Transformation Of BphC Gene Into Medicago Sativa L. Mediated By Agrobacterium Tumefaciens

The gene transformation acceptor selected in this experiment was Medicag o sativa L., the BphC gene which can degradate PCBs was introduced into the genome of alfalfa Gongnong No.1by Agrobacterium-mediated genetic transfer mation, in order to obtain the new breeding of alfalfa which has the ability of degrading polychlorinated biphenyls. In this experiment, a better method for the tissue cultuer of Medicago sativa L."Gongnong No.1"was established by horm one regulation,and in order to improve the conversion rate of the alfalfa, Agro bacterium-mediated genetic transformation system on alfalfa has been optimized. The ten transgenetic alfalfa expressing BphC were gained by identification.The main result were as following:An efficient method for the regeneration of Medicago sativa L."Gongnong No.l "has been developed.At the stage of callus induction, the orthogonal experi ment on different concentrations of2,4-dichlorophenoxyacetic acid(2,4-D), kineti n(KT), and Easein hydrolysate(CH) was used to study those influences on callu s induction, finally determined the best callus induction medium:MS+2,4-D2mg/L+KT0.1mg/L+CH1500mg/L.At the stage of embryo induction,1-Naphtal eneacetic acid(NAA),6-benzylaminopurine(6-BA) of different concentrations were added in MS medium,after fifteen days,the maximal percent of the somatic e mbryo per callus was observed on MS medium with0.1mg/L NAA and0.5mg/L6-BA.In order to improve the genetic transformation of alfalfa by Agrobacterium-mediated, an efficient genetic transformation system was established. Incubate the explants with OD6oo=0-4-0.6agrobacterium with BphC gene for lmin, aft er Id co-culture,the explants were transferred to callus induction medium contai ning2mg/L2,4-D,0.1mg/L KT,1500mg/L CH,2mg/L glufosinate and300 mg/L cefotaxime, after20days,the callus which can resist glufosinate was tr ansferred to the embryoid induction medium containing0.1mg/L NAA,0.5mg/L6-BA,2mg/L glufosinate and300mg/L cefotaxime. About four weeks later, some callus formated the embryoids,put the embryoids into MS antibacterial medium containing150mg/L cefotaxime, after about three weeks gained40con versionplants.After the screening of high concentrations glufosinate medium,gamed10tr ansgenic alfalfa plants,put the DNA which was extracted using the CTAB meth od through PCR analysis,the result shows that the bphc gene was integrated i nto the10transgenic plants. Put these10transgenic alfalfa plants into the aqu eous solution containing50mg/L of catechol, after10days, the non-transge nic plants appeared to yellow,decay and death, while the10transgenic plants a re growing well, it was showed that the transgenic Plant can tolerate catechol.