Developing a dependable, efficient and in vitro regeneration system is crucial for facilitating genetic transformation in rice, particularly for indica cultivars like MTU 1010, which are often resistant to tissue culture techniques. This study presents an optimized protocol that significantly improves regeneration efficiency in Oryza sativa cv. MTU 1010. Mature seeds dehusked, surface-sterilized, and cultured on Murashige and Skoog (MS) medium augmented at 2.5 mg/L Utilization of the 2,4-dichlorophenoxyacetic acid (2,4-D) at a concentration of 30 g/L maltose to induce callus development. Embryogenic calli, predominantly originating from the scutellum, had favorable morphological characteristics, including compactness and friability, within one week of incubation. Among the various treatments, a concentration of 2 mg/L 2,4-D produced the highest callus induction rate of 87.89%. And MS medium supplemented at 0.5 mg/L α-naphthaleneacetic acid (NAA) and 2 mg/L 6-benzylaminopurine (BAP) is used for shoot organogenesis. supported optimal regeneration, achieving an efficiency of 87.5%. Regenerated shoots exhibited healthy elongation and rooting, with a survival rate of 90% upon acclimatization. This reproducible protocol addresses cultivar-specific challenges and provides a valuable framework for future genetic engineering efforts, including Agrobacterium-mediated gene transfer and CRISPR/Cas9 applications aimed at improving stress tolerance and yield potential in rice.