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Session IV – Rice from the world
Session IV
Rice from the world
PLENARY LECTURES
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Session IV – Rice from the world
USING ANTHER CULTURE AND MICROSATELLITE MARKERS
TO GENERATE WEED COMPETITIVE RICES FROM Oryza sativa L.
AND Oryza Glaberrima STEUD. GENEPOOLS
Hahne Gunther, Monty Patrick Jones and Marie-Noelle Ndjiondjop
WARDA/ARDAO, 01 Bouake BP 2551 Republic of Côte d’Ivoire
Summary
Breeders at the West Africa Rice Development Association have developed new interspecific
progenies derived from crosses between O. sativa subsp. japonica and O. glaberrima through
backcrossing and doubled haploid breeding. The new interspecific progenies have shown good
ability to compete with weeds in rainfed rice ecosystems. Mapped microsatellite markers and
bulked segregant analysis strategy were used to generate graphical genotyping of the selected
lines.
Key Words
Anther culture, microsatellite markers, Oryza sativa, Oryza glaberrima, weed competitiveness.
Abstract
Introduction
Weed competition is the most important yield-reducing factor in rainfed rice environments in
sub-Saharan Africa. Many of the commonly grown O. sativa varieties have a high yield
potential, but they compete poorly with weeds. By contrast, the indigenous cultivated O.
glaberrima Steud. landraces are highly competitive due to high tillering ability, vigor and leaf
area during vegetative growth. But their yield potential is low because of O. glaberrima's
specific panicle type and tendency to lodge. This paper gives a brief description of an innovative
interspecific crossing program between O. sativa and O. glaberrima initiated in 1991 to develop
weed competitive and highly input responsive, but not input-dependent, rice types for various
water limited ecosystems in West and Central Africa.
Materials and Methods
The process of interspecific hybridization and field evaluation
On the basis of a morpho-agronomic characterization, eight O. glaberrima parents that had the
best combinations of traits and the best five O. sativa upland varieties, developed by WARDA,
were selected for wide hybridization. Forty-eight crosses between O. sativa and O. glaberrima
lines were made at WARDA. After two backcrosses, individuals from the BC2F1 populations
were subjected to pedigree selection. On a parallel track, anther culture was used to obtain fertile
plants and shorten the number of generations required for the fixation of particular traits. Anthers
from BC1F1 were removed from the spikelets and passed through the anther culture process as
described in Jones et al. (1997). In field experiments that followed, the parents and progenies
were characterized in term of weed competitiveness  a score of leaf droopiness, leaf area index
(LAI) and specific leaf area (SLA; leaf area/leaf dry weight) (Dingkuhn et al. 1996). At maturity
grain yield and yield components were measured.
Microsatellites analysis
Seventeen upland advanced breeding lines that performed well in farmers’ fields were selected
because they inherited some characteristics of O. glaberrima. Bulked segregant analysis
(Michelmore et al. 1991) was used to identify microsatellite markers linked to the major gene
responsible for droopy leaves that gave CG14 the ability to compete with weeds. The droopy
leaves (vs. no-droopy leaves) bulks consisted of 10 homozygous lines of BC2F8 plants from the
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CG14 (O. glaberrima) x WAB56-104 (O. sativa) cross. For all experiments, the seeds were
sown in a greenhouse and leaves were collected for DNA extraction 4 weeks after germination.
To generate simple sequence length polymorphism, we use mapped microsatellite markers as
described in Temnykh et al. (2001).
Results and Discussion
Advantages of doubled haploid breeding (DHB) and weed competitiveness
Backcrossing with the O. sativa parents increased fertility and helped combine the O. sativa and
O. glaberrima features. The use of DHB to generate large proportions of doubled haploids from
japonica x O. glaberrima BC2F1 hybrids, helped to overcome constraints associated with the
conventional breeding of these species, such as: (1) slow fixations of the lines, (2) frequent
partial sterility of the progenies and (3) low recovery of useful recombinants.
While the CG14 had two to three times LAI, and 1.5 to 2 times the tiller number of WAB 56104, some progenies had intermediate SLA and LAI (Fig. 1). They combine the superior vigor of
the O. glaberrima with the panicle structure of the O. sativa. Some progenies achieved a high
SLA and therefore early groundcover (O. glaberrima types) during the vegetative phase
followed by a low SLA (O. sativa type) during the reproductive phase, resulting in yields of up
to 5 tons ha-1 under improved management conditions and 3 tons ha-1 in traditional low input
systems.
Utilization of mapped molecular markers to characterize introgressed segment in advanced
breeding lines able to compete with weeds
One hundred and thirteen primers were tested on the parental lines: CG14 and WAB56-104. An
average of 87% showed codominant polymorphism between CG14 and WAB56-104. We
developed the graphical genotyping of the lines using 131 markers. The mean of O. glaberrima
allele across lines was 12% and the mean of O. sativa allele was 86%. On average, the
introgressed segments were small (average size 10 cM) and randomly distributed on the 12
chromosomes, indicating that recombination occurred frequently in interspecific combination
and that there was no genome-wide barrier to recombination during meiosis. The polymorphic
microsatellite markers were amplified in 10 lines constituting each bulk. The graphical
genotyping of each line was obtained. The relationship between the introgressed fragment from
O. glaberrima genome and the droopy leaves ability was analyzed.
References
 Dingkuhn M., Jones M.P. and Sow A. 1996. New high-yielding, weed competitive plant types drawing
from O. sativa and O. glaberrima genepools. WARDA annual Report for 1995. pp. 4-12.
 Jones P.M, Dingkuhn M, aluko G.K, and Semon M. 1997. Interspecific Oryza sativa L. x O. glaberrima
Steud. progenies in upland rice improvement. Euphytica 92: 237-246.
 Michelmore R.W, Paran I, Kesseli R.V 1991. Identification of markers linked to disease-resistance genes
by bulked segregant analysis : a rapid method to detect markers in specific genomic regions by using
segregating populations. Proc Natl Acad Sci U S A 88:9828-9832
 Temnykh S., DeClerck G., Lukashova A., Lipovich L., Cartinhour S., and McCouch S. 2001.
Computational and experimental analysis of microsatellites in rice (Oryza sativa L.): frequency, length
variation, transposon associations, and genetic marker potential. Genome Research 11:1441-1452.
Figure 1. Time courses of specific leaf area (SLA) for an
interspecific rice progeny and the parents WAB 56-104 (O.
sativa) and CG14 (O. glaberrima). The broken line indicates
the “ideal” SLA for a high yielding, weed competitive plant
types.
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Session IV – Rice from the world
DEVELOPING TRANSGENIC RICE FOR PLANT PROTECTION
AND IMPROVED NUTRITION
Swapan K. Datta, International Rice Research Institute, DAPO Box 7777, Metro Manila,
Philippines (S.Datta@cgiar.org)
Summary
Genetic engineering along with crop management practices offer tremendous potential to
reduce the yield gap and could accelerate the delivery of improved rice yield and economic
benefit to farmers. Besides plant protection as has been demonstrated in transgenic Bt, BB,
and PR-rice, it is also now possible to develop nutritious rice. High-value rice grown in a
pesticide-free environment will be an added attraction to the consumer market and will
improve human health.
Kew words
Nutrition rice, plant protection, genetic engineering, field performance, Bt , BB, PR
(pathogenesis related protein) rice.
Abstract
Genetic engineering, a powerful tool, is now being used to complement traditional
breeding efforts to improve crop yield, pest and disease resistance, and nutritive value of
crops. Studies on transgenic rice continue to explore possibilities to confer traits such as
resistance to bacterial blight1, stem borer2, and sheath blight3, and improved nutrition4.
Non-antibiotic (POSITECH selection with the pmi gene) and marker-free selection systems
have been achieved5. Field evaluation of the resistance of transgenic rice to bacterial blight
and stem borer showed excellent results6, 7. Such studies demonstrate the steady progress
in transgenic crop research and ensure a pesticide-free environment or reduced use of
agrochemicals in the environment.
Rice provides several essential micronutrients such as iron, iodine, and zinc. However,
polished milled rice contains few micronutrients and no provitamin A. Ye et al. (2000)8
introduced three genes, psy, crt1, and lyc, targeted to the endosperm of japonica rice T309,
which is referred to as “Golden Rice”. Goto et al. (1999)9 reported enhancement of the
iron content in japonica rice by using the ferritin gene. Following work on Golden Rice
and high beta-carotene canola, we have developed a large number of transgenic tropical
indica rice cultivars suitable to diverse ecosystems with three genes (psy, crt1, and lyc).
Furthermore, constructs have been modified with suitable endosperm-specific promoters to
enhance -carotene expression in rice endosperm. In addition to -carotene rice, we have
also developed transgenic indica rice with ferritin (iron storage protein gene) and FRO2
(iron chelate reductase gene expressed in iron-deficient soil) singly or in combinations.
Transgenic plants showed enhancement of iron and zinc in polished rice seeds.
Improvement of lysine in transgenic rice is also in progress. Transgenic rice with multiple
plant protection along with improved nutrition will have a tremendous impact on rice
improvement for the welfare of developing countries.
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References
 Tu J, Ona I, Zhang Q, Mew TW , Khush GS, and Datta SK (1998) Transgenic rice variety IR72 with
Xa21 is resistant to bacterial blight. Theor. Appl. Genet. 97:31-36.
 Datta K, Vasquez A, Tu J, Torrizo L, Alam MF, Oliva N, Abrigo E, Khush GS, and Datta SK (1998)
Constitutive and tissue-specific differential expression of cryIA(b) gene in transgenic rice plants
conferring resistance to rice insect pest. Theor. Appl. Genet. 97:20-30.
 Datta K, Tu J, Oliva N, Ona I, Velazhahan R, Mew TW, Muthukrishnan S, and Datta SK (2001)
Enhanced resistance to sheath blight by constitutive expression of infection-related rice chitinase in
transgenic elite indica rice cultivars. Plant Science 160:405-414.
 Datta SK and Bouis HE (2000) The potential of biotechnology in developing nutrient-dense rice
varieties. Food and Nutrition Bulletin 21:451-456.
 Datta SK (2000) Transgenic rice: development and products for environmentally friendly sustainable
agriculture. In: Proceedings of the Challenge of Plant and Agricultural Sciences to the Crisis of
Biosphere on the Earth in the 21st Century, Watanabe K, and Komamine A (eds.), Landes BioScience.
Georgetown, USA 22:237-246.
 Tu J, Datta K, Khush GS, Zhang Q, and Datta SK (2000) Field performance of Xa21 transgenic indica
rice (Oryza sativa L.), IR72. Theor. Appl. Genet. 101:15-20.
 Tu J, Zhang G, Datta K, Xu C, He Y, Zhang Q, Khush GS, and Datta SK (2000) Field performance of
transgenic elite commercial hybrid rice expressing Bacillus thuringiensis -endotoxin. Nature
Biotechnology 18:1101-1104.
 Ye X, Al-Babili S, Kloti A, Zhang J, Lucca P, Beyer P, and Potrykus I (2000) Engineering the
provitamin A (-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science
287:303-305.
 Goto F, Yoshihara T, Shigemoto N, Toki S, and Takaiwa F (1999) Iron fortification of rice seed by the
soybean ferritin gene. Nature Biotechnology 17:282-286.
 Datta K. Baisakh N, Thet KM, Tu J, and Datta SK (2002) Transgenesis - breeding for multiple plant
protection. Theor. Appl. Genet. (in press).
A
B
C
Fig. 1. Transgenic rice showing protection against bacterial
blight with less lesion length compared with the control
under field conditions (A); with less lesion length against
sheath blight (B); and protection against yellow stem borer
(C).
Fig. 2. Indica rice seeds (polished) with beta-carotene in the
endosperm showing yellow color (left) compared with
control seeds.
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GOLDEN RICE:
INTRODUCING
THE
BETA-CAROTENE
BIOSYNTHESIS PATHWAY INTO RICE ENDOSPERM BY
GENETIC ENGINEERING TO DEFEAT VITAMIN A DEFICIENCY
Paola Lucca1, Xudong Ye1, Nandadeva Yakandawala1, Salim Al-Babili2, Peter Beyer2 and
Ingo Potrykus1
1 Institute for Plant Sciences, Swiss Federal Institute of Technology, CH-8092 Zurich.
2 University of Freiburg, Center for Applied Biosciences, D-79104 Freiburg.
e-mail: paola.lucca@irb.unisi.ch
Summary
Rice (Oryza sativa), a major staple food, is usually milled to remove the oil-rich aleurone
layer that turns rancid upon storage, especially in tropical areas. The remaining edible part
of rice grains, the endosperm, lacks several essential nutrients, such as provitamin A.
Recombinant DNA technology was used to improve its nutritional value in this respect. A
combination of transgenes enabled biosynthesis of provitamin A in the endosperm.
Keywords
rice, provitamin A, -carotene, deficiency, food fortification
Abstract
Vitamin A deficiency is a serious public health problem concerning about 124 million
children world-wide (Humphrey 1992). Oral delivery of vitamin A is problematic (Pirie
1983) mainly due to the lack of infrastructure, so alternatives are urgently required. Since
rice represents up to 80% of the daily calorie intake in Southeast Asia, the nutritional
enhanchment of the rice endosperm tissue would be highly desirable. Rice is usually milled
to remove the oil-rich aleurone layer that turns rancid upon storage, especially in tropical
areas. The remaining edible part of rice grains, the endosperm, is filled with starch
granules and protein bodies but lacks several essential nutrients for the maintenance of
health, such as caroteinoids and other micronutrients.
A complementary intervention to existing strategies for reducing micronutrient
deficiencies is to fortify food staples through plant breeding by recombinant and/or
conventional technologies (Bouis 2000; Bouis 2002). To obtain a functioning provitamin A
(beta-carotene) biosynthetic pathway in rice endosperm, we introduced in a single,
combined transformation effort the cDNA coding for phytoene synthase (psy) and
lycopene beta-cyclase (beta-lcy) both from Narcissus pseudonarcissus and both under the
control of the endosperm-specific glutelin promoter together with a bacterial phytoene
desaturase (crtI, from Erwinia uredovora under constitutive 35S promoter control). This
combination covers the requirements for beta-carotene synthesis and, as hoped, yellow
beta-carotene-bearing rice endosperm was obtained in the T(0)-generation (Ye 2000).
Additional experiments revealed that the presence of beta-lcy was not necessary, because
psy and crtI alone were able to drive -carotene synthesis as well as the formation of
further downstream xanthophylls. Plausible explanations for this finding are that these
downstream enzymes are constitutively expressed in rice endosperm or are induced by the
transformation, e.g., by enzymatically formed products. Results using Narcissus
pseudonarcissus as a model system led to the development of a hypothesis, our present
working model, that trans-lycopene or a trans-lycopene derivative acts as an inductor in a
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kind of feedback mechanism stimulating endogenous carotenogenic genes (Al-Babili
1999).
In conclusion, in a proof-of-concept study we have shown that it is possible to establish a
biosynthetic pathway de novo in rice endosperm, enabling the accumulation of provitamin
A. The work now in progress aims to increase the provitamin A amount by identifying the
metabolic rate-limiting bottlenecks in Golden Rice. The hygromycin-seelectable marker
gene was exchanged in the plasmids used for rice transformation since the selection
procedure for PMI was established in rice (Lucca. 2001). One further approach aims to
unify high-iron rice lines (Lucca 2001) with provitamin A lines because it is known that
provitamin A is capable of increasing the bioavailability of iron.
References
 Al-Babili, S., Hartung, W., Kleinig, H., Beyer P. (1999). “CPTA modulates level of carotenogenic
proteins and their mRNAs and affects carotenoid and ABA content as well as chromoplast structure in
Narcissus psedonarcissus flowers.” Plant Biology 1: 607-612.
 Bouis, H. E. (2000). “Enrichment of food staples through plant breeding: a new strategy for fighting
micronutrient malnutrition.” Nutrition 16(7-8): 701-4.
 Bouis, H. E. (2002). “Plant breeding: a new tool for fighting micronutrient malnutrition.” J Nutr 132(3):
491S-494S.
 Humphrey, J. H., West Jr., K. P., Sommer, A. (1992) WHO Bull. 70, 225.
 Lucca, P., R. Hurrell, Potrykus, I. (2001). “Genetic engineering approches to improve the bioavailability
and the level of iron in rice grains.” Theoretical and Applied Genetics 102(2/3): 392-397.
 Lucca, P., X. Ye, Potrykus, I (2001). “Effective Selection and Regeneration of Transgenic Rice Plants
with Mannose as Selective Agent.” Molecular Breeding 7(1): 43-49.
 Pirie, A. (1983). “Vitamin A deficiency and child blindness in the developing world.” Proc Nutr Soc
42(1): 53-64.
 Ye, X., S. Al-Babili, et al. (2000). “Engineering the provitamin A (beta-carotene) biosynthetic pathway
into (carotenoid-free) rice endosperm.” Science 287(5451): 303-5.
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GENE TECHNOLOGY: EXPANDING GENETIC DIVERSITY AND
ADDING VALUE TO RICE
C.P.Martinez, P.Moncada, J.Lopez, A.Almeida, G.Gallego, J.Borrero, M.C.Duque, F.
Correa, C.Bruzzone, J.Tohme, and Z.Lentini.
CIAT. A.A. 6713. Cali, Colombia. E-mail: c.p.martinez@cgiar.org
CENICAFE. A.A. 2427. Manizales.E-mail: pilar.moncada@cafedecolombia.com.
Summary
Molecular mapping of regions in the rice genome associated with traits of interest jointly
with genetic transformation is being used at CIAT to enhance the genetic base of cultivated
rice in Latin America. QTLs with positive effects on grain yield and yield components
from O.rufipogon were identified in O.sativa/O.rufipogon crosses. Useful traits from
O.glaberrima have been transferred to improved varieties through backcrossing.
Keywords
Wild rice species, transgressive segregation, molecular markers, genetic diversity, rice
genome.
Abstract
Introduction
The assessment of diversity at the DNA level is providing information on potential new
sources of variability for broadening crop genetic base, and for linking diversity in-situ with
ex-situ. Currently at the International Center for Tropical Agriculture (CIAT), molecular
mapping of crop genome regions associated with traits of interest, jointly with genetic
engineering are used to direct the modification of the rice genome for Latin America. A
collaborative project between CIAT and Genoplante, France, is being established to identify
genes of interest for Europe and Latin America in a joint effort. Transgenic rice plants
developed at CIRAD –IRD carrying transposons or T-DNA inserts will be increased at CIAT
and will be used to identify potential genes of interest, while increasing knowledge of gene
expression and gene function. There is considerable scope for genetic improvement in yield,
added value traits including quality characteristics such as for human nutrition, as well as
resistance to pests and diseases and adaptation to environmental stresses. The trends in
biotechnology research integrate screenings at the phenotype and genotype in a way that is
enhancing a better used of the natural diversity already present in wild species, which was
overlooked in the past. A collaborative project with Cornell University, and WARDA aimed
at characterizing and utilizing wild rice species for the improvement of cultivated rice for Latin
America was initiated at CIAT in 1994. A set of QTLs associated with yield increase from O.
rufipogon was identified in interspecific hybrids between O. sativa / O. rufipogon. An
advanced breeding program has been established to broaden the genetic base of breeding lines
carrying these QTLs while adapted to the various Latin American ecosystems. A similar
scheme is being evaluated to combine QTLs analysis for introgressing other traits of interest
from the wild genome.
Materials and Methods
Two improved rice cultivars (Bg90-2 and Caiapo) were crossed to O. rufipogon and two
backcrosses to the improved cultivars were made. The resulting BC2F1 plants were
transplanted and evaluated based on negative phenotypic selection for undesirable agronomic
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traits. The best individuals were selected to generate BC2F2 seed; over 300 BC2F2 families
were derived per cross combination. Families derived from the Bg90-2/O.rufipogon cross were
transplanted and evaluated in a replicated yield trial in CIAT-Palmira whilst families derived
from the Caiapo/O.rufipogon cross were direct seeded and evaluated under upland conditions
in Villavicencio, Colombia. Data on 12 agronomic traits, including grain yield, were taken.
Results
Transgressive segregation was observed for grains yield and yield components. In the cross
Bg90-2/O.rufipogon, and compared to BG90-2, 16% of the BC2F2 families showed higher
grain yield while 22% of them had higher 1000-grain weight, 48% showed higher total grain
yield per plant, 43% had longer panicles, and 26% had increased grain length. The BC2F2
families were screened using 140 RFLP markers from the rice molecular framework linkage
map selected at 10-20 cM intervals and 78 microsatellite markers, developed at Cornell
University. Linkage analysis was conducted using Qgene with a threshold value of 0.01.
Molecular markers RM13, and RM242 located on chromosomes 5 and 9,respectively, were
associated with alleles derived from O.rufipogon affecting grain yield positively. Out of 69
QTLs identified in the cross Bg90-2/O.rufipogon 18(26%) were trait improving alleles derived
from O.rufipogon and showed no detectable negative effect on any measured trait. In the case
of the Caiapo/ O.rufipogon cross, and based on single-point, interval, and composite interval
mapping, two putative O.rufipogon derived QTLs were detected for yield, 13 for yield
components, four for maturity duration and six for plant height. From a breeding perspective,
these QTLs can be used immediately. Advanced breeding lines from the Bg90/O.rufipogon
cross having either consistent yield advantage or longer/slender translucent grain type than any
of the parents have been identified and sent to national programs for testing. In addition,
advanced lines resistant to rice blast and Rhizoctonia solani have been identified and NILs are
being developed to better assess the effects of QTLs.
O.glaberrima has been shown to be highly resistant to rice stripe necrotic virus, a disease
reported in Colombia for the first time in 1991. This resistance has been introgressed into
cultivated rice after 2-3 backcrosses to improved cultivars. As the knowledge of genes and
genomes increases, using molecular genetics and gene technology can greatly improve the
efficiency of breeding by overcoming heretofore essentially intractable limits to genetic
diversity.
References
 McCouch SR, Thomson MJ, Septiningsih EM, Moncada P, Li J, Xiao J, Ahn SN, Tai T, Martinez C,
McClung A, Lai XH, Moelpojawiro S, Yuan LP, Moon HP,Guimaraes E, Tohme J. 2001. Wild QTLs
for rice improvement. In: Rockwood WG, editor. Rice research and production in the 21st century:
Symposium honoring Robert F. Chandler, Jr. Los Baños (Philippines): International Rice Research
Institute. p. 151-169.
 Moncada P, Martínez C P, Borrero J, Chatel M, Gauch Jr H, Guimaraes E, Tohme J, McCouch SR.
(2001) Quantitative trait loci for yield and yield components in an Oryza sativa x Oryza rufipogon
BC2F2 population evaluated in an upland environment. Theoretical Applied Genetics 102: 41-52.
 Tanksley, S. D. and Nelson, J. C. (1996) Advanced backcross QTL analysis: a method for the
simultaneous discovery and transfer of valuable QTLs from unadapted germplasm into elite breeding
lines. Theoretical Applied Genetics 92: 191-203.
 Thomson, MJ. TH Tai, AC McClung, MH Hinga, KB Lobos, Y.Xu, C.Martinez, and SR McCouch.
(2002) Mapping quantitaive trait loci for yield, yield components, and morphological traits in an
advanced backcross population between Oryza rufipogon and the Oryza sativa cultivar Jefferson
(Submitted to Theor. Appl. Genet).
 Xiao J, Li J, Grandillo S, Nag Ahn S, Yaun L, Tanksley S and McCouch S. (October 1998)
Identification of Trait-Improving Quantitative Trait Loci Alleles From a Wild Rice Relative, Oryza
rufipogon. Genetics 150:899-909.
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