Optimizing Rhizobium rhizogenes-mediated hairy root transformation and elicitor treatments to enhance anthraquinone production in Rheum palmatum L.

Rheum palmatum L. is a major source of traditional medicinal rhubarb, valued for its anthraquinones and other bioactive compounds. However, the overexploitation of wild populations and inconsistent quality of cultivated material highlight the need for alternative production strategies. In this study, a Rhizobium rhizogenes -mediated hairy root culture system for R. palmatum was established and optimized to enhance biomass and secondary metabolite production. Four R. rhizogenes strains (ATCC15834, LBA9402, ACCC10060, and C58C1) were evaluated for transformation efficiency under varying bacterial densities and acetosyringone (AS) concentrations. The ATCC15834 strain, treated with 150 μM AS, achieved the highest hairy root transformation efficiency (47.78%). Further optimization of liquid culture conditions demonstrated that the 6,7-V medium favored secondary metabolite accumulation, whereas the N6 medium promoted higher biomass production. Sucrose was identified as the optimal carbon source. Five elicitors—methyl jasmonate (MeJA), silver nitrate (AgNO₃), yeast extract, β -cyclodextrin, and chitosan—were investigated to evaluate their effects on hairy root growth and secondary metabolite profiles. High Ag + concentrations promoted anthraquinone accumulation, whereas MeJA increased anthrone and tannin levels, despite reducing biomass. This study establishes an efficient and reproducible hairy root culture system for R. palmatum , providing a promising strategy for sustainable in vitro anthraquinone production and a valuable platform for functional genomics studies of secondary metabolism.