*2.5. Flow Cytometric Profile of Plants*

Nuclei isolated from leaf samples (approximately 100 mg) of regenerated plants and donor plants of *P. amboinicus* were used to generate flow cytometric profiles. As previously reported by Galbraith et al. [57], the nuclei were isolated by cutting leaf samples with a new surgical blade in micro-Petri dishes (35 × 15 mm2) containing 2.0 mL of precooled isolation buffer (20 mM MOPS, 45 mM MgCl2, 30 mM sodium citrate, 0.1% (*v*/*v*) Triton X-100; pH 7.0). Using a double-layered nylon membrane of 30-micron thickness, the homogenates were filtered with the help of a micro syringe to eliminate any remaining leaf debris from the mixtures. The filtered suspensions were transferred to labelled Eppendorf tubes containing 2.5 μL of 10 mg/mL DNAse-free RNAse and incubated for 10 min. After 30 min of staining with 50 μgml−<sup>1</sup> of propidium iodide (Sigma-Aldrich, Inc., St. Louis, MO, USA), the nuclei

samples were passed and analyzed using a flow cytometry machine (Muse™ Cell Analyzer, Merck KGaA, Darmstadt, Germany).
