Cell and Tissue Journal

Abstract Aim: Oregano (Mentha longifolia) is an important medicinal plant from the mint family, which is widely used in food and pharmaceutical industries due to its active compounds. As an efficient approach, the use of biological elicitors (yeast, bacterial, and fungal agents) as well as non-biological elicitors (inactive enzymes, ultraviolet rays, heavy metal salts, polysaccharides, salicylic acid, jasmonic acid, and melatonin) can provide a way to increase the production of secondary metabolites in plant cell cultures. Given the importance of Oregano in the pharmaceutical and therapeutic industries, this study was carried out to assess melatonin's effect on the percentage of cell viability and the level of important bioactive compounds of oregano.  
Materials and methods: This study was performed in a factorial experiment with a completely randomized design and three replications in the lab. Melatonin (0, 100, and 200 μM based on the previous studies) was added to the MS culture medium containing 2,4-D (1 mg.L^-1). Then, 24, 48, and 72 h after treatment, the percentage of cell survival and the level of important oregano compounds were measured by tetrazolium and HPLC tests, respectively. In SAS software version 9.4, analysis of variance (ANOVA) was used to analyze the data and the LSD method to compare the means.
Results: Based on the results of the tetrazolium test, there was no significant difference in the percentage of cell viability in various concentrations of melatonin. However, the survival percentage of cells decreased with the passage of time, so that the lowest survival percentage was observed 72 h after melatonin treatment. Adding melatonin to the culture medium increased the production of secondary metabolites. At the concentration of 100 µM, the amount of menthol, menthone, pulegone, and 1,8-cineole increased by 77, 108, 46, and 80%, respectively, after 48 hours in contrast to the control. The increase in the level of menthol, menthone, pulegone, and 1,8-cineole compounds in the concentration of 200 µM of melatonin was respectively 125, 130, 140, and 120%, after 48 hours when compared to the control. Therefore, increasing melatonin concentration from 100 to 200 µM in cell culture significantly increased the level of compounds compared to the control. The levels of menthol, menthone, pulegone, and 1,8-cineole increased up to 48 hours after treatment, but after that time, they showed a significant decrease. Since the ultimate goal is to increase the production of biologically active compounds and not the highest cell viability, therefore, 48 hours after applying the treatment of 200 μM melatonin provides the best conditions for obtaining a high level of valuable oregano metabolites. The above treatment increased menthol, menthone, pulegone, and 1,8-cineole by 125, 130, 140, and 120%, respectively.
Conclusion: In this study, for the first time, the effect of melatonin elicitor on important secondary metabolites of the Oregano plant was investigated to determine the potential of using this metabolic stimulant in research and commercial areas. Overall, The use of melatonin at a concentration of 200 μM along with the extraction of metabolites 48 hours after the treatment can enhance menthol, menthone, pulegone, and 1,8-cineol, while maintaining the viability of oregano cells

Abstract Plants produce a big group of secondary metabolites which are used as medicinal compounds. According to recent estimates, global market value of herbal medicines, including medicinal plants and their products, significantly has been increasing. Considering to the fact that most of the world market for medicinal plants, production and supply of secondary metabolites derived from these plants are concerned and the plant secondary metabolites are of high economic value. Chemical synthesis of these metabolites is an expensive process. So production of metabolites by different biotechnological methods such as cell culture is a useful alternative. Molecular recognition and elicitor-plant receptors interaction is a complex process requiring for signal transduction. Biotic elicitors induce secondary metabolites and hypersensitive responses by activation of defense mechanisms. Manipulation of cell culture media by elicitors is an important strategy for inducing secondary metabolism and production of valuable metabolites. Molecular recognition and elicitor-plant receptors interaction is a complex process requiring for signal transduction. Following perception of elicitor signals, rapid defense responses can be organized as follows: increase of ionic currents across the plasma membrane, reactive oxygen species (ROS) production, activation of defense gene expression, structural changes in the cell wall and phytoalexin production. In this study, different aspects of increasing the production of secondary metabolites in cell culture of plants by biotic elicitors is investigated.