Increasing the production of limonin 17-β-D-glucopyranoside in hairy roots of Citrus × paradisi. cv Duncan by overexpression of limonoid UDP-glucosyltransferase via Agrobacterium rhizogenes mediated transformation

Abstract

Limonin 17-β-D-glucopyranoside (LG) is a terpenoid compound with a high medicinal value produced in different Citrus species. This substance has high anti-cancer and antioxidant properties and protects the liver from damage. Although LG is tasteless, many compounds related to it are very bitter, although these bitter substances also have significant medicinal properties. Usually, in most Citrus genotypes, bitter-tasting limonoids are produced more than tasteless types, which are unpleasant despite their therapeutic value. The aim of our study was the in vitro production of LG at high concentrations. For this purpose, hairy roots were induced in ‘Duncan’ grapefruit (Citrus × paradisi) leaf explants. Also, the limonoid UDP-glucosyltransferase (LGTase) gene was transferred from Citrus aurantium to hairy roots by Agrobacterium rhizogenes and forced to overexpression. Two different sets of hairy roots were obtained. The wildtype group only received the Ri plasmid, while cotransformation with the LGTase overexpression constructs in the transgenic group. The gene expression analysis showed that the transcription of the LGTase gene in transgenic hairy roots dramatically increases compared with wildtypes. The overexpression of LGTase significantly increased the total concentration of limonoids in transgenic hairy roots compared with leaves and non-transgenic roots. Evaluation of HPLC chromatograms showed that transgenic hairy roots produced more LG than other samples. In some cases, the concentration of LG increases up to 11 times compared with leaves in some lines. In addition, the concentration ratio of LG to limonin increased in transgenic lines, and transgenic types produced the most LG. Enhancing LGTase gene expression can be a suitable approach to producing high amounts of LG in hairy roots.