Plant material
Leaf specimens and seeds of Silene schimperiana were collected from shrubs grown in Gebel Tennia, Saint Katherine, South of Sinai E: 33.90090 N: 28.57364 Alt: 1810 (Figs. 1 and 2). Identification of plant was carried out by Dr. Omran Ghaly, Head of Plant Taxonomy Unit, Desert Research Center, Egypt, given the voucher number CAIH-1003-R and voucher specimens were deposited in the Herbarium of Desert Research Center (CAIH).
In vitro experiments
In vitro germination
Collected seeds of Silene schimperiana were washed with running tap water and detergent, then surface sterilized by soaking in commercial bleach containing sodium hypochlorite (5.25%) under laminar airflow cabinet (Holten LaminAir HVR 2448, USA), at different concentrations (0.5, 1.0, and 1.5% sodium hypochlorite solution). After 15 min, seeds were taken out and thoroughly washed thrice with sterilized distilled water. Seeds were cultured on half-strength Murashige and Skoog (1/2 MS) [21] medium (Duchefa, Haarlem, the Netherlands) supplemented with 3% (w/v) sucrose and 5.77 μM gibberellic acid (GA3; Sigma Cell Culture, min. 90%, St. Louis, USA) (0.45 μm filter sterilized) for germination. The pH was adjusted to 5.7 ± 0.1, then medium was gelled with 0.3% (w/v) phytagel (Duchefa, Haarlem, the Netherlands) before autoclaving at a pressure of 1.06 kg/cm, and 121 °C for 15 min (Harvey Sterilemax autoclave, Thermo Scientific, USA). Cultures were incubated under cool fluorescent tubes at day-night regime of 16-h photoperiod with the light intensity of 2500-3000 lux (F140t9d/38, Toshiba) at a constant temperature of 25 ± 2 °C and 60-70% relative humidity. The produced seedlings were cut into shoot tips and stem nodal segments and were used as explants.
In vitro propagation
Establishment stage
Shoot tips and stem nodal segment explants of seedlings were transferred to MS medium supplemented with 3% (w/v) sucrose and 0.3% (w/v) phytagel and 2.89 μM GA3, 1.08 μM α-naphthalene acetic acid (NAA) and different concentrations of 6-benzyl adenine (BA) of 1.11, 2.22, and 4.48 μM or kinetin (Kin) of 1.16, 2.33, and 4.65 μM (Sigma Cell Culture, min. 90%, St. Louis, USA). MS medium without plant growth regulators (PGRs) served as a control. The pH of the medium was adjusted to 5.7 ± 0.1 and autoclaved at a pressure of 1.06 kg/cm and 121 °C for 15 min. Cultures were incubated at 25 ± 1 °C at a photoperiod of 16/8 h light/darkness under cool white fluorescent tubes of 2500-3000 lux. The survival and growth percentage (%), the mean number and length (cm) of axillary shoots/explant were recorded after 3 weeks of culture.
Multiplication stage
The in vitro produced axillary shoots were transferred to MS medium supplemented with 3% (w/v) sucrose and 0.3% (w/v) phytagel and different concentrations of cytokinins (Sigma Cell Culture, min. 90%, St. Louis, USA); BA (2.22, 4.48, and 8.97 μM), N6-(2-isopentenyl) adenine (2iP; 2.44, 4.83, and 9.65 μM); or thidiazuron (TDZ; 2.27, 4.55, and 9.1 μM), individually. MS medium without PGRs served as a control. The pH of the medium was adjusted, autoclaved, and cultures were incubated as mentioned in the shoot induction stage. The number and length (cm) of axillary shoots/explant were recorded after 3 weeks of culture. Subculturing was done every 3 weeks.
Rooting and acclimatization stages
The multiplied axillary shoot clusters were transferred for rooting on quarter-strength MS medium supplemented with different combinations of indole-3-butyric acid (IBA; Sigma Cell Culture, min. 90%, St. Louis, USA) at 4.92 and 9.85 μM and NAA at 5.38 and 10.75 μM. Quarter-strength MS medium without PGRs served as a control for rooting induction. The pH was adjusted, media autoclaved, and cultures were incubated as mentioned in the previous stages. The percentage of rooted shoots (rooting %) and the number and length (cm) of roots/explant were scored after 4 weeks of culture on the rooting medium.
The rooted plantlets were removed from the nutrient medium and washed thoroughly with distilled water to eliminate the residues of the medium, then were transferred to plastic pots in a mixture of sand and peat (1: 2 v/v) (Peat moos, PROMIX®). The pots were covered by translucent polythene plastic bags to maintain high humidity and prevent the dissection of the newly transferred plantlets. Transplants were irrigated and the plastic bags were pored (one pore/5 days for 5 weeks) to decrease the humidity and acclimatize the plants to the external atmosphere gradually. After 6 weeks, the plastic bags were removed completely and the plants allowed to grow under open conditions.
Experimental design and statistical analysis
In vitro experiments were subjected to the completely randomized design. At least ten replicates were cultured for each treatment and the experiments were repeated twice. One-way analysis of variance (ANOVA) was used to evaluate significant differences between the mean values of different treatments, using Duncan’s Multiple Range Test [22] as modified by Snedecor and Cochrane [23]. The differences between means were compared at p < 0.05.
DNA barcode analysis
Extraction and purification of DNA
Silene schimperiana leaf specimens were collected and ground to a fine powder in liquid nitrogen using a sterile mortar and pestle. For DNA extraction and purification, DNeasy Plant Kit (Qiagen, Germany) was used. The concentration and quality of extracted DNA were estimated by running on 1% agarose gel electrophoresis, using a DNA size marker (Lambda DNA Hind III digest Phi X 174/HaeIII digest).
PCR and gene sequencing
The PCR reaction was carried out as reported by Ibrahim et al. [24] in a total volume of 50 μL PCR master mixture consisted of the following: 1x buffer (Promega, USA), 15 mM MgCl2, 0.2 mM dNTPs (Promega, USA), 20 pcoml of each primer (Invitrogen, USA), 1 u of Taq DNA polymerase (GoTaq, Promega, USA), 40 ng DNA and ultra-pure water to the final volume. DNA barcoding was performed with the ncDNA ITS gene and two cpDNA genes, rpoC1 and rbcL. For PCR amplification and sequencing of rpoC1, rbcL, and ITS, the following primer pairs were used: ITS-F (5′-CCT TAT CAT TTA GAG GAA GGA G-3′), ITS-R (5′-TCC TCC GCT TAT TGA TAT GC-3′); rpoC1-F (5′-GGC AAA GAG GGA AGA TTT CG-3′), rpoC1-R (5′-CCA TAA GCA TAT CTT GAG TTG G-3′); and rbcL-F (5′-ATG TCA CCA CAA ACA GAA AC-3′), rbcL-R (5′-TCG CAT GTA CCT GCA GTA GC-3′). The average amplicon sizes/bp were 722, 520, and 693 for ITS, rpoC1, and rbcL, respectively.
The PCR was carried out with a Perkin-Elmer/GeneAmp® PCR System 9700 (PE Applied Biosystems, USA) programmed to fulfill 40 cycles after an initial denaturation cycle for 5 min at 94 °C. Each cycle consisted of a denaturation step at 94 °C for 30 s, an annealing step at 50 °C for 30 s, and an elongation step at 72 °C for 30 s. The primer extension segment was extended to 7 min at 72 °C in the final cycle.
The amplification products were determined by electrophoresis in a 1.5% agarose gel using ethidium bromide (0.5 ug/ml) in 1X Tris borate Edita (TBE) buffer at 95 volts. For PCR product size determination, a 100 bp DNA ladder (Promega, USA) was used as a molecular size standard. Gel images were visualized using a UV transilluminator and photographed using a Gel Documentation System (BIO-RAD 2000, USA).
Purification of PCR products was carried out by a QIAquick PCR Purification Kit (Qiagen, USA). The PCR product was sequenced using the dideoxynucleotide chain termination method with a DNA sequencer (ABI 3730XL, Applied Biosystems) (Microgen, Korea) and a BigDye Terminator version 3.1 Cycle Sequencing RR-100 Kit (Applied Biosystems, USA) according to the protocol supplied by the manufacturer.
Assignment of species
DNA barcoding of Silene schimperiana was carried out using the Basic Local Alignment Tool (BLAST) available on the National Center of Biotechnology Information (NCBI) website. All sequences were submitted to GenBank, USA. It provided MK628682, MK628685, and MK628687 accession numbers for the nucleotide sequences.
BLAST searches were applied to all produced sequences using the online databases (DDBJ/EMBL/GenBank), analyzed using BLASTN 2.9.0 program (http://www.ncbi.nlm.nih.gov/BLAST), and aligned using Align Sequences Nucleotide BLAST. The identification of species was considered successful when the highest similarity percentage included a single species scored more than 97% [20]. Phylogenetic analysis was conducted using MAFFTv6.864, http://www.genome.jp/tools-bin/mafft, and phylogenetic trees were generated.