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We previously reported an alfalfa half‐sib family, HS‐B, with improved salt tolerance, compared to parental plants, P‐B. In this study, we conducted additional experiments to address potential physiological mechanisms that may contribute to salt tolerance in HS‐B. Vegetatively propagated HS‐B and P‐B plants were treated with a nutrient solution (control) or a nutrient solution containing NaCl (EC = 12 dS/m). Shoots and roots were harvested at various time points after treatment for quantification of proline, soluble sugar, and H2O2 using spectrophotometers. Subcellular localization and quantification of Na in roots were conducted using a Na+‐specific dye under a confocal microscope. HS‐B produced 86 and 89% greater shoot and root dry biomass, respectively, compared to parental plants, P‐B, under salinity in the greenhouse. Under saline conditions the HS‐B shoots accumulated 115% and roots 55% more soluble sugars than P‐B counterparts. The non‐saline HS‐B shoots, however, showed 72% less soluble sugars than the non‐saline P‐B plants. Under saline conditions HS‐B accumulated 39% less proline in shoots, while roots accumulated 56% more proline, compared to their P‐B parents. HS‐B plants also showed a greater reduction of stomatal conductance under mild saline stress. HS‐B shoots and roots contained 3–4 times less reactive oxygen species (H2O2) after salt treatment compared to P‐B plants. Sodium localization and distribution analysis using Na+‐specific dye revealed HS‐B plants accumulated 88% and 48% less Na+ in stele and xylem vessels compared to P‐B. The study provides insights into the potential mechanisms that may contribute to salt tolerance in HS‐B: maintaining RWC by accumulating soluble sugars while reducing transpiration, maintaining healthy status by reducing oxidative stresses, and preventing salt toxicity by reducing accumulation of Na+ inside root cells and xylem.
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