<i>In vitro</i> regeneration of <i>Hypericum perforatum</i> L. using thidiazuron and analysis of genetic stability of regenerants

Abstract

In the present study, <i>in vitro</i> regeneration method for <i>Hypericum perforatum </i>L. using different plant growth regulators has been developed. Callus was induced from hypocotyl explants on MS medium using 0.5 mg L^-1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg L^-1 kinetin (Kn). Shoots were developed on the medium containing 1 mg L^-1 thidiazuron and rooting was achieved with 2 mg L^-1 indoleacetic acid (IAA). Random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) were used to assess the genetic stability of the regenerated plants. Among a large number of regenerated plantlets, 10 plants were randomly selected for molecular analysis. 15 RAPD primers generated monomorphic banding pattern among the mother and regenerated plants. PCR-RFLP profiles of nuclear ribosomal internal transcribed spacer (nrITS) region using <i style="">Eco</i>RV, <i style="">Cla</i>I and <i style="">Hpa</i>II also did not show any variation in banding pattern. The entire ITS region of the mother plant, and a randomly selected regenerated plant, when cloned and sequenced showed query coverage of 99% when analyzed through CLUSTAL W. These molecular analyses showed that the regenerated plants produced were similar to the mother plant both in phenotypic and genotypic characters.