This study was carried out at Tissue Culture Laboratory, Fac. Environ. Agric. Sci., Arish University, North Sinai, Egypt, during the period from 2016 to 2018. The main objective of this study was to study the effect of explants type, growth regulators and light on callus induction and growth as well as effect of mannitol as osmotic stress agent on production of active ingredients from Capparis spinosa L. callus culture. Callus cultures were initiated from in vitro leaves and stems which isolated from 6-weeks-old in vitro shoots and cultured on MS solid medium supplemented with various concentrations of (2, 4-D) combined with Kinetin (Kin) or 6-Benzyladenine( BA) . Cultures were maintained under darkness or continuous light conditions at 25 ± 2^oC. After 4 weeks from subculture of callus on free MS medium, calli were individually weighed and about l g of callus was placed on MS medium fortified with mannitol (0, 2, 4 or 8 gl^-1). All cultures were maintained under dark condition at 24°C for 6 weeks. Results indicated that either control treatment or cytokinin treatments couldn't induce callus on both explant types. However, 2, 4-D either alone or combined with different cytokinin treatments induced callus on both explant types without significant differences among these treatments in most cases. Callus dry weight was reached to the maxinaum values when leaf explants were cultured on MS medium supplemented  with  1.0 mgl^-1 2, 4-D + 2 mgl^-1 Kin or BA and when nodal segments were cultured on MS medium fortified with 2,4-D and kin at 2.0mgl^-1 for both of them under darkness. Also, the obtained results revealed that, addition of 4 gl^-1 mannitol gave the highest  values for each of callus fresh and dry weights (g) while, addition of 8 gl^-1 mannitol gave the highest content of proline compared with control treatment. Moreover, the GC–MS analysis revealed to identify of thirty two components some of these compounds were higher with mannitol at 4gl^-1 compared with control.