There is potential to accelerate cultivar development with a doubled haploid system for breeding line production. Anther culture methodology was evaluated for U.S.A. spring barley (Hordeum vulgare L.) breeding applications. Gelrite was found to be an acceptable replacement for ficoll in the induction medium to reduce costs while maintaining embryoid and plant production levels. Beneficial effects of 28 d cold pretreatment of donor spikes for anther culture were confirmed with Pacific Northwest USA barley genotypes. A 3 d mannitol solution pretreatment of fresh anthers was shown to be less effective for green plant production compared to 28 d cold pretreatment of donor spikes. Extended donor spike cold pretreatment from 28 to 42 d did not reduce anther culture productivity. Based on this research, anther culture techniques show promise for economical and convenient application in spring barley breeding.Abbreviations DH
doubled haploid
- LS
Linsmaier and Skoog basal medium
- BAP
benzylaminopurine
- GLM
Generalized Linear Model
- SAS
Statistical Analysis System 相似文献
Liquid Chromatography Mass Spectrometry (LC-MS) is a powerful and widely applied method for the study of biological systems, biomarker discovery and pharmacological interventions. LC-MS measurements are, however, significantly complicated by several technical challenges, including: (1) ionisation suppression/enhancement, disturbing the correct quantification of analytes, and (2) the detection of large amounts of separate derivative ions, increasing the complexity of the spectra, but not their information content. Here we introduce an experimental and analytical strategy that leads to robust metabolome profiles in the face of these challenges. Our method is based on rigorous filtering of the measured signals based on a series of sample dilutions. Such data sets have the additional characteristic that they allow a more robust assessment of detection signal quality for each metabolite. Using our method, almost 80% of the recorded signals can be discarded as uninformative, while important information is retained. As a consequence, we obtain a broader understanding of the information content of our analyses and a better assessment of the metabolites detected in the analyzed data sets. We illustrate the applicability of this method using standard mixtures, as well as cell extracts from bacterial samples. It is evident that this method can be applied in many types of LC-MS analyses and more specifically in untargeted metabolomics.
Virus-like particles (VLPs) of the recombinant hepatitis B virus (HBV) core protein (HBc) are routinely used in HBV diagnostics worldwide and are of potential interest as carriers of foreign peptides (e.g., immunological epitopes and targeting addresses, and/or as vessels for packaged diagnostic and therapeutic nanomaterials). Despite numerous reports exploiting different expression systems, a rapid and comprehensive large-scale methodology for purification of HBc VLPs from yeast is still lacking. Here, we present a convenient protocol for highly efficient production and rapid purification of endotoxin-free ayw subtype HBc VLPs from the methylotrophic yeast Pichia pastoris. The HBc gene expression cassette along with the geneticin resistance gene was transferred to the P. pastoris genome via homologous recombination. A producer clone was selected among 2000 transformants for the optimal synthesis of the target protein. Fermentation conditions were established ensuring biomass accumulation of 163 g/L. A simple combination of pH/heat and salt treatment followed by a single anion-exchange chromatography step resulted in a more than 90% pure preparation of HBc VLPs, with a yield of about 3.0 mg per 1 g of wet cells. Purification is performed within a day and may be easily scaled up if necessary. The quality of HBc VLPs was verified by electron microscopy. Mass spectrometry analysis and direct polyacrylamide gel staining revealed phosphorylation of HBc at at least two sites. To our knowledge, this is the first report of HBc phosphorylation in yeast. 相似文献
Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV) and Apple mosaic virus are economically important viruses infecting fruit tree species worldwide. To evaluate the occurrence of these pome fruit viruses in Latvia, a large‐scale survey was carried out in 2007. Collected samples were tested for infection by DAS ELISA and multiplex RT‐PCR. The accuracy of the detection of the viruses in multiplex RT‐PCR was confirmed by sequencing amplified PCR fragments. The results showed a wide occurrence of viruses in apple and pear commercial orchards established from non‐tested planting material. More than 89% of the tested apple trees and more than 60% of pear trees were infected with one or more pome fruit viruses. Analyses showed that the high occurrence of viruses in several apple cultivars is due to the propagation of infected clonal rootstocks and scions from infected mother trees. Sequence analyses targeting the 3′‐terminal region of the tested viruses showed various degrees of genetic diversity within respective virus isolates. This is the first report of the occurrence of ACLSV, ASGV and ASPV in apple and pear trees in Latvia and demonstrates their genetic diversity in different host genotypes. 相似文献
Approaches utilizing microlinearity between related species allow for the identification of syntenous regions and orthologous
genes. Within the barley Chromosome 7H(1) is a region of high recombination flanked by molecular markers cMWG703 and MWG836.
We present the constructed physical contigs linked to molecular markers across this region using bacterial artificial chromosomes
(BAC) from the cultivar Morex. Barley expressed sequence tags (EST), identified by homology to rice chromosome 6 between the
rice molecular markers C425A and S1434, corresponded to the barley syntenous region of Chromosome 7H(1) Bins 2–5 between molecular
markers cMWG703-MWG836. Two hundred and thirteen ESTs were genetically mapped yielding 267 loci of which 101 were within the
target high recombination region while 166 loci mapped elsewhere. The 101 loci were joined by 43 other genetic markers resulting
in a highly saturated genetic map. In order to develop a physical map of the region, ESTs and all other molecular markers
were used to identify Morex BAC clones. Seventy-four BAC contigs were formed containing 2–102 clones each with an average
of 19 and a median of 13 BAC clones per contig. Comparison of the BAC contigs, generated here, with the Barley Physical Mapping
Database contigs, resulted in additional overlaps and a reduction of the contig number to 56. Within cMWG703-MWG836 are 24
agriculturally important traits including the seedling spot blotch resistance locus, Rcs5. Genetic and physical analysis of this region and comparison to rice indicated an inversion distal of the Rcs5 locus. Three BAC clone contigs spanning the Rcs5 locus were identified.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
Bryophytes are the second largest taxonomic group in the plant kingdom; yet, studies conducted to better understand their chemical composition are rare. The aim of this study was to characterize the chemical composition of bryophytes common in Northern Europe by using elemental, spectral, and non‐destructive analytical methods, such as Fourier transform IR spectrometry (FT‐IR), solid‐phase 13C‐NMR spectrometry, and pyrolysis‐gas chromatography/mass spectrometry (Py‐GC/MS), for the purpose of investigating their chemotaxonomic relationships on the basis of chemical‐composition data. The results of all these analyses showed that bryophytes consist mainly of carbohydrates. Judging by FT‐IR spectra, the OH groups in combination of C? O groups were the most abundant groups. The 13C‐NMR spectra provided information on the presence of such compounds as phenolics and lipids. It was found that the amount of phenolic compounds in bryophytes is relatively small. This finding definitely confirmed the absence of lignin in the studied bryophytes. Cluster analysis was used to better understand differences in the chemical composition of bryophyte samples and to evaluate possible usage of these methods in the chemotaxonomy of bryophytes. 相似文献
The virus-neutralising domain III (DIII) of the West Nile virus glycoprotein E was exposed on the surface of RNA phage AP205 virus-like particles (VLPs) in mosaic form. For this purpose, a 111 amino acid sequence of DIII was added via amber or opal termination codons to the C-terminus of the AP205 coat protein, and mosaic AP205-DIII VLPs were generated by cultivation in amber- or opal-suppressing Escherichia coli strains. After extensive purification to 95 % homogeneity, mosaic AP205-DIII VLPs retained up to 11–16 % monomers carrying DIII domains. The DIII domains appeared on the VLP surface because they were fully accessible to anti-DIII antibodies. Immunisation of BALB/c mice with AP205-DIII VLPs resulted in the induction of specific anti-DIII antibodies, of which the level was comparable to that of the anti-AP205 antibodies generated against the VLP carrier. The AP205-DIII-induced anti-DIII response was represented by a significant fraction of IgG2 isotype antibodies, in contrast to parallel immunisation with the DIII oligopeptide, which failed to induce IgG2 isotype antibodies. Formulation of AP-205-DIII VLPs in alum adjuvant stimulated the level of the anti-DIII response, but did not alter the fraction of IgG2 isotype antibodies. Mosaic AP205-DIII VLPs could be regarded as a promising prototype of a putative West Nile vaccine. 相似文献
Leaf-specific thionins of barley (Hordeum vulgare L.) have been identified as a novel class of cell-wall proteins toxic to plant-pathogenic fungi and possibly involved in the defence mechanism of plants. The distribution of these polypeptides has been studied in the host-pathogen system of barley and Erisyphe graminis DC.f.sp. hordei Marchal (powdery mildew). Immunogold-labelling of thionins in several barley cultivars indicates that resistance or susceptibility may be attributed to the presence or absence of thionins at the penetration site in walls and papillae of epidermal leaf cells.All of the leaf-specific thionin genes are confined to the distal end of the short arm of chromosome 6 of barley. None of the genes for cultivarspecific resistance to powdery mildew which have previously been mapped on barley chromosomes are found close to this locus. 相似文献