except colour) may be suitable for the micronizing market. The micronising process produces a high protein feed for use in certain dried animal feedstuff and pet foods. Another criterion for marrowfat peas might be large, even-sized seed, which is tested through grading (Table 7.5b). It is necessary for a variety to be graded in only one large group and not to be divided into more size groups (a high number of size groups is analysed in Ukraine). 232 G. Engqvist et al. Peas for human consumption also should be tested for soakability, cookability, grain cooking uniformity, seed colour after cooking, taste (smell) and consistency. It is generally known that peas harvested at low moisture contents, or are over-dried, may fail soaking tests. 7.7.4 Chemical characters Seed protein content is strongly influenced by location and season and varies only slightly between varieties. Hungary is the only country where the content of four major amino acids is relevant for the registration process (Table 7.5c). Trypsin inhibitors are low molecular weight proteins that can bind to, and inhibit, the hydrolytic activity of pancreatic protease enzymes, leading to reduced protein digestibility and even pancreatic enlargement in rats. The presence of trypsin inhibitors in grain legume seed has been shown to decrease the nutritional value of the seed proteins. The trypsin inhibitor levels found in pea seeds are 5–20 times lower than those found in soybeans. Although the importance of the digestibility problems associated with trypsin inhibitors in peas is highlighted, this character is officially analysed only by the French and Czech testing authorities (Table 7.5c). Manipulating C. 8 Hedley Grain Legume Carbohydrates Strategies for Manipulating Grain Legume Carbohydrates 8 Editor: Cliff Hedley . . . the other is a conclusion, shewing from various causes why the execution has not been equal to what the author promised to himself and to the public. Boswell Life, vol. 1, p. 2 (1755) Samuel Johnson (1709–1784), English poet, critic and lexicographer 8.1 The Problems The main use of grain legume seeds is for human and animal nutrition and so improving the seeds nutritional components is of paramount importance to the development of legume crops. A major problem with improving the nutritional value of grain legume seeds, however, is the possible negative impact of such changes, especially on the plant or seed. This is a particular problem when dealing with so called antinutritional components within the seed. Many inhibitors that affect the digestion of nutrients by animals or humans are useful within the plant, where they may serve as protective agents against attack by insects or disease-forming microorganisms. They may also have a positive effect on the consuming organism. For example, some inhibitors are rich in the sulphur-containing amino acids that are often present in low amounts in legume seeds (Domoney, 1999). With regard to the carbohydrates, the raffinose family of oligosaccharides (RFO) poses such a problem. Although not antinutritional in the true sense of the word, they restrict the use of legume seeds in animal feed and, because they are associated with flatulence, they are often avoided in ©CAB International 2001. Carbohydrates in Grain and Legume Seeds (ed. C.L. Hedley) 233 234 C. Hedley the human diet (see Chapter 3). It is likely, however, that removal of these compounds by plant breeders would have an adverse affect on the growth and development of the plant and seed. Since the RFO are believed to be involved in protection against abiotic stress and may have an important role in germination (see Chapter 5). As with the protein inhibitors, the RFO also may have a positive affect in the diet by promoting beneficial changes in the gut flora, in particular increasing the proportion of Bifidobacteria, which have been implicated in protection against colon cancer (see Chapter 3). With the possible exception of the storage proteins, nutritionists have been more interested in the effect of the antinutritional, or non-nutritional components in legume seeds, rather than the nutritional components. The main nutritional carbohydrate in most legume seeds is starch. It is known that existing legume starches are digested more slowly than those from other species, in particular from cereals, and that this can have a positive effect in reducing the glycaemic index in humans, which is an advantage to those people with type 2 diabetes (see Chapter 3). The lack of information, in general, on starch digestion and, more specifically, on why legume starches are digested more slowly, however, creates the problem of not knowing the nutritional consequences of changing the starch content or composition in legume seeds. Also, there is very little information on the effect of manipulating starch on the growth and development of the plant or seed. Studies using pea seed mutants affected in starch content, composition (see Chapter 7) and granular structure (see Chapter 4) have given some information on the possible consequences of manipulating legume starches. For example, it is known from using these lines that blocking starch synthesis completely in peas, by introducing a mutation at the rug3 locus, appears to have a minimal affect on the growth of the plant and on the viability of the seed (Wang et al., 1998). There is also no evidence that changing the chemical composition and the granular structure of pea starches has any adverse affects on the seed biology. One interesting observation, following the introduction of mutations directly affecting starch, however, is that they have pleiotropic effects on other seed components, in particular, proteins (Casey et al., 1998a,b), lipids (Jones et al., 1995) and soluble carbohydrates (Jones, 2000). For example, introducing recessive alleles at the r and rb loci in pea gives a small percentage increase in the protein content, a large difference in the protein composition and a large percentage increase in the lipid content of the seed (Wang and Hedley, 1993). The third carbohydrate category, after the soluble carbohydrates and starch is the ‘fibre’ fraction, which contains a multitude of soluble and insoluble compounds, generally characterized by nutritionists as nondigestible elements in the diet. Many of the fibre components are looked on as beneficial within the human diet, mainly from a health point of view (see Chapter 3). Within the plant, most of these compounds are associated Manipulating Grain Legume Carbohydrates 235 with the cell wall and so reducing or manipulating particular fibre components could have a detrimental effect on the structure of plant cells and the development of the seed (see Chapter 6). Likewise, any genetic manipulation of plant or seed development that results in an effect on the content or composition of the cell walls could affect the nutritional usefulness of grain legumes in the diet. One component of the ‘fibre’ fraction, as recognized by nutritionists, that could be manipulated, however, is ‘resistant starch’, much of which results from the processing of native starches in food, prior to being consumed in the diet (see Chapter 4). There is little information on why some starches produce more ‘resistant starch’ when processed than others, although this has often been associated with the amylose content. There is some evidence for this from work using pea mutants with starches that are known to have different levels of amylose (Skrabanja et al., 1999). As mentioned earlier, there is no evidence that manipulating starch is detrimental to the seed (at least in pea) and so producing high-amylose starches would not be a problem from this point of view. There would, however, need to be a balance between the proportion of starch that can be digested, the rate of starch digestion and the proportion of the starch that becomes resistant to digestion. 8.2 Strategies for Overcoming the Problems Before discussing possible ways that grain legume seed can be improved with regard to their carbohydrates, one simple fact should be borne in mind. The thing that links all carbohydrates together, and all other organic components within the plant, is that they were all initially derived from simple sugars such as sucrose. It is very likely, therefore, that reducing or increasing one carbohydrate component will have an effect on others, either as a direct consequence of a change on partitioning of sugars, or because of an effect on the cellular environment of changing the concentration of a cellular component. 8.2.1 The soluble carbohydrates As mentioned above and in Chapter 3, the main problems with the soluble carbohydrates lie with the inability to digest the RFO, with the resulting reduction in nutritional value for animal feed and the problem of flatulence in humans. Any changes in the content and composition of the RFO would also need to take into consideration their role in the plant, mentioned above and in Chapter 5. The first requirement of any conventional breeding programme designed to genetically manipulate plant characteristics, such as the 236 C. Hedley content and composition of the RFO, is to identify suitable levels of genetic variation. As discussed in Chapter 7, papers have been published recently describing genetic variation for these compounds in pea, lentil and bean. In particular, lines with very low levels of verbascose have been identified. What is not known at present, however, is the importance of individual RFO components, either to the growth and development of the plant and seed, or as a contributory factor to the nutritional problems in animals or humans. These are two areas of research, therefore, that should be undertaken in the short term and preferably within a co-ordinated programme utilizing similar genetic material. If genetic variation cannot be found within existing germplasm then it will be necessary to create it, either by modifying existing genes using mutagenesis, or by using molecular techniques, either to manipulate gene action by reverse transcription, or by the introduction of novel genes from other species. The use of mutagenesis is a random technique and does not require information on the biochemistry of a particular pathway. On the other hand, the use of techniques based on molecular biology requires information on the biochemistry and may require the isolation of specific proteins controlling steps in a pathway. There is a developing literature on the biochemistry of the RFO and other associated cyclitols and galactosyl cyclitols (see Chapters 2 and 5). There are still many gaps in our knowledge, however, and very little information about specific steps in the pathways or the links between them. It is known that legume species differ for the range of these compounds produced within the seed and for the presence or absence of particular pathways. For example, pea only produces the RFO within its seeds, while lentil has, in addition, the D-pinitol pathway and can produce relatively high amounts of ciceritol. Pea lines that are verbascose minus accumulate more stachyose, the previous homologue in the RFO pathway, while lentil lines that are verbascose minus accumulate ciceritol, demonstrating a link between the two pathways in this species. It is important that studies are carried out to assess the effect of compounds such as ciceritol on human and animal nutrition, as well as their possible protective role in the plant. If ciceritol, for example, has a reduced adverse effect on nutrition while maintaining a positive role within the plant, then a strategy for manipulating these compounds can be developed. This could involve blocking the RFO pathway, or parts of the pathway, in species such as lentil, that possess both pathways. Assimilate then being diverted into the D-pinitol pathway. The D-pinitol pathway could be engineered to function in species such as pea, followed by blocking, or partly blocking, the RFO pathway. Much of the biotechnology required to carry out these procedures is in place, including the transformation systems (see Chapter 6). Manipulating Grain Legume Carbohydrates 237 The alternative strategy of manipulating the activity of α-galactosidase, to degrade the RFO during storage and prior to inclusion in the diet, should also be considered (see Chapter 6). 8.2.2 Starch Starch biosynthesis is relatively simple, compared with the complexity of the soluble carbohydrate pathways. This simplicity is reflected in the wealth of information in pea on starch biochemistry and the presence of well defined mutants at most of the steps in the pathway. Unlike the soluble carbohydrates, starch behaves as a relatively inert substance within the plant, with little evidence of major problems following its manipulation. The main problems with starch concern its use as a source of nutrition and as an important fraction of the non-digestible material in food. There is little knowledge as to why legume starches are digested more slowly than those from cereals, or why legume starches produce high levels of resistant starch when processed or cooked. To manipulate starches with regard to these characteristics demands knowledge of the relationship between starch chemical and granular structure and the starch nutritional characteristics, on the one hand, and the genetic control of starch chemical and granular structure, on the other. The genetic variation required to study these relationships exists in pea. This should be used to determine the effects of specific mutations on starch chemical and granular structure and the link between these characteristics of starch and starch digestibility in the native and processed condition. Although pea can probably be used as a model for other starch storing grain legumes, it will become important to extend the studies on legume starches to other species. This will entail either identifying genetic variation, similar to that now available for pea, in gene banks, creating the variation using a mutagenesis programme, similar to that carried out in pea, or using information gained from the pea studies to modify specific steps in starch biosynthesis using transformation. One or all of these alternatives should be initiated as soon as possible, particularly in those species that are commonly used for human nutrition (lentil, common bean and chickpea). 8.2.3 Fibre The complexity of the ‘fibre’ fraction makes it much more difficult to define strategies for improvement compared with the soluble carbohydrates and starch. In this case, it is a primary requirement to determine which compounds are the most important from a nutritional/health point 238 C. Hedley of view. It will then be necessary to determine what the consequences to the plant may be if the selected compounds are changed, reduced or increased. This process would require the selection or derivation of variation that would serve the dual purpose of helping to answer these fundamental questions and providing variation for future breeding programmes. The role of specific ‘fibre’ components within the plant cell and the production of sufficient material to utilize for nutritional tests could make use of the cell culture systems described in Chapter 6. 8.3 Conclusions Defining strategies for improving or manipulating grain legume carbohydrates will only result in increased scientific and technological activity if grain legume crops are considered worth developing from an economic point of view. As stated in the introduction to this book, in general the consumption of grain legumes has been declining for many years in the developed parts of the world. A shrinking market gives rise to decreased interest from producers and a reduction in available funding for research and development. This downward spiral can only be stopped and perhaps reversed by creating new opportunities for grain legume crops and by adding value to the raw materials derived from them. It is very important to get away from the ‘poor man’s meat’ identity that grain legumes have had in the past. The health benefits from consuming more grain legumes in the diet are well documented and with current awareness of diet and health issues in western populations this should be a good ‘selling point’. Another current concern of western populations is the effect that intensive farming is having on the environment. An increased acreage of grain legumes and reintroducing these species into farming rotation systems would reduce the chemical inputs required for other crops such as cereals. A more sustainable agricultural system would reduce the level of fertilizers reaching water supplies and have a positive effect on reducing chemical pollution. An alternative, or perhaps additional, strategy for increasing the use and production of grain legumes is to consider the seeds as a source of raw materials for the processing industry, rather than as an entity to be eaten as a vegetable. The three major constituents of the starchy legumes, protein, starch and fibre, all have useful functional properties that can be readily utilized in food products. Procedures have already been developed for isolating these three fractions (see Chapter 4) relatively easily, from pea. Considering the two carbohydrate fractions, pea starch has unique pasting properties, having a stable development and high end-point viscosity, compared with equivalent amounts of cereal and tuber starch. Most legume starches have good gelling properties, although this is usually accompanied by a high level of syneresis, which could be a negative property. As Manipulating Grain Legume Carbohydrates 239 mentioned above and in Chapter 4 the introduction of mutant genes affecting starch synthesis in pea has resulted in a spectrum of starches with properties that could and should be utilized within the food processing industry. Likewise, the insoluble fibre fraction extracted from the seed embryo has excellent water-holding properties that could be utilized within the processed meat industry as an alternative to inorganic salts, such as phosphates. Once the proteins, starch and fibre have been isolated from legume seeds these materials could also find alternative uses in non-food products. For example, some legume starches, in particular those with high amylose contents, have properties that make them an excellent raw material for thermoplastics. 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Plant Journal 7, 97–107. Index Index Index α-amylase 31, 46–49, 139 acid detergent fibre (ADF) 48–49, 57 adzuki bean 19–20, 32 α-D-galactosyl moiety 125 α-galactosidase genes 199–201 thermostable α-galactosidase gene 200 α-galactoside 17–19, 31, 63, 68, 71–73, 80 consumption 69–70, 82–83 animal diets 69–70 food 82–83 content in animal diet 70 content in legume dishes 83 content in seeds 63–64, 67–68 beans 63 chickpea 63–64 faba bean 63–64, 68 lentil 63–64, 68 lupin 68 pea 63, 68 losses during cooking 83 nutritional properties 71–74 antinutritional effect 61 elimination of negative effect 72–73 gas production 78 ileal digestibility 72–73, 80 intestinal transit 73 monogastric nutrition 72–73 nutrients absorption 73 see also unavailable carbohydrates α-galactoside biosynthesis gene(s) 199–201 galactinol synthase (GS) gene 200 stachyose synthase gene 200 α-D-glucose-6-phosphate 126 α-glucosidase 139–140 α-glucosidic bonds 140 aglycon 28, 30 agronomy 209–232 agronomic characters 228–231 chemical characters 232 technological characters 231 ajugose 17 aldoses 16, 53 amorphous 94, 96, 103–104, 106 β-amylase 139 amyloglucosidase 46 amylopectin 23–24, 130 amylose 23, 47–48, 130 arabinose 26–27 Arachis hypogaea 30 315 316 Index available carbohydrates 79, 84–85 classification 79 nutritional properties 84–85 glycaemic index 84 see also mono- and disaccharides, starch bean 8 adzuki 19–20, 32 broad 26, 122 brown 26 butter 30 common 3, 123 dry 20 faba 3, 8, 17, 122, 130 field 30 garden 20 green 29 haricot 29 jack 3 kidney 21, 29–30 lima 29, 123 mung 2, 20, 29–30, 32, 123 navy 29 pinto 29 runner 29 D-bornesitol 19 breeding 209–232 contradiction of goals 210 goals 209–211, 235 pedigree 211 physical selection 222 selection methods 220–222 Brabender viscograph 100, 103 broad bean 26, 123 brown bean 26 butter bean 30 Cajanus cajan 2, 20 calculation and statistical analysis 37, 40, 43 Canavalia ensiformis 3 capillary zone electrophoresis (CZE) 42 advantages 43 disadvantages 43 recommended method 43 carbohydrates 5, 12, 22, 26, 28, 30–31, 34, 37–43, 45, 49, 53–54, 56, 120 accumulation 122 biosynthesis 125–127 chemistry 15 extraction procedure 34 GC determination 35 physiological role 131–132, 134–138 unloading of 118 cell wall 25–28, 47, 49–50, 52, 54 preparation 50 cell wall components 127, 202 biosynthesis 159–160, 202–203 oligosaccharides 203–204 cellulose 25, 50, 52, 55 content in seeds 65 chemical analysis 31 chickpea 2, 8, 19–21, 30, 32, 122 D-chiro-inositol 18–19, 23, 32, 133 Cicer arietinum 2, 8, 20–21, 30, 32, 214 ciceritol 20–22, 32, 126, 236 content in seeds 63–64 chickpea 63–64 lentil 63–64 common bean 3 consumption of grain legumes 7–11 cooking of legume starch 112–113 extrusion 113 high pressure 112 low pressure 112 cowpea 3, 19–20, 122, 123 crude fibre 48 crystallinity 94, 96–97, 100–101 biaxial crystalline polymers 94 cyclitols 31–32, 127, 135–137, 143 occurrence of 32 debranching enzymes 140 delignification 54, 57 desiccation 131 injury 143 stress 124, 132 tolerance 131, 141 developmental stages 120 Index dietary fibre (DF) 25, 46, 48 differential scanning calorimetry DSC 98 digalactosyl myo-inositol 133 disaccharides 16, 45, 71, 124 see also sucrose dishes 62, 83, 86–87 amount of legumes 87 consumer acceptance 87 flatulence 62, 86 intake of α-galactosides 83 preventive effect 62 dry bean 20 embryo composition 119 role 119 endosperm degradation 119 development, role 119 enzymic method 45–46, 49 exoamylase 139 extraction 31 recommended procedure 34 solvents 31, 34 temperature 31 faba bean 3, 8, 17, 20, 29, 122, 130 fagopyritol B series 23 fagopyritol B1 19–20, 23, 32 fagopyritol B2 20, 23 fagopyritol B3 23 fibre 11, 25, 46, 48, 58, 65–67, 83, 85–86, 234, 237 composition 69 consumption in food 83 content in seeds 65–67 fibre fractions 65 genetic variation 216 physiological effect 85 see also non-starch polysaccharides, unavailable carbohydrates field bean 30 field pea 34 food application of legume starches 109–110 free radicals 136 fructose 16–17, 26–27, 31, 44 content in seeds 63–64 faba bean 63–64 lentil 63–64 see also monosaccharides 71 fucose 26 functional properties 98–101 gelatinization 98 melting 98 pasting 98 fungal disease(s) 228–230 galactinol 19–21, 32, 125–126 synthase 125 galactinol series 21 galactociceritol 20 galacto-cyclitols 20, 31 di-galacto-inositol 20 galactomannans 26, 135 galacto-ononitol 20, 32 galactopinitol A 19–22, 32 galactopinitol A series 22 galactopinitol B 22 galactopinitol B series 22 galactopinitols 19 galactose 19, 26–27, 31 galactoside moieties 140 galactosyl cyclitols 127 galactosyl ononitol 126 galactosyl ononitol series 21 galactosyl pinitol 126 D-galacturonic acid 26–27, 56 galacturonosyl residues 28 garden bean 20 gas chromatography GC 35, 57–58 advantages 37, 43 disadvantages 37, 43 recommended procedure 35 gelatinization 23–24 germplasm banks 213 Pisum 213 glucomannans 25, 54 glucose 16–17, 24–27, 31, 44–45 Glycine max 2, 20, 26, 30, 32, 214 glycosidic bond/linkage 16, 23–24, 26–27, 57 317 318 Index granular structure of legume starches 93–97 amylose 93–94, 97, 102–103, 106–108, 110–111, 116 amylopectin 94, 97, 103, 107, 111 double helix 94–95, 97, 103 A-type polymorph 95–97 B-type polymorph 95–97 C-type polymorph 95–97 green bean 29 haricot bean 29, 30 hemicellulose 16, 25, 28, 50, 52, 54–55 content in seeds 65 extraction 54 hexoses 16, 50 high performance anion chromatography (HPAC) 39 Dionex system 39 high performance liquid chromatography (HPLC) 38, 57–58 advantages 41, 43 affinity chromatography 40 disadvantages 41, 43 ion moderate partition 39 normal phase 39, 56 recommended method 40 reverse phase 39 inositol 18 invertase 16 in vitro cultures 146–148 cell suspension culture 201–208 isolation of native starch 89 dry processing 89–91 wet processing 89–90 jack bean 3 ketoses 16, 53 kidney bean 21, 29–30 lactose 16, 31, 43 LEA proteins 132 legume seeds 17–20, 28–29 Leguminosae 19, 36 Lens culinaris 3, 8, 30, 32, 214 lentil 3, 8, 17, 19–21, 29–30, 32, 122 leucaenitol 19 lignin 25, 28, 50, 54–55 content in seeds 65 determination 57 lima bean 29, 121, 123 lucerne 20, 21, 32, 135 lupin 2, 8, 17, 19–21, 26 Lupinus albus 2, 20, 26 Lupinus angustifolius 2 Lupinus luteus 2, 20, 45 Lupinus mutabilis 2 Lupinus spp. 8 Maillard’s reactions 136 maltase 16 maltose 16, 31 mannans 25 mannose 26–27, 54 Medicago sativa 20, 32 melezitose 31 methyl cyclitols 32 mid-infrared spectroscopy 225 mimositol 32 modified starch 101–109 biotechnological 108–109 fermentation 109, 114–115 germination 108, 114 hydrolysis 108 chemical 104–107 acetylation 104 cationization 107 cross-linking 106 hydroxipropylation 106 phosphorylation 105 physical 102–104 annealing 103 extrusion 103 gamma irradiation 103 steaming 102 monosaccharides 16, 31, 34, 57, 71 free monosaccharides 124 fructose 63–64, 120, 124, 136, 143 galactose 124, 136 glucose 124, 136, 139–140, 143 Index muco-inositol 18 mung bean 2, 20, 29–30, 32, 123 myo-inositol 18, 20–21, 31–32, 36, 125, 128 D-myo-inositol-1-phosphate 126 Nastar R 116 Nastar R Instant 116 native starch 102, 104, 106, 110, 116 navy bean 29 near-infrared (NI) spectroscopy 224 neutral detergent fibre NDF 48 non-food use 98 non-starch polysaccharides 69, 76–78 composition 69, 77 content in seeds 69–70, 76 nutritional properties 76–78 digestibility of energy 78 eliminate of negative effect 76 energetic effect 76 gas production 78 ileal digestibility 76–77 intestinal transit 77 nutrient absorption 77–78 see also fibre, unavailable carbohydrates oligomers 126 oligosaccharides 16, 25, 45, 71, 124 accumulation 121 degradation 140 α-galactosides 63, 68, 71–73, 80 rathinose 120, 123, 125, 132, 135, 137, 140, 143 RFO 125–126, 132–134, 136, 140 stachyose 120, 123, 125, 132, 135, 137, 140, 143 verbascase 120, 123, 132, 137, 140 ononitol 126 D-ononitol 19–21, 32 optical rotation 44–45 pea 3, 8, 20, 26, 29–30, 122, 123 breeding programmes 210, 211 hulls 29 319 mutant 17 production 209 peanut 30, 123 pectic acid 27 pectic substances 26–27, 55 occurrence of 29 pectin 16, 25–26, 28, 50, 52, 55 pectinic acid 27 pentoses 16, 50 Phaseolus aureus 30 Phaseolus coccineus 30 Phaseolus lunatus 30 Phaseolus vulgaris 3, 6, 20, 26, 30, 214 phenyl α-D-glucoside 31 photoassimilates, supply of 119 pigeon pea 2, 19–20, 122 D-pinitol 19–20, 22, 31–32, 126 pinto bean 29 Pisum sativum 3, 8, 20, 22, 26, 30, 214 plant genetic transformation, methods 181–195 Agrobacterium-mediated transformation 183 electroporation 184 microinjection 184 particle bombardment 183–184 plant regeneration 148–156 organogenesis 151–153 pea regeneration 149 somatic embryogenesis 150–151 polymerization, degree of 103, 108 polysaccharides 15–16, 22, 27, 45 cellulosic 25 non-cellulosic 25 protopectin 27 protoplasts culture 156–158 raffinose 16–19, 31–32, 36, 40–41, 63–65, 68, 71 antinutritional effect 71 content in seeds 63–64, 68 beans 63 chickpea 63–64 faba bean 63, 65, 68 lentil 63–64, 68 lupin 68 pea 63, 68 320 Index raffinose continued synthase 125 see also α-galactoside 63, 68, 71–73, 80 raffinose family of oligosaccharides (RFO) 11, 16–18, 31, 33, 40–42, 125, 233 rapid visco-analyser (RVA) 100 reducing sugar method 44 resistant starch (RS) 46, 80–83, 110–111, 235 Berry method 47 consumption in food 83 content in legume products 81–82 definition 80 determination 46 method of determination 81 nutritional properties 80 retrograded starch (RS III) 110–111 see also fibre, unavailable carbohydrates rhamnopyranose 28 rhamnose 26 round pea 36 rug3 17, 234 runner bean 29 saccharides 16, 42 sample clean-up 38 sample preparation 31 sapogenin 28 sapogenol 58 saponins 28, 58 determination 58 extraction from seeds 58 genetic variation 219 scyllo-inositol 18, 32 seed coat 34, 131 colour 210 components 117 desiccation tolerance 131–132, 135–136, 143 development 119–122, 225–227 full maturity 121, 130 germination 142 orthodox storage 137 quality 211 recalcitrant 138 stage of development 120 size 210 storage 137 structure 117 viability 137 vigour 137 yield 210 zygotic 131 seeds desiccation tolerance 131 mature 140 sequoyitol 19, 32, 128 soaking 112 soluble carbohydrates 11, 15–16, 31, 235 accumulation 122 biosynthesis 125–127 extraction from seeds 31 physiological role 131–132, 134–138 soluble sugars 63–64, 67–68 content in seeds 63–64, 67–68 beans 63 chickpea 63–64 faba bean 63–64, 68 lentil 63–64, 68 lupin 68 pea 63, 68 genetic variation 216, 219 see also oligosaccharides, α-galactoside somaclonal variation 162–181 biochemical changes 173 cytological instability 165, 170, 172–173 product quality changes 181 stress resistance 170–172, 180 yield characters 177–179 somatic embryos 135 soybean 2, 19–20, 26, 29–30, 32, 120, 122, 123 spectrophometric method 45 stachyose 16–19, 31, 33, 36, 40–41, 63–65, 68 content in seeds 63–64, 68 beans 63 chickpea 63–64 Index faba bean 63, 65, 68 lentil 63–64, 68 lupin 68 pea 63, 68 synthase 125 see also oligosaccharides, α-galactoside standards 31 starch 11, 16, 22, 63–65, 74–76, 80–83, 234, 237 accumulation 120, 128, 130 animal nutrition 74–76 biochemistry 130 calcium chloride method 46 consumption in food 82–83 content in seeds 63–65, 68, 121 beans 63 chickpea 63–64 faba bean 63–65, 68 lentil 63–64, 68 lupin 68 pea 63–64, 68 degradation 138–139 determination 45 feed processing 75–76 genetic variation 216–219 glucose hydrolysis method 46 granules 23, 90, 92–96, 98, 101–104, 106, 108, 111, 115, 130 size of 90 nutritional classification 80 rapidly digestible 80 resistant starch 80–81, 235 slowly digestible 80 nutritional properties 74–76, 80–81 energetic value 76 ileal digestibility 74–76, 81 rapidly digestible (RDS) 47 retrograded 47 role 128 slowly digestible (SDS) 47 see also available carbohydrates starch biosynthesis genes 195–199 ADP-glucose pyrophosphorylase gene(s) 195 genes influencing starch 198–199 invertase gene 198–199 321 pyrophosphatase gene 199 starch phosphorylation gene 198 sucrose synthase gene 198–199 starch synthase gene(s) 196–197 starch branching enzyme gene(s) 197–198 starch ethers 104 stress proteins 132 substitution 104, 106–107 sucrose 11, 16–19, 31, 33, 36, 41, 63–64, 67–68, 118 content in seeds 63–64, 67–68 beans 63 chickpea 63 faba bean 63, 68 lentil 63, 68 lupin 68 pea 63, 68 see also disaccharide sugar 16, 44, 56–57 sweet lupin 2 Swelite R 116 swelling properties of 92, 100–103, 105–106, 109–111 temperature stress 136 test kits glucose 44 medical/food 44 Megazyme 47 testa 34 role 117–118 thin layer chromatography (TLC) 42, 58 advantages 42 recommended method 42 transgenic grain legume plants, field trials 192–195 transgenic traits 184–195 herbicide tolerance 184–195 insect resistance 189–190 nutritional quality 191–192 virus resistance 190–191 tri-galactopinitol 20, 22 tri-galactopinitol A 22, 135 tri-galactopinitol B 22 322 Index trimethylsilylation TMS 35–36, 59 triterpene glycosides 30 unavailable carbohydrates 79–80, 85–86 classification 79–80 nutritional properties 85–86 cholesterol metabolism 85 glycaemic index 84 see also α-galactoside, fibre uronic acid 25–26, 50 verbascose 16–18, 31, 36, 40, 63–64, 68 content in seeds 63–64, 68 beans 63 chickpea 63–64 faba bean 63, 68 lentil 63–64, 68 lupin 68 pea 63, 68 see also α-galactoside Vicia faba 3, 8, 20, 26, 30, 45, 119, 120, 214 Vigna angularis 20, 32 Vigna radiata 2, 20, 32 Vigna unguiculata 3, 20 viscosity 97–98, 100–104, 106, 109–110, 116 water-insoluble cell wall components 78 content in seeds 78 nutritional properties 78 digestibility of energy 78 gas production 78 see also fibre, unavailable carbohydrates white lupin 2 xylans 25–26 xyloglucans 25–26 xylose 25–27 yellow lupin 2, 122, 132–134