Establishment Of Regeneration System And Transformation Of Bt Gene On Excellent Poplar Clones

Poplar is an important afforestation trees in China. Because of haven adaptability, fast growing, high yield and other characteristics, it is a major industrial timber species for short rotation in China , and occupy an important position in the ecological environment management and resolution of timber shortages . The pest is a major problem in modern forestry. Transgenic insect-resistant poplar is a recent development of a new pest control technology. Bacillus thuringiensis (Bt) is a very important entomopathogen which can produce specifically toxic crystal proteins against many agriculturally and forestry pests. Bt gene is a gene that is wildly used for controlling the pest in the world.In this research, the regeneration systems have been established from leaves explants of the Populus deltoides cv. Zhongwo1. Meanwhile we constructed the expression vector of pBI121 + Bt, and carried out Agrobacterium-mediated genetic transformation by Populus×euramericana cv. Nanlin 95.The integration of targeted gene was proved by PCR analysis. The main results are summarized as follow:1,In Zhongwo1 leaves for explant establish regeneration system, the best disinfection time of leaves is using 75 % of the alcohol for 15 s and 0.1 % HgCl2 for 4 min. It can reduce the rate of pollution to 15 %, and make the callus induction rate reach to 82 %.2,The best suitable medium of Zhongwo1 leaves for callus induction was MS + 6-BA 0.2 mg/L + NAA 1.0 mg/L; appropriate differentiation medium was MS + KT 0.1 mg/L + NAA 0.1 mg/L+6-BA 1.0 mg/L + sucrose 25 g/L; the best medium for rooting was 1/2MS+IBA 0.6 mg/L.3,The regeneration system of adventitious buds direct differentiation from leaves of Zhongwo1 was established, and the best medium was MS + NAA 0.2 mg/L + 6- BA 1.0 mg/L + sucrose 25 mg/L.4,Expression vector pBI121+ Bt have constructed by gene engeneering measure such as restriction enzyme digestion,purification,ligation and transformation. Then transformed it into A.tumefaciens EHA105 by liquid nitrogen freeze-thaw method.5,By using Agrobactirium tumefaciens mediated transformation method to genetic transformation with Nanlin 95 leaves of tissue culture. Eleven kanamycin resistant seedlings are obtained by screening the transgenic plants, and then detection by PCR, initially got four Bt-transgenic plants.