| Jatropha curcas L.is a member of the family Euphorbiaceae,belongs to a kind of shrub or small tree in nettle spurge,containing oil 60%in its seeds.In this study,an efficient plant regeneration and transformation with ipt gene mediated by Agrobacterium tumefaciens method for Jatropha curcas L.were achieved.Transgenic plants were identified.The establishment of efficient regeneration system and Agrobacterium tumefaciens-mediated transformation of Jatropha curcas L.will make substantial contribution as an essential and useful means to further fundamental study of breeding by genetic engineering technology.The main results were as follows:1.Establishment of high efficient regeneration system of Jatropha curcas L.The explants of 12 days cotyledon,30 days leavies,petiole of leavies from aseptic seedlings s of Jatropha curcas L.were used as the explants of callus inductivity and shoots regeneration on MS medium.The results indicated The most effective medium for callus induce and shoots regeneration of cotyledon was the MS medium containing 6-BA 3.0mg·L-1 and IBA 0.2mg·L-1. The maximum frequency of callus inductivity and differentiation were 100%and 95.8%;The most effective medium for callus inductivity and shoots regeneration of leavies was the MS medium containing 6-BA1.0mg·L-1 and IBA0.5mg·L-1.The maximum frequency of callus Inductivity and differentiation were 100%and 85.0%;The best medium of petiole of leavies was MS medium containing 6-BA1.0mg·L-1 and IBA 0.2mg·L-1.Callus inductivity was 100%, differentiation maximum frequency was 100%;The shoots were multiplicated and elongated on MS medium with 6-BA 1.0 mg·L-1,the rate of multiplication could reach 2.9 times,the optimum rooting was obtained on 1/2MS medium with IBA 0.5mg·L-1,rooting rate was 82.4%.The earth of transplanting plants was soil and perlite in ratio of 2:1,the survival rate of transplanting were 80%.2.Establishment the transformation System of Jatropha curcas L.mediated by Agrobacterium tumefaciensThe genetic transformation system of Jatropha curcas L.was optimum by using the cotyledon as receptors mediated by Agrobacterium tumefaciens.The concerntration of Kanamycin (Kan) selection pressures were determined as 30mg·L-1 for shoots regeneration of cotyledon,and 10mg·L-1 for rooting.We studied effect of Cefotaxime(Cef) at different concentration on cotyledon genetic transformation.Cef had a inhibited effection on the differentiation of cotyledon. The suitable content of Cef was the medium with 150 mg·L-1.The shoots formation rate was 70.2%.To monitor the induction rate of Kanamycin-resistant callus,the optimal parameters(such as preculture time,bacterium concentration,infection time,co-culture time.) for A.tumefaciens mediated transformation of Jatropha curcas L.were examined.The results showed that:12 days aged cotyledon using for infection explants,2d precuiture time on MS+6-BA 3.0 mg·L-1+IBA 0.2 mg·L-1 medium,0.6-0.8(OD600) bacterium concentration, 10min infection time,and 3d co-culture time on MS+ 6-BA 3.0 mg·L-1+IBA 0.2mg·L-1.It indicated that the ipt gene was integrated into Jatropha curcas L.genomes,confirmed by Gus assay and PCR.3.Detection select of transgenic Jatropha curcas L.and physiology charactersAnalysis of physiological characters at normal temperature and low temperature stress revealed that there isn't markedly difference between the main physiological characters transgenic Jatropha curcas L.and that of control.However,under low temperature stress compared with control,the chlorophyll content of leaf,soluble sugar content and soluble protein content of transgenic Jatropha curcas L.leaves increased,POD activity behave good exhibition.
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