Direct somatic embryogenesis from hypocotyl segments of Digitalis trojana Ivan and subsequent plant regeneration

This study describes, for the first time, an in vitro protocol for the direct development of somatic embryos and subsequent plant regeneration from hypocotyl segments excised from 21-days-old in vitro-germinated seedlings of Digitalis trojana Ivan (Helen of troy foxglove). Two sets of experiments were carried out, the first compared different concentrations of four cytokinins: N6-benzyladenine [BAP], 6-furfurylaminopurine [kinetin], 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea [TDZ], and 6-(4-hydroxy-3-methylbut-2-enylamino) purine [zeatin] alone, while the second set tested TDZ or BAP combinations with IAA (indole-3-acetic acid), IBA (indole-3-butyric acid) or NAA (α-naphthalene acetic acid). In the first set of experiments, TDZ was found the most effective at 1.0 mg/l concentration, producing a mean of 10.7 somatic embryos per explant. In the second set, a combination of 1.0 mg/l TDZ with 0.5 mg/l IAA produced significantly more somatic embryos per explant (13.8 embryos) than with BAP (8.8 embryos). During subculture on growth regulator-free half-strength MS medium, somatic embryos gradually developed into plantlets. Regenerated plantlets were successfully transplanted and grown in a greenhouse environment. The efficient regeneration protocol reported here provides an important method of micropropagation of this plant. Furthermore, this protocol may be used for a large-scale production of cardenolides and genetic transformation of this valuable medicinal plant for its further improvement.

► An effective protocol for direct somatic embryogenesis was developed.
► TDZ alone at 0.5 or 1.0 mg/l was more effective for somatic embryo induction.
► IAA was more effective than NAA or IBA for somatic embryogenesis.
► Combination of low IAA and high TDZ was more effective for somatic embryogenesis.
► Plantlet development was achieved after culturing on half-strength MSO medium.