The medicinally effective plant Achillea fragrantissima exhibits a magnitude of pharmacological activities. In this study, the effects of different ZnONP concentrations on antioxidant enzymes, bioactive secondary metabolites, redox potential, and molecular changes in A. fragrantissima callus cultures were investigated. First, the concentrations of the growth regulators 2,4-D and BA were optimized using Murashige and Skoog (MS) medium. The MS medium was then administered with 2,4-D and BA at its optimal dosage (1.0 mg.L⁻¹); afterward, different ZnONP supplements (0.0, 5.0, 10.0, 15.0, and 20.0 mg.L⁻¹) were added. ZnONPs resulted in many physiological and molecular responses. ZnONPs significantly increased POD, APX, and SOD activities. While 10.0 mg.L⁻¹ ZnONPs significantly increased POD and APX activities, 15.0 mg.L⁻¹ ZnONPs significantly increased SOD. However, CAT activity gradually decreased with ZnONPs. Metabolically, ZnONPs increased phenolics, flavonoids, alkaloids, and saponin levels. Phenolic levels peaked at 20.0 mg.L⁻¹, flavonoids at 15.0 mg.L⁻¹, and alkaloids and saponins at 10.0 mg.L⁻¹. Terpenoids were more prevalent at lower levels of ZnONPs. With 15.0 and 10.0 mg.L⁻¹ giving the maximum activity, ZnONPs enhanced the DPPH activity and TAC of the callus culture extracts, respectively. RAPD and ISSR fingerprinting were applied using 12 random and ISSR primers to evaluate the genetic stability of ZnONP-induced callus cultures. Six RAPD primers showed 83% polymorphism while the seven ISSR primers achieved 30% polymorphism. Consequently, DNA mutations may have been induced by ZnONPs and caused DNA fragments to either appear or disappear in RAPD and ISSR callus profiles. The dendrogram based on RAPD and ISSR combined data showed that by increasing ZnONP concentration the genetic differentiation among callus cultures was elevated. In conclusion, higher accumulation of secondary metabolites and redox activity were increased in A. fragrantissima callus cultures using low ZnONPs (10.0 mg.L⁻¹) concentration.

具有药用功效的植物蓍草表现出大量的药理活性。在本研究中，研究了不同浓度的ZnONP对香薰愈伤组织培养物中抗氧化酶、生物活性次生代谢物、氧化还原电位和分子变化的影响。首先，使用 Murashige 和 Skoog (MS) 培养基优化生长调节剂 2,4-D 和 BA 的浓度。然后向MS培养基中加入最佳剂量（1.0 mg.L ^-1）的2,4-D和BA；然后，添加不同的ZnONP补充剂（0.0、5.0、10.0、15.0和20.0mg.L ^{-1 ）。}ZnONPs 导致许多生理和分子反应。ZnONPs 显着增加 POD、APX 和 SOD 活性。10.0 mg.L ^-1 ZnONPs 显着增加 POD 和 APX 活性，15.0 mg.L ^-1 ZnONPs 显着增加 SOD。然而，CAT 活性随着 ZnONPs 的增加而逐渐降低。在代谢方面，ZnONPs 增加了酚类、类黄酮、生物碱和皂苷的水平。酚类含量峰值为20.0 mg.L ^-1，类黄酮含量为15.0 mg.L ^-1，生物碱和皂苷含量为10.0 mg.L ^-1。ZnONP 含量较低时，萜类化合物更为普遍。ZnONPs在15.0和10.0mg.L ^-1时具有最大活性，分别增强了愈伤组织培养物提取物的DPPH活性和TAC。使用 12 个随机引物和 ISSR 引物应用 RAPD 和 ISSR 指纹分析来评估 ZnONP 诱导的愈伤组织培养物的遗传稳定性。6 个 RAPD 引物显示出 83% 的多态性，而 7 个 ISSR 引物则显示出 30% 的多态性。因此，DNA 突变可能是由 ZnONP 诱导的，并导致 DNA 片段在 RAPD 和 ISSR 愈伤组织谱中出现或消失。基于 RAPD 和 ISSR 组合数据的树状图表明，通过增加 ZnONP 浓度，愈伤组织培养物之间的遗传分化得到提高。总之，使用低浓度 ZnONPs (10.0 mg.L ^-1 )的香薰愈伤组织培养物中次生代谢物的积累和氧化还原活性增加。