Determination of ploidy and nuclear DNA content in populations of Atriplex halimus (Chenopodiaceae)

Nuclear DNA contents were determined by flow cytometry for 20 populations of the perennial C₄ shrub Atriplex halimus L. (Chenopodiaceae) originating from the Mediterranean basin and Fuerteventura (Canary Islands). Two populations were also analysed for chromosome number: one (from Ibiza, Spain), with a 2C nuclear DNA content of 2.40 pg, was shown to be diploid (2 n  = 2 x  = 18), whilst the other (from Sicily, Italy), with 5.11 pg, was tetraploid (2 n  = 2 x  = 36). With respect to nuclear DNA content, two groups of populations were detected, diploids with 2.40–2.44 pg and tetraploids with 4.77–5.13 pg. The diploid populations were mainly from the western Mediterranean (Spain and France) and Fuerteventura, whereas tetraploids were generally, but not exclusively, from more arid areas in North Africa and the eastern Mediterranean. In general, the diploid and tetraploid populations corresponded to the subspecies halimus and schweinfurthii , respectively. For certain populations having morphologies intermediate between those considered typical of these two subspecies, nuclear DNA contents showed them to be tetraploid. There was significant variation in nuclear DNA content among the tetraploid populations, with greater values in the more easterly populations.  © 2005 The Linnean Society of London, Botanical Journal of the Linnean Society , 147 , 441−448.     

DNA ploidy and vimentin expression in primary breast cancer

DNA ploidy and vimentin expression in primary breast cancer
The DNA content of 50 breast cancers of varying tumour type, grade and stage was measured using static image cytometry, and correlated with vimentin expression in the tumour cells. A tendency to increased vimentin expression and aneuploidy was observed in high grade and late stage tumours¹. A statistically significant difference was observed in DNA index and ploidy balance between grade 1 and grades 2 and 3 carcinomas ( P <0.05) and between stage I and stage II carcinomas ( P <0.05). There was a significant difference in the expression of vimentin between grades 1, 2, 3 ( P <0,001), and stages I, II and III ductal carcinomas ( P <0.05). No significant difference was observed in the proliferation index and the degree of hyperploidy ( P >0.05). Clonal heterogeneity was observed in 25% of breast carcinomas, and was associated with increased vimentin expression. These changes may be indicative of genomic alteration and tumour aggressiveness.     

外来入侵植物和本地植物核DNA C-值的比较及其与入侵性的关系

用流式细胞仪测定了8科10属13种外来入侵植物、6种本地植物和1种外来非入侵植物的核DNAC-值。结果表明:作为整体,外来入侵植物的平均核DNAC-值显著低于本地种和外来非入侵种,但对同属不同类型植物进行比较,未发现一致的规律;在4个既包含外来入侵种又包含本地种的属中,泽兰属(Eupatorium)和鬼针草属(Bidens)外来入侵种的核DNAC-值显著低于同属本地种,莲子草属(Alternanthera)的2种外来入侵植物中仅有1个种的核DNAC-值显著低于同属本地种,而草胡椒属(Peperomia)外来入侵种的核DNAC-值显著高于同属本地种;表明核DNAC-值与外来植物入侵性无必然联系。     

Cytogeography of Solidago gigantea (Asteraceae) and its invasive ploidy level

Aim Newly formed polyploids experience problems of establishment and spread similar to those faced by newly introduced alien species. To understand the significance of polyploidy in biological invasions, we mapped the distribution of ploidy levels in Solidago gigantea Aiton in its native range in North America, and in Europe and East Asia where it is invasive. Location North America, Europe and East Asia. Methods Flow cytometry was used to measure ploidy levels in a total of 834 plants from 149 locations. Together with data from contributors and a literature review, ploidy‐level data were assembled for 336 locations. Cytogeographical maps from North America and Europe were prepared, incorporating new and previously published ploidy‐level data. Results In the native range, diploid, tetraploid and hexaploid plants were found, and also one triploid and one pentaploid plant (2n = 3x and 2n = 5x, respectively, each being new reports for this species). There was a high degree of geographical separation among the ploidy levels, and populations with mixed ploidy were rare. However, four zones were identified where plants of different ploidy could come into contact. In Europe and East Asia, only tetraploid plants were found. Main conclusions The geographical pattern in North America suggests that the ploidy levels are ecologically differentiated, although further investigations are needed to identify the nature of these differences. Alien populations appear to be exclusively tetraploid, but it is not clear whether this is because tetraploids were selectively introduced or because diploids were unsuccessful. In any case, comparisons between native and introduced populations need to account for ploidy level.     

Comparative analysis of DNA nuclear content by flow cytometry on strawberry plants propagated via runners and regenerated from meristem and callus cultures

ABSTRACT

DNA variation may occur in plant species grown either in vivo or in vitro. In this study flow cytometric analyses were undertaken on Fragaria x ananassa Duch. runner plants, and on plants regenerated from callus cultures of leaf explants and from meristem cultures. Our aims were to investigate DNA variation in runner plants of different cultivars, and to compare DNA content in plants of the same cultivar obtained by different propagation procedures (i.e. from meristems or callus cultures). Plants growing in vitro and in the greenhouse were also compared. A good regeneration ability was observed in all the cultivars, with different percentages of shoot formation. No significant differences were detected in multiplication rate and rooting percentage within cultivars. This work documents the occurrence of DNA variations in strawberry plants in vivo and in vitro. Flow cytometric measurements of DNA content showed the presence of 4C nuclei, besides 2C nuclei, in runner plants of cultivar Pajaro. DNA content variations (2C/4C nuclei) were observed in plants regenerated from callus cultures. These variations were lost after transfer of the plants to the greenhouse, except for cultivar Don. The extent of such DNA variations was influenced by genotype. Our study confirms earlier reports indicating that DNA variation induced by in vitro culture could be lost or retained after transfer of the plants to the greenhouse.     

p53 expression measured by flow cytometry. A comparison of three monoclonal antibodies and the relationship with grade and DNA ploidy in breast cancer

The aim of this study was to quantify p53 expression by flow cytometry. A panel of three monoclonal antibodies: NCL-p53-240, NCL-p53-1801 and NCL-p53-DO7, was tested on breast cell lines and primary breast cancers. The relationships between ploidy, tumour grade and p53 expression for each antibody, were examined. Methodology was assessed using a variety of breast cell lines. Staining patterns were confirmed and the quantification technique qualified. Cytokeratin-positive cells from 58 samples obtained from patients with breast cancer were assayed for DNA content and p53 expression. p53 quantification was performed using calibrated fluorescent beads on cytokeratin-positive cells. Bloom and Richardson grading revealed 20 grade I and 38 grade II/III breast cancers. Examination of fluorescence thresholds showed a positive correlation between grade and DO7 (P=0.003) at a level of 8900 molecules, 240 (P=0.005) at a level of 2900 molecules and 1801 (P=0.005) at a level of 1850 molecules. These levels equated with 34% (DO7), 43% (240) and 43% (1801) of the samples being classified as p53-positive. Examination of ploidy revealed 23 diploid and 35 aneuploid breast cancers. Application of p53 threshold levels on diploid and aneuploid tumours showed correlation between aneuploidy and p53 expression for DO7 at a level of 9000 molecules, 240 at a level of 1900 molecules and 1801 at a level of 1800 molecules. These levels equated with 34% (DO7), 52% (240) and 52% (1801) of the samples being classified as p53-positive. We conclude that measurement of p53 by flow cytometry may be of clinical importance by indicating levels of positivity using fluorescence thresholds. p53 expression has been shown to correlate with both grade and ploidy. Flow-cytometric measurement of p53 may be a useful prognostic assay.This study was supported by the North of England Cancer Research Campaign     

Increased variability in nuclear DNA content of testis cells and spermatozoa from mice with irregular meiotic segregation.

Variability in DNA content to testis cells and sperm from F₁ hybrids between the laboratory mouse (M. muscullus) and the tobacco mouse (M. poschiavinus), has been determined by flow cytometry (FMC). The F₁ hybrid mouse is known to be heterozygous for seven metacentric chromosomes produced by Robertsonian fusion. Enriched populations of nuclei from late pachytene spermatocytes and round spermatids were obtained by velocity sedimentation. These nuclei, as well as epididymal sperm nuclei and spleen cells, were stained by the acriflavin-Feulgen technique for DNA and measured by FCM. Peaks in the fluorescence intensity frequency distributions resulting from these measurements were analyzed to determine their mean fluorescence intensities and their widths (coefficients of variation). Because mean intensities of corresponding cell types from M. musculus and the F₁ hybrids were identical, the average DNA contents were taken to be the same. The average coefficients of variation of the peaks to fluorescence from the pachytene, spermatid, and sperm nuclei and spleen cells from M. muscullus animals were about 5%. While the peaks of fluorescence from spleen cells and pachytene nuclei from f₁ hybrids also had average coefficients of variation of 5%, post-meiotic nuclei from spermatids and spermatozoa had coefficients of variationof 8%. From these results we conclude that, in these F₁ hybrids, abnormal meiotic segregation causes an increased variability of 6% in the amount of DNA in the spermatozoa.     

Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants

Flow cytometric estimation of nuclear DNA content was performed in six plant species employing three fluorochromes showing different DNA base preferences: propidium iodide (no base preference), 4',6-diamidino-2-phenylindole (DAPI; AT preference), and mithramycin (GC preference). Nuclei isolated from human leukocytes were used as a primary reference standard. While nuclear DNA contents estimated using propidium iodide were in agreement with published data obtained using other techniques, the values obtained using fluorochromes showing base preference were significantly different. It was found that the differences were caused by the differences in overall AT/GC ratios, and by the species-specific differences in binding of these fluorochromes to DNA. It was concluded that nuclear DNA content estimations performed with fluorochromes showing base preference should be interpreted with caution even when AT/GC ratios of the reference and the sample are equal. The use of intercalting dyes (e.g. propidium iodide) is recommended for this purpose. On the other hand, comparison of the staining behaviour of intercalating dyes with that of dyes showing base preference may give additional information on chromatin structural differences and arrangement of molecule pairs in DNA.     

Intraspecific DNA content variability in Festuca pallens on different geographical scales and ploidy levels

BACKGROUND AND AIMS: Intraspecific genome size variability of Festuca pallens occurring on relict rocky steppes in Central Europe was studied on two ploidy levels and three geographical scales: (1) local scale of 24 populations, (2) landscape scale of three transects in river canyons or hill systems, and (3) global scale of 160 samples covering the whole distribution area. METHODS: DAPI flow cytometry of homogeneously cultivated samples (>or=1 year), measured randomly with two internal standards, Lycopersicon esculentum and Pisum sativum. Differences in DNA content were confirmed (1) by the double peaks of simultaneously measured samples, (2) based on measurements carried out in different seasons, and (3) by additional measurements with propidium iodide. KEY RESULTS: On a global scale, the relative DNA content ranged between 1.170-fold in diploids and 1.164-fold in tetraploids. A maximum difference of 1.088-fold between the mean relative DNA content of nearby populations was found. In 16 of 24 populations significant variability was shown (P<0.001, 1.121-fold as maximum). For both ploidy levels, the relative genome size had the same range and geographical pattern, correlated with geographical coordinates (P<0.01). Diploids with larger genomes occur on relict habitats (P<0.01), and in areas of periglacial steppes (20,000 years ago; P<0.02). In tetraploids, the relative DNA content differs among the three previously recognized geographical types (Alpine, Pannonian and Scabrifolia, P<0.001). Tetraploids have a relative DNA content smaller than twice that of the diploids (P<0.001). An influence of microhabitat on DNA content variation was not confirmed. CONCLUSIONS: Genome size variability occurs over all spatial scales: intrapopulation, landscape and global. Correlation between geographical coordinates and palaeovegetation type, concomitant with diploids and tetraploids, and no influence of microhabitat were found. Genome size decreases in tetraploids. Lower CVs, and thus higher accuracy, resolution and reproducibility, favour DAPI measurements for the study of intraspecific genome size variability.     

中国吊钟花属植物核DNA含量(2C-值)与倍性水平研究

植物核DNA含量(2C-值)与倍性水平是重要的植物学基本特征,是进行种群进化、物种分类和生态学等研究的有力证据.为确定中国吊钟花属(Enkianthus Lour.)各物种的核DNA含量与倍性水平并探究该属植物在种间、种内核DNA含量差异,该研究以6种中国吊钟花属植物共23个居群60个样品为试验材料,以水稻品种'日本晴...     

High frequency plant regeneration from Eucalyptus globulus Labill. hypocotyls: Ontogenesis and ploidy level of the regenerants

Up to 70% regenerating hypocotyls provided with 5 to 20 buds were obtained on MS medium containing 0.01 or 1 mg l^-1 NAA and 0.2 mg l^-1 BA or 0.2 mg l^-1 BA and 0.2 mg l^-1 TDZ. The ability to regenerate buds was correlated with the presence of oil glands at a stage in germination when oil secretion was not yet occurring. The regeneration of shoot meristems took place from the cells involved in the differentiation of these glands. Such glands could also appear during callus redifferentiation, giving rise to indirect regeneration. Rooting of the regenerants was efficient using a two-step procedure of induction under darkness in the presence of 3 mg l^-1 IBA, followed by root development on medium devoid of growth regulators under a 16-h photoperiod, the medium being solidified with Gelrite. Regenerated plants showed no phenotypic alterations. Nuclear DNA contents in mother-plant material and regenerants were analysed using flow cytometry. There was no evidence of polyploidy in any of the samples, indicating the absence of polyploidisation during cell differentiation and under in vitro conditions. No regeneration was obtained from leaf or stem explants from micropropagated plantlets. This revised version was published online in June 2006 with corrections to the Cover Date.     

Diagnostic and prognostic significance of image cytometric DNA ploidy measurement in cytological samples of cervical squamous intraepithelial lesions

D. Demirel, N. Akyürek and I. Ramzy
Diagnostic and prognostic significance of image cytometric DNA ploidy measurement in cytological samples of cervical squamous intraepithelial lesions Objective: To study the DNA ploidy pattern of uterine cervical squamous intraepithelial lesions (SILs) and its diagnostic and prognostic significance. Methods: The study included 31 cases of SIL: 11 low‐grade (LSIL) and 20 high‐grade (HSIL). Feulgen–pararosaniline staining was performed on previously Papanicolaou‐stained smears and a DNA image cytometric study was performed. An internal reference was used to calibrate the samples. Results: All 31 cases of SIL, either LSIL or HSIL, were non‐diploid. Of the 11 cases of LSIL, four were tetraploid and seven were aneuploid, whereas, of the 20 cases of HSIL, four were tetraploid and 16 were aneuploid. Stemline aneuploidy was not a significant discriminator between LSIL and HSIL (P = 0.32). Based on single‐cell analysis, HSIL cases had significantly higher DNA content than LSIL cases (P < 0.01). When a mean of 30% or more was used for the 6c‐exceeding event (6cEE) value, the sensitivity and specificity to indicate HSIL were 83% and 64%, respectively, with a positive predictive value (PPV) of 81% and negative predictive value (NPV) of 65%. All HSIL cases were cervical intraepithelial neoplasia grade 2 or worse (CIN2+) on biopsy. In addition, cases which showed recurrence had more DNA content by single‐cell analysis than those with an indolent clinical behaviour: P = 0.04 and P = 0.03 for LSIL and HSIL, respectively. Conclusions: Image cytometric DNA analysis is a useful technique for diagnostic and prognostic purposes in uterine cervical SIL when appropriate ‘c’ values are used in single‐cell analysis. We propose that a >6c DNA content of 30% is useful as a cut‐off level for predicting cases with CIN2+ in DNA image cytometry of cervical smears.     

Nuclear DNA C-values in 30 species double the familial representation in pteridophytes

Nuclear DNA C-values and genome size are important biodiversity characters with fundamental biological significance. Yet C-value data for pteridophytes, a diverse group of vascular plants with approx. 9000 extant species, remain scarce. A recent survey by Bennett and Leitch (2001, Annals of Botany 87: 335-345) found that C-values were reported for only 48 pteridophyte species. To improve phylogenetic representation in this group and to check previously reported estimates, C-values for 30 taxa in 17 families were measured using flow cytometry for all but one species. This technique proved generally applicable, but the ease with which C-value data were generated varied greatly between materials. Comparing the new data with those previously published revealed several large discrepancies. After discounting doubtful data, C-values for 62 pteridophyte species remained acceptable for analysis. The present work has increased the number of such species' C-values by 93 %, and more than doubled the number of families represented (from 10 to 21). Analysis shows that pteridophyte C-values vary approx. 450-fold, from 0-16 pg in Selaginella kraussiana to 72.7 pg in Psilotum nudum var. gasa. Superimposing C-value data onto a robust phylogeny of pteridophytes suggests some possible trends in C-value evolution and highlights areas for future work.     

Karyotype analysis and DNA content in some species of Chrysolaena (Vernonieae,Asteraceae)

Cytogenetic characterization by karyotyping and determination of DNA content by flow cytometry of five species of Chrysolaena (Vernonieae, Asteraceae) was performed. This is the first study of nuclear DNA content realized in the genus. The 2C-values were compared with the ploidy level and the total karyotype length (TKL) of each species. Mitotic analysis revealed a base chromosome number x = 10 for all entities and different ploidy levels, from diploid (2n = 2x = 20) to octoploid (2n = 8x = 80). All species showed bimodal karyotypes composed of metacentric and submetacentric chromosomes. The average chromosome size (ML) varied from 1.86 μm to 2.70 μm, while the TKL ranged from 18.65 μm to 80.55 μm. The intrachromosomal asymmetry index (A₁) varied from 0.27 to 0.38, while the interchromosomal asymmetry index (A₂) ranged from 0.19 to 0.25. A new cytotype is reported for the first time for C. propinqua. Accessory chromosomes found in C. verbascifolia, C. cognata, C. flexuosa, and C. propinqua are also reported as new.     

山东蔊菜属种子形态学研究及其系统分类学意义

对山东菜属植物的种子,从大小、翅、颜色和种子微形态等方面进行了研究;结果表明该属的种子为卵形或近圆形,呈扁平状;种子表面纹饰为网状或乳突状。与近缘属种子形态的研究表明,菜属和豆瓣菜属亲缘关系较近。因种子的形态特点比较稳定,所以菜属的种子特点可以为其分类、生药鉴定以及演化提供种子形态学方面的证据。     

Agrobacterium as a tool for gene transfer in woody species: The state of the art and practical perspectives. A review

Abstract

The Agrobacterium-mediated ability to transfer genes into organisms without sexual crossing provides breeders with new opportunities to improve the efficiency of plant production. Gene transfer offers advantages over classical genetic manipulation since plants are improved without disruption of the integrity of their genomes. Several useful genes isolated from microrganisms and affecting pest resistance, rooting ability, hormonal metabolism etc., are now available. These genes can be easily cloned into suitable Ti and Ri derived plasmid vectors and transferred into woody species. The scarce ability of most fruit trees to regenerate the whole plant from in vitro-cultured cells remains the main obstacle to a wider use of gene transfer technology.     

Fibrillarin, a nucleolar protein, is required for normal nuclear morphology and cellular growth in HeLa cells

Fibrillarin is a key small nucleolar protein in eukaryotes, which has an important role in pre-rRNA processing during ribosomal biogenesis. Though several functions of fibrillarin are known, its function during the cell cycle is still unknown. In this study, we confirmed the dynamic localization of fibrillarin during the cell cycle of HeLa cells and also performed functional studies by using a combination of immunofluorescence microscopy and RNAi technique. We observed that depletion of fibrillarin has almost no effect on the nucleolar structure. However, fibrillarin-depleted cells showed abnormal nuclear morphology. Moreover, fibrillarin depletion resulted in the reduction of the cellular growth and modest accumulation of cells with 4n DNA content. Our data suggest that fibrillarin would play a critical role in the maintenance of nuclear shape and cellular growth.     

Karyotype and nuclear DNA content of hexa-, octo-, and duodecaploid lines of Bromus subgen. Ceratochloa

The subgenus Ceratochloa of the genus Bromus includes a number of closely related allopolyploid forms or species that present a difficult taxonomic problem. The present work combines data concerning chromosome length, heterochromatin distribution and nuclear genome size of different 6x, 8x and 12x accessions in this subgenus. Special attention is paid to the karyotype structure and genomic constitution of duodecaploid plants recently found in South America. Hexaploid lineages possess six almost indistinguishable genomes and a nuclear DNA content between 12.72 pg and 15.10 pg (mean 1Cx value = 2.32 pg), whereas octoploid lineages contain the same six genomes (AABBCC) plus two that are characterized by longer chromosomes and a greater DNA content (1Cx = 4.47 pg). Two duodecaploid accessions found in South America resemble each other and apparently differ from the North American duodecaploid B. arizonicus as regards chromosome size and nuclear DNA content (40.00 and 40.50 pg vs. 27.59 pg). These observations suggest that the South American duodecaploids represent a separate evolutionary lineage of the B. subgenus Ceratochloa, unrecognized heretofore.