Transient expression of β-glucuronidase in Arabidopsis thaliana leaves and roots and Brassica napus stems using a pneumatic particle gun

Successful transient expression of -glucuronidase (GUS) in Arabidopsis thaliana leaves and roots and Brassica napus stems was obtained after gene delivery with a pneumatic particle gun driven by compressed air. Effects of the pneumatic pressure used to accelerate the particles (accelerating pressure; 85 to 200 kg/cm²) and of preculture periods of plant tissues (0 to 6 days) on the efficiency of gene delivery were studied. In A. thaliana leaves, best results were obtained at 115 kg/cm² of accelerating pressure and 3 days of preculture. In A. thaliana roots, the optimum was at 200 kg/cm² of accelerating pressure and 3 days of preculture. These results indicate that both preculture period and accelerating pressure are vital factors that determine the efficiency of gene delivery by particle gun.     

Influence of Blue-Light Signaling Components on the Regulation of Cytokinin-Dependent Arabidopsis thaliana Seedlings’ Greening

Russian Journal of Plant Physiology - De-etiolation of plant seedlings is controlled by a complex system of light and hormonal signaling. This process is accompanied by chloroplast development and...     

Characterization of a Brassica napus myrosinase pseudogene: myrosinases are members of the BGA family of β-glycosidases

Myrosinase isoenzymes are known to be encoded by two different families of genes denoted MA and MB. Nucleotide sequence analysis of a Brassica napus genomic clone containing a gene for myrosinase revealed it to be a pseudogene of the MA family. The gene spans more than 5 kb and contains at least 12 exons. The exon sequence of the gene is highly similar to myrosinase cDNA sequences. However, the gene displays three potential or actual pseudogene characters. Southern blot analysis using probes from the 3 portions of the genomic and B. napus MA and MB cDNA clones showed that MA type myrosinases are encoded by approximately 4 genes, while MB type myrosinases are encoded by more than 10 genes in B. napus. Northern blots with mRNA from seeds and young leaves probed with the MA-and MB-specific probes showed that the MA and MB myrosinase gene families are differentially expressed. Myrosinases are highly similar to proteins of a -glycosidase enzyme family comprising both -glycosidases and phospho--glycosidases of as diverged species as archaebacteria, bacteria, mammals and plants. By homology to these -glycosidases, putative active site residues in myrosinase are discussed on the basis of the similarity between -glycosidases and cellulases.     

Effect of “External” Superoxide Anion on Apoptosis in Coleoptiles of Wheat Seedlings

A derivative of phthalic acid, dibutylphthalate (DBP), which has gametocidal effect at the concentration of approximately 10(-4) M, increased apoptosis in coleoptiles of wheat seedlings. This was associated with activation of chromatin margination and generation of mitochondria-containing vesicles. At the same concentration, DBP activated the release by the coleoptiles of superoxide anion into the environment. Lower (10(-5) M) and higher (10(-3) M) concentrations of DBP virtually had no effect on either process. A probable mechanism of effect of the "external" superoxide anion on apoptosis within the plant cell is discussed.     

Impact of a resistance gene against a fungal pathogen on the plant host residue microbiome: The case of the Leptosphaeria maculans–Brassica napus pathosystem

Oilseed rape residues are a crucial determinant of stem canker epidemiology as they support the sexual reproduction of the fungal pathogen Leptosphaeria maculans. The aim of this study was to characterize the impact of a resistance gene against L. maculans infection on residue microbial communities and to identify microorganisms interacting with this pathogen during residue degradation. We used near-isogenic lines to obtain healthy and infected host plants. The microbiome associated with the two types of plant residues was characterized by metabarcoding. A combination of linear discriminant analysis and ecological network analysis was used to compare the microbial communities and to identify microorganisms interacting with L. maculans. Fungal community structure differed between the two lines at harvest, but not subsequently, suggesting that the presence/absence of the resistance gene influences the microbiome at the base of the stem whilst the plant is alive, but that this does not necessarily lead to differential colonization of the residues by fungi. Direct interactions with other members of the community involved many fungal and bacterial amplicon sequence variants (ASVs). L. maculans appeared to play a minor role in networks, whereas one ASV affiliated to Plenodomus biglobosus (synonym Leptosphaeria biglobosa) from the Leptosphaeria species complex may be considered a keystone taxon in the networks at harvest. This approach could be used to identify and promote microorganisms with beneficial effects against residue-borne pathogens and, more broadly, to decipher the complex interactions between multispecies pathosystems and other microbial components in crop residues.     

Sublethal effects of imidacloprid and pymetrozine on population growth parameters of cabbage aphid, Brevicoryne brassicae on rapeseed, Brassica napus L.

Efficiency of imidacloprid and pymetrozine on population growth parameters of cabbage aphid, Brevicoryne brassicae L. （Homoptera： Aphididae） was determined using demographic toxicology by leaf dip method. At first, bioassay tests were performed. The LC50 value and confidence limit for imidacloprid and pymetrozine were 1.61 × 10^-5 mol/L （0.74 × 10^-5-2.66 × 10^-5） and 2.14 × 10^-4 mol/L （1.24 × 10^-4-3.40 × 10^-4）, respectively. To evaluate the sublethal effect of two insecticides on population growth parameters of cabbage aphid, LC30 concentrations of imidacloprid and pymetrozine were used at 5 mol/L and 30 mol/L. The experiments were carried out in a incubator at 20±1℃, 60% ± 5% RH and 16：8 （L：D） photoperiod on canola seedlings, Brassica napus L. var. ＇PF＇. Net fecundity rate decreased in both insecticide-treated populations. Intrinsic rates of increase （rm） were lower in imidacloprid and pymetrozine treatments than in controls. Intrinsic birth rates also decreased in treated populations. There was a relative increase in intrinsic death rates of treated populations. The mean generation times and doubling time were also lower in populations treated with insecticides than in controls. There was a considerable reduction in the average numbers of nymphs reproduced per female as compared with the control. The average longevity of female adults in the control was significantly different from those treated with imidacloprid and pymetrozine. However, there was no significant differences in aphid life-table parameters between the two insecticide-treated populations （P 〉 0.01）.     

Transfer of a Dwarf Gene from Brassica rapa to Oilseed B. napus, Effects on Agronomic Traits, and Development of a ‘Perfect’ Marker for Selection

Lodging can be a serious problem in oilseed Brassica napus. Dwarf genes have been used to reduce lodging in other crops, and their use in wheat and rice was a major factor in the success of the ‘Green Revolution’. We previously reported on a single, semi-dominant, gibberellin insensitive dwarf mutant of B. rapa (Brrga1-d), and provided evidence for homology of this gene to the wheat ‘Green Revolution’ dwarf gene. In this paper, we report using interspecific hybridization of B. rapa and B. oleracea and embryo rescue to resynthesize B. napus containing the Brrga1-d dwarf gene. The dwarf gene was backcrossed into two parents of a commercial hybrid combination and evaluated as inbred and hybrid lines in field experiments. The Brrga1-d gene reduced plant height and lodging in inbred and hybrid lines of B. napus, even when present as a single dose in heterozygous genotypes. Seed yields of inbred lines homozygous for Brrga1-d were reduced compared to near isogenic wild-type inbreds, presumably due to damage by insects caused by a slight delay in flowering time. However, yields of hybrids that were homozygous or heterozygous for Brrga1-d were similar to those of near isogenic wild-type hybrids. In addition, we report on a ‘perfect’ PCR marker for selection of the Brrga1-d gene that is based on detecting the nucleotide mutation causing the dwarf phenotype.     

A Comparison Studies on Ultrastructure and Calcium Distribution in Fertile and Sterile Anthers of a Genic Male-sterile Rapeseed （Brassica napus L.）

It‘s common understanding that plant male sterility is closely related to cell ultrastructure or cell microstructure,plant physiological and biochemical metabolism during the generation and development of anthers. The materials used for the study were fertile and sterile anthers in various stages of a genic male-sterile rapeseed     

Agrobacterium tumefaciens-mediated transformation of rutabaga (Brassica napus var. napobrassica) cultivar “American Purple Top Yellow”

An efficient protocol for genetic transformation of rutabaga (Brassica napus var. napobrassica) cultivar ??American Purple Top Yellow?? was developed by optimizing several factors influencing gene delivery and plant regeneration. A two-step regeneration protocol, adapted from canola, was optimal for rutabaga regeneration using hypocotyl explants. Transient expression studies monitored by histochemical ??-glucuronidase (GUS) assays indicated that several factors, including Agrobacterium tumefaciens strain, cocultivation time, and cocultivation medium, affected gene delivery. For stable transformation, precultured hypocotyl explants were cocultivated with Agrobacterium cells on sterilized filter paper overlaid on callus induction medium containing 100???M acetosyringone for 6?d under a 16-h photoperiod. Selection and regeneration of transformed cells were conducted on media containing 50?mg?l^?1 kanamycin and 250?mg?l^?1 Timentin. Using this protocol, GUS- and PCR-positive transformants were obtained from 3.2 to 4.2?% of hypocotyl explants inoculated with each of the three Agrobacterium strains after 3?C5?mo. Most transformants exhibited a normal phenotype. Southern blot analysis confirmed stable integration of the gusA transgene in T₀ plants.     

“Connecting worlds – a view on microfluidics for a wider application”

From its birth, microfluidics has been referenced as a revolutionary technology and the solution to long standing technological and sociological issues, such as detection of dilute compounds and personalized healthcare. Microfluidics has for example been envisioned as: (1) being capable of miniaturizing industrial production plants, thereby increasing their automation and operational safety at low cost; (2) being able to identify rare diseases by running bioanalytics directly on the patient’s skin; (3) allowing health diagnostics in point-of-care sites through cheap lab-on-a-chip devices. However, the current state of microfluidics, although technologically advanced, has so far failed to reach the originally promised widespread use.In this paper, some of the aspects are identified and discussed that have prevented microfluidics from reaching its full potential, especially in the chemical engineering and biotechnology fields, focusing mainly on the specialization on a single target of most microfluidic devices and offering a perspective on the alternate, multi-use, “plug and play” approach. Increasing the flexibility of microfluidic platforms, by increasing their compatibility with different substrates, reactions and operation conditions, and other microfluidic systems is indeed of surmount importance and current academic and industrial approaches to modular microfluidics are presented. Furthermore, two views on the commercialization of plug-and-play microfluidics systems, leading towards improved acceptance and more widespread use, are introduced. A brief review of the main materials and fabrication strategies used in these fields, is also presented. Finally, a step-wise guide towards the development of microfluidic systems is introduced with special focus on the integration of sensors in microfluidics. The proposed guidelines are then applied for the development of two different example platforms, and to three examples taken from literature.With this work, we aim to provide an interesting perspective on the field of microfluidics when applied to chemical engineering and biotechnology studies, as well as to contribute with potential solutions to some of its current challenges.     

The introduction of CMS mitochondria to triazine tolerant Brassica napus L., var. “Regent”, by micromanipulation of individual heterokaryons

Cytoplasmic triazine tolerance and cytoplasmic male sterility traits were combined in the nuclear genomic background of the Brassica napus variety Regent, following protoplast fusion, selection of fusion products by manual micro-manipulation, and culture in a Nicotiana tabacum nurse system. Whole plant cybrid regenerants were morphologically normal and produced seed on pollination, demonstrating their potential for incorporation into a breeding program.     

Physical mapping and microsynteny of Brassica rapa ssp. pekinensis genome corresponding to a 222 kbp gene-rich region of Arabidopsis chromosome 4 and partially duplicated on chromosome 5

We constructed a bacterial artificial chromosome (BAC) library, designated as KBrH, from high molecular weight genomic DNA of Brassica rapa ssp. pekinensis (Chinese cabbage). This library, which was constructed using HindIII-cleaved genomic DNA, consists of 56,592 clones with average insert size of 115 kbp. Using a partially duplicated DNA sequence of Arabidopsis, represented by 19 and 9 predicted genes on chromosome 4 and 5, respectively, and BAC clones from the KBrH library, we studied conservation and microsynteny corresponding to the Arabidopsis regions in B. rapa ssp. pekinensis. The BAC contigs assembled according to the Arabidopsis homoeologues revealed triplication and rearrangements in the Chinese cabbage. In general, collinearity of genes in the paralogous segments was maintained, but gene contents were highly variable with interstitial losses. We also used representative BAC clones, from the assembled contigs, as probes and hybridized them on mitotic (metaphase) and/or meiotic (leptotene/pachytene/metaphase I) chromosomes of Chinese cabbage using bicolor fluorescence in situ hybridization. The hybridization pattern physically identified the paralogous segments of the Arabidopsis homoeologues on B. rapa ssp. pekinensis chromosomes. The homoeologous segments corresponding to chromosome 4 of Arabidopsis were located on chromosomes 2, 8 and 7, whereas those of chromosome 5 were present on chromosomes 6, 1 and 4 of B. rapa ssp. pekinensis.     

New cases of human dirofilariosis on the Balkan Peninsula – “Masked intruders” uncovered by a surgeon

Dirofilaria spp. nematodes are accidental parasites of humans causing mild to serious, superficial or visceral infections. Superficial dirofilariosis is rather common in Europe and is typically manifested as subcutaneous form. Herein we report 46 new cases of human dirofilariosis (19 patients with subcutaneous, 18 patients with ocular, 4 patients wih genital, 2 patients with submucosal, 2 patients with pulmonary and 1 patient with intramuscular form of infection) that were recorded from the beginning of 2015 to May 2021 on the Balkan Peninsula with a goal to update the prevalence of this parasitosis and point out potential problems in diagnosis and treatment. Besides, given the high possibility of misinterpretation as tumor, our second aim was to encourage the inclusion of this pathogen in the differential diagnosis of subcutaneous nodules. Although quite common forms, subcutaneous and ocular dirofilariosis can be very often misdiagnosed in clinical practice due to the absence of specific clinical manifestations. Therefore, raising awareness of clinicians about this zoonosis is needed as well as closer collaboration between physicians and veterinarians.     

Particulate Matter on Foliage of 13 Woody Species: Deposition on Surfaces and Phytostabilisation in Waxes – a 3-Year Study

Particulate matter (PM) as an air pollutant can be harmful for human health through allergic, mutagenic and carcinogenic effects. Although the main focus is on decreasing air pollution, after PM has been emitted to the atmosphere, one of the realistic options to decrease it's concentrations in urbanized area will be phytoremediation. This study compared the capacity to capture PM from air of seven tree species commonly cultivated in Poland (Catalpa bignonioides Walter, Corylus colurna L., Fraxinus pennsylvanica Marsh., Ginkgo biloba L., Platanus × hispanica Mill. ex Muenchh., Quercus rubra L., Tilia tomentosa Moench ‘Brabant’) and six shrub species (Acer tataricum subsp. ginnala (Maxim.) Wesm., Sambucus nigra L., Sorbaria sorbifolia (L.) A.Br., Spiraea japonica L.f., Syringa meyeri C.K. Schneid. ‘Palibin’, Viburnum lantana L.). Significant differences were found between species in mass of total PM accumulation for two PM categories and three size fractions determined and in amount of waxes. A positive correlation was found between in-wax PM of diameter 2.5–10 μm and amount of waxes, but not between amount of waxes and amount of total PM or of any size fraction.