Establishment of efficient Cannabis (Cannabis sativa L.) protoplast isolation and transient expression condition

There has been a significant increase in interest in Cannabis (Cannabis sativa L.) plants for the production of secondary metabolites (i.e., cannabinoids and terpenoids), which could have medicinal benefits for human health. Genetic engineering technology will allow us to unlock the potential uses of cannabis plants. Although a protoplast system is a powerful tool for gene function analysis and genome editing, the lack of an established protocol that may offer acceptable yield and quality of protoplast isolation from Cannabis is a major obstacle. In this study, we established a combination of digestion solutions for successful protoplast isolation from Cannabis. The highest yield (9.7 × 10⁶ per g of fresh leaf weight) was obtained using the protocol composed of optimal mannitol concentration (0.4 M), enzyme combination (1.5% cellulase, 0.4% macerozyme, and 1.0% pectolyase), and vacuum-permeating treatment. Notably, the transformation efficiency of Cannabis protoplast was approximately 55.3% when transformed with p35S: GFP construct. Using the protoplast preparation and transformation methods established in this study, we revealed that CsCBCAS, CsCBDAS, and CsTHCAS proteins exhibit punctate subcellular localization patterns that may arise from membrane-bound organelles.