In vitro regeneration of Hypericum perforatum L. using thidiazuron and analysis of genetic stability of regenerants

In the present study, in vitro regeneration method for Hypericum perforatum 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 EcoRV, ClaI and HpaIl 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.