Establishment of Hairy Root Culture System in Atractylodes chinensis for Enhanced Production of Medicinal Sesquiterpenoids

Atractylodes chinensis (DC.) Koidz., a pharmacologically significant medicinal plant with various metabolites, produces sesquiterpenoids like atractylodin and β -eudesmol that dictate its medicinal quality and underpin its clinical efficacy. While hairy root systems offer industrial potential for metabolite production, no such system existed for A. chinensis . Here, we report the successful establishment of an efficient hairy root induction system for A. chinensis using Agrobacterium rhizogenes ATCC15834 with leaf and petiole explants, achieving higher induction rates than strains R1000 30 and LBA9402. Optimal growth occurred on 1/2 MS solid medium supplemented with 0.5 mg·L-1 IAA, 0.1 mg·L-1 KT, and 5% sucrose, yielding maximal biomass and metabolite accumulation over a 52-day culture period. Among the established hairy root lines, those induced from petioles (particularly line P-6) exhibited superior biomass and significantly higher levels of key metabolites, including atractylodin, β -eudesmol, total polyphenolic acids, and total flavonoids at day 52, compared to the leaf-induced line (L-1). Transcriptomic analysis confirmed the key genes involved in β -eudesmol and atractylodin biosynthesis upregulated relative to wild plants. Furthermore, treatment of the hairy roots with melatonin (MT) or its chemical homologs (5- methoxyindole, 5-MI; 5-methoxytryptamine, 5-MT) demonstrated that only 0.5 mmol·L⁻¹ 5-MI significantly enhanced β -eudesmol production. Remarkably, the β -eudesmol content achieved at 168 hours post-treatment was 1.82-fold higher than that in wild 3-year-old A. chinensis roots. This study establishes a robust platform for A. chinensis hairy root culture and highlights 5-MI as a potent inducer, providing a valuable foundation for the industrial scale production of its medicinally active compounds.