Generating Marker-Free Transgenic Wheat Using Minimal Gene Cassette and Cold-Inducible Cre/Lox System

The precise elimination of selectable marker genes is highly desirable, when their function is no longer needed, because their presence raised worldwide public concerns against the release of genetically modified plants. This is the first report of simultaneous application of the minimal gene cassette and cold-inducible Cre/lox recombination system in wheat. The bar selection and cre-recombinase genes were eliminated from T-0 and T-1 transgenic lines with 44 and 51 % efficiency. This approach provides a new, reasonably effective technique to produce selection gene-free transgenic wheat lines either immediately after tissue culture, or from the subsequent transgenic generation. The advantage of this method is that it does not require any additional cold treatment during the plant regeneration/growing because the transgene elimination is ensured by the vernalisation. Application of this method prevents gene flow by pollen and seed, because the selection and recombinase genes are eliminated before pollen development, therefore reducing the risk of GM plants.