<Emphasis Type="Italic">In vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> inhibition of α-amylases of stored-product mite <Emphasis Type="Italic">Acarus siro</Emphasis>

The stored-product mites are the most abundant and frequent group of pests living on the stored food products in Europe. They endanger public health since they produce allergens and transmit mycotoxin-producing fungi. Novel acaricidal compounds with inhibitory effects on the digestive enzymes of arthropods are a safe alternative to the traditional neurotoxic pesticides used for control of the stored-product pests. In this work, we explored the properties of acarbose, the low molecular weight inhibitor of -amylases (AI), as a novel acaricide candidate for protection of the stored products from infestation by Acarus siro (Acari: Acaridae). In vitro analysis revealed that AI blocked efficiently the enzymatic activity of digestive amylases of A. siro, and decreased the physiological capacity of mites gut in utilizing a starch component of grain flour. In vivo experiments showed that AI suppressed the population growth of A. siro. The mites were kept for three weeks on experimental diet enriched by AI in concentration range of 0.005 to 0.25%. Population growth of A. siro was negatively correlated with the content of AI in the treated diet with a half-population dose of 0.125%. The suppressive effect of AIs on stored-product mites is discussed in the context of their potential application in GMO crops     

Effect of various culture conditions on shoot multiplication and GC–MS analysis of <Emphasis Type="Italic">Plumbago zeylanica</Emphasis> accessions for plumbagin production

Plumbago zeylanica, a pharmaceutically important medicinal plant, contains a wide range of phytocompounds. Culture parameters like carbon source, nitrogen source, and culture media are essential for the development and growth of explants. In this investigation, the influence of various carbon sources (sucrose, glucose, and fructose at 3% concentration), nitrogen source (ammonium nitrate, sodium nitrate, and potassium nitrate) and plant tissue culture media (MS medium, Gamborg’s B5 medium, White medium and Nitsch medium) on shoot multiplication of five different accessions was studied. Optimum growth of all five accessions was observed in MS media containing 3% sucrose and ammonium nitrate as a source of carbon and nitrogen. Out of five accessions, IC-524441 showed the highest shoot multiplication. Further, methanolic extracts of all accessions (grown in MS media containing 3% sucrose and ammonium nitrate as nitrogen source) were prepared and comparison of extracts in DPPH assay indicated that accession number IC-524441 was the most effective free radical scavenging agent. Total phenolic, flavonoid and tannin content ranges were from 20 to 70 µg/ml, 40 to 100 µg/ml and 55 to 120 µg/ml, respectively, and the highest amount was found in accession number IC-524441. Sucrose and ammonium nitrate content may be responsible for increased antioxidant activity, flavonoids content, phenolic content, and tannin content in accession number IC-524441. GC–MS of ethyl acetate extract of all five accessions of P. zeylanica was conducted (grown in MS media containing 3% sucrose and ammonium nitrate as nitrogen source). GC–MS analysis of the aerial part showed the presence of various phytocompounds, which include 1,4-naphthalenedione, 3-eicosene, 5-eicosene, phthalic acid, o-anisic acid, thioctic acid, 1-octadecene, 5-t-butyl-cycloheptene, 2-benzoyl-1,2-dihydro-1-isoquinolinecarbonitrile, octadecanal, silane, 3-methoxy-2-methyl-2-(1-phenyl-ethylamino)-propionic acid, and 1-nonadecene. Accession number IC-524441 contains the highest amount of plumbagin, i.e. 14.19?±?0.5 µg/ml as compared to the others.     

<Emphasis Type="Italic">In vitro</Emphasis>–<Emphasis Type="Italic">ex vitro</Emphasis> salt (NaCl) tolerance of cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz) plants

Three cassava clones (SOM-1, “05”, and “50”) were cultured in vitro on MS medium plus sucrose (30 g L⁻¹) and myo-inositol (100 mg L⁻¹) without plant growth regulators and with additions of 0 (control), 0.5, 1, 1.5, 2, 2.5, and 3 g L⁻¹ NaCl to test their salt tolerance. The same cassava clones were cultivated in greenhouse conditions on a sandy soil substratum and irrigated with 20% strength Hoagland solution, and additions of 0, 4, and 8 g L⁻¹ of NaCl. Salinity negatively affected the survival, development, leaf water content, and mineral composition (mainly by accumulation of Cl and Na) of both in vitro and ex vitro plants, but with different intensity in each clone. In both conditions of culture (in vitro and ex vitro) clone SOM-1, from a desert arid saline zone of Somalia, was the most tolerant and clone “05”, from a rainy region of Ivory Coast, the most sensitive. Clone “50” tolerance to in vitro salt treatments, although lower, was not significantly different from that of SOM-1 but the ex vitro response was similar to “05”. In general, there was a correlation between in vitro and ex vitro behavior of the cassava plant regarding salt tolerance, which would allow the in vitro culture method to be used for selection of salt-tolerant plants of this crop.     

High production of bioactive depsides in shoot and callus cultures of <Emphasis Type="Italic">Aronia arbutifolia</Emphasis> and <Emphasis Type="Italic">Aronia</Emphasis> × <Emphasis Type="Italic">prunifolia</Emphasis>

Methanolic extracts from calluses and shoots of Aronia arbutifolia and Aronia × prunifolia cultivated in vitro were quantitatively analysed for phenolic acids by DAD-HPLC. The cultures were grown on ten variants of Murashige–Skoog medium variants enriched with various concentrations of growth regulators (GRs), BA and NAA, in the concentration range 0.1–3.0 mg/L. The analysed extracts were confirmed to contain from four to six compounds (depsides—chlorogenic acid, neochlorogenic acid, and rosmarinic acid, and also protocatechuic acid, p-hydroxybenzoic acid, and 3,4-dihydroxyphenylacetic acid). The total amounts of the metabolites varied considerably, depending on the amounts of the GRs in the tested medium variants, and increased in the callus and shoot extracts, respectively, up to 1.7 and 3.2 times (A. arbutifolia), and 2.2 and 2.7 times (A. × prunifolia). Maximum total amounts were confirmed in shoot extracts of both plants (approx. 200 and 600 mg/100 g DW, respectively). The main compounds in A. arbutifolia cultures were the depsides—chlorogenic acid, rosmarinic acid, and neochlorogenic acid (max. 91.94, 77.03, 32.57 mg/100 g DW, respectively). The same depsides dominated quantitatively in the cultures of A. × prunifolia (max. 131.82, 206.62 and 257.39 mg/100 g DW, respectively).     

Brood cell size of <Emphasis Type="Italic">Apis</Emphasis> <Emphasis Type="Italic">mellifera</Emphasis> modifies the reproductive behavior of <Emphasis Type="Italic">Varroa</Emphasis> <Emphasis Type="Italic">destructor</Emphasis>

We undertook a field study to determine whether comb cell size affects the reproductive behavior of Varroa destructor under natural conditions. We examined the effect of brood cell width on the reproductive behavior of V. destructor in honey bee colonies, under natural conditions. Drone and worker brood combs were sampled from 11 colonies of Apis mellifera. A Pearson correlation test and a Tukey test were used to determine whether mite reproduction rate varied with brood cell width. Generalized additive model analysis showed that infestation rate increased positively and linearly with the width of worker and drone cells. The reproduction rate for viable mother mites was 0.96 viable female descendants per original invading female. No significant correlation was observed between brood cell width and number of offspring of V. destructor. Infertile mother mites were more frequent in narrower brood cells.     

Functional expression of the lipase gene Lip2 of <Emphasis Type="Italic">Pleurotus</Emphasis> <Emphasis Type="Italic">sapidus</Emphasis> in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.     

Micropropagation of <Emphasis Type="Italic">Clitoria ternatea</Emphasis> Linn. (<Emphasis Type="Italic">Fabaceae</Emphasis>)— An important medicinal plant

Summary An efficient protocol was established for in vitro shoot multiplication from nodal explants of Clitoria ternatea on semisolid Murashige and Skoog (MS) basal medium supplemented with 8.9μM 6-benzylaminopurine (BA). Inclusion of 1-naphthaleneacetic acid (NAA) in the culture medium along with BA promoted higher rates of shoot multiplication than BA alone. The rate of shoot multiplication was maximum (5.21) after 4 wk of culture on MS basal medium supplemented with 8.9μM BA and 1.34μM NAA. The elongated shoots rooted within 7–8d in half-strength MS basal salts supplemented with 1.34μM NAA and 2% (w/v) sucrose. About 85% of the rooted plantlets were acclimatized and transferred to the greenhouse.     

Rapid plant regeneration through <Emphasis Type="Italic">in vitro</Emphasis> axillary shoot proliferation of butterfly pea (<Emphasis Type="Italic">Clitoria ternatea</Emphasis> L.)—a twinning legume

This study describes a reproducible protocol for rapid mass propagation of a multipurpose legume, Clitoria ternatea L., using cotyledonary node explants derived from axenic seedlings. Multiple shoots were induced in Murashige and Skoog (MS) medium supplemented with N ⁶-benzyladenine (BA), zeatin riboside, or thidiazuron. N ⁶-Benzyladenine at 1.0 mg l⁻¹ (4.44 μM) was most effective for shoot proliferation. Multiple shoots were also induced in nodal segments of in vitro-raised shoots grown on MS medium containing 1.0 mg l⁻¹ (4.44 μM) of BA. Rooting was best induced in shoots grown on half-strength MS medium with 0.25 mg l⁻¹ (1.42 μM) of indole-3-butyric acid. Plants were acclimatized in vermicompost and established in soil where they flowered and formed mature seeds.     

<Emphasis Type="Italic">Chromobacterium violaceum</Emphasis> and its important metabolites — <Emphasis Type="Italic">review</Emphasis>

C. violaceum appeared as important bacterium in different applications and mainly these aspects are related to the production of violacein. This review discusses the last reports on biosynthetic pathways, production, genetic aspects, biological activities, pathological effects, antipathogenic screening through quorum sensing, environmental effects and the products of C. violaceum with industrial interest. An important discussion is on biological applications in medicine and as industrial products such as textile and in cosmetics.     

Enzymatic synthesis of alkyl glucosides using <Emphasis Type="Italic">Leuconostoc</Emphasis> <Emphasis Type="Italic">mesenteroides</Emphasis> dextransucrase

Alkyl glucosides were synthesized by the reaction of Leuconostoc mesenteroides dextransucrase with sucrose and various alcohols. Alkyl α-d-glucosides were obtained with a yield of 30% (mol/mol) with primary alcohols, but secondary alcohols or tertiary alcohols gave yields below 5%. The optimal yield was 50% using 1-butyl α-d-glucoside with 0.9 M 1-butanol. The acceptor products of methanol or ethanol were confirmed as methyl α-d-glucopyranoside and ethyl α-d-glucopyranoside via MALDI-TOF MS and NMR analysis. Thus, methyl or ethyl α-d-glucoside constituted half the emulsification activities of Triton X-100 as commercially available surfactants. Young-Min Kim and Byung-Hoon Kim contributed equally to this work.     

Induction of β-1,3-glucanase in callus cultures <Emphasis Type="Italic">in vitro</Emphasis>

Sodium salicylate (NaSA) increased induction of both intracellular and extracellular beta-1,3-glucanases in calluses of campion and duckweed. NaSA concentrations from 30 to 100 mM were optimal for induction of intracellular glucanase in the campion callus, and for induction of extracellular glucanase the optimal concentration varied from 5 to 100 mM. The glucanase activity in the duckweed callus was lower than in the campion callus, and co-cultivation of the campion callus with Trichoderma harzianum mycelium increased the production of intracellular and extracellular beta-1,3-glucanases and polygalacturonase in the callus. Biosynthesis by T. harzianum of glucanases, extracellular polygalacturonase and xylanase, and of intracellular galactosidase was increased. The co-cultivation was accompanied by increased activity of intracellular acidic isoform of glucanase Glu-3 secreted by the callus cells into the medium, whereas NaSA activated in the callus culture the extracellular acidic isoform Glu-1 and extracellular basic isoform Glu-5. These data indicate the induction of these isoforms and the specificity of protective response of plant cells to different factors.     

Effect of γ-radiation on development,yield and quality of microtubers <Emphasis Type="Italic">in vitro</Emphasis> in <Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.

Explants obtained from in vitro-propagated plantlets of two potato cultivars, Shepody and Atlantic, were treated with five doses of γ-radiation (0, 2, 4, 6 and 8 Gy) to investigate the stimulating effects of low irradiation on the production and quality of microtubers in vitro. Microtubers of both cultivars treated with γ-radiation initiated 5 d earlier than in the non-irradiated control. The whole period of microtuberization was prolonged by 10 – 15 d with 4, 6 and 8 Gy irradiation treatment for cv. Atlantic. Irradiation of the plantlets (4 Gy) led to a significant increase not only in the microtuber number (116.7 and 34.5 % over the control) but also in the fresh mass (77.6 and 23.2 % in Shepody and Atlantic, respectively). Low dose irradiation (2 – 4 Gy) increased the starch content of microtubers. High doses (6 – 8 Gy) enhanced ascorbic acid and reducing sugar contents. 4 – 6 Gy doses also effectively increased the protein contents of microtubers.     

Characteristics of β-lactamases and their genes (<Emphasis Type="Italic">blaA</Emphasis> and <Emphasis Type="Italic">blaB</Emphasis>) in <Emphasis Type="Italic">Yersinia intermedia</Emphasis> and <Emphasis Type="Italic">Y. frederiksenii</Emphasis>

Background  

The presence of β-lactamases in Y. enterocolitica has been reported to vary with serovars, biovars and geographical origin of the isolates. An understanding of the β-lactamases in other related species is important for an overall perception of antibiotic resistance in yersiniae. The objective of this work was to study the characteristics of β-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii, isolated from clinical and non-clinical sources in India.     

Rapid induction of <Emphasis Type="Italic">Agrobacterium</Emphasis> <Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transgenic roots directly from adventitious roots in <Emphasis Type="Italic">Panax</Emphasis> <Emphasis Type="Italic">ginseng</Emphasis>

Root segments from seedlings of Panax ginseng produced adventitious roots directly when cultured on 1/2 MS solid medium lacking NH₄NO₃ and containing 3.0 mg l⁻¹ IBA. Using this adventitious root formation, we developed rapid and efficient transgenic root formation directly from adventitious root segments in P. ginseng. Root segments were co-cultivated with Agrobacterium tumefaciens (GV3101) caring β-glucuronidase (GUS) gene. Putative transgenic adventitious roots were formed directly from root segments on medium with 400 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin. Kanamycin resistant adventitious roots were selected and proliferated as individual lines by subculturing on medium with 300 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin at two weeks subculture interval. Frequency of transient and stable expression of GUS gene was enhanced by acetosyringon (50 mg l⁻¹) treatment. Integration of transgene into the plants was confirmed by the X-gluc reaction, PCR and Southern analysis. Production of transgenic plants was achieved via somatic embryogenesis from the embryogenic callus derived from independent lines of adventitious roots. The protocol for rapid induction of transgenic adventitious roots directly from adventitious roots can be applied for a new Agrobacterium tumefaciens-mediated genetic transformation protocol in P. ginseng.     

<Emphasis Type="Italic">Carex</Emphasis> × <Emphasis Type="Italic">involuta</Emphasis> and <Emphasis Type="Italic">Carex juncella</Emphasis> in the flora of Slovakia

The paper brings information on an isolated occurrence and morphological characters of Carex × involuta and C. juncella populations in the Vel’ká Fatra Mts. Their presence has been known neither from the territory of Slovakia nor from the whole Western Carpathians till now.     

<Emphasis Type="Italic">In vitro</Emphasis> culture of <Emphasis Type="Italic">Drynaria fortunei</Emphasis>, a fern species source of Chinese medicine “Gu-Sui-Bu”

This study reports spore germination, early gametophyte development and change in the reproductive phase of Drynaria fortunei, a medicinal fern, in response to changes in pH and light spectra. Germination of D. fortunei spores occurred on a wide range of pH from 3.7 to 9.7. The highest germination (63.3%) occurred on ½ strength Murashige and Skoog basal medium supplemented with 2% sucrose at pH 7.7 under white light condition. Among the different light spectra tested, red, far-red, blue, and white light resulted in 71.3, 42.3, 52.7, and 71.0% spore germination, respectively. There were no morphological differences among gametophytes grown under white and blue light. Elongated or filamentous but multiseriate gametophytes developed under red light, whereas under far-red light gametophytes grew as uniseriate filaments consisting of mostly elongated cells. Different light spectra influenced development of antheridia and archegonia in the gametophytes. Gametophytes gave rise to new gametophytes and developed antheridia and archegonia after they were transferred to culture flasks. After these gametophytes were transferred to plastic tray cells with potting mix of tree fern trunk fiber mix (TFTF mix) and peatmoss the highest number of sporophytes was found. Sporophytes grown in pots developed rhizomes.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Codonopsis lanceolata</Emphasis> using the <Emphasis Type="Italic">γ-TMT</Emphasis> gene

Efficient transformation of leaf disc-derived callus of Codonopsis lanceolata was obtained using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector, pYBI121, that carries the neomycin phosphotransferase (npt II) gene as a selectable marker. The green shoots recovered from agroinfected explants on selection medium (containing 0.1 mg/l α-naphthaleneacetic acid (NAA), 1 mg/l 6-benzylaminopurine (BAP), 100 mg/l kanamycin, and 250 mg/l cefotaxime) were rooted on Murashige and Skoog (MS) medium supplemented with 2 mg/l IBA and 10 mg/l kanamycin. To optimize the transformation conditions, several factors were assessed, including the co-cultivation period, the duration of pre- and post-culture in darkness and light, the kanamycin concentration, and the Agrobacterium densities. We produced transgenic Codonopsis lanceolata overexpressing γ-tocopherol methyltransferase (γ-TMT) by this protocol. Moreover, the α-tocopherol content of the plants was enhanced by the overexpression of this gene. Bimal Kumar Ghimire and Eun Soo Seong contributed equally to this work.     

Gill cell toxicity of northern boreal scyphomedusae <Emphasis Type="Italic">Cyanea</Emphasis> <Emphasis Type="Italic">capillata</Emphasis> and <Emphasis Type="Italic">Aurelia</Emphasis> <Emphasis Type="Italic">aurita</Emphasis> measured by an in vitro cell assay

Scyphozoan medusae are very successful foragers which occasionally occur in high abundances in boreal waters and may impact many different groups in the marine ecosystem by means of a variety of toxins. A rainbow trout gill cell line, RTgill-W1, was tested for its suitability as quantitative indicator of the cytotoxicity of Cyanea capillata and Aurelia aurita; the major scyphozoan species in the North and Baltic seas. Cultures of rainbow trout gill cells were exposed to whole venoms extracted from fishing tentacles and oral arms at increasing protein concentrations. The venom caused detachment, clumping and lysis of cells, as well as a drop in vitality, in a dose-dependent manner. Morphological changes in the cells were evident within 1 h after venom addition. The damage to gill cells was quantified by measuring the metabolic activity of the cells by means of the fluorescence of resorufin derived from the nonfluorescent substrate, resazurin. In general, a decrease in the metabolic activity of the cells was detected at a venom (protein) concentration above 2.0 μg ml⁻¹ (corresponding to 0.2 μg 10⁴ cells⁻¹), and a total loss of activity was observed above 40.0 μg ml⁻¹ (corresponding to 4.0 μg 10⁴ cells⁻¹). C. capillata venoms had increased cytotoxic activity as compared to A. aurita venoms at the same concentration. Cnidocyst extracts from oral arms of A. aurita induced an 85% loss of gill cell viability at concentrations of 0.2 μg 10⁴ cells⁻¹, whereas crude venoms from fishing tentacles reduced cell viability by 18% at the same concentration. Gel electrophoresis of the venoms indicated that these consist of a large number of proteins in a fairly wide size range, from 6 to 200 kDa, including some that are the same size as those found in cubomedusae. It also appears that larger (i.e., older) medusae have more complex venoms and, in some cases, more potent venoms than smaller animals.     

Synthesis of Bacteriophage λ Proteins <Emphasis Type="Italic">in vitro</Emphasis>

λ DNA stimulates a low level of protein synthesis in extracts of E. coli supplemented with RNA polymerase. The synthesis is efficiently and specifically repressed by addition of purified λ repressor. About 90% of the protein product is from the rightwards, or tof, promoter (P _R ) and 10% from the leftwards, or N, promoter (P _L ). The main polypeptide product is a low molecular species from the tof operon.