A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques

Two barley transformation systems, Agrobacterium-mediated and particle bombardment, were compared in terms of transformation efficiency, transgene copy number, expression, inheritance and physical structure of the transgenic loci using fluorescence in situ hybridisation (FISH). The efficiency of Agrobacterium-mediated transformation was double that obtained with particle bombardment. While 100% of the Agrobacterium-derived lines integrated between one and three copies of the transgene, 60% of the transgenic lines derived by particle bombardment integrated more than eight copies of the transgene. In most of the Agrobacterium-derived lines, the integrated T-DNA was stable and inherited as a simple Mendelian trait. Transgene silencing was frequently observed in the T₁ populations of the bombardment-derived lines. The FISH technique was able to reveal additional details of the transgene integration site. For the efficient production of transgenic barley plants, with stable transgene expression and reduced silencing, the Agrobacterium-mediated method appears to offer significant advantages over particle bombardment.     

RFLP mapping of a Hordeum bulbosum gene highly expressed in pistils and its relationship to homoeologous loci in other Gramineae species

A cDNA sequence (Hbc8-2) isolated from pistils of the self-incompatible species Hordeum bulbosum was analysed for expression pattern and genetic map location. Hbc8-2 was expressed just prior to anthesis in mature pistils, and expression was maintained at a high level throughout anthesis. The same expression pattern was found in self-incompatible rye ( Secale cereale), but no expression was detected in the self-compatible cereals wheat ( Triticum aestivum) or barley ( Hordeum vulgare) at comparable stages of development. However, three wheat expressed sequence tags from a pre-anthesis library had high homology to Hbc8-2. Southern blot analyses using Hbc8-2 as a probe detected hybridising bands in the genomes of various Gramineae species including rye, barley, bread wheat, wild wheat relatives ( Aegilops tauschii and Ae. speltoides), oats ( Avena fatua and A. strigosa), rice ( Oryza sativa) and maize ( Zea mays). This suggests that Hbc8-2-like sequences are present in many species but that high levels of expression may be associated with self-incompatibility. Hbc8-2 was mapped on the long arms of chromosome 2H(b) of H. bulbosum, 2R of rye, and 2B and 2D of wheat and was assigned to chromosome 2H of barley using wheat/barley addition lines. On a H. bulbosum genetic map, Xhbc8-2 was located between Xbcd266 and Xpsr87, while in rye and wheat it was located in a 13.2-cM interval between Xpsr331 and Xpsr932, consistent with previous comparative mapping studies of these species. Mapping in rye suggested that Hbc8-2 is probably proximal to the Z self-incompatibility locus which was previously shown to be tightly linked to Xbcd266.     

Transgene behaviour across two generations in a large random population of transgenic rice plants produced by particle bombardment

The relationship between transgene copy number, rearrangement levels, inheritance patterns, expression levels, transgene stability and plant fertility was analysed in a random population of 95 independently transformed rice plant lines. This analysis has been conducted for both the selectable marker gene ( aphIV) and the unselected reporter gene ( gusA), in the presence or absence of flanking Matrix Attachment Regions (MARs) in order to develop a better understanding of transgene behaviour in a population of transgenic rice plants created by particle bombardment. In the first generation (T(0)), all the independently transformed plant lines contained and expressed the aphIV gene conferring resistance to hygromycin, but only 87% of the lines were co-transformed with the unselected gusA marker gene. Both transgenes seemed to be expressed independently. Most lines exhibited complex transgene rearrangements as well as an intact transgene expression unit for both aphIV and gusA transgenes. Transgene copy number was proportional to the quantity of DNA used during bombardment. In T(0) plants, high gusA copy number significantly decreased GUS expression levels but there was no correlation between expression level and transgene copy number across the entire population of lines. Four main factors impaired transgene expression in primary transgenic plants (T(0)) and their progeny (T(1)): (1) absence of transgene expression in T(0) plants (41% of lines), (2) sterility of T(0) plants (28% of lines), (3) non-transmission of intact transgenes to some or all progenies (at least 14% of lines), and (4) silencing of transgene expression in progeny plants (10% of lines). Transgene stability was significantly related to differences in transgene structure and expression levels. The presence of Rb7 MARs flanking the gusA expression unit had no effect on plant fertility or non-transmission of transgenes, but provided copy number-dependent expression of the transgene and improved expression levels and stability over two generations. Overall, only 7% of the plant lines without MARs and 17% of the lines with MARs initially generated, exhibited stable transgene expression over two generations.     

Simultaneous fluorescent gram staining and activity assessment of activated sludge bacteria

Wastewater treatment is one of the most important commercial biotechnological processes, and yet the component bacterial populations and their associated metabolic activities are poorly understood. The novel fluorescent dye hexidium iodide allows assessment of Gram status by differential absorption through bacterial cell walls. Differentiation between gram-positive and gram-negative wastewater bacteria was achieved after flow cytometric analysis. This study shows that the relative proportions of gram-positive and gram-negative bacterial cells identified by traditional microscopy and hexidium iodide staining were not significantly different. Dual staining of cells for Gram status and activity proved effective in analyzing mixtures of cultured bacteria and wastewater populations. Levels of highly active organisms at two wastewater treatment plants, both gram positive and gram negative, ranged from 1.5% in activated sludge flocs to 16% in the activated sludge fluid. Gram-positive organisms comprised <5% of the total bacterial numbers but accounted for 19 and 55% of the highly active organisms within flocs at the two plants. Assessment of Gram status and activity within activated sludge samples over a 4-day period showed significant differences over time. This method provides a rapid, quantitative measure of Gram status linked with in situ activity within wastewater systems.     

Transgene behaviour in populations of rice plants transformed using a new dual binary vector system: pGreen/pSoup

Transgene integration, expression level and stability have been studied, across two generations, in a population of rice plants transformed using a new dual binary vector system: pGreen/pSoup. pGreen is a small Ti binary vector unable to replicate in Agrobacterium without the presence of another binary plasmid, pSoup, in the same strain. We engineered both pGreen and pSoup to contain each a different T-DNA. Transformation experiments were conducted using a pGreen vector containing the bar and gusA expression units (no transgene in pSoup) or with a pSoup vector containing an aphIV and gfp expression units (no transgene in pGreen). High plant transformation frequencies (up to 40%) were obtained using herbicide resistance ( bar) or antibiotic resistance ( aphIV) genes. Around 80% of the independently transformed plants expressed unselected reporter genes ( gusA or gfp) present in the vectors. Backbone sequences transfer was frequent (45% of lines) and occurred often in multicopy lines. Around 15-20% of the rice plant lines contained a single T-DNA integration without backbone. Integration of additional transgene copies did not improve expression levels in either T(0) plants or T(1) progenies. Nearly all multicopy lines contained transgenes integrated at several loci in the plant genome, showing that T-DNAs from either pGreen or pSoup frequently integrated at unlinked loci. Precise determination of loci number required the analysis of transgene presence in progeny. Segregation of transgene phenotype was generally misleading and tended to underestimate the real number of transgenic loci. The contribution of this new dual-binary vector system to the development of high-throughput rice transformation systems and to the production of marker-free transgenic rice plants is discussed.     

An update of the Courtot × Chinese Spring intervarietal molecular marker linkage map for the QTL detection of agronomic traits in wheat

We made an update of the intervarietal molecular marker linkage map of the wheat genome developed using a doubled-haploid (DH) population derived from the cross between the cultivars "Courtot" and "Chinese Spring". This map was constructed using 187 DH lines and 659 markers. The genome was well covered (more than 95%) except for chromosomes from homoeologous group 4 and chromosomes 5D and 7D, which had gaps slightly larger than 50 cM. A core-map based on a set of 200 anchor loci (one marker each 18.4 cM) was developed. The total length of this map was 3,685 cM which is similar to the size of the international reference map of the ITMI population (3,551 cM). Map coverage was identical for the three genomes (A, B and D) and for the number of anchor loci, as well as for the size of the map. Using this map, QTLs for several agronomic traits were detected on phenotypic data from the population grown in Clermont-Ferrand (France) under natural field conditions over 6 years, and in Norwich (UK) in controlled conditions and under natural field conditions in 1 year. Almost all of the 21 chromosomes were involved in at least one trait. However, several regions seemed to contain gene clusters either for grain traits (and thus bread-making quality) or plant development traits.     

The eukaryotic translation elongation factor 1A regulation of actin stress fibers is important for infectious RSV production

Cellular protein eukaryotic translation elongation factor 1A (eEF1A) is an actin binding protein that plays a role in the formation of filamentous actin (F-actin) bundles. F-Actin regulates multiple stages of respiratory syncytial virus (RSV) replication including assembly and budding. Our previous study demonstrated that eEF1A knock-down significantly reduced RSV replication. Here we investigated if the eEF1A function in actin bundle formation was important for RSV replication and release. To investigate this, eEF1A function was impaired in HEp-2 cells by either knock-down of eEF1A with siRNA, or treatment with an eEF1A inhibitor, didemnin B (Did B). Cell staining and confocal microscopy analysis showed that both eEF1A knock-down and treatment with Did B resulted in disruption of cellular stress fiber formation and elevated accumulation of F-actin near the plasma membrane. When treated cells were then infected with RSV, there was also reduced formation of virus-induced cellular filopodia. Did B treatment, similarly to eEF1A knock-down, reduced the release of infectious RSV, but unlike eEF1A knock-down, did not significantly affect RSV genome replication. The lower infectious virus production in Did B treated cells also reduced RSV-induced cell death. In conclusion, the cellular factor eEF1A plays an important role in the regulation of F-actin stress fiber formation required for RSV assembly and release.     

Identifying wheat genomic regions for improving grain protein concentration independently of grain yield using multiple inter-related populations

Grain yield (GY) and grain protein concentration (GPC) are two major traits contributing to the economic value of the wheat crop. These are, consequently, major targets in wheat breeding programs, but their simultaneous improvement is hampered by the negative correlation between GPC and GY. Identifying the genetic determinants of GPC and GY through quantitative trait loci (QTL) analysis would be one way to identify chromosomal regions, allowing improvement of GPC without reducing GY using marker-assisted selection. Therefore, QTL detection was carried out for GY and GPC using three inter-connected doubled haploid populations grown in a large multi-environment trial network. Chromosomes 2A, 2D, 3B, 7B and 7D showed co-location of QTL for GPC and GY with antagonistic effects, thus contributing to the negative GPC–GY relationship. Nonetheless, genomic regions determining GPC independently of GY across experiments were found on chromosomes 3A and 5D and could help breeders to move the GPC–GY relationship in a desirable direction.     

Persistence of bactericidal antibodies following early infant vaccination with a serogroup B meningococcal vaccine and immunogenicity of a preschool booster dose

Background:

The multicomponent serogroup B meningococcal (4CMenB) vaccine was recently licensed for use in Europe. There are currently no data on the persistence of bactericidal antibodies induced by use of this vaccine in infants. Our objective was to evaluate serogroup B–specific bactericidal antibodies in children aged 40–44 months previously vaccinated at 2, 4, 6 and 12 months of age.

Methods:

Participants given 4 doses of 4CMenB as infants received a fifth dose of the vaccine at 40–44 months of age. Age-matched participants who were MenB vaccine–naive received 4CMenB and formed the control group. We evaluated human complement serum bactericidal activity (hSBA) titres at baseline and 1 month after each dose of 4CMenB.

Results:

Before a booster dose at enrolment, 41%–76% of 17 participants previously vaccinated with 4CMenB in infancy had hSBA titres of 4 or greater against 4 reference strains. Before vaccination in the control group (n = 40) these proportions were similar for strains 44/76-SL (63%) and {"type":"entrez-nucleotide","attrs":{"text":"M10713","term_id":"209124","term_text":"M10713"}}M10713 (68%) but low for strains NZ98/254 (0%) and 5/99 (3%). A booster dose in the 4CMenB-primed participants generated greater increases in hSBA titres than in controls.

Interpretation:

As has been observed with other meningococcal vaccines, bactericidal antibodies waned after vaccination with 4CMenB administered according to an approved infant vaccination schedule of 2, 4, 6 and 12 months of age, but there was an anamnestic response to a booster dose at 40–44 months of age. If 4CMenB were introduced into routine vaccination schedules, assessment of the need for a booster dose would require data on the impact of these declining titres on vaccine effectiveness. ClinicalTrials.gov, no. {"type":"clinical-trial","attrs":{"text":"NCT01027351","term_id":"NCT01027351"}}NCT01027351A vaccine against serogroup B meningococcus has recently been licensed for use in Europe¹ and is being considered for licensure in Canada. This vaccine, known as multicomponent serogroup B meningococcal (4CMenB) vaccine, consists of 3 recombinant proteins: factor H binding protein (fHbp), Neisseria adhesin A (NadA) and Neisseria heparin binding antigen (NHBA) combined with detoxified outer membrane vesicles from the strain responsible for an epidemic of serogroup B meningococcal disease in New Zealand (NZ98/254). Clinical trials of 4CMenB have shown it to be immunogenic against reference strains selected to speciScally express one of the vaccine antigens.^2–6 On the basis of these trials, the approved schedule for infants aged 2 to 5 months is 3 doses given at least 1 month apart, with a booster dose given at 12 to 23 months of age.⁷ The persistence of vaccine-induced antibodies throughout childhood following this booster dose is unknown, but it is particularly relevant because the incidence of invasive serogroup B meningococcal disease in children aged 1 to 4 years is second only to the incidence in children less than 1 year of age.⁸In this study, we assessed the persistence of these bactericidal antibodies in children aged 40–44 months who had previously received either 4CMenB or a vaccine containing the recombinant proteins alone (recombinant protein serogroup B meningococcal [rMenB] vaccine) at 2, 4, 6 and 12 months of age.³ We also assessed the immunogenicity and reactogenicity of a booster dose.