Protoplast isolation, transient transformation, and flow-cytometric analysis of reporter-gene activation in Cannabis sativa L.

摘要: Abstract Cannabis sativa L. is a valuable, up-and-coming industrial crop with substantially growing market. However, due to an extended period of legal restriction, research cannabis has been limited, particularly in laboratory settings. Expanding the application biotechnological techniques can facilitate addressing species-specific impediments improving traits and further fundamental understanding its intricacies. Here, we describe protoplast transformation for study transient gene expression low-THC cultivar. To produce explant tissue as source protoplasts, method hormone-free vitro micropropagation established. Protoplasts are isolated from young leaves micropropagated stocks transiently transformed plasmid DNA carrying fluorescent marker gene. This first report this species. A isolation yield achieved up 2 × 106 cells per gram leaf material, vitality staining shows that 82 % protoplasts viable, quantification expressing protein indicates 31 be successfully transformed. Additionally, auxin-responsive reporter reaction treatment indole-3-acetic acid quantified using flow cytometry. work demonstrates relatively minor modification standard used important emerging crop.