Highly efficient gene transfer in Agrobacterium tumefaciens LBA4404 by tri-parental mating and electroporation

Objective To explore effective transformation methods for Agrobacterium tumefaciens LBA4404.Methods Electroporation was performed to transfer plasmid DNA of pYG308 into A.tumefaciens LBA4404.A number of factors that affected transformation efficiency were investigated.The plasmid pYG308 harboring the functional vgb gene cluster was transformed by tri-parental mating into A.tumefaciens LBA4404.The PCR amplification of vgb was conducted.Results Optimized electroporation protocol that yielded highly efficient gene transfer in A.tumefaciens LBA4404.High transformation efficiency was obtained with an appropriate combination of field strength and pulse time.Transformation efficiency could also be elevated by using the host cell harvested in the middle exponential growth phase and increasing the density of the host cells.Two plasmids were isolated from the transformants growing on the LB plate (containing Rif100,Str300,Km50),and shown to have the right physical map.Conclusions These evidence and PCR amplification of vgb gene demonstrate the successful construction of a bi-plasmid recombinant strain.