# In Vivo Biosynthesis of Au and Ag NPs Using Two _Medicago sativa_ L. Genotypes
## 4 Discussion  
Metal NPs, especially Ag and Au, have very wide applications in medicine, the food industry, cosmetic product production, the improvement of plant protection products, and other industries [3]. As the demand for NPs increases, they are being synthesised in larger quantities [2]. Physical and chemical methods make NP synthesis faster and more easily performed on a large scale but often involve the use of chemical solvents that can further increase the toxicity of NPs [7]. Currently, one of the biggest challenges and urgent goals is to find a method of NP synthesis that is as efficient as possible but healthier for the environment and people [11; 12]. Biological synthesis of NPs is an efficient way to obtain NPs without excessive toxicity from chemical solvents. In contrast, biologically synthesised NPs have much better properties because plant biomolecules, as natural solvents, improve the properties of NPs, for example, strengthening the antibacterial properties of Ag NPs [36; 37] or making diagnostic examinations with Au NPs as contrast agents more harmless to the human body [22]. Among the methods of NP biosynthesis used in other studies, synthesis in which the precursor is dissolved with plant extract obtained from various plant parts, including leaves, flowers, and fruit, is most often used [38; 39]. NPs can be seeded very effectively in an extract obtained from plant callus cultures, as they also contain a high diversity and concentration of biomolecules [40]. NPs can also be synthesised biologically in the cells and tissues of independently growing plants (in vivo). The plant just needs to be able to take in the precursor and break it down to nano size. Until now, there have been very few studies in which such a synthesis has been demonstrated with different agricultural plants, including _Medicago sativa_ L. Existing studies claim that the in vivo method should be further researched to find the fastest, simplest, and cheapest method for in vivo NP biosynthesis [29; 41]. This study used the simplest and cheapest methods to provide and demonstrate the in vivo biosynthesis of Ag and Au NPs in the seedlings of two _Medicago sativa_ L. genotypes, _'Kometa_' and _'la Bella'_. One of the objectives was to determine which of the two precursor NPs were synthesised more efficiently and whether different selections of alfalfa genotypes influenced NP biosynthesis. Other studies have reported that Ag and Au NPs affect plant growth; the effect depends on the size, shape, and concentration of the NPs. Low and medium concentrations below 20 mg/L easily penetrate plants, reach leaves via vascular tissue, and increase the synthesis and concentration of nitrogen (N), phosphorus (P), and potassium (K) in leaves. However, if higher concentrations of Ag and Au accumulate in the leaves, the defence mechanism at the cell/tissue level is activated in the roots, the proteomic expression changes, antioxidant activity increases, metabolism and reactive oxygen species form, and the total amount of chlorophyll decreases [37]. The results of this study also confirmed that the control plants that did not receive the precursor were healthy; their leaves were bright green, and their roots were light grey in colour. In the experimental groups with any of the precursors at any concentration, the seedling leaves became paler, and plant growth suffered. This was especially evident in the samples that had received the highest concentrations of both precursors (AgNO\({}_{3}\), 1000 mg/L; HAuCl\({}_{4}\), 200 mg/L) for 48 h. Such negative morphological changes could indicate that Ag and Au NPs were successfully synthesised in the samples in this study. AgNO\({}_{3}\) is used in agriculture as a growth regulator, which mostly has a positive effect on plant growth, and Au compounds are involved in plant photosynthesis, but toxicity is characteristic only for NPs [33; 37]. Relatively high concentrations of the precursor were used in this study.