The coriander seeds were obtained from the Pakan Bazr company, Isfahan, Iran. The experiment was carried out in a greenhouse with a relative humidity of 65%-75% and a photoperiod of 16/8 (lightness/ darkens) in Islamic Azad University of Maragheh, Iran. The growing medium was cocopeat and perlite at a ratio of 2:1, which was applied in three-liter pots.
Experimental design and treatments
The Cd stress and Se-NPs, as treatments of the study, was carried out in a factorial based on completely randomized design (RCD) with three replicates. Each pot contains one plant of coriander. Three different Cd stress were 0 (control), 4 mg L-1, and 8 mg l-1 applied at 4-leaf growth stage. The irrigation with Cd solution (250 mL in each pot) was occurred for five times.
Se-NPs (Sigma-Aldrich, USA) were spherical with average particle size of 10-40 nm, specific surface area of 30-50 m2 g−1, true density of 3.89 g cm−3, CAS number of 7446-08-4, and purity of 99.9%. The Se-NPs at levels of 0 (control), 20, 40, and 60 mg L-1 were sprayed on plants two times in 6-leaf stage (first time) and after 15 days (second time). After the flowering stage, the leaves were sampled to measure the physiological and biochemical attributes.
ICP/MS (Agilent 4500 series, USA) was used to measure Cd concentration in coriander tissues. The 0.2 g of dry matter was mixed with 4 ml of 65% nitric acid (NO2) and kept at 25 °C for 24 hours. Thereafter, the sample was placed in an oven at 90°C until evaporating NO2. The volume was made up to 10 ml with distilled water (Khosropour et al. 2019).
Relative water content (RWC) determination
The developed leaves were applied to measure RWC. After measuring the fresh weight (FW), saturation weight (SW) was obtain by immersing the leaves in distilled water for 24 hours. After that the leaf samples were dried at 70°C for 24 hours to get their dry weight (DW). Eventually, RWC was calculated based on the following equation (Dhopte and Manuel 2002).
Malondialdehyde (MDA) measurement
To determine MDA, 0.2 g fresh leaf tissue was grinderd with 5 mL of trichloroacetic acid (TCA, 1%) and centrifuged for 5 min at 10,000 rpm. Moreover, 5 mL of TCA (20%) containing 0.5 mL thiobarbituric was added to 1 ml of supernatant and placed in a 95 °C water bath for 30 min, and followed by centrifuging for 10 min at 10,000 rpm. The absorbance wavelength was 520 nm (Heath and Packer 1968).
Chlorophyll (Chl) assay
To determine Chl content, 0.5 g of leaf tissue was mixed with 4 ml of acetone (80%.) The supernatant was prepared after centrifuging at 4000 rpm for 5 min. To measure total Chl, a spectrophotometer (T80 model) was calibrated and zeroed using acetone 80% in 630, 647, and 664 nm wavelengths. The absorbance of the solutions was read and the concentration Chl was calculated (Arnon 1949).
To determine the proline content, 0.1 g of fresh leaf tissue was mixed with 10 ml of sulfosalicylic acid (3% w / v) and centrifuged at 4000 xg for 20 min. Then, the mixture was supplied with 2 mL of ninhydrin acid and 2 mL of glacial acetic acid. Simultaneously, 2 ml of standard 0, 4, 8, 12, 16, 20 mg of proline were mixed with 2 ml of ninhydrinic acid and 2 ml of acetic acid. The samples were read at 520 nm using a spectrophotometer (Bates et al. 1973).
Total phenolic content (TPC)
The TPC was measured with Folin-Ciocalteu as a reagent and gallic acid (GA) as a standard. For this, 2 g of leaf sample was homogenized with 8 ml of 80% ethanol and centrifuged at 12000 × g for 20 min. After that, 0.5 mL of supernatant was transferred into 15 mL Falcons. Then 500 μl Folin-Ciocalteu was added to mixture and after 2 min, 1 mL sodium carbonate (7%) was added to the reaction mixture and the final volume was reached to 6 mL using distilled water. The mixture was remained in a 30 ° C (dark condition) bath for 90 min. The absorbance of the samples was measured in a 725 nm wavelength with a spectrophotometer (McDonald et al. 2001).
Total flavonoid content (TFC)
The amount of TFC was measured by aluminum chloride colorimetric method. In this method, 0.5 mL of the extract solution with mixed with 1.5 ml of 95% ethanol, 0.1 ml of 10% aluminum chloride, 0.1 ml of 1 M potassium acetate and 2.8 ml of distilled water. After keeping the samples at room temperature for 30 min, the adsorption of the mixture was read at 415 nm. The quercetin standard was used to draw the curve (Chang et al. 2002).
Essential oil (EO) content and composition
To obtain EO content, dried leaves were hydro-distillated for 3 h using a Clevenger-type apparatus (European Pharmacopoeia, 1983). The EO samples were dehydrated by placing them in dark glass bottles containing anhydrous sodium sulfate. The samples were maintained at 4 º C until they were analyzed by gas chromatography and/or mass spectroscopy. EO yield was determined as the amoun of EO extracted from total dry weight per pot. The EO compisition were analysed by Varian 3400 GC-MS system equipment with AOC-5000 auto injector and DB-5 fused silica capillary column (30 m × 0.25 mm i.d.; film thicknesses 0.25 µm).
All data was statistically analyzed SAS software in three replicates. The value of treatments was compared by Duncan’s multiple range tests. The data were statistically investigated at 5% probability level.