To predict early heterosis and inbreeding depression for some callus traits in 4 bread wheat hybrids produced from 7 parents, 2 different experiments were achieved by using tissue culture technique in order to study the response of the exposed populations to water stress due to adding mannitol to MS media. Thus, the 1st experiment was a preface to decide which concentration of 2 or 2.5 mg/l of 2,4 Dichloro phenoxy acetic acid hormone (2,4-D) beside 0.5 mg/l of naphthalene acetic acid (NAA) for each concentration, will be more suitable undertaken in this study. Consequently, regarding the 2nd experiment, the selected concentration was reliable to use in the main experiment, discriminations were callus induction % and callus fresh weight (mg) for the 1st one. While, for the 2nd which exhibited the effect of ascorbic acid (AsA) in the tolerance improvement to stress of the tested populations, callus growth index (CGI), Invitro tolerance (INTOL), relative tolerance percentage (Rt %) and reduction percentage (R%) were used as osmotic stress tolerance indices.