MAT (Multi-Auto-Transformation) Vector System. The oncogenes of Agrobacterium as positive markers for regeneration and selection of marker-free transgenic plants

We have developed a new transformation method called MATVS (Multi-Auto-Tran sformation Vector System). The oncogenes (ipt or rol genes) of Agrobacteriu m are used as selectable markers to regenerate transgenic cells and to sele ct marker-free transgenic plants in the MATVS. The chimeric ipt gene or the rol genes are combined with the site-specific recombination R/RS system to remove the oncogenes from the transgenic cells after transformation. We re port here the application of MATVS to transformation of tobacco, aspen, ric e and snapdragon. (1) The GST-MAT vector pMAT8 has the native ipt gene and the R gene with a chemical inducible promoter (GST-II-27). Use of the GST-M AT vector generated marker-free transgenic tobacco plants containing a sing le copy transgene at high frequency. (2) Use of the GST-MAT vector pRBII1 c ontaining the rbcS 3B-ipt gene produced transgenic marker-free hybrid aspen plants without crossing. (3) Use of the ipt-type MAT vector, pNPI30GFP, co ntaining the 35S-ipt and 35S-R genes, resulted in the regeneration of marke r-free transgenic rice plants directly from infected scutellum tissues at h igh frequency within 1 mo. (4) Use of the rol-type MAT vector pNPI702, cont aining the rol genes and the 35S-R gene, produced transgenic marker-free pl ants of tobacco and snapdragon at high frequency without crossing. Our resu lts show that the promoter of the ipt gene, the preculture periods of plant tissues and the culture medium are important factors to improve the genera tion efficiency of marker-free transgenic plants. We can rapidly produce ma rker-free transgenic plants without the production of ipt-shooty intermedia tes. Therefore, it is a most promising method to save time and work for the generation of marker-free transgenic plants in crops.