Establishment On Regeneration System Of Populus Deltoides Cl.’725’ And Studies On Transformation Of Bt Gene

Poplar is an important light industrial raw material whose growth speed. It plays a significant role in protection and maintain ecological environment. So the cultivated area of poplar in the increasing year by year, however, the insect pest of poplar has been restricted the development of poplar. The main features of Populus deltoides cl.’725’, which suit for planting in the south of HuaiHe river in AnHui area, is highly water-tolerant. However, the Populus deltoides cl.’725’ are seriously attacked by inserts. The crystal protein(Bacillus thuringiensis, Bt) contain the specific insecticidal function.The crystal protein gene segment of Bacillus thuringiensis inserted to poplar by genetic engineering can improve the insect resistance of poplar, as well as reduce the environmental pollution caused by the excessive chemical.The germplasm resources of poplar are rich in China. Poplar is an important light industrial raw material whose growth speed and adaptability are high. It plays a significant role in protection and maintain ecological environment and industrial raw material forest base construction. Poplar is also a model tree because of its biological characteristics.With the increasing of cultivated area of poplar, the incidence of insect pest of poplar is growing. The main features of Populus deltoides cl.’725’, which suit for planting in the south of HuaiHe river in AnHui area, is highly water-tolerant. However, the Populus deltoides cl.’725’ are seriously attacked by inserts. The specific insecticidal function of crystal protein(Bacillus thuringiensis, Bt) have been widely used in transgenic research. The crystal protein gene segment of Bacillus thuringiensis inserted to poplar by genetic engineering can improve the insect resistance of poplar, as well as reduce the environmental pollution caused by the excessive chemical.In this study, the regeneration systems had established from leave explants of Populus deltoides cl.’725’. Meanwhile, plasmids which contain the gene of Bacillus thuringiensis were transformed into LBA4404, then the Bt gene segment were transformed into the leaves of Populus deltoides cl.’725’ using the agrobacterium-mediated technique. In this process, we improved the system by activity vir gene. Resistant seedlings were screened using Hygromycin B. Finally, Transgenic plants of Populus deltoides cl.’725’ cultivar confirmed by molecular test. The main results are summarized as follow:(1) In Populus deltoides cl.’725’ leaves for explant establish regeneration system, the best disinfection time of leaves is using75%of the alcohol for8s and0.1%HgCl2for3 min. It can reduce the rate of pollution to5.7%, and make the callus induction rate reach to67.2%.(2) The best suitable medium of Zhongwol leaves for callus induction was MS+6-BA0.2mg/L+2,4-D1.5mg/L+sucrose25g/L+agar6g/L; appropriate induction differentiation medium was MS+6-BA1.0mg/L+NAA0.3mg/L+KT0.3mg/L+sucrose25g/L+agar6g/L; the best multiplication media was MS+6-BA0.3mg/L+NAA0.05mg/L+sucrose25g/L+agar6g/L; the best medium for rooting was1/2MS+NAA0.01mg/L+IBA0.7mg/L+sucrose20g/L+agar6g/L(3) The leaf and bud of Populus deltoides cl.’725’ were screened using Hygromycin B:the critical kanamycin sensitive concentrations for inducing shoots and roots of the poplar were2.5mg/L and1.5mg/L respectly.(4) Making the competent cell of A.tumefaciens LBA4404, then transformed the plasmid of pCAMBIA1300+CrylC*into A.tumefaciens LBA4404by liquid nitrogen freeze-thaw method.(5) Using Agrobactirium tumefaciens mediated transformation method to genetic transformation with Populus deltoides cl.’725’ leaves of tissue culture. We obtained14Hygromycin B resistant seedlings by screening the transgenic plants, and then molecular detection by PCR, obtained5Bt-transgenic plants.