The Effect of the Type and Concentration of Some Elicitors on the Biochemical Characteristics and Production of the Secondary Metabolites in the Callus Culture of Moringa oleifera L.

The Moringa (Moringa oleifera L.) is a medicinal plant with medicinal properties and many valuable metabolites including flavonoids. Due to the medicinal and therapeutic importance of this plant, this research was conducted with the aim of studying the effect of elicitors such as methyl jasmonate, salicylic acid, and phenylalanine on biochemical properties and the production of valuable secondary metabolites from its callus tissue in vitro. According to the results obtained, it was found that higher concentrations of salicylic acid and methyl jasmonate led to a significant increase in peroxidase enzyme activity in the treated calluses. The callus samples treated with methyl jasmonate and phenylalanine showed significantly higher catalase enzyme activity compared to the control treatment at most concentrations and treatment durations. The highest level of flavonoids (0.628 mg g-1) was related to the culture medium containing 200 mg L-1 methyl jasmonate for 96 h. Regarding the duration of treatment with plant growth stimulants, for most treatments, the amount of anthocyanin production increased with the growing duration of elicitors treatment. Based on the results, increasing the salicylic acid concentration from 50 to 100 and 200 mg L-1, particularly over a 48-h period, resulted in a rise in rutin production in the Moringa callus. On the other hand, the highest amount of quercetin (5.38 mg g-1) in the culture medium with 200 mg L-1 methyl jasmonate for 96 hours and the highest amount of kaempferol (3.45 mg g-1) in the culture medium containing 100 mg L-1 salicylic acid was observed for 96 h.

Moringa, Moringa oleifera L, is a member of the Moringaceae family (Mashamaite et al., 2022). Due to the high medicinal and food value of this plant, it is known as the tree of the century. All constituent parts of Moringa, encompassing its roots, stems, leaves, and seeds, bear both medicinal and dietary utility, serving as a pivotal reservoir of vitamins and minerals (Mohlala et al., 2023). The leaves, in particular, exhibit pivotal medicinal attributes, owing to the presence of notable bioactive compounds such as saponins, tannins, steroids, flavonoids, coumarins, quinones, phenolic compounds, and alkaloids, which are proficiently employed in cancer prevention and treatment endeavors (Kurtulbaş et al., 2022). Prominently, a comprehensive range of quercetins, rutin, kaempferol, gallic acid, moringin, and the extensive Niazimicin assemblage represents the principal assortment of compounds derived from the diverse organs of this botanical specimen (Syeda & Riazunnisa, 2020). By virtue of its potent antioxidant property, quercetin, abundantly discovered within the leaf and seed samples of M. oleifera, orchestrates metal chelation and free radical inhibition (Bhaskar et al., 2022). There are various methods to increase the production of secondary metabolites as valuable compounds in medicinal plants, which include the use of agents in cell and plant tissue culture in vitro. The induction of callus cells in M. oleifera plant is largely influenced by temperature, nutrients, pH, and the addition of ascorbic acid and plant growth regulators in the culture medium. Stress induction increases the production of plant secondary metabolites. Stimulants are divided into biological and non-biological categories (Arya et al., 2021). Among the widely used stimulating factors used in most tissue culture programs are fungal carbohydrates, yeast extract, methyl jasmonate, salicylic acid, and amino acids such as phenylalanine and Polyamines such as chitosan (Raj & Saudagar, 2019). Common plant hormones such as salicylic acid and jasmonic acid are key markers for the expression of genes involved in plant defense systems (Patel et al., 2020; Shafighi et al., 2022). Considering the unique properties of the M. oleifera plant and its importance in the pharmaceutical and food industries, as well as the importance of new methods of plant tissue culture to produce and increase the amount of plant secondary metabolites, the present research aimed at increasing the production of biochemical compounds and valuable secondary metabolites such as rutin, quercetin and kaempferol from the callus tissue obtained from the leaf explants of this plant, using methyl jasmonate, salicylic acid, and phenylalanine as stimulating agents in different concentrations and time durations.
 

Callus tissue samples of M. oleifera plant were obtained from leaf explants grown in an MS base medium containing 2 mg/L 2,4-D and 0.5 mg/L BAP (Riyathong et al., 2010). Plant growth stimulants (methyl-jasmonate, salicylic acid, and phenylalanine) in concentrations of zero (control), 50, 100, and 200 mg/l during time periods in an MS base culture medium containing 2 ml 2,4-D and 0.5 mg/liter of BAP were utilized. Biochemical compounds such as peroxidase (MacAdam et al., 1992), catalase (Chanes & Mahely, 1996), proline (Bates, 1973), flavonoid (Chang et al., 2002) and anthocyanin (Wagner, 1979) along with metabolites secondary substances such as rutin, quercetin, and kaempferol (Hurst et al., 1983) were measured in callus tissue samples collected after 24, 48, and 96 hours after applying the stimuli. The experiment was conducted in a completely randomized design with three replications. Data variance analysis and average data comparison using SPSS ver. 26 were done.

The results of variance analysis of the data demonstrated that the activity level of peroxidase and catalase enzymes, the amount of amino acid accumulation of proline, total flavonoid, anthocyanin, the amount of rutin, quercetin, and kaempferol in the callus samples of M. oleifera plant in vitro was significantly (p < 0.01) affected by the type of stimulus used, the duration of use, and the interaction between the type of stimulus and the duration of its use. The highest amount of peroxidase enzyme activity was related to the culture medium containing 100 or 200 mg/l of methyl jasmonate and 200 mg/l of phenylalanine for a period of 96 hours. According to the obtained results, by raising the concentration of plant growth stimulants (methyl jasmonate and salicylic acid) from 50 to 100 and 200 mg/liter in different periods of time, the peroxidase enzyme activity did not change significantly. On the other hand, the highest amount of peroxidase enzyme activity (455.886 protein-µmol H2O2 min-1 mg-1) was related to the culture medium containing 200 mg/liter of phenylalanine for a period of 96 hours. The obtained results are consistent with the results reported by Shabani et al., (2009). The highest amount of proline amino acid accumulation (2.19 μg/mg) was observed in the treatment of callus samples with 200 mg/L of methyl-jasmonate for a period of 96 hours. In regards to the temporal extent of utilizing stimulants in any and all culture mediums that encompass methyl jasmonate, phenylalanine, or salicylic acid, the quantity of proline accumulation detected in callus obtained subsequent to a treatment period of 96 hours is notably greater. This phenomenon becomes apparent within the time frames of 24 and 48 hours. The highest amount of flavonoid (0.628 mg/g of callus) was observed in the treatment of 200 mg/l of methyl jasmonate for 96 hours. Zhang et al. (2009) stated that the flavonoid content of blackberry callus tissue increases remarkably in the treatment with methyl jasmonate and salicylic acid, which is consistent with the results obtained in this study. Also, the highest amount of anthocyanin (12.42 μmol/g of callus weight) was related to the treatment of 200 mg/l phenylalanine for 96 hours. According to the results obtained in this research, utilizing methyl jasmonate, phenylalanine, and salicylic acid as stimulants (especially in higher concentrations and time durations) significantly increased the amount of rutin production in callus tissue samples, which is consistent with the results obtained by Ishikawa et al. (2007). The highest amounts of rutin, quercetin, and kaempferol were respectively (12.86, 5.38 and 3.45 mg per gram of callus fresh weight) related to the culture medium containing 50 mg/liter salicylic acid for 48 hours, the culture medium containing 200 mg/liter methyl-jasmonate for 96 hours, and the culture medium containing 100 mg/liter salicylic acid for 96 hours. 

Recent advances in various fields of biotechnology have made it possible to revive new methods of plant tissue culture and produce valuable compounds at a commercial level. The employment of plant biotechnological techniques, particularly the generation of plant compounds in a controlled environment, presents the potential to manufacture items possessing therapeutic properties, irrespective of the variability in climate and seasons, thereby ensuring year-round availability. Today, many studies have been conducted and are being conducted on the medicinal and nutritional value of the Moringa plant (M. oleifera). Due to the high value of this plant in terms of medicine and treatment of diseases such as cancer in the traditional medicine of different nations, suitable and alternative methods for producing its effective compounds are very important. The use of plant tissue culture and esters is an effective method to produce valuable compounds of this plant in vitro. According to the results obtained in this study, treating callus with higher concentrations (100 and 200 mg/L) of methyl jasmonate, salicylic acid, and phenylalanine increases the possibility of increasing the production of plant secondary metabolites (rutin, quercetin, and kaempferol) in this plant.