title: In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH).
abstract: 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.
authors.affiliation: 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, France.
authors.forename: Matthieu
authors.initials: M
authors.lastname: Bourdon
authors.forename: Olivier
authors.initials: O
authors.lastname: Coriton
authors.forename: Julien
authors.initials: J
authors.lastname: Pirrello
authors.forename: Catherine
authors.initials: C
authors.lastname: Cheniclet
authors.forename: Spencer C
authors.initials: SC
authors.lastname: Brown
authors.forename: Christel
authors.initials: C
authors.lastname: Poujol
authors.forename: Christian
authors.initials: C
authors.lastname: Chevalier
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Renaudin
authors.forename: Nathalie
authors.initials: N
authors.lastname: Frangne
mesh_terms: D002455:Cell Division; D049108:Cell Enlargement; D002467:Cell Nucleus / Q000235:genetics*; D048429:Cell Size; D022202:Chromosomes, Artificial, Bacterial; D005638:Fruit / Q000166:cytology / Q000235:genetics* / Q000254:growth & development; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D018551:Solanum lycopersicum / Q000166:cytology / Q000235:genetics* / Q000254:growth & development; D018087:Plastids / Q000235:genetics; D011003:Ploidies