Study On Insect-resistant Gene Transformation For Populus Davidiana Dodetumefaciens

(P. simonii X P. nigra) X P.15ACL is Artificial hybrid, of which female parent is populus simonii and male parent is populus nigra. This varietie (line) is characterized by well-developed root system, rapid growth, drought and cold endurable and strong adaptablity. It has become an excellent poplar lines widely planted in the Northeast of China such as Heilongjiang province. However, the poplar pests, the main biological factors affecting poplar growth, have caused a serious threat and the huge economic losses in forestry production, construction of urban greening and ecology. Some insect-resistant genes have been transformed into trees successfully by genetic engineering, which offers a new approach for sustainable forest pest control. In this stury, using an Agrobacterium mediated method, a foreign gene fused by insecticidal peptide of spider and the C-terminal of of Bt ICP gene was transformated into (P. simonii×P. nigra)×P.15ACL.1. An efficient tissue culture system for (P. simonii×P. nigra)×P.15ACL was established. The rooting culture medium was WPM+0.2mg/L IBA+20g/L saccharose+5.6g/L agar (pH5.6). The leaf differentiation culture medium was MS+0.3mg/L 6-BA+0.1mg/L NAA+ 20g/L saccharose+5.6g/L agar (pH 5.6).2. The appropriate concentrations of Kanamycin and Cefotaxime in transformation were 15mg/L and 600 mg/L for leaf discs culture and 25mg/L and 700mg/L for rooting culture, respectively, determined by the antibiotics sensitivity test for the poplar and the bacteriostatic test of Cefotaxime sodium on Agrobacterium.3. A genetic transformation system for (P. simonii×P. nigra)×P.15ACL was established, which included:pre-cultured 2-3d, OD600=0.1-0.2, incubated 3 min, co-cultured 2-3d and the co-cultivation medium plus acetosyringone 200μmol/L was the best combination, carry-forward selection-cultured 3d.4. A total of 638 healthy leaf discs from 20-day old cultured plantlet were used for Agrobacterium tumfaciens-mediated transformation. As a ruslut,8 shoots with resistance to Kanamycin were obtained. PCR assay showed that 3 of them gave the predicted DNA fragment band, with a transformation rate of 0.47%. PCR-Southern analysis also confirm the three possitive samples. these result showed that the insect-resistant gene has integrated into the genome of (P. simonii×P. nigra)×P.15ACL.