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 G1/G0 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.

Citation:
Roberts A.V. (2007) ‘The Use of Bead Beating to Prepare Suspensions of Nuclei for Flow Cytometry from Fresh Leaves, Herbarium Leaves, Petals and Pollen’ Cytometry Part A, 71A (12) 1039-1044.