Modulation of naphthodianthrone biosynthesis in hairy root-derived Hypericum tomentosum regenerants

Abstract

The results of the current study represent the first report on an efficient regeneration protocol for Hypericum tomentosum L. hairy root cultures. Six out of ten hairy root clones of H. tomentosum obtained by Agrobacterium rhizogenes-mediated transformation differentiated shoots on Murashige and Skoog medium containing a urea-based cytokinin thidiazuron in combination with the auxin inhibitor p-chlorophenoxyisobutyric acid. The whole plant regeneration of this species in vitro was achieved by further cultivation of shoots on medium containing benzyladenine. All transformed plants were successfully acclimated to ex vitro conditions. Most of the adapted clones exhibited typical hairy root phenotype with stunted growth, small wrinkly leaves and shortened internodes. Increased number of dark nodules, the sites of hypericins accumulation, was observed in the leaves of all transgenic clones. The capability of naphthodianthrone production was also modulated leading to a significant 28- and 5-fold increase of total hypericin content in two transgenic clones. The qPCR analysis revealed seven rolC integrations in two transgenic clones and one integration in four clones. The clones with multiple rolC copies synthesized the highest and the lowest amount of naphthodianthrones, respectively. The chromosome number in all analysed samples was determined as 2n = 18 suggesting a revision of the cytogenetic characterization of H. tomentosum.