Use of Embryos Extracted from Individual Cannabis sativa Seeds for Genetic Studies and Forensic Applications

Legal limits on the psychoactive tetrahydrocannabinol (THC) content in Cannabis sativa plants have complicated genetic and forensic studies in this species. However, Cannabis seeds present very low THC levels. We developed a method for embryo extraction from seeds and an improved protocol for DNA extraction and tested this method in four hemp and six marijuana varieties. This embryo extraction method enabled the recovery of diploid embryos from individual seeds. An improved DNA extraction protocol (CTAB3) was used to obtain DNA from individual embryos at a concentration and quality similar to DNA extracted from leaves. DNA extracted from embryos was used for SSR molecular characterization in individuals from the 10 varieties. A unique molecular profile for each individual was obtained, and a clear differentiation between hemp and marijuana varieties was observed. The combined embryo extraction-DNA extraction methodology and the new highly polymorphic SSR markers facilitate genetic and forensic studies in Cannabis.