Rapid induction of genomic demethylation and T-DNA gene expression in plant cells by 5-azacytosine derivatives.

We have optimized conditions for demethylation of the genome and induction of a silent, hypermethylated T-DNA gene (ipt) by 5-azacytosine (5-azaCyt) derivatives in a suspension culture of tobacco cells. In this system, 5-azacytidine (5-azaC) is more effective in causing genomic demethylation and ipt gene induction than 5-azaCyt or 5-azadeoxycytidine (5-azadC). A single treatment with 2.5 μM 5-azaC resulted in a maximal level of ipt gene induction without inhibiting cell growth. However, we could not reduce the level of genomic methylation below approximately 2/3 of that found in untreated controls, even after extensive 5-azaC treatment. Furthermore, remethylation of the genome occurred after removal of 5-azaC. The use of 5-azaC as an inducer of silent plant genes is discussed, along with differences in the response of plant and animal genomes to demethylating agents.