Optimizing cannabis cultivation: an efficient in vitro system for flowering induction

Cannabis sativa L. is a versatile medicinal plant known for its therapeutic properties, derived from its diverse array of secondary metabolites synthesized primarily in female flower organs. Breeding cannabis is challenging due to its dioecious nature, strict regulatory requirements, and the need for photoperiod control to trigger flowering, coupled with highly dispersible pollen that can easily contaminate nearby female flowers. This study aimed to develop a protocol for in vitro flowering in cannabis, investigate factors affecting in vitro flower production, and generate viable in vitro seeds, potentially offering a method for producing sterile cannabinoids or advancing breeding techniques. We show that the life cycle of cannabis can be fully completed in tissue culture; plantlets readily produce inflorescences and viable seeds in vitro. Our findings highlight the superior performance of DKW medium with 2% sucrose in a filtered vessel and emphasize the need for low light intensity during flower induction to optimize production. The improved performance in filtered vessels suggests that plants conduct photosynthesis in vitro, highlighting the need for future investigations into the effects of forced ventilation to refine this system. All tested lines readily developed inflorescences upon induction, with a 100% occurrence rate, including male flowering. We revealed the non-dehiscent trait of in vitro anthers, which is advantageous as it allows for multiple crosses to be conducted in vitro without concerns about cross-contamination. The current work developed and optimized an effective protocol for in vitro flowering and seed production in cannabis, potentially providing a platform for sterile cannabinoid production and an efficient tool for breeding programs. This system allows for the full and consistent control of plant growth conditions year-round, potentially offering the reliable production of sterile molecules suitable for pharmacological use. As a breeding strategy, this method overcomes the complex challenges of breeding cannabis, such as the need for large facilities, by enabling the production of hundreds of lines in a small facility. By offering precise control over factors such as plant growth regulators, light intensity, photoperiod, and temperature, this system also serves as a valuable tool for studying flowering aspects in cannabis.