Rice is one of the agronomically and nutritionally important cereal crops. It provides food for more than 50% of the world's population. However, the yield of rice crop is decreased by the salinity stress. This can be overcome by the development of salt-resistant rice varieties. In this study salt resistant callus cultures were developed by the somatic hybridization between Oryza sativa and Myriostachya wightiana. To optimize the isolation and fusion of protoplasts, the experiments were carried out with three different enzyme mixtures and different incubation periods. To know the salt tolerant ability, growth and total antioxidant activity were measured in the calli of Oryza sativa, Myriostachya wightiana, and hybrid at the different NaCl concentrations. In Oryza sativa high viable protoplasts yield was obtained by Enzyme mixture B (6.1 ± 0.2 × 10⁶) whereas, Myriostachya wightiana gives high viable protoplasts yield by Enzyme Mixture C (3.4 ± 0.2 × 10⁶). Optimum digestion time for the Oryza sativa was 12 h, which gave the highest number that is, 6.36 ± 0.15 × 10⁶ protoplasts/g FW and viability were 91.2 ± 1.05%. Optimum digestion time for the Myriostachya wightiana was 24 h, which gave the highest yield that is, 3.49 ± 0.08 × 10⁶ protoplasts/g FW and viability were 60.3 ± 1.72%. The fusion was performed with 50% (w/v) PEG-MW4000. The optimal incubation time was found as 30 and 45 min, which gave the highest number of fused viable protoplasts. A maximum number of binary fusions was achieved at 45 min incubation. The use of liquid culture medium resulted in a range of 5–20 calli per Petri dish which continued their growth. The hybrid calli show better growth in all NaCl concentrations. Hence, it was concluded that the hybrids have a better tolerance to salt stress. These results are useful in the development of salt tolerant rice varieties.