Artificial minichromosomes are exciting transgenic tools because they replicate and segregate autonomously and are not integrated into the host genome. They enable efficient delivery of an extremely large gene or multiple genes with high stability. There are two approaches to construct artificial minichromosomes: bottom-up and top-down. The construction of artificial minichromosomes addresses the basic questions of the structure and function of chromosomes. Advances in DNA synthesis technology will open a new era of generating artificial minichromosomes.
This study aimed to evaluate the genetic diversity and to characterize 11 Gossypium hirsutum L. (cotton) germplasms by RAPD and SSR markers. The genomic DNA from the 11 cotton germplasms was amplified with 10 oligonucleotide primers and five microsatellite primer pairs for RAPD and SSR assay, respectively. The 10 RAPD primers generated 335 distinct bands with 100% polymorphisms indicated highly diversed nature. In addition to polymorphism, 29 unique RAPD sequences were identified in 11 cotton germplasms. The five SSR primer pairs generated 69 distinct bands of which 39 were considered as polymorphic (56.52% polymorphisms). Moreover, four unique SSR sequences were identified in three germplasms, namely CB-1, CB-5 and CB-9. The combined RAPD and SSR dendrogram made CB-11 germplasm distinct from the rest and placed alone in a separate cluster that correlated with its phenotypic and agronomic features. Thus, by means of RAPD and SSR markers the genetic diversity was assessed and the 11 cotton germplasms could be characterized authentically.
An in vitro technique was connected with application to produce colchicine-induced hexaploid from the artificial-crossing triploid hybrid of Drosera rotundifolia and D. spatulata. The colchicine-treated plants of artificial-crossing triploid hybrid showed morphologically mutated characteristics. Three hexaploid strains by chromosome doubling were produced after screening of clones treated with 0.05 and 0.1% colchicine solutions for one or three days. Each of the strains had 2n=60 with 20 middle size and 40 small size chromosomes (2n=6x=20M+40S). The stomata guard cell sizes of colchicine-induced hexaploid and the wild hexaploid species D. tokaiensis were larger than that of the artificial-crossing triploid hybrid. Flow cytometry analysis showed that 2C DNA-values of D. rotundifolia (2n=2x=20M), D. spatulata (2n=4x=40S) and D. tokaiensis (2n=6x=20M+40S) were 2.73, 1.41 and 3.74 pg, respectively. In contrast, the artificial-crossing triploid hybrid (2n=3x=10M+20S) and colchicine-induced hexaploid (2n=6x=20M+40S) were 2.31 and 4.41 pg, respectively. The genome size of the artificial-crossing triploid hybrid (2.31 pg) was nearly half the size of the colchicine-induced hexaploid (4.41 pg). Compared to the colchicine-induced hexaploid, the genome size of D. tokaiensis was unexpectedly small, even though they contained the same genome constitutions.
Meiotic behaviour of plant chromosomes is influenced by both genetic and environmental factors. The present paper shows the effect of abnormal meiosis on pollen fertility. Meiotic restitution is considered to be a common mechanism of polyploidization in plants and hence is one of the most important processes in plant speciation. Pollen mother cells (PMCs) of cultivated potato from anthers at different stages of meiotic process were analyzed for their chromosomal behaviour and irregularities. Various meiotic irregularities; univalent and multivalent formation, chromosome bridges, lagging chromosomes, etc. were reported for the first time in India.
Meiotic studies carried out in the individuals of Sedum oreades (Decne.) R. Hamet (Crassulaceae), which exists at diploid level (n=11) revealed the presence of 0-4B-chromosomes, non-synchronous disjunction of bivalents, chromatin bridges, and laggards as well as some pollen sterility (8%) in the collection from Manimahesh Hills (Gauri Kund, 3930 m, Chamba district, Himachal Pradesh). The present diploid chromosome count of n=11 is the first report from India but agrees with the earlier report from Nepal Himalayas. The presence of B-chromosomes in the species is also the first record for the species. The paper here discusses in detail the course of meiosis and the effect of B-chromosomes and other meiotic abnormalities on meiotic course and pollen fertility.
Several bulbs of Scilla scilloides collected at two localities in Northern Taiwan were studied for their karyotypes. All plants had 2n=34 chromosomes and their karyotypes, including the characteristic chromosomes of A and B genomes, were considered to be AABB genome constitution. The karyotype of each plant had two, three or four secondary constricted chromosomes. In all plants, two chromosomes with clear secondary constriction having an elongated thread and satellite corresponded to the b1 chromosome of the B genome. In some plants, two or one a2 chromosomes of the A genome had secondary constriction at the proximal region in addition to the b1 chromosome. All plants formed, at maximum, four nucleoli with various sizes in a nucleus, indicating that they had four NORs in contrast with the number of secondary constrictions. Size variation of nucleoli seemed to correlate with incidence of secondary constriction. The variation of secondary constriction or NOR in AABB plants of Taiwan populations was discussed with AABB karyotypes in other localities.
Plant samples of Ipomoea aquatica Forssk. commonly growing in and around the industrial effluent affected areas were selected to examine the effect of industrial effluents at the chromosomal and DNA levels. Five different industrial areas such as (i) textile, (ii) pesticide, (iii) tannery, (iv) ceramic, and (v) pharmaceutical were selected for this study. We observed 2n=30 chromosomes in all plant samples of Ipomoea aquatica collected from different affected areas similar to control specimens. After comparison with control, the fluorescent karyotypes (CMA and DAPI) showed significant differences among the samples. Fluorescent banding revealed the probable genomic alteration by deletion and tandem duplication. The different plant samples of this species had characteristic RAPD finger printing. Missing DNA fragments in some sample plants (which is present in control plants) may correlate to the deletion of chromosomal parts. Similarly, some new fragments in the samples (which were not found in the control) may correspond to tandem duplication of the chromosomal parts. Therefore, the toxic substances present in the industrial effluents affected the chromosomes and DNA of Ipomoea aquatic grown in and around the above industrial areas.
Meiotic analysis performed in Indigofera tinctoria L. (Family: Fabaceae) reveals n=8 chromosomes with an average chromosomal association of 7.79II+0.44I at metaphase I (MI). Predominance of 8II formation (87.86%) has been noted. The bivalents form rods (mean per cell – rod: 5.88±0.21; ring: 1.87±0.19; association: 7.75II+0.50I) mostly at diplotene with a mean chiasma of 9.62±0.21 per cell. Anaphase I cells (94.12%) are mostly cytologically balanced (8/8) with an average pollen fertility of 93.60%. A persistent feature (89.39%) in MI cells has been the presence of secondary association of chromosomes and the chromosomes assort themselves into variable groups of two (6.04%), three (7.04%), four (25.50%), five (56.71%), six (4.02%) and seven (0.67%). Out of 25 different associations studied among the group classes, 3II(1)+2II(1)+1II(3) under the five group class (25.83%) was the most frequent. Statistical analysis of cytological data reveals that the probable basic chromosome number in the species is x=5, suggesting polyploid lineage.
The Rheum species (R. emodi, R. spiciforme, and R. webbianum) are endangered species and have vanished from lower altitudes. Meiotic studies of the three species show that the polyploidy is reported only in R. webbianum, as it depicts two cytotypes, i.e., diploid (2x) in population from Panzila Top and tetraploid (4x) from Parkhachik and Tangole, of which the diploid cytotype is reported for the first time in this species. Moreover, a diploid cytotype (2x) in R. spiciforme is reported for the first time in India. Further, meiotic course is abnormal in all the studied populations, as all show meiotic abnormalities in the form of cytomixis, chromatin stickiness, unoriented bivalents, chromatin bridges and laggards, leading to abnormal microsporogensis, ultimately affecting pollen viability. Ethnobotanical information depicts the use of different parts of all the Rheum species by local people to control many diseases like piles, constipation, skin eruptions, intestinal infection, wounds, joint pains and also used as food.
Dioscorea deltoidea Wall. (Dioscoreaceae), commonly called “Nepal Yam,” is distributed in the Himalayas, from Kashmir to Assam at altitudes of 450–3100 m. It is an important medicinal plant commercially exploited for the extraction of diosgenin, a pioneer for steroid drugs. This valuable species of the Indian Himalayas faces a serious threat of extinction due to over-exploitation of its tubers and gradual shrinkage of its natural habitat. The species shows considerable intraspecific morphological and cytological variations involving polyploidy and hybridization. In the present study, we examined the meiotic course, microsporogenesis, pollen fertility, and morphological characters of 12 populations of diploid (2x) and four populations of tetraploid (4x) cytotypes of Dioscorea deltoidea. The majority of populations exhibit normal course of meiosis with 100% pollen fertility. However, in six populations (four diploid cytotypes and two tetraploid cytotypes), the individuals show inter-PMC transfer of chromatin material at various stages of meiosis and associated meiotic irregularities such as chromosome stickiness, unoriented bivalents, laggards, micronuclei, and chromatin bridges at different stages of meiosis. Consequently, these populations exhibited varying degree of pollen sterility and heterogenous-sized pollen grains. Analysis of various morphological characteristics of diploid and tetraploid cytotypes revealed that increase in ploidy level in the species is correlated with gigantism of some vegetative characteristics.
Water temperature is one of the most important factors in the survival of aquatic organisms. We investigated the effects of increased water temperature on the physiology of Kumgang fat minnow Rhynchocypris kumgangensis, a small cold-water species of freshwater fish that is endemic to Korea. We examined how organ indices and oocyte histology differed by season and exposure to high water temperatures. Fish were collected from streams in Korea during three different seasons, maintained at the same temperatures at which they were collected for three weeks, and then exposed to either control temperatures reflecting the same conditions from which they were collected (15.0°C, August; 7.8°C, November; and 12.3°C, April) or experimental temperatures that were increased a total of 7°C over seven days (to 22.0°C, August; 14.8°C, November; and 19.3°C, April) and maintained at the final temperature for an additional three days. Among the studied organ indices, the gonadosomatic indexes (GSI) in both sexes differed by season, and were significantly decreased by exposure to increased water temperatures during the germ-cell-developing season. The histological features of oocytes also differed by season, and exposure to increased water temperatures during the relevant season markedly attenuated oocyte development. These findings highlight sensitivity of both GSI and oocyte histology on water temperature change, and the possible negative effects of high water temperature on reproduction in Kumgang fat minnow.
Male meiotic studies have been carried out on the wild plants of Tanacetum artemisioides Sch. Bip. ex Hook. f. collected from the cold desert regions of Ladakh Division of Jammu and Kashmir. The studied individuals, which existed at the 2x level (2n=18), showed structural heterozygosity for reciprocal translocations as indicated by the presence of multiple chromosomal associations of four to six chromosomes. This is the first report of structural heterozygosity for the species. The structural heterozygotes showed considerable amount of pollen sterility (30–35%) which could be attributed to the presence of ring/chain shaped multivalent in PMCs.
The nucleus wields undisputed control in the normal disposition of almost all cellular processes. It houses most, if not all, parts of the genetic information of an organism. A nuclear morphology deviant from one which is normally encountered is proof of genomic instability. The presence of micronuclei authenticates this phenomenon. The present study reports for the first time, the dose-specific occurrence of micronuclei and micro-cells during microsporogenesis in Crotalaria juncea L. in response to colchicine exposure. It also assesses various meiotic chromosomal abnormalities induced in response to the dose administered and the trends discernible.
Urginea indica Kunth. Hyacinthaceae, commonly called Indian Squill, represents a species complex. Intraspecies variation is common. Karyological studies were made in six accessions of Urginea collected from six different places in India, of which four accessions of Urginea indica were collected from Kanakapura, Trichendur, Kashmir valley, and Kerala. Two accessions of Urginea wightii were from Gubbi and Bellary. Variations in ploidy were noted. Among the Urginea indica accessions, diploids (2n=20) were found in Kanakapura, Trichendur, and Kashmir Valley; a pentaploid (2n=50) was noticed in the Kerala accession, whereas two different ploidies were noticed in the Urginea wightii accessions of Gubbi and Bellary (2n=20, 2n=40) within the same plant. There is close homology between Kanakapura and Tiruchendur with subtelocentric being a major component.
Heracleum pinnatum C. B. Clarke (Apiaceae) collected from the cold deserts of Ladakh Himalaya, Jammu and Kashmir, India, is cytologically analyzed for the first time, revealing a diploid chromosome count of 2n=22. During meiosis, the majority of the pollen mother cells (PMCs) exhibited 11 bivalents, equal segregation of chromosomes during anaphases, regular tetrads, and normal-sized pollen grain formation. Occasionally, two proximate PMCs fused during the early stages of prophase-I and resulted in the formation of syncytes. The frequency of such syncytic meiocytes was low (3.72–3.96%) but these could be easily detected in the preparations due to their large size compared to typical PMCs. Also, the syncytes depicted a meiotic chromosome count of 2n=44 as confirmed from the presence of 22 normal bivalents. Further, meiotic course in such tetraploid PMCs was also observed to be perfectly regular, leading to the formation of ‘2n’ or larger-sized pollen grains that are almost double the size of typical ‘n’ pollen grains. Such larger-sized ‘2n’ pollen grains could also be differentiated on the basis of their shape. Additionally, a significant frequency of PMCs also showed the phenomenon of cytomixis involving transfer of chromatin material resulting in aneuploid meiocytes. Whole chromatin transfer during cytomixis among neighboring PMCs at early stage of meiosis-I lead to the formation of PMCs with double the chromatin material. Syncytes resulted as a consequence of fusion of meiocytes during the early stages of meiosis-I could be attributed to low temperature stress conditions prevailing at the time when the plants enter the flowering stage. It is quite possible that such apparently fertile ‘2n’ pollen grains originating from syncytes might play a role in the origin of polyploids in the species.
In algae, vacuolar polyphosphate stores phosphate, regulates osmotic pressure and cytosolic pH, and buffers metallic cations. However, its metabolism is not well understood. Cyanidioschyzon merolae cells contain four to five vacuoles storing polyphosphate whose degradation and synthesis are induced by phosphate deprivation and resupplementation, respectively. To understand polyphosphate metabolism, we observed C. merolae cells cultured in different phosphate conditions by electron microscopy. In the phosphate-containing medium, the vacuoles contained electron-dense substances presumably consisting of polyphosphate and metallic cations. When phosphate was depleted, the vacuoles lost the electron-dense substances, and lipid bodies in close contact with vacuoles and starch grains appeared in the cytoplasm. When phosphate was added again, small, electron-dense granules or vesicles appeared in the cytoplasm. Some of them were in contact with the vacuoles and had been taken into the vacuolar lumen, suggesting the possibility that polyphosphate is synthesized and transported to the vacuoles in these structures.
Both male and female Coccinia grandis L. were studied cytogenetically using conventional and fluorochrome dyes. In addition, RAPD was carried out to distinguish the two sexes. In male plants, a pair of prominent satellites was observed, one in each member of pair III. Both male and female plants were found to possess 2n=24 metacentric chromosomes (including “Y-chromosome”). The length of individual chromosomes ranged from 0.97 to 5.03 µm (Y-chromosome) in the male and 0.78 to 1.32 µm in the female plant. The Y-chromosome found in this study was 5.03 µm, which is about 3.06 times bigger than the largest autosome. The interphase nuclei of male specimens possessed a number of thick, round and darkly stained heterochromatic blocks. A bigger and elongated area was also observed in the interphase nuclei of male plants, which indicated the probable appearance of heterochromatic Y-chromosome. A distinct Y-chromosome was observed at prophase after staining with orcein, CMA, and DAPI. The Y-chromosomes were fully fluoresced with both CMA and DAPI, revealing the tandem existence of GC- and AT-rich repeats along the length. The most striking feature is that the Y-chromosome with distinct centromeres was observed even in the interphase nuclei and prophase stages after orcein, CMA, and DAPI staining. It suggested that the Y-chromosomes contracted much earlier than those of autosomes. This feature of the Y-chromosome has not yet been reported and is a new unique feature of Y-chromosomes of C. grandis. Different types of CMA- and DAPI-banding patterns were found in male and female plants, which are characteristic features of each sex. The difference of GC- and AT-rich percentage was due to the Y-chromosome. Four different primers were used for RAPD profiling. Polymorphism of RAPD bands was distinct in male and female plants. Therefore, the compilation of the above information will be very useful for making multidimensional genetic information of male and female C. grandis L. found in Bangladesh.