(Figure 1
; Melamed et
al., 1990). The molecules measured are
often fluorochrome-conjugated antibodies or fluorescent dyes binding
specifically to DNA or proteins. Published in Probe Volume 5(1): February-March 1995
J.S. (Pat) Heslop-Harrison
Department of Cell Biology
John Innes Centre
Colney Lane, Norwich NR4 7UH
United Kingdom
Flow cytometry allows for fast and informative, quantitative and qualitative
analysis of objects including chromosomes and nuclei, normally by measuring
the fluorescence of molecules that are specifically bound to structures of
interest
(Figure 1; Melamed et
al., 1990). The molecules measured are
often fluorochrome-conjugated antibodies or fluorescent dyes binding
specifically to DNA or proteins.
Flow cytogenetics underpins much of the human genome project: the Department of Energy Human Genome Program reports that "among the resources most crucial to mapping progress are the libraries of clones representing each of the human chromosomes. This chromosome-specific clone library production from physically purified chromosomes depends on the unique chromosome-sorting facilities" at Los Alamos and Lawrence Livermore National Laboratories (Anon, 1993). Flow technology is also recognized as being critical to pig and bovine genome projects (Miller et al., 1992; Dixon et. al., 1992).
Joe Gray, from the University of California, San Francisco, and Scott Cram, Life Science Division Leader at Los Alamos Natonal Laboratory, described some of the advantages of flow cytometry for plant molecular cytogenetics and genome analysis. "The analysis and sorting of plant chromosomes is of considerable economic interest. As is the case for mammalian chromosomes, flow karyotyping and chromosome sorting provide the opportunity for gene mapping and the construction of chromosome-specific libraries" (Gray and Cram, 1990).
Since they wrote this, there have been successful applications of the methods in plants for genome size measurements (including the specific AT and GC base-pair content), cell cycle analysis, flow karyotyping (by measuring the DNA content of chromosomes), chromosome sorting and production of chromosome-enriched DNA libraries, although these analyses are not as yet extensively exploited.
In this short review, I aim to highlight potential and recent applications of flow cytometry to plant genomes; the literature on chromosome analysis has been reviewed recently by a group of European collaborators from the Czech Republic, Italy and Germany (Dolezel et al. 1994), while many techniques for flow analysis of plants are discussed in the same paper and elsewhere (e.g., Heslop-Harrison and Schwarzacher, 1995).
Genome size analysis - Changes between species, during differentiation and during the cell cycle
Analytical information about the physical size of plant genomes and their state of replication is easily obtainable from flow cytometry. Knowing the number of base pairs in a genome is valuable for studies of new species, and an extensive list based on flow cytometric estimates was published by Aru Arumuganathan, now at the University of Lincoln, Nebraska, and Lisa Earle, from Cornell University, in 1991.
Flow cytometry provides a fast and accurate way to look at changes in genome size during evolution and differentiation. Establishment of ploidy and aneuploidy changes during tissue culture, and examination of intra- and inter-specific variation of DNA content can all be important in plant hybridization, breeding, and genetic manipulation programs (Dolezel et al. 1994; Leitch et al. 1992). Perhaps, in contrast to animals, polyploidy often accompanies differentiation, and is an important part of plant development, with different cell types having characteristic ploidies (Galbraith et al. 1991; Bino et al. 1993). Such differentiation by polyploidization is important for understanding the regulation of gene expression in differentiated tissues, and for understanding the nature of tissues used for plant regeneration and transformation.
Flow cytometry provides an accurate method for determining the proportions of cells in G1, S and G2/M stages of the cell cycle. These data can be used to calculate cell cycle times, which are needed in studies of the genetics and control of this process, and are useful for analysis of aspects of crop growth and development.
Flow karyotyping
Flow karyotypes, giving the average sizes of chromosomes from mitotic cells,
are quick, accurate and quantitative. To make a flow karyotype, a suspension
of many thousands of chromosomes is made and stained with a fluorochrome
which binds quantitatively to DNA. The fluorescence of chromosomes is
measured as they pass individually through a cytometer, giving a histogram
(figure 2
) where each peak
represents one or a group of chromosomes.
Flow methods enable differences as small as 1.5 to 4 Mb to be analyzed in humans, and both aneuploidy and many chromosome deletions can be detected easily. In plants, eight species have been flow karyotyped so far (see Dolezel et al. 1994), including Haplopappus gracilis, a plant with only two pairs of chromosomes, four solanaceous species and Melandrium album (with sex chromosomes), wheat (Triticum aestivum), and field or broad bean (Vicia faba).
Chromosome sorting for gene mapping and library construction
After identification of a chromosome by its fluorescence, particular chromosomes can be sorted from others in a flow cytometer by either electrical (as in