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Abstract: A protocol was developed for direct shoot regeneration of Scrophularia takesimensis using shoot tip explants. The explants were excised from mature field grown plants and cultured on Murashige and Skoog (MS) medium with different concentrations of 2-isopentyl adenine (2-iP), 6-benzyl adenine (BA) or thidiazuron (TDZ). Among the three cytokinins studied, BA was found to be the most effective cytokinin for multiple shoot induction. Addition of auxin to the shoot regeneration medium significantly enhanced the percentage of shoot induction and number of shoots per explant. The greatest percentage of shoot induction was achieved when shoot tip explants were cultured on MS medium supplemented with 3.0 mg L-1 BA and 1.0 mg L-1 indole-3-acetic acid (IAA) with an average of 13 shoots per explant. The regenerated shoots were transferred to modified MS medium supplemented with 3% (w/v) sucrose, 1.0 mg L-1 indole-3-butyric acid (IBA), and solidified with 0.8% (w/v) agar for four weeks to induce the growth of shoots and roots. Plantlets were transferred to the greenhouse with 100% survival rate. High performance liquid chromatography analysis detected the presence of harpagoside in greenhouse-grown plants organs which were established from in vitro culture. The content of harpagoside was high in fruit followed by leaf, root and stem. This protocol could be utilized for conservation and clonal propagation and chemical analysis of this economically important plant.