# In Vivo Biosynthesis of Au and Ag NPs Using Two _Medicago sativa_ L. Genotypes
###### Abstract  
The nano size as well as physical and chemical properties of Au and Ag nanoparticles (NPs) allow them to be used in medicine or plant protection, but chemical solvents used during synthesis makes them toxic and pose a threat to the environment. Chemical NP synthesis can be replaced by in vivo synthesis in which independently growing plants such as alfalfa take up and then split the precursor in their cells down to nano size using synthesis-promoting solvent-biomolecules, which can break down materials without free radicals and have anti-inflammatory, antioxidant effects, making NPs environmentally benign. In this study, two-week-old seedlings of two _Medicago sativa_ L. genotypes, _'Kometa'_ and _'la Bella'_, were exposed to two precursors (AgNO\({}_{3}\), HAuCl\({}_{4}\)) for 24 and 48 h to determine whether in vivo synthesis is possible. Two-beam and certain wavelength spectrophotometry and confocal microscopy confirmed statistically significant (\(p<0.05\)) changes in light absorption and light fluorescence compared to the control. Confocal microscopy showed both precursors visible in the roots of both genotypes. Currently, NP synthesis and visualisation methods require a complex, expensive, and time-consuming sequence of methods. It is important to find an effective, environmentally friendly, and as cheap and simple as possible method for the biosynthesis of NPs. nanoparticles; alfalfa seedlings; reducing agents; spectrophotometry +
Footnote †: journal: Medical  
Although the size, shape, and other properties of NPs can be well controlled by physical and chemical methods, metal solvents are often toxic substances, and solvent toxicity can be transferred to synthesised NPs [8]. Due to their small size and ability to increase toxicity, Ag and Au NPs can be dangerously toxic to humans and animals [9; 10]. NP biosynthesis, as one of the bottom-up methods, allows the replacement of mechanical or artificially obtained solvents for the synthesis of Ag and Au and other NPs with natural solvents obtained from living organisms, such as plants, bacteria, algae, and animals [11; 12; 13; 14; 15; 16]. Plants are commonly used for NP biosynthesis due to the possibility of growing them easily or maintaining them more easily than bacteria and animals. Various agricultural plants, such as alfalfa (_Medicago sativa_ L.) [17; 18; 19] and _Aloe vera_ L. [20], are used in NP biosynthesis studies because they contain many biomolecules, such as phenols glucose, fructose, ascorbic acid, and citric acid [20; 21; 22; 23], which can divide metal precursors, for example, AgNO\({}_{3}\) and HauCl\({}_{4}\) to Ag and Au NPs [24; 23; 22]. Biosynthesis with plants can be performed using plant extracts obtained from plant parts, including seeds [25], leaves [19], and fruit [26]. Extracts can also be obtained from plant callus cells. It has been established that callus cultures have concentrated biomolecules in larger quantities than vascular plants [27]. The biosynthesis of Au [28], Ag [29], and other NPs [30] is also possible using the in vivo method when the metal precursors are split by plant biomolecules in the plant cells as the plant continues to grow [28]. The realisation of all biosynthesis methods takes time, especially the in vivo method [29]. All three NPs biosynthesis methods (synthesis with extracts from plant parts or callus cultures and in vivo biosynthesis) are much cheaper than physical or chemical methods because they do not use expensive chemical solvents and require only a minimum amount of advanced equipment [14; 25; 28]. Comparing all mentioned biological methods, the least studied method is the in vivo biosynthesis method [28; 29; 30]. In vivo biosynthesis needs to be studied further because, recently, a lot of environmental pollution has been discovered. Metals of various sizes, household chemicals, acids, nitrates, salts, and other substances enter ecosystems uncontrolled [9]. Plants can absorb these materials and chemicals and then synthesise NPs from them in their cells.