Effect of Green Synthesized Fe3O4NP Priming on Alfalfa Seed Germination Under Drought Stress

Drought stress is one of the key environmental factors restricting the germination of alfalfa seeds (<i>Medicago sativa</i> L.). Nanopriming is an innovative seed-priming technology able to meet economic, agronomic, and environmental needs in agriculture. However, the use of conventional nanomaterials is hampered by high costs, environmental risks, and biotoxicity. In this study, we synthesized iron oxide nanoparticles (Fe₃O₄NPs) using seasonal <i>Ginkgo biloba</i> leaf extracts (collected from August to November) obtained via an enzymatic ultrasonic-assisted method. The synthesized Fe₃O₄NPs were characterized using SEM, EDS, DLS, FTIR, UV-Vis, and XRD. To investigate the effects of Fe₃O₄NP priming on alfalfa seed germination under drought stress, germination and pot experiments were conducted with five Fe₃O₄NP priming concentrations (unprimed, 0, 20, 40, and 60 mg/L) and three PEG-6000 concentrations (0%, 10%, and 15%) to simulate normal, moderate, and severe drought conditions. The results showed that leaf extracts collected in November exhibited the highest flavonoid content (12.8 mg/g), successfully yielding bioactive-capped spherical Fe₃O₄NPs with a particle size of 369.5 ± 100.6 nm. Germination experiments revealed that under severe drought stress (15% PEG-6000), the 40 mg/L Fe₃O₄NP treatment most effectively enhanced seed vigor, increasing the germination rate, vigor index, and α-amylase activity by 22.1%, 189.4%, and 35.5% (<i>p</i> < 0.05), respectively, compared to controls. Under moderate drought stress (10% PEG-6000), the 20 mg/L Fe₃O₄NP treatment optimally improved germination traits, increasing the germination rate by 25.5% and seedling elongation by 115.6%. The pot experiments demonstrated morphological adaptations in alfalfa seedlings: under moderate drought stress, the 40 mg/L Fe₃O₄NPs significantly increased lateral root numbers, while under severe drought stress, the 60 mg/L Fe₃O₄NPs increased the root surface area by 20.5% and preserved the roots' structural integrity compared to controls. These findings highlight that Fe₃O₄NPs synthesized via Ginkgo leaf extracts and enzymatic ultrasonic methods exhibit promising agricultural potential. The optimal Fe₃O₄NP priming concentrations enhanced seed vigor, germination traits, and drought resistance by modulating root morphology, with concentration-specific efficacy under varying drought intensities.