The effect of fungal cellulase elicitor on antioxidant activity and enzymes in licorice cell suspension culture (Glycyrrhiza glabra L.)

Document Type : Research Paper

Authors

1 Department of Plant Breeding and Biotechnology. Agriculture Faculty. University of Zabol. Zabol. Iran

2 Department of Agronomy. Faculty of Agriculture. University of Zabol. Zabol. Iran

10.22092/ijrfpbgr.2024.363348.1447

Abstract

Background and objectives: 
Licorice (Glycyrrhiza glabra) is a perennial plant from the Fabaceae family rich in bioactive secondary metabolites used to treat many human diseases. Recent advances in plant tissue culture techniques have shown promising results in improving productivity and allowing the gradual replacement of whole plant culture as a source of valuable secondary metabolites. Today, various tissue culture methods are used to increase the performance of secondary metabolites by strengthening plant defense and stimulating stress response in plant cells with the help of elicitors. Plant pathogenic fungi produce cellulase, which is used as an elicitor to stimulate the production of secondary metabolites in plant cell suspension culture conditions. This research aimed to study the effect of cellulase enzyme obtained from Aspergillus nigar fungus on the amount of antioxidant activity (DPPH method) and antioxidant enzyme activity in licorice cell suspension culture conditions. 
Methodology: 
Licorice seeds were collected from the Semirom region, Isfahan province, Iran. Seeds were cultured in the ½ MS medium after disinfection. In order to induce callus, the hypocotyl and cotyledon were cultured in MMS medium containing growth regulators (2 mg/L BA and 0.5 mg/L NAA) with 3% sucrose and 0.7% agar. Then 0.5 g of fibrous callus produced under a sterile hood was transferred to Erlenmeyer flasks containing 50 cc of MMS liquid culture medium containing 2 mg/liter of BA hormone and 0.5 mg/liter of NAA hormone and was placed in a shaker incubator with a speed of 150 rpm, a temperature of 25oC and darkness. On the 19th day after cultivation, cellulase enzyme with a 200 μg/ml concentration was added to each Erlens under a sterile hood.
 Then, harvesting was done at zero-time intervals (control) 24, 48 and 72 hours after adding the elicitor. Late cultured calli were also considered as a treatment. Statistical analysis was carried out using a completely randomized design with three replications. Data were collected for DPPH and all antioxidant enzyme activities such as catalase, superoxide dismutase, polyphenol oxidase, ascorbate peroxidase and guaiacol peroxidase. A regression analysis was used to determine the relationship between DPPH and antioxidant enzyme activities at the time interval after adding the elicitor. 
Results: 
The effect of cellulase enzyme on all traits except superoxide dismutase was significant (P<0.01). The activity of antioxidant enzymes and antioxidant activity (DPPH method) within 72 hours of applying treatment showed the most significant increase compared to the control. Correlation between traits showed that catalase enzyme had a positive and significant correlation with polyphenol oxidase and guaiacol peroxidase enzymes. Similarly, the guaiacol peroxidase enzyme positively correlated with the polyphenol oxidase enzyme (P<0.01). Antioxidant activity (DPPH method) also showed a positive and significant correlation with catalase, polyphenol oxidase and catalase enzymes. The result of simple regression analysis using DPPH and all antioxidant enzyme activities as dependent variables and the time interval after adding the elicitor as independent variables showed that all DPPH and all enzyme activities (except superoxide dismutase and ascorbate peroxidase) had increased by 72 hours after adding the elicitor.
Conclusion:
 The use of cellulase enzyme derived from Aspergillus nigar fungus in the conditions of cell suspension culture increases the antioxidant activity (DPPH method) and the activity of oxidant enzymes. Therefore, it was concluded that the cellulase enzyme caused cell stress in the conditions of cell suspension, and this stress caused the stimulation of the plant's defense mechanisms, such as antioxidant enzymes. A positive and significant correlation was observed between the antioxidant activity and some antioxidant enzymes, such as polyphenol oxidase and catalase, which indicates the positive effect of the fungal elicitor in stimulating the production of secondary metabolites and increasing the potential of inhibiting free radicals in licorice plant cell suspension culture conditions. The DPPH and all enzyme activities (except superoxide dismutase and Ascorbate Peroxidase) were increased 72 hours after adding the elicitor.

Keywords

Main Subjects

References

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