A transformation vector for the production of marker-free transgenic plants containing a single copy transgene at high frequency

We represent here the GST-MAT vector system. The R recombinase gene of the site-specific recombination system R/RS from Zygosaccharomyces rouxii was f used to the chemical inducible promoter of the glutathione-S-transferase (G ST-II-27) gene from Zea mays. Upon excision, the isopentenyltransferase (ip t) gene that is used as a selectable marker gene is removed. When the cauli flower mosaic virus 35S promoter (CaMV 35S) was used to express R recombina se, 67% of the marker-free transgenic plants had more than three transgene copies. Because the CaMV 35S promoter transiently and efficiently excised t he 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 redu ced. In this study we show that the GST-MAT vector efficiently produced tra nsgenic ipt-shooty explants from 37 (88%) out of 42 differentiated adventit ious buds and marker-free transgenic plants containing the GUS gene from fi ve (14%) out of 37 ipt-shooty lines. Furthermore, the GST-MAT vector also i nduced two marker-free transgenic plants without the production of ipt-shoo ty 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 t ransgenic plants containing a single transgene.