MAT vector systems for asexual production of marker-free transgenic plants

Abstract:

We have developed efficient removal systems of marker genes, which are called MAT (Multi-Auto-Transformation) vectors, to increase the regeneration frequency of transgenic crops without using antibiotic selection and to reduce their environmental impact by removing selection marker genes. MAT vector system is designed to use the oncogenes (ipt, iaaM/H, rol) of Agrobacterium, which manipulate the endogenous levels of plant hormones and the cell responses to plant growth regulators, for differentiating transgenic cells and for selecting marker-free transgenic plants. The oncogenes are combined with the site-specific recombination system (R/RS). At transformation, the oncogenes regenerate transgenic plants and then the R/RS system removes them to generate marker-free transgenic plants. The choice of promoters of the oncogenes and the recombinase (R) gene, the state of plant materials and the tissue culture conditions greatly affect both the regeneration efficiency of transgenic plants and the generation efficiency of marker-free transgenic plants. We have evaluated these conditions at several plant species (aspen, eucalypts, rice, sesame, snapdragon, tobacco) for increasing their generation efficiency.
We report here improvements of MAT vector systems and their advantages. (1) Transgene stacking: A chemical inducible promoter is fused with a recombinase gene due to control excision events. New MAT vectors are very useful to stack multiple genes through re-transformation. (2) In vivo transformation: The ipt gene is combined with the iaaM/H genes to manipulate auxin/cytokinin ratio and increase the regeneration frequency of recalcitrant plant species. This approach has also great potential to develop non-tissue culture-based transformation through the autonomous differentiation of transgenic cells in vivo. (3) Single-step transformation: The ipt-type MAT vctor can induce rice embryogenic tissues and regenerate marker-free transgenic rice plants directly from them, without the production of ipt-shooty intermediates. This procedure is an effective shortcut to select marker-free transgenic rice plants with one copy of highly expressing transgene.