Importance of Media Composition and Explant Type in Cannabis sativa Tissue Culture

Producing uniform <i>Cannabis sativa (Cannabis)</i> for medicinal/recreational flower production through sexual propagation has been problematic, leading to dominance of clonal propagation from "mother plants" in the cannabinoid industry, which also faces significant limitations. <i>Cannabis</i> tissue culture (TC) methods have been developed to overcome these challenges, but the long-term health and maintenance of <i>Cannabis</i> explants in TC have been largely overlooked in previous studies. The current study focused on the development of an efficient and optimized micropropagation protocol covering the entire process, with a specific focus on the health and performance in the multiplication stage. Multiplication media were formulated hormone-free to avoid longer-term vitrification issues, resulting in single-main-shoot cultures rather than multiple-shoot cultures. This instigated the use of stage II explant types different from the standard shoot tips previously used for multiple shoot cultures. Multiplication media were further improved from the basal salt composition via nitrogen and calcium additives. The optimized protocol was used on eight diverse <i>Cannabis</i> cultivars to test its applicability across various genetic backgrounds. Results indicated that the protocol was effective for conservation purposes across all cultivars and achieved good long-term multiplication rates for some but not all. The outcomes of this study mark a significant stride towards an efficient <i>Cannabis</i> TC methodology ready for more comprehensive industrial applications.