Establishment Of Efficient Transformation System Of Chinese Cabbage And Brassicaparachinensis Mediated By Agrobacterium Tumefacien

To establish high frequency of shoot regeneration system in Brassica rapa, effects of genotype, seedling age, hormone combination,2,4-D and agar concentration on shoot regeneration from cotyledonary explants were studied with three varieties including’Beijing No.80’,’45Days Caixin’and’Four season Caixin’.The plasmid vector pB I Bast-gus-intro was transferrde into’45Days Caixin’and’Four season Caixin’. An efficient transformation mediated by Agrobacterium was developed. The factors affecting the transformation frequency, such as the concentrations of antibiotics to inhibit the growth of Agrobacterium, preculture time of cotyledon explants, concentration of Agrobacterium, infection time, concentration of PPT and Kan and co-infection time etc. were also discussed by GUS coloration analysis. The plasmid vectors PB I121-ATP β-orf138orf222were transferrde into’Four season Caixin’mediated by Agrobacterium. Research results are mainly as follows:1. A high effective regeneration system has been established, the best medium for’Beijing No.80’was MS+5mg/L6-BA+0.5mg/L NAA+2mg/L AgNO3and the frequency could reach84.1%. The regeneration rate of’45Days Caixin’and’Four season Caixin’could reach73.1%and83.2%respectively when cultured on the medium MS+0.2mg/L TDZ+lmg/L NAA+2mg/L AgNO3. Higher frequency was obtained when the explants were pre-cultured3d in the MS medium with0.5～lmg/L2,4-D before being transferred to the medium with5mg/L6-BA and0.5mg/L NAA, the frequency could reach more than84%. The explants from4-5days old seedling and the culture medium with12g/L Agar are more efficient to induce adventitious buds.2. An efficient transformation mediated by Agrobacterium was developed. The suitable antibiotics and their concentration are studied for the selection of transformants. The suitable concentration of Kanamicin is10mg/L, PPT is5mg/L; the suitable concentration of Carbenicillin disodium salt is500mg/L. The pre-culturing time is3days and the co-cultivation time is2days.The highest frequency is observed when explants are immersed in bacterium suspension with OD600value about0.8for10-15minutes.3. Mitochondrial targeted expression vectors for orf138and orf222, PB I121-ATPβ-orf138/orf222were constructed.4. The Mitochondrial targeted expression vectors for orf138and orf222are transformed into’Four season Caixin’with Agrobacterium-mediated method. The frequency of transformates were2.6%and3.1%.