Thymus species are aromatic plants with diverse applications in food industries and medicine. This study was conducted to evaluate the potential effect of ZnO nanoparticles (NPs) on callus proliferation and thymol and carvacrol production in three Thymus species, that is, T. vulgaris, T. daenensis, and T. kotschyanus, and Zataria multiflora. For this purpose, callus induction was performed on Murashige and Skoog (MS) medium containing different plant growth regulators (PGRs). After optimization of callus growth, the effects of different concentrations of ZnO NPs (100 and 150 mg L −1 ) were investigated. MS containing 2 mg L −1 of 2, 4-dichlorophenoxy acetic acid (2,4-D) and 1 mg L −1 of kinetin (Kin) revealed significantly highest fresh weight (0.18 g) of callus in T.kotschyanus. Callus growth rate (0.079 mm day −1 ) was found highest in T. vulgaris under similar conditions. Moreover, highest callus induction (92.50%) was achieved by T. kotschyanus in MS containing 2.5 mg L −1 of 2,4-D. Regarding the highest content of thymol (22.8 mg L −1 ) and carvacrol (0.68 mg L −1 ) evaluated by high-performance liquid chromatography, best results were achieved under 150 mg L −1 of ZnO NPs in T. kotschyanus and T. daenesis, respectively. This is simple and cost-effective method to be applied on industrial level for production of enhanced secondary metabolites content. potent chemical elicitors as signaling agents that effect a great diversity of physiological and biochemical processes in plant metabolism and plant secondary metabolites production [1,2]. NPs are capable of increasing cell membrane permeability, stimulating defensive mechanisms in plant cells, and subsequent increase in secondary metabolites content [2,3]. Zinc oxide (ZnO) NPs as an inorganic micronutrient have a beneficial effect on plant growth, cell division, and structure and function of membrane because of their high electron mobility, large surface area, and catalytic activity [4]. Most of the recent studies are focused on the effects of NPs on seed germination [5], plant growth rate [6] physiological responses [3][4][5][6], and secondary metabolites production [2][3][4][5]. Recent studies have shown that plant growth, development, and physiology are significantly affected by NPs; however, their role on plant secondary metabolism, especially regarding callus culture, is still obscure [2]. The inclusion of NPs to tissue culture media could enhance
