Plant materials and treatments
The apical buds were sectioned longitudinally into four sections. In order to induce callus formation, explants were transferred to MS basal medium [10] supplemented with 3 mg L−1 6-(dimethylallyl amino) purine (2iP), 30 mg L−1 naphthalene acetic acid (NAA), 1.5 g L−1 activated charcoal, and solidified with Agar-Agar at 7.0 g L−1. Cultures were kept under complete darkness at 27 ± 2 °C. The cultures were transferred to fresh media, with the same composition after every 6 weeks interval until the callus had initiated. For callus propagation, it was transferred and grown in jars containing 25 ml of the MS medium, equipped with 100 mg L−1 glutamine, 5 mg L−1 thiamine HCl, 1 mg L−1 biotin, 30 g L−1 sucrose, and solidified with agar at 7.0 g L−1 and 0.5 g L−1 activated charcoal, with the addition of NAA at 6 mg L−1 and 2iP at 2 mg L−1. Two Fe forms were included in the medium [FeEDTA (ferric ethylenediaminetetraacetic acid) and FeEDDHA (ethylenediamine di-2-hydroxyphenyl acetate ferric)]. The supplementation of iron sources at different concentrations in the growth medium was assessed. MS medium was modified at two concentrations of FeEDTA [(T1) 36.7 mg L−1 (5.6 mg L−1 Fe) (control), and (T2) 73.4 mg L−1 (11.2 mg L−1Fe)] or three concentrations of FeEDDHA [(T3) 93.5 mg L−1 (5.6 mg L−1 Fe), (T4) 187.0 mg L−1 (11.2 mg L−1 Fe), and (T5) 280.5 mg L−1 (16.8 mg L−1 Fe)]. The pH of the medium was adjusted to 5.7–5.8 before the addition of agar. Media dispensed into culture containers. All culture containers with media were autoclaved at 121°C and 1.04 kg.cm−2 for 20 min. Cultures were incubated in the culture room at 27 ± 2 °C and irradiated for 16 h with a diffuse light provided by cool white fluorescent lamps (μmol m−2 s−1). The weight of the callus was recorded after 6 weeks from culturing. For multiplication, the callus was divided and subcultured on regeneration media equipped as mentioned above, except for the plant growth regulators 1 mg L−1 (NAA) and 3.0 mg L−1 (2iP) [14]. It was also equipped with the same FeEDDHA and FeEDTA concentrations to study their effects on bud multiplication, and some changes in phytochemical traits are mentioned below. Cultures were incubated in the growth chamber at 25 ± 2°C under 16 h photoperiods. The experiments regarding the percentage of bud induction and bud number per jar were recorded after 12 weeks of culturing callus on the multiplication media.
Catalase activity (CAT, EC 1.11.1.6) was assayed, according to [15]. CAT has been verified in the l M of H2O2 g−1 FW min−1; the activity of the enzyme has been evaluated at 25± 2 °C. The total 3 mL solution mixture was 2.8 mL (25 mM, pH 7.0), 0.1 mL enzyme extract, and 0.1 mL (0.4%). H 2.8 mL phosphate buffer. Upon adding H2O2, the reaction began. The decline of H2O2 inhibition depends on the regulation of absorbance reduction at 240 nm.
Estimation of peroxidase activity (POD)
Shootlet enzyme extract was prepared as recommended by [16]. The leaf tissues have been grounded with 0.1 M sodium phosphate buffer at pH 7.1 (2 ml buffer/g of fresh tissue in a mortar). These triturated tissues have been strained through four layers of cheesecloth, and the filtrates were centrifuged at 3000 rpm for 20 min at 6 °C. For an estimate, the enzyme of the supernatant fluid was used. The activity of peroxidase was calculated by the methods of [17].
Effect of FeEDDHA and FeEDTA on in vitro rooting
Clusters of unrooted in vitro shoots of date palm cv. Barhee were collected in the elongation stage. Typical shoots were separated individually and cultured on MS medium [10]. The culture media consisted of MS salts, supplemented with 30 mg L−1 sucrose, 0.5 mg L−1 NAA, and 0.5 g L−1 activated charcoal, 7 mg L−1 agar, and different concentrations of FeEDTA (0, 50, 75, and 150 mg L−1) or FeEDDHA (0, 0.5, 2.5, and 5.0 mg L−1). The pH was adjusted to 5.7–5.8, and then, the media were autoclaved at 121°C for 20 min. All cultures were incubated under room temperature 25 ± 2 °C, with a 16 h photoperiod provided by white florescent light. The experiment results regarding the percentage of root induction and root number per shoot were evaluated 6 weeks after the inoculation of shoots on the culture media.
Mineral analysis of shoots
Elemental analysis was performed on content of potassium (K), calcium (Ca), and boron (B) in date palm shoots after 12 weeks of culture according to the method described by [18]. Before the analysis, the shoots were separated and washed with deionized water twice and dried at 60°C until reaching constant weight. Dried shoots (0.5 g) were digested with a mixture of sulfuric-perchloric acid under heating for 1 h. The digested solution was transferred into volumetric flask 50 cm3, and volumes were completed in size with distilled water. The extract of the samples was filtered and diluted to the volume of 50 ml. Ca and Mg content was determined by flame atomic absorption spectrophotometry. A UV-visible spectrophotometer measured the absorbance of the solution at a wavelength of 620 nm [19]. For B analysis, dried shoots were ash dried (60 °C for 1 h) and digested with 10 ml 0.36 N H2SO4 [20]. B was quantified by a spectrometer. There were three replicates of each treatment. The atomic absorption spectrophotometer determined the shoot samples’ iron content at the wave length of 324.8 nm.
Assessment of chlorophyll content
The chlorophyll content in the leaves was measured using the method of [21].
Extraction and measurement of auxins
Auxins were extracted and quantified according to [22]. Five grams of leaves after various treatments with Fe-EDDHA and FeEDTA were homogenized using 80% methanol. The extract has been filtered through the Whatman filter paper (no. 1) and evaporated at 4 °C in dark conditions under a vacuum. The supernatant was dried in a vacuum, withdrawn by a 0.1 M phosphate potassium (pH 8.1). Eleuate was obtained by using 1 N hydrochloric acid (HCI) and by using a partitioning (4x) with diethyl ether, in dryness, in water with a pH set to 2.5. The injection in reversed HPLC, C18 column, in the isocratic elution mode by the concentrate, determined phytohormones using a portable acetone step (26:74) with 30 mM of phosphoric acids. A UV detector (2996 PDA detector) with 280 nm was passed through the column eluants, and auxins were detected and quantified. Standard auxins were used as the source (IAA).
Experimental design and statistical analysis
The data were statistically analyzed by one-way analysis of variance (ANOVA) using statistical analyses with the SPSS packet software. Separation of means among treatments was determined using L.S.D. test at 5%.