A freeze-preservation of synchronously dividing cultured cells of Acer pseudoplatanus L

A suspension culture of sycamore (Acer pseudoplatanus L.) was synchronised in division by release from nitrogen starvation. Cell samples, taken during the lag phase and synchronous growth, were examined cytologically and subjected to a freeze-preservation protocol. A high positive correlation was found between mitotic index (percentage of cells showing mitotic figures) and cell survival, as measured by fluorescein diacetate staining and reduction of 2,3,5-triphenyl tetrazolium chloride. Specific staining of lethally damaged cells and subsequent examination of the surviving cells demonstrated that the latter had a lower mitotic index and more consistent, low value of nuclear DNA than the total population. This indicated that it is cells, newly entered into G₁ phase of the cell cycle, which are particularly resistant to the stresses imposed by the freeze preservation protocol.     

A TECHNIQUE FOR DETERMINING THE PROPORTION OF THE CLONOGENIC CELLS IN S PHASE IN EMT6 CELL CULTURES AND TUMORS

The survival of cultured EMT6 cells was examined after treatment with hydroxyurea (HU) or high specific activity tritiated thymidine (³H-TdR). The concentrations of the agents, duration of exposure to the agents, and post-exposure treatment of the cultures were found to influence the cell survival; the effects of these factors are reported. Conditions were defined under which the proportions of cells killed by HU and by ³H-TdR were the same and were also the same as the proportion of labeled cells seen on autoradiographs of cultures labeled with small doses of ³H-TdR. Under these conditions, either ³H-TdR or HU could be used to determine the proportion of the clonogenic cells in S phase. Single cell suspensions prepared from solid EMT6 tumors were treated in vitro with HU or ³H-TdR, using the conditions found optimal for each agent with cultured cells. The proportion of the tumor cells killed by treatment with HU in vitro was the same as the proportion killed by HU in vivo and as the proportion labeled by ³H-TdR in vivo, and incubation of tumor cell suspensions with HU in vitro appeared to provide a valid measurement of the proportion of clonogenic tumor cells in S phase. Incubation of tumor cell suspensions with ³H-TdR in vitro proved difficult to perform and the results were relatively unreliable because of severe problems with reutilization of ³H-TdR during the incubation for colony formation.     

The Metabolism of the Herbicide Diphenamid (N-N-dimethyl-2,2-diphenyl-acetamide) in Cell Suspensions of Soybean (Glycine max)

The fate of the herbicide diphenamid was determined in cell suspensions of soybean [Glycine max (L.) Merr. ‘Wilkin’] at different stages of cell growth: early log phase (3 to 7 d), log phase (7 to 14 d), and stationary phase (14 to 18 d). [Carbonyl-¹⁴C]-diphenamid was added to the suspensions as an acetone solution. Neither diphenamid (2 to 3 μM) nor acetone (0.5% v/v) was phytotoxic. The ¹⁴C-labeled products were identified tentatively by thin layer chromatographic comparison with reference compounds. The major metabolic products formed were N-hydroxymethyl-N-methyl-2,2-diphenylacetamide, N-methyl-2,2-diphenylacetamide, 2,2-diphenylacetamide, and two polar metabolites (0.9 to 25% of the applied ¹⁴C activity) that appeared to be glucose conjugates; one an acidic glucoside. All metabolites were found in both the cell extract and the culture medium, except for the acidic glucoside, which was recovered in small amounts only from the cell extracts. These products were the same as those recovered from intact plants. Similar results were obtained from cell suspensions of different ages. The rate of metabolism by log phase cells was slightly less than the rate for either young or old cells. The results indicated that soybean cell suspensions can be used to obtain reliable information on the fate of agricultural chemicals in soybeans.     

POLYMORPHISM OF THE DIATOM PINNULARIA BREBISSONII IN CULTURE AND A FIELD COLLECTION1,2

Two clones of Pinnularia brebissonii (Kütz.) Rabh. var. brebissonii were established and maintained in logarithmic phase of growth. Initial length of the cells was 37 μm. As cell division occurred, the mean length of cells in each population decreased as predicted by the MacDonald-Pfitzer hypothesis; however, the decrease in mean length was not uniform throughout the growth period. This nonuniformity is probably caused by nonrandom division of cells in the population or by a changing increment of size reduction due to division. The initial increment of size reduction was calculated as 0.7 μm/division. The smallest, cells observed were 8 μm long. As cells decrease in length, cell volume decreases and the proportion of cells with aberrant valve structure increases. More than 90% of the valves were abnormal in a population with mean length of 14 μm. The abnormalities of structure involved the raphe, the central area and the striae.     

红球菌R04细胞的不对称分裂及其在联苯胁迫下的分裂抑制

【目的】研究红球菌R04细胞的分裂方式及联苯对其形态和细胞分裂的影响。【方法】以一株多氯联苯降解菌株(Rhodococcus sp.R04)为研究对象,利用荧光显微镜、扫描电子显微镜及透射电子显微镜分析红球菌R04在不同培养条件下的细胞分裂。【结果】红球菌R04细胞表现出对称分裂(约占30%)和不对称分裂(约占70%)两种分裂方式,且培养条件不影响不对称分裂细胞所占的比例。细胞分裂过程中,隔膜主要分布于细胞长度的30%–50%。在联苯的分解代谢过程中,红球菌R04细胞的生长分裂会受到联苯的抑制,但不影响红球菌R04细胞的分裂方式,在联苯胁迫下,细胞形成丝状化,表现出异常分裂,随着培养时间的延长,在细胞生长指数后期至转换期,细胞能够进行正常分裂。【结论】环境异生型化合物联苯/多氯联苯对其降解菌株——红球菌R04细胞的生长和分裂有较强影响,但是并不影响其分裂方式。     

Growth, ageing and death of a photoautotrophic plant cell culture

 Batch cultures of photoautotrophic cell suspensions of Chenopodiumrubrum L., growing in an inorganic medium on CO₂ under a daily balanced light–dark regime of 16 : 8 h could be maintained for approximately 100 d without subcultivation. The long-lived cultures showed an initial cell division phase of 4 weeks, followed by a stationary phase of another 4 weeks, after which ageing and progressive cell death reduced the number of living cells and the cultures usually expired after another 3–4 weeks. These developmental phases of the cell culture were characterised with respect to photosynthetic performance, dark respiration, content of phytohormones and capacity of cell division. Cell division of the majority of the cells finished in the G1- or G0-phase of the cell cycle, caused by a pronounced decline in the endogenous levels of auxin and cytokinins. Supply of these growth factors to resting cells resulted in resumption of cytokinesis, at least by some of the cells. However, responsiveness to the phytohomones declined during the stationary phase, and subcultivation was no longer possible beyond day 60 when the phases of ageing and death commenced. Ageing was characterised by a further decline in the photosynthetic capacity of the cells, by a climacteric enhancement of dark respiration, but also by a slight increase in the level of IAA and cytokinins concomitant with a decrease in ethylene. Similarities and differences between the development of batch-cultured photoautotrophic cells of C. rubrum and that of a leaf are discussed with respect to using the cell culture as a model for a leaf. Received: 30 April 1999 / Accepted: 21 August 1999     

Effect of 12C+5 ion beam irradiation on cell viability and plant regeneration in callus,protoplasts and cell suspensions of Lavatera thuringiaca

Summary The biological effects of irradiation with¹²C⁺⁵ ion beam on plant cells have been analyzed. Protoplasts and cell suspensions ofLavatera thuringiaca, and a somatic hybrid callus (Hibiscus rosa-sinensis +Lavatera thuringiaca), were irradiated with doses from 0.05 to 50 Gy, and the effects on cell growth, cell division, cell viability and embryogenesis rates were analyzed. Irradiation with¹²C⁺⁵ ion beam at relatively very low doses (5.0 Gy) significantly inhibited cell division, yet the survival rate and regeneration capability of the cells through somatic embryogenesis were conserved in more than 70 and 50 %, respectively. These results indicate that cell division is the most sensitive parameter to irradiation, accounting for the inhibition of colony formation and callus growth. The potential use of the¹²C⁺⁵ ion beam in asymmetric protoplast fusion experiments is discussed.     

Photoautotrophic growth of cell suspension cultures fromChenopodium rubrum in an airlift fermenter

Summary This communication describes the construction and operation of an airlift fermenter for the photoautotrophic growth of cell suspension cultures fromChenopodium rubrum. The basic batch culture unit provides a culture of 1.51 volume, sufficient to permit frequent aseptic sampling. It can be maintained at any desired temperature and aerated to different extents. Using an initial cell density of about 400,000 cells per ml suspension, the increase in cell number is 270% after a 14 days' growth period, although the stationary phase of growth is not yet reached. The transfer of photoautotrophic cell suspensions fromChenopodium rubrum from stationary growth into the large volume of fresh culture medium in the airlift fermenter results in an immediate protein formation, followed by an exponential phase of cell division, whereas rapid chlorophyll accumulation is delayed by 2 days.The growth capacities of photoautotrophic fermenter cultures including protein and chlorophyll formation as well asin vitro activities of the ribulosebisphosphate carboxylase and the phosphoenolpyruvate carboxylase are greatly lower as compared to photoautotrophic cells propagated in standard two-tier culture vessels using 30 ml culture medium. However the pattern of change in the activities of carboxylation enzymes is quite similar in both culture systems.Photoautotrophic cell suspensions fromChenopodium rubrum grown in an airlift fermenter assimilate about 90 mol CO₂/mg chlorophyll × hour. Dark CO₂ fixation is about 1.5% of the light values.Abbreviations PEF phosphoenolpyruvate - RuDP ribulosebisphosPhate - NS ground glass joints of standardized size made from Duran glass, Schott, Germany     

Isolation and growth of protoplasts from cell suspensions of Pinus contorta dougl. ex loud

Cell suspensions were initiated from embryo derived calli of Pinus contorta. Some of these cell lines could be maintained in culture for at least one year without reduced growth. A high yield of protoplasts was obtained from the cell suspensions. The protoplasts started to divide after two days and cell clusters could be observed after about two weeks. The growth phase of the cell suspensions was very important for the division of protoplasts. Only protoplasts isolated from suspensions in an actively dividing phase were able to divide with a high frequency and to give rise to cell clusters.     

Mitochondrial function plasticity in <Emphasis Type="Italic">Acanthamoeba castellanii</Emphasis> during growth in batch culture

The alterations in mitochondrial bioenergetics during growth in a batch culture of Acanthamoeba castellanii were studied. The capacity of cytochrome pathway-dependent respiration measured in vitro decreased from the intermediary phase, when cell division slowed down. The pattern of the cytochrome pathway capacity changes was paralleled from the intermediary phase by alterations in the amount of total (and reducible) membranous ubiquinone. These changes were accompanied by a decrease in mitochondrial reactive oxygen species production in vitro (when no energy-dissipating system was active), and almost no change in superoxide dismutase activity and protein level, thus indicating an equivalent need for this enzyme in oxidative stress defence in A. castellanii culture. On the other hand, a decrease in the activity and protein level of alternative oxidase and uncoupling protein was observed in vitro, when cells shifted from the exponential growth phase to the stationary phase. It turned out that the contribution of both energy-dissipating systems in the prevention of mitochondrial reactive oxygen species generation in vivo could lead to its constant level throughout the growth cycle of A. castellanii batch culture. Hence, the observed functional plasticity insures survival of high quality cysts of A. castellanii cells.     

Somatic embryogenesis and plant regeneration in Acacia farnesiana and A. schaffneri

Summary Efficient plant regeneration systems via somatic embryogenesis have been developed for Acacia farnesiana and Acacia schaffneri [Leguminosae (Mimosoideae)]. The protocol used in this study consisted of placing immature, zygotic embryos of these species in Murashige and Skoog semi-solid basal medium supplemented with 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.65 μM kinetin to induce callus. Some parts of the callus were used for direct embryo differentiation and others for establishment of cell suspension cultures. In the first case, somatic embryos were produced on semi-solid differentiation media without growth regulators or with abscisic acid (ABA). The higher number of somatic embryos, 345 and 198 embryos per g callus in A. farnesiana and A. schaffneri, respectively was obtained in media without growth regulators, but adding ABA increased the percentage of embryos that reached more advanced differentiation stages. The production of somatic embryos was achieved starting from cell suspensions only when these suspensions were plated into the semi-solid differentiation medium. Somatic embryos germinated on medium containing 217 μM adenine sulfate with efficiencies of 69% in A. farnesiana and 47% in A. schaffneri. Some somatic embryos that developed into plantlets were acclimatized in the greenhouse, and they grew into normal plants.     

Are polyamines directly involved in silymarin production in the milk thistle [<Emphasis Type="Italic">Silybum marianum</Emphasis> (L.) Gaernt]?

The possible role of polyamines (PAs) in the regulation of silymarin (Sm) production in milk thistle [Silybum marianum (L.) Gaernt] cell suspension cultures was studied in a young cell culture line (H2 line) and in a synchronized cell line (>3 years; H1 line). The effect of two exogenous PAs, putrescine (Put) and spermidine (Spd), and a number of metabolic inhibitors (L-canavanine, DL-α-difluoromethylornithine, methylglyoxal-bis-guanylhydrazone, cyclohexylamine) on the production of Sm during the growth cycle were analyzed. The results suggest that PAs are not directly involved in the Sm synthesis pathway. In our cell culture system, Sm production and PA contents were determined by the age of the suspension culture cells: with increasing age, the suspension culture cells showed a decreasing capacity to reach the stationary phase during prolonged subculture that was associated with a decreased production of Sm, a steady increase in PA content, and a constant Put/Spd ratio. The synchronization of dividing cells from the S. marianum H1 line did not modify this behaviour. In young cell suspensions, maximum Sm production occurred in the stationary phase, concurrent with the cellular PA contents reaching their minimum value. At the start of the stationary phase, the high percentage of cells in the growth phases (G₀/G₁) and a transient increase in the Put/Spd ratio were accompanied by maximum Sm production and a blockade of cell division.     

Cell division arrest by gamma-irradiation in photoautotrophic suspension culture of Euphorbia characias: Maintenance of photosynthetic capacity and overaccumulation of sucrose

Gamma-irradiation (250 Gy) applied to photoautotrophic cell suspensions of Euphorbia characias L. in the exponential growth phase led to the arrest of cell division and to a subsequent overaccumulation of sucrose and dry matter. From the fourth day of culture, the chlorophyll content and gross photosynthesis were not depressed by gamma-treatment nor by sugar accumulation. In both cultures, no difference was observed between oxygen uptake in the light at CO₂ saturating concentration and in the dark, suggesting that no change in energy-dissipative reactions took place after irradiation. A slight increase in oxygen uptake in both light and dark was observed in irradiated cells during the first four days. However, in the absence of limiting factors, the photosynthetic capacities of the dividing and irradiated non-dividing photoautotrophic cells were identical but higher than that of the non-dividing cells in the stationary growth phase. This suggests that gamma-irradiation arrests cell division by a mechanism different to that occuring in stationary-phase cultures. This may be of value in investigating the metabolism of secondary products.     

Freeze-Preservation of Rice Cells Grown in Suspension Culture

A simple procedure has been worked out for the freeze-preservation of rice (Oryza sativa L.) cells grown in suspension culture. The protocol differs in some interesting aspects from those established for other organisms. A peculiar feature of this procedure is that growth of freeze-recovered rice cells resumes after an extremely short lag period of 2–8 days and proceeds with a growth rate identical to that of untreated cells. This, together with data obtained with viability tests, rules out the possibility that selection of freeze-resistant mutant cells may occur, as postulated with other plant cells where growth resumption was considerably delayed in time. The viability of freeze-recovered rice cells, when assessed at time zero after thawing by measuring the mitochondrial respiratory efficiency, was 60–65% of that of untreated cells. However, the limits of this and other viability tests in determining the efficiency of the freeze preservation methods and the percentage of surviving cells were shown by experiments in which cell viability and cell growth were followed in cultures initiated with freeze-recovered rice cells.     

Plating efficiency measurements and the experimental control of ageing of adult human skin fibroblasts in vitro.

Adult human skin fibroblasts were serially cultured by means of eleven protocols differing in inoculum size, duration of culture between passage and the ability of the medium to support cell division. Each protocol was terminated only when there were too few cells for further subculturing. The fraction of the cells of an inoculum adhering to the growth surface was unaffected by serial subculturing or by differences in protocol. The final cell count at the end of a period of culture and the plating efficiency for the next culture diminished progressively with serial subculturing. Nevertheless, the computed number of cell generations per culture period of those cells which divided was unaffected by serial passaging. The total number of cell doublings accruing during an entire protocol depended only on the duration of the period of culture between successive passages which was characteristic of that protocol. The observations can be accounted for quantitatively by the following assumptions. A cell which loses its ability to divide after a given period of culture nevertheless continues to grow in size during the next period of culture. The increase in volume of cell substance during any such period is the same whether or not a cell divides. The second postulate is that the probability of a cell being able to divide at the start of a period of culture is proportional to the probability that it will not lose this ability by the following period of culture.     

Selective loss of S-phase cells when making cell suspensions from lymphoid tissue.

The relative yield of S-phase cells when making cell suspensions from lymph nodes was determined by two different methods: by estimating the proportions of S-phase cells in sections and smears from lymph nodes undergoing a local graft-versus-host reaction, and by measuring the [³H]thymidine activity relative to DNA content in intact tissue and cell suspensions from normal lymph nodes. Both methods showed a large selective loss of S-phase cells in the process of making cell suspensions. The cell types preparing for division in the GVH nodes were then determined by light microscopic autoradiography combined with electron microscopy of neighboring ultrathin sections. The majority of dividing cells were lymphoid; some of these showed advanced signs of cell death.     

Using single cell cultivation system for on-chip monitoring of the interdivision timer in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> cell cycle

Regulation of cell cycle progression in changing environments is vital for cell survival and maintenance, and different regulation mechanisms based on cell size and cell cycle time have been proposed. To determine the mechanism of cell cycle regulation in the unicellular green algae Chlamydomonas reinhardtii, we developed an on-chip single-cell cultivation system that allows for the strict control of the extracellular environment. We divided the Chlamydomonas cell cycle into interdivision and division phases on the basis of changes in cell size and found that, regardless of the amount of photosynthetically active radiation (PAR) and the extent of illumination, the length of the interdivision phase was inversely proportional to the rate of increase of cell volume. Their product remains constant indicating the existence of an 'interdivision timer'. The length of the division phase, in contrast, remained nearly constant. Cells cultivated under light-dark-light conditions did not divide unless they had grown to twice their initial volume during the first light period. This indicates the existence of a 'commitment sizer'. The ratio of the cell volume at the beginning of the division phase to the initial cell volume determined the number of daughter cells, indicating the existence of a 'mitotic sizer'.     

PHOSPHATE UPTAKE KINETICS IN EUGLENA GRACILIS (Z) (EUGLENOPHYCEAE) GROWN IN LIGHT/DARK CYCLES. II. PHASED PO4-LIMITED CULTURES1

The characteristics of phosphate uptake and photosynthetic capacity were studied in P-limited populations of Euglena gracilis Klebs (Z), using both P-limited batch cultures in stationary phase and cyclostat cultures grown on 14:10 LD. P uptake obeyed Michaelis-Menten kinetics between 0 and 150 μM PO₄ under both growth conditions. The value of V_max was 35% lower in the dark than in the light in the stationary phase cells. The value of K₈ was not affected by light conditions, and uptake was completely inhibited in the presence of 1 mm KCN. P uptake (at 2.0 μM PO₄) and photosynthetic capacity showed diel periodicity with peak rates occurring just before the beginning of the dark period for P uptake, and 8 h into the light period for photosynthetic capacity. V_max for P uptake increased by a factor of 1.5 over the light period, whereas K₈ remained constant at 1.4 μM PO₄. These patterns were displayed by both nondividing stationary phase cells and populations in which less than a third of the cells divided each day, indicating that the rhythmicity is not coupled to cell division.