With the rising demand for new cultivars of carnation, efficient transformation protocols are needed to enable the bioengineering of new traits. Here, we established a novel and efficientAgrobacterium-mediated transformation system using callus as the target explant for four commercial carnation cultivars. Leaf-derived calli of all cultivars were inoculated withAgrobacterium tumefaciensstrain LBA4404 containing the plasmid pCAMBIA 2301 harboring genes for β-glucuronidase (uidA) and neomycin phosphotransferase (nptII). Polymerase chain reaction (PCR) and histochemical assays confirmed the presence ofuidAand β-glucuronidase (GUS), respectively in transgenic shoots. The effect on transformation efficiency of medium composition and the presence of antioxidants during inoculation and co-cultivation was investigated. The transformation efficiency was increased in Murashige and Skoog (MS) medium lacking KNO3and NH4NO3,and also in MS medium lacking macro and micro elements and Fe to 5% and 3.1% respectively, compared to 0.6% in full-strength medium. Transformation efficiency was increased dramatically to 24.4% across all carnation cultivars by the addition of 2 mg/l melatonin to nitrogen-depleted MS medium. Shoot regeneration was also doubled in this treatment. The establishment of this efficient and reliable transformation protocol can advance the development of novel carnation cultivars through molecular breeding approaches.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-023-04325-5.