Synergistic precursor–elicitor strategy maximizes gene expression and metabolite biosynthesis in Glycyrrhiza glabra L. hairy roots

Glycyrrhiza glabra L. is a medicinal plant valued for its bioactive triterpenoids and flavonoids. This study aimed to enhance the production of both in hairy root cultures via precursor feeding and elicitation. Hairy roots were induced using wild-type Rhizobium rhizogenes A4 and a transgenic R. tumefaciens strain carrying the squalene synthase ( AtSQS1 ) gene. Peak biomass occurred in the third week, prompting selection of superior wild-type and transgenic lines for further study. These were treated with squalene or pyruvic acid (0–10 mM) on day 14 (harvest: day 21), or with methyl jasmonate (0–200 µM) or coronatine (0–5.0 µM) on day 19 (harvest: 24–72 h). Optimal precursor concentrations (squalene: 5.0 mM; pyruvic acid: 7.5 mM) significantly boosted metabolite levels. Coronatine (1.0 μM, 48 h) was most effective elicitor, yielding peak biomass (9.22 ± 0.20 g FW per flask) and highest glycyrrhizic acid (49.19 ± 1.01 mg/g DW) and glabridin (4.54 ± 0.02 mg/g DW). A synergistic combination of pyruvic acid (7.5 mM) followed by coronatine (1.0 μM for 48 h) yielded the most profound enhancement, achieving the highest recorded levels of glycyrrhizic acid (56.12 ± 0.96 mg/g DW) and glabridin (5.78 ± 0.02 mg/g DW). The combined treatment strongly upregulated key biosynthetic genes including LUS (5.17-fold), CYP88D6 (5.00-fold), bAS (4.58-fold), and SQS1 (4.11-fold), demonstrating a clear gene-metabolite correlation. These findings demonstrate that sequential application of metabolic precursors and coronatine elicitation is a powerful biotechnological strategy for significantly enhancing the production of pharmaceutically important compounds in G. glabra hairy root cultures. • Metabolite production and gene expression in licorice hairy root culture was studied. • Peak biomass was observed in both wild-type and transgenic lines in week three. • Coronatine elicitation was more effective than methyl jasmonate. • Combined precursor–elicitor strategy maximized metabolite and gene expression. • Strong correlation observed between key gene expression and metabolite accumulation.