Systematic Optimization Of Callus Induction And Plant Regeneration From Mature Embryo Of Indica Rice

The rice is the most important food crop in the world, meanwhile, it is also a mode for studying genomics and functional genomics of monocotyledon. At present, genetic transformation play an increasingly important role in the improving rice varieties, detection of gene function and stress resistance research, and become an effective means to carry out rice genomics and functional genomics research.In Genetic transformation of rice, the most commonly used method is agrobacterium-mediated method. Compared with other transformation method, agrobacterium-mediated transformation has unique advantages, as agrobacterium is a natural transformation vector. However, there is a big difference of tissue culture and genetic transformation between indica and japonica rice, because of their genetic specificity and gene-dependent differences. And there are much lower regeneration frequency for indica rice than for japonica rice. So far, small amount of transgenic rice of indica is obtained mainly by doing a large number of repeated experiments. At present, there is no efficient genetic transformation system for all the indica varieties although indica rice is widely cultivated in China. Therefore, it is very important to improve the present technology for genetic transformation of indica rice.Honglian type cytoplasmic male sterility (HL-CMS) rice is one of the three major types of the cytoplasmic male sterile rice used in rice hybrid production in the world. In this experiment, mature embryo of HL-CMS line YTA and maintenance line YTB were selected as a receptor for a series of transgenic projects in our laboratory, and the systematic optimization of callus induction and plant regeneration from mature embryo of those two two varieties were carried out. The main results are as follows.1. The main factors affecting the induction and subculture of the mature embryo callus of YTA and YTB were analyzed. It is showed that a larger difference in temperature between high and low temperature treatment is in favor of the dormancy broken of rice seeds and the improvement of the callus induction. In seeds sterilization, using concentration of 1.5% sodium hypochlorite solution only and 3mg?L^-1 2,4-D is conductive to improve the rate of callus induction. Under these conditions, certain concentration of 6-BA has little effect on callus induction. Adding 100-500 mg.L^-1 of sodium thiosulfate and 30 mg.L^-1 putrescine etc. small organic molecules to the medium can improve the quality of callus and reduce the browning rate. Different media have a great impact on YTA and YTB rice mature embryo callus induction and subculture. three media N6, NB and MS can be used for YTA and YTB callus induction and subculture, for 16 days after induction, then 6 days subcultured, embryogenic callus is at the best state.2. the main factors affecting agrobacterium-mediated transformation and plantlet differentiation were studied, the results showed that there is a contradiction between the resistance callus rate and the resistance callus quality; a certain vacuum negative pressure treatment and proper drying can improve the conversion rate; v-filter lelection may improve the positive rate of transgenic plants. To solve the problems of resistance callus being green easy but at lower seedling rate, a improved process of facilitating resistance callus differentiation was detected. It is suggested that the callus cultured in MS medium at first differentiation and 1/2MS medium at second differentiation can significantly increase the plantlet-setting rate even up to 100%.3. By observing paraffin sections of the internal structure of callus from YTB mature embryos under different conditions, it is confirmed that the morphological changes of callus were well consistent with the anatomical detection.