Growth and development in relation to the cell cycle inPhysarum polycephalum

Summary In strain CL ofPhysarum polycephalum, multinucleate, haploid plasmodia form within clones of uninucleate, haploid amoebae. Analysis of plasmodium development, using time-lapse cinematography, shows that binucleate cells arise from uninucleate cells, by mitosis without cytokinesis. Either one or both daughter cells, from an apparently normal amoebal division, can enter an extended cell cycle (28.7 hours compared to the 11.8 hours for vegetative amoebae) that ends in the formation of a binucleate cell. This long cycle is accompanied by extra growth; cells that become binucleate are twice as big as amoebae at the time of mitosis. Nuclear size also increases during the extended cell cycle: flow cytometric analysis indicates that this is not associated with an increase over the haploid DNA content. During the extended cell cycle uninucleate cells lose the ability to transform into flagellated cells and also become irreversibly committed to plasmodium development. It is shown that commitment occurs a maximum of 13.5 hours before binucleate cell formation and that loss of ability to flagellate precedes commitment by 3–5 hours. Plasmodia develop from binucleate cells by cell fusions and synchronous mitoses without cytokinesis.Abbreviations CL Colonia Leicester - DSDM Dilute semi-defined medium - FKB Formalin killed bacterial suspension - IMT Intermitotic time - LIA Liver infusion agar - SBS Standard bacterial suspension - SDM Semi-defined medium     

Cell fusion competence and its induction in Physarum polycephalum and Didymium iridis

In heterothallic Myxomycetes, diploid plasmodia arise when haploid amoebae of two different mating types are cultured together. In this mating process, the amoebae fuse in pairs, and the resulting zygotes develop directly into plasmodia. It has been shown previously that plasmodia start to form in this fashion only when the growing amoebae in a mixed culture reach a critical density. We have investigated the cellular basis of this phenomenon by growing amoebae of different mating type separately from one another and then mixing them to test their mating ability. Amoebae from cultures above and below the critical density were, respectively, competent and incompetent to mate. Furthermore, both partners had to be competent in order for mating to occur. No binucleate cells were formed in mixtures of incompetent amoebae, indicating that they failed to fuse with one another. Incompetent amoebae growing at low density on filters with 0.2-μm pores became competent when the filters were placed on dense cultures of amoebae. We suggest that amoebae release a filter-transmissible material that accumulates during growth and induces the cells to become fusion competent.     

Micropropagation of kiwifruit using non-axenic shoot tips

Kiwifruit (Actinidia chinensis Planch.) shoot tips were subjected to a standard surface sterilization procedure and cultured on a Murashige and Skoog basal medium in the presence of two surviving bacterial contaminants. The fresh weight increase of the cultures and the number of shoots produced were greater in liquid medium than in medium solidified with 0.4 or 0.8% agar. A greater number of shoots was obtained with 125 ml than with 50, 250, or 500 ml Erlenmeyer flasks. A concentration of 2 mgl^-1 N⁶-benzylaminopurine (BAP) gave a greater increase in fresh weight than either 0 or 4 mgl^-1.Shoots cut from proliferating cultures were dipped in 0.05% indolebutyric acid (IBA) and rooted directly in a peat: vermiculite: perlite mix. Over 93 % of 907 plantlets produced were successfully acclimatized. The productivity of the method was comparable to that reported for the axenic culture of meristems. The contaminants which survived the initial surface sterilization procedure thus presented no major obstacle to the in vitro propagation of kiwifruit.     

Nuclear membrane behavior during mitosis in normal and heteroploid Myxomycetes

Summary Photomicrographic evidence is presented of the difference in behavior of nuclear membranes during mitosis in amoebae, zygotes and plasmodia of Myxomycetes. One of the species was cultured on bacteria and possessed a normal cycle of plasmogamy and karyogamy between the amoebal and plasmodial phases. The second species was axenically grown in liquid media and had become highly heteroploid and lacked the ability to develop into plasmodia, existing only in the amoeboid form. The significance of the amoeboid form of mitosis in the heteroploid axenically cultured strain is discussed in relation to the difference in nuclear membrane behavior and the possible significance of such behavior.     

Secretion of an antibacterial factor during resuscitation of dormant cells inMicrococcus luteus cultures held in an extended stationary phase

A high proportion ofMicrococcus luteus cells in cultures starved for 3–6 months in spent medium following growth to stationary phase in batch culture lost the ability to grow and form colonies on agar plates, but could be resuscitated from dormancy by incubation in liquid medium containing supernatant taken from the late log phase of viable cultures of the same organism (Kaprelyants et al. 1994). In the present work, we found that during the first 50–70 h of such resuscitation the dormant cells actually divide for 10–17 generations in lactate minimal medium containing yeast extract whilst remaining nonculturable on agar plates. Further incubation results in a decrease in the total cell number in liquid medium. The addition of viable (culturable)Micrococcus luteus cells in concentrations of up to 10⁴ ml^–1 to test tubes containing either resuscitating cells or supernatant from these cultures revealed the excretion of a factor or factors which inhibited the proliferation of otherwise viable cells. The maximum production of this factor took place after some 96 h of incubation of starved cells in resuscitation medium. Supernatant from late logarithmic phase batch cultures ofM. luteus abolished the antibacterial effect of starved cultures incubated in resuscitation medium. It is concluded that the stimulating effect of viable cells, and of supernatant taken from batch cultures, on the resuscitation of dormant cells might be connected in part with overcoming the activity of an antibacterial factor causing self-poisoning of dormant cells during their resuscitation.     

Isolation and culture of protoplasts from callus tissue of Hibiscus syriacus L.

Protoplasts were isolated from callus tissue of Hibiscus syriacus L. using a solution of 3% Onozuka cellulase, 1% Onozuka macerozyme, and 0.5% hemicellulase. Highest yields of viable protoplasts were obtained from friable, white or yellow callus 8–9 days after subculture on Murashige & Skoog medium with 0.5 mg l^-1 2,4-dichlorophenoxyacetic acid and 0.1 mg l^-1 kinetin. Protoplasts cultured in thin liquid layers of this medium with mannitol continued dividing for longer than those cultured in droplets or in an agar medium. Cultures were maintained until protoplasts had divided to form groups of more than ten cells. Cell groups developed into callus and continued to grow on an agar medium, but failed to differentiate on a regeneration medium with 2 mg l^-1 naphthalene acetic acid and 1 mg l^-1 benzylaminopurine.     

Host-tissue differences in transformation of pumpkin (Cucurbita pepo L.) by Agrobacterium rhizogenes

Agrobacterium rhizogenes (wild-type strains 8196 and 15834) transformation of pumpkin (Cucurbita pepo L.) intact seedlings grown in vivo, and 6–8-day-old excised cotyledons cultured in axenic conditions was investigated. Transformed (hairy) roots were successfully induced only on the excised cotyledons with the strain 8196, while intact seedlings failed to form hairy roots with either of the two different bacterial strains. Axenic hairy-root cultures established on MS medium without hormones grew vigorously. Mannopine was detected in all transgenic root clones examined. The peroxidase activity in transformed roots was higher compared with normal roots. Electrophoretic analyses of soluble proteins and isoperoxidases showed substantial differences between transformed and normal pumpkin roots.     

A simple rapid procedure for obtaining axenic cultures from monoxenic cultures of myxomycete plasmodia

Axenic culture of myxomycete plasmodia has been attempted from time to time by various authors, but with very little success. From over 500 known species of myxomycetes, fewer than 20 species have been reported in axenic culture to date, including axenic myxamoebal cultures. In these cultures, the plasmodia required either complex media, or a killed bacterial supplement for growth. Furthermore, the time required for attaining the axenic state varied from several months to years. In the present study, a simple, rapid procedure has been developed to render monoxenic plasmodial cultures axenic. This procedure is based on our discovery that plasmodia have certain unusual substrate preferences that are inhibitory to the associated bacteria using Physarella oblonga as a model. The presence or absence of the bacteria could be ascertained through incubation in four different bacteriological media and by the use of a differential staining technique.     

Carbohydrate,metabolic, and osmotic changes in scaled-up liquid cultures of <Emphasis Type="Italic">Narcissus</Emphasis> leaves

Summary The use of scaled-up liquid cultures could be an efficient system for mass propagation of Narcissus, as it can greatly reduce the costs involved with manual handling. Induction of hyperhydric meristematic leaf section clusters and proliferation were carried out in an ancymidol (ANC)-containing liquid medium in flasks and disposable presterilized plastic bioreactors. Non-hyperhydric bulblets started to develop from hyperhydric meristematic leaf section clusters after subculture on a 0.8% agar strength medium, and young bulbs formed after 10 mo. in vivo acclimatization with a 98% survival rate. The present study reveals that in Narcissus leaf sections cultured in liquid medium, morphogenetic changes in leaf sections were associated with metabolic changes. The changes in carbohydrate, protein, and water potential of the liquid media and leaf sections were found to be closely related to meristematic center initiation on Narcissus hyperhydric leaf sections. Starch, sucrose, and glucose were significantly higher in the hyperhydric leaf sections cultured in ANC medium. The water potential was signifieantly higher in ANC-treated leaf sections and significantly lower in the medium containing ANC, at the stage shortly before or after hyperhydricity and meristematic centers hegan forming on the leaf sections. A 30kDa protein was found to be present in the hyperhydric leaf sections. Based on the present study, a largescale micropropagation protocol of Narcissus in agar and liquid cultures is proposed.     

Regulation of proteolytic enzymes in axenically grown myxamoebae of Physarum polycephalum

The cultivation of Physarum polycephalum amoebae in two media with different protein contents revealed a regulation of aminopeptidases and proteases depending on the albumin content of the medium: in growing amoebae and plasmodia the aminopeptidases have similar isoenzyme patterns and relative activities against nitroanilides. One alanine and four leucine aminopeptidase isoenzymes were found within the slightly acid pH range. During growth amoebae secrete—different from plasmodia—leucine aminopeptidase into the medium with low protein content. In an albumin-rich medium additional alanine aminopeptidase activity was found. Out of nine plasmodial proteases four were found in amoebae too. Only one band (pI 3.6) was present in the protein-poor medium. No protease activity could be detected in the proteinrich medium.Abbreviations BSA bovine serum albumin - SD semi-defined (medium with low protein content; Table 1) The investigation formed a part of the Ph.D. thesis of A. Haars, Göttingen, 1976     

The compound internal pyrenoid in cultured cells of the green algaMonoraphidium griffithii (Berkel.) Komar.-Legner.

Summary The chloroplast ultrastructure ofMonoraphidium griffithii (Berkel.) Komar.-Legner. has been studied in axenic cultures of various ages. The algae have grown in a complete nutrient solution (illumination about 3,000 lx) and on its agar medium (illumination about 600 lx).The large parietal cup-shaped chloroplast of the cells includes a multiformed compound internal pyrenoid that is situated, especially in older cells, in the central part of the chloroplast opposite to the dictyosome and the nucleus. The chloroplast thylakoids either reach the edge of the pyrenoid or penetrate its matrix and run there parallel in more or less long bits. Starch grains were not found to form any sheath around the pyrenoid regions. The number of starch grains increased with the age of the cell.     

Axenic cultivation of Naegleria gruberi : Requirement for methionine

A simplified axenic medium for Naegleria gruberi strain NEG-M contains -methionine, dextrose, yeast extract, a macromolecular fraction of fetal calf serum, and phosphate buffer. Amoebae cultured in suspension in this medium grow with doubling times of 8–10 h (at 32 °C) to yield 2–4 × 10⁶ cells/ml. Amoebae from growing or early stationary phase cultures, transferred to nonnutrient buffer, differentiate synchronously into flagellates. Differentiation occurs reproducibly 80 min after initiation (time for 50% flagellates at 25 °C) if amoebae are taken from a culture maintained at pH 6.6.     

AXENIC CULTIVATION OF THE HETEROTROPHIC DINOFLAGELLATE PFIESTERIA SHUMWAYAE AND OBSERVATIONS ON FEEDING BEHAVIOR1

Pfiesteria shumwayae Glasgow et J. M. Burkh. [=Pseudopfiesteria shumwayae (Glasgow et J. M. Burkh.) Litaker, Steid., P. L. Mason, Shields et P. A. Tester] is a heterotrophic dinoflagellate commonly found in temperate, estuarine waters. P. shumwayae can feed on other protists, fish, and invertebrates, but research on the biochemical requirements of this species has been restricted by the lack of axenic cultures. An undefined, biphasic culture medium was formulated that supported the axenic growth of two of three strains of P. shumwayae. The medium contained chicken egg yolk as a major component. Successful growth depended on the method used to sterilize the medium, and maximum cell yields (10⁴ · mL^?1) were similar to those attained in previous research when P. shumwayae was cultured with living fish or microalgae. Additionally, P. shumwayae flagellate cells ingested particles present in the biphasic medium, allowing detailed observations of feeding behavior. This research is an initial step toward a chemically defined axenic culture medium and determination of P. shumwayae metabolic requirements.     

MORPHOGENESIS OF MONOSTROMA OXYSPERMUM (KÜTZ.) DOTY (CHLOROPHYCEAE) IN AXENIC CULTURE,ESPECIALLY IN BIALGAL CULTURE1

The typical morphology of Monostroma oxyspermum (Kütz.) Doty is lost in axenic culture. In synthetic media of the ASP type, it grows as a colony-like mass composed of round cells with numerous rhizoids. Such a mass is a fragile structure which falls apart upon shaking, or slight touch, into small cell-groups and single cells or cells with a long rhizoid. Only temporary saccate or monostromatic fronds appear and reach 1–2 mm in length when grown in enriched seawater media, but disintegrate and become a colony-like mass. The typical morphology is easily restored by adding at specific intervals filtrates of bacterial cultures and supernatant medium from axenic brown and red algal cultures to the basal medium (ASP7), or by reinfecting the Monostroma with an appropriate bacterial flora. Furthermore, the typical morphology in also maintained by bialgal cultures between Monostroma and other axenic strains of various species of seaweeds except the species belonging to the Chlorophyceae. Monostroma thus appears to utilize some substances released by most species of brown and red algae for its typical growth. Active substances released by bacteria, brown and red algae have not yet been identified and purified. However, it is demonstrated that in axenic cultures many species of seaweeds produce active extracellular substances which play an important role in growth and Morphogenesis of other species of seaweeds.     

A gene,imz, affecting the pH sensitivity of zygote formation inPhysarum polycephalum

Mating inPhysarum polycephalum involves the fusion of two haploid amoebae and the differentiation of the resulting diploid zygote into a multinucleate plasmodium. Mating proceeds optimally with amoebae growing on an agar medium at pH 5.0. At pH 6.2, the amoebae still grow normally, but mating is completely blocked. The barrier at pH 6.2 is not in the differentiation step, since preformed diploids readily convert to plasmodia at this pH. The barrier can be overcome by raising the ionic strength of the agar medium; the effect, moreover, is not ion-specific. We have discovered a genetic locus,imz (ionicmodulation of zygote formation), that affects the upper pH limit for mating; the respective limits associated with the two known alleles,imz-1 andimz-2, are pH 5.6 and pH 6.0 at low ionic strength. Animz-1×imz-2 mating displays the pH 6.0 limit;imz-2 is therefore “dominant”. We suggest that this new gene affects a cell component that is exposed to the exterior of the amoeba and is involved in the fusion step of mating.     

Synthetic oligonucleotide probes for detection of mercury-resistance genes in environmental freshwater microbial communities in response to pollutants

Mercury-resistance genes were detected byin situ hybridization using new synthetic oligonucleotide probes specific formerA andmerB genes according to the published sequences of the corresponding enzymes. These DNA probes were used for the detection of specific mercury-resistant microorganisms isolated from the Rhine River which had been polluted 3 years previously in 1986. Mercuric reductase and organomercurial lyase genes persist in the bacterial genome even after the disappearance of the pollutant but are absent in axenic amoebae. A total of 49 bacterial isolates showed DNA homologies with the³²P-labelled DNA probes and 15 free-living amoebae were selected due to their harboured symbiotic mercury-resistant bacteria.     

Establishment of axenic endosymbiotic strains of Japanese Paramecium bursaria and the utilization of carbohydrate and nitrogen compounds by the isolated algae

Cells of the green paramecium, Paramecium bursaria, contain several hundred endosymbiont cells. The properties of the symbionts are considered to vary depending on the collection site of the host. Difficulties in achieving axenic cells and maintenance of axenic strains for long periods have been reported for symbiotic algae from P. bursaria isolated in Japan. To establish axenic algal strains from such Japanese P. bursaria, symbionts were isolated carefully, and isolated axenic strains were grown on an agar medium containing organic nitrogen compounds. Symbiotic algal strains were obtained from three Japanese P. bursaria strains and their axenicity was confirmed by DAPI staining, cultural tests of bacterial contamination, and DGGE-PCR. These axenic strains have been maintained for over 2 years. Utilization of carbohydrates and nitrogen compounds by symbionts was examined. Monosaccharides (glucose and fructose) increased the growth of the symbiont but disaccharides (maltose and sucrose) did not. Japanese axenic symbionts were able to use ammonia and amino acids, but not nitrate or nitrite. While potent nitrite reductase activity was stimulated by nitrate induction, nitrate reductase activity was not. Nitrate utilization of Japanese symbionts differed from that reported for European and American symbionts.     

Cell type-dependent expression of tubulins in Physarum

Three alpha-tubulins and two beta-tubulins have been resolved by two-dimensional gel electrophoresis of whole cell lysates of Physarum myxamoebae or plasmodia. Criteria used to identify the tubulins included migration on two-dimensional gels with myxamoebal tubulins purified by self-assembly into microtubules in vitro, peptide mapping with Staphylococcus V8 protease and with chymotrypsin, immunoprecipitation with a monoclonal antibody specific for beta-tubulin, and, finally, hybrid selection of specific mRNA by cloned tubulin DNA sequences, followed by translation in vitro. Differential expression of the Physarum tubulins was observed. The alpha 1- and beta 1-tubulins were detected in both myxamoebae and plasmodia; alpha 2 and beta 2 were detected only in plasmodia, alpha 3 was detected only in the myxamoebal phase, and may be specific to the flagellate. Observation of more tubulin species in plasmodia than in myxamoebae was remarkable; the only microtubules detected in plasmodia are those of the mitotoic spindle, whereas myxamoebae display cytoplasmic, centriolar, flagellar, and mitotic-spindle microtubules. In vitro translation of myxamoebal and plasmodial RNAs indicated that there are distinct mRNAs, and therefore probably separate genes, for the alpha 1-, alpha 2-, beta 1-, and beta 2-tubulins. Thus, the different patterns of tubulin expression in myxamoebae and plasmodia reflect differential expression of tubulin genes.