Condition Filtration Of Shanxinyang Genentic Transformation And Chitinase Gene Transformation

Shanxinyang(Populus davidiana Dode×P.bollena Lanche) was used wild aspen from Heilongjiang province as female parent and Xinjiang poplar as the male parent to make artificial hybridization.Although Shanxinyang has certain disease resistances compared to other poplar varieties,but also suffer pathogen infection and it always cause large scale epidemic,result in huge loss for forestry production and ecological construction.According to the poplar diseases reality are more kinds,fast epidemic and serious damage,in this paper Shanxinyang used as the recipient material,chitinase gene used as the target gene,studied transgenic Shanxinyang with resistance to fungal disease by Agrobacterium-mediated leaf disc transformation.1,Used 1/2MS supplemented with 0.03 mg/L NAA and 0.05 mg/L 6-BA as leaf regeneration medium,1/2MS supplemented with 0.05 mg/L NAA and 0.05 mg/L 6-BA as stem regeneration medium,1/2MS supplemented with 0.25 mg/L NAA as root differentiation medium,Shaninyang tissue culture and rapid propagation have been done by these medium, and it provide an effective transgenic acceptors for genetic transformation.2,The sensitivity of explants to antibiotic was tested in order to decide a reasonable selective pressure for transformation,the result shows:Kanamycin 8 mg/L can be used as the critical concentration of selecting the transgenic shoots during the shoots differentiation;40 mg/L is the best critical concentration in the rooting phase.800 mg/L Cefotaxime sodium is the critical concentration to remove bacteria.3,Leaves were excised from Shanxinyang used as reception material for genentic transformation,the optimal bacteria concentration to infect is OD₆₀₀≤0.1,5min for the best infection time.By this combination,the differentiation rate increased over 1 times and the transformation rate increased nearly 1 times when co-culture medium removed CoCl₂·6H₂O.4,Confirmed the best combination of inoculums density,inoculate time,acetosyringone and co-culture time by orthogonal experimental designs which are influence the transformation efficiency.A,tumefacienws cultured with liquid 1/2 MS medium plus acetosyringone(50μmol/L) for 12～14h at 28℃with constant agitation(180 rpm);cuLtures were centrifuged and resuspended with liquid 1/2 MS medium to OD₆₀₀=0.1,the plant material was immersed in bacterial suspension for 2～5min;co-culture medium removed CoCl₂·6H₂O and plus 200μmol/L acetosyringone lasted 3d,is the best treatments combination.5,Shanxinyang explants were excised froml0- to 15-days-old in vitro propagated plants, 324 explants inoculated by this optimized Agrobacterium-mediated genetic transformation combination,18 kanamycin-resistant plantlets were obtained,then they were tested by PCR analysis,14 kanamycin-resistant plantlets appeared specific bands,the positive frequentcy was 77.78%,the positive plantlets were tested by PCR Southern,all of them presented the same hybridization band as positive CK,the positive frequentcy was 100%,the transformation rate was 4.3%,it is showed that the foreign chitinase gene was already introduced into genome of Shanxinyang.