The phytofabrication and characterization of AgNPs was carried out as previously described with several modifications38.
Preparation of Parthenium hysterophorus aqueous extract
Fresh disease-free leaves of locally collected PH were rinsed with distilled water and shadow dried for 5–7 days to remove residual moisture. The dry leaves were crushed to a fine powder with a kitchen blender. The crushed leaf powder (10 g) was mixed in 100 ml of distilled water and boiled in a water bath for 30 min at 60 °C. The aqueous mixture was filtered through Whatman filter paper, grade 1, and the filtrate was stored at 4 °C.
Synthesis of Parthenium hysterophorus AgNPs (PHAgNPs)
PHAgNPs were synthesized by the addition of 1 ml of PH aqueous extract to 9 ml of AgNO3 (1 mM). The solution pH was adjusted to pH 8 with 1 N NaOH and incubated in a water bath for 30 min at 60 °C. The AgNPs were recovered by centrifugation at 15000 rpm for 20 min, and the resulting pellet was air dried and stored in ambient conditions until further use.
Characterization of PHAgNPs
The biosynthesized PHAgNPs were initially confirmed by UV-visible spectroscopy (Cyber lab-100 spectrophotometer), by measuring the absorbance spectra between wavelengths of 300 and 700 nm at regular intervals.
FTIR analysis was carried out to identify the reduced biomolecules from PH leaf extract in PHAgNPs. The PHAgNPs were pelletized by mixing with KBr and subjected to FTIR analysis with a JASCO 460 PLUS FTIR spectrophotometer, and the spectra were recorded between 4000 cm−1 and 400 cm−1 wavelengths.
TEM images were obtained to visualize the morphological features of PHAgNPs, with a JEOL 3010 transmission electron microscope at an acceleration voltage of 100 kV.
XRD analysis was performed to determine the structure and dimensions of PHAgNPs. The PHAgNP samples were lyophilized and subjected to XRD analysis with an XRD 600 instrument, Shimadzu, Japan instruments (voltage 40 kV; current 30 mA; CuKα radiation).
The mean particle size (L) (PAN analytical X-pert PRO Model) of the AgNPs was calculated with the following formula: L = 0.9 λ/β cos θ, per the Debye-Scherrer equation.
Where λ is the wavelength of the X-ray; θ is Bragg’s angle; and β is the full width at half maximum.
MS medium39 supplemented with casein hydrolysate 500 mg l−1, proline 500 mg l−1, Gamborg B5 vitamins (HiMedia, India) and sucrose 3%, was used as the rice culture medium. PHAgNPs at different concentrations (5 mg l−1, 10 mg l−1, 15 mg l−1 and 20 mg l−1) suspended in water were supplemented into the culture medium, and the pH was adjusted to pH 5.8, this was followed by sonication of the medium for 30 min. The medium was then appropriately distributed into conical flasks (50 ml medium per150 ml conical flask), and finally 4 g l−1 CleriGel (HiMedia, India) was added before autoclaving. After autoclaving, the medium was allowed to cool to ~45 °C. The flasks were then swirled manually and kept at 4 °C to solidify quickly, avoiding agglomeration of AgNPs.
Influence of PHAgNPs on callus induction
Oryza sativa cv. IR64 seeds were obtained from TNAU, Tamil Nadu, India. The seeds were sterilized with 0.1% HgCl2 and 70% ethanol as previously described40. The sterilized IR64 seeds were inoculated in flasks containing callus induction medium (rice culture medium with 2,4-D, 2.5 mg l−1 and PHAgNPs) and cultured in a growth chamber at 28 ± 1 °C in the dark for 14 days. The callus induction frequency was calculated with the formula:
Callus induction frequency (%) = No. of seeds producing calli/No. of inoculated seeds
Influence of PHAgNPs on rice plant regeneration and rhizogenesis
The embryogenic calli were transferred to regeneration medium (rice culture medium with 0.1 mg l−1 NAA and 3 mg l−1 BAP) with different concentrations of PHAgNPs (0, 5, 10, 15 and 20 mg l−1), and were incubated in a dark growth chamber at 28 ± 1 °C for 7 days. The flasks were then transferred to a chamber with a 16/8 h, day/night cycle. After 14 days, the regenerating calli were sub-cultured onto fresh medium with appropriate supplements every 14 days, until the plants regenerated. After 45–50 days on regeneration medium, the plant regeneration frequency was determined with the following formula:
Regeneration frequency (%) = No. of calli regenerating into plants/No. of calli initially inoculated onto regeneration medium × 100.
The regenerated plants were then transferred to rice culture medium supplemented with different concentrations of PHAgNPs (0, 5, 10, 15 and 20 mg l−1), and incubated in a growth chamber. After 14 days, the root length and number of roots per plant were recorded.
Total RNA isolation and cDNA synthesis
Total RNA was isolated with a PureLink RNA Mini Kit (Ambion, USA) from 14 day old regenerating calli cultured on control and PHAgNP (5 mg l−1, 10 mg l−1, 15 mg l−1 and 20 mg l−1) supplemented medium. The isolated RNA was verified by UV absorption spectrophotometry at 260/280 nm (BioDrop duo, UK) and agarose gel electrophoresis.
cDNA was synthesized with 1 µg of RNA from individual samples with a SuperScript™ III First-Strand Synthesis Kit (Thermo Fisher Scientific, USA), according to the manufacturer’s instructions.
The primers (Table 2) were synthesized for the auxin responsive gene OsIAA141, cytokinin responsive gene RR242, gibberellic acid responsive gene PBZ143, ethylene responsive gene ERF06344 and ABA responsive gene OsRab1645 with the Primer quest tool, IDT (https://eu.idtdna.com/Primerquest/Home/Index).
The real-time PCR reaction mixture included cDNA (1 μl), corresponding primers (0.2 μM) and 2× IQ SYBR Green Super Mix (Bio-Rad) (10 μl), adjusted to a final volume of 20 μl with sterile water.
The qRT-PCR was run on a Roche Light cycler (USA) with initial denaturation for 7 min at 95 °C, followed by 40 cycles of 95 °C for 15 s, 56 °C for 15 s, and 72 °C for 15 s; melting curve analysis was performed. Three biological replicates were analysed for each gene control and each treatment. The expression levels of each gene were normalized to the expression level of UBQ546 on the basis of threshold cycle (Ct) values, with the 2−ΔΔCT method47.
MDA content was determined as previously described48 with a few modifications. One hundred milligrams of regenerating callus tissue was homogenized in 5 ml of 10% trichloroacetic acid (TCA). The homogenate was centrifuged at 10000 rpm for 30 min at room temperature. Two millilitres of supernatant was collected, and 4 µl of 0.6% thiobarbituric acid prepared in 10% TCA was added. The mixture was then incubated in a water bath at 80 °C for 40 min and immediately cooled in an ice bath and centrifuged at 10000 rpm for 30 min. The absorbance was read at 450, 532 and 600 nm, and the MDA content was calculated with the formula:
Hydrogen peroxide estimation
Hydrogen peroxide was quantified as previously described13. One gram of callus tissue was homogenized in 10 ml of aqueous TCA (0.1%, w/v). The mixture was centrifuged at 10000 rpm for 30 min at 4 °C, and the supernatant was retrieved. One millilitre of supernatant was mixed with 4 ml of 1 M potassium iodide reagent and 1 ml of potassium phosphate buffer. The solution was incubated in the dark for 1 h at room temperature. The absorbance was read at 390 nm and plotted against a standard H2O2 curve. The results are expressed as µM g−1 FW.
Total proline estimation
Proline content estimation was carried out as previously described49. One gram of callus tissue was homogenized in 10 ml of 5-sulphosalicylic acid (3%). The mixture was centrifuged at 10000 rpm for 15 min, and the supernatant (2 ml) was mixed well with 5 ml of glacial acetic acid and 5 ml of 140 mM acid ninhydrin, and heated for 1 h at 100 °C. The mixture was cooled and then extracted in a separating funnel with 10 ml toluene. The absorbance of the separated toluene containing the chromophore was read at 520 nm and plotted against the proline standard curve. The results are expressed as µg g−1 FW.
All the experiments were carried out in triplicate (n = 3). The data were analysed in Windows Microsoft Excel 2007 software. The differences in mean values were determined with Duncan’s multiple range test, with a significance level of p ≤ 0.05.