A procedure for the simultaneous isolation of mitochondrial and plastid nucleoids was first established in BY-2 cells. Biochemical analysis suggested the presence of nucleosome-like repetitive structural units in both of the plant organelle nucleoids. The isolated organelle nucleoids were also used for establishment of in vitro transcription/DNA synthesis systems, with which regulation of organelle genomes during proliferation and differentiation was investigated. The results revealed that transient and synchronous activation of DNA synthesis occurred in mitochondria and plastids in the initial phase of cell proliferation, which was caused by a transient activation of dually-targeted organelle DNA polymerase genes. Another series of investigations revealed a drastic difference in the transcription activity between BY-2 proplastids and leaf chloroplasts, which was brought about by differential use of bacterial- and phage-type plastid RNA polymerases. To investigate regulation of plastid gene expression further, a procedure to induce amyloplast formation in BY-2 cells was established. Various changes observed during this process were collectively similar to those observed during root cap cell differentiation. BY-2 cells were also used to develop a novel model system for programmed cell death during hypersensitive reaction (HR), in which we have succeeded in inducing programmed cell death with 100% efficiency by application of an elicitor. This system revealed that the HR cells activated anti-microbial defense mechanism by themselves and transmitted signal(s) to establish acquired resistance in neighboring cells, while executing cell-death program. Thus, BY-2 cells have taught us a lot about proliferation, differentiation, and death of plant cells.
Ultra-small unicellular algae provide information on the basic cellular mechanisms and essential genes that support the lives of photosynthetic eukaryotes, including higher plants. We have discovered the smallest free-living photosynthetic picoeukaryote to date, a green alga, “Medakamo hakoo” (provisional name) strain M-hakoo 311, which was isolated from freshwater. Based on its pigment composition, M. hakoo belongs to the Chlorophyta. Its cell size and nuclear genome size were compared with those of the primitive red alga Cyanidioschyzon merolae, which lives in freshwater, and with the green alga Ostreococcus tauri, which lives in seawater. The minor and major axes of M. hakoo, C. merolae and O. tauri in the G1 phase were 0.73 and 0.98 μm, 1.2 and 2.37 μm, and 0.76 and 0.96 μm, respectively. The cell size of M. hakoo was thus very similar to that of O. tauri. The nuclear genome sizes of the three algae in the G1 phase were examined using the video-intensified photon-counting system (VIMPCS). The nuclear genome size of C. merolae has previously been determined as 16.5 Mbp by sequencing. Using that value as a standard, the genome sizes of M. hakoo and O. tauri were determined as 9.2 Mbp and 20 Mbp, respectively.
Pseudolarix amabilis belongs to one of three monotypic genera in Pinaceae. This species had 2n=44 chromosomes in somatic cells and its karyotype was composed of four long submetacentric chromosomes and 40 short telocentric chromosomes that varied gradually in length, supporting previous reports by conventional staining. The chromosomes were stained sequentially with the fluorochromes, chromomycin A3 (CMA) and 4′,6-diamidino-2-phenylindole (DAPI). CMA-bands appeared on 12 chromosomes at near terminal region and proximal region. DAPI-bands appeared at centromeric terminal regions of all 40 telocentric chromosomes. The fluorescent-banded karyotype of this species was compared with those of other Pinaceae genera considering taxonomical treatment and molecular phylogenetic analyses reported. On the basis of the fluorescent-banded karyotype, the relationship between Pseudolarix amabilis and other Pinaceae genera was discussed.
The first chromosomal characteristic of nucleolar organizer regions/NORs and karyological analysis of the royal knifefish (Chitala blanci) from Mekong River, northeast Thailand, were studied. Kidney cell samples were taken from one male and one 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 C. blanci was 2n=42, the fundamental numbers (NF) were 42 in both male and female. The types of chromosomes were 12 large telocentric and 30 medium telocentric chromosomes. The Ag-NOR banding indicated that a single pair of NORs was observed on the long arm centromeric region of medium telocentric chromosome pair 10. The karyotype formula could be deduced as: 2n (diploid) 42=L12t+M30t
Karyological characteristics of the Barramundi, Lates calcarifer (Bloch 1790) from Andaman Sea, Thailand, were analyzed in the present study. Kidney cells of two male and two female fish were directly used in the mitotic chromosome preparation. The mitotic chromosome preparations were conducted directly from kidney cells. Chromosomes were stained by using conventional staining and Ag-NOR banding techniques. The results showed that the diploid chromosomes number of L. calcarifer was 2n=48 and the fundamental number (NF) were 54 in both sexes. The chromosome types comprise the 2 large metacentric, 2 large submetacentric, 2 large acrocentric, 24 large telocentric, and 18 medium telocentric chromosomes. No strange sized chromosomes related to sex were observed. The Ag-NOR banding technique indicated that the single pair of nucleolar organizer regions/NORs is located on the short arm telomeric region of large acrocentric chromosome pair 3. The karyotype formula could be deduced as: 2n (diploid) 48=L2m+L2sm+L2a+L24t+M18t
Gold nanoparticles (GNPs) shall be applied in cancer therapy, conceivably by using a simple injection of GNPs into human veins. This will bring them in contact with red and white blood cells of the blood stream before they reach their main target, the cancer cells. However, possible cyto- and/or genotoxic effects of GNPs on lymphocytes are not known in detail and are thus studied here. Cytotoxicity was determined by Trypan blue exclusion assay. For genotoxicity, Comet and Comet-FISH (=fluorescence in situ hybridization) assays were done. In the latter test two gene markers for DNA damage, TP53 as tumor suppressor gene and TNF-α as tumor necrosis factor gene, were investigated. The cells were incubated in the presence of different concentrations of polyethylene glycol–coated rod-shaped GNPs of 50 nm or 30 nm in diameter. GNPs induced cytotoxic effects in human lymphocytes. The effects could be observed in concentration- and size-dependent manner; 30 nm sized GNPs were more toxic than 50 nm sized ones. Using the comet assay, it was demonstrated that GNPs induce high rates of DNA damage, which are represented e.g. as high ratios of tail moments, compared to non-treated lymphocytes. The target genes (TP53 and TNF-α) were observed preferentially in comet tails indicating high rates of induced DNA damage in this DNA area. Our results suggest that rod-shaped GNPs interact with human blood lymphocytes, reduce cell viability and cause relevant DNA damage in a concentration dependent manner. The small sized GNPs were more cyto- and genotoxic than big sized GNPs. The low concentration of big sized rod-shaped GNPs could be safe for cancer photothermal therapy rather than small rod GNPs. However, further investigations are recommended to be able to minimize potential risks of application.
The first standardized karyotype and idiogram of Indochinese silvered langur (Trachypithecus germaini germaini) in Thailand was established in the present study. Blood samples were taken from two males and two females then subjected to standard whole blood T-lymphocyte culture. The samples were harvested by colchicine-hypotonic-fixation-air-drying technique and followed by conventional staining, GTG-banding and high-resolution techniques with Giemsa's. The results revealed that its diploid number was 2n=44 and fundamental number (NF) was 88 in both males and females. The autosomes comprise 6 large metacentric, 10 large submetacentric, 2 large acrocentric, 6 medium metacentric, 14 medium submetacentric, 2 small submetacentric, and 2 small acrocentric chromosomes. We found that nucleolar organizer regions (the representative of chromosome marker) are located on the long arms near centromeres of a pair submetacentric autosome 17. The X chromosome was a largest submetacentric chromosome while the Y chromosome was the medium submetacentric chromosome. The GTG-banding and high-resolution techniques demonstrated that the respective numbers of bands and locations in T. germaini germaini were 245 and 301 respectively. Each homologous chromosome pair appears clearly differentiated. The karyotype formula for T. germaini germaini could be deduced as: 2n (44)=L6m+L10sm+L2a+M6m+M14sm+S2sm+S2a+sex-chromosomes.
Our continuous investigations revealed that a coastal herb, Lysimachia mauritiana, exhibited a wide-ranging intraspecific karyotypic polymorphism (18 cytotypes) in the Ryukyu Archipelago of Japan. Recently it is suggested that the small island Takarajima including 11 cytotypes showed the highest intraspecific karyotypic diversity both within and among populations in the Ryukyus. To explore karyotypic variations on its adjacent three islands, Amamioshima, Kakeromajima and Tokunoshima, karyomorphological and cytogeographical analyses were conducted in 452 individuals from 27 localities throughout the islands. A total of five different chromosome numbers (2n=16, 17, 18, 19, 20) and 15 cytotypes were recognized in the area. Except for two, two to six cytotypes coexisted in every locality. The islands showed different dominant cytotypes, i.e. 16 (6m) in Amamioshima Is., unknown in Kakeromajima Is. and 18 (6m) in Tokunoshima Is. Furthermore, it is suggested that cytotypes in northeast area in Amamioshima Is. might be converged to 16 (6m) in the near future by dynamic analyses in cytotype.
Present cytomorphological surveys include meiotic studies and chromosome counts in 42 species under 93 accessions collected from the various localities of Solang Valley in Kullu district, Himachal Pradesh at different altitudes ranging from 2,400 to 3,100 m. Artemisia salsaloides (n=10), Dandranthema boreale (n=36), Ligularia fischeri (n=30) and Tussilago farfara (n=12) are recorded as the first ever chromosomal counts from India. Additional/variable cytotypes are recorded in Anaphalis nepalensis (2n=6x=42), A. triplinervis (2n=6x=42), Artemisia nilagirica (2n=4x=36; 2n=6x=54), Artemisia salsoloides (n=10), Brachyactis pubescens (2n=4x=36), Dandranthema boreale (2n=8x=72), Taraxacum officinale (2n=8x=64) and Tussilago farfara (2n=2x=24). Intraspecific polyploid cytotypes are reported in Anaphalis nepalensis (4x, 6x), Artemisia nilagirica (4x, 6x) and Taraxacum officinale (2x, 3x, 4x, 5x, 6x) from the valley. Existence of B-chromosome has been reported in Cnicus wallichii (2n=34+0-1B) which is the first ever record for the species at world level.
Successful induction of autotetraploidy has been achieved in five plants of B. fruticulosa Cyr. subsp. fruticulosa (2n=16, FF), a wild relative of cultivated brassicas. The diploid seedlings of B. fruticulosa were treated with different concentrations of aqueous colchicine using the cotton-swab method for 8–15 h for 2 to 3 d. The highest percentage of success was recorded when the seedlings were treated with 0.2% aqueous colchicine for 12 h within 2 d. The synthesized plants showed remarkable enhancement in several morphological and floral characters making them more robust. Cytologically, among all the associations, bivalent chromosome associations were observed more frequently (7.20 to 8.76 per cell) in various plants. However, quadrivalent frequency ranging from 2.28 to 3.14 and univalent frequency ranging from 3.44 to 4.09 per cell were characteristic of the colchicine-induced tetraploids. The number of chiasmata ranged from 15 to 36 with an average number of 21.28 and 23.60 and their terminalisation coefficient ranged between 0.73 and 0.86. The anaphase I and II disjunction of bivalents/chromosomes was leading more or less regularly and equally to the formation of seeds from the synthesized plants. Significant enhancement in morphological traits as revealed in colchicine-induced plants and normal meiotic behaviour leading to a good seed set may ultimately result in providing the plant breeder with more variability. The synthesized autotetraploids will have great opportunities to be utilized in hybridization with Brassica juncea and related species for introgression of desirable traits, especially tolerance for mustard aphid.
Seeds of Lens culinaris Medik (Lentil) were irradiated with various doses of gamma rays, (viz. 5 kR, 10 kR, 15 kR, 20 kR, 25 kR and 30 kR) and some seeds were also treated with different concentrations (0.1, 0.2, 0.3, and 0.4%) of Ethyl Methane Sulphonate (EMS) for 6 and 12 h in separate experiments. Two plants, one translocation heterozygote and one inversion heterozygote, were isolated from the population raised from 10 kR gamma ray-irradiated seeds and 0.2% EMS-treated seeds, respectively. The mutant plants displayed various types of chromosomal configurations at diakinesis/metaphase I, and anaphase/telophase I/II in meiosis. The translocation heterozygote exhibited the formation of ring/chain of four and six chromosomes in a majority of the PMCs at diakinesis/metaphase-I, and the inversion heterozygote was characterized by the presence of bridge and fragments at anaphase/telophase I/II because of various numbers and positions of crossovers in the inversion loop. Pollen fertility declined to 38% in the translocation heterozygote and 27.33% in the inversion heterozygote as compared to 96% in the control.
In this study, chromosome numbers and morphologies are presented for four species growing naturally in Turkey. These species are Inula peacockiana (Aitch. & Hemsl.) Korovin, Cousinia boissieri Buhse, Scorzonera renzii Rech. f., Lactuca intricata Boiss. Chromosome number of studied species are Inula peacockiana 2n=20, Cousinia boissieri 2n=26, Scorzonera renzii 2n=14 and Lactuca intricata 2n=18. Also, karyotype analyses were made using the Bs200Pro Image Analysis Software. In addition, the karyotype asymmetry of each taxa was evaluated by different methods; TF%, AsK%, Rec and Syi, A1, A2, A, AI and Stebbins classification. The correlation between the karyotype asymmetries of the different taxa was calculated.
Prairie cordgrass has recently gained attention as an important biotic component of stressed ecosystems. This polyploid species is distributed broadly across the U.S. More cytogenetic data are needed to investigate how increased ploidy levels influence chromosome changes and gene expression in order to understand the formation of stable cytotypes. Silver staining is the cytogenetic method commonly used to study the number and distribution of active nucleolar organizing regions (NORs) on chromosomes at metaphase, and the number and size of nucleoli in nuclei at interphase. Intensity and distribution patterns of silver stained NORs (AgNORs) reveal differential ribosomal gene expressions in plants and animals. The purpose of this study is to estimate the number of AgNORs, their locations, and their activities in metaphase chromosomes and to determine heteromorphic variation in size and number of nucleoli within interphase nuclei for tetra-, hexa-, and octoploid prairie cordgrass populations. Increases in mean numbers of AgNORs in each metaphase and interphase cell were observed as ploidy level increases. Although distribution patterns of silver stained NORs at metaphase cells of tetra- and octoploids reflect changes in ploidy, the neo-hexaploids did not follow the pattern, indicating that active NORs were not stable in early generation of formation of neo-hexaploidy. Collectively, these results suggest that distribution patterns of silver stained NORs in metaphase and number of silver stained nucleoli in interphase cells can be used as markers to detecting chromosome variations within and among ploidy levels and to determine when ploidy levels stabilize in prairie cordgrass.