1. 1. Plant Material And Elicitor Treatments
Phoenix dactylifera L. bud clusters have been routinely maintained in vitro through subcultures and transferred every 6–8 weeks into multiplication media, as previously reported in our research (Fki et al. 2011). In this study, a temporary immersion bioreactor container (RITA™, Cirad, France) with two separate parts was employed. Elicitation experiments were performed in triplicate. All of the aseptic manipulations were carried out under a laminar airflow cabinet. To this end, 20 g fresh weight of growing buds were transferred onto the upper part of the RITA bioreactor containing 200 ml of sterile fresh Murashige and Skoog (MS) liquid medium in the lower part to which various concentrations of Methyl jasmonate (MeJA) were added. MeJA (Sigma-Aldrich) solutions were freshly prepared on the day of application at the following concentrations (0 (Control), 50, 100 and 200 µM), according to Udomsin et al. 2020. The cultures were maintained at 26 ± 2°C under 16/8 h (light/dark) photoperiod. The pumped air traveled down to the medium storage tank through a small pipe.
The pressure that builds up in the media storage tank forces the nutrient solution up to the culture chamber for 30 minutes during the immersion phase. The in vitro culture samples were harvested after 7 days of temporary immersion cultivation. For each of the four MeJA concentrations tested, three independent experiments were conducted with triplicate samples of bud explants (n = 12). Changes in buds morphology and viability were recorded based on visual inspection and fluorescent microscopy after staining. Plant materials from tissue cultures were harvested, weighed and aliquots were stored at − 80°C until final extraction for further biochemical assays. Furthermore, other freshly harvested buds were oven dried and used for phytochemical analysis.
2.2. Estimation of cell viability in in vitro cultures: fluorescein diacetate (FDA), acridine orange/ethidium bromide (AO/EB) and DCFH-DA staining using fluorescent microscopy
Browning in plant tissue culture indicates the release of colored compounds from the upper part of the RITA to the medium during growth or subdivision of the explant, in such a way that both the media and explants progressively change into brown. The viability of tissues was estimated after staining with fluorescein diacetate (FDA) according to Cai et al. (2020) and Jones and Saxena (2013). To this aim, tow milligrams of fluorescein diacetate were dissolved in 1 ml acetone. Then, the buds were stained with a reaction mixture, containing FDA solution (60 µl/ml) in liquid culture medium. The blend was left to incubate for 10 min at 25°C and was subsequently washed three times with ultrapure water. Fluorescence staining with AO/EB was employed to estimate cell death (Rybaczek et al., 2015). We identified four types of cells based on the fluorescence emission: (1) Viable cells look green, (2) early apoptotic cells appear green yellow to yellow, (3) late apoptotic cells range from yellow-orange to brilliant orange whereas (4) necrotic cells appear as dark orange to bright red. The production of intracellular reactive oxygen species (ROS) was measured through 2′, 7′- dichlorofluorescin diacetate (H2−DCFDA) oxidation. After treatment with MeJA, buds were incubated with 20 µM H2−DCFDA at 37°C for 1 hour in darkness then washed twice with PBS. Buds stained with FDA, AO/EB and H2-DCFDA were visualized with a fluorescence microscope (B-383FL OPTIKA®, Italy). Data was quantitatively analyzed based on ImageJ v1.53K software.
2. 3. Biochemical Assays
2. 3. 1. Measurement of cellular ROS, Hydrogen peroxide (H 2 O 2 ), nitrite formation (NO), lipid peroxidation and total thiols
A 2’, 7’-dichlorofluorescein diacetate (H2-DCFDA) fluorescence probe was used for the monitoring of ROS formation in Phoenix dactylifera L. The probe is transformed by intracellular esterase and H2O2 into the polar, fluorescent 2', 7' -dichlorofluorescein (DCF) according to Li (2019). DCF intensity was assessed in a microplate-reader at excitation and emission wavelengths of 488 and 525 nm, respectively, via a CFX96 (Bio-Rad) fluorescence plate reader. The level is expressed as relative fluorescence Unit (RFU) per mg of protein (RFU/ mg of protein).
Hydrogen peroxide (H2O2) concentration was measured in bud extract by ferrous oxidation-xylenol orange (FOX) method adapted to microtiter plates according to Gay and Gebicki (2000). Absorbance was read at 570 nm using H2O2 as a standard and findings were presented as µmoles g− 1 FW.
Nitric oxide (NO) production was measured by a spectrophotometer at 540 nm according to Antoniou et al. (2018) using the Griess reagent. NaNO2 was used as a standard and results were expressed as µmoles g− 1 FW.
The level of lipid peroxidation was determined by assessing thiobarbituric acid reactive substances (TBARS) with reference to malondialdehyde (MDA) - a known marker of oxidative stress - (Kuk et al., 2003). Values were calculated using 1, 1, 3, 3- tetraethoxypropane as a standard at 532 nm and expressed as nmoles g− 1 FW.
Total thiols (TSH) content was measured by spectrophotometer at 405 nm using the 5 − 5’-dithiobis-2-nitrobenzoic acid (DTNB) according to the method of Anderson (1985). Glutathione was used as a standard and results were expressed as µM g− 1 FW.
2. 3. 2. Enzymatic Assays
Catalase (CAT) activity (U min− 1 mg− 1 protein) was determined by spectrophotometry at 240 nm according to Aebi (1984).
Superoxide dismutase (SOD) activity (U min− 1 mg− 1 protein) was determined by the inhibition on nitroblue tetrazolium (NBT) reduction spectrophotometrically at 560 nm using Giannopolitis and Ries method (1977).
Guaiacol peroxidase (GPOD) activity (U min− 1 mg− 1 protein) was determined through the rate of guaiacol oxidation at 436 nm, according to Pütter’s method (1974).
Phenylalanine ammonia-lyase (PAL) activity (U min− 1 mg− 1 protein) was defined as the variation of 0.01 in the assay medium by spectrophotometry at 290 nm based on Assis et al. method (2001).
Tyrosine ammonia-lyase (TAL) activity (U min− 1 mg− 1 protein) which is able to generate 1.0 µmol p-coumaric acid per min at 320 nm was calculated according to Wang et al. method (2006).
Polyphenol oxidase (PPO) activity (U min− 1 mg− 1 protein) was estimated according to the method of Soliva et al. (2001) by measuring absorbance at 410 nm.
Protein concentrations in the extracts were quantified by a BCA protein assay kit (Pierce™) using bovine serum albumin as a standard.
2. 4. Phytochemical Assays
Dried buds were ground into fine powder, then extracted with 70% aqueous ethanol (0.1g of extract per 5 mL of extraction solvent) through ultra-sonication for 30 min at room temperature (Ultrasonic bath, Velleman). After centrifuging the mixture at 10,000 g for 10 minutes at 4°C, the supernatants were filtered through a 0.22 µm filter membrane and stored at 4°C. The filtrates were used to analyse TPC, TFC, DPPH, ABTS, HPLC and FTIR.
2. 4. 1. Total Phenolic Content (Tpc)
The ethanolic extracts of TPC were quantified by the Folin Denis method, using gallic acid as standard (Ascacio-Valdés et al. 2014). 90 µL of Folin-Ciocalteu reagent (10%) were added in each well containing 20 µL of the samples in 96 well plates and kept without disturbing for another 5 minutes. Then sodium carbonate (90 µL) was added from a 6% stock solution and then incubated during 90 min at room temperature. Gallic acid (GA and ethanol (20 µL) were used as positive and negative control, respectively. The absorbance was measured at 725 nm using a Metertech M965 microplate spectrophotometer. Total phenolic concentrations were reported as milligrams of GA equivalent (GAE /g DW).
2. 4. 2. Total Flavonoids Content (Tfc)
The ethanolic extracts of TFC were calculated in line with Zhishen et al. (1999). A total 10 µL of Aluminum chloride (10%) and CH3COOK (1M) was blended with 20 µL of the sample, then, diluted with 160 µL of H2O and incubated during 30 min. The absorbance was measured at 415 nm with Metertech M965 microplate spectrophotometer. TFC values were calculated using a quercetin (QE) standard curve and represented as mg QE equivalents g− 1DW.
2. 4. 3. Antioxidant Capacity
DPPH (1, 1-Diphenyl-2-picrylhydrazyl), an artificial stabilized free radical, was used to determine antioxidant capacity, according to Brand-Williams et al. (1995).
The ABTS·+ (2, 2’-Azinobis (3-ethylbenzothiazoline-6-sulphonic acid) scavenging assay was performed according to Re et al. (1999).
2. 4. 4. Measurement Phenolic And Flavonoid Compounds By High Performance Liquid Chromatography (Hplc) Analysis
Phenolic and flavonoid compounds were quantified in accordance with the protocol of Zeb (2015) using Agilent 1200 Series column high performance liquid chromatography (HPLC) system fitted with Diode-Array Detector (DAD) run at 280 nm wavelength. The mobile phase solution was 0.1% H3PO4 (Movable Phase A), acetonitrile (Movable Phase B), and Flow-Type Gradient Solution. For each sample, three injections (10 µl) were provided..
2. 4. 5. Fourier-transform Infrared (Ftir) Analysis
Fourier transform infrared spectra (FTIR) of each sample were measured twice in a range of wave numbers 500–4000 cm− 1 with a resolution of 4 cm− 1 using FTIR spectroscope (Spectrum Two™, Perkin Elmer, USA).
2. 5. Statistical Analysis
All experiments were perfomed in triplicate and the results were presented as mean ± standard deviations (SD). The data for TPC and TFC are presented as the mean of three replicates. Linear regression analysis was employed to determine the IC50 values of DPPH and ABTS tests. One-way analysis of variance (ANOVA) was done for all treatments to study if the differences in morphological parameters and content of secondary metabolites between vitro cultures were stimulated by the presence of elicitor. Pearson test was adopted to correlate between variables. Post hoc testing for the ANOVAs was conducted using Tukey test with p < 0.05 being considered statistically significant. Our analysis was achieved using GraphPad Prism version 9 for Windows, GraphPad Software (San Diego, CA, USA).