Yield loss by virus infection has been an unsolved and emerging threat for canola (Brassica napus) crops under climate change scenarios, prompting application of modern technologies in canola breeding in recent years, such as CRISPR/Cas genome editing. The RNA targeting CRISPR/CasRx can be utilized to introduce RNA virus resistance into canola plants through transformation. Regeneration potential of three canola cultivars (Bravo, Cobbler, Crusher) and Westar was assessed on three culture media systems (P1, P2 and P3), from which Cobbler and P2 system were selected for transformation protocol establishment due to the high plant regeneration efficiency with this combination. The highest shoot formation rate (90%) and the average shoots produced per explant (12.75 shoots) were obtained from Cobbler cotyledons on P2. A comparative shoot regeneration was also obtained by using Cobbler explants and P1 system (8.5 shoots/explant). Transformation of Cobbler explants (cotyledons, hypocotyls and epicotyls) was carried out with Agrobacterium tumefaciens strain GV3101 containing CasRx vector with BlpR gene conferring phosphinothricin (PPT) resistance. By using P2 and selective medium containing PPT 5 mg.L-1, the highest transformation efficiency was obtained with epicotyl explants (4.5%), followed by hypocotyls (1.27%), while cotyledon explants were failed to regenerate after selection with PPT. The result suggests the potential for generation of canola plants expressing CRISPR/CasRx targeting RNA viruses such as TuMV.