Silver Nanoparticles Synthesized with Extracts of Leaves of Raphanus sativus L,Beta vulgaris L,and Ocimum basilicum and Its Application in Seed Disinfection

Abstract

Silver nanoparticles (AgNPs) were produced by green synthesis using Raphanus sativus (RsAgNPs), Beta vulgaris (BvAgNPs), and Ocimum basilicum (ObAgNPs) leaf extracts as reducing agents. Plant phytochemical composition analysis indicated that they contain phenolic compounds that can participate in the synthesis reaction as flavonoids. Synthesized AgNPs presented maximal absorption peak at 430 nm (RsAgNP), 440 nm (BvAgNP), and 420 nm (ObAgNP) in ultraviolet‐visible spectrophotometry analysis. Scanning electron microscopy and energy dispersive X‐ray spectroscopy analysis showed that RsAgNPs are 76 nm diameter spheres made of up to 54.1% silver, the BvAgNPs are 78 nm diameter spheres with 39.76% silver, and the ObAgNPs are cubes of 99 nm edges with 69.74% silver. The found Z potential values indicate that all the obtained AgNPs are stable in phosphate buffer. A disinfection of 100% and 90% was achieved for the in vitro culture of Arabidopsis thaliana and Psidium guajava (guava) seeds, respectively, with all AgNPs synthesized. Treatment with these AgNPs showed no negative effects on germination, and on the contrary, in guava, a higher germination percentage was observed when the seeds were exposed to RsAgNPs. Only at high concentration (10 mg/mL) of AgNPs, the growth of A. thaliana was decreased, while at low concentration (0.01 mg/mL) of ObAgNP and BvAgNPs, higher growth was showed, specifically 60 and 40% more than the control, respectively. All AgNPs showed antimicrobial activity against Escherichia coli and Klebsiella oxytoca which are bacteria of clinical interest, and against Agrobacterium tumefaciens, a bacterium used in the genetic transformation of plants.

1. Introduction

Silver nanoparticles (AgNPs) are currently used in different products such as textiles, cosmetics, medical supplies, household items, food additives, and for water treatment [1]. The synthesis of AgNPs can be carried out either by chemical reactions, using ascorbic acid or hydrazine as reducing agents or through green synthesis, replacing these chemicals with extracts from bacteria, fungi, algae, or plants [2]. Green synthesis is cheaper and does not generate toxic residues, and recently many publications have exposed the high efficiency of different plants in the synthesis of AgNPs (Table 1) [1–14]. Leaf extracts from cultivated plants with different purposes are used for the synthesis of AgNPs. For example, with the help of extracts of plants with nutritional elements like Cynara scolymus [3]; ornamental plants like Gleichenia pectinata [4] and Murraya paniculata [5]; or plants considered weeds like Cyperus rotundus [6], AgNPs with antimicrobial, anti‐inflammatory, or anticancer activity have been synthesized.

Table 1

Silver nanoparticles obtained by green synthesis.

Plants	Activity	Shape/size (nm)	Maximum absorbance peak (nm)	Extract compounds	Reference
Cynara scolymus leaves	Anticancer: breast cancer cells MCF7	Spherical 98.47 ± 2.04	434	Aromatic and aliphatic compounds	[3]
Gleichenia pectinata leaves	Antimicrobial: P. aeruginosa, E. coli, Klebsiell pneumoniae, and Candida albicans	Spherical 7.51 ± 2.88	460	Terpenoid, alcohol, and carbonyl group	[4]
Murraya paniculata leaves	Antimicrobial: Bacillus cereus, Staphylococcus aureus, Escherichia coli, Candida albicans cytotoxicity: human umbilical vein endothelial cell line	Spherical/14 ± 9	438	Alkaloids, phenolic, flavonoids, polysacchari des, and proteins	[5]
Cyperus rotundus complete plant	Antibacterial: Escherichia coli, Bacilus cereus and C. haemolyticum	Spherical 20.5 ± 9.6	446	Essential oils, alkaloids, flavonoids, terpenoids, chromones, phenylpropa noids, phenolic acids, and iridoides	[6]
Selaginella myosurus complete plant	Anti‐inflammatory: in vivo and in vitro	Spherical 58.81	420–480	Flavonoids, saponins, steroids, terpenoids, tannins, and phenols	[7]
Fenogreek seed seeds	Antibacterial: S. aureus, E. coli	Spherical 40	396	Phenolic compounds and flavonoids (apigenin‐7‐O‐glycoside and luteolin‐7‐O‐glycoside)	[8]
Opuntia ficus-indica fruit	Antimicrobial: microorganisms from wastewater treatment plant. E. coli var 1, Enterobacter aerogenes var 1, Citrobacter freudi var 2, atypical E. coli, and aerobic mesophilic microorganisms	Spherical 64.28 ± 11.82	435	Not reported	[9]
Baleria longiflora leaves	Antibacterial: Enterococcus sp., Streptococcus sp, B. megaterium, Pseudomonas putida, P. aeruginosa, Staphylococcus aureus	Spherical 2.4 ± 0.5	443	Amines and flavonoids	[10]
Phoenix dactylifera seeds	Antitoxoplasm a: in vivo in mice balb/c	Irregular in shape with some spherical NPs 20	Not reported	Not reported	[11]
Malus domestica fruit	Antibacterial: K. pneumoniae, E. coli, P. aeruginosa, Enterobacter aerogenes anticancer: Breast cancer cells MCF7, noncancerous kidney cells HEK29.3	Spherical 50	425	Not reported	[12]
Phyllantus emblica. fruit	Antibacterial: Bacteria that infect rice Acidovorax oryzae	Spherical 39.1	430	Amidas, aminas, and alquenos	[13]
Prosopis fracta fruit	Antibacterial: S. aureus, Streptococcus pneumonia, E. coli, Salmonella typhi	Spherical 10.26–14.65	425–438	Phenolic compounds and flavonoids	[14]

Organic compounds from plant material, such as flavonoids, saponins, steroids, terpenoids, tannins, and phenol, provide reducing and stabilizing agents for the synthesis of AgNPs (Table 1). Leaf extracts of Raphanus sativus L (radish), Beta vulgaris var. cicla (chard), and Ocimum basilicum (basil) have compounds that can participate in the synthesis of AgNPs. Leaves of chard contain flavonoids and carotenoids [15], leaves of basil contain monoterpenes, phenylpropanes, alkaloids, glycosides, tannins, saponins, and aromatic compounds [2, 16], and leaves of radish contain tannins and coumarins [17]. R. sativus is an annual or biennial plant belonging to the Brassicaceae family that is cultivated for its root, and it is destined for human consumption [18]; Beta vulgaris var. cicla is a plant of the Amaranthaceae family that is cultivated for the consumption of leaves [19]; and Ocimum basilicum is an annual plant the Lamiaceae family that is used in the kitchen as a spice [20]. Furthermore, these plants are easily grown home or in greenhouses and are available at any time of the year.

In vitro plant tissue culture consists of placing an isolated part of a plant (referred to as an explant) or seeds in a medium with a determined chemical composition and under controlled conditions. This can be carried out with different objectives such as for micropropagation, to obtain pathogen‐free plants, for genetic improvement, and for germoplasm conservation [21]. However, these culture conditions favor the development of bacteria and fungi; thus, in vitro culture must be performed under aseptic conditions, the material and medium must be sterilized, and the explants must be disinfected [21]. Traditional disinfection uses one or more of the following substances: ethanol, sodium hypochlorite, calcium hypochlorite, mercuric chloride, and surfactants such as Tween 20 and in some cases antibiotics [21]. The selection of disinfecting agents and the procedure depends on the type of explant or seed and must ensure the complete elimination of microorganisms without causing damage to plant tissue. AgNPs could be used as an alternative agent for the disinfection of plant material and its cultivation under axenic conditions.

The objective of this work is to obtain AgNPs by green synthesis from extracts of radish, chard, and basil leaves and to evaluate their effectiveness in the disinfection of seeds for in vitro cultivation. In addition, the possible negative impact of this AgNPs on plants was also analyzed.

2. Materials and Methods

2.1. Synthesis of Silver Nanoparticles

Fresh leaves of radish, chard, and basil were obtained from a local market (Aguascalientes, Ags. Mexico). The AgNPs were synthesized following the protocol described by Mahakham et al. [22] with slight modifications: 20 g of clean leaves were crushed in a blender for 20 s with 100 mL of distilled water. This preparation was first incubated in a boiling water bath for 1 h and then left overnight at room temperature. The aqueous extract was separated from the solid residues by centrifugation at 10,000g for 10 min and filtered on Whatman ^#1 paper. A volume of 10 mL of the aqueous extract was slowly mixed with 90 mL of a 1 mM AgNO3 solution (J. T. Baker™ reactive grade). This mixture was incubated protected from light, at room temperature, in an orbital shaker at 100 rpm (Shaker RK‐30) until a color change was observed (after about 7 days). This preparation was centrifuged at 10,000g for 10 min (Sorval ST16 centrifuge), and the supernatant was removed to recover the AgNPs in the pellet. The nanoparticles (NPs) were washed three times with sterile distilled water: the pellet was mixed with 100 mL of sterile distilled water, this mixture was centrifuged at 10,000g for 10 min, the supernatant was removed; once with ethanol, the sediment was mixed with 100 mL of 96% ethanol, the mixture was centrifuged at 10,000g for 10 min, and the supernatant was removed. This pellet was dried at 40°C for 24 h and disintegrated by sonication (Digital ultrasonic, VEVOR).

2.2. Characterization of Silver Nanoparticles

The reduction of Ag⁺ to Ag⁰ was monitored by analyzing the reaction mixture (leaf extract 97 and AgNO3) in the ultraviolet‐visible spectrophotometer (Genesys 10S ThermoScientific) in an absorption scan from 300 to 800 nm wavelength. The dry AgNPs were analyzed by scanning electron microscopy (SEM) and energy dispersive X‐ray spectroscopy (EDS) in a Joel JSM‐5900LV equipment to observe their geometry and to determine their element composition. The SEM and EDS analyzes were performed at the High Resolution Microscopy Laboratory of the Faculty of Biology of the Autonomous University of Aguascalientes (Mexico). The size of the AgNPs was calculated by measuring the images captured by SEM with Image J2 software [23]. One hundred NPs were measured to obtain the size distribution histogram. Their hydrodynamic diameter was determined by the dynamic light scattering technique (DLS) in a Zetasizer ZS90 (Malvem) equipment. The Z potential was obtained by electrophoretic mobility using the Smoluchowski model. The analyses were performed on AgNPs colloidal mixture prepared in ultrapure water (Milli‐Q) at a concentration of 100 mg/L. Measurements were taken in triplicate at times of 0, 72, 96, and 120 h.

2.3. Analysis of Anthocyanin in the Plant Extract

Anthocyanin content was calculated in triplicate with the following protocol [24]: 50 mg of 5 mm² leaf fragments were incubated in 10 ml of a methanol‐HCl solution (99: 1) (J. T Baker), overnight at 4°C. The absorbance of the extract was measured at a wavelength of 530 (A ₅₃₀) and 657 nm (A ₆₅₇) with a Genesys 10 s UV‐Vis de thermo‐scientific spectrophotometer. The relative anthocyanin units per gram of tissue (RAU/g) were calculated with the following equation: RAU/g = A ₅₃₀ − (0.25 × A657).

2.4. Phytochemical Screening

Thin‐layer chromatography (TLC) was performed on silica gel 60 RP18 F254s aluminum plates (Merk, Darmstadt, Germany) as adsorbent and water‐acetonitrile (7 : 3 v/v) as a mobile phase. TLC was sprayed with reagent specific for flavonoid identification; 1% methanolic 2‐amino‐ethyl diphenyl boric acid (Sigma‐Aldrich® Co.); and 5% ethanolic polyethylene glycol 4000 (Sigma‐Aldrich® Co.) and then examined by UV‐fluorescence (365 nm) [25]. The compounds were detected observing orange, yellow, and blue fluorescence bands, which are characteristic of phenolic compounds and flavonoids. Finally, the retention factor (Rf) was calculated for each compound, which was determined by dividing the displacement of the selected band by the distance traveled by the solvent. Rutin (Sigma‐Aldrich ®, U.S.A) was used as reference standard. The analyzed samples were obtained as follows: 20 g of clean leaves were crushed in a blender for 20 s with 100 mL of methanol (J. T. Baker) and were incubated at 4°C for one day in the dark. Methanol was removed at 50°C on a rotary evaporator.

2.5. Total Phenolic Content Determination

Total phenolic content was determined by the Folin–Ciocalteu method. A standard curve was prepared with gallic acid (n = 3) at concentrations of 0, 2, 4, 6, 8, 10, and 12 μg/mL. Next, 2.5 mL of deionized water and 0.1 mL of Folin–Ciocalteu (Sigma‐Aldrich) 1 N reagent were added to each tube. After 6 min, 0.25 mL of a 10% sodium carbonate (Baker analyzed) solution was added to each sample and vortexed. The absorbance of each sample was monitored at 760 nm using a spectrophotometer (Jenway, Genova). From these readings, a line equation (y = 0.0288x − 0.007) and the correlation coefficient (R ² = 0.997) were calculated to extrapolate samples. The same treatment was carried out with the methanolic extract of each plant to determine its content of phenolic compounds.

2.6. Evaluation of Seed Disinfection with AgNPs

Guava (P. guajava cv “media china”) seeds were obtained from fresh fruit, washed with distilled water and dried at 25°C for one week. Arabidopsis thaliana (Arabidopsis, Columbia ecotype) seeds were collected from plants grown in the Plant Molecular Biology Laboratory of the Autonomous University of Aguascalientes. Dry seeds of guava and Arabidopsis were treated in seven groups with different condition as follows: group 1 were disinfected with a solution of Tween 20 at 0.15% and sodium hypochlorite at 2%; group 2, 3 and 4 were mixed with 0.01 mg/mL each AgNPs in sterile distilled water; and group 5, 6 and 7 were mixed with 1 mg/mL of each AgNPs. For each group, 40 seeds of guava and 10 mg of seeds of Arabidopsis were treated. Seeds were incubated in the dark, at room temperature, with agitation for 24 h. Seeds were sown in MS‐agar culture medium [26], in the dark until germination, and afterward in a 16 h‐light photoperiod for 2 weeks. Germination, elongation of the seedlings, and the percentage of contamination cultures were measured.

2.7. Analysis of Seed Exposure to AgNPs

Guava and Arabidopsis seeds were disinfected with a solution of 0.15% Tween 20 and 2% sodium hypochlorite and incubated in water at 4°C in the dark for 24 h. Forty seeds were incubated in MS broth culture medium with 0, 0.01, 1, 2, 5, 10, and 20 mg/mL of each of the AgNPs and in MS‐broth culture medium with 1 mg/L of benzyladenine as a control. They were incubated at 25°C in 16‐h light photoperiods for two weeks.

2.8. Evaluation of Antimicrobial Activity

The antimicrobial activity of the synthesized AgNPs was analyzed following the next protocol: [27] bacterial suspensions with a titer of 1 × 10⁶ colony forming units per milliliter (CFU/mL) in 0.05 M potassium phosphate buffer pH 7 were exposed to 1 mg/mL AgNPs. An aliquot of each suspension was inoculated after 1, 2, 4, 8, and 24 h of exposure onto solid LB‐agar plates and incubated for 16 h. Grown colonies were counted to calculate CFU/mL after exposure to AgNPs as well as logarithmic inactivation. The bacteria mentioned in Table 2 was analyzed under the conditions indicated.

Table 2

Bacteria used in the evaluation of the antimicrobial activity of AgNPs.

Bacteria/plasmid	Antibiotic resistance	Incubatin	Interest
Escherichia coli DH5α/pUC18	Ampicilin	37°C	Clinical
Klebsiella oxytoca	No	37°C	Clinical
Agrobacterium tumefaciens EHA 105/pBI121	kanamycin, rifampicin, streptomycin	28°C	Agronomic

2.9. Statistical Analysis

Anthocyanin content and seedling growth were analyzed by comparison of means by one‐way ANOVA followed by Tukey’s test. The germination of the different treatments was compared using a Chi square test. In both analyzes, statistical significance was set at p ≤ 0.05. The statistical analysis was carried out with the statistic software StatSoft (version 10), and the graphs were made in Excel.

3. Results and Discussion

3.1. Green Synthesis of Silver Nanoparticles

AgNPs were synthesized using leaf extracts of radish (RsAgNPs), chard (BvAgNPs), and basil (ObAgNPs) as reducing agent. The plant extract mixtures with AgNO₃ showed a change in color after 7 days of incubation (Figure 1(a)). The chard extract changed from pale yellow to reddish brown; that of basil extract turned from intense yellow to dark brown; and that of the radish extract changed from pale yellow to metallic gray (Figure 1(a)). The first indication of the formation of AgNPs is the change in color, due to the reduction of Ag⁺ ions to Ag⁰ [5] and to an excitation of the free electrons of the silver particles [3, 7]. Generally, the AgNPs suspension is yellow or orange if the particles are not aggregated, and gray if they are aggregated [5]. The reaction mixture was analyzed by UV‐visible spectrophotometry. This analysis showed a characteristic plasmon and maximum peaks of 430 nm (RsAgNPs), 440 nm (BvAgNPs), and 420 nm (ObAgNPs) (Figure 1(b)), which is in the range reported for other AgNPs (Table 1). AgNPs interact with light through a phenomenon known as surface plasmon resonance; the point of maximum absorbance depends on the size and shape of the NPs, which in turn depend on the synthesis conditions [28].

Figure 1

Analysis of biosynthesized AgNPs from plant extracts of B. vulgaris, O. basilicum, and R. sativa. (a) AgNPs synthesis reaction mixture (AgNO₃ + plant extract), a color change was observed after 7 days of incubation. (b) UV‐visible spectrophotometry, the plasmon of each AgNPs showed a maximum peak at a different wavelength.

(b)

3.2. Phytochemical Screening

In green synthesis of AgNPs, AgNO₃ donates the silver ions and plant extracts provide the reducing and stabilizing agents [29]. Phenolic compounds reduce the silver salt to form NPs, while they themselves are oxidized.6 Flavonoids in the enol form are oxidized to their keto form [6]. Phenolic compounds, proteins, and chlorophyll adhere to the surface of the NPs [30]. These compounds also act as stabilizing agents covering and protecting the formed AgNPs, preventing their agglomeration [6, 30]. Alkaloids, flavonoids, saponins, and sugars are examples of plant extracts components that provide the necessary conditions for the biosynthesis of AgNPs [2]. Organic compounds bind to the metallic component of AgNPs through the functional groups ‐C=O‐,‐NH₂, and ‐SH‐ [29]. The plant extracts fulfill a double function, both for the reduction and to stabilize AgNP [30]. We analyzed the content of phenolic compounds in the plant extracts (Table 3). The results showed that basil extracts have a higher content of phenolic compounds, approximately 10 times more than that found in radish and chard (Table 3). Coumarins, lignins, lignans, tannins, phenolic acids, and flavonoids are phenolic compounds that are produced by plants as defense mechanisms against environmental conditions [30]. Anthocyanins are phenolic compounds from the flavonoids group [31], so it was decided to quantify them (Table 3). They protect the plant from the excess of reactive oxygen species and have antioxidant, antimicrobial, and medicinal properties [32]. Anthocyanins were detected in the three plant extracts analyzed, though in different amounts (Table 3). Anthocyanin content was similar in basil and radish leaves (0.14 RAU/g), while in the chard leaves a smaller amount was found, approximately half (0.07 RAU/g) (Table 3). Plant extract from each plant was analyzed by TLC for flavonoids. These compounds are observed as an orange, yellow, or blue fluorescence band (Figure 2). Rutin is another flavonoid found in many plants. This flavonoid has antioxidant, anti‐inflammatory, cardiovascular, neuroprotective, antidiabetic, and anticancer properties [31]. Results of TLC indicate that the chard and radish extracts contain rutin (Figure 2). The phenolic compounds of the extracts showed quantitative (Table 3), and qualitative differences (Figure 2), which leads us to suppose that the composition of the extracts determines the final characteristics of the AgNPs.

Table 3

Components of plant extracts.

Plants	Total phenolic content (mg GAE/g)	Anthocyanin (RAU/g)
B. vulgaris (chard)	26.773 ± 4.289	0.07 ± 0.008
R. sativus (radish)	33.192 ± 9.199	0.149 ± 0.037
O. bacilucum (basil)	240.83 ± 20.301	0.148 ± 0.053

mg GAE/G = mg of gallic acid equivalents/gram of dry material; RAU/g = relative anthocyanin units per gram of tissue.

Figure 2

Separation by thin layer chromatography: (1) chard extract, (2) rutin, (3) radish extract, and (4) basil extract. Stationary phase: silica gel 60 RP18 F254s and mobile phase: water/acetonitrile (70 : 40 v/v).

3.3. Characterization of Silver Nanoparticles

SEM analysis allowed to observe the morphology and size of biosynthesized AgNPs (Figure 3). The BvAgNPs had lamellar structures with a thickness on the nanometric scale and aggregates of NPs with an irregularly shaped, almost spherical (Figure 3(a)), with a diameter of 78 ± 17 nm (Figure 3(b)). ObAgNPs were obtained as nanometer‐thick sheets and cubes (Figure 3(c)) of approximately 99 nm per edge (Figure 3(d)). The RsAgNPs produced were spherical (Figure 3(e)) with an approximate diameter of 76 ± 16 nm (Figure 3(f)). These differences highlight the influence of the components of the plant extracts on the size and shape of the AgNPs, since other factors that also affect these characteristics, such as temperature, pH, AgNO3 concentration, and synthesis method, [33] were kept constant. There are many reports that AgNPs obtained by biosynthesis are spherical (Table 1), such as RsAgNPs. However, AgNPs with other morphology, like irregular, triangular, hexagonal, polyhedral, scaly, floral, rod, and pentagonal shapes have also been produced [28], such as those we observed in BvAgNPs and ObAgNPs.

Figure 3

Analysis of morphology and size of AgNPs. Scanning electron microscopy photographs and size distribution histograms of AgNPs synthesized from (a, b) B. vulgaris (BvAgNPs), (c, d) O. basilicum (ObAgNPs), and (e, f) R. sativa (RsAgNPs).

The EDS analysis showed that silver is the main component of the synthesized AgNPs, with making up the content of 39.76% for BvAgNPs (Figure 4(a)), 69.74% for ObAgNPs (Figure 4(b)), and 54.1% for RsAgNPs (Figure 4(c)). Other elements such as carbon, oxygen, chlorine, and calcium were found in the AgNPs obtained. Furthermore, iron was found in BvAgNPs, magnesium and sulfur in ObAgNPs, and boron and copper in RsAgNPs. In green synthesis, the NPs are generated as a metal core covered by organic matter from the plant extract. These elements are part of the molecules that cover the biosynthesized NPs and that are provided from the plant extract, which give them stability and increase their dimensions. [3, 4, 8] In other AgNPs, in addition to silver, other elements from the plant extract have been found, such as carbon [3, 8], oxygen [3, 8], nickel [8], silicon [4, 8], calcium [8], potassium [3, 4, 8], sodium [3, 8], and aluminum [4].

Figure 4

Dispersive X‐ray energy spectroscopy and elemental composition of (a) BvAgNPs, (b) ObAgNPs y, and (c) RsAgNPs.

(b)

(c)

The stability of the AgNPs was analyzed by determining the potential Z. The found Z potential values were −23 (ObAgNPs), −24 (BvAgNPs), and −32 mV, (RsAgNPs) (Table 4). A negative Z potential represents the electrical charge of the AgNPs surface and is indicative of stability [3]. The electrical charge is provided by the organic matter from the plant extract and that covers the AgNPs [33]. With these Z potential values, it can be affirmed that the AgNPs are stable, since they are between −18 and −31 mV, like those reported in AgNPs synthesized from P. guajava leaves [34]. The hydrodynamic diameter was calculated to determine the dimensions of the AgNPs. The hydrodynamic diameter values were 158.7 nm (BvAgNPs), 104.1 nm (ObAgNPs), and 118.8 nm (RsAgNPs) (Table 4). These values were determined in phosphate buffer, where AgNPs acquire a dipolar electrical layer on their surface that increases their dimensions. Therefore, these values were higher than those calculated by SEM, as has been reported in other works (Figure 3). For example, in AgNPs synthesized from Opuntia ficus-indica peel extract, an average size of 64 nm was determined by SEM and 220 nm by hydrodynamic diameter [9].

Table 4

Characteristics of biosynthesized AgNPs.

AgNPs	Shape/size	% Ag	Size/hydrodynamic diameter (nm)	Zeta potential (mV)
BvAgNPs	Sheets and almost spherical shape	39.76	78 ± 17 nm/158.683 ± 3.384	−23.383 ± 1.514
ObAgNPs	Sheets and cubes	69.74	99 nm ± 22.3/104.083 ± 1.685	−24.783 ± 0.354
RsAgNPs	Spherical	54.1	76 ± 16 nm/118.8 ± 2.244	−32.3 ± 0.565

3.4. Seed Disinfection for In Vitro Cultivation

The disinfection capacity of AgNPs in Arabidopsis and guava seeds was evaluated. The seeds were treated with the AgNPs and compared with a traditional disinfection treatment. The percentage of disinfection of P. guajava seeds was 90 (BvAgNPs), 89 (ObAgNPs), 95 (RsAgNPs), and 85% (traditional disinfection treatment). Arabidopsis seeds showed 100% disinfection with all the different treatments. With both seeds, no significant differences were observed between the treatments with the AgNPs and the traditional disinfection treatment. In another work, similar results were obtained, a 100% disinfection of Lamiacea seeds with AgNPs synthesized from extracts of Alkanna tinctorum rhizomes roots and Syzygium aromaticum flowers was achieved [35]. Our results support the idea that bisynthesized AgNPs could be used to disinfect seeds for in vitro cultivation.

3.5. Germination of Seeds Treated with AgNPs

The effect of AgNPs on seed germination was evaluated. A. thaliana and P. guajava seeds were exposed to different concentrations of the three types of AgNPs, and their germination was compared with those of the control group. A. thaliana germination was 94 ± 1.7% on average and remanded similar with all treatments. While with the P. guajava seeds treated with 1 mg/mL of AgNPs, a germination of 60% (RsAgNPs), 45% (BvAgNPs), 55% (ObAgNPs), and 37% (control) was obtained. (Figure 5). Statistical analysis indicates a significantly higher germination in the seeds treated with the RsAgNPs. Guava seeds have a very hard coat that limits the passage of water, so the percentage of germination under natural conditions is around 7%. [36] Therefore, we suppose that the RsAgNPs could be facilitating the access of water to the seeds of P. guajava. Something similar was observed in rice seeds treated with AgNPs synthesized from kaffir lime extracts where a higher germination was obtained because the NPs increased the enzymatic activity that favors the entry of water into the seed [22]. However, germination inhibition has been observed in the germination of lettuce [37] and Mimusops laurifolia [38] seeds due to exposure to AgNPs. These contradictory effects are related to the dimensions and concentration of the AgNPs applied to the seeds. [35, 36, 39] The RsAgNPs presented a more homogeneous spherical shape and a smaller size compared to the BvAgNPs and ObAgNPs. (Table 4), which may have caused the lower observed germination results.

Figure 5

Germination of P. guajava. Comparison of the germination rate of guava seeds treated with sodium hypochlorite as control and with BvAgNPs, ObAgNPs, and RsAgNPs. The reported treatment (^∗) showed a higher percentage of germination (α < 0.05).

3.6. Effect of AgNPs on Seedling Growth

Guava and A. thaliana seedlings obtained after seed exposure to AgNPs showed similar growth compared to controls (Figure 6(a)). AgNPs remain attached to the surface of the seed, so they did not produce effects on the growth or morphology of the seedling. This coincides with what was observed in Lamiaceae seeds exposed to biosynthesized AgNPs, which did not show effects on development [35]. In seeds of A. thaliana germinated in medium with 0.01 mg/mL of BvAgNPs and ObAgNPs, the seedlings showed greater elongation than the control, by 60% and 39%, respectively (Figures 6(b) and 6(c)), while with the RsAgNPs no effect was observed at that concentration. The BvAgNPs and ObAgNPs were observed to be less homogeneous than those of RsAgNPs, which together with others such as size and silver content (Table 4) could have contributed to the results obtained in plant elongation. (Table 4). It was also observed that the seedlings exposed to 1 mg/mL grew the same as the control, while those exposed to 10 mg/mL were 25% (BvAgNPs), 31% (ObAgNPs), and 45% (RsAgNPs) smaller in size than those of the control. (Figures 6(b) and 6(c)). The hormetic effect of AgNPs was previously observed in sugarcane explants grown in medium supplemented with AgNPs [40]. The authors reported an increase in the number and length of the shoots at concentrations lower than 0.1 mg/mL, and toxicological effects with 0.2 mg/mL of AgNPs [40]. At low concentrations, AgNPs provide N, Mg, and Fe as nutrients for the plant, without being harmed by silver, while at high concentrations of AgNPs, the plant is unable to counteract the oxidative stress induced by silver. [40] In addition, high concentrations of AgNPs affect plant metabolism [41]. Furthermore, AgNPs could affect the action of some phytohormones like indole acetic acid (IAA) and ethylene. IAA participates in the formation and elongation of stems, and ethylene is involved in the maturation of organs, at the beginning of flowering, and in the appearance of fruits [36]. The overproduction of ethylene causes seedlings to have short hypocotyls and roots [36]. Ethylene acts on receptors that use copper as cofactor, but Ag⁺ occupies the copper binding site, inhibiting the ethylene response [42]. The amount of Ag⁺ coming from a low concentration of AgNPs could cause an increase in the elongation of the seedlings by inhibiting the action of ethylene. The decrease in seedling growth at higher concentrations of AgNPs could be related to the induction of the efflux of IAA that facilitates Ag⁺ [42].

Figure 6

Growth of A. thaliana. (a) Seedlings from seeds exposed to 0.01 mg/mL of 357 AgNPs. (b) Average size of seedlings grown at different concentrations of AgNPs; those indicated showed higher (^∗) or lower (^∗∗) growth than the control (α < 0.05). (c) Seedling from seeds sown in medium with 0.01 mg/mL of AgNPs.

(b)

(c)

3.7. Antimicrobial Activity of AgNPs

The antimicrobial activity of AgNPs against the bacterium A. tumefaciens, which is used for the genetic transformation of plants, was evaluated. The results showed a 6 U log inactivation (99.9999% removal) after 1 h exposure with the BvAgNPs (Figure 7(a)) and RsAgNPs (Figure 7(b)). The genetic transformation of plants mediated by A. tumefaciens allows the insertion of DNA to provide advantages and greater agricultural productivity [43]. This bacterium must be eliminated for the explant to develop in a healthy way. Antibiotics are traditionally used; however, they can also damage the explant [44]. AgNPs could be an alternative for the elimination of this bacterium. The antimicrobial mechanism of action of AgNPs is related to the damage they cause to the cell wall and membrane [10] and applies to various species. However, it is the first time that its use is proposed in this area, since most of the investigations of AgNPs reported are against bacteria of clinical interest (Table 1). In addition, in this work, the activity of AgNPs against bacteria of clinical interest was also demonstrated (Table 2). A log inactivation of 6 U was observed with ObAgNP and RsAgNP against E. coli, and with BvAgNP and RsAgNP against K. oxytoca, after 1 h of exposure (Figures 7(b) and 7(c)). The RsAgNPs were the only ones of the biosynthesized NPs that had activity against all three evaluated bacteria after 1 h of exposure. This is probably due to the shape of the RsAgNPs since it has been seen that NPs with a spherical shape have increased antimicrobial properties.

Figure 7

Antimicrobial activity. Logarithmic inactivation of (a) BvAgNPs, (b) ObAgNPs, and (c) RsAgNPs against A. tumefaciens, E. coli and K. oxytoca.

(b)

(c)

4. Conclusions

An AgNPs production protocol was effectively established by means of green synthesis, from extracts of leaves of easily available plants: radish (RsAgNPs), chard (BvAgNPs), and basil (OmAgNPs). Obtained AgNPs showed different characteristics which may be related to the different chemical composition of plant extracts. These AgNPs had seed disinfection capacity as efficient as reagents traditionally used in disinfection for in vitro plant tissue culture. They were active against various bacteria their activity against A. tumefaciens is highlighted. This bacterium is used as a vector in the genetic plant transformation; however, it is difficult to eliminate it later. Therefore, treatment with AgNPs could be an option to keep transgenic plants free of A. tumefaciens. This work opens the way to use biosynthesized AgNPs for the benefit of in vitro plant cultivation.

Data Availability

The data that support the findings of this study are available from the corresponding author (JFMD), upon reasonable request.

Conflicts of Interest

The authors declare that there are no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Footnotes

Acknowledgments

The authors greatly acknowledge the Autonomous University of Aguascalientes for financial support of this work. The authors are also thankful to Yenny Adriana Gómez‐Aguirre, Julia Victoria Nava Carmona, María Fernanda Prado Fernández, and Ariel Alessandra Mosqueda Pérez for their support in the experimental development of this work and to Rogelio Garcidueñas Piña for his support in the statistical analysis. This work was supported by Autonomous University of Aguascalientes, Mexico.

ORCID

Cristina Garcidueñas-Piña

Roberto Carlos Valerio-García

José Francisco Morales-Domínguez

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