Regeneration of plantlets through somatic embryogenesis from root derived calli of Hibiscus sabdariffa L. (Roselle) and assessment of genetic stability by flow cytometry and ISSR analysis

Induction of somatic embryogenesis and complete plantlet regeneration from callus culture of Hibiscus sabdariffa L. var. HS4288 has been made. Leaf and root explants were cultured on Murashige and Skoog (MS) and Driver-Kuniyuki Walnut (DKW) basal media supplemented with different concentrations of synthetic auxins and cytokinins. Root explants on DKW medium supplemented with 2.26 mu M 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and 4.65 mu M kinetin (KIN) induced highest percentage (70%) of embryogenic calli. Average number of globular embryos per root derived callus produced within 6 weeks of culture initiation on MS media with different plant growth regulators (PGRs) ranged from 2.27 +/- 0.12 to 8.80 +/- 0.17 and that of cotyledonary embryos ranged from 0.00 to 2.53 +/- 0.20. On DKW medium comparatively more globular embryos (2.70 +/- 0.15 to 14.53 +/- 0.23) and cotyledonary embryos (0.00 to 8.90 +/- 0.17) were produced than that of MS medium. Regeneration of complete plantlets was highest (76.67%) when embryogenic calli with mature somatic embryos were grown on DKW medium containing 2.32 mu M KIN and 2.22 mu M 6-Benzyladenine (BA). Plants were primarily hardened in humidity, temperature and light controlled chamber and finally in a greenhouse showed 70% survival ability. Different stages of somatic embryogenesis process in the root derived embryogenic calli were elaborated in detail by morphological, histological and SEM study. The data were statistically analyzed by Duncan Multiple range test (p <= 0.05) and Principal component analysis (PCA). Flow cytometry and Inter-simple sequence repeats (ISSR) marker analysis confirmed that there was no genetic variation within the regenerated plants.