Carbohydrates in Grain Legume Seeds Improving Nutritional Quality and

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CARBOHYDRATES IN GRAIN LEGUME SEEDS

Improving Nutritional Quality and Agronomic Characteristics
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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
3
CABI Publishing is a division of CAB International
CABI Publishing CABI Publishing

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© 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.
4
Contents
Contents
Contents
Contributors xi
Preface xv
1 Introduction 1
Editor: Cliff Hedley
1.1 The Grain Legumes 1
1.2 Grain Legume Production 1
1.3 Grain Legume Consumption 7
1.4 Grain Legume Carbohydrates 11
2 Carbohydrate Chemistry 15
Editor: Pavel Kadlec
2.1 The Carbohydrates 15
2.1.1 Soluble carbohydrates 16
2.1.2 Polysaccharides 22
2.1.3 Other carbohydrate components 28
2.2 Chemical Analysis of the Carbohydrates 31
2.2.1 Soluble carbohydrates (monosaccharides, sucrose,
α-galactosides, cyclitols) 31
2.2.2 Polysaccharides 45
2.2.3 Other carbohydrate components 56
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vi Contents
3 Nutrition 61
Editor: Halina Kozlowska
3.1 Introduction 61
3.2 The Content of Carbohydrates in Grain Legumes Utilized in
Europe 62
3.2.1 The content of carbohydrates in grain legumes used for
human nutrition 62
3.2.2 The content of carbohydrates in grain legumes used for
animal nutrition 67
3.3 Physiological Effect of Grain Legume Carbohydrates in Animal
Nutrition 69
3.3.1 Consumption of grain legume carbohydrates in feed 69
3.3.2 Effect of mono- and disaccharides in animal nutrition 71
3.3.3 Effect of oligosaccharides in animal nutrition 71
3.3.4 Effect of starch in animal nutrition 74
3.3.5 Effect of non-starch polysaccharides (NSP) in animal
nutrition 76
3.3.6 Effect of grain legume carbohydrates in ruminant nutrition 78
3.4 Physiological Effect of Grain Legume Carbohydrates in Human
Nutrition 79
3.4.1 Nutritional classification of grain legume carbohydrates 79
3.4.2 Consumption of grain legume carbohydrates in food 82
3.4.3 Physiological effect of available carbohydrates from grain
legumes 84
3.4.4 Physiological effect of unavailable carbohydrates from grain
legumes 85
4 Processing 89
Editor: Bálint Czukor
4.1 Native Starch 89
4.1.1 Isolation 89
4.1.2 Granular structure 93
4.1.3 Functional properties 98
4.2 Modified Starch 101
4.2.1 Physical methods 102
4.2.2 Chemical methods 104
4.2.3 Biotechnological methods 108
4.3 Food Application of Native and Modified Legume Starches 109
4.4 Effect of Processing on Starch and Other Carbohydrates in
Foods 110
4.4.1 Resistant starch formation 110
4.4.2 Content, composition and digestibility 112
4.5 Legume Seeds as a Source of Raw Materials 116
6
Contents vii
5 Seed Physiology and Biochemistry 117

Editor: Ryszard J. Górecki
5.1 The Legume Seed 117
5.1.1 Seed components 117
5.1.2 Seed development 119
5.2 The Accumulation and Biosynthesis of Carbohydrates 122
5.2.1 Accumulation of soluble carbohydrates 122
5.2.2 Biosynthesis of soluble carbohydrates 125
5.2.3 Accumulation of starch 128
5.2.4 Biochemistry of starch 130
5.3 Physiological Role of Carbohydrates in Legume Seeds 131
5.3.1 During seed development 131
5.3.2 During temperature stress 136
5.3.3 During seed storage 137
5.3.4 During germination 138
6 Biotechnology 145
Editor: Nickolay Kuchuk
6.1 Introduction 145
6.2 In vitro Cultures and Plant Regeneration of Grain Legumes 146
6.2.1 Introduction to in vitro culture 146
6.2.2 Plant regeneration systems 148
6.2.3 Pioneering studies on pea regeneration 149
6.2.4 Regeneration via somatic embryogenesis 150
6.2.5 Regeneration via organogenesis and multiple shoot
formation 151
6.2.6 Recent studies to produce more efficient, fast and reliable
systems for regeneration 153
6.2.7 Factors effecting regeneration 154
6.2.8 Advantages of the different developmental pathways for
in vitro manipulation 155
6.3 Isolated Protoplasts from Grain Legumes 156
6.3.1 Introduction to protoplast cultures 156
6.3.2 Protoplast cultures from leguminous species 157
6.3.3 Application of grain legumes protoplasts to the study of
carbohydrates 158
6.4 Somaclonal Variation in Grain Legumes 162
6.4.1 Introduction 162
6.4.2 Factors causing variation 163
6.4.3 Mechanisms of somaclonal variation 163
6.4.4 Potential and disadvantages of somaclonal variation 164
6.4.5 Variation in grain legumes at the cell and tissue culture
level in vitro 165
6.4.6 Variation in grain legumes at the whole plant level 172
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viii Contents
6.5 Transformation Methods in Grain Legumes 181

6.5.2 Gene delivery systems used in agronomically important
legumes 182
6.5.3 Methods giving positive results – transgenic plants 183
6.5.4 Transgenic plants and useful genes/traits transformed
into grain legumes 184
6.5.5 Field trials with transgenic grain legume plants and
commercialized transgenic legume crops 192
6.5.6 Future prospects 193
6.6 The Availability and Possible Manipulation of Genes Involved
in Starch Biosynthesis 195
6.6.1 Biochemical pathways of starch biosynthesis 195
6.6.2 The availability of genes involved into starch biosynthesis 196
6.6.3 The availability of other genes influencing starch
biosynthesis and starch quality 198
6.7 The Availability and Possible Manipulation of Genes Involved
in α-Galactoside Accumulation and Degradation 199
6.7.1 Biochemical pathways of α-galactoside biosynthesis 199
6.7.2 The availability of genes involved in α-galactoside
accumulation and degradation and their possible
manipulation 199
6.8 Cell Suspension Culture as a Model for Studying Carbohydrate
Metabolism 201
6.8.2 Composition of plant cell walls 202
6.8.3 Biosynthesis of the cell wall components 202
6.8.4 Oligosaccharides as signals and substrates in the plant
cell wall 203
6.8.5 Plant cell suspension cultures – a powerful tool in
investigating cell wall metabolism 204
7 Breeding and Agronomy 209

Editor: Goran Engqvist
7.1 Current Breeding Goals 209
7.2 Breeding Techniques 211
7.2.1 Pedigree breeding 211
7.2.2 Bulk selection 212
7.2.3 Deviations from the pedigree and bulk methods 212
7.3 Access to Genetic Variation 213
7.3.1 Germplasm banks 213
7.3.2 Existing variation for the carbohydrates 214
7.3.3 Newly identified genetic variation 214
7.4 Selection Methods 220
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Contents ix
7.5 Physical Screening Methods 222

7.5.1 Near-infrared (NI) spectroscopy 224
7.5.2 Mid-infrared spectroscopy 225
7.6 Some Agronomic Considerations of Carbohydrates 225
7.6.1 During plant growth and development 225
7.6.2 During seed development 226
7.7 European Registration Requirements for New Varieties 227
7.7.1 Background 227
7.7.2 Agronomic characters 228
7.7.3 Technological characters 231
7.7.4 Chemical characters 232
8 Strategies for Manipulating Grain Legume Carbohydrates 233

8.1 The Problems 233
8.2 Strategies for Overcoming the Problems 235
8.2.1 The soluble carbohydrates 235
8.2.2 Starch 237
8.2.3 Fibre 237
8.3 Conclusions 238
References 241
Index 315
9
10
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
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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
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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
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
13
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
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
14
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
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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
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30 October 2000 14:56:14 A3867: AMA: Hedley: First Proof:30-Oct-00 Preface
1
C. Hedley
Introduction
Introduction 1
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 cate-
gorized 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
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14 November 2000 14:29:35

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.) Economically and agriculturally the most important legume in the world, providing protein
Merrill 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.
White (flowered) Lupinus albus L. Originating in the Mediterranean area and found widely in North America over 200 species
lupin are known. The name lupin (sometimes spelled as lupine) comes from the Latin for ‘wolf’
18
Yellow lupin Lupinus luteus L. and derives from the mistaken belief that the plant ‘wolfed’ minerals from the soil. The
C. Hedley
Lupinus mutabilis L. contrary is true, lupins aid soil fertility and are drought tolerant. Agriculturally they are grown
Sweet lupin Lupinus angustifolius L. for animal feed.
Chickpea Cicer arietinum L. Also called Bengal gram, boot, chana chola, chole, gram, hommes, pois chiche and
A3867: AMA: Hedley: First Proof:14-Nov-00

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
1
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.
Jack bean Canavalia ensiformis Also called sword bean. Grown in tropics, North and East Africa, Far East and India. Used as
(L.) D.C. 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.
30 October 2000 14:56:16

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 Vicia faba L. Also called broad, horse or tick bean. Harvested fresh for human consumption or used for
(field bean) canning or freezing. The seed is harvested dry for animal feed.
Lentil Lens culinaris One of the most ancient of cultivated foods. Of unknown origin, the lentil is widely grown
Medic. L. 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 Also called Poona pea, yard-long bean, catjang. An important food source in Africa (Nigeria),
(L.) Walp parts of Asia and Mediterranean area. Typically a dhal-type paste is made from the soaked
19
dehulled seeds.
Introduction
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
A3867: AMA: Hedley: First Proof:30-Oct-00

for human consumption or as a combining crop for animal feed. Coloured flowered varieties
are grown for animal feed.
1
3
4
30 October 2000 15:58:48

Table 1.2. Total cultivation area (ha) and production (t) of grain legumes in the World and Europe (FAO, 2000).
1997 1998 1999
Area Production Area Production Area Production
World total 68,324,137 55,512,759 66,913,556 56,143,600 68,320,568 57,514,450

Europe total 4,780,849 10,260,753 4,550,670 9,258,881 4,181,107 8,225,785
20
Albania 68,328,459 68,322,546 68,327,710 68,324,683 68,332,300 68,330,000
C. Hedley
Austria 68,327,783 68,386,282 68,327,043 68,385,254 68,327,333 68,385,600

Belarus 68,262,200 68,492,000 68,269,000 68,334,000 68,212,000 68,253,000
Belgium–Luxembourg 68,134,199 68,318,958 68,324,040 68,316,300 68,324,050 68,315,900
Bosnia and Herzegovina 68,312,620 68,314,740 68,312,620 68,314,740 68,312,620 68,314,740

Bulgaria 68,354,537 68,340,014 68,351,309 68,337,263 68,355,330 68,339,164
Croatia 68,311,471 68,324,039 68,327,417 68,324,684 68,328,150 68,326,050
1
Czech Republic 68,351,428 68,104,697 68,359,395 68,133,391 68,352,697 68,106,602

Denmark 68,397,800 68,387,000 68,106,800 68,387,857 68,106,800 68,357,857
Estonia 68,328,700 68,317,000 68,326,200 68,312,390 68,235,700 68,310,608
Finland 68,326,000 68,313,100 68,324,900 68,311,000 68,324,900 68,311,000
France 68,629,269 3,121,076 68,630,307 3,305,621 68,497,700 2,657,000
Germany 68,185,000 68,587,900 68,235,315 68,779,853 68,227,061 68,792,674
Greece 68,326,374 68,342,252 68,326,016 68,341,419 68,325,870 68,341,250
Hungary 68,361,936 68,118,436 68,363,294 68,140,907 68,362,365 68,140,907
Ireland 68,324,200 68,319,000 68,324,200 68,319,000 68,234,200 68,319,000
Italy 68,374,674 68,115,902 68,378,347 68,127,408 68,380,124 68,137,938
Latvia 68,323,200 68,321,650 68,324,946 68,321,270 68,324,946 68,321,850
30 October 2000 14:56:17

Lithuania 68,352,300 68,106,000 68,366,100 68,104,100 68,352,300 68,106,400

Macedonia 68,312,123 68,330,910 68,314,698 68,330,487 68,314,698 68,330,487
Malta 68,321,450 68,321,200 68,322,450 68,321,200 68,22,3450 68,321,200
Moldavia 68,346,563 68,363,880 68,353,483 68,371,027 68,353,100 68,372,511
Netherlands 68,324,200 68,316,800 68,324,200 68,316,800 68,324,200 68,316,800
Poland 68,144,929 68,260,052 68,148,928 68,289,017 68,148,928 68,289,017
Portugal 68,354,876 68,330,699 68,352,610 68,331,810 68,352,610 68,331,810
Romania 68,348,447 68,375,334 68,359,064 68,372,282 68,338,074 68,346,572
Russian Federation 1,239,840 1,802,220 1,068,850 68,888,550 1,037,700 68,877,000
Slovakia 68,339,038 68,397,807 68,341,443 68,103,855 68,343,676 68,397,735
Slovenia 68,324,267 68,236,614 68,324,280 68,327,513 68,234,280 68,327,513
Spain 68,613,100 68,390,000 68,532,400 68,382,800 68,504,100 68,291,400
21
Sweden 68,353,400 68,168,800 68,345,900 68,142,300 68,343,900 68,139,600
Switzerland 68,234,679 68,314,500 68,234,266 68,312,100 68,324,400 68,311,100
Introduction
United Kingdom 68,196,800 68,748,000 68,201,200 68,701,000 68,188,500 68,373,200

Ukraine 68,635,300 1,072,800 68,555,900 68,770,000 68,503,000 68,585,500
Yugoslavia 68,381,045 68,142,000 68,378,045 68,137,000 68,363,045 68,118,000
A3867: AMA: Hedley: First Proof:30-Oct-00 1

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
22
Introduction 7
Table 1.3. The proportion of cultivated land used for legume production (%).
Grain legumes Soybean Total legume production

Country (excluding soybean) production (including soybean)
Europe
Austria 2.1 3.0 5.1
Belgium 0.8 – 0.8
Bulgaria 1.4 0.4 1.8
Czech Republic 2.2 0.1 2.3
Denmark 4.6 – 4.6
Finland 0.4 – 0.4
France 3.6 0.4 4.0
Germany 0.7 – 0.7
Greece 0.8 0.1 0.9
Hungary 2.1 0.5 2.6
Ireland < 0.1< – < 0.1<
Italy 1.1 2.5 3.6
Netherlands 1.2 – 1.2
Poland 1.7 – 1.7
Portugal 6.6 – 6.6
Romania 0.8 1.3 2.1
Spain 1.4 0.1 1.5
Sweden 1.1 – 1.1
UK 3.5 – 3.5
USA 0.4 15.5 15.9
World total 0.5 0.4 0.9
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,
23
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 3.6 3.5 3.5 2.4 3.4 3.7

Belgium 4.5 3.8 4.1 4.4 4.4 4.6
Bulgaria 0.9 1.0 1.2 0.8 0.9 1.0
Czech Republic – – – 2.4 2.3 2.4
Denmark 4.8 4.2 2.6 3.8 3.7 3.3
Finland 2.9 2.5 1.8 2.4 2.2 2.2
France 5.1 4.7 4.7 5.0 5.0 4.7
Germany 2.6 3.1 2.5 2.7 2.7 –
Greece 1.4 1.5 1.6 1.5 1.6 1.6
Hungary 2.2 2.2 2.1 1.5 2.3 2.2
Ireland 4.4 4.7 4.7 4.7 4.7 –
Italy 1.3 1.7 1.7 1.6 1.6 1.6
Netherlands 4.4 3.6 4.2 4.2 4.0 4.0
Poland 1.9 2.1 1.1 1.9 1.4 1.8
Portugal 0.3 0.3 0.3 0.3 0.3 0.3
Romania 0.9 1.0 1.1 1.2 1.1 1.1
Spain 0.8 0.7 0.6 0.7 0.7 0.6
Sweden 2.5 2.5 2.5 2.5 2.4 –
UK 3.6 3.3 3.2 3.9 3.2 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).
Production Import Food use Feed use

Species (× 1000 t) (× 1000 t) (%) (%)
Pea (P. sativum) 4800 800 4 91

Faba bean (V. faba) 1020 350 17 80
Lupin (Lupinus spp.)a 19 300 2 97
Chickpea (C. arietinum) 40 105 95 –
Lentil (L. culinaris) 35 210 95 –
Beansb and other grain legumes 155 390 95 –
aLupins– sweet cultivars of L. albus, L. luteus and L. angustifolius.
bBeans – including; common bean (P. vulgaris), Lima bean (P. lunatus), mungo
bean (V. mungo) and others.
24
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 produc-
tion 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, com-
prising 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).
Grain legumes Other protein sources
Share in protein crops 25.0 75

Share of protein requirement 8.0 92
Animal consumption of grain legumes
Pea 76.8 –
Faba bean 18.8 –
Lupin 4.4 –
Table 1.7. Grain legumes used for animal feed in Europe (Gatel and Champ,
1998).
Total Compounded feed Share in feed

Country consumption (t) production (t) production (%)
Belgium 5,661 5,325 12.4

Denmark 5,222 5,666 3.9
France 2,114 21,998 9.6
Germany 5,859 19,326 4.4
Italy 5,598 11,700 5.1
Netherlands 5,586 16,495 3.6
Spain 5,969 15,215 6.4
UK 5,450 12,657 3.6
EU 5,409 1,248 5.4
25
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 consump-
tion 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 develop-
ment of vegetarian habits mainly in northern Europe, however, has stopped
this trend. The consumption of legumes is not necessarily affected by eco-
nomic 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).
Total Total
Region Peas Beans Other range mean
Northern and Western Europe 1.37 0.53 0.41 1.0–4.9 2.43

Central and Eastern Europe 1.65 0.54 0.06 0.2–9.3 2.32
Mediterranean countries 0.58 1.90 6.33 4.2–13.6 8.85
Middle East 0.58 2.75 7.03 3.0–12.8 10.42
Far East 0.48 1.33 1.15 1.0–11.4 2.88
North America 0.52 5.29 0.04 3.7–13.4 6.50
Central America 0.14 10.06 0.74 5.4–15.7 10.88
The Caribbean 0.44 3.13 7.31 2.1–13.6 10.71
South America 0.58 9.41 0.76 1.0–16.8 7.03
West Equatorial Africa 0.24 0.76 6.03 0.2–26.4 10.37
Eastern Africa 0.14 3.25 6.97 2.0–20.3 7.03
South Africa 0.58 2.62 0.89 2.9–11.0 4.08
Oceania 0.87 0.64 0.95 0.4–8.0 2.40
World 0.61 2.51 3.26 0.2–26.4 6.36
26
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 oligosaccha-
rides (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 character-
ized 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.
27
12 C. Hedley
Table 1.9. Protein, oil and carbohydrate composition (% of seed) of grain legume
seeds.a
Protein Oil Carbohydrates
Legume species Min. Max. Min. Max. Min. Max.
Soybean 38.4 19.7 32.5

G. max 35.1 – 42.0 17.7 – 21.0 30.2 – 35.5
Lupin
L. albus 38.1 11.1 36.5
34.3 – 44.9 8.0 – 14.5 31.0 – 42.0
L. luteus 41.7 5.3 32.0
39.0 – 47.0 4.0 – 7.1 26.0 – 37.0
L. angustifolius 34.1 5.7 41.5
28.0 – 37.9 4.6 – 7.0 36.0 – 47.0
Chickpea 21.8 5.2 65.3
C. arietinum 15.5 – 28.2 3.1 – 7.0 59.9 – 70.8
Mung bean 23.3 1.2 60.0
V. radiata 22.9 – 23.6 1.2 – 1.2 58.2 – 61.8
Pigeon pea 21.2 2.6 64.9
C. cajan 19.5 – 22.9 1.3 – 3.8 63.0 – 66.8
Jack bean 29.6 2.4 47.8
C. ensiformis 26.9 – 32.2 1.8 – 2.9 46.1 – 49.5
Common bean 23.4 1.5 61.3
P. vulgaris 20.9 – 27.8 0.9 – 2.4 58.2 – 63.4
Faba bean 29.0 2.0 59.8
V. faba 22.4 – 36.0 1.2 – 4.0 57.8 – 61.0
Lentil 26.8 1.4 64.4
L. culinaris 23.0 – 32.0 0.8 – 2.0 60.5 – 68.2
Cowpea 23.5 1.3 60.0
V. unguiculata
Pea 25.3 2.7 65.5
P. sativum 18.3 – 31.0 0.6 – 5.5 60.7 – 70.7
aSources 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
28
Introduction 13
Table 1.10. Carbohydrate composition (% of seed) of grain legumes.
Legume species Total Starch Sucrose Raffinose Stachyose Verbascose ‘Fibre’a
Soybean 32.5 1.5 6.2 0.9 4.3 0.1 20

Lupin spp. 36.7 0.4 2.5 0.7 6.8 0.6 26
Chickpea 65.3 44.4 2.0 1.5 5.5 3.0 9
Mung bean 60.0 45.0 1.1 1.7 2.0 3.0 7
Pigeon pea 64.9 44.3 2.5 1.0 3.0 4.0 10
Jack bean 47.8 35.0 1.5 0.7 1.5 0.1 9
Common bean 61.3 41.5 5.0 0.3 4.1 0.1 10
Faba bean 59.8 41.0 3.3 0.2 0.7 2.5 12
Lentil 64.4 46.0 2.9 0.5 2.4 0.9 12
Pea 65.5 45.0 2.1 0.9 2.4 3.2 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.
29
2
Carbohydrate
P. 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 alde-
hydes 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.

(ed. C.L. Hedley) 15
31
16 P. Kadlec et al.
Fig. 2.1. 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 mono-
saccharides 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).
32
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 quanti-
ties 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 oligo-
saccharides 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 α-D-galactopyranosyl-(1→6)-α-D-glucopyranosyl-(1→2)
-β-D-fructofuranoside
• stachyose α-D-galactopyranosyl-(1→6)-α-D-galactopyranosyl-
(1→6)-α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside
• verbascose α-D-galactopyranosyl-(1→6)-[α-D-galactopyranosyl-
(1→6)-]2-α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside
• ajugose 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
33
18 P. Kadlec et al.
Fig. 2.2. 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-1,2,3,5-
trans-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),
34
which is a precursor for the formation of D-pinitol, a methyl derivative of

D-chiro-inositol (3-O-methyl-D-chiro-inositol). Other methylation products of
myo-inositol are sequoyitol (5-O-methyl-myo-inositol) and D-bornesitol (1-O-
methyl-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 correspond-
ing 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 UDP-
galactose 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 galacto-
pinitols. 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-galacto-
pyranosyl-(1→2)-4-O-methyl-D-chiro-inositol) (Fig. 2.5) and B (O-α-D-
galactopyranosyl-(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-D-
chiro-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 buck-
wheat, Fagopyrum esculentum (Obendorf, 1997). Fagopyritol B1 (O-α-D-
galactopyranosyl-(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-methyl-
myo-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-D-
galactopyranosyl-(1→6)-O-α-D-galactopyranosyl-(1→2)-4-O-methyl-D-chiro-
35
20
Table 2.1. Occurrence of cyclitols, methyl-cyclitols and galacto-cyclitols in legume seeds.
14 November 2000 14:30:45

(D-pinitol)
(ciceritol)
(galactinol)
(D-ononitol)
di-galacto-chiro-
B1 (fagopyritol B1)
tri-galacto-pinitol
Species Latin name
galacto-chiro-inositol
methyl-scyllo-inositol
galacto-ononitol
methyl-chiro-inositol
galacto-pinitol A
inositol (fagopyritol B2)
di-galacto-pinitol A
galacto-myo-inositol
di-galacto-inositol
(galacto-ciceritol)
methyl-myo-inositol
galacto-pinitol B
myo-inositol
D-chiro-inositol
Lupin Lupinus luteus L. ++ ++ ++ + + + ++ ++ ++ ++
Lupin Lupinus albus L. ++ + + + + ++ ++ ++
Soybean Glycine max [L.] Merrill + ++ + + ++ ++ ++
Pigeon pea Cajanus cajan [L.] Millsp. ++ ++ + ++ ++ +++ +++
36
Cowpea Vigna unguiculata [L.] Walp + + + + +
Mung bean Vigna radiata [L.] Wilczek ++ ++ ++ +++ + + + + + ++ + +
P. Kadlec et al.
Faba bean Vicia faba L. ++ + +

Dry bean Phaseolus vulgaris L. ++ + +
Garden bean Phaseolus vulgaris L. ++ +
A3867: AMA: Hedley: First Proof:14-Nov-00

Pea (round) Pisum sativum L. +++ ++
Lentil Lens culinaris Medic. L. ++ ++ + ++ + + +++ + ++
2
Chickpea Cicer arietinum L. ++ ++ ++ +++ + + + +++ + ++
Lucerne Medicago sativa L. ++ + + ++ ++ + + ++ +++ ++ +

Adzuki bean Vigna angularis L. + + + + ++ ++
+, 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).
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-galacto-
pyranosyl-(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.
37
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
38
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 Amylose is essentially a linear chain of (1→4)-linked α-D-

glucopyranosyl 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
Fig. 2.7. Fagopyritol B series.
Fig. 2.8. Linear chain structure of amylose.
39
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 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 end-
group 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 organiza-
tion of starch granules, which contain both crystalline and amorphous
domains (Bogracheva et al., 1997; see Chapter 4).
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).
40
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 polysaccha-
rides 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 hemicellu-
loses, pectins and some associated components. In addition, the complex
structure of lignin is discussed, whereas the proteins and various amphi-
philic 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)-D-

glucans with a very high degree of polymerization resulting in molecular
weights ranging from about 0.5 to 1 million daltons. The fibrillar appear-
ance 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 inter-
and 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 back-
bones, 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
41
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, respec-
tively. 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.
PECTIN 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
42
composition and forms of occurrence of pectic substances in plants has

led to the development of several more restrictive definitions. The nomen-
clature 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 con-
sidered 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, L-
fructose 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
43
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, hemicellu-
loses) 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 com-
mon 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
44
Table 2.2. Occurrence of pectic substances and lignin in various legumes.

Legumes Pectic substances % Lignin % References
Faba beans seeds

Water-soluble 0.8 Shehata et al. (1985)
EDTA-soluble 0.4 Shehata et al. (1985)
NaOH-soluble 0.4 Shehata et al. (1985)
Green beans 15.4 Vazquez-Blanco et al. (1995)
Blanched 0.5 Margareta et al. (1994)
Canned 2.5 2.4 Ross et al. (1985)
Canned 0.4 Margareta et al. (1994)
Canned 8.1 2.6 Weightman et al. (1994)
Fresh from store 2.7 2.1 Ross et al. (1985)
Freshly cooked 3.8 1.4 Ross et al. (1985)
Frozen 5.0 4.4 Ross et al. (1985)
Microwave heating 0.4 Margareta et al. (1994)
Kidney beans, canned 5.3 3.4 Weightman et al. (1994)
Lima beans, canned 3.8 0.8 Weightman et al. (1994)
Mung beans 10.0–18.0 Goldberg et al. (1986)
Navy bean, dried cooked 7.8 1.2 Weightman et al. (1994)
Pinto beans
Dried, then cooked 7.5 2.7 Weightman et al. (1994)
Dried raw 8.2 1.6 Weightman et al. (1994)
Red kidney beans 12.0 Moscoco et al. (1984)
Runner beans 14.0 Selvendran and King (1989)
White beans
Lentils 17.7–18.1 1.2–1.7 Bhatty (1990)
Lentils 1.2–4.8 1.2–3.0 Vidal-Valverde et al. (1992a)
Peas
Black-eyed peas, canned 1.2 2.2 Weightman et al. (1994)
Green peas 3.7 0.6 Theander (1995)
Blanched 0.1 Margareta et al. (1994)
Canned 0.1 Margareta et al. (1994)
Microwave heating 0.9 Margareta et al. (1994)
Pea hulls 15.0 Weightman et al. (1994)
Soybean okara 22.0 Yamaguchi et al. (1996a)
Soybean okara 4.6 Yamaguchi et al. (1996b)
45
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 Saponin content (g kg-1)
Butter bean Phaseolus lunatus 1.0

Chick pea Cicer arietinum 2.3
Field bean Vicia faba 0.1
Green pea Pisum sativum 1.8
Haricot bean Phaseolus vulgaris 2.3
Kidney bean Phaseolus vulgaris 3.5
Lentil Lens culinaris 1.1
Mung bean Phaseolus aureus 0.5
Peanut Arachis hypogaea < 0.1<
Runner bean Phaseolus coccineus 3.4
Soybean Glycine max 6.5
Yellow split pea Pisum sativum 1.1
46
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
47
32
Table 2.4. Sources of standard of cyclitols, methyl cyclitols and their galactosides.
30 October 2000 14:57:09

Common name Source Reference
D-Pinitol Soybean seed (Glycine max) Schweizer et al. (1978)

D-Ononitol Lucerne leaves (Medicago sativa) Binder and Haddon (1984)
O-Methyl-scyllo-inositol Mung bean (Vigna radiata) Binder and Haddon (1984)
O-Methyl-muco-inositol Redwood (Sequoia sempervirens) Binder and Haddon (1984)
Sequoyitol Ginkgo biloba Binder and Haddon (1984)
scyllo-Inositol Chemical isomerization of myo-inositol Sasaki et al. (1988)
chiro-Inositol 1. Chemical isomerization of myo-inositol 1. Sasaki et al. (1988)
2. Buckwheat (Fagopyrum esculentum) 2. Horbowicz et al. (1998)
3. Demethylation of D-pinitol 3. Binder and Haddon (1984)
Galactinol 1. Cucumber leaves (Cucumis sativus) 1. Pharr et al. (1987)
48
2. Common bugle (Ajuga reptans) 2. Bachmann (1993)
3. Castor bean seeds (Riccinus communis) 3. Kuo (1992)
P. Kadlec et al.
4. Jojoba beans (Simmondsia chinensis) 4. Ogawa et al. (1997)

Galacto-ononitol Adzuki bean (Vigna angularis) Yasui (1980)
Galactopinitol A Soybean seeds (Glycine max) Schweizer and Horman (1981)

Galactopinitol B Soybean seeds (Glycine max) Schweizer and Horman (1981)
Fagopyritol B1 1. Soybean seeds (Glycine max) 1. Schweizer et al. (1978); Schweizer and Horman (1981)
2
2. Buckwheat (Fagopyrum esculentum) 2. Horbowicz et al. (1998)
3. Jojoba beans (Simmondsia chinensis) 3. Ogawa et al. (1997)

Ciceritol 1. Chickpea (Cicer arietinum), lentil (Lens culinaris) 1. Quemener and Brillouet (1983)
2. Lentil (Lens culinaris) 2. Bernabé et al. (1993)
Galacto-ciceritol Chickpea (Cicer arietinum) Nicolas et al. (1984)
Mimositol Seeds of Mimosa scabrella Ganter et al. (1991)
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 tem-
perature. 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 (%)
Method I (in 2 g of flour)

Sucrose 0.00 29.29 –
20.21 46.15 83.42
40.08 69.29 99.80
80.23 101.43 89.84
Raffinose 0.00 21.64 –
20.14 37.15 77.01
40.25 56.43 86.43
80.20 98.22 95.49
Stachyose 0.00 20.36 –
20.07 37.86 87.19
40.06 57.15 91.54
80.21 87.72 83.98
Method II (in 500 mg of flour)
Sucrose 0.00 8.10 –
5.21 13.70 107.49
10.30 18.26 98.64
20.18 27.32 95.24
Raffinose 0.00 2.48
5.22 7.30 92.34
10.19 11.36 87.14
20.36 20.38 87.92
Stachyose 0.00 27.38 –
5.04 34.06 132.54
10.19 37.46 98.92
20.09 45.52 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.
49
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.
RECOMMENDED EXTRACTION PROCEDURE 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.
50
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.
RECOMMENDED GC METHOD FOR QUANTIFYING SOLUBLE CARBOHYDRATES 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 work-
ing 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 cen-
trifuged. 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 deriva-
tives. 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,
51
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.
52
CALCULATION AND STATISTICAL ANALYSIS 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:
Area A
Concentration of component 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)
Area IS
Internal standard concentration = × 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
= ×100%
Sample weight (mg)
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.
53
38 P. Kadlec et al.
6. A wide range of stationary phases are available for columns to custom-

ize 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)
SAMPLE CLEAN-UP The water extract of soluble carbohydrates from biologi-

cal material, (containing an internal standard) should be cleaned before
HPLC analysis to improve reliability and resolution, using one of the follow-
ing procedures.
1. The extract is filtered through a Sep-Pak C18 cartridge, pre-wetted
with methanol and pure water. The effect of sample pre-treatment car-
tridges 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 pre-
cipitated 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 formu-
lated 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
54
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).
HPLC (NORMAL PHASE) 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).
RP-HPLC (REVERSE 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).
IMP-HPLC (ION MODERATE PARTITION) 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).
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).
55
40 P. Kadlec et al.
AC-HPLC (AFFINITY CHROMATOGRAPHY) Sugar-Pack 1 column (Waters Asso-

ciates 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).
CALCULATION AND STATISTICAL ANALYSIS Concentration of sugars is calcu-

lated from the peak height of detector response as:
H s R sW IS
Content of sugars (% dry matter) = × 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, correspond-
ing 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 For

routine analysis of individual RFOs:
• 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).
56
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 carbohy-
drates 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.
COMPARISON BETWEEN GC AND HPLC Bach Knudsen and Li (1991) deter-

mined 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:-
sucrose Y = 0.094 + 0.985 X ± 0.017 R 2 = 0.995
raffinose Y = −0.097 + 1.015 X ± 0.025 R 2 = 0.989
stachyose Y = −0.355 + 1.034 X ± 0.025 R 2 = 0.990
total Y = −0.262 + 1.006 X ± 0.026 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
57
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).
RECOMMENDED TLC METHODS FOR QUANTIFYING SOLUBLE CARBOHYDRATES F o r

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).
ADVANTAGES OF TLC METHOD 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).
Capillary zone electrophoresis (CZE)

The determination of soluble carbohydrates by capillary zone electro-
phoresis 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.
58
RECOMMENDED CZE METHODS FOR QUANTIFYING SOLUBLE CARBOHYDRATES The

following description and specification of two CZE systems is fairly
representative of the technique.
1. P/ACE 2210 Series capillary electrophoresis system (Beckman Instru-
ments (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).
CALCULATION AND STATISTICAL ANALYSIS 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.
ADVANTAGES AND DISADVANTAGES OF CZE COMPARED WITH GC AND HPLC

Advantages:
1. The method is easier to use and set up.
2. The equipment is less expensive as HPLC or GC equipment.
3. Uses non-toxic chemicals.
4. 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.
59
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
OPTICAL ROTATION 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-polar-
ized 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 concen-
trations of sugars.
REDUCING SUGAR METHOD 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:
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 condi-
tions is needed along with calibration using standards. Fehling originally
devised the most widely known test based on this method, in 1848.
MEDICAL/FOOD DIAGNOSTIC TEST KITS 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
60
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.
ENZYMATIC METHOD 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.
SPECTROPHOTOMETRIC 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).
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.
POLARIMETRIC METHODS This is termed the Ewers method (AOAC method

14.031) and is based on acid hydrolysis of starch, followed by the measure-
ment of the optical rotation of the resulting solution.
61
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:
50 × 34.66 × p
Starch (%) =
[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).
CALCIUM CHLORIDE METHOD 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 con-
sisting of the mean of ten separate optical rotation measurements) must
not differ by greater than 0.006 units.
METHODS IN WHICH THE STARCH IS FULLY HYDROLYSED INTO GLUCOSE 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.
DETERMINATION OF RESISTANT STARCH 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,
62
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 modi-
fied 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
63
48 P. Kadlec et al.
Table 2.6. In vitro and in vivo quantification of resistant starch (RS) as a propor-
tion of the total starch (TS).
Quantification of resistant starch (% RS/TS)
in vitro in vivo
Champ Englyst
Origin of the RS methoda methodb Ileostomyc Incubationd
Beans 17 – – 17
Raw green banana 61 71 68 84
Retrograded high amylose corn starch 30 34 – 49
aChamp (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.
MEGAZYME KIT 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.
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.
64
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 polysaccha-
rides/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 solu-
ble 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 mono-
saccharides is generally performed by GC, but other chromatographic
65
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).
PREPARATION OF PLANT CELL WALL MATERIAL 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 com-
ponents may be found in close association with the cell wall polysaccha-
rides, affecting the properties of the DF fraction. This apparent conflict of
interest must be taken into consideration when planning the study of the
66
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 fraction-
ation, 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 sam-
ple 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 intra-
cellular 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,
67
52 P. Kadlec et al.
fast and cheap to use supercritical fluid techniques (super critical extrac-
tion (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 hemi-
celluloses. 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.
68
after centrifugation is used for the extraction. The procedure converts

cellulose into a soluble fraction by heating in dry DMSO and paraformalde-
hyde. 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 non-
cellulosic 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 hemicellu-
lose. 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 sequen-
tially 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
69
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 hemi-
celluloses. 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: per-
chlorate, 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 inor-
ganic 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.
70
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 proce-
dure 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 desicca-
tor (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 possibili-
ties as also indicated in the preceding sections. It should be noted that the
results obtained are exclusively dependent on the actual fractionation
71
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.
DETERMINATION OF GALACTURONIC ACID (GA) 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 ampero-
metric detection for the determination of galacturonic acid, after acid
hydrolysis.
72
DETERMINATION OF NEUTRAL SUGARS The most widely used method for sugar
determination involves acid hydrolysis of the sample with 2 M trifluoro-
acetic 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 combi-
nation of enzymic hydrolysis and methanolysis, followed by HPLC separa-
tion 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.
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).
KLASON METHOD In this procedure lignin is the residue after all the
enzyme and chemical treatments of the Englyst DMSO method have been
completed.
DELIGNIFICATION METHOD The residue is delignified by treatment with

sodium chloride–acetic acid at 70°C for 4 h.
GOERING AND VAN SOEST METHOD The residue from the ADF is treated with
potassium permanganate solution, containing trivalent iron and mono-
valent silver as catalysts. Deposited manganese and iron oxides are dissolved
with an alcoholic solution of oxalic and hydrochloric acids, leaving
73
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 See Reeves (1988).
FT-IR ANALYSIS 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 water-
insoluble hemicellulose and cellulose (Jeltema and Zabik, 1980).
GRAVIMETRIC METHOD 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 hydro-
lyse 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).
Saponins
EXTRACTION 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 evapo-
rated 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).
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
74
(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), equiva-

lent 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 cholesteryl-
n-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 tempera-
ture 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.
HPLC ANALYSIS 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).
75
3
H. Kozlowska et al.
Nutrition
Nutrition 3
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 proces-
sors, 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 sev-
eral 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.

77
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 oligo-
saccharides 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 arie-
tinum) 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 oligosaccha-
rides 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.
78
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.
Beans Peas Lentils Chickpeas Faba beans
Fructose (mean) – – 0.1 – 0.4

(range) .30–0.2
Sucrose 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
Raffinose 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
Ciceritol – – 0.7 2.2
0.2–2.1 1.2–3.1
Stachyose 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
Verbascose 0.6 1.8 0.3 trace 1.8
0.1–1.8 .30–4.3 .30–6.4 trace–0.4 1.1–2.4
Total α-galactosides 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
Total soluble sugars 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
Starch 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%.
79
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.
80
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 gastro-
intestinal 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 compo-
nent 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 Chickpeas Faba beans
NDF 8.9–12.8 13.2–25.6 9.7–24.1 7.5–19.2 13.0–19.5

ADF 3.5–7.2 no information 2.0–6.8 3.8–14.7 10.3–11.4
Cellulose 3.2–13.1 0.9–13.3 3.5–14.8 1.1–13.7 8.3–14.3
Hemicellulose 0.5–5.6 0.9–12.4 1.2–15.7 0.6–16.0 1.6–8.9
Lignin 0.1–3.1 0.3–2.1 trace–2.6 trace–7.1 0.7–2.0
TDF 11.2–27.5 16.1–21.6 11.0–21.4 8.2–24.0 17.1–23.8
SDF 8.1–10.0 4.6–6.0 1.2–4.4 3.7 6.0–8.7
IDF 9.1–11.6 11.6–16.1 8.8–13.7 7.9 8.3–15.5
NSP 6.4–20.4 no information 6.9–14.7 5.5–35.4 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 polysaccha-
rides.
81
Table 3.3. Dietary fibre (DF) (as a percentage of dry matter, DM) content in beans
(Phaseolus vulgaris).
Type DF content (%DM) References
Great northern 20.7 Mongeau and Brassard (1994)

Light red kidney 14.3–18.1 Mongeau and Brassard (1995)
Red kidney (Goya) 20.7–21.3 Mongeau and Brassard (1994)
Red kidney (Townhouse) 21.3–21.9 Mongeau and Brassard (1994)
White navy 15.8–19.2 Mongeau and Brassard (1995)
No name 12.7–12.8 Mongeau and Brassard (1995)
Mottled 19.1 Lintas et al. (1992)
White 19.6 Lintas et al. (1992)
No name 11.6 Lintas et al. (1992)
Pinto 27.0 Chen and Anderson (1982)
No name 16.8 Paul and Southgate (1985)
No name 28.1 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).
82
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; Vidal-
Valverde 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 α-
galactosides was found in faba bean seeds (less than 2.5%), an intermediate
83
Table 3.4. Content of soluble carbohydrates (SC) in seeds of different legume

species (as a %).
Total Soluble
α-galacto- carbohy-
Sucrose Raffinose Stachyose Verbascose sides drates Starch
Peaa 1.8 0.8 2.5 1.7 4.9 6.8 47.5

Faba beanb 2.5 0.2 0.9 1.4 2.4 4.9 41.3
White 2.8 0.7 6.6 0.5 7.9 10.7 ND
lupinc
Yellow 1.1 2.2 6.9 2.8 11.9 13.0 ND
lupind
Narrow leaf 1.4 1.4 5.2 2.0 8.6 10.0 ND
lupine
Chickpeaf 3.5 0.6 1.9 ND 8.2 11.7 52.6
Lentilg 1.9 0.3 1.7 0.4 4.7 6.6 46.2
aFrejnagel et al. (1997); Gdala and Buraczewska (1997) (mean of 19 varieties).
bGdala 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.
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 impor-
tant 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.
84
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 Pea Faba bean Lupinus luteus Lupinus angustifolius
Rhamnose 0.9 0.8 0.5 0.8

Fructose 0.4 0.1 0.3 0.3
Arabinose 19.4 16.4 12.6 10.7
Xylose 7.3 13.0 11.3 8.3
Mannose 0.9 0.7 1.9 1.6
Galactose 8.6 18.3 16.5 34.9
Glucose 47.6 45.3 89.0 31.5
Uronic acid 15.0 14.6 12.3 12.0
Total NSP 172.4 208.6 309.0 365.0
DFa 179.2 229.6 325.0 392.0
aNSP + 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
85
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 con-
tent 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 Share in dietb GAL content in diet

(g kg−1) (%) (g kg−1)
CP GAL A B A B
Soybean meal 440 49.0 16 6–8 7.8 –

Pea 214 49.4 33 20 16.4 13.1
Faba bean 259 24.2 27 15 6.5 7.1
Yellow lupin 375 104.8 19 10 19.9 14.0
Narrow leaf lupin 328 75.5 21 10 15.8 12.4
White lupin 333 78.9 21 10 16.6 12.9
aData for soybean meal according to Seve et al. (1989) and Coon et al. (1990).
bGrain legumes share in diet: A – total substitution of soybean protein, B – partial
(about 50%) substitution of soybean protein.
86
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 oligosac-
charides are not digested in the stomach and small intestine may need to be
87
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 exoge-
nous 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 (%) − + References
Raffinose 70.2 97.4 Gdala et al. (1997b)

Stachyose 88.8 99.5
Verbascose 74.2 98.0 (26.7 g GAL kg−1)
Raffinose 40.7 95.7 Gdala et al. (1997b)
Stachyose 60.1 87.2
Verbascose 77.8 86.3 (37 g GAL kg−1)
Raffinose (whole lupin) 31.9 75.3
Stachyose (whole lupin) 10.4 37.3 Brenes et al. (1992)
Raffinose (dehulled lupin) 41.6 86.0
Stachyose (dehulled lupin) 23.2 65.0
88
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 fructo-
oligosaccharides 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 supple-
mented with fructo-oligosaccharides (FOS) or oligosaccharides (OS) from lupin
seeds, administered to rat small intestine (Zdu4czyk et al., 1999).
Control FOS FOS OS OS

fluid −4% −8% −4% −8%
Glucose 63.7 74.7 72.2 24.4 14.3

Methionine 44.2 37.2 25.8 17.2 18.7
Water 6.8 8.7 6.6 0.7 1.2
89
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 oligo-
saccharides 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, amylo-
glucosidase), sucrase (maltase–sucrase) and α-dextrinase (isomaltase). Glu-
cose, 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 Apparent digestibility (%) References
Pea seeds 84–92 Carré et al. (1987, 1991)

Pea starch 94.4 Yuste et al. (1991)
Wheat starch 97.6 Yuste et al. (1991)
Maize starch 97.8 Yuste et al. (1991)
Starch was extracted from seeds according to the methods of Faulkes et al. (1989).
90
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 digest-
ibility 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.
Share in diet Apparent

Source of starch (%) digestibility References
Pea 45.1 94.4–99.1 Bengala Freire et al. (1991)

Pea 66.1 88.9–92.9 Canibe and Bach Knudsen (1997)
Pea 73.7–83.1 85.2–87.3 Gdala and Buraczewska (1997a,b)
Faba bean 30.1 97.9 Van der Poel et al. (1992)
Faba bean 30.1 96.3 – 98.1 Jansman et al. (1993)
Faba bean 62.1 81.5 – 86.4 Gdala and Buraczewska (1997a,b)
91
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).
Meal (n = 25) Pellets (n = 11)
Digestibility of protein (%) 78.4 85.5

Digestibility of starch (%) 90.5 98.5
Metabolizable energy (kcal kg−1 DM) 2870.5 3150.5
DM, dry matter.
92
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 compo-
sition 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 nutri-
ent absorption (Low, 1985). Dietary fibre can be a negative factor, there-
fore, 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 36.3 43.7 39.2 23.6

Xylose 26.3 59.7 −8.0 −6.9
Mannose – – 86.0 99.8
Galactose 44.8 61.7 42.1 25.4
Glucose 4.7 32.8 0.3 26.5
Uronic acid 35.8 25.9 25.9 26.5
Total NSP 39.9 38.8 14.4 12.1
aAverage for two white flowered varieties.
bAverage for two coloured flowered varieties.
93
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 supple-
mentation 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).
Digestibility of energy Gas

Total production
sugarsa Starch WICWb (%) (MJ kg−1) (ml g−1 DM)
Pea 3.7 45.3 17.1 89.7 16.5 146.7

Faba bean 1.9 40.0 20.1 90.5 16.3 112.0
Yellow lupin 4.1 0.7 35.3 84.5 16.9 95.9
Narrow leaf lupin 4.5 0.8 38.0 82.0 16.5 104.6
aExpressed as saccharase (% dry matter, DM).
bWICW – water-insoluble cell wall components.
94
Nutrition 79
nitrogen values, which make grain legumes uncompetitive with other

protein sources (Gatel and Champ, 1998). The problem of ruminal degrad-
ability 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 non-
cellulosic 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 carbo-
hydrates, 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 oligosaccha-
rides 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 Physiological
Role in the plant Type of saccharide Site of digestion digestion classification
Source of energy Mono- and Small intestine Mono- and Available

disaccharides (enzymatic) disaccharides carbohydrates
Storage Starch; amylose Small intestine Mono- and Available
polysaccharides and amylopectin (enzymatic) disaccharides carbohydrates
Storage α-Galactosides Large intestine Short chain fatty Unavailable
oligosaccharides (microbial) acids: acetate, carbohydrates
propionate,
butyrate
Structural Non-cellulosic Large intestine Carbon dioxide, Unavailable
components of polysaccharides: (microbial) hydrogen carbohydrates
plant cell walls pectins, hemi- methane
cellulose,
cellulose
95
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 com-
pounds passing into the large intestine. These oligosaccharides are then
broken down to monosaccharides by bacterial enzymes, with the produc-
tion 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 degrada-
tion 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).

Digestibility in small
Starch type Source intestine
Rapidly digestible starch Freshly cooked starchy foods Rapid

Slowly digestible starch Most raw cereals Slow – but complete
Resistant starch Partially milled grain and seeds; Resistant
raw potato and banana; cooled
cooked potato, corn flakes and
bread
96
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. The content of resistant starch (RS) in legume products (g (100 g)−1 of
starch).
Methods of RS RS
Legume products determination content References
Raw pea starch Weight experiment on ratsa 1.0 Berggren et al. (1995)
Pre-cooked lentil Weight experiment on ratsa 11.0 Tovar et al. (1992)
Pre-cooked red bean Weight experiment on ratsa 8.0 Tovar et al. (1992)
Canned pea Colectomized rats 15.2 Hildebrandt and
Marlett (1991)
Canned peab Weight experiment on ratsa 27.8 Björck and Sijeström
and three methods in vitro (1992)
Boiled red lentil In vivo, in ileostomists 13.8 Steinhart et al. (1992)
In vivo, in ileostomists 13.6 Jenkins et al. (1987)
Boiled white beans In vivo, intubation experi- 16.5 Noah et al. (1998)
ments in healthy subjects
Autoclaved white In vivo, in ileostomists 5.7 Muir and O’Dea (1993)
beans
Autoclaved white In vivo, in ileostomists 20.9 Schweizer et al. (1990)
beans
Boiled lentil In vitro digestibility of 16.5 Cummings and Englyst
starch (1995)
Boiled red lentil In vitro based on chewing 23.1 Äkerberg et al. (1998)
Boiled white beans In vitro based on chewing 16.7 Äkerberg et al. (1998)
Autoclaved white In vitro based on chewing 13.8 Äkerberg et al. (1998)
beans
aRats treated with antibiotics to suppress hindgut fermentation.
bPea with low content of starch – 19% dry matter (DM).
97
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 pur-
poses. 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 physio-
logical 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
98
Nutrition 83
Table 3.17. Annual consumption of α-galactosides, starch, resistant starch and

dietary fibre in legumes, in Europe (data for 1996).
Beans Pea Lentil Chickpea Total
Annual consumption of legumes 1.14 0.67 0.43 0.19 2.43

(kg per person)
Average content of (g kg−1)
α-galactosidesa 36.8 49.4 28.4 47.0 39.6
Starcha 549.3 475.0 461.8 526.0 511.5
Resistant starch 82.4 72.3 69.3 78.9 77.0
b193.3b b187.9c b214.0d b245.5e
Dietary fibre 199.5
Annual consumption of (g)
α-galactosides 42.0 33.1 12.3 8.9 96.3
Starch 626.2 318.3 198.6 99.9 1243.0
Resistant starch 93.9 48.4 29.8 15.0 187.1
Dietary fibre 220.4 125.9 92.0 46.6 484.9
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).
Dry legumes Original GAL Losses on Final intake of
Dish (g) (g) cooking (%) GAL (g)
Pea soup 20–30 1.5–2.3 10 1.3–2.1

Lentil soup 13–20 0.7–1.0 10 0.6–0.9
Bean soup 20–30 0.6–0.9 10 0.5–0.8
Mashed peas 75–105 5.6–7.9 70 1.7–2.4
Cooked lentils 75–112 4.0–6.0 70 1.2–1.8
Cooked beans 75–110 2.5–4.0 70 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
99
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 mono-
gastric 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 69
Bread, wholemeal 72
Rice, white 66
Rice, brown 66
Spaghetti, white 50
Corn flakes 80
Root vegetables
Carrots 92
Potato, new 70
Potato, instant 80
Legumes
Beans, navy 31
Beans, kidney 29
Beans, soya 15
Peas 33
Lentils 29
100
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
101
(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 fermenta-
tion 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 immuno-
logical 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 compo-
nents (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
102
Nutrition 87
Table 3.20. Consumer acceptance of a selection of dishes with added legumes

(Dostalova et al., 1999a).
Sensory acceptancea (%)

Amount of
Legumes cooked Without With
Dish added legumes (%) legumes legumes
Frankfurter soup Beans 6.6 77 80

Tomato soup Soybeans 9.1 80 78
Cabbage soup Lentils 7.6 79 63
Salad with tunny Beans 9.3 71 78
Serbian salad Chickpeas 6.2 81 88
Serbian salad Beans 8.1 79 88
Vegetable salad Lentils 5.4 70 85
Mashed potatoes Peas 5.4 52 71
Mashed potatoes Peas 9.3 74 74
Potato salad Soybeans 6.2 59 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.
103
4
B. Czukor et al.
Processing
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 separa-
tion 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 applica-
tions. In addition, the wet process is necessary for scientific research on

105
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.,
106
Processing 91
Fig. 4.1. 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.
107
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 proper-
ties 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 exam-
ple, 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
108
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.
109
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 amylo-
pectin, 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 crys-
talline 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.
110
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 poly-
morph (Blanshard, 1987; Wang et al., 1998), the resulting starches being
Fig. 4.4. Continued.
111
96 B. Czukor et al.
Fig. 4.5. 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 poly-
morphs, then the properties would be intermediate between those of A-
and B-type starches. If, on the other hand, each granule contains both A-
and 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
112
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).
Amylose Crystallinity
contenta Tp ∆T ∆H
Genotype (% starch) total % %B %A B/A (°C) (°C) (J g−1)
Wild-type 35 20 45 59 0.8 61.8 13.4 10.8

rug4 33 23 39 57 0.7 65.4 12.5 9.8
rb 23 27 43 58 0.7 66.1 9.0 12.6
rug3 12 17 37 63 0.6 70.0 9.4 7.5
lam 8 22 69 29 2.4 58.6 8.4 6.8
rug5 43 20 52 45 1.2 49.0–57.0 30.0 5.1
r 65 19 73 b0b ∞ 52.5–60.0 34.0 2.4
aData from Bogracheva et al. (1997).
bA polymorphs not detected.
113
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 depend-
ent 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 mois-
ture 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 tempera-
ture 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 functional-
ity and is widely exploited in industry.
114
Processing 99
Fig. 4.6. 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.
115
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.
116
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.
117
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 micro-
scope (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 charac-
terized 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.
118
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 realign-
ment 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 order-
ing 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
119
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, ortho-
phosphoric acid, sodium or potassium orthophosphate or sodium tripoly-
phosphate, 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
120
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 differ-
ent temperatures for 7 days (Hoover and Sosulki, 1985a,b).
Extent of syneresis (%)
Starch source Degree of substitution* 4°C −15°C
Pinto bean 0.050 18.2 6.8

0.050 13.0 5.8
Navy bean 0.050 20.2 7.9
0.055 12.0 6.2
Black bean 0.050 30.1 15.3
0.053 11.7 10.4
0.095 8.9 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 disod-
ium 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
121
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 phos-
phorus 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 3.4 6.4 10.0 17 21 28 28
Faba bean 2.1 5.8 9.9 16 20 26 27
Field pea 1.9 5.3 9.2 12 19 23 23
Native (gelatinized)
Lentil 42.8 57.8 61.8 66 72 79 79
Faba bean 32.8 51.8 60.8 65 70 72 72
Field pea 30.8 45.8 55.8 62 68 68 70
Cross-linked (ungelatinized)
Lentil 3.2 6.0 10.0 16 20 27 27
Faba bean 2.0 5.5 9.4 15 19 25 26
Field pea 1.8 5.0 8.8 12 18 22 22
Cross-linked (gelatinized)
Lentil 38.8 54.8 58.8 63 69 76 76
Faba bean 29.8 49.8 58.8 63 68 70 70
Field pea 27.8 42.8 52.8 58 64 66 68
122
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 com-
pared 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).
123
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 Faba bean Kidney bean Chickpea Corn starch
Isolated starch
A 33.2 32.3 39.5
B 65.1 62.1 67.2
C 47.2 44.7 49.4
D 84.3 82.1 90.3
Cooked corn starch – – – 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.
124
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 applica-
tions (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 pro-
tein digestibility of wadies prepared from black-gram dhal (Yadav and Khetarpaul,
1994).
Temperature Fermentation Starch digestibility Protein digestibility

(°C) time (h) (mg maltose released g−1 meal) (%)
Control 0 35.7 53.0

25 12 56.1 68.1
18 56.9 70.0
30 12 60.6 71.9
18 61.2 73.8
35 12 66.6 77.3
18 67.2 79.3
125
• 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 func-
tion 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 condi-
tions 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),
126
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 car-
ried out on this subject, the correlation between starch structure and resis-
tance 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 relation-
ship 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 whole-
meal 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).
127
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 non-
reducing 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
128
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 attrib-
uted to hydrolysis of oligosaccharides bound to proteins or other macro-
molecules, 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 non-
starch polysaccharide content during cooking, the content of soluble non-
starch 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).
129
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 par-
tially 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
130
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 Lacto-
bacillus 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 impor-
tant 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 Lacto-
bacillus 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 micro-
organisms 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).
131
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 flatus-
inducing 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.
132
5
Seed
J. Górecki
Physiology
et al. and Biochemistry
Seed Physiology and 5

Biochemistry
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, endo-
sperm 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

133
118 J. Górecki et al.
Fig. 5.1. 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
134
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 carbo-
hydrate 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 exendo-
spermous. 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 photo-
assimilates is altered (Pate, 1984). The carbon that is used for the develop-
ment 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).
135
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, with-
out concentrating specifically on seed development. The system for soy-
bean 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 compar-
ing 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 One flower at any node

R2 Flower at node immediately below the uppermost node with a
completely unrolled leaf
R3 Pod 0.5 cm long at one of the four uppermost nodes with a
R4 Pod 2 cm long at one of the four uppermost nodes with a
R5 Beans beginning to develop (can be felt when pod is squeezed)
R5.5 At one of the four uppermost nodes with a completely unrolled leaf
R6 Pod containing full size green beans at one of the four uppermost
nodes with a completely unrolled leaf
R6.5 Beginning maturity
R7 Pods yellowing, 50% of leaves yellow, physiological maturity
R8 95% of pods brown, harvest maturity
136
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 dif-
ferent 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 pat-
tern 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
137
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 oligo-
saccharides (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 111.3 26.3 131.4 trace 1.7 288.6
Peac 69.7 19.6 65.5 45.6 5.7 200.5
Yellow lupina 20.3 23.9 199.6 59.5 5.8 248.0
Faba beanb 57.0 15.0 65.3 99.4 0.3 243.6
Lentilb 39.2 5.4 92.4 33.7 10.8 236.5
Pigeon peab 61.5 13.4 35.7 90.1 4.8 237.1
Cowpeab 45.3 12.2 110.0 11.2 0.9 184.7
Chickpeab 36.1 22.9 35.1 1.8 22.2 171.5
(b) Cotyledons
Soybeanb 76.1 11.8 43.5 trace 2.6 141.0
Peac 26.6 4.9 13.7 34.8 2.4 80.1
Yellow lupina 7.7 8.4 48.3 23.6 4.3 110.0
Faba beanb 19.3 3.4 11.1 52.5 0.0 88.4
Lentilb 11.5 2.4 25.4 18.0 33.0 85.2
Pigeon peab 18.8 9.4 16.5 34.6 11.4 95.4
Cowpeab 15.2 3.3 55.2 10.8 2.3 87.7
Chickpeab 20.6 4.3 26.7 1.0 34.5 94.2
aGórecki et al. (1997).
bHorbowicz and Obendorf (1994).
cLahuta (unpublished).
138
30 October 2000 14:59:09

Table 5.3. Distribution of soluble carbohydrates in dry seeds in various legumes (mg g−1 defatted meal) (Kuo et al., 1988).
Sucrose Raffinose Stachyose Verbascose Total RFO RFO + sucrose
Pea cv. Little Marvel 62.3 11.6 32.3 19.1 63.0 125.3
Soybean cv. Williams 82 72.7 12.6 43.4 trace 56.0 120.2
cv. Amsoy 71 64.2 11.6 41.0 trace 52.6 125.3
Cowpea 25.9 3.7 46.4 3.6 53.7 79.6
Mung bean cv. Berken 13.9 3.9 16.7 26.6 47.2 61.1
Lima bean cv. Fordhook 36.0 6.9 30.3 trace 37.2 73.2
139
Common bean cv. Top Crop 19.4 2.5 34.3 trace 36.8 56.2
cv. Blue Lake 29.0 2.2 28.1 trace 30.3 59.3
red kidney bean 21.5 3.1 31.6 trace 34.7 56.2
Pole bean cv. Kentucky 26.7 4.3 26.2 trace 30.5 57.2

Wonder
Seed Physiology and Biochemistry
Broad bean 20.7 2.3 10.7 11.4 24.4 45.1
5
Groundnut cv. Florunner 81.0 3.3 9.9 trace 13.2 94.2

123
Monosaccharides
The monosaccharides found in legume seeds are often involved as transi-
tory intermediates in the synthesis of higher polymers of carbohydrates.
They are also often found in the phosphorylated form. Free mono-
saccharides 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, how-
ever, the amounts can be relatively high early in development (Yazdi-
Samadi 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 carbo-
hydrates. 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 oligo-
saccharides (Table 5.4).
140
Table 5.4. Mono-O-α-D-galactosyl–sucrose derivatives (Dey, 1990).
α-D-Galactosyl linkage with Name of molecule
C-2 of D-glucose Umbelliferose

C-3 of D-glucose No name
C-6 of D-glucose Raffinose
C-1 of D-fructose No name
C-3 of D-fructose No name
C-6 of D-fructose 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 UDP-
glucose 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
141
Fig. 5.2. 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). Galactosylo-
nonitol 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
142
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 myo-
inositol (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+
143
Fig. 5.3. 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 myo-
inositol 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
144
Fig. 5.4. Proposed pathways of synthesis of the main cyclitols and methyl-
cyclitols 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
145
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 characteris-
tics 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-elonga-
tion 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 gran-
ules 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
146
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 pyrophosphory-
lase, 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 trans-
ferred 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; Finch-
Savage 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
147
set of heat-stable hydrophilic proteins accumulate during late seed matura-

tion. 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 acquisi-
tion 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
148
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
149
seeds coincides with an increase in the RFO and the acquisition of desicca-
tion tolerance, while in pea only the RFO accumulate (Fig. 5.6; Górecki
et al., 1997). Galactopinitol A, galactopinitol B and fagopyritol B1 accumu-
late 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 accumu-
late galactopinitols during slow drying (Obendorf et al., 1996). Galacto-
pinitol 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 germina-
tion. 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).
150
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 toler-
ance (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 carbohy-
drate 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 concentra-
tion 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 mem-
branes appears to be the main role of these compounds in conveying
151
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 carbo-
hydrate 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, includ-
ing 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 crystalliza-
tion 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 mono-
saccharides, 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 respira-
tion, 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
152
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 deple-
tion 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 non-
crystalline 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
153
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 cyto-
plasm 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
154
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 fragmenta-
tion 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, iso-
enzymes 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).
155
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-D-
glucosidically 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 malt-
ose, 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 amylo-
pectin and remain in the limit dextrins after amylolytic or phosphorolytic
attack (Beck and Ziegler, 1989). Starch phosphorylase releases glucose-
1-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).
156
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 yel-
low lupin seeds the activity of α-D-galactosidase increases only at the begin-
ning 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
157
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 five-
to 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).
158
oligosaccharides. Drying resulted in a small increase of raffinose in hypo-

cotyls and radicles, but this was not enough to protect seedlings from desic-
cation 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 desicca-
tion 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).
159
6
N. Kuchuk et al.
Biotechnology
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.

161
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 biotechnologi-
cal techniques and methods for genetic manipulation.
In vitro plant cells from excised tissues (explants) might undergo de-
differentiation and re-differentiation following several developmental path-
ways, 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, how-
ever, 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 morpho-
genesis 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
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Biotechnology 147
Table 6.1. Morphogenic responses of grain legumes cultured in vitro.
Species 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
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Table 6.1. Continued
Species References
C. arietinum Altaf and Ahmad (1990); Malik and Saxena (1992b); Brandt
and Hess (1994); Polisetty et al. (1997); Santangelo et al.
(1997)
G. max Parrott et al. (1992); Sharma and Kothari (1994); Kaneda
et al. (1997)
L. culinaris Malik and Saxena (1992b); Polanco and Ruiz (1997)
L. albus Sator (1990); Rybczynski and Podyma (1993b)
P. vulgaris Mohamed et al. (1992); Herselman and Mienie (1995)
P. sativum Jackson and Hobbs (1990); Kosturkova et al. (1997)
V. faba 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 impor-
tant for making the right choice of scheme to be applied. In organogenesis,
a group of cells forms bud and root primordia with subsequent develop-
ment into a leafy vegetative shoot and root, respectively. In somatic embryo-
genesis, 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.
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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 com-
position. 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 organo-
genic 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 regen-
erated 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
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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
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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 success-
fully 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 de-
differentiation 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 regenera-
tion 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 cotyle-
donary 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
167
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 forma-
tion 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 meta-
bolizable 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 regenera-
tion 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 high-
est 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 cytokinin-
like 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
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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 organogene-
sis 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 cytokinin-
like 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),
169
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 main-
tained 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 micro-
propagation, while young leaflets, epicotyl and hypocotyl have been used
less. The developmental stage of the explants is very important as it can
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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 con-
centration, can induce callogenesis or rhizogenesis. BA alone, or in combi-
nation 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
171
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 embryo-
genesis 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.
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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 detec-
tion 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 References
Pea
Cell division Landgren (1976); Jia (1982)
Callogenesis Constabel et al. (1973); Gamborg et al. (1975); von Arnold
and Eriksson (1976): Kuchuk (1989)
Embryogenesis, Puonti-Kaerlas and Eriksson (1988); Lehminger-Mertens and
organogenesis Jacobsen (1989a); Ochatt et al. (1998)
Plant regeneration Lehminger-Mertens and Jacobsen (1989b); Boehmer et al.
(1995); Sanago et al. (1996), Ochatt et al. (1998)
Soybean
Cell division Kao et al. (1970); Gamborg et al. (1983); Lu et al. (1983);
Tricoli et al. (1986); Hammat and Davey (1988)
Callogenesis Zieg and Outka (1980); Xu et al. (1982); Oelck et al. (1983);
Kuchuck (1989)
Plant regeneration Myers et al. (1989); Guo (1991); Dhir et al. (1991, 1992); Lu
et al. (1993)
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(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 sys-
tem 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 experi-
mental 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, regenera-
tion 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 mainte-
nance 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 proto-
plasts 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
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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 concen-
tration of the carbohydrate in the protoplast culture media (Lehminger-
Mertens 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 concen-
tration 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 synthe-
sis, 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 poly-
mers 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 proto-
plasts. 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, how-
ever, such linkages are less apparent. Williamson et al. (1976) studied the
distribution of carbohydrate residues on the plasma membrane of soybean
175
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 proto-
plasts, indicating a slight modification in cross wall formation during cyto-
kinesis. 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 micro-
tubule spindles, phragmoplast, incomplete cross wall and aberrant chromo-
some 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 abnor-
malities (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
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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 accu-
mulation 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 under-
going 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 hydro-
lases associated with the cell wall. Sucrose and hexose uptake into proto-
plasts 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
177
uptake by protoplasts is composed of three different mechanisms, a satu-

rated, 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 regener-
ate 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 pheno-
typically 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.
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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 de-
differentiation 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 geno-
type 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 mito-
chondrial 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
179
better understanding of the mechanisms and factors of determining varia-

tion 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 improve-

ment, 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).
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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 cytologi-
cal 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
181
166
30 October 2000 15:00:09

Table 6.3. Somaclonal variation in grain legumes on cell, tissue and organ culture level in vitro.
Species Type of in vitro culture Trait Response (value/description) References
Arachis hypogaea Anther-derived callus Chromosome number Mixoploid callus (haploid to Bajaj et al. (1981)
(peanut, groundnut) octoploid cells)
Cajanus cajan Anther-derived callus Chromosome number Haploid, diploid and Bajaj et al. (1980)
aneuploid cells
Cicer arietinum Callus, cell suspension Salt tolerance (NaCl) NaCl-tolerant cell lines Gosal and Bajaj (1984)
Callus Salt tolerance (NaCl) NaCl-tolerant cell lines Pandey and Ganapathy (1984)
Anther-derived callus Chromosome number Haploid to octoploid cells Bajaj and Gosal (1987); Gosal
and Bajaj (1988)
182
Glycine max Cell suspension Resistance (reaction) to Accumulation of a Ebel et al. (1976)
elicitor from Phytopthora phytoalexin glyceolin in
N. Kuchuk et al.
megasperma var. sojae elicitor treated cells

Callus Resistance (reaction) to Bioassay for screening Holliday and Klarman (1979)

P. megasperma var. resistant or susceptible
sojae genotypes in vitro culture
6
Callus; excised Resistance (reaction) to Bioassay for screening Gray et al. (1986); Willmot
cotyledons soybean brown stem rot resistant mutants in culture; et al. (1989)

(Phialophora gregata) high degree of correspondence
culture filtrate between reaction in tissue
culture and glasshouse tests
Callus Resistance to Diaporthe Identification of tolerant Simoni et al. (1995)
phaseolorum culture filtrate; soybean cultivars based on
dual culture with the fungus in vitro test
Phaseolus coccineus Suspensor-derived Chromosome number, DNA Great variability in Bennici et al. (1976)
(runner bean) callus content chromosome number
Phaseolus vulgaris Excised roots, callus, Resistance to Pseudomonas Differential growth inhibition Bajaj and Saettler (1968,
30 October 2000 15:00:10

cell suspension phaseolicola culture filtrate up to 77%; increase in 1970)

abnormal cells; 55-fold
increase in ornithine
Callus from different Ploidy level (DNA content) Mixoploid calli (2C–8C and Haddon and Northcote (1976)
plant parts even higher DNA content)
Anther-derived callus Chromosome number Haploid, diploid and Peters et al. (1977)
polyploid cells
Callus Salt tolerance (NaCl) Salt-sensitive calli and plants Smith and McComb (1981)
Callus Resistance to Pseudomonas Callus screening test; positive Hartman et al. (1986)
syringae pv. phaseolicola correlation between reaction
culture filtrate on callus and plant level
Callus Resistance to Sclerotinia Callus-weight assay; Miklas et al. (1992)
183
sclerotiorum culture filtrate screening of partial
physiological resistance
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within dry bean germplasm

Vigna radiata Callus, cell suspension Salt tolerance (NaCl) NaCl-tolerant cell lines Gosal and Bajaj (1984)

Seedling root Thioproline resistance; NaCl Fivefold increase endogenous Kumar and Sharma (1989)
6
tip-derived resistance levels of free
Callus Proline; elevated tolerance to

NaCl
Vigna sinensis Cell suspension Chromosome number Polyploidy (3n–6n); Ghosh and Sharma (1979)
(cowpea) multinucleate cells
Continued
167
Table 6.3. Continued.
168
Species Type of in vitro culture Trait Response (value/description) References

30 October 2000 15:00:10

Pisum sativum Callus Chromosome number; Polyploid cells (3n, 4n and Kallak and Yarvekylg (1968,
chromosome aberrations; more); multinuclear cells; 1971, 1976, 1977a,b)
mitotic abnormalities karyological abnormalities
Callus Chromosome number Polyploid (up to 12n) and Frolova and Shamina (1974)
aneuploid cells
Immature Chromosome number; Polyploid cells (up to 8n and Mikhailov and Bessonova
cotyledon-derived chromosome aberrations more); chromosomal (1975)
callus aberrations
Pollen callus Chromosome number Haploid and mixoploid Gupta (1975)
(mainly tetraploid) cell
populations
Cell suspension Chromosome number Multinuclear cells, aneuploidy Ghosh and Sharma (1979)
184
Callus culture Chromosome number Highly polyploid cells (32n or Knosche and Gunther (1980);
more) Knosche (1981)
N. Kuchuk et al.
Callus culture Tolerance to the herbicides Calli with improved tolerance Jakel et al. (1990)
Propham and Probanil to herbicides
(O-isopropyl-3-chlor-

phenylcarbamate)
6
Callus, cell suspension Salt tolerance (NaCl) NaCl-tolerant cell lines Gosal and Bajaj (1984)
Callus Chromosome number, Haploid, diploid and Kunakh et al. (1984)

chromosome aberrations polyploid cells (up to 8n)
Meristem-derived and Chromosome number Diploid or aneusomatic Natali and Cavallini (1987a,b)
embryo axis-derived (chromosomal mosaics) shoots
callus; regenerated
shoots
Mesophyll protoplasts Resistance to P. syringae pv. Model system for pea – Akpa and Archer (1994)
pisi Pseudomonas interaction
Vicia faba Suspension Chromosome number Polyploid (4n–8n) and Venketeswaran (1963)
aneuploid cells
Callus Chromosome number Diploid and polyploid cells Frolova and Shamina (1974,
30 October 2000 15:00:11

(mainly 4n) 1978); Shamina and Butenko

(1976)
Callus Chromosome number Polyploid (3n–8n) and Yamane (1975)
aneuploid cells
Callus Chromosome number Binuclear and multinuclear Cionini et al. (1978a,b);
(as DNA content) cells; diploid and polyploid D’Amato et al. (1980)
cells (up to 64C)
Callus Chromosome number Diploid, aneuploid and Papes et al. (1978); Jelaska
polyploid cells et al. (1981)
Callus and cell Chromosome number Diploid, polyploid and Roper (1979)
suspension aneuploid cells
Anther-derived callus Chromosome number Haploid, diploid and Hesemann (1980)
185
(ploidy level measured polyploid cells (more than
cytophotometrically as 16C)
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C-value)
Callus Chromosome number Polyploid and eneuploid cells Ogura (1982)
Callus culture Tolerance to the herbicides Calli with improved tolerance Jakel et al. (1990)

Propham and Probanil to herbicides
6
(O-isopropyl-3-chlor-
phenylcarbamate)

169
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, usu-
ally 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 bacte-
rium (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
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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) devel-
oped 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 for-
mulate 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
187
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, chromo-
some 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 chromo-
somes. 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 amplifi-
cations or loss of nuclear DNA, using diploid plants of two pea cultivars,
regenerated from meristem-derived calli. Cytogenetic analyses showed a
188
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 mito-
chondrial 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
189
Table 6.4. Somaclonal variation in grain legumes on the whole plant level (regenerants and their seed progenies).
174
Species Type of in vitro culture Plant regeneration via Trait Response (value/description) References
30 October 2000 15:00:14

Glycine max Immature zygotic Somatic Qualitative and Albinotic chimaeras (R0), Barwale and Widholm
embryo-derived embryogenesis, quantitative chlorophyll deficiency, (1987)
callus organogenesis traits abnormal leaf morphology,
dwarf plant habit (R1–R3)
Cotyledonary node Proliferation of shoot Qualitative and Plant height, sterility Graybosch et al. (1987)
meristems and de quantitative (R0, R1, R2)
novo shoots traits
(organogenesis)
Immature embryo Somatic Plant Increased variation in R1 Hildebrand et al. (1989)
embryogenesis morphology, (mainly leaf morphology;
lipid lipid composition); not
composition inherited to R2
190
Cotyledonary node- Organogenesis Qualitative Lanceolate leaves, leaf and Freytag et al. (1989)
and epicotyl-derived traits pod variegation, determinate
N. Kuchuk et al.
callus growth habit (R0, R1, R2)

Cotyledonary node- Organogenesis Atrazine Atrazine tolerant plants Wrather and Freytag

and epicotyl-derived tolerance (R0, R1, R2) (1991)
callus
6
Immature cotyledons Direct somatic Qualitative Complete and partial Amberger et al. (1992)
embryogenesis traits sterility, wrinkled and

curled leaves, chlorophyll
deficiency, reduced plant
height, determinate growth
habit, variation in malate
dehydrogenase, aconitase
and diaphorase (R0–R3)
Unknown Unknown Resistance to Resistant regenerants (not Song et al. (1994)
Septoria glycines proven)
culture filtrate
Embryogenic Somatic Resistance to Improved resistance to Jin et al. (1996)
30 October 2000 15:00:14

suspension embryogenesis Fusarium solani pathogen (R1–R3)

culture filtrate
Pisum sativum Root, leaf and Shoot organogenesis Chromosome Various ploidy of callus; Kunakh et al. (1984)
stem primary and number tetraploid plants (R0)
long-term callus
Shoot apical Proliferation of shoot Chromosome Variation in chromosome Ahmed et al. (1987)
meristem meristems number; number; chromosome
chromosome morphology; anaphase
aberrations abnormalities
Immature zygotic Shoot organogenesis Chromosome Diploid and aneusomatic Natali and Cavallini
embryo-derived callus number (chromosomal mosaics) (1987a)
191
plantlets (R0)
Immature zygotic Somatic Chromosome Tetraploid plants (R0) Kysely et al. (1987)
Biotechnology
embryo- and shoot embryogenesis number

apex-derived callus
Shoot apex-derived Shoot organogenesis Qualitative and Leaf mutations waxy and Gostimskij et al. (1985);

long-term callus quantitative chlorotica; more vigorous Ezhova et al. (1989)
6
traits habit; dark green leaves;
oblong leaflets; first flower

position (R1–R5)
Stem and Shoot organogenesis Qualitative Anthocyanin absence; leaf Lutova and Zabelina
leaf-derived callus traits type; plant habit (R0–R1) (1988)
Primary and Shoot organogenesis Seed proteins Increased legumin/vicilin Mikhailova-Krumova
long-term callus and amino acid ratio; altered amino acid et al. (1991)
composition balance (R1)
175
Continued
Table 6.4. Continued.
176
Species Type of in vitro culture Plant regeneration via Trait Response (value/description) References
30 October 2000 15:00:15

Shoot apex-derived Shoot organogenesis Nuclear DNA Genotype-dependent, Cecchini et al. (1992)
callus variation tissue culture induced
DNA content variation in
regenerated plants (R0)
Immature zygotic Somatic Qualitative and Significantly altered leaf Stejskal and Griga
embryo-derived embryogenesis, quantitative and flower morphology; (1992); Griga et al.
callus, young organogenesis traits sterility; lethality (R0); pods (1995); Griga and Létal
leaflet-derived callus and seeds per plant; crude (1995)
protein content (R1–R4)
Arachis hypogaea Cotyledon callus Unknown Resistance to Enhanced resistance of R2 Venkatachalam et al.
Cercosporidium plants (1998)
personatum
192
culture filtrate
N. Kuchuk et al.
Phaseolus vulgaris Shoot meristems Meristem Resistance to Resistant regenerants (not Gantotti et al. (1985)
proliferation phaseolotoxin proven)

Vicia faba (Shoot apical) Meristem Resistance to Correlation between low Thynn et al. (1989)
meristem culture proliferation/multiple Botrytis cinerea, to medium phytoalexin level
6
infected with shoot formation Phytophthora in regenerants and fungal

mycelial suspension megasperma resistance (R0)
and Rhizoctonia
solani
Vigna radiata ‘De-embryonated’ De novo Qualitative Chlorophyll and Mathews et al. (1986)
cotyledons organogenesis traits morphological mutations
(R0, R1, R2)
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 morpho-
logical 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,
193
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 (BA-
stimulated 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 cotyledon-
ary 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 other-
wise 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,
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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, how-
ever, 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 genera-
tion 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 qualita-
tive 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 chlo-
rophyll mutations included chlorina and xantha types, which were lethal
under field conditions. The viable mutations included those with penta-
foliate 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)
195
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.
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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 gen-
eration, 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 com-

plement 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).
197
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 medi-
ated 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), micro-
injection 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
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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 trans-
formation 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 germina-
tion 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 con-
fer 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 electro-
porated 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.
199
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 transfor-
mation 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 summa-
rized 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 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 bio-
synthesis, 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
200
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 (PPT) TOLERANCE 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 non-
transgenic 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 trans-
formants 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.
ATRAZINE 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
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186
30 October 2000 15:00:21

Table 6.5. Useful genes/traits transformed into grain legumes.
Transformation
Species Gene/trait incorporated method References
Glycine max 5-Enolpyruvyl-shikimate-3-phosphate synthase (EPSP); herbicide Agrobacterium Hinchee et al. (1988)
tolerance (glyphosate) – Roundup®
Phosphinotricin acetyl transferase (PAT, bar); herbicide tolerance (PPT) Bombardment Christou and Swain
– Basta® (1990)
202
Lysine-feedback-insensitive bacterial DHDPS/ dapA and AK/lysC; Bombardment Falco et al. (1995)
fivefold lysine increase in the seed
N. Kuchuk et al.
Methionine-rich 2S albumin from Brazil nut; high oleic acid; modified Agrobacterium James (1997)
oil; virus resistance bombardment
Synthetic cryIAc gene (B.t.) – resistance to insect larvae (four species) Bombardment Stewart et al. (1996)

Atrazine resistance (psbA) Direct injection of Yue et al. (1996)
6
DNA into ovary
Bean pod mottle virus (BPMV) coat protein gene – virus resistance Agrobacterium Di et al. (1996)

Soybean mosaic virus (SMV) coat protein gene – virus resistance Agrobacterium Xu et al. (1996)
Phaseolus vulgaris Coat protein from the bean golden mosaic virus (BGMV) – virus Bombardment Russell et al. (1993)
resistance; bar
Antisense sequence of AC1, AC2, AC3 and BC1 genes from bean Bombardment Arago et al. (1996)
golden mosaic geminivirus – virus resistance; methionine-rich 2S
albumin from Brazil nut
Pisum sativum α-amylase inhibitor from common bean (αAI-Pv) – resistance to three Agrobacterium Shade et al. (1994);
species of bruchid beetles; bar – phosphinothricin resistance (Basta®, Schroeder et al. (1995);
Buster®, Herbiace®) Grant et al. (1995);
30 October 2000 15:00:21

Bean et al. (1997)

Heat-stable amylase from Bacillus licheniformis, improved seed starch Agrobacterium Saalbach et al. (1997)
degradation
Chimaeric alfalfa mosaic virus (AMV) coat protein gene – partial Agrobacterium Grant et al. (1998a,b)
resistance to AMV
Cicer arietinum CrylA (c) gene from B. thuringiensis (resistance to pod-borer larvae) Bombardment Kar et al. (1997)
Vicia narbonensis Methionine-rich 2S albumin from Brazil nut (threefold increase of Agrobacterium Pickardt et al. (1995)
seed methionine content)
Heat-stable amylase from Bacillus licheniformis, improved seed Agrobacterium Saalbach et al. (1997)
starch degradation
Yeast invertase, changes in regulation of carbohydrate and protein Agrobacterium Weber et al. (1998)
metabolism during cotyledon development
203
Lupinus angustifolius Sulphur-rich sunflower seed albumin (enhanced methionine level in Agrobacterium Molvig et al. (1997);
Biotechnology
the seed), bar – phosphinothricin resistance (Basta®) Pigeaire et al. (1997)
A3867: AMA: Hedley: First Proof:30-Oct-00 6

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188
30 October 2000 15:00:22

Table 6.6. Transgenic plants in grain legumes.
Species Explant Transformation method Regeneration protocol References
Glycine max Seedling cotyledons Agrobacterium Organogenesis Hinchee et al. (1988)

Pisum sativum Shoot cultures epicotyls Agrobacterium Organogenesis Puonti-Kaerlas et al. (1990)
Phaseolus vulgaris Embryo axis Bombardment Organogenesis Russell et al. (1993)
Arachis hypogaea Embryogenic callus Bombardment Somatic embryogenesis Ozias-Akins et al. (1993)
204
Cicer arietinum Seed-derived embryos Agrobacterium Organogenesis Fontana et al. (1993)
Vicia narbonensis (narbon Epicotyl segments Agrobacterium Somatic embryogenesis Pickardt et al. (1995)
N. Kuchuk et al.
bean, French vetch)

Lens culinaris Nodal meristems in planta Electroporation in vivo Seed production without Chowrira et al. (1995)

in vitro culture
Vigna unguiculata Mature cotyledons Agrobacterium Organogenesis Muthukumar et al. (1996)
6
Lupinus angustifolius Maturing embryo axis Agrobacterium Organogenesis Pigeaire et al. (1997)

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 resis-
tance 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).
BACILLUS THURINGIENSIS ENDOTOXINS 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 bom-
bardment (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.
AMYLASE INHIBITORS 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.
205
Shade et al. (1994) and Schroeder et al. (1995) transferred the gene encod-
ing 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 resis-
tance 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 trans-
formed 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
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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 pig-
mentation. Within grain legumes, most reports relate to the improvement
of oils and proteins by genetic transformation. Weber et al. (1998), how-
ever, 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 embryo-
genesis 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 bio-
synthetic 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 trans-
formation 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 contrib-
uting 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,
207
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 albu-
min (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 herbicide-
tolerant varieties, has a leading position in this context. The progress in
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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 carbo-
hydrates, 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 1. Improved disease resistance

2. Hybrid technology 2. Improved disease resistance
3. Hybrid technology and
herbicide tolerance
Maize 1. Control of corn-borer 1. Control of Asian corn-borer
2. Herbicide tolerance 2. Control of corn rootworm
3. Insect protected/herbicide 3. Disease resistance
tolerance
4. Hybrid technology 4. Higher starch content
5. Hybrid/herbicide tolerance 5. Modified starch content
6. High lysine
7. Improved protein
8. Resistance to storage grain pests
9. Apomixis
Soybean 1. Herbicide tolerance 1. Modified oil
2. High oleic acid 2. Insect resistance
3. Virus resistance
Pea None 1. Insect resistance – pea weevil
2. Quality – sunflower albumin
3. High methionine protein
4. Antifungal genes
Lupin None 1. Insect resistance – pea weevil
2. Virus resistance (BYMV)
3. Herbicide tolerance
4. High methionine protein
Chickpea None 1. Herbicide tolerance
2. High seed protein
Lentil None 1. Herbicide tolerance
209
Table 6.8. Global area (millions of hectares) of transgenic crops grown in 1996
and 1997, by crop (James, 1997).
1996 1997
Increase
Crop Area % Area % 1996/7 ratio
Soybean 0.5 18 5.1 40 10.0

Maize 0.3 10 3.2 25 11.0
Tobacco 1.0 35 1.6 13 1.6
Cotton 0.8 27 1.4 11 1.8
Canola 0.1 5 1.2 10 9.5
Tomato 0.1 4 0.1 1 2.0
Potato < 0.1< < 1< < 0.1< < 1< 3.0
Total 2.8 100 12.8 100 4.5
Table 6.9. Global area (millions of hectares) of transgenic crops grown in 1996
and 1997, by trait (James, 1997).
1996 1997
Increase
Trait Area % Area % 1996/7 ratio
Herbicide tolerance 0.6 23.5 6.9 54 10.7

Insect resistance 1.1 37.5 4.0 31 3.8
Virus resistance 1.1 40.5 1.8 14 1.6
Insect and virus resistance – – < 0.1< < 1< –
Quality traits < 0.1< < 0.1< 0.1 < 1< 2.0
Total 2.8 100.5 12.8 100 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
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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 nec-
essary 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-6-
phosphate (Hill and Smith, 1991) in storage tissues. Most of the glucose-6-
phosphate 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 phosphory-
lated to fructose-6-phosphate by fructokinase and possibly by hexokinase
(Renz and Stitt, 1993). The hexose monophosphates are freely intercon-
verted by the action of phosphoglucomutase and phosphoglucoisomerase.
The carbon then enters the amyloplast at the level of hexose monophos-
phates (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
211
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 starch-
branching 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
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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 deter-

mining 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 incorpora-
tion of phosphate into starch-like glucans has been cloned and incorpo-
rated 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 accumula-
tion, 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 photo-
assimilates into the storage organs of major crop plants, sucrose meta-
bolism is of particular interest. Two classes of sucrose cleavage enzymes,
invertase and sucrose synthase, are present in plants.
213
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 accumula-
tion in legume species. The size of potato tubers was increased by over-
expression 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 bio-
synthesis 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 oligosac-

charides are covered elsewhere (see Chapters 2, 3 and 5). As with the sec-
tion on starch, it is necessary to present information on these compounds
here to make it easier to discuss their possible genetic manipulation.
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Biotechnology 199
The biosynthesis of the raffinose family of oligosaccharides (RFO) has

been fairly well characterized. The committed reaction of raffinose bio-
synthesis 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 micro-
organisms, plants and animals (Dey and Pridham, 1972). Genes encoding
a number of α-galactosidases have been cloned and sequenced. Many of
215
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 Lacto-
bacillus 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 (Green-
wood 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 commer-
cial 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.
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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 inter-
fering 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 polysaccha-
rides and N- and O-linked glycoproteins. Most plant cells have only a pri-
mary 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
217
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 micro-
fibrils, 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 polysac-
charides 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 polysac-
charides (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
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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 oligo-
saccharides 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 oligo-
saccharide fragments can be generated in vivo from plant cell wall poly-
saccharides, 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 frag-
ments 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
219
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 oligosac-
charides 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. Further-
more, 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
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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 metabo-
lites 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 polysaccha-
rides and starch biosynthesis and turnover. Photoautotrophic suspension
221
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 influ-
ence 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 func-
tion 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.
222
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).
223
7
Breeding
G. Engqvist
andet 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, there-
fore, 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

225
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 ‘semi-
leafless’ 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. Earli-
ness 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 ten-
dency 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.
226
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 compo-
nents 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
227
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 selec-
tion 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
228
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 predic-
tions 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 differ-
ent 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
229
Table 7.1. European central crop databases and management centre.
Species Database management centre
Vicia faba INRA, Le Rheu, France

Cicer arenatum National Plant Breeding Station, Elvas, Portugal
Glycine max N.I. Vavilov Research Institute of Plant Industry, St Petersburg,
Russia
Lens spp. Aegean Agricultural Research Institute, Izmir, Turkey
Pisum spp. John Innes Centre, Norwich, UK
Wiatrowo, Wagrowic, Poland
Phaseolus spp. Federal Office of Agrobiology, Linz, Austria
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 demon-
strated 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
230
Table 7.2. Management centres within Europe for the main grain legume databases.
30 October 2000 15:00:41

Organization/institute Acronym Country Number of accessions
Aegean Agricultural Research Institute ARA Turkey 88

Instituto del Germoplasma BAR Italy 4297
Institut für Pflanzenbau und Pflanzenzuchtung BRN Germany 1009
Centre for Genetic Resources CGN Netherlands 1008
Department of Agriculture and Fisheries for Scotland ECS Scotland 1980
Zentral Institut für Genetik und Kulturpflanzen-forschung GAT Germany 2478
Centre de Recherches Agronomiques de L’Etat GBX Belgium 348
International Centre for Agricultural Research in the Dry areas ICA Syria 3318
Instituto Nacional de Investigaciones Agrarias IMD Spain 256
John Innes Centre JII UK 3008
231
Institute of Plant Breeding JOK Finland 17
Agrobiologische Institute LIN Austria 33
Nordic Gene Bank for Agricultural and Horticultural Plants NGB Sweden 3055
Breeding and Agronomy
Pea and Lentil Breeding Programme RAB Morocco 65

Institute of Plant Introduction and Genetic Resources SAD Bulgaria 1598
Research and Breeding Institute of Technical Crops and Legumes SUM Czech Republic 937
7
Institute for Plant Production and Qualification TAP Hungary 934
Vegetable Crops Research Institute, Research Station Ujmajor UJM Hungary 748

Servicio de Investigacion Agraria VAL Spain 491
N.I.Vavilov All-Union Scientific Institute of Plant Industry VIR Russia 6518
Israel Genebank for Agricultural Crops, Volcani Centre VOL Israel 690
Pisum Gene Bank WTD Poland 2899
215
Table 7.3. Storage component analysis of 227 lines of round-

seeded peas expressed as a percentage of the dry weight.
Mean Range CV%
Starch 45.9 32.7–54.5 7.75

Soluble sugars 5.3 3.9–8.2 11.51
NDF 16.7 11.8–26.3 15.18
ADF 8.3 5.9–12.7 15.51
CV, coefficient of variation.
understanding the synthesis of starch have been made following the identi-
fication and development of mutants within the starch biosynthetic path-
way. 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 composi-
tion. 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
232
Fig. 7.2. 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 partic-
ular 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
233
Table 7.4. Pea loci and alleles affecting starch

synthesis and composition (Wang et al., 1998).
Loci Number of alleles Starcha Amyloseb
r 10 27–36 60–75
rb 8 30–37 23–32
rug3 5 1–12 12
rug4 3 38–43 31–33
rug5 3 29–35 43–52
lam 5 39–49 4–10
aStarchis given as a percentage of the dry weight.
bAmylose is given as a percentage of the starch
on a dry weight basis.
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.
234
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
235
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 differ-
ent 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 non-
destructive 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.
236
Fig. 7.3. Continued
237
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
238
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.
239
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 polychro-

matic beam from a tungsten lamp, which is passed through a monochroma-
tor 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 finger-
print. 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.
240
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 dur-
ing daytime periods of low photosynthesis, stored sucrose is made available,
241
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. Carbohy-
drate 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 carbo-
hydrate 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 devel-
oping 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
242
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
243
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
Table 7.5a. The registration requirements of several European countries for new
varieties of pea – agronomic characters.
Czech Republic
Denmark
Germany
Moldova
Romania
Hungary
Bulgaria
Ukraine
Sweden
Estonia
Poland
France
Spain
Italy
Agronomic character UK
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 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
244
Table 7.5b. The registration requirements of several European countries for new
varieties of pea – processing characters.
Czech Republic
Denmark
Germany
Moldova
Romania
Hungary
Bulgaria
Ukraine
Sweden
Estonia
Poland
France
Spain
Italy
UK
Processing character
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 ✓
Table 7.5c. The registration requirements of several European countries for new
varieties of pea – chemical characters.
Czech Republic
Denmark
Germany
Moldova
Romania
Hungary
Bulgaria
Ukraine
Sweden
Estonia
Poland
France
Spain
Italy
UK
Chemical character
Trypsin inhibitor activity (TIU) ✓ ✓

Amino acid content:
lysine, methionine, cystine, alanine ✓
Protein content of seed ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
Protein yield from seed ✓ ✓ ✓ ✓
245
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
246
(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 cater-
pillar 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).
247
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).
248
8
Manipulating
C. Hedley Grain Legume Carbohydrates
Strategies for Manipulating 8

Grain Legume Carbohydrates
. . . 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 impor-
tance to the development of legume crops. A major problem with improv-
ing 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 pro-
tective agents against attack by insects or disease-forming microorganisms.
They may also have a positive effect on the consuming organism. For exam-
ple, 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 oligosaccha-
rides (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

249
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 non-
digestible 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
250
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 con-
sumed 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 men-
tioned 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
251
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 com-
pounds 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, demon-
strating 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).
252
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 charac-
teristics, 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 carbohy-
drates and starch. In this case, it is a primary requirement to determine
which compounds are the most important from a nutritional/health point
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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 carbohy-
drates 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
254
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. The current knowledge of starch genetics, chemistry and
granular structure, based on pea, has opened the door to the development
of an almost infinite selection of starches that could be produced to suit a
wide range of food and non-food applications.
255
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Index
Index
Index
α-amylase 31, 46–49, 139 ileal digestibility 72–73, 80

acid detergent fibre (ADF) 48–49, 57 intestinal transit 73
adzuki bean 19–20, 32 monogastric nutrition 72–73
α-D-galactosyl moiety 125 nutrients absorption 73
α-galactosidase genes 199–201 see also unavailable carbohydrates
thermostable α-galactosidase gene α-galactoside biosynthesis gene(s)
200 199–201
α-galactoside 17–19, 31, 63, 68, 71–73, galactinol synthase (GS) gene
80 200
consumption 69–70, 82–83 stachyose synthase gene 200
animal diets 69–70 α-D-glucose-6-phosphate 126
food 82–83 α-glucosidase 139–140
content in animal diet 70 α-glucosidic bonds 140
content in legume dishes 83 aglycon 28, 30
content in seeds 63–64, 67–68 agronomy 209–232
beans 63 agronomic characters 228–231
chickpea 63–64 chemical characters 232
faba bean 63–64, 68 technological characters 231
lentil 63–64, 68 ajugose 17
lupin 68 aldoses 16, 53
pea 63, 68 amorphous 94, 96, 103–104, 106
losses during cooking 83 β-amylase 139
nutritional properties 71–74 amyloglucosidase 46
antinutritional effect 61 amylopectin 23–24, 130
elimination of negative effect amylose 23, 47–48, 130
72–73 arabinose 26–27
gas production 78 Arachis hypogaea 30
315
331
14 November 2000 14:32:47 A3867: AMA: Hedley: First Proof:14-Nov-00 Index
316 Index
available carbohydrates 79, 84–85 carbohydrates 5, 12, 22, 26, 28, 30–31,
classification 79 34, 37–43, 45, 49, 53–54, 56,
nutritional properties 84–85 120
glycaemic index 84 accumulation 122
see also mono- and disaccharides, biosynthesis 125–127
starch chemistry 15
extraction procedure 34
GC determination 35
bean 8 physiological role 131–132,
adzuki 19–20, 32 134–138
broad 26, 122 unloading of 118
brown 26 cell wall 25–28, 47, 49–50, 52, 54
butter 30 preparation 50
common 3, 123 cell wall components 127, 202
dry 20 biosynthesis 159–160, 202–203
faba 3, 8, 17, 122, 130 oligosaccharides 203–204
field 30 cellulose 25, 50, 52, 55
garden 20 content in seeds 65
green 29 chemical analysis 31
haricot 29 chickpea 2, 8, 19–21, 30, 32, 122
jack 3 D-chiro-inositol 18–19, 23, 32, 133
kidney 21, 29–30 Cicer arietinum 2, 8, 20–21, 30, 32,
lima 29, 123 214
mung 2, 20, 29–30, 32, 123 ciceritol 20–22, 32, 126, 236
navy 29 content in seeds 63–64
pinto 29 chickpea 63–64
runner 29 lentil 63–64
D-bornesitol 19 common bean 3
breeding 209–232 consumption of grain legumes 7–11
contradiction of goals 210 cooking of legume starch 112–113
goals 209–211, 235 extrusion 113
pedigree 211 high pressure 112
physical selection 222 low pressure 112
selection methods 220–222 cowpea 3, 19–20, 122, 123
Brabender viscograph 100, 103 crude fibre 48
broad bean 26, 123 crystallinity 94, 96–97, 100–101
brown bean 26 biaxial crystalline polymers 94
butter bean 30 cyclitols 31–32, 127, 135–137, 143
occurrence of 32
Cajanus cajan 2, 20
calculation and statistical analysis 37, debranching enzymes 140
40, 43 delignification 54, 57
Canavalia ensiformis 3 desiccation 131
capillary zone electrophoresis (CZE) 42 injury 143
advantages 43 stress 124, 132
disadvantages 43 tolerance 131, 141
recommended method 43 developmental stages 120
332
Index 317
dietary fibre (DF) 25, 46, 48 free radicals 136

differential scanning calorimetry DSC fructose 16–17, 26–27, 31, 44
98 content in seeds 63–64
digalactosyl myo-inositol 133 faba bean 63–64
disaccharides 16, 45, 71, 124 lentil 63–64
see also sucrose see also monosaccharides 71
dishes 62, 83, 86–87 fucose 26
amount of legumes 87 functional properties 98–101
consumer acceptance 87 gelatinization 98
flatulence 62, 86 melting 98
intake of α-galactosides 83 pasting 98
preventive effect 62 fungal disease(s) 228–230
dry bean 20
galactinol 19–21, 32, 125–126

embryo synthase 125
composition 119 galactinol series 21
role 119 galactociceritol 20
endosperm galacto-cyclitols 20, 31
degradation 119 di-galacto-inositol 20
development, role 119 galactomannans 26, 135
enzymic method 45–46, 49 galacto-ononitol 20, 32
exoamylase 139 galactopinitol A 19–22, 32
extraction 31 galactopinitol A series 22
recommended procedure 34 galactopinitol B 22
solvents 31, 34 galactopinitol B series 22
temperature 31 galactopinitols 19
galactose 19, 26–27, 31
galactoside moieties 140
faba bean 3, 8, 17, 20, 29, 122, 130 galactosyl cyclitols 127
fagopyritol B series 23 galactosyl ononitol 126
fagopyritol B1 19–20, 23, 32 galactosyl ononitol series 21
fagopyritol B2 20, 23 galactosyl pinitol 126
fagopyritol B3 23 D-galacturonic acid 26–27, 56
fibre 11, 25, 46, 48, 58, 65–67, 83, galacturonosyl residues 28
85–86, 234, 237 garden bean 20
composition 69 gas chromatography GC 35, 57–58
consumption in food 83 advantages 37, 43
content in seeds 65–67 disadvantages 37, 43
fibre fractions 65 recommended procedure 35
genetic variation 216 gelatinization 23–24
physiological effect 85 germplasm banks 213
see also non-starch polysaccharides, Pisum 213
unavailable carbohydrates glucomannans 25, 54
field bean 30 glucose 16–17, 24–27, 31, 44–45
field pea 34 Glycine max 2, 20, 26, 30, 32, 214
food application of legume starches glycosidic bond/linkage 16, 23–24,
109–110 26–27, 57
333
30 October 2000 15:02:01 A3867: AMA: Hedley: First Proof:30-Oct-00 Index
318 Index
granular structure of legume starches legume seeds 17–20, 28–29

93–97 Leguminosae 19, 36
amylose 93–94, 97, 102–103, Lens culinaris 3, 8, 30, 32, 214
106–108, 110–111, 116 lentil 3, 8, 17, 19–21, 29–30, 32, 122
amylopectin 94, 97, 103, 107, 111 leucaenitol 19
double helix 94–95, 97, 103 lignin 25, 28, 50, 54–55
A-type polymorph 95–97 content in seeds 65
B-type polymorph 95–97 determination 57
C-type polymorph 95–97 lima bean 29, 121, 123
green bean 29 lucerne 20, 21, 32, 135
lupin 2, 8, 17, 19–21, 26
Lupinus albus 2, 20, 26
haricot bean 29, 30 Lupinus angustifolius 2
hemicellulose 16, 25, 28, 50, 52, 54–55 Lupinus luteus 2, 20, 45
content in seeds 65 Lupinus mutabilis 2
extraction 54 Lupinus spp. 8
hexoses 16, 50
high performance anion chromatography
(HPAC) 39 Maillard’s reactions 136
Dionex system 39 maltase 16
high performance liquid chromatography maltose 16, 31
(HPLC) 38, 57–58 mannans 25
advantages 41, 43 mannose 26–27, 54
affinity chromatography 40 Medicago sativa 20, 32
disadvantages 41, 43 melezitose 31
ion moderate partition 39 methyl cyclitols 32
normal phase 39, 56 mid-infrared spectroscopy 225
recommended method 40 mimositol 32
reverse phase 39 modified starch 101–109
biotechnological 108–109
fermentation 109, 114–115
inositol 18 germination 108, 114
invertase 16 hydrolysis 108
in vitro cultures 146–148 chemical 104–107
cell suspension culture 201–208 acetylation 104
isolation of native starch 89 cationization 107
dry processing 89–91 cross-linking 106
wet processing 89–90 hydroxipropylation 106
phosphorylation 105
physical 102–104
jack bean 3 annealing 103
extrusion 103
gamma irradiation 103
ketoses 16, 53 steaming 102
kidney bean 21, 29–30 monosaccharides 16, 31, 34, 57, 71
free monosaccharides 124
fructose 63–64, 120, 124, 136, 143
lactose 16, 31, 43 galactose 124, 136
LEA proteins 132 glucose 124, 136, 139–140, 143
334
Index 319
muco-inositol 18 mutant 17
mung bean 2, 20, 29–30, 32, 123 production 209
myo-inositol 18, 20–21, 31–32, 36, 125, peanut 30, 123
128 pectic acid 27
D-myo-inositol-1-phosphate 126 pectic substances 26–27, 55
occurrence of 29
pectin 16, 25–26, 28, 50, 52, 55
Nastar R 116 pectinic acid 27
Nastar R Instant 116 pentoses 16, 50
native starch 102, 104, 106, 110, 116 Phaseolus aureus 30
navy bean 29 Phaseolus coccineus 30
near-infrared (NI) spectroscopy 224 Phaseolus lunatus 30
neutral detergent fibre NDF 48 Phaseolus vulgaris 3, 6, 20, 26, 30, 214
non-food use 98 phenyl α-D-glucoside 31
non-starch polysaccharides 69, 76–78 photoassimilates, supply of 119
composition 69, 77 pigeon pea 2, 19–20, 122
content in seeds 69–70, 76 D-pinitol 19–20, 22, 31–32, 126
nutritional properties 76–78 pinto bean 29
digestibility of energy 78 Pisum sativum 3, 8, 20, 22, 26, 30,
eliminate of negative effect 214
76 plant genetic transformation, methods
energetic effect 76 181–195
gas production 78 Agrobacterium-mediated
ileal digestibility 76–77 transformation 183
intestinal transit 77 electroporation 184
nutrient absorption 77–78 microinjection 184
see also fibre, unavailable particle bombardment 183–184
carbohydrates plant regeneration 148–156
organogenesis 151–153
pea regeneration 149
oligomers 126 somatic embryogenesis 150–151
oligosaccharides 16, 25, 45, 71, 124 polymerization, degree of 103, 108
accumulation 121 polysaccharides 15–16, 22, 27, 45
degradation 140 cellulosic 25
α-galactosides 63, 68, 71–73, 80 non-cellulosic 25
rathinose 120, 123, 125, 132, 135, protopectin 27
137, 140, 143 protoplasts culture 156–158
RFO 125–126, 132–134, 136, 140
stachyose 120, 123, 125, 132, 135,
137, 140, 143 raffinose 16–19, 31–32, 36, 40–41,
verbascase 120, 123, 132, 137, 140 63–65, 68, 71
ononitol 126 antinutritional effect 71
D-ononitol 19–21, 32 content in seeds 63–64, 68
optical rotation 44–45 beans 63
chickpea 63–64
faba bean 63, 65, 68
pea 3, 8, 20, 26, 29–30, 122, 123 lentil 63–64, 68
breeding programmes 210, 211 lupin 68
hulls 29 pea 63, 68
335
320 Index
raffinose continued quality 211

synthase 125 recalcitrant 138
see also α-galactoside 63, 68, 71–73, stage of development 120
80 size 210
raffinose family of oligosaccharides storage 137
(RFO) 11, 16–18, 31, 33, structure 117
40–42, 125, 233 viability 137
rapid visco-analyser (RVA) 100 vigour 137
reducing sugar method 44 yield 210
resistant starch (RS) 46, 80–83, zygotic 131
110–111, 235 seeds
Berry method 47 desiccation tolerance 131
consumption in food 83 mature 140
content in legume products 81–82 sequoyitol 19, 32, 128
definition 80 soaking 112
determination 46 soluble carbohydrates 11, 15–16, 31,
method of determination 81 235
nutritional properties 80 accumulation 122
retrograded starch (RS III) biosynthesis 125–127
110–111 extraction from seeds 31
see also fibre, unavailable physiological role 131–132,
carbohydrates 134–138
rhamnopyranose 28 soluble sugars 63–64, 67–68
rhamnose 26 content in seeds 63–64, 67–68
round pea 36 beans 63
rug3 17, 234 chickpea 63–64
runner bean 29 faba bean 63–64, 68
lentil 63–64, 68
lupin 68
saccharides 16, 42 pea 63, 68
sample clean-up 38 genetic variation 216, 219
sample preparation 31 see also oligosaccharides,
sapogenin 28 α-galactoside
sapogenol 58 somaclonal variation 162–181
saponins 28, 58 biochemical changes 173
determination 58 cytological instability 165, 170,
extraction from seeds 58 172–173
genetic variation 219 product quality changes 181
scyllo-inositol 18, 32 stress resistance 170–172, 180
seed yield characters 177–179
coat 34, 131 somatic embryos 135
colour 210 soybean 2, 19–20, 26, 29–30, 32, 120,
components 117 122, 123
desiccation tolerance 131–132, spectrophometric method 45
135–136, 143 stachyose 16–19, 31, 33, 36, 40–41,
development 119–122, 225–227 63–65, 68
full maturity 121, 130 content in seeds 63–64, 68
germination 142 beans 63
orthodox storage 137 chickpea 63–64
336
Index 321
faba bean 63, 65, 68 pyrophosphatase gene 199

lentil 63–64, 68 starch phosphorylation gene
lupin 68 198
pea 63, 68 sucrose synthase gene
synthase 125 198–199
see also oligosaccharides, starch synthase gene(s) 196–197
α-galactoside starch branching enzyme gene(s)
standards 31 197–198
starch 11, 16, 22, 63–65, 74–76, 80–83, starch ethers 104
234, 237 stress proteins 132
accumulation 120, 128, 130 substitution 104, 106–107
animal nutrition 74–76 sucrose 11, 16–19, 31, 33, 36, 41,
biochemistry 130 63–64, 67–68, 118
calcium chloride method 46 content in seeds 63–64, 67–68
consumption in food 82–83 beans 63
content in seeds 63–65, 68, 121 chickpea 63
beans 63 faba bean 63, 68
chickpea 63–64 lentil 63, 68
faba bean 63–65, 68 lupin 68
lentil 63–64, 68 pea 63, 68
lupin 68 see also disaccharide
pea 63–64, 68 sugar 16, 44, 56–57
degradation 138–139 sweet lupin 2
determination 45 Swelite R 116
feed processing 75–76 swelling properties of 92, 100–103,
genetic variation 216–219 105–106, 109–111
glucose hydrolysis method 46
granules 23, 90, 92–96, 98,
101–104, 106, 108, 111, temperature stress 136
115, 130 test kits
size of 90 glucose 44
nutritional classification 80 medical/food 44
rapidly digestible 80 Megazyme 47
resistant starch 80–81, 235 testa 34
slowly digestible 80 role 117–118
nutritional properties 74–76, thin layer chromatography (TLC) 42,
80–81 58
energetic value 76 advantages 42
ileal digestibility 74–76, 81 recommended method 42
rapidly digestible (RDS) 47 transgenic grain legume plants, field
retrograded 47 trials 192–195
role 128 transgenic traits 184–195
slowly digestible (SDS) 47 herbicide tolerance 184–195
see also available carbohydrates insect resistance 189–190
starch biosynthesis genes 195–199 nutritional quality 191–192
ADP-glucose pyrophosphorylase virus resistance 190–191
gene(s) 195 tri-galactopinitol 20, 22
genes influencing starch 198–199 tri-galactopinitol A 22, 135
invertase gene 198–199 tri-galactopinitol B 22
337
322 Index
trimethylsilylation TMS 35–36, 59 Vigna angularis 20, 32

triterpene glycosides 30 Vigna radiata 2, 20, 32
Vigna unguiculata 3, 20
viscosity 97–98, 100–104, 106, 109–110,
unavailable carbohydrates 79–80, 116
85–86
classification 79–80
nutritional properties 85–86 water-insoluble cell wall components
cholesterol metabolism 85 78
glycaemic index 84 content in seeds 78
see also α-galactoside, fibre nutritional properties 78
uronic acid 25–26, 50 digestibility of energy 78
gas production 78
see also fibre, unavailable
verbascose 16–18, 31, 36, 40, 63–64, 68 carbohydrates
content in seeds 63–64, 68 white lupin 2
beans 63
chickpea 63–64
faba bean 63, 68 xylans 25–26
lentil 63–64, 68 xyloglucans 25–26
lupin 68 xylose 25–27
pea 63, 68
see also α-galactoside
Vicia faba 3, 8, 20, 26, 30, 45, 119, 120, yellow lupin 2, 122, 132–134
214
338

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CARBOHYDRATES IN GRAIN LEGUME SEEDS

Carbohydrates in Grain Legume

CABI Publishing is a division of CAB International

CABI Publishing CABI Publishing

Tel: +44 (0)1491 832111 Tel: +1 212 481 7018

© CAB International 2001. All rights reserved. No part of this publication

Library of Congress Cataloging-in-Publication Data

SB177.L45 C27 2000

ISBN 0 85199 467 9

Typeset by AMA DataSet Ltd, UK.

5 Seed Physiology and Biochemistry 117

6.5 Transformation Methods in Grain Legumes 181

7 Breeding and Agronomy 209

7.5 Physical Screening Methods 222

8 Strategies for Manipulating Grain Legume Carbohydrates 233

Mr Mike Ambrose, John Innes Centre, Norwich Research Park, Colney,

Dr Jana Dostalova, Dept of Food Chemistry and Analysis, Prague

Dr Saima Kalev, Jogeva Plant Breeding Institute, EE 2350 Jogeva, Estonia.

Dr Paolo Ranalli, Istituto Sperimentale per le Colture Industriali, Via di

Grain legumes have been an important part of the social evolution of

1.1 The Grain Legumes

1.2 Grain Legume Production

©CAB International 2001. Carbohydrates in Grain and Legume Seeds

14 November 2000 14:29:35

Common name Latin name Description

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A3867: AMA: Hedley: First Proof:30-Oct-00

30 October 2000 15:58:48

1997 1998 1999

Area Production Area Production Area Production

World total 68,324,137 55,512,759 66,913,556 56,143,600 68,320,568 57,514,450

Austria 68,327,783 68,386,282 68,327,043 68,385,254 68,327,333 68,385,600

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Lithuania 68,352,300 68,106,000 68,366,100 68,104,100 68,352,300 68,106,400

United Kingdom 68,196,800 68,748,000 68,201,200 68,701,000 68,188,500 68,373,200

A3867: AMA: Hedley: First Proof:30-Oct-00 1

Grain legumes Soybean Total legume production

1.3 Grain Legume Consumption

most of the home-produced and imported seed (about 95%) of chickpea,

Country 1990 1991 1992 1993 1994 1995

Austria 3.6 3.5 3.5 2.4 3.4 3.7

Production Import Food use Feed use

Pea (P. sativum) 4800 800 4 91

Grain legumes Other protein sources

Share in protein crops 25.0 75

Total Compounded feed Share in feed

Belgium 5,661 5,325 12.4

Table 1.8. Consumption of grain legumes for human nutrition in regions,

Northern and Western Europe 1.37 0.53 0.41 1.0–4.9 2.43

1.4 Grain Legume Carbohydrates

Protein Oil Carbohydrates

Legume species Min. Max. Min. Max. Min. Max.

Soybean 38.4 19.7 32.5

Table 1.10. Carbohydrate composition (% of seed) of grain legumes.

Legume species Total Starch Sucrose Raffinose Stachyose Verbascose ‘Fibre’a

Soybean 32.5 1.5 6.2 0.9 4.3 0.1 20