The ploidy stability of the regenerants, derived from in vitro leaf-disc cultures, was determined by flow cytometry. For flow cytometry, the nuclear suspension from fresh plant material was prepared by chopping the tissues with scalpel in cold woody plant buffer. The suspension was then filtered through a 30 m nylon filter membrane (Millipore,USA) to remove fragments and large tissue debris. For staining nuclei, 50 gml−1 propidium iodide (PI; Fluka) was added to the filtered suspension. To prevent staining of double stranded RNA, 50 gml−1 of RNAase (Fluka) was also added. Samples were stirred for 20 min and analyzed thereafter. In vivo plants (from the open field) were used as the controls for ploidy comparison.