Investigation on the Effects of Carbon Nanotubes and Kinetin on Growth and Regeneration of Salvia nemorosa under in vitro Conditions

Introduction The use of medicinal plants has surged in recent times, with a substantial portion of modern medicines derived from botanical sources. This surge in demand underscores the potential of cultivating and producing medicinal plants to not only bolster public health but also significantly contribute to a society's economic well-being.Salvia nemorosa is an herbaceous, perennial, and medicinal plant. This valuable plant belongs to the Labiatae family. The aerial parts of the plant, especially the leaves, contain essential oils. Furthermore, S. nemorosa contains chemical compositions such as saponin, organic acids, diterpenes, triterpenes, polyphenols, and a bitter substance called picrosalvin. In the plant tissue culture, the preparation of culture medium and formulation compatible with the tissue of the tested plant is vital for efficient regeneration and plant growth. Generally, no specific culture medium can be recommended for the growth of explants, and is necessary to make changes in the culture medium in order to better respond to different types of explants. Carbon nanotubes are nanomaterials that can be added to the culture medium and increase the growth rate of different parts of the plant such as roots, stems, and branches. Cytokines also commonly participate in cell divisions and proliferate shoots by removing terminal dominance. Considering the mentioned importance, this study was conducted to investigate the effect of carbon nanotubes, kinetin, and their interactions on some morphophysiological and phytochemical properties of S. nemorosa under in vitro conditions.   Materials and Methods This experiment was carried out in the tissue culture and biotechnology laboratory of the Department of Horticultural Sciences, Faculty of Agriculture, University of Mohaghegh Ardabili. This study was conducted based on factorial design (completely randomized design) with five replications. The treatments included five levels of carbon nanotubes (0, 10, 20, 40, and 80 mg/L CNT) and three levels of kinetin (0, 2, and 4 mg/L CK). The seeds of S. nemorosa were sterilized with 70% ethanol for 40 seconds and then 2.5% hypochlorite sodium for 15 minutes (then the seed were washed with deionized water three times 3, 5, and 15 minutes). The sterilized seeds were planted in MS medium containing 30 g/L sucrose and 8 g/L agar, and then transferred to a growth chamber. After 40 days, the obtained seedlings were cut into single nodes by removing leaves and transferred to the main treatment medium. After that, some traits such as fresh weight, number of branches, number of leaves, number of roots, leaf area, root length, seedlings height, viability rate, germination rate, chlorophylls a, b, carotenoid content, phenol content, and flavonoid content were examined. The obtained data related to the experiment were analyzed with SAS software, the comparison of data means was done with Duncan's test, and the graphs were plotted using Excel software. Results and Discussion The results indicated that the interaction effect of carbon nanotubes and kinetin on the indices of fresh weight, number of branches, number of leaves, number of roots, leaf area, root length, seedling height, survival percentage, germination percentage, pigment content photosynthesis was not significant. On the contrary, the interaction effect of two treatments on the content of total flavonoid and total phenol showed a significant difference. The highest phenol content was obtained in the treatment combination of 80 mg/L of carbon nanotubes and 4 mg/L of kinetin. On the other hand, increasing the concentration of carbon nanotubes up to 80 mg/L, the average shoot production, number of leaves, leaf area, plant height, root number, root length, chlorophyll a, b, carotenoid, phenol and flavonoid content. increased significantly. Also, by increasing the concentration of kinetin (4 mg/liter), the number of branches, the content of chlorophyll a and b increased significantly. Conclusion The characteristics of the S. nemorosa plant, except for fresh weight, significantly increased under the influence of carbon nanotube treatments. However, under the influence of kinetin treatment, only the number of branches, chlorophyll a, b, phenol content, and flavonoid content showed a significant increase. Based on the results of this study, carbon nanotubes can be used for proliferation and increasing the secondary metabolites of S. nemorosa. Despite the results of this study, it is still possible to use higher concentrations of carbon nanotubes in future research in order to increase the phytochemical properties and productivity of other medicinal plants