Univrsity of Tehran Press Iranian Journal of Field Crop Science 2008-4811 56 4 2025 12 22 Methyl Jasmonate and Chitosan Enhanced the Antioxidant Content of Calotropis procera Hairy Root Methyl Jasmonate and Chitosan Enhanced the Antioxidant Content of Calotropis procera Hairy Root 105 120 105589 10.22059/ijfcs.2025.390829.655131 FA Fateme AliAskari Department of Agronomy and Plant Breeding, Faculty of Agriculture, and Natural Resources University of Tehran. karaj. Iran. Manizheh Sabokdast Department of Agronomy and Plant Breeding Faculty of Agriculture and Natural Resorces University of Tehran. karaj. Iran. 0000-0002-0403-2208 Mohammad Reza Naghavi Department of Agronomy and Plant Breeding Faculty of Agriculture and Natural Resources, University of Tehran. karaj. Iran. Journal Article 2025 05 08 Introduction. Calotropis procera, a prominent medicinal plant native to the dry regions of Asia and Africa, is widely recognized for its medicinal properties. The plant is a rich source of bioactive compounds, particularly flavonoids, phenolic acids, and alkaloids, which are known for their anti-inflammatory, antioxidant, and anticancer properties. Given the increasing demand for these valuable compounds, the development of efficient in vitro propagation systems, especially hairy root culture, is crucial for enhancing the production of secondary metabolites. This has led to significant interest in exploring sustainable methods to scale up production without relying on wild plant populations. In this study, we aimed to establish an effective method for inducing and maintaining hairy root cultures of C. procera, to evaluate the growth curve and assess the effects of different elicitors on secondary metabolite production, particularly focusing on phenolic and flavonoid content and antioxidant activity. Materials and Methods. The first step in the study involved seed germination and the preparation of explants. Seeds of C. procera were germinated in MS medium, and 30-day-old seedlings were used to prepare explants from different parts, including leaves, stems and, roots. The explants were then subjected to inoculation as pretreatment with Rhizobium rhizogenes (formerly Agrobacterium rhizogenes), a bacterium known for its ability to induce hairy roots in plants. The induction process was further optimized by analyzing the effect of bacterial strains and explant types on the efficiency of root induction. Results and Discussion. Among the bacterial strains tested, R. rhizogenes strain LBA9402 exhibited the highest efficiency in hairy root induction, with leaf explants showing the most significant response. Following inoculation, hairy roots were successfully induced and confirmed by PCR analysis, targeting the rolB gene, which is characteristic of transgenic hairy roots. PCR results showed that the rolB was amplified from the hairy roots, confirming the transgenic nature of the emerged roots. Results indicated that LBA9402 was the most effective strain, and leaf explants yielded the highest rate of root induction. The next phase of the study focused on the growth characteristics of the induced hairy roots. After several subcultures, the growth of the hairy roots was monitored in liquid MS medium containing 30 gL-1 sugar, and fresh and dry weights were measured at regular intervals. The growth curve indicated that the highest fresh and dry weights of the hairy roots were recorded on days 28 and 24, respectively. These measurements were used to draw a growth curve for the selected hairy root line, which will be crucial for optimizing the production of secondary metabolites. In parallel with growth studies, the effects of two elicitors, chitosan and methyl jasmonate, on the production of phenolic compounds, flavonoids, and antioxidant activity in hairy root cultures were evaluated. The elicitors were applied at different concentrations and time intervals, and the total phenolic and flavonoid content were measured using standard assays. The results showed that methyl jasmonate at a concentration of 10 µM significantly increased the production of flavonoid compounds, especially within the first 24 hours. The results of total phenolic content measurement within 48 hours in the presence of 50 µM methyl jasmonate elicitor were higher (2927.22 µg GAE/g FW) than other groups and the highest antioxidant activity, as measured by the DPPH assay, was observed in the methyl jasmonate-treated roots, suggesting that this elicitor has a strong potential for enhancing the antioxidant properties of C. procera hairy roots. Conclusion. This study contributes to the development of an efficient in vitro culture system for C. procera, providing insights into the optimization of conditions for hairy root induction and secondary metabolite production. The findings emphasize the role of bacterial strain selection, explant type, and elicitor treatments in enhancing the yield of valuable compounds. These results also suggest that methyl jasmonate is a potent elicitor for the production of phenolic and antioxidant compounds in C. procera hairy root cultures, making it a promising candidate for large-scale production of these bioactive metabolites. Further research is needed to explore the molecular mechanisms underlying the effects of elicitors on secondary metabolite biosynthesis and to optimize the production of other bioactive compounds in C. procera. These findings serve as an important prerequisite for future experiments in the field of hairy root cultivation, aimed at producing secondary metabolites in this valuable medicinal plant Introduction. Calotropis procera, a prominent medicinal plant native to the dry regions of Asia and Africa, is widely recognized for its medicinal properties. The plant is a rich source of bioactive compounds, particularly flavonoids, phenolic acids, and alkaloids, which are known for their anti-inflammatory, antioxidant, and anticancer properties. Given the increasing demand for these valuable compounds, the development of efficient in vitro propagation systems, especially hairy root culture, is crucial for enhancing the production of secondary metabolites. This has led to significant interest in exploring sustainable methods to scale up production without relying on wild plant populations. In this study, we aimed to establish an effective method for inducing and maintaining hairy root cultures of C. procera, to evaluate the growth curve and assess the effects of different elicitors on secondary metabolite production, particularly focusing on phenolic and flavonoid content and antioxidant activity. Materials and Methods. The first step in the study involved seed germination and the preparation of explants. Seeds of C. procera were germinated in MS medium, and 30-day-old seedlings were used to prepare explants from different parts, including leaves, stems and, roots. The explants were then subjected to inoculation as pretreatment with Rhizobium rhizogenes (formerly Agrobacterium rhizogenes), a bacterium known for its ability to induce hairy roots in plants. The induction process was further optimized by analyzing the effect of bacterial strains and explant types on the efficiency of root induction. Results and Discussion. Among the bacterial strains tested, R. rhizogenes strain LBA9402 exhibited the highest efficiency in hairy root induction, with leaf explants showing the most significant response. Following inoculation, hairy roots were successfully induced and confirmed by PCR analysis, targeting the rolB gene, which is characteristic of transgenic hairy roots. PCR results showed that the rolB was amplified from the hairy roots, confirming the transgenic nature of the emerged roots. Results indicated that LBA9402 was the most effective strain, and leaf explants yielded the highest rate of root induction. The next phase of the study focused on the growth characteristics of the induced hairy roots. After several subcultures, the growth of the hairy roots was monitored in liquid MS medium containing 30 gL-1 sugar, and fresh and dry weights were measured at regular intervals. The growth curve indicated that the highest fresh and dry weights of the hairy roots were recorded on days 28 and 24, respectively. These measurements were used to draw a growth curve for the selected hairy root line, which will be crucial for optimizing the production of secondary metabolites. In parallel with growth studies, the effects of two elicitors, chitosan and methyl jasmonate, on the production of phenolic compounds, flavonoids, and antioxidant activity in hairy root cultures were evaluated. The elicitors were applied at different concentrations and time intervals, and the total phenolic and flavonoid content were measured using standard assays. The results showed that methyl jasmonate at a concentration of 10 µM significantly increased the production of flavonoid compounds, especially within the first 24 hours. The results of total phenolic content measurement within 48 hours in the presence of 50 µM methyl jasmonate elicitor were higher (2927.22 µg GAE/g FW) than other groups and the highest antioxidant activity, as measured by the DPPH assay, was observed in the methyl jasmonate-treated roots, suggesting that this elicitor has a strong potential for enhancing the antioxidant properties of C. procera hairy roots. Conclusion. This study contributes to the development of an efficient in vitro culture system for C. procera, providing insights into the optimization of conditions for hairy root induction and secondary metabolite production. The findings emphasize the role of bacterial strain selection, explant type, and elicitor treatments in enhancing the yield of valuable compounds. These results also suggest that methyl jasmonate is a potent elicitor for the production of phenolic and antioxidant compounds in C. procera hairy root cultures, making it a promising candidate for large-scale production of these bioactive metabolites. Further research is needed to explore the molecular mechanisms underlying the effects of elicitors on secondary metabolite biosynthesis and to optimize the production of other bioactive compounds in C. procera. These findings serve as an important prerequisite for future experiments in the field of hairy root cultivation, aimed at producing secondary metabolites in this valuable medicinal plant

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