Optimization Of Wheat Transformation Mediated By Agrobacterium Tumefaciens And Development Of Wheat Transgenic Lines Resistant To Barley Yellow Dwarf Virus (BYDV)

Wheat immature embryo is considered to be the best explant material source for calli development and shoot regeneration because of its high cultural characteristics and better regeneration potential. But, in the Agrobacterium-mediated transformation of wheat, the transformed tissues become severely necrotic after co-cultivation, which induces the final transformation efficency. Therefore, it is very important for the improvement of wheat transformation to overcome Agrobacterium-induced tissue necrosis and optimise the transformation system of immature embryos. The yellow dwarf disease of wheat is caused by barley yellow dwarf virus (BYDV) and transmitted by aphids, and its outbreaks can result in devastating yield loss in wheat. BYDV-GAV is one of the main BYDV isolate in recent years in China. In order to get some wheat variety resistant for BYDV-GAV, The RNAi vector pWBVec-2B-hpGAVpol was constructed and then transferred to wheat mediated by Agrobacterium tumefaciens. The mian contents and conclusion were as follows:1Using wheat variety CB037as the explant, the selection effects on the wheat mature embryo calli with different mass concentrat ion of hygromycin (Omg/L,30mg/L,50mg/L,80mgLl,110mg/L) were studied. The results showed that the optimal mass concentrat ion of hygromycin was30mg/L for wheat embryo calli select ion and50mg/L for shoot regeneration selection, respectively. This contrites to wheat transformation mediated by Agrobacterium tumefaciens.2The wheat genetic transformation mediated by Agrobacterium tumefaciens was studied by using the transient expression of gus gene. Variety CB037as receptor was used to investigate the effects of different treatment on wheat transfrmation. The results showed that40g/L monnitol pre-treatment on calli and application of500mg/L L-Cyestein in tissue culture both contributed significantly to the improvment of wheat transformation system mediated by Agrobacterium tumefaciens, with the transient GUS expression efficency of94%, callus embryogenesis efficency of65.7%, regenation efficency of39.1%and transformation efficency of8.2%. Hygromycin resitance test agreed with PCR analyse of transgenic plant blades at a rate of92.6%, which proved that hpt gene is not only a good select maker, but olso a good reporter gene for Agrobacterium-mediated wheat transformation.3We develop some wheat transgenic lines resistant to barley yellow dwarf virus (BYDV). RNAi Vector pWBVec-2B-hpGAVpol was transferred to wheat elite variety Kenong199by the Agrobacterium-mediated transformation protocol established in this study. The results showed that14transgenic plants were developped with a transformation frequency of2.1%. T1progeny has been obtained by the self-pollination of To plants.