Study Of Agrobacterium Tumefaciens-mediated Transformation Of Shanxinyang For TCS Gene

The new aspen variety Shanxinyang(Populus davidiana DodexP.bollena Lanche)was bred by our country experts who cultivated nearly half a century,it was used Xinjiang poplar as the male parent and aspen as female parent,it has wildly promoted as an excellent species followed by Populus albaxP.Berolinensis was promoted,with the Shanxinyang has extensive planted,the occurrence of pests and diseases are more and more serious,its resistance breeding become more necessary.The research of transgenic poplar by genetic engineering technologies is an efficient way to breed the new variety to select new species with the aspects of stress tolerance,insect and disease resistance.The Tichosanthin(TCS) gene from Trichosanthes kirilowii with the function of disease and virus resistance was introduced to Shanxinyang by Agrobacterium mediated method,so that it obtain the ability of disease and virus resistance,become high-quality resistant species. The major research as follows:The plasmid pC-tPro-TCS-GUS and pC-tPro-TCS were transformed into Agrobactrium tumefaciensLBA4404 by the freeze-thaw method,and prepared a certain amount of engineering bacteria.The sensitive experiments show:500mg/L bacteriostatic antibiotics(Cefotaxime) is the appropriate concentration for genetic transformation of Populus davidiana DodexP.bollena Lanche;selective antibiotics(hygromycin)2mg/L can be used as the critical concentration of selecting the transgenic leaves during the leaves differentiation and the adventitious buds during the adventitious bud rooting;6mg/L hygromycin can be used in the shoots rooting phase.The Shanxinyang leaflets were excised from 20-days-old in vitro propagated plant as explants,discussed several factors affect transformation efficiency:pre-culture time,inoculum density and inoculate time,the results shows:Shanxinyang leaves were pre-cultures on 1/2MS medium for 2d,the plant material was immersed in bacterial solution for 5min at OD₆₀₀=0.2, co-cultivation in 1/2MS(pH5.8)plus acetosyringone(200μmol/L) for 3d,selective regeneration medium supplemented with AgNO₃ has better effect to stop the growth of A.tumefaciens,after pre-selection period of one week,the explants were transferred to selective regeneration medium. Using this optimum Agrobacterium tumfaciens-mediated transformation system for Shanxinyang,7resistant shoots were obtained;the resistant shoots were identified by PCR analysis,all of samples gave the predicted DNA fragment band,no DNA amplification wan detected in the samples from the control plants;then by PCR-Southern analysis,the differential hybridizing strips appeared and the positive frequentcy was 100 percent.The results showed that the foreign gene has been integrated into genome of Shanxinyang,the Northern hybridization and Western blot identification and anti-virus capabilities detection of transgenic plants need to be further studied.