Horticultural Science and Technology 원예과학기술지 ISSN:1226-8763(Print) 2465-8588(Online)

Endophytic and seed-borne microbial attacks represent major barriers leading to poor regeneration- powered micro-propagation of Cannabis sativa. Thus, a reliable sterile method during germination and in vitro plantlet preparation is important for the further successful micro-propagation of C. sativa plants. In the present study, we aim to develop a quick and efficient procedure for the preparation of pathogen-free plantlets using a hydrogen peroxide (H₂O₂) solution as a liquid germination medium. In this framework, all three phases, i.e., seed sterilization, germination, and early seedling development, were carried out in H₂O₂ solutions of varying concentrations (0, 1, 3, 5, and 10%). Among the different concentrations tested, the 1% H₂O₂ solution resulted in the fastest and the most successful germination for all tested genotypes of C. sativa seeds, in this case “Cheungsam,” “Cherry Blossom,” “Queen Dream,” and “Hot Blonde.” Higher concentrations of H₂O₂ (3, 5, and 10%) adversely affected the germination rate of C. sativa seeds compared to 0% or 1% H₂O₂. In addition, 5% and 10% H₂O₂ solutions significantly reduced seedling survival rates on MS media. Importantly, contamination rates of 88.4–96.7% were observed for C. sativa seedlings germinated with the 0% H₂O₂ solution and transferred to antibiotic-free MS media. However, a contamination rate of less than 4.0% was observed for seedlings prepared using 1% H₂O₂ as a germination medium. Moreover, the survival rates of seedlings germinated in 1% H₂O₂ were significantly higher than those of other seedlings. Our results suggest that a 1% H₂O₂ solution is a useful liquid germination medium for C. sativa seeds to enhance germination and survival rates while decreasing microbial contamination in antibiotic-free tissue culture media.