Abstract: Background and Objective: Indica rice genotypes are recalcitrant in nature to in vitro callus initiation and regeneration which causes less amenable to genetic modification in comparison with japonica rice. The present study was aimed to establish suitable callus initiation and regeneration system for 4 Indica rice cultivars i.e., Swarna, Kasalath, Lonabalam and Biron. Methodology: Murashige and Skoog (MS) media supplemented with different concentrations (0.0, 0.5, 1.0, 1.5, 2, 2.5, 3.0 mg L–1) of 2,4-dichlorophenoxyacetic acid (2,4-D) to prepare callus induction media (CIM) for callus initiation. In addition, L-proline in combination with 6-benzylaminopurine and 2,4-Dichlorophenoxyacetic acid (2,4-D), also implemented to investigate callus formation due to use amino acid as a supplement. Afterward, MS media fortified with different shoot and root inducing hormone at different concentration for regeneration purpose. Results: The results showed that Kasalath has highest callus induction frequency 76.47±2.12% at 1.0 mg L–1 2,4-D concentration whereas Swarna, Lonabalam and Biron callus induction frequency were 67.87±2.08 , 36.84±0.70 and 63.15±1.41% at 2.5, 2.0 and 1.0 mg L–1 2,4-D concentration, respectively. Addition of proline showed better callus induction frequency for Kasalath cultivar which is 86.66%. After subculture of primary calli, callus was inoculated into shoot induction media (SIM) and highest shoot induction frequency was observed in Lonabalam, 63.63±1.52% at 0.5 mg L–1 NAA, 2.0 mg L–1 BA, 3.0 mg L–1 KIN hormonal concentration. Duration of Shoot initiation was observed at 32 days, 27 days, 24 days and 33 days’ interval for Swarna, Kasalath, Lonabalam and Biron. Regenerated plantlets were then inoculated into rooting media for root induction. After root formation plantlets were transferred to the pots for acclimatization. Conclusion: Among different Indica rice, Swarna and Kasalath showed better callus frequency whereas Lonabalam was found better responsive to regeneration media. Besides conventional hormonal effect, L-proline has been proved effective only for Kasalath genotype for callus formulation.