Growth and siderophores production in <Emphasis Type="Italic">Alcaligenes faecalis</Emphasis> is regulated by metal ions

During stationary phase of growth under low stress of iron in succinic acid medium, Alcaligenes feacalis BCCM ID 2374 produced microbial iron chelators. Increase in iron concentration supported bacterial growth but suppressed siderophores production, 1 μM and 2 μM of iron was optimum for maximum siderophore yield, i.e. 354 and 360 μg/ml in untreated and deferrated medium, respectively. Threshold level of iron, which suppressed siderophores production in A. feacalis BCCM ID 2374, was 20 μM. Ten micromoles and above concentration of CuCl₂ and CoCl₂, and 20 μM of MgCl₂, MgSO₄, ZnCl₂ and ZnSO₄ severely affected siderophores production.     

Optimization of growth regulators and silver nitrate for micropropagation of <Emphasis Type="Italic">Dianthus caryophyllus</Emphasis> L. with the aid of a response surface experimental design

This paper reports on the optimum concentrations of naphthalene acetic acid (NAA) and 6-benzyladenine (BA) to stimulate callus growth and NAA; kinetin and silver nitrate (AgNO₃) for callus redifferentiation in Dianthus caryophyllus L. Meristems were excised and placed in MS medium with 30 g l⁻¹ sucrose and 9.0 μM 2,4-d. Callus clusters were transferred to MS medium containing NAA (0, 1.7, 3.3, and 5.0 μM) and BA (0, 1.7, 3.3, and 5.0 μM) for proliferation and to MS medium with 30 g l⁻¹ sucrose, 2.5 g l⁻¹ phytagel, kinetin (0, 33, and 66 μM); NAA (0, 7.95, and 15.9 μM) and AgNO₃ (0, 23.54 and 47.08 μM) for shoot and root induction. Treatments were applied according to a Box–Behnken design. After callus growth and redifferentiation, plants were incubated in the greenhouse at 18 ± 2°C for 4 wk and at 20–26°C for 4 wk. Finally, plants were changed to near-commercial greenhouse conditions with different day (30–35°C) and night (16–24°C) temperatures. Results showed better callus growth at higher NAA concentrations. A maximum callus weight was found with 5.0 μM NAA but without BA. A maximum of 78% calluses with shoots was obtained with 15.9 μM NAA, 47.08 μM AgNO₃, and 0.74 μM kinetin and 58% with roots with 15.7 μM NAA and 47.08 μM AgNO₃, but without kinetin. The shoots obtained showed little hyperhydricity. Vigorous plants were obtained after gradual acclimatization with an 80% survival rate under nursery conditions.     

Somatic embryogenesis and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Bixa orellana</Emphasis> L.

Establishment, maintenance, regeneration, and transformation of somatic embryos by both direct and indirect means (callus-mediated) was achieved for Bixa orellana, a tropical plant whose seeds produce commercially edible ‘annatto pigment,’ which mainly constitutes an apocarotenoid called bixin. Callus-mediated methodology was found to be efficient in producing a greater number of embryos in a short time. The maximum of 28 somatic embryos were produced in 16–18 weeks when immature zygotic embryonic stalks were inoculated onto Murashige and Skoog (MS) medium containing B5 vitamins supplemented with 0.44 μM benzyladenine (BA), 0.054 μM α-naphthaleneacetic acid (NAA), 2.89 μM gibberellic acid (GA₃), 0.02 μM triiodobenzoic acid (TIBA), and 0.011 μM triacontanol (TRIA). Callus initiation from hypocotyl explants was obtained on MS medium supplemented with 1.07–2.14 μM NAA and 10.2 μM BA. In 3 months, somatic embryos were produced when callus was inoculated onto MS medium supplemented with 4.44 μM BA, 40 μM AgNO₃, and 0.011 μM TRIA. Somatic embryos were efficiently regenerated on MS basal solid and liquid media supplemented with 0.44–4.4 μM BA, 0.54–2.69 μM NAA, 4.92 μM 2iP, 2.1 μM calcium d-pantothenate, 0.21 μM biotin, 227.7 μM cysteine HCl monohydrate, and 108.6 μM adenine sulfate. Agrobacterium tumefaciens GV 3101 harboring pCAMBIA 1305.2 binary vector-mediated stable transformation of somatic embryos exhibited a transformation frequency of 2.56%. As somatic embryogenesis in any perennial system is useful in terms of both commercial and scientific nature, this somatic embryo-based transformation protocol for the commercially important dye-yielding tropical plant B. orellana is useful for its improvement through genetic engineering.     

Rapid in vitro multiplication of <Emphasis Type="Italic">Melia azedarach</Emphasis> L. (a multipurpose woody tree)

An efficient regeneration protocol for rapid multiplication of Melia azedarach, an economically as well as medicinally important timber-yielding tree, was developed. Nearly 90% of the culture exhibited axillary bud sprouting and multiple shoot formation from nodal segments derived from 20-year-old candidate plus tree on Murashige and Skoog (MS) medium supplemented with 5 μM 6-benzyladenine (BA). The highest shoot regeneration frequency (92%), maximum number of multiple shoots (19.7 ± 0.31) as well as shoot length (4.9 ± 0.08 cm) was induced from nodal explants on MS medium amended with 5.0 μM BA, 0.5 μM indole-3-acetic acid (IAA) and 30 μM adenine sulfate (AdS). Addition of 250 mg l⁻¹ ammonium sulphate, (NH₄)₂SO₄, and 100 mg l⁻¹ K₂SO₄, prevented defoliation and tip burning without affecting the number of shoots. The explant harvest period also influenced the bud break and shoot sprouting from nodal segments. Repeated subculturing of nodal explants on fresh MS medium containing lower concentration of BA (2.5 μM) along with IAA (0.5 μM), AdS (30 μM) and additives was found most suitable growth regulator regime for achieving 1.2-fold increase in shoot multiplication rate. The percentage of shoot multiplication as well as the number of shoots per node remained the same during first three subculture passages, afterwards a decline was recorded. About 90% of the in vitro regenerated shoots were successfully rooted ex vitro by giving a pulse treatment of 250 μM indole-3-butyric acid for 15 min, followed by their transfer to thermocol cups containing soilrite. The raised plantlets were successfully acclimatized first under culture room conditions, then to green house with 85% survival rate.     

Induction of metal binding compounds and antioxidative defence in callus cultures of two black poplar (<Emphasis Type="Italic">P. nigra</Emphasis>) clones with different tolerance to cadmium

Callus cultures of two parental clones of Populus nigra L., Poli and 58-861, originating from contrasting environments, were exposed to different cadmium concentrations (0, 150 and 250 μM CdSO₄). Clones showed different growth responses to cadmium, evaluated by the tolerance index (Ti), with Poli being more tolerant to the metal at both concentrations. The cadmium concentration at the end of the treatment was very similar between clones at 150 μM CdSO₄, while a higher value in 58-861 compared to Poli was detected at 250 μM CdSO₄. The bioconcentration factor evidenced the lowest value in Poli at 250 μM CdSO₄. Unlike 58-861, cadmium provoked a strong induction of thiols and phytochelatins in clone Poli. In both clones, organic acid concentration differed notably in untreated calli and cadmium treatment induced a general lowering of these compounds. A notably higher antioxidant enzyme activity (ascorbate peroxidase, APX; catalase, CAT; guaiacol peroxidase, GPX) was measured in control calli of clone Poli compared to 58-861. Cadmium induced a remarkable enhancement of APX and CAT, but not GPX, activity at 150 μM CdSO₄ in Poli. Conversely, in 58-861 at 150 μM CdSO₄, and in both clones at 250 μM CdSO₄, a decrease in the antioxidant activity occurred. This investigation provided evidence that these two contrasting genotypes of P. nigra are characterised by a different response to cadmium in callus cultures. In particular, in Poli, the higher tolerance to cadmium is associated with a higher activity of antioxidative enzymes and the ability to strongly increase thiol and PC concentration in response to metal exposure.     

Hydrogen peroxide is a product of oxygen consumption byTrichomonas vaginalis

The amitochondriate sexually-transmitted human parasitic protozoanTrichomonas vaginalis (Bushby strain) grown anaerobically on complex medium containing cysteine and ascorbic acid consumed O₂ avidly (6.9 μM min⁻¹ per 10⁶ organisms) with an apparentK _m value of 5.1 μM O₂ : O₂ uptake was inhibited by O₂ > 120 μM. Spectrophotometric assays in the presence of microperoxidase (419-407 nm) indicated that H₂O₂ was produced and that inhibition by high O₂ concentrations was again evident. Hydrogenosomes oxidizing pyruvate in the presence of ADP and succinate showed similar patterns of O₂ consumption, H₂O₂ production (33.5 pmol min⁻¹ per mg protein), and O₂ inhibition. Cytosolic NADH oxidase gave no detectable H₂O₂, whereas the cytosolic NADPH oxidase produced H₂O₂ at a rate (43 pmol min⁻¹ per mg protein) greater than that of hydrogenosomes. These results are discussed in relation to the oxidative stress experienced by the pathogen in its natural habitat.     

Effects of cadmium stress on seed germination,seedling growth and antioxidative enzymes in <Emphasis Type="Italic">Achnatherum inebrians</Emphasis> plants infected with a <Emphasis Type="Italic">Neotyphodium</Emphasis> endophyte

The effects of cadmium (Cd) on germination, and antioxidative enzyme activity (AEA) involving superoxide dismutase, catalase, peroxidase, and ascorbate peroxidase, and on amounts of malondialdehyde and proline present within Achnatherum inebrians, were determined for specimens infected (E+) vs. non-infected (E−) by Neotyphodium gansuense, and cultivated in the presence of various concentrations of CdCl₂ (0, 50, 100, 200 and 300 μmol/l). Under high Cd concentrations (100, 200 and 300 μM), E+ (vs. E−) specimens exhibited a higher germination rate and index, and higher values for shoot length, root length and dry biomass, but there was no significant difference (P > 0.05) under low Cd concentrations (0 and 50 μM). AEA and the proline content increased, but malondialdehyde content declined in the E+ (vs. E−) specimens under high Cd concentrations (100, 200 and 300 μM). There was no significant difference (P > 0.05) under low Cd concentrations (0 and 50 μM). Endophyte infection was concluded to be of benefit to the germination and anti-oxidative mechanisms within A. inebrians under plant exposures to high CdCl₂ concentrations.     

Thidiazuron-induced high-frequency direct shoot organogenesis of <Emphasis Type="Italic">Cannabis sativa</Emphasis> L.

Induction of high-frequency shoot regeneration using nodal segments containing axillary buds from a 1-yr-old mother plants of Cannabis sativa was achieved on Murashige and Skoog (MS) medium containing 0.05–5.0 μM thidiazuron. The quality and quantity of regenerants were better with thidiazuron (0.5 μM thidiazuron) than with benzyladenine or kinetin. Adding 7.0 μM of gibberellic acid into a medium containing 0.5 μM thidiazuron slightly increased shoot growth. Elongated shoots when transferred to half-strength MS medium supplemented with 500 mg l⁻¹ activated charcoal and 2.5 μM indole-3-butyric acid resulted in 95% rooting. The rooted plants were successfully acclimatized in soil. Following acclimatization, growth performance of 4-mo-old in vitro propagated plants was compared with ex vitro vegetatively grown plants of the same age. The photosynthesis and transpiration characteristics were studied under different light levels (0, 500, 1,000, 1,500, or 2,000 μmol m⁻² s⁻¹). An increase in photosynthesis was observed with increase in the light intensity up to 1,500 μmol m⁻² s⁻¹ and then decreased subsequently at higher light levels in both types of plants. However, the increase was more pronounced at lower light intensities below 500 μmol m⁻² s⁻¹. Stomatal conductance and transpiration increased with light intensity up to highest level (2000 μmol m⁻² s⁻¹) tested. Intercellular CO₂ concentration (C _i) and the ratio of intercellular CO₂ concentration to ambient CO₂ (C _i/C _a) decreased with the increase in light intensity in both in vitro as well as ex vitro raised plants. The results show that in vitro propagated and hardened plants were functionally comparable to ex vitro plants of same age in terms of gas and water vapor exchange characteristics, within the limits of this study.     

Susceptibility of <Emphasis Type="Italic">Candida</Emphasis> biofilms to histatin 5 and fluconazole

Candida-associated denture stomatitis has a high rate of recurrence. Candida biofilms formed on denture acrylic are more resistant to antifungals than planktonic yeasts. Histatins, a family of basic peptides secreted by the major salivary glands in humans, especially histatin 5, possess significant antifungal properties. We examined antifungal activities of histatin 5 against planktonic or biofilm Candida albicans and Candida glabrata. Candida biofilms were developed on poly(methyl methacrylate) discs and treated with histatin 5 (0.01–100 μM) or fluconazole (1–200 μM). The metabolic activity of the biofilms was measured by the XTT reduction assay. The fungicidal activity of histatin 5 against planktonic Candida was tested by microdilution plate assay. Biofilm and planktonic C. albicans GDH18, UTR-14 and 6122/06 were highly susceptible to histatin 5, with 50% RMA (concentration of the agent causing 50% reduction in the metabolic activity; biofilm) of 4.6 ± 2.2, 6.9 ± 3.7 and 1.7 ± 1.5 μM, and IC₅₀ (planktonic cells) of 3.0 ± 0.5, 2.6 ± 0.1 and 4.8 ± 0.5, respectively. Biofilms of C. glabrata GDH1407 and 6115/06 were less susceptible to histatin 5, with 50% RMA of 31.2 ± 4.8 and 62.5 ± 0.7 μM, respectively. Planktonic C. glabrata was insensitive to histatin 5 (IC₅₀ > 100 μM). Biofilm-associated Candida was highly resistant to fluconazole in the range 1–200 μM; e.g. at 100 μM only ~20% inhibition was observed for C. albicans, and ~30% inhibition for C. glabrata. These results indicate that histatin 5 exhibits antifungal activity against biofilms of C. albicans and C. glabrata developed on denture acrylic. C. glabrata is significantly less sensitive to histatin 5 than C. albicans.     

Aviglycine and propargylglycine inhibit conidial germination and mycelial growth of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>f. sp. <Emphasis Type="Italic">luffae</Emphasis>

Two inhibitors, aviglycine and propargylglycine, were tested for their ability to suppress methionine synthesis thus inhibit conidial germination and mycelial growth of Czapek-Dox liquid medium grown Fusarium oxysporum f. sp. luffae μM. The linear inhibition range for mycelial growth was about 7.6–762.9 μM. Although aviglycine did not completely inhibit both conidial germination and mycelial growth, it showed significant inhibitory effect at 1.5 μM. The inhibition range for propargylglycine against conidial germination and mycelial growth were from 0.08 to 8841 μM and from 0.8 to 884.1 μM, respectively. Propargylglycine inhibited conidial germination and mycelial growth at a concentration of 8841 μM. The EC₅₀ values of aviglycine were 1 μM for conidial growth and 122 μM for mycelial growth, and the EC₅₀ values of propargylglycine were 47.7 μM for conidial growth and 55.6 μM for mycelial growth. Supplement of methionine released inhibition of aviglycine or propargylglycine to conidial germination. In addition, a mixture of aviglycine (1.5 μM) and propargylglycine (8841 μM) showed additive inhibitive effect than applied alone on 10 isolates. From these results, both aviglycine and propargylglycine exhibited inhibitory activity, and suggest that they can provide potential tools to design novel fungicide against fungal pathogens.     

Molecular characterization of phenylalanine ammonia lyase gene from <Emphasis Type="Italic">Cistanche deserticola</Emphasis>

We cloned the gene, CdPAL1, from Cistanche deserticola callus using RACE PCR with degenerate primers that were designed based on a multiple sequence alignment of known PAL genes from other plant species. The gene shows high homology to other known PAL genes registered in GenBank. The recombinant protein exhibited Michaelis–Menten kinetics with a K _m of 0.1013 mM, V _max of 4.858 μmol min⁻¹, K _cat of 3.36 S⁻¹, and K _cat/K _m is 33,168 M⁻¹ S⁻¹. The enzyme had an optimal pH of 8.5 and an activation energy of 38.92 kJ mol⁻¹ when l-Phenylalanine was used as a substrate; l-tyrosine cannot be used as substrate for this protein. The optimal temperature was 55°C, and the thermal stability results showed that, after a treatment at 70°C for 20 min, the protein retained 87% activity, while a treatment at 75°C for 20 min resulted in a loss of over 85% of the enzyme activity. Treatment with heavy metal ions (Hg²⁺, Pb²⁺, and Zn²⁺) showed remarkable inhibitory effects. Among the intermediates from the lignin (cinnamyl alcohol, cinnamyl aldehyde, coniferyl aldehyde, coniferyl alcohol), phenylpropanoid (cinnamic acid, coumaric acid, caffeic acid, and chlorogenic acid) and phenylethanoid (tyrosol and salidroside) biosynthetic pathways, only cinnamic acid showed strong inhibitory effects against CdPAL1 activity with a K _i of 8 μM. Competitive inhibitor AIP exhibited potent inhibition with K _i = 0.056 μM.     

The effect of exogenous and endogenous phytohormones on the in vitro development of gametophyte and sporophyte in <Emphasis Type="Italic">Asplenium nidus</Emphasis> L.

The fern Asplenium nidus L. is in great demand as an ornamental plant. The aim of this work was to investigate the influence of phytohormones in promoting a gametophytic and sporophytic growth in homogenized sporophytes tissue. Exogenous application of 0.5 and 5 μM N ⁶-benzyladenine, 0.05 and 0.5 μM indole-3-acetic acid (IAA), and 0.3 and 3 μM gibberellic acid (GA₃) favoured sporophyte regeneration, whereas gametophyte regeneration took place when plant material was cultured in a hormone-free liquid MS medium. The endogenous contents of the auxin IAA, the cytokinins trans-zeatin, trans-zeatin riboside, dihydrozeatin, dihydrozeatin riboside, isopentenyladenine and isopentenyladenosine, and the gibberellins GA₁, GA₃, GA₄, GA₇, GA₉ and GA₂₀ in growing gametophytes and sporophytes were evaluated. Similar levels of the auxin and cytokinins and qualitative differences in the gibberellins were found between both generations.     

Characterization of a multifunctional feather-degrading <Emphasis Type="Italic">Bacillus subtilis</Emphasis> isolated from forest soil

In this study, we isolated and characterized a novel feather-degrading bacterium that shows keratinolytic, antifungal and plant growth-promoting activities. A bacterium S8 was isolated from forest soil and confirmed to belong to Bacillus subtilis by BIOLOG system and 16S rRNA gene analysis. The improved culture conditions for the production of keratinolytic protease were 0.1% (w/v) sorbitol, 0.3% (w/v) KNO₃, 0.1% (w/v) K₂HPO₄, 0.06% (w/v) KH₂PO₄ and 0.04% (w/v) MgCl₂·6H₂O (pH 8.0 and 30°C), respectively. In the improved medium containing 0.1% (w/v) feather, keratinolytic protease production was around 53.3 ± 0.3 U/ml at 4 day; this value was 10-fold higher than the yield in the basal feather medium (5.3 ± 0.1 U/ml). After cultivation for 5 days in the improved medium, intact feather was completely degraded. Feather degradation resulted in free –SH group, soluble protein and amino acids production. The concentration of free –SH group in the culture medium was 15.5 ± 0.2 μM at 4 days. Nineteen amino acids including all essential amino acids were produced in the culture medium; the concentration of total amino acid produced was 3360.4 μM. Proline (2809.9 μM), histidine (371.3 μM) and phenylalanine (172.0 μM) were the major amino acids released in the culture medium. B. subtilis S8 showed the properties related to plant growth promotion: hydrolytic enzymes, ammonification, indoleacetic acid (IAA), phosphate solubilization, and broad-spectrum antimicrobial activity. Interestingly, the strain S8 grown in the improved medium produced IAA and antifungal activity, indicating simultaneous production of keratinolytic and antifungal activities and IAA by B. subtilis S8. These results suggest that B. subtilis S8 could be not only used to improve the nutritional value of feather wastes but also is useful in situ biodegradation of feather wastes. Furthermore, it could also be a potential biofertilizer or biocontrol agent applicable to crop plant soil.     

Biological activities of the essential oils and methanol extract of tow cultivated mint species (<Emphasis Type="Italic">Mentha longifolia</Emphasis> and <Emphasis Type="Italic">Mentha pulegium</Emphasis>) used in the Tunisian folkloric medicine

The composition of the essential oils and methanolic extracts of two cultivated mint species (M. longifolia and M. pulegium), as well as the in vitro antimicrobial and antioxidant activities of the essential oil and methanol extract of Mentha longifolia and Mentha pulegium were compared. GC-MS analysis of the essential oil identified 41 compounds constituting 96.66 and 96.13% of the total oil from M. longifolia and M. pulegium, respectively. The later oils were rich on pulegone (47.15 and 61.11%, respectively). Moreover, 1,8 cineole (11.54%), menthone (10.7%), α-pinene (3.57%), α-terpineol (3.17%) and d-cadinene (3.53%) were only present in M. longifolia oil, while isomenthone (17.02%), and piperitone (2.63%), were characteristic of M. pulegium oil. Shoot extract of the two species showed significantly different contents in total polyphenols (89.1 and 37.41 mg GAE/g DW), flavonoids (63.93 and 33.83 mg CE/g DW) and tannins (1.47 and 3.07 mg CE/g DW), respectively in M. longifolia and M. pulegium. The essential oils showed strong antimicrobial activity against all 16 microorganisms tested, whereas the methanol extracts were inactive. Moreover, the essential oils showed higher antioxidant activity than the methanolic extracts against the DPPH and superoxide radical scavenging. In fact, antioxidant activities of the oils were the same for both M. longifolia and M. pulegium against DPPH (IC₅₀ = 9 and 10 μg/ml, respectively) and 2-fold and 4-fold higher than shoot extracts (IC₅₀ = 20 and 48 μg/ml, respectively). Moreover, both oils showed the same antioxidative abilities as compared to the positive control (butylated hydroxytoluene). In the same way, the capacity to inhibit superoxide anion was very significant for the two oils (0.1 μg/ml for M. longifolia and 0.11 μg/ml for M. pulegium).     

Effect of trace metals on ethanol production from synthesis gas by the ethanologenic acetogen, <Emphasis Type="Italic">Clostridium</Emphasis><Emphasis Type="Italic">ragsdalei</Emphasis>

The effect of trace metal ions (Co²⁺, Cu²⁺, Fe²⁺, Mn²⁺, Mo⁶⁺, Ni²⁺, Zn²⁺, SeO₄ ⁻ and WO₄ ⁻) on growth and ethanol production by an ethanologenic acetogen, Clostridium ragsdalei was investigated in CO:CO₂-grown cells. A standard acetogen medium (ATCC medium no. 1754) was manipulated by varying the concentrations of trace metals in the media. Increasing the individual concentrations of Ni²⁺, Zn²⁺, SeO₄ ⁻ and WO₄ ⁻ from 0.84, 6.96, 1.06, and 0.68 μM in the standard trace metals solution to 8.4, 34.8, 5.3, and 6.8 μM, respectively, increased ethanol production from 35.73 mM under standard metals concentration to 176.5, 187.8, 54.4, and 72.3 mM, respectively. Nickel was necessary for growth of C. ragsdalei. Growth rate (μ) of C. ragsdalei improved from 0.34 to 0.49 (day⁻¹), and carbon monoxide dehydrogenase (CODH) and hydrogenase (H₂ase)-specific activities improved from 38.45 and 0.35 to 48.5 and 1.66 U/mg protein, respectively, at optimum concentration of Ni²⁺. At optimum concentrations of WO₄ ⁻ and SeO₄ ⁻, formate dehydrogenase (FDH) activity improved from 32.3 to 42.6 and 45.4 U/mg protein, respectively. Ethanol production and the activity of FDH reduced from 35 mM and 32.3 U/mg protein to 1.14 mM and 8.79 U/mg protein, respectively, upon elimination of WO₄ ⁻ from the medium. Although increased concentration of Zn²⁺ enhanced growth and ethanol production, the activities of CODH, FDH, H₂ase and alcohol dehydrogenase (ADH) were not affected by varying the Zn²⁺ concentration. Omitting Fe²⁺ from the medium decreased ethanol production from 35.7 to 6.30 mM and decreased activities of CODH, FDH, H₂ase and ADH from 38.5, 32.3, 0.35, and 0.68 U/mg protein to 9.07, 7.01, 0.10, and 0.24 U/mg protein, respectively. Ethanol production improved from 35 to 54 mM when Cu²⁺ was removed from the medium. The optimization of trace metals concentration in the fermentation medium improved enzyme activities (CODH, FDH, and H₂ase), growth and ethanol production by C. ragsdalei.     

Nitrogen and phosphorus utilization in the cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> isolated from Laguna de Bay,Philippines

Phytoplankton supports fisheries and aquaculture production. Its vital role as food for aquatic animals, like mollusks, shrimp, and fish cannot be overemphasized. Because of its contribution as a food source for fish, the growth kinetics of Microcystis aeruginosa, a dominant cyanobacterium in the lake, was studied. The regular occurrence of M. aeruginosa is experienced during the months of May to July or from September to November in Laguna de Bay, the largest freshwater lake in the Philippines. M. aeruginosa was collected from Laguna de Bay, isolated, and established in axenic conditions. Data on the growth kinetic parameters for nitrate-nitrogen and phosphate-phosphorus utilization by M. aeruginosa gave the following values: half-saturation constant (K _s ), 0.530 mg N. L⁻¹ and 0.024 mg P. L⁻¹ respectively; maximum growth rate (μ _max ), 0.671. d⁻¹ and 0.668. d⁻¹ respectively; maximum cell yield, 6.5 and 6.54 log, cells. ml⁻¹ respectively; nutrient level for saturated growth yield, 8.71 mg N. L⁻¹ and 0.22 mg P. L⁻¹ respectively; and minimum cell quota (Q ₀ ), 2.82 pg N. cell⁻¹ and 0.064 pg P. cell⁻¹ respectively. The low K _s value and high maximum growth rate (μ _max ) for phosphorus by M. aeruginosa would suggest a high efficiency of phosphorus utilization. On the other hand, the high K _s value for nitrogen indicated a low rate of uptake for this nutrient.     

Comparison of in vitro regeneration pathways in <Emphasis Type="Italic">Punica granatum</Emphasis> L.

When cotyledonary explants, excised from in vitro germinated seedlings, of pomegranate (Punica granatum L.) were incubated on solid Murashige and Skoog (1962) medium supplemented with 21 μM naptheleneacetic acid (NAA) and 9 μM 6-benzyladenine (BA), 80% of explants developed callus. A high frequency of shoot organogensis was obtained when explants were incubated on MS medium supplemented with 8 μM BA, 6 μM NAA, and 6 μM giberrellic acid (GA₃). However, adding 24 μM silver nitrate (AgNO₃) to this medium markedly enhanced shoot regeneration frequency (63%) and mean number of shoots per explant (11.26) and length of shoots (2.22 cm). Highest frequency of in vitro rooting, mean number of roots/shoot (4.32), and mean root length (2.71 cm) were obtained when regenerated shoots were transferred to half-strength MS medium supplemented with 0.02% activated charcoal. Well-rooted plantlets were acclimatized, and then transferred to soil medium. Moreover, when zygotic embryos of P. granatum, excised from seeds collected at 16 weeks following full bloom, were incubated on MS medium containing 30 g l⁻¹ sucrose, 15% coconut water, 21 μM NAA, and 9 μM BA, they developed the highest frequency of embryogenic callus, clumps with globular embryos, and mean number of both globular and heart-shaped embryos per callus clump. Subjecting zygotic embryo explants to six-week dark incubation period was essential for embryogenic callus induction, and these were subsequently transferred to 16 h photoperiod for further growth and development of somatic embryos. Germination of somatic embryos was observed when these were transferred to MS medium was supplemented with 60 g l⁻¹ sucrose.     

Characterization of zinc transport by divalent metal transporters of the ZIP family from the model legume <Emphasis Type="Italic">Medicago truncatula</Emphasis>

To understand how plants from the Fabaceae family maintain zinc (Zn) homeostasis, we have characterized the kinetics of three Zn transporting proteins from the ZIP family of divalent metal transporters in the model legume Medicago truncatula. Of six ZIP’s studied, MtZIP1, MtZIP5 and MtZIP6 were the only members from this family determined to transport Zn and were further characterized. MtZIP1 has a low affinity for Zn with a K_m of 1 μM as compared to MtZIP5 and MtZIP6 that have a higher affinity for Zn with K_m of 0.4 μM and 0.3 μM, respectively. Zn transport by MtZIP1 was more sensitive to inhibition by copper (Cu) concentrations than MtZIP5 and MtZIP6, because 3 μM Cu inhibited Zn transport by 80% in MtZIP1 while 5 μM Cu was required to achieve the same inhibition of Zn transport in MtZIP5 and MtZIP6. Cadmium (Cd) had a greater effect on the ability of MtZIP1 to transport Zn than MtZIP5 and MtZIP6, because at a concentration of 3 μM Cd, the Zn transport by MtZIP1 was inhibited 55% and the transport of Zn by MtZIP5 and MtZIP6 was inhibited by 20–30%. However, only MtZIP6 transported Cd at higher rates than those observed in the control plasmid pFL61, demonstrating a low affinity for Cd based on a K_m of 57 μM. These results suggest that Medicago truncatula has both high and low affinity Zn transporters to maintain Zn homeostasis and that these transporters may function in different compartments within the plant.     

Comparison of metabolite levels in callus of <Emphasis Type="Italic">Tecoma stans</Emphasis> (L.) Juss. ex Kunth. cultured in photoperiod and darkness

Tecoma stans is a tropical plant from the Americas. Antioxidant activity and both phenolic compound and flavonoid total content were determined for callus tissue of T. stans cultured in either a set photoperiod or in darkness. Callus lines from three explant types (hypocotyls, stem, and leaf) were established on B5 culture medium supplemented with 0.5 μM 2,4-D and 5.0 μM kinetin. While leaf-derived callus grew slower under a 16-h photoperiod (specific growth rate, μ = 0.179 d⁻¹, t _D = 3.9 d) than in darkness (μ = 0.236 d⁻¹, t _D = 2.9 d), it accumulated the highest amount (p < 0.05) of both phenolics (86.6 ± 0.01 mg gallic acid equivalents/g) and flavonoids (339.6 ± 0.06 mg catechin equivalents/g). Similarly, antioxidant activity was significantly higher (p < 0.05) when callus was cultured in period light than when grown in extended darkness. Antioxidant activity measured with a 2,20-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS)-based assay was 350.5 ± 15.8 mmol Trolox/g extract for callus cultured under a defined photoperiod compared to 129.1 ± 7.5 mmol Trolox/g extract from callus cultured in darkness. Content of phenolic compounds and flavonoids was in agreement with a better antioxidant power (EC₅₀ = 450 μg extract/mg 1,1-diphenyl-2-picrylhydrazyl) and antiradical efficiency. Results of the present study show that calli of T. stans are a source of compounds with antioxidant activity that is favored by culture under a set photoperiod.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of northern red oak (<Emphasis Type="Italic">Quercus rubra</Emphasis> L.)

In vitro propagation of northern red oak (Quercus rubra) shoots was successful from cotyledonary node explants excised from 8-wk-old in vitro grown seedlings. Initially, four shoots per explant were obtained on Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzylaminopurine (BA), 0.45 μM thidiazuron (TDZ), and 500 mg l⁻¹ casein hydrolysate (CH) with a regeneration frequency of 64.7% after 3 wk. Subculturing explants (after harvesting shoots) to fresh treatment medium significantly increased shoot bud regeneration (16.6 buds per explant), but the buds failed to develop into shoots. A higher percentage (73.3%) of the explants regenerated four shoots per explant on woody plant medium (WPM) supplemented with 4.4 μM BA, 0.29 μM gibberellic acid (GA₃), and 500 mg l⁻¹ CH after 3 wk. Explants subcultured to fresh treatment medium after harvesting shoots significantly increased shoot regeneration (16 shoots per explant). Shoot elongation was achieved (4 cm) when shoots were excised and cultured on WPM supplemented with 0.44 μM BA and 0.29 μM GA₃. In vitro regenerated shoots were rooted on WPM supplemented with 4.9 μM indole-3-butyric acid. A higher percentage regeneration response and shoot numbers per explant were recorded on WPM supplemented with BA and GA₃, than on MS medium containing BA and TDZ. Lower concentrations of BA and GA₃ were required for shoot elongation and prevention of shoot tip necrosis. Each cotyledonary node yielded approximately 20 shoots within 12 wk. Rooted plantlets were successfully acclimatized.