Study On Modified Cry3Aa Homologous Gene Transformation Of P.Deltoides XL-80, XL-77and XL-90

In this study, leaves of P.deltoides XL-80, XL-77and XL-90were used as experiment material, on the basis of the establishment of high frequency regeneration system, we adapted the method of Agrobacterium-mediated to introduce the separately two modified Cry3Aa homologous gene Cry3Aa1#and Cry3Aa2#into XL-77and XL-90.The main conclusion of this experiment was as following:1. Establishment of high frequency regeneration system of P.deltoides XL-80, XL-77and XL-90(1) The optimum differentiation medium of P.deltoides XL-80was MS+1.0mg·L-16-BA+0.1mg·L-1NAA+0.005mg·L-1TDZ,and the ration of adventitious buds differentiation reached95%;the optimum subculture medium for adventitious bud was MS+0.5mg·L-16-BA+0.1mg·L-1NAA+0.2mg·L-1GA3, and coefficient of proliferation reached to5.8; the optimum seedling rooting medium was1/2MS+0.3mg·L-1NAA, the rooting rate can reach100%, and the plantlet growth healthy with the roots looks very nice.(2) The optimum differentiation medium of P.deltoides XL-77was MS+1.0mg·L-16-BA+0.15mg·L-1NAA+0.005mg-L-1TDZ,and the ration of adventitious buds differentiation reached95.8%;the optimal medium for adbentitious buds elongation was with MS+0.5mg·L-16-BA+0.2mg·L-1NAA; the optimized medium for rooting was1/2MS+O.1mg·L-1NAA, and the height of the plant was3.5-6.5cm with developed roots and slender and green leaves.(3) The optimum differentiation medium of P.deltoides XL-90was MS+0.5mg·L-16-BA+0.1mg·L-1NAA+0.01mg·L-1TDZ,and the ration of adventitious buds differentiation reached96.3%;the optimum subculture medium for adventitious bud was MS+0.5mg·L-16-BA+0.1mg·L-1NAA+0.2mg·L-1GA3, and coefficient of proliferation reached to7.4, also the plantplet looks very strong; the optimized medium for rooting was1/2MS+2.0mg-L"1NAAr, the rooting rate can reach100%,and the plantlet grow healty with so many lateral roots.2. Determination of selective pressure of transformation of P.deltoides XL-77and XL-90The selectable marker gene of Cry3Aa1#and Cry3Aa2#were both neomycin phosphotransferase (NpTII), so adopting kmamycin as selective agent in transformation. Through contrast experiment, the results show that for the P.deltoides. XL-77it is advisable to supplemented with30mg-L"1Km in the differentiation medium and in the rooting stage to add a concentration of30mg-L"1Kmwill be more appropriate, and for the P.deltoides XL-90it is advisable to supplemented with20mg-L"1Km in the differentiation medium and in the rooting stage to add the concentration of30mg·L-1Km will be more appropriate.3. Establishment of the optimized system of transformation for the P.deltoides XL-77and XL-90we studied several factors affecting transformation effection mediated by agrobacterium tumefaciens with PBI12l-2k was developed, drawing the best transform condition for Cry3Aa1#gene:pre-culture time were3days, infection time were20minute, AS was not necessary during the cocultivation, coculture time were4days and for Cry3Aa2#gene:pre-culture time were3days, infection time were20minute, coculture time were4days and added AS160160umol·L-1in coculture medium.4. Obtaining transgenic plants and PCR analysis of P.deltoides XL-77and XL-90.By the strictly select of kmamycin, we obtained the Km resistant transformation plants of P.deltoides XL-77including14strains Kmr with Cry3Aa1#gene and7trains Kmr with Cry3Aa2#gene, and also obtained the Km resistant transformation plants of P.deltoides XL-90including5strains Kmr with Cry3Aa1#gene and3trains Kmr with Cry3Aa2#gene. PCR analysis showed that it had3positive plants with Cry3Aa1#gene (2plants of P.deltoides XL-77and1plant of P.deltoides XL-90) and3positive plants with Cry3Aa2#gene (2plants of P.deltoides XL-77and1plant of P.deltoides XL-90). The result of this research demonstrated primarily that Cry3Aa1#gene and Cry3Aa2#gene were introduced into genome of P.deltoides XL-77and XL-90.