# In Vivo Biosynthesis of Au and Ag NPs Using Two _Medicago sativa_ L. Genotypes
## 4 Discussion
It is possible that higher concentrations of HAuCl\({}_{4}\) and AgNO\({}_{3}\) stick around the roots of the seedlings and block the root vascular tissue even before the precursor is taken up by the plant. Confocal microscope images also showed that, in the samples that received precursors, their presence was not visible or visible very weakly in the leaves. Ag and Au particles were visible in the root samples of both alfalfa genotypes after 24 or 48 h of exposure tothe precursor. It is possible that the technical capabilities of the microscope interfered with obtaining good images; thus, it cannot be claimed that the precursor was not present in the leaves. The presence of the precursor in the plant leaves may be indicated by changes in the maximum fluorescence intensity in all experimental groups compared to the control. The penetration of both precursors into the green mass of both alfalfa genotypes was confirmed by the results of two-beam spectrophotometry. As the precursor concentration increased, light absorbance increased for almost all samples compared to the control but decreased for _'la Bella'_ samples that received AgNO\({}_{3}\). In general, the results were quite variable. The variability of the results was also confirmed by NanoDrop spectrophotometry, which showed that the presence of both precursors changed the light absorbance in all experimental groups, but both decreased and increased at different concentrations of the precursor. This study used very young seedlings (only 2 weeks old), while other studies used much older plants (48 days to several months old). Young plants may be more sensitive to precursor- or NP-induced toxicity. Therefore, NP biosynthesis can also be disturbed and difficult. Lower concentrations of precursors should be recommended for future studies [29; 41]. According to the confocal microscope images, HAuCl\({}_{4}\) penetrated the root tissues of both genotypes after exposure to the precursor for 24 h, but when the exposure was longer, the particles were clearly visible only in the _'la Bella'_ sample. AgNO\({}_{3}\) particles were not visible in _'Kometa'_ plants after 24 h of exposure but were visible after 48 h of exposure. In both _'Kometa'_ and _'la Bella'_ samples, the potential synthesis of Ag NPs could take longer than the biosynthesis of Au NPs. In the confocal microscope images, Au particles were visible in three different samples, while Ag particles were clearly visible in only two samples. These results indicate that the synthesis of Au NPs is not only faster but also more frequent. According to the confocal microscope results, one of the precursors was clearly visible three times in the _'la Bella'_ samples but only two times in the _'Kometa'_ samples. However, changes in light fluorescence and absorption were detected in almost all experimental samples; thus, these results did not show which alfalfa genotypes had more efficient biosynthesis. The particles often formed large agglomerates, which produced bright fluorescence. This study is significant in that it showed that particles from both HAuCl\({}_{4}\) and AgNO\({}_{3}\) precursors can penetrate and interact with the young root and leaf cells of two _Medicago sativa_ L. genotypes. Based on the impairment of plant growth after the uptake of precursors and fluorescence intensity changes in confocal microscopy, it can be theoretically stated that Ag and Au NPs might have been synthesised using the in vivo method. This interaction was detected using methods that consume a small number of resources and time. However, these results are not enough to say absolutely which substances were present in the plant cells, whether they were NPs or still a precursor. It is clearly understood that in vivo biosynthesis studies should be continued and an analysis of the chemical composition of plants should be carried out, which would confirm the presence of NPs.