Impact of Copper Oxide Nanoparticles on Adventitious Shoot Regeneration, Axillary Shoot Multiplication, Rooting, and Bioactive Compounds in Ajuga multiflora Bunge

The present study investigated the effects of copper oxide nanoparticles (CuO NPs) at concentrations of 0, 5, 10, 20, and 40 mg/L on micropropagation and the accumulation of lipophilic metabolites in <i>Ajuga multiflora</i>, a medicinally valuable ornamental species. The highest number of adventitious shoots (29.4 shoots per explant) was obtained on the shoot induction medium with 5 mg/L CuO NPs. Shoot production gradually decreased at higher CuO NPs concentrations, falling to just 1.1 shoots per explant at 40 mg/L CuO NPs. A similar pattern was seen in axillary shoot multiplication (22.4 shoots per explant at 5 mg/L CuO NPs). However, the maximum shoot fresh weight (0.269 g) was reached on the shoot multiplication medium containing 10 mg/L CuO NPs. Root induction was most effective at 5-10 mg/L CuO NPs, while higher concentrations (20 or 40 mg/L CuO NPs) suppressed or inhibited root formation and altered plantlet morphology. Notably, this study is among the first to assess CuO NPs' effects across multiple regeneration stages rather than focusing on just one morphogenic event. This emphasizes the importance of optimizing the dose not only for initial shoot induction but also for later multiplication and rooting, ensuring effective micropropagation. Metabolite analysis showed that both the type of organ (microshoots vs. leaves) and CuO NPs concentration significantly affected the levels of α-tocopherol, carotenoids, sterols, and fatty acids. Leaves had higher amounts of α-tocopherol and total carotenoids compared to microshoots. The phytosterol levels also varied, with leaves containing more 22-dehydroclerosterol and total phytosterols, while microshoots had more clerosterol. Treatment with 5 mg/L CuO NPs increased phytosterol accumulation in both organs. CuO NPs significantly influenced the fatty acid profiles. In microshoots, total polyunsaturated fatty acids (PUFAs) increased and total saturated fatty acids (SFAs) decreased with higher CuO NPs levels. Conversely, in leaves, higher CuO NPs concentrations led to increased SFAs and decreased PUFAs, along with a significant rise in the omega-6 (n-6)/n-3 PUFAs ratio. These findings suggest that controlled application of CuO NPs can serve as an elicitor to boost phytochemical production during micropropagation.