In vitro propagation of the endangered species Berberis aristata DC. via leaf-derived callus

Berberis aristata, an endangered plant, is an important component in different medicinal formulations. The low seed germination rate coupled with fructification at high altitudes during the rainy season makes the task of seed collection difficult. In order to increase the yield of relevant plant material for medicinal and conservation purposes, an efficient in vitro regeneration protocol is important. Regeneration of endangered B. aristata from leaf-derived callus may offer an efficient alternative to natural propagation through seeds. When compared with nodal segments taken from in vitro-germinated seedlings and inoculated onto the same medium, leaf-derived callus was found to be better for shoot induction and multiplication. In optimization experiments, the rate of shoot multiplication from leaf-derived callus was increased from 17.6 +/- 0.6 to 26.5 +/- 0.2 shoots per callus by supplementing Woody Plant (WP) medium with 0.5 mu M thidiazuron (TDZ) in place of 8.88 mu M benzyladenine (BA) + 2.68 mu M naphthalene acetic acid (NAA). Although shoot length was decreased when using TDZ, this could be alleviated by gibberellic acid (GA(3)) treatment. Rooting was achieved on half-strength WP medium supplemented with indole butyric acid (IBA) in either a one- or two-step rooting method. The two-step rooting procedure was found to be superior; under the best overall conditions, 55.6 +/- 2.1% of plantlets formed roots within 25.7 +/- 0.3 d. Well-rooted plantlets were successfully established in earthen pots under greenhouse conditions, with a 61.1% survival rate. This procedure is suitable for large-scale production of plants via in vitro propagation for this endangered plant species.