Factors influencing large-scale micropropagation of Sphagneticola calendulacea (L.) Pruski and clonality assessment using RAPD and ISSR markers

Abstract

Sphagneticola calendulacea (L.) Pruski (synonym Wedelia chinensis (Osbeck) Merrill) has great significance in traditional systems of medicine and is popularly known for its hepatoprotective properties. A comprehensive micropropagation technique for its conservation and season-independent, large-scale production was established. Initial culture was established in different basal media [Murashige and Skoog (MS), Schenk and Hildebrandt (SH), Gamborg’s (B5), and Woody Plant medium (WPM)] following a non-toxic, simple, and effective sterilization of ex vitro shoot tip (ST) and nodal segment (NS) explants. MS medium favored the highest rate (∼87.27%) of multiple (∼2.03) shoot bud initiation from NS explants that was further multiplied with supplementation of thidiazuron (TDZ), kinetin, N6-(Δ2-isopentenyl) adenine, or zeatin in combination with indole-3-acetic acid (IAA) or α-naphthaleneacetic acid (NAA). MS medium containing TDZ (0.2 mg L−1) and NAA (0.05 mg L−1) resulted in the maximum (∼98.82%) response with the highest number (∼33.12) and length (∼6.04 cm) of multiple shoots. The maximum number (∼12.18) and length (∼6.24 cm) of healthy roots per plant was achieved on ½ MS medium plus 1 mg L−1 IAA. In a simple acclimatization process of 6 wk, the survival rate of plantlets cultured in a combination of soil, sand, and vermicompost (1:1:1; v/v/v) was 82.00%. DNA fingerprinting of the in vitro-regenerated plantlets via analysis of random amplification of polymorphic DNA and intersimple sequence repeat markers showed clonal fidelity of the plantlets with the mother plant.