Plant material and growth condition
All experiments were implemented using in vitro multiple bud cultures of P. dactylifera cv. Barhee, which derived from juvenile leaf explants (Fki et al. 2011). Buds were cultured for 2 months on an MS medium. The media were supplemented with 2,4-D at different concentrations (0, 1, 5, 10, 20 and 40 mg L-1), 50 g L-1 sucrose and 8 g L-1 agar. The media were adjusted to pH 5.8 before autoclaving at 120°C for 15 min. All cultures were incubated at 25°C with a photoperiod of 16 h under fluorescent light.
In vitro shoot proliferation
After 2 months of incubation on MS medium enriched with different concentrations of 2,4-D, cultures were individually transferred into jars containing a PGR-free regeneration medium and incubated in 25°C in a culture room. Then they were maintained by sequential subculturing every 2 months.
After successive transfers on multiplication media, RITA® bioreactors were used for the proliferation of in vitro cultures using 200 mL of liquid MS medium. The pH was adjusted to 5.8 before autoclaving (120 °C/1 bar/ 20 min). The RITA® system is a simple bioreactor composed of two parts separated by a filter. In vitro cultures were placed in the upper compartment and the liquid medium in the basal compartment. The immersion cycle was 10 min every 24 h.
Elongated regenerated shoots derived from the RITA® system were divided into single or few shoots and then transferred to an MS agar-solidified medium for further elongation and root development.
Physiological and biochemical analysis
Growth rate estimation
To determine the effect of 2,4-D concentration, growth rates were calculated according to the following formula based on changes in fresh mass:
GR = t1(fresh weight after 2 months) / t0 (fresh weight of starting bud).
Estimation of chlorophyll content
For this experiment, 0.1 g of fresh leaves were homogenized in a mortar using 2.5 mL of 80% acetone (Scheer et al. 1989). The homogenate was transferred to a 1.5 mL tube, and centrifuged at 10,000 rpm for 10 min. The absorbance of the supernatant was measured at 663 nm for chlorophyll a (Chl a) and 645 nm for chlorophyll b (Chl b). Chlorophylls contents were determined using Arnon’s formula (1949).
Total protein and free proline contents
The quantitative measurement of total soluble protein was estimated by the Bradford method. Five microliters of the raw enzymatic extract were added to 795 μL of millipore water and 200 μL of Bradford reagent. The optical density was measured spectrophotometrically at 595 nm and the protein content was determined by reference to a range of calibration established with increasing amounts of bovine serum albumin from 0 to 15 μg.
Free proline was extracted and determined as described by Bates et al. (1973). Samples were ground in a mortar using liquid nitrogen then 1.5 mL of 3% sulphosalicylic acid were added to 150 mg of powder. After stirring the mixture, the homogenate was centrifuged at 13,000 rpm for 15 min at 4 °C. A 200 μL aliquot of the supernatant solution was added to 800 μL of reagent composed of ninhydrin (1.25 g of ninhydrin + 30 mL of glacial acetic acid + 20 mL of phosphoric acid (6M)) then heated for 1h in a water bath at 100°C. After cooling, 1 mL of toluene was added to the mixture before stirring. After 4 h of settling, two phases appeared and the upper phase (toluene) contained proline. The proline content was determined spectrophotometrically at 520 nm.
The browning of in vitro cultures of cv. Barhee treated with various 2,4-D concentration was investigated by visual observation. In this experiment, the degree of browning in relation to treatments was scored according to Abul-Soad (2012).
Determination of total phenolic content
The total phenolic content of plant material was determined using the Folin Ciocalteu method, as reported by Cicco et al. (2009). 500µL of Folin Ciocalteu reagent (10%) and 5 mL of Na2CO3 were added to 100µL of the extract. The reaction mixture was left standing for 30 min., then absorbance was measured at 727 nm. Gallic acid was used as a standard. Results are expressed as mg of gallic acid equivalent, GAE (mg of GAE/g of extract).
Antioxidative enzyme assays
CAT activity was determined according to the method described by Aebi (1984). The assay buffer contained 250 µL phosphate buffer (10 mM, pH 7), 50 µL H2O2 and 100 µL of enzyme extract (pH 7). The reaction was based on the consumption of H2O2 which was spectrophotometrically monitored at 240 nm.
SOD activity was measured by monitoring the inhibition of nitro blue tetrazolium (NBT) reduction at 560 nm (Beauchamp and Fridovich 1971). The reaction mixture contained 50 mM phosphate buffer (pH 7.5), 0.1 mM EDTA, 2 µM riboflavin, 10 mM methionine 75 µM NBT and crude enzymatic extract. The reaction was allowed to run for 10 min then stopped by turning off the light. Blanks and controls were run in the same manner. Blanks were without irradiation and controls were run without enzymatic extract.
Hydrogen peroxide level
Hydrogen peroxide content was measured according to Velikova et al. (2000) at 390 nm. The amount of H2O2 was calculated using a standard curve and expressed as µmol g-1 FW.
Estimation of lipid peroxidation
Lipid peroxidation was determined by estimating the total amount of malondialdehyde (MDA) contents, according to Hernández and Almansa (2002). The absorbance was measured at 532 nm.
DNA was extracted from the regenerated callus of Phoenix dactylifera using CTAB protocol following the method of Rogers and Bendich (1985). After purification, DNA concentrations were determined using a Nanodrop® (Thermo scientific) spectrophotometer. DNA integrity was checked by electrophoresis in 1% agarose gel (Sigma, Ultra-pure) in TBE buffer 1M (Tris; Borate; EDTA) at 70 V for 30 min to allow proper resolution. The gel was stained by 0.5 μg mL−1 ethidium bromide (Sigma) and observed under UV.
Primers and ISSR assay
A total of 8 arbitrary ISSR primers were evaluated for polymorphism in genomic DNA samples. Based on resolution and reproducibility of banding patterns, 4 primers were selected (Table 1).
For PCR amplifications, a 25 μL reaction mixture was used, which contained 50 ng of total DNA (1 μL), 10 µM of primer (1 μL), 2.5 μL of 10X Taq DNA polymerase reaction buffer, 0.5 unit of Taq DNA polymerase (Bio Basic) and 10 mM of dNTP mixture (DNA polymerization mix (Bio Basic). Amplifications were performed on a DNA amplification thermocycler (Techne TC-312) which was programmed using the following conditions: a denaturation step of 5 min at 94°C, followed by 35 cycles composed of 30 s at 94°C, 90 s at the annealing temperature, and 90 s at 72°C. A final extension of 72°C for 5 min was included. A negative PCR control sample devoid of DNA was used to verify the purity of amplification reactions. Amplifications were performed at least twice and only reproducible products were considered for further data analysis. Amplification products were separated on 1.8% agarose gel in 1X TBE buffer and detected by staining with ethidium bromide (0.5 μg mL-1) according to Reid (1991). The size of generated amplicons was estimated according to a 100-bp ladder (Bio basic INC).
DNA methylation study
Enzymatic hydrolysis of DNA
Enzymatic hydrolysis of total genomic DNA into nucleosides was performed using two enzymes: alkaline phosphatase and P1 nuclease (Jaligot et al. 2000). To this aim, DNA samples (5 µg) were added to 5 µL of a 0.5 U mL-1 solution of nuclease P1 (Sigma N8630) and 17.5 µL of a 0.0168 U mL-1 solution of alkaline phosphatase (Sigma P4252) and the reaction volume adjusted to 100 µL with the digestion buffer (30 mM NaCH3CO2, 0.1 mM ZnCl2, pH 5.3). The mixture was incubated for 4 h at 37°C with regular stirring every 30 min. The reaction was stopped by the addition of 245 µL of absolute ethanol to the hydrolyzed mixtures then centrifuged at 14000 rpm for 15 min in order to precipitate proteins in the pellet. The supernatant was transferred to a new tube. Ethanol was removed by evaporation under a laminar flow cabinet. Nucleosides were dissolved in 500 µL of sterile water then filtered through nylon filters (0.2 μm) prior to HPLC analysis.
HPLC analysis of nucleosides
An isocratic elution method was followed according to Cock et al. (2010). 50 mM KH2PO4, 8 % (V/V) methanol through a supelcosil LC-18S reverse-phase column (SUPELCO Inc; column length 25 cm; diameter 4.6 mm; particle diameter: 5 mm), with a flow rate of 0.8 mL min-1 and a run time of 30 min for clear separation of the peaks. The effluent was monitored at the wavelength of 285 nm with a photodiode array detector (Beckman Coulter).
GMRs were calculated as percentages using the following formula:
GMR =100 * (5mdC)/[(dC) +(5mdC)].
All assays were undertaken in triplicate and data were analyzed using the statistical package GraphPad Prism 4. HPLC analyses were evaluated via one-way ANOVA t-test using GraphPad Prism 4. 01 computer program. Standard error (S.E.) was used at p<0.05 in Tuckey and Duncan’s test to establish significant differences between treatments in all experiments.