Agrobacterium-mediated transformation of THC-containing Cannabis sativa L. yields a high frequency of transgenic calli expressing bialaphos resistance and non-expressor of PR1 (NPR1) genes

Abstract

We established transformation technologies using Agrobacterium tumefaciens (Smith & Townsend) Conn to insert foreign genes into high THC-containing cannabis (Cannabis sativa L.). The Arabidopsis non-expressor of pathogenesis-related protein 1 (AtNPR1) gene was selected as a potentially useful agronomic gene, which was linked to the bar gene from Streptomyces that encodes herbicide resistance. We investigated how Agrobacterium strains (EHA105 and GV3101), glufosinate concentrations, explant source, and light intensity affected transformation frequency (TF). Transformation was confirmed by RT-PCR with primers for the NPR1 or bar genes. Glufosinate at 0.5–1 mg/L inhibited growth of non-transformed calli within 8 weeks following A. tumefaciens infection. Strain EHA105 yielded a higher TF compared to strain GV3101. Whole leaflets yielded a higher TF compared to sectioned leaf explants with strain GV3101. However, this effect was not seen with EHA105. Petiole segments showed a higher TF than leaf sections with strain EHA105. Placing explants under light or dark conditions did not affect TF, which ranged from 5% to 95% in different experiments. This is the first report of successful transformation of two high THC-containing C. sativa genotypes with two foreign genes simultaneously——AtNPR1 and bar. The recovery of plantlets from transgenic calli was not attempted and awaits further research.