Synergistic effects of 2,4-D and PEG-induced osmotic stress on morphogenesis and physiological adaptation of date palm cv. Sukkari in vitro

Large-scale micropropagation of date palm ( Phoenix dactylifera L.) cv. 'Sukkari' is limited by oxidative and osmotic stress during in vitro culture. This study tested the hypothesis that moderate 2,4-dichlorophenoxyacetic acid (2,4-D) enhances callus competence under non-stress conditions, whereas high 2,4-D combined with polyethylene glycol (PEG-6000) induces physiological costs that reduce plantlet quality. A 3×3 factorial completely randomized design was used with three 2,4-D concentrations (50, 100, 150 μM) and three PEG-6000 levels (0%, 10%, 20%). The experimental unit was one culture jar containing three explants; jar means were used as biological replicates, n = 5. Callus traits, growth parameters, photosynthetic pigments, water relations, osmoprotectants, antioxidant enzymes, and molecular indicators were measured. Two-way ANOVA was performed with F-values, degrees of freedom, and P-values reported; assumptions were checked using Shapiro-Wilk and Levene’s tests. The interaction between 2,4-D and PEG was significant for most traits (P < 0.01). 100 μM 2,4-D without PEG optimized morphogenesis, reducing callus induction time by 34% to 23.2 days and increasing callus volume 6.3-fold to 3.21 cm 3 compared with 150 μM × 20% PEG. Dry weight reached 0.195 g and multiplication rate 4.48 shoots/explant under optimal conditions. Severe stress (20% PEG) reduced survival by 85% to 14.22%, inhibited shoot multiplication completely at 150 μM × 20% PEG (0.00 shoots/explant), and decreased relative water content to 59.3% and membrane stability index to 44.0%. Proline and soluble sugars increased 11.3-fold and 2.3-fold respectively at 20% PEG, with superoxide dismutase and peroxidase activities rising >200%. Gene expression of stress-related genes increased up to 8.2-fold under severe stress. DNA quality measured by OD260/280 ratio declined to 1.19, indicating reduced DNA purity. Claims of clonal uniformity require SSR/ISSR validation. 100 μM 2,4-D with 0–10% PEG is recommended for efficient micropropagation, while severe stress should be avoided commercially. Future research should validate these findings under ex vitro acclimatization conditions.