Indirect somatic embryogenesis and shoot organogenesis from cotyledonary leaf segments of Digitalis lamarckii Ivan., an endemic medicinal species

Abstract

This study describes, for the first time, an in vitro protocol for the development of somatic embryos and shoots from a callus derived from cotyledonary leaf segments excised from 3-week-old in vitro-germinated seedlings of Digitalis lamarckii Ivan. (dwarf foxglove), an endemic medicinal species of Turkey. The embryogenic callus was induced on Murashige and Skoog (MS) medium containing 0.54 \muM \mu-naphthalene acetic acid (NAA) and 2.22 \muM 6-benzylaminopurine (BAP). Somatic embryos developed readily when the embryogenic callus was transferred to MS medium containing BAP (4.44 or 8.87 \muM) alone or BAP combined with NAA (1.34, 2.69, or 5.37 \muM). The most effective hormonal combination for somatic embryogenesis was 1.34 \muM NAA and 8.87 \muM BAP, which produced a mean of 37.0 embryos per cotyledonary leaf explant. An organogenic callus was induced on MS medium containing 2.69 \muM NAA and 2.22 \muM BAP. Shoot development was observed when the organogenic callus was transferred to MS medium containing different concentrations of BAP alone (2.22, 4.44, or 8.87 \muM) or BAP combined with NAA (1.34 or 2.69 \muM). The highest mean number of shoots (5.67 shoots per explant) was obtained when the medium contained 8.87 \muM BAP and 2.69 \muM NAA. The regenerated shoots were readily rooted on MS medium containing 1.0, 2.5, or 5 \muM indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA). Rooted regenerants were then transferred to the pots, where they grew well and attained maturity. Over 90% of the regenerants survived the hardening process. The protocol described could be useful for germplasm conservation, commercial cultivation, genetic improvement, and cardenolide production studies in D. lamarckii.