This study delves the challenges and potential solutions associated with the in vitro culture of Sorghum bicolor, a plant species that has historically been difficult to regenerate in a laboratory due to its recalcitrant nature. The primary objective of this research was to evaluate the impact of various hormonal treatments on the induction of callus - a mass of undifferentiated plant cells and the subsequent regeneration of the plant from the callus using tissue culture techniques. The research utilized two types of explant sources: immature embryo and mature seeds. These were subjected to a range of concentrations (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg/L) of the hormones 2,4-Dichlorophenoxyacetic acidIndole-3-acetic acid, 6-Benzylaminopurine, Kinetin (6-furfurylaminopurine) and α-Naphthaleneacetic acid. The aim was to induce callus formation and stimulate the regeneration of six distinct sorghum genotypes. These included IS 3477, IS 33095, IS 7005 (non-pigmented), IS 2898, IS 7115, and IS 1202 (pigmented). Results showed that callus induction frequencies varied significantly among the different genotypes. IS 3477 and IS 33095 exhibited the highest rates of callus induction, while IS 7115 and IS 1202 showed the lowest. The most significant embryogenic callus induction was observed in IS 3477 (92%) when treated with 2,4-D at 2.5 mg/L and KN at 0.5 mg/L. In contrast, IS 1202 displayed the lowest induction rate (48%). Further investigation into the effects of various cytokinin concentrations revealed that a regeneration medium containing 2.0 mg/L BAP resulted in high regeneration frequencies. This was particularly evident in the non-pigmented IS 3477 genotypes, which produced the highest number of shoots (20). Additionally, mature seed explants displayed varying embryogenic callus induction frequencies. IS 3477 exhibited the highest rate (76%) when treated with 2,4-D at 2.5 mg/L and BAP at 0.5 mg/L, while IS 1202 displayed the lowest induction rate (36%). Optimal regeneration responses were observed with ZN at 1.5 mg/L, with IS 3477 producing the highest shoot count (11). This study elucidated the genotype-specific responses to in vitro protocols across diverse sorghum-pigmented and non-pigmented lines. The findings offer valuable insights into the development of high-frequency response profiles, crucial for advancing impending genetic transformation efforts. This research underscores the importance of understanding different genotypes' specific requirements to optimize in vitro culture techniques for plant regeneration.