Agrobacterium rhizogenes-mediated transformation of Gentiana utriculosa L. and xanthones decussatin-1-O-primeveroside and decussatin accumulation in hairy roots and somatic embryo-derived transgenic plants

Abstract

Production of innovative drugs from natural products in controlled conditions plays an important role in modern pharmacology in order to tackle global health challenges. The potential of Gentiana utriculosa hairy roots, obtained by transformation with Agrobacterium rhizogenes A4M70GUS, as well as that of the shoots regenerated from them via somatic embryogenesis, for xanthone production was investigated. Gentiana utriculosa was shown to be a new source of xanthones, medicinal raw materials for different pharmaceutical applications, among which decussatin has been recently recognized as a prospective hepatoprotective and antiulcer compound. Decussatin and decussatin-1-O-primeveroside were detected in both hairy roots and transgenic shoots, while mangiferin, present in nontransformed plants, was not detected. Quantitative HPLC analysis revealed up to 4.5-fold higher decussatin production in hairy roots line 9 compared with the plants from the nature, and this clone was selected. It showed stable growth after more than two years of continuous subcultivation. Cultivation of hairy roots on solid medium was favorable for biomass production, while liquid culture was beneficial for decussatin and decussatin-1-O-primeveroside accumulation. Cytokinins kinetin or N6-benzyladenine promoted somatic embryo maturation and germination as well as multiplication of obtained plantlets, while active charcoal reduced hyperhydricity. Plants regenerated from selected hairy root line 9 comprised two TL-DNA inserts, as confirmed by Southern blot analysis. They accumulated at least 2.5-fold more decussatin then nontransformed plants. Therefore, these plants could be valuable material to create xanthone high-yielding cultivars of G. utriculosa.