Embryogenesis, plant regeneration and cardiac glycoside determination in Digitalis ferruginea subsp ferruginea L

The present study reports, for the first time, an efficient in vitro plant regeneration protocol for Digitalis ferruginea subsp. ferruginea L. (rusty foxglove). We have used different concentrations of gibberellic acid (GA(3)) on Murashige and Skoog (MS) medium to assess the germination frequency of seeds. High frequency of germination was achieved on MS medium with 1.0 mg l(-1) GA(3). 6-Benzylaminopurine (BAP) combined with alpha-naphtaleneacetic acid (NAA) or 2, 4-dichlorophenoxy acetic acid (2, 4-D) in the induction MS medium induced both somatic embryogensis and shoot organogenesis. The highest percentage of callus growth (85 %) was obtained when hypocotyl explants were cultured on MS medium containing 0.5 mg l(-1) 2, 4-D plus 1.0 mg l(-1) BAP. The maximum mean number of somatic embryos (7.3 +/- A 1.3 embryos) or shoots (12.0 +/- A 1.1 shoots) per callus was obtained when medium contained 0.25 mg l(-1) NAA plus 1.0 mg l(-1) BAP or 0.5 mg l(-1) NAA plus 2.0 mg l(-1) BAP. The regenerated shoots easily rooted on MS medium. Higher amounts of lanatoside C [13.2 +/- A 0.5 mg 100 g(-1) dry weight (dw)] and digoxin (2.93 +/- A 0.31 mg 100 g(-1) dw) accumulation were obtained when shoots were obtained by indirect regeneration. We also investigated derivatives of cardenolides, i.e., digitoxigenin (730 +/- A 180 mg 100 g(-1) dw), gitoxigenin (50 +/- A 20 mg 100 g(-1) dw) and digoxigenin (490 +/- A 170 mg 100 g(-1) dw) from natural samples.