High frequency shoot proliferation from cotyledonary node of Lawsonia inermis L. and validation of their molecular finger printing

Abstract

An efficient and reproducible protocol for in vitro plant regeneration was developed for Lawsonia inermis L. using cotyledonary node explant derived from axenic seedlings. Highest shoot proliferation frequency (ca 96.6%) was achieved on Murashige and Skoog’s, 1962 (MS) basal medium supplemented with 8.88 μM 6-Benzyladenine (BA) + 2.68 μM Napthalene acetic acid (NAA). Up-scaling of shoots was carried out using in vitro nodes on MS medium supplemented with 4.44 μM BA. So overall, an average of 238 shoots was produced at 75 days. Of the four different forms of cotyledonary node explants evaluated, highest shoot multiplication was observed in cotyledonary node explant with two whole cotyledons. In vitro regenerated shoots were best rooted (ca 34.3 roots / shoot) on ½ MS medium devoid of any growth regulator. The plantlets were successfully acclimated in sand:soil:: 1:1and established in the garden soil. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis revealed a homogeneous amplification profile for all micropropagated plants validating the genetic fidelity of the in vitro-regenerated plants and supporting the regeneration protocol for economic commercial exploitation.