Transformation Of Anti-Freezing Gene KN2into Commencial Wheat Varieties By Microprojectile Bombardment And Agrobacterium Tumefaciens Infection

Low temperature freezing injury is one of the important meteorological disasters seriously affecting crop yield and quality, and causing a great loss to the agricultural production. In China, the wheat cultivation area is broad with a large north-south span and varied topography. Low temperature disaster situations such as frost and late spring cold are complex, heavily and frequently occurring, especially in the mid lower reaches of the Yangtze River and Huanghuaihai plain. Research on introducing excellent exogenous anti-freeze genes into wheat varieties using transgenic technology for developing new antifreeze varieties is feasible. In this experiment, a total of six wheat cultivars including Huamai2668, Huamai2152, etc. were used to study the green dot forming rate of callus under the condition of different particle bombardment parameters, and the effect of particle bombardment and Agrobacterium infection on wheat genetic transformation. The results were as follows:1. The optimization of different parameters of particle bombardment:When gold powder concentration per bombardment was250μg, wheat regenerable green dot rate was higher than that when500μg was applied. The higher the gold concentration was, the greater damage it caused to tissues and cells. The green dot rate of callus which was differentiated from immature embryo was higher by double shots than single shot for a bombardment. However, double shot also weakened the differentiation capacity of cells to some extent. In the standoff distance of6cm trial, the regenerable green dot rate of Hua2566reached46.72%, which is higher than that of9cm.2. Wheat genetic transformation mediated by particle bombardment:With the immature embryos of KeNong199, Zhengmai9023, Huamai2566, Huamai2152, Huamai2668varieties as explants, Bar as the selectable marker gene, KN2as the target gene, experiment of of these genes co-transformation into receptor cells was conducted. A total of20resistant plants were obtained, and the transformation efficiency was0.54%,0.49%,0.46%,0.34%,0.50%, respectively.3. Wheat genetic transformation mediated by Agrobacterium:The callus was differentiated from immature embryos of the tetraploid durum wheat Ldn and the common wheat Huamai2152as receptor material, and infected with Agrobacterium strain AGL1.Finally. Only2Ldn positive plants were obtained.