In Vitro Assessment of Salt Tolerance in ‘Balady’ Apple (Malus domestica) Seedling Clones

Soil salinization has become significant threat to some regions' apple production. This study aimed to improve the detection and selection process for salt stress tolerance in ‘Balady’ apple (Malus domestica) seedling clones using advanced tissue culture techniques. Open-pollinated mature seeds of the local Egyptian ‘Balady’ apple were collected from a single tree, and then each surface-sterilized seed was cultivated independently and assigned a distinct serial number. After three consecutive subcultures, salt stress was induced by supplementing the proliferation medium with graded NaCl concentrations (0, 1000, 2000, and 3000 ppm). The micro-propagated individuals were not identical according to similarity indices based on Inter Simple Sequence Repeats; the lowest percentage of 84.9% was scored between the mother plant and the MS5 clone. The MS5 clone exhibited superior growth parameters under elevated salinity levels compared to other tested clones, highlighting its resilience and adaptability. The MS5 clone, the most salt-tolerant clone, showed the lowest levels of reduced sugars and total phenols and minimal overall antioxidant activity. However, it demonstrated stimulated enzymatic antioxidant activity at the highest salt concentration. Data from three common differentially expressed genes revealed that protein phosphatases PP2C-77a and ABA-insensitive transcription factor ABI5-5b were downregulated. Also, the serine/threonine-protein kinase SAPK3 was overexpressed in the MS5 genotype under severe salt stress conditions. A unique 290 bp positive marker was identified in the salt-tolerant MS5 clone using the HB12 ISSR primer, providing molecular basis for its tolerance. This study confirmed the viability of in vitro screening for salt-tolerant genotypes within apple seedling populations.