2.1 Reagents

Silver nitrate (AgNO₃) was acquired from Loba Chemie Pvt. Ltd., and Indole-3-Acetic Acid (IAA), Indole-3-Butyric Acid (IBA), MS medium components, and Potato dextrose agar were obtained from Himedia Laboratories. Sodium hypochlorite (NaClO), Agarose, Ethidium bromide, potassium bromide (KBr), glass wares and other tissue culture materials were obtained from Sisco Research Laboratories (SRL). Fungicide was (Bavistin) purchased from a local fertilizer company. PCR mixture obtained from Amplicon QIII and RAPD marker (OPK-7-5′-AGC GAG CAA G-3′) was specifically obtained from Operon Technologies, Inc., USA. Deionized water (18 mΩ cm⁻¹) was used throughout the experiments.

2.2 Preparation of silver nanoparticles

In a typical experiment, 1 mM of silver nitrate solution was prepared by dissolving 16.9 mg of AgNO₃ in 100 ml of deionized water and the solution was kept in an ice bath for half an hour. 10 mM of both IAA and IBA was prepared, separately. At the beginning of the reaction, 5 ml of 1 mM ice cold silver nitrate solutions was taken in test tubes. Then IAA and IBA solutions were separately added and mixed in test tubes containing aqueous AgNO₃. The time of addition of IAA and IBA in a silver nitrate solution was considered as the starting of the reaction and both mixtures were subjected to continuous stirring in a water bath at 60–70 °C until the transparent solution changed into yellow color. The color change indicates the formation of colloidal nanoparticles stabilized by IAA (at 700 μl) and IBA (at 800 μl). The IAA- and IBA-stabilized AgNPs were characterized and carried out for in vitro and ex vitro experimental studies.

2.3 Characterization of IAA- and IBA-stabilized AgNPs

Nanoparticles characterization and analysis are very essential and quite critical task using sophisticated instruments which give statistical and structural data relevant to nanoparticles shape, size, size distribution, dispersion, surface charge and their overall physiochemical properties. In this experiment, all UV-Vis spectra of AgIAA, AgIBA, AgNO₃ solution, water-dissolved IAA and IBA were obtained from UV-Vis Spectrometer (Shimadzu UV-1601, Japan) with UV probe 2.33 version software. Further, the IAA- and IBA-stabilized AgNPs solutions were centrifuged at 10,976g for 10 min in 15 °C to remove the excess stabilizing agent from the colloidal solution and residue nanoparticles were diluted in ultra-pure distilled water for further analysis. To know the nanoparticles yield, it was calculated independently according to Mie theory and some quantitative experiments from the literature with the obtained UV-Vis spectral extinction coefficient data (Trutna et al., 2008; Paramelle et al., 2014). For transmission electron microscopy analysis, IAA- and IBA-stabilized AgNPs were diluted in 1:20 ratio with deionized water to avoid overloading from which 1.5 μL was loaded with 3 mm carbon-coated copper grid and allowed to air dry at RT. Then the sample was analyzed by TEM (HITACHI H-7650, Japan) operated at an accelerating voltage of 120 kV. Here, Dynamic light scattering (DLS; Malvern Instruments Ltd., Malvern, UK) was used to determine the hydrodynamic diameter of the silver nanoparticles. All DLS measurements were performed at a fixed angle of 90° at 25 °C. The zeta potential of a particle is the overall charge that the particle acquires in a particular medium and is also measured using the same zeta size equipment at 25 °C. To know the interaction of IAA and IBA with silver nanoparticles, the solutions were subjected to FT-IR (ABB MB 3000) analysis. The sample was prepared from the pellet of IAA- and IBA-stabilized AgNPs after centrifugation at 14,000 RPM for 10 min and the pellet was grained with potassium bromide (KBr) into a fine powder. The grained powder samples were subjected to FTIR spectra analysis from 400 to 4000 cm⁻¹ and the percentage of transmission was recorded and compared with the fine powders of pure IAA and IBA as the control. Cyclic voltammetry studies to comprehend the reduction of silver nanoparticles in the solution were triggered by the IAA and IBA, and change in the oxidation state of the metal ions. Cyclic voltammograms (CV) were recorded using a computer controlled 400A electrochemical analyzer using glassy carbon (GC) with 2 mm of surface diameter as working electrode, Pt wire as a counter electrode and Ag/AgCl as a reference electrode. The surface of the electrode was polished with alumina powder (0.05 μm) and rinsed carefully with double distilled water before use. Thermogravimetric Analysis (TGA) is a widely used tool for quantitative analysis of surface bound grafting densities (Benoit et al., 2012). TGA was performed with a TGA Q50 (TA instruments, New castle, Delaware). The instrument heat profile used was a heating step from 10 to 100 °C at 5 min followed by heating to 800 °C at 10 min for thermal degradation of the sample. A hormone control and AgIBA were run for TGA at gas atmosphere to determine the grafting density of hormone on silver nanoparticles which is a critical feature to consider when designing nanoparticles for biological and environmental applications.

2.4 In vitro tissue culture studies of IAA- and IBA-stabilized AgNPs

2.5 Ex vitro application and assessment of IAA- and IBA-stabilized AgNPs on stem cuttings

The present investigation deals with the ex vitro assay to assess both rooting ability and antifungal activity (indirect & direct) of the IAA- and IBA-stabilized AgNPs. Fungal strains of Curvularia lunata, Rhizoctonia solani and Colletotrichum gloeosporioides were obtained from Plant pathology laboratory, Center for advanced studies in Botany, University of Madras. The three fungal conidial suspensions (10⁶ conidia/ml) of the organisms were prepared in sterilized distilled water for 10-day old cultures. Freshly prepared suspension of inoculum from three cultures was mixed and 1 ml of pooled inoculums was inoculated into each plastic cup containing sterilized moistened soil with inside volume of 250 ml. Stem cuttings of Hibiscus rosa sinensis were collected from the youngest available woody stems in the vicinity of our university campus. Following tip cutting method, stems of 10–12 cm long were detached with 0.7–1.0 cm diameter and 2–3 nodes without adult leaves were obtained. For this study, we have taken six sets and each set contains four stem cuttings accordingly to be treated with IAA- IBA-capped AgNPs (approximately 0.02 mg/ml), native IAA-IBA (10 mM) test solutions and both 1% bavistin (systemic fungicide) and water treated kept as positive and negative controls. Each set of cuttings was soaked (basal long soak method) for 30 min with its respective solution. After treatment, the cuttings were tapped slightly to remove excess solutions and inserted one each into phytopathogenic fungi inoculated soil at 2.5 cm deep inside. The plastic cups with cuttings were kept in a tray chamber with translucent polyethylene enclosed in a glass-house. Either side of the tray chamber was left open during the day to prevent excessive heat build-up. No supplemented illumination was provided except on cloudy days. Water was sprinkled once daily depending upon the soil moisture status. Holes in the bottom of the cups allowed excessive water drainage. The cuttings were examined for adventitious root initiation and disease progress at 30th day and the data were recorded. The disease progress is assessed throughout the experiment by observing the roots, stems and leaves by discoloration and bodily damage. The results were expressed as mean values ± standard deviations (S.D.) from the five replications. Along with this study, the hormone AgNPs mechanism of action against phytopathogens was directly assessed by the quantitative micro spectrophotometric assay (Broekaert et al., 1990) and also morphological changes were studied by simple light microscopy. In this study, centrifuged and pelleted fungal conidia from three cultures were inoculated into fresh potato dextrose broth to make conidial suspensions in the concentration of 10⁶ conidia/ml. From that suspension, 100 μl of each suspensions was individually added in the 96 well microplate and incubated with 100 μl of either AgIAA or AgIBA (0.02 mg/ml) for 0–120 min at the natural light in accordance with our previously published work (Raja Muthuramalingam et al., 2015). For negative and positive controls, both water and fungicide bavistin at 1 mg/ml were used. Growth inhibition was measured at every 30 min up to 120 min on the range of 595 nm in a microplate reader. The rate of growth inhibition was determined based on the equation outlined by Broekaert et al., (1990).

3.1 Physico-chemical characteristic features of IAA- and IBA-stabilized silver nanoparticles

In Fig. 1a, UV-Vis absorption spectra for the pale yellow silver colloids obtained from both IAA and IBA prepared by heat mediated wet chemical reduction method showed a surface Plasmon absorption (SPR) band with a maximum of 450–460 nm indicating the presence of spherical and roughly spherical nanoparticles. The SPR bands of UV-Vis spectrum confirmed that the IAA and IBA hormones reduced the AgNO₃ to AgNPs. With standardized synthesis, both IAA- and IBA-stabilized AgNP colloids showed absorption (λ_max) at shorter and longer wavelength regions (above 420 nm) indicating the presence of smaller to larger particles which may be due to the complete reduction depending on rooting hormone concentration. And the calculated nanoparticles molar concentration of IAA was approximately at 5.9 × 10¹⁰ mol⁻¹ (approximately 0.02 mg/ml) and for IBA it was approximately at 5.7 × 10¹⁰ mol⁻¹ (0.02 mg/ml). The morphology of IAA- and IBA-stabilized AgNPs was revealed by a TEM bright field investigation. Due to sample dilution and silver containing high electron density the images were clearly captured in the TEM bright field. However, successful imaging of nanoparticles by TEM depends on the contrast of the sample relative to the background. Concentrated samples increase the contrast between the samples and surrounding could reduce the image quality. Diluted nanoparticle colloidal solution ideal for TEM analysis helps to take the desired micrograph with a large number of individual particles with good image quality. And metal nanoparticles with electron densities that are significantly higher than amorphous are easily captured by TEM including Silver, gold, copper, and aluminum (“TEM analysis of particles”, 2012). In Fig. 1b, at 100 and 50 nm scales, TEM images show the presence of uniform spherical shaped and monodispersed IAA- and IBA-stabilized silver nanoparticles in the size range of 20–150 and 6–70 nm, respectively. The pale yellow color of the solution mainly depends on the size of colloidal silver within the range of 10–150 nm. In agreement with that statement and TEM results, Fig. 1c of DLS analysis histograms of IAA-stabilized AgNPs showed that it has distributed particle ranges from 20 to 140 with the average size of 97.35 nm in diameter and IBA-stabilized AgNPs have distributed particle size ranges from 4 to 65 nm with the average size of 44.17 nm in diameter. The peak intensity indicates the multiple populations of spherical particles of diameter present in both of silver colloidal solutions. Electric potentials at the boundary of nanoparticles were examined by Malvern instrument, the mean zeta potential of IAA-stabilized AgNPs was −37.9 mV and that of IBA capped AgNPs was −40.4 mV (Fig. 1d). It concluded that both particles in colloidal solution attained overall surface negative charges which give opposite electrostatic repulsion between particles which was responsible for particle dispersion. Therefore, nanoparticles with zeta potential value greater than +25 mV and less than −25 typically have the high degree of stability. Indeed, hormone-stabilized AgNPs have greater stability up to 6 months at RT examined by UV-Vis spectra (data not shown). FTIR analysis was carried out to understand the interaction of IAA and IBA functional group with silver nanoparticles. Fig. 2a shows FTIR overlapped spectra of IAA and IAA-stabilized AgNPs. Spectra peak at 1045 and 1383 cm⁻¹ assigned to HC—CH, CH⚌CH stretches of the aromatic ring. The peak at 1686 cm⁻¹ corresponds to the carbonyl group (C⚌O) or C⚌C stretch and peak at 3448 cm⁻¹ was assigned to —OH group of IAA. After the synthesis of AgNPs using IAA, FTIR spectral peaks were found to shift toward shorter wavelength with higher intensity and sharp band. Particularly, peaks at 1383, 1625 and 3446 cm⁻¹ were found with increased intensity than IAA spectra. Small peak shift at 3448–3446 cm⁻¹ and significant shift at 1686–1625 cm⁻¹ were observed and these peak shifts were mainly due to the interaction of carbonyl and carboxyl groups with silver nanoparticles. Similarly, in Fig. 2b, FTIR spectra of indole 3-butyric acid show a broad peak at 3400 cm⁻¹ which corresponds to OH group and peaks at 1086 and 1383 cm⁻¹ were assigned for C—C and C⚌C stretch of the aromatic ring with the carbonyl group corresponding to peak at 1564 cm⁻¹. IBA capped silver nanoparticles show FTIR transmittance peaks shift toward long wavelength with increased intensity. Specifically, peaks at 1212, 1412, 1698 and 3387 cm⁻¹ correspond to IBA aromatic ring, carbonyl, and hydroxyl groups, respectively. These peak shifts and increased intensity of FTIR spectra are mainly due to the interaction of carboxyl and hydroxyl group of IBA with silver nanoparticles. It is clearly evident that carboxyl/ hydroxyl functional groups of IAA and IBA were involved in the reduction of silver ion in Ag metal and also acted as capping agent to control the nucleation growth in silver nanoparticles synthesis (Wade, 2003). Cyclic voltammetry is generally used to study the electrochemical properties of an analyte in solution. The redox potential of silver ions to AgNPs in the presence of rooting hormones in solution is verified by CV. Fig. 2c and d shows the cyclic voltammograms of AgNO₃, IAA and IBA, IAA- and IBA-stabilized AgNPs using platinum electrode with the fresh surface in LiClO4 (0.1 M) supporting electrolyte, at a scan rate of 100 mV/s. In Fig. 2c and d, an aqueous solution of AgNO₃ (curve a) exhibits one reduction peak at +0.27 V and a larger oxidation peak centered at +0.71 V in the potential range between −1 and +1.5 in both. The reduction peak is attributed to the electrodeposition of silver ions on the electrode surface, and the oxidation peak corresponds to the oxidation of silver from the electrode. In Fig. 2c and d, native IAA (curve b) exhibited no reduction peak and two oxidation peaks were observed at +0.92 V and +1.32 V in the aqueous medium. And native IBA exhibited no reduction peak and small oxidation peak at +0.72 V. Fig. 2c and d of (curve c) exhibits a complete reduction of the Ag⁺ ions to Ag° by IAA and IBA to AgNPs was indicated by the absence of cathodic and anodic peak current by AgNO₃ ions as shown in curve c. The reduction of AgNO3 may be due to the transfer of electrons from the IAA and IBA containing carboxyl/hydroxyl group to a metal ion, and the resulting AgNPs were stabilized. In Fig. 3a, we exhibit the scheme of AgNPs synthesis correlated with FTIR and CV analytical results. TGA also confirmed the presence of hormones on the silver nanoparticles as evidenced by a 94% weight loss corresponding to the grafted hormones at 300 °C. From TGA results, the hormone started denatured at 100 °C and completely denatured at 300 °C in 14.41 min. Hormone IBA synthesized Silver nanoparticles possessed 6% remain at the 300 °C in 14.41 min which concluded that 94% of hormone (Fig. 3b) was grafted on nanoparticles and the grafting density was also calculated using these data according to Benoit et al., (2012). The data revealed that 10,940 units of IBA chain are grafted on one particle and the density of hormone IBA unit in one particle is calculated as 17 unit/nm².

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Figure 1 (a) UV-visible excitation spectral data for IAA, IBA, AgNO₃, AgIAA and AgIBA. (b) Transmission electron microscopy images of AgIAA and AgIBA at 100 and 50 nm scales. (c) Dynamic light scattering histogram of particle size distribution corresponding to AgIAA and AgIBA. (d) Zeta potential measurement for AgIAA and AgIBA.

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Figure 2 (a) Fourier transform infrared overlaid spectra of native IAA and AgIAA and (b) native IBA and AgIBA, (c) cyclic voltammograms of IAA and (d) IBA reduced AgNPs: Curve-a: are AgNO₃, Curve-b: are pure rooting hormones of both IAA and IBA, Curve-c: are reduced form of AgNPs by both rooting hormones.

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Figure 3 (a) Schematic representation of AgNPs formation by IAA and IBA root promoting hormones. (b) TGA of bare IBA hormone and AgIBA.

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3.2 The effect of IAA-and IBA-stabilized AgNPs on in vitro treated micro cuttings

The result was obtained after the 25th day from the inoculation of basal dipped micro cuttings cultured on MS medium. Astonishingly, the visual and statistical analysis determined that hormone AgNPs proved as a potential root enhancer compared to native hormone-treated N. tabacum micro cuttings. Fig. 4d and e shows the highly promoted rhizogenic effect of IAA- and IBA-stabilized AgNPs treated plants, and it has two- and threefold higher stimulated root numbers and root hairs with elongated roots than native IAA and IBA hormone-treated plants (Fig. 4b and c). A negative control plant showed no sign of root initiation and two stunted leaves were observed in the nodal region as shown in Fig. 4a. From the treated plants, adventitious roots arose from the cambium nearby the mechanically damaged stem tissue at micro cuttings. The mean data of root numbers and length of roots were calculated and are represented in Table 1 with a bar graph of rooting percentage. This attempt with both IAA- and IBA-stabilized AgNPs, is certainly a successful one which was critically relevant to increase the root growth of in vitro cultured treated shoots compared to conventional native hormones treated. These results also pave the way to ex vitro studies in similar treatment method. The UV-Vis spectrometry analysis has given some potential information regarding hormone nanoparticles absorption into the treated plant. In Fig. 5a spectrum data show that absorbance intensities were decreased when the micro cutting incubated at 0–30 min with AgIAA. According to Mie theory calculation through absorbance intensities, during 30 min incubation, only 2% of hormone nanoparticles (AgIAA) was taken by micro cuttings revealed. It may vary by long time incubation and the size of the micro cuttings used to treat. The distribution of nanoparticles into treated plants was studied based on auxin hormone fluorescence properties, such that it is exposed to UV light, the native IAA and IBA hormones exhibited fluorescence, but IAA- and IBA-stabilized AgNPs did not have fluorescence (Fig. 6a). It assures that the time of AgNPs formation had quenched the fluorescence indole derivatives of native IAA and IBA through molecular energy transfer which was also confirmed by fluorescence spectrometry. In Fig. 6b, fluorescence spectra reveal that the wavelength at 364 nm native IAA exhibited excitation spectra at 316 nm and emission spectra at 392 nm with strong intensity level. IAA-stabilized AgNPs have excitation spectra at 224 nm and emission spectra at 280 nm with very low intensity (Fig. 6d). The native IBA also has exhibited excitation spectra at 324 nm and emission spectra at 404 nm with strong intensity level, but IBA-stabilized AgNPs exhibited decreased fluorescence intensity peak because of the static quenching of IBA fluorescence (Fig. 6c and e). This type of quenching requires molecular contact between fluorophore and quencher. Here Ag⁺ ions were employed as a quencher during the formation of AgNPs where the energy was transferred from fluorophore of hormone molecules. At this point, we anticipated the real fact of hormone-stabilized nanoparticles inside the treated plants. By inverted microscope analysis using the green excitation filter at 250–400 nm, we explored fluorescence spots inside the basal region not at the apical region in both the hormone-stabilized AgNPs treated plant specimen illustrated in Fig. 6f and g. The possibility of fluorescence of AgNPs treated plant might be the interaction between AgNPs and plant tissue. These interactions might lead to intracellular uptake, and for their part the biomolecules might induce phase transformations, free energy releases, restructuring and dissolution of hormone capped nanomaterial. Generally in a plant, uptake and accumulation occur only during the initial time of treating in an early pulse in the surrounding tissue at wounding region and were the sole reason for apical part not possessing fluorescence (Bennett et al., 1996). The kinetics of the AgNPs uptake of solution have been determined by its nanoscale range, which mimics like inorganic nutrient molecules could be easily actively transported through vascular structures of the plant at the wounding region. The SEM-EDS elemental analysis also revealed the presence of Ag metal elements in treating plant tissue and the bioaccumulation concentration of silver was detected by EDS only in trace level (0.54%) Fig. 5b. With this significant analysis, we also evidenced no morphological damages and growth inhibition like metal induced stress was observed in hormone-stabilized AgNPs treated plants. In RAPD analysis, the fluorogram of agarose gel banding pattern of PCR amplified fragments also reveals that no banding variation (0%) or no mutation at the DNA level between controls and both hormone-stabilized AgNP treated plants was observed (Fig. 7). Therefore, it is assumed that silver ion, a non-essential metal ion for plant growth either may have a role in plant growth or may have inhibited any complexes which involve a negative role in plant growth. Additionally, there are some precedential reports saying that Ag⁺ is able to promote rooting alone, without an addition of auxins to in vitro culture mediums (Kumar et al., 2009). All plants possess a certain tolerance to heavy metals, but at exceeded concentration and prolonged exposure to silver metal and any other metals definitely can cause toxic effects and disorder such as transpiration and photosynthesis inhibition, disturbance of metabolism and oxidative stress, all of which collectively yield a common effect on plant development and growth. Contrary to our results, we produce several lines of evidence that suggest the involvement of silver nanoparticles as a carrier of rooting hormone to deliver into the plant for the root development. Successful in vitro rooted plants treated with AgNPs were transferred to the culture tray containing soil then kept in the greenhouse. After one month of transformation, the N. tabacum plants were sustained by stimulated root growth and further planted in the field. The AgNPs treated field-grown plants have 90% of the success rate of acclimatization and completed its cycle after 4 months of producing viable seeds (Fig. 4f). Therefore, the hormone-stabilized AgNPs promoted the roots fully functional even after transfer to soil and very well acclimatized like normal plants.

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Figure 4 Rooting effect on in vitro cultured Nicotiana tabacum micro cuttings treated with: (a) control water treated, (b) IAA, (c) IBA, (d) AgIAA, (e) AgIBA and (f) acclimatization stages of hormone AgNPs treated plants.

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Table 1 Effect of native auxin hormones and hormone capped AgNPs on percentage of rooting, root number and root length in treated microcuttings of Nicotiana tabacum model plant grown at in vitro. The table data represent mean ± SD values and the bar diagram for a percentage of rooting made with standard error values.

Treated with	Root numbers per explant	Root length (cm)
IAA	7 ± 0.53	1.6 ± 0.7
IBA	9 ± 0.11	2.2 ± 0.2
AgIAA	13 ± 1.1	3.6 ± 1
AgIBA	15 ± 0.3	4.1 ± 0.5
Control	0	0

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Figure 5 (a) UV absorbance spectrum of hormone AgNPs (AgIAA) taken into the plant microcuttings at 0–30 min. (b) SEM-EDS analysis of BAF (bioaccumulation factor) into hormone AgNPs treated plant shows Ag element X-ray excitation peak with the weight of 0.54%.

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Figure 6 Uptake and location of hormones AgNPs into treated Nicotiana tabacum micro cuttings studied by fluorescence methods: (a) UV light exposure to (I) water (II) IAA (III) AgIAA (IV) IBA (V) AgIBA; fluorescence spectrum analysis of excitation and emission of (b) IAA (c) IBA (d) AgIAA (e) AgIBA; fluorescence spots inside the plant tissue treated with (f) AgIAA and (g) AgIBA.

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Figure 7 RAPD profile of in vitro treated plants: M - 100 kb Marker, Lane 1 - control, Lane 2 - IAA treated, Lane 3 - IBA treated, Lane 4 - AgIAA treated and Lane 5 - AgIBA treated.

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3.3 Effect of IAA- and IBA-stabilized AgNPs on ex vitro shoot cuttings

In this research work, rooting ability and antimicrobial activity (indirect and direct) of rooting hormone synthesized silver nanoparticles were investigated. After 30 days of surveillance, only those stem cuttings exogenously treated with native hormones and hormone capped AgNPs survived and regenerated. After 45 days of examination of cuttings, highest root number with elongated roots was observed only in AgIBA treated cuttings and AgIAA treated showed one fold reduced growth than AgIBA treated (Fig. 8d and e). Also, differences were observed between these two native hormones-treated plants shown in Fig. 8b and c. Both IAA and IBA native hormones treated showed a very low number of roots with short lengths. The detailed statistical data are expressed in Table 2. A timely comparison observed between in vitro and ex vitro native hormones treated, and ex vitro cuttings seems that root growth was inhibited by soil inoculated pathogens. Thus, pathogens might limit the nutrient uptake and water potential which were important for root growth. And the bavistin (positive control) treated did not show any root initiation, and after 40 days they started to dry and were finally lost (Fig. 8a-II). However, there was no visual evidence of inhibition of root development and were symptoms of soil inoculated pathogens in all treated stem cuttings except water (negative control) treated. It has some visual damping off and canker disease symptoms at the base of stem cuttings which were common symptoms for Rhizoctonia solani and Curvularia lunata (Fig. 8a-I). So it was determined that direct application of hormone-stabilized AgNPs at the base of root cuttings was an effective way to protect the cuttings from pathogen invasion. Through obtaining knowledge from in vitro study, we could firmly spell that basal end treated with hormone-stabilized nanoparticle has accumulated in the treated area and slowly gets oxidized to release rooting hormone and silver ion which facilitates prolonged root promoting activity and protects root inhibition activity from soil inoculated phytopathogens. It is concluded hormone capped silver nanoparticles have best inhibitory properties compared to fungicides against root disease causing pathogens and sustained with their potentiality to promote the root growth on treating Hibiscus stem cuttings. The percentage of the acclimatization rate of AgNPs treated (93%) was also higher than native hormone treated (47%). As per earlier reports, hormone IBA is an effective rooting hormone than IAA by many studies (Stefancic et al., 2005), such a way that, we achieved similar results from both in vitro and ex vitro studies. Here hormone-stabilized AgNPs especially Ag-IBA have better activity than Ag-IAA and native IAA, and IBA was confirmed through many observations. The direct antifungal assay and the micro spectrophotometric assay quantitatively revealed the inhibitory action of AgNPs against three plant pathogens of Curvularia lunata, Rhizoctonia solani and Colletotrichum gloeosporioides. The AgNPs (AgIBA) show better activity with various growth inhibition rates compared to controls (Fig. 9). And the light microscopic observations of direct antifungal activity have clear evident that all the three strains were very much susceptible to hormone AgNPs. It is observed disrupted cell structures and cellular leakage are due to its highly reactive potential activity (Fig. 9). Regarding phytotoxicity of hormone AgNPs, significantly, there were no morphological damages at treated plants. As far as propagation work is concerned, once the process and influence of hormone-stabilized nanoparticles are well understood, it should be possible to improve techniques considerably, thus enabling work to proceed with more confidence and with greater certainty of success in agriculture.

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Figure 8 Rooting effect on ex vitro cultured Hibiscus rosa sinensis stem cuttings treated with: (a) I & II-water and bavistin fungicide treated as controls, (b) IAA, (c) IBA, (d) AgIAA, (e) AgIBA and (f) Acclimatization stages of hormone AgNPs treated plants.

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Table 2 Effect of native auxin hormones and hormone capped AgNPs on percentage of rooting, root number and root length in treated stem cuttings of Hibiscus rosa sinensis model plant grown at ex vitro. The table data represent mean ± SD values and the bar diagram for a percentage of rooting made with standard error values.

Treated with	Root numbers per explant	Root length (cm)
IAA	1 ± 0.2	1 ± 0.3
IBA	1 ± 0.13	1.2 ± 0.55
AgIAA	7 ± 0.44	2.2 ± 1
AgIBA	9 ± 0.3	3.1 ± 1.2
Control	0	0

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Figure 9 Direct antifungal activity of Hormone stabilized AgNPs: Light microscopic images of (a) Curvularia lunata, (b) Colletotrichum gloeosporioides and (c) Rhizoctonia solani with control water and hormone AgNPs treated. Subset of chart shows growth inhibitory percentage obtained from micro spectrophotometric assay to the corresponding organism treated with AgNPs. Data were calculated by mean ± SD and presented in a chart.

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