In vitro callus of date palm cv. Ashgar cultured in the Tissue Culture Laboratory of Date Palm Research Centre, Basra University, Basrah, Iraq, were used as the experimental materials (Fig.1). The apical buds were sectioned longitudinally into four sections. To induce callus formation, explants were transferred to MS basal medium 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 (Fig.1, a and b). The cultures were transferred to fresh media, with the same composition after every 6 weeks interval until the callus had initiated (Fig.1, c and d).
Plant materials
Callus was excised and cultured on the MS basal medium. To study the effects of BA in combination with NAA or PAA on buds regeneration and changes in phytochemicals, supplementation of these growth regulators at various culture medium concentrations was assessed. MS medium was modified at two concentrations of BA (2.0, 5.0 mgL-1) alone or in combination with NAA or PPA at five concentrations (0.0, 0.05, 0.5, 1.0 and 2.0 mg L-1). Treatments were consisted of 12 media, as shown in Table I
Culture conditions
All media used in this study consisted of 100 mg L-1 glutamine, 5 mg L-1 thiamine HCl, 1 mg L-1 biotin, 30 g L-1 sucrose, 0.5 g L-1 activated charcoal and solidified with agar at 7.0 g L-1 . The pH of the medium was adjusted to 5.7 by adding HCL or NaOH before autoclaving at 121 °C for 20 min. All cultures were maintained at 25 ±2 °C under ambient light of 1500-2000 lx light by cool white fluorescent lamps and a photoperiod of 16 h light/8 h dark. In this experiment, the degree of callus growth and browning in relation to treatments was recorded as follows in Table II:
The results of the experiments regarding the percentage of shoot regeneration and shoot number per a jar were evaluated 12 weeks after the inoculation of callus on the media. There were twelve replicates of each treatment.
Rooting of shoots
Micropropagated shoots of date palm cv. Ashgar with no visible signs of root development were transferred to MS medium supplemented with PAA or IBA or NAA at different concentration (0.0, 0.5, 1.0, and 2.0 mgL-1). Each treatment included 12 replicated jars, incubated at room temperature 25 ± 2 °C, with a 16 h white florescent light photoperiod. The percentage of root induction and root number per shoot were evaluated six weeks after the inoculation of shoots on the media. There were ten replicates of each treatment
Total phenolic content. The total phenolics content was determined with the Folin- Ciocalteu reagent according to the method of Singleton and Rossi (1965). Gallic acid was used as a reference standard; results were expressed as milligram of Gallic acid equivalent (mg g-1).
Genetic stability among regenerated date palm plantlets
In order to study the genetic similarities, several regenerated plantlets were analyzed at the molecular levels using RAPD analysis.
RAPD analysis
Total genomic deoxyribonucleic acid (DNA) was isolated from regenerated date palm plantlets using the CTAB method as described in Rogers and Bendich (1985). Polymerase chain reaction (PCR) reactions were conducted using a set of four arbitrary 4-mer primers (Operon Technology, Inc., Alameda, CA, USA). These primers and their sequences are presented in Table III.
The PCR mixture
The reaction mixture (20 μl) contained 10 ng DNA, 200 μM deoxynucleotide triphosphates (dNTPs), 1 μM primer, 0.5 units of Red Hot Taq polymerase (AB-gene Housse, UK) and 10-X Taq polymerase buffer (AB-gene Housse, UK). For DNA amplification, a Perkin Elmer thermal cycler (2720) programmed as follow: Denaturing: 95°C for 5 min 94°C for 0.45 min. Then annealing (35cycles) 35°C for 1 min. This is followed by 72°C for 1 min and 30 s and finally Extension: at 72°C for 7 min (Adawy et al., 2004). The amplification products were separated in 1% (w/v) agarose gel in 1X Tris/Borate/Ethylenediaminetetraacetic_acid (TBE) buffer and visualized by staining with ethidum bromide. The reproducibility of DNA profiles was determined by replicating all RAPD reactions at least three times using DNA markers. The primers were evaluated from wise pair comparison for the proportion of shared bands amplified (Nei, 1978). The similarity coefficient was calculated by using the statistical software package STATISTICA-SPSS (Stat Soft Inc.).
Statistical analysis
In all experiments, each treatment consisted of 12 replicates. Data were statistically analyzed by analysis of variance (ANOVA). The least significant difference (LSD) method was used to test the difference between treatments, and p≤ 0.05 was considered statistically significant. Statistical analyses were performed with SPSS packet software.