Optimization Of The Tissue Culture Regeneration System And Genetic Transformation With TA29-Barnase Gene Of Bt Transformed Populus Nigra

Bt transformed Populus nigra cultivated by genetic engineering is a new insect-resistant varity of poplar. The progress on cultivation and promotion of the Bt transformed Populus nigra was slow, because of the protential biological security issue caused by the transgenic flow. The male sterile gene engineering of plant is used in preparing hybrid at the earliest, then it has been used to limit transgenic flow. In recent years, the male sterile gene engineering of plant have become a prospective method for solving the biological security issue of transgenic plants, and it is great significance for further promoting and commercial planting of the transgenic forest tree.In this study, we have optimized in vitro regeneration system with Populus nigra aseptic leaves as explant, and also explored the effect of different conditions on transformation, and then we have established the transformation system. The TA29-Barnase gene transformation by Agrobacterium-mediated aims to reduce the potential threat of the transgenic flow, and make it better applied in production. The main results obtaare as follows:1. When using water-cultured tender stem of one-year old seedings of Bt transformed Populus as explant, the optimal sterilizing methods was immersing into 75% alcohol for 30 sec, and then treating with 0.1% HgCl2 for 8min. the pollution rate was 15.0%, and death rate was 23.5%. The optimum axillary germination and proliferation medium of Bt transformed Populus nigra was MS+0.5 mg/L 6-BA+0.1 mg/L NAA+30 g/L sucrose+6 g/L agar, the axillary germination percentage was 86.7%, and proliferation coefficient was 3.10.2. The optimal buds induction medium of leaf was MS+0.5 mg/L 6-BA+0.05 mg/L NAA+0.01 mg/L TDZ+30 g/L sucrose+6 g/L agar, the differentiation rate can reach 100%, and average 18 shoots per explant were obtained. It is beneficial to leaf differentiation when leaf cultured without light for 10 days and then transferred into low light, and the differentiation rate was 90%; The best rooting medium was 1/2MS+0.05 mg/L NAA+0.2 mg/L IBA+20 g/L sucrose+6 g/L agar, with the maximum adventitious bud frequency(100%), shoots length(6.5 cm).3. When the concentration of Carb was 400 mg/L, it could restrain the growth of Agrobacerium and had a little effect on adventitious bud regenerations. The proper PPT concentration was 2 mg/L in selecting resistant bud after transformation and 1 mg/L in selecting rooting from resistant bud. The best transform condition was that the concentration of bacterial liquid was OD600 = 0.3, the infection time was 20 min, the optimum co-culture time was 2 days, and the transformation rate was 18.0%.4. With selecting by PPT, we obtained 9 PPT resistant plants. PCR analysis showed that there were 5 positive plants with the TA29-Barnase gene, and positive rate was 55.6%. The result of this research demonstrated the integration of TA29-Barnase gene into the genome of Bt transformed Populus nigra.