Gold nanoparticle-mediated elicitation enhances andrographolide biosynthesis via auxin signalling in optimized hairy...

Andrographis paniculata, a medicinal plant valued for its diterpenoid andrographolide, is limited by low yields obtained through conventional cultivation and extraction methods. Developing efficient in vitro systems for enhanced andrographolide biosynthesis remains a significant challenge. This study establishes an optimized Rhizobium rhizogenes-mediated protocol for including hairy root cultures (HRCs) in A. paniculata and evaluates biogenically synthesized gold nanoparticles (AuNPs) as elicitors to enhance andrographolide biosynthesis and auxin signalling. Hairy root induction was achieved from two-week-old A. paniculata cotyledon explants using R. rhizogenes A4 strain, with an optimal transformation efficiency of 79.9% under 2-day co-cultivation with 200 µM acetosyringone, 40 s sonication, and 2 min vacuum infiltration. Polymerase chain reaction (PCR) amplification of the rolA gene confirmed the transgenic nature of the HRCs. Maximum biomass (23.39 g FW; 2.37 g DW) and andrographolide content (37.8 mg g⁻¹ DW) were achieved on the 40th day of culture. Elicitation with various concentrations of AuNPs (20, 40, 60, and 80 mg L⁻¹) for different exposure periods (24, 48, and 72 h) revealed that treatment with 40 mg L⁻¹ for 48 h significantly enhanced andrographolide accumulation (8.8-fold; 372.17 mg g⁻¹ DW), as quantified by high-performance liquid chromatography (HPLC). Real-time quantitative polymerase chain reaction (RT-qPCR) analysis showed a significant upregulation of auxin-responsive factors ARF1 (9.2-fold) and ARF3 (6.09-fold), indicating activation of auxin-mediated transcriptional regulation. Collectively, these findings suggest that AuNPs are effective abiotic elicitors and establish a robust, scalable HRC platform for enhanced andrographolide production via modulation of phytohormonal signalling. Furthermore, the optimized HRC protocol provides a foundation for future CRISPR/Cas9-based metabolic engineering to improve yield and pathway efficiency.