Studies on the Production of Digitalis Cardenolides by Plant Tissue Culture

Abstract

Shoot-Forming Cultures. The shoot-forming cafli of D. purpurea L., which had been established in the previous study (5), were transferred into the liquid basal medium, supplemented with 1 mg/L BA and 1 mg/L IAA, and cultured in the light. Thus, green, shoot-forming cultures without root were established and subcultured every 3 weeks. White, shoot-forming cultures were obtained by sub-culturing a portion of the green, shoot-forming cultures in the dark.Culture Condition. Murashige and Skoog medium (9)-with 1 mg/L thiamin-HCl and without agar, edamin, IAA, and kinetinwas used as the basal medium. Approximately 1.5 g fresh weight of cells were inoculated into a 500-ml Erlenmeyer flask containing 100 ml of liquid medium and cultured in continuous light (fluorescent lamp: about 4 x 105 erg/s.cm2) or in the dark at 28°C on a reciprocal shaker (100 strokes/min; 2.0 cm in length).Measurement of Growth. Fresh weight was measured after removing culture medium by suction filtration. The harvested fresh culture was lyophilized, and its dry weight was determined.Assay for Digitoxin. Lyophilized cells (0.1-2 g) were homogenized with 50 ml ethanol in a glass homogenizer. The homogenate was heated at 74°C for 4 h and filtered. The filtrate was dried in vacuo, and the residue was taken up in 2 ml ethanol and diluted with 18 ml H20. When necessary, the extract was further diluted with H20 to a desirable level. Determination of digitoxin concentration of the extract was done by radioimmunoassays, as described (5).Assay for Chi. Chl content was determined spectrophotometric-

Formation of Cardenolides in Cell and Organ Cultures of Digitalis lanata

“…In addition, the chloroplast-containing nonembryoid globules described in Sect. 2 as well as "undifferentiated" green cell cultures of D. purpurea formed only very small amounts of cardenolides ( [16] own unpublished results). Hence the morphological organization of the tissues is of higher importance for cardenolide formation and accumulation than the development of chloroplasts.…”

“…Removal of BA from the medium and addition of gibberellic acid [16,17,32] or abscisic acid [17] also originated higher amounts of cardenolides in the cultures. In addition, growth and cardenolide content of the in-vitro cultivated shoots of D. lanata depended on the carbon and nitrogen sources, the phosphate level [17], as well as on the presence of inositol, thiamine, Mn2+, Zn 2 +, H3 B0 3, MoO~-, 1-, Fe 2 +, and EDTA in the medium (own unpublished results).…”

“…4.2, illumination is no prerequisite for cardenolide accumulation. Cardenolides were also detected in non-illuminated shoot cultures of D. purpurea (about 30% of the amount present in illuminated cultures, [16]) and considerable quantities of cardenolides were found in dark-grown cultures of a carotenefree cell line (strain S-2) derived from one of the embryogenic strains of D. lanata. In this strain illumination even reduced cardenolide accumulation [35].…”

Effects of Mineral Salts, Initial pH and Precursors on Digitoxin Formation by Shoot-forming Cultures ofDigitalis purpureaL. Grown in Liquid Media

“…We have already reported that of the green shootforming cultures, white shoot-forming cultures, undifferentiated green cells and undifferentiated white cells of Digitalis purpurea L., the green shoot-forming cultures accumulated the largest amounts of digitoxin. 1) We next examined the effects of cultural conditions to determine the best for digitoxin formation by the green shoot-forming cultures. We have reported the effects of carbon and nitrogen sources, phosphate, vitamines, natural extracts and some plant growth substances previously,2) and we report here the effects of the other major and minor elements of the Murashige-Skoog basal medium used, the initial pH of the medium and precursors of the digitoxin biosynthesis.…”

Vincamine production in multiple shoot culture derived from hairy roots of Vinca minor

“…Multiple shoot cultures of Digitalis lanata (Hagimori et al 1982), D. purpurea (Lui & Staba 1979) and Catharanthus roseus (Hirata et al 1987) have been established for the production of digoxin, digitoxin and indole alkaloids, respectively. These cultures were maintained for long periods without changes in morphology, growth characteristics or in secondary metabolites production (Hirata et al 1990).…”