Institute of Experimental Botany AS CR
We are Discovering the Plant World

Doležel, J., Kubaláková, M., Číhalíková, J., Suchánková, P., Šimková, H.
In: Birchler, J.A. (ed.): Plant Chromosome Engineering. Methods and Protocols. Pp 221-238. Springer, New York, 2011. : 221-238, 2011

Keywords: Accumulation of metaphases; cell cycle synchrony; cytogenetics stocks; flow karyotype; hydroponics; fluorescence in situ hybridization; mitotic synchrony; plant chromosome isolation; plant flow cytogenetics; root-tip meristem
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, 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.
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IEB authors: Jarmila Číhalíková, Jaroslav Doležel, Marie Kubaláková, Hana Šimková, Pavla Suchánková