Agrobacterium Tumefaciens-mediated Creeping Bentgrass And Tobacco Transformation With GshI And Pcs

The use of plants to clean-up soils contaminated with heavy metals could provide a cheap and sustainable technology for bioremediation. Field trials suggested that the using of hyperaccumulators in phytoremediation is limited because of its low rate of biomass production. The introduction of.novel traits into high biomass plants in a transgenic approach is a promising strategy for the development of effective phytoremediation technologies.High efficient system of plant regeneration has been development. In order to acquire a metal-hyperaccumulating turfgrass, gshl (from E.coli) and pcs (from Arabidopsis thaliana) was introduced into creeping bentgrass respectively by Agrobacterium tumefaciens.In order to develop an efficient procedure of Agrobacterium-mtdiaied transformation using mature seed-derived calli, we optimize several parameters to achieve higher transformation efficiency.The results obtained are as following:1. We isolated gshl from E. coli and pcs from Arabidopsis thaliana, and placed gshl and pcs into pCAMBlA13011 respectively.2. gshl gene and pcs gene were transformed into creeping bentgrass respectively mediated by Agrobacterium tumefaciens. 58 transgenic plants with gshl regenerated from resistant callus were obtained, 6 transgenic plants were found to be positive by PCR analysis. 16 transgenic plants with pcs regenerated from resistant callus were obtained, 1 transgenic plant were found to be positive by PCR analysis. Southern blot analysis of part of positive transgenic plants revealed that the exogenous gene has been integrated into the plant genome.3. Different protocols were used in order to evaluate their effects on transformation efficiency. Transient GUS expression was observed in calli following co-cultivation. The results showed that the most efficient treatment to increase the transient GUS expression is sonication. Pretreatment calli with 100u.M acetosyringone before co-cultivation could not increase the transformationefficiency. Infection callus with Agrobacterium in the dark is better than apply Agrobacteriwn suspension to each piece of callus4. gshI gene and pcs gene were transformed into tobacco respectively mediated by Agrobacterium lumefaciens. 13 independent transgenic plants with gshl were obtained, 11 transgenic plants were found to be positive by PCR analysis. 22 independent transgenic plants with/?cs were obtained, 18 transgenic plants were found to be positive by PCR analysis. Southern blot analysis of part of positive transgenic plants revealed that the exogenous gene has been integrated into the plant genome. Tl generation seeds have been obtained.