Study On The Transformation Of Populus Simonii×P.nigra Haploid Plants With The Choline Oxidase Gene (codA)

In this study, salt tolerance gene—choline oxidase gene (codA) was transformed into the Populus simonii × P. nigra haploid plants mediated by Agrobacterium tumefaciens. 4 transgenic plants was obtained, the highest transform efficiency was 1.8 percent. The results of PCR and Southern blot showed that the codA gene has been integrated into the genome of Populus simonii × R nigra and the results of reverse transcription-PCR and real-time quantitative PCR approved that the transformed gene can be transcrip general. In salt test, the transgenic plants have higher salt tolerance ability than those of control plants. The results it as follows:(1) Kanamycin was confirmed as select antibiotic in genetic transformation experiment. For reducing adventitious buds, the optimal concentration was 40 mg-L"1 and for rooting was 20 mg-L"1.(2) Effects of the four antibiotics on Agrobacterium and the formation rate of adventitious buds from leaves of populus simonii X R nigra were studied. The results showed that the Cefotaxime Sodium was the best among the three antibiotics and the optimal concentrations of Cefotaxime was 200 mg-L"1.(3) Set up the genetic transformation system which used the leaves of Populus simonii×P nigra as explants:Pre-culture: selected leaves which seeding age was 3 weeks or 4 weeks on differentiationmedium for 2 days. Infection: Cultivated agrobacterium (OD₆₀₀=0.5) in liquid medium which pH was 5.4 for4 hours 6 hours, then used asepisis water diluted to OD₆₀₀=0.1. Theinfection time was 5-10 minutes. Co-culture: Cultivated the leaves on differentiation medium for 2³ days withoutillumination. Select culture: Cultivated the leaves on differentiation medium which contained 40mg.L^-1 Kanamycin, used 200 mg.L^-1 Cefotaxime Sodium inhibitAgrobacterium at the same time. Rooting culture: In this study, we obtained 4 transgenic plants, cultured them on the selectrooting medium, then transplated the transgenetic seedings in the greenhouse. The average survived rate was 81.2 percent.(4) The resultes of salt test showed that all the transgenic plants have high salt tolerance ability than those control plants. In NaCl stress test, the highest concentration was 0.8 percent which the transgenic plants can toleranced, whereas to the control plants, the concentration was 0.8 percent. In NaHCO₃ stress test, the concentration was 0.08 percent and 0.06 percent. For 4 transgenic plants, the sequence of salt tolerance ability from high to low was T4, T1, T2 and T3.