Study On Insect-resistant Gene Transformation For Populus Davidiana Dodetumefaciens

In this study, Populus davidiana Dodetumefaciens was selected as target material for genetic transformation by Agrobacterium. Some factors that affected the efficiency of genetic transformation were studied. Agrobacterium mediated genetic transformation system of P. davidiana Dodetumefaciens was established. And the transformation regeneration plants were obtained. Stable integration of the goal gene (Spider insecticidal peptide and Bt) in the plant genome was confirmed by PCR detection and Southern blot analysis. The theory base was given for P. davidiana Dodetumefaciens transformation and fastness breeding. The conclusions were drawn as follows:1. A high frequency regeneration system of P. davidiana Dodetumefaciens has been established. The results shown that different Phytohormone proportion can greatly effect Poplar's growing. The best leaf regeneration medium is MS supplemented with 0.5 mg/L 6-BA, 0.01 mg/L NAA; the best root regeneration medium is WPM supplemented with 0.4 mg/L IBA.2. The appropriate concentration of Kanamycin and Cefotaxime, Sodium Salt for sensitivity experiment of the leaves and rooting was determined. The concentration of Kanamycin was 20 mg/L when screening transformed hybrid buds, while 20 mg/L inducing roots. The concentration of Cefotaxime, Sodium Salt was 700 mg/L when disposing Agrobacterium, and the concentration isn't any effect on growing.3. The main factors that affected the efficiency of genetic transformation were studied, the transformation procedure Agrobacterium -mediated of P. davidiana Dodetumefaciens was established successfully. Take the vigorous leaves that were cut to pre-culture for 3 days; centrifuge Agrobacterium when being developped to the logarithm growth time(OD₆₀₀=0.5), then dilute Agrobacterium to the concentration of OD₆₀₀=0.3～0.4 with asepsis distilled water for infection; infection time was 10～15 minute; after being co-cultured 2 days, inoculate on the selecting medium(containing Kanamycin 20 mg/L and Cefotaxime, Sodium Salt 700 mg/L) to be filtrated in succession during inducing unsureness buds. When the unsureness buds grow about 1.5～2 cm, cut them off and take them into the selecting medium (containing Kanamycin 30 mg/L and Cefotaxime, Sodium Salt 800 mg/L) for rooting.4. 1862 leaves were transformated, 56 Km-resistant buds were obtained after many selections, the rate of km-resistant buds is 3 %.13 Km-resistant plant were obtained after selection rooting, the rate of rooting is 23.2 %.5. After PCR analysis, three positive transformants were achieved, the rate of positive is 23.1 %. Then by the Southern blotting analysis, the differential hybridizing strips appeared and the positive frequentcy was 100 percent, the rate of transformation was 1.6‰.