New Zealand Journal of Botany abstracts

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

M. E. HOPPING

The Horticulture and Food Research Institute of
New Zealand Ltd
Mt Albert Research Centre
Private Bag 96 169
Auckland, New Zealand*
Present address: Levin Research Centre, Private
Bag 4005, Levin, New Zealand.

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 G(/ G[ nuclei relative to fluorospheres), which broadly B93028 Received 18 February 1993; accepted 16 July 1993 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 GQ/G] nuclei. Best agreement with published Hordeum DNA content was obtained when a standard with similar DNA content was used.

Keywords Actinidia deliciosa; nuclei; ribo- nuclease; propidium iodide; preservation; flow cytometry; nuclear DNA content

B93028 ; Received 18 February 1993; accepted 16 July 1993
New Zealand Journal of Botany, 1993, Vol. 31: 391-401
0028-825X/93/3104-0391 $2.50/0 © The Royal Society of New Zealand 1993

PDF file of entire paper: medium quality (820K); (scanned from paper original: notes about this process)

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