Studies Of Regeneration System Of Two Populus And Transformation Of Cry1C+9C Genes

SN04-31 and LH04-17 clones are fine lines of Fl generation by Self-selection Our research group. The former is F1 generation by 72 Poplar(Populusxeuramericana ’SanMartino’)×LiaoNing Poplar(Populus liaoningensis), the latter is F1 generation by 69 Poplar (Populus deltiodes’lux’)×63 Poplar (Populus deltides’Harvord’).SN04-31 and LH04-17 have a straight trunk, the tree beautiful, fast growing, adaptable, and other fine heterosis.In this study, Stems of SN04-31 and LH04-17 were used as experiment material, high efficient regeneration system of the two poplar were established. On the basis, factors of adventitious bud induction of leaves of LH04-17 were optimized, and the optimal medium for differentiation was defined. On this basis, effects of carbenicillin and cefotaxime in inducing adventitious buds of LH04-17 were studied, and the inhibition effects of the two bacteriostatic antibiotics on Agrobacterium EHA105 were also analyzed, and conditions for genetic transformation of LH04-17 were optimized, and a stable and efficient genetic transformation system of LH04-17 was established. Resistant plants were detected by PCR analysis, the main progress of this study was as following:1.Establishment of high efficient regeneration system of SN04-31 and LH04-17:The method of Optimal sterilization, Stem segments were sterilized in 70% alcohol about 20s and then in 0.1%HgCl2 about 1.5min～2.5min; The most optimal induction medium was MS+6-BA0.5mg/L+NAA0.1mg/L, and the differentiation rate of explants could be up to 90%; The most optimal proliferation medium of populus SN04-31 and populusLH04-17 was MS+6-BA0.3mg/L+NAA0.05mg/L and 1/2MS+6-BA0.3mg/L+NAA0.02mg/L;The most optimal seedling medium was MS-6-BA0.1mg/l+NAA0.02mg/l; the most optimal rooting medium of the two poplar was 1/2MS+IBA0.2mg/L.and the rooting rate is more 96%, average root number is 11.2, the above medium contain Sugar3%+agar 0.7%.2. Regeneration System of adventitious bud from leaves of LHO4-17The most optimal medium of adventitious bud regeneration from leaves of LH04-17 was MS+6-BA0.5mg/L+NAA0.1mg/L+Sugar3%+agar0.7%. and the frequ-ency of adventitious bud regeneration is 91%. The leaf explants of the second four top leaves of tube plants is more propitious to induce adventitious buds than others, and the Regeneration rate is 92%; the differentiation of Terminal leaf petiole is stronger than near leaf tip; the differentiation of rooted tube plants is stronger than those of non-rooted tube plants.3. Sensitivity determination of AntibioticResults showed that the best bacteriostatic antibiotics were cefotaxine, and the optimum concentrations ranged from200 to 250mg/L. Additionally, the kanamycin selection pressure of resistant buds at different stages was definited. The optimum concentrations of Kanamycin, was 15-20mg/Lfor shoot regeneration, and10-12.5mg/L for rooting of resistant buds.4. Establishment and Optimization of Genetic transformation system of LH04-17Results showed that the leaves were pre-cultured 2d; bacterial concentration OD600 0.4, and infection time were 8～12min; co-culture time were 2d; screening culture time were 3d. Based on the transformation system optimized, access rate of resistant shoots were highest.5. PCR analysis of resistant plant5 Kan resistant plants were analyzed by PCR. Results showed that it had 1 positive plant with Cry1C+9C gene. The result of this research proved primarily that Cry1C+9C gene were introduced into the populus genome.