Expression and purification of <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis> agglutinin in <Emphasis Type="Italic">Escherichia </Emphasis><Emphasis Type="Italic">coli</Emphasis>

Recombinant Zantedeschia aethiopica agglutinin (ZAA) was expressed in Escherichia coli as N-terminal His-tagged fusion. After induction with isopropylthio-β-d-galactoside (IPTG), the recombinant ZAA was purified by metal-affinity chromatography. The purified ZAA protein was applied in anti-fungal assay and the result showed that recombinant ZAA had anti-fungal activity towards leaf mold (Fulvia fulva), one of the most serious phytopathogenic fungi causing significant yield loss of crops. This study suggests that ZAA could be an effective candidate in genetic engineering of plants for the control of leaf mold.     

Expression and characterization of <Emphasis Type="Italic">Trichoderma virens</Emphasis> UKM-1 endochitinase in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

A gene encoding endochitinase from Trichoderma virens UKM-1 was cloned and expressed in E. coli BL21 (DE3). Both the endochitinase gene and its cDNA sequences were obtained. The endochitinase gene encodes 430 amino acids from an open reading frame comprising of 1,690 bp nucleotide sequence with three introns. The endochitinase was expressed as soluble and active enzyme at 20°C when induced with 1 mM IPTG. Maximum activity was observed at 4 h of post-induction time. SDS-PAGE showed that the purified endochitinase exhibited a single band with molecular weight of 42 kDa. Biochemical characterization of the enzyme displayed a near neutral pH characteristic with an optimum pH at 6.0 and optimum temperature at 50°C. The enzyme is stable between pH 3.0–7.0 and is able to retain its activity from 30 to 60°C. The presence of Mg²⁺ and Ca²⁺ ions increased the enzyme activity up to 20%. The purified enzyme has a strong affinity towards colloidal chitin and low effect on ethyl cellulose and D-cellubiose which are non-chitin related substrates. HPLC analysis from the chitin hydrolysis showed the release of (GlcNAc)₃, (GlcNAc)₂ and GlcNAc, in which (GlcNAc)₂ was the main product.     

<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.     

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.     

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.     

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.     

Regeneration and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of mature woody species <Emphasis Type="Italic">Photinia</Emphasis> × <Emphasis Type="Italic">fraseri</Emphasis> “Red Robin”

An efficient regeneration protocol for genetic transformation was developed from the leaves of an 11-year-old Phtinia × fraseri “Red Robin” tree. A high frequency of adventitious buds (88.63 ± 1.38%) and the highest maximum mean number of adventitious buds per explant (4.65 ± 0.48) were obtained in light conditions on Murashige–Skoog (MS) medium containing 2 mg l⁻¹ benzyladenine (BA) and 0.2 mg l⁻¹ α-Naphthaleneacetic acid (NAA). After preculturing for 6 days, over 95% of the shoots successfully rooted on 1/2 MS medium supplemented with 0.3 mg l⁻¹ indole-3-butyric acid (IBA) within 3 weeks. For genetic transformation, two crucial parameters (30 mg l⁻¹ kanamycin for selection and 30-min suspension time) were optimized. Taken together, a reliable transformation system with an efficiency of more than 5% was established. This genetic transformation protocol can be utilized for further genetic manipulation of the Photinia tree.     

Fertility and precocity of <Emphasis Type="Italic">Osmunda</Emphasis> × <Emphasis Type="Italic">intermedia</Emphasis> offspring in culture

The feasibility of later-generation hybrid production in ferns has not been previously studied, although it is a significant factor in relation to reproductive isolation. Osmunda × intermedia, a hybrid between O. japonica and O. lancea, is semifertile and has moderate spore germination rates. Under the artificial conditions of this study, F2 and F3 offspring were formed. Some of the F2 offspring showed precocity, and some of the F3 offspring also showed precocity. This fertility suggests that introgressive hybridization might be ongoing in nature. This also indicates a currently unknown genetic control over the timing of fertile frond production in Osmunda.     

In vitro propagation of seven <Emphasis Type="Italic">Daphne</Emphasis> L. species

Cultures of seven Daphne species: Daphne caucasica, D. cneorum, D. giraldii, D. retusa, D. jasminea, D. laureola and D. tangutica were established in vitro on MS/WPM based media. Five of the species responded best on MS-based media (D. tangutica, D. laureola, D. caucasica, D. retusa and D. giraldii), while the remaining two species performed best on WPM-based media (D. cneorum, and D. jasminea). Shoot proliferation was achieved from both apical and nodal explants. Shoots were sub-cultured from stock cultures, cut into nodal explants 3–5 cm long and place vertically on basal media supplemented with different concentrations and combinations of cytokinins and auxins. Individual species displayed different responses to the various cytokinins and auxins. Among species, D. jasminea produced the greatest proliferation rate with an average of 7.84 + 0.6 shoots per explant on WPM supplemented with 2.32 μM BA + 0.0045 μM TDZ + 0.054 μM NAA, while the best multiplication rate for the same species grown on the same media supplemented with a single cytokinin (BA) and no auxin was 2.60 + 1.3 shoots per explant. Following multiplication, new shoots transferred to the elongation trails and then 50–100 mm Shoots used for rooting experiments. Increased rooting efficiencies were observed on in vitro-generated shoots with the two-layer medium or dipping methods over when PGRs were uniformly incorporated into the medium. Maximum rooting frequencies (average) ranged from 59% in D. tangutica to 85% in D. jasminea. Following in vitro rooting, rooted shoots immersed in 0.01% solution of humates and planted into a standard horticultural substrate composed and watered weekly with a solution containing half-strength MS salts.     

In vitro growth response of <Emphasis Type="Italic">Phytophthora cactorum</Emphasis>, <Emphasis Type="Italic">P. nicotianae</Emphasis> and <Emphasis Type="Italic">P.</Emphasis> × <Emphasis Type="Italic">pelgrandis</Emphasis> to antibiotics and fungicides

The reactions of isolates of Phytophthora cactorum, P. nicotianae and P. × pelgrandis to metalaxyl, mancozeb, dimethomorph, streptomycin and chloramphenicol were tested to obtain information about the variability of resistance in these pathogens. Distinct genetic groups showed significant differences in resistance to all tested substances except streptomycin. In response to streptomycin, the growth inhibition rates of distinct groups did not differ significantly. The most remarkable differences were detected in the reactions to chloramphenicol and metalaxyl. Discriminant analysis evaluating the effect of all substances confirmed the differences among the groups, which are in agreement with the differences revealed by earlier DNA analyses.     

Cryopreservation of hairy root cultures of <Emphasis Type="Italic">Maesa lanceolata</Emphasis> and <Emphasis Type="Italic">Medicago truncatula</Emphasis>

To study the production of secondary metabolites of Maesa lanceolata and Medicago truncatula, hairy root cultures of both plant species were established. Because maintenance of large numbers of cultures is laborious and costly, we developed a cryopreservation protocol and stored different isolated lines over time. Using encapsulation-dehydration, high survival rates were observed for both Maesa and Medicago hairy roots. Root tips were isolated and encapsulated in calcium-alginate beads, containing 0.1 M sucrose. The encapsulated hairy roots were precultured for 3 days using basal medium containing high sucrose concentrations. Medicago root tip growth during the preculturing time lead to unwanted outgrowth which could be tempered by addition of plant growth inhibitors. After preculturing, the beads were dehydrated in the air flow of a laminar flow until 35–40% of the initial bead weight was reached. Dehydrated beads were plunged into liquid nitrogen and after different storage times thawed in a water bath at 40°C. The survival rates were 90% for Maesa and 53% for Medicago, which are sufficient to allow implementation in large storage experimental set-ups.     

Gibberellic acid promotes in vitro regeneration and shoot multiplication in <Emphasis Type="Italic">Lotus</Emphasis><Emphasis Type="Italic">corniculatus</Emphasis> L.

Shoots of Lotus corniculatus L., previously transformed with Agrobacterium tumefaciens LBA4404/pTOK233, were grown in gibberellic acid (GA₃)-containing media in an attempt to improve their growth and multiplication. Nodal stem segments of four poorly multiplying clones, a clone with very good multiplication, and two non-transformed clones were incubated in media containing GA₃ for 3 weeks, and then returned to a hormone-free medium for a further 3–5 subculture periods. Gibberellic acid increased the number of axillary buds from one in controls to 2–13 shoots during the first 3 weeks of subculture in GA₃-containing media. The multiplied buds, which apparently derived from additional axillaries, continued to multiply in hormone-free medium, producing bunches of more than 50 shoots after 9 weeks. In addition, many nodal segments also produced callus tissues at the basal end, or along the internode below the node, in which abundant shoot meristems formed de novo. Histological examination confirmed their origin via organogenesis. About 25% of regenerated shoots rooted spontaneously. The application of auxins significantly improved rooting in more than 75% of all clones. Well-developed plantlets were produced upon the transfer of rooted shoots to pots. No differences in responses to hormones were observed between LBA4404/pTOK233-transformed clones and non-transformed clones, indicating that the reaction to GA₃ was not the consequence of transformation events. The results point to a possible involvement of GA₃ in lateral branching. They might also be recommended as a procedure suitable for increasing the production of transformed plants in L. corniculatus.     

Sequence Analysis of the <Emphasis Type="Italic">Lactoferrin</Emphasis> Gene and Variation of <Emphasis Type="Italic">g</Emphasis>.<Emphasis Type="Italic">7605C</Emphasis>→<Emphasis Type="Italic">T</Emphasis> in 10 Chinese Indigenous Goat Breeds

Much attention has been focused on the study of lactoferrin at the protein or nucleotide level in mice, humans, and cattle, but little is known about it in goats. The goat LF gene from 5' UTR to exon 17 was amplified, and the variation of g.7605C→T in 10 Chinese indigenous goat breeds was analyzed. Among the three ruminant species (cattle, sheep, and goats), the intron-exon distribution pattern was similar, and all the exons had the same length, but the length of introns varied greatly due to insertions or deletions. The frequency of allele T at g.7605C→T (50.12%) was a little higher than that of allele C (49.88%), and the genotype distribution differed greatly between goat populations. The g.7605C→T site showed higher genetic diversity in goat populations. The genetic differentiation was 0.0783, and gene flow was 2.9433 among the 10 Chinese indigenous goat populations.     

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.     

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.     

<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     

Cryopreservation of cell suspension cultures of <Emphasis Type="Italic">Taxus</Emphasis> × <Emphasis Type="Italic">media</Emphasis> and <Emphasis Type="Italic">Taxus floridana</Emphasis>

Different lines of cell suspension cultures of Taxus × media Rehd. and Taxus floridana Nutt. were cryopreserved with a two-step freezing method using a simple and inexpensive freezing container instead of a programmable freezer. Four to seven days old suspension cell cultures were precultured in growth medium supplemented with 0.5 M mannitol for 2 d. The medium was then replaced with cryoprotectant solution (1 M sucrose, 0.5 M glycerol and 0.5 M dimethylsulfoxide) and the cells incubated on ice for 1 h. Before being plunged into liquid nitrogen, cells were frozen with a cooling rate of approximately −1 °C per min to −80 °C. The highest post-thaw cell viability was 90 %. The recovery was line dependent. The cryopreservation procedure did not alter the nuclear DNA content of the cell lines. The results indicate that cryopreservation of Taxus cell suspension cultures using inexpensive freezing container is possible.     

Somatic Embryogenesis and in vitro Flowering in <Emphasis Type="Italic">Saposhnikovia divaricata</Emphasis>

Efficient somatic embryogenesis (SE) and in vitro flowering and fruiting were achieved in Saposhnikovia divaricata (Turcz.) Schischk. Friable embryogenic callus developed from the root, internode, and leaf explants on Murashige and Skoog medium (MS) with 2.26 μM 2,4-dichlorophenoxyacetic acid (2,4-D), and subsequently developed into somatic embryos on MS medium containing 4–5% sucrose, 1.74 μM naphthaleneacetic acid (NAA), 4.44 μM 6-benzylaminopurine (BA), and 1.90 μM abscisic acid (ABA). Then the mature embryos were separated and transferred onto MS with 3% sucrose and 0.6% agar for further development and conversion to plantlets. In vitro flowering and fruiting were obtained when the subcultures were carried out for over 15 months. Paclobutrazol (PP333) or ethephon (ETH) at low levels promoted flowering significantly. Also, abnormal rootless somatic embryos of S. divaricata could form flowers and fruits in vitro.