A novel and rapid method for Agrobacterium-mediated production of stably transformed Cannabis sativa L. plants

The development of genetically transformed plants is an elusive landmark in Cannabis sativa L. breeding. Despite its economic interest, at present, protocols for producing transgenic C. are scarce. We studied the ability hypocotyl, cotyledon and meristem explants from six hemp varieties plant regeneration. For this, we firstly evaluated vitro regeneration rates hypocotyls cultured medium without growth regulators, cotyledons supplemented with 0.4 mg L?1 thidiazuron (TDZ) 0.2 ?-naphthaleneacetic (NAA). Subsequently, effect different kanamycin concentrations (50, 100, 200, 500 750 L?1) on hypocotyl rate was determined. Finally, assessed transformation after co-culture Agrobacterium tumefaciens strain LBA4404 carrying binary plasmid pBIN19 containing ?-glucuronidase (uidA) reporter gene resistance neomycin phosphotransferase (nptII) genes. Plant validated through culture regenerating shoots kanamycin-containing selective medium, by GUS histochemical assay uidA expression, PCR amplification nptII Our results showed that reached a higher (53.3 %) than (18.1 co-culture. On other hand, 100 proved to be best concentration terms (63.3 spontaneous rooting (12.2 %), which displayed 7.1 % albinism rate. After A. subsequent antibiotic-containing explant type achieving 23.1 rate, contrasts 1.0 detected cotyledons. Transgenic were obtained all types evaluated. Although there significant differences among evaluated, superior already-developed meristems, reaching 5.0 0.8 respectively. extremely low co-culture, cotyledon-derived analyzed successfully transformed. hormone-free protocol doubles shoots, also three times faster already published protocols. This has relevant implications breeding, enabling not only genetic transformation, but use new breeding techniques such as targeted genome editing using CRISPR/Cas systems. may foster specific biochemical profiles, or tolerant biotic abiotic stresses others.