Phytohormones on cell growth and taxol accumulation via cell culture of taxus wallichiana zucc

Abstract

Taxus wallichiana Zucc., an important medicinal plant, is the main source of paclitaxel, a diterpene alkaloid of commercial interest for pharmacological properties. Toward the main objective of in vitro production of paclitaxel, the specific objectives of this research were i) to investigate medium components for optimizing cell growth and paclitaxel accumulation via callus and cell suspension cultures ii) to enhance paclitaxel production using precursors and abiotic elicitors. For optimization of cell growth, high frequency of callus formation (100%) was obtained from young stem explants on WP solid medium, but B5 medium was the best one for callus growth supplemented with 2,4-D (4.0 mg/l). The friable and reddish, light brown callus masses collected for cell culture proliferated with high growth index (3.07) on medium B5 supplemented with 2,4-D (4.0 mg/l), NAA (2.0 mg/l), sucrose (30 g/l), glycine (10 mg/l) and Cw (10%). The growth rate of callus was highest between 14th and 28th day

of culture period, and the appropriate subculture time was day 35 favored optimal growth

of callus. Subsequently, cell suspension cultures were established by transferring selected friable calluses to liquid medium for their further growth and enhancement of paclitaxel biosynthesis. Cell growth of Taxus wallichiana Zucc in liquid medium supplemented with 2,4-D (3 mg/l), NAA (3 mg/l), sucrose (30 g/l), Cw (10 %), Glycine (10 mg/l), Casein hydrolysate (1000 mg/l) increased significantly with high growth coeffection. For elicitation of paclitaxel biosynthesis, the suitable concentration of phenylalanine

for paclitaxel production (0.518 mg/g DW) was 15 mg/l which was 1.254 times higher than the control. The addition of O-chi in cell cultures strongly promoted the biosynthesis of paclitaxel whereas Yeast extract, Salycilic acid showed little effect. O-chi was the most appropriate substrate for paclitaxel production and its optimal concentration was 5 mg/l for highest paclitaxel product (3.450 mg/g DW) which was 14.009 times higher than the control. Subsequently, MJ for elicitation of paclitaxel production at the suitable concentration was 10 mg/l (1.658 mg/g DW) which increased paclitaxel content to 6.215 times higher than the control. Moreover, time of elicitor addition on the culture medium determined was day 21 at stage of strong growth of cells and cell cultures were harvested on day 27. Finally, paclitaxel content was highest (0.0413% of dcw) after 6 days of elicitor

exposure.