The impact of ascorbic acid and salicylic acid elicitors on secondary metabolites production in callus of Hashemi and Shiroodi rice cultivars

Background and objectives: The rice plant belongs to the Poaceae family and is known by the scientific name Oryza sativa. This plant is one of the important cereals and is the main food of half of the world, especially in Asia. Rice is a herbaceous, annual plant for areas with hot climate. Rice contains a variety of components such as carbohydrates, fats, protein, fiber and mineral element which the amount of these compounds is different in each cultivar. It has been show that some powerful antioxidants including Ƴ-Oryzanol, tocopherols, tocotrinols and phenolic compounds have anti-aging effects. The purpose of current research was to investigate the possible effect of ascorbic acid and salicylic acid elicitors on secondary metabolites production, antioxidant potential and other biological activities of the extract obtained from callus cultures of two cultivars of Shirodi and Hashemi rice on Murashig and Skoog (MS) medium culture. Shiroodi is a high-yielding cultivar with medium seed quality, and Hashemi cultivar is a low-yielding cultivar with high seed quality.

Material and Methods: The current experiment was conducted at the Plant Physiology Laboratory of Golestan University in 2023. In order to produce callus, we used seed as an explant to induce callus. For this purpose, the peeled seeds of the target cultivars were placed on MS medium culture containing growth regulators 2, 4-D (20 mg/l), BAP (10 mg/l) and NAA (10 mg/l). In different treatments, ascorbic acid and salicylic acid were added to the culture medium at a concentration of 70 mg/l. The treatments included growth regulators without ascorbic acid and salicylic acid (control treatment), growth regulators with ascorbic acid, growth regulators with salicylic acid, and growth regulators with both elicitors. After six weeks, callus was collected and different growth and biochemical parameters were evaluated. The experiment was done in the form of completely randomized design and the whole measurements were done in four replications.

Results: The obtained data showed that the application of both elicitors, ascorbic acid and salicylic acid, caused a significant increase in the amount of fresh and dry weight in Hashemi cultivar, compared to Shiroudi cultivar, and the highest amount of callus formation was also observed in this treatment. Also, the presence of these elicitors caused a significant increase in the production of secondary metabolites and antioxidant properties. Accordingly, significant differences were observed in total phenol, total flavonoid, total anthocyanin, activity of PPO, POD, SOD enzymes, total protein content, soluble sugar, and photosynthetic pigments in Hashemi compared to Shiroudi cultivar. The highest antioxidant activity based on DPPH free radical scavenging percentage was also observed in the group treated by both ascorbic acid and salicylic acid.

Conclusion: The current data revealed that the application of combined elicitors, ascorbic acid and salicylic acid (70 mg/L), significantly increased biomass, secondary metabolites content and antioxidant activity in the both Hashemi and Shiroodi rice cultivars. However, Hashemi cultivar showed greater enhancement compared to shiroodi cultivar. It seems these elicitors increase secondary metabolites production by activating of biosynthetic pathways and consequently enhance the antioxidant potential of rice callus.