Nanlin 895 Young Insect-resistant Transgenic Research,

In this study, leaf disc of Populus×euramericana cv.'Nanlin895' was used as experiment material, on the basis of the establishment of high frequency regeneration system , adapting to Agrobacterium-mediated to introduce separately two insect-resistance genes of Bacillus thurigiensis (Bt) gene and cowpea trypsin inhibitor (CpTI) gene. The main conclusion of this experiment was as following: 1.Establishment of high frequency regeneration system: through the screening to the basic culture medium,the adjustment of hormone kind and proportion ,choice of the culture condition , the optimal medium for differentiation was MS +6-BA0.5mg/L + TDZ0.002mg/ L, obtain 100% differentiation frequency, each leaf disc produced a large number of multiplication of shoot, this offered solid foundation for genetic transformation. 2.Determination of selective pressure of transformation: the selectable marker gene of Bt and CpTI were both NpTⅡ, so adopting kanamycin as selective agent in transformation. Through contrast experiment, it is 20mg/L kanamycin as selective pressure. 3.Establishment of the efficient system of transformation: we studied several factors affecting Agrobacterium-mediated transformation, drawing the best transform condition: pre-culture time were 3 days, bacterial concentration OD₆₀₀ 1.0～1.3, infection time were 20 minute, added AS 120-200 mg/L in coculture medium, coculture medium adjusted pH 5.8 and did not removal of CoCl₂·6H₂O, cocultivation temperature settled 28℃, coculture time were 4 days. Under the transformation system optimized here, transformation frequency added to 28.7% from 4.5%, improving 6 times. 4.Obtaining transgenic plants and PCR analysis: to strictly select by kanamycin, we obtained 22 Kan resistant plants with Bt gene and 5 with CpTI gene regeneration plants. PCR analysis showed that it had 18 positive plants with Bt gene and 1 positive plant with CpTI gene. The result of this research demonstrated primarily that Bt gene and CpTI gene were introduced into genome of Populus×euramericana cv. 'Nanlin895'. 5.Transplanting transgenic plants: Test-tube shoots for one month plantleted 3～4 days before transplanting, 8 transfer to Bt gene, 1 transfer to CpTI gene and 1 contrast plant all survive, the survival rate was 100%.