Effective selection system for generating marker-free transgenic plants independent of sexual crossing

In a previous report, a novel selection protocol termed "the MAT-vector sys tem" for generating marker-free transgenic plants (MFTPs) was presented. Th e first stage of the system is visual selection of morphologically abnormal transgenic shoots, ipt-shooty, that have lost apical dominance and rooting ability. The second stage involves elimination of the ipt gene and the app earance of MFTPs free of ipt gene influence. The present report describes a practical MAT-vector in which removal of the ipt gene is efficiently media ted by the site-specific recombination system R/RS from Zygosaccharomyces r ouxii, in place of the maize transposable element Ac, used previously. This improved MAT-vector produced MFTPs from 39% of moderate ipt-shooty and 70% of extreme ipt-shooty lines. These results are superior to the previous MA T-vector which produced MFTPs from only 5% of ipt-shooty lines. The present novel system also induced direct development of MFTPs from adventitious bu ds without production of ipt-shooty intermediates. The presence of beta-glu curonidase (GUS) and neomycin phosphotransferase (NPTII) genes of interest, and the absence of the ipt gene were verified by a GUS histochemical assay , NPTII assay, and molecular analysis.