Heterogeneity in Cell Behaviour in Primordia ofVicia faba

The response of cells of small primordia ofVicia faba to³H-TdR and colchicine is discussed. The delayed uptake of³H-TdR shown by cells of small primordia appears to be a property of only 50% of the cells; the remaining never become capable of incorporating³H-TdR. Prom the labeled cells and polyploid cells induced by colchicine the shortest cycle time in small primordia is estimated to be 12 hours. Within a period equal to 1 mitotic cycle, 31–35% of all mitoses are tetraploid, following treatment with colchicine. The remainder are diploid and diploid mitoses were seen for up to 30 hours. These observations are indicative of a heterogeneity for mitotic cycle time in populations consisting of up to 1,500 cells. The percentage labeled mitosis curve of diploid cells was changed in primordia treated with colchicine; higher peaks were found. These results show that even small populations of cells, at the beginning of a morphogenetic system, are very heterogeneous for key cell properties. This research has been supported by the U.S.A.B.C. [AT (11-1) 1625-21].     

Colchicine effect on the chromosome number of barley embryos culturedin vitro

Summary The effect of colchicine upon the embryos of barley (Hordeum vulgare L.) culturedin vitro has been studied. Different concentrations of the drug as well as different times of culture in its presence have been tested, in order to ascertain the optimal conditions for the induction of non diploid cells. Tetraploid cells were always infrequent after the treatments, reaching the higher values during the first days of culture; aneuploid cells being frequent in some cases.     

Premature centromere division dominantly inherited in a subfertile family

An increased frequency of mitoses showing premature centromere division (PCD) in every chromosome was found in lymphocyte cultures from four members of a subfertile family. These cells were observed in both the presence and absence of colchicine. Cultured fibroblasts from the proband showed only normal diploid metaphases. PCD cells seemed to have a shorter cell cycle. The anomaly was transmitted in a way compatible with autosomal dominant inheritance in this family.     

Manipulation of ploidy for kiwifruit breeding: in vitro chromosome doubling in diploid <Emphasis Type="Italic">Actinidia chinensis</Emphasis> Planch.

Tetraploid plants were induced by colchicine treatment of in vitro leaf petiole segment cultures of five diploid Actinidia chinensis Planch. genotypes, including the commercially important, yellow-fleshed cultivar ‘Hort16A’, three female selections with red-fleshed fruit and one male pollinizer. Petiole segments were incubated on a shoot regeneration medium for a period of 4 weeks, and subsequently microshoots were treated with 0.05 or 0.1% colchicine. About one-third of the regenerated shoots were tetraploid following 0.05% colchicine treatment, more than with 0.1% colchicine treatment. Similar rates of tetraploid induction were achieved with all the genotypes tested. The efficiency of induction of polyploidy depended on the interaction between the types of in vitro culture chosen and the concentration of colchicine used. There are no previous reports of colchicine being used so successfully to induce polyploidy in Actinidia.     

Establishment and characterisation of a rapidly dividing diploid cell suspension culture of Arabidopsis thaliana suitable for cell cycle synchronisation

Polyploid plants often have altered gene expression, biochemistry, and metabolism compared to their diploid predecessors. Therefore cultured diploid cells have distinct benefits over cultured polyploid cells for the study of gene regulation and metabolism of the parent plant. Here we report methods for establishing and maintaining a rapidly dividing diploid Arabidopsis thaliana cell suspension culture, and subsequent cell cycle synchronisation. Rapid growth of homogeneous cell populations was achieved after 3 months of initiation of cultures from leaf calluses. The cells were grown in the dark on an orbital shaker (110 rpm, 50 mm orbit) at 24 °C. Continued maintenance of the culture required the use of late-exponential stage cells for subculture at weekly intervals using careful subculturing techniques to achieve accurate biomass transfer. Cell cycle synchronisation was achieved following sucrose starvation, phosphate starvation, hydroxyurea treatment, aphidicolin treatment, and a combination of phosphate starvation and aphidicolin treatment. Inhibition of the cell cycle and accumulation of cells in specific phases was monitored by microscopy to determine the metaphase/anaphase index, and by flow cytometry. The cell cycle was partially and reversibly blocked by sucrose or phosphate starvation and by hydroxyurea (2.5 mM) treatment. A complete block at G1/S interphase was achieved after aphidicolin treatment or phosphate starvation combined with aphidicolin treatment. Release from the aphidicolin block achieved ca. 78% cell cycle synchronisation in the cell population. Endoreduplication was evident after release from the block in all treatments but after one cycle (24 h) the cells returned to the diploid state. This diploid culture is currently being used in our laboratory for the genetic analysis of cell death.     

甜叶菊同源四倍体离体诱导及鉴定

用不同浓度秋水仙素溶液处理甜叶菊不定芽,诱导同源四倍体,并进行解剖学、染色体鉴定和流式细胞仪鉴定倍性。结果表明:(1)用0.20%的秋水仙素溶液浸泡甜叶菊不定芽12h,同源四倍体诱导率最高,可达32.14%。(2)同源四倍体植株与二倍体(对照)相比,其气孔、叶片等均表现巨大性,且叶片变厚、叶色浓绿、叶片皱缩。(3)对照植株染色体2n=2x=22,四倍体植株染色体2n=4x=44;流式细胞仪倍性鉴定结果显示,对照DNA相对含量为100,四倍体DNA相对含量为200。(4)该研究共鉴定出48株甜叶菊同源四倍体植株,为进行倍性植株的诱导奠定了技术基础,为进一步开展甜叶菊同源四倍体新品种的选育提供了实验材料。     

Ploidy stability of maize anther culture-derived callus lines

Summary Ploidy levels of 26Zea mays L. anther culture-derived callus lines of the F1 hybrids (H99 × Pa91, Pa91 × FR16, and H99 × FR16) were determined at various times after culture initiation using flow cytometry (for 21 lines) or chromosome counting of callus cells or regenerated plants (for the remaining 5 lines). Twenty of the lines remained haploid, whereas 6 were diploid. The results from flow cytometry, after examining the DNA content of 5000 nuclei of each callus line, show that each callus line consisted of homogenous haploid or diploid cells. Thus for diploid callus lines, spontaneous chromosome doubling must have occurred before or in the early stages of androgenesis, before the initiation of callus cultures. These long-term callus cultures (growing for up to 38 mo.) have stably maintained their ploidy levels so it is unlikely that the culture conditions have caused chromosome doubling. The restriction fragment length polymorphism pattern obtained with 52 to 58 markers for each diploid callus line shows that all the diploid lines are homozygous diploid so each originated from a microspore and not from diploid maternal F1 hybrid tissue.     

A plant culture (BY‐2) widely used in molecular and cell studies is genetically unstable and highly heterogeneous

Nicotiana tabacum (tobacco, 2n = 4x = 48) is an allotetraploid with 24 S‐genome chromosomes (from a diploid related to N. sylvestris) and 24 T‐genome chromosomes (from a diploid related to N. tomentosiformis). The BY‐2 suspension cell culture, derived from N. tabacum cultivar Bright Yellow 2, has been used extensively for research in molecular and cell biology for almost 40 years; a Web of Knowledge search reveals that it has been used over 150 times since 2008 alone, largely for cell cycle and plant physiology studies. However, we show that this culture is unstable and, as with other long‐term cultures, exists as a community of cells with different karyotypes reflected in different chromosome numbers, morphologies and distributions of satellite repeats, At least one rearranged chromosome type was found in all cells investigated in detail. In comparison with N. tabacum, one satellite repeat, NTRS, has become dispersed across several chromosomes and there is complete homogenization of 35S rRNA genes towards T‐genome type rDNA units. Karyotype divergence should be considered when using BY‐2 cells for plant physiology or cell cycle/development studies in the future. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170 , 459–471.     

Factors affecting the frequency of tetraploid cells in a predominantly diploid suspension culture ofDaucus carota

Summary Tetraploid clones were isolated from a predominantly diploid culture line ofDaucus carota by plating alone or after colchicine treatment. Although individually these tetraploid clones had similar growth rates to the diploid culture, they were progressively eliminated from mixtures with the diploid line. As the diploid culture line always contained a low frequency of tetraploids, the selective elimination of tetraploids must have been balanced by their continuous production. The frequency of endoreduplication detected was too low to maintain the equilibrium frequency of tetraploids and it is proposed that polyploidisation also occurred by endomitosis. The mean frequency of tetraploid metaphases within the diploid culture line was reduced by shortening the interval between subcultures from 14 to 7 days. This 7-day regime also eliminated linear growth and stationary phase periods, but did not alter the maximum growth rate or mitotic index of the culture. It is argued that the change in mean tetraploid metaphase frequency is indicative of a change in the number of tetraploid cells within the culture, brought about by an alteration in the equilibrium between production and elimination of tetraploid cells.     

Factors influencing changes in ploidy and nuclear DNA levels in cells from normal,crown-gall and habituated cultures ofHelianthus annuus L.

Summary Normal and crown-gall tissue cultures ofHelianthus annuus were initiated from diploid cells of the stem and diploid tumour cells respectively. For the first 16 months both normal and tumour isolates remained predominantly diploid, but subsequently many isolates showed varying degrees of polyploidy. It was concluded that cultures fromH. annuus were initially stable, but that this stability was lost during prolonged culture. There was no evidence that tumour or habituated isolates had any greater tendency to become polyploid than normal isolates grown in similar conditions. Furthermore normal isolates initiated and maintained on media supplemented with different auxins (2,4-dichlorophenoxyacetic acid, 1-naphthaleneacetic acid, 3-indoleacetic acid and 4-chloro-2-oxobenzothiazolin-3-yl acetic acid) remained chiefly diploid during the first 13 months in culture, but subsequently became polyploid. In addition, increasing the concentration of kinetin in the medium had no obvious effects on the occurrence of polyploid nuclei.     

PLOIDY ANALYSIS OF THE TWO MOTILE FORMS OF CHRYSOCHROMULINA POLYLEPIS (PRYMNESIOPHYCEAE)1

In some cultures of the flagellate Chrysochromulina polylepis Manton et Parke, established from cells isolated from the massive bloom in Skagerrak and Kattegat in 1988, we observed, two motile cell types. They were termed authentic and alternate cells and differed with respect to scale morphology. To investigate whether or not the two cell forms were joined in a sexual life cycle, the relative DNA content per cell and relative size of cells of several clonal cultures of C. polylepis were determined by flow cytometry. Percentages of authentic and alternate cells in the cultures were estimated by transmission electron microscopy. Pure authentic cultures (α) contained cells with the lowest level of DNA and were termed haploid. Two pure alternate cultures (β) contained cells with double the DNA content of authentic cells and were termed diploid. Other pure alternate cultures contained haploid cells only, or both haploid and diploid cells. Three cell types were observed, each capable of vegetative propagation: authentic haploid, alternate haploid, and alternate diploid cells. Both the haploid and diploid alternate cells were larger than the haploid authentic cells. Cultures containing diploid cells appeared unstable: cell type ratio and ploidy ratio changed during the experiment where this cell type was present, particularly when grown in continuous light. In contrast, cultures with only haploid cells remained unchanged at all growth conditions tested. Light condition may influence cell type ratio and ploidy ratio. Our attempt to induce syngamy by mixing different authentic haploid clones did not result in mating. Assuming that the authentic and alternate cell types are of the same species, the life cycle of C. polylepis includes three flagellated scale-covered cell forms. Two of the cell types are haploid and may function as gametes, and the third is diploid, possibly being the result of syngamy.     

Characterization of the cell cycle of cultured human diploid cells: Effects of aging and hydrocortisone

Age-related changes in the cytokinetics of human diploid cells in vitro have been compared in normal cultures and in cultures in which lifespan has been prolonged by the addition of hydrocortisone to the medium. For both cultures, with advancing age the fraction of cells in the actively proliferating pool decreased and the intercellular variation in cell cycle times increased. The average cell cycle time was prolonged during aging due almost entirely to changes in the duration of G₁. The duration of S remained constant, while a small delay in G₂ was observed in late passage cells near the end of their lifespan. Although the same pattern of change in proliferative parameters occurred in both control and hydrocortisone-treated cultures, the changes were somewhat delayed in the presence of the steroid. The results are interpreted in terms of several cell cycle models and suggest that the events controlling cell proliferation are sensitive to hydrocortisone modulation during the G₁ and possibly the G₂ periods.     

Haploid and diploid cell cultures from a haplo-diploid insect

Summary

This is the first report of haploid and diploid cell culture from the haplo-diploid parasitoid wasp, Mormoniella vitripennis. Cells were cultured from haploid and diploid wasps by collecting populations of eggs from virgin females (unfertilized, haploid, parthenogenetic eggs) and mated females (mostly fertilized, diploid eggs). Eggs were surface sterilized in 70% ethanol, followed by 50% Chlorox, and rinsed in phosphate buffered saline; larvae were allowed to hatch in culture. Larval cells were dissociated and cultured at 28°C in the presence of Grace's medium supplemented with fetal bovine serum. Most cells in the HMV (predominantly haploid) and DMV (predominantly diploid) cell cultures grew in suspension in the first week, formed monolayers of fibroblasts and epithelial cells by the second week in culture, and continued to grow in monolayers and vesicle-like structures for up to three months. Chromosome analysis of HMV. cells demonstrated over 70% haploid cells, with five chromosomes (N=5). The remainder were aneuploid. No diploid cells (2N= 10) were found in the HMV cell culture. Chromosome analysis of DMV cultures revealed 62% diploid, with ten chromosomes; 13% were haploid, with five chromosomes; the remainder were aneuploid. These data confirm that haploid and diploid cells can be cultured from a haplo-diploid insect species. The HMV cells which are predominantly haploid, and DMV cells which are predominantly diploid may be valuable models for the study of cellular and gene activity in haploid and diploid genetic milieux.     

The influence of progressive growth on the specific catalase activity of human diploid cell strains. I. Effect of cellular genotype: Homozygous strains

The specific catalase activity of human diploid cell strains increases with progressive growth of the culture, and falls again following subculture. Although the increase is small, it is readily demonstrable, and is exponential with time. The response of catalase activity to proggressive growth of the culture was studied in three abnormal human cell lines. A diploid cell strain, developed from a patient homozygous for the gene causing acatalasia I, had no detectable catalase activity throughout the life cycle of the culture. Another diploid cell strain, developed from a patient homozygous for the gene causing acatalasia II, had about 5% normal catalase activity, but the proportionate increase in specific activity as the culture grew was the same as for normal cells. Thus the mutation causing acatalasia II does not change the responsiveness of the cell in terms of catalase activity to progressive growth of the culture. The behavior of a heteroploid line was similar to that of the normal diploid strains, but when the growth of the heteroploid cultures reached a plateau, their population densities were four times higher than those of the diploid strains and they had about twice the specific catalase activity.     

Ploidy of small individual embryo-like structures from maize anther cultures treated with chromosome doubling agents and calli derived from them

Summary In order to determine the ploidy of individual embryo-like structures (ELSs) following chromosome doubling treatments, a method was developed to determine the DNA content (ploidy level) of nuclei from single ELSs weighing as little as 1 mg using flow cytometry. About half (53%) of the ELSs which formed during anther culture of the maize inbred line used in control medium were haploid, 27% mixoploid and 20% diploid. Gibberellic acid (GA₃) increased the diploid percentage to 52% without affecting the mixoploid frequency (26%). A four day treatment with the chromosome doubling agent colchicine (50M) increased chromosome doubling while oryzalin eliminated the diploidy and mixoploidy. When regenerable callus cultures were initiated from the ELSs none were found to be mixoploid but the haploid and diploid proportions were similar to that of the ELSs analyzed. Regenerable cultures could not be initiated from the colchicine treated ELSs, however. These studies show that with the genotype used here, GA₃ and colchicine increased the amount of chromosome doubling of the ELSs while oryzalin and pronamide did not. The mixoploidy which existed in about 25% of the ELSs was never observed in calli apparently because these structures do not initiate callus or cells of only one ploidy level grew.Abbreviations ELS embryo-like structure - GA₃ gibberellin A₃     

The effects of growth in vitro on the chromosome complement of Daucus carota (L.) suspension cultures

The chromosome number distributions and modal karyotypes of several suspension culture lines of Daucus carota L. have been analysed at various times after initiation. All lines had stable modal chromosome numbers and karyotypes, with small but significant variation about the modes. Some lines showed a predominance of diploid cells with a karyotype similar to the plant. Polyploid multiples of the modal chromosome number were present in all lines at low frequency. Variation of the 2,4-D concentration in the culture medium produced little alteration of the chromosome number distributions, but omission of 2,4-D produced a significant drop in the frequency of multipolar mitoses in those culture lines in which this treatment induced differentiation. There was no evidence of any direct effect of 2,4-D on general mitotic dynamics. Alteration of the frequency with which cultures were transferred to fresh medium showed that stationary phase was critical in the maintenance of the low frequency of tetraploids present in a predominantly diploid culture line. The results are explicable in terms of a competitive selection for cells with the dominant modal chromosome number in the presence of various mechanisms continuously producing polyploid, aneuploid and structurally altered karyotypes.     

Enhancement of paclitaxel content in induced tetraploid <Emphasis Type="Italic">Corylus avellana</Emphasis> L. cell suspension culture with regulating the expression of genes in paclitaxel biosynthetic pathway

During recent years, hazel cell suspension culture has been significantly considered as a new important source of paclitaxel. Artificial polyploidy alters different characters in plants which results in amplifying the secondary metabolites in medicinal plants. In this paper, the effects of tetraploidy induction on paclitaxel content and gene expression in hazel cell suspension culture were investigated. Various concentrations of colchicine and duration of exposure in solid and liquid media were examined and the ploidy level of cells was determined using flow cytometric analysis. The tetraploid cells were obtained from 0.2% colchicine in the exposure time of 5 and 6 days in solid medium and 0.3% colchicine in the exposure time of 3 and 4 days in liquid medium. Tetraploid cells were employed to prepare cell suspension. 3 µM of phenylalanine and 0.05 mM of vanadyl sulfate were added to both tetraploid and diploid (control) suspension to elicit paclitaxel induction. High performance liquid chromatography analysis demonstrated that the tetraploid cell suspensions produced paclitaxel of about [9.88 µg g^?1 (DW)] 1.7-fold compared with diploid cells [5.74 µg g^?1 (DW)]. The application of phenylalanine and vanadyl sulfate increased the concentration of paclitaxel in both diploid and tetraploid cells. Moreover, qRT-PCR analysis showed that the expression of GGPPS gene was significantly increased and PAL gene expression was altered after tetraploidization.     

Formation of a micronuclei-like structure induced by colchicine treatment in Saccharomyces cerevisiae

Minute nuclei named “smaller nuclei” were generated when the cells of Saccharomyces cerevisiae were treated with colchicine. The formation of “smaller nuclei” seemed to be related to nuclear division because those nuclei were only produced under conditions suitable for nuclear division. The fact that the average DNA content of “smaller nuclei” was almost one tenth of that of the isolated normal diploid nuclei showed that the “smaller nuclei” are not condensed nuclei but aneuploid nuclei like micronuclei in animal cells. It appeared therefore likely that a micronuclei-like structure could be produced by colchicine treatment in S. cerevisiae.     

Effects of colchicine on polyploidy induction of Buddleja lindleyana seeds

Buddleja lindleyana Fort. is a garden ornamental plant with great potential for development and also a commonly used medicinal plant. To enrich its germplasm resources, the seeds of B. lindleyana were treated with colchicine solution with concentration gradients of 0.5%, 1.0%, 1.5%, 2.0% and 3.0% for 12-, 24- and 48-h respectively, and the water treatment was set as the control group. The purpose was to explore the effects of colchicine on the germination and mutagenic effect of B. lindleyana seeds at different concentrations and different times, to screen the appropriate combination of mutagenic concentration and time, to provide guidance for the construction of B. lindleyana mutation population in future research. The results were as follows: (1) Colchicine had an inhibitory effect on seed germination and seedling height of B. lindleyana seeds, and the higher the concentration, the more obvious the inhibitory effect. (2) After colchicine treatment, 30 mutant plants showed morphological variations such as leaf malformation, leaf color macular, early leaf bud germination, uneven leaf surface and leaf hyperplasia, among which 3.0%?+?48-h treatment group had great potential to produce yellow-leaf plants. (3) Detection and analysis by flow cytometry revealed that among the 30 morphologically variant plants, there were 22 diploid plants, 3 tetraploid plants, and 5 chimera plants. Among them, tetraploids were mainly from colchicine concentration of 3.0% (2 plants) and 1.5% (1 plant), chimeras were mainly from colchicine concentration of 1.0% (2 plants), 1.5% (1 plant) and 3.0% (2 plants), and the seed soaking time was 48-h. (4) The length and width of guard cells and stomata were significantly different between diploid and tetraploid, and there were significant differences in leaf width and leaf shape index between tetraploid and diploid, but there were no significant differences in leaf length among diploid, tetraploid and chimera. In short, we got tetraploids and chimeras materials which were potentially useful cultivars of B. lindleyana and provided an effective identification method for polyploids of B. lindleyana.

     

Characterization of functionally active interleukin‐18/eGFP fusion protein expression during cell cycle phases in recombinant chicken DF1 Cells

The dependence of foreign gene expression on cell cycle phases in mammalian cells has been described. In this study, a DF1/chIL‐18a cell line that stably expresses the fusion protein chIL‐18 was constructed and the enhanced green fluorescence protein connected through a (G₄S)₃ linker sequence investigated the relationship between cell cycle phases and fusion protein production. DF1/chIL‐18a cells (1 × 10⁵) were inoculated in 60‐mm culture dishes containing 5 mL of media to achieve 50%–60% confluence and were cultured in the presence of the cycle‐specific inhibitors 10058‐F4, aphidicolin, and colchicine for 24 and 48 h. The percentage of cell density and mean fluorescence intensity in each cell cycle phase were assessed using flow cytometry. The inhibitors effectively arrested cell growth. The fusion protein production rate was higher in the S phase than in the G0/G1 and G2/M phases. When cell cycle progression was blocked in the G0/G1, S, and G2/M phases by the addition of 10058‐F4, aphidicolin, and colchicine, respectively, the aphidicolin‐induced single cells showed higher fusion protein levels than did the 10058‐F4‐ or colchicine‐induced phase cells and the uninduced control cells. Although the cells did not proliferate after the drug additions, the amount of total fusion protein accumulated in aphidicolin‐treated cells was similar to that in the untreated cultures. Fusion protein is biologically active because it induces IFN‐γ production in splenocyte cultures of chicken. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:581–591, 2016