The Use of Bead Beating to Prepare Suspensions of Nuclei for Flow Cytometry from Fresh Leaves, Herbarium Leaves, Petals and Pollen

摘要

‘‘Bead beating’’ is commonly used to release DNA from cells for genomic studies but itwas used here to prepare suspensions of plant nuclei for measurement of DNA amountsby flow cytometry. Plant material was placed in 2-ml screw-capped tubes containingbeads of zirconia/silica (2.5 mm diameter) or glass (2.5 or 1.0 mm diameter) and 1 mlof lysis buffer. The tubes were mechanically shaken with an FP120 FastPrep Cell Disrupterto release intact nuclei from plant tissue by the impact of the beads. The nucleiwere then stained with propidium iodide (PI) and analyzed by flow cytometry. Themethod was tested using fresh leaves, fresh petals and herbarium leaves of Rosa canina,leaves and pollen of R. rugosa, and fresh leaves of Petroselinum crispum, Nicotiana tabacum,and Allium cepa. Batches of 12 samples of fresh leaves were prepared, simultaneously,in 45 s by bead beating in the Cell Disrupter. In flow cytometry histograms,nuclei of fresh leaves gave G1/G0 peaks with CVs of less than 3.0% and nuclei from freshpetals and herbarium leaves of R. canina, and pollen of the generative nuclei ofR. rugosa gave peaks with coefficients of variation (CVs) of less than 4.0%. DNA amountsestimated from 24-month-old herbarium leaves, using P. crispum as an internal standard,were less than those of fresh leaves by a small but significant amount. Suspensions ofnuclei can be prepared rapidly and conveniently from a diversity of tissues by beadbeating. Exposure of laboratory workers to harmful substances in the lysis buffer isminimized.