Comparative effects of mixed saline-alkaline stress on seed germination and early seedling growth in proso millet: the dominant role of alkalinity.

Soil salinization and alkalization severely constrain crop production worldwide, yet the combined effects of mixed saline-alkaline stress on minor cereal germination have received limited investigation. This study sought to elucidate the effects of mixed saline-alkaline stress on seed germination and seedling growth of proso millet (Panicum miliaceum L.) under controlled laboratory conditions, and to comparatively assess the relative contributions of osmotic stress, ionic toxicity, and high-pH damage through analysis of different neutral-to-alkaline salt ratios at equivalent Na⁺ concentrations. Seeds were subjected to nine treatment combinations comprising three salinity levels (80, 160, and 240 mM Na⁺) and three neutral-to-alkaline salt ratios (3:1, 1:1, and 1:3), employing NaCl, Na₂SO₄, NaHCO₃, and Na₂CO₃. Germination characteristics, seedling morphology, and key physiological parameters were assessed over seven days. Mixed saline-alkaline stress markedly suppressed germination in a concentration-dependent manner, with germination percentage decreasing from 94.5% (control) to 23.8% at 240 mM Na⁺ under high-alkali conditions (P < 0.05). At equivalent Na⁺ concentrations, high-alkali treatments (pH > 9.5) diminished germination index by 35.6-52.3% relative to low-alkali treatments. The IC₅₀ values were 187.3 mM Na⁺ (95% CI: 171.2-205.8 mM; R² = 0.982), 142.6 mM Na⁺ (95% CI: 131.4-155.1 mM; R² = 0.976), and 98.4 mM Na⁺ (95% CI: 89.7-108.3 mM; R² = 0.991) for low-, medium-, and high-alkali ratios, respectively. High-alkali stress was associated with 67.4% greater electrolyte leakage and 2.3-fold higher MDA accumulation than neutral salt treatments at equivalent Na⁺ concentrations, indicating greater membrane disruption under high-pH conditions. Two-way ANOVA revealed significant main effects of both Na⁺ concentration and alkaline proportion, as well as a significant salinity × alkalinity interaction (P < 0.001; Supplementary Table S1). These results demonstrate that alkaline stress exerts stronger inhibitory effects on proso millet germination compared to neutral salt stress, with the magnitude of salinity effects being dependent on the pH level. While complete mechanistic separation of osmotic, ionic, and pH components was not achieved due to experimental design limitations (absence of iso-osmotic non-ionic controls and direct ion measurements), comparative analysis across salt ratio treatments suggests that high pH is associated with additional inhibitory effects beyond those observed in neutral salt treatments. These controlled-condition findings establish preliminary germination -stage thresholds that may inform screening protocols for saline-alkaline tolerance breeding in proso millet, pending validation under field conditions.