Advances in rice genetics and biotechnology

G.S. Khush, J. Bennet, S.K. Datta, D.S. Brar and Z. Li

International Rice Research Institute (IRRI), Manila, the Philippines

INTRODUCTION

Major advances have been made in increasing rice production worldwide as a result of large-scale adoption of modern high-yielding rice varieties and improved cultural practices. World rice production has more than doubled in the past 30 years, from 257 million tonnes in 1966, to 560 million tonnes in 1996. This was mainly achieved through the application of principles of classical Mendelian genetics and conventional plant breeding methods. The present world population of 5.8 billion is likely to reach 7.5 billion by 2020, and rice production must increase by 50 percent to satisfy the growing demand of these ever-increasing numbers. To meet this goal, rice varieties with higher yield potential, durable resistance to diseases and insects and tolerance to abiotic stresses are needed. Recent advances in rice genetics and biotechnology have provided tools that can advance the efficiency of breeding methods and allow unconventional approaches to rice improvement.

RICE GENETICS

Cultivated rice is a diploid with 24 or 12 pairs of chromosomes which have been numbered according to the decreasing order of length at the pachytene stage of sexual cell division. Thus, the longest chromosome is number 1, the second longest, number 2 and the shortest, number 12. Chromosomes of both the cultivated species and closely related wild species are similar and their genomes are designated as AA. The chromosomes of other wild species, however, differ from those of cultivated rice, and they belong to genomes designated as BB, CC, DD, EE, FF and GG. A few of the tetraploid species have BBCC, CCDD or HHJJ genomes.

Marker genes and linkage groups

Nearly 400 mutant genes affecting morphological, physiological and biochemical characters, disease and insect reaction, abiotic stresses and coloration of plant parts have been described. These genes have been assigned to different groups through genetic analysis, and 12 linkage groups corresponding to 12 chromosomes have been established. The linkage groups have been associated with respective chromosomes through genetic analysis using primary trisomics (Khush and Kinoshita, 1991). The orientation of the linkage groups and the position of centromeres on the linkage maps were determined through the use of secondary trisomics and telotrisomics (Singh, Multani and Khush, 1996).

Biochemical markers

Morphological markers, although useful in genetic studies, are of limited value in rice improvement. The second category of markers are isozymes, which are more useful than morphological markers. They are codominant and, thus, all genotypes can be distinguished in segregating populations. They have no deleterious effect on plant phenotype and a large number of samples can be scored in the laboratory at very early stages of growth. However, only a limited number of isozyme loci are known and the number is not sufficient to saturate the genetic maps (Khush and Kinoshita, 1991). In rice, for example, only about 50 isozyme loci are known.

DNA markers

The third category of markers, which includes random fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and microsatellites, have all the advantages of isozyme markers but are far more numerous, making it possible to prepare saturated maps. In rice, an RFLP linkage map was first prepared at Cornell University, the United States, in cooperation with the International Rice Research Institute (IRRI) (McCouch et al., 1988). This map consisted of 135 markers and was expanded to about 700 markers (Causse et al., 1994), half of which were converted to sequence-tagged sites (STS) by DNA sequencing (Robeniol et al., 1996). The molecular genetic map prepared at the Rice Genome Research Program (RGRP), at Tsukuba, Japan, is the most elaborate and consists of 2 275 markers (Harushima et al., 1998). The orientation and the positions of centromeres on the molecular linkage maps were determined by Singh et al. (1996b). Thus, the chromosome arm locations of the DNA markers are now known (Harushima et al., 1998). A complete linkage map of 306 microsatellite markers has recently been reported by McCouch et al. (1997).

Synteny among cereal genomes

In other cereals, such as sorghum, maize, oats, barley and wheat, high density molecular genetic maps have been prepared (Nagamura, Antonio and Sasaki, 1997). Molecular markers have been utilized to study genetic relationships among the cereal species. In particular, cDNAs with coding regions of expressed genes are very useful as common landmarks for comparative linkage mapping, because they are well conserved between distantly related species. The feasibility of using a common set of RFLP probes to develop comparative linkage maps in plants was first demonstrated with tomato and potato (Bonierbale, Plaisted and Tanksley, 1988). In cereals, the first comparative mapping was carried out in sorghum and related crops by means of maize DNA probes (Hulbert et al., 1990). The first publication that demonstrated the breadth of synteny among the cereals was by Ahn and Tanksley (1993) showing the relationship between rice and maize. Kurata et al. (1994) showed that the wheat genome could be aligned with that of rice. Moore et al. (1995) showed that all the cereal maps could be combined in a single synthesis. Rice is used as the base for comparative mapping simply because it has the smallest genome among the cereals analysed.

COMPARATIVE GENETICS

As discussed above, there is now overwhelming evidence for the existence of extensive regions of conserved colinearity among cereal species at genetic map level. This knowledge can be exploited to advance marker studies on all grass species and to extend our knowledge of key syntenic agronomic genes as they are placed on genetic maps. Comparative genome research is an excellent tool for gene isolation. The similarity in gene content and order allows the search for genes of interest using cloned genes of one species to look for similar genes in other species. For example, Kilian et al. (1995) used cloned barley gene Rpg 1 to search for a similar gene in the syntenic region of rice.

Molecular markers for gene tagging

Numerous genes of economic importance, such as those for disease and insect resistance, are repeatedly transferred from one varietal background to another by plant breeders. Most genes behave in a dominant and recessive manner and require time-consuming efforts to transfer. Sometimes the screening procedures are cumbersome and expensive and require large field space. If such genes can be tagged by tight linkage with DNA or isozyme markers, time and money can be saved when transferring them from one varietal background to another. The presence or absence of the associated molecular marker would indicate, at a very early stage, the presence or absence of the desired target gene. Codominance of the associated molecular marker allows all the possible genotypes to be identified in any breeding scheme, even if the gene for a particular economic trait cannot be scored directly. A molecular marker very closely linked to the target gene can act as a "tag" which can be used for indirect selection of the gene(s) in a breeding programme.
The availability of comprehensive molecular genetic maps in rice has allowed the tagging of many genes of economic importance with molecular markers (Table 1).

QTL mapping

Although a number of important characters are determined by loci that have a major effect on phenotype, many economically important traits such as yield, quality and tolerance to abiotic stresses are of a quantitative nature. Genetic differences affecting such traits are controlled by a relatively large number of loci, each of which can make a small positive or negative contribution to the final phenotypic value of the traits. These loci are termed quantitative trait loci (QTLs). Genes governing traits, which are called polygenes or minor genes, also follow Mendelian inheritance but are greatly influenced by the environment.

The advent of molecular markers has made it possible to map and tag QTLs. Molecular markers provide the opportunity to manipulate QTLs as Mendelian or quasi-Mendelian entities. Several QTLs for traits of economic importance, such as blast resistance (Wang et al., 1994), root length (Redona and Mackill, 1996), submergence tolerance (Nandi et al., 1997) and yield potential (Xiao et al., 1996), have been tagged with molecular markers.

BIOTECHNOLOGY

Breakthroughs in molecular and cellular biology have led to the development of novel tools for rice improvement and have speeded the pace of development of improved cultivars. Some of these advances are discussed below.

Marker-assisted breeding

DNA markers have several potential applications in the genetic improvement of rice, including characterization and protection of germplasm, assessment of genetic diversity, tracing of gene flow and marker-
assisted selection (MAS). Of these, marker-assisted backcrossing and gene pyramiding are the most important applications. Compared with traditional backcrossing by phenotypic selection, gene transfer by marker-assisted backcrossing is more accurate and faster, particularly when the trait is difficult to assess (Tanksley et al., 1989). In marker-assisted backcrossing or gene pyramiding, individuals carrying the target gene are selected by segregating populations based on tightly linked markers rather than on their phenotype. Thus, the populations can be screened at early seedling stage and under various environmental conditions. MAS can overcome interference from interactions between alleles of one locus or of different loci. MAS was successfully employed in pyramiding four different genes (Xa-4, Xa-5, Xa-13 and Xa-21) for bacterial blight resistance (Huang et al., 1997). Breeding lines with two, three or four bacterial blight resistance genes have been developed. The pyramided lines showed a wider spectrum and a higher level of resistance than the lines with single genes. These pyramided lines are being used as donors for transferring resistance genes into elite rice varieties developed at IRRI and by the national rice improvement programmes through MAS.

Low-cost DNA markers

Wide adoption of DNA marker technology depends on the availability of high-capacity protocols that are cheap, robust and rapid. Zheng et al. (1995) presented a MAS protocol that is based on the polymerase chain reaction (PCR). This protocol is one-tenth the cost of RFLP protocols and more than ten times faster. The chief requirements of the approach are polymorphic STS markers in the vicinity of the genes of interest. Once suitable polymorphisms are established between the two parental lines in a breeding programme, the marginal costs per plant are low, reliability and convenience are high, and populations of 1 000 can be analysed within two weeks using 5 cm segments of leaf. Start-up costs are modest. This protocol was developed and disseminated under the auspices of the Asian Rice Biotechnology Network, which has been in existence since 1993. Xu et al. (1998) developed a similar protocol for the classification of rice germplasm.

Gene cloning

The high-density molecular genetic map is also of great value in map-based cloning of agriculturally important genes. The possibilities of cloning rice genes based on map position have been greatly enhanced with the development of bacterial artificial chromosome (BAC) libraries. Wang et al. (1995b) used BAC libraries and identified clones carrying Xa-21 gene for bacterial blight resistance. Song et al. (1995) isolated Xa-21 by positioning cloning. The isolated gene has been introduced into several elite rice cultivars through transformation. Yang et al. (1997) constructed a BAC library consisting of 18 432 clones in IR64 background at IRRI. Clones carrying Xa-5 for bacterial blight resistance have been identified (Yang et al., 1998) and research to clone X-a5 is under way. Once cloned, this gene could be transferred to elite rice varieties through transformation.

Genetic engineering

Protocols for rice transformation have been developed which allow the transfer of foreign genes from diverse biological systems into rice. Direct DNA transfer methods, including protoplast-based (Datta et al., 1990), biolistic-based (Christou, Ford and Kofron, 1991) and Agrobacterium-mediated (Hiei et al., 1994), are being used for rice transformation. Major targets for rice improvement through transformation are: disease and insect resistance, abiotic stress tolerance, and enhancement of yield potential. Some examples of transgenic rice plants that carry agronomically important genes are given in Table 2.

Insect resistance

As early as 1987, genes coding for toxins from Bacillus thuringiensis (Bt) were transferred to tomato, tobacco and potato, where they provided protection against Lepidopteran insects. A major target for Bt deployment in transgenic rice is the yellow stem borer. This pest is widespread in Asia and causes substantial crop losses. Improved rice cultivars are either susceptible to the insect or have only partial resistance. Thus, Bt transgenic rice has much appeal for controlling the yellow stem borer. To date, 40 different nucleotide sequences of Bt genes have been determined. They are clearly related to each other and have been classified into 17 distinctly different crystal protein genes, the so-called cry genes. These genes encode proteins of 130 to 140 kDa or 70 kDa, which are first dissolved then proteolytically cleaved in the midgut of the larvae of Lepidopteran insects. Truncated cry genes (CryIA(b) or CryIA(c)) have been introduced into rice by several research groups (Fujimoto et al., 1993; Wunn et al., 1996; Ghareyazie et al., 1997; Datta et al., 1997; Alam et al., 1998). Bt rices show excellent levels of resistance in the laboratory and the transgenic greenhouse. When the major field symptoms of stem borer damage (dead hearts and whiteheads) were reproduced in the greenhouse on control plants, Bt rice was free of these symptoms (Ghareyazie et al., 1997). To minimize the possible development of resistance-breaking biotypes, attempts have been made to put the CryIA(b) gene under the control of tissue-specific promoters that direct the expression of the gene only in the pith or green tissues (especially leaf sheath), the primary target of stem borer (Datta et al., 1997).

As well as Bt genes, other genes for insect resistance, such as those for proteinase inhibitors, a-amylase inhibitors and lectins, are also beginning to receive more attention. Insects use diverse proteolytic or hydrolytic enzymes in their digestive guts for the digestion of food proteins and other food components. Plant-
derived proteinase inhibitors or a-amylase inhibitors are of particular interest because they are part of the natural plant defence system against insect predation. Xu et al. (1996a) reported transgenic rice carrying cowpea trypsin inhibitor (CpTi) gene that had enhanced resistance against striped stem borer and pink stem borer.

Disease resistance

Several fungal, viral and bacterial diseases cause serious yield losses. Sources of resistance to some diseases (blast, bacterial blight) have been identified within cultivated rice germplasm, and elite cultivars with resistance have been developed. However, sources of resistance to sheath blight are not available and only a few donors for resistance to tungro disease are known.

A highly successful strategy termed coat protein (CP) mediated protection has been employed against certain viral diseases such as tobacco mosaic virus in tobacco and tomato. When expressed in the transgenic crop, a chimeric gene, made by combining a strong promoter with the virus gene encoding the capsid protein, results in the accumulation of capsid protein in plant cells. Such plants are resistant to infection by the virus from which the gene was isolated.

CP genes for the two component viruses that cause tungro disease have been cloned (Hay et al., 1991) and efforts are under way to express these genes in rice plants. A CP gene for rice stripe virus was introduced into two japonica varieties by electroporation of protoplasts (Hayakawa et al., 1992). The resultant transgenic plants expressed a high level of CP and exhibited a significant level of resistance to virus infection, and this resistance was inherited by the progenies. Sheath blight of rice caused by Rhizoctonia solani, which has a wide host range, causes serious yield losses. Transgenic tobacco and canola plants with enhanced resistance to R. solani have been obtained by introducing the bean chitinase gene under the control of CaMV35S promoter (Broglie et al., 1991). Chitinases and glucanases degrade the major structural polysaccharides of the fungal cell wall. These enzymes have both a binding domain and a catalytic domain for their respective polysaccharides, alone or in combination; they attack the growing hyphal tip and are potent inhibitors of fungal growth. About six chitinase genes have been identified in rice and are being manipulated to increase the level of resistance to fungal diseases of rice (Zhu and Lamb, 1991). Lin et al. (1995) introduced a 1.1 kb rice genomic DNA fragment containing a chitinase gene. The presence of this chimeric chitinase gene in T₀ and T₁ transgenic plants was detected by southern blot analysis. Western blot analysis of transgenic plants and their progeny revealed the presence of two proteins that reacted with the chitinase antibody. Progeny from the chitinase-positive plants were tested for resistance to the sheath blight pathogen. The degree of resistance correlated with the level of chitinase expression.

Abiotic stress tolerance

A plant under stress responds in different ways depending on the development stage at which it is subjected to stress. Various biochemical, physiological and molecular events are associated with stress response, and different molecular strategies are required to overcome these problems. A limiting factor in any of the strategies, however, is the lack of well-defined structural genes and regulatory elements that contribute positively to stress tolerance. The following strategies have been identified to develop germplasm that is tolerant to abiotic stresses:

• an increase in osmolyte/osmoprotectant concentrations in the cytoplasm;
• constitutive expression of stress-responsive proteins;
• membrane manipulation through increased unsaturation;
• enhanced expression of stress-related enzymes.

Recently, Xu et al. (1996b) reported some success in using a Hva1 gene, producing late embryogenesis abundant (LEA) proteins controlling water and salinity stress in rice plants. It is believed that LEA proteins may play a protective role in plant cells under water stress conditions.

Improvement of starch biosynthesis

Starch biosynthesis plays a pivotal role in plant metabolism, both as a transient storage metabolite of leaf tissue and as an important energy and carbon reserve for sink organs such as seeds, roots, tubers and fruits. Starch is, therefore, a critical determinant of the sink strength of developing sink organs as well as a source. Several enzymatic steps are involved in starch biosynthesis in plants. Adenosine diphosphate-glucose phyrophosphorylase (ADPGPP) is a critical enzyme in regulating starch biosynthesis in plant tissues. Even when it occurs at high levels in storage organs, ADPGPP's activity is limiting. This limitation appears to be primarily at the level of allosteric regulation of the enzyme, at least in sink tissues. It should be possible to affect starch production in storage tissues by regulated expression of the gene encoding this enzyme (Kishore, 1994). Starch levels and dry matter accumulation were enhanced in potato tubers of plants transformed with glgc¹⁶ gene from Escherichia coli encoding ADPGPP (Stark et al., 1992). Transformed potato plants that were similar in growth and development to non-transgenic controls had tubers with higher dry matter and starch contents under both growth chamber and field conditions. The nature of starch produced by the tubers containing glgc¹⁶ gene was similar to that of controls. The glgc¹⁶ gene has been introduced into rice at IRRI and its expression is being investigated.

Gene silencing

The widespread occurrence of transgene inactivation in plants has been explained in terms of the triggering of defence systems that monitor and manipulate intrusive or aberrant DNA (Kumpatla et al., 1998). Studies on the events underlying transgene silencing are defining ways of minimizing the problem and, thus, increasing the likelihood of achieving stable expression in the field. One approach is to select transformants that contain very few copies (between one and three) of the introduced genes, because multiple copies often lead to events that trigger silencing. A related precaution is to avoid using transgenes that so closely resemble endogenous genes that either one gene or both are switched off in a phenomenon termed cosuppression. A third strategy is related to DNA methylation, which is common in plants (Kumpatla et al., 1997). Methylation of promoter sequences can prevent the binding of regulatory proteins and lead directly to gene silencing. Foreign genes should, therefore, be introduced into rice under the control of promoters that will function appropriately, even when the gene is methylated. Suitable promoters will most likely be found in rice or other cereals in association with genes that show regulatory behaviour similar to that desired for the transgene. For example, the maize C₄ PEP carboxylase promoter has provided expression of the CryIA(b) gene in green tissue (Ghareyazie et al., 1997) and expression has been stable for eight generations.

Wide hybridization

The genus Oryza, to which cultivated rice belongs, has 22 wild species and two cultivated species. The wild species are a reservoir of useful genes for rice improvement. Wild species with AA genome can be crossed routinely with cultivated species and useful genes can easily be transferred to cultivated rice. However, special techniques such as embryo rescue and hormone treatment are necessary to produce hybrids between cultivated rice and more distantly related species. Hybrids have been produced through an embryo rescue technique between elite breeding lines and cultivars and several accessions of 11 wild species representing BBCC, CC, CCDD, EE, FF, GG and HHJJ genomes (Brar, Elloran and Khush, 1991). A number of useful genes have been transferred from wild to cultivated species (Table 3).

Khush (1997) transferred a gene for grassy stunt virus resistance from O. nivara to IR24, an elite cultivar of rice, through backcrossing. Jena and Khush (1990) transferred genes for resistance to three biotypes of brown planthopper from O. officinalis into the elite breeding line IR31917-45-3-2. Some of the desired lines also showed resistance to brown planthopper populations in India and Bangladesh. Four breeding lines resistant to brown planthopper derived from this wide-cross have been released as varieties in Viet Nam. Genes for blast, bacterial blight and brown planthopper have also been transferred to rice from other wild species such as O. minuta, O. latifolia, O. australiensis and O. brachyantha.

Cytoplasmic diversification

Most of the commercial hybrids of indica rice are based on wild abortive (WA) source of cytoplasmic male sterility (CMS). Such cytoplasmic uniformity increases the genetic vulnerability of hybrid rices to diseases and insects. To overcome this problem, diversification of cytoplasmic male sterility source is essential. Forty-nine accessions of O. rufipogon were crossed as female parents with elite cultivars, IR54 and IR64, both of which can restore fertility of WA cytoplasm. Of all the backcross derivatives of these crosses, one line with cytoplasm of O. rufipogon (Acc. 104823) and the nucleus of IR64 was found to be stable for complete pollen sterility. This newly developed CMS line has been designated IR66707A (Dalmacio et al., 1995). Crosses of IR66707A with six restorers of WA cytoplasm also showed almost complete pollen sterility, indicating that this source of CMS is different from that of WA cytoplasm. Southern hybridization of IR66707A, O. rufipogon and IR66707B with eight mitochondrial DNA-specific probes was carried out. Of 40 combinations, 18 showed a monomorphic pattern while, in 22 polymorphic combinations, the banding patterns of IR66707A and O. rufipogon were identical. The results indicate that IR66707A has the same mitochondrial genome as the donor O. rufipogon and that CMS may not be caused by any major rearrangement or modification of mt DNA.

Molecular characterization of alien genetic variation

Both isozyme and RFLP markers detect extensive polymorphism between rice and wild species and have proved useful as genetic markers in the characterization of alien genetic variation. Introgression has been detected for isozyme loci from several wild species. RFLP analysis of the introgression lines derived from O. sativa x O. officinalis showed introgression of the chromosome segments of 11 of the 12 chromosomes of O. officinalis (Jena, Khush and Kochert, 1992). In the majority of cases, O. sativa alleles were replaced by O. officinalis alleles. Using molecular markers, introgression of small chromosome segments has also been detected from chromosomes 10 and 12 of O. australiensis into rice chromosomes (Ishii et al., 1994).

Cytogenetic and RFLP analysis of introgression lines derived from crosses of O. sativa and distantly related species showed genetic recombination between chromosomes of cultivated and wild species to be the cause of alien gene transfer. RFLP analysis of introgression lines showing reciprocal replacement of alleles of O. sativa with alleles of O. officinalis and O. australiensis supports the conclusion about alien gene transfer through crossing over rather than the substitution of a complete chromosome or arm of chromosome of a wild species (Jena, Khush and Kochert, 1992; Ishii et al., 1994). The rapid recovery of recurrent parent phenotypes in BC₂ and BC₃ progenies of O. sativa x O. officinalis, O. sativa x O. australiensis, O. sativa x O. brachyantha and O. sativa x O. granulata is an indication of very limited recombination between A genome, on the one hand, and C, E, F and G genomes on the other.

FUTURE PROSPECTS

Rice is now recognized as a model system for genetic analysis and biotechnology applications for improvement of monocots. Factors contributing to this situation include the comparatively small size of the rice genome, the synteny of its genome with those of other cereals, and its comparative ease of transformation. Rice is probably second only to the dicot Arabidopsis in terms of global public-sector investment in plant genetic research. The programme to sequence the Arabidopsis genome (about 100 million base pairs) is expected to be completed in 2002. A similar programme to sequence the genome of the rice cultivar Nipponbare (about 440 million base pairs) began in 1998 and will take about ten years to complete. These programmes are referred to as "structural genomics" and will provide sequences for the estimated 30 000 genes of both plants and for the intergenic DNA that plays important but poorly understood roles in gene expression, DNA replication, chromosome organization, recombination, speciation and evolution.

The public availability of the Arabidopsis and rice sequence data has already ushered in the era of "functional genomics". One challenge is to ascertain the function of previously unknown rice genes revealed by sequencing. A second is to understand the functions of apparently redundant rice genes that may have subtly different roles in different tissues or in response to different environments. Particularly important in this context are the regulatory genes that function in signal transduction pathways and serve to integrate the response of rice to growth factors, pathogens, stresses and other environmental cues. The Xa21 gene is a member of this group (Song et al., 1995). A range of biochemical and mutational approaches known as "reverse genetics" is meeting this challenge. Included are various techniques that use heterologous transposable elements to create mutations and provide data on patterns of gene expression (Sundaresan et al., 1995). The finding that the tobacco retrotransposon Tto1 undergoes autonomous transposition in rice (Hirochika et al., 1996) illustrates the applicability of these approaches to rice.

Another challenge is to understand how rice genes interact with one another and the environment to control developmental and defence responses. An important new technology here is the biochip. This is a micro-array of tens of thousands of oligonucleotides that enable the abundance of all messenger RNAs in a tissue to be determined simultaneously (Lockhart et al., 1996). Biochips are already used to follow changes in the expression of 6 000 genes of yeast in response to changes in nutrition or in response to mutation of specific genes (Wodicka et al., 1997).

The sequencing of the Nipponbare genome will greatly accelerate the isolation of useful alleles from other cultivars. Genes conferring a key trait in a cultivar A will be mapped to specific regions of the rice genome and then the Nipponbare database will be consulted to identify candidate genes that might contribute to the trait. Transformation with the allele of candidate genes will indicate the responsible gene.

In conclusion, it is worth mentioning a key feature of functional genomics of yeast that is not yet available in rice. This feature is targeted gene replacement which allows knock-out mutagenesis and allelic substitution at any locus in yeast (Burns et al., 1994). It is achieved by constructing a transformation vector that contains two regions of sequence identity with the target locus. Depending on the details of the construction, homologous recombination will result in either mutation or allelic substitution. A preliminary indication that such a facility may be possible in plants was provided recently for Arabidopsis (Kempin et al., 1997). As thousands of rice genes are discovered through genome sequencing, attempts will be made to discover their function through random mutagenesis. Direct knock-out would be much more efficient. In relation to rice improvement, transformation can already add a novel allele to the genome, but it is unable to direct this allele to replace an existing allele. As a result, the effect of the novel allele could frequently be masked. Targeted allelic substitution would eliminate this problem and justify the description of transformation as a "surgical" approach to rice improvement.

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Progrès de la génétique et des biotechnologies concernant le riz

Les principaux progrès en génétique du riz incluent la description de 400 gènes mutants affectant les maladies morphologiques, physiologiques et biochimiques et la réaction aux insectes, les stress abiotiques et la coloration de certaines parties du plant, ainsi que l'établissement de 12 groupes de liens correspondant à 12 chromosomes; l'utilisation d'isozymes comme marqueurs sur au moins 50 locus du riz; et l'application du polymorphisme de taille des fragments de restriction (RFLP), l'amplification aléatoire polymorphique de l'ADN (RAPD), le polymorphisme de taille des fragments amplifiés (AFLP) et les microsatellites pour préparer des cartes saturées.

La disponibilité de cartes génétiques moléculaires détaillées du riz a permis de marquer de nombreux gènes présentant un intérêt économique avec des marqueurs moléculaires, de façon à réduire considérablement le coût du triage des matériels de reproduction. L'avènement des marqueurs moléculaires a rendu possible la cartographie et le marquage des locus des caractères quantitatifs (QTL).

Un protocole MAS fondé sur la réaction en chaîne de la polymérase (PCR) récemment mis au point coûte 10 fois moins cher que les protocoles RFLP et est 10 fois plus rapide. Les possibilités de cloner les gènes du riz sur la base de la position sur la carte ont été sensiblement renforcées avec la mise au point de bibliothèques de chromosomes artificiels bactériens et des protocoles ont été élaborés pour transformer les plants de riz en vue d'améliorer leur résistance aux insectes, tels que le riz transgénique Bt résistant à la chenille mineuse de la tige et le riz transgénique portant un gène inhibiteur de la trypsine résistant à la pyrale et à la noctuelle du riz. Les gènes de la protéine d'enveloppe pour les deux virus causant la maladie de tungro ont été clonés et on cherche aujourd'hui à exprimer ces gènes dans les plants de riz.

Récemment, l'utilisation du gène Hval produisant des protéines LEA résistant au stress hydrique et aux effets de la salinité dans les plants de riz a permis d'obtenir de bons résultats; le gène glgc de E. coli codant ADPGPP a été introduit dans le riz pour améliorer la production d'amidon. Un programme de séquençage du génome du cultivar de riz Nipponbare a démarré en 1998. Une nouvelle technologie importante dans ce domaine est la biopuce: microsérie de dizaines de milliers d'oligonucléotides qui permettent de déterminer simultanément l'abondance de tous les ARN messagers dans un tissu.

Adelantos en la genética y la biotecnología del arroz

Los progresos principales en la genética arrocera comprenden la descripción de 400 genes mutantes que influyen en factores morfológicos, fisiológicos, bioquímicos, y de reacción a las enfermedades y a los insectos, las tensiones abióticas y la coloración de partes de plantas, así como el establecimiento de 12 grupos de ligamiento correspondientes a 12 cromosomas; la utilización de isozimas como marcadores por lo menos en 50 loci del arroz; y la aplicación de la técnica del polimorfismo de longitud de los fragmentos de restricción (PLFR), el ADN polimórfico amplificado al azar, el polimorfismo de longitud de los fragmentos de amplificación (PLFA) y los microsatélites para preparar mapas de saturación.

La posibilidad de disponer de mapas genéticos moleculares completos del arroz ha permitido identificar muchos genes de importancia económica con marcadores moleculares, con lo que se ha conseguido reducir considerablemente el costo de selección de materiales de mejoramiento genético. La aparición de los marcadores moleculares ha permitido cartografiar y etiquetar LRC (loci de rasgos cuantitativos).

Un protocolo MAS basado en la reacción en cadena de la polimerasa (RCP), que se ha desarrollado últimamente, tiene una décima parte del costo de los protocolos de PLFR y es más de 10 veces más rápido. Las posibilidades de clonar genes de arroz sobre la base de su posición cartográfica ha aumentado notablemente con la creación de colecciones de cromosomas artificiales bacterianos (CAB), y se han elaborado protocolos para transformar las plantas de arroz de suerte que resulten resistentes a los insectos, como el arroz transgénico Bt para luchar contra el barrenador amarillo del tallo y un arroz transgénico portador del gen inhibidor de la tripsina del caupí (Cpti) para hacerlo resistente a la piral y la noctuela del tallo. Se han clonado ya genes de la proteína del revestimiento (PR) para los dos virus componentes que causan la virosis de las hojas anaranjadas y se está tratando de expresar esos genes en las plantas de arroz.

Recientemente ha habido cierto éxito en la utilización del gen Hval que produce proteínas abundantes de la embriogénesis tardía que regulan las condiciones adversas por falta de agua y por salinidad en las plantas de arroz. Para mejorar la producción de almidón se ha introducido en el arroz el gen glgc proveniente de E. coli que codifica el ADPGPP. En 1998 se emprendió un programa para establecer la secuencia del genoma del cultivar Nipponbare. Una tecnología nueva e importante a este respecto es el biochip: un microdispositivo de decenas de millares de oligonucleótidos que permiten que pueda determinarse simultáneamente la abundancia de todos los ARN mensajeros de un tejido.