Evaluation location: Washington, United States
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 alfalfa plants grown in cone-tainers (size) and maintained in greenhouse. Ten plants were available for all but 2 accessions. Plants were bulked in groups of 5 for ploidy analysis, therefore, 2 analyses done for each accession. Material for analysis was cut from young, fully expanded leaflets. Leaf tissue was homogenized by chopping into fine particle with a razor blade for 30 to 60 seconds in extraction buffer, incubated for 1 and half to 2 min, filtered through 30 um nylon mesh, stain buffer added and incubated in dark for 1 min before analysis. Extraction and stain buffers were kept on ice, but procedures were done at room temperature. Before analyzing 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 Medicago (Study 196, Archer FDC-5) with known ploidy (tetraploid) was run, the gain (signal amplification) was adjusted to set the tetraploid peak at channel 100, and the configuration script saved. This script was used to run all subsequent samples. Sample speed was constant at 1.0 ul/sec. A minimum of 1000 particles per sample were analyzed.