Thidiazuron-induced shoot organogenesis and production of hepatoprotective lignan phyllanthin and hypophyllanthin in Phyllanthus amarus

Callus cultures from nodal and leaf explants of Phyllanthus amarus were established on Murashige and Skoog (MS) medium with various combinations of auxins and cytokinins. The leaf-derived callus induced on 2.26 mu M 2,4-dichlorophenoxyacetic acid (2, 4-D) + 2.32 mu M Kinetin (Kin) upon transfer to medium containing thidiazuron (TDZ) exhibited higher shoot regeneration (32.4 +/- A 1.3 shoots per culture). Four-week-old shoots rooted readily on 1.5 mu M Indol acetic acid (IAA)-containing medium and were successfully acclimatized with 98% survival. The lignans, Phyllanthin (PH) and Hypohyllanthin (HPH), of leaf extracts from naturally grown plants were identified by using TLC, HPLC and H1-NMR. The PH and HPH production in the regenerated shoots was compared to their production in callus cultures, plants under field conditions and in naturally grown plants. The regenerated shoots on MS + 2.27 mu M TDZ produced about two times higher PH and HPH than the leaves of naturally grown plant. The present study provides a useful system for further studies on in vitro morphogenesis, elicitor-assisted production of PH and HPH and A. rhizogenes-mediated genetic transformation in Phyllanthus amarus.