Unleashing the potential of carbon nanotubes for production of medicinal metabolites in Nigella sativa adventitious roots

Increased market demand for Nigella sativa products necessitates the implementation of strategies to enhance biomass production. In this study, adventitious root (AR) cultures were successfully established for the first time in Nigella sativa under the influence of carbon nanotubes (CNTs). During initial optimization experiments, alpha-Naphthaleneacetic acid (NAA) at 2.0 mg/l exhibited the highest growth parameters for leaf explants, resulting in 83% adventitious root (AR) induction frequency and 13.3 g/l fresh biomass. Application of CNTs at concentrations ranging from 5.0 to 20.0 mg/l significantly enhanced adventitious root growth on solid MS media. Wherein, treatment T4 (CNTs: 20.0 mg/l) showed remarkable results with 93% AR induction frequency and 15.5 g/l fresh biomass. Further investigations were carried out using shake flasks, where the concentration of CNTs and the duration of pre-treatment played a crucial role in morphogenetic potential and biomass accumulation. A 4-hour pre-treatment with CNTs at a concentration of 10.0 mg/l exhibited the highest growth proliferation, achieving 82% morphogenetic potential and 17 g/l fresh biomass. In the AR cultured in liquid media, the presence of CNTs also influenced the total phenolic and flavonoid content in adventitious roots, with 2-hour and 4-hour pre-treatments resulting in higher levels compared to the 8-hour pre-treatment. Particularly, the 4-hour pre-treatment with 25.0 mg/l CNTs demonstrated the highest total phenolic content (9.0 mg/gDW) and total flavonoid content (7.0 mg/gDW). DPPH antioxidant activity increased with higher CNT concentrations (ranging from 5.0 to 25.0 mg/l) and longer pre-treatment durations. Notably, the highest activity of 90% was observed in AR maintained at 25 mg/l CNTs after an 8-hour pre-treatment. Moreover, Phenyl alanine ammonia lyase (PAL) activity in AR exhibited a decrease with higher CNT concentrations and longer pre-treatment durations. Nonetheless, the application of CNTs led to an increased accumulation of essential oils in N. sativa AR culture. The CNT5 treatment showed elevated levels of alpha-thujene, beta-pinene, d-limonene, p-cymene, alpha-terpineol, carvone and beta-Elemene. Notably, this treatment also exhibited significant levels of thymoquinone (19.2%), thymol (6.4%) and carvacrol (2.3%). The findings of this study have significant implications for scaling up the industrial production of metabolite-enriched germplasm of Nigella sativa.