Integration of seed priming with nano-sized chitosan-proline and biochar application improves salt tolerance in differentially responding genotypes of alfalfa (Medicago sativa)

Context Salinity is one of the major abiotic stresses challenging alfalfa (Medicago sativa) production. Aims In this study, we evaluated the potential of nano-sized chitosan-proline (NsCP) seed priming and biochar application to enhance salt tolerance in alfalfa. Methods Seeds of two alfalfa genotypes (OMA-84, salt-sensitive; and OMA-285, salt-tolerant) were soaked for 18 h in aerated distilled water (hydropriming) or a solution of NsCP (100 mM) for seed priming. Seeds were then planted in plastic pots containing acid-washed pure sand supplemented with or without biochar (25 g kg−1 sand) and with or without salt stress (120 mM). Key results Both genotypes showed significant reduction in root and shoot growth, biomass production, and carbon assimilation under salinity stress, with more pronounced effects on OMA-84. However, applying both NsCP seed priming and biochar significantly improved the biomass production and plant photosynthetic assessment traits. Notably, this combined approach proved more effective in enhancing salt tolerance than individual treatments. Biochar amendment increased the Na+ and Cl− concentration but it also contributed to salt tolerance by elevating K+ level, promoting proline accumulation, and antioxidant activities. Conclusions NsCP seed priming enhanced the salinity stress tolerance in alfalfa genotypes by facilitating osmotic adjustment (proline accumulation), maintaining ionic homeostasis (higher K+ and lower Na+ concentration), and increasing the levels of α-tocopherol, flavonoids, and the activities of antioxidant enzymes. Implications Integrated application of NsCP and biochar significantly enhanced salt tolerance in alfalfa, demonstrating practical strategies for sustainable agriculture in saline environments by promoting ionic homeostasis, osmotic adjustment, and antioxidant defence mechanisms.