Response of <i>Melissa officinalis</i> subsp. <i>officinalis</i> seedlings to Fe ₃ O ₄ ‐ <scp>NPs</scp> under <i>in vitro</i> conditions: physiological, biochemical and molecular analyses

Application of iron oxide nanoparticles (NP) (Fe₃O₄-NPs) in plant biotechnology presents new opportunities for enhancing metabolic activity of medicinal plants; however, their specific effects on Melissa officinalis subsp. officinalis remain poorly understood. This study examined effects of Fe₃O₄-NPs at 0, 25, 50, 75, and 100 mg L^-1 on morphological traits, phenolic compound accumulation, antioxidant activity, enzyme inhibition, and expression of PAL, TAT and RAS genes under in vitro conditions. Seeds were germinated on Murashige & Skoog medium and cultured for 30 days. Morphological characteristics were measured, total phenolics and flavonoid content were quantified spectrophotometrically, and phenolic profiles determined via HPLC. Antioxidant activity (CUPRAC, DPPH, ABTS), enzyme inhibition (AChE, MAO-A, urease), and gene expression (qRT-PCR) were also assessed. Treatment at 25 mg L^-1 yielded the highest content of total phenolics (41.68 mg GAEg^-1 plant) and rosmarinic acid (22.10 μg mg^-1 DW), together with improved antioxidant, MAO-A (2.95 mg plant mL^-1) and urease (6.71 mg plant mL^-1) inhibition activity. Higher concentrations (75-100 mg L^-1) increased AChE inhibition but reduced antioxidant capacity. PAL, TAT, and RAS expression was upregulated in all treated groups: PAL peaked at 25 mg L^-1, RAS at 100 mg L^-1, and TAT at 75 mg L^-1. There was no direct correlation between gene expression and phenolic levels, suggesting involvement of post-transcriptional or alternative regulatory mechanisms. These results demonstrate that Fe₃O₄-NPs act as dose-dependent modulators of secondary metabolism and bioactivity in M. officinalis, offering promising tools for nanoparticle-based elicitation strategies in medicinal plant biotechnology.