Flow cytometric measurement of nuclear DNA content inCapsicum (Solanaceae)

Nuclei were isolated from leaf tissue of differentCapsicum species and the relative fluorescence intensity was measured by flow cytometry after propidium iodide staining.Pisum sativum nuclei with known nuclear genome size (9.07 pg) were used as internal standard to determine nuclear DNA content of the samples in absolute units. The 2C DNA contents ranged between 7.65 pg inC. annuum and 9.72 pg inC. pubescens, and the general mean of the genus was 8.42 pg. These values correspond, respectively, to 1C genome size of 3.691 (C. annuum), 4.690 (C. pubescens) and 4.063 (general mean) Mbp. In general, white-flowered species proved to have less DNA, with the exception ofC. praetermissum, which displayed a 2C DNA content of 9.23 pg. It was possible to divide the studied species into three main groups according to their DNA content, and demonstrate differences in DNA content within two of the three species complexes established on the basis of morphological traits.     

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.     

Flow cytometric analysis of nuclear genome of the Ethiopian cereal tef [Eragrostis tef (Zucc.) Trotter]

Flow cytometric analysis of nuclear DNA content was performed by using nuclei isolated from young leaf tissue of tef (Eragrostis tef). The method was very useful for rapid screening of ploidy levels in cultivars and lines of tef representing the phenotypic variability of this species in Ethiopia. The results of the analysis showed that all cultivars were tetraploid. Flow cytometry was also used to determine nuclear DNA content in absolute units (genome size) in four tef cultivars. Nuclei isolated from tomato (Lycopersicon esculentum, 2C=1.96 pg) were used as an internal reference standard. The 2C DNA content of individual tef cultivars ranged from 1.48 to 1.52 pg (1C genome size: 714 Mbp-733 Mbp), the differences among them being statistically nonsignificant. The fact that the nuclear genome of tef is only about 50% larger than that of rice should make it amenable for analysis and mapping at the molecular level.     

Genome size variation inCajanus cajan (Fabaceae): A reconsideration

A recent investigation of genome size in certain samples of the pigeonpea,Cajanus cajan, indicates values from 1.55 pg to 1.99 pg (1C level), which is 1.29-fold variation between accessions. In the present analysis those of these accessions which had particularly high or low DNA contents in that study were subjected to a reanalysis using propidium iodide and DAPI flow cytometry and Feulgen densitometry. Only minor differences in genome size, not more than 1.047-fold, were found with flow cytometry, and no significant differences were obtained with Feulgen densitometry. The previously reported genome size cannot be confirmed. It is about half as large and was determined in the present study as 0.825 pg (1C, propidium iodide flow cytometry,Glycine max as standard) and 0.853 pg (1C, Feulgen densitometry,Allium cepa andPisum sativum as standards), respectively.     

Estimation of nuclear DNA content of various bamboo and rattan species

We determined the nuclear DNA content (genome size) of over 35 accessions each of bamboo and rattan species from Southeast Asia. The 2C DNA per nucleus was quantified by flow cytometry. The fluorescence of nuclei isolated from the leaves and stained with propidium iodide was measured. The genome size of the bamboo species examined was between 2.5 and 5.9 pg DNA per 2C nucleus. The genome size of the rattan species examined ranged from 1.8 to 10.5 pg DNA per 2C nucleus. This information will be useful for scientists working in diverse areas of plant biology such as biotechnology, biodiversity, genome analysis, plant breeding, physiology and molecular biology. Such data may be utilized to attempt to correlate the genome size with the ploidy status of bamboo species in cases where ploidy status has been reported.     

Nuclear DNA content of the Solanum spp. in the series Etuberosa as determined by laser flow cytometry

Nuclear DNA content was determined in three accessions of Solanum brevidens, three accessions of S. etuberosum, and one accession of S. fernandezianum, which are diploid (2n = 2×= 24), closely related, non tuber-bearing wild potato species belonging to the series Etuberosa (Solanaceae). The plants were grown in vitro at 18°C or at 25°/22°C (day/night). S. brevidens was also grown in soil in the glasshouse at 25°/19°C (day/night), and in growth chambers at 18°C or 32°C. Leaf nuclei were isolated using a chopping method and stained with propidium iodide. Chicken red blood cells (CRBC; 2.33 pg) were added to the samples of nuclei as internal standards. The fluorescence of plant nuclei relative to CRBC was measured with an EPICS PROFILE flow cytometer. The 2C values of in vitro-grown S. brevidens and S. etuberosum were similar (1.48–1.54 pg, depending on the accession), but they were smaller than the 2C value of S. fernandezianum (1.63 pg). The 2C values of S. brevidens and S. etuberosum were generally smaller than those of the diploid species S. berthaultii (1.60–1.61 pg) and the diploid clones of S. tuberosum (1.60–1.72 pg). A similar relative difference of nuclear DNA content was found also between tetraploid S. brevidens and tetraploid S. tuberosum (2C = 3.15–3.16 pg and 3.50–3.62 pg, respectively). High (32°C) and low (18°C) growth temperatures caused abnormal changes in morphology and reduced fertility in S. brevidens in the growth chamber. The 2C values of S. brevidens grown at 25°/19°C (day/night) or at 32°C were similar, whereas the 2C values were c. 10% lower at 18°C.     

Nuclear DNA content of some important plant species

Nuclear DNA contents of more than 100 important plant species were measured by flow cytometry of isolated nuclei stained with propidium iodide.Arabidopsis exhibits developmentally regulated multiploidy and has a 2C nuclear DNA content of 0.30 pg (145 Mbp/1C), twice the value usually cited. The 2C value for rice is only about three times that ofArabidopsis. Tomato has a 2C value of about 2.0 pg, larger than commonly cited. This survey identified several horticultural crops in a variety of families with genomes only two or three times as large asArabidopsis; these include several fruit trees (a pricot, cherry, mango, orange, papaya, and peach). The small genome sizes of rice and the horticultural plants should facilitate molecular studies of these crops.     

Cytophotometric and autoradiographic evidence for functional apomixis in a gynogenetic fish,Poecilia formosa and its related,triploid unisexuals

Summary Amounts of DNA in individual Feulgen-stained nuclei from squash preparations of ovaries and testes from wild-caught and laboratory-reared stocks of Poecilia spp. were determined with an integrating microdensitometer. The DNA content of primary spermatocytes (4C) at zygotene, pachytene, or at metaphase I (3.3–3.4 pg) was approximately twice that found in secondary spermatocytes (2C) and four times that found for young spermatids (1C). Rarely, mature sperm were found with 2C DNA amounts. Nuclei from follicular epithelium and oogonia from both bisexual and diploid unisexual fish contained about 1.6–1.7 pg DNA; whereas, the DNA content of primary oocyte nuclei was about 3.5–3.7 pg DNA, indicating that just one cycle of chromosomal replication had occurred in these cells during the period of DNA synthesis before the visible onset of meiotic prophase. Similar results were obtained for triploid unisexuals whose 6C primary oocyte nuclei contained 5.0–5.1 pg DNA, which was twice the DNA content of 3C oogonia and follicular epithelial cells (2.4–2.5 pg DNA). Autoradiographic studies, designed to monitor the incorporation of ³H-thymidine by oogonia and primary oocytes in vivo and in vitro, also showed that there is no additional synthesis of DNA during the course of meiotic prophase in these unisexual fish. Therefore, we conclude that apomixis, not endoreduplication, is the cytological basis of reproduction in Poecilia formosa and its related, triploid biotypes.     

Nuclear DNA amount during sporogenesis and gametogenesis in sexual and aposporous buffelgrass

Nuclear DNA amounts (C values) were measured in Feulgen-stained sections of anthers and ovules of sexual plant B-2s (genotype aaaa) and aposporous cultivar Higgins (genotype AAaa) of buffelgrass (Pennisetum ciliare). The mass of the unreplicated nuclear genome of a gamete equals 1C DNA. In both lines, pollen mother cell nuclei were 4C before leptotene; anther wall, dyad, 1-nucleate pollen, and generative cell nuclei were 2C; microspore tetrad, enlarging microspore, and sperm nuclei were 1C. The tapetum persisted as uninucleate cells with 4C DNA. Archespores (2-4C) of both lines initiated meiosis to form megaspore tetrad nuclei with 1-2C DNA. In B-2s, chalazal megaspores (2-4C) formed reduced 8-nucleate Polygonum type embryo sacs, and sacs at 2- and 4-nucleate stages showed distributions with peaks near C1 and C2, corresponding to G1 and G2 cell cycle phases; this is characteristic of active mitosis. Nuclei of 8-nucleate sacs and of eggs and polars were 1C, indicating chromosomes were not duplicated before fertilization. Antipodal nuclei had levels from 1 to 36C, possibly due to polyteny or endopolyploidy. In Higgins, aposporous initials and 2-nucleate embryo sacs showed bimodal distributions of 2n nuclei with peaks at 2C and 4C DNA. Nuclei of newly formed 4-nucleate Panicum type aposporous sacs and of polars were 2C; aposporous eggs stained too faintly for reliable measurement.Names of products are included for the benefit of the reader and do not imply endorsement or preferential treatment by USDA     

Analysis of the cell cycle in an arbuscular mycorrhizal fungus by flow cytometry and bromodeoxyuridine labelling

Summary The cell cycle of an arbuscular mycorrhizal fungus,Glomus versiforme, was determined by flow cytometric analysis of nuclei isolated from spores and mycorrhizal roots of leek, and by immunogold staining after bromodeoxyuridine (BrdU) uptake by DNA. The aims of our work were to establish: (i) whether there are changes in ploidy during fungal growth and morphogenesis, (ii) when and where the cell cycle is activated. Our results demonstrate that nuclei isolated from quiescent spores ofG. versiforme are arrested in the GO/G1 phase (99.2%), whereas fungal nuclei from mycorrhizal roots are in the synthetic (S) (10.1%) and G2/M phase (3.9%). Nuclei undergoing DNA synthesis were detected in situ after BrdU uptake. Labelled nuclei were observed in intercellular hyphae and in large arbuscular trunks. This paper demonstrates that colonization of an arbuscular mycorrhizal fungus is linked to activation of its cell cycle.Abbreviations AM fungi arbuscular mycorrhizal fungi - BrdU 5-bromo-2-deoxyuridine - PI propidium iodide - DAPI 4,6-diamidino-2-phenylindole     

Patterns of endopolyploidy and 2C nuclear DNA content (Feulgen) inScilla (Liliaceae)

The Feulgen-DNA content of 3558 nuclei from 21 different tissues and organs ofScilla decidua (Liliaceae) was measured by a scanning cytophotometer interfaced to a computer. The basic nuclear DNA content (2 C value) was 13.62 pg, and 71 per cent of the nuclei were polyploid. The highest DNA values were found in the antipodal cells of the ovule, and the elaiosomes of the seeds (512 C). In addition to polyploidy, the 2 C values exhibited tissue-specific variation which was statistically significant (0.05% level of probability), It is suggested that differential DNA replication and endopolyploidization may be basic factors in the complex mechanism of cell and tissue differentiation.Dedicated to Professor Dr.Lothar Geitler in honour of his 80th birthday.     

Genome size and environmental factors in the genus Pinus

Nuclear 1C DNA content in haploid megagametophyte tissue of 18 North American and one exotic Pinus species was determined using scanning microspectrophotometry. The nuclear DNA content in root meristematic cells of Zea mays L. ssp. mays, inbred line Va35 (4C = 10.31 pg) was used as a standard. DNA content measured by microspectrophotometry was verified using laser flow cytometry with two additional standards, Hordeum vulgare cv. Sultan (2C = 11.12 pg) and P. eldarica (2C = 47.30 pg). DNA values obtained by both methods were significantly correlated (r = 0.987). The 1C nuclear DNA content ranged from 21 pg to 31 pg. The ratio of DNA content in embryo tissue of P. eldarica to that in megagametophyte tissue was 1.72 by scanning microspectrophotometry and 1.74 by laser flow cytometry. To date, this is the most comprehensive data set available for North American Pinus species. Relationships between genome size of 18 North American Pinus species and climatic factors and indices of growth were investigated using regression and correlation analyses. Positive correlations were observed between nuclear DNA content and growth indices, minimum seed-bearing age, and seed dimensions. Strong negative correlations were observed between nuclear DNA content and two climatic factors, the lowest mean annual and monthly precipitation (excluding January) and the highest mean monthly spring air temperature. These correlations suggest that the large genome size and its variation in Pinus are adapted responses to the habitats of these species.     

Nuclear DNA content and the control of chloroplast replication in wheat leaves

During development of the first leaf of breadwheat (Triticum aestivum L.) the number of chloroplasts per mesophyll cell increases between three- and four-fold. To establish if chloroplast replication is accompanied by endoreduplication, the nuclear DNA content of the cells was determined by chemical assay of isolated nuclei from mesophyll protoplasts and by microdensitometry of nuclei in mesophyll tissue. The DNA content of the nuclei was constant (27 to 32 pg) at each phase of chloroplast replication. Approximately 93% of the cells had a nuclear DNA content close to the 2C value of 32 pg. It is concluded that chloroplast replication is not dependent on nuclear endoreduplication in seedling leaves of wheat.     

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.     

Nuclear DNA contents in four primitive angiosperms

The basic (2 C) nuclear DNA content has been determined for the first time in four primitive angiosperms by means of scanning densitometry of Feulgen-stained nuclei. The mean values obtained are the following:Liriodendron tulipifera L. (2n = 38): 1.58 pg;Magnolia soulangiana Soul-bod. (2n = 76): 11.95 pg;Cinnamomum camphora T. Nees (2n = 24): 1.18 pg;Illicium anisatum L. (2n = 28): 6.72 pg. These values do not represent extremes, but rank among low DNA amounts. All species display at least low degress of endopolyploidy.     

Genome size variation and C-band polymorphism inAlstroemeria aurea, A. ligtu andA. magnifica (Alstroemeriaceae)

Among a total of 43 accessions ofAlstroemeria aurea, A. ligtu andA. magnifica nuclear DNA amounts (2C-values) showed significant intraspecific variation, 1.09, 1.21 and 1.15 fold, respectively, when determined through flow cytometric measurements of fluorescence of propidium iodide (PI) stained nuclei. After staining with another fluorochrome, 4,6-diamidino-2-phenylindole (DAPI), an intraspecific variation of 1.10, 1.11 and 1.12 fold, respectively, was found. C-band polymorphisms were present among and within the accessions of all three species. In some cases only very small differences in C-banding pattern were observed. In other cases, however, differences were more prominent. Besides C-band polymorphism, there were also instances of chromosome length polymorphism, which concerned the total chromosome complement or single chromosomes. The variation in nuclear DNA amount inA. aurea andA. ligtu was more or less continuous, except for one accession ofA. ligtu subsp.simsii. Artificial selection and possibly introgression of chromosomes from other species may have moulded the karyotypes of some of the accessions ofA. aurea, a species that has been under cultivation for more than 160 years. The variation as observed inA. magnifica subsp.magnifica was discontinuous and could be due to a broad species concept.     

DNA CONTENT AND HETEROCHROMATIN VARIATIONS IN VARIOUS TISSUES OF PEANUT (ARACHIS HYPOGAEA)

The average 2C DNA amount for the peanut (Arachis hypogaea L.) genome is 4.21 pg, and 73% of the dormant peanut cotyledon nuclei displayed 8C DNA amounts or higher, as compared to 0 to 4% in root-shoot apices and leaf tissue. Thermal melt profiles and heterochromatin values indicated replication of the whole genome. Cotyledon nuclear DNA declined in the percent of polyploid nuclei as well as DNA amounts within ploidy classes during germination. The presence of high DNA C levels in cotyledons generated during embryogeny is interpreted to increase the protein-synthesizing capacity and subsequently supplies a ready source of nucleosides and phosphates during early embryo growth as a result of DNA degradation. However, the later DNA decline at the onset of cotyledon senescence was age related similarly to leaf senescence. The change in proportion of heterochromatin was related to the metabolic state of the tissue and not to the DNA content, as dormant and senescing nuclei contained a higher proportion of heterochromatin as compared to nuclei from metabolically active tissue such as germinating roots. The shift in heterochromatin is interpreted to be involved in gene expression.     

Morphogenic and genetic stability in longterm embryogenic cultures and somatic embryos of Norway spruce (Picea abies {L.} Karst)

Embryogenic cultures were initiated from mature zygotic embryos of Picea abies. The somatic embryos in the embryogenic cultures were first stimulated to mature and then either to develop further into plantlets or to differentiate new embryogenic cultures. The procedure was repeated three times during two years. The ability to give rise to new embryogenic cultures or to develop into plantlets was similar for all somatic embryos irrespective of how long they had been cultured in vitro. The nuclear DNA content, measured in a flow cytometer, was estimated at 32 pg/G1 nuclei in seedings developed from zygotic embryos. Nuclei isolated from embryogenic cultures and from plantlets regenerated from somatic embryos had the same DNA content as those isolated from seedlings.Abbreviations N⁶-benzyladenine BA - 2,4-dichlorophenoxyacetic acid 2,4-D - abscisic acid ABA     

QUANTITATIVE CHANGES IN NUCLEAR DNA ACCOMPANYING POSTGERMINATION EMBRYONIC DEVELOPMENT IN VANDA (ORCHIDACEAE)

Nuclear DNA was measured cytophotometrically in sections and isolated nuclei of the developing embryo of Vanda. The data were interpreted in terms of developmental stage and spatial relationships of the nuclei within the embryo. An equal amount of DNA was found in all meristematic nuclei regardless of the developmental stage of the embryo and was taken as the 2C value. Most of the nuclei in the parenchymatous region fell into the discrete DNA classes, 2C, 4C, and 8C. A significant number, however, had DNA contents above 8C but could not be grouped into classes based on a whole-number multiple of 2C. Nuclear size was found to vary in direct proportion to DNA content through 8C. Above 8C, correlation between nuclear size and DNA content was poor. The amount of DNA in the nuclei of the parenchymatous region was shown to increase in direct proportion to the distance of the nucleus from the meristem. The average amount of DNA in parenchymal nuclei above 8C was found to increase with the developmental stage of the embryo. Mechanisms which might account for the observed changes in DNA per nucleus are discussed.     

Genome size and base composition of five<Emphasis Type="Italic"> Pinus</Emphasis> species from the Balkan region

The 2C DNA content and base composition of five Pinus (2n=24) species and two Pinus subspecies from the Balkan region have been estimated by flow cytometry. P. heldreichii (five populations) and P. peuce (one population) were assessed for the first time, as also were subspecies of P. nigra (three populations—two of subspecies nigra and one of subspecies dalmatica) along with P. sylvestris, and P. mugo from the same region. The 2C DNA values of these Pinus ranged from 42.5 pg to 54.9 pg (41.7–53.8×10⁹ bp), and the base composition was quite stable (about 39.5% GC). Significant differences were observed between two subspecies of P. nigra and even between two populations of subsp. nigra. The two other species (P. sylvestris and P. mugo) had 2C values of 42.5 pg and 42.8 pg, respectively, while that of P. peuce was 54.9 pg. These genome sizes are in accordance with published values except for P. sylvestris, which was 20% below estimates made by other authors.Communicated by M. Beckert