Fluorescence in situ hybridization (FISH) is an experimental technique used in chromosome analysis. In situ hybridization (ISH) for chromosomes has been used for over 40 years. The development of a non-radioactive fluorescent detection system enabled the identification of multiple specific nucleotide sequences on chromosomes. Multi-probe detection further increased the speed of chromosome analysis. Here, we describe the use of FISH in plant chromosomes, from the labelling of the probes to multi-color detection and the creation of multi-color FISH images.
RAPD analysis was carried out using 1200 decamer random sequences to clarify the whole fingerprint of DNA fragment appearance patterns among three Japanese Drosera species: D. tokaiensis, D. rotundifolia and D. spatulata. The RAPD analysis results showed that D. tokaiensis had not only magnificent bands common to other two species, but also many specific bands, although D. tokaiensis is of amphiploidal origin between D. rotundifolia and D. spatulata. The specific bands of D. tokaiensis might generate after the speciation or amphiduplication. Therefore, the relationship of genome compositions among the three species suggested that RAPD fragments were preferentially amplified from 20 middle-sized chromosomes in D. rotundifolia and D. tokaiensis. Thus, the chromosome information and the RAPD profiling suggested that alloploidal genome formation during Drosera speciation might obtain new beneficial genetic characters to survive and adapt to certain environments.
Somatic chromosomes (2n=26) of Araucaria araucana, Araucariaceae were observed for the first time by the fluorescent banding method using chromomycin A3 (CMA) and 4′,6-diamidino-2-phenylindole (DAPI). The karyotype was composed of nine pairs of long metacentric chromosomes and four short chromosomes and was similar to previous works. Large and thick CMA-bands appeared at the proximal region or secondary constriction of two long metacentric chromosomes. Several weak DAPI-bands appeared at the interstitial and/or centromeric regions on some chromosomes. Most chromosome pairs were identified by chromosome shape and fluorescent banding pattern.
The distribution and composition of C-heterochromatin in four species of Mictini (Coreinae), viz., Anoplocnemis compressa (Dallas, 1852), Anoplocnemis binotata (Distant, 1918), Ochrochira nigrorufa (Distant, 1889) and Prionolomia sp., have been analyzed by C-banding and DAPI/CMA3 sequence specific staining. Cytogenetically, the possession of holokinetic chromosomes and a pre-reductional type of meiosis for sex chromosomes characterize these four species. The C-banding pattern has been found to be species-specific. In A. compressa, C-bands are thick and terminal, whereas in A. binotata, very thin C-bands are seen interspersed throughout the length of chromosomes. In Ochrochira nigrorufa, thick C-bands are present at terminal and interstitial regions. In Prionolomia sp., two conspicuous terminal C-bands are observed only on the largest autosomal pair while the rest of the complement is completely C-negative. This unique pattern can serve as a powerful cytological marker. Constitutive heterochromatin has been found to be rich in both AT and GC base pairs in all the studied species.
A population of cultivated Haworthia limifolia displayed various types of chromosomal configurations at anaphase/telophase-I and -II due to inversion heterozygosity. It was characterized by the presence of bridge and fragment because of various numbers and positions of crossovers in the inversion loop. The heterozygous condition led to reduced pollen fertility.
Polyploidy was induced by colchicine in Allium cepa L. Five plants of Allium cepa (AC1–AC5) were isolated from the 0.2% colchicine treated inflorescences. The meiotic stages were analyzed in treated buds. Various cytological features like chromosomal associations (quadrivalents, bivalents and univalents), chiasmata frequency and terminalization coefficient were recorded at diakinesis/metaphase-I. Anaphase/telophase-I/II were studied in all the cholchitetraploids. In all these plants, the anaphase/telophase-I were abnormal showing unequal distribution, laggards, micronuclei and multipolar at telophase-II. Pollen mitosis was studied in these plants. The chromosomes (n=16) were found at metaphase. Pollen fertility was low (40.9 to 56.9%) in all colchitetraploids.
Climate change requires breeders and pre-breeders to harness new sources of genetic variation, as having a narrow genetic base is detrimental for continued food production targets projected for 2050 globally. Finding tolerance across alien resources is not an obstacle. It’s where that tolerance resides and the nature of the alien source as to its genetic make-up. From the mid-1950s the interest on wild wheats has existed and numerous academic findings emanated, but the most impacting potential that has now become a forte for progress is seen in the closely related diploid (2n=2x=14) progenitor species that are genome-wise homologous to the wheat genome like D (Aegilops tauschii) and A (Triticum urartu ssp. boeoticum, monococcum). Hence from the mid-1980s, the wheat progenitor accessional diversity exploitation emerged on the scene. This allowed researchers to target their time based practical productivity returns by selecting the most related alien resources around wheat/alien homologous chromosome pairing to affect a maximum recombination output and exploit the primary Triticeae gene pool. The complementation of just one extensively used resource (Ae. tauschii) has since the 1990s opened up a resource to capture allelic diversity for many stresses, enrich the molecular diagnostic tools and provide outputs that are also extremely valuable for the environmental shifts that are coming up, notably like salinity, drought, and more vital, the “heat” regimen changes. These new novel wheats known as “synthetic wheats” based upon the cultivated wheat’s D-genome accessional genetic diversity have shown superb promise. This experiment with the same Ae. tauschii accession male parent crossed with different durum cultivars as the female parent (78 entries) is designed to study the inheritance of different genes and also to identify the effect of cytoplasmic inheritance, if any. The total of 78 entries was screened against two biotic stresses (Karnal bunt and stripe rust), phenologically characterized and analyzed with SSRs for molecular characterization.
Tussilago farfara of family Asteraceae has been worked out cytologically for the first time from India. The presently studied individuals collected from Solang Valley, Kullu District, Himachal Pradesh exist at triploid level with 2n=24 against the earlier highly polyploidy counts of 2n=60 and 2n=72 from outside of India. These individuals depict abnormal meiosis characterized by the presence of trivalents and univalents, unequal distribution of chromosomes and laggards during anaphases/telophases resulting into abnormal sporads and high pollen sterility. The triploid individuals might have originated from a hybridization process involving tetraploid and diploid as putative parents.
Three varieties of Glycine max (L.) Merr. (soybean) released by Bangladesh Agricultural Research Institute (BARI), viz., BARI-5, BARI-6 and Shohag, were investigated cytogenetically and at the molecular level using RAPD-markers for authentic characterization. The three varieties represented variation for several phenotypic and agronomic traits. Although the varieties were all found to possess 2n=40 metacentric chromosomes, they differed in other karyotypic features such as total length of 2n chromosome complements, range of relative length, centromeric index, etc. Five to eight CMA-positive bands were found at different locations in the metaphase chromosomes of the three soybean varieties. Entirely DAPI-fluoresced chromosomes were frequent in these varieties. The number, location and distributions of GC- and AT-rich repeats are specific for each variety. Fluorescent banding revealed the occurrence of genomic alterations within these varieties. Five RAPD primers generated 36 distinct bands with 66.67% polymorphisms indicating a highly diversed nature. In addition to polymorphism, seven variety specific RAPD fragments were identified in the three soybean varieties. The dendrogram based on RAPD analysis made variety Shohag distinct from the other two varieties and placed it in a separate cluster that correlated with its phenotypic, agronomic and cytogenetical features. Therefore, each variety has been characterized authentically by cytogenetical and molecular analysis.
Three varieties of Lathyrus sativus L. released from Bangladesh Agricultural Research Institute (BARI), viz., BARI Khesari-1, BARI Khesari-2 and BARI Khesari-3, were studied cytogenetically and at the molecular level by using RAPD for genomic characterization. The three varieties were found to posses 2n=14 chromosomes. The karyotype formulae of BARI Khesari-1 and BARI Khesari-2 are 8m + 6sm, while it is 10 m + 4sm in BARI Khesari-3. After orcein and CMA staining, one to two very small chromosome-like bodies were found in some cells of BARI Khesari-1, whereas no such body was observed after DAPI staining. Due to their unique features these can be considered as GC-rich heterochromatic “B-chromosomes.” In BARI Khesari-2, two pairs of satellites were found, while only one pair was present in BARI Khesari-3. The inverted position of satellited regions indicated the probable occurrence of homozygous inversion. An indication of paracentric inversion regarding DAPI-banding pattern was found in pair V of BARI Khesari-2 and thus could be considered as marker chromosomes for this variety. The three varieties have distinct CMA- and DAPI-banding patterns. In BARI Khesari-2, a total of 12 C-bands were found on 10 chromosomes (out of 14 chromosomes). On the basis of band position and length, a deep correlation between C-banding and DAPI-banding pattern was found suggesting the heterochromatic nature of DAPI bands. Each variety showed different RAPD fingerprinting with 59.09% polymorphisim. In addition, a number of variety-specific unique RAPD bands was observed. Therefore, each variety could be characterized on the basis of karyotype and RAPD analysis.
The aim of this study was to evaluate the possible hepatoprotective effects of green tea extract (GTE) against formaldehyde (FMD) induced hepatotoxicity in albino mice with biochemical and histopathological approaches. Swiss albino mice were randomly divided into six groups, each consisting of six animals. Serum aspartate aminotransferase, alanine aminotransferase, malondialdehyde, glutathione and advanced oxidized protein product levels, and histopathological changes were also investigated in liver tissues of mice. The results indicated that FMD-induced oxidative damage caused a significant decrease in aspartate aminotransferase, alanine aminotransferase, glutathione levels, and a significant increase in malondialdehyde and advanced oxidized protein product levels of the liver tissues. Each dose of GTE provided significant protection against FMD-induced toxicity and the strongest effect was observed at a dose of 150 mg kg−1 bodyweight histopathological studies showed that FMD caused some structural damages such as hepatocyte degeneration and necrosis. In vivo results showed that GTE extract is a potent protector against FMD-induced hepatotoxicity.
We used a microplate-based method to quantify microalgal lipids with Nile Red staining and a fluorescence microplate reader. However, a method to quantify starch that combines microplates with staining has not been reported. Therefore, we examined microplate-based quantification of lipids using Nile Red staining and of starch using Lugol staining. Neither starch nor lipids accumulated during the zero phase of cultured Parachlorella kessleri, only starch accumulated during the starch phase, and starch was subsequently lost and lipids accumulated during the oil phase. The quantities of starch and lipids were measured using a microplate-based method, which indicated linear production of starch and lipids within limited ranges (lipids, 0.071–0.380 g mL−1; starch, 155–404 mg mL－1) when standard curves were prepared for lipids extracted with methyl tertiary-butyl ether and for starch extracted with anthrone. The concentration of starch produced during the starch phase was 0.59 mg mL−1 and that of lipids during the oil phase was 2.49 mg mL−1. The concentration of starch produced was 0.05–0.13 mg mL−1 during phases other than the starch phase, and lipids were not detected other than during the oil phase because lipid contents were approximated based on the quantity of triacylglycerol, which stains with Nile Red.
The effective potentiality of anticancerous drugs, namely, cisplatin, etoposide and vinblastine, as well as the ethanolic extract of Piper betle L. leaves, is evaluated (concentrations used: 0.001, 0.01, 0.1, 1.0 and 10.0 µM for vinblastine; 25, 50, 75, 100, 125 and 150 µM for other test materials) on germinating grass pea (Lathyrus sativus L.) seedlings in relation to radicle length and mitotic index. Furthermore, inhibition in the frequency of dividing polyploid cells induced due to prior treatments of colchicine (0.5%, 8 h) and in vitro callus growth are also assessed following treatments with different concentrations of the test materials. The objective of the present study is to determine the effectivity of the test materials on a plant species with the view to develop a plant system (convenient to use and cost effective) as a model for preliminary screening of novel anticancerous drugs as well as plant extract(s) possessing such potentiality for further exploration.
We present the karyotypes of three Japanese alpine Taraxacum species (Taraxacum alpicola, T. yatsugatakense and T. yuparense) in order to provide insight into their origins and taxonomic relationships. T. alpicola and T. yatsugatakense were triploid plants with similar karyotypes (2n=24=18(M+m)+4mcs+2smcs), while T. yuparense was a diploid plant with the karyotype 2n=16=12m+4mcs. The karyotypes of both T. alpicola and T. yatsugatakense were considered a combination of two chromosome sets of 6m+2mcs and one chromosome set of 6m+2smcs. Our study reveals that T. alpicola and T. yatsugatakense are autoallopolyploids.
In this study the effect of different colchicine concentrations (0.025, 0.05, 0.1 and 0.2%) and treatment time (8, 24 and 48 h) on in vitro polyploidy induction from shoot tips was investigated as a factorial experiment based on completely randomized design with three replications. Polyploidy induction was confirmed by chromosome counts, size and number of stomata and other morphological characters. By assessment of different media, it was revealed that the suitable medium for regeneration of shoot tips is MS medium supplemented with 0.1 mg L−1 IBA (indole-3-butyric acid) and 1 or 2 mg L−1 BAP (6-benzylaminopurine) or 2 mg L−1 TDZ (thidiazuron). In addition, MS medium supplemented with 1 mg L−1 NAA (1-naphthaleneacetic acid) and 0.5 or 1 mg L−1 IBA led to the maximum rooting of plantlets. With increasing colchicine concentration and its treatment duration, explants survival and their rooting considerably decreased. The results revealed that 0.1% concentration of colchicine with 48 h of treatment established the maximum amount of the in vitro induced tetraploid plantlets. The derived tetraploid plantlets had bigger stomata with lower density. Chlorophyll content in tetraploid plantlets was significantly higher than diploids, and they also showed an elevated level of antioxidant enzymes, protein and soluble carbohydrate contents as compared with diploids.
Micronuclei are extra nuclear structures, formed by the exclusion of chromosomes during cell division. The genotoxicity of a compound and its impact on the genome can be assessed via micronuclei formation. The present study has been undertaken to evaluate the mutagenic effect of the alkylating agent EMS on the meiotic behavior of pollen mother cells of Phaseolus vulgaris L. The cytological analysis revealed the induction of micronuclei at all the three administered concentrations of EMS, viz. 0.1, 0.3 and 0.5%. Besides micronuclei formation, other meiotic abnormalities associated with chromosomal segregation, such as stickiness, laggard, bridge, multivalent, etc., were also reported at metaphase I/II and anaphase I/II. The micronuclei formed would have two fates—either it remains until the tetrad stage or is expelled out in the form of microcytes which will give rise to sterile pollen grains affecting the pollen fertility. The present study also documents the first instance of nuclear budding in Phaseolus vulgaris L. It is contemplated that these nuclear buds will later on convert into micronuclei. The micronuclei formation results in partial elimination of the genome, which could be efficiently utilized in breeding programmes for the production of addition and substitution lines. Similarly, haploid lines could also be produced by the complete loss of genome. It is therefore essential to understand the biological aspects of micronuclei formation, its consequences on the cell carrying it and other factors related to it so that in the future, it could be used as a marker of genetic damage and also be utilized in breeding programmes.
Taxonomic studies of the spondylomoracean genus Pyrobotrys have remained at traditional levels due to the lack of molecular phylogenetic analyses and available culture strains for reexaminations. Here, we established new culture strains of Pyrobotrys using novel culturing methods, and classified them into three species, namely, P. casinoensis, P. elongata, and P. squarrosa, based on comparative microscopic observations and molecular phylogeny. Although P. elongata is characterized by its enormous stigma in cells of the most posterior tier in a colony, such a species has not been reported since its original description in 1938. In the present study, the presence of an enormous stigma was confirmed based on light microscopy in three P. elongata strains originating from two localities. Based on transmission electron microscopy, the large stigma of P. elongata was essentially identical to the stigma of other species of Pyrobotrys in having a single layer of stigma globules. Our molecular phylogeny demonstrated that P. elongata was robustly separated from other species of Pyrobotrys.