Construction of the binary vector with bi-selectable markers for generating marker-free transgenic plants

摘要

Plant transformation typically involves antibiotic or herbicide resistance genes as selection markers to identify the transformed plants. However, there have been public concerns over the safety of the marker genes that remain in transgenic plants. It is therefore desirable to remove marker genes prior to the release of transgenic plants. In this study, we used the co-transformation strategy to develop a binary vector with bi-selectable markers, pGA2TNH. Such strategy enables the generation of marker-free transgenic plants and increases utilization of additional plant species, especially crops with natural resistance or tolerance to kanamycin. One plasmid carrying two separated T-DNAs was adopted in order to construct multiple selectable markers in one of the T-DNAs. Markers used were the hpt gene for hygromycin resistance and the nptIl gene for kanamycin resistance. pGANP-CP1/pBin19 and pGA2T-CP1 were constructed to evaluate and compare their efficacy in generating marker-free transgenic plants. The former consisted of two individual plasmids carrying separate T-DNA of the target and marker genes, while the latter contained a single plasmid carrying two T-DNAs for the target and marker genes. In pGA2TNH system, the co-transformation frequency of the R-0 transgenic Nicotiana benthamiana plants with both selection markers and target gene was 50%, which was as efficient as the other two systems. As expected, segregation of the two T-DNAs was observed in progeny. In one marker gene copy of transgenic plants, the elimination of marker gene was found at a ratio of 17.5% in pGA2TNH system, and 18.6% and 24.1% in pGA2T-CP and pGANP-CP1/pBin19, respectively. This demonstrated that the binary vectors we constructed were efficient and feasible in eliminating marker genes. It also provides a practical and simple tool for generating marker-free transgenic crops, which will have a significant impact on their acceptance by the public.