An-Najah Staff

Laser flow cytometry (FC) is used to determine nuclear DNA content (genome size) and ploidy level in many organisms including plants. Genome size (the total amount of DNA contained within one copy of a genome =1C-value) of some plants is not determined yet. Information on the genome size is needed in molecular genetic studies including genetic modification and transformation of novel traits to improve the performance of certain plant species e.g. the highly embryogenic Alamo cultivar of switchgrass (Panicum virgatum L.), and copaiba diesel tree (Copaifera officinalis L.) as potential bioenergy crops for bioethanol and biodiesel production. For FC analyses, intact nuclei were isolated from young leaves and roots and stained with propidium iodide using the CyStain PI absolute P- Partec^® kit, with the following advantageous modifications: (1) 1% (w/v) polyvinylpyrrolidone (PVP-40) was added to the extraction buffer making the suspension more suitable for FC. It removes phenolic impurities and cytoplasmic compounds from plant nuclei, and reduces the crystalline calcium oxalate and other metabolites that block the fluidics system of the FC. (2) Before staining the nuclei, their filtrates were centrifuged at 120g for 10 min at 4°C to increase the nuclei number in the final solution. DNA content was obtained using nuclei from reference standards that were isolated, stained and analyzed simultaneously, and the FC mean fluorescence intensity was calculated using a linear scale from a region encompassing the G0/G1 peak. The 1C-value of switchgrass Alamo tetraploid ecotype was ca. 2,958 Mbp (=3.02 pg DNA/nucleus). It was about half of that of tobacco. The 1C-value of copaiba diesel tree was ca. 1,184 Mbp (=1.21 pg DNA/nucleus) and it was close to that of oilseed rape and soybean.