Establishment of hairy root culture of Rubia yunnanensis Diels: Production of Rubiaceae-type cyclopeptides and quinones

Rubia yunnanensis is an important medicinal plant with various bioactive secondary metabolites. In order to reduce the dependence on wild populations of the species, we aim to establish in vitro culture system that can produce Rubiaceae-type cyclopeptides (RAs) and quinones. Agrobacterium rhizogenes-mediated transformation of stem segments of in vitro grown R. yunnanensis plants using four A. rhizogenes strains was studied and transformation conditions were optimized. Hairy roots appeared with the highest frequency (68.89%) when stem segments (with leaves) without pre-culture were immersed in A. rhizogenes A4 strain bacterial suspension for 30 min, co-cultured on Murashige and Skoog (MS) solid medium in the dark for three days, and afterwards incubated in darkness. PCR analysis of rolB and rolC genes confirmed transformed nature of six hairy root clones. The hairy roots grew rapidly, especially showing the highest accumulation of biomass in MS liquid medium compared to in vitro grown plants and calli. Histological observation of hairy root revealed anatomical difference in vascular cylinder, where the cells exhibited high mitotic activity characterized by vigorous growth. The UPLC-MS/MS analysis revealed that the amount of RAs in the hairy roots grown in ½MS liquid medium (4.611 μg g-1 DW) was higher than that in in vitro grown plants (0.331 and 4.096 μg g-1 DW for shoots and roots respectively) and calli (1.082 μg g-1 DW), but still far lower than that in the roots of seed-borne plants (80.296 μg g-1 DW). However, the hairy roots accumulated high level of quinones (2320.923 and 5067.801 μg g-1 DW for MS and ½MS liquid media respectively), of the same order of magnitude as the roots of seed-borne plants (7409.973 μg g-1 DW). Hairy root culture of R. yunnanensis, with high accumulation of biomass and production of quinones, may offer an attractive perspective for the production of the RAs and quinones that could be further optimized for pharmaceutical use.