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An experiment was planned to explore variability of durum wheat toward salinity stress in randomised block design with two tolerant and one sensitive check under three different environments, i.e., control, ECiw—6 dS m−1 and ECiw—10 dS m−1 in three replications. Different physico-biochemical traits were measured in roots as well as shoots at the reproductive stage. Both roots and shoots showed MI of 8.5% on mean basis under normal condition which increased to 32.78% in roots and 31.44% in shoots at the extreme level of salinity ECiw—10 dS m−1. Significant increase was noted in proline content in roots as well as in shoots under stress environment and found approximately 2.5 times higher mean proline accumulation in roots and in shoots. TSS content increased in roots while reverse trend was noted for shoots. Shoots had higher accumulation of soluble proteins in comparison with roots, but the shoot soluble protein decreased with salinity while roots showed the reverse trend, i.e., content increased under salinity stress. KRL 99, KRL 3–4, MACS 3949 maintained their root as well as shoot Na+/K+ below 1 under the severe stress of ECiw—10 dS m−1. Results showed that genotypes exhibited a significant differential response and the antioxidative enzyme activity increased both in roots and shoots under mild and severe salinity. Overall, it was noted that individual genotypes displayed variability in terms of physico-biochemical traits toward salinity stress that confers their ability to survive under stress environment and could serve as a genetic source for salt tolerance breeding programmes.

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