Flow analysis and sorting of microchromosomes (<3 Mb)

BACKGROUND: The analysis and isolation of high numbers of chromosomes smaller than 3 Mb in size (microchromosomes) with good purity is dependent primarily on the detection sensitivity of the flow cytometer and the precision of the sort unit. The aim of this study was to investigate the capability of using a conventional flow cytometer for the detection and sorting at high purity microchromosomes with an estimated size of 2.7 Mb. METHODS: Chromosomes were isolated from a human cell line containing a pair of X-derived microchromosomes, using a modified polyamine isolation buffer. The chromosome preparation was labeled with Hoechst and Chromomycin and analyzed and purified using a MoFlo sorter (DAKO) configured for high-speed sorting. The purity of the flow-sorted microchromosomes was assessed by reverse chromosome painting. RESULTS: Improved resolution of the peak of microchromosomes in a bivariate plot of Hoechst versus Chromomycin fluorescence was obtainable after discriminating clumps and debris based on gating data within a FSC versus pulse width plot. CONCLUSIONS: Chromosomes of smaller size, less than 3 Mb, can be detected with high resolution and flow-sorted with high purity using a conventional flow sorter.     

Flow cytometric analysis of the chromosomes and stability of a wheat cell-culture line

A rapidly growing, long-term suspension culture derived from Triticum aestivum L. (wheat) was synchronized using hydroxyurea and colchicine, and a chromosome suspension with chromosomes was made. After staining with the DNA-specific fluorochromes Hoechst 33258 and Chromomycin univariate and bivariate flow-cytometry histograms showed 15 clearly resolved peaks corresponding to individual chromosome types or groups of chromosomes with similar DNA contents. The flow karyotype was closely similar to a histogram of DNA content measurements of Feulgen-stained chromosomes made by microdensitometry. We were able to show the stability of the flow karyotype of the cell line over a year, while a parallel subculture had a slightly different, stable, karyotype following different growth conditions. The data indicate that flow cytometric analysis of plant karyotypes enables accurate, statistically precise chromosome classification and karyotyping of cereals. There was little overlap between individual flow-histogram peaks, so the method is useful for flow sorting and the construction of chromosome specific-recombinant DNA libraries. Using bivariate analysis, the AT:GC ratio of all the chromosomes was remarkably similar, in striking contrast to mammalian flow karyotypes. We speculate about a fundamental difference in organization and homogenization of DNA sequences between chromosomes within mammalian and plant genomes. Received: 24 April 1996 / Accepted: 24 May 1996     

High-resolution flow karyotyping and chromosome sorting in Vicia faba lines with standard and reconstructed karyotypes

Flow cytometric analysis has been performed on chromosomes isolated from formaldehyde-fixed root tips in a Vicia faba (2n = 12) line with a standard (wild-type) karyotype and in six V. faba translocation lines with reconstructed karyotypes. The resolution of individual chromosome types on histograms of chromosome fluorescence intensity (flow karyotypes) depended on the type of fluorochrome used for chromosome staining. The highest degree of resolution was achieved with 4,6-diamidino-2-phenylindole (DAPI). The lower resolution obtained after staining with mithramycin A (MIT) and propidium iodide (PI) was probably due to the sensitivity of these stains to changes in chromatin structure induced by formaldehyde fixation. After the staining with DAPI, only 1 chromosome type could be discriminated in the line with a standard karyotype. In the translocation lines, the number of chromosome types resolved on flow karyotypes ranged from 2 in the G and the ACB lines to all (6) chromosome types in the EFK and EF lines. Refined flow karyotyping permitted the sorting of a total of 15 different chromosome types from five of the translocation lines. It is expected that flow sorting of chromosomes from reconstructed karyotypes will become a powerful tool in the study of nuclear genome organisation in V. faba.     

Flow sorting of mitotic chromosomes in common wheat (Triticum aestivum L.)

The aim of this study was to develop an improved procedure for preparation of chromosome suspensions, and to evaluate the potential of flow cytometry for chromosome sorting in wheat. Suspensions of intact chromosomes were prepared by mechanical homogenization of synchronized root tips after mild fixation with formaldehyde. Histograms of relative fluorescence intensity (flow karyotypes) obtained after the analysis of DAPI-stained chromosomes were characterized and the chromosome content of all peaks on wheat flow karyotype was determined for the first time. Only chromosome 3B could be discriminated on flow karyotypes of wheat lines with standard karyotype. Remaining chromosomes formed three composite peaks and could be sorted only as groups. Chromosome 3B could be sorted at purity >95% as determined by microscopic evaluation of sorted fractions that were labeled using C-PRINS with primers for GAA microsatellites and for Afa repeats, respectively. Chromosome 5BL/7BL could be sorted in two wheat cultivars at similar purity, indicating a potential of various wheat stocks for sorting of other chromosome types. PCR with chromosome-specific primers confirmed the identity of sorted fractions and suitability of flow-sorted chromosomes for physical mapping and for construction of small-insert DNA libraries. Sorted chromosomes were also found suitable for the preparation of high-molecular-weight DNA. On the basis of these results, it seems realistic to propose construction of large-insert chromosome-specific DNA libraries in wheat. The availability of such libraries would greatly simplify the analysis of the complex wheat genome.     

Flow karyotyping of human melanoma cell lines

Single-laser flow cytometry has been used to study the feasibility of flow karyotyping of human solid tumors. As a model, seven human melanoma cell lines have been used with varying numerical chromosome composition as verified by FCM DNA content measurements and chromosome countings. For all seven cell lines, flow karyotypes that showed a variety of consistent deviations from the normal diploid flow karyotype could be obtained although the resolution of the flow system and varying debris continuum limited the number of resolvable peaks. The predominant changes observed involved the regions normally representing chromosomes 3-8, 9-12, and 13-16. It is concluded that at present the preparation procedure is the main limiting factor for exploring the full potential of flow karyotyping for cytogenetic analysis of solid-tumor cell lines.     

Chromosomes in the flow to simplify genome analysis

Nuclear genomes of human, animals, and plants are organized into subunits called chromosomes. When isolated into aqueous suspension, mitotic chromosomes can be classified using flow cytometry according to light scatter and fluorescence parameters. Chromosomes of interest can be purified by flow sorting if they can be resolved from other chromosomes in a karyotype. The analysis and sorting are carried out at rates of 10(2)-10(4) chromosomes per second, and for complex genomes such as wheat the flow sorting technology has been ground-breaking in reducing genome complexity for genome sequencing. The high sample rate provides an attractive approach for karyotype analysis (flow karyotyping) and the purification of chromosomes in large numbers. In characterizing the chromosome complement of an organism, the high number that can be studied using flow cytometry allows for a statistically accurate analysis. Chromosome sorting plays a particularly important role in the analysis of nuclear genome structure and the analysis of particular and aberrant chromosomes. Other attractive but not well-explored features include the analysis of chromosomal proteins, chromosome ultrastructure, and high-resolution mapping using FISH. Recent results demonstrate that chromosome flow sorting can be coupled seamlessly with DNA array and next-generation sequencing technologies for high-throughput analyses. The main advantages are targeting the analysis to a genome region of interest and a significant reduction in sample complexity. As flow sorters can also sort single copies of chromosomes, shotgun sequencing DNA amplified from them enables the production of haplotype-resolved genome sequences. This review explains the principles of flow cytometric chromosome analysis and sorting (flow cytogenetics), discusses the major uses of this technology in genome analysis, and outlines future directions.     

Flow sorting and microcloning of maize chromosome 1

Flow sorting maize chromosome 1 and construction of the first chromosome 1 DNA Lambda library are described. Maize metaphase chromosome suspensions were prepared from synchronized seedling root tip cells of the maize hybrid line Seneca 60 and stained with propidium iodide for flow karyotyping and sorting. The observed flow karyotype was very similar to the predicted flow karyotype constructed based on published values for the relative chromosome sizes of Seneca 60. The estimated size of chromosomes from the peak for the chromosome 1 matched the expected size of maize chromosome 1. The peak for the chromosome 1 was well resolved from other peaks on the flow karyotype. An average of 7 x 10(3) chromosomes of chromosome 1 could be produced from 10 root tips. About 0.6 million chromosomes of maize chromosome 1 were sorted and pooled based on the cytogram of fluorescent pulse area Vs fluorescent pulse width and stored at -20 degrees C in the freezer. DNA isolated from sorted chromosomes was good quality of more than 100 kb in size. Chromosome 1 DNA was partially digested with BamHI, dephosphorylated and ligated with arms of BamHI digested Lambda Dash vector. A total of 1.2 x 10(5) independent recombinants with the average insert size 12.6 kb was obtained. This library covered approximately 90% of maize chromosome 1. Hybridization of cloned fragments with labeled maize genomic DNA showed that the high, middle, or low copy number DNA sequences presented in the different phage clones. PCR (polymerase chain reaction) using chromosome-specific primers confirmed the specificity of this library. The individual chromosome library is useful in plant genome mapping and gene isolation.     

Purification of the chromosomes of the Indian muntjac by flow sorting.

Metaphase chromosomes were isolated from a male Indian muntjac cell line, were stained with ethidium bromide and were analyzed by flow microfluorometry to establish a deoxyribonucleic acid (DNA)-based karyotype. Five major peaks were evident on the chromosomal DNA distribution corresponding to the five chromosome types in this species. The amount of DNA in each chromosome was confirmed by cytophotometric measurements of intact metaphase spreads. The five chromosome types were separated by flow sorting at rates up to several hundred chromosomes per second. The sorted chromosomes were identified by morphology and by Giemsa banding patterns. The automsomes, Numbers 1, 2 and 3, and the X + 3 composite chromosome were separated with a high degree of purity (90%). The centromere region of the X + 3 chromosome was fragile to mechanical shearing, and during isolation a small proportion of these chromosomes broke into four segiments: the long arm, the short arm, the short arm plus centromere and the centromere region. A large fraction of the constitutive heterochromatin of this species is present in the centromere region of the X + 3 chromosome and in the Y chromosome; these two regions possess similar amounts of DNA and therefore sort together. Chromosome flow sorting is rapid, reproducible and precise; it allows the collection of microgram quantities of purified chromosomes.     

Flow cytometric analysis and sorting of plant chromosomes from Petunia hybrida protoplasts

A method to obtain a high metaphase index and thereafter a plant chromosome suspension is described for Petunia hybrida (2n = 14). Mesophyll protoplast cultures have been used, giving easily disrupted cell walls and a high percentage of dividing cells after 42 h. On 2.5 mM colchicine-treated cells, metaphase indexes reaching 10% were routinely obtained. The lysis medium in which the protoplast-derived cells were disrupted was a simplified culture medium. After chromosome release, samples were stained with Hoechst 33342 dye and analysed by flow cytofluorometry. The histogram of fluorescence intensities included three peaks of metaphase chromosomes and a duplication of this flow karyotype provoked by "monochromatid chromosome." This interpretation was established after flow sorting; micronuclei could also be observed and sorted. Of the 7 chromosomes, only the largest formed a distinct peak while the others were incompletely resolved, due to the similar DNA content of various chromosomes. Model distributions of Petunia hybrida chromosomes have been computed according to the relative chromosome length. The theoretical histograms indicated that low variability is indispensable for resolving distinctive chromosome peaks. The experimental flow karyotype was consistent with one of the models having CV of 2.5%.     

Anti-kinetochore staining for single laser, bivariate flow sorting of Indian muntjac chromosomes.

Flow cytometric chromosome sorting typically relies upon dual-laser, bivariate analysis after staining with two different base pair-specific dyes for resolution of chromosomes with similar DNA content. The availability of FITC-conjugated antibodies offers the possibility of single-laser bivariate analysis when combined with propidium iodide (PI) DNA staining, but requires exploitable antigenic differences between chromosomes of interest. A technique was developed for indirect immunofluorescent anti-kinetochore staining of Indian muntjac chromosomes in suspension. Primary antibody binding within permeabilized whole cells minimized centrifugation-induced loss of chromosomal integrity. Subsequent FITC-conjugated second antibody binding was not affected by concurrent PI-counterstaining. Anti-kinetochore staining facilitated resolution of chromosomes No. 2 and X, which formed a doublet peak upon univariate DNA content analysis, as well as recognition of the Y2 peak which was indistinguishable from debris by univariate analysis. The technique allowed greater than 90% purification of each Indian muntjac chromosome.     

Chromosome sorting in tetraploid wheat and its potential for genome analysis

This study evaluates the potential of flow cytometry for chromosome sorting in durum wheat (Triticum turgidum Desf. var. durum, 2n = 4x = 28). Histograms of fluorescence intensity (flow karyotypes) obtained after the analysis of DAPI-stained chromosomes consisted of three peaks. Of these, one represented chromosome 3B, a small peak corresponded to chromosomes 1A and 6A, and a large peak represented the remaining 11 chromosomes. Chromosomes sorted onto microscope slides were identified after fluorescence in situ hybridization (FISH) with probes for GAA microsatellite, pSc119.2, and Afa repeats. Genomic distribution of these sequences was determined for the first time in durum wheat and a molecular karyotype has been developed for this crop. Flow karyotyping in double-ditelosomic lines of durum wheat revealed that the lines facilitated sorting of any arm of the wheat A- and B-genome chromosomes. Compared to hexaploid wheat, flow karyotype of durum wheat is less complex. This property results in better discrimination of telosomes and high purities in sorted fractions, ranging from 90 to 98%. We have demonstrated that large insert libraries can be created from DNA purified using flow cytometry. This study considerably expands the potential of flow cytogenetics for use in wheat genomics and opens the possibility of sequencing the genome of this important crop one chromosome arm at a time.     

Chromosome analysis and sorting in <Emphasis Type="Italic">Vicia sativa</Emphasis> using flow cytometry

Procedures were developed for flow cytometric analysis and sorting of mitotic chromosomes (flow cytogenetics) of common vetch (Vicia sativa L., 2n=12). Suspensions of intact chromosomes were prepared from root tips after cell cycle synchronization, formaldehyde fixation, and mechanical homogenization. On average, 3 × 10⁵ morphologically intact chromosomes could be isolated from 25 root tips. Flow cytometric analysis of DAPI-stained chromosomes resulted in histograms of relative fluorescence intensity (flow karyotypes) containing four peaks, representing particular chromosomes and/or pairs of chromosomes with similar relative DNA content. Peaks I and II were assigned to chromosomes 6 and 5, respectively. These chromosomes could be sorted with a purity exceeding 90 %. The two remaining peaks on the flow karyotype were composite, each of them representing a pair of chromosomes. Chromosomes 1 and 3 were assigned to composite peak III while chromosomes 2 and 4 were assigned to composite peak IV. The chromosomes could be sorted with a purity of 99 % from both composite peaks. Bivariate flow karyotyping after simultaneous staining of chromosomes with DAPI and mithramycin was not found helpful in discriminating additional chromosomes. This study extends the number of legume species for which flow cytogenetics is available and provides a new tool for targeted and effective analysis and mapping of common vetch genome.     

Analysis of bivariate flow karyotypes

Bivariate flow karyotype analysis is performed using data from chromosomes stained with two fluorescent dyes, typically chromomycin A3 and Hoechst-33258, and measured in a flow cytometer or cell sorter (Carrano et al.: Proceedings of the National Academy of Sciences of the United States of America 76:1382-1384, 1979; Gray et al.: Proceedings of the National Academy of Sciences of the United States of America 72:1231-1234, 1975; Langlois et al.: Proceedings of the National Academy of Sciences of the United States of America 79:7876-7880, 1982). In the resulting bivariate histogram, most chromosome types appear as individual peaks. In sorting of chromosomes to purify a specific chromosomal type, its corresponding peak in the bivariate histogram is delineated by a rectangular region which surrounds it. All events (objects) that fall within this region trigger the sorting process. In most cases, peaks for different chromosomal types overlap to some extent, and in addition there is always an underlying background due to chromosome fragments and clumps. Thus the sorted population will not be pure; it may include more than one chromosome type and will include debris. To determine the purity of a sort, i.e., the percentage of the sorted material that is of the actual chromosomal type desired, two methods of mathematical analysis have been developed. In the more general method, the bivariate data within an analysis region that includes the sort region, are fit with a series of bivariate Gaussian functions, one for each peak. In a simplified method, the data within the analysis region are transformed into a univariate distribution of either chromomycin A3 or Hoechst-33258 fluorescence. The peaks in these univariate distributions are fit with univariate Gaussian functions. In both methods the purity is determined mathematically. The results of both methods agree well with independent methods of analysis.     

Analysis and sorting of rye (Secale cereale L.) chromosomes using flow cytometry.

Procedures for chromosome analysis and sorting using flow cytometry (flow cytogenetics) were developed for rye (Secale cereale L.). Suspensions of intact chromosomes were prepared by mechanical homogenization of synchronized root tips after mild fixation with formaldehyde. Histograms of relative fluorescence intensity obtained after the analysis of DAPI-stained chromosomes (flow karyotypes) were characterized and the chromosome content of the DNA peaks was determined. Chromosome 1R could be discriminated on a flow karyotype of S. cereale 'Imperial'. The remaining rye chromosomes (2R-7R) could be discriminated and sorted from individual wheat-rye addition lines. The analysis of lines with reconstructed karyotypes demonstrated a possibility of sorting translocation chromosomes. Supernumerary B chromosomes could be sorted from an experimental rye population and from S. cereale 'Adams'. Flow-sorted chromosomes were identified by fluorescence in situ hybridization (FISH) with probes for various DNA repeats. Large numbers of chromosomes of a single type sorted onto microscopic slides facilitated detection of rarely occurring chromosome variants by FISH with specific probes. PCR with chromosome-specific primers confirmed the identity of sorted fractions and indicated suitability of sorted chromosomes for physical mapping. The possibility to sort large numbers of chromosomes opens a way for the construction of large-insert chromosome-specific DNA libraries in rye.     

Flow Karyotyping of Wheat Addition Line “T240” with a Haynaldia villosa 6VS Telosome

To construct a chromosome-specific DNA library of chromosome 6VS from Haynaldia villosa, a wheat alien chromosome addition line T240 with a 6VS chromosome arm and its parental common wheat cv. CA921 were used to optimize protocols for preparing chromosome suspension amenable to flow sorting of the 6VS chromosome. Our results showed that root tips incubated sequentially in Hogland’s solution containing 1.25 mM hydroxyurea (DNA synthesis inhibitor) for 18 h, a hydroxyurea-free period for 2 h, and 1 μM trifluralin for 4 h (metaphase blocking reagent) increased the metaphase index (MI) by up to 62 % . Many metaphase chromosomes were released to form a chromosome suspension when root tips fixed in 2 % paraformaldehyde were treated in a homogenizer at 9,500 rpm for 10–15 s. Most of the released chromosomes had intact morphology. The background solution of chromosome suspension was clear and relatively few of cell debris and chromosome clumps. Univariate flow karyotypes were established with chromosome suspension flow through FACS Vantage 2000 SE flow cytometer. The flow karyotype of CA921 consisted of four chromosome peaks, whereas that for T240 had a fifth peak. This fifth peak in T240 contained the telosome, which was further confirmed to be 6VS through fluorescence in situ hybridization.     

Optimising human chromosome separation for the production of chromosome-specific DNA libraries by flow sorting

Summary A number of cell lines, some containing chromosomes with distinctive heteromorphisms, have been flow karyotyped using a single laser flow sorter in an attempt to select those suitable for sorting all human chromosomes individually. Using the non-base-specific DNA stain ethidium bromide, chromosomes 3,4,5, and 6 form individual peaks in practically all normal subjects, while the right combination of heteromorphisms enables chromosomes 1, 2, 8, 9, 13, 16, 17, 18, 19, 20, 21, 22, and Y to be sorted separately. Two male cell lines, one containing a duplication and one a deletion of the X, produce flow karyotypes suitable for sorting chromosomes 7 and 8. The use of numerical chromosome abnormalities to enrich the sex chromosomes and the autosomes 18 and 21 is also illustrated. The DNA stain Hoechst 33258 binds preferentially to AT base pairs. Flow karyotypes produced with this fluorochrome separate some chromosomes not well separated with ethidium bromide. Chromosomes 5, 6, 8, 13, 14, 15, 17, and 20, and Y can be sorted individually with Hoechst 33258 with the right combination of heteromorphisms. Using these techniques, all human chromosomes apart from 10, 11, and 12 have been found as individual flow karyotype peaks, suitable for sorting with a high degree of purity.     

Construction of a DNA Library Enriched with Mouse 4xChromosome of T(X;4)37H Translocation

Normal mouse chromosomes are routinely separated into only 5 peaks by the current flow cytometry. Since this limited resolution hindered isolation of the normal mouse X chromosome with an appropriate purity, we attempted to sort the mouse 4^x chromosome, a larger translocation chromosome of T(X;4)37H, consisting of nearly the entire chromosome 4 and chromosome X by flow cytometry. To obtain a large number of cells having 4^x chromosome for flow sorting, we isolated a somatic hybrid cell line MHH-1 formed between S194 myelome cell line and normal splenocytes from a male mouse carrying T(X;4)37H. Flow karyotyping of propidium iodide-stained chromosomes from MHH-1 cell line revealed an additional peak containing 4^x chromosomes at about 80%. DNA purified from sorted 4^x chromosomes was cloned into phage lambda gtWES after complete digestion with EcoRl restriction endonuclease. Thus a 4^x chromosome-enriched library of about 4.4 × 10⁴ recombinant phages was made and 13 single copy DNA clones specific to the X chromosome were isolated from the library so far.     

Flow cytometric sorting of maize chromosome 9 from an oat-maize chromosome addition line

Large numbers of maize chromosome 9 can be collected with high purity by flow cytometric sorting of chromosomes isolated from a disomic maize chromosome addition line of oat. Metaphase chromosome suspensions were prepared from highly synchronized seedling root-tips of an oat-maize chromosome-9 addition line (OM9) and its parental oat and maize lines. Chromosomes were stained with propidium iodide for flow cytometric analysis and sorting. Flow-karyotypes of the oat-maize addition line showed an extra peak not present in the parental oat line. This peak is due to the presence of a maize chromosome-9 pair within the genome of OM9. Separation of maize chromosome 9 by flow cytometric sorting of a chromosome preparation from a normal maize line was not possible because of its size similarity (DNA content) to maize chromosomes 6, 7 and 8. However, it is possible to separate maize chromosome 9 from oat chromosomes and chromatids. An average of about 6×10³ chromosomes of maize chromosome 9 can be collected by flow-sorting from chromosomes isolated from 30 root tips (ten seedlings) of the oat-maize addition line. Purity of the maize chromosome 9, sorted from the oat-maize chromosome addition line, was estimated to be more than 90% based on genomic in situ hybridization analysis. Sorting of individual chromosomes provides valuable genomic tools for physical mapping, library construction, and gene isolation. Received: 28 February 2000 / Accepted: 14 July 2000     

On-line sorting of human chromosomes by centromeric index,and identification of sorted populations by GTG-banding and fluorescent in situ hybridization

Summary Using slit-scan flow cytometry, the shape of human metaphase chromosomes, as expressed in their centromeric index (CI), and the DNA content of the chromosomes have been used as parameters in bivariate flow karyotyping. The resolution of the DNA vs CI flow karyogram of the larger chromosomes up to chromosome 13 is much higher than the resolution obtained in the DNA-based monovariate flow karyogram. Chromosome length appears to be an important factor in the resolution of the DNA vs CI-based flow karyogram. A method has been developed to obtain chromosomes in suspension that are long enough for adequate analysis. Several chromosomes that cannot be distinguished or are difficult to discriminate in the DNA-based karyogram can now be distinguished as individual peaks, e.g., chromosomes 1 and 2. The peak of chromosomes 9–12 can be separated into two peaks formed by chromosomes 9 and 11, and 10 and 12, respectively. The advantage of the system applied in this study is that the DNA vs CI analysis is performed on-line, allowing chromosomes to be sorted on the bases of their CI. Pulse shapes of the selected chromosomes can be recorded simultaneously with the transmission of the sorting command. The purity of the sorted fraction can be estimated from the offline inspection of these pulse shapes. Fractions of chromosome 1 have been sorted out on the basis of the CI information, centrifuged on slides, fixed and subsequently banded with trypsin and Giemsa or hybridized with the chromosome 1 specific probe, pUC 1.77. The observed purity under the selected conditions ranges from 80%–99% and is in accordance with the estimates of the purities made on the basis of the simultaneously recorded pulse shapes. Fixation of the chromosome suspension prior to flow cytometric analysis and sorting appears to be essential for the preservation of their morphology and has no adverse influence on the resolution of Giemsa banding or on the quality of in situ hybridization.     

Preparation and flow cytometric analysis of metaphase chromosomes of tomato

Summary A procedure for the preparation of tomato chromosome suspensions suitable for flow cytometric analysis is described. Rapidly growing cell suspension cultures of Lycopersicon esculentum cv VFNT cherry and L. pennellii LA716 were treated with colchicine to enrich for metaphase chromosomes. Metaphase indices between 20 and 35% were routinely obtained when cultures were exposed to 0.1% colchicine for 15–18 h after 2 days of subculture. Mitotic cells were isolated by brief treatment with cell wall digesting enzymes in a medium with low osmolarity (325 mOsm/kg of H₅₂O). The low osmolarity medium was needed to avoid the chromosome clumping and decondensation seen in standard media. Suspensions of intact chromosomes were prepared by lysing swollen protoplasts in various buffers (MgSO₄, polyamines, hexylene glycol, or KCl-propidium iodide) similar in contents to the buffers used to isolate mammalian chromosomes. For univariate flow cytometric analysis, chromosome suspensions were stained with a fluorescent DNA-binding stain (propidium iodide, Hoechst 33258, mithramycin, or chromomycin A3) and analyzed using an EPICS flow cytometer (Profile Analyzer or 753). Peaks for the chromosomes, chromatids, clumps of chromosomes, nuclei, and fluorescent debris were seen on a histogram of log of fluorescence intensity, and were confirmed by microscopic examination of the objects collected by flow-sorting. Chromosome suspensions prepared in MgSO₄ buffer have the highest frequency of intact chromosomes and the least fluorescent cellular debris. Peaks similar to theoretical univariate flow karyotypes of tomato chromosomes were seen on the observed univariate flow karyotypes, but were not as well resolved. Bivariate flow analysis of tomato chromosome suspension using double-stain combination, Hoechst 33258 and chromomycin A3, and two laser beams showed better resolution of some chromosomes.