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Kaspar Kirstein Nielsen - One of the best experts on this subject based on the ideXlab platform.
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comparative analysis of transgenic tall fescue festuca arundinacea schreb plants obtained by agrobacterium mediated transformation and particle bombardment
Plant Cell Reports, 2008Co-Authors: Caixia Gao, Danfeng Long, Ingo Lenk, Kaspar Kirstein NielsenAbstract:Agrobacterium-mediated transformation and particle bombardment are the two most widely used methods for genetically modifying grasses. Here, these two systems are compared for transformation efficiency, transgene integration and transgene expression when used to transform tall fescue (Festuca arundinacea Schreb.). The bar gene was used as a selectable marker and selection during tissue culture was performed using 2 mg/l Bialaphos in both callus induction and regeneration media. Average transformation efficiency across the four callus lines used in the experiments was 10.5% for Agrobacterium-mediated transformation and 11.5% for particle bombardment. Similar transgene integration patterns and co-integration frequencies of bar and uidA were observed in both gene transfer systems. However, while GUS activity was detected in leaves of 53% of the Agrobacterium transformed lines, only 20% of the bombarded lines showed GUS activity. Thus, Agrobacterium-mediated transformation appears to be the preferred method for producing transgenic tall fescue plants.
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a rapid and efficient transformation protocol for the grass brachypodium distachyon
Plant Cell Reports, 2005Co-Authors: Pernille Christiansen, Claus H Andersen, Thomas Didion, Marianne Folling, Kaspar Kirstein NielsenAbstract:A fast and efficient microprojectile bombardment-mediated transformation protocol is reported for the grass species Brachypodium distachyon, a proposed alternative model plant to Oryza sativa for functional genomics in grasses. Embryogenic calli derived from immature embryos were transformed by a construct containing the uidA (coding for β-glucuronidase) and bar (coding for phosphinothricin acetyl transferase) genes, and Bialaphos, a non-selective herbicide, was used as the selection agent throughout all phases of the tissue culture. Average transformation efficiencies of 5.3% were achieved, and for single bombardments transformation efficiencies of up to 14% were observed. The time frame from the bombardment of embryogenic callus to the harvesting of transgenic T1 seeds was 29 weeks and 25 weeks for the diploid and two tetraploid accessions used, respectively. Since the seed-to-seed life cycle is 19 weeks for the diploid and 15 weeks for the tetraploid accessions, our B. distachyon transformation system allows testing of both the T0 and the T1 generation as well as production of T2 seeds within 1 year.
Caixia Gao - One of the best experts on this subject based on the ideXlab platform.
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comparison of three selectable marker genes for transformation of tall fescue festuca arundinacea schreb plants by particle bombardment
In Vitro Cellular & Developmental Biology – Plant, 2011Co-Authors: Danfeng Long, Klaus K Nielsen, Zhimin Yang, Ingo Lenk, Caixia GaoAbstract:A variety of selection systems have been developed for transformation of forage crops. To compare the most frequently used systems, we tested three selectable marker genes for their selection efficiency under four selection procedures for the production of transgenic tall fescue. Embryogenic calluses initiated from mature embryos were bombarded with three constructs containing either the phosphinothricin acetyltransferase (bar) gene, the hygromycin phosphotransferase (hpt) gene or the neomycin phosphotransferase II (nptII) gene. Transformation efficiency was strongly influenced by the selectable marker gene, selection procedure and genotype. The highest transformation efficiency was observed using the bar gene in combination with Bialaphos. Average transformation efficiencies with Bialaphos, phosphinothricin (glufosinate), hygromycin and paromomycin selection across the two callus lines used in the experiments were 9.4%, 4.4%, 5.2% and 1.6%, respectively. Southern blot analysis revealed the independent nature of the tested transgenic plants and a complex transgene integration pattern with multiple insertions.
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comparative analysis of transgenic tall fescue festuca arundinacea schreb plants obtained by agrobacterium mediated transformation and particle bombardment
Plant Cell Reports, 2008Co-Authors: Caixia Gao, Danfeng Long, Ingo Lenk, Kaspar Kirstein NielsenAbstract:Agrobacterium-mediated transformation and particle bombardment are the two most widely used methods for genetically modifying grasses. Here, these two systems are compared for transformation efficiency, transgene integration and transgene expression when used to transform tall fescue (Festuca arundinacea Schreb.). The bar gene was used as a selectable marker and selection during tissue culture was performed using 2 mg/l Bialaphos in both callus induction and regeneration media. Average transformation efficiency across the four callus lines used in the experiments was 10.5% for Agrobacterium-mediated transformation and 11.5% for particle bombardment. Similar transgene integration patterns and co-integration frequencies of bar and uidA were observed in both gene transfer systems. However, while GUS activity was detected in leaves of 53% of the Agrobacterium transformed lines, only 20% of the bombarded lines showed GUS activity. Thus, Agrobacterium-mediated transformation appears to be the preferred method for producing transgenic tall fescue plants.
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generation of large numbers of transgenic kentucky bluegrass poa pratensis l plants following biolistic gene transfer
Plant Cell Reports, 2006Co-Authors: Caixia Gao, Marianne Folling, Li Jiang, Liebao Han, Klaus K NielsenAbstract:A very efficient transformation system, using biolistic bombardment, has been developed for the production of transgenic plants of Kentucky bluegrass (Poa pratensis L.). Embryogenic calli, initiated from immature embryos, were transformed either with pAct1IHPT-4 containing the hygromycin phosphotransferase (hpt) gene or with pDM803 containing the phosphinothricin acetyltransferase (bar) gene and the β-glucuronidase (uidA) gene. In total 119 independent transgenic plants were recovered from 153 hygromycin-resistant lines. Bialaphos selection yielded a total of 99 Bialaphos-resistant lines and from these 34 independent transgenic plants were recovered. Southern blot analysis demonstrated the independent nature of the transgenic plants and also revealed a complex transgene integration pattern with multiple insertions.
Haruo Seto - One of the best experts on this subject based on the ideXlab platform.
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studies on the biosynthesis of Bialaphos biochemical mechanism of c p bond formation discovery of phosphonopyruvate decarboxylase which catalyzes the formation of phosphonoacetaldehyde from phosphonopyruvate
The Journal of Antibiotics, 1997Co-Authors: Hideo Nakashita, Tomomi Hidaka, Osamu Hara, Kei Watanabe, Haruo SetoAbstract:The biosynthetic step following the phosphoenolpyruvate (PEP) phosphomutase reaction which forms a C-P bond of Bialaphos was proven by the identification of phosphonopyruvate (PnPy) and phosphonoacetaldehyde (PnAA) as intermediates in the culture broth of Streptomyces hygroscopicus, a producing organism of Bialaphos, and by detection of enzymatic decarboxylation of PnPy to PnAA. Purified PnPy decarboxylase turned out to require thiamine diphosphate and Mg2+ as cofactors. PnPy decarboxylase drives the unfavorable forward reaction to form PnPy catalyzed by PEP phosphomutase and is suggested to be essential to C-P compound biosynthesis.
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Sequence of a P-methyltransferase-encoding gene isolated from a Bialaphos-producing Streptomyces hygroscopicus.
Gene, 1995Co-Authors: Tomomi Hidaka, Makoto Hidaka, Tomohisa Kuzuyama, Haruo SetoAbstract:The nucleotide sequence of the Streptomyces hygroscopicus gene encoding P-methyltransferase, catalyzing the formation of a carbon-phosphorus bond, involved in Bialaphos biosynthesis, has been determined. The amino-acid sequence deduced from the nt sequence, shows homology with those of magnesium-protoporphyrin IX monomethyl ester oxidative cyclase (Mg-ProtoMe cyclase) of Rhodobacter capsulatus and the enzyme catalyzing the methylation of the aldehyde carbon of phosphonoacetaldehyde in fosfomycin biosynthesis.
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The carboxyphosphonoenolpyruvate synthase-encoding gene from the Bialaphos-producing organism Streptomyces hygroscopicus.
Gene, 1995Co-Authors: Sang-hyeob Lee, Tornomi Hidaka, Hideo Nakashita, Haruo SetoAbstract:The nucleotide (nt) sequence of the Streptomyces hygroscopicus gene encoding carboxyphosphonoenolpyruvate (CPEP) synthase, that catalyzes a transesterification between phosphoenolpyruvate (PEP) and phosphonoformate (PF) in the Bialaphos biosynthetic pathway, has been determined. The amino-acid sequence deduced from the nt sequence is similar to several eukaryotic 2-phospho-d-glycerate hydrolases (EC 4.2.1.11).
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effects of Bialaphos on sterility glutamine synthetase and free amino acids of rice
Phytochemistry, 1993Co-Authors: Atsumi Shimada, Haruo Seto, Takeo Nagai, Yasuo KimuraAbstract:Abstract Bialaphos, l -2-amino-4-[(hydroxy) (methyl) phosphinoyl]butyryl- l -alanyl- l -alanine, induced a high level of sterility in rice, when applied
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Nucleotide sequence of a carboxyphosphonoenolpyruvate phosphonomutase gene isolated from a Bialaphos-producing organism, Streptomyces hygroscopicus, and its expression in Streptomyces lividans
Molecular and General Genetics MGG, 1992Co-Authors: Tomomi Hidaka, Makoto Hidaka, Takeshi Uozumi, Haruo SetoAbstract:The carboxyphosphonoenolpyruvate (CPEP) phosphonomutase gene of Bialaphos-producing Streptomyces hygroscopicus , which encodes a C-P bond forming enzyme was cloned into Streptomyces lividans and sequenced. The amino acid composition of the protein coded in an open reading frame of 295 codons and its calculated molecular mass, 32,800 Da, coincided well with those of the purified enzyme. Introduction of the CPEP phosphonomutase gene, the expression of which is controlled by the promoter of the aph gene, into S. lividans resulted in the production of this enzyme at a level almost equivalent to that in the parent strain.
Masahiro Mii - One of the best experts on this subject based on the ideXlab platform.
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Bialaphos resistant transgenic soybeans produced by the agrobacterium mediated cotyledonary node method
Journal of Agricultural Science and Technology, 2014Co-Authors: S C Liu, Masahiro Mii, G C Zhang, L F Yang, J Y Gai, Y L ZhuAbstract:A stable Agrobacterium -mediated transformation system was established using Bialaphos as the selective agent in soybeans [ Glycine max (L.) Merr.]. The cotyledonary node explants of the soybean cultivar ‘NY-1001’ were inoculated with the Agrobacterium tumefaciens strain EHA105, harboring the vector pCAMBIA3301 containing the gus gene as the reporter gene and the bar gene conferring Bialaphos resistance. The highest frequency of GUS transient expression (92%) was obtained after inoculation and 4-day co-cultivation with A. tumefaciens strain EHA105. Efficient GUS expression was observed in regenerated shoots from explants after 4-day co-cultivation combined with culturing on shoot induction medium (SIM) without Bialaphos for 7 days followed by 4 mg.L -1 Bialaphos for 2 weeks. Bialaphos (4 mg.L -1 in SIM; 2 mg.L -1 in shoot elongation medium (SEM)) effectively selected the transformants. The putative transformants and escapes could be exactly distinguished by using a half-leaf GUS assay method to detect GUS expression in the elongated resistant shoots, which resulted in the shortening of culture period for the early detection of transformed shoots. The transformation efficiency of this system was 1.06%. The transgenic plants were verified by polymerase chain reaction (PCR), Southern blotting, and herbicide-resistant responses. All four T 0 transgenic plants were fertile and transmitted the phenotypes of both gus and bar in a 3:1 ratio to their progeny. These results indicate that the established system is suitable for further breeding of herbicide-resistant transgenic cultivars, as well as for functional genomics studies of soybeans.
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production of Bialaphos resistant nierembergia repens by electroporation
Plant Biotechnology Reports, 2008Co-Authors: Yoshiaki Shizukawa, Masahiro MiiAbstract:Transgenic plants with the herbicide-resistance gene (bar gene) were obtained via organogenesis from isolated mesophyll protoplasts of Nierembergia repens after applying electroporation. Transient β-glucuronidase (GUS) activity of electroporated protoplasts assayed 2 days after applying an electric pulse showed that optimum condition (transient GUS activity 319 pmol 4 MU/mg per min and plating efficiency 2.43%) for electroporation was 0.5 kV/cm in field strength and 100 μF in capacitance. The protoplasts electroporated with the bar gene at this condition initiated formation of microcolonies on medium after 2 weeks. After 4 weeks of culture, equal volume of fresh 1/2-strength Murashige and Skoog (MS) medium containing 0.2 mg/l Bialaphos was added for selection of transformed colonies. After 6 weeks of culture, growing colonies were transferred onto regeneration medium containing 1.0 mg/l Bialaphos, on which they formed adventitious shoots 1–2 months after electroporation. The adventitious shoots rooted easily after transfer onto MS medium with Bialaphos lacking plant-growth regulators. Transformation of these regenerants with the bar gene was confirmed by Southern analysis. Some of the transformants showed strong resistance to the application of Bialaphos solution at 10.0 mg/l.
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transgenic Bialaphos resistant snapdragon antirrhinum majus l produced by agrobacterium rhizogenes transformation
Scientia Horticulturae, 1998Co-Authors: Yoichiro Hoshino, Ismail Turkan, Masahiro MiiAbstract:Abstract Transgenic herbicide-resistant snapdragon ( Antirrhinum majus L.: Scrophulariaceae) plants were obtained by co-cultivation of leaf explants with Agrobacterium rhizogenes harboring Ri plasmid and pARK5. The T-DNA region of pARK5 contains bar gene encoding phosphinothricin acetyltransferase under the control of the cauliflower mosaic virus 35S promoter, and nptII gene encoding neomycin phosphotransferase II under the control of nopaline synthase promoter. Transformed hairy roots induced from leaf explants inoculated with A. rhizogenes strain A13 (pARK5) could grow on 1 mg l −1 Bialaphos-containing half-strength MS medium, although hairy roots transformed by the wild type strain of A. rhizogenes A13 could not survive at the same concentration of Bialaphos. Shoot regeneration from the hairy roots integrating T-DNA of pARK5 was also stimulated at 1 mg l −1 Bialaphos. Regenerated shoots readily developed roots on 2 g l −1 gellan gum-solidified half-strength MS medium. The transgenic nature of the plants was confirmed by opine, PCR and Southern analyses. Regenerated plantlets were successfully established in the growth chamber. Acclimatized plants exhibited altered phenotypes known as Ri syndrome such as reduced apical dominance with highly branched stems and short internodes, and also showed resistance to an applied standard level of commercial herbicides.
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Bialaphos stimulates shoot regeneration from hairy roots of snapdragon antirrhinum majus l transformed by agrobacterium rhizogenes
Plant Cell Reports, 1998Co-Authors: Yoichiro Hoshino, Masahiro MiiAbstract:Hairy roots of snapdragon (Antirrhinum ma-jus L.: Scrophulariaceae) induced by a wild-type strain of Agrobacterium rhizogenes were cultured on media containing various concentrations of a phosphinothricin-based herbicide, Bialaphos, or plant growth regulators (PGRs). Adventitious shoot regeneration from hairy roots was observed with a low frequency (10%) on half-strength Murashige and Skoog medium. Addition of α-naphthalene-acetic acid in combination with 6-benzylaminopurine, thidiazuron, or zeatin to the medium had no effect on shoot regeneration from hairy roots. Although Bialaphos at 0.9 mg l–1 or more was toxic to hairy roots, it significantly increased the shoot regeneration frequency up to 56% at 0.5 mg l–1. In contrast, non-transformed roots and leaves regenerated no shoots on media with or without Bialaphos. Regenerated shoots detached from host roots readily developed roots on gellan-gum-solidified medium. Regenerated plants were successfully transferred to the greenhouse, but did not produce seed.
Charles J Thompson - One of the best experts on this subject based on the ideXlab platform.
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global changes in gene expression related to antibiotic synthesis in streptomyces hygroscopicus
Molecular Microbiology, 1992Co-Authors: Julian Davies, T G Holt, C Chang, Christine Laurentwinter, T Murakami, James I Garrels, Charles J ThompsonAbstract:Two-dimensional gel electrophoresis was used to follow changes in gene expression associated with antibiotic (Bialaphos) biosynthesis in Streptomyces hygroscopicus. Cultures were pulse-labelled with [35S]-methionine before, during, and after the switch from primary to secondary metabolism in order to compare kinetic profiles of Bialaphos (antibiotic) production (bap) genes during this metabolic transition. Separation of gene products on two-dimensional gels revealed that 27 were dependent on brpA for optimal expression and were activated as the culture approached stationary phase. Genes which encoded 10 brpA-dependent proteins were mapped to a 10 kb SstI fragment of the 35 kb bap gene cluster by expressing them in Streptomyces lividans using the thiostrepton-inducible tipA promoter. N-terminal amino acid sequences of two brpA-dependent proteins, obtained by direct microsequencing of protein spots excised from two-dimensional gels, identified them as gene products mapping to the same region and involved in secondary metabolic conversions of the bap pathway. The kinetics of synthesis of 16 brpA-dependent gene products were characterized using QUEST computer software. Cluster analysis performed on the kinetics of synthesis of 346 of the most highly expressed gene products of HP5-29, including 16 brpA-dependent ones, identified 75 families having distinct patterns of expression. Many brpA-dependent proteins were clustered together; 10 were found in one kinetic family. These kinetic families also included brpA-independent gene products perhaps subject to similar regulatory mechanisms and thus possibly involved in Bialaphos biosynthesis. The activation/derepression of bap expression took place as cultures approached stationary phase and was temporally related to synthesis of ppGpp.
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nucleotide sequence analysis reveals linked n acetyl hydrolase thioesterase transport and regulatory genes encoded by the Bialaphos biosynthetic gene cluster of streptomyces hygroscopicus
Journal of Bacteriology, 1991Co-Authors: A. Raibaud, T G Holt, M Zalacain, R Tizard, Charles J ThompsonAbstract:Nucleotide sequence analysis of a 5,000-bp region of the Bialaphos antibiotic production (bap) gene cluster defined five open reading frames (ORFs) which predicted structural genes in the order bah, ORF1, ORF2, and ORF3 followed by the regulatory gene, brpA (H. Anzai, T. Murakami, S. Imai, A. Satoh, K. Nagaoka, and C.J. Thompson, J. Bacteriol. 169:3482-3488, 1987). The four structural genes were translationally coupled and apparently cotranscribed from an undefined promoter(s) under the positive control of the brpA gene product. S1 mapping experiments indicated that brpA was transcribed by two promoters (brpAp1 and brpAp2) which initiate transcription 150 and 157 bp upstream of brp A within an intergenic region and at least one promoter further upstream within the bap gene cluster (brpAp3). All three transcripts were present at low levels during exponential growth and increased just before the stationary phase. The levels of the brpAp3 band continued to increase at the onset of stationary phase, whereas brpAp1-and brpAp2-protected fragments showed no further change. BrpA contained a possible helix-turn-helix motif at its C terminus which was similar to the C-terminal regulatory motif found in the receiver component of a family of two-component transcriptional activator proteins. This motif was not associated with the N-terminal domain conserved in other members of the family. The structural gene cluster sequenced began with bah, encoding a Bialaphos acetylhydrolase which removes the N-acetyl group from Bialaphos as one of the final steps in the biosynthetic pathway. The observation that Bah was similar to a rat and to a bacterial (Acinetobacter calcoaceticus) lipase probably reflects the fact that the ester bonds of triglycerides and the amide bond linking acetate to phosphinothricin are similar and hydrolysis is catalyzed by structurally related enzymes. This was followed by two regions encoding ORF1 and ORF2 which were similar to each other (48% nucleotide identity, 31% amino acid identity), as well as to GrsT, a protein encoded by a gene located adjacent to gramicidin S synthetase in Bacillus brevis, and to vertebrate (mallard duck and rat) thioesterases. The amino acid sequence and hydrophobicity profile of ORF3 indicated that it was related to a family of membrane transport proteins. It was strikingly similar to the citrate uptake protein encoded by the transposon Tn3411. Images