Effects of 5-azacytidine on transformation and gene expression inNicotiana tabacum

Abstract

Protoplasts ofNicotiana tabacum var. Xanthi were incubated with liposomes containing the plasmid plGVneo23 encoding kanamycin resistance. Transformed protoplasts and calli and plants derived from transformed protoplasts were treated with the demethylating agent 5-azacytidine. Three lines of evidence indicate that 5-azacytidine can increase NPT II activity in transformed cell lines and plants: a) Addition of azacytidine to the protoplast medium increased the proportion of kanamycin-resistant transformants recovered. b) NPT II activity could not be detected in approximately 50% of calli derived from transformed protoplasts although such calli grew slowly on medium containing kanamycin. Treatment of NPT-negative calli with 5-azacytidine restored detectable gene activity and increased the growth rate of the callus in the presence of kanamycin. c) Shoot tips regenerated from transformed calli were either NPT-positive or NPT-negative. When shoots were NPT-negative, treatment with 5-azacytidine restored detectable gene activity and improved growth in the presence of kanamycin.