Japonica Rice Genetic Transformation System

Rice is one of the main food crops in the world, providing food and energy to more than one third of the population of the world, the level and quality of rice production is directly related to many people's health, In recent years, it creates a new pathway for the conventional breeding and heredity improvement to obtain transgenic rice by gene engineering. The tissue culture of rice as the basis technology for rice gene engineering, plays an important role in rice improvement. It is necessary to establish stable, high frequency riceregeneration system in rice transgenic technology in order to realize ideal and better transformation. With the popularity of plant genetic engineering technology, more and more laboratories hope to build a set of efficient, economic and simple system of transgenic technology.The result of the study showed as following.Japonica rice mature embryos vitro culture regeneration technology system were established and optimized using Songjing I Japonica rice. In the experiment, we found seed treatment (75% alcohol 3min +0.1% mercuric chloride for 10 min+ 10% sodium hypochlorite 15min) is better for seed sterilization. The NB (N6 macronutrients, B5 microelements and inorganic elements) medium was used as the basic medium of calli induction. Inducing medium using by adding 2.0mg/L of 2,4-D with 0.5 mg/L of 6-BA is the best, and adding the appropriate amount of hydrolyzed casein and Glutamine can improve the quality of callus. Following generations with MS-N6 basic medium used interchangeably, and adding 2mg/L2,4-D +1mg/L 6-BA +0.2mg/L NAA is in favor of embryo callus subculture; subculture add the proper amount of vitamin C and activated carbon can reduce the occurrence of browning callus; callus subculture time should be controlled in the 10 d, and not more than 14 d, and the callus should be differentiation in 3 subculture times. Best green plantlet differentiation is MS+6-BA2.0+NAA0.2+ TDZ0.1 (mg/L). Agrobacterium-mediated genetic transformation technology system were optimized using mature embryo callus of Songjingâ… Japonica rice. Using Songjingâ… mature embryo callus as transformed receptor, the effects of the agrobacterium-mediated genetic transformation were studied on influence of the concentration of screening agent Kan, co-culture time, washing agrobacterium, drying treatment etc.The results showed that 200 mg/L was Kan-resistant concentration. Callus and Agrobacterium co-culture 3d can obtain the most resistant callus, the resistant callus rate was the highest. Before the callus inoculating filtration medium, drying treatment can inhibit the reproduction and growth of Agrobacterium tumefaciens effectively, and increase resistant rate of callus.Using Songjingâ… mature embryo callus as transformed receptor, by Agrobacterium tumefaciens and Kan resistance filtrating,73 transgenic rice plants resistant to Kan were obtained. And the results of PCR amplification showed us that transgenic rice plants obtained with the special bands at 997bp. Preliminary proof that APX4 gene had being integrated into the genome of Japonica rice Songjingâ… .