Availability of Hydrogen Peroxide Solutions as a Germination Liquid Medium for Contamination-free in vitro Seedling Development of Cannabis sativa

Abstract

Endophytic and seed-borne microbial attacks represent major barriers leading to poor regenerationpowered 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 (H 2 O 2 ) 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 H 2 O 2 solutions of varying concentrations (0, 1, 3, 5, and 10%). Among the different concentrations tested, the 1% H 2 O 2 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 H 2 O 2 (3, 5, and 10%) adversely affected the germination rate of C. sativa seeds compared to 0% or 1% H 2 O 2 . In addition, 5% and 10% H 2 O 2 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% H 2 O 2 solution and transferred to antibiotic-free MS media. However, a contamination rate of less than 4.0% was observed for seedlings prepared using 1% H 2 O 2 as a germination medium. Moreover, the survival rates of seedlings germinated in 1% H 2 O 2 were significantly higher than those of other seedlings. Our results suggest that a 1% H 2 O 2 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.