Micropropagation of mature <Emphasis Type="Italic">Cornus mas</Emphasis> ‘Macrocarpa’

Sprouting axillary buds sampled from a mature 27-year-old shrub of Cornus mas ‘Macrocarpa’ were used as starting material for in vitro culture establishment. Multiple shoot cultures, grown on basal woody plant medium with the pH adjusted to 5.6–5.7 and supplemented with 6-benzylaminopurine in combination with 1-naphthaleneacetic acid, were capable of continuous axillary and adventitious shoot proliferation up to 1 year. Later on, growth ceased, shoot tip necrosis appeared and shoot cultures died. Transfer of living shoots onto modified woody plant medium with the pH adjusted to 6.8–7.0 led to vigorous growth of multiple shoot cultures without any loss of multiplication rates or decreased vitality for several years. The use of 6-benzylaminopurine in combination with 1-naphthaleneacetic acid proved superior to the application of thidiazuron which induced a frequent formation of short and fasciated shoots. 1-naphthaleneacetic acid promoted in vitro adventitious rooting frequency up to 73.3%, whereas indole-3-butyric acid was not effective. Ex vitro acclimatized plants did not show any visually detectable morphological variation.     

Pre-anthesis CPPU Treatment Modifies Quality and Susceptibility to Post-harvest Berry Cracking of <Emphasis Type="Italic">Vitis vinifera</Emphasis> cv. ‘Thompson Seedless’

Plant growth regulators (PGRs), especially gibberellic acid (GA), are often used in table grape (Vitis vinifera) production to increase berry size. However, overuse of this PGR (for example, more than 200 g·ha^?1·season^?1 of GA) may result in problems with berry condition at the post-harvest stage. A synthetic cytokinin, florchlorfenuron (CPPU), could be considered an alternative to GA. Thus, our objective was to study the effects of pre-anthesis CPPU treatment on berry quality and susceptibility to post-harvest cracking of table grapes. CPPU applied to ‘Thompson Seedless’ inflorescences 48 or 36 days prior to anthesis increased ovary size, number of cells in the ovary pericarp at bloom and berry size at harvest. It also increased the amount of soluble solids and decreased titratable acidity of the fruit, thus allowing an earlier harvest. Finally, if GA was applied during berry growth, CPPU at pre-anthesis improved berry potential tolerance to post-harvest cracking and spoilage. This work demonstrates that application of CPPU at the pre-anthesis stage modifies grape development, improving berry size and potential post-harvest fruit performance facilitating also an early harvest.     

Transgenic plants from shoot apical meristems of <Emphasis Type="Italic">Vitis vinifera</Emphasis> L. “Thompson Seedless” via <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation

Shoot apical meristem explants of Vitis vinifera “Thompson Seedless” were used for Agrobacterium-mediated genetic transformation. It was determined that the meristems had to be subjected to a dark growth phase then wounded to obtain transgenic plants. Morphological and histological studies illustrated the role of wounding to expose apical meristem cells for transformation. A bifunctional egfp/nptII fusion gene was used to select kanamycin resistant plants that expressed green fluorescent protein (GFP). Kanamycin at a concentration of 16 mg L⁻¹ in selection medium resulted in recovery of non-chimeric transgenic plants that uniformly expressed GFP, whereas 8 mg L⁻¹ kanamycin allowed non-transgenic and/or chimeric plants to develop. Polymerase chain reaction (PCR) and Southern blot analyses confirmed the presence of transgenes and their stable integration into the genome of regenerated plants. Up to 1% of shoot tips produced stable transgenic cultures within 6 weeks of treatment, resulting in a total of 18 independent lines.     

Establishment of embryogenic cultures and plant regeneration in the Portuguese cultivar ‘Touriga Nacional’ of <Emphasis Type="Italic">Vitis vinifera</Emphasis> L.

‘Touriga Nacional’ is the most important Portuguese grapevine cultivar used for Port wine, table wine and varietal wine production. In order to obtain a reproducible plant regeneration system that allows the application of biotechnological tools to grapevine breeding, embryogenic cultures were induced from immature flowers of three Touriga Nacional selected clones. Gynoecia and anthers were cultured on Nitsch and Nitsch (Science 163:85–87, 1969) basal medium supplemented with four combinations of the growth regulators 6-benzylaminopurine (BAP), 2,4-dichlorophenoxyacetic acid (2,4-D) and indole-3-acetyl-l-aspartic acid (IASP), at 28°C, in the dark. Primary callus, observed on anthers and gynoecia in all media, produced embryogenic callus when cultured on differentiation medium, at 24°C under light. The efficiency on induction of embryogenic callus ranged from 1.2 ± 4.7% to 7.9 ± 13.8% in anthers, and from 17.9 ± 24.9% to 25.3 ± 22.9% in gynoecia. Seven lines of embryogenic cultures were established from the three clones. Multiplication of embryogenic calluses was successfully obtained in maintenance medium, at 26°C, in the dark. These embryogenic calluses produced somatic embryos when subcultured on differentiation medium, under a 16 h photoperiod. Somatic embryos were isolated and cultured on germination medium to achieve conversion which ranged from 35.3 ± 48.5% to 72.7 ± 45.6%. The plantlets obtained were cultured in medium without growth regulators. Secondary embryogenesis was also frequently observed in the hypocotyl-root transition region of somatic embryos. Although some morphological variation occurred between somatic embryos, the regenerated plantlets had a normal phenotype. Maintenance of embryogenic cultures has been achieved since 2002.     

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.     

Changes in contents and antioxidant activity of phenolic compounds during gibberellin-induced development in <Emphasis Type="Italic">Vitis vinifera</Emphasis> L. ‘Muscat’

Gibberellic acid (GA₃) is a plant growth regulator commonly used for increasing the productivity of seedless fruit. However, little is known about the effect of GA₃ on phenolic compounds in grapevine. In the present study, the effect of GA₃ application on contents and antioxidant activities of phenolic compounds in different tissues of Muscat grapevine (Vitis vinifera L. cv. Muscat) was investigated. An application of 100 mg/L GA₃ could successfully induce seedless grape berry, enhance berry size and accelerate the development of berries, resulting in earlier ripening of seedless berry. The contents of total phenolics and total flavonoids, and antioxidant activities in leaf, stem and tendril obviously increased, while these remarkably decreased in berry skin and flesh after GA₃ application. In addition, the grapevine leaf, stem, tendril and skin extracts were shown to contain high amounts of phenolics and significant antioxidant activities. Thus, these findings indicate that GA₃ application causes different effect on phenolic compounds and antioxidant activities, depending on grapevine tissues.     

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.     

Proteins responding to drought and high-temperature stress in <Emphasis Type="Italic">Populus </Emphasis>× <Emphasis Type="Italic">euramericana</Emphasis> cv. ‘74/76’

Proteomic analysis provides a powerful method of studying plant responses to stress at the protein level. In order to study stress-responsive molecular mechanisms for Populus × euramericana cv. ‘74/76’, one of the most important forest plantation tree species in subtropical and temperate regions, we analyzed the response of 2-year-old cuttings of P. × euramericana cv. ‘74/76’ to drought and high temperature using two-dimensional gel electrophoresis. More than 1,000 reproducible leaf proteins were detected in the controls and treatments, and 26 proteins were found to change notably in abundance. We identified 13 proteins affected by drought stress and 11 proteins affected by high temperature. These proteins are mainly involved in photosynthesis such as ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and putative photosystem I reaction center subunit II precursor, and detoxification (manganese superoxide dismutase and methionine sulfoxide reductase A). Furthermore, the level of the photosynthesis proteins affected greatly by the imposed stress conditions was consistent with the observed noticeable decrease in net photosynthesis rate. These studies provides a fundamental data for future research on responses to drought and high temperature, two major factors limiting the growth of forest trees during summer under recent climatic warming.     

<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.     

Distinct <Emphasis Type="Italic">rpsC</Emphasis> single nucleotide polymorphism lineages of Flavescence Dorée subgroup 16SrV-D phytoplasma co-infect <Emphasis Type="Italic">Vitis vinifera</Emphasis> L.

During a survey on grapevine yellows disease complex in vineyards of Lombardy region (northern Italy), phytoplasmas associated with Flavescence dorée disease were identified in symptomatic grapevines. Polymerase chain reaction and restriction fragment length polymorphism (RFLP) analyses of 16S rDNA revealed the prevalence of phytoplasmal subgroup 16SrV-D. Bioinformatic analyses of nucleotide sequences of rplV and rpsC genes, amplified from 16SrV-D phytoplasma infected grapevines and cloned, underscored the presence of five confirmed rpsC single nucleotide polymorphism (SNP) lineages, determined by different combination of SNPs at nucleotide positions 29, 365, 680, and 720 of rpsC gene. Virtual and actual RFLP analyses with the enzyme TaqI validated the presence of these SNPs. Co-infections by up to four distinct rpsC SNP lineages of 16SrV-D phytoplasma were found in grapevines. These results could open new perspectives for the study of the ecology and the epidemiology of Flavescence dorée.     

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.     

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.     

The effects of phenolic acids on the photosynthetic characteristics and growth of <Emphasis Type="Italic">Populus</Emphasis> × <Emphasis Type="Italic">euramericana</Emphasis> cv. ‘Neva’ seedlings

Populus × euramericana cv. ‘Neva’ is an important tree species in northern China. In the study, we used its potted oneyear- old seedlings as experimental material and established three treatments (CK, 0.5X, and 1.0X) according to the concentrations of phenolic acids in order to examine the effects of different concentrations on the photosynthetic characteristics and growth of poplar. With increasing concentrations of phenolic acids, the net photosynthetic rate, stomatal limitation, transpiration rate, apparent quantum yield, photochemical quenching coefficient, electron transport rate, chlorophyll content, and total biomass decreased significantly. The intercellular CO₂ concentration, light-compensation point, nonphotochemical quenching, malondialdehyde content, and root/shoot ratio increased significantly. Peroxidase and superoxide dismutase activities initially decreased and then increased. We concluded that phenolic acids significantly inhibited poplar’s photosynthesis and the higher phenolic acid concentration, the greater inhibition of photosynthesis occurred. This inhibition effect was mainly caused by nonstomatal factors. Phenolic acids induced noticeable photoinhibition, resulted in the irreversible damage of membrane structure, and then changed intracellular metabolic processes. To cope with phenolic acid stress, poplar seedlings increased dissipation of excess light energy and distributed relatively more biomass to underground parts within carbon allocation.     

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.     

Micropropagation and β-ecdysone content of the Brazilian ginsengs <Emphasis Type="Italic">Pfaffia glomerata</Emphasis> and <Emphasis Type="Italic">Pfaffia tuberosa</Emphasis>

The aim of this study was to investigate both a mass in vitro propagation system and the β-ecdysone content in roots and aerial parts of Pfaffia glomerata and Pfaffia tuberosa. Nodal segments of two genotypes (BRA and JB-UFSM) of P. glomerata, originated from aseptically grown plants, were cultivated on hormone-free Murashige and Skoog medium. For the proliferation of P. tuberosa shoots, nodal segments, originated from aseptically grown plants, were either cultivated on hormone-free Murashige and Skoog (MS) medium or were supplemented with 1.0 μM thidiazuron (TDZ); the elongation and rooting of these plants were carried out on MS medium without TDZ. Plantlets of both species were acclimatized and transferred to field conditions. The β-ecdysone content in the plants was determined by high performance liquid chromatography. The BRA genotype showed a greater in vitro proliferation rate and β-ecdysone content than that of the JB-UFSM genotype. The culture of nodal segments of P. tuberosa on medium with 1.0 μM TDZ with subsequent subcultivation of shoots on hormone-free medium was shown to be a suitable method for micropropagation due to the high multiplication rate and good plant development. Both species showed good adaptation to ex vitro conditions. The β-ecdysone content in micropropagated P. tuberosa was similar to that found in field-grown plants. For both species, the aerial parts accumulated higher β-ecdysone content than roots. These results reveal that micropropagation is a successful, alternative method for rapid plant multiplication of both species of Brazilian ginseng. Furthermore, this study demonstrates that these two species have a potential for cultivation that is associated with high β-ecdysone production.     

Somatic hybridization between the diploids of <Emphasis Type="Italic">S.</Emphasis> × <Emphasis Type="Italic">michoacanum</Emphasis> and <Emphasis Type="Italic">S. tuberosum</Emphasis>

Interspecific somatic hybrids between a diploid potato clone DG 81-68 susceptible to Phytophthora infestans (Mont.) de Bary and a resistant diploid tuber-bearing species Solanum × michoacanum were generated and analyzed. About 30 regenerants displaying an intermediate morphology were obtained as a result of three separate PEG-mediated fusion experiments. The RAPD analysis confirmed the hybridity of all the regenerants. About 50% of the hybrid plants exhibited vigorous growth and were stable in culture, while the rest of them rooted poorly and grew slowly in vitro. Most of the hybrid clones were at the tetraploid level (70%), while 30% of the clones examined were at the hexaploid level. The S. × michoacanum (+) DG 81-68 hybrids with growth anomalies were aneuploid. The variation in late blight resistance of the hybrid clones was found in detached leaflet tests, with enhanced resistance characteristic for three tetraploid hybrids.     

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.     

Association of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma asteris’ with yellowing and phyllody of <Emphasis Type="Italic">Plantago lanceolata</Emphasis>

Long plantains (Plantago lanceolata L.) with symptoms resembling those associated with phytoplasma infection were observed repeatedly during the period 2000–2008 in southern Bohemia (Czech Republic). The symptoms of the plants were leaf yellowing, stunted growth, flower phyllody and lack of seed production. Transmission electron microscopy showed phytoplasmas in the sieve cells of affected plants but not in healthy ones. Association of phytoplasmas with the disease was confirmed by polymerase chain reaction using phytoplasma-specific universal ribosomal primers R16F2n/R16R2. An amplification product of the expected size (1.2 kb) was observed in all samples of the symptomatic long plantains. The restriction profiles obtained from digestion of the PCR products with three endonucleases (AluI, HhaI, MseI) showed that the phytoplasmas infecting long plantains in the Czech Republic were indistinguishable from those belonging to the aster yellows group (subgroup 16SrI-B). Sequence analysis of 1748 bp of the ribosomal operon indicated that the closest related phytoplasma was that associated with ‘Rehmannia glutinosa var. purpurea’, originating also in Bohemia. This is the first report of the natural occurrence of ‘Candidatus Phytoplasma asteris’ in plants of P. lanceolata.