A Meristem-based, genotype independent transformation system for Cannabis sativa has been established at the Wisconsin Crop Innovation Center (WCIC). DNA delivery in this system is primarily via Agrobacterium, however a biolistics approach has also been demonstrated. Germline transformation, with transmission of stable traits to T1 progeny, has been confirmed empirically via tdTomato imaging, RUBY expression, GUS staining of T1 progeny and further validated via PCR assays for several other genes of interest. Hundreds of independent T0 events, along with their multi-generational progeny, have been produced and analyzed from multiple genotypes. We have clearly demonstrated our germline transformation protocol has enabled both engineering and a CRISPR/Cas9 approach to successfully knockout or alter key genes in the cannabinoid, terpene, and flavonoid pathways in Type III cannabis plants. In addition to these pathways, work in powdery mildew and hop latent resistance, trichome density, yield, and other traits have also progressed. Transgenic or edited lines showing elimination, reduced, and increased amounts of cannabinoids, terpenes, and flavonoids, and putative lines for powdery mildew resistance will be discussed. Our successful engineering and gene editing protocols open the door for future metabolic engineering of Cannabis sativa to fully leverage the potential of this emerging crop species.

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