An Efficient Selection Scheme for Agrobacterium-mediated Co-transformation of Rice Using Two Selectable Marker Genes hpt and bar

A stable, efficient, and reliable selection system using bar and hpt as selectable markers for Agrobacterium mediated co-transformation of rice with two separate vectors was developed. Two plasmids, pMJC-GB-IFS2 and pMJC-GH-CHR carrying bar or hpt gene as selectable markers and isoflavone synthase 2 (IFS2) gene or chalcone reductase (CHR) gene as target genes, respectively, were used for co-transformation. Between the two selection schemes, either the selection first for hygromycin B resistance during the callus proliferation stage and later for phosphinothricine (PPT) resistance at the shoot regeneration stage or vice versa, the former selection scheme was far more effective obtaining co-transformed transgenic plants (100%) than the latter (12.8%), meaning that the PPT selection during the callus proliferation stage resulted in producing many escapes without transformed bar gene. Therefore, it was suggested that the hygromycin B selection must precede the PPT selection for efficient Agrobacterium mediated co-transformation of rice.