TAXUS CALLUS-CULTURES - INITIATION, GROWTH OPTIMIZATION, CHARACTERIZATION AND TAXOL PRODUCTION

Callus was induced from Taxus baccata cv. Repandens Parsons ex Rehd., T. brevifolia Nutt., T. cuspidata Sieb. and Zucc., and T. x media cvs. Hicksii and Densiformis Rehd. using different concentrations of 2,4-d -(2,4-dichlorophenoxyacetic acid), IBA (indole-3-butyric acid), or NAA alpha-naphthalene acetic acid in combination with kinetin. All cultur es grew slowly following the first subculture, and a majority turned b rown and ceased growth within the next six to twelve months. The callu s cultures which lived, continued to grow very slowly for one to two y ears before the growth rate improved. Initiation of roots and shoot pr imordia-like structures occurred on some cultures maintained in the da rk, and 16 h light/8 h dark, respectively. A fast-growing, habituated callus line (CR-1) derived from T. x media Rehd. cv. Hicksii was estab lished from callus initiated in 1986. Supplementing the medium with ca sein hydrolysate and both fructose and glucose enhanced the growth rat e. A great deal of heterogeneity was found among and within the callus , with respect to the amount of taxol produced. The callus exhibited l evels of taxol ranging from 0.1 to 13.1 mg kg(-1) (0.0001 to 0.0131%) on a dry weight basis. Overall, the older brown-colored callus produce d more taxol than the younger pale yellow-colored callus. The presence of taxol in callus samples was established by high performance liquid chromatography, its biological activity confirmed by a microtubule-st abilizing bioassay and its structure confirmed using one- and two-dime nsional H-1 and C-13 nuclear magnetic resonance spectroscopy.