Plant material and achene germination
The mature achenes of T. insularum intended for evaluation as explants were supplied from the seed collection of Dr. Huseyin Inceer deposited at 4°C at the Karadeniz Technical University, Department of Biology, Trabzon (Çanakkale, Gökçeada, 30 m a.s.l. 17 April 2009, Inceer 717).
Initially, the achenes were washed with tap water for 30 min and then treated with 70% (v/v) ethanol for 30 s. After removal of the ethanol, the achenes were disinfected with 3% sodium hypochlorite (NaOCl) for 10 min. Finally, the disinfected achenes were washed with sterile distilled deionized water three times for 15 min, and then cultured in Murashige and Skoog (MS) (Murashige and Skoog 1962) (Duchefa), and Gamborg’s B-5 (B5) (Gamborg et al. 1968) (Duchefa) basal media, each containing 4.7 µM kinetin (KIN) (Sigma). At the end of the 30th day, the plantlets were evaluated for germination percentage to determine which of these two main basal media was better suited to T. insularum.
In the shoot multiplication studies, nodal segments obtained from seedling shoots after the third subculture were used as an explant. MS basal medium including vitamins and containing 2% (w/v) sucrose (Duchefa) and 0.8% (w/v) phyto agar (Duchefa) was selected as the most suitable medium for shoot proliferation studies. 6-benzylaminopurine (6-BA, 4.4 µM), KIN (4.7 µM), 6-(y,y-dimethylallylamino)-purine (2iP, 4.9 µM), thidiazuron (TDZ, 4.5 µM) and zeatin (ZEA, 4.6 µM) in combination with indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) or α-naphthalene acetic acid (NAA) (0.5 µM) were added to the medium to support the basic basal medium and increase the shoot multiplication rate. All plant growth regulators (PGRs) used in this study and supplied by Sigma were filter-sterilized with 0.22-µm filters, except for 6-BA and NAA, and were added to the cooled media after autoclaving. The pH of the media was adjusted to 5.8 with 1 M HCl or 1 M NaOH before autoclaving. All cultures were maintained at 24 ± 2°C under a 16/8 h photoperiod at a photosynthetic flux of 50 μmol m−2 s−1, provided by cool daylight fluorescent lamps. The subculturing protocol was performed every four weeks. Multiplication rates were calculated by assessing the number of shoots per explant, length of shoots, the number of leaves on each shoot, callus formation, and plant quality (internode length and shoot thickness).
Nuclear DNA content
Flow cytometry was used to check for the stability of nuclear DNA content as well as ploidy level in in vitro derived shoots of T. insularum. The leaves of 30-day-old seedlings and of shoots multiplied on MS media supplemented with cytokinins (KIN, 2iP, TDZ, 6-BA and ZEA) and auxins (IBA, IAA, and NAA) were used for flow cytometric analysis. Leaf fragments of the sample plant and the standard plant (Zea mays L.) were chopped up using a razor blade in 1 mL of woody plant buffer (Loureiro et al. 2007, 0.2 M Tris HCl, 4 mM MgCl2.6H2O, 2 mM EDTA, Na2.2H2O, 86 mM NaCl, 10 mM K2S2O5, 1% PVP-10, 1% (v/v) Triton X-100, pH 7.5), supplemented with 50 μg mL-1 propidium iodide and 50 μg mL-1 DNase-free RNase, filtered through a 30-μm mesh and stored on ice, in the dark, until measurement. Three independent samples were extracted, filtered and then measured using a BD Accuri™ C6 instrument. Usually 10.000 nuclei per sample were analysed for nuclear DNA content and absolute values (Inceer et al. 2016). DNA content was then calculated from mean values of G1 peaks according to the following formula:
MS media, each individually supplemented with 2.5 µM IBA, 2.9 µM, IAA and 2.7 µM NAA and without growth regulators, were again selected for rooting the well-proliferated and sufficiently elongated shoots (≥ 20 mm). Four weeks after the transfer of shoots to the root media, the rooting percentage, number of roots per shoot, root length and secondary root number (lateral roots on the longest root) were calculated to evaluate the rooting success. Each experiment was carried out in triplicate.
After rooting studies, rooted and well-developed plants were selected to determine their survival rates under greenhouse conditions as well as in the botanical garden. The greenhouse humidity and temperature were adjusted to 80-85% and 26 ± 2°C, respectively. After 30 days, the plants were moved to a field in the botanical garden. While 100% peat was employed in the greenhouse, 1:1 (v/v) peat and forest soil was used for transferring the plantlets to the botanical garden.
For all germination experiments, five achenes were placed into each Magenta vessel, and four vessels were prepared per treatment. Each treatment was performed in triplicate. Statistical differences between obtained from shoot proliferation, root induction and flow cytometric studies were calculated on Statistical Package for the Social Sciences software (SPSS version 21). Duncan’s multiple range test (DMRT, 95% confidence level) from one-way analysis of variance (ANOVA) was used to detect the statistical significance of differences among the mean values in shoot multiplication and root induction. Nuclear DNA data were evaluated using ANOVA and Dunnett’s test at P = 0.05.