Petunia plants escape from negative selection against a transgene by silencing the foreign DNA via methylation

Transgenic Petunia hybrida clones harbouring the T-DNA gene 2 of Agrobacterium tumefaciens were used to test a strategy for the trapping of plant transposable elements. In the Petunia line used, floral variegation is due to the presence of the non-autonomous transposable element dTph1 at the An1 locus. The gene 2 product converts the auxin precursor indole-3-acetamide and its analogue 1-naphthalene acetamide into the active auxins indole-3-acetic acid and 1-naphthalene acetic acid. Plant cells that express gene 2 can use a low concentration of the precursors as auxins and become sensitive to the toxicity of high concentrations of these compounds. By selecting protoplast-derived microcalli or seedlings able to grow on medium with high precursor concentrations, variant plants were obtained in which gene 2 was no longer expressed. Southern analysis, using gene 2-specific probes, revealed that in one variant the T-DNA was deleted. For 30 other variants no alteration in gene 2 structure was observed, indicating that transposable element insertion was not responsible for the inactivation of gene 2. Analysis with restriction enzymes allowing discrimination between methylated or non-methylated DNA sequences showed that the inactivated gene 2 sequences were methylated. Addition of the in vivo methylation inhibitor 5-azacytidine to the medium led to reactivation of gene 2 expression in some of the variants. These observations demonstrated that reversible DNA methylation was the main cause of silencing of gene 2 in this system.