Establishment And Optimization Of Multi-gene Genetic Transformation And Regeneration Of Indica Rice Minghui 86

Rice is one of the most important food crops in China,and its stable production is important guarantee for national food security.However,drought,rice pest insects and diseases,herbicides and other factors will seriously restrict the yield of rice,and also affect the quality of rice to varying degrees.Therefore,the most effective way to obtain new rice materials bearing high yield,resistance,insect resistance and disease resistance is genetic transformation with plant expression vectors harboring multiple genes.Based on the existing genetic transformation system in our laboratory,this study is expected to establish a more rapid and efficient genetic transformation system by improving and optimizing the culture conditions.The high-yield gene RRM2,drought resistant gene HS1,herbicide resistant gene EPSPS,insect resistant gene BT,and apoptosis inhibiting gene IAP and P35 were constructed at two plant expression vectors,namely P5 and P8 respectively.The indica rice restorer line Minghui86 was transformed with P5 and P8 multi-gene vectors respectively.The main results of this study are as follows:1.Under the same induction culture conditions,the immature embryos and mature embryos of MingHui 86 were induced.The results showed that the calli induced in the immature embryos of MingHui 86 were in good condition,the calli were bright yellow,large,exuberant,and the induction rate was high.The texture and structure of calli derived from mature embryos of Minghui 86 were smaller,the growth rate of callus was slower,and the induction rate was relatively low.2.The growth comparison of callus under different gradient concentrations of screening agent glyphosate and G418 showed that MingHui 86 calli were insensitive to glyphosate screening,and the high concentration of glyphosate was required to be added in the screening process.The suitable screening concentrations of glyphosate and G418 are 800mg/L and 150mg/L,respectively.3.The concentration of Agrobacterium and the time of inoculation and co-culture are the main factors affecting the transformation efficiency.The results showed that the high concentration of bacterial liquid,and the long period of inoculation and co-culture made excessive reproduction of Agrobacterium tumefaciens,resulting in toxicity and inhibition of normal growth of callus,and consequently reduction of induction rate.Therefore,the proper transformation process of the callus could be summarized as bellows:the OD600 of the infection liquid of Agrobacterium tumefaciens is 0.4?0.6,the infection time is strictly controlled in the 15?20min,and the calli after infection are co-cultured at 26 ? in dark for 2?3d.4.Exogenous hormone 2,4-D is the most commonly used growth regulator in plant tissue culture.The formation and differentiation of callus of indica Rice Minghui 86 are influenced by different exogenous and endogenous hormones,but the proper amount of exogenous hormone 2,4-D are necessary for callus growth of indica rice.The results showed that the best concentration of 2,4-D for calli induction of Minghui 86 was 2 mg/L,and the calli induced by this concentration of 2,4-D were of good quality and strong growth,which was beneficial for subsequent callus transformation.