Shoot organogenesis, molecular analysis and secondary metabolite production of micropropagated Rehmannia glutinosa Libosch.

Abstract

This study is the first description of a procedure for shoot organogenesis from calli derived from various seedling explants. The frequency of shoot induction and number of shoots per explant were measured on Murashige and Skoog (MS) medium with 0.2–3.0 mg L−1 6-benzylaminopurine (BAP) alone or combined with auxin [0.1 mg L−1 indole-3-acetic acid (IAA) or 1-naphthalenacetic acid]. The highest organogenic response where 90 % of explants formed a regenerative callus with a mean number of nine shoots was achieved after 6 weeks of culture when hypocotyl explants and 1.0 mg L−1 BAP was used together with 0.1 mg L−1 IAA. The shoots regenerated from hypocotyl-derived calli were rooted for 6 weeks on MS medium lacking growth regulators or containing auxin (IAA or indole-3-butyric acid). The plantlets were successfully acclimatized in a greenhouse. Micropropagated plants subjected to random amplified polymorphic DNA and inter simple sequence repeat (ISSR) marker based profiling revealed a uniform banding pattern identical with that of donor plants. The iridoid and phenylethanoid glycoside content of the organogenic callus culture and in vitro-raised plants was determined using UHPLC. In the tested samples catalpol, aucubin, harpagid, harpagoside, verbascoside, isoverbascoside and traces of loganin and catalposide were identified. The leaves produced the highest levels of catalpol (43–45 mg g−1 DW). Organogenic callus and 6-week-old in vitro grown plantlets were characterized by the highest accumulation of aucubin, verbascoside and isoverbascoside. The work also demonstrated the regenerative ability of hypocotyl-derived calli and production of bioactive metabolites to be stable for 4 years of culture.