Development of an efficient callus production protocol for Amsonia orientalis: A critically endangered medicinal plant

Background: The plant blue star, rich in cardioactive and anti-cancer glycosides and glycoalkaloids and also used as an ornamental plant, is evaluated as "critically endangered" since it is nearly extinct in nature. In the present study, we describe a rapid and efficient callus production protocol for further studies that can be conducted on Amsonia orientalis such as secondary metabolite production and in vitro propagation. Materials and Methods: Mature nodal explants were cultured on Murashige and Skoog media including 0.5 mg L 1 6-benzylaminopurine. Obtained shoots were subcultured on same fresh media supplemented with different (0.5, 1.0, 2.0 or 4.0 mg L 1 ) 6-benzylaminopurine concentrations. Explants from multiplied shoots were used for callusing experiments. Effects of various combinations of 6-benzylaminopurine, kinetin, indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid on callus production were tested. At the end of the incubation period calli were weighted and means were compared using Duncan's multiple range test. Results: When compared to others, all concentrations of 2,4-dichlorophenoxyacetic acid in combination with 6-benzylaminopurine were found to be the most effective on callus induction. Maximum mean callus weight of 0.327±0.07 g/callus was found at media supplemented with 0.5 mg L 1 2,4-dichlorophenoxyacetic acid and 0.5 mg L 1 6-benzylaminopurine in combination. Furthermore, higher kinetin concentrations caused production of fragile calli which are frequently used to initiate cell cultures. Conclusions: The results have shown that media enriched with indole-3-acetic acid+kinetin combinations are more suitable for fragile calli production while media supplemented with 2,4-dichlorophenoxyacetic acid+6-benzylaminopurine combinations provide large but compact callus tissues. The findings will contribute to the establishment of cell suspension cultures.