Plant material and growth conditions
M. suaveolens cuttings were taken from the botanical garden of the Faculty of Science and Technology, Fez. Each cutting included at least two nodes. Culturing was carried out in plastic pots (3 kg capacity) containing 5 plants per pot and grown in a greenhouse.
Cu and SA treatments
After 30 days of plants transplantation (DAT), watering was performed with Cu. This Cu dose was chosen because a preliminary experiment showed that from 40 mM Cu induced growth inhibition of M. suaveolens. Cu was applied weekly over five-weeks (200 ml per pot). SA (0.5 and 1 mM) was applied by foliar spray at 60 DAT (These SA concentrations were chosen based on a preliminary screening experience). Three SA sprays were performed at a weekly interval using a hand sprayer (100 ml per pot).
This experiment included total six treatments: control, 0.5 mM salicylic acid (SA 0.5 mM), 1 mM salicylic acid (SA 1 mM), 40 mM Cu (Cu), 40mM Cu + 0.5 mM salicylic acid (Cu+ SA 0.5 mM) and 150 mM Cu + 1 mM salicylic acid (Cu+ SA 1 mM). Experiment was performed in five replicate and sampling was done at 100 DAT.
Cu and nutrients (K, P and Ca) content
Content of Cu and minerals (K, P and Ca) was determined according to Cottenie et al., . The root and shoot samples of M. suaveolens were dried at 100 °C for 48 hours. 100 mg of dried plant was calcined at 450 °C for 12 hours in a muffle furnace. The ash obtained was dissolved in 3 ml of nitric acid (0.1 N) and then filtered through Whatman filter paper 540 hardened ashes. The volume was adjusted to 20 ml with distilled water. Based on this solution, the assay was performed by inductively coupled plasma emission spectrometry (ICP-AES) to determine the content of Cu and minerals (K, P and Ca).
Hydrogen peroxide content
The extraction and determination of hydrogen peroxide are carried out according to the method described by Sergieve et al., . 1 g of leaves and roots are ground in 15 ml of trichloroacetic acid, the ground material is centrifuged for 15 min at 12,000 rpm. The assay is carried out by adding to 0.5 ml of supernatant, 0.5 of 10 mM phosphate buffer pH 7 and 1 ml of 1 mM potassium iodide then the optical density of the tubes is read at 390 nm.
Lipid peroxidation was determined as 2-thiobarbituric acid (TBA) reactive substances, i.e. malondialdehyde (MDA). Briefly, 0.25 g tissue sample was homogenized in 5 ml of 0.1% trichloroacetic acid (TCA) followed by centrifugation at 10 000g for 5 min. To 1 ml aliquot of the supernatant, 4 ml of 20% TCA containing 0.5% TBA was added. The mixture was heated at 95oC for 15 min and cooled immediately. The developed colour was extracted with 2 ml n-butanol, and the absorbance was measured at 532 nm followed by the subtraction of the value of nonspecific absorption at 600 nm. The level of lipid peroxidation was expressed as nmol of MDA g–1fresh weight at an extinction coefficient of 155 mM cm–1 . The results were presented as the average of three replicate estimations for each treatment.
Environmental scanning electron microscope
Microscopic observations of the fresh leaves were made using an environmental scanning electron microscope (Quanta 200, FEI Company). The microscope was equipped with a tungsten electron gun. Analyses were carried out under partial pressure of water vapour.
Count of peltate glandular trichome
Peltate glands counting was performed on the ventral side of the fresh leaves on a 1 mm2 area, taking into consideration the basal (near the petiole), central and apical areas of the leaf. For each treatment, the glands number represents the average of glands of five plants and three leaves per plant .
Essential oil extraction
Essential oil extraction was carried out by hydrodistillation (Clevenger apparatus) of 100 g of parts aerial of S. officinalis dried in the free area. The extraction was carried out in 2 liters of distilled water for 180 min. The essential oil was collected, dehydrated with sodium sulfate and stored at 4°C. Essential oil yield was calculatedby the following formula :
YEO (ml/100 g DM) = (V/DM x100)
YEO: essential oil yield of DM.
V: the volume of essential oil collected (ml).
DM: dry plant weight (g).
Essential oil analysis by GC–MS
Essential oil content was determined by gas chromatography (GC) (Agilent 7890A Series) coupled to mass spectrometry (MS) and equipped with a multimode injector, a BD-ASTMD 6584 column (15 m × 0.320 mm × 0.1 μm) and ionization by electronic impact. The protocol consists of: 1 μl of a solution of HE solubilized in chloroform was injected into the column by split mode 1: 2 using helium as carrier gas at 4 ml min−1. The ion source and quadruple temperatures were 230 °C and 150 °C respectively. The oven temperature program was started at 30 °C and maintained 1 min then increased at 2 °C min−1 until 75 °C and maintained one minute then increased by 8 °C min−1 until 210 °C and kept constant for 1 min. The composition of the essential oil determined from the peak areas was calculated as a percentage of the total compounds existing in the sample detection using full scan mode between 30 and 1050 m z−1 with gain factor 5 and the identification was performed using NIST 2014 MS Library.
One-way analysis of variance was carried out for each parameter studied. Tukey’s post hoc multiple mean comparison test was used to test for significant differences between treatments (P ≤ 0.05). Univariate analysis was used to test significant differences in treatments, accessions, and their interaction for an individual parameter. All statistical analyses were performed with IBM. SPSS statistics, Version 22. The results of each experiment (biochemical assays) were repeated three times (20 times for morphological assays).