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.     

Microbiological cultivation ofMycoplasma hyorhinis from cell cultures

The failure of many cell culture isolates ofMycoplasma hyorhinis to grow on microbiological media has stressed the need for alternate assays to detect these organisms. The use of freshly prepared yeast extract in mycoplasmal media together with incubation in 5% CO₂/air successfully detectedM. hyorhinis in 12 of 12 infected cultures. These were not detected by the use of conventional mycoplasmal media using aerobic or anaerobic incubation. This assay may also be helpful in detection of other mycoplasmal species commonly isolated from cell cultures. This study was supported by grant AI 15748 from NIH. We thank Ronnie Vanaman and Judi Sarama for their technical assistance.     

Ultrafiltration to remove endotoxins and other cytokine-inducing materials from tissue culture media and parenteral fluids.

The presence of small amounts of endotoxins are often undesirable when investigating cytokines such as interleukin-1 and tumor necrosis factor alpha. Polymyxin B, widely used to block endotoxins, does not block several forms of endotoxins, and at high concentrations, polymyxin B itself stimulates interleukin-1 production. Human peripheral blood mononuclear cells are highly sensitive to endotoxins; they respond with cytokine production to endotoxins at concentrations of 10-50 pg/ml and detect pyrogenic materials nonreactive in the Limulus test. In the present study, ultrafiltration using polysulfone filters was found to remove all interleukin-1- and tumor necrosis factor alpha-inducing substances produced in E. coli cultures. Interleukin-1- and tumor necrosis factor alpha-inducing substances derived from Pseudomonas aeruginosa cultures were also rejected by the filters. Ultrafiltration is therefore a convenient and effective procedure to remove interleukin-1 and tumor necrosis factor alpha-inducing substances from parenteral fluids and solutions that come in contact with blood such as fluids used in hemodialysis. This technique is also applicable for the large-scale production of culture media for mammalian cell expression of recombinant, pyrogen-free proteins intended for use in humans.     

Development and germination of pecan somatic embryos derived from liquid culture

Aggregates of globular and pre-globular stage somatic embryos from suspension cultures of pecan (Carya illinoensis Koch) were cultured on solidified media for embryo development. Embryo aggregates and pre-globular stage embryo masses were given various treatments to further ontologic development. A 2- to 4-wk mild dehydration of the embryo aggregates suppressed recurrent embryogenesis, promoted development of globular embryos into cotyledonary stage embryos, and enhanced plant development beyond germination. Fine embryogenic tissue masses filtered from suspension formed cotyledonary-staged embryos when the collection filters were plated on solified medium. The embryogenic capacity of preglobular stage embryo masses was compared between media supplemented with varying concentrations of polyethylene glycol (molecular weight 8 000) vs. filter overlays. The filter paper overlays were not necessary for embryo development. An inverse relationship was found between the number of embryos that developed and the concentration of polyethylene glycol in the medium. However, this relationship was reversed for ability of embryos to germinate and develop into a plant.     

Nitrogen fixation activity of a filamentous,nonheterocystous cyanobacterium in the presence and absence of exogenous,organic substrates

Gallionella ferruginea is able to utilize Fe(II) and the reduced sulfur compounds sulfide and thiosulfate as electron donor and energy source. Tetrathionate and elemental sulfur, on the other hand, are not metabolized. In sulfide-O₂ microgradient cultures G. ferruginea grows at the interface between the oxidizing and the reducing zones. Optimal growth depends on low oxygen and sulfide concentrations. Establishing within the gradient protects the bacterium from too high sulfide concentrations. G. ferruginea excretes extracellular polymeric substances (EPS). While in FeS-gradient cultures 2×10⁶ cells/ml were obtained the bacterial mass could be increased to 1–3×10⁸ cells/ml in shaken batch cultures using thiosulfate as substrate. A further increase of bacterial mass by adding an organic carbon source was not possible confirming that G. ferruginea is an obligate autotrophic organism. When growing on sulfide or thiosulfate the otherwise characteristic twisted stalk consisting of ferric hydroxide is lacking. It is thus shown to be a metabolic end product of Fe(II) oxidation rather than metabolically active cellular material.     

On the performances of noise filters in the restoration of oscillatory behavior in continuous yeast cultures

Continuous flow microbial fermentations under industrial conditions are subject to the influx of noise, mainly through the feed stream. Noise upsets the normal deterministic behavior. For continuous cultures of Saccharomyces cerevisiae exhibiting oscillatory responses, four kinds of commonly used noise filters, three algorithmic and one neural, have been compared for their ability to restore noise-free oscillations. An auto-associative neural filter was the best, similar to earlier observations for other organisms under non-oscillatory conditions. This enhances the general applicability of neural filters for industrial scale fermentations.     

Fed-batch hairy root cultures within situ separation

Fed-batch cultures ofL. erythrorhizon hairy root were carried out by controlling sucrose concentration and media conductivity in a shake flask and a modified stirred tank reactor. For the efficient product recovery from the culture,in situ adsorption by XAD-2 was also conducted. When sucrose was used as a carbon source, the highest shikonin production and hairy root growth were obtained. When glucose or fructose was used instead, the growth was severely inhibited. In addition, it was found that alternating feeding of sucrose could be used as an effective strategy for enhancing the productivity of shikonin derivatives., As the XAD-2 amount was increased up to 1.5 g/L, shikonin production was enhanced by removing shikonin produced and other products which might be inhibitory to cell growth. Most amount of shikonin produced was successfully recovered in XAD-2 (Over 99%). Using hairy root culture in a modified stirred tank reactor, the shikonin productivity and hairy root growth rate on the average were 9.34 mg/L day and 0.49 g DCW/L · day, respectively.     

Morphactin-induced changes in the cytokinin effect on tissue and organ cultures ofNicotiana tabacum

Summary The influence of a morphactin, chlorflurenol-methylester (CFM), on the growth, the morphogenesis and the isoelectric peroxidase pattern was investigated in both callus cultures (two different tissue culture strains) and multiple bud cultures ofNicotiana tabacum var.Wisconsin. CFM (range of concentration between 10^–6g/ml and 10^–4g/ml) was applied singly, or in combination with a cytokinin, benzylaminopurine (BAP), or with an auxin, indoleacetic acid (IAA), or with IAA plus BAP.In general, the callus growth was inhibited under the influence of CFM. In some of the experiments carried out in hormone-free media, growth stimulation was observed. Even minimal inhibition or stimulation of the callus growth was always accompanied by characteristic changes in the peroxidase patterns.The following results show the influence of the morphactin CFM on cytokinin effects (endogenous cytokinin or equally the exogenously applied cytokinin, BAP). (1) In the multiple bud cultures, BAP and CFM (both substances combined with IAA) similarly caused inhibition of root formation and stimulation of bud formation. The bands in the peroxidase patterns, characteristic of cytokinin action, were accentuated also of those bud cultures which had been treated with BAP or with CFM. (2) In the callus cultures, the cytokinin characteristics appeared under CFM influence in the peroxidase patterns of one of the tissue culture strains only when CFM was applied in combination with BAP and not in combinations of CFM with IAA.The observed morphactin-induced increase in the cytokinin effects could occur via changes in the hormone level of the tissue.     

Inhibitory effects of filter-sterilized media on microspore development and embryogenesis in <Emphasis Type="Italic">Brassica napus</Emphasis> and <Emphasis Type="Italic">Triticum aestivum</Emphasis>

Summary Plant tissue culture media, sterilized through commercial filter membrane units, inhibited plant cell growth and development. Embryogenesis was suppressed in microspore cultures of Brassica napus and anther cultures of Triticum aestivum, and in vitro pollen development was suppressed in B. napus. Inhibition of growth and development occurred on media that had been filter-sterilized in low volumes through non-washed filter membrane units of all brands tested, including Nalgene, Corning, Millipore, and Sartorius membrane products. Similar results were obtained with Costar Transwells and Millipore inserts (Millicell-CM.-HA, and-PCF). These deleterious effects were eliminated by washing the membranes with water followed by a small volume of the medium. Media sterilized by filtration through washed filters produced significant improvements of growth and development of embryos and pollen.     

Production of podophyllotoxin from Podophyllum hexandrum: a potential natural product for clinically useful anticancer drugs

Podophyllum hexandrum Royle of family Berberidaceae is an endangered medicinal plant. Rhizome ofP.hexandrum contains several lignans which posses antitumor activity. Podphyllotoxin is the most active cytotoxic natural product. It is used as starting compound for the synthesis of anticancer drug etoposide and teniposide. Podophyllotoxin acts as an inhibitor of microtubule assembly. These drugs are used for lung cancer, testicular cancer, neuroblastoma, hepatoma and other tumors. Besides this, it also shows antiviral activities by interfering with some critical viral processes. Availabilityof podophyllotoxin from plants has its limitations because of its intense collection from nature and lack of organized cultivation. The chemical synthesis of podophyllotoxin is considered to be very complicated as yet. The use of biotechnological approaches for the production of podophyllotoxin using cell cultures, organ cultures, and biotransformation route or by manipulating biosynthetic pathway proves to be an attractive alternative for production of podophyllotoxin. The present paper discusses the current status of research, limitations and future prospects for theproduction of podophyllotoxinin vitro.     

Effect of light on the formation of atypical fruiting structures inGanoderma lucidum

Ganoderma lucidum develops atypical fruiting structures (AFSs) with non-basidiocarpous basidiospores during the incubation under light on nutrient agar media. To examine the light quality effective in inducing AFSs, 17 isolates ofG. lucidum were incubated on agar media under light from different colored fluorescent lamps. Of the 17 isolates, 13 isolates produced AFSs and basidiospores under fluorescent lamps. Nine isolates formed AFSs in a broad light region from P-B (pure blue) to P-R (pure red) lamps. The remaining 4 isolates produced AFSs under different colored fluorescent lamps. No isolates formed AFSs in the dark or under BLB (black light blue) illumination. The mycelial growth was inhibited by light illumination, especially BLB light. Although the AFSs were induced at a very low light intensity such as 0.5µmol m^–2s^–1, the optimum light intensity for the AFS formation varied depending on the kind of fluorescent lamp and the isolate. The AFS formation inG. lucidum isolates was also tested under monochromatic light produced by the combination of interference filters and colored glass filters.G. lucidum isolates were separable into various types in the responses of AFS formation to monochromatic light, indicating thatG. lucidum is heterogeneous in its photo-response with regard to AFS formation.     

Somatic embryogenesis in the endemic black iris

Apple shoot cultures accumulate phenolic acids, flavonols, catechins, and procyanidins. Increasing the sucrose content and reducing the macronutrient content of culture media both resulted in an enhanced content of phenolic substances. The qualitative composition of the substances was affected as well. Morphology of the shoots, preculture and time of sampling in the subculture interval influenced the reaction pattern.     

Initiation and growth characterization of some hop cell suspension cultures

Cell suspension cultures of some hop (Humulus lupulus L.) cultivars were initiated from corresponding callus cultures induced on different media. Dissimilation curves were determined to characterize the growth of the suspension cultures maintained in Gamborg's B5 and in Murashige and Skoog's medium. Both media proved to be suitable; a comparison of the curves obtained for suspension cultures of the hop cultivar Wye Northdown grown in both media did not reveal striking differences. For four hop cell lines, the concentration of nitrate and sugar in the culture medium was analysed by HPLC during the growth of their suspension cultures. The cell suspension cultures of the various hop cultivars were also screened for the presence of bitter acids by HPLC and of volatile compounds by capillary GC. However, neither bitter acids nor volatile compounds could be detected; the addition of a non-toxic lipophylic phase (XAD-2, XAD-1180 or Miglyol 812) to the culture media did not help to induce the formation of volatile compounds.This paper will also appear as a chapter of the Ph.D. thesis of the first author; this thesis will be distributed among colleagues only.     

Interspecific differences in differentiation and dedifferentiation patterns in the genusNicotiana

Differentiation on hormoneless media, habituation ability and crown gall induction inNicotiana tissue cultures have been used as physiological parameters of evolutionary differentiation between species. Some of them on hormone free media differentiated whole plantlets, others produced only shoots or roots or showed undifferentiated growth (habituation), some eventually died. Moreover, the same genotypes showed a differential behaviour as far as tumor formation byAgrobacterium tumefaciens was concerned. Particularly, the competence for crown gall transformation inNicotiana species seems negatively correlated with differentiation capacity and may be ascribed to differences in the plants capacity to synthesize growth regulators. The correlation between the results obtained and the phylogenetic position of the genotypes tested is finally discussed.     

Production of pigments byMonascus purpureus in solid culture

Summary The effect of physical and nutritional factors on the production of pigments byMonascus purpureus FRR 2190 was studied using cultures grown on both rice and a synthetic medium that was solidified with carrageenan and extruded into rice-like particles. Pigment yield was highly sensitive to physical parameters. Optimal pigment formation in rice cultures occurred at an initial pH of 6 and an initial moisture content of 56%. Lower moisture contents led to a large decrease in pigment concentration. Red and yellow pigment production on solidified gel media was increased up to three-fold compared to that of liquid cultures of the same medium composition, particularly when peptone was used as the sole nitrogen source. Solid state rice cultures gave the highest pigment yields.     

Use of a solid support in the study of photosynthetic activity of the cyanobacterium Spirulina platensis

Oxygen evolution activity of Spirulina platensis cells ttached to nitro-cellulose filters or glass fiber filters (GF/C) was measured using the leaf disc electrode (LD-2 Hansatech Ltd, Kings Lynn, U.K.), originally designed for its use with leaves of higher plants. Measurements were performed in saturating (CO₂) as described previously for leaf discs and pieces. Photoinhibition could be induced in cells on the solid support as indicated by a significant increase in their quantum requirement (from 11 to 33 after 25 min exposure to a photon flux density of 2500 μE m^-2s^-1 and a smaller effect on the photosynthetic rate at light saturation. Photoinhibited cells showed recovery from the photoinhibitory treatment when illuminated under dim light.     

Comparison of 2,4-D and picloram for selection of long-term totipotent green callus cultures of sugarcane

Long-term regeneration of sugarcane (Saccharum spp. hybrid and Saccharum spontaneum L.) callus cultures was achieved by selection of green callus on MS agar medium containing 0.5 mgl^-1 picloram or 2,4-D. Newly initiated sugarcane callus cultures were a complex mixture of different tissue types including white, nonregenerative and green, regenerative tissues. The proportion of the tissue types changed as a function of time in culture, genotype, and amount and kind of auxin. Green callus on picloram media always regenerated green plants. Nine hybrids and ten wild relatives of sugarcane produced green calli on picloram media whereas only three hybrids were grown as green calli on 2,4-D media in long-term culture. Green calli were inoculated into liquid MS medium with 0.5 mgl^-1 picloram for suspension culture. These cultures were totipotent after 19 months. For routine culture, we initiated callus cultures on modified MS medium with 3 mgl^-1 2,4-D, then in two to three weeks we subcultured callus on MS medium with 0.5 mgl^-1 picloram and selected for green callus. Green calli regenerated large numbers of green plants after more than four years.     

Chitinase induction in onion tissue cultures

Chitinase activity in Allium cepa (onion) tissue cultures was investigated using a radioactive assay and polyacrylamide gel electrophoresis (PAGE). Increased chitinase activity was detected in callus cultured on agar-solidified media for 72 h with 1 mM salicylic acid or 0.05 M ethrel, and in callus cultured in liquid suspension for 72 h with 0.1 mM salicylic acid or 0.1 M ethrel. Non-denaturing PAGE analysis of callus suspension cultures treated with salicylic acid revealed a new chitinase activity which accumulated in the fluid of the suspension cultures.     

Proteins produced by barley microspores and their derived androgenic structures promote in vitro zygotic maize embryo formation

Maize zygotes formed in planta were isolated and co-cultured with barley microspores. Evidence suggests that culture with microspores and/or conditioned media in barley promoted zygotic embryo formation. In order to characterise active substances present in the conditioned media, we collected medium at various times after the initiation of culture. We showed that proteins appeared over time and that their quantity increased during the course of the culture. Some proteins were glycosylated as revealed by the ConA peroxidase test. The use of the antigen -glucosyl Yariv reagent has shown that arabinogalactan-proteins (AGPs) were present. In addition, conditioned medium samples were analysed for their oligosaccharide content. New oligosaccharides appear in the course of the culture but they do not seem to affect development. We discuss in details the results in the context of understanding cell-cell interactions between embryo and nurse cells and the possible parallel with that occurs in ovulo between endosperm and embryo.     

Development of suspension culture of Podophyllum hexandrum for production of podophyllotoxin

A suspension culture of Podophyllum hexandrum was established. As the cultures grew, reduction in cell viability, biomass and product yield were associated with browning of culture medium, clumping of cells and drop in medium pH. Supplementation of the medium with both polyvinylpyrrollidone (PVP) and pectinase eliminated these problems. PVP at 10 g l^–1 was optimum for both growth of and product formation in P. hexandrum suspension cultures.