Transgene stacking in plants in the absence of sexual crossing

A transgene stacking system is a prerequisite for the introduction of multi ple genes and for the modification of complex metabolic pathways in plants. We demonstrate here that the MAT-vector system previously used for generat ing marker-free transgenic plants is also an efficient and reliable transfo rmation system for the repeated introduction of multiple transgenes indepen dent of sexual crossing. We previously reported that the GST-MAT vector sys tem, in which excision of the yeast site-specific recombination R/RS system is regulated by the maize GST-II-27 promoter, could generate marker-free t ransgenic plants containing a single transgene with high frequency. Here we show that the GST-MAT vector can be used successfully to introduce a secon d transgene (GFP) into a marker-free transgenic tobacco line containing sin gle copies of the first transgenes (nptII and uidA genes). The transgene-st acked marker-free transgenic tobacco plants were generated from ca. 20% of excision-positive ipt-shooty explants within 5 months of Agrobacterium infe ction. The presence of uidA, nptII, GFP genes and the absence of the ipt ge ne were verified by PCR analyses. Furthermore, Southern blot analysis showe d that no chromosomal rearrangements were introduced between the first and second transformations.