Effect of elicitor on some biochemical traits of Withania coagulans (Stocks) Dun. in cell suspension culture conditions

Medicinal plants produce bioactive compounds with high antioxidant value. Due to the importance of Withania coagulans</em> in the pharmaceutical industry and also due to the cutting of this shrub species and its use in the pharmaceutical industry using traditional methods, the risk of extinction of this valuable species will increase, so we should look for the method - to be alternatives to increase the production of secondary metabolites of this plant. Using elicitors as a technique in vitro increases plant production of effective compounds (secondary metabolites). It has been reported that fungal stimulants are also used to induce the production or increase of secondary metabolites in medicinal plants. Therefore, in this study, the effect of cellulase elicitor on the growth index and biochemical and physiological traits of Withania coagulans</em> (Stocks) Dunal) was investigated in cell suspension culture.

Seeds of Withania coagulans</em> were collected from the Mehrestan region of Saravan city, and after disinfection, they were cultivated in ½ MS medium to produce explant. Production explants were cultured in an MS culture medium containing auxin and cytokinin hormones (2mg/l 2, 4, D, and 0.5 mg/l Kinetin) to produce callus. Three sub-cultures of production called into MS culture medium containing transfer previous regulating hormones were done. Investigated treatments include six elicitor treatments: 1: Control 2: Elicitor with a concentration of 200 μg/ml and 24 hours of exposure to the Elicitor, 3: Elicitor with a concentration of 200 μg/ml and 48 hours of exposure to the Elicitor, 4: Elicitor with a concentration of 200 μg/ml and 72 hours of exposure Elicitor, 5: Elicitor with a concentration of 7.5 μg/ml and 14 days of exposure to the Elicitor and 6: Elicitor with a concentration of 10 μg/ml and 14 days of exposure to the cellulase elicitor in a completely randomized design with three replications in the central laboratory of the university of Zabol was applied. Investigated traits were: callus growth index, antioxidant activity, proline content, soluble carbohydrates, malondialdehyde, total alkaloid, and activity of antioxidant enzymes (catalase, ascorbate peroxidase, guaiacol peroxidase, superoxide dismutase, and polyphenol oxidase).

The effect of different cellulase treatments on all traits was significant except polyphenol oxidase enzyme activity. In general, different elicitor treatments improved all the measured traits except the content of total carbohydrates. However, the increase in the trait depended on the concentration and period of exposure to the Elicitor. So, in most of the measured traits, the elicitor treatment with a concentration of 10 micrograms/ml and a period of 14 days caused an increase in all treatments. All cellulase elicitor treatments increased the antioxidant activity of Withania coagulans</em> cells in cell suspension culture. The highest antioxidant activity was observed in the sixth and fifth treatments and the lowest in the second and first treatments (control). The addition of Elicitor in the early stage of cell growth in cell suspension culture (eighth day) despite the lower concentrations of Elicitor (7.5 and 10 μg/ml) leads to a further increase in callus growth, antioxidant activity, Proline, and total alkaloid increased then the addition of Elicitor in exponential phase, despite the high concentration of Elicitor (200 μg/ml). A higher concentration of elicitors causes a hypersensitive response, which leads to cell death; hence, an optimal Elicitor level is required to induce growth.

Cellulase elicitor improved the growth index and antioxidant potential of Withania coagulans </em>through physiological and biochemical traits. According to the obtained results, the use of cellulase elicitor can be considered an important strategy to increase the growth, effective compounds, and antioxidant properties of Withania coagulans</em> for commercial production.