Quantification of taxol by high-performance thin layer chromatography in Taxus wallichiana callus cultivated in vitro

In the present study, the induction of callus, callus biomass growth and the yield of taxol were investigated in Taxus wallichiana. This is the first report of a quantification study of taxol in in vitro grown tissues of Taxus obtained from• Jammu and Kashmir provinces, India. For callus induction, three different explant types (leaf, cone and stem) were cultured in media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). The stem was most responsive to callusing, and the maximum callus induction frequency of 71.5% was noted on medium amended with 2.0 mg/l 2,4-D. The medium amended with 2,4-D and ascorbic acid (AA) showed better callus growth and the maximum biomass (0.082 g, fresh weight). The yield of taxol was quantified in the callus grown on a medium supplemented with different plant growth regulators (PGRs) by using high-performance thin layer chromatography (HPTLC). The taxol yield was maximum (1.053 μg/g dry weight) in 2,4-D-stimulated callus, followed by callus treated with NAA in which 0.896 μg/g dry weight taxol was detected. The maximum taxol yield (56.6%) was obtained in the callus grown on the medium amended with 2,4-D, and a 33.3% increased yield was noted on the NAA-supplemented medium. As a stress marker, the activities of the antioxidant enzymes superoxide dismutase (SOD) and ascorbate peroxidase (APX) and the level of proline were measured in an auxin (2,4-D and NAA)-supplemented medium. The callus grown on the 2,4-D-supplemented medium had high levels of SOD (3.91 min^-1 •mg^-1 protein), APX (1.61 min^-1 •mg^-1 protein) and proline (6.57 mg/g), thus suggesting a higher stress level; the callus grown on the NAA-supplemented medium had slightly lower levels of SOD and APX enzyme activities and proline content (3.01 min^-1 •mg^-1 protein, 1.04 min^-1 •mg^-1 protein and 5.90 mg/g, respectively). BAP had little influence on stress parameters. The present study thus indicates a good taxol yield in the callus cultured in 2,4-D, which functioned as a signalling element and a stressor. The taxol yield in response to PGRs was analysed in in vitro cultivated tissues.