Enhancing in vitro production of bioactive compounds in callus cultures of Achillea fragrantissima (Forssk.) Sch. Bip. via utilization of ZnO nanoparticles

Nanoelicitors are substances, both biological and non-biological, that enhance secondary metabolites productivity in medicinal plants. Achillea fragrantissima (Forssk.) Sch. Bip. is a medicinal plant known for its healing properties, including parthenolide with anticancer effects and essential oils. This study aimed to evaluate how various levels of zinc oxide nanoparticles (ZnONPs) at 5, 10, 15, and 20 mg/L affect the expression of three key biosynthetic genes: chalcone synthase (CHS), phenylalanine ammonia lyase (PAL), and 1-deoxy-D-xylulose 5-phosphate. Callus induction was initially optimized using MS medium enriched with 0.5 mg/L BA and 1 mg/L 2,4-D. Following ZnONPs supplementation, secondary metabolite accumulation significantly increased, as estimated by high-performance liquid chromatography. The overall phenolic and flavonoid content increased significantly, with the highest levels observed at 15 mg/L ZnONPs (120.72 µg/mg DW), including notable increases in chlorogenic acid (16.70 µg/mg), rosmarinic acid (16.88 µg/mg), catechin (27.93 µg/mg), and naringenin (16.44 µg/mg). Gene expression analysis via qRT-PCR revealed a concentration-dependent increase in CHS, PAL, and DXR. At 15 mg/L ZnONPs, maximum gene expression was observed for CHS (4.75-fold), PAL (4.24-fold), and DXR (3.54-fold). The gene expression levels at 10 mg/L ZnONPs were significantly higher than the control (CHS: 3.92-fold; PAL: 3.24-fold; DXR: 3.84-fold). Conversely, gene expression decreased at 5 and 20 mg/L, indicating a threshold beyond which ZnONPs cause phytotoxic effects. These findings suggest that 15 mg/L ZnONPs is optimal for enhancing secondary metabolite biosynthesis and gene expression in A. fragrantissima, highlighting their potential as affordable and effective elicitors for boosting the production of therapeutically valuable compounds in vitro. Zinc oxide nanoparticles (15 mg/L) significantly enhanced secondary metabolites in Achillea fragrantissima callus culture by upregulating key biosynthesis genes (CHS, PAL, and DXR), confirming their role as nano-elicitors.