HighlightsSuccessful expression of human cytokine GM-CSF in two industrial tobacco cultivars is achieved.Rapid cloning in two binary destination vectors is accomplished by using Gateway approach.Agrobacterial infiltration procedure is optimized and shown to require the surface tension lowering agent Silwet L-77.Accumulation of recombinant protein was confirmed by using high affinity monoclonal hemagglutinin tag antibodies.Production of hGM-CSF has been achieved without plant codon usage optimization.