Leaf Anatomical Traits as Candidate Biomarkers for Salt Tolerance Screening in Rice (Oryza sativa L.) ‘Tubtim Chumphae’ Identified by Discriminant Analysis

Rice cultivation faces major environmental challenges due to climate change, particularly soil salinity, which limits plant growth and productivity. Salt tolerance in rice is typically evaluated using physiological and biochemical traits, whereas leaf anatomical traits combined with advanced statistical analyses remain underexplored. This study investigated leaf anatomical characteristics of the rice cultivar Tubtim Chumphae at the seedling stage under different salinity levels (0, 25, 50, 75, and 100 mM NaCl). Seedlings were cultivated in a soil-based pot system for 42 days prior to treatment, and salinity stress was applied for 4 weeks. Data were analyzed using the Kruskal–Wallis test and multivariate approaches, including Discriminant Analysis of Principal Components (DAPC) and Partial Least Squares Discriminant Analysis (PLS-DA). The results revealed that several anatomical traits significantly varied with salinity, including vertical epidermal cell size of long cells (Epi-VL-LC), major vascular bundle size in the lamina (MVB-la-HL), major vascular bundle size in the midrib (MVB-mid-HL and MVB-mid-VL), as well as stomatal size (St-HL and St-VL) and stomatal density (StD) (p < 0.01). DAPC effectively distinguished salinity levels based on leaf anatomical traits, and the PLS-DA results further supported the robustness of the classification. Epidermal cell size, cell wall and cuticle thickness, stomatal traits, and vascular bundle dimensions were identified as key candidate anatomical biomarkers of salt tolerance. S75 (75 mM NaCl treatment) was suitable as a screening level and S100 (100 mM NaCl treatment) as a confirmation level. The findings provide a useful reference for evaluating salt tolerance in this rice cultivar and may be integrated with morphological, physiological, and biochemical traits to support future rice breeding programs. These findings provide a reference for evaluating salt tolerance in this cultivar and may complement morphological, physiological, and biochemical traits in future rice breeding programs.