In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH).
The Plant Journal. 2011-04-05; 66(6): 1089-1099
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1. Plant J. 2011 Jun;66(6):1089-99. doi: 10.1111/j.1365-313X.2011.04568.x. Epub 2011
In planta quantification of endoreduplication using fluorescent in situ
Bourdon M(1), Coriton O, Pirrello J, Cheniclet C, Brown SC, Poujol C, Chevalier
C, Renaudin JP, Frangne N.
(1)Institut National de la Recherche Agronomique (INRA), Unité Mixte de Recherche
1332 Biologie du Fruit et Pathologie, BP 81, F-33883 Villenave d’Ornon Cedex,
Endopolyploidy, i.e. amplification of the genome in the absence of mitosis,
occurs in many plant species and happens along with organ and cell
differentiation. Deciphering the functional roles of endopolyploidy is hampered
by the fact that polyploid tissues generally comprise cells with various ploidy
levels. In some fleshy fruits (amongst them tomato fruit) the ploidy levels
present at the end of development range from 2C to 256C in the same tissue. To
investigate the temporal and spatial distribution of endopolyploidy it is
necessary to address the DNA content of individual nuclei in situ. Conventional
methods such as fluorometry or densitometry can be used for some tissues
displaying favorable characteristics, e.g. small cells, small nuclei,
organization in a monolayer, but high levels of varying polyploidy are usually
associated with large sizes of nuclei and cells, in a complex three dimensional
(3-D) organization of the tissues. The conventional methods are inadequate for
such tissue, becoming semi-quantitative and imprecise. We report here the
development of a new method based on fluorescent in situ bacterial artificial
chromosome hybridizations that allows the in situ determination of the DNA ploidy
level of individual nuclei. This method relies on the counting of hybridization
signals and not on intensity measurements and is expected to provide an
alternative method for mapping endopolyploidy patterns in mature, 3-D organized
plant tissues as illustrated by the analysis of ploidy level and cell size in
pericarp from mature green tomato fruit.
© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.
PMID: 21418357 [Indexed for MEDLINE]