Evaluation location: Connecticut, United States
Ploidy level determined through University of Connecticut (Dr. Mark H. Brand). Base number is n=17; 2n=34; 4n=68. Fresh leaves were harvested and nuclei suspensions were prepared by chopping approximately 50 mg of young leaf tissue with a fresh razor blade in 55 mm plastic Petri dishes containing extraction buffer prepared according to Arumuganathan and Earle (1991). The procedure was modified in accordance with Meng and
Finn (1999) by adding 2 g of PVP-10 per 50 ml of extraction buffer and fluorescently staining released nuclei with propidium iodide after filtering, rather than during the chopping process. Relative fluorescence of total DNA (FL2) for each stained nucleus was determined with a Becton-Dickinson FACS Calibur Dual Laser Flow Cytometer (Becton, Dickinson and Co., Franklin Lakes, NJ) located at the Flow Cytometry and Confocal Imaging Facility at the
University of Connecticut in Storrs, CT. The cytometer was equipped with an argon ion laser emitting radiation at 488 nm. For each sample 10,000-20,000 particles were measured. Fluorescent emission data was collected and displayed by BD CellQuestTM software (Becton, Dickinson and Co., Franklin Lakes, NJ) in histograms of nuclei number according to fluorescence intensity, which was proportional to DNA content. The peaks of
test samples were compared to peaks derived from control plants containing 2N DNA to determine relative ploidy levels as either diploid, triploid or tetraploid.