Carbon nanotubes (CNTs) as novel promising nanomaterials have attracted considerable interest with excellent potential to boost the production of bioactive substance in plants. In the present study, we assessed morphological, physiological and phytochemical properties of Thymus daenensis callus culture upon exposure to various concentrations of multi-walled CNTs (MWCNTs) in vitro. Aseptic explants were cultured on 2, 4- dichlorophenoxy acetic acid (2, 4-D) and benzyl amino purine (BAP) containing Murashige and Skoog (MS) media. Results showed that callus exposure to MWCNTs may stimulate cells to absorb more water, grow faster and ultimately assist T. daenensis callus in accumulating higher fresh and dry biomass (maximum 100 mg ml1 ). The application of MWCNTs resulted in higher values of antioxidant capacity, total flavonoids and phenolic compounds. High-Performance Liquid Chromatography (HPLC) analysis revealed that MWCNTs at specific concentration stimulate rosmarinic acid (25.25 mg g1 DW), ferulic acid (1.97 mg g1 DW), caffeic acid (1.02 mg g1 DW), catechin (1.99 mg g1 DW), vanilin (0.91 mg g1 DW) and hesperedin (2.83 mg g1 DW) biosynthesis. This increase followed a dose-dependent manner and was associated with changes in phenylalanine ammonia lyase enzyme activity (PAL). Maximum levels of antioxidant enzymes were obtained in callus grown on MS media supplemented with 500 and 1000 mg ml1 of MWCNTs. Transmission electron microscopy (TEM) and optical microscopy micrographs revealed that MWCNTs entered the cell wall, cytoplasm and chloroplast, thereby increasing the cell size through mechanisms of action. The results revealed that MWCNTs can cross cell walls, enter the cytoplasm and other cellular organelles and facilitate water absorption. More biomass production and antioxidant biosynthesis were associated with higher levels of secondary metabolite production. Overall, the results indicated that MWCNTs can be used as a valuable elicitor in vitro for producing the highest economic values of rosmarinic acid, ferulic acid, caffeic acid, catechin, vanilin and hesperedin for pharmaceutical, therapeutic and food purposes.
