Plant mediated fabrication of silver nanoparticles, process optimization, and impact on tomato plant

Nanotechnology is one of the fastest-growing markets, but developing eco-friendly products, their maximum production, stability, and higher yield is a challenge. In this study, silver nanoparticles were synthesized using an easily available resource, leaves extract of the Neem (Azadirachta indica) plant, as a reducing and capping agent, determined their effect on germination and growth of tomato plants. The maximum production of silver nanoparticles was noted at 70 °C after 3 h of reaction time while treating the 10 ml leaf extract of Neem plant with 10 ml of 1 mM silver nitrate. The impact of the extract preparation method and solvent type on the plant mediated fabrication of silver nanoparticles was also investigated. The UV-spectrophotometric analysis confirmed the synthesis of silver nanoparticles and showed an absorption spectrum within Δ420–440 nm range. The size of the fabricated silver nanoparticles was 22–30 nm. The functional groups such as ethylene, amide, carbonyl, methoxy, alcohol, and phenol attached to stabilize the nanoparticles were observed using the FTIR technique. SEM, EDX, and XRD analyses were performed to study the physiochemical characteristics of synthesized nanoparticles. Silver nanoparticles increased the germination rate of tomato seeds up to 70% while decreasing the mean germination time compared to the control. Silver nanoparticles applied at varying concentrations significantly increased the shoot length (25 to 80%), root length (10 to 60%), and fresh biomass (10 to 80%) biomass of the tomato plant. The production of total chlorophyll, carotenoid, flavonoids, soluble sugar, and protein was significantly increased in tomato plants treated with 5 and 10 ppm silver nanoparticles compared to the control. Green synthesized silver nanoparticles are cost-effective and nontoxic and can be applied in agriculture, biomedical, and other fields.

synthesized AgNPs also have been reported to show a negative impact on plant growth.AgNPs inhibit root growth, cause leaf damage, and result in oxidative stress, producing reactive oxygen species (ROS), which harms the overall health of the plant, especially when used in high concentrations.AgNPs also release toxic silver ions (Ag+) into the environment, which are taken up by plants and accumulate in their tissues.These negative effects interfere with the plant's ability to take in nutrients, hinder photosynthesis, and impede overall plant growth.
Solanum lycopersicum L. is one of the most consumed vegetables worldwide.It is also a source of healthpromoting biochemicals, including several carotenoids and phenolic compounds 44 .The current study reports the plant mediated fabrication of silver nanoparticles using the leaf extract of Azadirachta indica (Neem plant).The study aimed to optimize some important factors such as time, temperature, pH, volume of plant extract, concentration of reagent, method of extraction and solvent type to obtain the maximum yield of stable AgNPs.The synthesized nanoparticles were subjected to various techniques, including UV-spectrophotometry, FTIR, XRD, EDX, and SEM to study the characteristics of prepared AgNPs.It also determines their impact on seed germination and growth of Tomato plants under laboratory and greenhouse conditions.

Effect of temperature, time, pH, reagent concentration, and plant extract volume
To determine the effect of temperature, the resulting solutions of leaf extracts were incubated at room temperature at 30, and 40 °C and did not show any SPR peak within the range of 400-500.The synthesis of AgNPs started upon increasing the temperature up to 50-70 °C.The solutions of S1E1 and S1E2 (details have been mentioned in the materials and methods) extracts kept at 70 °C gave more absorption rates of 0.342 and 0.279, respectively, than 60 °C as shown in Fig. 1a (maceration method) and 2a (boiling water method) and selected for further procedure.When both methanolic leaf extracts (S2E1 and S2E2) were reacted with AgNO 3 at 50 °C, the synthesis of AgNPs was observed, and the UV spectrum gave a clear absorption peak.The increase in temperature increased the yield of AgNPs, and 70 °C was used for further procedure.The solutions of S3E1, S4E1, and S4E2 extracts also gave maximum production at 70 °C, while S3E2 exhibited a maximum absorption rate of 0.278 at 60 °C, further increase in temperature (70 °C) triggered the agglomeration in the reaction mixture and a decrease in absorption rate, therefore 60 °C was selected for further experiment.
To determine the effect of time, the leaf extract of each solvent was mixed with 1 mM silver nitrate solution in different vials and kept in an incubator at the selected temperature.The minimum absorption rate was noted after 1 h of reaction in the case of each solvent.The absorption rate of resulting solutions of S1E1 was more after 3 h (0.682) than 1 (0.342), 2 (0.589), 4 (0.325), 5 (0.273), and 6 h (0.225).A sharp and clear characteristic peak was observed within the range of 416-422 nm.The absorption rate of resulting solutions of S1E2 extract increased by increasing the time to 4 h (0.63) and then decreased.But 3 h time was selected with an absorption rate of 0.556 because the peak was shifted to a higher wavelength at 4 h, suggesting the production of larger AgNPs.Similarly, the methanolic extracts (S2E1 and S2E2) showed a maximum absorption rate after 3 h of reaction and minimum absorption at 6 h.The solutions of S3E and S3E2 extracts gave maximum production of AgNPs after 2 h (0.524 and 0.355, respectively) of reaction as the solutions exhibited higher absorption.In the case of S4E1 absorption rate was also increased by increasing the timing of the reaction up to 2 h but started to decrease after 3-6 h of reaction.In contrast, the resulting solution of S4E2 gave the maximum production of AgNPs after 4 h [Fig.1b (maceration method) and Fig. 2b (boiling method)].
To determine the effect of pH, the leaf extracts reacted with silver nitrate solution in an incubator, making the pH of solution 5, 7, 9, 11, and 13.No color change was observed at pH 5 and 7, and the UV spectrum showed no characteristic peak.The aggregation of molecules in the mixture could be seen clearly.The production of AgNPs started with an increase in the pH of the solution up to pH 13.The solution kept at pH 13 gave a broad peak indicating the synthesis of bigger AgNPs.The absorption rates of 0.387, 0.361, 0.227, 0.328, 0.376, and 0.283 were given at characteristic peak within 420-435 nm by S1E1, S1E2, S2E1, S3E1, S4E1, S4E2, respectively.The maximum absorption rate (0.237) was shown when the solution mixture of S2E2 and silver nitrate was kept at pH 9 instead of 11.While in the case of S3E2, pH 13 gave the maximum absorption rate of 0.586 and a sharp characteristic peak [Fig.1c (maceration method) and Fig. 2c (boiling method)].
To determine the effect of various reagent concentrations i.e., 1, 2, 3, and 4 mM of AgNO 3, were reacted with considered leaf extracts of the Neem plant.The leaf extract (S1E1), when treated with 1 mM and 2 mM silver nitrate solution, exhibited a color change from yellow to dark brown.The SPR peak was observed at 424 and 426 nm wavelengths, respectively.The peak was shifted to 412 nm at 3 mM concentration of silver nitrate, but the reaction mixture changed slightly in color.The maximum absorption rate (0.697) was presented when 1 mM solution of silver nitrate was reacted with S1E2 to synthesize nanoparticles.The leaf extract (S2E2), when treated with 1 mM and 2 mM silver nitrate solution, exhibited the characteristic peak at 432 and 420 nm wavelengths, respectively.A more absorption rate of 0.812 was shown in the 2 mM solution than in 1 mM.The maximum AgNPs were produced when 1 mM silver nitrate was reacted with S3E1.UV-vis spectrum displayed a characteristic peak at 424 and maximum absorbance (1.162).The leaf extracts S3E2 exhibited maximum absorption in a 2 mM reagent solution.The leaf extract of the Neem plant S4E1 and S4E2, when treated with 1 mM and 2 mM silver nitrate solution, exhibited a color change, and the characteristic peak was observed within 420-430 nm.The absorption rate again decreased at 3 mM concentration of AgNO 3, the color change was also slight, and aggregation occurred due to a higher concentration of the reagent in the mixture.The reaction mixture's color didn't alter, and the expected peak still appeared when 4 mM concentration was used, indicating no synthesis of AgNPs due to higher concentration of salt as compared to the presence of biomolecules in the extract [Fig.1d (maceration method) and Fig. 2d (boiling method)].
To determine the effect of plant extract volume, the silver nitrate solution was added in 10, 20, 30, 40, and 50 mL of each leaf extract in different vials and kept in an incubator.The lowest absorbance and slight color change were shown when 10 mL reagent was reacted with 40 and 50 mL of each plant extract, and a characteristic peak was observed in the UV spectrum.When the volume of plant extract was decreased, the absorbance was increased, suggesting the reduction of silver into silver nanoparticles.The maximum absorption rate was shown when the reagent was reacted with plant extracts S1E1 and S1E2, respectively, in a 1:2 ratio.The maximum

Effect of solvent type
The extracts were reacted with silver nitrate solution to synthesize AgNPs at optimized conditions for each solvent.The UV-spectrum of extract S1E1 showed a sharp and clear peak at 426 nm wavelength giving a maximum absorption rate of 0.372 more than S2E1, S3E1, and S4E1.The characteristic peak for S2E1 was shifted to a higher wavelength than other extracts, suggesting the synthesis of larger AgNPs.Among the other four extracts, S1E2, S2E2, S3E2, and S4E2, the S1E2 and S2E2 extracts gave a more absorption rate of 0.563 and 0.566, respectively, than other solutions.Still, the S1E2 extract was selected for further use because the peak of S2E2 was slightly shifted to a higher wavelength suggesting the synthesis of larger-sized NPs [Fig.3a (maceration method) and Fig. 3b (boiling water method)].

Physiochemical characterization of silver nanoparticles
The AgNPs were synthesized using aqueous extracts, S1E1 and S1E2, of leaves powder of Neem plant prepared via maceration and boiling, respectively.The S1E1 extract was reacted in a 1:1 ratio with 2 mM silver nitrate solution, kept at 70 °C, and after 3 h of reaction, the solution was taken in Eppendorf 's tube to purify the NPs.Similarly, the AgNPs synthesized using 20 mL of S2E1 extract of Neem plant reacted with 10 mL of 1 mM silver nitrate soliton at 70 °C for 3 h and were purified and stored.The pH of the solutions for both extracts was maintained at 11.The purified AgNPs using both extracts were subjected to various techniques given below.
The purified AgNPs of both extracts were dissolved in distilled water and subjected to UV-spectrophotometry within 300-800 nm to measure the absorption spectrum.A clear but broad characteristic peak for AgNPs-1 and a sharp one for AgNPs-2 can be seen in Fig. 4a due to the surface plasmon resonance phenomenon of the nanoparticles.AgNPs-2 exhibited a sharp peak as compared to AgNPs 1.
The particle size of sample AgNPs-1 using the Zeta sizer was recorded as 10-30 nm, shown in Fig. 4c.The maximum number of NPs was between 15 and 30 nm, and the minimum count was between 10 and 15 nm.While the maximum particle size in sample AgNPs-2 was distributed within the range of 22-30 nm, as shown in Fig. 4d.
FTIR analysis was performed to examine the attached functional groups of biomolecules on the surface of AgNPs acting as capping/stabilizing agents.The FTIR spectrum of AgNPs-1 showed absorption bands at 675.08 cm −1 , 16,335.6 cm −1 , and 2154.6 cm −1 , while the FTIR spectrum of AgNPs-2 showed bands at 678.9 cm −1 , 1639.5 cm −1 , 2144.8 cm −1 , 3298.2 cm −1 and 3518 cm −1 (Fig. 4b).The peaks near 675.08 cm −1 and 678.9 cm −1 assigned to CH out of plane bending vibrations are substituted ethylene systems -CH=CH (cis).The bands at 1635.6 and 1639.5 cm −1 correspond to amide-I arising due to carbonyl stretch (-C=O) in proteins.The peaks at 2154.6 cm −1 and 2144.8 cm −1 are assigned to the stretching vibration of -C-N of amide I, while the band at 3298.2 cm −1 is assigned to C-H (methoxy compounds) and stretching vibration of aromatic compounds and 3518 cm-1 corresponds to -OH stretching vibration indicating the presence of alcohol and phenol involved in capping and stabilizing of AgNPs.
The XRD analysis indicates the synthesis of crystalline nature silver nanoparticles using both extracts (Fig. 4e,f).The size of both samples was determined using the Debye-Scherrer formula.The average size of AgNPs in sample 1 was 32 nm and 24.98 in sample AgNPs-2, determined by the Debye-Scherrer formula close to the values observed in the zeta sizer.A single sharp peak recorded at 2θ degrees of 30.07 for AgNPs-1 and 30.06 for AgNPs-2 is assigned to plane 111 suggesting both samples' face-centered crystalline structure.
Energy Dispersive Xray (EDX) analysis of both samples, AgNPs-1 and AgNPs-2, shows the peaks in the reign of silver due to its surface plasmon resonance, which is generally at 3 keV, verifying the synthesis of AgNPs.EDX profile of AgNPs-1 showed signals (Fig. 7 the signal for AgNPs was stronger in sample AgNPs-2 than in the AgNPs-1.Table 1 presents the elemental composition and concentration of two samples of Siver Nanoparticles (AgNPs), labelled as AgNPs-1 and AgNPs-2.
The surface morphology of green synthesized silver nanoparticles was studied using a scanning electron microscope (SEM).Smooth spherical to oval shaped AgNPs nanoparticles in aggregated form were synthesized using plant extract S1E1, prepared in distilled water via maceration technique, as shown in Fig. 5a.Silver nanoparticles were spherical with a smooth surface when prepared with plant extract (S1E2) in distilled water using www.nature.com/scientificreports/ the boiling method (Fig. 5b).The size was estimated to be less than 20 nm in sample AgNPs-1 and less than 30 nm in sample AgNPs-2.The germination percentage started to decline as compared to the control when NPs concentration increased up to 25 and 50 ppm.The germination rate index was significantly higher in seeds treated with AgNPs than in control except at 50 ppm suggesting a substantial increase in germination rate due to NPs, as shown in Fig. 6.Silver nanoparticles also improved the seed vigor to a concentration of 20 ppm.The highest seed vigor was recorded by Nadar (1131.50)and Naqeeb (1253.67) at 10 ppm, indicating the optimal concentration (Fig. 6).

Effect of silver nanoparticles on growth parameters of tomato plants
Various growth parameters were studied to understand the effects of AgNPs on the growth of Tomato plants.The shoot and root length of Nadar plants was 31.87 cm and 21.40 cm, respectively, in the control group.Plant length increased when treated with 5, 10, 15, and 20 ppm AgNPs.The maximum shoot and root lengths were measured at 10 ppm for the Nadar variety and 5 ppm for the Naqeeb variety.Furthermore, fresh and dry matter of shoot and root in control plants of Nadar varieties were 24.50 g and 9.40 g and 6.79 g, and 3.09 g, respectively, which increased to 44.4 g and 14.82 g and 12.07 g, and 4.67 g when plants were treated with 10 ppm silver nanoparticles.Naqeeb plants followed a similar pattern, as shown in Table 2. Increased AgNPs concentration to 50 ppm reduced fresh and dry matter in both verities (Fig. 7).Quantification of Photosynthetic Pigments, Alkaloids, Flavonoid, Total Soluble Sugar and Protein Content Photosynthetic pigments are essential parameters, and their amount in leaves reveals the overall health of plants.This study determined the effects of AgNPs concentrations on the Tomato plant's total chlorophyll and carotenoid content compared to the control group.The total chlorophyll and carotenoid content increased significantly with increasing AgNPs concentrations to 20 ppm.Table 3 suggests an increase in AgNPs to 25 and 50 ppm reduced the amount of chlorophyll and carotenoid restricting the plant growth.Significant effects on alkaloids concentration of silver nanoparticles treated plants could be seen.Plants treated with 5 ppm and 10 ppm produced more alkaloids than control and other concentrations of silver nanoparticles.The total flavonoid concentration in the leaves was also determined.The results revealed an increase in the amount of flavonoids in plants exposed to    3 shows the considerable change in soluble sugar as well.The sugar content was increased from 0.406 ± 0.109 (control) and 0.433 ± 0.104 to 0.635 ± 0.061 (Nadar), and 0.644 ± 0.156 at 10 ppm in AgNPs treated plants.

Discussion
Nanomaterials have gained a lot of interest and application in various fields.Scientists have used traditional synthesis methods of nanomaterials like the physical, chemical, thermal, and hydrothermal that are extremely expensive, hazardous, and use toxic chemicals [45][46][47] .Therefore, the green synthesis of nanoparticles is found to be a more advanced, widespread, and remarkable area of nanotechnology.Using plants to synthesize nanoparticles is easy, single-step, nonpathogenic, cost-effective, non-toxic, and sustainable as it uses renewable resources and is environment friendly 9,48 .Various physical factors control the silver nanoparticles' size, shape, and stability during green synthesis 49 .Silver nanoparticles acquire unique optical properties due to surface plasmon resonance (SPR) generated from free electron movement.Therefore, interacts with a specific wavelength of visible light reported between 400 and 500 nm and develops a brown color in the solution depicting the synthesis of silver nanoparticles 50,51 .
The present work synthesized silver nanoparticles using Neem plant leaves extract and optimized the factors affecting the green synthesis protocol.Temperature affects the size and shape of silver nanoparticles and the rate at which silver nanoparticles are synthesized 52 .It is suggested that low temperature delays the synthesis of silver nanoparticles.Silver nanoparticles start synthesizing at 40 °C, while better and faster development of silver nanoparticles requires an elevated temperature of about 60-80 °C53 .Smaller-sized silver nanoparticles are synthesized because of higher temperatures in a short time, and the UV spectrum of such nanoparticles moves to a lower wavelength (blue shift).In contrast, a broad peak at a higher wavelength (red shift) is attributed to agglomeration or an increase in the size of the particles at low temperatures 54 .Kredy et al. 55 showed that lower reaction temperatures resulted in larger nanoparticles, whereas high temperatures produced small nanoparticles.Furthermore, it was found that Vitex agnus-castus leaf extract could lead to a fast reduction of Ag+ ions, even at a low temperature of 40 °C.In contrast, the efficient production of AgNPs was observed in another study when the temperature was between 60 and 80 °C53 .The AgNPs of 17 nm were synthesized using Ocimum sanctum (Tulsi) leaf extract, the yield of production was increased after 15 min and continued to increase with increasing the time up to 3 h 56 .The reduction of silver nanoparticles starts after adding silver nitrate to the plant extract, as evidenced by the color change from light green to brown 57 .After an optimum time for the formation of stable silver nanoparticles, silver nanoparticles start to agglomerate, resulting in larger particle sizes 58 .The formation of silver nanoparticles is also concerned with the optimum amount of metal precursors and the reducing agents in the plant extract.The availability of metal ions in the reaction mixture influences the yield of nanoparticles.The higher the metal precursor concentration and the reducing agent, the greater the possibility of obtaining AgNPs 59 .According to previous studies, pH is another important parameter affecting nanoparticle synthesis.The higher pH increases the availability of biochemicals for reducing and capping silver nanoparticles, producing highly stable and smaller-sized silver nanoparticles.The acidic pH prevents the formation of silver nanoparticles and contributes to particle instability 23,54 .
FTIR analysis makes us understand the role of biomolecules in plant leaf extract for reducing silver to silver nanoparticles and their stability in acting as capping agents 60 .The FTIR analysis reveals the involvement of alkanes, alkynes, amines, carboxylic groups 61 , methylene 62 , and various other biomolecules in reducing and stabilizing green synthesized silver nanoparticles 63 .Biomolecules extracted in different solvents make changes in size, shape, stability, and peaks of FTIR spectra, demonstrating the significance of the extraction method and solvent type 64 .XRD pattern and SEM analysis of AgNPs synthesized by Neem and other plants suggested the synthesis of crystalline structure spherical silver nanoparticles [65][66][67] .
Green synthesized nanoparticles have tremendous biological applications as antibacterial, antifungal, and anti-cancerous agents in medicine and agriculture 68,69 .The silver nanoparticles promote crop yield with better plant growth even at low concentrations 70 .Metallic nanoparticles improve the efficiency of photosynthetic systems in plants, increasing the content of photosynthetic pigments, which improves plants' growth and weight.Foliar application of silver nanoparticles on Fenugreek plants gradually increased all growth parameters compared to untreated plants using 40 mg/L and decreased at 60 mg/L but still higher than the control 71 .Silver nanoparticles (9-35 nm size) synthesized using leaves of Ocimum basilicum L., and Mangifera indica L. increased the shoot length, fresh and dry biomass, chlorophyll, carbohydrates, and protein content of wheat plants at 20 and 40 ppm.Inhibitory effects were also observed beyond these concentrations 72 .

Conclusions
Nanotechnology plays a vital role in introducing tools for disease management, enhancing disease diagnostics, and developing new measures.Green synthesis uses simple procedures, readily available raw materials, environment-friendly, cost-effective, and easily scaled up for large-scale synthesis of nanoparticles.Furthermore, there is no need to use high temperatures, pressure energy, and toxic chemicals.The optimization of different factors resulted in the formation of stable nanoparticles and gave maximum yield making sure to utilize the resources well.The silver nanoparticles improved the germination rate and plant growth of Tomato plants, enhancing the production of chlorophyll, carotenoids, alkaloids, and flavonoids.So, green synthesized silver nanoparticles have a noticeable effect on the agricultural world in minimizing environmental contamination and promoting plant growth.However, the physicochemical characteristics of the generated AgNPs may exhibit specificity towards a particular crop species and the prevailing climatic conditions of a particular locality.Therefore, further research is required to assess the antimicrobial efficacy of silver nanoparticles (AgNPs) in practical agricultural settings.Additional investigation should be focused on determining the optimal threshold concentrations of silver nanoparticles (AgNPs) that effectively reduce the occurrence or severity of diseases in plants, while avoiding any negative effects on non-target organisms and soil fertility.

Plant material, preparation of leaf extract and reagent
Azadirachta indica (Neem plant) leaves were collected from the botanical garden, University of the Punjab, Lahore, and washed using tap water several times to remove all the dust.Then, leaves were dried at room temperature under shade, ground with the help of an electric grinder, and stored.Seeds of Tomato varieties were brought from Ayyub Agriculture Research Institute, Faisalabad.All the required chemicals, i.e., silver nitrate (AgNO 3 ), methanol, dimethyl sulfoxide (DMSO), dichloromethane (CH 2 Cl 2 ), and potassium bromide (KBr), acetone, n-hexane, acetic acid, sulphuric acid, and phosphate buffer were bought from Sigma-Aldrich through Science Traders.
The plant extracts were prepared using maceration and boiling methods.Firstly, powdered material of Neem leaves was added to each solvent with a 1:10 ratio, respectively.The mixtures were kept overnight at room temperature, followed by filtration through Whatman's No. 1 filter paper, and the filtrate was used to synthesize silver nanoparticles.Then using the same ratio of leaves powder in mentioned solvents, the mixtures were boiled for 30 min, cooled down, and filtered with Whatman's no. 1 filter papers.The extracts were labeled as S1E1, S2E1, S3E1, S4E1, S1E2, S2E2, S3E2, and S4E2 representing the solvent type (distilled water S1, methanol S2, DMSO S3, and DCM S4) and extraction method (maceration E1and boiling method E2) respectively.Molar solutions of silver nitrate, i.e., 1, 2, 3, and 4 mM, were prepared by mixing the required amount of silver nitrate in distilled water.

Effect of temperature, time, pH, reagent concentration and plant extract volume
The temperature of the reaction mixture was optimized by adding 10 mL of freshly prepared extracts (mentioned in Sect.2.2) with 10 mL of 1 mM AgNO3 solution in glass vials.An incubator was used to set up the experiment for an hour to check the effect of various temperatures (30 °C, 40 °C, 50 °C, 60 °C, 70 °C, and RT room temperature).Firstly, the synthesis of AgNPs was indicated by the reaction mixture's color change due to the reduction of Ag+ to Ag0.Then each solution was taken in Eppendorf 's tube and centrifuged.The pellet www.nature.com/scientificreports/ was dissolved in distilled water and centrifuged again.The centrifugation process was repeated several times to purify the nanoparticles.Each solution was subjected to UV-spectrophotometer (Shimadzu UV-1800) within the 300 nm to 800 nm wavelength to observe the spectrum.The temperature producing maximum AgNPs was chosen for the further optimization procedure.
The incubation time was optimized using 10 mL of each freshly prepared leaf extract with 10 mL of 1 mM AgNO3 solution in glass vials.The solutions were incubated in the dark at the selected temperature for 1, 2, 3, 4, 5, and 6 h.The change in color of the reaction mixture indicated the synthesis of AgNPs.The color change in the reaction mixture was observed.Purified silver nanoparticles were subjected to UV-spectroscopy for further confirmation of synthesis after each time interval.The incubation time showing the maximum production of AgNPs was used for the further optimization procedure.
Various pH values were considered to find the optimum value for synthesizing silver nanoparticles using the Neem plant's dried leaves.The freshly prepared leaf extract was added to 1 mM silver nitrate solution in a respective vial.The pH of the solution was maintained at 5 and kept in an incubator at the selected temperature.The color change was observed at first for the synthesis of AgNPs, and then the solution was subjected to UVspectrophotometry after an optimized time.The process was repeated at pH 7, 9, 11, and 13.NaOH and HCl were used to maintain the pH of the solutions.The pH at which the maximum yield of nanoparticles was given was selected for further procedures (Fig. 8).
Various concentrations of silver nitrate, i.e., 1 mM, 2 mM, 3 mM, and 4 mM were used to optimize the reagent concentration for the synthesis of AgNPs.Prepared plant extracts were reacted with different concentrations of AgNO3 in a 1:1 ratio at a selected temperature and time.After observing the color change, the resulting solutions were subjected to UV-vis spectroscopy to produce a spectrum and monitored.The concentration of silver nitrate solution producing the maximum AgNPs was selected for further procedure.
Optimization of used plant extract volume was performed using different amounts of plant extract, i.e., 10 mL, 20 mL, 30 mL, 40 mL, and 50 mL treated with 10 mL of AgNO 3 solution.Color change of the solutions was observed after reaction completion to confirm the synthesis of silver nanoparticles.The synthesized AgNPs were centrifuged several times until AgNPs were purified and assessed using a UV-vis spectrophotometer within 300-800 nm.The volume of Neem leaf extract producing maximum AgNPs was selected for further procedure [73][74][75][76][77] .

Effect of solvent type
The prepared plant extracts using two different methods were used to synthesize AgNPs at already optimized conditions to select the maximum NPs producing solvent for each technique.The resulting solutions were subjected to UV-vis spectroscopy to monitor the maximum production of AgNPs.The prepared plant extracts using two different methods were used to synthesize AgNPs at already optimized conditions to select the maximum NPs producing solvent for each technique.The resulting solutions were subjected to UV-vis spectroscopy to monitor the maximum production of AgNPs.www.nature.com/scientificreports/Characterization of green synthesized silver nanoparticle Green synthesized silver nanoparticles at optimized conditions were subjected to the following techniques to determine their size, surface structure, morphology, and other characteristics.

UV-spectrophotometric analysis
This technique was used to determine the formation of silver nanoparticles.The purified silver nanoparticles were dissolved in 5 mL of distilled water using a vortex meter and subjected to UV-vis analysis.The absorption spectrum within the wavelength range of 300-800 nm was taken on a UV-vis spectrophotometer (Shimadzu UV-1800) 78 .
Fourier transform infrared (FTIR) and particle size analysis Fourier transform infrared spectroscopy (FTIR) was used to study the organic functional groups attached to the surface of AgNPs.The nanoparticles were purified and dried at 60 °C.The dried samples were mixed with a fine powder of potassium bromide (KBr) and analyzed by FTIR (Bruker Alpha Platinum ATR).The size surface charge of silver nanoparticles was determined using a zeta sizer.Particle size measurement was based on the time-dependent fluctuation of laser light scattering by the nanoparticles undergoing Brownian motion 78 .
X-ray diffraction and energy dispersive X-ray (EDX) analysis X-ray diffraction (XRD) was used to examine the overall oxidation state and crystal structure of AgNPs.The energy-dispersive X-ray technique was used to study the elemental composition of synthesized AgNPs 78 .

Scanning electron microscopic analysis
Scanning electron microscopy (SEM) was performed to visualize synthesized particles' morphology at submicron scale and elemental information at the micron scale (FEI Nova 450 NanoSEM).Thin films of the sample were prepared on a carbon-coated copper grid.A drop of AgNPs was placed on carbon-coated copper grids and allowed to stand for two min, and the excess solution was removed using a blotting paper, and then the film on the grid was allowed to dry at room temperature and exposed to an electron beam 79 .

Effect of silver nanoparticles on seed germination of tomato
The effect of green synthesized AgNPs on seed germination of the Tomato was determined in Petri plates.Seeds were exposed to 5% sodium hypochlorite solution for 2-3 min, repeatedly washed with distilled water, and soaked in prepared concentrations (5, 10, 15, 20, 25, and 50 ppm) of silver nanoparticles for approximately 2 h.Seeds were transferred to sterilized Petri plates and placed on three layers of filter paper with 1 cm distance between them.The Petri plates were covered, sealed with a tap, and incubated at room temperature for two weeks.The seed germination rate, mean germination time, and percentage were measured using the formulas below.
Calculations of the germination rate index show the percentage of germination per day, so the higher the percentage and the shorter the duration, the higher the GRI.
Ni is the number of germinated seeds until the ith day, Di is the number of days from the experiment until the ith counting, and n is the total number of germinated seeds.

Effect of silver nanoparticles on tomato under greenhouse conditions
Experimental design and application of silver nanoparticles A pot experiment was performed to determine the effect of green synthesized AgNPs on the growth and physiological response of Tomato plants during the season of 2020-2021.Seeds of two Tomato varieties, Nadar and Naqeeb, were brought from the Vegetable Research Institute of Ayyub Agricultural Research Institute (AARI), Faisalabad, and used to prepare seedlings.The cleaned clay pots filled with loamy soil were placed in a wireenclosed area and arranged according to a particular variety, respective treatment, and replicate number following a randomized complete block design (RCBD).Already prepared healthy seedlings were transplanted into pots and kept one plant per pot.Standard agricultural practices were used to irrigate and maintain the plants.Solutions of silver nanoparticles in various concentrations, i.e., 5, 10, 15, 20, 25, and 50 ppm, were applied on plants using the foliar spray until run-off at 15 days intervals.Tap water was given to plants taken as control.

Plant growth analysis
Growth parameters, including shoot length, root length, and fresh weight of the plants, were recorded after 55 days of transplantation.The plants were removed carefully from the pots, packed in labeled bags, and brought

Figure 1 .
Figure 1.Effect of various (a) temperature after 1 h of reaction (b) time at 70 °C (c) pH (d) reagent concentrations (e) volume of plant extract on synthesis of AgNPs using leaf extract of Neem (Azadirachta indica) in distilled water using maceration method.

Figure 2 .
Figure 2. Effect of various (a) temperature after 1 h of reaction (b) time at 70 °C (c) pH (d) reagent concentrations (e) volume of plant extract on synthesis of AgNPs using leaf extract of (Azadirachta indica) in distilled water using boiling method.

Figure 3 .
Figure 3.Effect of different solvents on the synthesis of AgNPs using leaf extract of Neem (Azadirachta indica) using (a) maceration (b) boiling method.

Figure 4 .
Figure 4. Physiochemical characteristics of green synthesized silver nanoparticles using aqueous extract of Neem plant (a) UV spectrum (b) FTIR spectrum (c) size distribution of sample 1 (d) size distribution of sample 2 (e) XRD spectrum of sample 1 (f) XRD spectrum o of sample 2.

Figure 6 .
Figure 6.Effect of different concentration of green synthesized silver nanoparticles on (a) germination percentage (b) germination rate index (c) mean germination time and (d) vigor index.
www.nature.com/scientificreports/AgNPs.The total flavonoid content of the Nadar and Naqeeb plants in the control group was 2.548 ± 0.149 and 2.433 ± 0.368 mg QE/g, respectively.The minimum level of total flavonoids in plants of both varieties exposed to 50 ppm silver nanoparticles was 1.936 ± 0.135 and 1.841 ± 0.120 mg QE/g.A similar alkaloid production trend was observed in plants treated with AgNPs.Silver nanoparticles also greatly affected the protein content accumulation in both varieties of Tomato plants.The protein content started increasing in plants treated with AgNPs at 20 ppm.Further increase in the concentration of AgNPs reduced the protein amount.The maximum protein content of 3.154 ± 0.122 in 10 ppm treated Nadar plants and 2.951 ± 0.549 in Naqeeb plants was found.Table

Table 1 .
Elemental composition and concentration of silver nanoparticles.Effect of silver nanoparticles on seed germination of tomatoThis study shows how tomato seeds germinate when exposed to various concentrations(5, 10, 15, 20, 25, and 50 ppm) of green synthesized AgNPs.Silver nanoparticles significantly affected the germination percentage, rate, meantime, and vigor index at 5, 10, 15, and 20 ppm.There was no significant effect on germination time, but the seeds treated with 5 ppm AgNPs took lesser time than other treatments.The maximum germination rate of 83.33% and 86.67 was achieved by Nadar and Naqeeb seeds, respectively, subjected to 10 ppm AgNPs with MGT of 8 days.

Table 2 .
Effect of green synthesized silver nanoparticles on growth parameters of Tomato plants.Treatment mean for each treatment is the average of the three replicates; Mean values with different lower-case alphabets are significantly different at p ≤ 0.05 (Duncan's multiple range test).± values represent standard error (SE).

Table 3 .
Effect of green synthesized silver nanoparticles on physiological parameters of Tomato plants.Treatment mean for each treatment is the average of the three replicates; Mean values with different lower-case alphabets are significantly different at p ≤ 0.05 (Duncan's multiple range test).± values represent standard error (SE).