DNA replication and the development of preprophase bands in soybean protoplast cultures

Nuclear DNA replication and the development of preprophase bands (PPBs) are two chronologically close processes during the higher plant cell cycle. However, it is not clear whether occurrence of PPBs is coupled with DNA replication. A soybean protoplast culture with a high frequency of PPBs was used to study the relationship between the two processes when treated with aphidicolin, a potent and specific inhibitor of eukaryotic DNA polymerase-α. When DNA replication was partially inhibited by 10 mg l^-1 aphidicolin, both the percentage of cells with PPBs and the mitotic index (MI) decreased in absolute terms, but there were proportionately more PPBs than mitoses. Since PPBs change in appearance as they develop, they were divided into categories of early (interphase associated) and late (prophase associated). The increased PPB/MI ratio was associated with an increased proportion of early stage PPBs relative to late stage PPBs. When DNA replication was completely blocked by 50 mg l^-1 aphidicolin, both MI and the percentage of cells with PPBs were close to zero. These results suggest that development of PPBs was to a large extent coupled DNA replication. We propose that the increased PPB/MI ratio at 10 mg l^-1 aphidicolin was due to a linkage between the duration of interphase and the time period in which early stage PPBs are visible. The increased duration of early PPBs partially compensates for the reduced number of nuclei reaching the stage of PPB initiation. Furthermore, in cultures containing aphidicolin, the percentage of PPBs with simultaneous perinuclear fluorescence (PNF, accumulation of microtubules on nuclear envelope) was reduced and whenever PNF was prominent and dense on the nuclear envelope the nucleus showed chromatin condensation. These observations indicated that the transition from PPB to PNF and then to the prophase spindle is closely related to the progress of the nuclear cycle.     

Effects of cycloheximide on preprophase bands and prophase spindles in onion (Allium cepa L.) root tip cells

Summary Effects of cycloheximide (CHM) on preprophase bands (PPBs) of microtubules (MTs) and on prophase spindle MTs in root tip cells of onion (Allium cepa L.) were examined. When root tip cells were treated with 36 M CHM for 0.5–4 h, the population of cells with a PPB did not decrease markedly although the population of mitotic cells and that of prophase cells with a PPB gradually decreased to half of the control root tips. In prophase cells treated with 11 and 36 M CHM for 2 h, the width of the PPB was 1.4 times broader than that in the prophase PPB without CHM. Electron microscopic observation on the cross section of the PPB showed that the number of MTs and the distance between adjacent MTs in prophase PPBs treated with CHM were similar to those in the early developmental stage of PPBs without CHM. The bipolar spindle, that appeared in late prophase was not seen in prophase cells treated with 11 M or higher concentrations of CHM for 2 h. In order to examine differences of perinuclear MT arrangement between CHM treated and non-treated prophase cells, arrangement of perinuclear MTs was examined by confocal laser scanning microscopy. In control cells without CHM, MTs appeared on the nuclear surface with several branched or cross over type MT foci in the cytoplasm when broad PPB formation started. These MT foci were replaced by the aster type MT foci, from which several MTs radiated along the nuclear surface. The aster type MT foci gradually gathered to form a bipolar spindle. MTs connecting the spindle pole region and the PPB were seen in late prophase. In CHM-treated cells (11-360 M for 2 h), branched and cross over type MT foci were prominent, even in prophase cells with well condensed chromosomes. Neither linkages of MTs between the spindle pole region and the PPB nor aster type MT foci were seen. These observations showed that CHM prevents the bundling of MTs in the PPB and also inhibits the formation of aster type MT foci that is essential for bipolar spindle development.     

Response to auxin by cells ofRiella helicophylla during reversible arrest in different cell-cycle phases

When cells of the unistratose meristem ofRiella helicophylla (Bory et Mont.) Mont. are reversibly arrested at G1/S transition by treatment with the inhibitor of thymidylate synthase 5-fluorodeoxyuridine, with the inhibitor of DNA polymerase , aphidicolin, or with an inhibitor of late DNA synthesis, 5-aminouracil, they continue to expand. Simultaneous supply of auxin enhances cell expansion, while simultaneous addition of the auxin antagonistp-chlorophenoxyisobutyric acid prevents cell enlargement. When the meristematic cells are reversibly arrested during G1 phase by treatment with chlorsulfuron, an inhibitor of acetolactate synthase, cell size remains unchanged, but it increases when auxin is supplied simultaneously. Simultaneous application of chlorsulfuron during treatment with 5-fluorodeoxyuridine, aphidicolin or 5-aminouracil, prevents cell expansion. After recovery from 5-fluorodeoxyuridine, aphidicolin or 5-aminouracil treatment, the cellular pattern of the meristem is severely disturbed, while in combination withp-chlorophenoxyisobutyric acid or chlorsulfuron, meristem differentiation is almost unaffected. During reactivation of divisional functions in mature cells induced by isolation of tissue fragments, blockage of DNA synthesis by aphidicolin causes an augmentation of rhizoid initials which are characterized by enhanced RNA synthesis. Exogenous supply of auxin is required for outgrowth of these rhizoid initials, while, in untreated fragments, auxin for rhizoid growth is provided probably by the dividing cells. When reactivation of divisional functions in tissue fragments is reversibly inhibited by chlorsulfuron, no changes in the cells are discernible and application of auxin has no effect; after release from blockage the cells regenerate like those in untreated fragments. The results suggest that the phases of the cell cycle differ with regard to auxin synthesis and competence to respond to auxin. Probably, during cycle inhibition at G1/S or S a rising auxin level causes disintegration of cell-cycle events.Abbreviations APH aphidicolin - 5-AU 5-aminouracil - CS chlorsulfuron - 5-FdUrd 5-fluorodeoxyuridine - PCIB p-chloro-phenoxyisobutyric acid Part of doctoral thesis, University of Kassel, GermanyI thank Professor Luise Stange (this Institute) for her suggestions and many stimulating discussions. This research was supported by a scholarship of the Otto-Braun-Fonds and by a grant of the Deutsche Forschungsgemeinschaft to Professor Stange.     

The pyridine-nucleotide cycle in tobacco

In order to elucidate the NAD-recycling pathway the following enzyme activities have been characterized in different tobacco tissues and in tomato root: NAD pyrophosphatase, nicotinamide mononucleotide (NMN)/nicotinic acid mononucleotide (NaMN) glycohydrolases, nicotinamidase and nicotinic acid phosphoribosyltransferase. The investigations were performed with protein extracts purified by gel filtration and enzymatic activities were determined by high-performance liquid chromatography methods. The kinetic parameters of the different enzymes from tobacco root and their specificity are reported. The data are in favor of the so-called pyridine-nucleotide cycle VI (NADNMNnicotinamidenicotinic acidNaMNnicotinic acid adenine dinucleotideNAD). In the nicotine-producing tobacco root a further direct route leading from NaMN to nicotinic acid is proposed. These data are reconciled with the assumption that it is nicotinic acid which is provided by the pyridine-nucleotide cycle for the synthesis of nicotine.Abbreviations HPLC high-performance liquid chromatography - Na nicotinic acid - NaAD nicotinic acid adenine dinucleotide - NaMN nicotinic acid mononucleotide - NMN nicotinamide mononucleotide - PRPP 5-phosphoribosyl-1-pyrophosphate This contribution is dedicated to Professor Augustin Betz on the occasion of his 65^th birthday     

Nuclear and microtubular cycles in heterophasic multinuclearTriticum root-tip cells induced by caffeine

Summary Nuclear and microtubular cycles were studied in large heterophasic multinuclear cells induced in root tips ofTriticum turgidum by caffeine treatment. Multinuclear cells and cells with polyploid nuclei exhibited various configurations of multiple and complex preprophase microtubule (Mt) bands (PPBs), including helical ones. The developmental stages of PPBs in some heterophasic cells did not comply with the cell cycle stages of the associated nuclei, a fact indicating that these events are not directly controlled by the associated nuclei. The heterophasic cells exhibited asynchronous nuclei at different stages of mitosis. In cells displaying prophase and interphase nuclei, the prophase spindle was either absent or developed around both of them or developed around the prophase nuclei earlier than around the interphase ones. During prometaphase-metaphase of the advanced nuclei the lagging interphase nuclei were induced to form prematurely condensed chromosomes (PCCs) along with spindle formation around them. These observations suggest that the mitotic transition in heterophasic cells is delayed but is ultimately achieved due to the effect of the advanced nuclei, which induces a premature mitotic entry of the lagging nuclei. Although kinetochore Mt bundles were found associated with PCCs, their metaphase and anaphase spindles were abnormal resulting in abnormal or abortive anaphases. In some heterophasic cells, metaphase-anaphase transition did not take place simultaneously in different chromosome groups, signifying that the cells do not exit from the mitotic state after anaphase initiation of the advanced nuclei. Asynchronous pace of mitosis of different chromosome groups was also observed during anaphase and telophase. Implications of these observations in understanding plant cell cycle regulation are discussed.Abbreviations cdk cyclin dependent kinase - Mt microtubule - PCC prematurely condensed chromosome - PPB preprophase band     

The organization of F-actin in root tip cells ofAdiantum capillus veneris throughout the cell cycle

Summary The patterns of F-actin in relation to microtubule (Mt) organization in dividing root tip cells ofAdiantum capillus veneris were studied with rhodamine-phalloidin (RP) labelling and tubulin immunofluorescence. Interphase cells display a well organized network of cortical/subcortical, endoplasmic and perinuclear actin filaments (AFs), not particularly related to the interphase Mt arrays. The cortical AFs seem to persist during the cell cycle while the large subcortical AF bundles disappear by preprophase/prophase and reappear after cytokinesis is completed. In some but not all of the preprophase cells the cortical AFs tend to form a band (AF-PPB) coincident with the preprophase band of Mts (Mt-PPB). In metaphase and anaphase cells AFs are localized in the cell cortex, around the spindle and inside it coincidently with kinetochore Mt bundles. During cytokinesis AFs are consistently found in the phragmoplast. In oryzalin treated cells neither Mt-PPBs, spindles and phragmoplasts exist, nor such F-actin structures can be observed. In cells recovering from oryzalin, AF-PPBs, AF kinetochore bundles and AF phragmoplasts reform. They show the same pattern with the reinstating respective Mt arrays. In contrast, in cells treated with cytochalasin B (CB), AFs disappear but all categories of Mt arrays form normally.These observations show that F-actin organization in root tip cells ofA. capillus veneris differs from that of root tip cells of flowering plants examined so far. In addition, Mts seem to be crucial for F-actin organization as far as it concerns the PPB, the mitotic spindle, and the phragmoplast.Abbreviations AF actin filament - CB cytochalasin B - MBS m-male-imidobenzoyl-N-hydroxysuccinimide ester - MSB microtubule stabilizing buffer - Mt microtubule - PBS phosphate buffered saline - PPB preprophase band - RP rhodamine phalloidin     

Nuclear ploidy is contingent on the microtubular cycle responsible for plant cytokinesis

Summary. Division of the plant cell relies on the preprophase band of microtubules (PPB)-phragmoplast system. Cells of onion (Allium cepa L.) root meristems were rendered binucleate by preventing the consolidation of cell plate formation in telophase with 5mM caffeine. These binucleates developed either a single PPB around one of their two nuclei or two PPBs, one per nucleus, in the prophase of the ensuing mitosis. Prophase cells developing one single PPB were shorter in length (42.3±4.1µm) than those developing 2 PPBs (49.8±4.1µm), and interphase duration was inversely related to cell length. Cells whose length was less than or equal to 42µm, i.e., which had not even reached the mean size of the small binucleates in prophase, were followed throughout mitosis. In metaphase, they always assembled two mitotic spindles (one per nucleus). However, the cells that had assembled a single PPB also developed a single phragmoplast in telophase, leading to polyploidization. As these meristematic cells were not wide enough to accommodate the midzones of both mitotic spindles in any single plane transversal to the cell elongation axis, the spindles tilted until their midzones formed a continuum where the single common phragmoplast assembled. Its position was thereby uncoupled from that of the preceding PPB. Subsequently, the chromosomes from two different half-spindles were included, by a common nuclear envelope, in a single tetraploid nucleus. Finally, the cytokinetic plate segregated the two tetraploid nuclei formed at each side of the phragmoplast into two independent sister cells.Correspondence and reprints: Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.     

Immunocytochemical study of cell cycle control by cytokinin in cultured soybean cells

An immunocytochemical method was used to determine the proportion of cells in the DNA synthesis (S phase) of the mitotic cycle in suspension cultures of soybean (Glycine max (L.) Merr. cv. Acme) callus of cotyledonary origin, the stably cytokinin-dependent tissue used in the cytokinin bioassay devised by Carlos O. Miller. A standard cell synchronization protocol involving hydroxyurea was used to demonstrate the applicability of the immunocytochemical method to this cell culture. Cells were brought to mitotic arrest by cytokinin withdrawal, and the cell division cycle was restarted by the addition of cytokinin. We have followed the pattern of resumption of S phase after the readdition of cytokinin. This pattern reveals the existence of three subpopulations of cells in cytokinin-starved cultures, consistent with the occurrence of three cytokinin-requiring events in the cell cycle: one in mitosis, one in S phase, and one in the G₁ phase.Abbreviations BrdU 5-bromo-2-deoxyuridine - DI deionized water - FITC fluorescein isothiocyanate - HU hydroxyurea - l-AOPP l--aminooxy--phenylpropionic acid - LI labeling index - PA polyamine - PI propidium iodide     

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.     

Parameters influencing transient and stable transformation of barley (Hordeum vulgare L.) protoplasts

Cat gene expression has been investigated following PEG-mediated plasmid uptake into barley protoplasts. The uptake conditions optimised for transient expression were employed for stable transformation. Transformed protoplast-derived calli of the cvs. Dissa and Igri, were selected on medium containing G418 at 40 g ml^–1 or kanamycin sulphate at 250 g ml^–1. Absolute transformation frequencies of 28.9×10^–5 and 21.3×10^–5 were recorded for Dissa with kanamycin sulphate and G418 selection, respectively. The frequency for Igri was 11.5×10^–5 with G418 selection. Antibiotic resistant protoplast-derived colonies expressed NPTII activity; Southern hybridisation confirmed integration of the nptII gene into barley genomic DNA.Abbreviations ABA abscisic acid - AC-CAP acetylated chloramphenicol - BAP 6-benzylaminopurine - cat chloramphenicol acetyltransferase gene - CAT chloramphenicol acetyltransferase activity - CaMV cauliflower mosaic virus - CAP chloramphenicol, 2,4-d-2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - G418 Geneticin - gus -glucuronidase gene - HEPES (N[2-hydroxyethyl] piperazine-N-[2-ethanesulphonic acid]) - IAA indole acetic acid - MES 2-N-morpholinoethane sulphonic acid - NAA -naphthaleneacetic acid - npt II neomycin phosphotransferase gene - NPTH neomycin phosphotransferase activity - PEG polyethylene glycol - SCV settled cell volume     

Rearrangements of Microtubules Involved in Establishing Cell Division Planes Start Immediately after DNA Synthesis and Are Completed just before Mitosis

This work concerns an aspect of spatial regulation of cell division, the development of the preprophase band (PPB) of microtubules. The PPB is significant in plant development because its position in the dividing cell indicates where the new cell wall will be inserted[mdash]an important site for control of histogenesis. We have categorized and determined the durations of stages in the development of PPBs, and have established their timing relative to the S-, G2-, and mitotic phases of the cell cycle. Roots of wheat seedlings were supplied with bromodeoxyuridine in continuous and pulse-chase treatments. Cells that were in the S-phase were identified and changes in their microtubule arrays were monitored by double immunolabeling. PPB initiation was detectable as early as the end of the S-phase as a narrowing of the preceding interphase array of microtubules. Development continued throughout G2 to a mature, narrow PPB, which existed only briefly and then eroded during the transition to the prophase mitotic spindle. The microtubule rearrangements of PPB development showed that preparation of the future site and plane of division in higher plant cells begins just after DNA replication and is completed just before mitosis.     

Study of mitosis in root-tip cells ofTriticum turgidum treated with the DNA-intercalating agent ethidium bromide

Summary This work examines mitosis in root-tip cells ofTriticum turgidum treated with the RNA synthesis inhibitor ethidium bromide, using tubulin immunolabeling and electron microscopy. The following aberrations were observed in ethidium bromideaffected cells: (1) incomplete chromatin condensation and nuclear-envelope breakdown; (2) delay of preprophase microtubule band maturation; (3) preprophase microtubule band assembly in cells displaying an interphase appearance of the nucleus; (4) prevention of the prophase spindle formation, caused by inhibition of perinuclear microtubule (Mt) formation and/or inability of the perinuclear Mts to assume bipolarity; (5) organization of an atypical metaphase spindle which is unable to arrange the chromosomes on the equatorial plane; (6) formation of an atypical perinuclear metaphase spindle in cells in which nuclear-envelope breakdown has been almost completely inhibited; (7) inhibition of the anaphase spindle formation as well as of anaphase chromosome movement; (8) disorganization of the atypical mitotic spindle during transition from mitosis to cytokinesis. The observations favor the following hypotheses. Nucleation of prophase spindle Mts is related to the mechanism that causes nuclear-envelope breakdown. The mitotic poles lack Mtnucleating and -organizing properties, and their function does not account for prophase and metaphase spindle assembly. The organization of the prophase spindle is not a prerequisite for the formation of the metaphase spindle; the metaphase spindle seems to be formed de novo by Mts nucleated on the nuclear envelope and/or in the immediate vicinity of chromosomes.Abbreviations 5-AU 5-aminouracil - EB ethidium bromide - EM electron microscopy - k-Mt kinetochore microtubule - Mt microtubule - MTOC microtubule-organizing center - NE nuclear envelope - NEB nuclear-envelope breakdown - PPB preprophase band of microtubules     

Hydrogen ion dependence of carrier-mediated auxin uptake by suspension-cultured crown gall cells

1. The dependence on external hydrogen ion concentration of the carrier-mediated components of the uptakes of indol-3-yl acetic acid (IAA) and 2,4-dichlorophenoxyacetic acid (2,4D) by suspension-cultured crown gall cells from Parthenocissus tricuspidata Planch. is examined. The initiall rates of uptake of IAA and 2,4 D from 0.30 mol/l solutions exhibit pH optima of approximately pH 5.0 and pH 4.0 respectively.-2. The inherent difficulties, especially at low pH, of estimating the carrier-mediated uptake component in the presence of the large diffusive uptake of undissociated acid are discussed.-3. At pH 5.0, the uptakes of the two auxins are characterised by similar Michaelis Constants (0.65 and 0.80 mol/l) and Maximum Velocities (0.87 and 1.0 pmol/min/mg cells) for IAA and 2,4D respectively.-4. A dependence of carrier-mediated auxin uptake on cations other than hydrogen ions could not be demonstrated. It is suggested that uptake of auxin anions may occur by co-transport with hydrogen ions.Abbreviations IAA indol-3-yl acetic acid - 2,4 D 2,4-dichlorophenoxyacetic acid     

Growth kinetics of the Golgi apparatus during the cell cycle in onion root meristems

In onion root meristems, the number of dictyosomes per cell shows a kinetics of growth strongly related to the cell cycle. During the interphase of steady-state proliferative cells, the volume density and numerical density of the Golgi apparatus decrease to reach minimum values in late-interphase cells, characterized by their greatest length. This pattern is also found in the total volume occupied by Golgi apparatus. Once in mitosis, the above-mentioned parameters begin to increase reaching maximum mean values in telophase. After the experimental uncoupling of chromosome and growth cycles by presynchronization with hydroxyurea, we found a similar behaviour pattern in the Golgi apparatus: decreasing values during interphase and a triggering of Golgi-apparatus growth in prophase independently of the bigger cell sizes reached in mitosis as an effect of pretreatment with hydroxyurea. These results indicate a cyclic kinetics of this subcellular component in higher-plant meristems, coupled with early mitotic events.     

Microtubule pattern and the occurrence of pre-prophase bands in embryogenic cultures of black spruce (Pieca mariana Mill.) and non-embryogenic cultures of jack pine (Pinus banksiana Lamb.)

Summary The organization of microtubules during interphase and prophase in embryogenic cultures of black spruce (Picea mariana) was investigated by indirect immunofluorescence. Somatic embryos of black spruce possessed an extensively branched and interconnecting network of fine interphase cortical microtubules. The development of pre-prophase bands (PPBs) in embryogenic black spruce cultures was compared with that in non-embryogenic cell cultures of jack pine (Pinus banksiana). PPBs in both species were initially arranged as a very broad array of microtubules, later (early to mid-prophase) becoming narrower and more intensely fluorescent. The occurrence of pre-prophase bands in relation to the number of phragmoplasts (i.e. PPB index) of black spruce somatic embryos was significantly higher (p<0.01) than that found for jack pine cells.     

Plant tyrosine decarboxylase can be strongly inhibited by l-α-aminooxy-β-phenylpropionate

The effect of tunicamycin, an inhibitor of N-glycosylation of proteins, on growth and on synthesis of DNA and protein was studied in suspension cultures from Nicotiana tabacum and Catharanthus rosea. In the presence of 0.1–1 g · ml^-1 tunicamycin, cell division and DNA synthesis stopped in cells which had been proliferating logarithmically, but protein formation continued. Cytophotometric determination of the nuclear DNA content in Catharanthus cells showed that a cell-cycle arrest had occurred in G1 phase. Metabolic labelling of cells with the glycoprotein precursors glucosamine or mannose was inhibited, too. The results indicate that one or more glycoproteins are needed for the cell to pass through the G1 phase, as was recently postulated for animal and yeast cells.Abbreviations TCA trichloroacetic acid - TM tunicamycin     

Is the preprophase band of microtubules a marker of organization in suspension cultures?

Summary To date it has been accepted that preprophase bands of microtubules (PPBs) either do not precede cell division or do so inconsistently in suspension cultures, the assumption being that such cultures proliferate in an unorganized state in which placement of cell plates is not regulated by the PPB system that is widespread in organized tissues. Using indirect immunofluorescence microscopy with antitubulin, the relative frequency of occurrence of PPBs in enzymatically separated cells from root tips and suspension cultures of carrot and tobacco, was quantified by taking the ratio of the number of PPBs: phragmoplast. This ratio was termed the PPB index.One carrot suspension culture proliferated in a medium containing 2,4-Dichlorophenoxyacetic acid (2,4-D), and recognizable stages in somatic embryogenesis formed when 2,4-D was removed from the medium. Another carrot suspension culture was nonembryogenic and removal of 2,4-D resulted in a reduction of cell division and increase in cell elongation. The tobacco culture was a cytokinin habituated cell line and also required 2,4-D to maintain cell division. It ceased proliferation, and cell elongation took place if 2,4-D was removed.The PPB index in the root tips from both species, and in both types of carrot suspension culture was approximately the same but was approx. 15-fold lower in the tobacco suspension. PPBs in the tobacco suspension were atypical in structure as well as sparse in numbers. The PPB index allows quantitative comparisons between different tissues to be made. The low PPB index and the irregular PPBs in the tobacco suspension correlates with its inability to undergo organized morphogenesis and generate spatially defined cell lineages upon 2,4-D removal. In contrast, the high PPB index in the carrot suspension cultures correlates with their potential for organized embryo formation, whether or not that potential is realized by withdrawal of 2,4-D. However, their high PPB index is not obligatorily coupled to embryogenesis.     

Life cycle of Macrothrix flabelligera Smirnov, 1992 (Cladocera, Macrothricidae), recently reported in the Neotropical region

Information on the life cycle of individual species is vital to an understanding of their ecology, particularly their distribution. In this investigation, aspects of the Macrothrix flabelligera (Cladocera, Macrothricidae) life cycle were studied in laboratory cultures under controlled conditions. Neonates from parthenogenetic females previously acclimatized were isolated and grown at constant temperature, in a 12-h/12-h light/dark cycle. Organisms were fed with a suspension of Scenedesmus bijugus at a concentration of 10⁵ cells ml^–1. Daily measurements of body length and age were made, to obtain the growth rate, size and age of primipara. M. flabelligera had high fecundity rates, producing broods of 13.6 eggs female^–1. At 23.0°C, the duration of embryonic and post-embryonic development was 35.9 h and 4.9 days, respectively. The mean longevity was 12.7 days. A length–weight relationship was established and used to convert measured length into weight, in order to plot weight growth curves, as subsidiary information for future secondary production calculations.     

Cell cycle duration in antheridial filaments ofChara spp. (Characeae) with different genome size and heterochromatin content

The relationship between nuclear 1 C DNA content and cell cycle progression throughout successive stages of antheridial filaments were studied among five taxa ofChara: two dioecious species (n = 14):C. aspera (7.2 pg DNA),C. tomentosa (7.4 pg DNA), and three monoecious species (n = 28):C. vulgaris (13.5 pg DNA),C. fragilis (19.3 pg DNA), andC. contraria (19.6 pg DNA). With the use of double³H-thymidine labelling and morphometry a number of characteristics common to all of the investigated species were determined within the proliferative periods preceding spermiogenesis. These include: (1) simplified type of the cell cycle (S + G₂ + M), due to complete lack of G₁ intervals, (2) constant duration of S phase, (3) progressive shortening of G₂ + M periods, and (4) gradual reduction of cell lengths at successive mitotic divisions. Nucleotypic dependence was found between genome size and several time parameters estimated for consecutive stages of antheridial filaments: the higher the DNA C-value, the longer the cell cycles, their component phases, the total duration of the proliferative period, as well as the lower the rate of growth of interphase cells. Differential Giemsa staining of late G₂ phase nuclei revealed that the higher content of C-heterochromatin is connected with prolonged cell cycle durations in species with similar DNA C-values.