University of Isfahan Journal of Plant Biological Sciences 3041-9603 16 3 2024 09 22 The effect of plant growth regulators and biostimulants on callus formation of Catharanthus roseus The effect of plant growth regulators and biostimulants on callus formation of Catharanthus roseus 79 94 29788 10.22108/ijpb.2025.145277.1409 FA Arezoo Sabahi Ojnaz Department of Production Engineering and Plant Genetics, Faculty of Agriculture and Natural Resources, University of Mohaghegh Ardabili Nasser Zare Department of Production Engineering and Plant Genetics, Faculty of Agriculture and Natural Resources, University of Mohaghegh Ardabili Roghayyeh Hazrati Department of Production Engineering and Plant Genetics, Faculty of Agriculture and Natural Resources, University of Mohaghegh Ardabili Rasool Asghari‑Zakaria Department of Production Engineering and Plant Genetics, Faculty of Agriculture and Natural Resources, University of Mohaghegh Ardabili Parisa Sheikhzadeh Department of Production Engineering and Plant Genetics, Faculty of Agriculture and Natural Resources, University of Mohaghegh Ardabili Journal Article 2025 05 14 Catharanthus roseus is a medicinal plant that is of great importance due to its abundant production of the alkaloids vincristine and vinblastine and their use in the production of anticancer drugs. Given the high price of these compounds and their small amounts in this plant, efforts to increase the production of these compounds have led to extensive research on this plant. The aim of the present study is to investigate and test various factors affecting callus formation in Catharanthus roseus, such as plant hormones (2.4-D (0.5, 1, 1.5 mg/L), NAA (0.5, 1, 3 mg/L), and BAP (0.5, 1, 3 mg/L)), ascorbic acid (50, 100, and 150 mg/L), melatonin, and yeast extract (50, 100, and 150 mg/L), as well as to investigate the best method for disinfecting Catharanthus roseus seeds. The results of the study showed that for disinfection of Parvansh seeds, the best method with the highest percentage of survival and the lowest percentage of fungal and bacterial contamination is related to the anti-infection of sodium hypochlorite at 2.5%. Also, the appropriate culture medium for callus growth is MS culture medium containing 1 mg/L NAA and 1 mg/L BAP. Also, in the ascorbic acid treatment, the highest fresh weight of callus was observed in the 150 mg/L ascorbic acid treatment (0.0872), which had the least color change (browning). Also, the results of examining the morphological characteristics of calluses treated with melatonin and yeast extract showed that the highest percentage of callus formation and the highest fresh weight of callus were related to yeast extract at a concentration of 150 mg/L. Introduction Medicinal plants have played a vital role in human life since ancient times, and today, about 80% of the medicines we consume have plant roots. Catharanthus roseus, native to Madagascar and tropical regions, is one of these natural treasures that has attracted attention due to its valuable medicinal compounds, especially anticancer alkaloids such as vinblastine and vincristine. For the sustainable production of these compounds, modern cultivation methods such as in vitro culture (tissue culture) are used. In this method, different parts of the plant, such as callus, hairy roots, stem, and leaves, can be used to produce seedlings and control the amount of alkaloids. Plant hormones, especially auxin and cytokinin, play a key role in growth and development, as well as the process of callus formation. Research has shown that the appropriate composition and concentration of these hormones can significantly affect the dry weight of callus and the production of alkaloids; For example, the combination of BAP and NAA has yielded positive results. In addition to hormones, substances such as ascorbic acid (vitamin C), yeast extract, and melatonin also help improve callus formation and plant resistance. Ascorbic acid, as a potent antioxidant, plays a role in cell division and growth, and yeast extract increases metabolic activity by providing nutrients and natural hormones. Melatonin, with its antioxidant properties, also reduces oxidative stress and stimulates cell division. The primary objective of this research is to identify the most effective method of seed disinfection and to examine the impact of varying concentrations of plant hormones, ascorbic acid, melatonin, and yeast extract on the callus formation process in the Parvanesh plant, aiming to optimize the production of valuable medicinal compounds. Materials and Methods In this study, Parvansh seeds were obtained from Pakan Seed Company, Isfahan. Seed disinfection and initial cultivation: For seed disinfection, 70% alcohol and three different concentrations of sodium hypochlorite (1.5%, 2.5%, and 5%) were used. After washing and drying, the seeds were cultivated in MS culture medium (without hormones) and first in the dark (temperature 24 ± 2 °C) and then in controlled light conditions (16 hours of light, 8 hours of darkness) for germination. Investigation of hormones for callus formation: When the seedlings reached the 8-leaf stage, the leaves were cut into small pieces (0.5 to 1 cm) and placed as explants on MS culture medium containing different combinations of plant hormones 2.4-D, NAA, and BAP. After 4 weeks, the percentage and fresh weight of calluses were measured. Effect of ascorbic acid: After determining the best hormone combination (NAA 1 mg/L + BAP 1 mg/L), different concentrations of ascorbic acid (50, 100, and 150 mg/L) were added to this culture medium (using a syringe filter for sterilization). Then, leaf explants were cultured on these media, and the percentage and fresh weight of calluses were evaluated. Effect of melatonin and yeast extract: In the next step, melatonin and yeast extract were also added in three similar concentrations (50, 100, and 150 mg/L) to the culture medium containing NAA and BAP hormones (this time before autoclaving). Callus growth was examined after 20 days. Statistical analysis: All experiments were performed in a completely randomized design with three replications, and data were analyzed using SPSS software and Duncan's test. Graphs were also drawn using Excel. Results and Discussion The results showed that the seed disinfection method plays a vital role in the success of cultivation. 2.5% sodium hypochlorite gave the highest percentage of seed viability, indicating its high efficiency in eliminating bacterial and fungal contamination while maintaining seed health. In contrast, 5% sodium hypochlorite had the lowest viability, and the use of 70% alcohol and 1.5% sodium hypochlorite resulted in the highest contamination. Different combinations of plant hormones were tested to stimulate callus formation from leaf explants. The results showed that MS medium containing 1 mg/L NAA and 1 mg/L BAP, as well as MS medium containing 1 mg/L 2,4-D and 0.5 mg/L BAP, produced the highest callus fresh weight. The highest callus formation percentage (95.23%) was also observed in the same hormonal combination (1 mg/L NAA and 1 mg/L BAP), indicating the importance of hormonal balance in the callus formation process. Adding ascorbic acid to the culture medium significantly improved callus formation and callus quality. A concentration of 150 mg/L ascorbic acid not only resulted in the highest callus formation percentage (86.19%) and callus fresh weight, but also significantly prevented callus browning. This indicates that ascorbic acid plays an essential role in reducing oxidative stress and maintaining callus health. In investigating the effect of yeast extract and melatonin, the results showed that yeast extract at different concentrations, especially 150 mg/L, was able to produce 100% callus formation and the highest callus fresh weight (0.633 g). In contrast, melatonin at a concentration of 150 mg/L resulted in the lowest callus percentage and weight. This finding confirms the importance of yeast extract as a rich source of nutrients and growth regulators in improving callus formation. Conclusion The best method of seed disinfection is the use of 2.5% sodium hypochlorite, which provides high viability and low contamination. The optimal composition for callus production includes MS medium with 1 mg/L NAA and 1 mg/L BAP. An ascorbic acid concentration of 150 mg/L has the highest percentage of callus formation. Yeast extract at a concentration of 150 mg/L increases callus formation, but melatonin at higher concentrations inhibits callus growth. Catharanthus roseus is a medicinal plant that is of great importance due to its abundant production of the alkaloids vincristine and vinblastine and their use in the production of anticancer drugs. Given the high price of these compounds and their small amounts in this plant, efforts to increase the production of these compounds have led to extensive research on this plant. The aim of the present study is to investigate and test various factors affecting callus formation in Catharanthus roseus, such as plant hormones (2.4-D (0.5, 1, 1.5 mg/L), NAA (0.5, 1, 3 mg/L), and BAP (0.5, 1, 3 mg/L)), ascorbic acid (50, 100, and 150 mg/L), melatonin, and yeast extract (50, 100, and 150 mg/L), as well as to investigate the best method for disinfecting Catharanthus roseus seeds. The results of the study showed that for disinfection of Parvansh seeds, the best method with the highest percentage of survival and the lowest percentage of fungal and bacterial contamination is related to the anti-infection of sodium hypochlorite at 2.5%. Also, the appropriate culture medium for callus growth is MS culture medium containing 1 mg/L NAA and 1 mg/L BAP. Also, in the ascorbic acid treatment, the highest fresh weight of callus was observed in the 150 mg/L ascorbic acid treatment (0.0872), which had the least color change (browning). Also, the results of examining the morphological characteristics of calluses treated with melatonin and yeast extract showed that the highest percentage of callus formation and the highest fresh weight of callus were related to yeast extract at a concentration of 150 mg/L. Introduction Medicinal plants have played a vital role in human life since ancient times, and today, about 80% of the medicines we consume have plant roots. Catharanthus roseus, native to Madagascar and tropical regions, is one of these natural treasures that has attracted attention due to its valuable medicinal compounds, especially anticancer alkaloids such as vinblastine and vincristine. For the sustainable production of these compounds, modern cultivation methods such as in vitro culture (tissue culture) are used. In this method, different parts of the plant, such as callus, hairy roots, stem, and leaves, can be used to produce seedlings and control the amount of alkaloids. Plant hormones, especially auxin and cytokinin, play a key role in growth and development, as well as the process of callus formation. Research has shown that the appropriate composition and concentration of these hormones can significantly affect the dry weight of callus and the production of alkaloids; For example, the combination of BAP and NAA has yielded positive results. In addition to hormones, substances such as ascorbic acid (vitamin C), yeast extract, and melatonin also help improve callus formation and plant resistance. Ascorbic acid, as a potent antioxidant, plays a role in cell division and growth, and yeast extract increases metabolic activity by providing nutrients and natural hormones. Melatonin, with its antioxidant properties, also reduces oxidative stress and stimulates cell division. The primary objective of this research is to identify the most effective method of seed disinfection and to examine the impact of varying concentrations of plant hormones, ascorbic acid, melatonin, and yeast extract on the callus formation process in the Parvanesh plant, aiming to optimize the production of valuable medicinal compounds. Materials and Methods In this study, Parvansh seeds were obtained from Pakan Seed Company, Isfahan. Seed disinfection and initial cultivation: For seed disinfection, 70% alcohol and three different concentrations of sodium hypochlorite (1.5%, 2.5%, and 5%) were used. After washing and drying, the seeds were cultivated in MS culture medium (without hormones) and first in the dark (temperature 24 ± 2 °C) and then in controlled light conditions (16 hours of light, 8 hours of darkness) for germination. Investigation of hormones for callus formation: When the seedlings reached the 8-leaf stage, the leaves were cut into small pieces (0.5 to 1 cm) and placed as explants on MS culture medium containing different combinations of plant hormones 2.4-D, NAA, and BAP. After 4 weeks, the percentage and fresh weight of calluses were measured. Effect of ascorbic acid: After determining the best hormone combination (NAA 1 mg/L + BAP 1 mg/L), different concentrations of ascorbic acid (50, 100, and 150 mg/L) were added to this culture medium (using a syringe filter for sterilization). Then, leaf explants were cultured on these media, and the percentage and fresh weight of calluses were evaluated. Effect of melatonin and yeast extract: In the next step, melatonin and yeast extract were also added in three similar concentrations (50, 100, and 150 mg/L) to the culture medium containing NAA and BAP hormones (this time before autoclaving). Callus growth was examined after 20 days. Statistical analysis: All experiments were performed in a completely randomized design with three replications, and data were analyzed using SPSS software and Duncan's test. Graphs were also drawn using Excel. Results and Discussion The results showed that the seed disinfection method plays a vital role in the success of cultivation. 2.5% sodium hypochlorite gave the highest percentage of seed viability, indicating its high efficiency in eliminating bacterial and fungal contamination while maintaining seed health. In contrast, 5% sodium hypochlorite had the lowest viability, and the use of 70% alcohol and 1.5% sodium hypochlorite resulted in the highest contamination. Different combinations of plant hormones were tested to stimulate callus formation from leaf explants. The results showed that MS medium containing 1 mg/L NAA and 1 mg/L BAP, as well as MS medium containing 1 mg/L 2,4-D and 0.5 mg/L BAP, produced the highest callus fresh weight. The highest callus formation percentage (95.23%) was also observed in the same hormonal combination (1 mg/L NAA and 1 mg/L BAP), indicating the importance of hormonal balance in the callus formation process. Adding ascorbic acid to the culture medium significantly improved callus formation and callus quality. A concentration of 150 mg/L ascorbic acid not only resulted in the highest callus formation percentage (86.19%) and callus fresh weight, but also significantly prevented callus browning. This indicates that ascorbic acid plays an essential role in reducing oxidative stress and maintaining callus health. In investigating the effect of yeast extract and melatonin, the results showed that yeast extract at different concentrations, especially 150 mg/L, was able to produce 100% callus formation and the highest callus fresh weight (0.633 g). In contrast, melatonin at a concentration of 150 mg/L resulted in the lowest callus percentage and weight. This finding confirms the importance of yeast extract as a rich source of nutrients and growth regulators in improving callus formation. Conclusion The best method of seed disinfection is the use of 2.5% sodium hypochlorite, which provides high viability and low contamination. The optimal composition for callus production includes MS medium with 1 mg/L NAA and 1 mg/L BAP. An ascorbic acid concentration of 150 mg/L has the highest percentage of callus formation. Yeast extract at a concentration of 150 mg/L increases callus formation, but melatonin at higher concentrations inhibits callus growth.

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