Yields of paclitaxel decreased with repeated subculturing of Taxus media cells. We used minimal growth conservation and manipulation of genome methylation to sustain paclitaxel production by Taxus media cell cultures. The subculture period of Taxus cells can be prolonged to 180 d by incubating them at a low temperature (5 degrees C). Paclitaxel levels increased in the cells after conservation and during the first recovery subculture cycle, and then decreased during the subsequent recovery subculture cycle. Analysis of genetic variations in these cultures using amplified fragment-length polymorphism (AFLP) technology identified only two polymorphic bands associated with the second and sixth recovery cycle cultures. However, the results of high-performance liquid chromatography indicated that DNA methylation increased during the course of repeated subculturing. A decrease in DNA methylation level caused by treatment with 5-Aza-2'-deoxycytidine coincided with an increase in paclitaxel levels. Simultaneous exposure to both methyl jasmonate and 5-Aza-2'-deoxycytidine increased paclitaxel levels to 320.43 mu g g(-1) (dry weight), which is more than six times the paclitaxel content before conservation. To our knowledge, this is the first report about improving paclitaxel production by ensuring sustainable use of Taxus cells. (C) 2013 Elsevier Ltd. All rights reserved.