Mitotic Root Tip Cells Research Articles

Effect of Household Detergent on Mitotic Root Tip Cells of Vigna unguiculata (Cowpea)

Effect of Household Detergent on Mitotic Root Tip Cells of Vigna unguiculata (Cowpea)

Karyotype study on 15 populations of Eremostachys laciniata Bunge in Iran

A karyotype study was performed on 15 populations of Eremostachys laciniata Bunge from Iran in order to quantify the extent of cytological variation for use in breeding programmes. Ten mitotic root tip cells at the metaphase stage were prepared from each population by the squash method. The chromosomes of suitable mitotic cells were counted and various parameters, including long and short arm lengths, chromosome lengths, total length of the haploid chromosome complement, arm ratios, and the centromeric index, were measured. All 15 populations were diploid with 2n = 2x = 22 and were differentiated by their karyotypic parameters. All 15 E. laciniata populations occupied Class 1A of Stebbins’ karyotype classification, indicating the presence of a primitive symmetrical karyotype. The size of the mitotic chromosomes was medium, and mean chromosome lengths ranged from 3.84 to 4.77 μm. The shortest chromosome lengths were observed in the Ajabshir population and the longest in the Heydarabad population. The Heydarabad population also had the longest total chromosome length and the Ajabshir population had the shortest total chromosome length. The centromeric indices of the chromosome complements varied from 39.3% to 43.7%. Metacentric chromosomes were the most common in all populations, whereas submetacentric chromosomes were rare. The A1 index varied from 0.215 (Areshtanab) to 0.337 (Til). The A2 index ranged from 0.116 (Til) to 0.143 (Kaleybar), and the karyotype asymmetry index varied between 0.051 and 0.097. The results of a principal component analysis (PCA) showed that the main elements of the first three main principal components accounted for 97% of the total variance. Cluster analysis by Ward’s method indicated that all 15 populations could be classified into three clusters. Finally, the results of the karyotypic parameters showed that the Til, Heydarabad, and Marand populations had higher levels of karyotypic heterogeneity and may be useful when developing breeding programmes.

Assessment of genotoxicity and acute toxic effect of the imatinib mesylate in plant bioassays

Imatinib mesylate (IM) is at present one of the most widely used cytostatic drugs in developed countries but information on its ecotoxicological activities is scarce. This article describes the results of the first investigation in which genotoxic and acute toxic properties of the drug were studied in higher plants. IM was tested in two widely used plant bioassays namely in micronucleus (MN) assays with meiotic tetrad cells of Tradescantia (clone #4430) and in mitotic root tip cells of Allium cepa. Additionally, acute toxic effects (inhibition of cell division and growth of roots) were monitored in the onions. Furthermore, we studied the impact of the drug on the fertility of higher plants in pollen abortion experiments with three wildlife species (Chelidonium majus, Tradescantia palludosa and Arabidopsis thaliana). In MN assays with Tradesacantia a significant effect was seen with doses ⩾10μM; the Allium MN assay was even more sensitive (LOEL⩾1.0μM). A significant decrease of the mitotic indices was detected at levels ⩾10μM in the onions and reduction of root growth with ⩾100μM. In the pollen fertility assays clear effects were observed at doses ⩾147.3mgkg−1. Data concerning the annual use of the drug in European countries (France, Germany, Slovenia) enable the calculation of the predicted environmental concentration (PEC) values which are in the range between 3.3 and 5.0ngL−1. Although comparisons with the genotoxic potencies of other commonly used cytostatic drugs and with highly active heavy metal compounds show that IM is an extremely potent genotoxin in higher plants, it is evident that the environmental concentrations are ⩾5 orders of magnitude lower as the levels which are required to cause adverse effects.

LITTLE NUCLEI 1 and 4 Regulate Nuclear Morphology in Arabidopsis thaliana

The morphology of plant nuclei varies among different species, organs, tissues and cell types. However, mechanisms and factors involved in the maintenance of nuclear morphology are poorly understood. Because nuclei retain their shapes even after cytoskeletal inhibitor treatments both in vivo and in vitro, we assumed involvement of the nuclear lamina, which plays a critical role in the regulation of nuclear morphology in animals. The crude nuclear lamina fraction isolated from Arabidopsis thaliana leaves was analyzed by mass spectrometry, and putative nuclear lamina proteins were identified. Among their T-DNA insertion lines, nuclei of little nuclei1 (linc1) and linc4 disruptants were more spherical than those of wild-type plants. Because A. thaliana harbors four LINC genes, we prepared all single and linc1/4 and linc2/3 double disruptants. In leaf epidermal cells, the circularity index of the nucleus in all linc disruptants except linc3 was significantly higher than that in the wild-type plants. The extent of the effects of LINC1 and/or LINC4 disruption was significantly higher than that of the effects of LINC2 disruption. The nuclear area was significantly smaller in the linc1, linc4 and linc1/4 disruptants than in the wild-type plants. Regardless of the defects in nuclear morphology, all linc disruptants exhibited a normal ploidy level. In interphase cells, LINC1 and LINC4 were mainly localized to the nuclear periphery, whereas LINC2 was in the nucleoplasm and LINC3 was detected in both regions. From prometaphase to anaphase in mitotic root tip cells, LINC1 was co-localized with chromosomes, whereas other LINCs were dispersed in the cytoplasm.

Disturbance of reactive oxygen species homeostasis induces atypical tubulin polymer formation and affects mitosis in root‐tip cells of Triticum turgidum and Arabidopsis thaliana

In this study, the effects of disturbance of the reactive oxygen species (ROS) homeostasis on the organization of tubulin cytoskeleton in interphase and mitotic root-tip cells of Triticum turgidum and Arabidopsis thaliana were investigated. Reduced ROS levels were obtained by treatment with diphenylene iodonium (DPI) and N-acetyl-cysteine, whereas menadione was applied to achieve ROS overproduction. Both increased and low ROS levels induced: (a) Macrotubule formation in cells with low ROS levels and tubulin paracrystals under oxidative stress. The protein MAP65-1 was detected in treated cells, exhibiting a conformation comparable to that of the atypical tubulin polymers. (b) Disappearance of microtubules (MTs). (c) Inhibition of preprophase band formation. (d) Delay of the nuclear envelope breakdown at prometaphase. (e) Prevention of perinuclear tubulin polymer assembly in prophase cells. (f) Loss of bipolarity of prophase, metaphase and anaphase spindles. Interestingly, examination of the A. thaliana rhd2/At respiratory burst oxidase homolog C (rbohc) NADPH oxidase mutant, lacking RHD2/AtRBOHC, gave comparable results. Similarly to DPI, the decreased ROS levels in rhd2 root-tip cells, interfered with MT organization and induced macrotubule assembly. These data indicate, for first time in plants, that ROS are definitely implicated in: (a) mechanisms controlling the assembly/disassembly of interphase, preprophase and mitotic MT systems and (b) mitotic spindle function. The probable mechanisms, by which ROS affect these processes, are discussed.

Ploidy Levels of Hardy Actinidia Accessions in the U.S. Determined by Flow Cytometry

The hardy Actinidia species represent a source of genetic diversity for improving A. deliciosa (kiwifruit) as well as for creating new economically important cultivars through intra- and interspecific crosses. Attempts at breeding in Actinidia have been complicated by the existence of intraspecific as well as interspecific variation in ploidy. The haploid chromosome number in Actinidia is 29 and diploid (2n=2x=58), tetraploid (2n=4x=116), and hexaploid (2n=6x=174) levels have been identified. Because of the problems encountered when crossing parents differing in ploidy level, it is desirable to know the ploidy levels of plants to be used in breeding. We determined the ploidy levels of 61 Actinidia accessions currently available in the U.S., including primarily accessions of relatively winter-hardy species. The 61 accessions, representing eight species and three interspecific hybrids, were screened for ploidy using flow cytometry. Mitotic root tip cells from one plant from each putative ploidy level were examined microscopically to confirm the ploidy level derived from flow cytometry. There were 17 diploids, 40 tetraploids, and 4 hexaploids. Intraspecific variation was not found among accessions of the species arguta, callosa, deliciosa, kolomikta, melanandra, polygama, or purpurea. All kolomikta and polygama accessions were diploid. All arguta, callosa, melanandra, and purpurea accessions were tetraploid. Actinidia deliciosa was hexaploid. One chinensis accession was tetraploid. Two accessions (NGPR 0021.14 and 0021.3), acquired as chinensis, were hexaploid and may, in fact, be A. deliciosa based on their morphology. `Issai' (arguta × polygama) was hexaploid and `Ken's Red' and `Red Princess' (both melanandra × arguta) were tetraploid.

The morphology, cytology, and C-banded karyotypes of Brassica campestris, B. oleracea, and B. napus plants regenerated from protoplasts

The behaviour of Brassica campestris (2n=20, AA), B. oleracea (2n=18, CC), and B. napus (2n=38, AACC) were studied during a tissue-culturing process. Hypocotyl-protoplasts were cultivated into calli from which new plants were regenerated. The regenerated plants were compared, and mitotic root-tip cells were C-banded and karyotyped. A majority of the plants were tetraploid. The meioses were studied in the PMCs. A number of abberations were observed, mainly due to faulty spindle function. There was a difference between the three species in that B. campestris performed the most poorly with many fewer regenerated plants. These plants were more morphologically disturbed and had more problems during pollen production than B. oleracea and B. napus plants.

Identification of ‘Amigo’ and ‘Kavkaz’ translocations in Ohio soft red winter wheats (Triticum aestivum L.)

One cultivar ('GR876') and two advanced Ohio soft red winter wheat lines ('OH413' and 'OH414'), with 'Kavkaz' in their pedigrees, were examined for the presence of the 'Kavkaz,' 1RS/1BL rye/wheat chromosome translocation. Another advanced line ('OH416'), with 'Amigo' in its pedigree, was examined for the presence of the 'Amigo,' 1RS/1AL translocation. Only two satellited chromosomes were observed in most mitotic root-tip cells from 'GR876,' 'OH413,' and 'OH414,' compared to four in most cells from 'OH416.' Heteromorphic bivalents were observed in most PMCs from hybrids produced by crossing 'GR876,' 'OH413,' and 'OH414' as females to 'Chinese Spring.' No heteromorphic bivalents were observed in PMCs from 'OH416' x 'Chinese Spring' hybrids. When 'GR876' and the Ohio lines were hybridized with 'Chinese Spring' dimonotelosomic-1B, telosomic trivalents, consisting of the short- and longarm telosomes paired with chromosome 1B, were only observed in PMCs from 43-chromosome hybrids involving 'OH416.' The long-arm telosome paired with the translocation chromosome, while the short-arm telosome remained unpaired in all other 43-chromosome hybrids. Separation of gliadin proteins from 'GR876' and the Ohio lines by PAGE revealed that secalin bands for 'GR876,' 'OH413,' and 'OH414,' migrated similarly to the secalins for 'Kavkaz.' Bands for 'OH416,' identified as possible secalins, migrated similarly to those for 'Amigo.' Cultivar 'GR876' and advanced Ohio soft red winter wheat lines 'OH413' and 'OH414' carry the 'Kavkaz' translocation, while 'OH416' carries the 'Amigo' translocation.

Diploid perennial intergeneric hybrids in the tribe Triticeae. IV. Hybrids among Thinopyrum bessarabicum, Pseudoroegneria spicata, and Secale montanum

Diploid intergeneric hybrids among Thinopyrum bessarabicum, Pseudoroegneria spicata, and Secale montanum were synthesized with the aid of embryo rescue. Karyotype analyses of mitotic root-tip cells revealed that all satellited chromosomes in the J, S, and R genomes were present in their hybrid combinations, making it possible to identify these hybrids at the seedling stage. Spikes of these hybrids were intermediate to, but distinctly different from, those of the parental species. Meiotic chromosome associations at metaphase I in the pollen mother cells averaged 4.34 I + 2.77 rod II + 1.42 ring II + 0.24 III + 0.14 IV for P. spicata × T. bessarabicum; 11.05 I + 1.22 rod II + 0.04 ring II + 0.13 III + 0.01 IV for T. bessarabicum × S. montanum; and 12.98 I + 0.52 rod II + 0.01 III for P. spicata × S. montanum. These meiotic data suggest that the S genome of Pseudoroegneria and the J genome of Thinopyrum are more closely related to each other than they are with the R genome of Secale. The R genome is slightly closer to the J genome than to the S genome. Since these synthetic hybrids represent genomic combinations that may not exist in nature, their induced amphiploids should be created and evaluated.Key words: intergeneric hybrids, genome, phylogeny, Thinopyrum, Pseudoroegneria, Secale.

USE OF REPEATED DNA SEQUENCES AS CYTOLOGICAL MARKERS

The majority of DNA that is found in most of the flowering plants appears to be non‐coding DNA. Much of this excess DNA consists of nucleotide sequences which exist as multiple copies throughout the genome and are designated as repetitive sequences. Those sequences which are found in moderately high to high numbers of copies are observed to be of the greatest value as cytological markers. Moderately high copies may exist as sequences which are dispersed throughout the chromosomes of some species and not dispersed in other more distantly related species. By taking advantage of this characteristic and the technique of in situ hybridization with biotinylated probes, breakpoints of chromosomal translocations may be observed between species such as wheat and rye. Many of the high copy number repetitive sequences are organized in a tandem fashion in specific loci in the chromosome. Chromosomal identification may be accomplished by using the in situ hybridization technique. Upon in situ hybridization with a repetitive sequence isolated from Aegilops squarrosa, the patterns of the sites of hybridization allowed the D‐genome chromosomes to be identified. The sequence was also observed only on the D‐genome chromosomes of several polyploid species indicating its usefulness as a genome specific marker. Using this genome specificity, assessment of the orientation of the D‐genome chromosomal segments of hexaploid wheat carrying the sequence during interphase and prophase of mitotic root tip cells was possible. Repetitive DNA sequences, therefore, provide cytological markers necessary for studies of chromosomal identification, genome allocation, and genome orientation. The use of biotin‐labeled DNA probes allows the technique of in situ hybridization to be performed much more rapidly and with a greater degree of safety and reliability.

Use of Repeated DNA Sequences as Cytological Markers

The majority of DNA that is found in most of the flowering plants appears to be non-coding DNA. Much of this excess DNA consists of nucleotide sequences which exist as multiple copies throughout the genome and are designated as repetitive sequences. Those sequences which are found in moderately high to high numbers of copies are observed to be of the greatest value as cytological markers. Moderately high copies may exist as sequences which are dispersed throughout the chromosomes of some species and not dispersed in other more distantly related species. By taking advantage of this characteristic and the technique of in situ hybridization with biotinylated probes, breakpoints of chromosomal translocations may be observed between species such as wheat and rye. Many of the high copy number repetitive sequences are organized in a tandem fashion in specific loci in the chromosome. Chromosomal identification may be accomplished by using the in situ hybridization technique. Upon in situ hybridization with a repetitive sequence isolated from Aegilops squarrosa, the patterns of the sites of hybridization allowed the D-genome chromosomes to be identified. The sequence was also observed only on the D-genome chromosomes of several polyploid species indicating its usefulness as a genome specific marker. Using this genome specificity, assessment of the orientation of the D-genome chromosomal segments of hexaploid wheat carrying the sequence during interphase and prophase of mitotic root tip cells was possible. Repetitive DNA sequences, therefore, provide cytological markers necessary for studies of chromosomal identification, genome allocation, and genome orientation. The use of biotin-labeled DNA probes allows the technique of in situ hybridization to be performed much more rapidly and with a greater degree of safety and reliability.

Cytogenetic analysis of plants regenerated from tissue explants and mesophyll protoplasts of winter rape, Brassica napus L.

Plants were regenerated from seedling tissue explants of four lines of winter rape (Brassica napus L.) including a cytoplasmic male sterile line carrying Raphanus sativus L. cytoplasm, and from leaf mesophyll protoplasts of the cytoplasmic male sterile line. Chromosome number variability was investigated in 102 regenerated plants. Mitotic root-tip cells were scored initially; those plants exhibiting mixoploidy or an altered chromosome number were reexamined at meiosis to confirm the presence of alterations in germ line cells. Plants regenerated from seedling explants included 2n = 38 diploids (87.0%) similar to the parental line, monosomics (7.8%), trisomics (2.6%), and 2n = 76 tetraploids (1.3%). The germ line number was not resolved in one mixoploid (1.3%). Protoplast-derived regenerated plants were diploid (44%), hypodiploid (20%), and tetraploid or hypotetraploid (36%). Meiotic analysis of regenerated plants showed a range of multiple chromosome associations with no plants consistently exhibiting bivalent formation only. Chromosomal alterations other than number may have been induced by culture, but could not be substantiated since multiple chromosome associations were also frequent in control plants grown from seed.Key words: plant regeneration, protoplast regeneration, Brassica napus, cytogenetics.

On the origin of labeled RNA in the cytoplasm of mitotic root tip cells of Vicia faba

On the origin of labeled RNA in the cytoplasm of mitotic root tip cells of Vicia faba