2.6.1. Flow cytometry
Leaf fragments from in vitro-grown plants derived from the
anther cultures were chopped with a razor blade in Lysis
buffer (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; pH
7.5) to release the nuclei. The entire sample was filtered through
a 25 m nylon mesh and stained with 2 l DAPI. Analysis of
nuclei was conducted using a Partec CA II flow cytometer (Partec
GmbH, Germany). Tissue from a diploid plant of P. forbesii was
used as an internal standard. The ploidy level of each tested plant
was determined based on a comparison between the fluorescence
peak value of the tested plants with that of the diploid control.