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Elimination of calcium ions from the medium of undifferentiated cell cultures of Digitalis thapsi increased cardenolide production and induced extracellular H # O # accumulation, as measured by the quenching of pyranine fluorescence. The addition of catalase reduced the response and the inclusion of superoxide dismutase enhanced the loss of fluorescence. This suggested that, besides H # O # , the superoxide anion was also formed before dismutating to H # O # . Additionally, exogenous H # O # or superoxide dismutase stimulated cardenolide production whereas the addition of catalase markedly reduced it. These results point to a connection between H # O # and cardenolide formation. The absence of calcium did not alter the levels of lipid peroxidation products ; however, changes in the antioxidant system of D. thapsi cells were observed. Catalase activity was extremely low in control cultures and remained unaltered upon calcium elimination. Ascorbate peroxidase activity was not modified in calcium-free cultures. By contrast, calcium deprivation stimulated superoxide dismutase activity and strongly inhibited glutathione reductase activity. Also, a significant decrease in reduced glutathione was observed. These responses were emulated by treatment of the cultures with the glutathione biosynthesis inhibitor buthionine sulfoximine and by ethyleneglycol-bis-β-aminoethyl ether and LaCl $ . All these results indicate that the depletion of extracellular calcium induces changes in the redox state of cells and suggest that this alteration stimulates cardenolide formation in D. thapsi cultures.

Production of silymarin and the effect of the elicitor, methyl jasmonate (MeJA), was monitored in cell cultures of Silybum marianum over 4 years. Silymarin concentrations gradually declined after prolonged subculture, making the success of elicitor strategy limited in long-term cultures. The continuous presence of MeJA in cultures for an extended period was necessary for induction of silymarin accumulation. A repeated elicitor strategy was not a good option for improving silymarin productivity in batch cultures. Removal of medium from elicited cultures and addition of fresh medium avoided the toxic effects of elicitor accumulation, allowing the system to respond to a repeated MeJA treatment without loss of productivity.