Cardiolipin (CL) is widely distributed in various prokaryotes and eukaryotes as a membrane phospholipid. In eukaryotes, it is localized in the inner membrane and at the contact sites between the inner and outer membranes of mitochondria. CL is suggested to be involved in many mitochondrial functions. We previously identified the CLS gene for CL synthase in Arabidopsis thaliana and showed it to be a useful tool for understanding the physiological roles of CL in higher plants. In this study, we isolated homozygous cls mutants (cls-1 and cls-2) of A. thaliana, in which the CLS gene was disrupted by T-DNA insertion, by observing the development of embryos obtained after self-fertilization of heterozygous cls mutants. Both embryogenesis and growth of the homozygous cls mutants were substantially retarded relative to wild type, and additional phenotypes were observed, namely slow root growth, an abnormal veining pattern in cotyledons, and a low yield of seed production. The delayed embryogenesis and growth of homozygous cls mutants were recovered by introduction of CLS into the mutants. These findings demonstrate that CLS plays important roles in development of A. thaliana, presumably due to the biosynthesis of CL in the mitochondria.
A discovery of polymorphic nucleolar organizer regions (NORs) and karyological analysis in the dusky leaf monkey (Trachypithecus obscurus) from Thailand were studied. Blood samples were taken from 2 male and 1 female leaf monkeys. After standard whole blood lymphocytes had been cultured at 37°C for 96 h in presence of colchicine, metaphase spreads were performed on microscopic slides and air-dried. Conventional, GBG-, CBG-banding and high-resolution techniques were applied to stain the chromosomes. The results showed that the diploid chromosome number of T. obscurus was 2n=44 and the fundamental number (NF) for both sexes was 88. The types of autosomes were 6 large metacentric, 12 large submetacentric, 2 large acrocentric, 4 medium metacentric, 10 medium submetacentric, 2 small metacentric, 4 small submetacentric, and 2 small acrocentric chromosomes. The X chromosome was a large submetacentric chromosome and the Y chromosome was a small submetacentric chromosome. In addition, the long arm near centromere of chromosome pair 17 showed clearly observable NORs. This is the first report on nature polymorphism in NORs in T. obscurus. The results showed that a heteromorphism of 1 female had different size of NORs for chromosome pair 17s, while 2 males had an equal size of both chromosome pair 17s with a homomorphism. From the GTG-banding and high-resolution techniques, the numbers of bands and locations in the T. obscurus were 235 and 314, respectively, and each chromosome pair could be clearly differentiated. CBG-banding showed C-positive (dark bands) on the centromeres of all autosomes but showed C-negative (light bands) on the Y chromosome. Moreover, we also found interstitial dark bands on the telomeres of long arms which result from centric fusion or tandem fusion. The karyotype formula for T. obscurus could be deduced as: 2n (diploid) 44 = L6m + L12sm + L2a + M4m + M10sm + S2m + S4sm + S2a + sex chromosomes.
Localization of nucleolar organizer regions (NORs) of four gibbon species in Thailand was studied. Blood samples were taken from 1 male each of the following 3 species: white-handed gibbon (Hylobates lar), pileated gibbon (H. pileatus), and white-cheeked gibbon (Nomascus leucogenys): and 1 female of the dark-handed gibbon (H. agilis). These were subjected to standard whole blood T-lymphocyte culture. The samples were harvested by colchicine-hypotonic-fixation-air-drying technique and were followed by the Ag-NOR banding technique. The results showed that the H. lar had NORs on chromosome pair 13 (submetacentric chromosomes) while the H. agilis had NORs on chromosome pair 10 (submetacentric chromosomes) and H. pileatus had NORs on chromosome pair 15 (metacentric chromosomes). In addition, the N. leucogenys showed 5 positions with NORs on chromosomes pairs 23, 24 (acrocentric chromosomes) and the Y-chromosome (submetacentric chromosome). These results are useful to confirm marker chromosomes of gibbons and also comprise basic genetic information for these animals.
Three commercial varieties of Cucurbita maxima Duch. (Sweet Gourd or Pumpkin) such as Baromashi (Local), Solid Gold and BARI Mistikumra-2 were investigated karyotypecally and at molecular level by RAPD analysis for authentic characterization. Three varieties were found to possess 2n=45–48, very small chromosomes ranging from 0.2–1.0 μm in length. The chromosomes of Baromashi (Local) and BARI Mistikumra-2 could be grouped in 4 classes on the basis of chromosomal length whereas the Solid Gold variety has only 3 such classes. The smallest chromosomes of 3 varieties were so minute that each karyotype showed at least 2 sharp modalities. Each variety showed distinct RAPD fingerprinting with 7 different primer combinations. The variety Baromashi (Local) and BARI Mistikumra-2 showed exactly similar banding pattern in primer OPA-3 and OPA-6. Maximum common band was found in OPD-3 suggesting the sharing of similar sized fragments among these 3 varieties. No common band was found in OPA-6 indicating the highest polymorphism (100%). The average polymorphism among these 3 varieties in different primer combinations was 64.35%. Solid Gold showed 8 unique bands in different sizes whereas Baromashi (Local) and BARI Mistikumra-2 had 7 and 5 unique bands, respectively. Karyotype and RAPD fingerprinting were almost similar in Baromashi (local) and BARI Mistikumra-2 whereas Solid Gold completely different from the other 2 in these parameters. These results were supported by different phenotypic or morphological features. Therefore, with the help of karyotype and RAPD analysis, it was possible to characterize 3 varieties of Cucurbita maxima Duch.
Five commercial varieties of Trichosanthes anguina (Snake Gourd), namely Turag, Dhaka Green, Super Long Green, Anika and Apurbo, were investigated cytogenetically and at the molecular level for authentic characterization. The variety Super Long Green was found to possess 2n=23 chromosomes and considered as a primary trisomic. 2n=22 chromosomes were found in the other 4 varieties. The total length of the 2n chromosome complement was largest in Anika and smallest in Apurbo. Only one extra large chromosome was found in Turag and Anika suggesting it had either been transferred from different genome or was a case of repeated duplication. No decrease in chromosomal length was found in Turag, a moderate decrease was found in Dhaka Green and Anika, and a sharp decrease in Super Long Green and Apurbo. A pair of satellites was found in Turag. Only a stain specific satellite was found in Dhaka Green. No CMA band was found in Turag and Super Long Green. The other 3 varieties showed distinct CMA banding pattern in respect of number, size, location and percentage of GC-rich area. The 5 varieties showed distinct RAPD bands with 6 different primer combinations. In addition, the Apurbo and Turag varieties showed unique DNA fragments with the primer combinations OPA-3 and OPA-4 respectively. A combination of cluster and karyotytpe analysis clearly indicated that Turag and Apurbo were distinctly related from the other 3 varieties. Therefore, with the help of cytogenetical and RAPD analysis, it was possible to characterize each variety.
Spergularia diandra(Guss.) Heldr. & Sart. (Family: Caryophyllaceae), worked out presently from the valleys of Ganjul and Hangrang in the Kinnaur district of Himachal Pradesh, exhibits intraspecific diploid (2x=n=9) and tetraploid (4x=n=18) cytotypes. The species has been worked out chromosomally for the first time from India and adds a new 4x cytotype to the already existing 2x cytotype known from outside of India. The individuals of 2x cytotype are restricted to the Ganjul Valley while those of 4x cytotype are present in Hangrang valley. The individuals of the 2x and 4x cytotypes could be distinguished on the basis of habit, plant size, number of branches, bushy nature, and fleshiness and size of leaves. The diploid plants showed spindle abnormalities during meiosis resulting into heterogeneous sized fertile/stained pollen grains and some pollen sterility (10.00–12.00%). On the other hand, some plants of 4x cytotype showed the phenomenon of cytomixis involving chromatin transfer among proximate meiocytes, and chromatin stickiness resulting into 5.00–8.00% unstained/sterile pollen grains. The cytomixis in the 4x cytotype is the first ever record for the species and seems to be a natural phenomenon under direct genetic control as the plants with and without cytomixis grow under the same environmental conditions.
Cytological investigations carried out on a population basis in Astragalus chlorostachys Lindl. which exists at diploid level (n=8) revealed the presence of quadrivalents, the late disjunction of 1–4 bivalents and some pollen sterility (34%) in 1 accession collected from Koksar, at 3140 m in the Lahaul Valley (Lahaul-Spiti, Himachal Pradesh). The presence of quadrivalents in this diploid species seems to be a consequence of reciprocal translocations. The occurrence of structural heterozygosity in the species has been reported here for the first time. All of the other populations showed normal bivalent formation and regular sporad generation and 100% pollen fertility. The paper here discusses in detail the course of meiosis and the effect of multivalent formation and non-synchronous disjunction of bivalents on meiotic course and pollen fertility.
The aim of the study was to determine possible detrimental effect of nickel and zinc supplements added at various concentrations into commercial rabbit diet during 3 months of permanent feeding in an ad libitum system. The following concentrations of heavy metals were examined: P1—17.5 g NiCl2, P2—35 g NiCl2, P3—17.5 g NiCl2+30 g ZnCl2, P4—35 g NiCl2+30 g ZnCl2 to 100 kg of complete feed. Chromosome preparations were obtained from blood lymphocytes after metaphase arrest using colcemid solution. Nickel (NiCl2) presence at concentration of 35 g/100 kg of rabbit feed after 3 months of systematic feeding had a negative effect on rabbit chromosomes according to the detected aneuploidy occurence. On the other hand, ZnCl2 at concentration 30 g/100 kg of complete feed partially neutralized the negative NiCl2 effect. Significant differences in chromosomal aneuploidy were found between the P2 testing group and control (p<0.001) and between the P4 and control groups (p<0.05). In conclusion, the negative effect of selected heavy metal was clearly demonstrated through cytogenetical study based on the action of nickel element intake on an increase of aneuploidy, via a supplemented commercial rabbit diet using NiCl2 compound. Hence, it is important to point to the zinc role as a partial protector against the detrimental nickel effect on chromosomal status.
This paper presents cytomorphological diversity covering the chromosome number, meiotic behavior and pollen study of 5 species on an accession basis under the family Verbenaceae from North India. Six new chromosome reports have been made for the first time which involve 2 varied chromosome counts; n=18 for Caryopteris odorata and n=6 for Verbena officinalis; 3 new euploid cytotypes of diploid (2n=2x=14) for V. officinalis and hexaploid (2n=6x=42) and octaploid (2n=8x=56) for Verbena bonariensis; and B-chromosome for Vitex negundo with 2n=32+0-2B. Morphotypes are identified for 2 species; Caryopteris odorata and Lantana camara, and cytomorphotype for Verbena officinalis. All the investigated species show abnormal meiotic behavior.
At present, meiotic studies have been carried out on a population basis covering 23 species belonging to 13 genera and 10 families of Polypetalae from District Kangra of Himachal Pradesh. New reports in the form of varied chromosome numbers have been marked for the first time on a world-wide basis for 13 species, namely Bupleurum lanceolatum (n=16), Impatiens bicolor (n=8), I. racemosa (n=7), I. sulcata (n=7), Medicago polymorpha (n=16), Fumaria indica (n=8), Geranium lucidum (n=13), G. ocellatum (n=13), Hypericum elodeoides (n=8), H. japonicum (n=16), Malva verticillata (n=21), Epilobium roseum (n=9), Geum roylei (n=14), Rosa macrophylla (n=14) and Triumfetta pilosa (n=16). Six species, namely Boenninghausenia albiflora (n=10), Circaea alpina (n=11), E. palustre (n=18), Rosa brunonii (n=7) and R. indica (n=7), have been cytologically worked out for the first time from India, along with additional cytotypes for 3 Indian materials, namely Abutilon indicum (n=7), E. cylindricum (n=9) and Oxalis corniculata (n=7). Amongst these species, the course of meiosis varies from normal to abnormal in different populations of Malva neglecta, Rosa brunonii and R. indica whereas all the studied accessions of Bupleurum lanceolatum, Geum roylei, Impatiens sulcata, Oxalis corniculata and Rosa macrophylla have been found to be abnormal. The meiotic abnormalities include various phenomenon such as cytomixis, chromatin stickiness, unoriented bivalents, laggards, chromatin bridges and multipolarity at different stages of meiosis along with abnormal microsprogenesis ultimately leading to reduced pollen fertility and formation of heterogenous sized pollen grains.
We examined the chromosome number of Taraxacum laevigatum naturalized in Japan using 35 plants collected from a total of 12 localities distributed throughout Hokkaido, Akita, Tochigi, Toyama, Gifu, Aichi, Osaka, Okayama and Okinawa Prefectures in Japan. All plants had 2n=24 chromosomes (triploid), but we identified 2 strains with different karyotypes formulated as 2n=24=1M+15m+5sm+3smcs and 2n=24=2M+19m+2mcs+1smcs.
Isabgol is an annual herb cultivated as a medicinal plant. In the present study, karyotypic analysis of 12 Iranian endemic Isabgol (Plantago ovata Forsk.) ecotypes was carried out. The chromosome number was 2n=2x=8 with 4m+4st karyotype formula. The mean value of total chromosome length (TL) was 2.86 μm, ranging from 2.44 μm (E4) to 3.26 μm (E12). All ecotypes had 2 pairs of satellites (3S, 4S), varying in size from 0.34 to 0.36 μm. Most of the chromosomal parameters were significantly different among the various ecotypes. In general, karyotypes were symmetrical and fell in the Stebbins 2A category. The scatter diagram of A1 and A2 asymmetry indicated 4 groups of ecotypes while PCA analysis together with cluster analysis and scatter diagram of TL and CI, showed 3 groups, containing 9, 2 and 1 ecotypes, respectively. C-Banding patterns and fluorescence in situ hybridization (FISH) with 45S rDNA probe provided landmarks for chromosome identification in somatic preparations.
This research was carried out to evaluate the cytogenetic diversity of Persian beans with using an image analysis system. Ploidy levels of genotypes were determined from x=9 (2 diploids), x=10 (2 diploids) and x=11 (9 diploids). The highest chromatin length belonged to genotype 5 (12.501 μm) and the lowest to genotype 13 (5.042 μm). With regards to the Stebbins two-way table, I, L, and F genotypes divided to group a 2A; A, B, and N genotypes divided to group 1B; and the rest of the genotypes divided to group 2B. The most asymmetric karyotype between and within chromosomes was genotype N. Five important karyotype indexes that are related to the cytogenetic diversity of Persian beans were studied in Completely Randomized Design (CRD) with 3 replications, at the University of Tehran in 2010. The results of ANOVA showed significant differences among genotypes in all karyotype indexes (A, %Syi, %TF, SC, A2 and A1). Principal components analysis classified the 5 karyotype indexes in to 2 new components that explained 96.41% of total variations. The first component explained 65.34% of total variations that mainly included diastatic arm ratio, CI, A1, TF, Syi and A. The second component explained 31.28% of total variations that mainly included TL, SA, LA and A2. Cluster analysis by the Ward method classified characters in to 3 groups.The results showed that genotypes were significantly different in all characters; therefore analysis of these clusters showed the most genetic distance to be between N and H.
Karyotype and RAPD analysis were studied in 3 forms of climbing perch, Anabas testudineusviz. wild (native, non-spotted), Thai (introduced from Thailand, spotted) and Thai (a spotted-released form from local hatcheries). 2n=46 chromosomes were found in the spotted-released form. The total length of 2n chromosome complement was 89.96 μm with a chromosomal length ranging from 0.92 and 2.96 μm. The centromeric formula of this spotted-released form was 15m+9sm+22t. This form did not show any CMA-bands. The karyotypic features of this spotted-released form were totally different from those given by previous reports on spotted forms. Four primers, namely OPA-2, OPA-4, OPA-7 and OPA-8, were tested on these 3 forms. The wild form showed unique bands in the OPA-2 (1300, 550, 350 bp) and OPA-7 (280 bp) primers. The spotted and spotted-released form showed different RAPD banding patterns in primers OPA-4, OPA-7 and OPA-8. The wild form was found to be separate from the other 2 forms at 13.5 linkage distance. The 2 Thai forms have 11.0 linkage distances indicating that these 2 forms are not genetically very close. The karyotype and RAPD results indicate that either the Thai spotted-released form is not developed exactly from the same stock as the Thai spotted form or that due to the application of physical stress or chemical treatment this spotted-released form has been modified.
The genus Ipomoea with about 500 species is distributed in the tropical and subtropical regions of the world. The species are not only among the most beautiful ornamental plants, they are also important because of their medicinal value. The present study deals with the karyotype analysis of 10 species of Ipomoea growing in Maharashtra state of India. All the species analysed are diploid, showing 2n=30. I. aquatica has the smallest chromosomes, 1.25 to 2.67 μm, with a mean length of 1.99(±0.38), and the total length of the haploid component as 29.88 μm, while I. carnea has the longest chromosomes, at 2.13 to 4.79 μm, with mean length as 3.39(±0.74) and total length of the haploid component at 50.83 μm. The karyomorphological investigations revealed that none of the species had a symmetrical karyotype. The degree of asymmetry was, however, low. Amongst the several parameters studied, that of Stebbins (1971) and the AI values were found to be the most reliable for assessing asymmetry. The species falling under group 2B of Stebbins had higher AI values as compared to those of group 1B, proving that they are more evolved. The heterogeneity within the 10 species could be traced by AI values.
Stem cells are multipotent and have the potential to play an important role in the treatment of many diseases. However, limited sources of stem cells restrict their clinical application. To solve this problem, a simple in vitro epigenetic reprogramming technique to differentiate skin fibroblasts into multipotent cells has been established in our center. In the present study, human skin fibroblasts were induced into multipotent stem cells in vitro by epigenetic reprogramming. The differentiation potential of the induced multipotent stem (iMS) cells in vitro and in vivo was also explored. After transient reprogramming, stem cell markers, including Oct-3/4, Nanog, Sox-2, SSEA-4 and C-myc, were activated in the reprogrammed cells. When these cells were treated with β-mercaptoethanol (β-ME) and all-trans retinoic acid (RA), they were differentiated into neural cells in vitro. These reprogrammed cells were multipotent as demonstrated by their abilitiy to differentiate and form teratomas. Neural cells were found in the teratomas. The results indicated that the iMS established in our laboratory are multipotent and can grow into neural tissues. iMS may be effective in regenerative medicine in order to treat tissue damage, genetic disorders, and neuronal degenerative diseases.
Centromeres of human chromosomes contain highly repeated sequences of DNA including alphoid DNA. Because of the complicated genomic organization of the centromere, the distribution of alphoid DNA in chromosomes has not been fully investigated. We conducted fluorescence in situ hybridization using a synthetic peptide nucleic acid as a sensitive probe (PNA–FISH) to detect chromosomal sites of alphoid DNA. As a result, the size variation of centromeric alphoid DNA among chromosomes was visualized with hybridization times as short as 1–2 h. In addition to the inter-chromosomal variation, we detected possible inter-individual variation in the size of alphoid DNA sites, which had been difficult to precisely analyze by conventional molecular and cytogenetic methods. We then applied this sensitive and rapid detection method to evaluate the yield of multicentric chromosomes induced in cultured human peripheral blood lymphocytes by high-dose gamma-irradiation. This PNA–FISH allows us to unequivocally determine centromeres in complexly rearranged chromosomes, confirming its usefulness in biological radiation dosimetry.
Cytomictic behavior (assessed from meiotic studies performed during July to August—period A and August to September—period B; out of 7 plants—designated as P1 to P7, cytomixis is recorded in P1 and P7 in both the periods, while P4 only in period B) was noted in a wild species of Corchorus (C. fascicularis Lamk.; Family: Tiliaceae, important genetic resource for Jute) under acclimatization /cultivation (3rd generation raised from bulk seeds of previous generation plants) in the experimental field plots of University of Kalyani, West Bengal, India (latitude 22°50′ to 24°11′N, longitude 88°09′ to 88°48′E, altitude 9.75 m, sandy loamy soil, pH −6.89). The intensity of cytomixis varied among the plants (P1 showed high degree of cytomictic behavior; while it was incipient in P4 and P7) and between the periods. Cytomixis was evidenced at early prophase including diplotene-diakinesis and metaphase I and rare, often in anaphase I cells resulting in aneuploid (hypo-: n=1–6, hyperploid: n=8 to 14 and 18) variations in chromosome number at meiosis I. These results may suggest that the phenomenon of cytomixis is a natural cellular process controlled genetically and to an extent influenced by environmental factor(s) and the gene(s) involved in the process, and that it may be differentially expressed or repressed under a given set of condition(s).
The first chromosomal characteristic of nucleolar organizer regions (NORs) and karyological analysis of the grey featherback fish (Notopterus notopterus) from Chi basin, northeast Thailand, were studied. Kidney cell samples were taken from 4 male and 4 female fish. The mitotic chromosome preparations were done directly from kidney cells. Conventional and Ag-NOR staining techniques were applied to stain the chromosomes. The results showed that the diploid chromosome number of N. notopterus was 2n=42, the fundamental number (NF) was 42 in both males and females. The types of chromosomes were 8 large telocentric, 12 medium telocentric and 22 small telocentric chromosomes. The secondary constriction (NORs) was observed at the region adjacent to the centromere of the long arms of a pair of telocentric chromosome. The karyotype formula for N. notopterus is as follows: 2n(diploid)42=L8t+M12t+S22t