Evaluation location: England, United Kingdom
Ploidy level and cellular DNA content were determined by flow cytometry (DeLaat et al. 1987) using the CyStain UV Precise P or the CyStain UV Absolute P kits (Partec). For ploidy level determination, small amounts (0.5 cm2 maximum) of leaf tissue were cut into segments and placed in 0.5 ml nuclei extraction buffer for 1 min. The suspension was passed through a 50 μm CellTrics disposable filter (Partec) and stained for 2 min with 2 ml 4′,6-diamidino-2-phenylindole (DAPI) staining buffer. Samples were then excited by UV irradiation from a mercury arc lamp and analysed in the blue fluorescence channel of a flow cytometer (Partec-ROBBY). O. viciifolia accession 1127, corresponding to PI 212241 in Kidambi et al. (1990), which is known to be tetraploid, was used as a control. The cellular DNA content for O. viciifolia 1001 was estimated by DAPI staining as described above (GC content dependent quantification), and also by staining with propidium iodide after ribonuclease treatment, followed by analysis in the red fluorescence channel of the flow cytometer (GC content independent quantification). A range of plant species with known 2C values (Doležel et al. 2007) were assessed as standards for measuring the DNA content of O. viciifolia. Zea mays L. CE-777 (2C = 5.43 pg) was identified as the most appropriate of these references.
Somatic chromosome counts were carried out with conventional squashes of root tip cells. Seeds were placed between moist filter paper at 20 °C and young roots harvested when approximately 1 cm in length, usually after three or four days. The excised roots were immediately placed in 2 mM hydroxyquinoline for 4-5 h at room temperature. The roots were then fixed overnight at 5 °C in Carnoy’s solution (60 % ethanol, 30 % chloroform, 10 % glacial acetic acid). Hydrolysis was carried out in 1 M HCl at 60 °C for 8-9 min, and the roots stained for 30 min in Feulgen solution (leuco basic fuchsin). The meristematic tips were then removed, placed on a slide in a drop of 45 % acetic acid, and observed with a phase contrast microscope (Carl Zeiss Axioskop 40) equipped with a camera and image analysis software (Improvision OpenLab 4.0.2).