Analysis Of Nuclear DNA Content Research Articles

Multi-scale tissue fluorescence mapping with fiber optic ultraviolet excitation and generative modeling

Cellular imaging of thick samples requires physical sectioning or laser scanning microscopy, which can be restrictive, involved, and generally incompatible with high-throughput requirements. We developed fiber optic microscopy with ultraviolet (UV) surface excitation (FUSE), a portable and quantitative fluorescence imaging platform for thick tissue that enabled quick sub-cellular imaging without thin sections. We substantially advanced prior UV excitation approaches with illumination engineering and computational methods. Optical fibers delivered <300nm light with directional control, enabling unprecedented 50× widefield imaging on thick tissue with sub-nuclear clarity, and 3D topography of surface microstructure. Probabilistic modeling of high-magnification images using our normalizing flow architecture FUSE-Flow (made freely available as open-source software) enhanced low-magnification imaging with measurable localized uncertainty via variational inference. Comprehensive validation comprised multi-scale fluorescence histology compared with standard H&E histology, and quantitative analyses of senescence, antibiotic toxicity, and nuclear DNA content in tissue models via efficient sampling of thick slices from entire murine organs up to 0.4×8×12mm and 1.3 million cells per surface. This technology addresses long-standing laboratory gaps in high-throughput studies for rapid cellular insights.

Phenotypic Evaluation and Nuclear DNA Content Analysis of Oryzalin-Induced Echeveria Mutant Cultivars

Phenotypic Evaluation and Nuclear DNA Content Analysis of Oryzalin-Induced Echeveria Mutant Cultivars

Characterization of nuclear DNA content and chromosome numbers of Tulipa luanica Millaku, T. kosovarica Kit Tan, Shuka & Krasniqi and T. albanica Kit Tan & Shuka

The Balkan Peninsula is considered an important centre of native tulip species. Tulipa kosovarica and Tulipa luanica are new species recently discovered in Kosovo, and Tulipa albanica in Albania. The current study aims at the investigating the nuclear DNA content and chromosome number of these three tulipa species in order to provide for the first time data on their genome size and differences among these three Tulipa species. Analysis of nuclear DNA content was performed by flow cytometer (Partec CyFlow Space) in mature fresh leaves for each Tulipa species. Samples for chromosome analysis were taken from the root tip meristem of the bulbs. Results showed significantly higher amounts of nuclear DNA (2C) in T. luanica compared to T. kosovarica and T. albanica. The chromosome number for these three species was 2n = 2x = 24, while the chromosome sizes of T. luanica resulted larger, compared to that of T. kosovarica and T. albanica. A correlation between the nuclear DNA content and chromosome size was found among these tulipa species. Moreover, nuclear DNA content and chromosome sizes of T. luanica, T. kosovarica and T. albanica showed clear differences among these species.

Female Reproductive Potential and 3x by 2x Breeding Approach for Hybrid Seeds Development in Sukali Ndizi Genotype (Musa genome AAB)

Sukali Ndizi is one of the most popular local desert banana cultivars in Uganda. The crop is affected by a number of diseases and pests. Genetic improvement of the crop by hybridization may be hindered by very low seed set and poor seed germination. The main characteristics for assessing the female reproductive potential (seed set, seed quality and embryo germination of hybrid seeds) and 3x by 2x breeding approach were assessed in crosses of eleven different male diploid parents either wild or improved diploids with Sukali Ndizi landrace as the female parent for a period of 5 consecutive years planted in pollination blocks at National Agricultural Research Laboratories Kawanda (NARL). The month of pollination did not show any pattern throughout the pollination period but the crosses showed considerable variability in seed set, seed quality, and seed germination for different male diploids used. The water gravity test which differentiated the extracted seeds as sunken/viable and floating/nonviable seeds showed that a substantial amount of seed was floating and the sunken externally characterized by black hard integuments was only 39% with a range = 24%–60% which contained embryos, of which 22% (range = 0–37%) germinated. The increase in non-viable seed suggested that ovule abortion in Sukali Ndizi which mainly involved embryo and endosperm abortions was the cause of limited seed germination and the paternity of the zygotes was a major factor underlying abortions. Flow cytometric analysis of nuclear DNA content was used to estimate ploidy levels of the progenies with results showing that using 3x by 2x breeding approach differing levels of ploidy were achieved [3x (288), 4x (61), 5x (14) and 2x (2)] signifying the predominance of 3x progenies and the presence of 5x and 4x conforming to the possibility of sexual polyploids. Although pollination of Sukali Ndizi can be done all around the year, the seed set both quantity and quality and germination were very poor, necessitating the development of an efficient regeneration protocol for zygotic embryos at varying maturity stages to increase the recovery of hybrids.

Nuclear DNA Content Estimation of Seaweed by Fluorimetry Analysis.

Fluorimetry analysis of nuclear DNA content allows identification of genome size and ploidy levels of different life phases, tissues, and populations in seaweed species. It is an easy method that saves time and resources compared to more complex techniques. Here we describe the methodology for measuring nuclear DNA content in seaweed species by DAPI fluorochrome staining and its comparison with the standard Gallus gallus erythrocytes nuclear content, one of the preferred internal standards. With this methodology, up to a thousand nuclei can be measured in a single staining session, allowing for a quick analysis of the studied species.

Analysis of Nuclear DNA Content and Karyotype of Phaseolus vulgaris L.

The common bean (Phaseolus vulgaris L.), whose annual production is 26 million tons worldwide, is one of the main sources of protein and is known as one of the most important food sources. In this study, the karyotype variations and the genome size of four common bean genotypes in Turkey were investigated to determine whether the geographic variables in these regions affected the genome size and the karyotype parameters. In addition, it is known that as that the cytological and chromosomal parameters change under the influence of the climatic conditions of each region, appropriate and stable cytological methods for each plant facilitate and enable the determination of the chromosomal structure and the identification of specific chromosomes in the genotypes of the relevant region. Correct and valuable information such as this enables breeders and researchers to determine the correct shape and actual size of chromosomes. The genome size of the genotypes was measured with a flow cytometer, and chromosome analyses were performed with the squash method. For each genotype, the karyotype parameters, such as the number of somatic chromosomes, the Mean Total Chromosome Length (MTCL), the Mean Centromere Index (MCI), and the Mean Arm Ratio (MAR), were measured. The results showed that the highest and the lowest amounts of DNA per nucleus (3.28 pg and 1.49 pg) were observed in the Bitlis and Elaziğ genotypes. In addition, all genotype chromosome numbers were counted to be 2n = 2x = 22. The Mean Total Chromosome Length varied from 15.65 µm in Elaziğ to 34.24 µm in the Bitlis genotype. The Mean Chromosome Length ranged between 1.42 µm and 3.11 µm in the Elaziğ and Bitlis genotypes. The Hakkari and Van genotypes consist of eleven metacentric chromosomes, while the Bitlis and Elaziğ genotypes consist of ten metacentric chromosomes and one sub-metacentric chromosome. However, the Mean Centromere Index and Arm Ratio differed considerably among the genotypes. The highest (46.88) and the lowest (43.18) values of the Mean Centromere Index were observed in the Hakkari and Elaziğ genotypes, respectively. On the other hand, the lowest (1.15) and the highest (1.36) values of the Mean Arm Ratio were obtained in the Bitlis and Elaziğ genotypes, respectively. Eventually, intraspecies variations in genome size and chromosomal parameters were observed, and it was determined that the changes in nuclear DNA content and different chromosomal parameters among the four Phaseolus genotypes from four different regions of Turkey indicate the effect of climate change in the regions on these parameters. Such information in these areas can be used as useful information for the improvement of this plant and breeding programs.

Establishment of an Efficient In Vitro Propagation of Cnidium officinale Makino and Selection of Superior Clones through Flow Cytometric Assessment of DNA Content.

Cnidium officinale is a valuable medicinal plant cultivated in Asia for its rhizomes. This study reports the in vitro regeneration of Cnidium officinale plants and the induction of rhizomes from microshoots. The rhizomatous buds of Cnidium officinale induced multiple shoots on Murashige and Skoog (MS) medium supplemented with 0.5 mg L−1 BA, which led to the regeneration of plants within four weeks of culture. After four weeks of culture, the plants were assessed for fresh weight, the number of leaves, the number of roots, and the length of roots to compare the performance of the different clones. The clones with good growth characteristics were selected with the aid of a flow cytometric analysis of 2C nuclear DNA content. The plants bearing high DNA values showed better growth characteristics. Various factors, namely, sucrose concentration (30, 50, 70, and 90 g L−1), ABA (0, 0.5, 1.0, and 2.0 mg L−1), the synergistic effects of BA (1.0 mg L−1) + NAA (0.5 mg L−1) and BA (1.0 mg L−1) + NAA (0.5 mg L−1) + ABA (1.0 mg L−1) with or without activated charcoal (1 g L−1), and light and dark incubation were tested on rhizome formation from microshoots. The results of the above experiments suggest that MS medium supplemented with 50 g L−1 sucrose, 1.0 mg L−1 ABA, and 1 g L−1 AC is good for the induction of rhizomes from the shoots of Cnidium officinale. Plantlets with rhizomes were successfully transferred to pots, and they showed 100% survival.

Susceptibility of Hepatic ProgenitoInhibition by acetazolamide of reactivated carbonic anhydrase activity in a receptor protein tyrosine phosphatase γ (RPTPγ) mutantr Cells to Lipotoxicity as Contributor for Impaired Liver Regeneration and Disease Advancement In Pediatric Nonalcoholic Fatty Liver Disease (PNAFLD)

BackgroundIn PNAFLD mechanisms underlying early onset and rapid disease progression have been unclear. Active liver growth in preadolescent children imparted by facultative progenitor cells would increase genome damage following oxidative stress and lipotoxicity, leading to DDR and further hepatic injury events. To reveal this we considered hTERT‐FH‐B hepatocytes expressing progenitor properties will be ideal for modeling growth‐related lipotoxicity and DDR of PNAFLD.Methods/ResultsThe FH‐B cells were derived from late‐gestation liver with doubling time of 24h, including 20‐30% in S phase, which was similar to pH3 histone +ve proliferating liver cell fractions in 5‐8‐yr old healthy children. Moreover, FH‐B cells express hepatobiliary markers (G6P, Glycogen, GGT, DPPIV) along with progenitor markers (SOX9, FOXA2, Cx43, EpCAM) and P450 profiles indicating hepatic immaturity. To induce lipotoxicity in cultured cells 50 μM palmitic acid (PA) was added with and without oxidized collagen matrix or 2,5‐10 ng/ml TNFa. This dose‐dependently caused oxidative stress (dihydrorhodamine dye for ROS) and lower mitochondrial membrane potential (MMP, JC‐1 dye) in 1‐4h and onset of steatosis (Oil red O or Bodipy staining) and decreased cell viability (MTT assay) in 18‐24h. The contribution of ATM signaling during lipotoxicity was apparent from pNBS1 accumulation, which marks DNA strand break sites. Moreover, lipotoxicity and DNA damage greatly increased in presence of ATM‐specific kinase inhibitor, KU‐60019. Additionally, ATM insufficiency exacerbated cell growth‐arrest through downstream p53‐dependent p21 activation, which increased polyploidy indicated by Hoechst dye‐stained nuclear DNA content analysis, and inhibited cell number increases in growth assays over few days. To test therapeutic feasibility, we studied obeticholic acid (OCA) and found this drug decreased ATM insufficiency‐related exacerbation of lipotoxicity. The improvements included steatosis, ROS, MMP. DDR, cell viability, as well as less growth‐arrest or polyploid accumulations and continued cycling.ConclusionsThe successful modeling of PNAFLD in hTERT‐FH‐B cells establishes the critical susceptibility of actively growing facultative progenitor hepatocytes to mitochondrial dysfunction and impaired DDR related to ATM insufficiency. The potential of OBC in improving lipotoxic outcomes will be relevant for PNAFLD. These pathophysiology‐based models offer opportunities to advance therapy in PNAFLD.

Inoculation with Bacillus amyloliquefaciens and mycorrhiza confers tolerance to drought stress and improve seed yield and quality of soybean plant.

The present study aimed to evaluate the effect of Bacillus amyloliquefaciens and/or Arbuscular Mycorrhizal Fungi (AMF) as natural biofertilizers on biomass, yield, and seed nutritive quality of soybean (Giza 111). The conditions investigated include a well-watered (WW) control and irrigation withholding at the seed development stage (R5, after 90 days from sowing) (DS). Co-inoculation with B. amyloliquefaciens and AMF, resulted in the highest plant biomass and yield under WW and DS conditions. The nuclear DNA content analysis suggested that co-inoculation with B. amyloliquefaciens and AMF decreased the inhibition of drought stress on both the size and granularity of seed cells, which were comparable to the normal level. The single or co-inoculation with B. amyloliquefaciens and AMF increased the primary metabolites content and alleviated the drought-induced reduction in soluble sugars, lipids, protein and oil contents. Plant inoculation induced the expression of genes involved in lipid and protein biosynthesis, whereas an opposite trend was observed for genes involved in lipid and protein degradation, supporting the observed increase in lipid and protein content. Plant inoculated with B. amyloliquefaciens showed the highest α-amylase and β-amylase activities, indicating improved osmolyte (soluble sugar) synthesis, particularly under drought. Interestingly, single or co-inoculation further strengthen the positive effect of drought on the antioxidant and osmoprotectant levels, i.e. phenol, flavonoid, glycine betaine contents, and glutathione-S-transferase (GST) activity. As a result of stress release, there was a decrease in the level of stress hormones (abscisic acid, ABA) and an increase in gibberellin (GA), trans-zeatin-riboside (ZR), and indole acetic acid (IAA) in the seeds of inoculated plants. Additionally, the ATP content, hydrolytic activities of plasma membrane H+ -ATPase, Ca2+ -ATPase, and Mg2+ -ATPase were also increased by the inoculation.

Polystichum setiferum at the Northeastern Limit of Its Distribution Range

Abstract During field studies in 2019 in the Beskid Śląski mountain range in southern Poland, one individual morphologically resembling Polystichum setiferum was found. Nuclear DNA content analysis confirmed the species identity. The new stand of P. setiferum corresponds to historical literature reports and herbarium specimens from the Czech Republic and Poland, and extends the northeastern limit of the distribution of this species. As the historical stands in both countries have not been confirmed, the newly discovered stand is at present strongly isolated, as the nearest extant locations of P. setiferum in Austria, southern Hungary, and western Romania are nearly 500 km away. Therefore, this unique stand deserves special attention and protection.

Quantitative nuclear DNA content and cell cycle analysis of a mixotrophic dinoflagellate by image cytometry

AbstractThe goal of this work was to develop and demonstrate the utility of microscope‐based image cytometry (ICM) as a method for quantifying nuclear DNA content and cell cycle phase distribution in microalgae both in culture and in natural blooms, as an alternative to flow cytometry (FCM). To do so, aliquots from the same samples of the dinoflagellate Karlodinium veneficum were examined using both ICM and FCM. ICM specimen preparation and data acquisition methods were optimized to improve precision and agreement between the two techniques. Accuracy and precision of DNA measurements by ICM were significantly higher using the DNA fluorophore DAPI compared to SYBR® Green I. Milli‐Q H2O was found to be superior to Tris‐EDTA as a staining and slide preparation solution for ICM analyses. Lower‐powered objective magnification (10x, 20x) in image acquisition for ICM produced higher precision in nuclear DNA measurements. Overall precision of ICM analysis of DAPI‐stained K. veneficum cells was comparable to FCM, with respective 1C DNA peak coefficients of variation as low as 6.2%. Cell cycle distributions of mid‐log culture samples analyzed by both ICM and FCM were in agreement (two‐way ANOVA; p = 0.93); while distributions analyzed in a field sample were similar but not identical (Z‐test; p < 0.001). Overall, the results show the feasibility of ICM as a useful tool for microalgal cell cycle analysis, with the potential for more flexible application to mixotrophic/phagotrophic species and complex field populations.

Induction and evaluation of tetraploid plants of Eucalyptus urophylla clones

Eucalyptus urophylla is an important species of the genus Eucalyptus that is used across the world, especially in Brazil. It is used for pulp and paper production, and charcoal production for iron and steel industry, thus making the development of biotechnological approaches essential for the breeding programs of this species. The polyploidy induction is one such approach that may introduce phenotypic characteristics of interest to the market and industry. This is the first study that shows the production, evaluation, and development of tetraploids (4x = 44) from a clone of E. urophylla. For this, the shoot apical meristems were cultured for elongation using in vitro conditions for 15 with 5 or 10 µM colchicine, trifluralin, or oryzalin. Following this, every 30 days, the elongated plantlets were transferred to JADS medium for multiplication using tufts as explants. Thereafter, four tufts containing six to eight buds each were inoculated in flasks followed by six subcultures and were then transferred to the greenhouse for acclimatization. The experiment was performed in a completely randomized design in a factorial scheme (3x2+1: antimitotic substance x concentrations + control), with 12 replicates. Analysis of nuclear DNA content and chromosome counting by flow cytometry, photosynthetic pigment quantification, stomatal size and density, survival, and rooting were performed for 110 days after acclimatization. Data were submitted for analysis of variance followed by Scott & Knott’s test (P ≤ 0.05). As a result, we observed that the oryzalin and trifluralin treatments were efficient in the induction of tetraploid plants, while colchicine proved to be ineffective. Also, trifluralin treatment at 5 µM provided a higher polyploidy induction (18.2% tetraploids) in comparison to the others. Diploid plants displayed 1.41 pg of nuclear DNA content with 22 chromosomes, while tetraploid plants showed 2.86 pg with 44 chromosomes. In conclusion, the tetraploid plants, as compared to the diploids (control), were found to show higher chlorophyll content, larger but fewer stomata, and lower rooting and survival, which can be mitigated nonetheless through silvicultural techniques. This study shall bring new perspectives for the breeding programs of E. urophylla

Arabidopsis nucleoporin CPR5 controls trichome cell death through the core cell cycle regulator CKI.

The Arabidopsis trichome is a polyploid epidermal cell resulting from multiple rounds of endocycles. The CYCLIN-DEPENDENT KINASE INHIBITOR (CKI) family proteins are core cell cycle regulators that promote the endocycle. CONSTITUTIVE EXPRESSION OF PR GENES 5 (CPR5) is a plant-specific nucleoporin. It has been found that two Arabidopsis CKI, SIAMESE (SIM) and SIAMESE-RELATED 1 (SMR1), function downstream of CPR5 to activate plant effector-triggered cell death. The sim smr1 double mutants form multicellular and clustered trichomes, while the cpr5 mutants produce dead and branchless trichomes. This study explored roles of the CPR5-CKI signalling pathway in trichome cell cycle transition. To examine the underlying mechanism of how cell cycle transition is regulated in plant trichomes, Trypan blue staining, flow cytometry, scanning electron microscopy (SEM) and nuclear DNA measurement were conducted. The native promoter-driven CKI and GUS fusion reporter showed that both SIM and SMR1 proteins were preferentially expressed in trichomes. The cpr5-induced dead and branchless trichomes were fully suppressed by the sim smr1 double mutant, suggesting that SIM and SMR1 function downstream of CPR5 in trichome development. Flow cytometry analysis showed that as compared to the number of 2C (C=DNA content in a haploid nucleus) cells, the number of 4C cells significantly increased, whereas that of polyploidy cells (8C and 16C) dramatically decreased in the cpr5 mutant. The elevated 4C/2C ratio in the cpr5 mutant is consistent with de-repression of pro-endocycle regulators SIM and SMR1. The polyploidy cells (8C and 16C) may be selectively targeted to cell death, which is therefore attributed to the branchless trichomes in the cpr5 mutant. Nuclear DNA content analysis demonstrated that the nuclear DNA content of trichomes in the cpr5 sim mutant was significantly higher than in the sim mutant, indicating that CPR5 is a negative endocycle regulator in trichomes. This study reveals that the CPR5-CKI signalling pathway controls trichome cell cycle transition and excessive endocycles are required for cell death in plant trichomes.

Morphological and Cytological Comparisons of Eight Varieties of Trailing Lantana (Lantana montevidensis) Grown in Florida

Trailing lantana (Lantana montevidensis) is a popular low-growing ornamental plant valued for its heat and drought tolerance and continuous purple or white flowering throughout much of the year. Recently, trailing lantana was predicted to be invasive by the University of Florida/Institute of Food and Agricultural Sciences (UF-IFAS) Assessment of Non-Native Plants in Florida, and therefore not recommended for use. All cultivars fall under this designation unless proven otherwise. Eight trailing lantana varieties were obtained from wholesale growers and naturalized populations found in Texas and Australia. Plants were propagated vegetatively, finished in 4-inch pots, and planted under field conditions to determine morphological and cytological differences among varieties. Australian trailing lantana differed morphologically from the other varieties in its smaller habit, leaves (which had serrate-crenate leaf margins, and fewer appressed hairs), heavy fruiting, and cold sensitivity (observational reduced growth and flowering during winter months). Nuclear DNA content analysis suggests that Australian trailing lantana is likely a tetraploid and all other varieties evaluated were likely triploids with high levels of sterility. Pollen stainability of Australian trailing lantana was moderately high (58.83%), whereas pollen production was rarely observed in all other varieties. Results support that there are two forms of trailing lantana, the U.S. varieties distinguished by their leaf and flower morphology, ploidy level, and the absence of fruit and viable pollen.

Analysis of nuclear DNA content and ploidy levels in 3x × 4x Lilium (Lilium × elegans L.) progenies by flow cytometry

Interploidy crosses between Lilium lancifolium (3x) and Asiatic lily cultivar ‘Brunello’ (4x) were attempted for creating genetic variability and to analyse the progenies for different ploidy levels. Experimental results revealed that most of the crosses attempted were developed into fruits, confirming that male-sterile triploid lilies can be used as the female parent for crossing with a suitable male parent. Wide variation in chromosome numbers (28 to 38) was obtained in different plant progenies, indicating that aneuploidy is generated by 3x × 4x crosses. The nuclear DNA content analysis of 13 plant progenies showed that the 2C nuclear DNA content has increased (range = 32.60 pg to 41.32 pg) as compared to Lilium lancifolium, while it was found lower than the cultivar ‘Brunello. Further, morphological characterization of different plant progenies revealed significant differences among themselves, which confirmed the dependence of these traits on cultivars ploidy level. Therefore, present findings will be instrumental for development of new Lilium cultivars with high aesthetic value and utility.

Resynthesis of <i>Brassica juncea</i> for resistance to <i>Plasmodiophora brassicae</i> pathotype 3

The oilseed crop Brassica juncea carries many desirable traits; however, resistance to clubroot disease, caused by Plasmodiophora brassicae, is not available in this species. We are the first to report the clubroot resistant resynthesized B. juncea lines, developed through interspecific crosses between a clubroot resistant B. rapa ssp. rapifera and two susceptible B. nigra lines, and the stability of the resistance in self-pollinated generations. The interspecific nature of the resynthesized B. juncea plants was confirmed by using A- and B-genome specific SSR markers, and flow cytometric analysis of nuclear DNA content. Self-pollinated progeny (S1 and S2) of the resynthesized B. juncea plants were evaluated for resistance to P. brassicae pathotype 3. The S1 and S2 progenies of one of the resynthesized B. juncea lines were resistant to this pathotype. However, resistance was lost in 6 to 13% plants of the S2 progenies derived from the second resynthesized B. juncea line; this apparently resulted from the loss of the genomic region carrying resistance due to meiotic anomalies.

Analysis of relative nuclear DNA content in carnation (Dianthus caryophyllus) accessions reveals ploidy levels by flow cytometry

Analysis of relative nuclear DNA content in carnation (Dianthus caryophyllus) accessions reveals ploidy levels by flow cytometry

УРОВНИ ПЛОИДНОСТИ И ОТНОСИТЕЛЬНОГО СОДЕРЖАНИЯ ДНК В КУЛЬТУРЕ КЛЕТОК И ТКАНЕЙ РАСТЕНИЙ IN VITRO

<p>We presented results of variations in the ploidy level and the genome size of the <em>R. acetosa</em> regenerants. These regenerants was obtained by indirect and direct morphogenesis in in vitro culture. Explants were prepared from seedlings on the three-leaf stage of plant development. More than 100 explants were used to stimulate the indirect and direct morphogenesis. Mesophilic explants were cultured on the MS nutrient medium containing auxin to callus proliferation (2 mg/L NAA, 1 mg/L BA). Cultivation of the callus was maintained for 4 weeks followed by an indirect morphogenes. Indirect morphogenesis stimulated on the MS medium with cytokinin and gibberellic acid predominance (0.5 mg/L BA, 0.2 mg/L GA3). Direct stimulate morphogenesis from the apical meristem of seedlings on nutrient media with a predominance of cytokinins (1 mg/L BA, 0.25 mg/L NAA). Rhizogenesis have stimulated by transferring of the regenerants to the ½MS medium supplemented with 0.2 mg/L of NAA. Research of a ploidy level and genome size was performed by flow cytometry used propidium iodide staining with <em>Vicia faba</em> cv “Innovec” (2C=26.90 pg) as internal DNA standard. We calculated the relative DNA content (2C) for <em>R. acetosa</em> equal to 6,98 pg. Cytogenetical analisis showed that the maximum genome size variation recorded for regenerants obtained through the indirect morphogenesis. Variations in the genome size of the regenerants obtained by direct morphogenesis deviates from the control group to 0.30 pg (2С=7.28 pg) and after indirect morphogenesis to 1.04 pg (2С=8.2 pg). Cytogenetical analysis of the regenerated plants showed the presence of different somatic chromosome numbers ranging from 2n = 14 to 2n = 28. The relative DNA content of tetraploid forms was 11.87 pg. In our study was shown, that the most effective method of plant conservation in the <em>in vitro</em> culture is a direct morphogenesis. Analysis of the relative nuclear DNA content and chromosome counts of regenerants obtained by indirect morphogenesis from the callus cultures showed significant variations in the DNA content, as well as the appearance of polyploid forms. Therefore, long-term cultivation of callus cultures increases the probability of genomic aberrations, which reduces the stability of the plant genome.</p>

Taxonomic remarks on the genus Sternbergia L. (Amaryllidaceae) in Turkey based on leaf anatomy, karyosystematic analysis and nuclear DNA content

Anatomy of leaf cross sections, karyological studies, and nuclear DNA content analysis by flow cytometry were carried out on the six Sternbergia species from Turkey: S. candida (endemic to the country), S. clusiana, S. colchiciflora, S. lutea, S. sicula and S. vernalis. In karyological analysis, number and morphology of the somatic chromosomes, the ploidy level and karyotype formula of the specimens used in the study were investigated in detail. The measurements of somatic chromosomes were calculated with formula of the relative variation in chromosome lenght (CVCL) and mean centromeric asymmetry (MCA). According to the results of this study, there are two different basic chromosome numbers. i.e. x = 10 (S. candida, S. clusiana, S. colchiciflora and S. vernalis) and x = 11 (S. lutea and S. sicula). Results of karyotype analysis corroborated results of previous studies. The smallest chromosome length was measured as 3.05 μm in S. clusiana while the longest length of 20.89 μm was observed in S. sicula. This is the first study reporting nuclear DNA content results for Sternbergia. Based on results of the nuclear DNA content analysis, there is a great variation within the genus in the interspecific level as 2C DNA content varies between 54.03 and 80.42 pg among the species investigated in the study. Anatomical features of the leaves were good taxonomic markers, especially concerning their palisade cells, ribs, cuticle, laticifer canals, vascular bundles, crystals, stomatal index, and stomata structure. Analytical drawings were presented. Six noteworthy leaf characters were quantified, organized in a data matrix, and analysed with UPGMA methods. In conclusion, cytological characteristics of the Sternbergia species together with their leaf features proved to be quite useful in identification and therefore can provide more clarity for taxonomic revision of the genus.

Equisetum ×moorei Newman (Equisetaceae) – a ‘new’ nothotaxon in the Polish flora

Abstract To investigate present and historical distribution of Equisetum ×moorei in Poland and its habitat requirements, field studies at sites of potential occurrence of this hybrid taxon as well as literature and herbarium search were performed. As a results of these investigations, E. ×moorei was found at five contemporary and at a few historical localities in the present territory of Poland. Since the Equisetum populations near Olkusz (S Poland) showed phenotype similar, to some extent, to triploid hybrid E. ×ascendens, we performed nuclear DNA content analysis of these populations. However, it turned out that the investigated individuals belonged to a diploid taxon that can be ascribed to E. ×moorei.