| The article studies the transformation of non-heading chinese cabbage (Brassica campestris ssp.chinensis Makino) with insect-resistant Cpu gene; cotyledons with petiole were used as transformation recipients from the AB-Naihan line and Duan-126 line. The effects of explants, the medium and other factors influencing the transformation were all explored ;on MS medium ,a great many of adventitious buds were obtained. Being treated 3 day on preculture medium containing 0.1mg/i 2,4-D and 0.1mg/i NAA,the inductive rate of adventitious buds from AB naihan line increased from6.0% up to 44.6%;but there is no obvious effect on Duan-126 line.Kanamycin can strongly inhibited the induction of cotyledon of non-heading chinese cabbage; 100% of explants turned white on the selection medium when adding Kanamycin up to 15mg/i. Not inhibiting the inductive rate of cotyledon petioles, Carbeniciilin markedly shorten the induction time of adventitious buds ,from control 20 days to current 14 days. The concentration of agroinfection has little effect on cotyledon petioies induction,but with the incrcasement of coculture days,inductive rate of adventitious buds sharply reduced. Based on the above results,the transformation procedures were as follows: used as transformation recipients ,the cotyledon petioles of AB-Naihan line were treated 3 days on pretreat medium supplemented with 2,4-D 0.1mg/i and NAA 0.1mg/I ,and cocultured 1-2 days with agrobacterium ,then transferred to MS induction medium containing 6-BA Smgll and NAA 0.lmg/1;After 20 days of induction ,these shoots were screened twice on selection medium containing 15 mg/i kanamycin;at Iast,we obtained some kananiycin-resistant shoots; These shoots were propagated ,rooted and transplanted to field, flowered and seeded. Taking Ti plasmid as positive control,non-transformant as negative control,we did PCR and PCR-Southern analysis on transformated plants;The results showed that the CpTI genes has been intergrated into genome of transformated plants.
|
PDF Full Text Request