Improved Protocol for Transformation of Maize (Zea mays L.) Mediated by Agrobacterium tumefaciens

Maize inbred H99 and A188 generally respond well in tissue culture, but only A188 had been efficiently transformed by Agrobacterium tumefaciens by the method previously described. The problem in transformation of H99 appeared to be a negative impact of infection with A. tumefaciens. Various conditions and additives to the culture media to promote callus induction from infected immature embryos were screened. Two major modifications, 1) addition of silver nitrate to the media for selection of transformed cells and 2) use of carbenicillin in place of cefotaxime to eliminate bacteria after infection, turned out to be most effective. A large number of transgenic plants of H99 were produced by the improved protocol based on these modifications, whereas no transformants were created by the original protocol in these genotypes. Integration and inheritance of the transgenes were confirmed by Southern hybridization and genetic analysis. Even the frequency of transformation of A188 was further improved by the new protocol.