The calli cultures of Guizotia abyssinica (niger) cultivars IGP 76 and GA 10 were exposed to different levels of salt treatments (0, 30, 60, and 90 mM NaCl), in order to evaluate growth, physiological, and biochemical responses. A significant decrease in relative growth rate and tissue water content of GA 10 calli than IGP 76 under salt-stress conditions was associated with higher sodium ion accumulation. Osmotic adjustment revealed by the osmolytes (proline, glycine betaine, and total soluble sugars) accumulation was significantly higher in IGP 76 salt-stressed calli as compared to GA 10. The sustained growth and better survival of IGP 76 calli was correlated with lower malondialdehyde content and increased superoxide dismutase, ascorbate peroxidase, and catalase activities and higher ${alpha}$-tocopherol content in comparison to GA 10. The higher osmolytes accumulation and presence of better antioxidant system suggested superior adaptation of IGP 76 calli on salt-containing medium for prolonged periods in comparison to GA 10. The regeneration frequency, organogenesis, and acclimatization response of the plants derived from salt-adapted calli was comparatively lower than the plants derived from control calli of IGP 76. The growth, physiological, and biochemical characterization of the salt-tolerant regenerated plants exposed to stepwise long-term 90 mM NaCl treatment revealed no significant changes in comparison to the control. Thus, our results suggests the development of an efficient protocol for in vitro selection and production of salt-tolerant plants in self-incompatible crop, niger, and an alternative to traditional breeding programs to increase the abiotic stress tolerance.