Using Medicago sativa L. Callus Cell Extract for the Synthesis of Gold and Silver Nanoparticles

Gold (Au) and silver (Ag) nanoparticles (NPs) are used for drug transport and plant protection due to their insoluble nature and unique properties. To produce health-friendly NPs, toxic solvents should be replaced with plant-based synthesis. Plants, such as alfalfa (<i>Medicago sativa</i> L.), release biomolecules that reduce metal ions and form nanoclusters without free radicals, showing anti-inflammatory and antioxidant properties. In this study, callus cultures of two <i>M. sativa</i> genotypes, 'Kometa' and 'La Bella Campagnola', were exposed to two precursors (AgNO₃ and HAuCl₄) for 24 and 48 h to assess the feasibility of biological NP synthesis. Spectrophotometry showed significant (<i>p</i> ≤ 0.05) changes in light absorbance compared with the control. Dynamic light scattering and zeta potential measurements indicated a change in the composition of the liquid compared with the control. To improve image quality and obtain more accurate data, transmission electron microscopy (TEM) analysis was repeated, confirming the presence of quasi-spherical nanoparticles with diameters in the range of 5-25 nm for both AuNPs and AgNPs in the callus culture extracts of both genotypes. Nanoparticle Tracking Analysis demonstrated that the AgNPs and AuNPs from both genotypes displayed polydisperse size distributions, with a mean particle size ranging from 220 to 243 nm. Elemental analysis provided clear evidence that Ag and Au were present only in treated samples, confirming effective NP biosynthesis and excluding contamination. X-ray diffraction (XRD) analysis was performed to characterise the crystalline structure; however, due to the very small particle size (5-25 nm), no clear diffraction patterns could be obtained, as nanocrystals below ~20-30 nm typically produce signals below the detection limit of standard XRD instrumentation. The novelty of this research is the cost-effective, rapid biosynthesis of quasi-spherical AuNPs and AgNPs with diverse sizes and enhanced properties, making them more eco-friendly, less toxic, and suitable for antibacterial and anticancer studies.