Study On Establishment Of Indica Rice Regeneration System Based On Mature Embryos And Transformation Of Anti-sense Waxy Gene Into Indica Rice

Rice is one of main food crops in China, its planting area accounts for 30% in all food crops and its yield accounts for 40% in total food yield. Because of the continuous increase of food yield and improvement of living standard, people require the better quality of rice rather than the yield of rice. People increasingly attach importance to eating quality of rice grain. Hunan is an important production district of rice in China. Many varieties of rice have higher amylose content and worse quality in Hunan and this seriously affects the rice export. Therefore, the improvement of the amylase content becomes a very important and urgent task. It creates a new pathway for the conventional breeding and heredity improvement to obtain transgenic rice by gene engineering. The induction and regeneration system of calli suited for the genetic transformation was found by mature embryos of three indica varieties. The system of A. tumefaciens-mediated genetic transformation was optimized by the study on physiological and chemical characters of calli in indica rice in course of tissue culture. Then, the calli of Xiang Zao indica 33 and 01 Zao 5202 were co-transformed by anti-sense waxy and hygromycin-resistant gene. the co-transformed plants was obtained by the hygromycin selection, plant regeneration and PCR analysis. The results are as follow:1. The induction and regeneration system of calli in indica rice mature embryos was found. Three basic media for rice tissue culture have little effect on the induction rate of calli in indica rice. But the quality of calli cultured on the N₆ medium is better and calli cultured on the CC medium have higher rate of differentiation. In our study, the N₆ medium was used as the basic medium of calli induction and the CC medium was used as the basic medium of calli differentiation. 2,4-D is a common revulsant of calli for the tissue culture. The calli of tested rice mature embryos can be effectively induced by adding 1.5²mg·L^-1 2,4-D on N₆ medium, but higher or lower concentration of 2,4-D is disadvantageous to the induction of rice calli. The state of calli can be improved by adding 0.2⁰.4 mg·L^-1 6-BA on the medium and plant regeneration rate of calli increases, but higher concentration of 6-BA can poison calli.2. Growth, differentiation and orientation of morphogenesis of cell depend on their changes of physiology and biochemistry. Change of the soluble protein contentis regular during tissue subculture of calli in indica rice. The protein content and their bands of electropherogram obviously increases in course of active growth of calli. After calli were inoculated on the differentiation medium, their synthesis of protein increases and the soluble protein content appears two peak values at 6d and 12d. The shapes of calli have obvious change in two peaks. This shows that the active synthesis of protein is material foundation of organ morphogenesis. Changes of protective enzyme activity and their isoenzyme electropherogram reflect the rule of free radical reaction in course of the calli culture. PPO activity is relative to the browning degree of calli. POD and SOD activity shows the state of cell metabolism. The calli provided with higher activity of POD is more sensitive to A. tumefaciens and appropriate to genetic transformation. Change of POD, CAT, SOD activity is relative to keep the dynamic balance of active oxygen in cell during the differentiation culture of calli. Cell metabolism is most active when protective enzyme activity arrives at the peak value. Change of isoenzyme electrophorogram is closely relative to the differentiation course of calli in indica rice. The symboled bands of protective isoenzyme will appear during differentiation culture of calli in indica rice. Therefore, Character of isoenzyme electrophorogram is a biochemical sign during tissue culture of plants.3. All changes of cells and tissues are concerned with kinds, levels and ratios of endogenous hormones and their physiological effects result from the interaction of all kinds of hormones during plant tissue culture. Absorption peak of all hormones is different under different wavelength. Wavelength scanning shows that absorption wavelength of IAA is similar to GA3, ABA similar to Z. They can be separated effectively by HPLC under the schemed chromatogram conditions. The measuring results show that endogenous phytohormones to maintain at higher level are an important factor to keep the cells embryonic capacity and active metabolism. Change of IAA, GA3, ABA and Z content is basically similar to the variational trend of calli growth velocity. The endogenous hormones content of calli in different stage is different and the calli containing higher level cytokinin and keeping growth metabolism active is advantageous to A. tumefaciens-med'iated genetic transformation. Higher ABA level may be relative to startup and expression of calli differentiation. With the increase of IAA content, embryonic cells start to form and IAA content arrives at the peak value when formation of the embryonic cells comes into itsfastigium during the differentiation culture of calli. Cytokinin content is relative to seedling differentiation and GA is relative to seedling growth. Appropriate ABA content is an important factor of calli differentiation and high level ABA is advantageous to formation of differentiation seedling. Ratio of ABA/IAA and ABA/GA3 is relative to calli differentiation.4. Transformation efficiency of calli in different stage for indica rice is different by A. tumefaciens. As the tested material, the calli that were subcultured on the medium for 12d have the most transformation efficiency. Treatment time of A. tumefaciens have an bigger impact on genetic transformation of rice and it is proper to treat for 20min. The co-cultivation time and desiccation treatment can affect the genetic transformation of rice, pi 300 and pl3W8 plasmid is transferred into the tested calli by A. tumefaciens-mediated method. The regeneration plants were obtained by two hygromycin-resistant selections and differentiation of calli. PCR analysis proves that exogenous genes have intergrated into the rice genome. There are either hygromycin-resistant gene or anti-sense waxy gene in some regeneration plants and this shows that they are co-transformation plants.