Protoplast culture and paclitaxel production by Taxus yunnanensis

Viable protoplasts of Taxus yunnanensis were isolated from friable, light y ellow callus. Protoplast yield was dependent on callus age, with a maximum from 20-day-old callus. Protoplasts were induced to undergo sustained divis ions and to form cell colonies when cultured in medium consisting of B5 sal ts, KM vitamin and organic components, 0.45 M fructose, 3.0 mg l(-1) 2,4-di chlorophenoxyacetic acid and 0.1 mg l(-1) kinetin. The planting density was 2.5-3.0x10(5) protoplasts per ml of culture medium. Cell-free extract from callus enhanced protoplast division and the highest plating efficiency was about 7%. Protoplast-derived colonies showed significant variations in bot h growth and paclitaxel content. A negative correlation existed between pac litaxel accumulation in colonies and their growth to some extent (r = -0.44 85). Among 70 colonies isolated from the heterogeneous protoplast cultures, colony TY-7 accumulated the highest paclitaxel content. Paclitaxel accumul ation in colony TY-7 was not great enough to produce paclitaxel for commerc ial purposes, however, success in inducing colony formation from T. yunnane nsis protoplasts provides an opportunity to obtain cell lines with high pac litaxel productivity from mutagenized protoplast cultures.