Regeneration competence of an endangered orchid, Vanda cristata Wall. ex Lindl. using leaf explants: A study in vitro

Abstract

In the present paper, a protocol for plant regeneration was developed for micropropagation of an epiphytic orchid, Vanda cristata Wall. ex Lindl., by using whole leaf explants procured from five months old in vitro grown cultures. The regeneration competence of leaf explants was tested on two different nutrient media (M and KC) supplemented with auxins (IAA, IBA, NAA) and cytokinins (BAP, KN, TDZ) at different concentrations. The regeneration response was invariably initiated mostly at the bases of the leaf explants via shoot bud, protocorm like bodies (PLBs) and callus formation whereas leaf tips (apical side) showed very less response. KC medium supplemented with NAA(10.6 µM)+BAP(8.8 µM), acted synergistically; cent per cent explants initiated regeneration response via formation of callus, PLBs and shoot buds at the basal part of leaf explant and complete plantlets were obtained. Highest number of regenerants (6) was observed in this combination as compared to the control. The genetic stability analysis of in vitro regenerated plantlets of V. cristata was performed by using inter-simple sequence repeats (ISSR) primers; 6.67% polymorphism and 93.33% of monomorphism was calculated using five ISSR primers, that showed a high degree of genetic stability to obtain true-to-type plantlets. The protocol developed in this study may be useful for conserving V. cristata including other orchid species using different biotechnological approaches.