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Bananas and plantains (Musa spp.) are among the most critical socioeconomic crops globally, being a staple food for millions of people in the tropics and an essential component for the export market, including the subtropics. Besides conventional breeding, genetic improvement of bananas and plantains could be performed through genetic engineering and new breeding techniques. Furthermore, plant tissue culture is essential for these technologies, including developing embryogenic cell suspensions and in vitro plants. The transient and stable genetic transformation could be performed from in vitro plants, shortening Musa transgenic lines development compared to genetic transformation while using embryogenic cell suspension. In this study, a genetic transformation protocol was established from banana apical meristems for the ‘Williams’ cultivar (genotype AAA). The protocol was based on the co-cultivation of the explants (whole in vitro plants or bisected meristematic tissues derived from in vitro plants) with Agrobacterium tumefaciens strain LBA4404 harboring two binary vectors denominated pLVCIBE1 (cassette: MabHIPP promoter::luc2::Tnos, P35S::hpt::Tnos) and pLVCIBE2 (cassette: P35S::luc2::Tnos, P35S::hpt::Tnos), independently. The stable genetic transformation was obtained by subculturing in vitro banana plants in selection medium (12.5µg/mL of hygromycin) for 8 weeks from bisected meristematic tissue transformation. Genetic transformation was confirmed in vivo with the use of the luciferase reporter gene system. Furthermore, PCR was performed on DNA extracted from leaves of regenerated transgenic in vitro plants after 8 weeks of selection, confirming stable genetic transformation. Therefore, genetic transformation was achieved in the apical meristematic tissue of in vitro banana plants with co-cultivation of Agrobacterium tumefaciens.