Thèse de doctorat en Sciences de la vie
Sous la direction de Catherine Bergounioux.
Soutenue en 1988
à Paris 11 , en partenariat avec Université de Paris-Sud. Faculté des sciences d'Orsay (Essonne) (autre partenaire) .
Pas de résumé disponible.
Plant chromosome analysis using flow cytometry : purification of chromosome from Petunia hybrida (Hort.)
This work is devoted to Petunia hybrida (2n=14) and Nicotiana plumhaginifolia (2n=20) plant chromosome classification by flow cytometry. Chromosome suspensions for flow analysis were prepared from mesophyll protoplast cultures which exibited substantial synchrony during the early days of culture, giving easily disrupted cell walls. Metaphase chromosomes were dispersed breaking the protoplasts-derived cells into a suspension buffer elaborated in order to stabilize both the chromosome structure and DNA content. Chromosomes were stained with Hocchst 33342 dy (HO) or with both HO and ethidium bromide (Bet), and classified according to their relative fluorescence intensities. The Petunia hybrida and Nicotiana plumbaginifolia flow karyotypes are in good correlation with their theoretical distribution generated on the basis of the relative chromosome lengths. A simple device useful to obtain local concentrations of sorted chromosomes further morphological analysis is described. Using a two colour methodology (HO+Bet) it was shown that the longest chromosome from petunia corresponded to a well isolated peak and could be purified, the purity of the sorted fraction being higher than 96%. Modeling of flow cytometric karyotypes was realized in order to determine whether the chromosomes of a given species can be accurately classified using flow cytometry. Moreover, if the aim is to sort one chromosome type, models will indicate the feasible targets and predict interactions between purity and yield. Theoretical flow karyotypes show that it should be possible to separate chromosomes from Zea mays (2n=20) and Arabidopsis thaliana (2n=10).