Method of molecular docking
The computational (in-silico) technique has been widely used as an efficient instrument for virtual biological screening. Using this method, the biological activities and estimated affinities of natural products, synthetic compounds, and semisynthetic molecules have been evaluated (El-Sherbiny and El-Baz, 2015). In this study, chitosan was docked against Ethylene Response Factor1 (ERF-1) to study their inhibitory activity against (ERF-1) active site. (ERF-1) has a main role in the transcription of auxins by activation of (ASA1) to binding with their promoter, leading to auxin accumulation which has the upper hand in the inhibition of root growth (Tang et al., 1992). By inhibiting (ERF-1) action, the root can grow up and elongate normally (Hasançebi et al., 2011). At first, the protein was generated by Uniport (proteins Id: AF-P35614). Protein and chitosan were prepared, and energy was minimized by the MMFF94 force field. The molecular docking was done, twenty poses were generated then the best orientations were captured, and affinity scores and RMSD values were collected in table (2) (Eissa et al., 2022).
Synthesis and Characterization of Chitosan Nanoparticles
Chitosan (CAS number: 9012-76-4) has been purchased from Sigma-Adrich. Nanoparticles were created using ionotropic chitosan gelation with TPP anions. Chitosan (0.2%) was combined with an ascorbic acid (1%) solution and agitated at room temperature for 1 hour (1000 rpm). A TPP stock solution was generated by disintegrating 0.03 g of TPP in 11 mL of water. Once one ml of TPP stock solution was added dropwise to the chitosan solution while stirring at room temperature, chitosan nanoparticles formed spontaneously (1000 rpm, 1 h). The chitosan nanoparticles were sonicated for an hour after that to ensure their small size. The particle size and shape were determined using a transmission electron microscope (TEM), a JOEL-JEM 2100 with a 200 kV operating voltage. The size distribution of as-prepared Chitosan NPs was measured using a Malvern zeta-sizer nano ZS instrument with He/Ne laser (633 nm) at an angle of 173° collecting backscatter optics and the surface charge properties were measured using zeta-potential measurements. Furthermore, the surface properties of the as-prepared nanoparticles were investigated using an FT-IR JASCO 6300 spectrometer in the 4000 − 400 cm-1 range.
Plant samples and germination of seed
Astragalus chrysochlorus seeds were collected and taxonomized by Professor Zeki Aytaç, Sertable, Karaman, Turkey. Astragalus chrysochlorus seeds have been saved at the Herbarium of the Faculty of Science, Istanbul University (ISTF). The seed surface was disinfected with 70% ethanol for 1 minute, then 5% Domestos for 15 minutes, followed by three 15-minute washes with distilled sterile water.
In Petri plates containing 25 ml of MS medium without plant growth regulators, ten samples were supplemented with 3% (w/v) sucrose. Solidify using 0.8% agar (w/v) in the basal medium. After adjusting the pH to 5.8, it was autoclaved in a chamber at 121° C for 20 minutes (Hereaus, Vötsch, No. 440/0026/86). For germination, seeds incubated in single growth were illuminated with fluorescent light (1400 Em-2s-1) at 25 ± 2°C with a 16 h/daylight cycle. The growth chamber conditions were applied for micropropagation, besides the steps of indirect organogenesis described next (Saharan et al., 2016).
Micropropagation
Three cytokinins [transzeatin riboside] mentioned as (ZR), beside kinetin as (K) beside 6-benzyl amino purine as (BA)], included in a base medium incorporating two various auxins like [α-naphthalene-acetic acid that recorded as (NAA) and indole-3butyric acid that recorded as (IBA)], monitored during multiplication of shoot. During the initial step of the Instinct Enhancement Test, different five concentrations (0.1, 0.5, 1.0, 1.5 and 2.0 mg/L) of each hormone were prepared. In the 2nd sentence, the concentration of 0.5 mg/L binds to 5 different cytokinins for each cytokinin concentration (0.1, 0.3, 0.5, 0.7 and 1.0 mg/L) from auxins. A base medium lacking regulator of growth was used in the wild experiments. Seedlings (approximately 1 cm long) isolated from 15-day-old growing seedlings were used as explants bud culture sources. They were placed on the Surface and developed on the above medium within the growth room. Cultures were transferred to fresh medium three- weeks apart. For each treatment, ten mock plant samples were tested, and each treatment was duplicated three times. shoot multiplier and multiplied by the number of shoots per hypocotyl measured at day forty culture. Propagated shoots were then used in rooting testing (Faizan et al., 2021).
In-direct organogenesis
Actively dividing ex-plants hypocotyl for about (~ 0.5cm) from fifteen-day-old in outside grown germinated seedlings that used as starter for stimulation of callus. Different Four concentrations from 2,4-Di-chlorophenoxy-acetic acid (2,4-D) (0.5, 1.0, 1.5, and 2.0 mg/L) were experimented with for callus growth initiation. About Ten explants were placed in the horizontal direction on the surface of the petri dish medium and then incubated in the dark condition at around 25°C. For each treatment, 5 replicates were used. The frequency of the formation of the Callus and a growth index was measured through [(last a fresh weight callus - callus initial fresh weight) / initial fresh weight of callus after thirty-day culture. The actively golden calli were already divided into small growing parts on the 30th day of culture beside callus material was cultured on basal medium MS containing 0.5 mg/L ZR. Residual callus material grown on trees without growth regulators MS base medium used for bud generation. Samples of crops were incubated within a growth chamber (Steel et al., 1997).
Subculture
Subculture was done within 3 weeks, ten Calli were used, and five repetitions were performed for each treatment. On the 40th cultivation day, the weed shoots a number of per callus was measured. Next, the regenerated shoots were isolated from the Calli and then used in rooting tests. Roots propagate buds Micropropagation shoots (minimum length 1 cm) and unexpected scenes from calluses were used for rooting experiments. Three auxins [NAA, indole-3-acetic acid (IAA) and IBA] at three different concentrations as (0.1, 0.5 and 1.0 mg/L) beside different three concentrations of sucrose (1%, 2% and 3%) on MS medium without plant growth regulators was examined their effect on rooting. The crops were incubated within the growth chamber. For every root experience, ten micropropagation and weed shoots were used and each one was repeated 3 times. Percentage results of the shoots formed from roots, number of roots/shoots beside the average root length noted on the 40th day from cultivation.
Acclimatization
Seventy-five healthy seedlings have a well-developed shoot and root system selected, isolated from the culture medium, washed with water for removing agar, followed by transferring in plastic tubs including sterile soil supplemented with perlite 15D44. The transplanted samples were covered using a transparent plastic bag to keep on moisture, placed in an acclimatization chamber, and irrigated by mineral salt MS solution for four days apart. After two weeks, plastic bags were opened, and then sheltered plants were kept in natural conditions daylight at 19 to 23°C, at the laboratory. The survival ratio in propagated plants grown in vitro was assessed at end of the 10th -week migration step.
Root cultures Establishment
Root tips (1 to 1.5 cm) were cut after thirty days in seedlings and germinated in vitro in parallel with culture on the original MS medium to study root cultures. Tips were initially inoculated into a 50 ml Erlenmeyer flask containing 15 ml of liquid MS basic medium with concentrations of either 0.5 or 1.0 mg/L of NAA, either IAA or IBA. Cultures were incubated in a rotary shaker adjusted at 120 rpm in dark, and the root cultures’ fresh weight was recorded at 15, 30, 45 and 60 days. Ten repetitions were used for each treatment (Steel et al., 1997).
Statistical Analysis
The statistical analysis was performed using two-way ANOVA in SPSS, version 25. (IBM Corp., Armonk, NY, USA, Released 2013). According to Steel et al. (1997), data were treated as if they were in a complete randomization design [Steel et al., 1997]. The Duncan test was used to perform multiple comparisons. The level of significance was set at 0.05 (Turgut-Kara and Ari, 2006).