Chromatin tagging systems are based on bacterial operator/repressor systems combined with fluorescence proteins. They can visualize spatiotemporally the specific loci where the tandem array of an operator sequence is inserted and three-dimensionally analyze chromatin dynamics in a nucleus of living cells. This method in combination with RNA imaging or an induction system of DNA breaks make it possible to analyze chromatin dynamics at RNA transcription and DNA repair. Chromatin tagging systems are also applied to dynamic analyses of organelle genomes. In this review, we introduce their fundamental principles and recent applications.
“Híbrido de Timor” (HT) is a natural interspecific hybrid of Coffea arabica L. with Coffea canephora Pierre ex Froehner that has played a substantial role in breeding programs as source of resistance genes. The original HT plant is represented by the anorthoploid accession ‘CIFC 4106’, an allotriploid plant with 3x=33 chromosomes and 2C=2.10 pg. From this HT plant, other accessions have been obtained and used in crossings with C. arabica to provide resistant plants to the main pathogens. However, each HT accession and their derivates show a particular karyotype due to irregular anaphasic disjunction, which results in a few reproductive cells with an unbalanced number of chromosomes. Regarding this fact and the crop relevance of HT, this study aimed to develop a direct somatic embryogenesis system for the clonal propagation of this germplasm, using the first HT plant as explant donor. To accomplish this purpose, disinfested leaf explants of greenhouse-cultivated HT ‘CIFC 4106’ plants were inoculated in medium supplied with 0.001 g L−1 6-benzylaminopurine. Somatic embryogenesis process was asynchronous, with distinct developmental stages occurring simultaneously. Besides, emergence of secondary embryos from primary ones was observed. Mature embryos were germinated, and well-rooted embryos were selected for plantlet regeneration. The nuclear DNA content and the karyotype showed that allotriploidy was conserved in all regenerated plantlets. Considering these results, the direct somatic embryogenesis protocol adopted in the present work was imperative for the accurate clonal propagation of HT, being relevant for multiplication and conservation of elite accessions that show an irregular meiosis.
Five species (13 ecotypes) belonging to three subgenera of ornamental-medicinal Iranian Fritillaria were karyotypically studied, using a standard squash technique. All species were diploid (2n=2x=24) having mean chromosome lengths of 15.8 µm (15.2–16.7 µm). Their satellites varied in number (1–3 pairs) and in size (1.2–2.6 µm), mostly being located on long arms. Four chromosome types (“m”, “sm”, “st”, “T”) formed 10 different karyotype formulas: “T” type chromosome is reported for the first time in most species (with the exception of S4, Fritillaria. reuteri Boissi). ANOVA confirmed significant intra- and inter-specific chromosomal variation across the Iranian Fritillaria species. Twelve different methods were used to assess the degree of karyotype asymmetry. Among those, one qualitative parameter (Stebbins classification) and eight quantitative (CVTL, DI, A1 & A2, AI, A, AsK%, MCA, CVCI) parameters verified that S2 (F. gibbosa Boiss.) and S5 (F. zagrica Stapf.) species represented the most asymmetrical and symmetrical karyotypes, respectively.
Chromosome numbers of seven Carex taxa collected from the Korean peninsula are reported. This study is the first observations of meiotic chromosomes for Carex species from Korean populations. A univalent is observed in C. ischnostachya Steud. var. fastigiata T. Koyama (n=26II+I), and two different chromosome numbers are observed in an individual of C. leucochlora Bunge (n=27II, 29II). For the first time, the chromosome number for C. polyschoena H. Lév. & Vaniot (n=26II) is reported here. The results of diverse chromosome numbers suggest that polyploidy and aneuploidy including agmatoploidy and symploidy (increases and decreases in chromosome number due to fission or fusion, respectively) have played an important role for Carex species diversity in Korea.
Bulbs of Allium cepa L. were treated with 0.2% ethyl methane sulphonate (EMS) for various durations: 6, 12, 18 and 24 h. Two mutants were isolated in a population raised from 0.2% EMS for 12 h (translocation heterozygote) and 18 h (inversion heterozygote) treated bulbs of Allium cepa. The translocation heterozygote exhibited the formation of a chain of four chromosomes in majority of the pollen mother cells (PMCs) at diakinesis/metaphase-I. At anaphase-I, the translocation heterozygote displayed various abnormalities such as laggards, unequal distribution, micronuclei, etc. The inversion heterozygote plant displayed various types of chromosomal configurations at anaphase/telophase-I and II in meiosis. It was characterized by the presence of bridge and fragment because of various numbers and the position of crossovers in the inversion loop. Pollen fertility was found to be very low in both the translocation heterozygote and inversion heterozygote.
In the present investigation, we examined the morphological and ecological adaptations, and male meiosis and pollen fertility in an endemic species of genus Astragalus, A. rhizanthus Royle ex Benth. from cold deserts of Lahaul–Spiti district of Himachal, Pradesh, India. The three populations from Lahaul Valley and one from Spiti Valley depicted the same meiotic chromosome number of n=8 in all the accessions. In three populations (Lahaul, Sissu, 3170 m, Chandartal Lake, 4300 m; Spiti, Lossar, 3900 m), meiosis was normal resulting into cent percent pollen fertility. However, in the accession from Lahaul Valley (Koksar, 3140 m) majority of the pollen mother cells (PMCs) showed normal chromosome behaviour during meiosis-I and II. While, 12.09% PMCs showed the occurrence of chromatin material transfer involving 2–5 meiocytes during prophase-I, metaphases-I/II, anaphases-I/II and telophases-I/II. Consequent to this, hypo- and hyperploid PMCs resulted. In certain cases, PMCs fused together to form syncytes which had two complements in the same cell. Pollen sterility was observed to be 9%. The paper also discusses morphological and ecological adaptations that the species has adopted to cope with the harsh and hostile environmental conditions prevailing in the cold deserts of Lahaul–Spiti.
The present population-based cytological studies were conducted in Angelica glauca collected from the northwestern Himalayan region of India. For the first time we report the occurrence of various meiotic abnormalities viz. cytomixis, chromosomal stickiness, unoriented bivalents, laggards, bridges and other spindle irregularities in the species that exist at diploid level (2n=2x=22). The phenomenon of cytomixis (17.35%), involving the transfer of chromatin material between 2–4 adjacent pollen mother cells (PMCs) during male meiosis through broad and narrow cytomictic channels, was observed from early prophase stages to tetrad stage. Cytomixis results in the formation of hypo-, hyperploid and enucleated PMCs. The severe chromosomal stickiness restricts chromosome separation during anaphase. Further, the spindle abnormality produces monads, dyads, triads, polyads and micronuclei in sporads during microsporogenesis. All these abnormalities affect the pollen grain size and reduce the pollen fertility (64.23–84.25%) in such populations. In the present paper, the consequences of cytomixis, chromatin stickiness and other anomalies have been discussed in detail for different populations sampled from different altitudes.
Colocasia esculenta (L.) Schott, a commonly growing plant in industrial effluent affected areas, was selected for observation of the effects of industrial effluents on chromosomal and DNA levels. Five different industries—(i) Textile (ii) Pesticide (iii) Tannery (iv) Ceramic and (v) Pharmaceutical—in and around Dhaka City were selected for this study. The diploid chromosome number 2n=28 was found in the specimens collected from textile, pesticide and tannery effluent-affected areas similar to the control. However, 2n=38 were observed in the plant sample collected from Ceramic and Pharmaceutical areas. The probable reason for increasing chromosome number was non-disjunction of certain chromosomes at anaphase due to inhibition of spindle formation. The fluorescent karyotypes of the plant showed significant differences between the samples and the control. Fluorescent banding (CMA and DAPI) revealed the occurrence of probable genomic reshuffling by deletion and tandem duplication. The different plant samples of the species had characteristics RAPD fingerprinting. Lacking of some DNA fragments in some sample plants may correlate to the deletion of chromosomal parts. Similarly, some new fragments in the sample plants may correspond with the tandem duplication of the chromosomal part. Therefore, the substances present in the industrial effluents affected on the chromosome and DNA level of Colocasia esculenta.
Neurobasis chinensis chinensis of the family Calopterygidae is the only species of this genus present in India. This species was collected from Andretta and Mcleodganj areas of Himachal Pradesh, India. The species possesses 2n=23 which is less than the type number (2n=25) of the family. The chromosome complement shows the presence of large pair of autosomes originated by the fusion of two autosome pairs and is responsible for the reduction in the chromosome number. This fusion has been confirmed by C-banding with the presence of two interstitial and terminal C-bands on the largest autosomal bivalent having two chiasmata while remaining autosomal bivalents possess terminal C-bands and single chiasma which is the characteristic feature of Odonata. Ag-NOR staining shows the presence of terminal NOR bands in seven autosomal bivalents and the X chromosome is rich in NORs. C-banding and Ag-NOR staining have been performed for the first time on this species.
This study aimed to develop molecular markers for fluorescence in situ hybridization (FISH) to examine microsatellite regions of chromosomes in four Allium species: A. ampeloprasum, A. ascalonicum, A. cepa and A. sativum using (CAA)10, (GAG)10, (GAA)10 and (CA)15 probes. The probes of (GAG)10, (GAA)10 and (CA)15 yielded no signal. The microsatellite probe of (CAA)10 hybridized to chromosomes in each species. Signals of the FISH signals were distributed across the chromosomes in A. ampeloprasum, A. ascalonicum and A. sativum but were located at chromosome termini in A. cepa.
In the present work, detailed male meiotic studies were performed on 24 dicot species from the hills around Shimla within the altitudinal range of 1880–3140 m and which include the first ever chromosome count for Polygonum polystachyum (n=11) from India. Further, a new intraspecific aneuploid cytotype of 2n=18 for Impatiens brachycentra and B-chromosomes have been reported in Pimpinella acuminata. The phenomenon of cytomixis involving chromatin transfer among neighbouring PMCs along with associated meiotic irregularities and pollen malformation has been detected in some individuals of Clematis grata (n=8) and Ranunculus laetus (n=14). For the rest of the species, the study confirms the earlier chromosome counts by other workers. Analysis of chromosomal data on the worked out species reveals that aneuploidy and polyploidy has a role to play in evolution.
In this study, the karyotype of the Acrida ungarica species (chromosome number, chromosome morphology and chrosome lengths) belonging to the Acridinae subfamily of the Acridiae family was examined. As a result of these examinations, the species number of chromosomes was determined to be 2n♂=23 (XO). It was found that all autosomal chrosomes as well as the X chrosome had an acrocentric structure. As a result of counting carried out on five individuals, the mean chiasma frequency was found to be 16.39.
Cytological studies were conducted on five tetraploid species plus two varieties of Magnolia subgenus Yulania and a hybrid between two of these species. Two different chromosome configurations in meiosis are observed in this article. The first group was typical for M. acuminata, M. liliiflora and their relatives which share several cytological characteristics including chromosome configurations and behavior in meiosis. The cytological evidence indicate autotetraploid origin for these species, despite the homologous chromosomes likely having changed their structures causing the formation of heteromorphic multivalents, chromosome bridges and fragments during meiosis. The second group of chromosome configuration was found in M. cylindrica and M. concinna. These species were characterized by very rare trivalents and tetravalents. These observations suggest these taxa as allotetraploids which may have been formed from hybridisation between hexaploid and diploid parents as deduced from trivalents observed at some rate. Multivalent formation and possible translocations and inversions of chromosomes in M. liliiflora influence its propagation by causing low fertility. M. liliiflora is probably a comparatively new species with an unstable chromosome constitution and is growing only in small populations at low elevation. It is vulnerable, and the species could already be extinct in nature. On the other hand, M. acuminata shows a more regular chromosome behavior and fewer abortion spores than M. liliiflora, and should be a more stable species with longer evolution time. Thus, the superior fertility of M. acuminata has enabled it to develop a stable and extensive distribution from NE to SE USA.
In this project, the effects of Ginkgo biloba L. leaf extract (GbLE) on the seed germination, seedling growth (radicle length, radicle number and fresh weight), mitotic activity and chromosomal aberrations of Allium cepa L. germinated under both normal conditions and salt stress were studied. The radicle length of seeds germinated in the medium with GbLE alone increased in comparison with ones of the control seeds germinated in distilled water medium while their fresh weight reduced according to the control. However, the germination percentage and radicle number of the mentioned seeds were statistically the same as ones of the control seeds. Furthermore, the mitotic index and the frequency of chromosomal aberrations in root tip meristems of A. cepa seeds germinated in the medium with GbLE alone demonstrated a significant increase compared to the control seeds germinated in distilled water medium. On the other hand, salt stress considerably inhibited the seed germination and seedling growth of A. cepa. In addition, it markedly decreased the mitotic index in root tip meristems of the seeds and increased the number of chromosomal aberrations. Whereas, the inhibitive effect of salt on the seed germination, seedling growth and mitotic activity was dramatically alleviated in varying degrees by GbLE application. But, the detrimental effects of salt on the chromosomal aberrations greatly increased with this treatment.
Using field emission scanning electron microscopy (FE-SEM) and fluorescence microscopy, the respective relations between the arrangements of the gamete cell fusion site and the inheritance pattern of chloroplast DNA (cp-DNA) as a sex-specific marker were studied for the isogamous dasycladalean alga Acetabularia caliculus. The gamete had two flagella elongated from the cell anterior. One oval-shaped eyespot situated on the cell posterior was visualized using FE-SEM. When the gametes belonging to the opposite mating types were mixed together, the two gametes aligned side-by-side at their lateral side and fused to form a quadriflagellate planozygote. In the planozygote, the two flagella from the opposite mating type gametes lay side-by-side and two eyespots aligned on the same side of the cell, suggesting opposite positioning of the cell fusion sites between two parental gametes. To confirm whether the gamete fusion pattern observed in the planozygotes was a result of sex-specific fusion or not, the inheritance pattern of cp-DNA was observed using fluorescence microscopy after staining with DAPI concomitantly with the cell fusion pattern. When the eyespots were used as positional marker and were viewed from the surface that included the eyespots, the chloroplast of one parental gamete was found to contain cp-DNAs, although cp-DNAs were not detected in the chloroplast derived from another parental gamete. These results suggest that the gamete fusion pattern observed by FE-SEM was a result of sex-specific fusion.
Lepidagathis lutea Dalzell is an endemic species to the northern Western Ghats, of which cytology has not been reported until present. In this investigation, basic chromosome count is reported to be eight for the first time for the genus Lepidagathis Willd. The study reveals its closeness to the genus Barleria L. which is shown to have basic chromosome counts 8, 9 and 10. Karyotype and somatic chromosome counts (2n=16) for L. lutea are reported here for the first time. According to Stebbins classification, karyotype is of 4B asymmetric category and the karyotypic formula was observed to be 2n=16=10m+6sm.
Cell wall thickness is a quantity that must be known in order to calculate the stress applied to a cell wall along its plane by turgor pressure. Measuring the width of a cell wall section with transmission electron microscopy (TEM) is one of the typical methods of measuring cell wall thickness. However, the possible occurrence of artifacts in this method has not been tested so far. In the present study, the cell walls of the giant cylindrical cells of the xanthophycean alga Vaucheria frigida (Roth) C. Agardh were examined by TEM from thin sections made perpendicular to the longitudinal cell axis, and their morphology and thickness were compared under different preparation methods in order to evaluate the reliability of each method of measuring cell wall thickness. Cell wall morphology was not significantly different between specimens dehydrated with acetone and embedded in hydrophobic Spurr’s resin and those dehydrated with ethanol and embedded in hydrophilic LR White. However, the cell walls of the former were significantly thinner (ca. 250 nm) than those of the latter (ca. 450 nm). In addition, cell wall thicknesses measured using an atomic force microscope (as the difference in the levels between the top of an isolated cell wall and the surface of the substratum) were about 130 nm or less, remarkably smaller than those measured by TEM from thin sections. These results indicate that cell wall thickness measurements from thin sections may be affected by artifacts, and other reliable methods are needed for measuring wall thickness correctly.
Several chromosome numbers are reported for Stachys taxa but little information is available about chromosome morphology. Thus, this study aims to investigate the karyotypes of six endemic Stachys species from Turkey. Two different chromosome numbers are reported as 2n=30 (Stachys chasmosericea and S. pinardii) and 2n=34 (S. euadenia, S. buttleri, S. pseudopinardii and S. longiflora). The chromosome types of the studied taxa are metacentric and submetacentric. Karyological features and asymmetry indices (MCA, CVCI and CVCL) of the six endemic species are identified for the first time, and karyological relationships are determined. Also we presented published chromosome data of Stachys along with new counts. These data can be used for chromosome number evolution for the genus and helpful for understanding its evolution.
We analyzed the karyotypes of laboratory strains of Mongolian gerbils showing agouti, black and albino coat colors using conventional and differential staining methods. Of these, three strains have been kept in the author’s laboratory over 30 years and one strain was obtained from a supplier. The conventionally stained karyotypes of the gerbils were fundamentally identical to those in previous studies, consisting of 2n=44 (FNa=74) with 42 autosomes: 16 pairs of bi-armed (metacentrics, submetacentrics, and subtelocentrics) and 5 pairs of uni-armed chromosomes (acrocentrics), a large-sized metacentric X chromosome and a small-sized submetacentric Y chromosome. In addition, the G- and C-banded karyotypes were identical to those in previous studies. Notably, our strains carry no intraspecifical karyological variation and their karyotypes are quite stable without a polymorphic state, except for heteromorphic variations caused by size differences as a usual phenomenon. Moreover, the unique C-bands were found in Nos. 5 and 13 chromosomes but fluorescent staining results (using quinacrine mustard and chromomycin A3) at the unique C-band regions differed between Nos. 5 and 13 chromosomes. This fact indicates that such a difference has been caused by higher molecular structures containing proteins rather than nucleotide contents.
Specimens consisting of two tandem internodal cells were prepared using Chara corallina in order to analyze the electrical response upon wounding. When one cell (victim cell) was killed by severing, another cell (receptor cell) generated a sharp spike followed by a long-lasting depolarization at the nodal end. It has been reported that K+ released from the victim cell is involved in generation of the long-lasting depolarization (Shimmen 2006). In the present study, the trigger to induce the sharp spike was studied. When the turgor pressure of the victim cell was decreased by adding 1 M methanol to the external medium, a sharp spike similar to that induced by wounding was generated at the nodal end of the receptor cell. It is suggested that the sudden disappearance of the turgor pressure of the victim cell due to severing is a trigger to induce the sharp spike of the receptor cell.