• TABLE OF CONTENTS
HIDE
 Front Cover
 Title Page
 Copyright
 Table of Contents
 Foreword
 Opening address
 Welcoming statement
 I. Farmer participatory breeding...
 II. Gene discovery and novel...
 III. Germplasm characterization...
 IV. Physiological approaches
 V. QTL indentification
 VI. Marker-assisted selection
 Annex A : program
 Annex B : list of participants






Group Title: Resilient crops for water limited environments : proceedings of a workshop held at Cuernavaca, Mexico, 24-28 May 2004
Title: Resilient crops for water limited environments
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Permanent Link: http://ufdc.ufl.edu/UF00077531/00001
 Material Information
Title: Resilient crops for water limited environments proceedings of a workshop held at Cuernavaca, Mexico, 24-28 May 2004
Physical Description: xvi, 286 p. : ill. (some col.) ; 28 cm.
Language: English
Creator: International Maize and Wheat Improvement Center
Publisher: CIMMYT
Place of Publication: Mexico D.F
Publication Date: c2004
 Subjects
Subject: Crops -- Drought tolerance -- Congresses   ( lcsh )
Crops -- Genetic engineering -- Congresses   ( lcsh )
Genre: bibliography   ( marcgt )
conference publication   ( marcgt )
non-fiction   ( marcgt )
 Notes
Bibliography: Includes bibliographical references.
Statement of Responsibility: D. Poland ... et al., editors.
 Record Information
Bibliographic ID: UF00077531
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: oclc - 58563826
lccn - 2005359166
isbn - 9706481249

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Table of Contents
    Front Cover
        Front cover
    Title Page
        Page i
    Copyright
        Page ii
    Table of Contents
        Page iii
        Page iv
        Page v
        Page vi
        Page vii
        Page viii
        Page ix
        Page x
    Foreword
        Page xi
    Opening address
        Page xii
        Page xiii
        Page xiv
    Welcoming statement
        Page xv
        Page xvi
    I. Farmer participatory breeding and economic studies
        Page 1
        Breeding for improved drought tolerance in maize adapted to southern Africa
            Page 1
        Rice seed systems in Southern China : Views from institutions and farmers
            Page 2
            Page 3
        Adaptability of 16 upland rice varieties to two moisture regimes
            Page 4
            Page 5
        Farmers' participatory varietal selection at target drought prone area of Tamil Nadu
            Page 6
            Page 7
        Farmers' participatory plant breeding technique : An effective tool for the early selection and adoption of rice varieties in rainfed rice ecosystems
            Page 8
            Page 9
        Participatory maize variety evaluation for increased adoption
            Page 10
        Economic costs of drought and rice farmers' coping mechanisms : A synthesis of cross-country comparative analysis
            Page 11
            Page 12
        Development and dissemination of drought tolerant rice varieties through on-farm, farmer-oriented approaches
            Page 13
            Page 14
        Drought and cropping pattern change in Tamil Nadu, India : Needed technological transformation in rice farming
            Page 15
            Page 16
            Page 17
            Page 18
        Varietal adoption and farmers' coping strategies in rainfed rice ecosystems of Tamil Nadu
            Page 19
            Page 20
            Page 21
            Page 22
        Application of maize mega-environments in seed systems in the Southern African development community
            Page 23
        Breeding drought tolerant varieties of rice through participatory plant breeding for the rainfed uplands
            Page 24
            Page 25
            Page 26
        Reaching the poor maize farmers in the hills of Nepal : Experiences and achievements of the Hill Maize Research Project (HMRP)
            Page 27
            Page 28
            Page 29
        An international partnership for the breeding and delivery of drought-tolerant rice varieties by market-oriented plant breeding and marker-assisted selection
            Page 30
            Page 31
    II. Gene discovery and novel approaches
        Page 32
        Heritability of rice yield under reproductive-stage drought stress, correlations across stress levels, and effects of selection: Implications for drought tolerance breeding
            Page 32
            Page 33
            Page 34
        Empowering rice drought gene discovery activities through bioinformatics
            Page 35
            Page 36
        Functional analysis of plant hydrophilins
            Page 37
            Page 38
        Rice SNP map between indica and japonica subspecies : DNA marker resolution on the kilobase scale
            Page 39
            Page 40
        XVSAP1 from Xerophyta viscosa improves salinity and water deficit stress tolerance in Arabidopsis and tobacco
            Page 41
            Page 42
        Molecular dissection on rice photosynthesis-related traits at reproductive stage in irrigated and drought conditions
            Page 43
            Page 44
        Identification of SSR markers linked to candidate genes for drought tolerances in rice
            Page 45
            Page 46
        Monitoring the transcriptome changes of 14013 rice unigenes in responses ti drought by cDNA microarray
            Page 47
            Page 48
        Molecular responses to dehydration and sailinity in rice : differences and cross-talk between two stress signaling pathways
            Page 49
            Page 50
        The primary studies on gene expression in drought tolerance or sensitive rice cultivars in different water conditions
            Page 51
            Page 52
        Effects of the brachytic-2 dwarf gene on maize (Zea mays) root systems and grain yield under moisture stress
            Page 53
            Page 54
        Towards the improvement of abiotic stress tolerance in maize using genes isolated from the monocotyledonous resurrection plant Xerophyta viscosa
            Page 55
        Gene mining of African rice germplasm (Oryza glaberrima and Oryza sativa) to improve drought resistance in rainfed production systems for resource poor farmers of Africa
            Page 56
        Structural-function relationships in the middle region of S. cerevisia HSP104 protein
            Page 57
            Page 58
        Differential gene expression in cell cultures and plants of chili pepper (Capsicum annuum L.) under water stress
            Page 59
            Page 60
        Development of functional markers for drought tolerance in rice : Identification and validation of candidate genes and SNPs
            Page 61
            Page 62
            Page 63
            Page 64
        A complexity of genes underlie the response to drought tolerances in maize at flowering
            Page 65
            Page 66
            Page 67
        Genetic analysis of IR64 introgression lines of rice under irrigated and water stressed field conditions
            Page 68
            Page 69
        Search for molecular markers in cereals : An approach by 'intron scanning' and genome complexity reduction using DOP-PCR
            Page 70
            Page 71
        Over-expression of exogenous superoxide dismutase gene (MnSOD) and its effect on stress resistance in maize
            Page 72
            Page 73
            Page 74
        EcoTILLING candidate genes for drought tolerance in rice
            Page 75
            Page 76
        Genetic transformtion and testing of stress responsible candidate genes on improving drought tolerance in rice
            Page 77
            Page 78
        An Arabidopsis gain-of-function mutant with enhanced drought tolerance by activation tagging
            Page 79
            Page 80
        Generation challenge programme : 'cultivating plant diversity for the resource-poor"
            Page 81
            Page 82
    III. Germplasm characterization and improvement
        Page 83
        Introduction
            Page 83
        Methods
            Page 83
            Page 84
        Hertiability of rice yield under reproductive-stage drought stress, correlations across stress levels, and effects of selection : implications for drought tolerance breeding
            Page 85
            Page 86
            Page 87
        Breeding approaches to develop drought tolerant maize hybrid
            Page 88
            Page 89
        Improvement of maize populations for drought stress tolerance in Mozambique
            Page 90
        Screening for drought resistance in Oryza glaberrima varieties under floating lowland conditions in Mali
            Page 91
        The importance of experiment design and statistical analysis for genetic studies under water-limited conditions
            Page 92
            Page 93
        Development of drought tolerant lines for upland rice ecologies in the tropics of Africa : preliminary results
            Page 94
        Assessment of maize hybrids for drought tolerance in Mozambique
            Page 95
            Page 96
        A manual for breeding for drought tolerance in rice : Feedback and capture of experience by practitioners
            Page 97
        Genetic improvement for drought tolerance in rice (Oryza sativa L.)
            Page 98
            Page 99
        Genetic and physiological basis of breeding productive and drought tolerant genotypes in upland rice
            Page 100
            Page 101
        Screening and selection of rice lines for drought tolerance in target production environment
            Page 102
            Page 103
        Evaluation of early maturing maize hybrids for the low altitude areas of Malawi
            Page 104
            Page 105
        Advanced evaluation yield trials of drought and low-N tolerant maize varieties for midaltitude areas of Tanzania
            Page 106
            Page 107
        Evluation of rice lines for drought tolerance in target production environment
            Page 108
            Page 109
        Characterization of maize testing environments in the Southern Africa Development Community (SADC) region
            Page 110
            Page 111
        Diallel analysus of tropical maize inbreds under stress and optimal conditions
            Page 112
            Page 113
        Two years of selecting for drought and low-N tolerance in two populations of maize and two hybrid groups (early and intermediate in maturity)
            Page 114
            Page 115
        Early drought and low nitrogen tolerant double top cross maize hybrids for the dry midaltitude ecology of Kenya
            Page 116
            Page 117
        Development of maize varieties under drought stress and non-stress conditions
            Page 118
            Page 119
        Meeting challenges of breeding for improved drought tolerance and other traits in maize in Kenya
            Page 120
            Page 121
        Breeding maize cultivars for drought tolerance in Malawi
            Page 122
            Page 123
        Development of highland banana cell suspension system; a critical stage in genetic improvement of the banana
            Page 124
            Page 125
        Direct selection for grain yield under on-session natural moisture stress condition : results from a large-scale screening nursery
            Page 126
            Page 127
        Breeding for reproductive stage drought tolerance through development of near flowering lines in rice
            Page 128
            Page 129
        Population structure of O. glaberrima Steud and its implications for breeding drought tolerance in cultivated rice
            Page 130
            Page 131
        Role of stress tolerant germplasm in increasing maize productivity in drought-prone areas of Angola
            Page 132
            Page 133
        Guinea sorghum hybrids : Bringing the benefits of hybrid technology to a staple crop of Sub-Saharan Africa
            Page 134
        Managed drought stress environments in Mexio and their association with global wheat growing environments
            Page 135
            Page 136
        Comparative genetic variability in rice accessions for water limited environments
            Page 137
            Page 138
            Page 139
        Evaluation of advanced backcross population for non-flooded, irrigated conditions and other associated stresses
            Page 140
            Page 141
            Page 142
        Challenges and opportunities for maize improvement for drought stressed areas of Ethiopia
            Page 143
            Page 144
        Using near-isogenic introgression lines to map rice genes conferring drought tolerance
            Page 145
            Page 146
    IV. Physiological approaches
        Page 147
        Root growth responses to water: Interaction between hydrotropism and gravitropism in Arabidopsis wild-type and no-hydrotropic response mutant
            Page 147
        Carbohydrate accumulation and remobilization of upland and lowland rice in response to water deficit at various development stages
            Page 148
            Page 149
        Relation between carbohydrate metabolism and drought resistance in 'Pinto Villa,' a drought resistant common bean variety
            Page 150
            Page 151
        A potentially new screening method for tolerance of plants to limiting growing conditions
            Page 152
        Improving drought resistance in rainfed rice for the Mekong region...
            Page 153
            Page 154
            Page 155
        Improving drought resistance in rainfed rice for the Mekong region : the experiences from Laos...
            Page 156
            Page 157
            Page 158
            Page 159
        Effects of drought stress during reproductive stages of grain tield and quality of different genotypes in rice (Oryza sativa)
            Page 160
            Page 161
        Improving drought resistance in rainfed rice for the Mekong region : Experience from Thailand...
            Page 162
            Page 163
            Page 164
        Mapping root traits using a RI population of rice under well-watered conditions
            Page 165
            Page 166
        Evaluation of rapid drought stress protocol to predict field performance of rice under drought stress conditions
            Page 167
            Page 168
        Response to direct selection for yield under reproductive stage stress backcross populations
            Page 169
            Page 170
        Studies on phenotypes of Zhenshan 97B/IRTA 109 RILs population in field screen facility
            Page 171
            Page 172
        Phenotypic diversity of Southern Africa maize landraces
            Page 173
            Page 174
        Breeding aerobic rice adapted to non-flooded irrigated conditions
            Page 175
            Page 176
            Page 177
            Page 178
        Improving drought resistance in rainfed lowland rice for the Mekong region : the experience from Cambodia...
            Page 179
            Page 180
            Page 181
        Screening method and phenotypic evaluation of rice genotypes to drought in rainfed drought prone environments in North and Northeast Thailand
            Page 182
            Page 183
            Page 184
        Using "smart" physiological-trait based crossing strategies accumulate drought-adaptive genes
            Page 185
            Page 186
            Page 187
        Morphological traits for vegetativ stage drought tolerance in rice (Oryza sativa)
            Page 188
            Page 189
            Page 190
        Root study of traditonal rice cultivars and IR64 introgressed lines for vegetative stage drought
            Page 191
            Page 192
            Page 193
        Genetic analysis of physio-morphological traits associated with drought tolerance in rice across the environments
            Page 194
            Page 195
        Proteomic analysis of drought responsiveness in rice
            Page 196
            Page 197
        Aquasporins during osmotic stress : regulation and evidence for endomembrane trafficking
            Page 198
            Page 199
        Controls of leaf growth and stomatal conductance under water deficit : combining genetic and ecophysiological analyses
            Page 200
            Page 201
        The capacity and genetic base of canopy temperature (CT) as indivator on drought tolerance (DT) in tice reproductive stage
            Page 202
            Page 203
    V. QTL indentification
        Page 204
        Mapping quantitative trait loci for drought tolerance in rice: Comparison and across environments, genetic backgrounds and validation
            Page 204
            Page 205
        Highly efficient fine mapping of QTLs for drought tolerance using overlapping introgression lines of rice
            Page 206
            Page 207
        Isogenization and characterization of root-ABA1, a major QTL affecting root traits and leaf ABA concentration in maize
            Page 208
            Page 209
            Page 210
        Validation of QTLs for drought resistance in near isogenic introgression lines of rice
            Page 211
            Page 212
            Page 213
            Page 214
        High resolution mapping of a genomic region harboring several QTLs and sd1 in rice
            Page 215
        Mapping quantitative trait loci (QTL) for important agronomic traits in wheat under rainfed and well irrigated conditions
            Page 216
            Page 217
        Mapping QTLs and ESTs associated with drought tolerance in rice toward discovery of candidate genes
            Page 218
            Page 219
        Identifying quantitative trait loci (QTL) for drought tolerance in bread wheat (Triticum aestivum L.)
            Page 220
            Page 221
        Developing high yield and drought tolerant rice cultivats and discovering the complex genetic network underlying drought tolerance in rice
            Page 222
            Page 223
            Page 224
        Discovery of drought tolerant (DT) gene/QTLs and development of DT rice cultivars
            Page 225
            Page 226
        Molecular dissection of drought tolerance (DT) related quantitative rait loci (QTL) in ZhenshanB/IRAT109 RIL population
            Page 227
            Page 228
            Page 229
            Page 230
        Genetic analysis of drought tolerance in tropical maize
            Page 231
            Page 232
            Page 233
        Mapping QTLs associated with root traits related to drought resistance in Vietnamese upland rice
            Page 234
            Page 235
            Page 236
        Integrating molecular approaches in breeding for drought tolerance of maize in India
            Page 237
            Page 238
        Quantitative trait loci identification under drought conditions in maize
            Page 239
            Page 240
        Quantitative trait loci (QTL) associated with drought tolerance at the reproductive stage in rice
            Page 241
            Page 242
        Progress in developing drought tolerant rice cultivars for Eastern India
            Page 243
            Page 244
        Identification of QTLs for flowering time in maize under water-stressed conditions
            Page 245
            Page 246
            Page 247
        Identification of QTLs for drought tolerance in a set of random introgression lines of rice
            Page 248
            Page 249
            Page 250
        Genetic and molecular understanding of drought tolerance for the improvement or irrigated rice under drought stressed conditions in Central and Southern China
            Page 251
            Page 252
        Mapping QTLs affecting rice stigma exsertion in a RIL population with and without water stress conditions
            Page 253
            Page 254
        QTL mapping of drought-resistance at late growing stage in rice
            Page 255
            Page 256
    VI. Marker-assisted selection
        Page 257
        Comparison of conventional and molecular-based techniques in cassava mosaic disease reisistance screening of new cassava genotypes tested in dry and humid zones of Nigeria
            Page 257
        Marker-aided breeding for development of drought tolerant IR64 lines
            Page 258
            Page 259
        Marker-aided breeding for development of drought tolerant IR64 lines
            Page 260
        Genetic respone to strong directional selection on rice tolerance to critial stress
            Page 261
            Page 262
        Impact of moisture stress as different reproductive stages of plant growth on grain yield parameters and marker assisted QTL pyramiding for root length in rice
            Page 263
            Page 264
        Molecular marker assisted selection for drought tolerance in a maize population bred for semi-arid Kenya
            Page 265
            Page 266
        Marker-assisted selection in tropical maize based on consensus map, perspectives, and limitations
            Page 267
            Page 268
        Marker-assisted farmer participatory breeding for drought resistance in rice (Oryza sativa L.)
            Page 269
            Page 270
        Stress protein (SDS-PAGE) for MAS-breeding : seed characteristic and vigour to detect stable QTLs using seed protein markers in developing drought rolerance in rice (O. sativa L.)
            Page 271
            Page 272
        Improving drought resistance in rainfed lowland rice for the Mekong region : concept and practical use of MAS to develop cultivars with high quality and drought tolerance
            Page 273
            Page 274
            Page 275
        Association mapping of loci for drought tolerance and non-target traits using introgression lines in rice
            Page 276
            Page 277
    Annex A : program
        Page 278
        Page 279
        Page 280
        Page 281
        Page 282
        Page 283
    Annex B : list of participants
        Page 284
        Page 285
        Page 286
        Page 287
Full Text




Resilient Crops

for Water Limited

Environments:

Proceedings of a Workshop
Held at Cuernavaca Mexico

24 -28, Moy 2004- -
D. olaAdM. Sawkins, J.-M Ribaut1 and D. Hoisington +
Editors -' -




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Resilient Crops for Water
Limited Environments:

Proceedings of a Workshop
Held at Cuernavaca Mexico

24 28 May 2004

D. Poland, M. Sawkins, J.-M. Ribaut, and D. Hoisington
Editors


CIMMYT.

























































CIMMYT (www.cimmyt.org) is an internationally funded, not-for-profit organization that conducts research and training related to maize
and wheat throughout the developing world. Drawing on strong science and effective partnerships, CIMMYT works to create, share, and
use knowledge and technology to increase food security, improve the productivity and profitability of farming systems, and sustain natural
resources. Financial support for CIMMYT's work comes from many sources, including the members of the Consultative Group on
International Agricultural Research (CGIAR) (www.cgiar.org), national governments, foundations, development banks, and other public
and private agencies.

International Maize and Wheat Improvement Center (CIMMYT) 2004. All rights reserved. The designations employed in the present
tion of materials in this publication do not imply the expression of any opinion whatsoever on the part of CIMMYT or its contributory
organizations concerning the legal status of any country, territory, city, or area, or of its authorities, or concerning the delimitation of its
frontiers or boundaries. CIMMYT encourages fair use of this material. Proper citation is requested.

Correct citation: Poland, D., M. Sawkins, J.-M. Ribaut, and D. Hoisington (eds.). 2004. Resilient Crops for Water Limited Environments: Proceedings
of a Workshop Held at Cuernavaca, Mexico, 24-28 May 2004. Mexico D.F.: CIMMYT

AGROVOC descriptors: Cereals; Rice; Maize; Wheats; Hybrids; Plant breeding; Arabidopsis; Genotypes; Loci; Quantitative genetics;
Molecular genetics; Disease resistance; Injurious factors; Water; Drought resistance; Yield increases; Developing countries; Research
projects
AGRIS category codes: F30 Plant Genetics and Breeding; H50 Miscellaneous Plant Disorders
Dewey decimal classification: 631.53
ISBN: 970-648-124-9

Design and Layout: Marcelo Ortiz S.


Printed in Mexico









Contents


Page

xid FcreuuCrd
xii Opainrg dkhress (Gordcn Oaruw~ PR-esidert, The Rockefeller FoLadaticrl
xv VWlonrirn SLaterrent (Vbsa Iufrfaga, erectdr General, QIVIVVI)

1. Farrter P artidpatcry Eedirng and EaorrricStLales
1 Breeding for improved drought tolerance in maize adapted to southern Africa
M. Banziger, P.S. Setimela, D. Hodson, and B. Vivek

2 Rice seed systems in southern China: Views from institutions and farmers
S.Ding, X. Gu, F Chen, C. Chen, S. Pandey, Q. Zhang, and L. Luo

4 Adaptability of 16 upland rice varieties to two moisture regimes
J. Imanywoha, P. Kibwika, M. Walusimbi, G. Bigirwa, and J. Lamo

6 Farmers' participatory varietal selection at target drought prone area of Tamil Nadu
P. Jeyaprakash, S. Robin, R. Pushpa, S. Manimaran, R. Chandrababu, and P. Balasubramaniyan

8 Farmers' participatory plant breeding technique: An effective tool for the early selection and adoption of rice varieties in
rainfed rice ecosystems
S. Mahendran, L. Mahalingam, T. Sivakumar, M. Hemalatha, N. Chitra, R. Chandra Babu, P. Shanmugasundram, and S. Robin

10 Participatory maize variety evaluation for increased adoption
M.S. Mwala, J. de Meyer, P. Setimela, and M. Banziger

11 Economic costs of drought and rice farmers' coping mechanisms: A synthesis of cross-country comparative analysis
S. Pandey, H. Bhandari, R. Sharan, S. Taunk, D. Naik, P. Prapertchob, and S. Ding

13 Development and dissemination of drought tolerant rice varieties through on-farm, farmer-oriented approaches
A. Prasad, J.S. Gangwar, V. Singh, S.C. Prasad, A. Chaudhary, D.N. Singh, D.S.Virk, K.A. Steele, and J.R. Witcombe

15 Drought and cropping pattern change in Tamil Nadu, India: Needed technological transformation in rice farming
C. Ramasamy, K.N. Selvaraj, and R. Chandra Babu
19 Varietal adoption and farmers' coping strategies in rainfed rice ecosystems of Tamil Nadu
K.N. Selvaraj, C. Ramasamy, and R.C. Babu
23 Application of maize mega-environments in seed systems in the Southern African Development Community
P.S. Setimela, M. Banziger, and M. Listman
24 Breeding drought tolerant varieties of rice through participatory plant breeding for the rainfed uplands
D.N. Singh, M. Chakraborty, M.K. Chakravarty, P. Singh, M.K. Baranwal, B. Kumar, R. Kumar, A. Prasad, V. Singh, S.C. Prasad, A. Choudhary, D.S. Virk,
K.A. Steele, and J.R. Witcombe
27 Reaching the poor maize farmers in the hills of Nepal: Experiences and achievements of the Hill Maize Research Project (HMRP)
C.A. Urrea, T.P. Tiwari, N.P. Rajbhandari, and D.P. Sherchan

30 An international partnership for the breeding and delivery of drought-tolerant rice varieties by market-oriented plant breeding
and marker assisted selection
D.S. Virk, K.A. Steele, D.N. Singh, S.C. Prasad, A. Prasad, J.S. Gangwar, V. Singh, and J.R. Witcombe


S* iii *










II. Gene Discoery and N el Approadces
32 Heritability of mean grain yield under reproductive-stage drought stress and correlations across stress levels in sets of
selected and unselected rice lines in the Philippines, Thailand, and India: Implications for drought tolerance breeding
G.N. Atlin, R. Lafitte, R. Venuprasad, R. Kumar, and B. Jongdee

35 Empowering rice drought gene discovery activities through bioinformatics
R. Bruskiewich, V. Bartolome, A. Cosico, A. Kathiresan, L. Mansueto, A. Portugal, J.Prayongsap, M. Raveendran, M.A. Sallan, N. Sharma, X. Wang,
T. Ulat, V. Ulat, J. Bennett, R. Lafitte, K. McNally, and G. McLaren.

37 Functional analysis of plant hydrophilins
A.A. Covarrubias, J.L. Reyes, Y. Olvera-Carrillo, F Campos, M. Battaglia, R.E. Quiroz, and A. Garciarrubio

39 Rice SNP map between indica and japonica subspecies: DNA marker resolution on the kilobase scale
F Alex Feltus, Jun Wan, S.R. Schulze, N. Jiang, and A.H. Paterson

41 XVSAP1 from Xerophyta viscosa improves salinity and water deficit stress tolerance in Arabidopsis and tobacco
D. Garwe, J.A. Thomson, and S.G. Mundree

43 Molecular dissection on rice photosynthesis-related traits at reproductive stage in irrigated and drought conditions
S.P.H. Hu, W. Mei, H.Y. Liu, G.H. Zou, G.L. Liu, X.Q. Yu, J. Li, P. Long, and L. Luo

45 Identification of SSR markers linked to candidate genes for drought tolerance in rice
J. Hussain, K.L. McNally, and H.R. Lafitte

47 Monitoring the transcriptome changes of 14013 rice unigenes in response to drought by cDNA microarray
S.K. Katiyar, L.T. Pazhamala, V.A. Poroyko, and H.J. Bohnert

49 Molecular responses to dehydration and salinity in rice: Differences and cross-talk between two stress signaling pathways
S.K. Katiyar, V.A. Poroyko, and H.J. Bohnert

51 The primary studies on gene expression of drought tolerance or sensitive rice cultivars in different water conditions
Z.C. Liu, H.H. Tong, H.Y. Liu, L. Chen, L.J. Luo

53 Effects of the brachytic-2 dwarfing gene on maize (Zea mays) root systems and grain yield under moisture stress
K. Mashingaidze and E.C. Chinhema

55 Towards the improvement of abiotic stress tolerance in maize using genes isolated from the monocotyledonous resurrection
plant Xerophyta viscosa
S.G. Mundree and J.A. Thomson

56 Gene mining of African rice germplasm (Oryza glaberrima and Oryza sativa) to improve drought resistance in rainfed
production systems for resource poor farmers of Africa
M.N. Ndjiondjop and H. Gridley

57 Structural-function relationships in the middle region of S. cerevisiae HSP104 protein
J. Nieto-Sotelo, F Zarate, L.M. Martinez, and B.L. Arroyo

59 Differential gene expression in cell cultures and plants of chili pepper (Capsicum annuum I.) under water stress
N. Ochoa-Alejo and Y.M. Camacho-Villasana

61 Development of functional markers for drought tolerance in rice: Identification and validation of candidate genes and SNPs
A.R. Reddy, A.C. Sekhar, P.R. Babu, G. Markandeya, L.S. Prasad, L.V.B. Reddy, N. Seetharama, N.P. Saxena, A.M.M. Reddy, V.K. Kumar, and B.C. Shekhar

65 A complexity of genes underlie the response to drought tolerance in maize at flowering
M.C. Sawkins, M. de la Luz Gutierrez, J. Habben, C. Zinselmeier, C. Martinez, E. Huerta, M. Moreno, and J.-M. Ribaut


. iv *










68 Genetic analysis of IR64 introgression lines of rice under irrigated and water stressed field conditions
V.N. Singh, A.K. Singh, B.B. Singh, G.S. Chaturvedi, and G. Atlin

70 Search for molecular markers in cereals: An approach by 'intron scanning' and genome complexity reduction using DOP-PCR
H.P. Singh, FA. Feltus, S.R. Schulze, T. Silva, and A.H. Paterson

72 Over-expression of exogenous superoxide dismutase gene (MnSOD) and its effect on stress resistance in maize
L. Wanchen and D. Juan

75 EcoTILLING candidate genes for drought tolerance in rice
H.H. Wang, Ma. Elizabeth Naredo, J.L. Wu, B.J. Till, E.A. Greene, S. Henikoff, L. Comai, H. Leung, and K.L. McNally

77 Genetic transformation and testing of stress responsible candidate genes on improving drought tolerance in rice
B.Z. Xiao, X. Chen, H.H. Hu, X. Hou, C.B. Xiang, Q. Zhang, and L. Xiong

79 An Arabidopsis gain-of-function mutant with enhanced drought tolerance by activation tagging
H. Yu, Y. Hong, P. Zhao, X. Chen, and C. Xiang

81 Generation Challenge Programme: "Cultivating Plant Diversity for the Resource-Poor"
R. Zeigler


III. GerrplasmC waraterizaticn and Irrpraeraurt
83 Evaluation of discriminant analysis as a tool for rapid identification of markers associated with drought resistance in rice
G.K. Aluko and J.H. Oard

85 Heritability of rice yield under reproductive-stage drought stress, correlations across stress levels, and effects of selection:
Implications for drought tolerance breeding
G.N. Atlin, R. Lafitte, R. Venuprasad, R. Kumar, and B. Jongdee

88 Breeding approaches to develop drought tolerant maize hybrids
F.J. Betran, M. Banziger, D. Beck, J.-M. Ribaut, and G.O. Edmeades

90 Improvement of maize populations for drought stress tolerance in Mozambique
P.S. Chauque, P. Fato, and M. Denic

91 Screening for drought resistance in Oryza glaberrima varieties under floating and lowland conditions in Mali
F Cisse and Y. Doumbia

92 The importance of experiment design and statistical analysis for genetic studies under water-limited conditions
J .Crossa, J-M. Ribaut, M. Vargas, W. Federer, K. Mathews, and J. Burgueno

94 Development of drought tolerant lines for upland rice ecologies in the tropics of Africa
A.A. Efisue, H.E. Gridley, F Cisse, W. de Milliano, M. Laing, and P. Shanahan

95 Assessment of maize hybrids for drought tolerance in Mozambique
P. Fato, P.S. Chauque, and M. Denic

97 A manual for breeding for drought tolerance in rice: Feedback and capture of experience by practitioners
K. Fischer, S. Fukai, and R. Lafitte

98 Genetic improvement for drought tolerance in rice (Oryza sativa L.)
S. K. Ganesh, P. Vivekanandan, N. Nadarajan, R.C. Babu, P. Shanmugasundaram, PA. Priya, and A. Manickavelu

100 Genetic and physiological basis of breeding productive and drought tolerant genotypes in upland rice
N.G. Hanamaratti, P.M. Salimath, H.D. Mohankumar, and Shailaja Hittalmani


** V *0










102 Screening and selection of rice lines for drought tolerance in target production environment
P. Jeyaprakash, R.C. Babu, P. Shanmuagsundaram, S. Marimuthu, R. Sundararajan, S. Robin, N. Subbaraman, and P. Balasubramaniyan

104 Evaluation of early maturing maize hybrids for the low altitude areas of Malawi
K.K.E. Kaonga

106 Advanced evaluation yield trials of drought and low-N tolerant maize varieties for midaltitude areas of Tanzania
K.M. Kitenge, Z.O. Mduruma, PR. Matowo, and T. Semuguruka

108 Evaluation of rice lines for drought tolerance in target production environment
L. Mahalingam, S. Mahendran, T. Sivakumar, M. Hemalatha, N. Chitra, R.C. Babu, P Shanmugasundram, and S. Robin

110 Characterization of maize testing environments in the Southern Africa Development Community (SADC) region
F. Maideni, M. Banziger, P. Setimela, and J. Betran

112 Diallel analysis of tropical maize inbreds under stress and optimal conditions
D. Makumbi, M. Banziger, J.-M. Ribaut, and F.J. Betran

114 Two years of selecting for drought and low-N tolerance in two populations of maize and two hybrid groups
(early and intermediate in maturity)
P.R. Matowo, Z.O. Mduruma, K.Kitenge, and Z. Mrinji

116 Early drought and low nitrogen tolerant double top cross maize hybrids for the dry midaltitude ecology of Kenya
W.N.P. Muasya and A.O. Diallo

118 Development of maize varieties under drought stress and non-stress conditions
C. Mungoma and K. Mwansa

120 Meeting challenges of breeding for improved drought tolerance and other traits in maize in Kenya
C. Mutinda, A.O. Diallo, and F Manyara

122 Breeding maize cultivars for drought tolerance in Malawi
W.G. Nhlane

124 Development of highland banana cell suspension system: A critical stage in genetic improvement of the banana
P. Namanya, G. Mutumba, S.M. Magambo, and W. Tushemereirwe

126 Direct selection for grain yield under on-season natural moisture stress condition: Results from a large-scale screening nursery
R. Pushpa, R. Manimaran, S. Robin, P Jeyaprakash, S. Mahendran, L. Mahalingam, S. Shanmugasundaram, R.C. Babu, and K. Thiyagarajan

128 Breeding for reproductive stage drought tolerance through development of near flowering lines in rice
S. Robin, R. Manimaran, R. Pushpa, P. Jeyaprakash, S. Mahendran, L. Mahalingam, S. Shanmugasundaram, R.C. Babu, H.R. Lafitte, G. Atlin,
and K. Thiyagarajan

130 Population structure of 0. glaberrima Steud and its implications for breeding drought tolerance in cultivated rice
M. Semon, M.R Jones, M.R. Nielsen, and S.R. McCouch

132 Role of stress tolerant germplasm in increasing maize productivity in drought-prone areas of Angola
F.R Sito, E. Gaspar, and D. Nginamau

134 Guinea sorghum hybrids: Bringing the benefits of hybrid technology to a staple crop of sub-Saharan Africa
A. Toure, FW. Rattunde, and E. Weltzien

135 Managed drought stress environments in Mexico and their association with global wheat growing environments
R. M. Trethowan, M.P. Reynolds, W.H. Pfeiffer, K. Ammar, M. van Ginkel, and J. Crossa


. vi *










137 Comparative genetic variability in rice accessions for water limited environments
O.2 Verma, B.B. Singh, G.S. Chaturvedi, A.K. Singh, V.N. Singh, S. Prasad, R. Kumar, and G. Atlin

140 Evaluation of advanced backcross populations for non-flooded, irrigated conditions and other associated stresses
C.H.M. Vijayakumar, S.R. Voleti, B. Mishra, M. Sreenivas Prasad, R. Mahender Kumar, S. Rama Krishna, K.V. Rao, I.C. Pasalu, J.S. Prasad, and B.C. Viraktamath

143 Challenges and opportunities for maize improvement for drought stressed areas of Ethiopia
D. Wegary, G. Seboksa, and M. Nigussie

145 Using near-isogenic introgression lines to map rice genes conferring drought tolerance
S.B. Yu, H.J. Zhou, J.Q. Mu, S.J. Zhao, YF. Hu, C.G. Xu, and Q. Zhang


IV. Physidol ca Approaches
147 Root growth responses to water: Interaction between hydrotropism and gravitropism in Arabidopsis wild-type and no-
hydrotropic response mutant
G.I. Cassab, D. Eapen, M.L. Barroso, M.E. Campos, G. Ponce, and M. Saucedo

148 Carbohydrate accumulation and remobilization of upland and lowland rice in response to water deficit at various development stages
G.S. Chaturvedi, B.B. Singh, A.K. Singh, M.K. Singh, and V.N. Singh

150 Relation between carbohydrate metabolism and drought resistance in 'Pinto Villa,' a drought resistant common bean variety
S. Cuellar Ortiz, M. de la Paz Arrieta Montiel, J.A. Gallegos, and A.A. Covarrubias Robles

152 A potentially new screening method for tolerance of plants to limiting growing conditions
W.A.J. de Milliano

153 Improving drought resistance in rainfed rice for the Mekong region: Defining target population of environments (TPE), charac-
terizing the available water and breeding for better adaptation to the variable water supply including an overview of the
project "Improving drought resistance in rainfed lowland rice for the Mekong region"
S. Fukai, K. Fischer, J. Basnayake, B. Jongdee, G.Pantuwan, O.Makara, M. Tsubo, and P. Inthapanya

156 Improving drought resistance in rainfed rice for the Mekong region: The experience from Laos in the selection of drought
tolerant donor lines for the target population of environments (TPE) based on yield and on leaf water potential (LWP),
flowering delay, and drought response index (DRI)
P. Inthapanya, S. Jhay, J. Basnayake, Ch. Boulaphan, M. Changphengsay, S. Fukai, and K. Fischer

160 Effects of drought stress during reproductive stages on grain yield and quality of different genotypes in rice (Oryza sativa)
D. Jin, J. Zhang, S. Cheng, C. You, G. Zhou, and Q. Zhang

162 Improving drought resistance in rainfed rice for the Mekong region: The experience from Thailand with a focus on the use of
leaf water potential (LWP) and spikelet sterility as indirect drought tolerant traits
B. Jongdee, G. Pantuwan, S. Jearakongman, S. Rajatasereekul, J. Basnayake, S. Fukai, and K. Fischer

165 Mapping root traits using a RI population of rice under well-watered conditions
C. Kehui, Y. Bing, X. Weiya, X. Yongzhong, J. Deming, and Q. Zhang

167 Evaluation of rapid drought stress protocol to predict field performance of rice under drought stress conditions
R. Kumar, G. Atlin, and R. Lafitte

169 Response to direct selection for yield under reproductive stage stress in rice backcross populations
H.R. Lafitte, S. Yan, Y.M. Gao, and Z.-K. Li


S* vii *










171 Studies on phenotype of Zhenshan 97B/IRTA109 RILs population in field screen facility
H.Y. Liu, H.W. Mei, G.H. Zou, S.P. Hu, G.L. Liu, X.Q. Yu, J. Li, P. Long, and L. Luo

173 Phenotypic diversity of southern Africa maize landraces
C. Magorokosho, M. BAnziger, and J. Betran

175 Breeding for aerobic rice adapted to non-flooded irrigated conditions
B. Mishra, C.H.M. Vijayakumar, and S.R. Voleti

179 Improving drought resistance in rainfed lowland rice for the Mekong region: The experience from Cambodia and on the use of
drought resistance index (DRI) as an integrative drought tolerance trait
0. Makara, M. Sarom, S. Khan, J. Basnayake, S. Fukai, and K. Fischer

182 Screening method and phenotypic evaluation of rice genotypes resistance to drought in rainfed drought prone environments in
north and northeast Thailand
G. Pantuwan and B. Jongdee

185 Using "smart" physiological-trait based crossing strategies to accumulate drought-adaptive genes
M.P. Reynolds, Rubeena, and R. Trethowan

188 Morphological traits for vegetative stage drought tolerance in rice (Oryza sativa)
A.K. Singh, S. Prasad, V.N. Singh, G.S. Chaturvedi, and B.B. Singh

191 Root study of traditional rice cultivars and IR64 introgressed lines for vegetative stage drought
B. Singh, A.K. Singh, G.S. Chaturvedi, S. Prasad, V.N. Singh, A. Singh, and R. Kumar

194 Genetic analysis of physio-morphological traits associated with drought tolerance in rice across the environments
R. Suresh, P. Shanmugasundaram, R.C. Babu, P. Jayaprakash, S. Michael Gomez, S. Satheeshkumar, and P. Chezhian

196 Proteomic analysis of drought responsiveness in rice
M. Raveendran, R. Bruskiewich, and J. Bennett

198 Aquaporins during osmotic stress: Regulation and evidence for endomembrane trafficking
R. Vera-Estrella, B.J. Barkla, H.J. Bohnert, and 0. Pantoja

200 Controls of leaf growth and stomatal conductance under water deficit: Combining genetic and ecophysiological analyses
F. Tardieu, B. Muller, M. Reymond, W. Sadok, and T.H. Simonneau

202 The capacity and genetic base of canopy temperature (CT) as indicator on drought tolerance (DT) in rice reproductive stage
G.H. Zou, H.W. Mei, H.Y. Liu, S.P. Hu, G.L Liu, X.Q. Yu, J. Li, P. Long, and L. Luo


V. QFI. Idertficaticn
204 Mapping quantitative trait loci for drought tolerance in rice: Comparison across environments, genetic backgrounds, and validation
R.C. Babu, P Shanmugasundaram, P. Chezhian, P. Jeyaprakash, P. Balasubramaniyan, V. Chamarerk, M.S. Pathan, V. Babu, and H.T. Nguyen

206 Highly efficient fine mapping of QTLs for drought tolerance using overlapping introgression lines of rice
B.Y. Fu, Y.Z. Jiang, J.L. Xu, Y.M. Gao, C.H.M. Vijayakumar, J. Ali, J.R. Domingo, R. Maghirang, and Z.K. Li

208 Isogenization and characterization of root-ABA a major QTL affecting root traits and leaf ABA concentration in maize
S. Giuliani, P. Landi, M.C. Sanguineti, M. Bellotti, S. Salvi, S. Stefanelli, S. Vecchi, and R. Tuberosa

211 Validation of QTLs for drought resistance in near isogenic introgression lines of rice
S. Jearakongmana, T. Toojindab, B. Jongdeec, and S. Rajatasereekuld


. viii *










215 High resolution mapping of a genomic region harboring several QTLs and sdl in rice
Y.Z. Jiang, P. Bagali, Y.M. Gao, R. Lafitte, B.Y. Fu, J.L. Xu, R. Maghirang, R. Domingo, S. Hittalmani, D. Mackill, and Z.K. Li

216 Mapping quantitative trait loci (QTL) for important agronomic traits in wheat under rainfed and well irrigated conditions
R. Jing, X. Chang, Z. Hao, N. Gao, and J. Jia

218 Mapping QTLs and ESTs associated with drought tolerance in rice towards discovery of candidate genes
S.K. Katiyar, M.S. Dudhare, L.T. Pazhamala, R. Mushtaq, and R. Pratibha

220 Identifying quantitative trait loci (QTL) for drought tolerance in bread wheat (Triticum aestivum L.)
F. Kirigwi, A.K. Fritz, G.L. Brown-Guedira, B.S. Gill, G.M. Paulsen, and M. van Ginkel

222 Developing high yield and drought tolerant rice cultivars and discovering the complex genetic network underlying drought
tolerance in rice
Z.K. Li, R. Lafitte, C.H.M. Vijayakumar, B.Y. Fu, Y. M. Gao, J.L. Xu, J. Ali, M.F. Zhao, S.B. Yu, J. Domingo, R. Maghirang, G.S. Khush, and D. Mackill

225 Discovery of drought tolerant (DT) gene/QTLs and development of DT rice cultivars
L. Luo, H.W. Mei, X.Q. Yu, H.Y. Liu, G.H. Zou, S.P Hu, G.L. Li, P. Long, Z.C. Liu, L. Chen, and H. Zeng

227 Molecular dissection of drought tolerance (DT) related quantitative trait loci (QTL) in ZhenshanB/IRAT109 RIL population
H.W. Mei, H.Y. Liu, G.H. Zou, S.P. Hu, G.L. Liu, X.Q. Yu, J. Li, P. Long, and L. Luo

231 Genetic analysis of drought tolerance in tropical maize
R. Messmer, Y. Fracheboud, M. BUnziger, P. Stamp, and J.-M. Ribaut

235 Mapping QTLs associated with root traits related to drought resistance in Vietnamese upland rice
D.T. Nguyen, T.K.L. Nguyen, Q.C. Pham, T.H. Nguyen, Q.T. Tran, X.H. Dao, and H.T. Nguyen

237 Integrating molecular approaches in breeding for drought tolerance of maize in India
B.M. Prasanna, A.H. Beiki, R. Kumar, A. Garg, N.N. Singh, and J.-M. Ribaut

239 Quantitative trait loci identification under drought conditions in maize
G. Shibin, F Zhilei, and L. Wanchen

241 Quantitative trait loci (QTL) associated with drought tolerance at the reproductive stage in rice
T. Toojinda, J.L. Siangliw, A. Vanavichit, B. Jongdee, and G. Pantuwan

243 Progress in developing drought tolerant rice cultivars for eastern India
S.B. Verulkar, A. Khillare, A. Raisagar, M.S. Dudhare, and M.N. Shrivastava

245 Identification of QTLs for flowering time in maize under water-stressed conditions
X. Li, M. Li, and S. Zhang

248 Identification of QTLs for drought tolerance in a set of random introgression lines of rice
J.L. Xu, R. Lafitte, Y.M. Gao, B.Y. Fu, and Z.K. Li

251 Genetic and molecular understanding of drought tolerance for the improvement of irrigated rice under drought stressed
conditions in central and southern China
B. Yue, Z.L. Zhang, D. Jin, K.H. Cui, W.Y. Xue, L. Xiong, and Q. Zhang

253 Mapping QTLs affecting rice stigma exsertion in a RIL population with and without water stress conditions
X.Q. Yu, H.W. Mei, H.Y. Liu, G.H. Zou, S.P. Hu, G.L Liu, J. Li, R Long, and L. Luo

255 QTL mapping of drought-resistance at late growing stage in rice
B. Yue, W. Xue, Y. Xing, D. Jin, L. Xiong, and Q. Zhang


. ix *










VI. IVlrker-Assisted Selecticn
257 Comparison of conventional and molecular-based techniques in cassava mosaic disease resistance screening of new cassava
genotypes tested in dry and humid zones of Nigeria
O.A. Ariyo, A.G.O. Dixon, G.I. Atiri, and S. Winter

258 Marker-aided breeding for development of drought tolerant IR64 lines
D.K. Dwivedi, Y.M. Gao, R. Lafitte, C.H.M. Vjayakumar, B.Y. Fu, J.L. Xu, J. Ali, J. R. Domingo, R. Maghirang, D. Mackill, and Z.K. Li

260 Marker-assisted selection for drought tolerance in maize
R.P. Ganunga, M. Banziger, J.-M. Ribaut, K. Kaonga, and J. Betran

261 Genetic response to strong directional selection on rice tolerance to critical stress
Y.M. Gao, B.Y. Fu, J.L. Xu, C.H.M. Vjayakumar, M.F Zhao, J. Domingo, R. Maghirang, M. Arif, R. Lafitte, and Z.K. Li

263 Impact of moisture stress at different reproductive stages of plant growth on grain yield parameters and marker assisted
QTL pyramiding for root length in rice
S. Hittalmani, B.G. Hanamaeddy, H.E. Shashidhar, T.N. Girish, S. Grace, A. Selvi, M.G. Vaishali, K. Nagabhushan, A. Gowda,
N.S. Rudresh, and S. Kumar

265 Molecular marker assisted selection for drought tolerance in a maize population bred for semi-arid Kenya
K. Ngugi and Z. Muthamia

267 Marker-assisted selection in tropical maize based on consensus map, perspectives, and limitations
J.-M. Ribaut, M.C. Sawkins, M Banziger, M. Vargas, E.Huerta, C. Martinez, and M. Moreno

269 Marker-assisted farmer participatory breeding for drought resistance in rice (Oryza sativa I.)
H.E. Shashidhar, K.H. Bindu, R. Venuprasad, N. Sharma, A, Kanbar, K. Manjunatha, M.S. Vinod, H.R. Prabuddha, T.C. Kabeda, J. Madhu,
H.S. Ramachandrappa, B.K. Shailaja Hittalmani, and M.V. Ravi

271 Stress protein (SDS-PAGE) for MAS-breeding: Seed characteristics and vigour to detect stable QTLs using seed protein
markers in developing drought tolerance in rice (0. sativa L.)
H.P. Singh, B.B. Singh ,and G.S. Charturvedi

273 Improving drought resistance in rainfed lowland rice for the Mekong region: Concept and practical use of MAS to develop
cultivars with high quality and drought tolerance
T. Toojinda, B. Jongdee, G. Pantuwan, J.L. Siangliw, and N. Pa-ln

276 Association mapping of loci for drought tolerance and non-target traits using introgression lines in rice
TQ. Zheng, E.A. Ramos, J.L. Xu, B.Y. Fu, Y.M. Gao, Y.Z. Jiang, R. Lafitte, R. Maghirang, R. Jessica, S. Verulkar, D. Dwivedi, C.H.M. Vijayakumar,
M.F. Zhao, S.S. Virmani, D. Mackill, and Z.-K. Li

278 Annex A: Program

284 Annex B: List of Participants


** x **










Foreword


In May of 2004, more than 150 scientists from around the world-mostly from Asia and Africa-met in
Cuernavaca, Mexico to present and discuss their research on one of the world's most intractable agricultural
problems: drought tolerance in crop plants. The meeting, entitled the "Resilient Crops for Water-Limited
Environments Program Workshop," was supported by The Rockefeller Foundation and the International Maize
and Wheat Improvement Center (CIMMYT).

A truly global critical mass of expertise participated in the workshop, originating from more than 20 countries
and three of the world's international agricultural research centers; CIMMYT, IRRI, and WARDA. Of particular
interest were maize, rice, and wheat, which account for more than half of the calories consumed by people in the
developing world, and are the basis for their food security and livelihoods. Scientists comprehensively
addressed the physiology, biochemistry, and genetics of plant response to water stress. In addition, they looked
at drought tolerance from the ground-level perspective of incorporating farmer participation into varietal
development, to the heights of molecular genetics/genomics/bioinformatics, and even how plant gene
regulation pathways interact and respond to water deficits in a growing developing crop plant. Indeed our
information and hence knowledge base in this subject is growing at a rapid rate.

Specific goals of the workshop were information sharing among Rockefeller Foundation grantees from Africa
and Asia; updating participants on knowledge generation, breeding technologies and capacity building, and
seed delivery systems; and finally planning of collaborative research among the participants. These objectives
were achieved beyond the expectations of the organizers and all are to be congratulated for their outstanding
progress since our previous workshop held May 2002 at IRRI, Philippines.

The ultimate aim of this program's outputs is to stabilize food crop production through genetic improvement of
cereals, primarily maize and rice, for drought tolerance, thereby decreasing the shock of drought on the
livelihoods of poor farm households in Africa and Asia. This program's vision of success-the creation and
delivery of new drought tolerant crop varieties to farmers-in turn is closely tied to parallel efforts to stem the
decline of soil fertility in much of the developing world, and build up input and output markets in rural areas.
Taken together, these broad initiatives offer a strong and viable approach to lifting millions from crushing
poverty through creation of more sustainable rural and agricultural development.

These proceedings contain, in condensed form, hundreds of thousands of research hours dedicated to
developing crops that can help farmers withstand the destabilizing impact of drought in some of the most needy
farm communities of Africa and Asia. Although we have not yet reached our full potential in this effort,
exceptional progress is clearly being made, indeed it seems to be accelerating, and the pipeline of research and
collaboration is just beginning to deliver new varieties to the people who need them most. Specific examples of
new varieties actually being delivered and utilized on large areas were reported from both southern Africa and
eastern India, providing realistic "proof of concept" and motivating all concerned to redouble their efforts. The
conference participants are to be commended for their exceptional work and information sharing at the
workshop, as is the CIMMYT organizing committee, for the superb preparations and support for the meeting
and this publication.



John O'Toole
Associate Director, Food Security
The Rockefeller Foundation


S* Xi *










Opening Address


The War on Drought: Technologies, Markets, and Partnerships





Gordon Conway
President
The Rockefeller Foundation 1913



I am very happy to be here today with all of you, having just completed my first-ever visit to CIMMYT. The visit
helped set in my mind the context for our current emphasis on drought and agriculture, and the objectives of the
scientists attending this workshop.

Our story begins with Norman Borlaug and the Green Revolution. His work and vision, supported by The
Rockefeller Foundation at that time, continues to inspire us. Yield ceilings of staple crops increased dramatically,
especially in well-favored, well-irrigated lands. Production grew faster than population, and the real price of staple
foods decreased. In short, the Green Revolution fed the world. Wheat productivity rose 200 percent from 0.9 tons
per hectare in 1962, to 2.7 tons in 2002. Rice productivity went from 1.8 tons per hectare to 3.9 tons, an increase of
117 percent. And maize went from 1.2 tons per hectare in the early 1960s to 3 tons at the turn of the millennium.

The Green Revolution also fed the world by bringing down the price of staple grains. The real price for rice
decreased from $800 per metric ton in 1975 to around $200 in 1995. Less dramatic but still highly significant,
wheat went from a little over $300 per metric ton in 1975 to $200 in 1995. Without a Green Revolution, the price
of cereals would have increased year by year to meet demand in Japan and Europe, mainly for livestock feed.

That said, today 1.2 billion people remain in poverty, with 90 percent of them in Africa and Asia, and 70 percent
in rural areas. Critics of the Green Revolution say that we need to tackle poverty first. But the reality is that only
through agriculture will those people work their way out of poverty, a daunting task. The best lands have been
fully utilized and the poor are often forced to farm on less-favored lands. Furthermore, the soils have low native
fertility and water resources are frequently inadequate at best.

In Africa, large areas are at risk for drought. There are two ways that drought impacts on farmers' livelihoods. It
can affect everyday stability, as with fluctuations in production. Drought also threatens sustainability, through
"drought shock," when production collapses.

We are making some progress addressing water stress and its impact on stability, as will be reported at this
meeting. In response to drought shock, we will have to reach even further, perhaps into genes of plants like the
so-called "resurrection plant."

Drought's impact cannot be overstated: it reduces tropical maize yields 17 percent annually, or by $2.2 billion. In
two regions where Foundation grantees are concentrated, China and eastern India, annual losses to drought total
4.4 million tons and 2.9 million tons, respectively. Globally, $3.6 billion of rice production is lost each year to
drought. Annual losses for these two crops approach $6 billion. But to really put these losses in perspective, we
must recall that these impacts are on the economies of mainly agriculture-based developing countries of Africa
and Asia. If researchers like you are successful, you will save some of the world's neediest people $6 billion a year.

But drought and responses to it must also consider a host of non-technical issues. Farmers have a range of coping
mechanisms, which are often effective, but place a heavy, long-lasting burden on the family. For instance, in
Africa families frequently sell their livestock, or sell or mortgage their land. In the shorter term, farmers purchase


* xii *










less nutritious foods, delay needed health care, and curtail their children's education for lack of school fees.
Migration, and all that it entails, is another common response to drought. Taken individually, or one by one,
these coping mechanisms impose a significant burden on farm families.

Given the complex nature of the problem, there is a diversity of strategic responses we can pursue, both technical
and social. On the social side, we can develop roads, irrigation systems, provide crop insurance, or create
opportunities for non-agricultural employment. The enormity of that type of infrastructure and civic
development renders it impractical for the Foundation's grantmaking.

On the technical side, we can opt for genetic improvement or breeding for drought tolerance, an approach that
builds on the progress we've made to date and is compatible with our mission in that it addresses a global concern.
Forecasts are that global warming's impact on Africa's maize production will be especially severe, making the
situation that much more dire. These needs are consistent with The Rockefeller Foundation's history and
reputation. We accept risk, we lead the way, and we stay the course. Given the competence of the Foundation's staff
and their experience and familiarity with new scientific advances, we have confidence that we will continue to
move forward.

To move forward, I believe that what Africa and to a lesser extent Asia need is a Doubly Green Revolution-one
that is equally successful to the first Green Revolution, but that is more equitable, sustainable, and
environmentally friendly than its predecessor. And it is critical that the poor literally reap its benefits. This is not
simply an altruistic wish-it is emphasized in the Foundation's mission statement that declares: "a commitment
to enrich and sustain the lives and livelihoods of poor and excluded people." We must meet these criteria to have
projects funded.

The Rockefeller Foundation's food security goal is simple: to promote sustainable livelihoods in areas bypassed
by the Green Revolution. How and where are we investing? In Asia and Africa, we are investing in more resilient
crop varieties. But given the severity and nature of the agricultural problems in Africa, we are going further and
supporting efforts to enhance soil productivity and develop markets to improve incomes of farmers. Globally we
are supporting the development of international public goods for poor farmers.

Improved crop varieties and better soil fertility lead to higher productivity. When coupled with better markets,
this results in increased incomes for farmers and improved livelihoods for their families. It's a neat, concise
model, but The Rockefeller Foundation only provides the funding. Researchers like you are doing the work.

Now let me say a few words about nutrients and markets. Africa is losing soil nutrients at an alarming rate. In
western Kenya, for instance, farmers are losing 125 kg of nitrogen per hectare per year. This is what Europeans
add to their land annually. A quick look at the figures reveals the dire need for fertilizer and the cost
constraints that limit its use in Africa in particular. Average fertilizer application in Africa is 3.3 kg per hectare
as compared to 42.9 kg per hectare in Asia. The yields reflect this inequity; the average yield in Africa is 1.1
tons per hectare, while in Asia it is nearly 3 tons. We quickly see that cost is the major determinant in use or
lack of use. In Europe you can buy a ton of urea fertilizer for about $90. In Mombassa that same quantity of
fertilizer will cost about $400; in western Kenya, $500; and in Malawi, an astronomical $770. This is an
enormous barrier to improved agriculture in Africa.

It is absolutely critical to provide affordable nutrients to farmers, which requires efficient input markets. I have
some direct experiences with this end of the farm business. My grandfather was an input vendor for agricultural
products. He would go door-to-door selling seed and other inputs and tools to farmers. We need something like
that in Africa and Asia; small vendors selling small amounts of fertilizer to small-scale farmers.

The other side of the equation is output markets. In western Kenya, I recently came across a very large grain silo,
similar to the type seen throughout the Midwest of the United States. This one, however, had been sitting empty
for nearly five years. Clearly, the market is not working.

The Rockefeller Foundation is experimenting with small-scale cereal banks as a way to create market
opportunities for resource-poor farmers. Now we even see farmers using mobile phones to access regional
markets in order to get better prices for their maize. In East Africa, over a four-month period, a group of local


* Xiii *










cereal banks sold 393 tons of maize to the largest millers in the region at a premium price. Compared to the
previous year, the villagers have doubled the income from their grain sale. And for the first time, villagers
have sufficient cash to provide for their food and medical needs, as well as the children's school fees.

However, none of this works without partnerships! If the scientist can't access proprietary technologies, and in
turn, can't develop them and get them to the farmers, progress breaks down. Furthermore, the technologies
must be affordable when they arrive at the farmgate. In particular, the key to food security in Africa is the
creation of genuine partnerships, starting and ending with the farmers and involving the public and private
sectors, the National Agricultural Research Systems (NARS), the International Agricultural Research Centers
(IARCs), such as WARDA, CIMMYT, and IRRI, and the advanced labs of Japan, China, Europe, and the USA.

Partnerships can be seen as a three-legged stool-the public sector, private sector, and the communities, all brought
together by civil society. The public sector-and to a lesser degree the private sector-provide money and scale. The
private sector provides entrepreneurship, and the community provides participation and sustainability. We've seen
this work in pharmaceuticals as well as crop varieties. This system is what will bring greater prosperity to those in
need. Key to the success of African breeders and public research is access to patents.

The Rockefeller Foundation has initiated two programs in an effort to get these technologies to African
breeders and farmers. The first, the African Agricultural Technology Foundation (AATF), is an African-led and
based, freestanding, not-for-profit organization focused on bringing proprietary technologies from both public
and private sectors to bear on African agricultural development, while remaining responsive to smallholder
needs. We are looking to the AATF to procure licensing agreements for existing technologies, underwrite
adaptive research and development, promote regulatory consent, and stimulate new technologies and delivery
to the farmers. One reason this approach is needed is that it can tailor solutions to specific situations required
to win the war on drought. Because, as we all know, one size does not fit all. Local adaptation is imperative
different varieties will be needed for numerous and diverse target populations.

Second is the Public Intellectual Property Resource for Agriculture (PIPRA) program, which will help
American universities profit from their patented discoveries while making those technologies available for
humanitarian goals.

Both of these new programs focus on the farmers' needs as a starting point; for the old model of the scientist
telling the farmer what to do and how to do it is finished. Today, the farmer increasingly tells the scientist what to
do. We have reached the stage where we are at least consulting with farmers over characteristics like taste and
acceptability. We need to further shift the emphasis to having farmers tell us what they want us to do.

So in the battle against drought, what would success look like? First, we would see that small-scale maize and
rice farmers have access to drought-tolerant varieties. These would provide the buffer against the shocks of
drought years. This sustainability would lead to increased productivity and stability of crop yields, which in
turn would generate more income for farmers and all that entails, including better nutrition and health care,
and money for their children's education. All this is incumbent on participation in serviceable input and
output markets.

And what would failure look like? Above the Grand Canyon there is an abandoned Puebla village that was
inhabited around the 12th century AD for about 30 years. We don't know what occurred there to drive the
people out, but most likely it was drought-which leads me to a recent exchange I had with a Hopi Indian in
America's dry Southwest. He told me that according to Hopi tradition, one day their culture, along with their
maize, will save the world. It reminds me that in this war on drought and water stress, we must look to
indigenous people-not just as those we must help, but also to see how we can learn from them.

Thank you.


. Xiv *










Welcoming Statement


Masa Iwanaga
Director General -
International Maize and Wheat Improvement Center (CIMMYT) CIMMYT.




Bienvenidos, or for those unfamiliar with Spanish, welcome, to Mexico and thanks to you all for coming such
great distances to be here today with us. I'd also like to extend a special greeting to our guests from The
Rockefeller Foundation, notably Gordon Conway, the president of the Foundation, John O'Toole, and Gary
Toenniesen. It is an honor to have you in attendance.

This is not the first time CIMMYT has shared the stage or an important mission with The Rockefeller
Foundation. We share a long and productive history of collaboration going back to the mid 1940s when The
Rockefeller Foundation and the governments of the United States and Mexico formed the Office of Special
Studies-CIMMYT's predecessor-to address food shortage issues in Mexico. CIMMYT was officially
established in 1966 with support from The Rockefeller Foundation and the Mexican government, but
Rockefeller's involvement did not end there. The Foundation has continued to initiate and promote international
agriculture research collaboration worldwide. Closer to home, today the Foundation supports numerous
CIMMYT strategic projects.

Two recent and important examples of Rockefeller/CIMMYT collaboration concern soil fertility and Quality
Protein Maize (QPM). Recognizing the worrisome and continuing decline in soil fertility in the developing
world, CIMMYT and Rockefeller have made long-term efforts to promote sustainable soil management and
identify "best bet" technologies for resource-poor farmers. Extensive partnering through efforts such as the
Southern Africa Drought and Low Soil Fertility project (SADLF) and the Soil Fertility Network (SoilFertNet)
have lent focus to research and the diffusion of knowledge in this critical area. The success of SoilFertNet will
serve as the foundation for the new Consortium for Soil Fertility, a Rockefeller-supported network with 200
members, that will extend partnering and ground level impact in the region.

The diffusion of QPM is another important project on which we collaborate. QPM promotes better livelihoods in
two ways: directly through human consumption and via better feed for poultry and pigs. The Rockefeller
Foundation currently funds a CIMMYT project to develop and disseminate locally adapted QPM varieties in sub
Saharan Africa, which possess a range of biotic and abiotic stress tolerances and are well liked by local consumers.
Putting QPM into locally adapted backgrounds is accomplished with the help of National Agricultural Research
Systems (NARS), which the project also supports through capacity-building initiatives. Socioeconomic impacts
assessment are integrated into the project to assure that the target farmers are being well served.

These successful examples of partnering make me very optimistic about the endeavor that those of us here are now
engaged in. Drought presents probably the most important agricultural problem for small-scale farmers. The
complex challenge of drought tolerance is a high priority for both The Rockefeller Foundation and CIMMYT
because growing demand for food, climate change, competition for fresh water, and expansion to marginal lands
have intensified the need for drought tolerant plants in many parts of the world. CIMMYT is already fully engaged
in this effort through breeding for water-stress resistant wheat and maize and by screening the extensive genetic
resources in our genebank collections. With our extensive global network of NARS and advanced research institute
partners, we continue to work on developing new breeding strategies for drought, including molecular techniques,
and study ways to ensure that drought tolerant varieties get out to farmers and meet their criteria.


* xv *










As I mentioned earlier, in eastern and southern Africa, we have strengthened research capacity to help NARS
breed drought tolerant varieties. Through smallholder involvement, the process of participatory maize variety
selection, and "Mother/Baby" trials, we are fostering community-based seed production and delivery systems in
those regions.

To understand drought tolerance in staple food crops we must first look at genetic bases. CIMMYT is examining
these bases in 30 different environments, all under water-stress conditions. This background information
provides the data needed to effectively use marker-assisted selection for drought resistance in maize.

Turning to wheat, CIMMYT is now testing transgenic wheat we produced that carries the dehydration responsive
element-binding protein (DREB) gene, which comes from the Arabidopsis thaliana, and was provided by the Japan
International Research Center for Agricultural Sciences (JIRCAS). These trials provide a basis for comparing genetic
reactions to water stress and have shown very encouraging results. The trials also represent the first transgenic
wheat field trials in Mexico, and are the most advanced level of testing for food crops containing DREB to date. The
trials will be evaluated regarding their performance in both water-stress and normal conditions.

Another encouraging avenue of CIMMYT wheat research involves crossing wheat with its wild relatives to
incorporate various resistance traits, including drought tolerance. We are also developing wheat varieties adapted
to bed planting and resource conservation technologies and selecting for improved drought resistance traits, taking
germplasm x environment (G x E) interactions into account in the experimental strategies and data analysis.

In addition, CIMMYT is undertaking a massive study of wheat landraces at our research station in Obregon,
Mexico. Researchers have identified about 50 varieties showing superior drought responses. The desired drought
tolerance traits will be moved into modern wheats during pre-breeding projects. One can only imagine the good
we could do if we combined three sources of drought tolerance for wheat-the DREB gene, wild relative crosses,
and preferred alleles culled from landraces!

We at CIMMYT, and many of you here at this conference, recognize that since drought is one of agriculture's
most intractable problems, it will take innovation and an integration of many diverse approaches to overcome
it-at the breeding level as well as in farmers' fields where the ultimate impact will be measured. The new
CIMMYT strategy is designed in such a way that we can do just that. Our new strategic plan focuses on
improving livelihoods-a feat that could be achieved with the help of drought tolerant crops. Through new
partnerships and revamped programs, CIMMYT plans to make a difference in the lives of those who need us
most. We are happy to have The Rockefeller Foundation and all of you as partners in this effort.

Thank you all very much for your attention. I'm sure this week will prove to be an important and productive
event, and I look forward to meeting with all of you and benefiting from our shared knowledge. Again, welcome
to Mexico. I hope you enjoy your time here.


* xvi **






1. Farme Patptr Seh adEoo~ td


Breeding for improved drought tolerance in maize adapted

to southern Africa

MARIANNE BANZIGER1, PETER S. SETIMELA1, DAVID HODSON2, AND BINDIGANAVILE VIVEK1
1 International Maize and Wheat Improvement Center (CIMMYT), P.O. Box MP 163, Mount Pleasant Harare, Zimbabwe
2 International Maize and Wheat Improvement Center (CIMMYT), Apdo. Postal 6-641 06600, Mexico, D.F., Mexico

Corresponding author: Marianne Banzinger; E-mail: m.bazinger@cgiar.org


In 1997, CIMMYT initiated a product-oriented
breeding program targeted at improving maize for the
drought-prone midaltitudes of southern Africa. Maize
varieties were selected in Zimbabwe using
simultaneous selection in three types of environments,
recommended agronomic management/high rainfall
conditions, low-N stress and managed drought.
Between 2000 and 2002, 41 hybrids from this approach
were compared with 42 released and prereleased
private seed company hybrids in 36-65 trials across
eastern and southern Africa. Average trial yields
ranged from less than 1 t/ha to above 10 t/ha.
Hybrids from CIMMYT's stress breeding program


showed a consistent advantage over private company
check hybrids at all yield levels. Selection differentials
were largest between 2 to 5 t/ha and they became less
significant at higher yield levels. An Eberhart-Russell
stability analysis estimated a 40% yield advantage at
the 1 ton yield level, which decreased to 2.5% at the 10
ton yield level. We conclude that including selection
under carefully managed high priority abiotic stresses,
including drought, in a breeding program and with
adequate weighing can significantly increase maize
yields in a highly variable drought-prone environment
and particularly at lower yield levels.


I. Farmer participatory breeding and economic 1






1. Farme Patptr Seh adEoo~ td


Breeding for improved drought tolerance in maize adapted

to southern Africa

MARIANNE BANZIGER1, PETER S. SETIMELA1, DAVID HODSON2, AND BINDIGANAVILE VIVEK1
1 International Maize and Wheat Improvement Center (CIMMYT), P.O. Box MP 163, Mount Pleasant Harare, Zimbabwe
2 International Maize and Wheat Improvement Center (CIMMYT), Apdo. Postal 6-641 06600, Mexico, D.F., Mexico

Corresponding author: Marianne Banzinger; E-mail: m.bazinger@cgiar.org


In 1997, CIMMYT initiated a product-oriented
breeding program targeted at improving maize for the
drought-prone midaltitudes of southern Africa. Maize
varieties were selected in Zimbabwe using
simultaneous selection in three types of environments,
recommended agronomic management/high rainfall
conditions, low-N stress and managed drought.
Between 2000 and 2002, 41 hybrids from this approach
were compared with 42 released and prereleased
private seed company hybrids in 36-65 trials across
eastern and southern Africa. Average trial yields
ranged from less than 1 t/ha to above 10 t/ha.
Hybrids from CIMMYT's stress breeding program


showed a consistent advantage over private company
check hybrids at all yield levels. Selection differentials
were largest between 2 to 5 t/ha and they became less
significant at higher yield levels. An Eberhart-Russell
stability analysis estimated a 40% yield advantage at
the 1 ton yield level, which decreased to 2.5% at the 10
ton yield level. We conclude that including selection
under carefully managed high priority abiotic stresses,
including drought, in a breeding program and with
adequate weighing can significantly increase maize
yields in a highly variable drought-prone environment
and particularly at lower yield levels.


I. Farmer participatory breeding and economic 1











Rice seed systems in southern China: Views from

institutions and farmers


SHIJUN DING1, XIAOJUN Gu2, FENGBO CHEN1, CHUANBO CHEN1, SUSHIL PANDEY3, QIFA ZHANG1, AND LIJUN LUo1' 2

1 Huazhong Agricultural University, Wuhan, 430070, China
2 Shanghai Agro-biological Gene Center, SAAS, Shanghai, 201106, China
3 International Rice Research Institute (IRRI), DAPO Box 7777, Metro Manila, Philippines

Corresponding author: Shijun Ding; E-mail: dingsj@public.wh.bh.cn


Introduction
Agents involved in rice seed systems in China include
agricultural administrations, research institutes,
universities and colleges, seed companies, and farmers.
Although farmers may request locally adapted seed
and direct delivery channels, the other agents, who
have more power over the seed systems, have different
views about the rice seed systems (Lin, 1992; Huang et
al., 2000). The farmers do not have much say on the seed
systems, which adversely affects rice farmers in southern
China, especially marginalized (or subsistence) farmers.
Farmers and other agents are required to work together
towards establishment of comprehensive rice seed
systems. By doing so, the understanding and views of
farmers and other agents, and the differences between
them, can be investigated.


Methods
Institutional interviews and a rice farm
household survey were carried out.
Institutional interviews from the supply
side of the rice seed systems were
mainly carried out with officials and
scientists at various agricultural
administrative offices (AAO), public
agricultural research institutes and
universities (PARI), and agricultural
technology extension stations (ATES).
The rice farm household survey from
the demand side was then implemented.
Huopai village from Xiangyang county
of Hubei province in south central
China, which represents the main rice
production area, and Yueli village from
Nandan county of Guangxi province in
southwest China, which represents


marginalized rice production areas, were selected as
fieldwork sites. Officials and scientists from AAOs,
PARIs, ATESs, and seed companies in two provinces,
were interviewed; 61 rice farm households from Yueli,
Guangxi, and Huopai, Hubei were surveyed from
March to May 2002.


Results
A rice seed system based on data from interviews in
two provinces was identified (Figure 1). "Rice yield"
was rates as the highest priority breeding target by staff
from four PARIs in two provinces; "rice quality"
followed. From farmers' perspectives (Tablel), the
largest proportion of rice farmers (56.5 %) in Hubei
chose "rice quality" as the first criterion when


Research Production Marketing Adoption




R&D Dept Breeding Dept Marketing Dept

Seed Company ([
SSeed Dealer






PARI: Public Agricultural Research Institutes / Universities.
GOC: Companies organized by former staff from agricultural administrative office.
ATES: Governmental Agricultural Technologies Extension Stations at county and township.

Figure 1. Rice seed systems identified based on data from institutional interview in
provinces of Hubei and Guangxi.


* 2 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004










Table 1. Percentage distribution of farmers' answers to "why do you
adopt the rice varieties currently grew in the field" by village

Huopai Yueli
Good quality 56.5 27.8
High yield 30.5 16.7
High ratio of milled rice 8.7 2.8
Local adaptability 4.3 33.3
No other choices 0 19.4
No. of answers 23 36



purchasing seed, while the largest proportion of rice
farmers (33.3%) in Guangxi chose "local adaptability"
as the first criterion. Farmers in Huopai have a wide
range of seed dealers available for seed purchases,
while seed can only be purchased in township ATES as
dealers, in Yueli. Furthermore, 19.4% of farmers in Yueli
reported that the rice they grew in the field was the
only variety that could be found in the market.



Conclusions

Seed systems can be horizontally divided into four
processes: research, production, marketing, and
adoption. The systems can also be vertically separated
into several participating agents: public agricultural
research institutes, seed companies, agricultural
technologies extension stations, seed dealers, and rice
farmers. Seed companies play an increasingly
important role in providing rice seed to farmers, while
government seed provision, such as the agricultural


technology extension station's network, still work.
Private companies closely cooperated with public
agricultural research institutes and government
agricultural administrative offices, although some
government officials and scientists from research
institutes were "jumping into the sea" to start their
own seed business. Different views of farmers and
other agents involved in seed systems obviously
could be found (Chen et al., 2004). While farmers in
Huopai village requested rice seed with "good
quality" and farmers in Yueli village looked for more
"local adaptability," research institutes and companies
took "high yield" as the primary target for their
breeding strategies, with "good quality" next. Locally
adapted rice seed breeding was not yet considered
important by breeders, implying that the
marginalized rice farmers'needs were neglected.
Public and private research institutes and companies
should listen to farmers while preparing their seed
development strategies.



References
Chen, F, P Liu and S. Ding. 2004. Farmers' understanding of rice varieties in three villages
in China, China Rice, No 2.
Huang, J., R. Hu, L. Zhang and S. Rozelle. 2000. Economics of Investment on Agricultural
Sciences and Technologies in China, China Agriculture Press.
Lin, J. Y. 1992. Hybrid Rice Innovation: A Study of Market-Demand Induced Technological
Innovation in A Centrally Planned Economy, the Review of Economics and Statistics, Vol.
LXXIV, No. 1, February.


I. Farmer participatory breeding and economic 3 *











Adaptability of 16 upland rice varieties to two

moisture regimes


J. IMANYWOHA 1, P. KIBWIKA2, M. WALUSIMBI1, G. BIGIRWA1, AND J. LAMO1

1 Cereal Program NAARI, Uganda
2 Makerere University, Kampala, Uganda

Corresponding author: J. Imanywoha; Email: Imanywoha@yahoo.co.uk


Introduction
Despite its history in the region dating back to 1904
(Jameson, 1970), rice did not become a major crop
until the early 1990s (Agricultural Secretariat Report,
1992). Its importance is significantly increasing due to
urbanization, changing eating habits of the new
generation, and increasing number of public
institutions; as such, per capital consumption quickly
jumped from 300 grams in 1980 to the present 10.5 kg
in 2002. The increase in demand has stimulated
production, especially in the in the wetlands, and
increased imports from the Far East. Rice imports
stand at US$120 million annually. Production of rice
in the wetlands is raising environmental concerns
among the government and other organizations (State
of the Environment Report for Uganda, 1994). A
project on upland rice was initiated with the aim of
increasing rice production in general and
productivity of rice under upland conditions in
particular. This would attract both rice farmers in
lowlands and non-rice growers to upland rice that is
easy to produce, thus increasing rice production.
Consequently, this would reduce the amount of
imported rice and protect wetlands.


Methods
Sixteen upland rice varieties selected in preliminary
yield trial were evaluated in separate trials under
optimum and drought conditions. Both trials were
conducted at Namulonge Agricultural and Animal
Research Institute (NAARI). Treatments were
arranged in a randomized block design with four
replicates. Individual plots consisted of 8 rows, 5 m
long at spacing of 30 cm between rows and 5 cm
between plants. The trial under optimum conditions


was planted at the onset of rains and received 678
mm of well distributed rainfall. The drought stressed
trial received one month's rain and an irrigation 40
days after planting and another at milk stage. Both
trials received N45P30 applied at planting.

From the above experiments, ten varieties were
selected for participatory selection on-farmers' fields.
The selection was based on maturity period,
resistance to rice blast, yield, and drought tolerance.
The ten varieties were planted in five districts, and
the top five varieties subsequently selected for further
evaluation on-farm. The five were then reduced to
three and these were released for commercial
production in Uganda. Farmers' selection criteria
included grain yield and maturity period, followed
by resistance to lodging and diseases, grain size, and
culm length.


Results and discussion
All the varieties tested flowered in a narrow range of
nine days (71 and 80 days) under adequate moisture,
but under moisture stress, the range increased to 19
days (76 and 96.3 days). WAB 189 out-yielded all the
other varieties under optimum conditions, but was
observed to be the most sensitive to drought. Its
percent productive tillers was reduced by 96.5%;
panicle length was reduced by 40.7%. Days to flower
increased from 79 to 96.3 days under drought
compared to optimum conditions (Table 1). Varieties
WAB 450, ITA 325, ITA 257, and Sikamo had
significantly higher yields than the local check and
were also observed to be least affected by drought on
the traits studied.


* 4 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004











References


Varieties that performed well under drought stress at
Namulonge continued to perform well on farmers'
fields and three of them-ITA 257, ITA 325, and WAB
450-were finally selected by farmers in all the districts
they were evaluated. They have been released under
the names: NARIC 1, NARIC 2, AND NARIC 3,
respectively. NARIC 3 has become so popular that
many seed companies, NGOs, and government
institutions are trying to produce more of its seed to
meet the demand. This suggests that the farmers have
discovered that upland rice can be produced more
easily than the tedious swamp rice


Bank of Uganda Agricultural Secretariat, 1992. Report on Agricultural Input Situation.
Jameson, J.D. 1970. Agriculture in Uganda. London: Oxford University Press. Pp 395
State of the Environment Report for Uganda. 1994. Ministry of Natural Resources: National
Environment Information Center. Pp 15-47.


Table 1. Performance of 16 upland rice varieties under drought stress and normal conditions

DAY TO FLOWER CULM LENGTH PANICLE LENGTH YIELD
VARIETY UNDER STRESS NORMAL UNDER STRESS NORMAL UNDER STRESS NORMAL UNDER STRESS NORMAL

IRAT 233 89.3* 79* 51.1* 79.6 21.1 24.5* 756 4831
IDSA-17 80.5 75 39.4 71.9 19.3 22.3a 592 3935
BR-1890-1-1-2 90.3* 80-* 49.3-* 86* 19.2 23.6* 467 4530
WAB 3299 91.7* 78* 44.2 85.1* 19.5 24.1* 147 4879
IRAT 240 89.2* 77 39.5 86.1* 18 23.5* 1010 5109
WAB 332 89.2* 78* 46.1 89.7* 18.2 23.1* 263 4321
SIKAMO 77.3 73 41.2 86.2* 18.3 25 1765* 4882
IITA 257 76 71 44.7 71.6 20.2 23 1789 4420
WAB 189 96.3* 79* 36.2* 81.5 16.2 27.3* 275 5215
WAB 450 81 77 46.6 76.3 23 23 1915* 4536
Check 80.7 74 44.3 79.3 22 23 882 3868
ITA 325 79.3 74 53.2* 94.3* 20.1 22.1a 1802* 4392
UK 2 78.7 74 43 72.3 19.1 22.5 952 3192
NP 4 87.4* 74 39.5 82.1 17.2 24.5* 427 4935
NP2 80.3 73 42.1 79.6 19.5 23.6* 1030 3775
NP3 78.3 74 41.3 75.2 18 24.1* 79.9 3370

MINIMUm 77.3 71 36.2 71.6 16.2 22.1 147 3192
MMAXMUM 96.3 80 53.2 94.3 23 27.3 1915 5215
MEAN 84.1 75.6 43.6 81 878.6 1473.6
LSD (0.05) 4.1 3.8 3.1 4.2 929.4 4386.9

a = Significantly below the Check
*= Significantly above the Check


I. Farmer participatory breeding and economic 5 *


Conclusions











Farmers' participatory varietal selection at target drought

prone area of Tamil Nadu

P. JEYAPRAKASH1, S. ROBIN2, R. PUSHPA2, S. MANIMARAN2, R. CHANDRABABU3, AND P. BALASUBRAMANIYAN1

1 Agricultural Research Station, Paramakudi-623 707, Tamil Nadu, India
2 Department of Rice, Tamil Nadu Agricultural University, Coimbatore-641 003, India
3 Centre for Plant Molecular Biology, Tamil Nadu Agricultural University, Coimbatore 641 003, India

Corresponding author: P. Jeyaprakash; E-mail: agri jp@yahoo.com


Introduction
Success of new crop varieties depends upon quick
adoption. Often it is rather slow in marginal
environments. Many times the improved varieties from
the researcher fail due to the reason that farmers'
perceptions change over time. Lines selected from such
networked Participatory Varietal Selection (PVS) trials
are likely to be broadly adapted, stress tolerant, and
acceptable to farmers (Atlin et al., 2002).To determine
the acceptability of and the preferences for the newly
developed rice cultivars, a PVS was carried out.


Methods
PVS by the local farmers was conducted on-station
during the 2002-2003 cropping season at the
agricultural research station, Paramakudi. A total of 61
progressive farmers were asked to select the genotypes
by scoring for selected traits. Scoring was between "1"
and "10" based on their preferences. Highly preferred
was scored "10" while the a very low preference was
scored "1." Plant height, duration, grain quality,
drought tolerance, and grain yield were surveyed apart
from overall acceptability of the genotypes. Thirty rice
genotypes comprising 19 rice cultures in advanced
stage and 11 released varieties were presented for
selection. The identity of the cultures/varieties was
hidden and a random code number was assigned for
each genotype. Each farmer was provided with a
spreadsheet, with numbers of genotypes, to give scores
based on their phenotypic evaluation for the five traits
and for overall acceptability. The farmers scored for the
traits and the scores were consolidated by summing up.


Results
Farmers enjoyed scoring the genotypes and the
response was good. This being their furst such
experience, they showed great interest in scoring. The
total scores for individual traits are presented in table 1.
Among the genotypes, the culture IET 17458 and PM
9106 scored high in terms of overall acceptability.
Besides overall acceptability, the culture PM 9106
bagged up top score for duration, plant height, grain
quality, and grain yield. The rice variety TRY (R) 2
scored the highest rank for drought tolerance.


Conclusion
The culture IET 17458 and PM 9106 were highly
preferred by the farmers, indicating that sthe
advanced stage rice cultures are highly acceptable
and have potential for introduction as new varieties
in farmers' fields. PVS paved the way for need
based selection by the farmers and thereby helps
promote quicker adoption of useful varieties in the
farming community.


* 6 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004











Table 1. Overall acceptability by the farmers on the major traits in participatory evaluation

Lines PAS Plant height Duration Grain quality Drought tolerance Grain yield

IET-17458 1 5 6 5 8 3
PM 9106 2 2 1 1 3 1
TRY-2 3 12 11 4 1 4
PM 2 K 022 4 6 8 7 4 5
TKM-11 5 4 15 2 2 2
PMK-2 6 1 4 8 5 7
TM 97032 7 7 5 6 6 6
PM2K019 8 3 3 9 7 10
ASD-20 9 8 9 3 12 9
ADT-36 10 11 2 10 9 8
IET-17450 11 5 17 12 10 11
IR-36 12 15 13 13 17 16
PM2K020 13 9 12 11 11 13
IR-20 14 19 10 14 22 15
IET-17455 15 20 24 17 16 19
IET-17448 16 13 18 16 20 17
IET-16704 17 26 21 18 18 12
IET-17441 18 21 19 15 14 14
IET-14567 19 17 23 21 13 21
RM 96019 20 24 16 20 23 18
IET-17446 21 22 27 26 28 25
IET-17434 22 27 22 23 25 20
CO-45 23 16 25 25 19 28
TKM-10 24 14 24 22 21 22
TM-97198 25 28 26 28 24 26
IET-16820 26 25 14 24 26 24
PM2K017 27 23 28 27 27 27
TKM-12 28 10 15 19 15 23
CO-47 29 29 30 29 30 29
IET-17433 30 30 29 30 29 30


References
Atlin G.N., T.R. Paris, B. Linquist, S. Phengechang, K. Chongyikangutor, A. Singh, V.N. Singh,
J.L. Diwevedi, S. Pandey, P. Lenas, M. Lazer, PK. Sinha, N.P. Mandal, and Swarno.2002.
Breeding rainfed rice for drought prone environments in integrating conventional and
participatory plant breeding in South and Southeast Asia.


I. Farmer participatory breeding and economic 0 7











Farmers' participatory plant breeding technique: An

effective tool for the early selection and adoption of rice

varieties in rainfed rice ecosystems


S. MAHENDRAN1, L. MAHALINGAM1, T. SIVAKUMAR1, M. HEMALATHA1, N. CHITRA1, R. CHANDRA BABU2,
P. SHANMUGASUNDRAM2, AND S. ROBIN3

1 Coastal Saline Research Centre, Tamil Nadu Agricultural University (TNAU), Ramanthapuram 623503, Tamil
Nadu, India, TNAU, Coimbatore-641003, India
2 Department of Biotechnology, Centre for Plant Molecular Biology, TNAM, Coimbatore-641003, India
3 Department of Rice, TNAU, Coimbatore-641003, India

Corresponding author: S. Mahendran; E-mail: profmahe@Rediffmail.com


Introduction
The shadow of drought loomed heavily on this year's
harvest of kharifcrops. Rice production in the current
year is likely to be drastically reduced and there is a
large decline in the production of coarse cereals
(Economic Survey, 2003-04). Ramanathapuram district
situated in the southeast coast of Tamil Nadu, India
falls in the rain shadow region and therefore is a highly
drought prone and most backward in development.
The district's average rainfall is 827 mm, and of this,
60% is received during the North East Monsoon
(October to December). Rice is the predominant crop
cultivated in an area of 104,223 ha under semi-dry and
rainfed conditions. The crop suffers either early
drought or late drought as a consequence of erratic and
uneven distribution of rainfall during the cropping
season. This often resulted in low crop yields or at
times complete crop failure. For instance, during 2003
04 season, due to the deficit of rainfall in December,
complete crop failure has been recorded in an area of
100,017 ha, accounting for losses of 2.51 lakh tons rice
production and 90% of the total rice production in
Ramanathapuram district. Thus, drought is threatening
food security in this region of Tamil Nadu. To sustain
the rice production and to achieve higher yield, it is
vital to develop drought tolerant varieties in addition
to other moisture stress management techniques. Crop
improvement for drought resistance must be
considered within the broader context of a total
agricultural research and extension strategy (Nix, 1982).
Breeders instinctively look for new sources of variation
when attempting to improve plants, but such empirical


selection in the case of drought resistance has been
difficult. Also, selection under controlled
environments rarely correlates with performance in
the field and the consistency of response to drought
also varyies year after year due to variability in the
environment. Embarking on a participatory plant
breeding (PPB) approach may have many motivations,
among them, increased and more stable productivity,
faster release and adoption of varieties, better
understanding of farmers' varietal criteria, enhanced
biodiversity, increased cost effectiveness, facilitated
farmer bearing, and empowerment of farmers
(Sperling et al., 2001).


Methods
Four blocks of Ramanathapuram district were selected
for this programme based on their representation of
the environments targeted in the breeding work,
diversity and range of ecological conditions,
involvement of women in farm activities, availability
of previous survey data, and easy access to the site. As
a part of this study, a participatory rural appraisal was
also conducted with the objective to characterise the
farmers' crop management practices, gender roles, and
farmers' preferences for traits in improved varieties to
suit the local needs. Farmers were selected based on
their primary occupation and degree of literacy. Five
rice varieties were selected and raised in an area of
50% of the farmers' fields and managed by the farmers
under the supervision of our scientists. In addition,
farmers were involved in the participatory varietal
selection process at our research farm.


* 8 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004











Conclusions


The participatory rural appraisal results revealed that
the awareness about high yielding varieties was as high
as 93%. However, the farmers preferred low yielding
landraces to high yielding varieties for their tolerance
to drought, low input cost, and readily available seed
materials. Men (74%) decide upon the variety to be
cultivated, despite the fact that the women spent more
(4 times more) time than men in the field. Among the
five varieties tested in the participatory varietal
selection (PVS) programme in the farmers fields, three
varieties (Ashoka 228, Ashoka 200F, and RM96019)
provided yields and were liked by farmers due to the
short duration of the variety, which is a drought escape
mechanism. Thus, yield and duration were important
traits considered by farmers. The on-farm participatory
breeding programme (PBP) (Table 1) revealed that
farmers' preference rankings were not always
correlated with scientists' ranking. However, the lines
selected by farmers were almost tallies at 75% with
scientist selection, indicating that farmers too are
capable of identifying varieties by considering
important plant growth characteristics like duration
and plant stature and grain quality of their choice. This
reflects their great experience, which also mattered
when selecting rice varieties at PBP. This shows that
there is a strong agreement among farmers and
between farmers and scientists' in selecting best
varieties suited to their environment.


A participatory plant breeding programme and
participatory varietal selection programme are
essential in unfavorable rainfed environments and
with diverse socioeconomic groups that depend on
rice for their livelihoods, such as Ramanathapuram
district of Tamil Nadu. Although it is too early to
show the impact of PBP and PVS through the spread
of materials generated, farmers' participation in the
coming years will become vital in the selection of
suitable varieties of their choice for the complex
rainfed environment. This is because farmers will be
in a better position to screen new varieties at their
own levels of management with the assistance of
scientists involved in this programme. This approach
will not only promote the identification of suitable
varieties for this environment, but will also create
confidence among the farming community, which
will result in early and easy spread of seed materials
in the shortest time possible.



References
Economic Survey 2003-04. Ministry of Finance and Company Affairs, Government of
India, New Delhi.
Nix, H.A. 1982. In: Drought Resistance in Crops with Emphasis on Rice. Philippines: IRRI.
pp. 3-15.
Sperling et al. 2001. Euphytica. 122(3): 537-50.


Table 1. On-farm farmers participatory varietal selection

Lines selected by Grain yield Rank given
Parentage scientist in order (kg/ha) by farmers

IR 64 x Azucena 17 and 23 4266 8 and 5
IR 64 x Azucena 18 4178
IR 64 x Azucena 15 4089 6
IR 64 x Azucena 21 and 19 4000 1 and 7


I. Farmer participatory breeding and economic 9 *


Results











Participatory maize variety evaluation for

increased adoption


M.S. MWALA1, J. DE MEYER2, P. SETIMELA3, AND M. BANZIGER3

1 University of Zambia, P.O. Box 32379, Lusaka, Zambia
2 Maize Program, International Maize and Wheat Improvement Center (CIMMYT), Apdo. Postal
6-641, 06600, Mexico D.F., Mexico
3 Maize Program, International Maize and Wheat Improvement Center (CIMMYT), Zimbabwe,
P.O. Box MP163, Mount Pleasant, Harare, Zimbabwe

Corresponding author: M.S. Mwala; E-mail: m.mwala@yahoo.com


Introduction
Development and transfer of improved maize varieties
has often been found to take too much time, resulting in
little impact on the farmers. National maize programs in
the Southern Africa Development Community (SADC),
in collaboration with CIMMYT sub-regional office in
Zimbabwe, adopted a participatory variety evaluation
method, the Mother/Baby scheme, through which
evaluation of the varieties was done under conditions
representing those of the farmers. The scheme allows
for the selection of appropriate varieties for the target
environments.


Methods
The scheme uses a network of partners within each
country, through which environments, representative
of a range of biophysical and socioeconomic aspects
of the target environments, are sampled (Banziger
and de Meyer, 2000). Local counterparts-who could
be an extension officer, a local researcher, an
agricultural teacher or an NGO staff-supervises the
trials while farmers evaluate the varieties in their
fields, providing feedback on what they perceive as
important characteristics for their conditions.


Results
The result has been that selected varieties possess
important characteristics that conform to farmers'
production and utilization expectations. Farmers, as
beneficiaries, have a major say in the development of
the varieties, thus uptake and use of these new


varieties is quicker. Other benefits include cost
effectiveness, i.e., time from evaluation to utilization as
partners disseminate the varieties, and
comprehensiveness resulting from partners
contributing their expertise during implementation.

During the last four years, the Mother/Baby scheme
has contributed to the rapid release of ZM 421 and ZM
521 in RSA, Malawi, Tanzania, and Zimbabwe, and ZM
621 in Malawi. Seed production of these varieties is
currently in excess of 100,000 tons, a significant input
of improved technology for farmers.


Conclusions
The evaluation of maize varieties in the target
environments, which involved the beneficiaries, has
provided an opportunity for the identification and
selection of appropriate varieties for the small-scale
farmers. Quicker use of improved technology could
have a direct and positive impact on the poverty and
food security coping strategies of the farmers


References
Blnziger, M., and J. de Meyer, 2002. Collaborative maize variety development for stress-
prone environments in southern Africa. In D.A. Clevelend, and D. Soleri (eds.), Farmers,
Scientists and Plant Breeding. CAB International. Pp. 269-96.


* 10 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004











Economic costs of drought and rice farmers' coping

mechanisms: A synthesis of cross-country

comparative analysis


S. PANDEY1, H. BHANDARI2, R. SHARAN3, S. TAUNK4, D. NAIK5, P. PRAPERTCHOB6, AND S. DING7

1 International Rice Research Institute, Makati City, Philippines
2 International Rice Research Institute, Makati, Philippines
3 Ranchi University, Ranchi, India
4 Indira Gandhi Agricultural University, Raipur, India
5 Orissa University of Agriculture and Technology, Bhubaneswar, India
6 Khon Kaen University, Khon Kaen, Thailand
7 Huazhong Agricultural University, Wuhan, China

Corresponding author: S. Pandey; E-mail: sushil.pandey@cgiar.org


Drought is a major problem affecting agricultural
production in the humid/subhumid South and
Southeast Asia, where rice is a major crop. The
economic costs of drought include not only the
production loss of rice, but also the loss in production
of post-rice crops that are grown on residual soil
moisture. Additional economic and social costs result
from the choice of production practices that are
designed to reduce the losses from drought and from
a longer-term decline in production capacity resulting
from the depletion of productive assets due to distress
sale during drought years. A good understanding of
these coping mechanisms are essential for designing
technological and policy interventions for a more
effective drought mitigation and drought relief.

For this paper, estimates of economic losses resulting
from drought in rainfed and partially irrigated areas
of southern China, eastern India, and northeastern
Thailand were obtained and farmers' drought coping
mechanisms were analyzed. These three regions
capture a range of climatic conditions, income levels,
and institutional set-ups. Through a comparative
analysis, deeper insights on factors that moderate or
amplify the effect of drought on the welfare of farmers
differentiated by socioeconomic strata are obtained.
Previous contributions to this literature are based on
arid and semi-arid zones and have seldom used the
analytical framework that involves a cross-country
comparative analysis.

Production losses due to drought were estimated
using provincial and district level time series data.


Production, area, and yield were regressed on time
trend and drought dummy variables representing
different intensities of drought. The coefficients
associated with the drought dummy variables provide
estimates of the effect of drought on production, area
and yield. These were suitably weighted by the
probability of drought estimated from the long-term
rainfall data to obtain the expected loss. In addition,
the elasticities of production, area, and yield with
respect to rainfall were estimated using the district
level data. The variations in elasticities across districts
were explained using a set of independent variables
that describe the district characteristics.

The average value of production loss resulting from
drought was estimated to be 8-10% of the value of
agricultural output, although the production loss
during the drought years was found to be as high as
40%. The loss in output was not just due to yield loss
but also due to a contraction in the area planted and
harvested. The effect of this production loss on the
employment and income of the poor were also
estimated. This second round effect of drought was
found to be substantial in India, where production
losses were also high. In southern China and
northeastern Thailand, the production losses were
found to be relatively lower.

Farmers drought coping mechanisms and the impact
of drought at the household level were analyzed using
a survey of more than 1,500 farmers from these three
regions. Information on both ex-ante (strategies that
reduce the losses from drought) and ex-post


I. Farmer participatory breeding and economic 11 *










(strategies that are designed to maintain consumption
in the face of production losses) strategies were
collected. Farm households were found to employ
elaborate strategies that involve careful choice of rice
varieties, planting date, planting method, and weeding
and fertilization practices to minimize the effect of
drought. In addition to these adaptations in rice
production, farmers were also found to make temporal
adjustments in cropping patterns based on drought
incidence. These adjustments were analyzed through a
comparison of production practices of households
during drought and normal years. The economic costs
of these ex-ante adjustment mechanisms were
measured by comparing the farm incomes of
households experiencing different intensities of
drought. The results indicate that farmers with smaller
farm size and poorer resource base bear a higher cost
relative to farmers with larger farm size and better
resource base.

The survey data were also used to analyze the ex-post
coping mechanisms. Increased dependence on wage
income, asset depletion, and public relief were found
to be the major mechanisms used to meet the shortfall


in income. The relative importance of these strategies
was found to vary across the region with asset
depletion and public relief being more important in
India than in China and Thailand. Despite these
mechanisms, most farmers were unable to maintain
their pre-drought level of consumption, especially in
India. Women and children were found to bear the
burden of drought disproportionately. This indicates
that the coping strategies currently employed by
farmers are not fully effective in protecting their
consumption. A better market integration and increased
opportunities for off farm employment resulted in a
lower consumption loss in China and Thailand.

The overall implications of the findings of the study
for technology design and for policy improvements
for drought mitigation and drought relief are derived.
Attention is drawn to improve drought forecasting,
encourage income diversification, increase moisture
availability to crops through irrigation and moisture
conservation, develop drought-tolerant crop varieties,
and improve the targeting of drought relief programs.


* 12 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004











Development and dissemination of drought tolerant rice

varieties through on-farm, farmer-oriented approaches


A. PRASAD1, J.S. GANGWAR1, V. SINGH1, S.C. PRASAD1, A. CHAUDHARY1, D.N. SINGH2, D.S.VIRK3,
K.A. STEELE3, AND J.R. WITCOMBE3

1 Gramin Vikas Trust (GVT), 280-Kanke Road, Ranchi-834008, Jharkhand, India
2 Birsa Agricultural University (BAU), Kanke, Ranchi-834006, Jharkhand, India
3 Center for Arid Zone Studies (CAZS), University of Wales, Bangor, UK

Corresponding author: Amar Prasad; E-mail: honoida@gvfindia.org


Introduction
The Gramin Vikas Trust (GVT) manages two rainfed
farming projects, funded by DFID, UK, for improving
the food security and livelihoods of poor men and
women in nearly 1,500 villages in six states in India.
The GVT uses a participatory development approach
facilitated by villagejankars (skilled, trained village
motivators) and self help groups (SHGs).

Poor farmers in the GVT villages cultivate marginal
lands much of which (about 40%) is rainfed upland.
Farmers have traditionally grown low-yielding
landraces in the upland because of a lack of suitable
improved cultivars. A participatory varietal selection
(PVS) programme was started in 1995 to allow farmers
to test and select suitable varieties and this was followed
by a participatory plant breeding (PPB) programme.


Methods
In PVS, Kalinga III was identified as the most preferred
variety. However, it is prone to lodging and has a poor
root system. It was used as a parent in a PPB
programme started in 1997 as a collaboration between
GVT, BAU, and CAZS with funding from the Plant
Science Research Programme of DFID. This used
collaborative methods (farmers selected in F4 bulks on
their farms) and consultative methods (farmers selected
among F4 bulks on research station) (Virk et al., 2003).

Attempts were made to encourage private seed
producers, but the low profit margin from the sale of
seed to poor upland farmers was unattractive.
Therefore, community-based seed production was
initiated by GVT in the winter season of 2001-02 in


Orissa, through SHGs that were motivated to
undertake seed production. GVT procured and
distributed this seed to other SHGs, NGOs, and GOs.


Results
The PPB resulted in the release of two drought tolerant
varieties, Ashoka 200F and Ashoka 228, in 2003 in
Jharkhand. In surveys in December 2002, most farmers
perceived that the new varieties were superior for many
traits including drought tolerance (Figure 1). Farmers
were increasing the area under the Ashoka varieties and
spreading them through the informal seed sector.


Higher grain yield ?
Higher straw yield?
Earlier maturity ?
S Better drought tolerance ?
No Yes
Better weed suppression?
Higher market price?
Preferred overall ?
Grow Ashoka 200F again?
Grow Ashoka 228 again?

-20 0 20 40 60 80 100
Corresponding farmers (%)
Three states; n = 159

Figure 1. Farmers' perception (expressed as % of farmers) for Ashoka
228 and Ashoka 200F rice varieties in comparison to the local cultivars.
Based on a survey of 159 households sampled over all three states
(Orissa, Jarkhand, and West Bengal) in December 2002.


I. Farmer participatory breeding and economic 13 *











Farmers sold between 2 and 50 kg of seed to farmers
within the village and outside the villages up to a
distance of 300 km. The number of SHGs and villages
and the quantity of seed produced in the winter
seasons has increased over years (Table 1, Figure 2).


Table 1. Distribution of Ashoka 200F and Ashoka 228 varieties in the
rainy season of 2003

Agency (number) No. of farmers Quantity (t)

GVT East 2465 30.2
GOs (7) 129 19.5
NGOs(13) 312 18.8
GVT West 32.0

Based of 5 GOs in Orissa as the number of farmers from 2 GOs in Jharkjand is not available.
- Not available.


160

140
120

8100

g 80

60

40

20


2001 2001-02 2002-03
Season


Conclusions

The community-based seed production has removed
the conventional five-year gap between release and
dissemination. Although informal spread from farmer
to farmer is an effective means of spread, seed
production and distribution by NGOs and GOs will
greatly accelerate the spread. To gain maximum
benefit from the new varieties, the effectiveness of
informal spread increases with the number and wider
distribution of primary adopters. Hence seed
production and distribution needs to be continued for
some years, and the seed needs to be widely
distributed outside GVT villages, particularly in states
not served by GVT (i.e., Chhattisgarh and Bihar). This
will be done through internationally funded projects
(e.g., DFID-funded Western Orissa Livelihood Project),
private seed agencies, and other NGOs.


- PPB is a continuous process and efforts are in place to
7) replace Ashoka varieties with superior alternatives. A
number of potential varieties with better drought
tolerance developed at CAZS, through marker
assisted selection for root traits are in the advanced
stages of testing (Prasad et al., 2003). Efforts on PPB
continue, with success most likely to come from
crosses involving Ashoka 200F and Ashoka 228.



References
-Prasad, A., et al. 2003. 3rd Rockefeller Found. Workshop 'Drought Tolerance in Rice'
2003-04 Bangalore (Absts.)
Virk, D.S., et al. 2003. Euphytica 132: 95-108.


Figure 2. Quantity of seed produced (t) by GVT from 2001 main season
to projected production in 2003-04 winter season. The number of self
help groups with number of villages in brackets involved in seed
production are above the line.


* 14 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004











Drought and cropping pattern change in Tamil Nadu, India:

Needed technological transformation in rice farming


C. RAMASAMY, K.N. SELVARAJ, AND R. CHANDRA BABU

Tamil Nadu Agricultural University
Coimbatore-641003, Tamil Nadu, India

Corresponding author: R. Chandra Babu; E-mail: chandrarc@hotmail.com


Introduction
Demand for rice in India is projected at 128 million
tons for 2012 and will require a production level of
3,000 kg/ha, significantly greater than the present
average yield. After a long period of technological
breakthroughs and adoption, the yield gap still exists
(Siddiq, 2000). Of the total area under rice in Tamil
Nadu State, nearly 7% of the area is under dry and
semi-dry conditions. Dry and semi-dry types of
cultivation are predominant in the districts such as
Ramnad, Sivagangai, and Thiruvallur. Studies
identified several production constraints including
insects/pests, diseases, adverse soils, genetic/
physiological and adverse climatic and environmental
factors, which contribute to major yield losses in rice
and indicated the possibility of biotechnological
approaches to solve such problems (Ramasamy et.al.,
1996; Ramasamy and Jatileksono, 1996). Studies
revealed that the average yield was about 30-35%
higher in the irrigated ecosystem than in the rainfed
ecosystem (Janaiah et al., 2000). This paper highlights
the impact of drought on cropping pattern change
and income distribution in Tamil Nadu. The paper
emphasizes the importance of crop breeding for water
limiting environments in order to cope with drought.
While attempting to develop such varieties, it is
crucial to consider the traits of the varieties that are
predominantly grown by the farmers.


Methods
Effect of drought on cropping pattern change was
assessed by construction of the Herfindahl index; the
factors affecting the diversification were analyzed
using the log linear equations. Factors such as rainfall,
irrigation intensity, fertilizer consumption per hectare,
wholesale price index, and productivity index were


included in the model to examine their influence on
acreage diversification. Influence of rainfall and
productivity of rice on area allocation decisions and
influence of rainfall distribution on paddy productivity
was also assessed. Weakening of the trickle down
effect of agriculture on poverty reduction due to
drought was estimated using a system of equations.
An inequality index was employed to analyze impact
of drought on income distribution.


Results
Rice yield recorded a compound growth rate of 2.13%
during 1965-66 and 2001-02 in Tamil Nadu. Sub-period
growth rates indicate that rice productivity witnessed
a high growth rate of 4.69% during the 1980s.
However, productivity of rice registered a negative
growth rate of 0.38% in 1990s. Growth of rice in terms
of area, production, productivity varied among the
various rice production environments such as rainfed
tank, tank, tank cum well, canal (river), and canal
(reservoir). Productivity growth in the rainfed tank
environment was stagnant, only 0.12% from 1984-85 to
2001-02. This included the large tract of dryland
regions with less dependable water resources. In the
state, nearly 50% of the districts are drought prone.
The average rainfall in the state decreased by more
than 40% during the drought period and the cultivated
area has not been extended due to lack of water. There
was a decline in gross sown area due to drought and
the estimated reduction was 4 lakh hectares (7%
decline as compared to normal period). The effect of
drought was also reflected in the expansion of area
under current fallow and other fallow lands. Current
fallow increased to 15.6 lakh hectares during the
drought period from 12.2 lakh hectares in the normal
period. Net area irrigated in the state was 28.01 lakh
hectares and gross area irrigated was 34.12 lakh


I. Farmer participatory breeding and economic 15 *










hectares, which constitute almost 55% of the net and
gross cropped area in the state during 2001-02. There
was a decline in area under irrigation in the drought
period; the extent decline was 8% due to drought.
Failure of the southwest monsoon affected the storage
capacity of Tamil Nadu's major reservoirs and water
supplies from surface and groundwater sources for
irrigation during the first half of the monsoon was a
major problem. Area in Tamil Nadu irrigated by canals
declined by 9.62%, tanks by 10.38%, ordinary wells by
4.19%, and tube wells by 7.55% due to drought.

Based on the rainfall distribution and availability of
groundwater, farmers' change their cropping pattern
and mitigate the drought impact. In Tamil Nadu,
shortfalls in rainfall occurred during 1974-75, 1980-81,
1986-87, 1988-89, 1990-91, 1995-96, and 2002-03. There
were seven drought years during the last three
decades. Diversification of cropping patterns,
particularly from high water consuming crops like
paddy to other lower water consuming crops, is not
reflected strongly, as the Herfindahl index registered
almost equal figures during both the drought and
normal periods. However, to some extent, crop
diversification was noticed in the Ramnad district, as
the index worked out to 0.24 and 0.37 during the
drought and normal periods, respectively (Table 1).
Reduction in area under paddy was observed due to
drought and crops like groundnut, gingelly, caster,
tobacco, blackgram, greengram, and redgram gained
area during the drought period. Area under paddy
declined, while area under blackgram, greengram, and
gingelly increased in the Thiruvallur district during
the drought period. Similarly, in Coimbatore district,
the area under paddy, maize, sorghum, pearl millet,
and vegetable crops declined during the drought
period, while groundnut, cotton, bengalgram,
redgram, horsegram, finger millet, and kodo millet
increased, implying that there was a shift from water
intensive crops to rainfed crops due to rainfall failure.
Reduction in paddy area and shift to other rainfed
crops was also exhibited in Ramanad district.


Table 1. Drought and crop diversification (Herfindahl Index)

Particulars Drought period Normal Period

Tamil Nadu State 0.14 0.15
Ramad 0.24 0.37
Thiruvallur 0.40 0.41
Coimbatore 0.14 0.15



* 16 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004


The econometric results showed that rainfall had a
negative effect on crop acreage, revealing that good
rainfall is expected to discourage diversification in
the state and in most of the selected districts.
Irrigation intensity (ratio of gross irrigated area to net
irrigated area) had a positive and significant effect on
acreage diversification, suggesting that availability of
irrigation water year round is expected to promote
crop acreage diversification. Coefficients of the whole
price index and productivity index reveal that
farmers prefer cultivating the same crops if they fetch
higher income either through increases in
productivity or product price. Rice-yield boosting
technology for water limited environments is
construed as more of an instrument promoting the
risk-taking function of the farmers than anything else.
It is imperative to maximize risk-taking ability of the
farmers and several alternative solutions are
proposed in this regard by several studies. There is a
clamor for reducing input prices and an equal urge
for raising output prices. Fixing higher prices for
output will lead to the large farmers getting an extra
ordinary level of profit and a further perpetuation of
income inequalities. Real price of paddy was similar
both during the drought and normal periods (Rs
39.27 and 38. 67 per quintal of paddy -1970-71 base)
indicating that yield risk has not been compensated
by product price.


1 3 5 7 9 11 13 15 17 19 21 23 25 27 29
Years
-- NSDP (80- 81 price) Drought -- Poverty ratio

Figure 1. Poverty and NSDP.










It was estimated that an increase in productivity of
paddy by one ton would replace paddy area by 1.89
lakh hectare in Tamil Nadu. Similarly, area expansion
under paddy was also noticed in the selected districts,
revealing that infusing high productivity traits in the
drought tolerant rice varieties will enable the farmers to
allocate some amount of land to other crops. Such
diversification can generate adequate income to
alleviate poverty in the rainfed areas. Rainfall
influences the risk-taking function of the farmers to a
very large extent in drought prone and dryland areas,
as evident from the estimated results. Rainfall has the
positive influence on area expansion of paddy and it is
found that for every additional increase in rainfall by
one mm, about 208 hectares of additional area would be
brought under paddy. The risk due to drought is
reflected in the level of investment made in modern
inputs such as fertilizers and pesticides. The current
level of fertilizer consumption in the state in terms of
NPK was 51.36, 20.71 and 24.76 kg/ha. Further, it was
noticed that there was a marginal decline in per hectare
fertilizer consumption in the state during the drought
period. Nitrogenous fertilizer consumption in the state
decreased to 4.09 lakh tons in the drought period from
4.68 lakh tons in the normal period. Similarly, there was
a reduction in consumption of phosphatic and potash
fertilizers during the drought period. It is also evident
from the econometric results that agricultural growth
has a trickle down effect and such effect weakened
during the drought period (Table 2). Income inequality
(paddy) was found less in the drought period statewide
and in selected districts as drought affected paddy



Table 2. Effect of drought on poverty reduction log linear estimates
Variables Equation 1 Equation 2 Equation 3 Equation 4
Net State Domestic -0.811*** -0.204 -0.200 -0.460
Product (-20.914) (-0.813) (-0.7621) (-1.588)
Net State Domestic 0.002 -0.001 -0.001 -0.001
Product (dummy) (0.488) (-0.304) (-0.3019) (-0.208)
Net State Domestic -0.634*** -0.629*** -0.644***
Product (lagged one year) (-2.506) (-2.301) (-2.462)
Consumer Price Index -0.005 -0.162
(-0.055) (-1.308)
Time -- -0.024*
(1.804)
Intercept 11.179*** 11.426"** 11.383)*** 14.532***
(31.503) (34.197) (13.236) (7.531)
R2 0.94 0.95 0.96 0.96

(Figures in parentheses denote t ratios)
*** Significant at one per cent level of probability and significant at10 per cent level of
probability


cultivation in the state irrespective of the size groups
and regions (Table 3). The government launched the
Calamity Relief Fund Scheme, which provides relief for
crops based on the extent of damage. Current area
under rice in the state is 20.60 lakh hectares, of which
ADT 43 constitutes nearly 21% followed by Improved
White Ponni (16%), ADT39 (14%), ADT36 (8%), C043
(7.5%), ADT38 (6.73%) and IR20 (6%). It is imperative
that the traits '.-. i. ii. and marketability) of these
varieties are considered in breeding varieties for water
limiting environment, thereby income realization from
paddy can be sustained.


Table 3. Inequality of income distribution (Gini Index rice)

Period Chengalpet Coimbatore Ramnad Tamil Nadu

Normal 0.3712 0.3539 0.4037 0.3639
Drought 0.4824 0.3063 0.3439 0.2621

Period: 1970-71 to 2000-01



Conclusion
Productivity growth in the rainfed tank environment,
which includes the large tract of dryland regions with
less dependable water resources, was stagnant. There
was a decline in gross sown area due to drought. The
effect of drought was also reflected in the expansion of
area under current fallow and other fallow lands.
Diversification of cropping pattern, particularly from
high water consuming crops like paddy to other lower
water consuming crops, was not strongly reflected.
However, to some extent, crop diversification is noticed
in Ramnad district. Rainfall had a negative effect on
crop acreage revealing that good rainfall is expected to
discourage the diversification in state and almost in all
the selected districts. Irrigation intensity had a positive
and significant effect on acreage diversification,
suggesting that availability of irrigation water on a year
round basis may be expected to promote crop acreage
diversification. Rice-yield boosting technology for water
limiting environments is construed as more of an
instrument for promoting risk-taking functions of the
farmers than anything else. Real price of paddy was
similar during both the drought and normal periods,
indicating that yield risk has not been compensated by
product price. Infusing high productivity traits in the
drought tolerant rice varieties will enable farmers to
allocate some amount of land to other crops; such
diversification can generate adequate income to
alleviate poverty in the rainfed areas.


I. Farmer participatory breeding and economic 17 *











Agricultural growth has a trickle down effect in
reducing poverty and such effects weakened during
the drought period. Varieties like ADT 43, Improved
White Ponni, ADT39, ADT36, C043, ADT38, and IR20
are the predominant varieties grown in the state. It is
imperative that the traits (genetic and marketability)
of these varieties should be considered in breeding
varieties for water limited environments, thereby
income realization from paddy can be sustained.


References


Janaiah, A., Manik L Bose, A.G. Agarwal. 2000. Poverty and income distribution in rainfed
and irrigated ecosystems, Economic and Political Weekly, December, Vol. 35 (52, 53).
Ramasamy C, T.R.Shanmugam, and D.Suresh Kumar. 1996. Constraints to higher rice yields
in different rice production environments and prioritization of rice research in southern
India. In Rice Research in Asia: Progress andPriorities. R.E.Evenson, R.W.Herdt, and M.
Hossain (eds.). Wallingford, UK: CAB International and IRRI.
Ramasamy, C., and T.Jatileksono. 1996. Intercountry comparison of insects and disease
losses. In Rice Research in Asia: Progress and Priorities. R.E.Evenson, R.W.Herdt, and M.
Hossain (eds.). Wallingford, UK: CAB International and IRRI.
Siddiq, E.A. 2000. Rice: Yawning Productivity Gaps. Survey of Indian Agriculture. The Hindu.
Pp.39.


* 18 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004











Varietal adoption and farmers' coping strategies in

rainfed rice ecosystems of Tamil Nadu


K.N. SELVARAJ, C. RAMASAMY, AND R. CHANDRA BABU

Tamil Nadu Agricultural University, Coimbatore-3, Tamil Nadu, India

Corresponding author: R. Chandra Babu; E-mail: chandrarc@hotmail.com


Introduction
Drought is one of the most important constraints in
rainfed rice production. Lack of high yielding varieties
and low rates of their adoption are the major reasons
for low productivity in water limiting environments.
Therefore, raising productivity in these areas is crucial
for alleviating poverty and ensuring food security of
the rural poor (Hossain, 1990). Although productivity
of food grains grew faster in the rainfed areas (1.66%
between 1985-86 and 1998-99 in Tamil Nadu), the
average productivity of food grains in irrigated areas
is higher by 50% than rainfed areas (Selvaraj et.al.,
2002)). Rainfed areas are poor in resource endowments
and infrastructure; the marginal impact of HYVs on
production is much higher in high and low potential
rainfed areas than irrigated areas (Fan and Hazell,
2000). Various safety nets are employed by the farmers
during the stress years in order to cushion the adverse
impacts of stress. The effectiveness and economic cost
of these coping mechanisms vary depending upon the
intensity of drought and the nature of production
system (Pandey et al., 2000)


Methods
Secondary data on rainfall, production, area under
HYVs and landraces were used to estimate probability
of occurrence of drought in the rainfed districts and
the effect of drought on area and paddy production.
Estimates were derived from the field survey
conducted in selected districts of Tamil Nadu covering
200 sample farms. Yield loss due to drought and
curtailment in use of modern inputs was analyzed
using the regression decomposition framework. Land,
labour, fertilizer, and plant protection chemicals were
considered in the decomposition framework in order
to estimate the yield loss due to these factors apart
from drought. Impact of risk of drought on yield of
HYVs and landraces was assessed by log linear


equation. The risk was measured in terms of deviation
between the recommended and actual use of
fertilizers. Transcendental production was used to
estimate the optimal level of yield and cost. Factor
shares (land, fertilizer, labour, and plant protection
chemicals) under different technologies (HYVs and
landraces) and proportionate change in the factor
shares were estimated based on the elasticity
coefficients following Hicks analytical tool.


Results
There were six, seven, and eight drought years in
Ramnad, Tiruvallur, and Coimbatore districts,
respectively, over the last three decades. In Tiruvallur
district, the shortfall in rainfall was 60%, while it was
53%, and 47% in Coimbatore and Ramnad districts,
respectively, during the drought years as compared to
the normal years. Some of the landraces are still
popular due to their tolerance and resistance to biotic
and abiotic stresses. Area under high yielding varieties
during the drought years decreased by 2.47 lakh
hectares. Area under landraces in Ramnad and
Tiruvallur district increased during the drought
period. Increase in area under landraces was 0.4 lakh
hectares in Ramnad, while the increase was minimal
in Tiruvallur district. Due to severity of drought, there
was a reduction in production of about 39% in the
Ramnad district, 32% in Tiruvallur, and more than
50% in Coimbatore district. Actual yield reduction was
much higher in Ramnad district 618 kg/ha) than in
Thiruvallur district (338 kg/ha).

Most farmers (40%) cultivated landraces (Mattai) in
Ramnad district, followed by HYV like MDU 5 and
IR36. High yielding varieties like TRY2, PMK2,
ASHOKA2007, ASHOKA228, MDU5, and RM96019
were distributed to the farmers of Ramnad district by
TNAU on trail cultivation in the farmers field for
subjective assessment of the varieties by the farmers.


I. Farmer participatory breeding and economic 19 *










Varieties like ADT36, Chellaponni, Jothi, Culture
Ponni, IR20, and MDU5 are predominantly grown in
the Sivagangai district. In Coimbatore district, the
majority of the farmers cultivate ADT39, ADT36,
IR20, C043, and CO42. Short duration varieties
ADT39 and C043 are the drought tolerant rice
varieties with a yield potential ranging from 3,000 to
3,500 kg/ha. ADT 43 and Bapatla are the most
popular varieties in Thiruvallur district. Average
marketable surplus was 70% for HYVs, while it was
56% for landraces in Ramnad, implying that farmers,
for consumption purposes, mostly prefer local races,
particularly in Ramnad district. According to the
majority of the farmers, the probability of occurrence
of drought was more than 30% in Ramnad district
and it varied among the districts. Probability of
occurrence of drought is highest in Coimbatore
district (more than 50%).

Farmers in the rainfed production environment are
operating at a sub-optimal level of production. Further,
comparison of actual and optimal cost of cultivation
reveals that for the production level realized by the
farmers, they incurred higher cost due to risk and
adoption of varieties with less response to technological
inputs. Farmers incurred an additional cost of Rs 899
for the realized production, whereas the scope exists to
increase yield by 228 kg/ha with the available
technology and resources. About 90% of the farmers in
the rainfed environment were found to be inefficient
since actual yields were lower than the optimal yield on
those farms. Econometric results indicate that yield loss
due to risk of rainfall failure was higher in the case of
HYVs as compared to landraces. Even a 10% increase in
risk resulted in 5.4% decline in yield of HYVs in
Ramnad district. However, yield reduction in landraces
was minimal and it was found that a 10% increase in
risk could cause yield to decline by only 0.2% (Table 1).
In order to cope with the risk, farmers cultivated
alternate crops like sorghum, blackgram, groundnut,
finger millet, and vegetables like ladies finger. Similarly,
the farmers sold livestock and borrowed loans from
both the formal and informal sources as drought
mitigating measures. Farmers also reduced
consumption expenditures during the drought period.
Share of income from paddy, other crops, other sources
(labour income, both off farm and non-farm) and
livestock were estimated during the normal and
drought period. Extent of reduction in consumption
expenditure was also estimated to understand farmers'
coping behaviour and the cumulative effect of drought
on livelihood of the farmers.


* 20 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004


On average, farmers realized 4.2 t/ha of (paddy)
from the high yielding varieties in Ramnad district
during the normal period, while they realized 3.3 t/
ha from the local varieties. Although yield realization
from the local varieties was less during the normal
period (0.9 t/ha), farmers cultivated landraces due to
assurance of a minimum level of yield during the
drought period. It was estimated that the farmers
realized an incremental benefit of Rs 5,783 per
hectare by cultivating landraces during the drought
period as compared to HYVs. (Based on the
difference in the yield reduction of high yielding
varieties and landraces due to drought. Although
yield reduction is lower in landraces, productivity of
high yielding varieties is higher in a drought period.
However, due to reduction in cost, landraces fetch
marginally higher return.) Cultivation of HYVs
during the normal period fetches an incremental
benefit of Rs. 2,165 per hectare as compared to
landraces (Table 2). Non-system tank forms the major




Table 1. Impact of risk (drought) on yield of paddy log linear estimates

Co-efficients t-value

Ramnad
HWs -0.540* -2.746
Land Races -0.016 -0.110
Sivagangai
HYV -0.190* -1.736
Thiruvallur
HW -0.062* -2.442

* significant at 5 per cent level, significant at 10 per cent level




Table 2. Comparative analysis of high yielding and local varieties (Rs/ha)

Land Races- High yielding varieties -
Drought years Normal years

Debit Credit Debit Credit
Cost Added Cost Reduced Cost Added Cost Reduced Cost

2563.21 2563.21
Return Reduced Return Added Return Reduced Return Added Return
3220.00* 4727.89
Total 5783.21 2563.21 4727.89
Incremental
benefit (Rs) 5783.21 2164.68

* Based on the difference in the yield reduction of high yielding varieties and land races due to
drought. Though yield reduction is lower in land races, productivity of high yielding varieties is higher
in drought period. However, due to reduction in cost, landraces fetch marginally higher return.










source of irrigation in Ramnad district, which
depends on rainfall. Therefore, it is imperative that
the varieties meant for water limiting environment
should ensure a minimal level of yield during the
stress period and that could induce the farmers to go
for higher levels of adoption.

However, yield reduction of HYVs in other selected
districts, namely Sivagangai and Thiruvallur
districts, was found to be less, despite the occurence
of variability in rainfall, because of supplementary
sources of irrigation. Yield reduction of HYVs would
be 2.0-0.6% in Sivagangai and Thiruvallur districts, if
risk of drought were to increase by 10%.
Technological change in rice cultivation is land
saving but fertilizers, labour, and plant protection
chemicals are also factors (Table 3). Farmers are of
the opinion that HYVs need high quantities of
fertilizers due their responsiveness and due to weeds
growing easily and profusely in the wake of fertilizer
application. This, in turn, requires more labour for
weeding as compared to landraces. Further, labour
requirement for other operations like planting,
harvesting, and threshing is higher in the case of
HYVs as compared to local varieties. Use of capital
for chemical fertilizers and insecticides was also
higher in the cultivation of HYVs, while local
varieties have much more resistance to pests and
diseases. However, the production elasticity of
fertilizer (0.319) and labour (1.051) implies that
marginal return from application of fertilizer and
labour is higher in HYVs as compared to landraces.

Use of higher doses of fertilizers in the cultivation of
land races affects the standing crop. The benefits of
the new technology can be derived if it assure
minimal level of yield during the drought period
because the farmers curtail the use of modern inputs
during the drought period, which may result in
further decline in productivity. Nitrogen
consumption per hectare of gross cropped area in the


Table 3. Estimates of factor share under different technologies and
proportionate change in the estimated factor shares

Factor Share

Factor Inputs Land Races HYVs Proportionate Change

Land 1.498 0.567 -0.621
Fertilizer 0.096 0.484 0.802
Labour -0.172 0.943 6.468
Plant Protection Chemical -0.606 -0.810 0.252


state was 3.248 kg in irrigated area, while it was
0.998 kg in rainfed area during the period between
1985-86 and 1998-99. Similarly, phosphorus
consumption was 1.186 kg per hectare of gross sown
area in irrigated areas, while it was 0.377 kg per
hectare in rainfed areas. In the case of potash, per
hectare consumption in irrigated area was 1.43,
whereas it was 0.517 per hectare in the rainfed areas
during the same period (Selvaraj et al. 2002). Since
high yielding varieties require higher doses of
fertilizers to realize their yield potential, fertilizer
consumption is higher in the irrigated areas.
Fertilizer consumption in rainfed areas is still less
than half the rate used in irrigated areas. Results of
decomposition analysis reveal that reduction in yield
due to curtailment of input usage accounts for only
3-10 percent and this could be attained through
creation of awareness among the farmers and more
than 90 percent of yield reduction is due to water
stress. Estimated yield loss due to drought is 1400 kg
per hectare in the case of HYVs, while it was 840 kg
per hectare in the case of landraces. Therefore,
breeding varieties with drought tolerance is crucial.
Varieties should possess the characteristics of
assuring a minimum level of yield and thus enable
the farmers to realize additional yield by 1,260 kg of
paddy per hectare from the present level of yield
obtained during the drought period.



Conclusion
Average marketable surplus was 70% for HYVs,
while it was 56% for landraces, implying that
farmers, for consumption purposes, mostly prefer
local races, particularly in Ramnad district.
According to the majority of the farmers, the
probability of occurrence of drought was more than
30% in Ramnad district and it varied among the
districts. Probability of occurrence of drought is
highest in Coimbatore district (more than 50%).
Farmers in the rainfed production environment are
operating at sub-optimal levels of production.
Furthermore, comparison of actual and optimal cost
of cultivation reveals that for the production level
realized by the farmers, they incurred higher cost
due to risk and adoption of varieties with less
response to technological inputs. Econometric results
indicate that yield loss due to risk of rainfall failure
was higher in the case of HYVs as compared to
landraces. Technological change in rice cultivation is
land saving, but fertilizers, labour and plant


I. Farmer participatory breeding and economic 21 *











protection chemicals are also factors. Estimated yield
loss due to drought is 1,400 kg/ha for HYVs, and 840
kg/ha for landraces. It was observed that the
cumulative effect of drought on the livelihoods of the
farmers was high if the ex-ante and ex-post coping
mechanisms are taken into account. Therefore,
breeding varieties with drought tolerance is crucial.
Varieties meant for rainfed areas should possess the
characteristics of assuring a minimum level of yield,
thus enabling the farmers to realize additional yield of
1,260 kg of paddy per hectare above the present level
of yield obtained during the drought period.


References
Fan Shenggen, and P Hazell 2000. Should developing countries invest more in less favoured
areas? An empirical analysis of rural India. Economic and Political Weekly. 35(17):
1455-64.
Hossain, M.1990. Factors affecting adoption of modern varieties of rice in Bangladesh.
Bangladesh Journal of Agricultural Economics. 13 (1&2):93-106.
Pandey, S., Dev Dutt Behura, Renato Villano, and Dibakar Naik. 2000. Economic cost of
drought and farmers' coping mechanisms: A study of rainfed rice systems in Eastern
India, Discussion Paper 39. Manila, Philippines: IRRI.
Selvaraj, K.N, C. Ramasamy, Anil Kuruvila, and A. Rohini. 2002. Productivity technology,
infrastructure growth and investment assessment for poverty reduction in dryland
aAgriculture. Asia-Pacific Journal of Rural Development. 12(1): 76-88.


* 22 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004











Application of maize mega-environments in seed systems

in the Southern African Development Community


P.S. SETIMELA1, M. BANZIGER1, AND M. LISTMAN2

1 Maize Program, International Maize and Wheat Improvement Center (CIMMYT), P.O. Box MP 163, Mount
Pleasant, Harare, Zimbabwe
2 Maize Program, International Maize and Wheat Improvement Center (CIMMYT), Apdo. Postal 6-641, 06600,
Mexico D.F. Mexico

Corresponding author: P.S. Setimela; E-mail: p.setimela@cgiar.org


Introduction
New open pollinated maize varieties that mature
earlier, are stress tolerant, and higher yielding are now
available. With the development of new varieties
farmers and relief organizations still find it difficult to
select an appropriate variety for their environment.
Variety selections are usually based on availability of
seed and price, ignoring adaptation factors such as
rainfall, minimum and maximum temperatures, and
the length of growing season. Identification of maize
mega-environments offers farmers, relief
organizations, and others an opportunity to select
appropriate varieties for their environments. Based on
maize regional yield trial data and geographical
information system (GIS) parameters, maize mega
environments were identified.


Methods
Using the revised mega-environments (Setimela et al.,
2002), a basis for choosing the right open pollinated
maize variety for a given environment was developed.
Similar areas across the SADC region were given the
same color code based on classification of the mega
environment. By determining one's location on the
map, color code (yellow, orange, red, and green), and
maturity group (early, intermediate, and late maturing
variety) one selects an appropriate group. Once an
appropriate group has been chosen, varieties within
the group are then assessed for their appropriateness
in the given environment. A description of important
characteristics for each variety is also given. Based on
these combinations of factors, farmers are guided to
the optimum variety.


Results
A clear and easy to follow system for choosing
appropriate varieties has been developed. A booklet to
guide farmers on choosing appropriate varieties for
their environments has been published and is now
available and widely distributed in the SADC region.


Conclusions
The use of mega-environments in combination with
descriptions of varieties has provided a quick and
transparent basis of choosing maize varieties in the
SADC region. When appropriate varieties are clearly
identified their seed production and provision is
facilitated..


References
Setimela, P.S. et al. 2003. Book of Abstracts Arnel R. Hallauer International Symposium on
Plant Breeding. Mexico D.F.: CIMMYT.


I. Farmer participatory breeding and economic 23 *











Breeding drought tolerant varieties of rice through

participatory plant breeding for the rainfed uplands


D.N. SINGH1, M. CHAKRABORTY1, M.K. CHAKRAVARTY1, P. SINGH1, M.K. BARANWAL1, B. KUMAR1, R. KUMAR1, A.
PRASAD2, V. SINGH2, S.C. PRASAD2, A. CHOUDHARY2, D.S. VIRK3, K.A. STEELE3, AND J.R. WITCOMBE3

1 Birsa Agricultural University, Kanke Ranchi -834006, Jharkhand, India
2 Gramin Vikas Trust, 280 -Kanke Road, Ranchi -834008, Jharkhand, India
3 Center for Arid Zone Studies, University of Wales, Bangor, UK

Corresponding author: D.N. Singh; E-mail: dnsingh ban@rediffmail.com


Introduction
Drought is the most important abiotic stress that
inhibits plant growth and reduces productivity.
Varieties developed through the conventional plant
breeding approach have not been adopted by the
farmers of rainfed uplands because they not only lack
in drought resistance but also in many of the farmer
preferred traits such as high fodder yield and cooking
qualities. Farmers of rainfed uplands thus cultivate
old and unimproved cultivars and obtain low yields.
Of the 141 varieties released for the uplands in India,
only a few have become popular with farmers. In
Jharkhand, most of farmers either grow the Brown
Gora landrace, or its derivative, Birsa Gora 102, that
have low yield and poor grain quality.


Materials and methods
A collaborative project on rice improvement was
undertaken between BAU, Gramin Vikas Trust (an
NGO), and the Centre for Arid Zone Studies,
University of Wales, Bangor, UK with funding from
the DFID bilateral project managed by GVT and Plant
Sciences Research Programme of DFID in 1997 to
develop farmer preferred drought tolerant varieties
through participatory plant breeding (PPB). GVT has
a network of farmers in Jharkhand, Orissa and West
Bengal and thus provided means of farmer field
testing and selection.

As a first step, a participatory varietal selection (PVS)
was undertaken with GVT adopted farmers who
identified Kalinga III. However, it lacked in lodging
resistance. Its improvement was initiated by crossing
it with a diverse variety IR64. Collaborative PPB



* 24 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004


(farmers selected in the F4 bulk in their own field)
and consultative PPB (farmers selected among F4
progeny bulks on station) resulted in the
development of Ashoka 200F and Ashoka 228 that
were released in 2003 by BAU as BVD 109 and BVD
110. Since these varieties were developed with the
participation of farmers who widely experimented
with them, they excel in farmer preferred traits.
Similar methods will be followed in development of
drought tolerant varieties of rice for the rainfed
upland in The Rockefeller Foundation supported
project to BAU. Formal seed production of these
varieties has not been possible because of lack of
their notification by the government of India.
However, GVT has undertaken their dissemination
through community based seed production and
these varieties are becoming very popular.


Results
Screening of local germplasm and other pure
breeding lines among a large collection of upland
germplams at BAU is being undertaken for drought
tolerance and characterization for yield and root
traits. The other important traits of interest are: early
maturity, more productive tillers, longer panicles
with more grains or grain weight. Among the root
traits attention is being given to longer roots, more
roots, higher root volume and weight and a higher
root-shoot ratio. Lines with superior traits for
various quantitative traits will be identified for use
as donors in the improvement of upland varieties for
drought tolerance and higher yielding ability.
Varieties Ashoka 200F and Ashoka 228 that have
high drought resistance will also be used as parents.











Development of backcross
populations
Backcrosses of Vandana x IR72 and Vandana x
IR72975 crosses will be made with Vandana as a
recurrent parent. Effort will be made to incorporate
the superior grain quality traits of IR72 and IR72975
into Vandana's background since Vandana has been
reported to be a drought tolerant variety with
longer roots but inferior grain quality.

The research aims at putting farmer preferred traits
into a drought tolerant variety for the rainfed
uplands; work includes

* identification of drought tolerant genotypes;
* development of genotypes with early vigour for
better weed suppression in the direct seeded
rainfed uplands;
* breeding for early maturity to impart this
drought escape mechanism;.
* higher yielding ability under direct seeding (2.5
to 3.0 t/ha);
* breeding tall varieties (95 to 100 cm) for higher
straw yield;
* breeding varieties with straw colour husk and
white kernels; and
* breeding varieties with superior cooking quality.


Conclusions

Keeping farmers' preferences in mind, a number of
crosses for developing drought tolerant varieties are
in the advanced stages of breeding (F6, F). These
are Vandana x IR72, Vandana x IR72975, WARDA 45
x A 157, Sathi 85-3 x A 162, PBRC78 x Komal 13,
PBRC x A 228, CH 45 x MT1, and bulks derived
from marker assisted selection programme for
drought tolerance from Kalinga III x Azucena cross.

Five to 10 lines from each cross have been selected
by scientists and farmers for drought tolerance and
higher yield (Figure 1). These will be tested in All
India Coordinated Trials and State Trials along with
on-farm testing. Simultaneous seed production and
dissemination will be undertaken for the farmer
preferred varieties through GO-NGO collaboration
to bridge the gap between release and


dissemination. Advanced generation four bulks
developed through marker assisted selection for root
traits to impart drought resistance were tested in on
farm trials in West Bengal in kharif2003. Differences
between bulks were significant for grain yield (Table 1,
Figure 2); they yielded 11 to 56% more than the check
variety Kalinga III. Bulk 5 also has an aroma QTL and
was the most preferred. This bulk will be proposed for
release and wider dissemination.



Table 1. Analysis of variance for grain yield (kg/ha) of marker assisted
selected bulks for root traits from Kalinga III x Azucena cross tested in
on-farm mother trials in West Bengal in kharif2003

Source df MS F P

Between varieties 5 227731 4.94 0.01
Between villages 7 507592 11.02 0.00
Between farmers/villages 11 59910 1.30 0.32
Error 14 46053




9--nn


2000


c 1500
'

1000


500


Komal 6 Komal 9


Komal 13 Kalinga III


Figure 1. Grain yield superiority of pipeline varieties (Komal series)
developed from Kalinga III x Vandana cross in BAU trilas in kharif2003.


I. Farmer participatory breeding and economic 25 "


64%
52% II
41%




6iiiiiii


U -IL-











/ARIi .


References


56% Singh, D.N., R. Kumar, D.S. Virk, S.C. Prasad, J.S. Gangwar, J.R. and Witcomb. 2000.
Proceeding in international symposium held at Mexico.
Singh, D.N., R. Kumar, S.K. Singh, D.S. Virk, J.R. Witcomb, S.C. Prasad, and J.S. Gangwar.
1500- 26% 23% 18% 2001. Proceeding in diamond jubilee international symposium, ISGPB. 81
11% Singh, D.N., M. Chakraborty, M.K. Chakravorty, R. Kumar, J. Ghosh, V. Singh, and D.S. Virk.
2003. Proceeding in national seminar on advances in genetics and plant breeding,
Impact of DNA revolution. Pp. 137.
s 1000- y Steel, K.A., D.N. Singh, R. Kumar, S.C. Prasad, D.S. Virk, J.S. Gangwar, and J.R. Witcomb.
2002. Proceeding in workshop on breeding rainfed rice for drought prone environment
D organized at IRRI. Pp. 20-22.
Virk, D.S., D.N. Singh, R. Kumar, S.C. Prasad, J.S. Gangwar, and J.R. Witcomb. 2002.
500 Proceeding in workshop on breeding rainfed rice for drought prone environment
organized at IRRI. Pp. 5-7.
Virk, D.S., D.N. Singh, S.C. Prasad, and J.S. Gangwar. 2002. Euphytica 132:95-108.



Bulk1 Bulk2 Bulk3 Bulk5 Bulk6 Kalinga Ill

Figure 2. Grain yield of four marker assisted selected bulks of Kalinga III
x Azucena cross in on-farm trials in West Bengal in kharif2003.


* 26 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004











Reaching the poor maize farmers in the hills of Nepal:

Experiences and achievements of the Hill Maize Research

Project (HMRP)

CARLOS A. URREA, T.P. TIWARI, N.P. RAJBHANDARI, AND D.P. SHERCHAN

Corresponding author: Carlos A. Urrea; E-mail: c.urrea@cgiar.org


Background
Maize in the hills of Nepal is cultivated in a wide range
of environmental regimes. The variations in altitude,
aspects, uncertainty of pre-monsoon and/or monsoon
rainfall leading to yearly variations in planting time and
maize growth, cropping systems, management
practices, indigenous knowledge systems, traditions,
input regimes, socioeconomic ground realities, farmers
specific preferences and needs influence what is
currently practiced and what will likely be adopted by
farmers. The above variations across the mid-hills of
Nepal are so overwhelming that it is a tremendous
challenge to focus meaningful maize research so as to
have any bearing on technology generation that has
potential for large-scale extrapolation and impact.
About 95% of the farmers have less than one hectare of
land and 72% of households have food sufficiency for
less than six months (Gurung, 1999). Maize is staple
food and very little goes to market. Most maize farmers
in the remote areas, inaccessible by roads and devoid of
any development infrastructures, are so poor that they
cannot afford to buy external inputs. Under such
circumstances, it is a daunting task to reach those
farmers with maize production technology and to make
an impact. The Hill Maize Research Project (HMRP) is
trying to do just that and has made progress by
developing technology for and with poor farmers. This
paper highlights the experiences and achievements of
the project and methodologies followed.


The project
The HMRP, a collaborative project between CIMMYT
and the Nepal Agriculture Research Council narcC),
funded by the Swiss Agency for Development and
Cooperation (SDC), started in 1999 with the overall goal
to improve food security and livelihoods of farming
families through the increased productivity and


sustainability of maize-based cropping systems in the
hilly areas of Nepal. In these chronically food-deficit
hills, maize is the most important food crop, grown
under rainfed conditions, mainly on a small scale by
resource-poor farmers in an area covering
approximately 1.8 million hectares. It is reasonable to
infer that food security will reduce conflict in the
society and thus enhance harmony in the households
and thereby in the community. The majority of the hill
population, especially children and women, suffer from
malnutrition because their diets lack protein. Because of
this, HMRP is working on the development and
promotion of Quality Protein Maize (QPM). To reach to
the resource-poor farmers in the remote areas, HMRP
has been expanding its research and development
activities through its partners. Participatory variety
selection (PVS) and community based seed production
(CBSP) through various partners such as NARS and
non-government organizations (NGOs) is making the
difference. This has helped in disseminating results
generated by the project.


Approach
The project strategy is based on working and
supporting research, training, and dissemination in a
holistic and concerted way through effective partners.
The strategy deals aims to promote an efficient
research system and the development and
dissemination of research products, through
increasing capacity building for maize researchers
under NARC and extension workers under the
Department of Agriculture (DoA). Linking research
and dissemination institutions and monitoring their
activities at outreach research sites with key players
like NGOs and community based organizations
(CBOs) for the client-centered programs are key
elements of the project.


I. Farmer participatory breeding and economic 27 *










To achieve the goals of the project, several approaches
are employed: enhancement of institutional
effectiveness of NARC to develop and deliver maize
technology; organization of short and long-term
training; incorporation of gender and equity
awareness and analysis into the research process;
strengthening of the capacity of the National Maize
Research Program (NMRP) to conduct participatory
research; and improvement of planning, monitoring,
evaluation, and reporting in NMRP. Expanded
collaboration with partners; implementation of a small
grants scheme; support partnerships at the agriculture
research station level and annual partnership
meetings; development of improved maize
technologies and assessment of the adoption of
technologies are the major areas of focus.



Varietal development and testing
Developing and maintaining open pollinated varieties
(normal and QPM) with stress tolerance (biotic and
abiotic stresses) is continuing. Screening for turcicum
resistance and drought tolerance is also continuing.

Testing of germplasm is undertaken in four steps: the
Observation Nurseries/Trials (ON), the Intermediate
Yield Trial (IYT), the Coordinated Variety Trials (CVT),
and the Farmer's Field Trial (FFT). The project has
continued to support the improvement, maintenance
and multiplication of currently released hill maize
varieties as well as the development and testing of
new cultivars. As a result, Hill Pool Yellow, ZM-621,
Pop 44 cl0, Pop 45 cl0 and Hill Pool White as a full
season; and Arun-4, Pool 15E, Pool 17E and Pool 16 as
early and extra early genotypes, have been identified
as promising genotypes.



Participatory approach
Participatory methods in varietal testing and
dissemination of maize production technology have
been initiated with partners in outreach research for
technology verification through agriculture research
stations, DoA I/NGOs and through small grant
schemes. PVS has been used as an effective tool to
work for and with the farmers to verify and
disseminate promising technologies.

The project has expanded considerably on-farm
testing with relatively good stocks of seed of the
newest varieties that includes coordinate farmers



* 28 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004


field trials, front line demonstration, mother/baby
trials and farmers acceptance test (FAT). Thirty-five
mother trials were established following PVS
methodology along with 625 baby trials involving
670 farmers throughout the country in 2003. In
addition, 4,500 sets of FAT consisting of released and
pipeline varieties were distributed to farmers
through the agriculture research stations, Agriculture
Development Office (ADOs) and NGOs. Similarly
about 230 front line demonstrations were
distributed in areas where there is no OR activities.



CBSP

Once a suitable maize variety has been identified,
HMRP encourages its partners to multiply it locally
as a community based seed production (CBSP) effort.
It has shown a positive impact in adoption of
improved maize in the hills of Nepal and has been
successful in empowering poor and women farmers
in organizing them as maize seed producing groups.
The impact of these programs has started to show
sustainable positive feedback in other communities
also. The maize seed marketing system, however,
remains weak and needs to be strengthened. In order
to strengthen innovative partnerships that will enable
NARC scientists to organize seed production in
remote areas, partnership with CBOs at grassroots
level are encouraged. About 259 tons of improved
maize seeds of different varieties were produced in
2003 in collaboration with NARC ARSs, DoA, and
CBOs (Figure 1).


1nnn


I 60

E
S 40


21
0-


-0-



0 5


2001 2002


50 '
E!
00 -


0


2003


Figure 1. Community based seed production under HMRP from 2000-2003.










Agronomic and other studies
To develop crop management practices, research has
continued on the effect of organic inputs and lime on
maize productivity; seed priming; nitrogen efficiency
and soil limiting factor verification; minimum tillage;
fertilizer management in maize based cropping
system; identification of legumes for suitable
intercropping; long-term soil fertility management in
maize/millet systems, and soybean intercropping in a
double plant per hill system. Given our preliminary
work, we envision this research having a significant
impact in the hills.


Research focused on reducing losses due to insect
pests is continuing in 2004. Monitoring postharvest
losses of maize, and developing strategies to control
white grubs is being supported under a small grant
scheme wherein Institute of Agriculture and Animal
Sciences and NARC scientists are isolating and
culturing indigenous pathogens for the control of
white grub in maize. Gender, equity, and poverty
awareness are seriously considered in all maize
improvement activities.


References
Gurung D.B. 1999. Potential and Constraints of the Maize Based Croping System in the Mid
and Far Western Hill of Nepal: A Survey Report HARP PP01/98, Report no 2. pp.68
HARP. Nov 1999.
Tiwari, TP, et al. 2003. Guidelines on the conduct of Mother-Baby trials of Participatory
Variety Selection (Draft) HMRP


I. Farmer participatory breeding and economic 29 *











An international partnership for the breeding and

delivery of drought-tolerant rice varieties by market-

oriented plant breeding and marker-assisted selection

D.S. VIRK1, K.A. STEELE1, D.N. SINGH2, S.C. PRASAD3, A. PRASAD3, J.S. GANGWAR3, V. SINGH3, AND J.R. WITCOMBE1

1 Center for Arid Zone Studies (CAZS), University of Wales, Bangor, UK
2 Birsa Agricultural University (BAU), Kanke, Ranchi-834006, Jharkhand, India
3 Gramin Vikas Trust (GVT), 280 Kanke Road, Ranchi-834008, Jharkhand, India

Corresponding author: D.S. Virk; E-mail: d.s.virk@bangor.ac.uk


Introduction
Farmers of the Chhotanagpur plateau of eastern India
predominantly grow rice landraces in the rainfed
uplands. To better address the needs of the market,
on-farm and farmer-oriented approaches were used in
a collaborative programme between GVT (NGO),
BAU (GO), and CAZS, an advanced research institute
(ARI), using funds from a DFID bilateral project
managed by GVT and from the DFID Plant Sciences
Research Programme. This collaborative research has
led to the development and delivery of farmer
accepted varieties in less than six years.


Methods
In participatory varietal selection (PVS), farmers
preferred Kalinga III for higher yield, earlier maturity,
and good grain quality. However, it has weak straw
(giving lodging susceptibility) and poor roots (giving
poor drought resistance). The improvement of Kalinga
III was undertaken by crossing it with IR64, and by
marker-assisted backcrossing (MABC) for root traits
from a Philippines variety Azucena (Price et al., 2002)
to transfer improved root growth that imparts drought
resistance (Fukai and Cooper, 1995). We used two
methods of involving farmers more closely in
breeding, called participatory plant breeding (PPB).
Either farmers selected in heterogeneous bulk (a form
of collaborative participation; Biggs, 1989 ) or farmers
made selections among materials grown on-station by
researchers (a form of consultative PPB; Biggs, 1989).
In the MABC progenies, six bulks were selected from
the BC2 generation for selection in 1999, having
various target root QTL. In addition, pure-breeding
lines have been developed with individual and

* 30 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004


pyramided QTLs. These bulks/lines have been
provided to farmers for testing and selection in on
farm trials since 2001. The PPB and MABC varieties
were evaluated at GVT and BAU farms in 2002 and
2003 in rainfed trials.

Adoption and impact of two varieties from the PPB
programme (Ashoka 200F and Ashoka 228) in
Jharkhand, Orissa, and West Bengal was studied
through surveys in 2002 and 2004. Bioeconomic
modelling was done to assess the longer-term impact
of these varieties on poverty alleviation.


Results
PPB resulted in the rapid release of two varieties
(Ashoka 200F and Ashoka 228) that were preferred by
farmers in Jharkhand in 2003 (Virk et al., 2003). In
rainfed trials, coarse-grained varieties Vandana and
BG 102 (both released for uplands) yielded
significantly less and matured later than Kalinga III.
New varieties, Ashoka 200F, Ashoka 228, Bulk 4, Bulk
6, P 82 showed significantly higher yield (7 to 17%
more than Kalinga III) and were either earlier or
similar to Kalinga III in days to flowering. Bulk 5 from
the MABC had 25% higher grain yield but was not
later flowering (Table 1) than BG 102 or Vandana. It
also has superior grain quality and aroma.

The adoption and testing of these varieties was
simultaneous and their scaling up with community
seed production has resulted in a wider dissemination
(Prasad et al., 2004). The share of upland rice area
allocated by farmers to the Ashoka varieties has
shown a remarkable increase (Figure 1).











Table 1. Mean grain yield (t ha-1) and days to 50% flowering for some
selected PPB varieties and marker assisted backcross bulks having root
growth QTL in rainfed trials conducted at two sites in Jharkhand in
2002 and 2003. P<0.05; **P<0.01.

Significance Days Significantly Significantly
Grain yield from to 50% earlier than later than
Variety (t ha-1) Kalinga III flowering Kalinga III Kalinga III

A200F 1.92 62.0
A 228 2.02 62.8
Bulk 3 1.85 62.1
Bulk 4 1.91 60.4
Bulk 5 2.24 69.7
Bulk 6 2.09 62.9
P81 1.94 64.6
P82 2.04 63.1
Kalinga III 1.79 63.3
Vandana 1.68 67.8
BG 102 1.65 67.6
lsd5% (1%) 0.10 1.1
Isdl% 0.14 1.5


100


, 80

- .--
S60


S 40


8 20


0


Conclusions

Varieties developed through PPB and MABC have
better drought resistance as they yield more than BG
102 and Vandana (both coarse grained), and Kalinga
III (slender grained) varieties with earlier or similar
maturity. New material is still being produced both
from MABC progenies, and from new crosses
involving Ashoka 200F and Ashoka 228 as parents.


Average area of upland rice per household (ha)

State N 2001 2002 2003 2004

Orissa 14 0.72 0.80 0.81 0.87
WB 15 0.28 0.28 0.30 0.33
Jhar 7 0.26 0.36 0.21 0.29
Overall 36 0.70 0.78 0.79 0.85


2001 2002 2003 2004


Figure 1. Adoption patterns of Ashoka varieties in the rice uplands per household in three states. Survey of February 2004.



References
Biggs, S.D., 1989. OFCOR Comparative StudyPaper No. 3. The Hague: Intl. Ser. Nat. Agric.
Res.
Fukai, S., and M. Cooper. 1995. Field Crops Research. 40: 67-86.
Prasad, et al. 2004. Rockefeller Foundation Workshop, 24-28 May 2004, Mexico.
Price, A.H., et al. 2002. Field Crops Research. 76: 25-43.
Virk, D.S., et al. 2003. Euphytica 132: 95-108.


I. Farmer participatory breeding and economic 0 31 *






11 en isoer n N vl proce


Heritability of rice yield under reproductive-stage drought

stress, correlations across stress levels, and effects of

selection: Implications for drought tolerance breeding

G.N ATLIN1, R. LAFITTE1, R. VENUPRASAD1, R. KUMAR2, AND B. JONGDEE1

1 International Rice Research Institute, Los Banos, Philippines
2 Indira Gandhi Agricultural University, Raipur, India

Corresponding author: Gary Atlin; E-mail: g.atlin@cgiar.org


To be useful in variety development, screening methods
for tolerance to reproductive-stage drought stress need
to be repeatable, applicable at a reasonable cost to large
breeding populations, and predictive of grain yield
under stress. Secondary anatomical and physiological
parameters have generally not fulfilled these criteria in
rice. As a result, there has been increasing interest in
screening for yield under stress, either in easily-drained
fields in the wet season or in managed-stress nurseries
in the dry season. However, little guidance is available
regarding how to incorporate such screens into cultivar
development programs, and there are few reports on
the effectiveness of direct selection for yield under
stress. We present experimental evidence that direct
selection for improved yield under severe reproductive
stage stress is likely to be effective, based on
information from the evaluation of populations of
selected and unselected lines at IRRI and elsewhere.
The results of a selection experiment conducted under
severe upland stress are also presented, and the
integration of drought tolerance screening in IRRI's
aerobic rice breeding program is described.


Heritability within and
correlations across stress levels in
unselected breeding populations
If selection for yield under drought stress is to be
effective, yield in the stress treatment must be
repeatably measurable. The stress treatment should
also provide information about cultivar differences that
is not available from screening under non-stress
conditions. Information on this question has been
assembled for 6 populations of unselected lines
evaluated in a total of 10 experiments at locations in
India, Thailand, and the Philippines under both well
watered conditions and severe stress. Broad-sense
heritabilities (H) within water regimes and genetic
correlations across them are presented in table 1. Yield
under stress averaged 35% relative to the well-watered
controls across these experiments. H estimates for grain
yield in the stress environments were similar to those
for yield in the non-stressed controls, indicating that
selection for yield under stress is likely to be as effective
as selection for yield under favorable conditions.


Table 1. Repeatability (H) of grain yield estimates in well-watered and moisture-stressed treatments, and genetic correlations across stress levels
(rG), in trials evaluating unselected populations
Relative H
Stress Non-stress yield under rG Data
Location Year environ-ment environ-ment Population stress Stress Non-stress provided by:
Bet Dagan, Israel 1997 Upland Upland CT9993/IR62266 0.26 0.81 0.63 0.35 A. Blum
Coimbatore, Tamil Nadu 1999 Upland Upland CT9993/IR62266 0.31 0.60 0.56 0.86 R. Chandra Babu
Paramakudiamil Nadu 2000 Upland Upland CT9993/IR62266 0.41 0.76 0.23 0.91 R. Chandra Babu
Ubon, Thailand 2000 Upland Upland CT9993/IR62266 0.30 0.50 0.54 0.71 G. Pantuwan
Raipur, India 2000-02 Lowland Lowland CT9993/IR62266 0.21 0.37 0.45 0.80 R. Kumar
Los Banos,Philippines 2003 Upland Lowland Vandana/IR64 0.67 0.42 0.27 0.69 R. Venuprasad
Los Banos,Philippines 2003 Upland Lowland Apo/IR64 0.13 0.24 0.45 0.35 R. Venuprasad
Los Banos, Philippines 2003 Upland Lowland Apo/IR72 0.29 0.67 0.30 0.64 R. Venuprasad
Los Banos, Philippines 2003 Upland Lowland Vandana/IR72 0.31 0.07 0.42 0.78 R. Venuprasad
Los Banos, Philippines 1998-9 Upland Upland IR64/Azucena 0.56 0.68 0.74 0.62 B. Courtois
Mean 0.35 0.51 0.46 0.67


* 32 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004






11 en isoer n N vl proce


Heritability of rice yield under reproductive-stage drought

stress, correlations across stress levels, and effects of

selection: Implications for drought tolerance breeding

G.N ATLIN1, R. LAFITTE1, R. VENUPRASAD1, R. KUMAR2, AND B. JONGDEE1

1 International Rice Research Institute, Los Banos, Philippines
2 Indira Gandhi Agricultural University, Raipur, India

Corresponding author: Gary Atlin; E-mail: g.atlin@cgiar.org


To be useful in variety development, screening methods
for tolerance to reproductive-stage drought stress need
to be repeatable, applicable at a reasonable cost to large
breeding populations, and predictive of grain yield
under stress. Secondary anatomical and physiological
parameters have generally not fulfilled these criteria in
rice. As a result, there has been increasing interest in
screening for yield under stress, either in easily-drained
fields in the wet season or in managed-stress nurseries
in the dry season. However, little guidance is available
regarding how to incorporate such screens into cultivar
development programs, and there are few reports on
the effectiveness of direct selection for yield under
stress. We present experimental evidence that direct
selection for improved yield under severe reproductive
stage stress is likely to be effective, based on
information from the evaluation of populations of
selected and unselected lines at IRRI and elsewhere.
The results of a selection experiment conducted under
severe upland stress are also presented, and the
integration of drought tolerance screening in IRRI's
aerobic rice breeding program is described.


Heritability within and
correlations across stress levels in
unselected breeding populations
If selection for yield under drought stress is to be
effective, yield in the stress treatment must be
repeatably measurable. The stress treatment should
also provide information about cultivar differences that
is not available from screening under non-stress
conditions. Information on this question has been
assembled for 6 populations of unselected lines
evaluated in a total of 10 experiments at locations in
India, Thailand, and the Philippines under both well
watered conditions and severe stress. Broad-sense
heritabilities (H) within water regimes and genetic
correlations across them are presented in table 1. Yield
under stress averaged 35% relative to the well-watered
controls across these experiments. H estimates for grain
yield in the stress environments were similar to those
for yield in the non-stressed controls, indicating that
selection for yield under stress is likely to be as effective
as selection for yield under favorable conditions.


Table 1. Repeatability (H) of grain yield estimates in well-watered and moisture-stressed treatments, and genetic correlations across stress levels
(rG), in trials evaluating unselected populations
Relative H
Stress Non-stress yield under rG Data
Location Year environ-ment environ-ment Population stress Stress Non-stress provided by:
Bet Dagan, Israel 1997 Upland Upland CT9993/IR62266 0.26 0.81 0.63 0.35 A. Blum
Coimbatore, Tamil Nadu 1999 Upland Upland CT9993/IR62266 0.31 0.60 0.56 0.86 R. Chandra Babu
Paramakudiamil Nadu 2000 Upland Upland CT9993/IR62266 0.41 0.76 0.23 0.91 R. Chandra Babu
Ubon, Thailand 2000 Upland Upland CT9993/IR62266 0.30 0.50 0.54 0.71 G. Pantuwan
Raipur, India 2000-02 Lowland Lowland CT9993/IR62266 0.21 0.37 0.45 0.80 R. Kumar
Los Banos,Philippines 2003 Upland Lowland Vandana/IR64 0.67 0.42 0.27 0.69 R. Venuprasad
Los Banos,Philippines 2003 Upland Lowland Apo/IR64 0.13 0.24 0.45 0.35 R. Venuprasad
Los Banos, Philippines 2003 Upland Lowland Apo/IR72 0.29 0.67 0.30 0.64 R. Venuprasad
Los Banos, Philippines 2003 Upland Lowland Vandana/IR72 0.31 0.07 0.42 0.78 R. Venuprasad
Los Banos, Philippines 1998-9 Upland Upland IR64/Azucena 0.56 0.68 0.74 0.62 B. Courtois
Mean 0.35 0.51 0.46 0.67


* 32 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004










Genetic correlations across water regimes were
consistently positive, averaging 0.67. This indicates that,
within segregating populations of lines generated from a
single cross, nearly half the genetic variation for yield
under severe stress is accounted for by factors that also
affect yield potential. The relatively high correlations
observed across establishment methods and stress levels
indicate that plant type and partitioning differences that
are also expressed in non-stress environments are
causing much of the yield variation observed under
stress in unselected breeding materials. These results
show that direct selection for yield under stress is likely
to result in yield gains under stress, and that
improvement in yield under stress can be combined with
improvement in yield potential.



Heritability within and correlations
across stress levels in advanced
cultivar testing
The results presented above refer to unselected breeding
lines from single crosses. In more diverse cultivar sets
exhibiting a wide range of maturity, the correlation of
genotype means across stress levels is affected by the
magnitude of the relative yield in the stress and non-stress
environments. This was observed in an experiment
conducted at IRRI, wherein a diverse set of upland and
lowland cultivars, breeding lines, and landraces was
evaluated for two years under 4 water regimes: (1) full
lowland irrigation, with transplanted management; (2)
favorable upland conditions in the wet season; (3)
moderate dry-season continuous stress; (4) severe dry
season continuous stress. Means in the four environments
were 2.7, 2.2, 1.1, and 0.5 t ha1, respectively. Genetic
correlations stress levels are presented in table 2. Yields
were positively and highly correlated among the non
stress and moderate stress treatments, but yields under
the severest stress level were not associated with yields
under non-stress conditions.


Table 2. Genetic correlations across water regimes for 44 upland and
lowland cultivars evaluated under 4 water regimes at IRRI, 2000-2003.

Moderate Upland Lowland
Environment upland stress non-stress non-stress

Severe upland stress 0.51 -0.08 -0.20
Moderate upland stress 0.78 0.85
Upland non-stress 0.97


Sources of variation in trials
measuring yield under stress
Although the heritability of yield is similar in stress and
non-stress environments, it is rather low. To plan
effective screening programs, an understanding of the
sources contributing to the variance of cultivar means is
needed. Major potential sources are within-trial field
variability, which may be exacerbated by water stress
treatments, and genotype x environment interaction.
The latter source is likely to be especially important in
drought screening trials conducted under natural stress
in the wet season. This was illustrated in a set of 39
advanced Thai breeding lines and released varieties
evaluated under stress and non-stress conditions in
transplanted and direct-seeded trials at Ubon and
Chumpae in 2003. Stress was applied by draining
paddies 2 weeks before the onset of flowering and not
re-watering. Very high genotype x environment
interactions were observed, due to differences in stress
timing and blast disease pressure among sites (Table 3).
Residual within-trial variances were also very high
resulting. This resulted in very low predicted
heritabilities for both stress and non-stress trials.

By contrast, managed-stress screens for drought tolerance
can have relatively high repeatabilities by controlling
genotype x trial interaction. This is illustrated in the
variance component and H estimates derived from the 44
entry cultivar set (described above) evaluated under
upland and lowland management at IRRI. Moderate and
severe stress treatments were applied in the relatively
uniform dry season. In this experiment, genotype x
environment interaction in the stress treatments was low
relative to the genotypic variance, and relative to GxE
interaction in the wet season. The main source of variation
in the stress trials was within-trial residual field variation,
indicating that useful levels of H can be achieved in a
single managed-stress screening trial if it is well-replicated
and designed (Table 4).


Table 3. Variance component and broad-sense heritability (H) estimates
for the combined analysis over Ubon and Chumpae in northeast Thailand
for 4 types of screening trial. (Wet season 2003)

Variance components H

Genotype x 2 sites, 1 site,
REGIME Genetic environment Residual 3 replicates 3 replicates

Direct-seeded: non-stress 0 983 3205 0.00 0.00
Direct-seeded: stress 32 393 966 0.08 0.04
Transplanted: inon-stress 161 0 1264 0.43 0.28
Transplanted:Stress 27 97 355 0.20 0.11


II. Gene Discovery and Novel Approaches 33 *











Selection experiment
The results reported above indicate that direct selection
for yield under stress is likely to be effective if
conducted in a well managed screening environment.
To test this hypothesis, a direct selection experiment
was conducted in two populations at IRRI in 2003-4.
Populations were derived without selection from the
crosses Apo/IR64 and Vandana/IR64. IR64 is an elite
irrigated lowland variety. Apo (IR55423-01) is a high
yielding upland rice variety with moderate tolerance to
continuous water stress in the field. Vandana is a
highly drought-tolerant eastern Indian upland variety
derived from a cross between an aus traditional variety
and an improved Philippine tropicaljaponica. Two
hundred and twenty-five (225) random F2 derived F3
lines from each cross were evaluated under severe
intermittent upland stress as well as under non-stress
irrigated lowland management in the dry season of
2003. Evaluation was conducted in 2 replicate alpha
lattice trials in which the experimental unit was a single
plot, 2 m in length in the upland and 5.25 m in length in
the lowland. The 25 highest-yielding lines were selected
from each screening environment (giving a selection
intensity of 12.5%) and compared with 25 random lines.


Table 4. Variance components for 44 upland and lowland cultivars
evaluated under 4 water regimes at IRRI, 2000-2003

Environment ,2 G2 2 H(one 4-rep trial) H(two 4-rep trials)

Severe upland stress 58 20 86 0.48 0.58
Moderate upland stress 86 36 138 0.46 0.55
Upland non-stress 337 168 184 0.76 0.61
Lowland non-stress 481 299 163 0.75 0.59



Table 5. Effects of direct selection for yield under upland stress in 2
populations at IRRI: 2003-4

Vandana/IR64 Apo/IR64
Evaluation environment
Entry Upland Lowland Upland Lowland

Upland-selected population 68.9 182 16.7 191
Lowland-selected population 57.8 214 12.8 224
Random population 54.8 184 18.1 193
IR64 3.6 286 5.8 293
Apo 14.8 240 17.8 242
Vandana 104.6 146 83.2 51

LSD o5 for checks 59.0 49.4 21.4 91
LSD.s5 for populations 11.8 24.0 4.3 18



* 34 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004


Results of the selection experiment are presented in
Table 5. Under severe upland stress, the tolerant upland
parent, Vandana, yielded 104.6 g m2, whereas Apo and
IR64 yielded only 14.8 and 3.6 g m2, respectively. The
selected Vandana/IR64 population outyielded the
random set of lines by nearly 20% under severe stress.
The selected population derived from Apo/IR64,
however, exhibited no improvement over the random
set under severe stress. Neither upland-selected
population exhibited reduced mean yield relative to the
checks under lowland conditions.

It is important to note that, on average, random lines
derived from Vandana/IR64 outyielded random lines
from Apo/IR64 by 300%. The effect of using a highly
tolerant donor on yield under severe upland stress was
greater than the effect of selection.

In general, this experiment demonstrates that direct
selection for yield under severe stress in small plots can
be highly effective in populations where adequate
genetic variability for the trait exists. It also illustrates
the importance of using highly tolerant donors when
tolerance to severe stress is the breeding objective.



Implications for drought-tolerance
breeding
Yield under drought stress is a moderately heritable trait
with repeatability similar to that of yield in non-stress
environments. Direct selection for yield under stress is
effective, if stress tolerance screening is done in replicated
field trials that effectively control within-trial field
heterogeneity. This means that selection for yield under
stress is best initiated after pedigree breeding lines have
been fixed for plant type, disease resistance, quality, and
other high-heritability traits, as is the case for selection for
yield potential. This was demonstrated in a selection
experiment. The moderate correlation usually observed
between yields in stress and non-stress environments
indicates that improvements in stress tolerance can be
generated while maintaining high yield potential.
Highly-tolerant donors are likely to be much more
efficient in generating progeny tolerant to severe stress
than donors with only moderate levels of tolerance. Such
donors, however, are unlikely to be agronomically
acceptable in high-productivity environments. This is a
strong argument for pre-breeding highly drought-tolerant
donor lines with acceptable agronomic traits under non
stress conditions. Such donors could be directly used as
sources of drought tolerance in cultivar development
programs that must generate cultivars combining high
yield potential with moderate stress tolerance.











Empowering rice drought gene discovery activities

through bioinformatics


RICHARD BRUSKIEWICH, VIOLETA BARTOLOME, ALEXANDER COSICO, ARUMUGAM KATHIRESAN, LOCEDIE MANSUETO, ARLLET PORTUGAL,
JUTHARAT PRAYONGSAP, MUTHURAJAN RAVEENDRAN, MAY ANN SALLAN, NAVEEN SHARMA, XUSHENG WANG, TERESA ULAT,
VICTOR ULAT, JOHN BENNETT, RENEE LAFITTE, KENNETH McNALLY, AND GRAHAM MCLAREN

International Rice Research Institute, DAPO 7777, Metro Manila, Philippines

Corresponding author: Richard Bruskiewich; E-mail: r.bruskiewich@cgiar.org


RF scholars and their research
The Rockefeller Foundation funded six dissertation
fellowships for rice drought bioinformatics research at
the International Rice Research Institute. The scholars
funded are noted in the following table:


Scholar


Scholarship University Affiliation


Country


Xusheng Wang M.Sc. Zheijiang University China
Locedie Mansueto M.Sc. University of the Philippines, Diliman Philippines
Jutharat Prayongsap Ph.D. Kasetsart University Thailand
Naveen Sharma Ph.D. Kuvempu University India
Muthurajan Raveendran Postdoc-toral Tamil Nadu Agricultural University India
Ravindra Babu Postdoc-toral University of Hyderabad India


The scholars' research focuses upon the application of
bioinformatics methodologies to mine rice structural
and functional genomics information for candidate
gene loci and associated alleles potentially conferring
drought tolerance in rice. There are estimated to be
more than 50,000 predicted genes in the rice genome.
Identification of the candidate subset of genes involved
in drought stress requires a strategy of intersecting
gene position, function, expression, and allele selection
evidence. Bioinformatics provide an effective means for
achieving such integration. Each scholar in the project
was asked to focus on subsets of drought experimental
data, for integration into the International Rice
Information System (IRIS; Bruskiewich et al. 2003;
www.iris.irri.org).


Integration of QTL data with rice
genome sequence data
Initial project efforts focused on anchoring Quantitative
Trait Locus (QTL) data on a limited set of genetic maps


to publicly available rice genome sequences and
expressed sequence tags (EST) from an IRRI
commissioned IR64 drought stress panicle library
(work undertaken by X. Wang, Wang, Zhu, Mansueto,
and Bruskiewich, submitted). Further work along this
theme (by J.Prayongsap) compiled additional drought
QTL maps in a comparative manner, for anchoring to
the rice genome, for publication in IRIS.


The role of ABA in drought stress
The phytohormone ABA regulates many important
physiological and developmental processes in
plants (Leung and Giraudat, 1998). ABA regulates
stomatal responses, stress tolerance responses, and
growth (Zeevaart and Creelman, 1988) in addition
to preparing the seed for dormancy and
germination (McCarthy, 1995). It also mediates
stress responses such as environmental stress
adaptation to salinity, low temperature and water
deficiency (Ingram and Bartels, 1996). In spite of its
role in stress tolerance and dormancy, ABA appears
to interfere with panicle development and
emergence. For the above reasons, ABA has become
a primary research target for the scholars.


ABA and root elongation
Roots penetrating deeper soil layers contribute to
maintaining plant water potential when the topsoil
dries up. Drought tolerant rice genotypes grow roots
deeper under low-water potential when compared to
roots of susceptible plants grown in similar conditions.
Abscisic acid (ABA) and ethylene play major roles as
primary elicitors or secondary messengers for inducing
genes during biotic and abiotic stress by plants (Bray,
1993). These phytohormones interact with each other to


II. Gene Discovery and Novel Approaches 35 *










promote longer roots in tolerant genotypes during low
water potential (Spollen et al., 2000). One scholar (N.
Sharma) is initiating gene expression studies with
drought stressed root tissues probing whole genome
arrays to shed light on the genes, regulatory pathways,
and overall mechanisms by which ABA and ethylene
interact in promoting longer roots. Allele mining of the
promising candidate genes identified from the
expression studies will give insight into the genetics
and function in the germplasm and possible use of the
candidate genes as markers (MAS) for maximum root
length and drought tolerance.


ABA and yield under stress
Although drought survival may be enhanced by deeper
roots, it is yield under drought stress that is of primary
agronomic concern. Another scholar (J. Prayongsap) is
therefore studying the impact of ABA on the rice panicle
by integrating QTL mapping data with gene expression
data obtained form the IR64 drought stressed panicle
EST library. In addition, IR64 mutant stocks are being
screened for lesions in ABA pathway genes.


Proteomics of ABA catabolism
The ABA level in plant is simultaneously regulated by
opposing forces of catabolism and/or biosynthesis
(Zeevaart, 1999). The genes involved in ABA catabolism
have not been identified or characterized in rice. The
identification of genes encoding for proteins in ABA
catabolism may be a crucial element in a breeding
program for reproductive stage drought tolerance. One
RF scholar (M. Raveendran) is striving to study the
effect of ABA on rice plant growth paniclee emergence,
spikelet fertility, biomass, photosynthesis,
transpiration, etc.) using proteomic analysis of total leaf
proteins and microsomal extracts from plants subjected
to exogenous application of ABA under well watered
conditions. He is assaying ABA content by HPLC-based
assay of ABA catabolism and ABA-8' hydroxylase
activity in the microsomal preparation.


Bioinformatic integration of
drought gene information
Integration of all the above datasets is being greatly
facilitated through software engineering efforts of one
of the scholars (L. Mansueto), who comes to the project
with a computing science background. This latter


* 36 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004


scholar also played a major role in the analyses of the
-8,000 clone drought stress panicle EST library, in the
commissioning of a microarray laboratory
information management system currently used by
the project, and in the integration diverse project data
with publicly available pathway and network
information. These activities have included Java
software engineering activities as part of an ongoing
effort to develop a new informatics platform for crop
gene discovery.



Project status
X. Wang obtained his M.Sc. from Zeijiang University
last July 2003 and a paper is submitted for
publication. L. Mansueto is expected to defend his
M.Sc. in the near future and continue in the project
for a short while as a non-RF computer consultant at
IRRI. J. Prayongsap, N. Sharma, and Dr. Raveendran
are expected to complete the RF-funded component
of scholarship later this year. Ms. Prayongsap intends
to return to Thailand to complete her PhD; however,
as a doctoral scholar, N. Sharma is planning to
continue his work at IRRI under the auspices of the
new CGIAR Genetic Resources Challenge Program
(www.GenerationCP.org). Dr. Babu has only recently
joined the project. His research will focus upon the
characterization of regulatory networks using whole
genome analysis and large-scale integration of
bioinformatics data generated within the project.


References
Bray, E.A. 1993. Molecular responses to water deficit. Plant Physiology 103:1035-40.
Bruskiewich R., Cosico A., Eusebio W., Portugal A., Ramos L.R., Reyes T, Sallan M.A.B., Ulat
V.J.M., Wang X., McNally K.L., Sackville Hamilton R. and McLaren C.R. 2003. Linking
Genotype To Phenotype: The International Rice Information System (IRIS).
Bioinformatics 19(Suppl.1): i63-i65
Ingram, J., Bartels, D. 1996. The molecular basis of dehydration tolerance in plants. Ann.
Rev. PI. Physiol. PI. Mol. Biol. 47:377-403.
Leung, J., Giraudat, J. 1998. Abscissic acid signal transduction. Ann. Rev PI. Physiol. PI.
Mol. Biol. 49:199-222.
Spollen W.G., LeNoble M.E., Samuels T.D., Bernstein N., Sharp R.E. (2000) Abscisic acid
accumulation maintains maize primary root elongation at low water potentials by
restricting ethylene production. Plant Physioll22: 967-76.
Wang X., Zhu J., Mansueto L., and Bruskiewich R. 2004. Identification of candidate genes
for drought stress tolerance in rice by the integration of a genetic (QTL) map with the
rice genome physical map. Submitted to Theoretical and Applied Genetics.
Zeevaart, J.A.D. 1999. Abscissic acid metabolism and its regulation. In: PJJ Hooykaas, MA
Haall, KR Libbenga, eds. Biochemistry and Molecular Biology of Plant Hormones.
Elsevier Science, Amsterdam, Pp 189-207.
Zeevaart, J.A.D., Creelman, R.A. 1988. Metabolism and physiology of abscissic acid. Ann.
Rev. PI. Physiol. PI. Mol. Biol, 39:439-73.











Functional analysis of plant hydrophilins


ALEJANDRA A. COVARRUBIAS, JOSE L. REYES, YADIRA OLVERA-CARRILLO, FRANCISCO CAMPOS,
MARINA BATTAGLIA, ROSA E. QUIROZ, AND ALEJANDRO GARCIARRUBIO

Depto. Biologia Molecular de Plantas, Instituto de Biotecnologia-UNAM
Av. Universidad 2001, AP 510-3 Cuernavaca, Mor. 62250, Mexico

Corresponding author: Alejandra A. Covarrubias; E-mail: crobles@ibt.unam.mx


Introduction
The Late Embryogenesis Abundant (LEA) proteins are
plant polypeptides synthesized at the onset of
desiccation in maturing seeds and in vegetative
organs exposed to water deficit. We have shown that
most LEA proteins are comprised in a more
widespread group denominated 'hydrophilins'. The
defining characteristics of 'hydrophilins' are high
glycine content and a high hydrophilicity index. By
database searching, this criterion specifically selects
most known LEA proteins, as well as additional
proteins from different taxons (Garay-Aroyo et al.,
2000). Thus, 'hydrophilins' might represent an
analogous adaptation to a common problem in
organisms as diverse as plants, bacteria, and fungi.

To gain insight into the function of different
hydrophilins, including LEA proteins, we developed
an in vitro partial dehydration assay wherein the
activity of malate dehydrogenase (MDH) and lactate
dehydrogenase (LDH) is measured in the presence or
absence of a putative protecting protein. Since a
number of LEA proteins are also accumulated upon
low temperatures in vivo, we have adapted a second
in vitro test where freezing is applied as a mean to
limit water availability (Lin and Thomashow, 1992).
Under the conditions of the partial dehydration assay,
where the progressive loss of water occurs in the
absence of other perturbing factors, such as heating or
freezing, we show that hydrophilins are able to
protect these enzymatic activities. Under these
conditions, the compatible osmolyte trehalose needs
to be in a 105 fold excess over LDH to exhibit the
same protective level as hydrophilins. Our data show
that group 2 LEA proteins (dehydrins), as well as
different LEA proteins from groups 3 and 4 (LEA76
and LEA D113) are capable of protecting both MDH
and LDH when exposed to a controlled desiccation


process, as well as during freezing conditions.
'Hydrophilins' from S. cerevisiae as well as from E.
coli also present protective characteristics in these in
vitro assays.


Results
The ubiquity of hydrophilins, their responsiveness to
water deficit, and their capacity to protect enzymatic
activities from inactivation due to in vitro partial
water removal have led us to pursue their function in
vivo as well as the mechanisms involved in the
regulation of their gene expression. One approach
has been to analyze one of the smallest Arabidopsis
LEA protein families, the LEA-IV family, which is
predicted to consist of three members: Atlea-IV1,
AtleaIV2, and Atlea-IV5. In addition to the
transcript accumulation patterns, using a specific
polyclonal antibody, we also obtained the protein
accumulation patterns for AtLEA-IV 5, which
showed that this protein accumulates in dry seeds
and, in vegetative organs in response to drought,
osmotic and ionic stress, but not in response to low
temperatures. Transgenic Arabidopsis plants
overexpressing Atlea-IV5 gene did not show
advantage compared to the wild-type when seeds
are germinated in hyperosmotic (500 mM mannitol)
or high salt (250 mM NaC1) media. However, a
significant resistance to dehydration/rehydration
treatment was observed in adult plants, as indicated
by the higher survival percentage and dry weight of
overexpressing lines compared to non-transgenic
plants. To address the function of these proteins
during water limitation and seed development, we
are silencing either Atlea-IV-5or the complete Atlea
IVgene family using RNA interference.


II. Gene Discovery and Novel Approaches 37 *










-9.5 -30.5
0 85-98


-58 ->
- 99-99.4


Osmotic potential (bars)


SdF


.4-.


Jose Luis Reyes unpublished


I ..-r^^^ y ^


water binding

Organization of water molecules


substrate binding

AND Binding to macromoleculares
or to cellular structures


Figure 1. A schematic model to explain the protective effect showed by hydrophilins during in vitro partial water loss. It is proposed that
hydrophilins are constituted by two domains, one able to bind and/or organize water molecules and other, which identifies particular targets
macromoleculess or cellular structures). Their flexible structure (random-coil) may be important for optimal protection of their targets, they may be
unstructured in solution but structured when completed with some macromolecule.


Conclusions
These findings lead us to suggest that, at least, some
'hydrophilins' preserve enzymatic activities during
partial dehydration by a mechanism involving
hydrophobic interactions, which would prevent
enzyme denaturation. Our results provide basis to
understand the role of hidrophilins in the adaptive
response to water stress.


References
Garay-Arroyo, A., Colmenero-Flores J.M., Garciarrubio A. and Covarrubias A.A. 2000. Highly
hydrophilic proteins in prokaryotes and eukaryotes are common during conditions of
water deficit. 1. Biol Chem. 275: 5668-74.
Lin, C., and Thomashow M.F 1992. A cold-regulated Arabidopsis gene encodes a polypeptide
having potent cryoprotective activity. Biochem. Biophys. Res. Comm. 183: 1103-08.
Reyes J.L., Rodrigo M.J., Colmenero-Flores J.M., Gil J.V., Campos F., Garay-Arroyo A., Salamini
F., Bartels D. and Covarrubias A.A. Hydrophilins from distant organisms can protect
enzymatic activities from water limitation in vitro. (Submitted)


* 38 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004


% Partial water loss


0
LDH






Hydrophilin


r


~'~===










Rice SNP map between indica and japonica subspecies:

DNA marker resolution on the kilobase scale

F. ALEX FELTUS1, JUN WAN1, STEFAN R. SCHULZE1, NING JIANG2, AND ANDREW H. PATERSON1'2'3
1 Plant Genome Mapping Laboratory, University of Georgia, Athens, Georgia 30602, USA
2 Department of Plant Biology, University of Georgia, Athens, Georgia 30602, USA
3 Department of Genetics, University of Georgia, Athens, Georgia 30602, USA

Corresponding author: F. Alex Feltus; E-mail: feltus@uga.edu


Introduction
Marker-assisted selection (MAS) will be an invaluable
tool for the development of rice varieties with a higher
tolerance to abiotic and biotic stresses. However, many
heritable, agriculturally-relevant, stress-tolerance
phenotypes are modified in a complex, polygenic
manner. This is evident in the large number of stress
relevant rice QTLs in the literature, and the genetic
difficulties faced by the breeder attempting stress
tolerance allele introgression. In order to design flexible
and efficient MAS strategies, the rice breeder will
require genetic markers that are reliable, low-cost, and
localized to multiple genomic regions. However, due to
coverage gaps in current rice genetic marker collections
and the cost/time commitment in marker development
for uncharacterized genotypes, there is an urgent need
for alternative marker solutions. We have aligned the
indica (cv. 93-11) andjaponica (cv. Nipponbare) rice
genomes for the purpose of discovering single
nucleotide polymorphism (SNP)/insertion-deletion
(INDEL) markers offering potentially high genetic
resolution, and which should be informative in crosses
involving the two rice subspecies. These SNP/INDEL
markers could be used for marker-assisted selection,
LD scans, and other applications in the pursuit of
higher stress-tolerant rice varieties.


Methods
The International Rice Genome Sequencing Project
(IRGSP) has used a BAC based strategy to sequence the
rice genome (ssp.japonica cv. Nipponbare). The Beijing
Genomics Institute (BGI) used a shotgun strategy to
sequence a separate subspecies of rice (ssp. indica cv. 93
11). All sequence information is publicly available. We
used BLAST to align the BGI indica contigs (Rice GD,
http://btn.genomics.org.cn:8080/rice/) with the


Institute for Genomic Research (TIGR)japonica
pseudomolecule assembly (vl.0; the TIGR Rice
Genome Project, http://www.tigr.org/tdb/e2kl/
osal). Prior to alignment, all indica shotgun contigs
were masked for repetitive DNA. SNPs and single
base INDELs were extracted from the BLAST
alignments using in-house Perl scripts after filtering
repetitive DNA, paralogous DNA, and polymorphisms
of low quality.


Results
After applying the stringent set of filters, the total
number of polymorphisms remaining was 408,898
(94% SNPs). On average, this resulted in a
polymorphism rate of 1.02 polymorphisms per
kilobase based on a rice genome size of 400 megabases.
The number of polymorphisms per chromosome is
shown in figure 1. In order to determine the quality of


60,000

, 50,000

S40,000

E
. 30,000

" 20,000

10,000

0


'7









Fl


1 2 3 4 5


6 7 8 9 10 11 12
Chromosome


Figure. Total number of indica-japonica SNP/INDEL polymorphisms by
chromosome.


II. Gene Discovery and Novel Approaches 39 *










this dataset, we sequenced random loci containing
SNPs from each chromosome. PCR primers were
designed from the pseudomolecule and used to
amplify Nipponbare and 93-11 genomic DNA.
Approximately 80% (87/109) contained SNPs that
matched our dataset. To test whether these
polymorphisms would extend to other indicajaponica
experimental crosses, we tested for overlap with the
SNP dataset from Nasu et al., which is the largest
collection of published rice SNPs. We examined a
subset of their SNPs for overlap with our dataset in
which all three japonica genotypes shared the same
nucleotide and two indica genotypes shared a different
nucleotide. Forty-eight percent (41/86) showed exact
overlap with our indica-japonica dataset.


Conclusions
On average, we estimate that 38% of the discovered
polymorphisms will be informative in other indica
japonica crosses. This corresponds to approximately
152,000 SNP/INDELs or 0.38 polymorphisms/kilobase
based on a genome size of 400 megabases. Therefore,
the number of discovered markers should be sufficient
for most genetic studies involving indicajaponica
crosses. This work was supported by The Rockefeller
Foundation initiative on 'Resilient Crops for Water
Limited Environments,' USAID Comparative Cereal
Genomics Initiative, and US National Science
Foundation Plant Genome Research Program.


References
Altschul, S. F., etal. (1990) 1Mol Bio215, 403-10.
Nasu, S., et al. (2002) DNA Res 9, 163-71.
Sasaki, T. and Burr, B. (2000) Curr Opin Plant Biol3, 138-41.
Yu, J., Hu, S., etal. (2002) Science 296, 79-92.


* 40 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004











XVSAP1 from Xerophyta viscosa improves salinity and

water deficit stress tolerance in Arabidopsis and tobacco


DAHLIA GARWE, JENNIFER A. THOMSON, AND SAGADEVAN G. MUNDREE

Environmental Stress Research Unit, Department of Molecular and Cell Biology,
University of Cape Town, Private Bag, Rondebosch, 7701, South Africa

Corresponding author: S. Mundree; E-mail: mundree@uct.ac.za


Introduction


Results


It has been estimated that two-thirds of the yield
potential of major crops are routinely lost due to
unfavourable growing environments (Bajaj et al., 1999).
In recent years, genetic engineering has been used to
improve stress tolerance in plants. A potential rich
source of genes that could confer tolerance to abiotic
stresses is a small group of angiosperms known as the
resurrection plants. These plants can lose more than
90% of their relative water content, survive in their
dried state for prolonged periods, and then resume
active life when water becomes available again (Bartels
et al., 1990; Sherwin and Farrant, 1996). Genes that
could potentially improve the drought tolerance of
agriculturally important plants such as maize and
wheat have been isolated from C. pantagenium
(Itturiaga et al., 1992) and X viscosa (Garwe et al., 2002;
Mundree et al., 2000;).


Methods
The strategy of complementationn by functional
sufficiency" was used to isolate a cDNA designated
XVSAP1 from a cDNA library constructed from X.
viscosa leaves dehydrated to 85%, 37%, and 5% relative
water content. Following molecular characterization,
the induction of XVSAP1 under water deficit
conditions was analysed using semi-quantitative RT
PCR. To confirm the functional role of the gene in stress
tolerance, XVSAP1 was transformed into Arabidopsis
thaliana and Nicotiana tabacum by Ti plasmid mediated
transformation under the control of a cauliflower
mosaic virus 35S promoter, a nos terminator and bar
gene selection.


Analysis of the cDNA sequence indicated a highly
hydrophobic protein with six transmembrane regions.
The deduced amino acid sequence showed 49% identity
to WCOR413, a low temperature regulated protein from
wheat. The protein also showed 25% to 56% identity to
WCOR413-like proteins from A. thaliana. Southern blot
analysis revealed that there are at least two copies of
XVSAP1 in X viscosa. It was shown that XVSAP1 is
induced by dehydration, salt stress (100 mM), both low
(4 C) and high temperature (42 C) and high light
treatment (1500 mmol m-2 s1). Expression of XVSAP1
in both Arabidopsis and tobacco plants led to constitutive
accumulation of the corresponding protein in the leaves.
Transgenic Arabidopsis grown in tissue culture and
tobacco grown hydroponically were more tolerant to
salt and osmotic stress. Non-transgenic plants had
shorter roots, leaf expansion was inhibited, and leaves
were more chlorotic than those of the transgenic plants.
In addition, transgenic tobacco plants attained a higher
fresh and dry weight than the untransformed controls.
Transgenic tobacco showed greater tolerance to drought
stress when grown in soil.


Conclusion
The study demonstrated that XVSAP1 is involved in
the response to abiotic stress in X viscosa and confers
tolerance to heat, drought, salt, and osmotic stresses
when expressed in heterologous plant systems. The
expression of XVSAP1 in crops such as maize and
wheat could lead to increased crop production in
southern Africa.


II. Gene Discovery and Novel Approaches 41 *











100 mM NaCI


100


f 75


S50


25


0


0 2 4 6 8
Time (d)


4*.tWKAZSKl,.*104 1G


0 2 4 6 8
Time (d)


A-It a,-ZKK"1"'C*21Q


Figure 1. Comparison of the relative root growth of Arabidopsis plants transformed with XVSAP1 (6K, 10C, 21G), the azygous control (AZ) and the
untransformed control (WT) on salt stress media. Five-day-old seedlings were transferred to plant nutrient agar without sucrose (PNA) or to PNA
supplemented with the indicated concentrations of NaCI. Root length was determined from eight plants.


0 7-.

0.20

0.15

S0.1

0.0.



KE1 AZ A7 A5

Figure 2. Dry weight comparison of tobacco plants transformed with
XVSAP1 (A5, A7) and untransformed controls azygouss, AZ; wild type,
WT) after salt and osmotic stress. Six-week old tobacco plants grown
hydroponically in one-fourth-strength MS (MS-4) solution were
transferred either to fresh MS-4 medium (MS, white bars) or MS-4
supplemented with 200 mM NaCI (dark grey bars) or MS-4 supplemented
with 9% polyethylene glycol (PEG, avg mol. wt. 3.350, light grey bars)
for one week. Measurements were taken one week after recovery in
fresh MS-4 solution. Error bars represent standard deviation based on the
mass of eight plants.



References
Bajaj S., et al. 1999. Mol Breeding 5: 493-503
Bartels D, et al. 1990. Planta 181:27-34.
Garwe D., etal. 2003. J.Exp. Bot 54:191-201
Itturiaga G., et al. 1992. Plant Mo Biol. 20, 555-58
Mundree S.G., et al. 2000. Planta 211: 693-700.
Sherwin H., and J.M. Farrant 1996. Ann. Bot 78: 703-10.


* 42 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004


0 mM NaCI











Molecular dissection on rice photosynthesis-related traits

at reproductive stage in irrigated and drought conditions


S.P. Hu, H.W. MEI, H.Y Liu, G.H Zou, G.L Liu, X.Q. Yu, J. Li, P. LONG, AND LIJUN Luo

Shanghai Agrobiological Gene Center, Shanghai 201106, China

Corresponding author: Lijun Luo; E-mail: lijun@sagc.org.cn


Introduction
Photosynthesis is an essential physiological process in
plants' growth and dry matter production. There is
great genetic diversity in photosynthesis among rice
germplasm (Cao et al., 2001). Usually, the plant with
high photosynthesis can produce more biomass and
grain yield. Recent studies on paddy rice under
irrigated conditions indicates that the photosynthetic
rate (PR), chlorophyll content (CC), stomatal resistance
(SR), and transpiration rate are quantitative traits and
affected by several QTLs (Teng et al., 2004). In this
paper, a RIL population from a paddy rice and upland
rice cultivar were investigated for PR, CC, SR, TR, as
well as water use efficiency (WUE) in both irrigated
(normal) and drought (stress) conditions. The
molecular dissection was also conducted to map the
related QTLs. The results will help us understand the
genetic bases of photosynthesis under drought
occurrence in rice production.


Methods
A set of 195 F10
recombinant inbred lines
were developed from
Zhanshan 97B (paddy
rice with the largest
growing area in China)
and IRAT 109 (drought
tolerant upland rice) and
its parents was planted
in the drought screening
facility, which can create
gradients of soil water
content, at Shanghai
Agrobiological Gene
Center. Each genotype
was seeded directly in
two-rows plots in


random block design with three replications. Each row
in a plot consisting of 15 plants with a spacing of 18
cm. After stress at the reproductive stage, two
representative plants in different soil water status in
each plot, were selected as normal and stress
treatments to measure the PR, SR, TR, WUE, and CC
by using the BAU System and SPAD 502, respectively.

Standard analyses of variance were performed to check
the genetic variance among the RI Lines for the
investigated traits. The phenotype correlation was
calculated using S-PLUS statistics software. The
genotyping was conducted according the published
procedures (Luo et al., 2001). An integrated genetic
linkage map with 186 SSR markers was constructed
using Mapmaker version 3.0 (Lincoln and Lander
1992). The putative quantitative trait loci linked to the
traits were identified using Windows QTL
Cartographer V2.0 (Basten et al., 2001) with a threshold
LOD score of 2.0.


Table 1. Putative QTLs for photosynthetic rate, stomatal resistance, chlorophyll content, transpiration rate and
water use efficiency in a Zhenshan 97B/IRAT109 RIL population

Trait QTL Water status Chr. Marker interval LOD a R2/
Photosynthetic Rate (PR) QPr12 normal 12 RM101-RM179 2.83 0.994 6.96
QPr2 stress 2 RM263-RM526 2.15 0.785 4.69
Chlorophyll content (CC) QCc3a normal 3 RM22-RM231 3.26 -0.896 7.04
QCc3b 3 RM16-RM426 3.03 -0.971 8.57
QCc3c stress 3 RM203-RM520 2.26 0.784 5.66
QCcc7 normal 7 RM134-RM248 3.66 0.931 7.87
stress 7 RM134-RM248 2.51 0.795 5.86
Stomatal resistance (SR) QSr2a stress 2 RM110-RM211 2.04 0.247 5.19
QSr2b 2 RM279-RM555 2.52 0.252 5.38
QSr9 9 RM215-RM245 2.46 0.252 5.43
QSsrlO 10 RM311-RM467 2.22 0.358 6.7
TranspirationRate(TR) QTr5 normal 5 RM274-RM480 3.04 -65.495 7.34
QTr6 6 RM30-RM340 2.03 48.899 4.3
QTr12 stress 12 RM4A-RM19 3.9 -75.42 11.85
Water Use Efficiency (WUE) QWuel normal 1 RM472-RM104 2.4 -4.095 8.03
QWuell 11 RM206-RM144 2.31 -3.315 5.15
QWue7a stress 7 RM500-RM320 2.62 -3.355 7.52
QWue7b 7 RM351-RM505 2.67 2.861 5.73
QWue9 9 RM434-RM410 2.04 -2.519 4.35


II. Gene Discovery and Novel Approaches 43 *










Results
1. There is significance difference in five
photosynthesis-related traits between the parents.
Under water stress conditions, IRAT109 has larger
PR, TR, WUE, and CC, but less SR than Zhenshen
97B.Both parents have higher WUE and PR, but
lower TR in stress. All the traits were normally
distributed with transgressive segregation in the
population.
2. Two QTLs related to PR were located on
chromosomes 2 and 12, respectively. One (Qpr2)
was detected under stress conditions, explaining
4.69% of total variation. The allele from Zhenshan
97B has positive effects. Another (Qprl2) in marker
interval RM101-RM179 was detected under
normal conditions.
3. Three QTLs on chromosome 3 were found to
associate with CC, but only one was detected
under stress conditions. A QTL in marker interval
RM134-RM248 on chromosome 7 was detected
under both stress and normal conditions.
4. Four QTLs influencing SR were located on
chromosomes 2, 9, and 10, respectively, under
stress. Together, these QTLs explained about 26%
of total variation. No significant QTL was detected
under normal conditions.
5. Three QTLs affecting TR were located on
chromosomes 5, 6,12, respectively. The QTL on
chromosome 12 (Qtrl2) has the largest


contribution to total variation. The allele from
Zhanshan 97B has negative additional effect.
6. Five QTLs underlying WUE were detected. Among
them, two (Qwuel, Qwuell) were detected under
normal conditions and located on chromosome 1
and 11, respectively. Three (Qwue7a, Qwue7b, and
Qwue9) were detected under stress and mapped on
chromosomes 7 and 9, respectively.


Conclusions
The photosynthesis-related traits performed
differently under normal irrigation and drought
conditions. As the water stress increased, the
transpiration rate of the plant decreased significantly,
but the water use efficiency as well as photosynthetic
rate increased. A total of 18 putative QTLs were
detected that showed association with photosynthesis
related traits; they were located on 10 chromosomes,
and not found on chromosomes 4 and 8. Only one
QTL, on chromosome 7, was detectable under both
water condition treatments.



References
Basten etal. 2001. QTL Cartographer, version 1.15, pp 28-152
Cao et al. 2001. Chinese 1. Rice Sci. 15:29-34
Luo L.J., etal, 2001. Genetics 158:1755-71
Teng et al. 2004. Euphytica 135:1-7.


* 44 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004











Identification of SSR markers linked to candidate

genes for drought tolerance in rice


J. HUSSAIN, K.L. McNALLY, AND H.R. LAFITTE

International Rice Research Institute (IRRI), Los Banos, Philippines

Corresponding author: K.L. McNally; E-mail: k.mcnally@cgiar.org


Microarray analysis of drought-stressed rice panicles
identified a number of ESTs showing significant
changes during water stress in a set of three diverse
rice cultivars growing in the field (Kathiresan, 2004;
Lafitte et al., 2004). We identified 96 of these drought
responsive ESTs that were annotated as having
drought-related functions, and were not distant from
QTLs reported for growth or yield under drought. The
QTLs used were primarily those reported for a rice
mapping population derived from upland-adapted
parents (Lafitte, Price et al., 2004), with supporting
information from another population (Babu, Nguyen
et al., 2003). We used 350 SSR primers located on the
same BAC/PAC clones as these drought-responsive
ESTs to study polymorphism in 11 rice cultivars. The
markers were not evenly distributed across the
genome, but were clustered. The cultivars surveyed
have been used as parents in mapping populations,
and they include indica and japonica subspecies as well
as upland and lowland-adapted cultivars. Specific
allelic patterns were generated from 325 of the
markers. The pattern analysis constructed from the
banding patterns clustered the cultivars by subspecies,
with the exception of the japonica/indica intermediate
CT9993, but not by adaptation (Figure 1). Some of the
regions associated with drought QTLs may reflect
allelic differences that are characteristic of indica vs
japonica contrasts, but ample variation for drought
responsive EST regions exists within subspecies as
well. Among the non japonica cultivars, Co39 showed
little similarity to other cultivars. The subtropical
high-yielding line from southern China, Teqing,
unexpectedly showed similarity to early maturing
Bala from eastern India.

Groups of 40 to 60 markers per population were
identified as being clearly polymorphic between
parents of two additional mapping populations
(IR64/Azucena and Vandana/Moroberekan). Each
population was genotyped, and single marker
analysis was conducted for performance under


Figure 1. Principal coordinate analysis of 11 rice cultivars on the basis
of 325 SSR markers. The SSR markers were selected as being both
tightly linked to drought-responsive ESTs in panicles at heading and also
lying near drought-related QTLs for plant growth and yield.


managed drought stress in field experiments.
Populations were screened under upland stress
conditions with stress applied during the reproductive
stage. A number of interesting regions contained QTLs
across both populations, and the ESTs, or other genes on
the BAC clone, may be considered putative candidate
genes for drought tolerance (Table 1). These included
protein kinases, genes associated with ubiquitin
mediated degradation of proteins, genes associated with
protection from oxidative damage, signaling or defense
genes, and cell cycle genes. In some cases, the EST
sequence hit several locations in the genome, but
significant marker effects were found only at a single
location. Co-localization of the significant marker effects
with the drought-responsive ESTs across diverse
populations supports the hypothesis that allelic variation
in these genes results in differences in crop performance
under field conditions. Further steps to confirm the
identity of these candidate genes will include gene
expression studies in contrasting sets of lines from the
mapping populations.


II. Gene Discovery and Novel Approaches 45 *











Table 1. Details of drought-responsive ESTs, the number of QTLs reported in that region (QTLs), and the number of traits for which significant marker
effects were observed in this study using 2 mapping populations grown with mild or severe reproductive stage drought (# traits)

EST: AF53-Rpf Annotation SSR Loci CHR cM QTLs # traits


01B E06 T7.abl

05 K18T 7 077.abl


05 M18 T7 071.abl

03 G07T 7 028.abl
07 D17T 7 073.abl

10 C02 T7 009.abl

05 K16 T7 062.abl

01C J19 T7.abl
11 H20 T7 076.abl
10 K19 T7 078.abl


06 B16 T7 050.ab1


03 C05T 7 025.abl
06 D13 T7 057.abl
08 D07 T7 026.abl


07 P19 T7 080.ab1
02 D19 T7 074.abl
06 J09 T7 037.abl
02 N02 T7 007.abl
09 M23 T7 088.abl

06 M03 T7 008.abl

05 C16 T7 058.abl


Casein kinase

Serine/threonine protein kinase


Calcium-dependent protein kinase

COP9 signalosome complex sub 2
Vacuolar membrane ATPase sub G

Ubiquitin-conjugating enzyme

Cullin 1

26S proteasome regulatory particle
26S proteasome regulatory particle
RCI2B (Low temp/salt responsive)


Glutathione peroxidase


Glycolate oxidate
Aldose reductase
Brassinosteroid signalling positive regulator-related


Ras-related GTP binding protein
Dihydroflavonol-4-reductase DFR1
Dihydroflavonol-4-reductase DFR1
Diadenosine 5',5"'-P1,P4-tetraphosphate hydrolase
Senescence-related proteins

Myotubularin

Ribosomal protein L10
Ribosomal protein L10; nucleoside hydrolase
Ribosomal protein L10; nucleoside hydrolase
Ribosomal protein L10


RM3360
RM1183, RM7643
RM6277, RM7466, RM8081
RM1220, RM6167, RM6651, RM8077
RM1356
RM1342, RM5305
RM4266, RM6013
RM572, RM8045, RM8046
RM1168, RM3212, RM5833, RM8248
RM3217, RM5503
RM186, RM6329
RM124, RM3332, RM3333
RM6864
RM592
RM5454, RM6024
RM5454, RM6024
RM3627
RM1024,RM5374
RM1209,RM5455
RM6366
RM1367
RM124, RM3332, RM3333
RM234
RM3295, RM3759, RM7084
RM151,RM118
RM3187
RM5508
RM2857, RM5946
RM2256
RM25, RM4085, RM5432
RM124, RM3332, RM3333
RM3917
RM409, RM5122, RM3700
RM3120, RM3496
RM281, RM3155, RM7400
RM3917
RM3120,RM3496
RM281, RM3155, RM7400
RM477, RM5545


References
Babu, R.C., B.D. Nguyen, et al. 2003. Genetic analysis of drought resistance in rice by
molecular markers: Association between secondary traits and field performance. Crop
Science 43: 1457-69.
Kathiresan, A., H. Lafitte, J. Chen, J. Bennett. 2004. Expression Microarrays and their
Application in Drought Stress Research. Field Crops Research. In press.
Lafitte, H., A. Price, and B. Courtois. 2004. Yield response to water deficit in an upland rice
mapping population: associations among traits and genetic markers. Theoretical and
Applied Genetics. In press.


* 46 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004


27.3
127.3
28.9
37.4
82.5
118
7.9
52.4
121
100.7
127.4
128.8
22.5
26
67.5
67.5
54.3
12.5
99.3
110.6
110.9
128.8
93.9
103
28.4
73.2
81.05
116.2
49.7
35.7
128.8
145.3
55.3
119.6
119.9
145.3
119.6
119.9
120.4


11
8
0
8
0
15
0
28
5,11,24
0
6
8
23
10
15
15
5
6,20
0
0
8
8
12
2,5
0
0
4
21
0
26
6
0
7
4
5
0
4
5
0











Monitoring the transcriptome changes of 14013 rice

unigenes in response to drought by cDNA microarray


SANJAY K. KATIYAR1' 2, LEKHA T. PAZHAMALA1' 2, VALERIY A. POROYKO1, AND HANS J. BOHNERT1

1 Department of Plant Biology and Crop Sciences, University of Illinois, Urbana 61801, USA
2 Department of Biotechnology, Indira Gandhi Agricultural University, Raipur 492 006, India


Introduction
Environmental stresses, such as drought, high
salinity, and low temperature, have adverse effects on
plant growth and seed production. Plants have
evolved a number of mechanisms to cope with
different abiotic stresses. One important step in the
control of the stress responses appears to be the
transcriptional activation or repression of genes.
Expression of many genes has been demonstrated to
be induced by these stresses, including those
encoding transcription factors; some have been
identified have been shown to be essential for stress
tolerance. Transcriptome analyses using microarray
technology is very powerful and useful tool in
identifying several genes that are induced by
dehydration stress and these genes can further be
classified into different functional groups (Katiyar et
al., 2003, 2004). We describe here the assembly of
EST-based microarrays, which include transcripts
from 14,103 rice unigenes, predominantly from
drought and salt stressed rice plants, leaves, and
roots. These microarrays were used to examine global
transcript abundance changes of rice to drought
stress. The results (i) provide a detailed
characterization of the changes in transcript
abundance following drought shock treatment, and
(ii) support the concept of a succession of gene
expression changes that fit into a logical framework
of sensing, signaling and response networks, and
identify numbers of genes that are correlated with the
drought shock response. Identification of a
succession of gene expression changes begins to trace
the underlying regulatory networks of drought
responsive genes and should allow us to define a
response hierarchy that reflects mechanisms of
tolerance or avoidance of drought stress.


Method
To identify the drought responsive transcripts during
vegetative growth, expression profiles were established
for different rice cultivars using microarrays containing
14,013 rice unigenes, mostly from cDNAs libraries of
plants challenged by abiotic stresses, harvested at
different developmental stages. The behavior of the
shoot transcriptome was monitored in a time course
experiment. For selected transcripts, behavior was also
tested by real-time RT PCR.


Results
The behavior of the shoot transcriptome was
monitored in a time course from 3 h to 9 h after
imposition of drought stress (shock by removing
water completely). The transcript abundance of
hundreds of genes was changed after drought shock
treatments of different durations. The analyses



Metabolism &Energy



Unknown
Transcription

Protein synthesis
Protein folding & modification "" '


Transposons

Figure 1. Global changes in transcript expression (> 2.5 folds) in rice
after drought treatment.


II. Gene Discovery and Novel Approaches 47 *










identified hundreds of transcripts, significantly
induced and repressed for the gene expression at
various time points. For selected transcripts, behavior
was also tested by real-time RT-PCR. Categorization of
drought-regulated transcripts and clustering revealed a
structured response of cellular and biochemical
activities characterized by functions in metabolism,
energy, transcription, protein synthesis, defense and
cell rescue, transport facilitation, and signal
transduction pathways.


Conclusions
The massive analysis of gene expression at genome
level will accelerate rice research, not only for the
understanding of gene networks, but also for the
development of new varieties with novel agronomic
traits for water-limited environments.



References
Katiyar, et al. 2003. Biotechnology for drought tolerance in rice: lessons from QTLs to high-
throughput gene expression analysis. Third workshop on drought tolerance in rice. UAS,
Bangalore, India 22-25th July' 2003.
Katiyar, et al. 2004. Gene discovery by functional genomics: an approach to identify genes,
functions and inter-relationships for drought stress tolerance in rice. IX National Rice
Biotechnology Network Meeting, National Agriculture Science Complex, IARI, New
Delhi-12 April 15-17, 2004.


* 48 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004











Molecular responses to dehydration and salinity

in rice: Differences and cross-talk between two

stress signaling pathways

SANJAY K. KATIYAR1' 2, VALERIY A. POROYKO1, AND HANS J. BOHNERT1

1 Department of Plant Biology and Crop Sciences, University of Illinois, Urbana 61801, USA
2 Department of Biotechnology, Indira Gandhi Agricultural University, Raipur 492 006, India


Introduction
Drought and salt loading are environmental
conditions that cause adverse effects on the growth of
rice plants and productivity. Plants respond to these
stresses at molecular and cellular levels, as well as the
physiological level. Expression of a variety of genes
has been demonstrated to be induced by these
stresses. The products of these genes are thought to
function not only in stress tolerance but also in the
regulation of gene expression and signal transduction
in stress response. Recently, cDNA microarray
analysis has been developed for quantitative global
analysis of expression profiles. Microarray technology
is a powerful tool for identifying genes induced by
environmental stimuli or stress and for analyzing
their expression profiles in response to environmental
signals. This technology is also useful in identifying
target genes for stress-related transcription factors,
opening a way to analyze gene networks in abiotic
stress responses (Shinozaki et al., 2003). Salt and
drought stress signal transduction consists of ionic
and osmotic homeostasis signaling pathways,
detoxification response pathways and pathways for
growth regulation (Zhu, 2002). Salt and drought stress
signaling has largely remained a mystery until
recently. Now the molecular identities of some
signaling elements have been identified. The
challenge in the near future remains to identify more
signaling elements. Once, more components are
known, signaling specificities and cross talks can be
properly addressed (Zhu, 2002). We prepared a rice
cDNA microarray containing 14,013 rice unigenes and
analyzed the expression profiles of these genes under
salt and drought stress and identified hundreds of
genes responding to each stress including several
transcription factors. A significant number of


drought-inducible genes are also induced by high
salinity treatments, indicating the existence of
significant crosstalk among the drought and high
salinity responses.


Methods
To identify the stress (both salt and drought)
responsive transcripts during vegetative growth,
expression profiles were established for different rice
cultivars using microarrays containing 14,013 rice
unigenes, mostly from cDNAs libraries of plants
challenged by abiotic stresses, harvested at different
developmental stages. The behavior of the shoot
transcriptome was monitored in a time course
experiment.


Results
The transcript abundance of hundreds of genes was
changed after drought-, and salt-stress treatments.
The cDNA microarray analysis showed that many
genes were induced after drought and high salinity
stress treatments, and that there is cross-talk
between drought and salinity responses. The gene
products are of two types: the first group includes
proteins that probably function in stress tolerance,
and the second group contains the protein factors
involved in further regulation of signal transduction
and gene expression and probably functions in stress
response. Many stress-inducible genes include those
that encode signaling molecules and transcription
factor genes, suggesting that many transcriptional
regulatory mechanisms exist in stress signal
transduction pathways.


II. Gene Discovery and Novel Approaches 49










References


Our gene expression profiling using a 14K cDNA rice
microarray provides evidence of differences between
dehydration-signaling and salt stress-signaling
cascades, and of cross-talk between them. Functional
analysis of stress-inducible transcription factors
identified in this study should provide more
information on the complex regulatory gene networks
that are involved in response to drought and high
salinity stresses.


Shinozaki, et al. 2003. Curr. Opinion in Plant Biology 6: 410-17.
Zhu. 2002. Annu. Rev. Plant Biol. 53: 247-73.


* 50 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004


Conclusions











The primary studies on gene expression of drought

tolerance or sensitive rice cultivars in different

water conditions


Z.C. Liu, H.H. TONG, H.Y. Liu, L. CHEN, L.J. Luo

Shanghai Agrobiological Gene Center, Shanghai 201106, China

Corresponding author: L.J. Luo; E-mail: lijun@sagc.org.cn


Introduction
Plants respond and adapt to abiotic stresses to acquire
stress tolerance in order to survive (Bray et al., 2000).
The response of plants to abiotic stress involves
hundreds of genes with different functions
(Crookshanks et al., 2001). In Arabidopsis, many stress
inducible genes have been cloned and some of them
have been used to improve the stress tolerance of
plants by gene transfer (Yamaguchi-Shinozaki et al.,
1993; Liu et al., 1998). Drought tolerance related genes
and their function in indica rice have yet to be studied
thoroughly (Dubouzet et al., 2003). It is important to
clone and analysis the functions of drought-inducible
genes in rice, especially in drought tolerant rice, in
order to understand the molecular mechanisms of
drought tolerance and to further improve the drought
tolerance of rice by gene manipulation. In this study, a
primary effort was made to understand the expression
patterns of four rice cultivars in both drought-stressed
and non-stress conditions by using the mRNA
differential display (mRNA DD-PCR) approach.


Materials and methods
Two paddy field rices (Oryza sativa L., indica cv.
Zheng Shan 97B, Zhong 413, drought sensitive) and
two upland rice (Oryza sativa L., japonica cv. Zhong
Han 3, and IRAT 109, drought tolerance) were used in
this study. Plants were grown in the drought
screening house of SAGC. Drought-stress treatment
began at microspore stage and lasted for four weeks,
while the control plants were grown in the nearby
paddy field house. Total RNA was extracted from
leaves as described by Puissant and Houdeline (1991).
Reverse transcription was carried out using AMV
reverse transcriptase as descripted in the Promega's


technical bulletin with slight modifications. Three
different anchored primers HT1M (H means HindIII
site, M may be C, A, or C), abbreviated as AP1-AP3,
were applied in the reverse transcription. Eight
arbitrary primers (as ABP1-ABP8) were designed as
reverse primer for PCR reactions. PCR amplification
of each reverse transcription products was carried out
in combination with one of eight arbitrary primers.
The amplified cDNA products were size fractionated
by a 3.5% denaturing polyacrylamide electrophoresis
(PAGE) using Bio-Rad Sequence Cell. 4 tl of each
PCR sample were incubated with 2 tl of loading dye
(95% formamide, 10 mM EDTA, pH 8.0, 0.01%
bromophenol blue) at 950C for 5 min and then kept
on ice for at least 2 min before immediately loading to
the Sequence cell. Silver staining of the
polyacrylamide gel was carried out to identify the
specific expression pattern.


Results
Amplified transcription products of RNA extracted
from drought-stressed and non-stressed leaves of
rice were clearly visualized from the agarose gel
electrophoresis. A number of drought specific
fragments that were bigger than 500bp were
detected from the primer combinations of AP1/
ABP2, AP1 /ABP6 (Figure 1). Drought tolerance cv.
Zhong Han 3 produced more specific bands than
drought sensitive cv. Zhong 413. After gel extraction
and PCR amplification, three fragments of about
500-700bp were isolated from drought-stressed
leaves of cv. Zhong Han 3 through the combinations
of anchored primer AP1/ ABP2, AP1/ ABP6, AP2
and ABP7. Several other drought-specific fragments
were also isolated but failed to be amplified again in
further PCR.


II. Gene Discovery and Novel Approaches 51 *







































Figure 1. Expression patterns of rice plants cv. Zhonghan 3 and Zhong
413 by mRNA DD-PCR on agarose gel electrophoresis. M 1kb DNA
ladder, lanes 1, 2 were Zhonghan 3 in drought-stressed and non-
stressed condition, lanes 3, 4 were Zhong 413 in drought-stressed and
non-stressed condition.


M I 2 3 4 AM 1 23 4


AP1/AP2 AP1/ABP6


Almost the same expression patterns as in agarose gel
electrophoresis were clearly visualized from the result
of PAGE with silver staining, except that the number
of drought-specifically expressed bands from PAGE
was much more abundant (Figure 2). In the
combination of primer AP 1 and arbitrary primer
ABP1-8 from cv Zhong 413, some 31cDNA fragments
were drought-specific transcription products, while
two fragments disappeared in drought-stressed
conditions. In the combinations of primer AP2/ABP1-
8 and AP3/ABP1 8, drought-specific expression
fragments were 34 and 66, respectively, while
expression fragments disappeared in drought-stressed
conditions were 5 and 3, respectively; the same trend
as in the combination of primer AP1/ABP1-8. The
expression patterns by mRNA differential display for
other cultivars were similar to those of cv. Zhong 413.



Conclusions
The abundant specific expression bands from drought
stressed rice leaves verified that rice plants respond to
drought-stress by altered gene expression. There is
some difference among cultivars in their response
patterns to drought stress. DT rice showed more
expression patterns than drought sensitive rice. These
drought-stress specific cDNA fragments could be
isolated and used to clone drought tolerance related
genes, and then further improve the drought tolerance
of rice by genetic engineering. Both agarose
electrophoresiss and PAGE with silver staining could
be used to investigate the gene expression patterns in
mRNA differential display approach. PAGE could
produce many more differential bands.



References
Bray, E.A., J. Bailey-Serres, E. Weretilnyk. 2000. Responses to abiotic stresses. In: Buchanan
B, Gruissem W., Jones R. (eds.), Biochemistry & Molecular Biology of Plants. American
Society of Plant Physiologists. Pp 1158-76.
Crookshanks, M., et al. 2001. FEBS Letters. 56:123-26.
Dubouzet, J.G., et al. 2003. The Plant Journal. 33: 751-63.
Liu, Q., et al. 1998. The Plant Cell. 10: 1391-1406.
Puissant, C., and L.-M. Houdeline. 1990. BioTechniques8: 148-49.
Yamaguchi-Shinozaki, K., and K. Shinozaki. 1993. PlantPhysiol. 101: 1119-20.


Figure 2. Expression patterns of rice plants cv. Zhong 413 by mRNA
DD-PCR with primers AP3/ABP1-ABP8 on polyacrylamide gel
electrophoresis. Lanes 1, 3, 5, 7, 9, 11, 13, 15 were patterns under
drought-stress; Lanes 2, 4, 6, 8, 10, 12, 14, and 16 were patterns in
normal paddy field condition. Black arrows (66) indicate cDNA
expressed only in drought-stress condition, while white arrows (3)
indicate cDNA disappeared under drought-stress.


* 52 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004











Effects of the brachytic-2 dwarfing gene on maize (Zea

mays) root systems and grain yield under moisture stress


K. MASHINGAIDZE AND E.C. CHINHEMA

ARC-Grain Crops Institute, P. Bag X1251, Potchefstroom 2520, South Africa

Corresponding author: K. Mashingaidze; E-mail: king@igg2.agric.za


Introduction
Drought resistance is a complex of many
morphological, physiological, and biochemical
characteristics. Maize cultivars adapted to marginal
rainfall areas of Zimbabwe should have deep and
dense rooting systems in order to reduce the effects of
frequent and intermittent droughts during the
growing season. Deep and dense rooting systems were
associated with drought resistance in wheat (Cholick
et al., 1977; Hurd and Spratt, 1975) and rice (Chang et
al., 1982; O'Toole, 1982; Yoshida and Hasegawa, 1982).
Dwarf plants were found to have a more extensive and
deeper rooting system than normal height wheat
cultivars (Hurd, 1974; Lupton et al., 1974; Cholick et
al., 1977). On the other hand, Mackay (1973) reported
that root growth occurred in a mirror image manner of
the aerial plant in spring wheat. The recessive
brachytic-2 (br-2) gene reduces plant height without a
corresponding decrease in the size of the major plant
parts of maize (Anderson and Chow, 1963; Scott and
Campbell, 1969). This study was conducted to
investigate the effects of the br-2 gene on maize root
systems and grain yield under moisture stress, in
order to determine whether it can be exploited for
improving drought tolerance in maize.


Methods
Dwarf inbred lines (namely, dw2N3d, dwK64r, dwSC
and dw211DR) were developed by transferring the
brachytic2 gene to elite tall inbred lines (namely, 2N3d,
K64r, SC and 211DR), by six backcrosses. The dwarf
and tall inbred lines were used to develop six pairs of
near-isogenic dwarf and tall single-cross hybrids. For
root studies, the six pairs of near-isogenic tall and
dwarf hybrids were planted in plastic pots (20 cm
diameter and 1 m deep) filled with washed river sand,
and arranged in a randomised complete block design


with four replications. The experiment was carried
out during the hot dry season, and each pot received
500 ml of nutrient solution, five times a week. Root
length, volume and dry mass (DM) were measured
at the mid-silking stage. For field trials, the six pairs
of tall and dwarf hybrids were planted at three sites
for two summer seasons (1993/94 and 1994/95).
Two sites [Makoholi (MES) and Matopos (MTP)
Research Stations] were located in marginal (450-650
mm) rainfall areas, characterized by periodic
seasonal droughts. The third site, University of
Zimbabwe farm (UZ farm), was located in a high
yield potential area characterized by fairly well
distributed high (750-1000 mm) rainfall. The hybrids
were arranged in a randomised complete block
design with three replications, at a population of 53
333 plants ha 1. Compound D (8N: 14P2Os: 7K2O)
fertilizer was applied at 300 kg ha 1 as a basal
dressing at planting. Ammonium nitrate (34.5% N)
was applied as a top dressing at 200 kg ha 1. Plots
were kept weed free.


Results
There were no significant (P>0.05) differences in root
length between near-isogenic hybrid pairs (Table 1).
Two dwarf hybrids, dw211DR x dwSC and dw2N3d
x dwSC, had significantly (P< 0.05) smaller root
volume and less dry mass than their tall
counterparts (Table 1). Dwarf hybrid dw211DR x
dw2N3d had a significantly (P< 0.05) smaller root
volume than its tall counterpart (Table 1). The roots
of all the dwarf hybrids were thinner and more
fibrous than those of the tall hybrids. There were no
significant differences in grain yield between the tall
and dwarf hybrid pairs at both marginal and high
rainfall sites (Table 1).


II. Gene Discovery and Novel Approaches 53 *











Table 1. Mean grain yield and root length, volume and dry mass of six
pairs of near-isogenic tall and dwarf maize hybrids grown in Zimbabwe
during the 1993/94 and 1994/95 summer seasons

Grain Grain
Near-isogenic yield at yield at
hybrid RootDM Rootlength Rootvolume MESandMTP UZ farm
pair (g) (cm) (cm3) (t ha-1) (t ha-1)

2N3d x SC 275.8a# 106.1a 428.8a 2.15a 4.84a
dw2N3d xdwSC 201.3b 101.0a 316.3b 1.77a 4.60a

211DR x 2N3d 247.0a 103.5a 498.3a 2.16a 6.93a
dw211DR x dw2N3d 222.3a 97.2a 371.3b 2.14a 7.54a

2N3d x K64r 208.8a 109.5a 402.0a 2.28a 7.54a
dw2N3d x dwK64r 229.5a 104.2a 403.8a 2.25a 6.69a

211DRxSC 326.8a 101.2a 542.5a 1.95a 6.08a
dw211DR x dwSC 204.5b 111.0a 423.8b 1.43a 5.81a

SC x K64r 242.8a 105.8a 428.5a 2.05a 6.43a
dwSC x dwK64r 257.3a 104.5a 406.0a 2.01a 7.06a

211DRx K64r 163.0a 113.0a 392.0a 2.53a 7.61a
dw211DR x dwK64r 180.1a 100.2a 321.0a 1.99a 6.76a

SEM' 24.3 5.1 32.1 0.24 0.69
LSD( 5) 69.8 15.0 92.3 0.70 2.02
CV(%) 21.1 9.8 15.6 24.00 18.02

# For each characteristic of a specific near-isogenic hybrid pair, means followed by the same letter
are not significantly different at the 5% level.
SEM = standard error of the mean.


Conclusions

The br gene had no effect on root length but it
induced the maize root system to become finer and
more fibrous irrespective of the genetic background
to which the gene was incorporated. A fine and
fibrous rooting system is expected to be more
efficient in extracting immobile nutrients and water
because there will be more root surface area in
contact with the soil. Root volume and dry mass
were either reduced or not affected depending on the
genetic background to which the gene was
incorporated. The br 2gene did not negatively affect
any root characteristics of dwarf hybrids where
dwK64r was used as one of the parents. The
superiority in root volume and dry mass of some tall
hybrids was a result of thicker roots. The br gene
did not cause the dwarf hybrids to be better or less
adapted to marginal or high rainfall areas than tall
hybrids. Thus, dwarf hybrids can be developed for
both marginal and high rainfall areas.



References
Anderson, J.C., and RN. Chow. 1963. Crop Science 3: 111-13.
Chang, T.T., et al.1982. Drought Resistance in Crops with Emphasis on Rice. Manila,
Philippines: IRRI. pp. 217-44.
Cholick, EA., et al. 1977. Crop Science 17: 637-39.
Hurd, E.A. 1974. Agricultural Meteorologyl4: 39-55.Hurd, E.A., and E.D. Spratt. 1975.
PhysiologicalAspects of Dryland Farming. New York: IBH. pp 167-235.
Lupton, FG.H., etal. 1974. Annals of Applied Biology 77: 129-44.
Mackay, J. 1973. Proc. 41 Int. Wheat Genetic Symposium. University of Missouri. pp 827-42.
O'Toole, J.C. 1982. Drought Resistance in Crops with Emphasis on Rice. Drought Resistance
in Crops with Emphasis on Rice. Manila, Philippines: IRRI. pp. 195-213.
Scott, G.E., and C.M. Campbell. 1969. Crop Science 9: 293-95.
Yoshida, S., and S. Hasegawa. 1982. Drought Resistance in Crops with Emphasis on Rice.
Manila, Philippines: IRRI. Pp 97-114.


* 54 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004










Towards the improvement of abiotic stress tolerance in

maize using genes isolated from the monocotyledonous

resurrection plant Xerophyta viscosa

SAGADEVAN G. MUNDREE AND JENNIFER A. THOMSON

Department of Molecular and Cell Biology, University of Cape Town, Private Bag, Rondebosch, 7701, South Africa


Corresponding author: Sagadevan G. Mundree; E-mail: mundree@science.uct.ac.za


Water is a major limiting factor in world agriculture. In
general, most crop plants are highly sensitive to even a
mild dehydration stress. There are, however, a few
genera of plants unique to southern Africa, called
"resurrection plants," which can tolerate extreme water
loss or desiccation. We have used Xerophyta viscosa, a
representative of the monocotyledonous resurrection
plants, to isolate genes that are associated with abiotic
stress tolerance. Several genes that are differentially
expressed, and that confer functional sufficiency to
osmotically-stressed Escherichia coli (srl::TnlO) are being
studied at the molecular and biochemical levels. Some
of these genes include ones that code for a novel
antioxidant, XVPerl, a subunit c"-like protein of the


vacuolar H'-adenosine triphosphatase, VcVHA-c", a
galactinol synthase, XVGols, an aldose reductase,
ALDRXV4, a cell membrane binding protein, XVSAP1
and a transcription factor, DREB1A. To determine the
effects of the expression of these genes in monocots,
they are first introduced into our model system,
Digitaria sanguinalis, for which we have developed an
efficient transformation system. Thereafter, if results are
positive, the genes are transformed into crops such as
maize (Zea mays). To do the same for dicots we first
introduce them into Arabidopsis thaliana and Nicotiana
tobacum. This presentation will focus on the latest
developments towards the improvement of abiotic
stress tolerance using some of the above genes isolated
from X. viscosa.


II. Gene Discovery and Novel Approaches 55 *










Gene mining of African rice germplasm (Oryza

glaberrima and Oryza sativa) to improve drought

resistance in rainfed production systems for resource

poor farmers of Africa

MARIE-NOELLE NDJIONDJOP AND HOWARD GRIDLEY
WARDA The Africa Rice Center, Samanko Research Station, ADRAO/ICRISAT BP 320, Bamako, Mali

Corresponding author: Marie-Noelle Ndjiondjop; E-mail: m.ndjiondjop@cgiar.org


Rice has been cultivated in West and Central Africa for
centuries and is now considered as one of the region's
staple foods. In these regions, drought is one of the
major constraints as it severely depresses yield in
upland and rainfed lowlands, where the majority of
producers are resource-poor farmers. Drought
resistance, however, is a complex trait, controlled by
the interaction of many genes, as it involves several
physiological, phenological, and morphological
mechanisms, and because of the polygenic nature of
resistance. Consequently, conventional breeding for
drought resistance in Africa has had limited success.
DNA markers and genetic mapping are expected to
provide impetus, not only in gaining a better
understanding of the traits associated with drought,
but also contribute to enhanced selection efficiency.

The project seeks to (i) characterize drought in
different environments and identify the most
important traits associated with drought tolerance; (ii)
select and characterize sources of drought resistance
for genetic mapping and QTL analysis; and (iii)
develop advanced lines combing drought resistance
with heavy yield and agronomic and quality


characteristics acceptable to farmers and consumers.
To achieve these objectives, the project will exploit a
core germplasm pool of (i) drought resistant O.
glaberrima accessions, collected and screened in Mali
by the Institut d'Economie Rurale (IER); (ii) drought
tolerant interspecific breeding lines developed by
WARDA from crosses between 0. sativa and 0.
glaberrima; and (iii) a range of traditional O. glaberrima
and 0. sativa accessions from WARDA's gene bank.
Confirmed sources of resistance amongst this core
germplasm will be crossed with elite, but drought
susceptible, 0. sativa lines to develop interspecific and
intraspecific populations segregating for drought
resistance. These populations will be phenotyped in
replicated field trials in different environments in
Mali and Nigeria. QTLs analysis will be performed to
identify across environment, drought-improving
alleles for future breeding purposes. In other
populations, selection will be conducted to generate
agronomically superior, drought resistant lines.


* 56 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004











Structural-function relationships in the middle region of

S. cerevisiae HSP104 protein


J. NIETO-SOTELO, F. ZARATE, L.M. MARTINEZ, AND B.L. ARROYO

Institute de Biotecnologia, Universidad Nacional Autonoma de Mexico, 62210 Cuernavaca, Mor., Mexico

Corresponding author: J. Nieto-Sotelo; E-mail: jorge@ibt.unam.mx


Introduction
High temperatures affect growth and development,
causing the accumulation of aggregated proteins
within the cell. Hspl00/ClpB chaperones facilitate the
resolubilization of aggregates in conjunction with
Hsp70 and Hsp40 (Glover and Lindquist, 1998).
Hspl00/ClpB are found in bacteria, protozoa, fungi,
and plants where their expression is inducible by heat
stress (Schirmer et al., 1995). In yeast, Arabidopsis, and
maize, Hspl00/ClpB play major roles in the
acquisition of thermotolerance, i.e., the ability to
survive very high temperatures following acclimation
to mild heat shock treatments (Sanchez and Lindquist,
1990; Queitsch et al., 2000; Nieto-Sotelo et al., 2002).
Expression of HsplOl in maize causes a reduction in
the growth rate of primary roots (Nieto-Sotelo et al.,
2002). Both the primary and secondary structure of the
middle region of Hspl00/ClpB proteins have been
conserved during evolution (Nieto-Sotelo et al., 1999).
The length of the middle region is quite different or
absent in other members of the Clp family (ClpA,
ClpC, ClpD), whose functions seem to be unrelated to
those of ClpB (Schirmer et al., 1995). The middle
region has the propensity to form a coiled-coil (Nieto
Sotelo et al., 1999). To understand the mechanism of
action of Hspl00/ClpB, we studied the structure
function relationship within their middle region.


Methods
Homozygous maize mutants hspl01 m4::Mul and
hspl01-m5::Mul were grown in a greenhouse where
maximal daily temperatures reached close to 400C).
Their growth was compared relative to their
corresponding wt sibling lines (Figure 1). Analysis of
cell proliferation activity was made by FACS analysis
of nuclei from leaf samples. Analyses of coiled-coils
were made with the COILS program. Site-directed


mutagenesis of HSP104 middle region was used to
obtain amino acid substitutions that changed the
hydrophobic face of the amphipatic helices 1, 2 or 3
(hspl04 ml, hspl04-mll, and hspl04mIIll, respectively).
Both wt and HSP104 mutants were used to transform
Dhspl04 cells to evaluate their function in vivo by
means of induced thermotolerance assays. Partially
purified HSP104 protein preparations from both wt
and each mutant protein were tested for their ability to
form hexamers on an ATP-dependent fashion by size
exclusion chromatography Protein conformations
were evaluated by protease sensitivity assays and the
western blot procedure using an anti-HSP104 specific
antibody. The T-COFFE, SWISS MODEL, and Deep
View Swiss-PdbViewer programs were used to model
the structure of HSP104 middle region using the
known structures of T thermophilus ClpB and E. coli
Hsc20 co-chaperone proteins as templates.


140

120

100

80

..60


hsp10l-m4 hsp101-m5


Figure 1. Height of two-month old maize plants grown in a greenhouse
where daily maximum temperatures fluctuated between 36 and 40TC.
Values between wt an mutants are statistically different at P=0.05.


II. Gene Discovery and Novel Approaches 0 57 *











Results and discussion


Hspl01 negatively affects growth of stems and leaves
in adult plants. In leaves, this effect is at least due in
part to a decrease in cell proliferation. Thus, maize
Hspl01 plays an important role in restraining growth
under moderately high temperatures, suggesting that
slow growth rates are more convenient to achieve a
stress tolerant state. Because a full-length maize
cDNA clone encoding ZmHSP1O1 did not
complement the thermotolerant deficient phenotype
of the yeast Dhspl04 mutant, we studied HSP104 of
Saccharomices cerevisiae per se. Both in yeast Hsp 104
and plant Hspl01 the middle region seems to form 4
amphipathic a-helices. Site-directed mutagenesis of
the middle region of yeast Hspl04 showed that this
domain is very important for function. Mutations that
changed the amphipathic character of helices 2 and 3,
but not those of helix 1, caused the loss of function of
Hspl04 (Figure 2) suggesting that helices 2 and 3 play
an important role in the maintenance of a coiled-coil
structure. Moreover, these mutations abolished
hexamer formation and changed the conformation of
the complex. Our experimental data are consistent
with a homology-model for yeast Hspl04 middle
region resembling the C-terminal domain of E. coli
Hsc20 co-chaperone (Cupp-Vickery and Vickery,
2000) and inconsistent with a model based on the
middle region of the recently published structure of
T thermophilus ClpB (Lee et al., 2003). Helix 2 and
helix 4 seem to form a parallel coiled-coil, whereas
helix 3 interacts with both helix 2 and 4 through their
hydrophilic face.


1


0.1 -----------------------


0.01
0 10 20 30 40 50
Time (min)
* Dhsp104 [e. vector] 0 Dhsp104 [pHSP104] A Dhsp104 [phsp104-mll]


0 wt [e. vector]


O Dhsp104 [phsp104-ml] A Dhsp104 [phsp104-mlll]


Figure 2. Induced thermotolerance assay of Dhspl04 mutant transformed
with plasmids encoding wt or middle-region mutant versions of HSP104.


References
Cupp-Vickery, J.R. and Vickery, L.E. 2000. Mol. Biol. 304, 835-845.
Glover, J.R., and Lindquist, S. 1998. Cell 94, 73-82.
Lee, S. etal, 2003. Cell 11775, 229-240.
Nieto-Sotelo, J. etal. 2002. Plant Cell 74, 7621-1633.
Nieto-Sotelo, J. etal. 1999. Gene 230, 187-195.
Queitsch, C., etal. 2000. Plant Cell 72, 479-492.
Sanchez, Y., and Lindquist, S. 1990. Science 248, 777112-7774.
Schirmer, E.C., etal. 1995. TIBS 2, 289-295.


Acknowledgements
CONACYT grants: 25303-N and 2002-C01-39935 to J.N.-S.


* 58 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004











Differential gene expression in cell cultures and plants of

chili pepper (Capsicum annuum I.) under water stress


N. OCHOA-ALEJO AND Y.M. CAMACHO-VILLASANA

Plant Genetic Engineering Department, Biotechnology and Plant Genetic Engineering Unit, Center for Research
and Advanced Studies (CINVESTAV, Irapuato Unit), National Polytechnic Institute, Km 9.6 Libramiento Norte
Carretera Irapuato-Leon, Apartado Postal 629; 36500-Irapuato, Gto., Mexico

Corresponding author: N. Ochoa-Alejo; E-mail: nochoa@ira.cinvestau.mx


Introduction
Chili pepper (Capsicum spp.) is a very important
horticultural crop in Mexico and worldwide (FAO,
2004). Chili pepper is very sensitive to water stress
and different physiological, biochemical, molecular,
and genetic approaches are necessary to increase its
resistance to drought. In order to study drought
tolerance mechanisms at the cell level, different cell
lines of chili pepper resistant to drought imposed by
the presence of polyethylene glycol (PEG) in the
culture medium were selected (Santos-Diaz and
Ochoa-Alejo, 1994). These cell lines exhibited more
negative osmotic potential, and accumulated higher
levels of proline, glycine betaine, and potassium than
the non-selected cell cultures. Since metabolic
adjustment to drought is very often accompanied by
gene expression changes, a comparative gene
expression study was carried out by differential
display with a PEG resistant cell line (T7) grown in
the absence (PO) or in the presence of 15% PEG (P15),
and with a PEG sensitive cell line (ST).
Approximately 124 cDNA fragments were
differentially expressed in the cell cultures as
revealed by differential display (Verastegui-Pena,
1999). Initially, eight cDNA fragments were cloned
for expression studies and sequenced. Comparison of
sequences of cDNA fragments with those of GenBank
(NCBI) revealed that ODE1 and ODE2 (Osmotically
Differentially Expressed) cDNAs exhibited high
homology with an ELIP (Early Light Induced
Protein) and with an ARF (Auxin Responsive Factor),
respectively. Further analysis of these two cDNAs
and 23 additional cloned cDNA fragments were
carried out in this work.


Methods


cDNA fragments were cloned in the PCR TOPO 2.1
vector (Invitrogen). After restriction with EcoR1, the
cDNAs were radiolabeled with 32P-dCTP and used as
probes for Northern analysis. Cell cultures were
maintained in the MS liquid medium (Murashige and
Skoog, 1962) as described by Santos-Diaz and Ochoa
Alejo (1994). Biomass of the cell cultures collected by
filtration on day 7 after subculture, and tissues of chili
pepper plants subjected to water withholding for 0, 3,
6, 9, and 12 days were used for total RNA extraction
according to the protocol reported by Camacho
Villasana et al. (2002). Northern analysis was
performed as described by Sambrook et al. (1989). In
order to try to get the whole encoding regions of the
differentially expressed cDNAs, the 5' RACE system
(Promega) was used.


Results
Six of 23 cDNA cloned fragments, namely ODE11,
ODE12, ODE13, ODE14, ODE15, and ODE16 showed
differential expression in the cell suspension lines by
Northern analysis (Figure 1). Higher expression of
these cDNA fragments was observed in the PEG
resistant cell line T7 grown in 0% PEG (PO) and only
ODE11 exhibited higher expression in 15% PEG (P15).
Sequence analysis of the cDNA fragments revealed no
homology with known sequences from the GenBank,
except for ODE12 that showed 93% nucleotide
homology with the mitochondria rRNA 26S of maize,
sugarbeet and rice. In the case of expression analysis
in tissues from water-stressed chili pepper plants,
only four cDNA fragments were tested (ODE1, ODE2,
ODE9, and ODE14) and a different pattern of
expression was observed as compared to cell cultures.
It was observed also a tissue-dependent differential


II. Gene Discovery and Novel Approaches 59 *










ST P1 P15 ST PC P15


it~l~


00E11


ST PO P11


001E14


oe12


Bta13


ST PO pis


-"


ODEi5


ST PO P15

16m:00]ti


Figure 1. Northern analysis of chili pepper cell cultures using six different
cDNAs (ODE11 to ODE16) as probes. ST, PEG-sensitive cell line cultured in
0% PEG; PO, PEG-resistant cell line T7 grown in 0% PEG; P15, cell line T7
in 15% PEG: 28s, ribosomal RNA (load control).
Supported by CONACYT (Mexico), project 35427.


BT PO P15


* 60 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004


expression of these fragments. Amplification of
the 5' end (RACE) was tried with ODE1, ODE2,
ODE9, and ODE14 and only with ODE14 a 936
bp cDNA was amplified and its nucleotide
sequence showed 81 to 84% homology with a
ketoacyl-CoA thiolase from five different plant
species.


Conclusions
A comparative gene expression study was carried
out by differential display with a PEG resistant
cell line T7 and with a PEG sensitive cell line.
Approximately 124 cDNA fragments were
differentially expressed. After cloning and
northern blot analysis 6 cDNA fragments were
selected and sequenced. The expression of these
fragments was also studied in tissues of chili
pepper plants subjected to water stress and
differential expression was observed. One of the
cDNAs was amplified by 5' RACE and showed
high homology with a ketoacyl-CoA thiolase.


References
Camacho-Villasana, Y.M., etal. 2002. Plant Molecular Biology Reporter20: 407-14.
FAO. 2004. http://faostat.fao.org/.
Murashige, T., and F. Skoog 1962. Physiologia Plantarum 15: 473-97.
Sambrook, J., et al. 1989. New York: Cold Spring Harbor Laboratory Press.
Santos-Diaz, M.S., and N. Ochoa-Alejo. 1994. Plant Cell, issue and Organ Culture
37: 1-8.
Verastegui-Pefa, Y.M. 1999. Master degree thesis. CINVESTAV-Irapuato Un it.











Development of functional markers for drought

tolerance in rice: Identification and validation of

candidate genes and SNPs

ARJULA R. REDDY1, A. CHANDRA SEKHAR1, P.R. BABU1, G. MARKANDEYA1, L. SIVARAMA PRASAD1, L.VIJAYA BHASKAR
REDDY1, N. SEETHARAMA2, N.P. SAXENA3, A. MADANA MOHAN REDDY1, V. KIRAN KUMAR1, AND B. CHANDRA SHEKHAR 1

1 Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500 046 A.P., India
2 National Research Center for Sorghum, Rajendranagar, Hyderabad, A.P., India
3 International Crop Research Institute for Semi Arid Tropics (ICRISAT), Patancheru, Hyderabad, A.P., India

Corresponding author: Arjula R. Reddy; E-mail: arjulsl@uohyd.ernet.in


Introduction
Drought is a major constraint to rice productivity.
Genetic improvement of drought tolerance by
conventional breeding has been rather slow due to
lack of proper phenotypic selection criteria, high
genotype X environment interaction, and low
heritability. Recent advances in molecular mapping
and functional genomics contributed significantly to
the identification of QTLs and genes associated with
drought tolerance. However, lack of precise
information on genes that are directly associated with
drought tolerance phenomenon in rice is still a major
limitation. Developing indepth coverage of rice coding
sequences through large-scale EST generation is
beginning to play a major role in quality annotation.
These resources are of potential use in gene discovery,
comparative genomic analysis, and SNP identification.
Furthermore, with near completion of a finished high
quality rice genome sequence, these tools will be
routinely used for dissecting complex traits such as
drought tolerance. Towards this end, we have
generated large scale ESTs from a cDNA library made
from drought stressed indica rice (N22) seedlings and
have identified a large number of putative candidate
genes and mapped to genetically anchored BAC/PAC
clones. A number of SSRs and QTLs associated with
drought tolerance have been identified using the rice
mapping population CT9993 X IR62266, DHLs.
Microarray experiments and validation of candidate
gene functions through transgenics are in progress.


Materials and methods


ESTs and candidate gene discovery
We generated -6,000 Expressed Sequence Tags (ESTs)
(accession Nos BI305180 to BI306756; BU 672765 to BU
673915 & CB964418 to CB967504) from a normalized
cDNA library constructed from drought stressed
Oryza sativa cv. Nagina22 (Reddy et al., 2002).
Standard sequence processing tools PHRED, Phrap,
and Crossmatch were used with Codoncode
InterPhace. Homology search was done against non
redundant (nr) nucleotide and protein sequence
databases using BLASTN 2.2.2 and BLASTX 2.2.2
versions of the BLAST programs (Altschul et al., 1997)
through BLAST 2.0 network client software with the
DNA tools interface (http://www.crc.dk/dnatools).
The BLASTN program was also used to identify rice
EST hits on High Throughput Genomic Sequences
(HTGS) and the Chinese WGS (whole genome
shotgun contigs) draft sequence of indica rice genome
available in GenBank. The results of the BLAST
analysis were manually checked for similarity in the
aligned region. Genscan, GeneMark HMM, RiceHMM,
Glimmer R, FGENESH, Rice Genome Automated
Annotation System (Rice GAAS) were used for
accurate gene prediction. Plant CARE and PLACE
Databases were utilized for identifying cis acting
elements in the promoter regions.

The ESTs associated with stress response were
identified from multiple sources based on the
compiled list of stress regulated genes documented or
presumed to be relevant to abiotic stress tolerance in
more than one plant species (http://stress
genomics.org/stress.fls/expression/expression.html).


II. Gene Discovery and Novel Approaches 61 *










Further, it is based on the microarray expression
profiles of possible candidate gene sequences, which
include 650 from Arabidopsis (Seki et al., 2001, 2002;
Kreps et al., 2002), 150 from barley (Ozturk et al., 2002)
and 100 from rice (Matsumura et al., 1999; Kawasaki et
al., 2001; Rabbani et al., 2003). All the stress responsive
gene sequences were retrieved from the above studies
and a local database was constructed and utilized for
BLAST analysis using TBLASTX with E-value >le-20.
The identified putative candidate ESTs were mapped
onto genetically anchored BAC/PAC clones to
identify the possible candidate genes at the QTL
associated with drought tolerance (Babu et al., 2003;
Price et al., 2002; Price and Courtois, 2000; Zhang et
al., 2001).

Currently we are analyzing the expression profiles of
these ESTs using microarrays (in collaboration with
University of Georgia). Poly A+ RNA from various
stages of field drought stressed N22 plants has been
prepared for this study using a rain out shelter with
regulated water supply.

SNP detection and analysis
Allelic variations in protein kinases, phosphatases,
and other genes associated with stress response signal
transduction pathways are identified using ESTs.
BAC/PAC clones sequences of Nipponbare, indica
WGS scaffold sequences of Guangluai 4 and the
Unigene clusters of corresponding hits have been
obtained using NCBI BLASTN tool. We developed a
new cvCluster vl.0 script for clustering ESTs using
xsact (Malde et al., 2003) engine of suffix array
construction. Assembling was done based on cultivar
specific data available in public domain to score for
SNPs and to discern true allelic variation from that of
sequencing errors, and to generate more informative
SNP data. These sub clusters were evaluated using
Polybayes (Marth et al., 1999) SNP scoring program.

SSR marker addition, map construction and QTL
identification
A set of 250 rice microsatellite primer pairs (Research
Genetics Inc USA.) were used to amplify the simple
sequence length polymorphic (SSLPs) DNA between
parental lines according to Chen et al., (1997).
Polymorphic primer pairs were used to amplify SSLPs
in complete DHL population, CT9993/IR62266.
Markers were assigned to 12 rice linkage groups at
LOD >3 to anchor markers (Nguyen et al., 2002) using
MAPMAKER/EXE V.3 program. The assigned makers
were ordered using three-point analysis at LOD 3.


* 62 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004


Ripple command was used to verify the order of
markers on each chromosome. This revised genetic
linkage map of was used to identify QTLs controlling
plant morphological (leaf), phenological and yield
related characters under control and field drought
stress conditions using MAPMAKER/QTL program.
The putative QTLs were declared significant when
LOD score was > 2.3.

Rice-sorghum syntenic mapping was carried out with
127 ESTs as RFLP probes. Test genotypes of rice (6),
sorghum (6), pearl millet (2) finger millet (2) and
maize (1) were screened for RFLPs. Population
screening was done in rice mapping population
(CT9993 X IR62266, DHLs) and sorghum mapping
population (N13 X E36-1, RILs).

Construction ofplant expression vectors
The cDNA clone encoding rice anthocyanidin
synthase (Ans) was cloned in sense and antisense,
downstream to a constitutive promoter in the plant
expression vector pE1806 that is known to enhance
gene expression by about 150-fold. The construct was
mobilized into Agrobacterium tumifaciens strain
LBA4404 through electroporation. The scutellar callus
derived from rice line Nootripathu was transformed
with LBA4404 harboring pE1806+Ans and the
transgenic plants were regenerated with hygromycin
selection.


Results
Annotation of high-quality ESTs through homology
search in the NCBI nr nucleotide and protein databases,
using BLASTN and B LASTX programs resulted in the
identification of putative genes (78% of the ESTs) and
novel genes (22%), which have no significant homology
in nr sequence database and dbEST division of
GenBank. Homology search of non-redundant ESTs in
rice dbEST division showed 1157 hits and 912 ESTs do
not have significant similarity to rice ESTs. These 912
ESTs constitute a novel 3' sequence resource for
accurate gene annotation. The functional distribution of
the identified putative stress responsive genes
represented those belonging to diverse pathways
associated with stress adaptation process (Figure 1).
Further, a number of stress responsive genes were
localized to the described QTL regions

Table 1 shows that a total of 1,620 ESTs representing
protein kinases, phosphatases, and other signal
transduction pathways genes were grouped in to 39










clusters and 157 cultivar specific contigs using
cvClusterl.0. A total of 60 SNPs were identified using
Polybayes with the probability greater than 0.9. These
include 29 transitions, 22 transversions, and 9 indels.
Screening of non-synonymous substitution and the
development of an automated pipeline for high
throughput SNP detection and analysis are in progress

New SSR markers and QTLs
A total of 54 new microsatellite markers and three
EST PCR markers were added to the existing genetic
linkage maps and subsequently used for QTL
identification. The map length has increased covering


0I Celular metabolism

EJ Cell structure

El Detoxification

E Hormone response

SHeat shock proteins

SOsmotic protectanats


4% 6%

1 Protein synthesis

SSecondary metabolism

'1 Signal transduction

STranscription factors

J Transport

1 Protein degradation


[] Protein kinases and phosphatase 0 Other functions


Pathogen response
SPhotosynthesis


E Unclassified


Figure 1. Functional distribution of putative stress response genes (580).


1,978 cM in length on the basis of Kosambi function with
an average distance of 5.8 cM between adjacent markers.
A total of 67 QTLs with a LOD score of> 2.3 were
identified for leaf, phenological and yield related traits
under control and field drought conditions (Table 2). The
number of QTLs identified for each trait varied from one
to nine with percent variance explaining 6.8 to 19.2.
Major QTLs for leaf, phenological and yield traits under
field drought stress were localized to a genetic region on
Chromosome 1 and 4. Of the 127 rice ESTs screened for
synteny, 41 showed polymorphism in rice and 19 in
sorghum. Work is in progress to map informative EST
RFLPs in the mapping populations of rice and sorghum.


Table 1. Putative candidate genes within drought tolerance QTL regions
uncovered from mapping experiments

Putative function Nearest marker QTL location

Signaling;
Mitogen-activated protein kinase homolog MMK 2 Chromosome 10
Putative receptor-like protein kinase
Small GTP-binding protein (rab5a) R2292, S13561 Chromosome 12
14-3-3 protein homolog GF14-12 S4036S Chromosome 8
Signal recognition particle receptor-like protein S4036S
Calcium dependent protein kinase C2161 Chromosome 5
1-aminocyclopropane-l-carboxylate oxidase C60626SB Chromosome 7
Transcription Factors:
EREBP-like protein S2769 Chromosome 3
AP2 domain containing protein S2769
Ethylene responsive protein S2769
Helicase-like transcription factor R78 Chromosome 4
OSMYB1 C308 Chromosome 5
Homeodomain leucine zipper protein S4036S Chromosome 8
Metabolism:
Sucrose-6F-phosphate phosphohydrolase SPP3 S3382S, R1547 Chromosome 1
Beta-oxyacyl-[acyl-carrier protein] reductase E61384S Chromosome 3
Putative anthocyanidin reductase R78
HMG protein Chromosome 1
RNase S-like protein S10578, S955 Chromosome 9
Membrane proteins:
Photosystem I chain IV precursor R658 Chromosome 7
Mitochondria FO ATP synthase D chain S4036S Chromosome 8
Water channel protein C735 Chromosome 7


Table 2. QTL's identified in CT9993 X IR62266 population in control and
field stress condition

S.No. Trait No. of QT's identified

1 Leaf characters (specific leaf area and specific leaf weight) 22
2 Phenological characters 19
3 Spikelet sterility 5
4 Biomass 5
5 Grain weight 11
6 Harvest index 5


II. Gene Discovery and Novel Approaches 63 *










Molecular analysis of transgenics
A total of twenty-one (21) regenerants (To) were
obtained on the selection medium from different
independent transformation experiments. The
integration of the transgene was confirmed by PCR
and Southern blot analysis. Of these, five regenerated
seedlings exhibited a strong purple pigmentation in
the leaf sheath and internode. TLC and Proton-NMR
spectroscopy confirmed the nature of pigment.
Northern analysis of transgenic plants showed
increased levels of Ans transcript. Western analysis of
the transgenic leaf extracts revealed the presence of
detectable levels 41KDa band while control non
transgenics did not show this detectable protein.



Conclusions

This EST library formed a rich source of drought
stress-related genes represented in GenBank for the
first time from indica rice seedlings subjected to
progressive drought. Annotation and mapping of the
ESTs onto the genomic sequences resulted in
identifying putative functions and corresponding
genomic regions for large number of ESTs.

The probable candidate genes of the stress-response
transcriptome in rice were uncovered by comparing
with the expression profiles from cDNA microarrays
of different plants subjected abiotic stress treatments.
The newly developed cvCluster tool identified the
true SNPs through sub clustering of cultivar specific
ESTs. The inclusion of new SSRs to the existing maps


allowed precision mapping of tightly linked QTLs.
These functional marker resources developed through
discovery of candidate genes and SNPs are useful in
eventual deployment in crop breeding for genetic
improvement of rice for water limited environments.
These syntenic maps will be used to identify common
genetic segments controlling drought tolerance in rice
and sorghum. Currently, T2 Ans transgenic plants are
being analyzed and tested for their response to biotic
and abiotic stress. Further, microarray data on drought
stress responsive gene expression pattern will be
described.



References
Altschul, et al. 1997. Nucleic Acids Res. 25: 3389-340.
Babu, R.C, etal. 2003. Crop Sci 43: 1457-69.
Chen, X., etal. 1997. TheorAppi Genet. 95: 553-67.
Ewing, B., et al. 1998. Genome Research. 8:175-18.
Kawasaki, S., etal. 2001. Plant Cell. 13:889-905.
Kreps, J.A., etal. 2002. Plant Physiol. 130: 2129-41.
Malde, K., etal. 2003.19:1221-26.
Marth, G., et al. 1999. Nature Genetics. 23: 452-56.
Matsumura, H., et al.1999. Plant 20: 719-26.
Nguyen, V.T., et al. 2002. Mol. Genet. Genomics. 267: 772-80.
Ozturk, Z.N., et al. 2002. Plant Mol Biol. 48: 551-73.
Price, A.H., and Courtois B. 2000. Plant Growth Regul. 29: 123-33.
Price, etal. 2002. Plant Mol Biol. 48: 683-95.
Rabbani, M.A., etal. 2003. PlantPhysiol. 133:1755-67.
Reddy, A.R., et al. 2002. Genome 45: 204-11.
Seki, M., et al. 2001. Plant Cell13: 61-72.
Seki, M, et al. 2002. Plant 31: 279-92.
Zhang J., etal. 2001. Theor. Appl. Genet. 103:19-29.


* 64 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004











A complexity of genes underlie the response to

drought tolerance in maize at flowering


M.C. SAWKINS1, M. DE LA LUZ GUTIERREZ1, J. HABBEN2, C. ZINSELMEIER2, C. MARTINEZ1, E.HUERTA1, M. MORENO1,
AND J.-M. RIBAUT1

1 CIMMYT Int., Apdo. Postal. 6-641, 06600 Mexico D.F., Mexico
2 Pioneer Hi-Bred International, Inc, Johnston, IA 50131-1004, USA

Corresponding author: M.C. Sawkins; E-mail m.sawkins@cgiar.org


Introduction
Water stress occurring at any plant developmental
stage is undesirable. However, in crop plants, and in
particular maize (Zeamaysssp. mays), drought during
flowering can be catastrophic in terms of productivity
(grain yield). During this time, growth of both the ears
and silks slows and a delay in silking in relation to
pollen shed, commonly referred to as the anthesis
silking interval (ASI) (Bolanos and Edmeades, 1996;
Ribaut et al., 1996) is observed. Selection for reduced
ASI has been successfully used to increase yield under
drought in maize (Bainzinger et al., 2000). The response
of maize to drought involves several genes, involved
in yield components and other morphological traits.
The overall objective of the drought work at CIMMYT
is to explain why some genotypes of maize are able
withstand drought during anthesis, and can maintain
a good yield performance under field conditions. To
do so, we adopted a multidisciplinary approach to
elucidate the mechanisms underlying the genetic
response of maize in the field under water stress, with
the hope of identifying the most important genes
conferring drought tolerance during stress at anthesis.
Results are presented from the application of two
technologies based on the expression of genes to
determine how particular genes are responding
during this phase of growth and under a particular
abiotic stress. These two activities are (i) the use of
microarrays in collaboration with Poineer Hi-Bred Int.,
to provide a global picture of changes in gene
expression in ears and silks under water stress during
flowering, and (ii) a more focused approach to study
the expression of candidate genes selected from the
microarray results, literature, and other sources using
quantitative RT PCR.


Materials and methods


Field design
Plants were grown in a replicated randomized block
design in the field (Tlatizapan, Mexico, 2003). Field
trials consisted of genotypes of both parental lines
(Ac7643 and Ac7729/TZSRW) and contrasting families
from the "tails" of the distribution of the segregating
populations (the most drought tolerant and
susceptible families). These were selected on the basis
of contrasting ASI and grain yield and that they had a
pollen shed date no greater than two or three days
before or after the mean male flowering time, in order
to minimize the difference in gene expression due to
differences in stress intensity. Irrigation was stopped
three weeks before flowering, thus ensuring that
plants would be stressed during pollen shed.

Tissue collection
Ear tips, bases, and silks from Ac7643, Ac7729/
TZSRW, and the eight segregating genotypes were
collected over the three replicates. Ears were bagged
before pollen shed to ensure that the experiments were
not compounded by the effects by pollination. Length
of ears were measured and 2 cm from the tip and the
base removed and collected. Approximately 40
samples from all genotypes and tissues were collected.
All tissues were immediately frozen in liquid nitrogen
in the field.

Microarray experiment
RNA extraction, hybridization to slides, and analysis of
results were performed at Pioneer Hi-Bred Int. 60 mer
custom in situ oligonucleotide microarrays (22K) were
used (Agilent Technologies). Two slides (44K) were
used in each comparison and probes selected from the
gene databases at Pioneer. We first compared Ac7643


II. Gene Discovery and Novel Approaches 65 *











(P1) and Ac7729/TZSRW (P2) from ear tips and silks.
Future experiments will compare the genotypes of the
segregating population to one of the parents.

RT-PCR
Although RT PCR is a complex assay, it has the
advantages of being able to detect rare transcripts, is
more sensitive than other established methods (e.g.,
Northern blotting), and requires less tissue. RT PCR
has been used to examine the expression of candidate
genes over the same tissues and genotypes, but over a
greater range of times and in different environments.
As considerable care needs to be taken to optimize all
parameters for RT-PCR, we have recently moved to
kinetic, or real-time, fluorescence RT PCR, using the
ABI PRISM 7000 Sequence Detection System
(Applied Biosystems). The results presented here will
be based on quantitative RT PCR. We initially opted to
study genes in sucrose regulation as sucrose
metabolism is a major component under drought.



Results

Microarray
Comparisons generated between P1 and P2 showed a
large number of changes in gene expression. This
number was reduced when the three replicates were
considered together and when only genes common to
silks and ear tips were considered. The silks showed
the greatest changes in gene expression with 1,491
genes showing significant up or down-regulation. The
ears showed 1,332 genes with significant changes in
expression. Combining this data reduced the dataset to
751 genes. Results were highly consistent across
replicates. Pearson's product-moment and Spearman's
rank-order correlation coefficients were calculated on
the log (ratio) values. The results show that there is
remarkable consistency among replicates for each gene
(see Table 1), with all comparisons highly significant.




Table 1. Correlations across field harvested replicates in changes in
gene expression using microarrays. Correlations were calculated using
the log(ratio) values.

Ear Tips Silks

Comparison Pearson's Spearman's Comparison Pearson's Spearman's

Repl vs. Rep2 0.981262 0.955404 Rep1 vs. Rep2 0.971110 0.933336
Repl vs. Rep3 0.987445 0.969333 Rep1 vs. Rep3 0.957953 0.900847
Rep2 vs. Rep3 0.967657 0.925731 Rep2 vs. Rep3 0.973611 0.947272


* 66 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004


In terms of the types of genes being differentially
expressed, the 751 genes can be classified into many
classes. Many are those that are normally found in
large-scale expression studies under abiotic stresses
(molecular chaperones, water and ion movement,
detoxification, osmoprotection, signal perception, and
transduction and transcription control). Genes
involved in hormone metabolism (ABA, cytokinin,
ethylene, and IAA), starch and sucrose metabolism
and cell growth (expansins, cellulose synthesis) also
show differences in expression between the two
parental lines.

RT-PCR
We studied the expression of six carbohydrate
metabolism genes; Sucrose Phosphate Synthase (SPS),
Sucrose Phosphate Phosphatasel (Sppl), Sucrose
Phosphate Phosphatase2 (Spp2), Cell Wall Invertase2
(Incw2), Vacuolar Invertase2 (Ivr2), and Sucrose
Synthase (Sus). We found differences in the expression
of these genes under drought and well watered
conditions in the ears when a comparison was made
between the drought tolerant and drought susceptible
material. A number of these genes showed no
differences in expression under drought. Although
some correlation was found with the microarray
results, there are some interesting differences
particularly in the silks (which were not used in the
RT-PCR assays), where there was a difference in the
types of carbohydrate metabolism genes showing


1.


S0.7

I 0.6
0-
0.5

S0.4

S0.3

0.2

0.1

0.0


----- ---------







Genotypes


Figure 1. Example of a gene that shows differences in gene expression
during drought but not in well-watered conditions.










differential expression. The results for one gene using
RT PCR, Cell Wall Invertase2, is shown in figure 1
under drought and well-watered conditions. We aim to
study these genes in more detail over the three tissues
in the near future using instead real-time RT PCR.


Conclusion
By combining a number of different approaches such as
QTL detection with functional genomics tools will
generate useful information and a way of validating
those genes important to drought tolerance in maize
during anthesis. This knowledge will be used to
improve germplasm under drought through marker
assisted selection (MAS) experiments.


References
Banzinger, M, etal. 2000. Proceedings of a Symposium, June 21-25, 1999, CIMMYI El
Batan, Mexico, 2000. Pp. 69-72.
Bolanos, I., and G.O. Edmeades. 1996. Field Crops Research 48:65-80.
Ribaut, 1.M., etal. 1996. Theoretical and Applied Genetics 92:905-14.


II. Gene Discovery and Novel Approaches 67 *











Genetic analysis of IR64 introgression lines of rice under

irrigated and water stressed field conditions


V.N. SINGH1, A.K. SINGH1, B.B. SINGH1, G.S. CHATURVEDI1, O.P. VERMA, AND G. ATLIN2

1 Narendra Deva University of Agriculture & Technology, Kumarganj, Faizabad India
2 International Rice Research Institute Manila, Philippines

Corresponding author: V.N. Singh; E-mail: hnsingh@sancharnet.in


Rice is the primary and staple food for more than 60%
of the world's population. In India, rice is grown on
45 million hectares, with an annual production of 89
million tons. Large upland areas of eastern Uttar
Pradesh are still sown to traditional rice varieties due
to the unavailability of drought and stable yield per
forming genotypes. Drought is major constraint,
which limits rice production in eastern Uttar Pradesh.
Major rainfed area lines are under the northern plane
zone (NEPZ), where rainfall is erratic. Therefore,
major rice lines encounter multiple stresses like
drought, submergence at crop establishment at the
vegetative stage, etc., which are major limiting factors
in rice production (Fukai and Cooper, 1995).


Methods
IR64 introgression (21 lines) received from the Central
Rice Research Institute, Cuttack, India were grown in
two sets of environment: irrigated and water stressed
(23 days duration). The lines trial was conducted
under upland conditions at the Crop Research Station,
Masodha Faizabad (U.P.), during the wet season 2003.
A 1 m wide buffer zone was kept between irrigated
and stress plots. Water stress was created by
withholding irrigation in stressed plots and plant
growth morpho-physiological traits like days to 50%
flowering, days to maturity, plant height, and effective
tillers per plant were studied (70 to 93 days). Flag leaf
length and breath, panicle length, grains/panicle,
fertile and sterile grain/panicle test weight, and grain


yield per plant and per plot were recorded 23 days
after water stress. Drought scoring data were observed
on the basis of SES scoring (IRRI, 1996). Evaluation of
various quantitative traits like general mean,
genotypic co-efficient of variation (GCV), phenotypic
co-efficient of variation (PCV), heritability (broad
sense), genetic advance as percent of mean, and
correlation with grain yield were analysed under both
irrigated and stress conditions.


Results
Significant genotypic variation existed among the
introgression lines for all the traits. There was delay in
days to 50% flowering and maturity under stress
conditions. Significant reductions in all traits were
noticed under stress conditions in relation to the
control plot. Yield reduction under stress conditions
was mainly due to reduction in yield contributing
traits. Under stress, high heritability (Table 1) was
found in traits like grain yield and yield contributing
traits, suggesting that importance of these traits as
criteria for improving overall yield under stress.
Moderate habitability was found for panicle length,
and low heritability for flag leaf breadth and test
weight; significant and positive correlation was
observed between grain yield per plant and yield
contributing traits, effective flag leaf breadth and total
grains per panicle. A negative correlation in was found
between grain yield and plant height (Table 1).


* 68 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004











Table 1. Estimates of genetic variability parameters and correlation for fourteen traits in rice, IR64 introgression line (21) under well-watered and
stress (23 days) at heading stage


Correlation with
General mean GCV PCV Heritability(B.S.) GA as % of mean grain yield

C S C S C S C S C S C S


Characters


Days to 50% flowering (days)
Days to maturity (days)
Plant height (cm)
Effective tillers / plant
Flag leaf length (cm)
Flag leaf breadth (cm)
Panicle length (cm)
No. of total grains / panicle
No. of fertile grains / panicle
No. of sterile grains / panicle
Grain yield / plant (g)
Test weight (1000 grain), g.
Grain yield / plot (g)


80.14 90.09 7.03 8.49
107.38 115.80 5.25 6.98
90.01 82.60 13.23 13.93
8.22 5.86 16.06 26.17
31.60 25.94 19.18 15.48
1.30 1.07 6.07 7.56
25.09 21.80 7.45 10.78
85.95 63.69 20.20 25.40
78.58 49.24 22.08 35.56
11.50 14.22 59.80 35.81
15.52 8.59 32.60 37.10
24.40 23.13 3.35 4.93
513.09 215.20 33.95 41.71


8.11 9.14 75.15 86.10
6.06 7.58 75.16 84.68
13.55 14.30 95.20 94.89
16.43 26.62 95.65 96.74
19.71 16.45 94.74 88.55
10.65 12.96 32.51 34.00
9.74 12.97 58.63 69.11
20.41 25.71 97.93 97.63
22.28 35.88 98.23 98.26
60.41 36.09 97.99 98.42
32.78 37.64 98.81 97.12
6.37 7.72 27.72 40.90
33.98 41.85 99.79 99.35


C= Irrigated, S= Stress at heading stage (23 days drought)
D/S= 26.07.2003
h2 (bs) = heritability in broad sense


Conclusion


References


We suggest that when selecting superior genotypes for Fukai S, and Cooper M. 1995. Development of drought resistant cultivars using physio-
rainfed conditions, grain yield, effective tillers, fertile morphological traits in rice. Field Crop Res. 40: 67-86.
IRRI. 1996. International Network for Genetic Evaluation of Rice: Standard Evaluation
grains, and yield per plant should be considered as System forRice. Los Banos, Philippines: International Rice Research Institute. Pp. 52.
important criteria for the breeding programme.


II. Gene Discovery and Novel Approaches 69 *


12.56 16.23
9.38 13.23
26.57 27.96
32.36 53.07
38.92 29.99
7.07 9.13
11.75 18.46
41.18 51.71
45.07 72.62
79.30 73.20
66.75 70.62
3.60 6.48
69.85 82.73


0.35 0.03
0.41 0.001
-0.33 -0.24
0.47* 0.45*
0.27 0.04
0.47* 0.46*
0.25 0.14
0.44* 0.47*
0.34 -0.09
-0.02 -0.21
0.84** 0.55**
0.22 0.03











Search for molecular markers in cereals: An approach

by 'intron scanning' and genome complexity reduction

using DOP-PCR


H.P. SINGH1' 3, F.A. FELTUS1, S.R. SCHULZE1, T. SILVA2, AND A.H. PATERSON1

1 Plant Genome Mapping Laboratory, University of Georgia, Athens GA 30602, USA
2 Department of Plant Sciences, University of Colombo, P.O. Box 1490, Colombo 3, Sri Lanka
3 N.D. University of Agriculture & Technology, Kumarganj, Faizabad 224 264 (U.P.), India

Corresponding author: H.P. Singh; E-mail: hpsing@uga.edu


Introduction
In crop plants, molecular markers are useful for the
creation of genetic maps, map-based cloning, and
many breeding applications including marker assisted
selection, backcross conversion, and genotyping
(Bhattramakki and Rafalski, 2001).There is a transition
beginning in the use of DNA markers, from the types
such as RFLPs, AFLPs, and SSRs, that have dominated
plant genomics in the past decade, to single nucleotide
polymorphism (SNP) based markers. A SNP is a single
base mutation in DNA and is the most simple
polymorphism and most common source of genetic
polymorphism in the human genome (Kruglyak, L.
1997; Collins et al., 1998). Unlike the human genome
for which information has accumulated on the
frequency, nature, and distribution of SNPs, SNP
discovery in cereal genomes is just beginning (Zhu et
al., 2003). To address this problem, the approaches of
'intron scanning' and DOP-PCR were adopted for the
discovery of DNA polymorphisms in cereals. Intron
scanning involves the design of conserved PCR
primers that amplify specific intron-containing loci in
order to unravel the DNA polymorphisms across the
grass species while DOP-PCR involves use of a single
degenerate primer to amplify multiple loci spread
throughout a genome (Jordan et al., 2002). Use of
polymorphism discovery methods such as these will
accelerate marker development in particular and
strengthen efforts toward gene discovery in general.


Methods
The intron scanning primers sets have been designed
by blasting sorghum/buffelgrass (Pennisetum ciliare)
EST's against the TIGR rice pseudomolecule for


* 70 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004


chromosomes 1-12 (www.tigr.org). The DOP PCR
protocol was followed according to Jordan et. al.(2002).
The degenerate primer used in the study was DOP 1B
(5' CTCGAGNNNNNNAAGCGATGW 3'). PCR
products obtained from intron scanning were direct
sequenced while DOP products were cloned and
sequenced. The sequencing has been done on an ABI
3700 sequencer (Applied Biosystems, Foster City,
California, USA). We have sequenced intron scan/DOP
products from seven rice, three sorghum, two millets,
and two bermuda grass genotypes. Sequencing data
were analyzed with Phred/ Phrap/ Consed system
(Ewing et al., 1998). The genomic loci were scanned for
SNP's using Polybayes (Marth et al., 1999).


Results
A significant number of SNP's were identified from
intron-scan and DOP PCR techniques (Table 1). DOP
PCR resulted in a higher total number of SNPs while
the success rates for sequencing were comparable for


Table 1. The DNA polymorphism identification of grass species by intron
scanning and DOP PCR approaches

Taxon Monomorphic loci Polymorphic loci SNP's

Intron Scan

Pennisetum glaucum 18 6 7
Oryza sativa 17 22 54
Sorghum spp. 4 23 69

DOP-PCR

Oryza sativa 419 142 273
Sorghum spp. 360 28 164











both approaches (Table 2). Intron scanning was found
to be significantly more cost effective than DOP PCR
(Table 3), with a 33% reduction in the cost incurred per
SNP identified.

Table 2. The comparative success rates for Intron scan and DOP-PCR
approaches

Primer PCR success Successful Sequencing
Taxon Genotypes sets rate Reads reads success rate

Intron-Scan
Oryza saliva 3 96 67.70% 144 115 79.86%
Sorghum spp. 7 96 66.70% 336 230 68.45%
DOP-PCR
Oryza saliva 2 1 100.00% 1152 869 75.43%
Sorghumspp. 7 1 100.00% 2496 2060 82.53%



Table 3. Analysis of performance and cost incurred for Intron scan and
DOP-PCR techniques (based on rice)

(a)
Performance
Technique Genotypes Primer set Reads Loci Polymorphisms
Intronscan 7 96 336 39 70
DOP PCR 7 1 2496 561 164

(b)
Cost incurred ($)
Technique aPrimers bPCR Reactions CSequencing Total Polymorphisms
Intron scan 622.08 336.00 840.00 1798.08 25.69
DOP PCR 3.24 3.50 6240.00 6246.74 38.09

a 0.18/nt; b 0.5/rxn; c2.50/read (including cleanup and sequencing)


Conclusions

Much effort has been made to study SNPs in the
human genome, but studies on the grass genome are
scarce and limited. This study has led the way to
scanning polymorphisms economically and
effectively. The intron-scanning approach was found
to be highly acceptable and can also can be employed
to identify SNPs at corresponding loci across diverse
taxa, detect evidence of selection by virtue of its close
relationship to genes, and discover conserved non
coding (CNC) regions in closely related genomes. The
SNPs detected in this study are under validation
process. After successful genotyping, these SNP's
could be then used as molecular markers for
molecular breeding and other purposes in diverse
cereal taxa.



References
Bhattramakki and Rafalski. 2001. Plant Genotyping: the DNA Fingerprinting of Plants (ed.).
R.J. Henry.
Collins, FS., et al. 1998. Genome Res. 8(12): 1229-31.
Ewing B., et al. 1998. Genome Research 8(3): 175-85.
Jordan, Barbara, et al. 2002. PNAS 99(5): 2942-47.
Kruglyak, L. 1997. Nat. Genet. 17(1): 21-24.
Marth, G.T., et al. Nat Genet. 1999.23(4): 452-56.
Zhu, Y.L., et al. 2003. Genetics 163(3): 1123-34.


II. Gene Discovery and Novel Approaches 71 *











Over-expression of exogenous superoxide dismutase

gene (MnSOD) and its effect on stress resistance in maize


LI WANCHEN AND Du JUAN

Maize Research Institute, Sichuan Agricultural University, China

Corresponding author: Li Wanchen; E-mail: aumdyms@sicau.edu.cn


In this study, the MnSOD gene from wheat was
designed to be promoted by maize ubiquitin promoter
and the expression product to be located at
mitochondrial membrane by mitochondrial transport
polypeptide. Expression vector for monocotyledon
was constructed with this expression structure and
embryonic calli of elite maize inbred lines were
transformed with microprojectile bombardment. It
was attempted to improve tolerance of maize to
drought and other stress by overexpression of the
MnSOD gene and the product accumulation in
mitochondrion.


Materials and methods

Construction of expression vector of MnSOD gene
for monocotyle
Mitochondrial transport polypeptide gene TPand
MnSOD gene MnSODwere cut off by BamHI and Clal
from pBSOD3.1 and integrated into pUGFPocs. This
produced an intermediate plasmid pUSOD3.locs,
which contained the complete expression structure 'P
Ubi MnSOD3.1- Tocs'. This structure was then cut off
by KpnI and integrated into pCAMBIA1300 with
hygromycin phosphotransferase gene hpt. The
structure of MnSOD expression vector pC 1300SOD3.1
is shown in figure 1.

Transformation and screening
After four hours of subculture on highly osmotic
medium, the embryonic calli were transformed with
the expression vector by microprojectile bombardment
model PDS 1000/He, using system pressure of
1300psi, vacuum degree of 27^28 inch mercury, and
vector DNA of 1g for each bombardment. The


LB- polyA hpt P-35S -nos ISOD P P-Ubi RBB

Figure 1. Structure of MnSOD expression vector pC1300SOD3.1

* 72 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004


transformed calli were cultured one week on
regulatory subculture medium for resume and three
weeks on each gradient of hygromycin concentration
of 5, 10, and 15 mg/1 for screening. Differentiation and
rooting media also contained hygromycin (5 mg/1).

PCR and Southern hybridization identification
DNA fragment of hpt gene was amplified as marker of
exogenous gene with DNA template extracted from the
leaf of the regenerated plants and specific primers (P1:
5'-TACACAGC CATCGGTCCAGA-3', P2: 5'
TAGGAGGGCGTGG ATATGTC-3') designed
according to hptsequence. 10 g of total DNA was
digested with HindIII, separated with 0.7% agarose gel
and transferred to Hybond N' membrane (Amersham,
UK). Full length sequence of hpt gene was radiolabeled
with 32P dCT and used as probe to be hybridized with
the template DNA on the membrane. The signal was
detected after washing with 0. lxSSC and 0.1%SDS, and
radioautography on X-ray film.

MnSOD activity detection
SOD enzyme was extracted from the leaf of the
regenerated plants with the method introduced by
Luo Guanghua and Wang Aiguo (1983), separated
with nondenatured polyacrylamide gel
electrophoresis of gradient concentration, treated and
stained with the method introduced by Beauchamp
and Fridovich (1971).

Antioxidant capacity detection of transgenic
plant lines
Leaf discs of 1.0 cm2 were sampled from the fourth
leaf of the transgenic offspring plants, weighted (m),
and treated with 3 ml methyl viologen (MV) solution
of 1.0 and 1.5 mol/1 under the conditions of 21C and
darkness for 16 hours, room temperature and
illumination for 2 hours, and 30C and darkness for 16
hours. The methyl viologen solution was collected for
each treated sample, ddHO2 was added to 3 ml and
electrolyte seepage rate (C1) was detected with electric










conductometer model DDS-11A (specific conductivity
= 0.98). The solution was collected and the leaf discs
were put back. Electrolyte seepage rate (C2) was
detected again after boiling 15 minutes, cooling to
room temperature, and adding ddHO2 to 3 ml. The
detection was repeated four times for each plant line.
The electric conductivity of 35 mg sample wass
calculated as: Electric conductivity = 35C1/mC2.
Significance test was made by variance analysis and
least significant difference (LSD).



Results and analysis

Restriction analysis of expression vector
The MnSOD expression vector pC1300SOD3.1 was
digested with SacI, KpnI, XbaI and SacI + Clal,
respectively. A 13.5 kb band of the linear vector, a 4300
bp band of the complete expression structure 'P-Ubi
MnSOD3.1 T-ocs', a 1800 bp band of ubiquitin, and a
1600 bp band of terminator fragment were identified
by agarose gel electrophoresis (Figure 2). This result
indicated that the expression vector followed the
designed structure.

PCR and Southern identification of
transgenic plants
After transformation and strict screening, 49 plants
were regenerated and 35 of them reproduced fertile
seeds. Specific hpt fragment of 832 bp was amplified
from nine of the fertile regenerated plants (Figure 3).
HindIII was used to digest total DNA of the nine
plants, because this enzyme had no recognition site
during hpt sequence. It was shown by Southern
hybridization with radio-labeled full length sequence
of hpt gene that the exogenous gene had integrated
into maize genome (Figure 4).


1 2 3 4 5


MnSOD activity detection
The plants identified as positive through PCR and
Southern blots were planted to reproduce plant lines
of T1 generation and used for MnSOD activity
detection. SOD had three isozymes: FeSOD, Cu/
ZnSOD, and MnSOD. 5 mmol/1 H202 inactivated
FeSOD completely, reduced activity of Cu/ZnSOD,
and had no significant influence on MnSOD. 5 mmol/
L H202, therefore, was used to inhibit endogenous
activities of FeSOD and Cu/ZnSOD in maize. Activity


1 9 A a r


Q a in 11 19


M. DNA marker, 1. positive control, 2. negative control, 3. un-transgenic plant, 4-12. transgenic plants.


Figure 3. PCR identification of transgenic plants.


1 2 3


=^n .....


4 5 6


1. Sac I 2. Kpn I 3.1 Kb marker 4. Xbal 5. Sac l+Cla I

Figure 2. Restriction analysis of expression vector pC1300SOD3.1.


1. un-transgenic plant, 2-6. transgenic plants.

Figure 4. Southern hybridization identification of transgenic plants.


II. Gene Discovery and Novel Approaches 73 *











of MnSOD was detected after nondenatured
polyacrylamide gel electrophoresis of gradient
concentration. All the transgenic plant lines showed
higher activity than the negative control, while plant
line 3 displayed the highest activity (Figure 5).

Antioxidant capacity of transgenic plant lines
After treatment with 1.0 mol/1 methyl viologen, electric
conductivity of all the transgenic plant lines, except
number 6, was less than the non-transgenic control,
while plant line 3 showed greatly significant difference
(Figure 6 a). After treatment with 1.5 mol/1 methyl
viologen (MV), electric conductivity of plant lines 2, 3,
4, and 5 was less than the control, while numbers 2, 3,
and 5 showed greatly significant difference (Figure 6 b).
This result indicated that antioxidant capacity of
cellular membrane of the transgenic plants was
increased more significantly than the control while
oxidation damnification was increased.


1 2 3 4 5 6


23 MnSOD


1. un-transgenic plant, 2-6. transgenic plants.

Figure 5. Profile of MnSOD enzyme of transgenic plants.


1 2 3 4 5 6
(a) 1.0? mol/L MV

5.

4"

3-
T 3

2-

1-


1 2 3 4 5 6
1.5? mol/L MV
1. un-transgenic plant, 2-6. transgenic plant lines.

Figure 6. Electric conductivity of transgenic plant lines.


* 74 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004











EcoTILLING candidate genes for drought tolerance in rice


HE HE WANG1, MA. ELIZABETH NAREDO1, JIANLI Wu1, BRADLEY J. TILL2, ELIZABETH A. GREENE2, STEVEN HENIKOFF2,
LUCA COMAI3, HEI LEUNG1, AND KENNETH L. MCNALLY1

1 International Rice Research Institute (IRRI), DAPO Box 7777, Metro Manila, Philippines
2 Fred Hutchinson Cancer Research Center, Seattle WA, USA
3 Department of Biology, University of Washington, Seattle WA, USA

Corresponding author: Kenneth L. McNally; E-mail: k.mcnally@cgiar.org


Introduction
EcoTILLING of diverse germplasm allows the
discovery of SNPs and the delineation of haplotypes
at loci of interest (Comai et al., 2004). This technique
relies on the enzymatic cleavage of heteroduplex
molecules formed between reference and query lines
by an S1 type single-strand endonuclease from celery,
CELl. Dual-labeling of the PCR amplicons by
different fluorescent tags allows the detection of the
cleavage products on denaturing PAGE with
automated genotyping. The appearance of new
bands against the background products allows the
detection of single nucleotide polymorphisms.
Furthermore, the banding patterns across a range of
diverse germplasm can be grouped into haplotypes.
This combination of haplotyping and identifying
SNP loci reduces the cost associated with SNP
discovery so that only those germplasm carrying
SNP differences need be sequenced in order to
establish the identity of the nucleotide difference. As
such, EcoTILLING is a powerful, inexpensive tool for
the detection of natural variation.



Materials and methods
At IRRI, we have begun employing this technique to
characterize SNPs and determine haplotype structure
at candidate genes for drought tolerance in diverse
germplasm selected from the International Rice
Genebank Collection (IRGC). This mini-core
collection of 1,546 cultivated, diverse Oryza sativa
accessions and contains diverse landrace, traditional,
and advanced varieties representative of the variety
groups and cultural types or non-sativa Oryza
species, 48 accessions covering the genome types
were chosen. Overall, these two sets represent about
1.5% of the genebank collection.


Candidate genes putatively involved in drought
tolerance in rice were identified through converging
evidence taking into account functions implicated in
drought tolerance, altered expression, co-localization
with drought QTLs, and/or shifts in allele
frequencies under selection. The co-localization with
QTLs was largely done to QTLs for yield components
under field stress. Our initial target loci for
implementing EcoTILLING are protein phosphatase
2a-4, DREB1, trehalose 6-phosphatase, and 9-cis
epoxycarotenoid dioxygenase (Table 1). Allelic
variation at pp2a4 was identified in pilot studies at
the University of Washington; this locus has served
as a positive control for implementing EcoTILLING in
rice at IRRI. Additional candidate gene loci have
already been identified, and primers to detect regions
of these genes are being designed.





Table 1. Drought candidate gene targets and primers

Gene Function Primer Sequence (5'to 3'
Left (IRD700), RGP Prod.
Right (IRD800) Chr. (cM) (bp)
pp2a4 protein pp2a4L ggTTggggCATA TCTCCTCgTggT CH10 30.2 928
phosphatase pp2a4R TCCTAggAgCTggTTCAAACTgCAA CHO3 16.8 668
drebl drought
response DREB1L CCgTTgATTgCTgATAgCCTCCITgA CH01 16.1 969
binding DREB1R TgAAATATTCCTATTgACCCgCAgCA
protein 1
tps trehalose TPSL ggCACACTgTCgCCTATTgTggATg CH02 109.3 997
phosphatase TPSR gTTTACgAgCCgTgCgACCAgTTTC
vp14 viviparousl4
(9-cis-epoxy-
carotenoid
dioxygenase) VP14L TggCAAgAAgAAggATgggCTgAAC CH12 105.1 1013


II. Gene Discovery and Novel Approaches 75 *











Results


EcoTILLING has been successfully implemented at
IRRI. Figure 1 shows the results for pp2a4 locus on
22 germplasm contrasted to the reference lines, IR64
and Nipponbare. Figure 2 shows the result for this
locus on 24 wild species contrasted to IR64. This
may well be the first example of the use of
EcoTILLINg to detect inter-specific differences. We
are in the process of testing whether or not pooling
levels up to 16-fold are possible. If so, then one run
of the automated genotyper with 96 lanes will
accommodate 1,536 samples for first pass screening.
Recent results of pooling tests and EcoTILLING at
other loci will be presented.



References

Comai, L., Young K., Till B.J., Reynolds S.H., Greene E.A., Codomo C.A., Enns L.C.,
Johnson J.E., Burtner C., Odden A.R., and Henikoff S. 2004. Efficient discovery of
DNA polymorphisms in natural populations by ecotilling. Plant. 37: 778-86.


JC~JI L'h....J*4~ squ


II I~ii,~


IRMx





irIoF[,


Figure 1. EcoTILLING of 22 accessions at the pp2a4 locus contrasted
against IR64 (left) and Nippon-bare (right). Circles indicate possible
polymorphisms.


i I

L


I ;


ii
~1
rl. I


4-,

I .


Ei,
I i


iil


Fu .' IIL llflh&


Figure 2. EcoTILLING of 24 wild accessions at the pp2a4 locus
contrasted against IR64. These species AA, CC, CCDD, EE, and GG
genome types.


* 76 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004











Genetic transformation and testing of stress responsible

candidate genes on improving drought tolerance in rice


B.Z. XIAO1, X. CHEN2, H.H. Hul, X. Hou1, C.B. XIANG2, Q. ZHANG1, AND L. XIONG1

1 Huazhong Agricultural University, Wuhan, China;
2 University of Science and Technology of China, Hefei, China

Corresponding author: Lizhong Xiong; E-mail: lizhongx@mail.hzau.edu.cn


Methods


Rice is the most important crop in China and in many
developing countries. Drought stress is one of the
most important constraints in rice production, mostly
due to variation in the rainfall patterns from one year
to another and also uneven distribution of rainfall and
increasing shortage of water resources in the rice
growing areas. A project supported by The Rockefeller
Foundation has been initiated to engineer rice plants
with improved drought tolerance, targeting the
drought stress at late stage of the growing season
under field conditions in the agroecosystem of central
and southern China. The long-term goal of the
proposed project is to generate new rice cultivars and
hybrids with significantly improved tolerance to
major abiotic stresses such as drought and salinity.


The specific objective for the current phase of this
project is to evaluate the effects of 10 candidate genes
from different functional categories on improving
drought tolerance in rice under field conditions of
central and southern China at the late stage of the
growing season. These 10 genes (Table 1), mostly from
Arabidopsis (CBF3, LOS5, SOS2, HVA1, ZATI0,
NCED1, NHX1, CodA) or from other species (NPK1,
TPS), were recommended and/or provided by a panel
of worldwide leading experts in stress biology invited
by The Rockefeller Foundation. All these candidate
genes were constructed under the control of
constitutive promoter or stress inducible promoter
and transformed, mediated by Agrobacterium, into a
drought-sensitive rice cultivar Zhonghua 11. As an
important addition to the project, rice homologues to


Table 1. Transformation progress of candidate genes and rice homologues

Priority Gene Gene product or description Transformation status

1 CBF3 AP2 type transcription factor Resistant callus selection
3 rice homologues 85%-90% of similarity to CBF3 T1 seeds available
2 LOS5 Enzyme in ABA biosynthesis Resistant callus selection
3 SOS2 Protein kinase Resistant callus selection
Rice homologue 75% of similarity to SOS2 Resistant callus differentiation
4 TPS Trehalose-6-phosphate synthase Similar work already reported
5 HVA1 (rice homologue) LEA protein, 93% of similarity to HVA1 To plants (more than 100 lines each construct)
HVA22 (rice homologue) 93% of similarity to HVA22 To plants
6 NPK1 MAPKKK Resistant callus selection
Rice homologue 76% of similarity to NPK1 Resistant callus differentiation
7 ZAT10 Zinc finger transcription factor Resistant callus selection
8 NCED3 9-cis-epoxycarotenoid dioxygenase Resistant callus differentiation
9 NHX1 Vacuolar Na+/H+ antiporter Resistant callus selection
Rice homologue 78% of similarity to NPK1 Resistant callus differentiation
10 CodA Choline oxidase Gene source unavailable

a The priority was determined by the discussion from a panel of leading experts in stress biology invited by Rockefeller Foundation.


II. Gene Discovery and Novel Approaches 77 *


Introduction










above candidate genes were also chosen for rice
transformation and testing. For each construct, at least
30 independent single copy transgenic lines, were
planned to be generated, and 20 plants each line will
be tested for drought tolerance following the
treatment and evaluation protocol provided by Dr A.
Blum (personal communication).


Progress and perspective
At present, all candidate genes, except TPS and CodA,
and 8 rice homologous genes have been constructed in
a high throughput binary vector under the control of a
constitutive promoter (rice Actl promoter) and a stress
inducible promoter (from a rice HVA22homologue
that is strongly induced by drought), respectively.
Each construct has been transformed into the rice


cultivar Zhonghua 11. The current status of
transformation is summarized in table 1. Transgenic
plants in To generation have been generated for five
rice genes and the transformation for remaining genes
is expected be finished before July, 2004. T1 or T2 seeds
will be available in the next rice grown season for
drought testing.

The completion of this work may create many new
opportunities for the development of drought tolerant
rice crosses or varieties and will also contribute to the
elucidation of functional conservation of the candidate
genes used in this project, which in turn will provide
insight into the mechanisms of adaptation of plant
cells to dehydration stresses.


S* 78 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004











An Arabidopsis gain-of-function mutant with enhanced

drought tolerance by activation tagging


HONG Yu, YUANYUAN HONG, PING ZHAO, XI CHEN, AND CHENGBIN XIANG

School of Life Sciences, University of Science and Technology of China, Hefei, Anhui Province 230026, China


Introduction
Drought is a worldwide problem. Although drought
tolerance is widespread in nature, the underlying
mechanisms are still not well understood, but
significant progress is being made (Xiong et al., 2002;
Xiong and Zhu, 2002; Zhu, 2002; Chinnusamy et al.,
2003; Seki et al., 2003; Shinozaki et al., 2003; Xiong and
Yang, 2003). To study drought tolerance mechanisms
and mine drought tolerance genes, we have taken
advantage of the model plant Arabidopsis by isolating
gain-of function mutants with enhanced stress
tolerance. From an activation tagging library of 55,000
individual lines that we created, one mutant with
enhanced drought tolerance was isolated. Initial
characterization showed that this mutant has
enhanced drought tolerance under greenhouse
conditions, a well-developed root system, and
enhanced tolerance to oxidative stress compared with
the wildtype. In addition, this mutant was also found
to have enhanced tolerance to salt stress. The results
from this study demonstrate that accelerated
evolution by activation tagging is a feasible approach
to obtain gain-of function mutants with enhanced
stress tolerance and altered expression level and
pattern can afford new functions to certain genes.


Methods
To isolate gain-of-function mutants with enhanced
stress tolerance, an Arabidopsis activation tagging
library of 55,000 independent lines was created as
described (Weigel et al., 2000). The mutant library was
initially screened for individuals with growth vigor.
Several such putative mutants were isolated and
tested for their tolerance to drought, salt, and
oxidative stresses. Drought tolerance test was done
with mutant and wild-type plants grown in soil under
greenhouse conditions while salt and oxidative
stresses were tested on media containing NaCl or
paraquat. Co-segregation analysis was carried out


after stress tolerance test by examining the resistance
to glufosinate herbicide of each individual plant using
leaf paint method.


Results
An Arabidopsis gain-of function mutant with enhanced
drought tolerance was isolated (Figure 1). The mutant
phenotype co-segregates with herbicide resistance,
thus confirming the drought tolerance phenotype was
directly resulted from the T DNA insertion. This was
further confirmed by recapitulation study. Molecular
analysis further revealed that the mutant phenotype
was caused by the elevated expression level and
altered expression pattern of the tagged gene. In
addition to drought tolerance, this mutant also shows
enhanced tolerance to salt stress and oxidative stress.
Physiologically, the mutant leaves show slower rate of
water loss in contrast to wildtype leaves.
Morphologically, this mutant shows growth vigor and
a well developed root system compared with wild
type. Apparently, the well-developed root system,
slower rate of water loss from leaves, and oxidative
stress tolerance collectively contribute to the enhanced
drought tolerance of this mutant.


Figure 1. Drought tolerance test. Wild-type and mutant plants were
grown side by side under identical conditions in the same tray.
Watering was withheld for two weeks when plants were 4 weeks old.
The wilted plants were wild-type.


II. Gene Discovery and Novel Approaches 79 *











Conclusions

The results from our study with this mutant
demonstrate that accelerated evolution by activation
tagging is a feasible approach to obtain gain-of
function mutants with enhanced stress tolerance.
Altered expression level and pattern can afford new
functions to certain genes. The mutant we isolated
should be a valuable resource for studying the
underlying mechanisms of drought tolerance. The
tagged gene has the potential of crop improvement
for enhanced stress tolerance.



References
Chinnusamy, V., K. Schumaker, and J.K. Zhu. 2003. Molecular genetic perspectives on
cross-talk and specificity in abiotic stress signalling in plants. I Exp Bot.
Seki, M., A. Kamei, K. Yamaguchi-Shinozaki, and K. Shinozaki. 2003. Molecular responses
to drought, salinity and frost: common and different paths for plant protection. Curr
Opin Biotechnol 14: 194-99.
Shinozaki, K, K. Yamaguchi-Shinozaki, M. Seki. (2003) Regulatory network of gene
expression in the drought and cold stress responses. Curr Opin Plant Biol6: 410-417.


Weigel, D., J.H. Ahn, M.A. Blazquez, J.O. Borevitz, S.K. Christensen, C. Fankhauser,
C. Ferrandiz, I. Kardailsky, E.J. Malancharuvil, M.M Neff, J.T. Nguyen, S. Sato, Z.Y.
Wang, Y. Xia, R.A. Dixon, M.J. Harrison, C.J. Lamb, M.F. Yanofsky, J. Chory. 2000.
Activation tagging in Arabidopsis. Plant Physio122:1003-1013.
Xiong, L., K.S. Schumaker, and J.K. Zhu. 2002. Cell signaling during cold, drought, and salt
stress. Plant Cell14 Suppl: S165-183.
Xiong, L., and Y. Yang. 2003. Disease resistance and abiotic stress tolerance in rice are
inversely modulated by an abscisic acid-inducible mitogen-activated protein kinase.
Plant Cell15: 745-759.
Xiong, L., and J.K. Zhu. 2002. Molecular and genetic aspects of plant responses to osmotic
stress. Plant Cell Environ 25: 131-139.
Zhu, J.K. 2002. Salt and drought stress signal transduction in plants. Annu RevPlant Biol53:
247-273.


* 80 0 CIMMYT / Drought / Rockefeller Foundation Workshop 2004











Generation Challenge Programme: "Cultivating plant

diversity for the resource-poor"

ROBERT ZEIGLER (DIRECTOR)

Hosted by the International Maize and Wheat Improvement Center (CIMMYT), Apdo. Postal 6-641, 06600
Mexico D.F., Mexico

Corresponding author: R. Zeigler; Email: r.zeigler@cgiar.org


Introduction
Rich genetic resources, whose diversity could benefit
the global community, are kept in trust for humanity
by CGIAR Centres. National genebanks, too, hold
immense reserves of untapped riches. Advances in
molecular biology can put this diversity to work in
powerful and exciting ways. But at present the power
of modern biology is rarely used to improve crops
grown by resource-poor people in the developing
world. The Generation Challenge Programme
(formerly the Challenge Programme for Unlocking
Genetic Diversity in Crops for the Resource-Poor) aims
to create a public platform that will use molecular
biology to unlock genetic diversity and put it to use in
bettering crops for the world's poorest farmers.


Organization
The Generation Challenge Programme brings
together three sets of partners. The CGIAR Centres
keep vast amounts of plant diversity in trust for
humanity and have expertise in molecular research
and global breeding programmes. The National
Agricultural Research Systems of developing
countries bring expertise in the assessment and
breeding of plants under specific conditions, with the
participation of farmers. Advanced Research
Institutes develop novel techniques and strategies to
decode genetic diversity. Together, these three
constituents can vastly improve the productivity of
crucial crops in marginal environments. The
Generation Challenge Programme is composed of five
subprogrammes, detailed below. A major objective of
the GCP is to apply genomics tools and technologies
to a better understanding of drought tolerance
mechanisms in the 22 mandate crops of the CGIAR.


Drought tolerance: The ultimate
challenge in a world of climate
change
One of the most difficult challenges faced by resource
poor farmers and scientists alike is drought. The
Generation Challenge Programme is focused on
harnessing the genomics revolution to enhance
drought tolerance in the staple crops of developing
countries. Plants' responses to drought are complex
and involve interactions between many different
molecular, biochemical, and physiological processes.
Moreover, the nature of drought stress itself varies by
crop, cropping system, region, and year. Using
comparative genomics, scientists can begin to dissect
the intricate relationships and pathways at work at
various plant component levels and across species.
Intense investigation of drought tolerance in one
species or plant type may well provide the vital clues
needed to unlock it in other species.


Subprogramme 1: Genetic
diversity of global genetic
resources
Using a selection of known genes as probes, this
subprogramme's goal is to assay the diversity in
existing collections. The assays are of two types.
Structural characterization comprises the sequences,
markers, and other genomic information that enables
researchers to identify particular genes. Functional
characterization links genetic information to the
performance of the plant; for example, it can relate
specific sequences to the plant's ability to withstand
low temperatures.


II. Gene Discovery and Novel Approaches 81 *




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