Sugarcane, an important agro-industrial crop, has narrow genetic base, low fertility and is difficult to cross, making crop improvement through conventional breeding a difficult task. Genetic transformation is an ideal approach for incorporating resistance against lepidopteran insects that adversely affect sugarcane production. Establishment of somatic embryogenic cultures is a pre-requisite for transformation as genetically uniform somatic embryos lead to true-to-type plant production. In this study, medium for direct somatic embryogenesis and plantlet regeneration from spindle leaf segments was optimized for CoJ 64 variety. A total of 41 media were tested, wherein effect of various auxins, cytokinins, agar, amino acids, adjuvants, carbon sources on induction of somatic embryos and regeneration from the segments was evaluated. Maximum somatic embryogenesis (97.61%) and plantlet regeneration (93.74%) was achieved in one-step on C6 medium [MS + NAA (5 mgL−1) + Kin (0.5 mgL−1) + maltose (20 gL−1) + agar (1.6%)]. Optimum levels of glutamine (100 mgL−1), citric acid (100 mgL−1) and activated charcoal (1000 mgL−1) for increasing percent somatic embryogenesis and regeneration were identified. The segments (468 in number) bearing somatic embryos were used as target tissue for particle gun-mediated transformation using cry1Ac, thereafter the segments were selected and cultured on C6 medium leading to generation of putative transgenic plantlets in a period of 11 weeks. Molecular analysis confirmed the insertion of cry1Ac in 24 plantlets (5.13% transformation frequency), which exhibited normal growth under transgenic glasshouse conditions. The study resulted in broadening the genetic base of a highly polyploid crop. The one-step medium can be used for obtaining transgenic plants for different agronomic traits in sugarcane.