This research was aimed at determining the effects of multi-walled carbon nanotubes (MWCNTs) on seed germination, seedling growth parameters and secondary metabolite (SM) production of Thymus daenensis in vitro. Seeds were aseptically cultured in Murashige and Skoog medium (MS) with various concentrations of MWCNTs (0, 125, 250, 500, 1000 and 2000 mg ml1). Seed germination and morphological changes in seedlings were measured. The measurements were aimed at quantifying the total phenolic contents (TPC) and flavonoids (TFC), antioxidant activities and the activity of polyphenol oxidase (PPO), L-phenylalanine ammonia-lyase (PAL), dehydrogenase (DHA) and peroxidase enzyme (POD) of the seedling extract. Seedling biomass and seedling height grew significantly as the MWCNTs level increased. The biomass and height peaked at 250 mg ml1 (0.41 ± 0.01 gr FW, 5.99 ± 0.55 cm) and then rapidly decreased to 0.040 ± 0.1 gr FWand 1.42 ± 0.24 cm in response to 1000 mg ml1, 30 days after the treatment. Additionally, SM and the analyses of enzyme activity revealed that the highest amounts of TPC (6.70 ± 0.06 mg GAE g1 DW), TFC (8.19 ± 0.01 mg QUE g1 DW), antioxidant activities (73.88 ± 0.47%) and maximum PAL activity (1.25 ± 0.08 mM cm g1 FW) were detected in plants grown on MS media fortified with 250 mg ml1 MWCNTs. The results reveal that MWCNTs in low doses (250 mg ml1) can encourage the production of biomass, elicit more SM from seedlings and enhance the biosynthesis of antioxidants. TEM images showed that MWCNTs could cross the plant cell wall and enter the cellular cytoplasm.
