Catálogo de publicaciones - libros

Solid calli and derived liquid cell cultures were initiated from one-year-old roots of CA Meyer. Half-strength Murashige and Skoog medium supplemented classically with an auxin and a cytokinin did not appear favourable for biomass accumulation nor for a high ginsenoside content. Changes in the levels of mineral nutrients, sucrose and growth regulators were preliminary investigated here to improve growth and ginsenoside production in liquid cultures. The hypothesis that ginseng cells released growth inhibitors in the medium was not supported by the results obtained in experiments involving frequent transfers to fresh growth medium.

An efficient method for regeneration of entire plants somatic embryogenesis in using tuber-derived callus has been developed. Primary callus was obtained by culturing tuber explants on Murashige and Skoog’s (MS) (1962) medium supplemented with 2.0 mg l N-benzyladenine (BA) and 0.5 mg l α-naphthaleneacetic acid (NAA) in darkness for one month. Somatic embryos were induced by subculturing the primary callus on MS medium supplemented with 0.5–4.0 mg l BA, kinetin, or zeatin, within 2 weeks of culture in light. Rooting in the embryos with well developed cotyledonary leaves was achieved by transferring them to half-strength liquid MS medium supplemented with 1.0 mg l zeatin riboside for three weeks. Converted somatic embryos were cultured on half-strength MS medium supplemented with 6% (w/v) sucrose, and with 0.5–10.0 mg l abscisic acid (ABA), paclobutrazol, or ancymidol, 0.5–5.0 mg l gibberellic acid (GA) and 15–100 mg l polyethylene glycol (PEG) 4000 for further development of plantlets and tuber formation . Recurrent somatic embryogenesis has been observed in the region at the junction of cotyledon and root when the converted somatic embryos were cultured on MS basal medium supplemented with 0–10 mg l ABA. The use of liquid MS basal medium with 0.1 mg l GA enhanced embryo conversion within fifteen days of culture. Phenotypically normal plants were recovered from converted somatic embryos and the plants showed well-developed tuber formation and flowering after 4 months of culture. Also, the effects of 0.5–5 mg l ABA, paclobutrazol and 0.5–2 mg l ancymidol, 0.5–5 mg l GA and 15–100 mg l PEG 4000 supplemented in half-strength MS medium on the production of the two major protoberberine-type alkaloids (D,L-tetrahydropalmatine and D-corydaline) by the tubers of somatic embryo-derived plants of were examined. The high performance liquid chromatography (HPLC) analysis revealed that the contents of D,L- tetrahydropalmatine and D-corydaline in the tubers of somatic embryo-derived plants were greater than the marketed crude drug and varied with growth regulator / PEG-4000 treatment and the age of the plant.

The selection and cloning of cell lines for the establishment of long-term cell cultures of L. is important for the prospective biotechnological production of taxanes. Callus cultures were established from young stems of adult trees and aseptically grown seedlings. All parts of young seedlings were more suitable for cell proliferation on callus-induction media as compared to young stems of adult trees. The best growing calli successfully achieved an average 8-fold increase in fresh weight after 20 months of culture. The growth characteristics of two seedling-derived cell lines were determined. The best growing calli were used for establishment of suspension cultures. Gamborg’s B5 medium supplemented with 3 mg l 2,4-dichlorophenoxyacetic acid, 0.5 mg l kinetin and 1.5% polyvinylpyrrolidone, a phenolic-binding agent, was used as agar-solidified and liquid medium. A 20-month-old callus culture of (VI/Ha) produced paclitaxel up to 0.0109 ± 0.0037 % of extracted dry weight basis. The content of taxanes was determined by high performance liquid chromatography or a competitive inhibition enzyme immunoassay system (CIEIA). A kinetic study of callus growth and taxane production was performed.