Abstract
The organogenic competence of different explants of Rhaponticum carthamoides was investigated on MS agar medium supplemented with BA, IBA or NAA at concentrations of 0.2 and 0.5 mg L−1. Adventitious shoot formation was obtained through direct organogenesis using leaves of in vitro cultures as explants and through indirect organogenesis when seedling explants (hypocotyl, cotyledon and root) were used for regenerative callus initiation. The shoots were rooted on half-strength MS medium (½ MS) without auxin or containing IBA (0.2–2.0 mg L−1). The plantlets regenerated through direct and indirect organogenesis were transferred into pots and grown in the greenhouse for 3 months. Significant differences in morphology, accumulation of chlorogenic acid and 20-hydroxyecdysone (20-HE) as well as in genetic profile were observed between these plants. UHPLC analysis showed that the highest level of chlorogenic acid (12 mg g−1 DW) was found in leaves of plants developed directly from explants, whereas leaves of plants developed via callus tissue accumulated the highest amount of 20-HE (7.4 mg g−1 DW). Its level exceeded that detected in leaves of 3-month-old plants obtained from seeds (2.4 mg g−1 DW). Genetic variations of R. carthamoides regenerated plants were evaluated by flow cytometry and RAPD and ISSR methods. Flow cytometry confirmed similar ploidy level in the mother plant and plants regenerated through direct and indirect organogenesis. Genetic similarity values calculated on the basis of RAPD and ISSR data among regenerated in vitro plants to the mother plant were ranged from 0.765 to 0.941 and 0.647 to 0.947, respectively.
Highlights
Rhaponticum carthamoides (Willd.) Iljin is an endemic, perennial, herbaceous plant belonging to the family Asteraceae
Values within a column followed by the same letter are not significantly different from each other at a 5 % level according to the Kruskal–Wallis test and multiple comparisons of average ranks
We included in the analysis genomic DNA isolated from fresh roots of the mother plant (MP; a field grown plant which was the source of seeds used for in vitro culture initiation), roots of ten randomly-selected plants obtained through clonal propagation (CP; plants obtained from axillary buds) and root-callus, which was used for induction of shoot organogenesis (RC-line maintained on Murashige and Skoog (MS) medium supplemented with 0.5 mg L-1 NAA and 0.2 mg L-1 BA for one year)
Summary
Rhaponticum carthamoides (Willd.) Iljin (known as ‘‘maral root’’ or Russian leuzea) is an endemic, perennial, herbaceous plant belonging to the family Asteraceae. The roots and rhizomes of R. carthamoides have been used for a long time in Siberian folk medicine in the treatment of overstrain and weakness after illness They have adaptogenic, antioxidant, immunomodulatory, anticancerogenic and antimicrobial properties (Kokoska et al 2002; Biskup and Lojkowska 2009; Kokoska and Janovska 2009). Of several molecular markers used for assessment of genetic fidelity of in vitro regenerated plants, random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) marker analyses are simple, quick and cost-effective methods. The present study describes the regeneration of R. carhamoides plants through direct (from leaves of in vitro culture) and indirect organogenesis (from hypocotyl, cotyledon and root of 3-week-old seedling). Chlorogenic acid is a free radical and metal scavenger; it may interfere with glucose absorption and has been shown to modulate gene expression of antioxidant enzymes (Clifford 1999; Olthof et al 2001). 20-HE is known to have antioxidant and anti-inflammatory properties (Zhou et al 2012), and to exert a stimulatory effect on protein synthesis and increase metabolic activity (Slama and Lafont 1995)
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