Evaluation of Seedling-stage Salt Tolerance in USDA Spinach (Spinacia oleracea L.) Accessions

Abstract

Soil salinity is a major constraint on crop production, and improving salt tolerance requires phenotyping methods that capture multiple components of the plant stress response. Salt tolerance was evaluated in 150 US Department of Agriculture spinach ( Spinacia oleracea L.) accessions at the seedling stage under 300 mM NaCl and a nonsaltwater control (no added NaCl) using a completely randomized design with three replications. Visual leaf injury, leaf chlorophyll content, and seedling height were measured, and both absolute and proportional stress indices were calculated. Salt-tolerant accessions were defined as those that maintained lower leaf injury, retained more leaf chlorophyll, and showed less reduction in seedling height under salt stress relative to the control. Genotypic effects were significant for all traits ( P < 0.0001), and broad-sense heritability was high, ranging from 89.70% to 99.12%. Leaf injury score correlated moderately with chlorophyll reduction ( r = 0.70), whereas no statistically significant correlation was detected between chlorophyll reduction traits and seedling height reduction traits. These results indicate that visual injury, chlorophyll reduction, and growth inhibition provide complementary information for evaluating seedling salt tolerance. An average salt-tolerance rank (ASR) integrating the leaf injury score (LIS), absolute decrease in chlorophyll (AD_C), inhibition index of chlorophyll (II_C), absolute decrease in seedling height (AD_SH), and inhibition index of seedling height (II_SH) identified PI 177082 as the most tolerant accession and PI 648962 as the most sensitive accession. PI 177082 had an ASR of 11.50, with an LIS of 1.67, an AD_C of 8.67 soil plant analysis development units, an II_C of 26.94%, an AD_SH of 7.33 cm, and an II_SH of 26.51%. Hierarchical clustering broadly supported the ASR-based classification while showing phenotypic diversity among the tolerant accessions. These findings indicate that seedling salt tolerance in spinach was better characterized by the combined descriptors than by a single trait. The contrasting accessions identified in our study provide a phenotypic resource for future validation, breeding, and association mapping studies aimed at improving salt tolerance in spinach.