Document Type : Research Paper
Author
Research Division of Natural Resources , East Azarbaijan Agricultural and Natural Resources Research and Education Center , Agricultural Research, Education and Extention Organization (AREEO), Tabriz, Iran
Abstract
Background and objectives: Milk thistle (Silybum marianum) is an annual or biennial herbaceous plant belonging to the Asteraceae family. The key bioactive compounds of this medicinal plant are flavonoids, which are primarily stored in the fruit (seed) and appear yellow. Among them, silymarin exhibits potent anti-inflammatory properties and plays a role in cancer prevention by affecting the vascular structure of prostate, breast, ovarian, liver, and leukemia cancer tissues. Biotechnological approaches, particularly tissue and cell culture techniques, serve as valuable complementary methods for the commercial production of plant-derived compounds. This study aimed to determine the optimal method for producing sterile S. marianum seedlings and to evaluate the effects of genotype, explant type, and growth regulators on callus induction, growth, and embryogenic callus formation.
Methodology: In this study, five genotypes (Hungary, Borazjan, Fereydon Kenar, Jolgeh Khalaj, and Moghan) were evaluated using three different seed disinfection methods in a factorial experiment based on a completely randomized design (CRD) with three replications. Seed germination and sterile seedling production were conducted in water and agar culture media without growth regulators, under dark conditions at 25°C. Subsequently, an experiment was performed using two genotypes (Hungary and Borazjan), cotyledon and hypocotyl explants, Murashige and Skoog (MS) medium, and growth regulators 2,4-D (1, 2.5, and 5 mg/L) and BAP (0.25 and 0.5 mg/L) in a factorial experiment based on completely random design under dark conditions. After 30 days, the induced callus was transferred to MS medium with half the initial hormone concentrations. Callus diameter was assessed using the Hochrony Berz method, while callus fresh weight, the number of embryogenic calluses, and the number of somatic embryos per callus in cotyledon explants were measured 30 days post-culture. Calluses were freeze-dried, and their dry weight was also recorded.
Results: The optimal method for producing sterile S. marianum seedlings involved treatment with Tween-20 solution, 70% ethanol, hydrogen peroxide, and sterile distilled water, followed by cultivation in water and agar medium without growth regulators. Analysis of variance for callus diameter, fresh weight, and dry weight indicated that the best conditions for callus induction were the Borazjan genotype, cotyledon explant, 1 mg/L 2,4-D and 0.5 mg/L BAP. Embryogenic callus formation occurred exclusively in cotyledon explants. Variance analysis of embryogenic callus production and the number of somatic embryos per callus revealed that optimal conditions for embryogenic callus formation were achieved using the Hungarian genotype with 5 mg/L 2,4-D and 0.25 mg/L BAP.
Conclusion: Based on the results, the most effective disinfectants for S. marianum seeds were Tween-20 solution, 70% ethanol, hydrogen peroxide, and sterile distilled water. For optimal callus production, cotyledon explants and low concentrations of auxin, such as 2,4-D, were preferable. In embryogenic callus formation, improved cultivars like Hungary outperformed native genotypes. Additionally, cotyledon explants were more suitable than hypocotyl explants, and high concentrations of auxin (2,4-D) combined with low concentrations of cytokinin (BAP) provided the best results.
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