We have developed a convenient method to visualize triacylglycerol-filled lipid droplets (LDs) in some species of bacteria, algae and fungi by staining with borondipyrromethene difluoride (BODIPY). When BODIPY was excited by blue light, LDs emitted green fluorescence, which was distinguished easily from the red autofluorescence of chloroplasts. This makes BODIPY staining suitable for the identification of small amounts of LDs, especially in plants. We first ensured that in Chlamydomonas reinhardtii cells growing in nitrogen-replete (+N) and -deficient (-N) media, the spots of BODIPY-stained LDs coincided with those of Nile Red-stained LDs. In addition, it was shown that the LD content per cell in N-starved cells was 200-fold higher than those of the control (+N) using a video-intensified microscope photoncounting system (VIMPCS). BODIPY staining was applied to visualize LD in bacteria, algae and fungi, and included those algae regarded as non-oleaginous. We identified LD spots in unicellular and multicellular bacteria and eukaryotes, namely Cyanidioschyzon merolae, Cyanidium caldarium delta, Chlamydomonas reinhardtii, Klebsormidium nitens and Penicillium sp., but not in Anabaena flos-aquae. We also examined the relationship between the contents of LDs and the genome size in the algae and fungi using VIMPCS but were unable to find a strong relationship between genome size and production of LDs. Finally, the location of LDs was considered in relation to organelles including the endoplasmic reticulum and chloroplasts, which are related to the formation of LDs.
Intraspecific hybrids of Momordica charantia produced by both direct and reciprocal crosses were characterized for plant morphology, cytology, foliar epidermal and palynological features in comparison with cultivated and wild varieties. The parents and F1 hybrids were evaluated for a total of 56 morphological characters including 17 qualitative and 39 quantitative traits. Both the parents and the F1 hybrids showed significant variations in most of the quantitative characters. The hybrids were intermediate in characters like color of the ovary, color and shape of fruit, number of seeds and in the distribution of trichomes on the stem and leaves, although they exhibited most of the parental characters. Unweighted Pair Group Method with Arithmetic Mean (UPGMA) dendrograms and Principal Coordinate Analysis (PCoA) scatter plots revealed the grouping of cultivated varieties together with the F1 hybrids. The most important loading traits as per the Principal Component Analysis (PCA) were pedicel length, fruit weight, fruit length, fruit diameter , fruit color, and the number of seeds. Cytological, foliar epidermal and palynological features are used to discuss the interrelationship of the 2 varieties of M. charantia.
In the present study, 5 species of Lygaeidae and 1 each of Berytidae and Malcidae have been cytologically investigated for the first time; the diploid numbers in them vary from 12 to 20. Out of the 5 lygaeids, male diploid number is 12=8A+2m+XY in Dieuches leucoceras and Polycrates nexus while being 14=10A+2m+XY in Aphanus orientalis, Lanchnophorus sp. and Mizaldus sp. A pair of extremely large autosomes is found in both the species with 2n=12 while it may or may not present in species with 2n=14. One species each of Berytidae (Metacanthuspulchellus) and Malcidae (Malcus flavidipes) show 2n=20=18A+XY and 2n=16=12A+2m+XY respectively, and both lack an extremely large pair of autosomes. Microchromosomes are present in Lygaeidae (Rhyparochrominae) and Malcidae but absent in Berytidae. The sex determining mechanism in all the species of 3 families is XY/XX (♂/♀). Fragmentations seem to have played a pivotal role in the origin of new species irrespective of family.
The F1 hybrids obtained and cytologically investigated from 2 reciprocal cross-combinations in the genus Antirrhinum–A.litigiosum–A.subbaeticum and A. pulverulentum–A. subbaeticum were diploids 2n=2x=16, as their progenitors. In the hybrids pollen mother cells (PMCs) parallel with variously shaped symmetric and asymmetric bivalents with one or more chiasma formation, positioned in different chromosome regions, univalent and multivalent associations, were presented. The lower values of the chiasma frequency and number of ring type of bivalents in F1 plants indicated a certain degree of differentiation between homologous chromosomes of the parent's components and suggested different activities of genes controlling chiasma formation and position. The meiotic irregularities in F1 progeny could be caused from karyotypic dissimilarities between the wild Antirrhinum genomes on the one hand and chromosome rearrangements (presence of multivalent and univalents, late bivalent disjunction, bridge formation) on the other. The evaluation of the chromosome pairing affinities, the abnormalities in chromosome behaviour at anaphase and telophase stages, and the pollen fertility in the F1 hybrids suggested a higher expressed phylogenetic distance between A. litigiosum and A. subbaeticum than the one between A. pulverulentum and A. subbaeticum genome, as the karyotype remoteness between A. litigiosum and A. subbaeticum cytologically seems to be expressed more strongly than that between A. pulverulentum and A. subbaeticum species. The cytological results confirmed the systematic relationships between the studied species in the genus Antirrhinum.A. pulverulentum and A. subbaeticum are closer to each other than to A. litigiosum and are included in the subsection Kickxiella, while A. litigiosum belongs to subsection Antirrhinum.
Cytological investigations have been carried out on the 9 wild accessions of Solanum nigrum L., a medicinally important species having wide-range therapeutic properties. Male meiotic studies performed in 9 accessions revealed the presence of 3 intraspecific cytotypes, 2x (n=12), 4x (n=24) and 6x (n=36) in the species. The course of meiosis in 8 of the 9 accessions among the 2x, 4x, 6x cytotypes was noticed to be perfectly normal resulting in very high pollen fertility (95–100%). However, 1 accession of 4x cytotype scored from Palchan (Solang Valley, Kullu, Himachal Pradesh) depicted irregular chromosomal behaviour during meiosis due to the presence of all 48 chromosomes as univalents. The further course of meiosis in this individual was highly irregular with the presence of large number of lagging and unoriented chromosomes, eccentric positioned univalents in the PMCs and unequal distribution of chromosomes at anaphase-I. Microsporogenesis was also abnormal which is characterized by the presence of irregular sporads, such as dyads, triads and polyads. Lagging chromosomes constituted micronuclei at sporad stages. Consequent to all these meiotic irregularities, 95% of the pollen grains were observed to be sterile. Pollen grains were also recorded to be of different sizes. In meiotically abnormal 4x accession, pollen grains were of 2 different types compared to normal 4x plants showing perfectly normal meiosis. The large pollen grains, which were measured to be 1.25–1.5 times bigger than normal sized pollen grains, were considered as unreduced or 2n pollen grains. The role of such 2n (unreduced) pollen grains in producing polyploid genotypes through sexual polyploidization in a chromosomally variable S. nigrum (2x, 3x, 4x, 5x, 6x, 8x, 9x, 12x) has also been discussed.
Chromosome and detailed karyotype information (number, shape, relative length, arm ratio, centromeric index) are described for E. alacris alacris of Chandoli National Park, India, belonging to the subfamily Eyprepocnemidinae. The diploid number of the chromosome was found to be 2n=23 with all metacentric pairs. The X chromosome is metacentric.
Among radiation-induced chromosome aberrations, multicentric chromosomes, as represented by dicentric chromosomes (dicentrics), are regarded as sensitive and specific biomarkers for assessing radiation dose in the 0 to 5 Gy range. The objective of this study was to characterize chromosome aberrations induced in vitro by a higher dose of radiation. Peripheral blood lymphocytes were exposed to 15 Gy gamma rays at a dose rate of 0.5 Gy/min and harvested at 48, 50, 52, 54, 56 and 72 h. The first mitotic peak appeared at 52–54 h, showing about a 6 h mitotic delay as compared with nonirradiated control cultures. Cell-cycle analysis of parallel and simultaneous cultures by sister-chromatid differentiation staining suggests that metaphase cells examined in 48–56 h cultures were in the first mitosis after culture initiation. The mean dicentric equivalent counts ranged from 9.0 to 9.3 in consecutively harvested cultures with no significant differences among them. At 72 h, about 20% of dividing cells were tetraploid, persisting with faithfully replicated unstable chromosome aberrations. The non-random distribution of replicated chromosome pairs, deduced from multicolor fluorescence in situ hybridization analysis, led us to surmise that the predominant mechanism underlying the induction of tetraploid cells is endoreduplication. These findings suggest that a high-dose in vitro irradiation applied to peripheral blood lymphocytes may affect on the replication process, in addition to structural chromosome damage.
Anthocyanins and flavones are biosynthesized from flavanones as a common intermediate, which is the common metabolic branch point for anthocyanin and flavone biosynthesis. The conversion of dihydroflavonols from flavanones for anthocyanin biosynthesis and the conversion of flavones from flavanone for flavone biosynthesis are catalyzed by flavanone 3-hydroxylase (F3H) and flavone synthase (FNSI, II), respectively. Therefore, to elucidate the molecular genetic mechanism of copigmentation between anthocyanins and flavones, the F3H and FNS genes and their transcriptional factors should be characterized, and in this study, 3 cDNA clones encoding F3H named IhF3H1, IhF3H2, and IhF3H3 were isolated and characterized from the flower buds of Dutch iris, Iris × hollandica. Nucleotide sequence analysis showed that IhF3H1 (Genbank accession no. AB183826) is 1,313 bp long and contains an open reading frame (ORF) encoding 370 amino acids with a calculated molecular mass of 40,995 Da and an isoelectric point (pI) of 5.47. IhF3H2 (Genbank accession no. AB265225) is 1,295 bp long and contains an ORF encoding 372 amino acids with a calculated molecular mass of 41,139 Da and a pI of 6.42. IhF3H3 (Genbank accession no. AB265226) is 1,335 bp long and contains an ORF encoding 372 amino acids with a calculated molecular mass of 41,117 Da and a pI of 5.69. The soluble crude protein extracts of Escherichia coli cells expressing IhF3H1, IhF3H2, and IhF3H3 were subjected to flavanone 3-hydroxylation assays in the presence of naringenin as a substrate and 2-oxoglutarate, ascorbate, and FeSO4 as cofactors. Heterologous expression demonstrated that each IhF3H cDNA encodes functional flavanone 3-hydroxylase, which catalyzes 3-hydroxylation from naringenin to dihydroflavonol.
The objective of this study is to analyse the karyotype to determine the chromosomal asymmetry and its relationship with the evolution of species of Eryngium: E. elegans Cham et Schlecht, E. ebracteatum Lam., E. horridum Malme and E. coronatum Hook & Arn. in Argentina. E. elegans, E. ebracteatum and E. horridum have 2n=16, and E. coronatum has 2n=48, with relatively symmetric karyotypes. It is suggested that the studied species had great adaptation to diverse environmental conditions reaching a great invasive power and aggressiveness as weeds, keeping their karyotype through the evolution.
Coreinae, the largest subfamily of Coreidae, is distributed worldwide but is most abundant in the tropics. Its 2 tribes, Homoeocerini and Petascelini, are specifically found in the Eastern Hemisphere. In 118 species of Coreinae cytogenetically analysed so far, male diploid number ranges from 15 to 29 and variations are reported in chiasma frequency, metaphase arrangement pattern and anaphasic movement of sex chromosomes during meiosis. In view of this, meiotic behavior of chromosomes in 2 North Indian species of Coreinae viz. Homoeocerus signatus Walker (Homoeocerini) and Petillopsis patulicollis (Walker) (Petascelini) has been described for the first time and characteristic features are discussed in the present paper. The male diploid chromosome complement of H. signatus Walker is 2n=21=18A+2m+X0 while that of P. patulicollis (Walker) is 2n=28=24A+2m+X1X20. The general course of meiosis is fairly uniform and is typical for heteropteran types. During diplotene, a single chiasma per bivalent is observed in H. signatus Walker whereas 2 chiasmata are observed in 2 to 3 bivalents in P. patulicollis (Walker). In both the species, the arrangement of chromosomes at metaphase I is typical of coreid types. In P. patulicollis (Walker), however, a few metaphase I plates are observed with an alternate arrangement.
Chromosome counts were carried out in root tip cells of Nepenthes khasiana (Nepenthaceae), a threatened insectivorous plant of Northeast India. N. khasiana has become threatened in its natural habitat due to overexploitation for its medicinal uses as well as its ornamental importance. Plantlets of Nepenthes khasiana collected from Jarain, Meghalaya were cytologically analyzed. All the root tip cells analyzed showed the chromosomenumber of 2n=80 without any variations. Karyomorphological studies were not plausible in this species due to the relatively small size of the chromosomes.
Male meiotic studies have been conducted in a synaptic variant of Mantisia wengeri, a critically—endangered and endemic rhizomatous herb of north-east India. Studies reveled synaptic variation of the chromosomes and the only 10% of pollen mother cells (PMCs) showed the normal meiotic pattern, while 90% of PMCs demonstrated abnormal meiotic behavior by showing a spectrum of chromosome associations. A total of 747 bivalents along with a good number (506) of univalents were recorded with moderate chiasma frequency (14.94±9.20) with the terminalization coefficient of 0.82 only. About 40% of PMCs showed anomalous distribution patterns of chromosomes, including unequal distribution and/or presence of laggards in the form of univalents/bivalents resulting in low pollen-stainability (57.33±8.40). A comparative analysis of synaptic behavior of chromosomes during meiosis in M. wengeri and M. spathulata was carried out. The role of genomes vis-à-vis the chromosome behavior of M. spathulata and M. radicalis in the development of the crossbreed M. wengeri has been discussed. Unbalanced chromosome associations with partial synaptic failure are considered as causative factors for sustained diversity in critically-endangered and endemic genus Mantisia.
The first chromosomal characteristics of nucleolar organizer regions (NORs) of the clown knife fish, Chitala ornata (Gray 1831) from the Chi River in Roi-Et Province, northeast Thailand, were studied. Blood samples were taken from 4 male and 4 female fish. Standard whole blood lymphocytes were cultured at 27°C for 96 h in the presence of colchicines. Metaphase spreads were performed on microscopic slides and then air-dried. Conventional and Ag-NOR staining techniques were applied to stain the chromosomes. The results showed that the diploid chromosome number of C. ornata was 2n=42, and the fundamental number (NF) was 44 in both male and female. Chromosomes types were present as 8 large telocentric, 2 medium acrocentric, 14 medium telocentric, and 18 small telocentric chromosomes. No strange sized chromosomes related to sex were observed. The region adjacent to the short arm near the centromere of chromosome pair 11 showed clearly observable secondary constriction/NORs. The karyotype formula for C. ornata is as stated: 2n (diploid) 42 = Lt8 + Ma2 + Mt14 + St18
Karyological analysis and morphometrics of the lesser Asiatic house bat (Scotophilus kuhlii) from Thailand were studied. Blood samples were taken from 5 male and 5 female bats, lymphocytes were cultured at 37°C for 72 h in presence of colchicine, and metaphase spreads were performed on microscopic slides and air-dried. Conventional staining and GTG-banding techniques were applied to stain the chromosomes. The results showed that the diploid chromosome number of S. kuhlii was 2n=36, and the fundamental number (NF) was 52 in both male and female. The types of autosomes were 6 large metacentric, 4 large submetacentric, 6 large telocentric, 2 medium submetacentric, 10 medium telocentric, 2 small metacentric, and 4 small telocentric chromosomes. The X chromosome was a large submetacentric chromosome and the Y chromosome was the smallest acrocentric chromosome. The GTG-banding technique, indicated that the number of bands and locations in S. kuhlii was 167, and that each chromosome pair could be clearly differentiated. In addition, a pair of long arms near the centromere of chromosome pair 17 showed clearly observable nucleolar organizer regions (NORs). In terms of morphological measurement, the following measurements were taken 6 external morphological characters as well as 13 cranial and dental characters. The karyotype formula of S. kuhlii was as follows: 2n (36)= Lm6+Lsm4+Lt6+Msm2+Mt10+Sm2+St4+XY