The use of bead beating to prepare suspensions of nuclei for flow cytometry from fresh leaves, herbarium leaves, petals and pollen.
"Bead beating" is commonly used to release DNA from cells for genomic studies but it was used here to prepare suspensions of plant nuclei for measurement of DNA amounts by flow cytometry. Plant material was placed in 2-ml screw-capped tubes containing beads of zirconia/silica (2.5 mm diameter) or glass (2.5 or 1.0 mm diameter) and 1 ml of lysis buffer. The tubes were mechanically shaken with an FP120 FastPrep Cell Disrupter to release intact nuclei from plant tissue by the impact of the beads. The nuclei were then stained with propidium iodide (PI) and analyzed by flow cytometry. The method 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 G(1)/G(0) peaks with CVs of less than 3.0% and nuclei from fresh petals and herbarium leaves of R. canina, and pollen of the generative nuclei of R. rugosa gave peaks with coefficients of variation (CVs) of less than 4.0%. DNA amounts estimated 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 of nuclei can be prepared rapidly and conveniently from a diversity of tissues by bead beating. Exposure of laboratory workers to harmful substances in the lysis buffer is minimized.
Roberts
Andy V
AV
School of Health and Bioscience, University of East London, London E15 4LZ, United Kingdom. a.v.roberts@uel.ac.uk
D002467:Cell Nucleus / Q000737:chemistry* / Q000235:genetics; D002469:Cell Separation / Q000379:methods; D018744:DNA, Plant / Q000032:analysis* / Q000302:isolation & purification; D005434:Flow Cytometry / Q000379:methods*; D019684:Magnoliopsida / Q000737:chemistry* / Q000235:genetics; D019697:Onions / Q000737:chemistry / Q000235:genetics; D028528:Petroselinum / Q000737:chemistry / Q000235:genetics; D018515:Plant Leaves / Q000737:chemistry / Q000235:genetics; D011058:Pollen / Q000737:chemistry / Q000235:genetics; D027826:Rosa / Q000737:chemistry / Q000235:genetics; D014026:Nicotiana / Q000737:chemistry / Q000235:genetics
D018744:DNA, Plant