Gentiana clusii Perr.&Song.: Enhanced production of secondary metabolites by in vitro propagation

Shoot and root in vitro culture of endemic European species Gentiana clusii was established for the first time. The effects of different concentrations of benzyl adenine (BA), 6-phurphurylaminopurine (KIN), indole-3-butyric acid (IBA) and naphthalene acetic acid (NAA) on shoot propagation and rooting of G. clusii were investigated. The optimal in vitro conditions for shoot propagation and long-term maintenance were achieved using woody plant medium (WPM) supplemented with 0.5 mg l−1 KIN, and subsequent application of IBA at 0.5 mg l−1 significantly improved rooting of these shoots. Root culture was established from excised root tips cultured in ½ MS liquid media with increasing concentrations of IBA (0.1–1.0 mg l−1). A high root growth rate and considerable biomass yield were obtained by addition of 1.0 mg l−1 IBA. HPLC analysis revealed that in vitro culture considerably promoted the production of secondary metabolites in G. clusii. The selected protocol for shoot propagation (WPM + 0.5 mg l−1 KIN) increased the content of sweroside, gentiopicrin and norswertianin-1-O-primeveroside (N-1-P) for more than 2-fold compared with the wild plants. IBA promoted N-1-P and norswertianin production in root cultures; their contents were enhanced 6.4- and 18.6-fold, respectively, compared with the wild plants. The extract of these roots displayed the highest antioxidant capacity (IC50 = 66.57 μg ml−1). The established shoot and root propagation protocols facilitate in vitro conservation of G. clusii, and provides a promising tool for the large scale production of valuable secoiridoids and xanthones.