DNA Content Of Nuclei Research Articles

Triploid Wild Rice (BKK) Strain Found in Bangkok Originated from Hybridizations among Three Parental <i>Oryza</i> Species

A wild rice (BKK) strain showing sterile spikelet and big leaves inhabited at the basin of the Chao Phraya river of Bangkok city, Thailand. The BKK strain was found as a natural triploid and thus its origin has been interested long time. Three different-sized fragments were amplified in RNA polymerase I largest subunit (PolA1) gene, which is a single-copy nuclear gene per haploid genome. Short type (0.14 kb) intron 20 sequence of BKK strain was identical to that of O. rufipogon and O. sativa. Phylogenetic analysis showed that long type (1.5 kb and 1.8 kb) intron 20 sequences of BKK strain were closely related to that of O. longistaminata and O. officinalis, respectively. We analyzed protein tag (Ptag) sequence encoded by exons 19 to 21 of PolA1 gene. Determined three Ptag sequences of BKK strain were identical to that of O. rufipogon, O. longistaminata, and O. officinalis, respectively. Relative DNA content of nuclei in O. officinalis and BKK strain was 1.5 and 1.75 times than that in O. sativa, respectively. And BKK strain contained CentO-C1 repeats, which were unique to O. officinalis. These results indicated that BKK strain comprised three genomes of O. rufipogon, O. longistaminata, and O. officinalis.

Уровень плоидности представителей семейства Chenopodiaceae, выявленный по размеру генома и числам хромосом

The article presents the results of measuring the genome size (DNA content in nuclei) by flow cytometry and determining the ploidy level for 30 species of the Chenopodiaceae family in 50 natural populations from Russia, Armenia, Belarus, Kazakhstan, Tajikistan and South Korea. Genome size of 12 species was determined for the first time; they are: Atriplex patens, A. pedunculata, A. sibirica, A. verrucifera, Axyris amaranthoides, Camphorosma songorica, Ceratocarpus arenarius, Chenopodium vachellii, Corispermum declinatum, Oxybasis gubanovii, Salsola collina, Spirobassia hirsuta. Along with flow cytometric analysis, ploidy level of 13 species (Atriplex patens, A. prostrata, Bassia prostrata, Chenopodium album, C. betaceum, C. frutescens, C. karoi, C. luteorubrum, C. novopokrovskianum, C. strictum s. l., C. vachellii, Krascheninnikovia ceratoides, and Oxybasis gubanovii) from 23 populations was determined by direct chromosome counting. Genome size of 11 species was studied in two or more populations. It was shown that differences in the genome size of samples in populations from different part of the area did not exceed 5 % in diploids (Atriplex sagittata, A. sibirica) and tetraploids (Atriplex patens, Chenopodium album, C. betaceum, C. novopokrovskianum, C. strictum and Krascheninnikovia ceratoides). Two cytotypes were identified in Bassia prostrata: diploid in the Republic of Altai and tetraploid in Novosibirsk Region. A tetraploid cytotype of Chenopodium sosnovskyi was revealed in Armenia. It was shown that the genome size can be a reliable criterion to determine the ploidy level in related taxa of the Chenopodiaceae.

B Chromosomes of the Asian Seabass (Lates calcarifer) Contribute to Genome Variations at the Level of Individuals and Populations.

The Asian seabass (Lates calcarifer) is a bony fish from the Latidae family, which is widely distributed in the tropical Indo-West Pacific region. The karyotype of the Asian seabass contains 24 pairs of A chromosomes and a variable number of AT- and GC-rich B chromosomes (Bchrs or Bs). Dot-like shaped and nucleolus-associated AT-rich Bs were microdissected and sequenced earlier. Here we analyzed DNA fragments from Bs to determine their repeat and gene contents using the Asian seabass genome as a reference. Fragments of 75 genes, including an 18S rRNA gene, were found in the Bs; repeats represented 2% of the Bchr assembly. The 18S rDNA of the standard genome and Bs were similar and enriched with fragments of transposable elements. A higher nuclei DNA content in the male gonad and somatic tissue, compared to the female gonad, was demonstrated by flow cytometry. This variation in DNA content could be associated with the intra-individual variation in the number of Bs. A comparison between the copy number variation among the B-related fragments from whole genome resequencing data of Asian seabass individuals identified similar profiles between those from the South-East Asian/Philippines and Indian region but not the Australian ones. Our results suggest that Bs might cause variations in the genome among the individuals and populations of Asian seabass. A personalized copy number approach for segmental duplication detection offers a suitable tool for population-level analysis across specimens with low coverage genome sequencing.

Influence of sex hormones on DNA content and cystathionine-γ-lyase expression in rat myocardium

Till now sexual features of cystathionine-γ-lyase/H 2 S system functioning in heart and its relationship to DNA content in myocardial cells nuclei depending on the level of sex hormones. The aim of this work was to evaluate the effect of sex hormones different levels on DNA fragmentation, indicators of cell cycle, cystathionine-γ-lyase expression and H 2 S content in heart in male and female rats. Materials and м ethods. The experiments were performed on 40 white laboratory rats of both sexes. Deficiency of sex hormones in the organism of rats was created by castration (ovariectomy and testectomy respectively to female and male rats) with surgical method. Estradiol and testosterone in blood plasma of the animals were determined by immunoenzyme method. DNA level in myocardial cells nuclei was determined by flow DNA cytometry. Cystathionine-γ-lyase gene expression was determined by real time polymerase chain reaction in (Real-time PCR). H 2 S content in heart was evaluated by spectrophotometry. Statistical processing of the obtained results was performed using standard methods by applying the software package MS Excel and Statistica SPSS 10.0 for Windows. Results . It has been found that in male rats expression of cystathionine-γ-lyase and H 2 S content in myocardium were significantly lower, by 46.9 and 16.1 % (p < 0.05) respectively, than in female rats. Along with this, males had greater activity of apoptosis, proliferation and polyploidization compared to females. Gonadectomy in males reduced expression of cystathionine-γ-lyase (47.0 %, p < 0.05), content of H 2 S in myocardium (by 22.2 %, p<0.05), activity of apoptosis, proliferation and polyploidization in heart, whereas castration of females caused opposite changes in relation to the control group. Gonadectomy in the animals changed the vector of sex differences in the studied parameters: cystathionine-γ-lyase expression and H 2 S content in myocardium of castrated males was significantly higher, whereas activity of apoptosis, proliferation, and polyploidization were less than in the relevant group of females. Conclusions. System of cystathionine-γ-lyase/H 2 S in myocardium is an important molecular target through which multidirectional sex hormones influence on DNA content in the nuclei of myocardial cells of rats is implemented.

Irises: A practical tool for image-based analysis of cellular DNA content.

The DNA content of nuclei is a valuable measure of cell cycle status. Irises is a software tool to facilitate systematic in situ determination of DNA content for cell cycle analysis at single-nucleus resolution within complex tissues. We demonstrate the utility of the tool with analysis of DNA content in germline nuclei of C. elegans. Compared with results obtained by manual analysis, we find the tool greatly facilitates analysis by improving speed at least 5-fold while maintaining accuracy. The source code and instruction manual (including installation for both Mac and PC) are provided.

Matrine Reduces Proliferation of Human Lung Cancer Cells by Inducing Apoptosis and Changing miRNA Expression Profiles

Matrine, a main active component extracted from dry roots of Sophora flavecens , has been reported to exert antitumor effects on A549 human non-small lung cancer cells, but its mechanisms of action remain unclear. To determine effects of matrine on proliferation of A549 cells and assess possible mechanisms, MTT assays were employed to detect cytotoxicity, along with o flow cytometric analysis of DNA content of nuclei of cells following staining with propidium iodide to analyze cell cycle distribution. Western blotting was performed to determined expression levels of Bax, Bcl-2, VEGF and HDAC1, while a microarray was used to assessed changes of miRNA profiles. In the MTT assay, matrine suppressed growth of human lung cancer cell A549 in a dose- and time- dependent manner at doses of 0.25-2.5 mg/ml for 24h, 48h or 72h. Matrine induced cell cycle arrest in G0/G1 phase and decreased the G2/M phase, while down-regulating the expression of Bcl2 protein, leading to a reduction in the Bcl-2/Bax ratio. In addition, matrine down regulated the expression level of VEGF and HDAC1 of A549 cells. Microarray analysis demonstrated that matrine altered the expression level of miRNAs compared with untreated control A549 cells. In conclusion, matrine could inhibit proliferation of A549 cells, providing useful information for understanding anticancer mechanisms.

Comparison of clinical staging of benign and malignant ovarian tissues with DNA flow cytometry

The prevention, diagnosis and treatment of ovarian cancer are major issues. The outcome of patients with advanced ovarian cancer is poor despite aggressive therapy including surgery, combination drug chemotherapy and radiation treatments. From the literature, the direct correlation between DNA ploidy and survival is greatly enhanced using high resolution DNA measurements, which results in a coefficient of variation (CV) range between 1%-2% (1.42 ± 0.19, n=66) for trout red blood cells (TRBC) and 2%-3% (2.18 ± 0.46 SD, n=22) for tonsil nuclei derived from formalin-fixed, paraffin-embedded tissues (deparaffinated). DNA nuclear determinations from 50 ovarian cancer and 21 benign patients is presented. This was accomplished by measuring the DNA content of nuclei simultaneously isolated from deparaffinated tissues, stained with the fluorescent DNA specific dye, 4’, 6-diamidino-1-phenylindole (DAPI) and analyzed on a high resolution flow cytometer. A high percentage of aneuploidy (92.0%) was determined from the ovarian cancer patients, especially of the aneuploid DNA histogram types, such as hypodiploid, multiploid and hypertetraploid (64.0%), which have shown poor prognosis in a variety of cancers including ovarian. Furthermore, aneuploidy was detected in 23.8% of the benign patients. DNA flow cytometry may complement pathological assessment of ovarian cancer to better determine malignancy, thus justifying a closer follow-up with more specialized approaches to treatment in clinical trials that involve new therapies including the use of chemically defined, natural products, which may help offset the overall poor prognosis seen in ovarian cancer.

Nuclear DNA content variation associated with muscle fiber hypertrophic growth in fishes

Muscle fiber hypertrophic growth can lead to an increase in the myonuclear domain (MND), leading to greater diffusion distances within the cytoplasmic volume that each nucleus services. We tested the hypothesis that hypertrophic growth in the white muscle of fishes was associated with increases in the mean DNA content of nuclei, which may be a strategy to offset increasing diffusion constraints. DAPI-stained chicken erythrocytes standards and image analysis were used to estimate nuclear DNA content in erythrocytes and muscle fibers from 17 fish species. Mean diploid (2C) values in fish erythrocytes ranged from 0.78 to 7.2pg. Erythrocyte 2C values were used to determine ploidy level in muscle tissue of small and large size classes of each species. Within each species, mean muscle fiber diameter was greater in the large size class than the small size class, and MND was significantly greater in larger fibers for 11 of the 17 species. Nuclear DNA content per species in muscle ranged from 2 to 64C. Fiber-size dependent increases in ploidy were observed in nine species, which is consistent with our hypothesis and indicates that endoreduplication is occurring during fiber growth. However, two species exhibited significantly lower ploidy in the larger size class, and the mechanistic basis and potential advantage of this ploidy shift is unclear. These results suggest that increases in ploidy may be a common mechanism to compensate for increases in MND associated with fiber hypertrophy in fishes, although it is likely that other factors also affect ploidy changes that occur in muscle during animal growth.

Loss of Krüppel-Like Factor 4 (KLF4) Leads to Increased Self-Renewal Under Stress Conditions and Improved Survival of Hematopoietic Stem Cells

Abstract 861Hematopoiesis is a highly regulated process in which a small number of hematopoietic stem cells (HSC) generate all mature blood cells. In order to preserve homeostasis of the hematopoietic system throughout lifetime, this pool of HSC must be maintained by the processes of self-renewal and survival. Self-renewal requires a coordination of survival signals and control of proliferation uncoupled from differentiation. Even though extrinsic signals from the microenvironment and cell-intrinsic regulators are required for self-renewal of HSCs, the intricate transcriptional machinery that selectively regulates HSC self-renewal and survival is still poorly understood. Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor that regulates proliferation, differentiation, apoptosis, and self-renewal. The role of KLF4 in reprogramming adult somatic cells into pluripotent stem cells along with OCT3/4, c-Myc and SOX2 suggests that KLF4 is required for preservation of an undifferentiated state. To investigate the function of KLF4 in HSC maintenance, we used conditional Klf4 knockout mice (Klf4fl/flVav-iCre+) to specifically delete KLF4 gene in hematopoietic cells. We first analyzed the frequency of HSC and progenitor cells in the bone marrow (BM) of Klf4fl/flVav-iCre– (control) and Klf4fl/flVav-iCre+ (knockout) by flow cytometry. We found that KLF4 deficiency leads to a 2.4-fold increase in the number of long-term HSC (Lin–Sca-1+c-Kit+ CD150+ CD48–) and a 2.2-fold increase in short-term HSC compartements (Lin–Sca-1+c-Kit+ CD150+ CD48+) whereas no significant changes were found in myeloid and lymphoid progenitor cells. Consistent with this phenotypic analysis, KLF4 expression in HSC is higher than hematopoietic progenitor cells and mature lineages (n=3; P<0.05). Even though ablation of Klf4 gene does not affect multi-lineage potential of HSC upon transplantation, its deletion leads to a reduction of monocytes and T cell expansion. To assess the effect of Klf4 ablation in self-renewal, we performed serial competitive repopulation assays using a 1:1 mixture of BM cells from control (Klf4fl/flVav-iCre–; CD45.2+) or knockout (Klf4fl/flVav-iCre+; CD45.2+) with B6.SJL (CD45.1+) mice. In primary transplants, the contribution of knockout BM cells in peripheral blood was similar to controls. Interestingly, loss of KLF4 led to enhanced contribution to peripheral blood in secondary transplants (4.5-fold; P<0.005) and tertiary transplants (2.6-fold; P<0.005). Consistent with this result, we found a significant increased number of colony forming units only in the third replating on methylcellulose (P<0.0005). To further characterize the role of KLF4 in HSC proliferation, we determined expression of Ki-67 and DNA content in nuclei of LSK CD150+ cells. The fraction of G0 cells defined as Ki-67– within 2n DNA in Klf4-knockout LSK CD150+ cells was similar to control (control 74.3 ± 0.7% vs 73.2 ± 2.3%). However, Annexin V staining revealed a 2.4-fold increased survival of LSK CD150+ cells in Klf4-knockout mice compared to control mice but not in myeloid progenitor cells (Lin–c-Kit+Sca-1–) suggesting that KLF4 selectively regulates the survival of HSC. These studies indicate that KLF4 controls steady state HSC survival and self-renewal under stress conditions. Disclosures:No relevant conflicts of interest to declare.

Hormonal control of endoreduplication in sugar beet (Beta vulgaris L.) seedlings growing in vitro

The effect on endoreduplication in sugar beet (Beta vulgaris L.) seedlings of five plant hormones in MS medium, ethylene, 24-epibrassinolide (EBR), gibberellic acid (GA(3) ), kinetin and 1-naphthaleneacetic acid (NAA), as well as a combination of kinetin and NAA at two different concentrations, was studied using flow cytometry. Analyses of DNA content in nuclei of the root, hypocotyl and cotyledons of seedlings growing in vitro were performed during their early development, starting from when the root was 0.5-1.0 cm long until expansion of the first pair of leaves. The proportions of nuclei with different DNA contents were established and the mean C-value calculated. The presence of exogenous plant hormones changed endoreduplication intensity, although to different extents, depending on the organ and developmental stage. Ethylene and NAA stimulated the process, while EBR and kinetin suppressed it and GA did not clearly affect it.

Flow cytometric analysis of somatic embryos, shoots, and calli of the cactus Copiapoa tenuissima Ritt. forma monstruosa

Flow cytometry was used for analyzing DNA contents of nuclei isolated from in vitro grown somatic embryos, shoots and calli, as well as mammillae of in vivo grown shoots of cactus, Copiapoa tenuissima Ritt. forma monstruosa. Endoreduplication was detected in both in vitro grown somatic embryos, shoots and calli and in mammillae derived from in vivo grown shoots. However, the lowest ploidy levels ranged from 2C to 4C in somatic embryos, and reaching up to 32C for in vitro grown shoots and calli from mammillae. Whereas, ploidy levels of in vivo-derived mammillae ranged from 2C to 16C. The presence of 2,4-dichlorophenoxyacetic acid (2,4-D) in the culture medium had no influence on levels of ploidy of regenerated tissues. The mean genome size of cactus was calculated as 2C = 2.87 ± 0.05 picogram (pg).

Genotypic variation and aging effects on the embryogenic capability of Kalopanax septemlobus

In an attempt to obtain insight into the differential responsiveness of different genotypes of Kalopanax septemlobus regarding their embryogenic capacity, several parameters such as endogenous hormonal levels, DNA content, embryogenic callus proliferation and somatic embryogenesis were studied in several genotypes of this plant. Also, to understand the effect of the age of the explants on their embryogenic capacity, the same parameters were studied in two embryogenic cell lines of different ages in the selected genotype. In the present study, it was observed that the cytokinins/abscisic acid (ABA) ratio plays an important role in embryogenic capacity in the studied genotypes of K. septemlobus species. A decrease in embryogenic capacity of callus was observed with increasing age, along with a marginal decrease in the DNA content of nuclei. Further, it can be suggested from our results that young embryogenic callus is a better choice for somatic embryo formation than the long-term-maintained callus in K. septemlobus.

Cytological study of a fern Cystopteris fragilis in Mongolian Altai

Cystopteris fragilis collected in Mongolian Altai showed three cytotypes with chromosome numbers of tetraploid (2n=c.168), pentaploid (2n=c.210) and hexaploid (2n=c.252). Flow cytometry analysis also showed three different peaks of DNA contents of nuclei in fronds. Meiotic metaphase chromosomes of the spore mother cells in the tetraploid and hexaploid C. fragilis showed all bivalents with c.84II and c.126II, while those of the pentaploid C. fragilis showed many univalents and bivalents that produced mostly sterile spores. The pentaploid C. fragilis was suggested to be a hybrid between the tetraploid and the hexaploid C. fragilis.

Effects of aqueous root extracts and hydrophobic root exudates of cucumber (Cucumis sativus L.) on nuclei DNA content and expression of cell cycle-related genes in cucumber radicles

Allelopathic interactions implicate the inhibition of cell division by allelochemicals. To examine the effects of autotoxic agents on cell cycle and plant growth, germinated cucumber seeds (Cucumis sativus L.) were incubated in solutions containing the aqueous root extracts of cucumber at 1:100, 1:50, 1:25 and 1:10 (w:v), or the hydrophobic root exudates of cucumber at 25, 50 and 100 mg·L−1. Aqueous root extracts and hydrophobic root exudates inhibited radicle elongation by 36.47–60.18% and 38.24–62.50%, respectively. The mitosis-specific genes were down-regulated in roots exposed to aqueous root extracts and hydrophobic root exudates. Meanwhile, exposure to either aqueous root extracts or hydrophobic root exudates decreased the proportion of 2C (C-value) and increased the proportion of 8C, leading to an increased mean C-value. We conclude that autotoxic agent-induced inhibition of radicle growth was partly attributed to the down-regulation of cell cycle-related genes and endoreduplication was enhanced under our experimental condition.

Androgenic response of genotypes selected from Capsicum annuum L. × C. chinense Jacq. hybrids

In research on androhaploids in the progeny of interspecific hybrids within the Capsicum genus, three genetically stable lines of F7 generation, selected from the C. annuum L. × C. chinense Jacq. hybrid, were used. In the first line, only callus tissue was formed as a reaction to the conditions of culture. Cytometric analysis of this tissue revealed the presence of cells with DNA content in nuclei at the level of 1C to 16C. The tissue was mixoploid and non-embryogenic. Anthers of the other line did not respond. In the third one, nine embryos were obtained, and they developed into plants. By means of cytometric analysis, the 1C DNA level was found in eight of them and these were androgenic plants. The origin of one of the diploid plant was not established due to the homozygous character of the donor plants. The experiment results confirm the already known diversity of genotype reaction to the conditions of culture. It moreover point to the possibility of selection of the forms with an androgenic potency from interspecific hybrids.

Multiparametric study and cluster analysis of transplantable human cell cultures

Using the methods of scanning cytophotometry, cytochemistry, and cytomorphometry, cells of hepatocarcinoma (HEp-G2), an adenocarcinoma of the large intestine (Caco-2), an embryonic kidney (HEK-293), a neuroblastoma (SH-SY5Y), a rabdomyosarcoma (RD), and larynx cancer (HEp-2) were studied 48, 72, and 96 h after the beginning of cultivation. The density of the monolayer, proliferative activity, the number of dead cells, the DNA content in nuclei and distribution of the cells in the population for this parameter, the total DNA content in nucleoli (in the perinucleolar chromatin), the number of nucleoli in nuclei, the distribution of cells by their number, the volume and area of the nuclear surface, and the total volume and area of the nucleolar surface were determined. The obtained data were used for a treelike cluster analysis of the cultures by the Pierson correlation. As a result of the analysis, the SH-SY5Y culture was separated into an individual cluster, while Caco-2, HEp-G2, HEK 293, HEp-2, and RD cultures were located in the tree of another cluster. The significance (weight) of parameters in the formation of a general pattern of cell cultures is not equal. It also turned out that the least transformed culture of neuroblastoma (SH-SY5Y) had no relationship with other cultures that show various degrees of similarity to one another. The cultures HEK 293, HEp-2, and RD were found to be close to one another in all parameters. Parameters had different significances in the formation of the general pattern of cell cultures. The parameters that characterized the population as a whole were of the greatest significance and included the following: density of monolayer, mitotic coefficient, and the number of dead cells. Additionally, the nuclear DNA content, the total area of the nucleolar surface, and the ratio of the nucleolar to nuclear DNA and of the total nucleolar to the nuclear areas are also of great importance. Other parameters were less significant.

Apomixis in Eulaliopsis binata: characterization of reproductive mode and endosperm development

Apomixis represents an alteration of classical sexual plant reproduction to produce seeds with essentially clonal embryos, stimulating wide interest from biologists and plant breeders for its ability to fix heterosis. Eulaliopsis binata (Poaceae), is identified here as a new apomictic species. Embryological investigation indicates that the developmental pattern of embryo sac formation in E. binata represents gametophytic apospory, the embryo originating from an unreduced cell, without fertilization and the mode of endosperm development was autonomous. Sexual embryo sacs were found with a frequency of 1–4% depending on the biotype. The DNA content of nuclei (C-value) in mature seeds was screened by flow cytometry (FCSS) and demonstrated that the endosperm was derived autonomously without fertilization and the three biotypes of E. binata showed varying degrees of apomixis. The Wide-leaf type showed obligate apomixis whereas the Slender-leaf and the Red-haulm type displayed facultative apomixis. In addition, adventitious embryos were observed on the wall of ovary, integument and nucellus cells, indicating that E. binata produces embryos via a mixture of apospory and adventitious embryony.

Detection of tumor cells in body cavity fluids by flow cytometric and immunocytochemical analysis

Measurement of electronic volume versus DNA content of nuclei can be used to discriminate between normal and malignant cells. Epithelial membrane antigen immunocytochemistry (EMA-ICC), a helpful ancillary test in body cavity fluids, is not universally accurate for detecting malignancy in effusions. The current study was undertaken to determine if multiparametric flow cytometry (based on simultaneous analysis of light scatter, nuclear volume, DNA, and nuclear protein content) in combination with (EMA-ICC) could be used for the detection of malignant cells in peritoneal and pleural fluids. We studied 130 body cavity fluids (68 peritoneal and 62 pleural fluids) by conventional cytology and multiparametric laser flow cytometry. EMA-ICC was performed using EMA antibodies and L-SAB detection system (DakoCytomation, Carpinteria, CA). EMA-ICC had significantly higher sensitivity than conventional cytology (79% versus 59%, P = 0.016) and ploidy (79% versus 38%, P = 0.001). Cytology had significantly higher specificity than ploidy (97% versus 82%, P = 0.012). The differences in specificity between EMA-ICC and ploidy (87% versus 82%, P= 0.607) or EMA-ICC and cytology (87% versus 97%, P = 0.109) were not statistically significant. However, assuming serial testing, sensitivity increased significantly for the combinations of cytology and EMA-ICC (79.4%, P = 0.016) and cytology and ploidy (73.5%, P = 0.004) as compared to cytology alone (58.8%). Also, the combination of cytology and ploidy had a higher sensitivity than ploidy alone (73% versus 38%, P < 0.0001). However, the sensitivity associated with the three tests used in serial (85.3%) was not significantly different from the sensitivities corresponding to the combination of cytology and EMA-ICC (79%) or cytology and ploidy (73%). Multiparametric flow cytometry utilizing high resolution DNA, nuclear volume, protein measurement, and ICC, in combination with cytomorphology, may be a valuable tool for rapid identification of malignant cells in body cavity fluids.

When Hymenopteran Males Reinvented Diploidy

In most plants and animals, a consistent relationship exists between the DNA content of a cell and its metabolic activity. The male-haploid sex determination of Hymenoptera and other arthropods may therefore impose a particular selective pressure upon males, which must evolve adaptations to cope with a genomic DNA reduced by half compared with that of females. Here, we show that a nuclear DNA content similar to that of females is restored in muscles of males in all hymenopteran lineages tested except the most basal one (Xyelidae). This doubling of DNA content in males does not occur in other haplodiploid insects, such as thrips (Thysanoptera) and whiteflies (Sternorrhyncha). These results indicate that this adaptation probably occurred early in hymenopteran history, possibly because males acquired strong flying and dispersal abilities.

CROSS-COMPATIBILITY IN INTERSPECIFIC AND INTERGENERIC HYBRIDIZATION AMONG THE COLCHICACEOUS ORNAMENTALS, GLORIOSA SPP., LITTONIA MODESTA AND SANDERSONIA AURANTIACA

Interspecific and intergeneric cross-pollination and subsequent ovule culture were carried out among 6 genotypes of Gloriosa spp., 1 genotype of L. modesta and 2 genotypes of S. aurantiaca in order to widen their variations and to develop novel cultivars. Ovules with placental tissues prepared from ovaries 14 days after pollination were cultured at 25°C in the dark on a medium containing 0.01 mg l -1 α α α α -naphthaleneacetic acid (NAA) and 0.01 mg l -1 benzyladenine (BA), on which rhizome-like structures were produced from the ovules in 49 out of 61 cross-combinations. Following transfer to a medium containing 0.25 mg l -1 NAA and 2.5 mg l -1 BA under a 16-h photoperiod, some of the rhizome-like structures produced shoots and/or root-like structures. These shoots developed into plantlets and produced tubers on a plant growth regulator-free medium. In some of the plantlets derived from intergeneric cross-pollination, early confirmation of the hybridity was accomplished by flow cytometry analysis of relative DNA content of nuclei.