Leaf fragments from in vitro-grown plants derived from the anther cultures were chopped with a razor blade in Lysisbuffer (15 mmol/l Tris, 2 mmol/l Na2EDTA, 0.5 mmol/l spermine,80 mmol/l KCl, 20 mmol/l NaCl, and 0.1% (v/v) Triton X-100; pH7.5) to release the nuclei. The entire sample was filtered througha 25 m nylon mesh and stained with 2 l DAPI. Analysis ofnuclei was conducted using a Partec CA II flow cytometer (PartecGmbH, Germany). Tissue from a diploid plant of P. forbesii wasused as an internal standard. The ploidy level of each tested plantwas determined based on a comparison between the fluorescencepeak value of the tested plants with that of the diploid con-trol.