Optimization Of Agrobacterium Mediated Transgenic System Of Wheat Mature Embryos And TaATG8Genetic Transformation

Wheat is one of China’s major grain crops, which is important source of phytoproteinfor human, playing an important role in the food industry and agriculture production.Genetic improvement of wheat varieties had been widely appreciated. Since traditionalbreeding methods couldn’t meet the demand of wheat yield and quality in the modernsociety, coupled with pests and diseases and some other adverse factors, it resulted in theproduction and quality decreased of wheat. Application of biological technology aiming toimprove the resistance to diseases and pests, quality and yield of wheat varieties attractedmore and more attention. Therefore, that establishes stable and efficient tissue culturesystem to improve the efficiency of wheat transformation,it will lay the foundation formolecular breeding.At the moment, in the Agrobacterium-mediated genetic transformation of wheatmature embryos transformation system, the conversion rate decrease resulted in thedecrease of callus regeneration rate during the tissue culture period. It was very necessaryto found excellent Agrobacterium-mediated wheat mature embryos regeneration system. Inthis study, wheat cultivar5389was used as acceptor material. After the exploration of theeffect of different mediums, the concentration of2,4-D on the embryogenic callusformation, and the effect of different concentration anti-oxidants, seedling ages of explant,hormone combinations, co-culture times and bacteriostatic agent on callus differentiation,the optimized wheat mature embryos transformation system were concluded.Autophagy is an important defense and protection mechanism in which the cell digestits aging long-lived proteins, damaged cells and organelles to provide essential material forreconstruction, regeneration and repair in the condition of nutritional deficiencies or stressduress. The ATG8was the core protein in the process of autophagy. The result of our studyshowed that in affinity and non-affinity interactions of wheat and rust, the expressions ofTaATG8in transcription and translation level were significantly different, suggested thatTaATG8might have raltion with wheat leaf rust infection.In this study, we constructed TaATG8gene over-expression and RNAi binaryexpression vector, and transformed them into Agrobacterium tumefaciens strain EHA105, respectively, then used optimized Agrobacterium-mediated wheat mature embryos togenetic transformation in order to study the function of TaATG8and the relationshipbetween TaATG8and autophagy. In this study, we obtained the following results:1. The optimum concentration of2,4-D was2mg L-1and MS medium was selectedfor embryogenic callus induction.2. The optimum explant age for wheat transformation was20d, the optimumconcentration of vitamin C was2mg L-1and the optimum coculture time was3d. Theoptimum hormone combination was ZT1.0mg L-1, BA0.5mg L-1and IAA0.5mg L-1.The antibacterial agents100mg L-1Timentin,200mg L-1Cef could effectively inhibit thegrowth of Agrobacterium as well as significantly improved the callus’s resistance andshoot regeneration rate.3. The positive GUS gene overexpression plants were obtained by detection ofDNA-PCR and RT-PCR.4. We used the fragment of TaATG8which didn’t have stopped codon combining withGFP to construct the plant express vector and inject the tobacoo leafs. Through theobvation of Confocal, we discovered the expression of TaATG8in cytoplasm.5. We constructed the TaATG8over-expression and RNAi vector and used optimizedtransformation system of wheat mature embryos to transform5389with TaATG8andtransform L10with RNAi vector, and then, we acquired the PCR positive plants. It laid thefoundation for the further research to defining the function of ATG8.