Phytotoxicity and Biostimulant Potential of Graphene Acetic Acid and Nitrogen-Doped Graphene in Tomato Seed Priming

Graphene-based nanomaterials are increasingly investigated as seed treatments and agricultural delivery platforms, yet their biological effects vary with material chemistry, concentration, and growth environment. This study evaluated the phytotoxicity and biostimulant potential of Graphene Acetic Acid (GAA) and Nitrogen-Doped Graphene (N-DG) applied through seed priming to tomato (Solanum lycopersicum L. cv. Rio Grande) and generated baseline evidence relevant to future nanocarrier applications. Seeds were water-primed or primed with 50, 350, or 800 mg L⁻¹ GAA or N-DG. In Petri-dish experiments, seed-surface interactions were examined using environmental scanning electron microscopy and energy-dispersive X-ray spectroscopy, and seedling length was measured after 4 and 6 days. GAA was subsequently assessed in a 23-day germination-tray experiment using agronomic and organic soils. Leaf biomass, root-system characteristics, photosynthetic pigments, metabolic markers, hydrogen peroxide (H₂O₂), soluble protein, and antioxidant enzyme activities were evaluated. Seed-surface analyses revealed treatment-associated morphological changes and differences in surface carbon content but did not demonstrate internal uptake. GAA significantly increased seedling length relative to the water-primed control at 800 mg L⁻¹ on Days 4 and 6 and at 350 mg L⁻¹ on Day 6, whereas N-DG produced no significant effect. In the germination-tray experiment, GAA did not affect leaf fresh or dry weight. However, all GAA concentrations significantly increased total root length, projected area, surface area, average root diameter, root volume, and the numbers of root tips and forks in agronomic soil; comparable responses were absent in organic soil. Photosynthetic pigments, total phenolics, total flavonoids, and soluble sugars were not significantly altered. Free amino acids, H₂O₂, soluble protein, guaiacol peroxidase, and superoxide dismutase displayed soil- and concentration-dependent responses without consistent increases in malondialdehyde, pigment loss, or reduced leaf biomass. Overall, GAA showed context-dependent biostimulant potential, particularly for early seedling growth and root-system development in agronomic soil, with little evidence of generalized phytotoxicity under the tested conditions. The contrasting responses between soils demonstrate that the post-priming growth environment strongly influences the biological response to GAA. Further evaluation across soil types, concentrations, crops, and longer growth periods is required before GAA or N-DG can be considered for agricultural biostimulant or nanocarrier applications.