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

Ewelina Piątczak,Łukasz Kuźma,Przemysław Sitarek,Halina Wysokińska

doi:10.1007/s11240-014-0620-3

Ewelina Piątczak, Łukasz Kuźma + Show 2 more

Open Access

https://doi.org/10.1007/s11240-014-0620-3

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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.

Highlights

Rehmannia glutinosa Libosch., a member of Orobanchaceae family, naturally occurs in China, Japan and Korea
The most efficient regeneration of shoots occurred with hypocotyl explants (Fig. 1a), with as many as 90 % of the explants being capable of forming organogenic calli, depending on the growth regulator used
The findings of the present study indicate the number of regenerated shoots to be highest in R. glutinosa seedling explants when the ratio of cytokinin to auxin was 10:1

Summary

Introduction

Rehmannia glutinosa Libosch., a member of Orobanchaceae family, naturally occurs in China, Japan and Korea. Plant Cell Tiss Organ Cult (2015) 120:539–549 et al 2008), increases brain angiogenesis (Zhu et al 2010), possesses extensive ischemic neural protection (Li et al 2005) and attenuates apoptosis in the ischemic brain (Li et al 2006) Another important class of natural products detected in the roots of R. glutinosa are phenylethanoid glycosides. Verbascoside (known as acteoside, kusaginin or orobanchin) exhibits a wide spectrum of biological activities, including antileukemic and cytotoxic activity against a murine cell line (Pettit et al 1990) It demonstrates anti-inflammatory activity through inhibition of cyclooxygenase-2 (COX-2) expression and inhibition of complement activity in human serum (Gyurkovska et al 2011), and possesses diuretic (Herbert et al 1991) and antibacterial properties (Pennacchio 2005). The phytochemical part of the present study compares the production of iridoid and phenylethanoid glycosides in the shoot regenerating calli and micropropagated plants with that of seed-derived R. glutinosa plants

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