Preparation and preservation of nuclei from plant tissues for quantitative DNA analysis by flow cytometry

Abstract

Abstract Large numbers of nuclei were routinely prepared and rapidly analysed by flow cytometry to obtain information on cell cycling and nuclear DNA content. Nuclei were extracted from herbaceous and woody plant species by chopping in ice-cold buffer. Extracted nuclei were stained with propidium iodide, which intercalates into double stranded nucleic acids, and then analysed in a flow cytometer equipped with a low powered laser. Latex fluorospheres were added to nuclei samples to standardise flow cytometer operation. For Actinidia deliciosa, high numbers of nuclei and well-resolved histograms with low coefficients of variation were obtained from shoot apices prior to flowering, from flower buds, and from developing axillary buds. More differentiated tissues yielded fewer nuclei and less well resolved histograms. Treatment of nuclei with ribonuclease lowered channel numbers by up to 10% while improving histogram definition by reducing histogram peak coefficients of variation. Ribonuclease treatment of nuclei from rapidly dividing fruit tissues lowered channel numbers by up to 20%. Nuclei preserved in 30% glycerol at -18°C were recovered undamaged even after 9 months of storage, but channel numbers were 5-7% lower than those from fresh tissue. Fluorescence ratios (fluorescence intensity of G0/ G1 nuclei relative to fluorospheres), which broadly indicate DNA content, were compiled for a range of herbaceous plants and woody fruit trees. A 28-fold range in values was obtained between Pyrus pyrifolia and Allium cepa. The nuclear DNA content of a seedline of Hordeum vulgare was determined by co-chopping leaves with known standards and comparing relative channel numbers of G0/ G1 nuclei. Best agreement with published Hordeum DNA content was obtained when a standard with similar DNA content was used.