Endopolyploidy in Diploid and Tetraploid Maize (Zea mays L.)

Nuclei from different tissues such as stem, mesocotyl, nodalroot and root tip of diploid and tetraploid maize were isolated,stained with propidium iodide and passed through an EPICS-751flow-cytometer cell sorter. Variations in flow histograms wereobserved in different tissues. Stem tissues of both the diploidand tetraploid had two peaks representing G1 and G2 somaticnuclei. The remaining tissues in both the diploids and tetraploidsexhibited three peaks. The first peak observed in these tissuesrepresents G1 somatic nuclei of the lowest ploidy level. Thesecond peak represent G2 somatic nuclei of the lowest ploidylevel+G1 somatic nuclei of the next ploidy level. The thirdpeak represents G2 of the higher ploidy level+G1 somatic nucleiof the next higher ploidy level. Statistically significant differenceswere observed between the diploid and tetraploid maize tissueswith respect to nuclei distribution in the higher ploidy levelpeaks implying variation in the degree of endopolyploidy inthe diploid and tetraploid maize. The results of this studysuggest that the amount of endopolyploid observed in maize genotypeshas an effect on their overall agronomic performance under thefield conditions.Copyright 1993, 1999 Academic Press Zea mays L., maize, endopolyploidy, diploid, tetraploid, flow cytometry     

Plant Genome Size Measurement with DNA Image Cytometry

To test the reliability of DNA image cytometry for the measurement of nuclear DNA content in plant material, we conducted independent experiments in two laboratories using different image analysis instruments for densitometric measurement of nuclear DNA amount in Feulgen-stained squash preparations of root tips. The 2C nuclear DNA content of the nine species studied spanned a 100-fold range (approx. 0.3–33 pg). The estimates of nuclear DNA content measured with image cytometry methods were comparable to values obtained previously using both photometric cytometry and flow cytometry. Image cytometry methods showed little variation among repeated experiments within each laboratory or among different operators using the same instrument. Furthermore, the interphase-peak method (measurement of several hundred interphase nuclei per slide) was comparable to the classical prophase/telophase approach (measurement of ten early prophase and ten late telophase nuclei per slide). Hence, DNA image cytometry gives accurate and reproducible results and may be used as an alternative to photometric cytometry in plant nuclear DNA content measurements. In the present study, we propose that two standards for quality control of nuclear DNA content measurement are used in plant DNA image cytometry: (1) the coefficient of variation of the peak should be lower than 6%, and (2) the 4C/2C ratio should be between 1.9 and 2.1.     

利用流式细胞光度术鉴定苹果倍性的研究

利用流式细胞光度术测定了苹果１２个二倍体,５个三倍体细胞ＤＮＡ含量。结果表明：二倍体细胞核ＤＮＡ含量平均为２．２７ｐｇ,三倍体细胞核ＤＮＡ含量平均为３．１３ｐｇ。     

Nuclear DNA content in species ofEleusine (Gramineae): A critical re-evaluation using laser flow cytometry

Interest in dinitroaniline herbicide resistant biotypes ofEleusine indica, and an as yet undetermined taxon ofEleusine, necessitated a revaluation of reported nuclear genome size estimates for available species in the genus. Laser flow cytometry showed that the nuclear DNA content of six of the seven species examined had 15 to 50% less DNA than reported previously. It was also determined that roots, as contrasted to leaves, possessed a large fraction of nuclei at the 4C or 8C DNA content level, in diploid or tetraploid species, respectively (i.e. the G2/M peak). Two major reasons for the previously reported overestimation may include sampling only of root tissues where endopolyploid and normal diploid nuclei both occur and the inappropriate choice of onion nuclei as an internal standard.     

VARIATION OF NUCLEAR DNA CONTENT IN HELIANTHUS ANNUUS (ASTERACEAE)

Nuclear 2C DNA content was determined by laser flow cytometry for 13 diploid (2n = 34) lines (cultivated varieties and inbred lines) of Helianthus annuus. Mean DNA amount of second leaf nuclei varied from 6.01 to 7.95 pg (32%) among lines. Mean DNA content varied up to 19% within lines. Variability in mean DNA content exceeding 27% and 48% was detected among leaves from different nodes of plants of the open-pollinated variety, Californicus, and the inbred line, RHA 299, respectively. The root tip and shoot tip nuclei of H. annuus have been reported to consist of a mixture of aneuploid (17 to 33 chromosomes) and diploid (34 chromosomes) cells, a condition called aneusomaty. Chromosome counts of root tips and an analysis of the distribution of DNA content of large numbers of nuclei from leaves indicate that aneusomaty either does not occur, or is not common, among the lines investigated. The intraspecific, intraline, and intraplant variation in DNA content in H. annuus support the concept that a sizable portion of a plant genome is unstable and subject to rapid changes in DNA amount.     

COMPARISON OF PLANT DNA CONTENTS DETERMINED BY FEULGEN MICROSPECTROPHOTOMETRY AND LASER FLOW CYTOMETRY

An improved procedure is reported for determining DNA amounts of plant nuclei. Nuclei stained with propidium iodide, isolated from chopped plant leaves, were passed through an Ortho Cytofluorograph with a Lexel model 95 argon laser (514 nm) and the fluorescence measured, integrated, and recorded using an Ortho 2140 Data Acquisition computer. All nuclear samples were mixed with nuclei of Sultan barley (2C DNA content = 11.12 pg [picogram]) as an internal standard. DNA contents of ten plant species, ranging from 2C = 1.7 pg to 36.1 pg measured by flow cytometry, correlated strongly (r = 0.99, slope = + 1.00) with DNA contents determined from Feulgen-stained nuclei of the same species using microspectrophotometry. The flow cytometric procedures were sufficiently sensitive to detect differences in DNA content between inbred lines of corn and their F1 hybrids. Our results obtained with improved procedures, specifically using propidium iodide as a fluorochrome and plant nuclei instead of chicken erythrocytes as an internal standard, demonstrate that laser flow cytometry can be a precise, rapid, and reliable method for determining nuclear DNA content of plants.     

Analysis of protein expression in pure cell nuclei populations isolated from human breast cancer tissue by DNA flow cytometric sorting

In this study, cell nuclei from aneuploid breast cancer samples were sorted with respect to DNA content into pure diploid and aneuploid fractions using flow cytometry. The nuclear proteins were then separated by one-dimensional gel electrophoresis (1D-PAGE) and differences in protein expression patterns, between diploid and aneuploid nuclei from the same tumours, were compared. Using a combination of peptide finger printing and peptide identification by MALDI-TOF mass spectrometry, we identified proteins and confirmed that the proteins were of nuclear origins. The results in this study add further information to the knowledge about the breast cancer disease complexity and heterogeneity at molecular level. For some of the tumours studied different nuclei protein patterns were obtained, in the diploid respective aneuploid nuclei populations, whilst other tumours did not show these differences.     

NASA/American Cancer Society High-Resolution Flow Cytometry Project - III. Multiparametric analysis of DNA content and electronic nuclear volume in human solid tumors

BACKGROUND: The NASA/American Cancer Society (ACS) flow cytometer can simultaneously measure electronic nuclear volume (ENV) and DNA content of nuclei. The preceding articles in this volume ("NASA/American Cancer Society High-Resolution Flow Cytometer Project-I") described the schematics, performance, and procedures used for the preparation of nuclei for analysis on this unit. In the present article, we describe the analysis of selected human tumors using the ratio of ENV/DNA content (nuclear packing efficiency [NPE]). METHODS: Tumor specimens (frozen) were minced with scalpels and stained with 1-10 microg/ml of 4',6-diamidino-2-phenylindole (DAPI) dihydrochloride at pH 6.0-7.2. Trout erythrocytes were used as internal standards. Data on ENV and DNA content were collected in list mode files. Propidium iodide-stained nuclei, analyzed on a Coulter XL cytometer, were used for comparison. RESULTS: Simultaneous measurement of ENV and DNA makes it possible to discriminate between hypodiploid or hyperdiploid tumor cells, as well as to differentiate between near-diploid aneuploid and diploid cells on the basis of their increased ENV. The NPE ratio is a valuable parameter for the detection of small quantities of tumor cells, separating overlapping diploid and aneuploid populations for cell cycle analysis and characterizing the level of differentiation in some tumors. CONCLUSION: NPE analysis provides unique measuring capabilities for the study of human solid tumors by flow cytometry.     

Two new nuclear isolation buffers for plant DNA flow cytometry: a test with 37 species

Background and Aims: After the initial boom in the application of flow cytometryin plant sciences in the late 1980s and early 1990s, which wasaccompanied by development of many nuclear isolation buffers,only a few efforts were made to develop new buffer formulas.In this work, recent data on the performance of nuclear isolationbuffers are utilized in order to develop new buffers, generalpurpose buffer (GPB) and woody plant buffer (WPB), for plantDNA flow cytometry. Methods: GPB and WPB were used to prepare samples for flow cytometricanalysis of nuclear DNA content in a set of 37 plant speciesthat included herbaceous and woody taxa with leaf tissues differingin structure and chemical composition. The following parametersof isolated nuclei were assessed: forward and side light scatter,propidium iodide fluorescence, coefficient of variation of DNApeaks, quantity of debris background, and the number of particlesreleased from sample tissue. The nuclear genome size of 30 selectedspecies was also estimated using the buffer that performed betterfor a given species. Key Results: In unproblematic species, the use of both buffers resulted inhigh quality samples. The analysis of samples obtained withGPB usually resulted in histograms of DNA content with higheror similar resolution than those prepared with the WPB. In morerecalcitrant tissues, such as those from woody plants, WPB performedbetter and GPB failed to provide acceptable results in somecases. Improved resolution of DNA content histograms in comparisonwith previously published buffers was achieved in most of thespecies analysed. Conclusions: WPB is a reliable buffer which is also suitable for the analysisof problematic tissues/species. Although GPB failed with someplant species, it provided high-quality DNA histograms in speciesfrom which nuclear suspensions are easy to prepare. The resultsindicate that even with a broad range of species, either GPBor WPB is suitable for preparation of high-quality suspensionsof intact nuclei suitable for DNA flow cytometry.     

Fluorescence in situ hybridization to interphase cell nuclei in suspension allows flow cytometric analysis of chromosome content and microscopic analysis of nuclear organization

Summary Fluorescence hybridization to interphase nuclei in liquid suspension allows quantification of chromosome-specific DNA sequences using flow cytometry and the analysis of the three-dimensional positions of these sequences in the nucleus using fluorescence microscopy. The three-dimensional structure of nuclei is substantially intact after fluorescence hybridization in suspension, permitting the study of nuclear organization by optical sectioning. Images of the distribution of probe and total DNA fluroescence within a nucleus are collected at several focal planes by quantitative fluorescence microscopy and image processing. These images can be used to reconstruct the three-dimensional organization of the target sequences in the nucleus. We demonstrate here the simultaneous localization of two human chromosomes in an interphase nucleus using two probe labeling schemes (AAF and biotin). Alternatively, dual-beam flow cytometry is used to quantify the amount of bound probe and total DNA content. We demonstrate that the intensity of probe-linked fluorescence following hybridization is proportional to the amount of target DNA over a 100-fold range in target content. This was shown using four human/hamster somatic cell hybrids carrying different numbers of human chromosomes and diploid and tetraploid human cell lines hybridized with human genomic DNA. We also show that populations of male, female, and XYY nuclei can be discriminated by measuring their fluores-cence intensity following hybridization with a Y-chromosome-specific repetitive probe. The delay in the increase in Y-specific fluorescence until the end of S-phase is consistent with the results recorded in previous studies indicating that these sequences are among the last to replicate in the genome. A chromosome-17-specific repetitive probe is used to demonstrate that target sequences as small as one megabase (Mb) can be detected using fluorescence hybridization and flow cytometry.     

Determination of ploidy levels in Ipheion uniflorum (R. C. Graham) Rafin (Liliaceae)

In this study, chromosome number and ploidy levels of Ipheion uniflorum cv. "Wisley Blue" (spring starflower) were determined. In meristematic root tip cells, chromosome number was found as 2n = 12 and 4n = 24. The ratios of diploid and tetraploid cells were found as 80.74% and 19.26%, respectively. In differentiated root tissues and mature leaf tissues ploidy levels were analysed by flow cytometry and polysomaty were found in both organs. In differentiated root tissues, ploidy levels were found as 2C, 4C, 8C and 16C DNA. In root tissues percentages of 2C, 4C, 8C and 16C nuclear DNA content were observed as 57.2%, 33.1%, 2.47% and 7.23%, respectively. In mature leaf tissues, ploidy levels were determined 2C, 4C, 8C and 16C DNA. In this tissue the frequency of 4C DNA was found very higher (74.3%) and 2C DNA content was determined as 19.2%. In mature leaf tissue, 8C and 16C nuclear DNA contents were observed as 2.72% and 3.78%, respectively. When nuclear DNA contents in leaves and roots were compared, an apparent difference in 2C and 4C DNA contents was found.     

Proliferative characteristics and ploidy of human brain tumors by DNA flow cytometry

We studied nuclear DNA distribution by flow cytometry in 59 human brain tumors. Samples were frozen at -20 degrees C immediately after surgery and unicellular suspensions were obtained with a mechanical dissociation technique. Nuclear DNA was stained with propidium iodide. Normal human brain tissue was used as a diploid reference standard. In 86.3% of benign tumors an unimodal DNA distribution with a DNA index usually within the diploid range was found. Among malignant tumors, 64% had un unimodal DNA distribution with diploid or near-diploid modal DNA content. The remaining 36% showed an additional cell peak with a DNA index ranging from 1.15 to 1.92. The percentage of S-phase cells was higher in malignant (median = 3.8) than in benign tumors (median = 1.9) (p less than .001), without correlation to histological tumor subtype.     

Nuclear changes in pathogen-infected tomato roots.

We have investigated nuclear changes induced in tomato (Lycopersicon esculentum) plant roots by two different pathogens, a phytoplasma of the stolbur group and the soil-borne fungus Phytophthora nicotianae var. parasitica, using light and electron microscopy as well as flow cytometry. Our results show that both pathogens strongly influenced nuclear structure, causing pyknosis and chromatolysis, and induced variations in proportion of nuclear populations with different DNA content, increasing the 2C and decreasing the 4C nuclear populations. These latter results suggest a block of the endoreduplication process in the presynthetic phase of the host cell cycle. Moreover, in pathogen-infected roots, nuclei of the same DNA content were less fluorescent, and therefore had a lower DNA content, compared to those of controls, as confirmed by morphological analyses. Our findings suggest that different stimuli may evoke similar nuclear changes, and confirm the usefulness of flow cytometry as a tool to recognize the stress conditions related to the presence of a pathogen.     

DNA content of Hurthle cell nodules in autoimmune thyroiditis

Hurthle cells are found in thyroid neoplasms and in reactive nodules in thyroiditis or goitrogenic processes. Cytometric studies have evaluated Hurthle cell neoplasms but not their reactive counterparts. DNA content of Hurthle cells in 22 cases of autoimmune thyroiditis was measured by flow cytometry and image content of Hurthle cells in 22 cases of autoimmune thyroiditis was measured by flow cytometry and image processing using nuclei extracted from paraffin-embedded tissue after microdissection of the Hurthle cell nodules. All 22 autoimmune thyroiditis Hurthle cell nodules were diploid, including 16 without associated neoplasms and six with associated malignant neoplasms (four papillary carcinomas, one follicular carcinoma and one follicular adenoma with papillary carcinoma). Concordance between flow cytometry and image processing was 100%. These findings indicate that the markedly atypical Hurthle cells in autoimmune thyroiditis are diploid by DNA quantitation. This suggests that atypia in Hurthle cells due to reactive or neoplastic processes may be differentiated by quantitative DNA analysis.     

Is There a Relationship between Infection by Rhizobia and Occurrence of Disomatic Nuclei in Nodules of Alfalfa (Medicago sativa L.)?

Cytological examination of nodules from diploid, tetraploid, and octoploid alfalfa (Medicago sativa L.) plants revealed that the proportion of nodule cells infected by rhizobia was not significantly affected by nuclear ploidy of the host plant. Flow cytometry was used to determine the influence of host plant nuclear ploidy on the nuclear ploidy of infected cells. In nodules from diploid plants, most of the nuclei were tetraploid, whereas in nodules from tetraploid plants, about half of the nodule nuclei were tetraploid and half were octoploid; in octoploid plants, most of the nodule nuclei were octoploid. The occurrence of disomatic nuclei was independent of infection of nodule cells by rhizobia, because diploid plants had mostly disomatic nodule nuclei, and octoploid plants had mostly monosomatic nodule nuclei, whereas all nodules maintained a constant proportion of infected to uninfected cells. These results do not support the earlier hypothesis that infected nodule cells contain disomatic nuclei.     

Modal DNA values of normal and malignant urothelial cells of the bladder in relation to nuclear size

In a study of the correlation between mean nuclear size and DNA content in urinary bladder carcinoma, the modal DNA values of cell suspensions from 125 biopsies, obtained from 86 patients with malignant or normal urinary bladder epithelium, were analyzed by flow cytometry (FCM). Light microscopic measurements of nuclear size were carried out on smears from the same material. The results were correlated to the histopathologic stage and grade. The mean nuclear volumes were significantly larger in diploid tumor cells than in cells of normal epithelium. Aneuploid tumors showed significantly larger nuclei than did diploid tumors. Although there was a significant correlation between increases in the nuclear volume and in the DNA content, there was some overlapping between various grades of malignancy: mean nuclear volumes in aneuploid grade 2 tumors did not differ from those in aneuploid grade 3 tumors. A combination of FCM and morphometry discriminated all but 16% of the tumors from the normal cases. It is concluded that FCM and morphometry are complementary and can be used for the objective characterization of urinary bladder carcinomas.     

Genome size and genome evolution in diploid Triticeae species.

One of the intriguing issues concerning the dynamics of plant genomes is the occurrence of intraspecific variation in nuclear DNA amount. The aim of this work was to assess the ranges of intraspecific, interspecific, and intergeneric variation in nuclear DNA content of diploid species of the tribe Triticeae (Poaceae) and to examine the relation between life form or habitat and genome size. Altogether, 438 plants representing 272 lines that belong to 22 species were analyzed. Nuclear DNA content was estimated by flow cytometry. Very small intraspecific variation in DNA amount was found between lines of Triticeae diploid species collected from different habitats or between different morphs. In contrast to the constancy in nuclear DNA amount at the intraspecific level, there are significant differences in genome size between the various diploid species. Within the genus Aegilops, the 1C DNA amount ranged from 4.84 pg in A. caudata to 7.52 pg in A. sharonensis; among genera, the 1C DNA amount ranged from 4.18 pg in Heteranthelium piliferum to 9.45 pg in Secale montanum. No evidence was found for a smaller genome size in annual, self-pollinating species relative to perennial, cross-pollinating ones. Diploids that grow in the southern part of the group's distribution have larger genomes than those growing in other parts of the distribution. The contrast between the low variation at the intraspecific level and the high variation at the interspecific one suggests that changes in genome size originated in close temporal proximity to the speciation event, i.e., before, during, or immediately after it. The possible effects of sudden changes in genome size on speciation processes are discussed.     

An efficient method for flow cytometric analysis of pollen and detection of 2n nuclei in Brassica napus pollen

A simple and reliable method was developed for isolating pollen nuclei from Brassica napus and Triticum aestivum for DNA analysis using flow cytometry. The nuclei were released from pollen by ultrasonic treatment. The isolated nuclei following filtration through nylon mesh and a purification procedure were suitable for flow cytometric analysis as well as for isolating genomic DNA. Ultrasonic treatment time was optimized for B. napus pollen at different developmental stages. The method is effective and suitable for the preparation of many samples. We analyzed the nuclear DNA levels in pollen of B. napus at three major developmental stages as well as in mature wheat pollen. Only a single 1C peak representing the haploid DNA level was detected in the nuclei isolated from Brassica uninucleate microspores as well as in mature Triticum pollen. Interestingly, diploid nuclei were detected in both binucleate and mature pollen of B. napus. The possible origins of the diploid nuclei are discussed.Abbreviations DAPI 4,6-Diamidino-2-phenylindole - NIB Nuclear isolation buffer     

Flow cytometric determination of DNA content in malignant and benign bone tumours

The nuclear DNA content of 38 malignant and 25 benign bone tumours was measured by flow cytometry. The specimens were taken either from biopsies or from surgical specimens. Seventeen of 26 primary malignant bone tumours were aneuploid, 15 had a single aneuploid DNA content, and 2 had a biclonal abnormality. Thirteen of 15 osteosarcomas were aneuploid, but only 2 of 6 chondrosarcomas showed an aneuploid DNA content. Six of 12 metastatic malignant bone tumours were also aneuploid. All 25 benign tumours had a diploid DNA content. Cell cycle analysis showed that the proportion of cells in S- and G2M-phases was higher in the malignant compared to benign tumours, indicating a higher proliferative activity. The increase was statistically significant (p less than 0.05) both in diploid and in aneuploid tumours. Among five tumours studied after chemotherapy, four displayed a marked hyperdiploid abnormality. Aneuploidy and high proliferative activity both were highly associated with malignant bone tumours, suggesting that DNA flow cytometry may be an adjunct in the assessment of malignancy of bone tumours.     

A new whole-mount DNA quantification method and the analysis of nuclear DNA content in the stem-cell niche of Arabidopsis roots

A semi-automated method to quantify fluorescence intensity of objects in intact organs and tissues, composed of several cell layers, has been designed. The method has been developed on whole-mount propidium-iodide stained Arabidopsis thaliana (Arabidopsis) root tips, in which the DNA content of individual nuclei could be quantified. A diameter of less than 150 μm makes this organ most appropriate for whole-mount imaging. Further advantages are the lack of chlorophyll and transparent cell walls, with only a little background fluorescence. The method has a great advantage over flow cytometry, as the information regarding the positions of nuclei is maintained, and nuclei with aberrant DNA content can be re-assessed individually, which facilitates the efficient distinction between technical artefact and aberrant DNA content. Our averaging 3D method calculates the average of the summed fluorescence intensities of all sections of a nucleus and interpolates the missing sections, thereby allowing for the correction of detection problems. Furthermore, this method has the advantage of detecting objects in tissues covering multiple cell layers. The results of our method in Arabidopsis root tips showed that the quiescent centre cells, which rarely divide, are diploid, and are arrested in G1 or G0. Most stem cells, with the exception of those of the vascular tissue, are diploid cells, and their rather low division rate is caused by an elongated G1 phase. In contrast, the majority of the vascular stem cells are tetraploid.