Optimizing ex-vitro one-step RUBY-equipped hairy root transformation in drug- and hemp-type Cannabis

Abstract

Using synthetic biology techniques to engineer secondary metabolic pathways through hairy root transformation is one of the most advanced approaches used in research. In this study, we optimized an ex-vitro one-step hairy root transformation of the RUBY system in both drug- and hemp-type cannabis, shedding light on its potential applications in secondary metabolite production. Three different strains of A. rhizogenes including (A4, ARqual, and K599) were used. Significant variation in HR induction and transformation efficiency (TE) was observed based on A. rhizogenes strains and seed types. Drug-type seedlings exhibited the highest hairy root induction, increasing by 58.8% compared to hemp-type seedlings. Also, the A4 strain consistently demonstrated the highest transformation efficiency (75%) irrespective of genotype, while the ARqual strain yielded the lowest one (8.33%). In conclusion, our study is the first to present an ex-vitro one-step transformation of both hemp- and drug-type cannabis. In comparison to the in vitro method, our ex-vitro method is simpler, faster, and has a lower risk of contamination, making it an excellent choice for the efficient production of secondary metabolites in cannabis using the CRISPR/Cas system.