An efficient multiple shoot induction and genetic fidelity assessment of Exacum bicolor Roxb., an endemic and endangered medicinal plant

Exacum bicolor, an endemic and endangered medicinal plant, belongs to the family Gentianaceae. A rapid protocol has been developed for efficient multiple shoot induction by testing nodal explants on Murashige and Skoog (MS) medium supplemented with various cytokinins. The cytokinins 6-benzyladenine (BA), 6-furfurylaminopurine (Kn), 2-isopentenyladenine (2-iP) and zeatin (Zn) were used individually and in combination at different concentrations (0.5, 1.0, 2.0, 5.0, and 10.0 mu M). The maximum number of shoots (19.33 +/- 1.09 per explant) as well as their fresh weight (5.1 +/- 0.68 g) and dry weight (216.83 +/- 2.84 mg) were obtained with 10.0 mu M BA + 2.0 mu M Kn. After 4 wk, the multiple shoots from agar culture were subcultured into liquid medium containing the same growth regulator combinations. After 8 wk of liquid culture, the best treatment had about tenfold increase in shoot number (199.5 +/- 1.14 per explant). The fresh weight (13.76 +/- 0.14 g) and dry weight (909.33 +/- 1.92 mg) were highest with full strength MS medium supplemented with 3% sucrose and containing 10.0 mu M BA + 2.0 mu M Kn. Maximum root development was observed after 30 d with 0.5 mu M indole-3-butyric acid (IBA) supplementation. Regenerated plants were successfully transferred to pots containing coco peat:perlite mixture and showed a 75% survival rate. Genetic fidelity of in vitro plantlets compared to mother plant were assessed using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers. All the regenerated plants were genetically identical to their mother plant, showing no detectable genetic variation in the regenerated plantlets. Thus, this protocol could be successfully used for mass multiplication and germplasm conservation of E. bicolor.