تاثیر نوع و غلظت عوامل محرک بر ویژگی‌های بیوشیمیایی و تولید متابولیت‌های ثانویۀ بافت کالوس گیاه مورینگا (moringa oleifera l.)

گیاه مورینگا (moringa oleifera l.)، گیاهی دارویی است که بسیاری از ترکیبات دارویی و متابولیت‌های ارزشمند ازجمله فلاونوئیدها را دارد. باتوجه‌به اهمیت دارویی و درمانی این گیاه، پژوهش حاضر با هدف بررسی تاثیر محرک‌هایی مانند متیل جاسمونات، سالیسیلیک‌اسید و فنیل‌آلانین بر ویژگی‌های بیوشیمیایی و تولید متابولیت‌های ثانویۀ بافت کالوس آن در شرایط درون‌شیشه انجام شد. نتایج نشان دادند غلظت‌های بیشتر سالیسیلیک اسید و متیل‌جاسمونات سبب افزایش معنادار میزان فعالیت آنزیم پراکسیداز در کالوس‌های تیمار‌شده می‌شود؛ همچنین کالوس‌های تیمارشده با متیل‌جاسمونات و فنیل‌آلانین در بیشتر غلظت‌ها و مدت زمان‌ها در مقایسه با تیمار شاهد به‌طور معنادار فعالیت آنزیم کاتالاز بیشتری نشان دادند. بیشترین مقدار فلاونوئید (628/0 میلی‌گرم‌بر‌گرم وزن کالوس) به محیط‌کشت حاوی 200 میلی‌گرم‌در‌لیتر متیل‌جاسمونات برای زمان 96 ساعت تعلق داشت. از نظر مدت زمان تیمار، در بیشتر تیمارها میزان تولید آنتوسیانین با افزایش مدت زمان استفاده از محرک‌ها افزایش یافت. بر اساس نتایج، افزایش غلظت سالیسیلیک‌اسید از 50 به 100 و 200 میلی‌گرم‌در‌لیتر به‌ویژه برای مدت زمان 48 ساعت سبب افزایش میزان تولید روتین در کالوس‌های گیاه مورینگا شد. بیشترین میزان کوئرستین (38/5 میلی‌گرم‌بر‌گرم وزن تر کالوس) در محیط‌کشت حاوی 200 میلی‌گرم‌در‌لیتر متیل‌جاسمونات به‌مدت 96 ساعت و بیشترین میزان کمپفرول (45/3 میلی‌گرم‌در‌گرم وزن تر کالوس) در محیط‌کشت حاوی 100 میلی‌گرم‌در‌لیتر سالیسیلیک‌اسید به‌مدت 96 ساعت مشاهده شد.

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.introductionmoringa, 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. materials and methodscallus 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.results and discussionthe 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