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Direct regenerations by using mature cotyledonary node as a explants has been shown to be time-saving and convenient strategy for micropropagation of soybean. So we have evaluated regeneration protocol through single shoot using cotyledonary node as a rapid and efficient protocol for two soybean cultivars and one mutant line. Cotyledonary nodes explants obtained from 7-days-old <em>in vitro</em> seedlings. After 28 days, the percent of regeneration and after 42 days, regeneration area were calculated. The results showed that percent of regeneration and regeneration area of mutant line was significantly more than two cultivars, L17 and Williams. After shoot induction, plants were transferred to shoot elongation medium followed by transferring plants to rooting medium. The results showed that the percent of rooting was not significantly affected by genotypes. In another experiment to test kanamycin sensitivity of regenerated shoots, it was found that kanamaycin with 150 mg/L concentration is lethal for regeneration of soybean shoots from cotyledonary node explants. The results showed that regeneration efficiency of mutant line was significantly more than two other cultivars. Kanamaycin sensitivity of regenerated shoots showed that kanamycin at 150 mg/L or above can be used as a selective agent for all three tested cultivars transformation.