Insect-Spinach Waste Synergy: Enhancing Cyamopsis tetragonoloba under Copper Stress

Fe₃O₄ nanoparticles (NPs) coated with hyperbranched spermine derivatives and functional groups such as glucose and folic acid, synthesized via green methods using rosemary extracts, represent a novel strategy to improve plant resistance to heavy metal stress. Various formulations, including FSM-coated (spermine), FHS-coated (hyperbranched spermine), FSMPF-coated (FSM-polyethylene glycol-folic acid), FSMPG-coated (FSM-P-glucose), FHSPF-coated (FHS-P-folic acid), and FHSPG-coated (FHS-P-glucose), were tested for their ability to improve the quality and stress resistance of Guar beans under copper oxide stress (CuO stress, CS). Six formulations (FSM, FHS, FSMPF, FSMPG, FHSPF, FHSPG) were synthesized and characterized by NMR, FTIR, TEM, SEM, DLS, and VSM. Compatibility and DNA protection were confirmed via electrophoresis. Greenhouse experiments showed that FSMPG and FHSPF significantly improved seedling growth, antioxidant enzyme activities, and nutrient uptake, particularly iron and nitrogen, under CuO stress. Cell suspension cultures of Guar were established to assess cytotoxicity of FHSPF2 and FHSPG1 NPs via trypan blue assay, revealing >80% cell viability up to 500 μg/mL. MTT assays on MCF-7 human breast cancer cells demonstrated excellent biocompatibility of FHSPF2 and FHSPG1 (>85% viability at 1000 μg/mL), while other variants showed moderate toxicity. Antibacterial tests against Escherichia coli and Staphylococcus aureus indicated mild activity only at the highest doses, with minimal impact at 500 μg/mL. Image analysis confirmed that callus morphology and growth were preserved, compared with the control. These results highlight the safe use of FHSPF2 and FHSPG1 NPs in agricultural biotechnology and biomedical applications, providing a sustainable approach to mitigate heavy metal stress and enhance plant resilience.