Study On Transformation Of β-1,3-Glucanases(BG2) Gene Into Pumpkin Mediated By Agrobacterium Tumefaciens

Squash has abundant nutrition and broad use, and its edible value and economic value were recognized. Heilongjiang province is a major seedy pumpkin production area in both china and abroad, and the cultivation area of seedy pumpkin in Heilongjiang can be up to 3,000,000 mu per year. The seedy pumpkin is also one of the industries which exporting goods to earn foreign currency. But serious fungal diseases greatly decreased the production and quality of pumpkin. Epidemic disease causes the seedling of pumpkin die or pumpkin itself go rot, widely spreaded epidemic disease in specific rainy years may cause the crop die out in local areas. The powdery mildew may cause plants premature senility which reduce the yield and affect the plumpness of seed. Traditional methods of breeding played an important role in the improvement of pumpkin, but they also had many disadvantages ,such as long period of breeding,huge investment,low efficiency and the shortage of disease-resistant materials.With recently rapid progresses in tissue culture and genetic transformation of goal genes to create new materials,biological technique became a new approach to breed new varieties with the ability of disease resistance.In this study, using JingHui 1# as the trial material , a high frequent regeneration system was established, and explannts were transformed byβ-1,3-glucanase(BG2) gene using the methods of Agrobacterium tumefaciens-mediated Transformation. We also studied the effect of inside, outside factors on the regeneration system as well as the efficiency of genomic transformation, and fixed on the best condition of regeneration and gemomic transformation. It provided the basis for breeding new variety of Anti-Fangal squash. The main results were summarized as follows:(1) Squash Regeneration system of JinHui 1# was established: 0.8% agar is the best culture medium for germ-free plant, and smoothly put it in the medium; Germ-free plant which have two vertical and green Cotyledons is the best physical state of explant; Half of the hypocotyle is the best explant; MS basic medium supplemented with 1.0 mg/L 6-BA is the best differentiation medium for inducing the appearance of adventitious buds, and MS+0.1mg/ L6-BA +0.1 mg/L GA3 is the medium for the elongation of bud; The most appropriate culture medium for generating root is MS+0.1mg/L NAA.(2) Concerntration of antibiotic for screening was fixed on: There is NPTⅡgene in the vector, therefore, the transformed plant have the resistance of Crab. By two screenings we made it certain that the concerntration of Crab was 100mg for the bud in differentiate stage.(3) The effect of time of pre-culturing, concerntration of bacterium, time of infecting, time of co-culturing, style of adding AS and concerntration of As on genomic transformation was found, and the best way was: pre-culturing for 36h in the condition of OD600(0.3~0.5) firstly, then infecting for 10min, co-culturing for 2d(add 100 mg/LAS to the cultlure) at last.(4) The resistant plant was examined by PCR and Southern Blot, 5 positive plant were gained.