Optimization Of Regeneration System For Stylosanthes Guianensis And The Transformation Of Bar Gene

Stylosanthes is a very important tropical legume forage. They are capable of nitrogen fixation and are capable of improving soil fertility in addition to providing high protein stock feed. Due to the slow growth of early age of Stylosanthes, the weeds problem has always been one of the factors which restricted the production and promotion of Stylosanthes. Transforming the herbicide-resistant genes into the Stylo, and cultivating herbicide resistant crop by genetic engineering is useful means to improve the production and utilization of range Stylo. In this paper, Reyan5Stylo (Stylosanthes guianensis cv.Reyan No.5) regeneration and genetic transformation system was studied. We researched the suitable conditions of tissue culture, then optimized the regeneration system Stylosanthes.The condition of transforming the herbicide resistant Bar gene to S.guianensis cv. Reyan No.5by Agrobacterium-mediated was investigated. The genetically modified material had been detected and identified by molecular tools. All the results showed as fellows:(1)The suitable concentration range of2,4-D is0.2-1.5mg/L,6-BA is0.5-2.0mg/L for callus culture of S.guianensis cv. Reyan No.5. Either2,4-D or6-BA used alone can induce callus well.(2)The best callus induction medium of S.guianensis cv. Reyan No.5is MS+2mg/L6-BA+0.5mg/L2,4-D+3%sucrose+0.75%agar powder, pH5.8, the percentage of callus could be reached100%; The best bud differentiation medium is MS+2mg/L6-BA+3%sucrose+0.7%agar powder, pH5.8, the percentage is83.3%; The root inducible medium is1/2MS+0.5mg/L IAA+0.5mg/L IBA+0.8g/L actived carbon+2%sucrose+0.7%agar powder, pH5.8, the percentage is100%; And the best explant is first hypocotyl; The best hardening-seedling and transplant method is open the bottle step by step then keep watering, the survival percentage could be reached96%.(3)For transformation of gene to S.guianensis cv. Reyan No.5, the optimal concentration of carbenicillin is200mg/L; In the early time the optimal concentration of Basta is0.15mg/L, after9-10days change it to0.3mg/L; The preincubate time of explant is36hours; And LBA4404Agrobacterium could more functionally infect S.guianensis cv. Reyan No.5.(4)By the above transformation system, Bar gene has been transformed to S.guianensis cv. Reyan No.5, parts of transgenic plants was detected by PCR and fluorescence detection, the result preliminary shows that gene Bar and GFP has been transformed to genome of plants. But further molecular detection is still needed.