Evaluation location: Washington, United States
Materials and Methods: Ploidy was measured on fresh leaf material by flow cytometry with a Partec CyFlow Ploidy Analyzer 'DAPI' using Partec CyStain UV precise P extraction buffer and staining buffer. Leaf material was taken from germinated seed on filter paper in glass Petri dishes and/or recently cloned greenhouse grown plants. Leaf material for analysis was taken from a minimum of 4 (maximum of 8) different plants or germinated seed of each accession. Leaf tissue was homogenized by chopping with a razor blade for 30 to 60 seconds in extraction buffer, incubated for 1.5 to 2 minutes, filtered through 30 um nylon mesh, stain buffer added and incubated in dark for 1 minutes before analysis. Extraction and stain buffers were kept on ice, but procedures were done at room temperature.
Before analyzing Phalaris samples the CyFlow Ploidy Analyzer was checked for DNA-DAPI quality control by running a sample tube of Partec Calibration Beads UV. Following this a sample of Phalaris arundinacea with known ploidy was run, the gain (signal amplification) was adjusted to 486, and the configuration script saved. This script was used to run all subsequent samples. Sample speed was constant at 1.0 ul/second A minimum of 1000 particles per sample were analyzed.
Phalaris arundinacea: Tetraploids are 2n=4x=28, Hexaploids are 2n=6x=24