Optimizing The Regeneration Of Hemarthria Compressa And Transformation Of SpTPS1Gene

Hemarthria compressa, a rhizomatous perennial grass in Hemarthria Robea Brown of Gramineous(Panicoideae). Being a fast growth, generativity, high production, high quality, adaptability, strong resistance and perennial evergreen forage, Hemarthria compressa is one of the most important pasture grass in southwest china. It plays a significant role in livestock development and restoration and reconstruction of ecological environment. But its resistance to frost damage and drought is not so strong that need to increase resistance by new variety breeding in order to generalize and ampliative growing area. Pollen enerage of Hemarthria compressa is very low caused the natural fruit set is very low; It is difficult to update through sexual reproduction, but can be solved by genetic engineering. Trehalose is one of the important osmotic adjustment material related to plant stress resistance. It maybe improve the accumulation of trehalso to increases the stress resistance by introduction of TPS gene into plant.Younger stems and immature inflorescences of ’Guangyi’were used as explants to optimization the plant regeneration in this experiment. Then the callus induced by young stems and immature inflorescences were used to be material with Agrobacterium-mediated transformation way, eatablish the TPS gene transformation system of Hemarthria compressa. The main techniques systems were as follows:(1) Callus induction stage for immature inflorescences of Hemarthria compressa ’Guangyi’, MS+2,4-D1.0mg/L+6-BA0.1mg/L was the best callus induce medium, the inducing rate of embryogenic was97.48%, the callus proliferation faster. The higtest inducing rate of embryogenic callus was98.27%by taking the middle period of development(0.5-2.0cm). The appropriate time were June and July for inoculate by taking immature inflorescences as explants, the inducing rate of embryogenic callus were97.15%and99.16%.(2) Callus differation stage:The highest rate of callus differation for callus induced by immature inflorescences happened in the medium of MS+6-BA1.0mg/L+NAA0.2mg/L, the rate was91.39%, this processing had more green buds, adventitious bud in clusters and growth healthy. The appropriate subculture time of callus induced by yong stems and immature inflorescences were both2-3times when used for genetic transformation. After subculture2time, the callus differation of immature inflorescences and young stems were91.01%and76.14%, the average number of green buds were16.19and15.28; after subculture3time, the callus differation of immature inflorescences and young stems were92.13%and76.40%, the average number of green buds were18.07and16.92.(3) The choice of antibiotics:300mg/L ceftaxime could elimination the Agrobacterium, but carbenicillin need450mg/L. The weight rate of callus induced by immature inflorescences and young stems were80.83%and76.86%,when added300mg/L ceftaxime. So ceftaxime300mg/L should be selected based on the action concentration and their effects on callus propagation.(4) The concentration of Bialaphos:We selected Bialaphos as selection according the marker gene, the optimum concentration of Bialaphos for resistant callus of immature inflorescences and young stems were2.5mg/L and2.0mg/L; for resistant green shoots,the optimum concentration of Bialaphos were both2.5mg/L for immature inflorescences and young stems.(5) Genetic transformation sytem:In the treatment OD6oo=0.4, T=30min and OD6oo=0.6, T=20min after4days co-culture, we acquired bialaphos-resistant callus of immature inflorescences as transgenic receptor. We got7bialaphos-resistant plants through differentiation, and got3positive plant by PCR. But we could notobtained any bialaphos-resistant callus of young stems.(6) With3transgenic plants and3non transgenic plants, relative water content and electronic conductivity of leaf were evaluated drought tolerance using25%(m/v) PEG-6000to simulating water stress method. After5days, the relative water content of transgenic plants remained as high as71.65～81.9%, the relative electronic conductivity was8.11～9.81%. But to the non transgenic plants, the average relative water content was only44.93%, the average relative electronic conductivity was48.82%. Both proved that drought resistance of the transgenic plants increased.