BackgroundThe base editors can introduce point mutations accurately without causing double-stranded DNA breaks or requiring donor DNA templates. Previously, cytosine base editors (CBEs) containing different deaminases are reported for precise and accurate base editing in plants. However, the knowledge of CBEs in polyploid plants is inadequate and needs further exploration.ResultsIn the present study, we constructed three polycistronic tRNA-gRNA expression cassettes CBEs containing A3A, A3A (Y130F), and rAPOBEC1(R33A) to compare their base editing efficiency in allotetraploidN. benthamiana(n= 4x). We used 14 target sites to compare their editing efficiency using transient transformation in tobacco plants. The sanger sequencing and deep sequencing results showed that A3A-CBE was the most efficient base editor. In addition, the results showed that A3A-CBE provided most comprehensive editing window (C1~ C17could be edited) and had a better editing efficiency under the base background of TC. The target sites (T2 and T6) analysis in transformedN. benthamianashowed that only A3A-CBE can have C-to-T editing events and the editing efficiency of T2 was higher than T6. Additionally, no off-target events were found in transformedN. benthamiana.ConclusionsAll in all, we conclude that A3A-CBE is the most suitable vector for specific C to T conversion inN. benthamiana. Current findings will provide valuable insights into selecting an appropriate base editor for breeding polyploid plants.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-023-04322-8.