Studies On Indica Rice Transformation Mediated By Agrobacterium Tumefaciens

Rice (Oryza saliva) is one of the most important grain crops in the world, about 40% Of the World's population take it as their staple food. In China, rice accounts for about fifty percent of cereal consumption, the importance of which is only next to wheat. Although the conventional breeding methods have played important role in the increase of rice yield and improvement of quality, there exist many problems in further genetic improvement of rice due to shortage of rice genetic resources, variations of ecological environment and limitation of routine breeding methods. With further development of the research in molecular biology, the techniques and methodologies of gene isolation, cloning and DNA recombination have become more and more mature, and now genetic transformation has become a useful method in rice genetic improvement. The research in . functional genomics of rice and the following large-scale genetic engineering will have to depend on high efficient genetic transformation. Therefore genetic transformation technology of rice has become important foundation of molecular biological research and genetic improvement of rice. Among all transgenic methods, Agrobacterium-mediated transformation is the most preferential in consideration of simplicity of operational means, small copy number of foreign genes inserted and good integrity of gene structures.At present Agrobacterium-mediated genetic transformation systems for Japonica rice, Indica rice and Javanica rice have been established, and some useful genes in insect resistance, disease resistance, adverse environmental endurance and quality improvement of rice have been introduced into deferent rice varieties. Nevertheless, genetic transformation of rice has been made complicated and random since there exists wide genetic diversity among rice varieties and responses to Agrobacterium-mediated genetic transformation deeply depend on rice genotype. Especially, there is much difficulty in Indica rice transformation. Although some examples have been reported to make successful genetic transformation of indica rice, they were limited to special varieties and transgenic frequency was very low. Common transformation techniques suitable for all Indica varieties have not be found so far. Therefore many problems will have to be solved through further investigation in order to establish stable and highly efficient transformation system. Among all rice varieties, more than 80% are Indica, and the main varieties planted in most south rice-producing provinces of China are Indica rice so that it is of social and economic significance to make genetic improvement of Indica rice.In this paper, 5 Indica rice varieties, JH10, JH35, JHIOI, SH527 and R21, which are important in rice breeding in Chongqing area, were selected to investigate the key factors influencing genetic transformation of Indica rice. The Agrobacterium-mediated genetic transformation system that optimized for the above Indica varieties was primarily established and 51 putative transgenic plantsof 2 varieties were obtained. PCR assay and histochemical examination showed primarily that foreign genes (GUS and NPTII) had already been integrated into rice. The main results were summarized as follows.1. The factors affecting induction of callus and regeneration for indica rice were investigated, among which type of medium, combination of phytohormones, genotype of rice and water stress significantly influenced induction and regeneration of calli. The regeneration system with immature embryos of rice was established and optimized for JH10, JH35 and 527.(1) In callus induction of Indica rice, 2, 4-D of a certain concentration was absolutely necessary, optimal concentration was 2-2.5mg/L and single 2, 4-D without other hormones could have good effect. The combination of 2, 4-D with other hormones, such as BA, KT, decreased induction frequency, but improve the quality of the obtained calli. The best combination of hormones needs further investigation.(2) The type and compositions of medium played a very important role in callus induction, subculture and differentiation of Indica rice, but different genotypes responded differently to type of medium. Thus, each genotype can be found to match its specific medium. Generally, MS and NB basal media are optimal medium types for Indica rice. All the tested genotypes had comparatively high inducing rates on these two media.(3) The combination of different hormones influenced the regeneration frequency of indica rice. As far as SH527, JH10, and JH35 were concerned, combination of 3mg/L BA with 0.5mg/L NAA was optimal hormone condition, under which high regeneration frequencies of these 3 varieties could be attained.(4) Treatments of water stress, including increase of sucrose concentration, supplementation of osmotic agents, such as mannitol and sorbitol, increase of agar concentrateion, and partly desiccation, could significantly increase differentiation frequencies of Indica rice. In our study, regeneration frequency of calli of JH35 treated by partial desiccateon was up to 59.4%, while that of control was 43.5%.(5) The regeneration frequencies varied with the genotypes. Among 5 tested varieties, JH101 and R21 had comparatively low regeneration frequencies, 5% and 8.3%, respectively and SH527, JH10 and JH35 had comparatively high regeneration, 33.3%, 30% and 35%, respectively, which can generally meet the demands of the following genetic transformation.2. The key factors influencing genetic transformation of Indica rice were investigated by using JH10 and JH35 as recipient material and Agrobacterium strain EHA105 harboring plasmid plG121 as transformation vector. The transformation protocol mediated by Agrobacterium Tumefaciens for JH10 and JH35 was primarily established and totally 51 putative transgenic plants were obtained.