title: Chromosome analysis and sorting using flow cytometry.
abstract: Chromosome analysis and sorting using flow cytometry (flow cytogenetics) is an attractive tool for fractionating plant genomes to small parts. The reduction of complexity greatly simplifies genetics and genomics in plant species with large genomes. However, as flow cytometry requires liquid suspensions of particles, the lack of suitable protocols for preparation of solutions of intact chromosomes delayed the application of flow cytogenetics in plants. This chapter outlines a high-yielding procedure for preparation of solutions of intact mitotic chromosomes from root tips of young seedlings and for their analysis using flow cytometry and sorting. Root tips accumulated at metaphase are mildly fixed with formaldehyde, and solutions of intact chromosomes are prepared by mechanical homogenization. The advantages of the present approach include the use of seedlings, which are easy to handle, and the karyological stability of root meristems, which can be induced to high degree of metaphase synchrony. Chromosomes isolated according to this protocol have well-preserved morphology, withstand shearing forces during sorting, and their DNA is intact and suitable for a range of applications.
authors.affiliation: Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Olomouc, Czech Republic. dolezel@ueb.cas.cz
authors.forename: Jaroslav
authors.initials: J
authors.lastname: Doležel
authors.forename: Marie
authors.initials: M
authors.lastname: Kubaláková
authors.forename: Jarmila
authors.initials: J
authors.lastname: Cíhalíková
authors.forename: Pavla
authors.initials: P
authors.lastname: Suchánková
authors.forename: Hana
authors.initials: H
authors.lastname: Simková
mesh_terms: D002453:Cell Cycle; D032461:Chromosomes, Plant; D003582:Cytogenetics; D018744:DNA, Plant / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007621:Karyotyping; D018519:Meristem / Q000166:cytology; D059828:Plant Cells; D010944:Plants / Q000235:genetics*; D012639:Seeds / Q000254:growth & development
chemical_list: D018744:DNA, Plant