SummaryWe represent here the GST-MAT vector system. The R recombinase gene of the site-speci®c recombination system R/RS from Zygosaccharomyces rouxii was fused to the chemical inducible promoter of the glutathione-S-transferase (GST-II-27) gene from Zea mays. Upon excision, the isopentenyltransferase (ipt) gene that is used as a selectable marker gene is removed. When the cauli¯ower mosaic virus 35S promoter (CaMV 35S) was used to express R recombinase, 67% of the marker-free transgenic plants had more than three transgene copies. Because the CaMV 35S promoter transiently and ef®ciently excised the ipt gene before callus and adventitious bud formation, the frequency of emergence of the ipt-shooty explants with a single T-DNA copy might be reduced. In this study we show that the GST-MAT vector ef®ciently produced transgenic ipt-shooty explants from 37 (88%) out of 42 differentiated adventitious buds and marker-free transgenic plants containing the GUS gene from ®ve (14%) out of 37 ipt-shooty lines. Furthermore, the GST-MAT vector also induced two marker-free transgenic plants without the production of ipt-shooty intermediates. Southern blot analysis showed that six (86%) out of seven marker-free transgenic plants had a single GUS gene. This result suggests that the GST-MAT vector is useful to generate high frequency, marker-free transgenic plants containing a single transgene.

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Agrobacterium-mediated transformation is highly dependent upon competency of the target plant tissues. It is important to develop the capacity of transformed cells to include cell proliferation and differentiation. A system which results in cell proliferation and differentiation only of transformed cells is highly desirable for plant transformation. We report here a new GST-MAT vector system (MATIMH), in which the ipt gene combined with iaaM/H genes was used as the selectable marker gene and the GST-II promoter was used as the promoter of the R gene in a site-specific recombination system. In tobacco transformation, the combination of the ipt gene and the iaaM/H genes can result in the production of both auxin and cytokinin in transformed tissues and induce regeneration of transgenic shoots exhibiting an ipt-shooty phenotype more efficiently than the ipt gene alone. When we transformed 20 tobacco leaf discs with the MATIMH vector, markerfree transgenic plants were produced from five (41.6%) out of 12 ipt-shooty lines. These results indicated that the combination of the iaaM/H genes and the ipt gene can more efficiently produce both transgenic plants and marker-free transgenic plants.

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