Age and Physiological Condition of Donor Plants Affect in vitro Morphogenesis in Leaf Explants of Passiflora edulis f. flavicarpa

The regeneration potential of D.alata L. germplasm preserved in vitro was compared with the micropropagation of fresh material. Nodal cuttings were conserved for 9 months in different treatments based on D-571 culture medium modified, using several variable components (mannitol, benzylaminopurine and activated charcoal). Regeneration at 8 weeks, assessed by means of percentage of explant regenerating and the multiplication at 5 weeks through the shoot length and de novo bud count formation per explant were determined. The results showed high rates (100 and 98%) of explant regeneration and micropropagation from in vitro material maintained in D-571 medium with 1.5% of mannitol + 0.1 or 1 mg l^–1 of benzylaminopurine + 2 g l^–1 of activated charcoal, respectively.     

The production of ethanol from lactose in a tubular reactor by immobilized cells of Kluyveromyces fragilis

Summary An immobilized-cell tubular reactor for the continuous fermentation of lactose by Kluyveromyces fragilis was developed. Two types of supporting media were successfully tested; beechwood cubes and activated charcoal pellets. Ethanol productivity of 17.2 g/l/h was achieved from a 15% whey-lactose solution using K. fragilis immobilized on charcoal pellets, with a final ethanol concentration of 18 g/l. The use of two reactors in series demonstrated that it is possible to obtain up to 50 g/l of ethanol in the final product. No decrease in biological activity of the immobilized yeast cells occurred over a period of up to 31 days of continuous operation.     

Practical methodology for gametophyte proliferation and sporophyte production in green penny fern (Lemmaphyllum microphyllum C. Presl) using mechanical fragmentation

Propagation of gametophytes and sporophytes using mechanical fragmentation has been considered a suitable method for mass production of ferns. This study aimed to develop a practical propagation method for Lemmaphyllum microphyllum C. Presl, which is a fern of significant ornamental and medicinal value. Gametophytes were obtained through in vitro spore germination and used for propagation experiments. The gametophyte was mechanically fragmented using a scalpel into small fragments, which were then used to investigate gametophyte proliferation. In addition, the gametophyte was fragmented using a blender and then used to study sporophyte formation. Optimal proliferation conditions of the gametophyte were determined using Murashige and Skoog (MS) basal medium (double-, full-, half-, quarter-strength), Knop medium, and medium components (sucrose, nitrogen sources, activated charcoal), at various concentrations. The fresh weight of the gametophyte was 14-fold higher than that of gametophytes (300 mg) used as culture material, when cultured on double-strength MS. Moreover, 1 g of the gametophyte fragmented in 25 mL of distilled water formed more than 430 sporophytes in a soil mixture in an area of 7.5 cm². The sporophytes were successfully cultivated in the greenhouse after acclimation. A large-scale production method for L. microphyllum that can be easily implemented in a fern production farm is outlined.

     

A methodology for large-scale Athyrium sheareri gametophyte proliferation and sporophyte production using tissue culture

Tissue culture methods using gametophytes are considered the easiest ways to mass-produce fern sporophytes. The aim of this study was to develop a practical propagation method for the ornamental fern, Athyrium sheareri. The gametophytes obtained from in vitro spore germination were used as experimental materials. We used the chopping method to investigate the culturing conditions for proliferating gametophytes and the blending method for evaluating the mass production of sporophytes in mixed soil. Gametophyte proliferation was determined via Knop medium, various concentrations of Murashige and Skoog (MS) basal medium (1, 1/2, 1/4), and media components (sucrose, nitrogen source, and activated charcoal). The fresh weight of the gametophytes increased by more than 24-fold in 1/2 MS medium. In addition, 1 g of gametophyte could produce a maximum of 255.3 sporophytes in a mixed soil of 7.5 cm² area. Treating gametophytes with exogenous plant growth regulators promoted the formation and growth of sporophytes. The cultivated young sporophytes were acclimated and successfully grown in greenhouses. We developed a mass production protocol for A. sheareri sporophytes suitable for field application, which is expected to have commercial value.

     

Recovery of c-phycocyanin from the cyanobacteriumSpirulina maxima

Spirulina biomass was separated into two fractions which may have various uses. A phycocyanin fraction may provide a food colourant and biomarkers, and a protein-rich leftover may be useful as aquaculture feed. Activated charcoal adsorption, ultrafiltration and spray drying were used effectively to produce a high quality colourant grade phycocyanin, while activated charcoal adsorption, ammonium sulphate precipitation, dialysis and chromatography were effective in preparing reagent grade phycocyanin.     

The Use Of Lead Tetraacetate,Benzidine, O-Dianisidine and A “Film Test” In Investigating The Periodic-Acid-Schiff Technic

In order to obtain a better understanding of the “periodic-acid-Schiff” reaction, also known as the “periodic-acid fuchsin-sulfurous-acid” reaction, three types of investigations were carried out

1) The Schiff reagent was replaced by other aldehyde reagents: benzidine or o-dianisidine. There was no significant change in the histological distribution and intensity of the reactions occurring after periodic acid oxidation.

2) Periodic acid was replaced by another oxidizing agent: lead tetraacetate (dissolved in acetic acid). There was no significant change in the histological distribution of the reactions with the Schiff reagent, but some change in their intensity. It was concluded that 1,2-glycols and a-amino alcohols play the main role in the reactions with both oxidants. The presence of α-hydroxy acids in some types of mucous cells is suggested by the results with lead tetraacetate.

Incidently, glycogen and starch are not sufficiently oxidized by lead tetraacetate (in acetic acid) at room temperature to give positive reactions with the Schiff reagent, while cellulose and other periodic-acid-Schiff reactive substances are.

3) The staining of films of presumed reactive substances with the periodic-acid-Schiff technic C O the intense reactivity of many polysaccharides, mucopolysaccharides and mucoproteins, but not of ordinary proteins. (Hyaluronic and chondroitin sulfuric acid are, however, not reactive in vitro).

In conclusion, the periodic-acid-Schiff technic consists of an oxidation of 1,2-glycols and a-amino alcohols to produce aldehyde groups, which are then stained by the Schiff reagent. The “film test” reveals that these radicals are present in certain polysaccharides, mucopolysaccharides and mucoproteins.     

Feeding Preferences in Greylag Geese and the Effect of Activated Charcoal

ABSTRACT Greylag geese (Anser anser) can cause serious damage to agricultural fields near wetlands that are attractive for resting and nesting but not for feeding. Alternative plantings or spraying fields may prevent goose damage. We randomly designed 64 plots in spring 2004 and prepared plantings of white clover (Trifolium repens), white clover with perennial ryegrass (Lolium perenne; mixture), fertilized perennial ryegrass (grass), or unfertilized perennial ryegrass. We measured goose-dropping densities in plots as a measure of feeding preference in autumn 2004 (7 weeks), spring 2005 (6 weeks), and autumn 2005 (7 weeks) following removal of a protective fence and vegetation sampling for content analysis in 2004. We also sprayed activated charcoal (20 kg/ha) in a suspension on 32 plots (8/planting) to deter geese in autumn 2004 only. In a second experiment we examined pairs of greylag geese in cages for preferences between grass treated with or without activated charcoal. Charcoal did not deter geese in either experiment. However, dropping density averaged highest for clover (1.01/m²), followed by the mixture (0.65/m²), then fertilized (0.23/m²) and unfertilized grass (0.16/m²). Preferences were consistent in all 3 experimental periods. Fertilized grass reached 31.8 cm in height on average in spring, whereas clover measured 15.4 cm. Crude protein and water-soluble carbohydrate content (g/kg dry matter) was 294 and 49, respectively, in white clover and 183 and 139, respectively, in fertilized grass. We found a positive partial correlation independent of vegetation type between dropping densities and crude protein and a negative correlation with water-soluble carbohydrate content. Thus, to prevent grazing damage to agricultural fields, we recommend planting white clover, strongly preferred by feeding geese, in areas (fallow agricultural or nonagricultural) adjacent to their habitat and not in agricultural fields under production.     

New Reagent for Protein-DNA Contacts Footprinting

Abstract

We have found, that the reaction of o-bromobenzoic acid with Cu²⁺ ions can be used as a source of activated oxygen species capable of cleaving DNA. Possibility to apply this reaction for footprinting the nucleosome core in the reconstituted chromatin was demonstrated.     

Multiplication of juvenile black wattle by microcuttings

The influence of mineral formulation, growth regulators and activated charcoal on micropropagation of juvenile black wattle (Acacia mearnsii) was studied. Nodal segments of one-month-old seedlings were used as starting material. After 30 to 45 days, they developed into shoots, which were divided into microcuttings and transferred to fresh media. The addition of 2 g l⁻¹ of activated charcoal to basal medium (3/4 strength MS salts and MS vitamins) improved the multiplication phase, reducing leaf chlorosis and raising the percentage of elongated shoots. The multiplication rate varied between 1.7 and 3 every month. Rooting percentage too was higher in the presence of charcoal, even when auxin was not added to the culture medium. The addition of 8.87 μM of benzyladenine and/or 1.44 or 2.88 μM of gibberellic acid did not influence significantly the multiplication, nor modifications in the FeSO₄, MnSO₄, CaCl₂ content of the medium. The system described allows the multiplication, elongation and rooting of black wattle in one step. This revised version was published online in June 2006 with corrections to the Cover Date.     

Formation of benzaldehyde by Pseudomonas putida ATCC 12633

Aromatic and heterocyclic aldehydes may be produced by the mandelate pathway of Pseudomonas putida ATCC 12633 via the biotransformation of benzoyl formate and substrate analogues. Under optimised biotransformation conditions (37 °C, pH 5.4) and with benzoyl formate as a substrate, benzaldehyde may be accumulated with yields above 85%. Benzaldehyde is toxic to P. putida ATCC 12633; levels above 0.5 g/l (5 mM) reduce the biotransformation activity. Total activity loss occurs at an aldehyde concentration of 2.1 g/l (20 mM). To overcome this limitation, the rapid removal of the aldehyde is desirable via in situ product removal. The biotransformation of benzoyl formate (working volume 1 l) without in situ product removal accumulates 2.1 g/l benzaldehyde. Benzaldehyde removal by gas stripping produces a total of 3.5 g/l before inhibition. However, the most efficient method is solid-phase adsorption using activated charcoal as the sorbant, this allows the production of over 4.1 g/l benzaldehyde. Addition of bisulphite as a complexing agent causes inhibition of the biotransformation and bisulphite is therefore is not suitable for in situ product removal. Received: 16 March 1998 / Received revision: 20 May 1998 / Accepted: 21 May 1998     

First case of human protothecosis in Canada: Laboratory aspects

A case of bursitis due to Prototheca wickerhamii is briefly reported. In histological sections the organism stained well with fungal stains, grey with silver methanamine and red with periodic acid Schiff reagent. This unicellular achlorophyllous alga was studied on common laboratory media. The characterization of the Prototheca sp. depends largely on wet mount microscopic examination from broth or agar cultures which ensures the observation of endosporulation and a consistent absence of budding. Otherwise the growth rate and the pasty white colonies may lead to an erroneous identification, most likely as a Cryptococcus sp. P. wickerhamii lends itself very well to standard physiological tests used for the identification of yeasts. The strain was found insensitive to 5-fluorocytosine. The MIC of amphotericin B was 0.15 g/ml.     

L'Azione dell'Ossigeno Liquido Sulla Germinabilità dei Semi Duri di Ginestrino (Lotus Corniculatus L.) e di Ladino (Trifolium Repens L.)

Abstract

Comparative researches on morphology and physiology of PICEA and LARIX. Fresh weight and chlorophyll content in seedlings kept at various light intensities. — The fresh weight and the chlorophyll content of lots of seedlings from Larix decidua and Picea excelsa grown on sand for 12 days in climatic cell at 25 [ddot]C with 86% relative humidity and a light intensity of 90, 250, 500, 1.000, 2.000 and 4.000 lux were determined.

The fresh weight of Picea seedlings is not significantly affected by all light intensities except for 4.000 lux, where it is 20% higher. Even in dim light (90 lux) the fresh weight of Picea seedlings is only 7% inferior to that of the lot kept at 2.000 lux.

The results obtained in Larix are remarkably different; its fresh weight is more influenced by the light intensity: at 4.000 lux, e. g., the fresh weight is considerably higher (more than 20%) than the arithmetical mean of all the lots, while at 90 lux it appears greatly inferior (30%) to the lot kept at 2.000 lux.

No correlation exhists between fresh weight and chlorophyll content variations.

In Larix only the difference between seedlings kept at 250 lux and 90 lux is very strong. In the latter the chlorophyll content for g. f. w. is 40% inferior to the average of all the lots. At the maxime intensities the chlorophyll content of Larix seedlings appears to be particularly increased, while that of Picea seedlings is slightly inferior to that observed at 2.000 lux.

These figures are in agreement with the special ecology of the two plants and particularly with the light need of Larix, as it is clearly demonstrated by the fresh weight and chlorophyll content per g. f. w. and by the different ratio in chlorophyll contents of the lots of seedlings kept at 2.000 and 4.000 lux.     

The Production of Catechols from Benzene and Toluene by Pseudomonas Putida in Glucose Fed-Batch Culture

Catechol and 3-methylcatechol were produced from benzene and toluene respectively using different mutants of Pseudomonas putida. P. putida 2313 lacked the extradiol cleavage enzyme, catechol 2,3-oxygenase, allowing overproduction of 3-methylcatechol from toluene to a level of 11.5 mM (1.27 g·1^-1) in glucose fed-batch culture. P. putida 6(12), a mutant of P. putida 2313, lacked both catechol-oxygenase and catechol 1,2-oxygenase, and accumulated catechol from benzene to a level of 27.5mM(3g·1^-1).

In both biotransformations product formation ceased within 10 hours of feeding the aromatic substrate, and this was due to product inhibition by the catechols. The primary site of catechol toxicity was inhibition of the aromatic dioxygenase. Neither cis-toluene dihydrodiol cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene), nor cis-benzene dihydrodiol (cis-l,2-dihydroxy-3-methylcyclohexa-3,5-diene) dehydrogenase was significantly inhibited by catechol overproduction whereas both ring activating dioxygenases were inhibited within 4-6 hours of the maximum product concentration being attained.

3-Methylcatechol overproduction from toluene was also studied using a continuous product removal system. Granular activated charcoal removed 3-methylcatechol efficiently and was easily regenerated by washing with ethyl acetate. Using P. putida 2313, it was shown that the final product concentration increased approximately fourfold. Additional products were formed and the significance of these are discussed.     

Effects of Activated Charcoal on Growth and Morphogenesis in Cell Cultures

The effects of activated charcoal on growth and morphogenesis in plate cultures of different plant cells have been studied. It was shown that medium containing charcoal induced embryogenesis in cultures of Daucus carota in which embryo formation could not be brought about by omitting auxin from the medium. Charcoal-medium also induced abundant root formation in older cultures of Allium cepa, which normally did not produce roots. The growth of cultures of Glycine max and Haplopappus gracilis was totally inhibited by charcoal. It is thought that activated charcoal removes substances from the medium, one of which might be auxin.     

Micropropagation of Cypripedium flavum through multiple shoots of seedlings derived from mature seeds

Cypripedium flavum, known as the rare lady’s slipper orchid, is one of the endemics with a yellow flower in China. Due to its conservation and commercial requirement, establishment of an efficient method for micropropogation is urgently needed. Multiple shoots were obtained by placing seedlings from seeds of C. flavum on Harvais media supplemented with two cytokinins (BAP or KIN) used alone or in addition to different concentration of potato homogenate. The effect of BAP was better than that of KIN on shoot multiplication. The Havais media supplemented with BAP (2.22 μM) and potato homogenate (20 g l⁻¹) was the most effective, providing high shoot multiplication frequencies (95%) associated with a high number of shoots per explant (2.55 shoots/plant). For root formation, high rooting and survival were achieved using 1/2 Harvais media supplemented with 0.6 g l⁻¹activated charcoals. High-level activated charcoal increased the number and the length of roots because the activated charcoal could absorb BAP in the media. This study demonstrated that C. flavum could be micropropagated by using multiple shoots of seedlings derived from mature seeds.     

Micropropagation of an Endangered Orchid Anoectochilus formosanus

A rapid and efficient procedure is outlined for in vitro clonal propagation of an elite cultivar of jewel orchid (Anoectochilus formosanus). Multiple shoot proliferation was induced in shoot tip explants on Hyponex (H3) media supplemented with 1 mg dm^–3 benzyladenine or 1 – 2 mg dm^–3 thidiazuron (TDZ). Addition of activated charcoal (1 g dm^–3) to the TDZ containing medium promoted multiple shoot formation (11.1 shoots per explant). However, the regenerated shoots had slow growth rate and failed to elongate. This problem was overcome by transferring the shoot clumps to a hormone free H3 medium supplemented with 2 % sucrose and 0.5 g dm^–3 activated charcoal. Rooting was induced in 100 % of the regenerated shoots in the same media. The plantlets were acclimatized and established in greenhouse.     

Morphogenetic responses obtained from a variety of somatic explant tissues of data palm

Shoot tip, bud, leaf, stem and root explants from bearing trees, offshoots, seedlings, and asexual plantlets ofPhoenix dactylifera L. were cultured on modified Murashige and Skoog nutrient media containing 3 g/l activated charcoal, 100 mg/l 2,4-dichlorophenoxyacetic acid, 3 mg/l N ⁶-(Δ²-isopentyl)adenine to obtain callus. Differential morphogenetic responses were obtained from calli dependent on the explant type and parent source. Subcultured shoot tips and leafy lateral buds callus on nutrient media devoid of charcoal and supplemented with 0.1 mg/l α-naphthaleneacetic acid (NAA) produced adventitious plantlets. Subcultured leaf calli produced roots only. Root callus failed to exhibit any morphogenetic response upon subculturing. Undifferentiated non-leafy buds and stem tissues did not give rise to callus, regardless of the parent source. Generally, the best callus and embryogenetic responses from explants were obtained from seedling and plantlet parent sources. Similarly, organogenetic responses such as root formation and shoot development from shoot tips cultured on media containing 10 mg/l NAA were also related to the parent explant source. Mention of a trademark or proprietary product in the paper does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be available.     

Effect of Sucrose Concentration, Charcoal, and Indole-3-Butyric Acid on Germination of Abies Numidica Somatic Embryos

The dependency of radicle elongation in Abies numidica somatic embryos on germination media has been studied. No significant differences were detected between the Murashige and Skoog (MS) and Schenk and Hildebrandt (SH) medium. The addition of 10 g dm^–3 activated charcoal or 0.05 mg dm^–3 indole-3-butyric acid (IBA) into both media had positive influence on embryo germination. Difference between activated charcoal and IBA effects were significant. The high rooting percentage (85 %) was recorded on half SH medium with 10 g dm^–3 sucrose and activated charcoal. After IBA addition rooting percentage was increased to 95 %. During 7 months 73 % of plantlets survived transfer to soil and in 54 % of plantlets shoot growth was observed.     

New approaches to improve the efficiency of somatic embryogenesis in oil palm (Elaeis guineensis Jacq.) from mature zygotic embryos

We developed an efficient and simple system for inducing somatic embryogenesis and regenerating plantlets from mature zygotic embryos of oil palm. Embryogenic calli were induced from mature zygotic embryos of oil palm on modified Murashige and Skoog medium with 2,4-dichlorophenoxyacetic acid or picloram, alone or in combination with activated charcoal. The greatest frequency of embryogenic callus induction (97.5%) was obtained by culturing mature zygotic embryos on callus induction medium with 450 μM picloram and 2.5 g?L^?1 activated charcoal. Embryogenic calli proliferated on a medium with a reduced concentration of picloram. Embryogenic calli were then subcultured on a medium supplemented with 12.3 μM 2-isopentenyladenine and 0.54 μM naphthaleneacetic acid, with subcultures at 4-wk intervals. Somatic embryos were regenerated on a medium with Murashige and Skoog macro- and micronutrients at half-strength concentrations supplemented with 20 g?L^?1 sucrose, 2.5 g?L^?1 activated charcoal, and 2.5 g?L^?1 Phytagel. Detailed histological analysis revealed that somatic embryogenesis followed an indirect pathway. Primary calli were observed after 4–6 wk of culture and progressed to embryogenic calli at 12 wk. Embryogenic cells exhibited dense protoplasm, a high nucleoplasmic ratio, and small starch grains. Proembryos, which seemed to have a multicellular origin, formed after 16–20 wk of culture and successive cell divisions. Differentiated somatic embryos had a haustorium, a plumule, and the first and second foliar sheaths. In differentiated embryos, the radicular protrusion was not apparent because it generally does not appear until after the first true leaves emerge.     

Characteristics of induction of virulence factor expression by activated charcoal in Listeria monocytogenes

Activated charcoal has been previously shown to induce in vitro expression of virulence factors by Listeria monocytogenes. In trying to elucidate the nature of the charcoal action, we found that the treatment of brain heart infusion medium with activated charcoal followed by charcoal removal does not result in an increase of virulence factor expression. At the same time, the addition of fresh charcoal to the charcoal-treated medium induces expression, suggesting that the effect of activated charcoal cannot be explained only by changes in medium composition. In addition, we observed that activated charcoal induced expression of virulence factors even when L. monocytogenes was physically separated from charcoal particles by either a nitrocellulose membrane or a thin layer of agar. We propose that the interaction of charcoal with some listerial product(s) might be responsible for the effect observed.