Electromagnetic exposure system
Anechoic chamber has been used to conduct the experiments and to isolate the experimental place from external HF (High frequency) and LF (Low frequency) radiations. fire proof, non-corrosive and fine woven Y-shield V4A03 stainless steel mesh has been used (thickness 0.16 mm, diameter 0.08 mm, open area 54% ,0.03 ohm conductivity, attenuation +55dBm). Chamber base has been provided with plywood (9 mm thickness) for its unreactive and non-conductive nature. PU (polyurethane) foam absorbers have been embedded inside the chamber to absorb additional radiation, extra noise cancellation, less super imposition and to avoid internal reflection in certain range (80MHz to 40GHz). Two pyramidal S-band horn antennas have been used working most efficiently in 2-4GHz with +15 dB gain and Maximum VSWR 1.3. One is emitter and other is receiver. Both are placed exactly facing each other with proper alignment to create a line of sight, where materials for experimentation was kept. Two co-axial adapters have been connected to both the antennas having resonating frequency of about 2-4 GHz and VSWR 1.3, which establish connection between waveguide and co-axial cables. Two low loss co-axial cables having length of about 3 meter have been used and connected through SMA connectors. Synth NV RF signal generator from Windfreak Technologies, LLC, USA, has been used and can be operated over a wide range of frequencies i.e. 34 MHz- 4.4 GHz. RF power also can be adjusted with that. LAB VIEW virtual programming platform has been used as control system. The seed materials were kept in the middle with Petri plates to maintain appropriate distance between two antennas and sample. The distance from both the sides of antenna to seed material is ~40.48 cm i.e. exact half of the length of radiation path which is more than ~1.5 times the material distance from source. The average power density was measured about 7.32 mW/m2. The frequency used was 2.7 GHz, which is equivalent to 4G cellular radiation intensity. The exposure inside the chamber is horizontal and the materials were placed in a height exact to the line of sight. The radiation was given for almost 48 h continuously.
Heat exposure system
Heating stress was given in a controlled environmental growth chamber with 42 ⁰C to the seeds for 48 h.
Seed germination, radicle growth study
Seed germination was recorded for 6 ,12, 18, 24, 30, 36, 42 and 48 h after exposure. Radicle length and breadth were also measured.
Enzymatic analysis
The action mixtures contained 1 milliliter of 10% soluble starch, 1 milliliter of 0.5 milliliters of sodium acetate buffer (pH 6.0), and 3 milliliters of enzyme solution that was appropriately diluted with water. The action was stopped by cooling the mixture after 30 minutes of incubation at 60°C (Hyun & Zeikus,1985).
Seedlings were grown in controlled green house chamber up to 10 days (28± 2 °C, 16/8 h light/dark photoperiod of 250 µmol m−2 s −1 photon flux density and 75± 2% relative humidity). Growth study was done by shoot length, root length, leaf area calculations.
Seedling biochemicals
Seedlings harvested after 12 days and biochemicals were done.
Protein, carbohydrate
After extracting soluble protein from fresh 500 mg plant extract by 10 mL of Phosphate buffer (pH=7.4), the sample was centrifuged in 13000 rpm for 20 min, 1 mL supernatant added to 5 mL of alkaline copper reagent (1:2, CuSO4, 5 H2O: C4 H 4 KNaO4,), reaction mixture added to 0.5 mL Folin-Ciocalteu reagent. BSA standard made and after 30 min incubation spectrometric reading done for development of light to dark blue colour at 520 nm (Lowry et al., 1951).
100 mg of plant sample extracted in 5 mL of 2.5 N HCL in 95˚C hot water bath for 3 hours. Acids neutralized by Na2CO3 until reaction stops. Addition of anthrone reagent (Anthrone, H2SO4 conc. 95%) after centrifugation of sample for 15 min in 7000 rpm. Reaction again heated for 5 min in 95˚C boiling water bath, reading for spectrophotometric analysis is done at 625 nm. Dark green to light green colour determines the presence of carbohydrate (Hedge and Hofreiter, 1962).
H2O2 determination
The H2O2 content was ascertained by applying the (Ansari et al., 2018) approach. Five milliliters of 50 millimolar sodium phosphate buffer (pH 6.5) was used to homogenize leaf tissues (0.2 g). After 3 ml of supernatant and 1 ml of 0.1% (w/v) titanium sulfate in 20% (v/v) H2SO4 were combined, the mixture was centrifuged for 25 minutes at 4,000 × g. With the use of an ultraviolet spectrophotometer at 410 nm.
Lipid peroxidation
MDA as the final product of lipid peroxidation determined by TBRS method. 100 mg plant sample macerated in mortar pestle using 0.1% TCA (tricarboxylic acid), centrifuged at 12000g for 30 min, supernatant was taken for the estimation. Added 0.5% TBA (tribarbituric acid) in 20% TCA of 4 mL to 1mL of supernatant. After heating in hot water bath for 30 min in 95˚C, the formation of bright sunset red colour was determined by spectrophotometric method in 532 nm and 600 nm range. The determination will come in nM unit (Stocks and Dormandy, 1971).
Calculation; 6.45 x (A532 – A600)/155
Antioxidant capacity
SOD, POD, CAT, GR
For SOD (superoxide dismutase) 1g of sample macerated in 10 mL ice cold 50 mM potassium phosphate buffer in pre chilled mortar pestle, centrifugation done at 10000g for 10 min. reaction mixture contains in 3mL 50 mM buffer, 13 mM methionine, 2 μM riboflavin, 0.1 mM EDTA, 75 mM NBT, 50 μM crude enzyme extract . 4x100 W bulbs were lighted near the mixture for 15 min, then immediately reading in spectrophotometer was taken at 560 nm, calculated the percentage inhibition. ( Beauchamp and Fridovich, 1971).
In POD (peroxidase) determination, the fresh tissue maceration of 0.5 g in 0.05 mm buffer, centrifugation at 15000g for 30 min, collected supernatant. The reaction mixture of 3 mL contains 0.01 M phosphate buffer, 0.05 mL guiaiacol, 100 μL enzyme extract. H2O2 was added 0.03 mL of 12.3 mM H2O2. Increased absorbance recorded at 30 sec interval up to 3 min, 470 nm (Zhou et al., 2006)
For determination of catalase (CAT), 100 mg of plant sample homogenized, in 2.5 ml phosphate buffer of 0.05 M, at 15000g centrifugation done for 30 min. 1.9 mL of 0.05 M phosphate buffer, 0.1 mL enzyme extract, 1 mL of H2O2. Then decreased absorbance recorded in different time intervals at 240 nm (Tseng et al., 2007).
Glutathione reductase (GR) determination spectrophotometrically done by taking 200 mg plant sample, maceration in phosphate buffer and mercapto ethanol, in 12000g for 15 min centrifugation done, supernatant taken for reaction mixture. 0.4mL of phosphate buffer, 0.2 mL of EDTA (0.2 mM), 0.2 ml of MgCl2 (1.5 Mm), 0.4 ml of NADPH (0.025 mM), 0.8 mL of enzyme extract. Addition of 0.4 ml of oxidized glutathione will start the reaction (0.25mM). The decreased absorbance at 340 nm will determine the enzyme activity recorded in 30 sec interval (Yannarelli et al., 2007).
Calculation:
SOD:
% inhibition of NBT reduction by SOD = control OD- treatment OD/ control x 100 =X% inhibition.
50% inhibition is equal to 1 unit of enzyme. then X% is equal to 1/50 x X= Y unit.
POD, GR, CAT:
Calculations of time change during enzyme action i.e. ΔAbs
Enzyme activity (Units/L) = (ΔAbs × Total assay volume) / ( Δt x ε x l x Enzyme sample volume).
Phenol
Phenol as an antioxidant determined by spectrophotometric method. 0.5-1.0gm of plant material was macerated in a mortar and pestle with 80% ethanol. After 10000 rpm 20 min centrifugation supernatant was collected and again re extracted. After the collection evaporation done. Distilled water added, 0.5 mL folin phenol reagent added. After 3 min Na2CO3 added, after boiling for 1 min absorbance taken at 650 nm. Catechol standard was prepared for reference (Malick and Singh, 1980).
Proline
Proline as dissolved solute provides osmotic tolerance against any kind of water imbalances. About 0.5 g of plant taken macerated with 10 mL of 3% sulphosalicyclic acid. 2 mL of filtrate in a test tube and 2 mL of glacial acetic acid and 2 mL of acid ninhydrin. After heating for 1 hr in boiling water bath, 4ml of toluene was added to create layer of polar and non-polar solvents, proline get separated in the below layer with bright scarlet red colouration which get detected by 520 nm of wave in spectrophotometer (Bates et al., 1973).