subsp. J. Holub.: Shoot Propagation In Vitro and Effect of Sucrose and Elicitors on Xanthones Production.

In vitro shoot culture of the endangered medicinal plant was established from epicotyl explants cultured on MS basal medium with 0.2 mg L 6-benzylaminopurine (BA) and evaluated for xanthones content for the first time. Five shoot lines were obtained and no significant variations in multiplication rate, shoot elongation, and xanthones profile were found among them. The highest rooting rate (33.3%) was achieved by shoots treated for 2 days with 5 mg L indole-3-butyric acid (IBA) followed by cultivation in liquid PGR-free ½ MS medium for 60 days. HPLC analysis revealed the lower content of xanthones-mangiferin, bellidifolin, demethylbellidifolin, demethylbellidifolin-8--glucoside and bellidifolin-8--glucoside-in in vitro cultured shoots compared to wild growing plants. The increasing concentration of sucrose, sorbitol and abiotic elicitors salicylic acid (SA), jasmonic acid (JA) and methyl jasmonate (MeJA) altered shoot growth and xanthone production. Sucrose and sorbitol applied at the highest concentration of 233.6 mM increased dry matter percentage, while SA at 100 μM promoted shoot growth 2-fold. The increased sucrose concentration enhanced accumulation of xanthones in shoot cultures 2-3-fold compared to the control shoots. Elicitors at 100-300 μM increased the accumulation of mangiferin, demethylbellidifolin-8--glucoside, and bellidifolin-8--glucoside almost equally, while MeJA at the highest concentration of 500 μM enhanced amount of aglycones demethylbellidifolin and bellidifolin 7-fold compared to the control. The obtained results facilitate conservation of and pave the way for further research on large-scale shoot propagation and production of pharmacologically active xanthones.