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Napier grass (Pennisetum purpureum) is a key forage crop in Kenya. However, its yield and quality are often hindered by headsmut and stunt disease. Genetic improvement through mutation breeding, particularly using colchicine to induce polyploidy, offers a potential solution for improving Napier grass. The experiments were carried out as a factorial experiment in a complete random design (CRD). This study aimed to evaluate the response of embryogenic calli to different colchicine concentrations (0, 0.05, 0.1, and 0.2%) over 24, 48, and 72 hours duration to induce polyploidy in South african and Bana napier grass germplasms. The most suitable media for shoot regeneration was Murashige and Skoog (MS) medium supplemented with 0.2 mgL-1 Benzyl Adenine (BAP), 0.1mgL-1 dichlorophenoxyacetic acid (2, 4-D) and 0.1mgL-1 Indole-3-Butyric Acid (IBA) while media with 1mgL-1IBA, 1mgL-1 2, 4-D and 0.5mgL-1 BAP was more suitable in inducing embryogenic calli in all genotypes. Chromosome doubling was confirmed through chromosome counting and stomatal size, and number. Notably, we recommend use of flow cytometry to confirm ploidy level. Results showed that a 0.1% colchicine concentration with a 48-hour treatment was most effective for producing mutant plantlets, while higher concentrations were toxic. Significant genetic and agronomic variations were observed between the mutants and controls, indicating that the selected mutants are valuable genetic resources and recommended for characterization across representative agro-ecologies for large-scale production and used in Penniseturm purpureum breeding programs.