Studies On Tissue Culture And Genetic Transformation Of Soybean And Stem Mustard

Tissue culture of soybean (Glycine max L) and stem mustard (Brassica juncea Mao) in vitro and genetic transformation of soybean by Agrobacterium tumefaciens were studied in this experiment.To build an efficient soybean regeneration system, the factors affecting soybean regeneration were tested. From the results, the highest frequency of regeneration was achieved on B5 medium supplemented with 2 mg/L 6-benzyladenine (BA ) and 0.2 mg/L indole-3-butyric acid (IBA).Cotyledon and true leaves of stem mustard were cultured on MS medium supplemented with different concentrations of various cytokinins ( BA, CPPU, TDZ or KT) alone or in combination with NAA or IBA, and the adventitious buds regeneration rate was evaluated.. The result showed that the cytokinin ability in shoot induction was in order of TDZ>CPPU>BA>KT, and the optimal concentration of BA, CPPU- TDZ- KT for bud differentiation on cotyledon was 4 mg/L- 0.4 mg/L. 1 mg/L, 1.5 mg/L respectively. Addition of Img/L TDZ and Img/L NAA resulted in the highest bud regeneration rate (67.9%) and number (35) of buds per explant. Regeneration rate could be improved apparently in the medium with AgNO3. It was observed that regeneration rate of cotyledon was highest when regeneration ability of cotyledon, true leaf, hypocotyl and root were compared. However, only callus formed from hypocotyls, neither shoot differentiation nor callus formation was noticed in root explants.A plant expression vector was constructed by following method: S gene of 1BV and 35S promoter was cut from recornbinant pBI121, and then the fragment was inserted into the multiclonal sites Hind /BamH I in the plasmid pCAMBIA1305.1. The recornbinant vector pCAMBIA 1305.1 containing S gene was confirmed by double digestion of restriction endonuclease. Agrobacterium tumefaciens EHA105 with the recornbinant vector pCAMBIA 1305.1 was obtained by tri-parental mating method.Soybean cotyledonary nodes were transformed with S gene of IBV via A..tumefaciens-med\ated transformation. The highest transformation rate of 6.7% was detected in the present experiment.