Integrative analysis of salt tolerance in wheat using multi-gradient phenotyping and molecular/physiological validation.

Introduction: Salt stress is a major constraint on wheat growth and productivity, necessitating efficient methods for identifying salt-tolerant germplasm. Methods: In this study, 120 wheat varieties were evaluated at the seedling stage under four NaCl concentrations (0, 50, 100, and 150 mM). Key traits, including plant height, root length, germination rate, and salt tolerance index (STI), were measured across two independent replicates. Statistical analyses, including ANOVA, correlation analysis, principal component analysis (PCA), hierarchical clustering, and TOPSIS, were applied to assess phenotypic variation and rank salt tolerance. Representative tolerant and sensitive varieties were further validated through physiological and molecular analyses. Results: Increasing salinity significantly inhibited growth and germination, with the greatest variation observed at 100-150 mM NaCl. Salt concentration, genotype, and their interaction significantly affected all traits. STI showed strong positive correlations with growth traits. Multivariate analyses revealed stable phenotypic groupings and identified tolerant and sensitive varieties. Physiological and molecular validation demonstrated that tolerant varieties maintained better ion homeostasis, higher antioxidant activity, lower membrane damage, and favorable K⁺/Na⁺ ratios under salt stress. Discussion: This study establishes an integrated framework combining multi-gradient phenotyping, statistical analysis, comprehensive ranking, and mechanistic validation. The approach provides a reliable and scalable strategy for identifying salt-tolerant wheat germplasm and supports future genetic research and breeding programs.