Imaging techniques with split fluorescent proteins are based on the reconstruction of separated asymmetric protein fragments. Although each fragment alone has no fluorescence, a reconstructed protein after the association of its constituent fragments recovers its fluorescence. In the case of the split-GFP assay, GFP is divided between the 10th and 11th β sheets to generate two asymmetric fragments, GFP1–10 and GFP11. GFP11 is a small tag that minimally disturbs the localization and dynamics of a fused protein. Thus, the split fluorescent protein assay enables analyses of protein localization patterns, protein–protein interactions and the topology of membrane proteins.
This story began in October, 1913, with a letter from Konstantin Flaksberger, Bureau of Applied Botany, Russia, wrote to Yuzo Hoshino, Hokkaido University, Japan, requesting seeds of Japanese cereals. Handed his letter by Hoshino, Takajiro Minami, Director of the University’s Experimental Farm, responded by sending Flaksberger seeds of oats and barley in December, 1913, and of barley and wheat in September, 1915. Minami then asked Flaksberger for Russian wheat and other cereals, and acknowledged their receipt in May, 1916, after which their correspondence ceased. Tetsu Sakamura had been a graduate student in the Faculty of Agriculture, Hokkaido University since 1913, carrying out chromosome studies of leguminous species, but he switched to wheat in 1916, asking Minami to provide wheat seeds. Using material that originated from Flaksberger, Sakamura determined the correct chromosome numbers of eight wheat species, among which he discovered polyploidy of 2n=14 (2x), 28 (4x), and 42(6x); these results were published in July, 1918, and in December he left to study overseas for two years. When Hitoshi Kihara entered the graduate course of Hokkaido University in August, 1918, he received Sakamura’s materials and started genome analytical work, demonstrating that the set of seven chromosomes of common wheat, which did not exist in tetraploid wheat, was the unit in chromosome transmission as well as in fertilization, thus proposing the concept of the genome. He extended genome analysis to most species of the Triticum–Aegilops complex, determining their genome constitution. His interest then turned to interaction between the nucleus and cytoplasm, and he found that the cytoplasm of Ae. caudata caused male sterility, leading to the discovery of cytoplasmic male sterility in wheat. In 1973, Tsunewaki’s group in Kyoto University started an investigation on differentiation of plasmon in the Triticum–Aegilops complex; plasmons of this complex were introduced into 12 wheat genotypes, including three of Flaksberger’s stocks, as a result of which 552 alloplasmic lines were produced; their phenotypes and restriction fragment patterns of organellar DNAs were compared. Combining the results of their plasmon analysis and Kihara’s genome analysis, the maternal and paternal lineages of most species in this complex were finally determined.
Present study explores the meiotic chromosome numbers in seven species of the genus Elymus from Himachal Pradesh and Uttarakhand, India. The study adds the first chromosome number of E. gangotrianus (n=14+0-2B), besides adding chromosome numbers for Indian taxa of E. himalayanus (n=21), E. jacquemontii (n=14), E. schrenkianus (n=21) and E. schugnanicus (n=14). In the genus Elymus polyploidy might have a role in its chromosome evolution.
The present study deals with chromosome variability and meiotic abnormalities present in Asplenium trichomanes L., a well-known medicinal fern. The meiotic analysis of spore mother cells (SMCs) on population basis covering wide altitudinal habitats revealed diploid (n=36) and tetraploid (n=72) cytotypes. The observation of meiotic abnormalities, their relative comparison of spore size and stomatal size, and their distribution in Kashmir Himalayas revealed a difference of these cytotypes. Significant differences in the two cytotypes were found in the sizes of spore and stomata, and distributions. Compared to the restricted distribution of the diploid cytotype, the tetraploid cytotype is common in distribution and found at various altitudes in Kashmir forests. Irregular meiotic behaviors in some SMCs were observed as the presence of stickiness, unoriented bivalents, laggards, bridges, interbivalent connections, non-synchronous disjunction in both cytotypes. The formation of triads and polyads seem to lead spore sterility.
It is widely accepted that a symbiosis involving a cyanobacterium with a peptidoglycan wall led to the origin of plastids of photosynthetic eukaryotes. Recently, we visualized plastid peptidoglycan in a moss species by epifluorescence microscopy using metabolic labeling with click chemistry. In the present study, we applied the same method to visualize plastid peptidoglycan in the filamentous charophyte alga Klebsormidium nitens, as its genome contains a set of genes capable of synthesizing peptidoglycan. To visualize peptidoglycan, the generation of D-alanyl-D-alanine (DA-DA) must be blocked to allow the incorporation of ethynyl-DA-DA (EDA-DA) into peptidoglycan. Because a gene-targeting technique has not been established for K. nitens, we used D-cycloserine, which is an inhibitor of D-Ala : D-Ala ligase. Treatment with 500 µM D-cycloserine arrested cell division and inhibited chloroplast division. The addition of 1 mM EDA-DA restored the rate of cell division to that observed in medium without D-cycloserine, suggesting that EDA-DA can integrate plastid peptidoglycan instead of DA-DA, thus restarting chloroplast division and therefore cell division. Subsequently, cells were subjected to click chemistry to attach an azide-modified Alexa 488 fluorophore to the EDA-DA probe. Microscopic observation indicated that both sides of chloroplasts have strong Alexa 488 fluorescence. Under conditions of no cell growth, the chloroplast peptidoglycan that existed prior to treatment with D-cycloserine might remain in the cell. Therefore, the fluorescence must be indicative of the location of newly synthesized peptidoglycan. Our observations suggest that chloroplast peptidoglycan is built along the chloroplast division plane and serves as an essential system for chloroplast division in K. nitens.
Mutagenic sensitivity of seeds irradiated with gamma rays at various doses was investigated in Coriandrum sativum. The attributes studied were seed germination rate, seedling growth, mitotic and meiotic abnormalities, field germination rate and survivability, pollen fertility and seed yield. Gamma irradiations induced both physiological and cytological disturbances such as reduction in seed germination rate and seedling growth as well as decrease in mitotic index and enhancement of mitotic and meiotic aberration frequencies at metaphase, anaphase and resting cells and in metaphase I (M I), anaphase I (A I) and anaphase II (A II) respectively. Results suggested that doses of gamma irradiations monitored are sub-lethal in nature as evinced from lethal dose, 50% (LD50) but can also potentially bring about cytogenetical variations in the seeds of treated plants (M1) causing mutation in subsequent generation.
In C4 plants, mesophyll cells (MCs) are engaged in CO2 pumping into bundle sheath cells (BSCs) to allow ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) to work under CO2-rich conditions, leading to various properties specific to C4 photosynthesis. Maize (Zea mays) is a representative C4 plant, but its senescing leaves exhibit C3-like photosynthetic properties. Under our culture conditions, first leaves of maize finished maturation by day 9 after sowing and then began to exhibit a considerable degree of O2 inhibition, a C3-like photosynthetic property. During senescence, the number and size of chloroplast nucleoids decreased in both MCs and BSCs, but the degradation of chloroplast-nucleoids, reportedly one of the earliest signs of senescence in photosynthetic organs, started earlier and proceeded faster in MCs than in BSCs, suggesting preferential senescence of MCs. Based on these results, we propose that preferential senescence in MCs may cause insufficient CO2 pumping into BSCs and lead to C3-like photosynthetic properties in senescing maize leaves.
The experiment was aimed to study the effect of colchicine concentrations (0.025, 0.05, 0.1 and 0.2%) and treatment times (8, 24 and 48 h) on polyploidy induction and morphological characteristics in Petroselinum crispum. Polyploidy induction was carried out in two independent experiments; seed treatment under in vivo, and node treatment under in vitro condition. Polyploids were confirmed by chromosome counts, density and size of stomata and other morphological characters. The survival rate for nodes and seeds decreased significantly with increasing concentration of colchicine and its treatment duration. Moreover, the induction of polyploidy was better in the node explants compared with the seeds. The maximum amount of tetraploidization of plants from the seeds was achieved at 0.05% colchicine after 24 and 48 h incubation. The most induction of the tetraploid plants was observed 24 h after treatment with 0.05% and 0.1% colchicine under in vivo and in vitro conditions, respectively. Furthermore, morphological measurements revealed that the size of the stomata and leaves in tetraploid plants was larger than those in diploid plants. However, the density of stomata was decreased in tetraploid plants as compared to the diploids.
Plant protoplasts are valuable in biotechnology, enabling from plantlet regeneration to the determination of gene function. Viability tests are required in protoplast applications to determine the rate of viable cells, allowing to decide on the most adequate isolation and purification procedures and to verify whether sufficient cells are available for subsequent steps. Fluorescence microscopy is usually employed for this purpose. However, obstacles have pointed out: the long time required to count a relatively small number of protoplasts, cell clumps preventing their observation, and the observer’s subjective visual perception of the fluorescence. Therefore, this study aimed to establish procedures for the viability test adapted for flow cytometry (FCM), Muse cell analyzer (Muse) and Comet Assay (CA). Capsicum annuum L. was chosen as a study species based on the recalcitrant morphogenic nature of its protoplasts. After isolation and purification, the FCM and Muse allowed assessing large numbers of protoplasts, and the CA the protoplast nuclei within a short period. Using the adjusted procedures, various cytological characters could be evidenced by FCM and Muse, and different levels of genomic damage evaluated by CA, allowing the discrimination and measurement of the viable protoplasts. Considering these results, the present study introduces improved quantitative procedures for viability tests. Moreover, aiming at plantlet regeneration, distinct applications should employ to measure the protoplast viability and thus define the most adequate isolation and purification procedures. Contributing with this purpose, guidelines present here to adequate and standardize the laboratory conditions for protoplast viability test using FCM, Muse and CA.
The chromosome number and karyotype analysis of sixteen taxa of genus Cirsium Mill. distributed in Turkey were analyzed. This taxa are, Cirsium steriolepis Petr., C. bulgaricum DC., C. polycephalum DC., C. trachylepis Boiss., C. sommieri Petr., C. osseticum (Adams) Petrak, C. leucocephalum (Willd.) Spreng ssp. tenuilobum (K. Koch) Greuter, C. haussknechtii Boiss., C. ekimianum Yildiz & Dirmenci, C. handaniae Yildiz, Dirmenci & Arabacı, C. davisianum Kit Tan & Sorger, C. hypoleucum DC., C. pseudopersonata Boiss & Bal. ssp. pseudopersonata, C. pseudopersonata Boiss. & Bal. ssp. kuznezowianum (Somm. & Lev.) Petrak, C. canum (L.) All., C. obvallatum (Bieb.) Fischer, and nine of them are endemic species (C. steriolepis, C. polycephalum, C. trachylepis, C. sommieri, C. leucocephalum ssp. tenuilobum, C. davisianum, C. handaniae, C. ekimianum and C. pseudopersonata ssp. pseudopersonata). The somatic chromosome number of all studied taxa were determeined as 2n=2x=34. Total chromosome lengths varied 68.00 to 147.92 µm among species. Karyotype asymmetry indices other parameters were also calculated. Karyotype analysis indicated that Cirsium taxa generally have median (m and M) chromosomes. In addition, only C. davisianum species has one submedian (sm) chromosome. The chromosome number and karyotype of C. polycephalum, C. handaniae, C. davisianum and C. ekimianum are newly reported.
The scope of the present study is to contribute to future breeding studies by determining the relationship between chromosome number and morphology of naturally occurring 18 genotypes of Turkish Lolium perenne. The chromosome number for all the studied genotypes was concurrently determined as 2n=14 (x=7). Their karyotypes showed a predominance of metacentric chromosomes. In the karyotype of one genotype, submetacentric chromosomes were dominant. A total of quantitative asymmetric indices were used to evaluate karyological features of the species. A meaningful dendrogram was carried out to assess the karyotype-symmetry conditions and described the karyotype relationships between different cytotypes. Chromosome lengths in a karyotype were between 1.52–2.95 µm and there was a broad variation in asymmetric index karyotype formula. It may be proposed that the populations of the Turkish perennial ryegrass have quite an important potential relevant to chromosomal variation, and therefore this variation gives essential opportunities to agronomists to develop new cultivars in the future.
Four successful interspecific hybrid plants were obtained through sexual hybridization between Brassica juncea cv. Rohini, Laxmi, and Varuna (2n=4x=36, AABB), and an autotetraploid B. fruticulosa (2n=4x=32, FFFF) induced by colchicine using the latter as a pollen parent. Morphological and cytological analyses were carried out to confirm the hybrid nature of F1 plants. The F1 plants (2n=34) were intermediate for most of the morphological attributes. Although, the F1s showed poor pollen fertility, nevertheless, few seeds were obtained from open pollination. Meiotic analysis of F1 plants showed a predominance of univalents, a typical feature of hybrids. The occurrence of bivalents, trivalent, and quadrivalent in PMCs of the F1s indicated homeologous pairing among the three genomes. The study suggests that B. fruticulosa has partial genome homeology with B. juncea which could be exploited in crop improvement programmes, particularly breeding for biotic stress especially, tolerance/resistance to the mustard aphid.
Meiotic studies were made in 23 populations referable to nine species included in two genera Atrichum and Pogonatum of the family Polytrichaceae. Presently, four species (P. microstomum, A. flavisetum, A. pallidum, and A. subserratum) are observed to occur as diplomonoecious and the remaining species (P. himalayanum, P. neesii, P. perichaetiale, P. urnigerum and A. undulatum) as haplodioecious. In P. neesii aneuploid (n=8) and triploid (n=21) numbers are recorded for the first time, and in Atrichum n=14 is reported for the first time in A. flavisetum and A. pallidum.
The karyotypes and NOR condition of the secondary constrictions of chromosome pair 1 in two populations of Crotalaria incana from the Atlantic and Pacific slopes of Mexico are described. Both populations exhibited the same chromosome number of 2n=14 and karyotypes which shared a secondary constriction on the short arm of chromosome pair 1. Differences were found in the position of the centromere on chromosome pair 5 and in the size of the proximal region of the short arm of chromosome pair 1. In addition, marked differences were observed in total haploid chromosome length (THC) and mean chromosome size (MC), without this affecting asymmetry indexes. Atlantic population exhibited chromosome fragments and fragile sites at prometaphase. These data revealed differences in karyotypes between two populations.
Hymenocallis littoralis was previously reported various chromosome numbers of 2n=44, 46, 48, 49 and 68 by a conventional staining. In order to analyze these chromosomes in detail, karyotype analysis by the conventional staining and a fluorescence in situ hybridization (FISH) using probes of 45S and 5S rDNA, and telomere repeats of human-type (TTAGGG)n and Arabidopsis-type (TTTAGGG)n was performed on chromosomes of H. littoralis from Thailand. The plants have 26 chromosome pairs (2n=52), and this chromosome number was not consistent with any of the previous reports. The 45S rDNA signals were localized at the terminal region of short arms of two chromosome pairs. Heteromorphic signals of 5S rDNA were detected at the proximal region of the short arms of one long chromosome, and at the interstitial region of the long arm of one short chromosome. The signals of human-type telomere repeat were detected at all chromosome ends, but FISH with Arabidopsis-type telomere repeat showed no signal.
Three populations of Cyperus tenuispica Steud. belonging to family Cyperaceae were collected from different localities of North India. The meiotic behavior, chromosome number, morphological analysis and pollen fertility were studied. The investigations revealed the existence of two cytotypes with n=8 in two populations and with n=7 in one population. Basic chromosome number of the Cyperus is n=8 and C. tenuispica shows same chromosome number. The plants of the population with n=7 is an aneuploid. The population with aneuploid chromosome number n=7 showed abnormal meiosis i.e., chromatin bridges, laggards, stickiness, and reduced pollen fertility. Three populations exhibit a difference in plant height, length of leaves, number of rays, number of spikelets and number of glumes per spikelet.
cytological analyses were made on three species and one variety of the genus Fissidens. Six populations of F. bryoides had n=10 and one had n=13. F. cristatus showed n=12 chromosome number. The aneuploid number (n=13) was also observed in F. bryoides, F. bryoides var. bryoides and F. involutus. F. involutus with n=13 chromosome number have a minute chromosome in the complement. Some meiotic abnormalities were observed in F. bryoides var. bryoides and F. involutus.