Abstract: Background and Objective: Soybean growth and productivity remain adversely affected by various biotic and abiotic stress factors like pests and drought. This study tested in vivo and in vitro pretreatment of seeds with colchicine to establish a tissue culture-based protocol for soybean polyploidisation, with the benefit of potentially conferring inherent stress resistance in this crop. Materials and Methods: Seeds of 2 soybean cultivars viz. Peking and Dundee were pretreated with different concentrations of colchicine (0.0, 0.01, 0.05 and 0.1%) and sub-cultured for germination followed by in vitro shoot induction and plantlet regeneration. Results: The findings indicated that seed germination and shoot proliferation were inhibited by both in vivo and in vitro pretreatment of seeds with 0.05 and 0.1% colchicine, particular in Peking, compared to Dundee, followed by 0.01% used for the same cultivars. These effects were accompanied by severe tissue senescence and callus formation. Conclusion: In vivo pretreatment of soybean seeds with colchicine was more efficient and suitable for multiple shoot proliferation and regeneration under plant tissue culture conditions than in vitro application of colchicine for this purpose.