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Session I - Rice: traditional breeding and biotechnology innovation
Session I
Rice: traditional breeding and biotechnology innovation
PLENARY LECTURES
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Session I - Rice: traditional breeding and biotechnology innovation
RICE FOR TOMORROW : PRIORITIES AND NEEDS
Jean Chataigner, agro-economist INRA
Summary
For European rice researchers, it is important to organise a debate on the suitability
between researches and rice economy needs in the world in European and Mediterranean
areas. To introduce this debate we will examine : changes in consumption and production
world rice, main features of the diversity in Mediterranean and European areas and some
questions about rice researches.
Key words
rice, economy, research, world, Europe.
Abstract
1. Rapid slowdown in world rice consumption and consequences
Since sixties, the green revolution, in a cold war context, faced demographic explosion
and, by improving food consumption, contributed to develop conditions of the
industrialization in Far East countries. Now, several factors, such as income growth,
changing in lifestyles and urbanization, are contributing to the rapid slowdown in world
rice consumption through the diversification in food consumption patterns. For many
Asian countries rice has become an inferior good i.e. rice consumption declines as income
rises. For some of them, the total consumption is stabilized (Thailand) or decreasing
(Taiwan, Japan, South Korea…)
In contrast, for the same reasons of diversification in food consumption patterns, rice
consumption rises elsewhere in the world ;
Given the importance of Asian countries, the global world rice consumption is increasing
more and more slightly : 60% from 1980 to 2000 and only 20% expected to 2020.
Consumption per capita is expected to 59 k. in 2020 from 65,7 in 2000 and 55 in 1970.
Main outcomes are expected :
- diversification involves more quality differentiation
- given absence of rice competitiveness for feed production, areas harvested only
for human consumption is projected to increase slightly and may decline if yield
annual growth rate achieves 1% (0,93 expected to the next ten years).
- Stabilization of world rice price excepted weather accident.
2. Post green revolution challenges
Green revolution was supported by strong food security policy and important state
investments. In these conditions biological innovations applied to homogeneous labour
farming systems, have been efficient. But they have been very efficient only where a good
water management existed. Spectacular results were observed elsewhere each time that a
clear policy, investment and good management of development have been applied, i.e. in
Russia in seventies, in Egypt in the last fifteen years, in developed countries where a
consistent farm income protection system exists.
It is too early to appreciate the influence of the multilateral trade agreement (WTO). Its
impact remains low for developing countries.
But for all Asian countries the competition between cities and rural areas for water using is
becoming very hard.
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More important is the dramatic change in farm labour with the industrialisation. From 1980
to 1990, in Indonesia, for example, the farm labour per hectare in hours decreased from
1600 to 800.
Main outcome
- These important changes are leading to spend a particular attention to new
emerging farming systems patterns, their competitiveness, and how to
accompany their modernization : mechanization, crop management, training, …
3.Main features of the European and Mediterranean area
In the ecological Mediterranean area from Portugal to Eastern Europe and Near East,
characterised by traditional japonica production, the main change is the increasing of total
and per capita consumption, accompanied by the diversification of qualities. These areas
are becoming one of the most important places of the international market.
But, at the same time, the production remains scattered, in many small areas of production.
However some trends are appearing, for example in the performance with high yield in the
Southern and standardisation in the Northern. Nevertheless, exception made for Egypt, rice
policies remain uncertain.
Main outcome
- An important effort of research cooperation, through the FAO medrice network
which offers opportunity to adapt production to market, especially with the
diversity of genetic resources.
4. Some questions for European Rice Research
According to a recent evaluation there are about 120 to 130 researchers in Europe, and may
be 200 with our partners in other parts of the Mediterranean area, associated in the medrice
network. High level of science is recognised in biotechnology, breeding, technology….and
others. European research is also a partner of international programs.
Is it possible to produce an economic rice feed production to accompany the
transformation of the diet in Asian countries ?
Which fields of competence is it necessary to develop first in Europe, taking into account
the potential existing ?
How to organise relations and financing between research, growers and industry to obtain
optimal efficiency in European rice economy ?
References
 CHATAIGNER J. 1995. Riz du monde : consommations de pauvres, consommations de riches. INRA
Sciences Sociales N°4
 Chan Ling Yap 1994. Supply and demand for rice in the medium and longer term. Eighteenth session of
the international rice commission. FAO
 WAILES, E.J., G.L. CRAMER, E.C. CHAVEZ, and J.M. HANSEN 2000. Arkansas Global Rice
Model :International Baseline Projections for 2000-2010. University of Arkansas, Agricultural
Experiment Station Report 200.
 CHATAIGNER J. Ed., 1997 Rice quality : a pluridisciplinary approach. Proceedings of the
international Symposium held in Nottingham, UK, 24-27 November 1997. Cahiers Options
Méditerranéennes Vol. 24, n°3.
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CONVENTIONAL AND BIOTECHNOLOGICAL APPROACHES FOR
DEVELOPING DISEASE AND INSECT RESISTANCE RICE
Gurdev S. Khush
Rice crop suffers serious yield loses from the attack of diseases and insects. According to
FAO estimates losses caused by diseases and insects exceed 25% of total production.
Most important diseases of rice are; blast, bacterial blight, sheath blight, tungro disease
caused by a complex of two viruses and grassy stunt virus. Most damaging insects are;
brown planthopper, green leafhopper, gall midge and three species of stem borers e.g.
striped borer, yellow stem borer and white stem borer. Major emphasis has been put on
developing rice germplasm with multiple resistance to these diseases and insects at the
International Rice Research Institute (IRRI) in the Philippines. Large germplasm
collections maintained in the germplasm bank at IRRI were screened for resistance to these
diseases and insects and donors for resistance were identified. Through genetic analysis of
these donors, genes for resistance were identified. These donors were tall traditional and
low yielding cultivars or landraces. Genes for resistance from these donors were
transferred to high yielding but susceptible varieties or breeding lines through conventional
breeding approaches. Breeding lines with resistance to different diseases and insects were
intercrossed and resistance to several diseases and insects was incorporated into the same
variety. Thus numerous varieties and breeding lines with multiple resistance were
developed. The germplasm with multiple resistance was shared with the national rice
improvement programs. Several multiple resistant varieties have been widely grown in
Asia and elsewhere. For example IR36 with resistance to blast, bacterial blight, tungro,
grassy stunt, brown planthopper, green leafhopper, gall midge and stemborers was planted
to 11 million hectares of rice land during 1980s. It became the most widely grown variety
of rice ever. IR 64, another multiple resistant variety is now grown to about 10 million
hectares of rice land.
We are now applying innovative techniques of cellular and molecular biology for
developing disease and insect resistant varieties. Many genes for disease and insect
resistance have been tagged with molecular markers. Molecular marker aided selection
(MAS) is being used to move genes for resistance from one varietal background to the
other. MAS has also been used to pyramid several genes for resistance to bacterial blight
into the same variety. Wide crosses have been accomplished between elite breeding lines
and varieties on one hand and several wild species on the other through embryo rescue
technique. These wide cross hybrids have permitted the transfer of useful genes for
disease and insect resistance from wild species to cultivated rice.
Genetic engineering techniques have been employed to introduce novel genes for
resistance into the elite germplasm. For example Bt gene from Bacillus thuriengensis was
introduced in several cultivars and into restorer parents of rice hybrids. Transgenic rices
with Bt show high level of resistance to stem borers and leaffolders. Similarly a chitinase
gene was introduced into rice thorough genetic engineering and transgenic rices are
moderately resistant to sheath blight.
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Session I - Rice: traditional breeding and biotechnology innovation
RICE BIOTECHNOLOGY RESEARCH IN INDIA: FUTURE NEEDS
Usha Barwale Zehr and Bharat Char
Mahyco, Sardar Patel Road, Jalna, India
Uzehr@mahyco.com, brchar@lsrc.mahayco.com
Summary
Rice biotechnology research in India in the future must focus on sustainable food security.
Asia accounts for 90% of the world’s rice production and consumption. Although there is
some reduction in per capita rice consumption in Asia, the Indian trend is still positive and
the likely demand for rice is likely to go up by 70% by 2030. Biotechnology approaches
offer research tools that can address the more complex problems, such as those associated
with abiotic and biotic stresses.
Keywords
Rice transformation, Drought stress, MAS
Abstract
The gains achieved in rice production in India in the 1960s and 1970s cannot be replicated
due to limited land resources and decreasing soil health. Sustainability in rice production
can only be achieved by addressing the major yield constraints imposed by drought, floods,
salinity, soil degradation and pests and diseases. Addressing these problems requires a coordinated approach using the best available research tools at our disposal. These include
the isolation of novel genes that confer tolerance or resistance to particular abiotic/biotic
stresses as well as the use of molecular markers in breeding programs to achieve greater
effectiveness and use of transgenics. Hybrid rice in India is at an introductory state and
has promise to enhance production by 20+ %. Molecular markers have been used
extensively in hybrid rice breeding program to ensure genetic purity as well as in breeding
programs for improving germplasm.
Using naturally tolerant crop species such as sorghum and pearl millet, we have identified
multiple genes which are induced during drought conditions. Based on sequence analysis
and evaluation in model species, potential candidate genes that confer stress tolerance will
be deployed in rice through genetic transformation. Similarly, from diverse microbial
populations, genes that enhance phosphate utilisation have also been isolated. Successful
utilisation of these technologies would allow us to cultivate rice in environments, which
are less than optimal.
Gene transformation systems in rice have been very well established for a number of years.
However, given consumer sensibilities, future transgenic products must make use of
marker-free systems which eliminate antibiotic resistant selectable markers from a plant
carrying a useful transgene. This would also remove the cause of concern of an antibioticresistance gene spreading amongst wild relatives of rice through pollen dispersal. We have
established an efficient Agrobacterium-mediated marker-free transformation system in rice
and other crops.
The brown plant hopper (BPH) biotype of the Indian subcontinent is a highly destructive
pest of rice causing substantial yield losses. We have used PCR based DNA markers to tag
genes for BPH resistance. A RAPD marker has been developed for a gene conferring
resistance to the Indian biotype of BPH. Microsatellite and ISSR markers for fine mapping
of this gene are under development and map-based cloning of the gene has been initiated.
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Pyramiding known BPH resistance genes is also in progress to achieve durable resistance.
Similar efforts are also on for other disease resistance genes.
Transgenic and non-transgenic biotechnology tools are available and will help in achieving
sustainable rice production, thus meeting the demand of the growing population. The focus
of the next 2-3 years has to be in areas of immediate crisis such as combating
environmental stresses and pests/diseases, but in the coming 5-10 years the focus will shift
to added nutritional value.
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Session I - Rice: traditional breeding and biotechnology innovation
AGRONOMICAL CONSTRAINTS IN RICE CULTURE: ARE THERE
ANY POSSIBLE SOLUTIONS FROM BIOTECHNOLOGY?
A. Ferrero1, M. Tabacchi2
1
Dipartimento Agroselviter, Via Leonardo da Vinci 44, 10095 Grugliasco (Italy),
ferrero@agraria.unito.it
2
Ente Nazionale Risi, Strada per Ceretto 4, 27030 Castello d’Agogna (Italy), m.tabacchi@enterisi.it
Summary
The most important constraints that affect rice productivity include poor crop establishment, water
scarceness, biotic and environmental stresses, low nutrient efficiency, low yielding capacity and
unsatisfactory grain quality. Both traditional and biotechnological research, should deal with all these
issues and develop new high yielding rice varieties requiring few inputs.
Keywords: rice establishment; biotic stress; soil and environmental stress, nutrient efficiency.
Abstract
By the year 2025 it will be necessary to produce 50% more rice. This goal has to be achieved despite
the numerous agronomical, economical and social issues that can affect crop productivity. A great
challenge for the research will be that of increasing rice yield and quality by using fewer inputs, less
land, less water and less labour. It is also necessary to reach these objectives while by saving natural
resources, lowering food cost supplies to consumers and alleviating poverty. The aim of this paper is
to examine the main agronomical constraints which affect rice productivity and require a possible
solution through the biotechnological research.
Crop establisment
Most water-seeded rice usually shows a poor crop establishment. The causes that can affect crop
establishment are mainly related to the anaerobic conditions in which germination occurs and the low
temperatures during the planting period. The constraint of the poor crop establishment could be
overcome by planting rice in dry soil, whenever possible, and developing new varieties with early
vigour and good tolerance to low temperatures during germination.
Environmental stress (low and high temperatures, water availability etc.)
As rice plants originate from sub-tropical and tropical zones, they are easily damaged by low
temperatures at any growth stage from germination to ripening (Kaneda & Beachell, 1974). For
example, if the heading of rice plants is delayed due to cold temperatures at the seedling stage, they
will also be subjected to a cool autumn and this results in poor ripening. Furthermore, their pollen
cells die due to freezing at the meiosis stage, they will become sterile.
Even high temperatures may result in crop damage, which can vary according to the plant growth
stage; the worst negative effect on rice yield is caused by spikelet sterility.
Main water problems are related to a looming water shortage, uneven distribution, nitrate and
pesticide pollution, waterlogging in heavy soils and the increasing costs of irrigation systems. The
water problems can be tackled by developing more efficient water management strategies and
providing new rice varieties that are more suitable for various water management conditions. The
availability of varieties with a high early vigour could allow the seeding of early rice directly in the
soil and the use rainwater in rainfed systems in much more efficient way. In these conditions rice can
withstand weed competition and sudden submergence by early rains. New varieties that are suitable
for a reduced use of water are also required in irrigated systems. The availability of short-cycle and
high-yielding rices could be a way of significantly curbing the irrigation water in a continuously
flooded cultivation. A more consistent reduction of water consumption could be obtained by
introducing profitable varieties that are suitable for discontinuous irrigation in all rice cultivation
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areas. These water management conditions could also contribute to the mitigation of methane
emission due to ricefield submergence. The new varieties should however also show a great capacity
to suppress weed growth and tolerate soil salinity, as the cultivation in non-flooded conditions could
result in an increased competition of the weeds and a rise of soil salinity due to upward salt migration.
Pest, disease and weed control
According to Oerke et al. (1994), the average rice losses in the world caused by diseases, animal pests
and weeds, despite given crop protection, account for about 50% of the crop potential. The main rice
noxious organisms are brown planthopper (Nilaparvata lugens) (BPH), Striped stemborers (Chilo
suppressalis) among the insects and blast (Pyricularia oyzae), tungro virus (Nephotettix virescens)
and sheath blight (Rhizoctonia solani) among the diseases and Echinochloa spp., Cyperus difformis
and C. iria, Ischaemum rugosum, Monochoria vaginalis and weedy rice forms, among the weeds.
All these species are usually controlled with pesticides. The use of these products may result in the
appearance of resistant species, cause environmental pollution and risk disrupting the precarious
balance of the natural enemies to pests. A solution to these issues could be the development of rice
cultivars that are resistant to pests and diseases, highly competitive against weeds, with allelopathic
traits and tolerant to safe and wide spectrum herbicides (Christou, 1994). The use of these varieties
combined with prophylactic measures could be a sound strategy to prevent damage or their spreading
to rice.
Lodging
Lodging resistance has been a key target trait to rise yield potential and is associated with many
component traits such as plant height, stem strength, thickness, etc. Lodging-resistant rice cultivars
usually show slow grain filling when nitrogen is applied in large amounts.
Soil stress
Adverse soil conditions create serious problems in rice cultivation, especially in developing countries
(Matsuo et al., 1997). From the physiological point of view, soil stress can be divided into excess of
toxic substances (aluminium, reduced iron, salts, etc.) and deficiency of mineral nutrients (zinc, iron,
phosphorus, etc.). Soil stress problem should be tackled by means of an interdisciplinary approach
with soil chemists, plant physiologists, plant breeders and geneticists.
Low yielding ability and nutrient use efficiency
Yield-related or yield-determining traits, such as plant-type and photosynthesis-related traits, could be
included in the category of yield components that could be improved through biotechnological
interventions. The main targets of this work are the increase of the final yield of the rice crop through
the modification of the source and sink capacity of rice plants and the improvement of nutrient use
efficiency, especially nitrogen. Another research goal for rice and as with many other crops, is the
development of varieties that are able to fix their own nitrogen; main aim of these studies is that of
reducing production costs and environmental pollution.
Grain quality problems
Reduced or variable milling yield, grain fissuring, grain shedding and non-contemporaneous maturity
are all rice grain characteristics that could be improved through the use of conventional and
biotechnological breeding methods. All of these problems are also related to other agronomic
constraints, such as cold temperature and lodging, but are sometimes closely linked to the genetic
features of the rice varieties.
References
 Christou P., 1994. Biotechnology of Food Crops – Rice Biotechnology and Genetic Engineering. Technomic
Publishing Company, Lancaster (USA), 201 pages.
 Kaneda C., Beachell H.M., 1974. Response of Indica-Japonica hybrids to low temperatures. SABRAO J. 6:17-32.
 Matsuo T., Futsuhara Y., Kikuchi F., Yamaguchi H., editors (1997). Science of the Rice Plant – Volume Three
(Genetics), Food and Agriculture Policy Research Center, Tokyo, Japan, 1008 pages.
 Oerke E.C., Dehene H.V., Schoenbeck F., Weber A., 1994. Rice losses. In “Crop Production and Crop Protection.
Estimated Losses in Major food and Cash crops”. Published by Elsevier Science B.V. Amsterdam, 808 pages.
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REDUCING
THE
NEED
OF
RICE
FOR
SYNTHETIC
NITROGENOUS FERTILIZERS: A MAJOR CHALLENGE FOR
BIOTECHNOLOGY
Edward C. Cocking, Centre for Crop Nitrogen Fixation, University of Nottingham,
Nottingham NG7 2RD, U.K.
Tel: +44 115 9513056; Fax: +44 15 9513240; E-mail: edward.cocking@nottingham.ac.uk
Key words
Azohrizobium caulinodans; Gluconacetobacter
endophytic colonization; rice.
diazotrophicus;
nitrogen
fixation;
Synthetic nitrogenous fertilizer is the single most important purchased material input in
rice production. Rice needs 1 kg of nitrogen to produce 15-20 kg of grain. Unless the
biotechnological challenge is met, increased demand for rice will entail increased
application of fertilizer N. To achieve food security and protection of the environment the
requirement for fixed nitrogen must be increasingly met by biological nitrogen fixation
rather than industrial nitrogen fixation. This paper reviews the prospects of achieving in
planta nitrogen fixation in rice.
The Challenge for Biotechnology
Nitrogen is an essential element for the crops that feed the World’s 6 billion people. But a
surfeit of nitrogen such as nitrates in water systems and oxides of nitrogen in the
atmosphere from chemically produced synthetic nitrogenous fertilizers is harming
ecosystems and threatening public health. An important reason for the success of the
Green Revolution in the last century was the extensive use of such nitrogenous fertilizers –
between 1961 and 1996 their use more than quadrupled, rising from 31 million metric tons
to 135 million metric tons. In 1992 the International Rice Research Institute organized a
workshop to assess the feasibility of developing nitrogen-fixing capability in rice (1). The
experts at this meeting were of the opinion that much progress could be made using
molecular and cell biology to enhance the level of nitrogen fixation during rice cultivation
and that this would probably be achieved through an endophytic process. The great
progress in our understanding of nitrogen-fixing symbiosis suggested that further exciting
discoveries in plant science would assist in reaching this goal.
What has already been achieved
The most efficient systems of endophytic biological nitrogen fixation are the rhizobialegumes and Frankia-woody plant non-legume symbioses in which bacteria fix nitrogen
within specialised plant organs, called nodules, resulting in considerable assimilation of
fixed nitrogen by the host plants. In comparison certain diazotrophs (called associative
diazotrophs) colonize mainly the surface of plant roots, including rice, where they are in
competition with other rhizosphere root inhabiting microorganisms, and very little of the
biologically fixed nitrogen benefits the plant. However, because of the inefficiency of
such associations, it is now a primary aim to establish endophytic nitrogen-fixing
associations in rice and other cereals and major non-legume crops in which diazotrophic
bacteria grow and fix nitrogen within the plant. Large populations of endophytic
diazotrophs have been found in sugarcane, a member of the Gramineae which also includes
rice, maize and wheat. These bacteria intercellularly colonize the plant, including the
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xylem, and seem to be major contributors to the observed high levels of biological nitrogen
fixation (2). This suggests that endophytic nitrogen fixation, without nodule formation, is
likely to be possible in other members of the Gramineae such as rice, wheat and maize and
in other non-legume crops.
Major Future Challenges
Using the lacZ reporter gene it has been shown in rice inoculated with Azorhizobium
caulinodans that the xylem of their roots is colonized by azorhizobia (3). It has also been
shown that the xylem of the root system of the model non-legume Arabidopsis thaliana is
also colonized when inoculated with Azorhizobium caulinodans (4). Using the ability to
induce xylem colonization by azorhizobia in rice and Arabidopsis investigations can now
be undertaken of factors influencing the extent of xylem colonization, and also the extent
to which inter and intra cellular endophytic colonization by azorhizobia and other
diazotrophs, including Gluconacetobacter diazotrophicus (endophytic in sugarcane), will
provide a niche for non nodular symbiotic nitrogen fixation in rice (5). The cells of
legume nodules are internally colonized by rhizobia present intracellularly in vesicles in
the cytoplasm. The presence of rhizobia in these vesicles provides a symbiotic niche
within cells for the fixation of nitrogen by enzymes present in the rhizobia, with energy
supplied from the plant’s photosynthesis. The major challenge for biotechnology is to
establish nitrogen fixing bacteria in vesicles in the cytoplasm of cells of the meristem and
cortex of roots of non-legume crops, including major cereals such as rice. If accomplished
this would provide an opportunity for endophytic symbiotic nitrogen fixation comparable
to the type of intracellular nitrogen fixation that occurs naturally in cells of legume root
nodules.
References
 Khush GS, Bennett J Editors 1992. Nodulation and nitrogen fixation in rice: potential and prospects.
Los Baňos (Philippines), IRRI.
 Boddey RM (1995) Biological nitrogen fixation in sugarcane: a key to energetically viable biofuel
production. Critical Reviews in Plant Sciences 14: 263-279.
 Gopalaswamy G, Kannaiyan S, O’Callaghan KJ, Davey MR and Cocking EC (1999) The xylem of rice
(Oryza sativa) is colonized by Azorhizobium caulinodans. Proc. R. Soc. London B. 267: 103-107.
 Stone PJ, O’Callaghan KJ, Davey MR and Cocking EC (2001) Azorhizobium caulinodans ORS571
colonizes the xylem of Arabidopsis thaliana. Molecular Plant-Microbe Interactions 14: 93-97.
 Cocking Edward C. Xylem colonization of rice and Arabidopsis by Azorhizobium caulinodans
ORS571. pp 141-148, In: the Quest for Nitrogen Fixation in Rice (Editors J.K. Ladha and R.M.
Boddey) 2000, IRRI.
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CAPACITY OF PRODUCTION OF RECOMBINANT HUMAN
INTERFERON-GAMMA PROTEIN IN TRANSGENIC RICE
Tzy-Li Chen1, Ning-Sun Yang1, Yi-Ling Lin2 and Ming-Tsair Chan1*
1. Institute of BioAgricultural Sciences, Academia Sinica, Taipei, 115, Taiwan
2. Institute of BioMedical Sciences, Academia Sinica, Taipei, 115, Taiwan
E-mail: mbmtchan @ ccvax.sinica.edu.tw
Summary
The expression vectors harboring a human IFN- cDNA driven by various promoters were
successfully transferred into rice by Agrobacterium-mediated transformation to evaluate
the expression of the recombinant human interferon-gamma (IFN-γ). The results of
ELISA and in vivo anti-virus analyses suggest that recombinant IFN- proteins expressed
in transgenic rice are biologically functional.
Key words
recombinant human interferon-gamma, transgenic rice plants, transgenic rice suspension
cells.
Abstract
Engineering plants for the production of valuable proteins is now known by name of
“molecular farming”. In recent years, production of mammalian pharmaceutical proteins
in plants has also been drawn attention in many research fields. IFN-, a potent cytokine,
has been shown to moderate some biological activities including anti-proliferative,
immuno-regulatory and anti-viral properties. IFN- was aggressively used for treatment of
HIV or other viral infections. A number of clinical studies have indicated that recombinant
human IFN- may be useful in treatment against human malignant mesothelioma, oral
submucous fibrosis, ovarian cancer, granulomatous slack skin, multiple myeloma, atopic
dermatitis. Moreover, a combination of cyclosporin-A (CsA) and recombinant IFN-
could induce significant apoptosis in four types of human gastric carcinoma cells.
The IFN- cDNA was constructed into various vectors and driven by different promoters.
These promoters include constitutive expression promoter CaMV35S, maize ubiquitin and
the tissue specific promoters, rice glutelin and riceα-amylase3 promoter (Chan and Yu,
1998a, 1998b). Apart from the use of various promoters, to enhance the IFN- protein
expression level, we introduce the human or riceα-amylase 3 signal peptide at the 5’ end of
the human IFN- sequence, and added a endoplasmic reticulum (ER) retention signal,
KDEL, at the 3’ end of the IFN- sequence to stabilize the translated protein. For this
purpose, we have constructed 18 plasmids including various promoters, leader sequences,
targeting sequences, with or without KDEL.
After Agrobacterium-mediated transformation (Chan et al., 1993), the rice cultivar TNG67
transformed explants were selected by hygromycin. We got about 1000 putative rice
transgenic plants. Southern and Northern blot analyses have confirmed these transgenic
plants. Moreover, the results of ELISA showed that the IFN- production level was ranged
from 0.4-1.3 mg IFN- per g rice leave.
We also set up two expression systems of rice suspension cells. The IFN- gene in the rice
suspension cells was driven by maize ubiquitin or rice α-amylase3 promoter, with a rice α-
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Session I - Rice: traditional breeding and biotechnology innovation
amylase3 signal peptide at the 5’ end of IFN- gene and carried a 6 his-tag sequence at the
3’ end of this coding region (Chan et al., 1994). Northern blot analysis indicated that
sucrose starvation treatment strongly induced the expression of IFN- gene inα-amylase3
promoter derived lines. The ELISA results also confirmed the highly expression levels.
The results of ELISA showed that IFN- protein levels in suspension medium ranged from
0.06-17.4 g IFN- per ml culture medium and around 950 g IFN- per g rice cell in
transgenic cell lines. The recombinant IFN- proteins were purified by metal-chelate
affinity chromatography (Ni-NTA). The western blot analysis showed the recognition of
anti-6his antibody of a 25 kDa molecular weight of rice synthesized recombinant IFN-.
Furthermore, the purified IFN- protein also showed a positive anti-viral activity.
Most therapeutic proteins are glycoproteins. The N-glycosylation is often essential to
maintain their stability and biological activity of recombinant glycoproteins. Although
plants inherit the N-glycosyltransferase, the N-linked glycan profile was different between
plants and mammals. This might affect the application of the expressing mammalian
glycoproteins in transgenic plants. Keeping this in view, we reconstructed a human -1,4
galacosyltransferase (hGT) cDNA as well as an IFN- cDNA. In the near future, we hope
to transfer this gene into rice and analyze the glycosylation profile and biological activity
of hGT modified IFN-.
References
 Chan MT, Chang HH, Ho SL, Tong WF, Yu SM (1993) Agrobacterium-mediated production of
transgenic rice plants expressing a chimeric alpha-amylase promoter/beta-glucuronidase gene. Plant
Mol Biol 22: 491-506
 Chan MT, Chao YC, Yu SM (1994) Novel gene expression system for plant cells based on induction
of alpha-amylase promoter by carbohydrate starvation. J Biol Chem 269: 17635-17641
 Chan MT, Yu SM (1998a) The 3' untranslated region of a rice alpha-amylase gene mediates sugardependent abundance of mRNA. Plant J 15: 685-695
 Chan MT, Yu SM (1998b) The 3'-untranslated region of a rice alpha-amylase gene functions as a
sugar-dependent mRNA stability determinant. Proc Natl Acad Sci USA 95: 6543-6547
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RICE RESEARCH IN PORTUGAL: PRESENT AND PERSPECTIVES
M.M. Oliveira(1,2), M. Vasconcelos(1,3), A.P. Farinha(1,4), S. Negrão(1), M. Romera(1),
C.P.Ricardo(1,5), M. Pagés(1,4), S.K. Datta(1,3)
(1) IBET/ITQB, Quinta do Marquês, 2784-505 Oeiras, Portugal
(2) Dep. Biologia Vegetal, Fac. Ciências de Lisboa, 1749-016, Lisboa, Portugal
(3) Plant Breeding, Gen. & Biochemistry Div., IRRI, P.O. Box 933, 1099 Manila,
Philippines
(4) Dept. Genetica Molecular, CSIC, Jordi Girona Salgado 18, 08034 Barcelona, Spain
(5) Instituto Superior de Agronomia, Tapada da Ajuda, 1349-017 Lisboa, Portugal
M.M.O.:
mmolive@itqb.unl.pt;
M.V.:
m.de-vasconcelos@cgiar.org;
A.P.F.:
apfarinha@yahoo.com.br; S.N.: snegrao@itqb.unl.pt; M.R.: maidarb@yahoo.com; C.P.R.:
ricardo@itqb.unl.pt; M.P.: mptgmm@cid.csic.es; S.K.D.: sdatta@cgiar.org
Summary
For a long time almost no investments were made in Portugal for the improvement of
traditional rice varieties. Presently, only imported seed, with higher yields and resistance to
diseases, is being used. We aim to improve rice for Portugal and Mozambique and develop
molecular studies of rice response to abiotic stress. To reach such goals and to give training
to researchers, we are establishing the necessary collaborations with teams working in the
field.
Keywords
Abiotic-stress response, Genetic engineering, Iron-deficiency, Nutritional rice, Rice
biotechnology.
Abstract
Portugal is the EU country with higher rice consumption per capita: 15.3 kg/year (17.3 in
2000). Various researchers and Institutions in Portugal have conducted work in terms of
field testing, weed and disease control and germplasm conservation; however, rice research
has not been a major priority in what concerns plant improvement. Portuguese traditional
rice is medium grain (japonica). In spite of the grain quality of several traditional varieties,
these have been gradually substituted by imported varieties with higher yields and
increased resistance to pests and diseases. Among the imported varieties, indica type rice is
also being introduced, mainly for Southern regions, with much acceptance by the farmers
due to higher yields. Presently Portugal produces 60 % of what it consumes, in an area of
about 27,000 ha (maximum allowed 34,000 ha), mainly distributed on 4 river beds
(Mondego, Tejo, Sorraia and Sado). The total production/year reaches 162,510 ton (6.01
ton/ha). We are still importing 50,100 ton from EU and 30,000 ton from outside EU.
Due to historical reasons, Portugal has interest in collaborating with ex-colonies in areas
that extend from research to development. Mozambique is one of the countries deserving
more attention due to the very poor nutritional conditions of the population.
Financial support from the Foundation for Science and Technology (FCT) and from the
Institute for International Scientific and Technological Cooperation (ICCTI), allowed our
Institute (IBET/ITQB) to start working in this area in 2000. This has been done in
collaboration with IRRI for the improvement of rice in Portugal and Mozambique using a
biotechnological approach, and with CSIC for fundamental studies addressing rice
response to abiotic stress. Within these collaborations, we aim to: (1) form researchers with
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Session I - Rice: traditional breeding and biotechnology innovation
knowledge in the area of rice molecular biology and biotechnology; and to (2) develop rice
varieties with improved nutritional quality for Mozambique. In a recent visit to rice
production areas in Mozambique, and after meetings with Mozambican rice researchers
and government staff, some priority areas have been identified which include the
improvement of rice management and rice varieties and disease control (Fig. 1).
Salinity, dehydration and cold are some constraints affecting rice culture both in Portugal
and Mozambique, what justified the initiation of fundamental studies addressing these
problems. Using the large experience developed at CSIC, on maize abiotic stress response,
a parallel for rice is being carried out, aiming to identify genes involved in the rice
response to stress. The genes focused include LEA genes, transcription factors and others
that may, or may not, share homology with genes previously described.
At IRRI, work is presently being focused on the improvement of iron content of a
commercially important rice variety, IR68144. Iron deficiency is the most common
nutritional problem in the world, since more than 3.5 billion people are anaemic. The iron
content in whole rice is insufficient to supply the recommended dietary daily allowances,
moreover, after milling, the iron content in the seed is dramatically reduced to very low
values. By genetic engineering, the ferritin gene, driven by an endosperm specific
promoter, was introduced in rice, aiming at enhancing iron accumulation in the rice seed.
Moreover, since young rice plants are severely affected by the lack of sufficient iron in the
soil (iron-deficient soils account to about 30% of the world’s arable land), the ferric chelate
reductase gene (involved in Fe-uptake from iron-deficient soils in dicotyledonous plants)
was also introduced. With this strategy we aimed to check if a monocotyledonous plant
such as rice could also profit from this gene.
At IBET/ITQB, where work on rice was only started in 2000, we are establishing
techniques and protocols in order to manipulate Portuguese rice varieties by in vitro culture
and genetic engineering and to express herbicide tolerance. Agrobacterium-mediated
transformation and particle bombardment are being tested for transformation efficiency.
We are also looking for extending collaborations at national level, and to join the expertise
of other colleagues in the recovery of rice culture and rice research.
In the European context, Portugal is now gathering the tools and the knowledge to perform
diversified work for rice improvement and also profiting from the sequencing of the rice
genome to develop studies of functional genomics. In a worldwide context, however, this
work can only be relevant if a strong collaboration with different institutions is effective
and if goals are set in a realistic manner.
Figure 1. Rice field in Beira (Mozambique) showing symptoms of bacterial blight and blast.
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Session I
Rice: traditional breeding and biotechnology innovation
SHORT COMMUNICATIONS AND POSTERS
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Session I - Rice: traditional breeding and biotechnology innovation
INTERACTION OF DRY AFTER-RIPENING AND INCUBATION
TEMPERATURE IN RED RICE
A. Gianinetti1 and M.A. Cohn2
1
Experimental Institute for Cereal Research, 29017 Fiorenzuola (PC), Italy;
agianinetti@tin.it
2
Dept. Plant Pathology and Crop Physiology, Louisiana State University Agricultural
Center, Baton Rouge, LA 70803 USA; mcohn@lsu.edu
Summary
Red rice is a weedy rice whose seed shows a variable degree of dormancy, which can be
overcome by dry afterripening. Here we show that temperature of incubation in water not
only influences germination of red rice seed, but also its dormancy status, with a
contrasting action at intermediate (15 °C) and low (1 °C) temperatures, which had,
respectively, a dormancy inducing and a stratification effect, particularly evident after
partial afterripening.
Keywords
Red rice, seed dormancy, germination, temperature, afterripening.
Abstract
Red rice, a noxious weed growing in rice fields, is widespread in the U.S.A., Latin
America and Europe. Red rice is the same species as cultivated rice (Oryza sativa L.) but
possesses unfavorable agronomic characters such as shattering, red pericarp and seed
dormancy (Noldin et al., 1999). The shattered dispersal units remain in the soil and
perpetuate the weed in subsequent years (Goss and Brown, 1939). Environmental factors
triggering its field germination are not known, but induction of secondary dormancy and
dormancy cycling can occur, and the magnitude of red rice soil seed bank suggests that
such mechanisms should be important for field survival.
In rice, induction into secondary dormancy at temperatures between 8 and 19 °C
(particularly at 10 and 15 °C) has been reported (Miura and Araki, 1996). However,
secondary dormancy was only partially induced at 8 °C, and at 5 °C no dormancy
developed (Miura and Araki, 1996), suggesting a steep increase in the induction of
secondary dormancy between 5 and 10 °C. Roberts (1962) also observed that stratification
at 3 °C broke primary dormancy of some cultivated rice varieties. Therefore, we
investigated the effect of incubation temperature on red rice dispersal units having different
intensities of dormancy (i.e. with different degrees of afterripening). We further attempted
to verify whether afterripening increases the range of temperatures suitable for red rice
germination as reported in other species (Baskin and Baskin, 1998).
To study the effects of incubation temperature on dormancy and germination of red rice
seeds following different times of afterripening, a two-step incubation experiment was
performed. Fully dormant seeds were dry afterripened at 30 °C for 1, 2, 3, 4, 6, 8 and 10
weeks and then were wet incubated for two weeks at 1, 5, 15, 20, 25, 30 and 35°C. All the
seeds that did not germinate during this initial incubation step were transferred to a second
incubation step at 30 °C (optimum temperature for germination) for two additional weeks.
The gradual loss of dormancy in a seed population is commonly associated with a
widening of the range of temperatures suitable for germination (Baskin and Baskin, 1998),
i.e. an opening of the "temperature window" for such process. As an effect of dry
afterripening, germination percentages obtained at the end of the first incubation step (Fig.
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Session I - Rice: traditional breeding and biotechnology innovation
1) increased faster at temperatures near the optimum for germination, so that the
‘temperature window’ for germination initially opened at high (optimum) temperatures and
only subsequently at low temperatures (15 °C). This is just the reverse of the trend
exhibited by barley, oat and wheat.
During the first incubation step, cold stratification (1 °C) had a consistent promotive effect
on the subsequent germination at 30°C, particularly after a short period of dry afterripening
(1-2 weeks at 30 °C). On the contrary, intermediate temperature (15 °C) had a dormancyinducing effect. For each incubation temperature tested during the first step, afterripening
times to 50 % germination, obtained either at the end of first or second incubation steps,
were utilized as relative dormancy indices to monitor effects of incubation temperature.
Comparison of the plots obtained for these indices either at the end of first or second steps
suggested that temperature acts independently on germination and dormancy and that the
final germination percentage is a result of the balance of these two opposite processes.
100
80
10
3
8
60
6
40
2
20
1
0
15
Germination after I step (%)
4
16
14
12
0
30
Temperature (°C)
Figure 1. effects of dry afterripening (at 30°c) upon germination after 14 days of wet incubation at different
temperatures in darkness (numbers in the plot are weeks of afterripening).
References
Baskin C.C. and Baskin J.M., 1998. Seeds: Ecology, biogeography and evolution of dormancy and
germination. San Diego, Academic Press Inc.
Goss W.L. and Brown E., 1939. Buried red rice seed. Journal of the American Society of Agronomy 31, 633637.
Miura K. and Araki H., 1996. Low temperature treatment during the imbibition period for the induction of
secondary dormancy in rice seeds (Oryza sativa L.). Breeding Science 46, 235-239.
Noldin J.A., Chandler J.M. and McCauley G.N., 1999. Red rice (Oryza sativa) biology. I. Characterization of
red rice ecotypes. Weed Technolgy 13, 12-18.
Roberts E.H., 1962. Dormancy in rice seed. III. The influence of temperature, moisture, and gaseous
environment. Journal of Experimental Botany 13, 75-94.
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Session I - Rice: traditional breeding and biotechnology innovation
RICE GROWING IN RUSSIA: PRESENT AND PERSPECTIVES
Yevgeny Kharitonov
All Russia Rice Research Institute, 350921, Krasnodar, Belozernoye, Russia
Key words
Russian rice growing, seed production, breeding strategy, gene resourses
Abstract
A powerful rice growing complex including reservoirs, hydrotechnician constructions, 235
thousand ha of engineer rice systems, elevators, several tens of rice growing farms was
created in Kuban area during 70s-80s last century.
About 110 thousand ha and more than 63% of rice sown area in Russian Federation are
sown with rice in Kuban region every year.
The main scientific Centrum of rice growing is All Russia Rice Research Institute
(ARRRI). The main branches of ARRRI activity are: developing resources-saving rice
growing technologies, high quality seed production, breeding of high productivity
cultivars, scientific and industrial collaboration with other rice growing areas of Russia.
ARRRI has more than 180 author’s patents on scientific technologies; a lot of them have a
world priority.
For the period of its activity ARRRI has developed more than 80 rice cultivars. Nowadays
about 90% of the Russian rice sown area is under the cultivars developed by ARRRI. They
predominate in: Ukraine, Kazakhstan, Uzbekistan, they are being grown in Bulgaria,
Rumania, Hungary, Turkey.
The main breeding method at the institute is hybridization. The domestic germplasm as
well as samples from world rice collection are included in this process. The next step is
selection. The third one is wide ecological testing in different regions of Russia. The
mutual Russian-French programme on ecological testing of Russian rice cultivars in
France has been started this year. The prior directions of breeding are developing cultivars
with good crop capacity, tolerant to unfriendly environments.
ARRRI is carrying out the 2-unit system of seed production: the elite seeds are produced
by ARRRI and by seed production farm “Krasnoye”. The industrial seed production is
made directly in rice growing farms. The main problem is the presence of red phenocopies.
That’s why the seed quality control becomes increasingly important. The new method for
identification of latent red rice has been developed in biotechnology laboratory. It is based
on Ultrathin-layer isoelectric focusing (UTLIEF) of rice seed protein.
The genotyping of 18 Russian cultivars by microsatellite markers has been carried out
according to the programme of scientific collaboration with CIRAD (France). These DNEfingerprints are important for the protection of ARRRI breeder’s rights.
ARRRI forms and studies the bank of rice gene resourses, exchanges the rice gene plasma
with leading world rice gene collections (IRRI). There are more than 3000 samples in
stock collection lab. In the nearest future ARRRI is going to develop and introduce the
modern biotechnologies: transgenesis and molecular marking into classical breeding
programmes in order to increase the efficiency of breeding and seed production processes.
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THE EFFECT OF VARIETY MAINTENANCE SYSTEMS ON THE
SEED PURITY OF RICE
D.A. Ntanos1, S.D. Koutroubas2
1
National Agricultural Research Foundation, Cereal Institute, 57001 Thermi-Thessaloniki,
Greece (rice.ci@nagref.gr)
2
School of Agricultural Development, Democritus University of Thrace, 68200 Orestiada,
Greece (skoutrou@agro.duth.gr)
Summary
Field experiments were carried out during a three years’ period (1994-96) to investigate the effect
of variety maintenance systems on the seed purity of rice. Plants coming from the breeders’ seed of
the variety L-202 were grown using two seed maintenance systems with low and high plant-toplant distance. Results indicated that a large distance between plants is preferable during the seed
multiplication process in order to conserve the varietal purity.
Keywords: breeders’ seed, low density, high density, conservation, varietal purity
Introduction
The maintenance of seed purity of a variety is an important and necessary process for a plant
breeder, although it is a secondary activity for most of the breeders. In rice, the multiplication of
varieties without special care leads with time passing by to a distortion of their stability and as a
consequence to the reduction of their productivity. The main factors which contribute to the
heterogeneity of long-established rice varieties are the natural mutations (Nagai, 1962), natural
crossing and mechanical mixture (Chandraratna, 1964). Breeders’ seed, which is the first stage in
the seed production process of a variety, is usually sown densely. Fasoulas (1993) reported that the
maintenance of the productivity and stability of a variety is accomplished by seeding under lowdensity conditions, without competition between plants. The purpose of this study was to examine
the effect of variety maintenance systems, especially the plant-to-plant distance, on the seed purity
of rice.
Materials and methods
Four field experiments were carried out at the farm of the Cereal Institute of Thessaloniki, Greece
and at a rice producers’ farm in the village of Chalastra, 35 km west of Thessaloniki in the years
1994, 1995 and 1996. Plants coming from the breeders’ seed of the variety L-202 were
transplanted in the field in 1994 at a distance of 80 cm (low density) and 15 cm (high density) using
the UNR-0 honeycomb design (Fasoulas and Fasoula, 1995). A total of 1209 and 1240 plants were
grown in the low density and high density, respectively. Plant height, sowing to heading duration,
lodging, length and type of panicle, flag leaf angle, plant habit and awning were determined. Based
on the grain yield, 20 and nine plants were selected from the low and high density sowing,
respectively. The seeds of selected plants from the low density sowing were sown in 1995 at a
distance of 80 cm using the R-21 honeycomb design with 31 replications and those of the high
density sowing were sown densely in plots 12 m long consisting of nine lines, 15 cm apart. In
1996, the two highest yielding and the two lowest yielding lines of the low density sowing, the two
highest yielding lines of the high density sowing and the variety L-202 (breeders’seed) as check
were grown in two experiments (one in each location) under dense sowing, using a randomized
compete block design with four replications. Data deriving from the honeycomb experiments were
analyzed according to Batzios and Roupakias (1997).
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Session I - Rice: traditional breeding and biotechnology innovation
Results and discussion
Grain yield of the variety L-202 ranged from 3 g/plant to 150 g/plant and from 3 g/plant to 67
g/plant under low and high density sowing conditions, respectively. Mean grain yield was much
greater under low density sowing conditions (43.8 g/plant) compared to that of the high density
sowing conditions (18 g/plant). The coefficient of variation (CV) was high and similar in both
cases (55.5% for the low density and 55.8% for the high density). Plants selected by the two
sowing densities differed in grain yield, plant height and time to heading, but not in the other traits
examined. The 20 lines of the variety L-202 selected by the low density sowing showed similar
grain yield and morphophysiological traits when they were evaluated under low density sowing.
Similar results were obtained for the nine lines selected by the high density sowing when they were
evaluated under high density sowing.
Table 1. Means for grain yield and eight agronomic traits of the lines of the variety L-202 coming from
the low and high density sowing when evaluated under high density sowing conditions in 1996
(Values are means over two locations).
Grain Plant Time to
Panicle Panicle Flag leaf Plant
yield height heading Lodging length type
angle
type Awning
Line
(1)
24-13.17
19-17.20(1)
24-04.07(2)
06-30.04(2)
20-22.04(3)
05-09.06(3)
L-202(4)
(kg/ha)
(cm)
(days)
(1,3,5,7,9)
(cm)
(1,5,9)
(1,3,5,7,9)
(1,3,5,7,9)
(1,3,5,7,9)
7520 a
7290 a
7430 a
7150 a
6960 a
6440 a
7530 a
91
90
89
90
92
92
91
89
90
88
89
88
86
90
1
1
1
1
1
1
1
20
20
19
20
19
19
21
5
5
5
5
5
5
5
3
3
3
3
3
3
3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
(1)
: Low density sowing, highest yielding line; (2): Low density sowing, lowest yielding line;
: High density sowing, highest yielding line; (4): Check (breeders’seed)
(3)
The two highest yielding and the two lowest yielding lines of the low density sowing, the two
highest yielding lines of the high density sowing and the check variety had similar grain yield and
morphophysiological traits when evaluated under high density sowing (Table 1). No off-type
plants were found in the lines coming from the low density sowing. On the contrary, in the lines
coming from the high density sowing (20-22.04 and 05-09.06) two off-type plants were found,
number that corresponds to a percentage of 0.008% of the total plants of this line in both
experiments. These two plants had for the “plant type” trait score 5, while the normal plants had
score 1. The off-type plants had greater possibility to lodge, particularly when they are not
harvested on time. This has as result a decreased grain yield and a degraded grain quality. The
verification of off-type plants is practically possible when the plants are developed under low
density sowing only. Therefore, breeders’seed of rice varieties is recommended to be sown in
relatively large distances during the seed multiplication process in order to conserve the varietal
purity.
References
Batzios, D. P, and D. G. Roupakias. 1997. HONEY: A microcomputer program for plant selection and
analyses of the honeycomb designs. Crop Sci. 37:744-747.
Chandraratna, M. F. 1964. Genetics and breeding of rice. Longmans, Green and Co LTD, London, 389 pp.
Fasoulas, A. C. 1993. Principles of crop breeding. Thessaloniki, Greece, 127 pp.
Fasoulas, A. C. and V. A. Fasoula.1995. Honeycomb selection designs. Plant Breed. Rev. 13:87-139.
Νagai, I. 1962. Japonica rice its breeding and culture. Yokendo LTD Tokyo, 843 pp.
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COMPARATIVE ANALYSIS OF QTLS CONTROLLING ROOT TRAITS IN
MAIZE AND RICE: PRESENT STATUS AND FUTURE PERSPECTIVES FOR
QTL CLONING USING RICE AS A MODEL
S. Salvi*, M.C. Sanguineti, P. Landi, M. Maccaferri, S. Giuliani, R. Tuberosa
Department of Agroenvironmental Science and Technology, Via Fillippo Re 6, 40126,
Bologna, Italy. (* For correspondence: salvi@agrsci.unibo.it)
Summary
The availability of the complete genomic sequence, a number of genomic tools and the
recognized synteny between rice and the other cereals make rice an excellent model
species for supporting gene mapping and cloning among all monocots. This is particularly
true for QTLs controlling agronomic traits such as roots characteristics. We describe the
mapping and aligning of a number of QTLs for root traits among maize and rice maps.
Keywords
maize, QTL, QTL cloning, rice, synteny
Abstract
The growing instability of seasonal rainfall patterns possibly linked to global warming has
prompted greater attention towards the improvement of traits which stabilize yield under
conditions of water deficit. Breeding for drought resistance, particularly through avoidance
mechanisms, most likely involves root characteristics. Significant genetic variation for root
traits has been reported in maize (Landi et al., 2001) and upland rice. Despite this, little
progress has so far been achieved in the utilization of root traits as a selection criteria to
indirectly improve yield in these two species due to the difficulty in properly investigating
roots in a large number of plants and the scanty information available on the genetic control of
root traits. A better knowledge of the genetic determinants of root traits and how they influence
yield would allow for more targeted approaches (e.g. marker-assisted selection) within
breeding programs and, eventually, for the fine mapping and cloning of the gene/s underlying
such QTLs. Because of the large investment of resources necessary to clone the gene/s
underlying a QTL (Salvi et al., 2002) it is advisable to focus on major QTLs affecting the trait
of interest in a number of genetic backgrounds within the same species and, when comparative
mapping is possible, across species, particularly when one of the investigated species has a
genome of small size. Among the cereal crops, rice has already been recognized as the model
species. Extensive synteny between rice and the other cereals provides a powerful means to
exploit the genetic information available in rice. Our long-term goal is to identify QTLs
controlling root traits in maize and rice and clone the genes underlying such QTLs using rice as
a model species. As a preliminary step, we have analysed the QTL data available in the
literature for root traits in maize and rice to identify a number of syntenic regions which could
be targeted with a cloning approach. Herein, we present a summary of this comparative
analysis and summarize the possible approaches for the cloning of such QTLs in rice.
In maize, four mapping populations have so far been investigated for QTLs for root traits
under controlled conditions and/or in the field (Tuberosa et al., 2002a, b). Several chromosome
regions affected root traits in two or even three populations. A number of these regions also
affected grain yield under well-watered and/or drought-stressed conditions. The most
important QTL effects were detected on chromosome bins 1.03, 1.06, 1.08, 2.03, 2.04, 7.02,
8.06 and 10.04. Exploiting the syntenic information available for maize and rice, we compared
the maize QTL results with those available from five studies describing QTLs in rice. Synteny
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Session I - Rice: traditional breeding and biotechnology innovation
information was based on data retrieved from http://ars-genome.cornell.edu/rice. Twenty
morphological root traits (e.g. total root weight, maximum root length, root/shoot ratio, etc.)
were considered among the five rice mapping populations, for a total of 166 QTLs. Sixty-three
(out of 100) maize bins were identified as syntenic to one or more regions with QTLs for root
traits in rice. The five maize bins most frequently identified were: bin 2.04 (syntenic to regions
carrying QTLs for 16 out of 20 traits, in all five populations), bin 5.03 (14 out of 20 traits; four
out of five populations), bin 8.05 (12 out of 20 traits; four out of five rice populations), bin 5.04
(11 out of 20 traits; four out of five populations) and bin 4.05 (10 out of 20 traits; three out of
five populations). The full comparative list is available upon request. It should be noted that
bin 2.04 is important for controlling variation in root traits in maize. Six rice QTLs for root
traits map in regions syntenic to maize bin 1.06, another region influencing variation in root
traits in maize. Based on these findings, it is tempting to envisage that, similarly to what has
already been shown for a number of Mendelian loci, a set of orthologous genes may also
control, at least in some cases, quantitative traits (and therefore root traits) in rice and maize. If
this hypothesis is substantiated in future experiments, then it will lead the way to the
exploitation of genomic and sequence information in rice to identify and clone the
corresponding agronomically important genes in maize and, possibly, in other cereals.
Presently, positional cloning appears to be the main strategy toward QTL cloning in cereal
species. All the requirements for the positional cloning of Mendelian genes are also needed for
the positional cloning of the gene underlying a QTL. Additionally, a much larger effort is
needed for the development of genetic material and for phenotypic scoring. After the QTL is
confined to a region <1-2 cM in the source cereal species, the availability of the rice genome
sequence and established information on synteny relationships among cereals allow to use the
molecular markers closest to the QTL to cross-reference the genetic map to the rice genome
sequence. The rice genome will then serve as source of new markers to increase the mapping
resolution and will provide candidate genes at the target QTL, eliminating or strongly reducing
the need of establishing contigued BAC/YAC libraries. Along with positional cloning, a
number of genomic tools now available in rice can be used to increase the efficiency of
identifying genes underlying quantitative traits. The opportunities toward QTL cloning offered
by the insertional mutant collections based on transposons and T-DNA will be discussed. The
possibility of identifying allelic series of known genes for studying quantitative phenotypes
provided by TILLING provides further opportunities.
Although QTL analysis and cloning remains a resource-demanding undertaking, its integration
with genomics and post-genomics approaches will play an increasingly important role for
harnessing the favourable allelic variation at loci affecting root characteristics in maize and
rice.
References
 Landi P., Giuliani MM., Darrah LL., Tuberosa R., Conti S., Sanguineti MC. 2001. Variability for root
and shoot traits in a maize population grown in hydroponics and in the field and their relationships with
vertical root pulling resistance. Maydica 46:177-182.
 Salvi S., Tuberosa R., Chiapparino E., Maccaferri M., Veillet S., van Beuningen L., Isaac P., Edward
K.J., Phillips R.L. (2002). Toward positional cloning of Vgt1, a QTL controlling the transition from the
vegetative to the reproductive phase in maize. Plant Molecular Biology 48:601-613.
 Tuberosa R., Sanguineti MC., Landi P.,Giuliani M.M., Salvi S., Conti S. (2002a). Identification of
QTLs for root characteristics in maize grown in hydroponics and analysis of their overlap with QTLs
for grain yield in the field at two water regimes. Plant Molecular Biology. 48, 697-712.
 Tuberosa R., Salvi S., Sanguineti MC., Landi P., Maccaferri M., Conti S. (2002b). Mapping QTLs
regulating morpho-physiological traits and yield in drought-stressed maize: case studies, shortcomings
and perspectives. Annals of Botany (In press).
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Session I - Rice: traditional breeding and biotechnology innovation
ERRATA CORRIGE
CONSTRUCTION OF A RICE YELLOW MOTTLE VIRUS-BASED
VECTOR FOR RICE FUNCTIONAL GENOMIC AND TO ASSESS
GENES INVOLVED IN RESISTANCE, TOLERANCE AND
SUSCEPTIBILITY MECHANISMS BETWEEN RICE AND RYMV
C. Sire1, M. Bangratz1, C. Sallaud2, E. Guiderdoni2, D. Fargette3, A. Ghesquiere1 , C. Brugidou1
1
-UMR 5096 UP-IRD-CNRS, Centre IRD, BP 64501 34394 Montpellier cedex 5 France,
sire@mpl.ird.fr
2
- CIRAD-Amis, TA 40/03, av Agropolis, 34398 Montpellier cedex 5, France.
3
- UMR DGPC, Centre IRD, BP 64501 34394 Montpellier cedex 5 France.
Summary
Rice is a major crop and a plant model for monocotyledons genomic, and especially for
cereals. A new field has to be developed with the functional genomic, to take advantage of
the growing number of genomic sequences. The potential of plant virus-based transient
expression vectors is substantial in fundamental virology but also for plant biology. Indeed,
this system can be used as a tool to investigate gene function by over-express (gain of
function mutant) or suppress (loss-of-function mutant) gene expression. In the aim to
investigate mechanisms involved during Rice yellow mottle virus (RYMV) infection, but
also for rice functional genomic, we need to construct a viral expression vector.
Keywords
Rice yellow mottle virus, rice, plant virus-based expression vector, gene expression, virus
induced gene silencing.
Abstract
At present plant virus vectors offer number of benefits for the expression of foreign genes. The
potential of such vectors for plant molecular biology is substantial particularly to identify previous
unknown genes. As rice is used as a reference for cereals, one of its associated virus, Rice yellow
mottle virus can be regarded as a model system for construction of transient expression vectors for
rice. Rice yellow mottle virus is a single-stranded-positive-sense RNA virus that specifically infect
rice leaves and causes serious disease in irrigated rice systems in East and West Africa. The purpose
of this work is to develop for rice a transitory expression vector based on an infectious full-length
cDNA clone of RYMV (Brugidou et al., 1995) to assess gene function. On one hand, gain of
function mutant will be generated by over-expressing an interesting gene identify through genome
sequencing. On the other hand, we can take advantage of the virus induced gene silencing (VIGS)
phenomenon (Baulcombe, 1999; Ratcliff et al., 2001) to generate loss-of-function mutant of a
particular gene. Such a vector will be used to better understand mechanisms involved in resistance,
tolerance and also susceptibility of rice toward RYMV.
Three main RYMV-based vector constructs are being generated. The first approach consists in
producing a fusion between a reporter gene (encoding GUS or GFP) and the viral coat protein (Fig.
1a). With such construct, gene expression can be monitored in planta. In a second approach, to overexpress a foreign gene, we consider duplicating sub-genomic mRNA promoter of the coat protein
(Fig. 1b) in order to improve gene expression thanks to an additional stem-loop structure. The third
approach is the construction of an interesting virus-based vector for gene complementation, a
construct devoid of its infectious elements like genes encoding viral coat protein and/or movement
protein.
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Session I - Rice: traditional breeding and biotechnology innovation
As RYMV is mechanically transmissible, transcripts (after in vitro transcription) will be
assessed in vivo at cell level but also on the entire plant. Actually, cell suspensions are
wounded by particle bombardment with infectious RYMV-vectors transcripts or plants are
mechanically inoculated. Then the accumulation of recombinant virus is evaluated by RTPCR or western blotting analysis. All the recombinant vectors described previously are
constructed and are being tested in vivo. Accordingly, after the in vivo functionalitychecking, all these constructs will be cloned under the control of constitutive promoters in
order to improve gene expression.
At last, as we also want to better understand molecular mechanisms involved in gene
silencing induce by RYMV (Voinnet et al., 1999), we consider studying inhibition of gene
silencing. Actually, we possess plants exhibiting gene silencing on both gfp and uidA
reporter genes. The purpose of the study is to estimate how the inhibition of gene silencing
by RYMV occurs. With this aim in view, we will inoculate these transgenic rice lines with
different RYMV-isolates (Fargette et al., 2002), showing variability on the movement
protein, in order to highlight the isolate that generates the main effect on silencing
inhibition.
a-
sg RNA promoter
T7
ORF 1
5’
0 pb
Gene of interest or
reporter gene
ORF2a
CP
3’
4452 pb
ORF2b
b-
Gene of interest
Figure 1 : The genome organization of RYMV. ORF are illustrated as boxes. ORF1, P1-encoded
protein or movement protein; ORF2, polyprotein; CP, coat protein. represents the sub-genomic
(sg) mRNA promoter.
a- Transcriptionnal fusion between the viral CP and a gene of interest. b- Cloning of a gene of
interest depending on the RYMV sgRNA promoter.
References
 Baulcombe, D.C. (1999). Fast forwards genetics based in virus-induced gene silencing. Current
Opinion in Plant Biology 2, 109-113.
 Brugidou, C., Holt, C., Yassi, M.N., Zhan,g S., Beachy, R. & Fauquet, C. (1995). Synthesis of an
infectious full-length cDNA clone of rice yellow mottle virus and mutagenesis of the coat protein.
Virology 206 (1), 108-115.
 Fargette, D., Pinel, A., Halimi, H., Brugidou, C., Fauquet, C. & Van Regenmortel, M. (2001).
Comparison of molecular and immunological typing of isolates of Rice yellow mottle virus. Archives of
Virology 147, 583-596.
 Ratcliff, F.G., Montserrat, Martin-Hernandez, A. & Baulcombe, D.C. (2001). Tobacco rattle virus as a
vector for analysis of gene function by silencing. The Plant Journal 25 (2), 237-245.
 Voinnet, O., Pinto, Y.M. & Baulcombe, D. (1999). Suppression of gene silencing : a general strategy
used by diverse DNA and RNA viruses of plants. Proceedings of the National Academy of Sciences 96,
14147-14152.
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APPLICATION OF SENSORY ANALYSIS TO DISCRIMINATE THE
ORIGIN OF S. ANDREA RICE
G. Zeppa, L. Rolle, V. Gerbi
Department of Exploitation and Protection of the Agricultural and Forestry Resources Laboratory of Food Technology, University of Turin, Via Leonardo da Vinci 44, 10095
Grugliasco TO Italy; email zeppa@agraria.unito.it
Summary
The aim of this study was to highlight the chemical and sensory distinction of S. Andrea
rice for the request of a Brand Denomination. Unlike chemical analysis, sensory analysis
showed a notable difference between S. Andrea rice produced in the Brand Denomination
area and that obtained from other Piedmont rice-growing areas.
Keywords
Sensory analysis, rice, paired comparison test, Protected Origin Denomination, S. Andrea
Abstract
On the European and Italian scene, Piedmont rice-growing has a significant position not
only from the point of view of quantity accounting for about 50% of the national
production, but also and most importantly for its quality. From this viewpoint the S.
Andrea rice obtained from the Rizzotto (Lady Wright xP6) with a long large grain is very
important. It does not overcook and is therefore particularly indicated for risotto and rice
salads.
The S. Andrea rice is tilled typically in the ‘baraggia’, an area at the foothills of the Alps
characterised by a compact, impermeable terrain and cool rainy climate and has created a
wide market especially abroad where it is much appreciated and requested for its high
quality.
However, at the current time, the surface area where S. Andrea rice is cultivated is
decreasing to the advantage of expensive varieties such as Arborio, Baldo, Carnaroli or
Roma and other minor rice varieties.
In order to increase production and limit the reduction of the growing zone, a request for
‘Production Origin Denomination’ (POD) for the Piedmont S. Andrea rice was submitted
in May 2000 according to the European Economic Community law 2081/92.
The aim of this study was to highlight the chemical and sensory specifications for the
request of this Brand Denomination and if it were possible to distinguish the S. Andrea rice
produced in the Brand Denomination area to that produced in other rice-growing areas of
Piedmont. Then eight samples of rice produced in the year 2000 coming from the same
number of firms were polished in a pilot plant and subjected to chemical and sensory
analysis.
Five of these samples came from firms inside the Brand Denomination area and three from
firms located outside this area.
Results of the chemical analysis (Table 1) does not show any statistical difference between
the S. Andrea rice produced in the POD area and that produced in other zones.
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Table 1- Mean (X) and standard deviation ( ) of major components of S. Andrea rice produced in the POD
area and in others areas
Water (%)
Proteins (% dry matter)
Amylose (% dry matter)
Ether extract (% dry matter)
POD area
X
14.28 0.47
6.79 0.67
17.2
0.9
0.17 0.07
Other areas
X
13.29 0.74
5.93 0.84
18.6 0.81
0.28 0.13
For the sensory analysis the samples of the two areas were mixed together in order to
carry out paired comparison tests (ISO 5495) on only two products. The rice was cooked
in still water (rice:water ratio 1:5) for 14 minutes and it was served with no seasonings in
white porcelain cups of about 100 mL.
The testing panel was made up of 20 trained assessors. Sensory analysis was performed in
the Department’s sensory room with 8 booths designed according to ISO 8589. The ² test
show a significant difference (P<0.05) between the two samples because the S. Andrea rice
produced in the POD area was attributed greater firmness than that produced in other areas
(Table 2).
Since the area is distinguishable for its soil and its climatic characteristics, there are
subsequently two productive areas within the POD production (the ‘baraggia’ and the
‘non baraggia’), thus a new series of paired comparison tests was carried out to compare
the S. Andrea rice produced in these production zones.
The obtained results show a high statistical difference (P<0.01) between the rice produced
in these two areas and again the product from the ‘baraggia’ was attributed a better
texture.
Table 2 - Significant values in the comparison between the S. Andrea rice produced in the ‘baraggia’ area
and in the ‘non baraggia’ area (ns: not significant)
Comparison
p
POD area vs other areas
‘Baraggia’ area of POD vs ‘non baraggia’ area of POD
‘Baraggia’ area of POD vs other areas
Other areas vs ‘Non baraggia’ POD area
0.04
< 0.01
< 0.01
ns
The results obtained for the S. Andrea rice shows the need to use objective parameters such
as chemical and sensory characteristics to define the production area within a POD and the
importance that sensory analysis can have to discriminate and characterise agri-food
products. Hence sensory analysis, also in the case of rice can be an effective tool for a fast
and full characterisation of products and thus integrate if not substitute longer and
laborious chemical analysis.
References
 Meilgaard M., Civille G.V., Carr B.T. 1991. Sensory evaluation techniques. CRC Press, Boston.
 Paule C.M., Powers J.J. 1989. Sensory and chemical examination of aromatic and nonaromatic rices. J.
Food Sci., 54(2), 343-346.
 Piggott J.R. 1988. Sensory analysis of foods. Elsevier Applied Science, London.
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