Establishment Of Rice Immature Embryo Regeneration System By High-temperature Culture And Waxy-Gt1 Fusion Gene Introduced Into Rice By Agrobacterium-mediated Transformation

Through many years` uninterrupted development, tissue culture technique has been a powerful tool for biology science research. Agrobacterium tumefaciens- mediated transformation is the preferred method of rice gene transformation, and the effective means of rice genetic improvement. Rice regeneration system and Agrobacterium tumefaciens-mediated transformation are the important basic work of rice gene engineering technique and molecular biology research, and we have got a lot of experience and achievements in the past few years. This paper summarizes the advances in rice regeneration system and Agrobacterium tumefaciens-mediated transformation in rice: influential factors of rice regeneration system and gene transformation mediated by Agrobacterium tumefaciens; theory,merit,problems and perspectives of rice transformation mediated by Agrobacterium tumefaciens and so on.The establishment and optimization of rice regeneration system play an important role in the studies on improving rice qualities and rice molecular biologics by transgenic technology. In order to probe whether rice calli could be induced and cultured in higher temperature environments, and establish regeneration system for the high-yield rice cultivar Chao 2-10, we designed four temperature environments: 26℃,36℃,38℃and 40℃for inducing and subculturing calli derived from cultivar Chao 2-10 rice immature embryos as explants, and completed the whole regeneration progress in this study. The results show that the immature embryos of Chao 2-10 rice can successfully produce calli, and the calli induction rates are all above 90%, statistical analysis show there are no significant differences on the induction rates of calli cultured in the four temperature environments; Calli induced and subcultured at 26℃and 36℃can normally grow, differentiate and regenerate, while calli induced and subcultured at 38℃and 40℃can grow but their differentiation and regeneration are all affected; Even though the weight amplitude of the calli cultured at 36℃is much lower than that of the calli cultured at 26℃during subculture progress, the regeneration rate of the calli cultured at 36℃is much higher than that of the calli cultured at 26℃, registering an average increase of 40.81%. Developing this study can lay the foundation to establish an efficient genetic transformation system for the high-yield cultivar Chao 2-10.Two experiments have been completed in order to explore if it is possible to restrain the growth of Agrobacterium through raising the culture temperature to the callus in the antibiotic selection period. First, Agrobacteria with certain quantities was cultured at 26℃,30℃,34℃,36℃,38℃and 40℃respectively. The result showed that Agrobacterium couldn't grow at 38℃and 40℃. Second, rice calli induced at 26℃were cultured at the temperatures that Agrobacterium couldn't grow (38℃and 40℃) for four different length (7d,14d,21d and 28d) respectively, and those cultured at the 26℃were used as the controls, for researching the influence of high temperatures on regeneration of rice callus. The result showed that rice calli cultured for all time at 38℃and part time at 40℃during simulative antibiotic selection period could grow normally, and the regeneration rate of the calli cultured at 38℃for 14d was higher greatly than that of the calli cultured at 26℃, increasing 63.17%, having significant difference with that of the calli cultured at 26℃at the 5% level. This research theoretically indicates that it is feasible to add less or no antibiotics to kill the Agrobacterium in the selection medium by culturing calli after co-culture with Agrobacterium at high temperature in the process of Agrobacterium-mediated transformation of rice.Increasing the nutrition content of rice is very important to strengthen our citizens` constitution. Protein content is one of the most important guide lines used for estimating rice nutrition quality. In order to increase the protein content in the center of rice endosperm and improve rice nutrition quality, we transfor Waxy-Gt1 gene constructed in our own lab into the high-yield rice cultivar Chao 2-10 mediated by Agrobacterium tumefaciens. The regeneration rate was 37.26%, and 125 transgenic plantlets was obtained. Developing this study can lay the foundation to select rice plantlets whose protein content is much higher caused by the high expression of the exogenous gene in the center of rice endosperm in future.