Development of an inducible excision system of a visual marker gene from the genome of transgenic cells.

In the plant genetic transformation process, single selection by a chemical-resistant marker gene occasionally allows the proliferation of non-transgenic cells, escaping selection pressure. The additional use of a visual marker gene is effective for accurate selection. For instance, R2R3-MYB genes are used for regulating anthocyanin biosynthesis; however, constitutive expression in transgenic plants is not always desirable and may cause developmental abnormalities due to excess anthocyanin accumulation. To overcome the remaining problems in the use of as a visible marker, we developed T-DNA. () and expression cassettes were inserted between two sequences, and the () and () expression cassettes were located outside of the --- region. In the developed system, and were excised from the genomes of transgenic cells using heat-inducible - recombination. Upon heat treatment in a general incubator, green shoots emerged from purple tobacco transgenic calli that were pigmented with expression. The excision of from the genome of green shoots was confirmed using polymerase chain reaction (PCR) and sequencing. GFP expression was observed in the roots of the obtained green transgenic plants. We report that the system developed here operated successfully in tobacco, showing the potential to provide an easier and cheaper visual selection of transgenic cells in the genetic transformation process.