Micropropagation and Evaluation of Genetic Variability Among in Vitro Regenerants of Naravelia zeylanica (L.) DC

An efficient protocol was developed for the regeneration of plantlets via direct and indirect organogenesis from stem nodal segments of Naravelia zeylanica (L.) DC. (Ranunculaceae). The interaction of 6-benzyl aminopurine (BAP) with -naphthalene acetic acid (NAA) in Murashige and Skoog (1962) medium induced a varied morphological response. A maximum of 17.6 ± 1.5 shoots per explant were obtained in the presence of 4.0 mg l BAP and 0.6 mg l NAA on MS medium. A higher concentration of BAP (5.08.0 mg l) and NAA (1.0-2.0 mg l) favoured callogenesis while a lower range (2.0-3.0 mg l BAP and 0.25-5.0 mg l NAA) induced adventitious shoot buds and 11.7 ± 1.3 plantlets were obtained from stem calli. The excised microshoots rooted well on MS basal medium without plant growth regulators. Morphologically direct organogenesis regenerants were similar to in vivo plants while the callus regenerants exhibited abnormal development after acclimatization. The survival rate of the plantlets derived from direct organogenesis was 96% whereas in callus regenerants it declined to 72%. Random Amplified Polymorphic DNA (RAPD) markers were employed to analyze the level of genetic variation among the plantlets of direct and indirect organogenesis. Five groups of Operon primers – B 11-20, C 01-20, D 01-10, E 01-10, and F 11-20 – were screened, from which six were selected, among which OPF-17 and OPF-19 gave clear and distinguishable bands. A dendrogram was constructed using Euclidean distances by Ward’s method. RAPD marker fingerprinting allowed a rapid assessment of the level of genetic variation among the regenerants. _____________________________________________________________________________________________________________