Secoiridoid glycosides production in hairy roots of Gentiana pneumonanthe L.: optimization of cytokinin concentration in the culture medium

Gentiana pneumonanthe L. (fam. Gentianaceae) is a herbaceous perennial growing on wet heathlands, grasslands and hay meadows of Europe and Asia. Like other Gentiana species, G. pneumonanthe is characterized by the production of bitter secoiridoid glycosides (SGs) widely used in the pharmaceutical industry. Hairy roots (HRs) of G. pneumonanthe, as an alternative source of these phytochemicals, have been induced by agropine type Agrobacterium rhizogenes strain (A40M70GUS), and were characterized by genetic stability, sizable biomass production, and increased biosynthetic capacity compared to the native plant roots and above ground parts. Although HRs of G. pneumonanthe showed intensive growth without the application of phytohormones, the effect of varying concentrations of cytokinins (BAP – benzylaminopurine; KIN – kinetin) on the morphogenesis and on the content of SGs has been investigated in the present study, with the aim of optimizing in vitro conditions for enhanced production of targeted secondary metabolites. Morphogenetic changes, such as the formation of calli, were induced by both phytohormones. Significant reduction of HR biomass occurred only on treatments with high BAP and KIN concentrations (3 and 4 mg l-1). The content of SGs, especially gentiopicrin and sweroside, was affected by both KIN and BAP. The lowest applied concentration of these phytohormones (0.1 mg l-1) showed a stimulatory effect on the production of gentiopicrin and sweroside. Further increase in both cytokinins concentrations decreased the content of these SGs. Being genetically stable and fast-growing, HR cultures of G. pneumonanthe could provide a constant and highly productive source of valuable secondary metabolites, and could further be implemented in large-scale bioreactors. Although many challenges still exist for the commercial implementation of this alternative production system, the results presented here demonstrate the opportunities for enhanced SGs production by the application of in low concentrations of both cytokinins and thus highlight the benefits of optimizing growth conditions.