Ocatin. A Novel Tuber Storage Protein from the Andean Tuber Crop Oca with Antibacterial and Antifungal Activities1

PubMed Central

Flores, Teresita; Alape-Girón, Alberto; Flores-Díaz, Marietta; Flores, Hector E.

2002-01-01

The most abundant soluble tuber protein from the Andean crop oca (Oxalis tuberosa Mol.), named ocatin, has been purified and characterized. Ocatin accounts for 40% to 60% of the total soluble oca tuber proteins, has an apparent molecular mass of 18 kD and an isoelectric point of 4.8. This protein appears to be found only in tubers and is accumulated only within the cells of the pith and peridermis layers (peel) of the tuber as it develops. Ocatin inhibits the growth of several phytopathogenic bacteria (Agrobacterium tumefaciens, Agrobacterium radiobacter, Serratia marcescens, and Pseudomonas aureofaciens) and fungi (Phytophthora cinnamomi, Fusarium oxysporum, Rhizoctonia solani, and Nectria hematococcus). Ocatin displays substantial amino acid sequence similarity with a widely distributed group of intracellular pathogenesis-related proteins with a hitherto unknown biological function. Our results showed that ocatin serves as a storage protein, has antimicrobial properties, and belongs to the Betv 1/PR-10/MLP protein family. Our findings suggest that an ancient scaffolding protein was recruited in the oca tuber to serve a storage function and that proteins from the Betv 1/PR-10/MLP family might play a role in natural resistance to pathogens. PMID:11950978

Ocatin. A novel tuber storage protein from the andean tuber crop oca with antibacterial and antifungal activities.

PubMed

Flores, Teresita; Alape-Girón, Alberto; Flores-Díaz, Marietta; Flores, Hector E

2002-04-01

The most abundant soluble tuber protein from the Andean crop oca (Oxalis tuberosa Mol.), named ocatin, has been purified and characterized. Ocatin accounts for 40% to 60% of the total soluble oca tuber proteins, has an apparent molecular mass of 18 kD and an isoelectric point of 4.8. This protein appears to be found only in tubers and is accumulated only within the cells of the pith and peridermis layers (peel) of the tuber as it develops. Ocatin inhibits the growth of several phytopathogenic bacteria (Agrobacterium tumefaciens, Agrobacterium radiobacter, Serratia marcescens, and Pseudomonas aureofaciens) and fungi (Phytophthora cinnamomi, Fusarium oxysporum, Rhizoctonia solani, and Nectria hematococcus). Ocatin displays substantial amino acid sequence similarity with a widely distributed group of intracellular pathogenesis-related proteins with a hitherto unknown biological function. Our results showed that ocatin serves as a storage protein, has antimicrobial properties, and belongs to the Betv 1/PR-10/MLP protein family. Our findings suggest that an ancient scaffolding protein was recruited in the oca tuber to serve a storage function and that proteins from the Betv 1/PR-10/MLP family might play a role in natural resistance to pathogens.

Agrobacterium tumefaciens supports DNA replication of diverse geminivirus types.

PubMed

Selth, Luke A; Randles, John W; Rezaian, M Ali

2002-04-10

We have previously shown that the soil-borne plant pathogen Agrobacterium tumefaciens supports the replication of tomato leaf curl geminivirus (Australian isolate) (TLCV) DNA. However, the reproducibility of this observation with other geminiviruses has been questioned. Here, we show that replicative DNA forms of three other geminiviruses also accumulate at varying levels in Agrobacterium. Geminiviral DNA constructs that lacked the ability to replicate in Agrobacterium were rendered replication-competent by changing their configuration so that two copies of the viral ori were present. Furthermore, we report that low-level replication of TLCV DNA can occur in Escherichia coli containing a dimeric TLCV construct in a high copy number plasmid. These findings were reinforced by expression studies using beta-glucuronidase which revealed that all six TLCV promoters are active in Agrobacterium, and two are functional in E. coli.

Agrobacterium-delivered virulence protein VirE2 is trafficked inside host cells via a myosin XI-K–powered ER/actin network

PubMed Central

Yang, Qinghua; Li, Xiaoyang; Tu, Haitao; Pan, Shen Q.

2017-01-01

Agrobacterium tumefaciens causes crown gall tumors on various plants by delivering transferred DNA (T-DNA) and virulence proteins into host plant cells. Under laboratory conditions, the bacterium is widely used as a vector to genetically modify a wide range of organisms, including plants, yeasts, fungi, and algae. Various studies suggest that T-DNA is protected inside host cells by VirE2, one of the virulence proteins. However, it is not clear how Agrobacterium-delivered factors are trafficked through the cytoplasm. In this study, we monitored the movement of Agrobacterium-delivered VirE2 inside plant cells by using a split-GFP approach in real time. Agrobacterium-delivered VirE2 trafficked via the endoplasmic reticulum (ER) and F-actin network inside plant cells. During this process, VirE2 was aggregated as filamentous structures and was present on the cytosolic side of the ER. VirE2 movement was powered by myosin XI-K. Thus, exogenously produced and delivered VirE2 protein can use the endogenous host ER/actin network for movement inside host cells. The A. tumefaciens pathogen hijacks the conserved host infrastructure for virulence trafficking. Well-conserved infrastructure may be useful for Agrobacterium to target a wide range of recipient cells and achieve a high efficiency of transformation. PMID:28242680

Advances in Agrobacterium tumefaciens-mediated genetic transformation of graminaceous crops.

PubMed

Singh, Roshan Kumar; Prasad, Manoj

2016-05-01

Steady increase in global population poses several challenges to plant science research, including demand for increased crop productivity, grain yield, nutritional quality and improved tolerance to different environmental factors. Transgene-based approaches are promising to address these challenges by transferring potential candidate genes to host organisms through different strategies. Agrobacterium-mediated gene transfer is one such strategy which is well known for enabling efficient gene transfer in both monocot and dicots. Due to its versatility, this technique underwent several advancements including development of improved in vitro plant regeneration system, co-cultivation and selection methods, and use of hyper-virulent strains of Agrobacterium tumefaciens harbouring super-binary vectors. The efficiency of this method has also been enhanced by the use of acetosyringone to induce the activity of vir genes, silver nitrate to reduce the Agrobacterium-induced necrosis and cysteine to avoid callus browning during co-cultivation. In the last two decades, extensive efforts have been invested towards achieving efficient Agrobacterium-mediated transformation in cereals. Though high-efficiency transformation systems have been developed for rice and maize, comparatively lesser progress has been reported in other graminaceous crops. In this context, the present review discusses the progress made in Agrobacterium-mediated transformation system in rice, maize, wheat, barley, sorghum, sugarcane, Brachypodium, millets, bioenergy and forage and turf grasses. In addition, it also provides an overview of the genes that have been recently transferred to these graminaceous crops using Agrobacterium, bottlenecks in this technique and future possibilities for crop improvement.

Improvement of Agrobacterium-mediated transformation and rooting of black cherry

Treesearch

Ying Wang; Paula M. Pijut

2014-01-01

An improved protocol for Agrobacterium-mediated transformation of an elite, mature black cherry genotype was developed. To increase transformation efficiency, vacuum infiltration, sonication, and a combination of the two treatments were applied during the cocultivation of leaf explants with Agrobacterium tumefaciens strain EHA105...

Lauric Acid Stimulates Mammary Gland Development of Pubertal Mice through Activation of GPR84 and PI3K/Akt Signaling Pathway.

PubMed

Meng, Yingying; Zhang, Jing; Zhang, Fenglin; Ai, Wei; Zhu, Xiaotong; Shu, Gang; Wang, Lina; Gao, Ping; Xi, Qianyun; Zhang, Yongliang; Liang, Xingwei; Jiang, Qingyan; Wang, Songbo

2017-01-11

It has been demonstrated that dietary fat affects pubertal mammary gland development. However, the role of lauric acid (LA) in this process remains unclear. Thus, this study aimed to investigate the effects of LA on mammary gland development in pubertal mice and to explore the underlying mechanism. In vitro, 100 μM LA significantly promoted proliferation of mouse mammary epithelial cell line HC11 by regulating expression of proliferative markers (cyclin D1/3, p21, PCNA). Meanwhile, LA activated the G protein-coupled receptor 84 (GPR84) and PI3K/Akt signaling pathway. In agreement, dietary 1% LA enhanced mammary duct development, increased the expression of GPR84 and cyclin D1, and activated PI3K/Akt in mammary gland of pubertal mice. Furthermore, knockdown of GPR84 or inhibition of PI3K/Akt totally abolished the promotion of HC11 proliferation induced by LA. These results showed that LA stimulated mammary gland development of pubertal mice through activation of GPR84 and PI3K/Akt signaling pathway.

Novel compounds that enhance Agrobacterium-mediated plant transformation by mitigating oxidative stress.

PubMed

Dan, Yinghui; Zhang, Song; Zhong, Heng; Yi, Hochul; Sainz, Manuel B

2015-02-01

Agrobacterium tumefaciens caused tissue browning leading to subsequent cell death in plant transformation and novel anti-oxidative compounds enhanced Agrobacterium -mediated plant transformation by mitigating oxidative stress. Browning and death of cells transformed with Agrobacterium tumefaciens is a long-standing and high impact problem in plant transformation and the agricultural biotechnology industry, severely limiting the production of transgenic plants. Using our tomato cv. MicroTom transformation system, we demonstrated that Agrobacterium caused tissue browning (TB) leading to subsequent cell death by our correlation study. Without an antioxidant (lipoic acid, LA) TB was severe and associated with high levels of GUS transient expression and low stable transformation frequency (STF). LA addition shifted the curve in that most TB was intermediate and associated with the highest levels of GUS transient expression and STF. We evaluated 18 novel anti-oxidative compounds for their potential to enhance Agrobacterium-mediated transformation, by screening for TB reduction and monitoring GUS transient expression. Promising compounds were further evaluated for their effect on MicroTom and soybean STF. Among twelve non-antioxidant compounds, seven and five significantly (P < 0.05) reduced TB and increased STF, respectively. Among six antioxidants four of them significantly reduced TB and five of them significantly increased STF. The most efficient compound found to increase STF was melatonin (MEL, an antioxidant). Optimal concentrations and stages to use MEL in transformation were determined, and Southern blot analysis showed that T-DNA integration was not affected by MEL. The ability of diverse compounds with different anti-oxidative mechanisms can reduce Agrobacterium-mediated TB and increase STF, strongly supporting that oxidative stress is an important limiting factor in Agrobacterium-mediated transformation and the limiting factor can be controlled by these

Transformation of medicinal plants using Agrobacterium tumefaciens.

PubMed

Bandurska, Katarzyna; Berdowska, Agnieszka; Król, Małgorzata

2016-12-20

For many years attempts are made to develop efficient methods for transformation of medicinal plants via Agrobacterium tumefaciens. It is a soil bacteria which possess a natural ability to infect plants in places of injures which results in arise of cancerous growths (crown gall). This is possible thanks a transfer of fragment of Ti plasmid into plant cells and stable integration with a plant genome. Efficiency of medicinal plant transformation depends on many factors for example: Agrobacterium strain, methods and procedures of transformation as well as on plant species, type and age of the explants and regeneration conditions. The main goal of plant transformation is to increase the amount of naturally occurring bioactive compounds and the production of biopharmaceuticals. Genetic plant transformation via bacteria of the genus Agrobacterium is a complex process which requires detailed analysis of incorporated transgene expression and occurs only in the case when the plant cell acquires the ability to regenerate. In many cases, the regeneration efficiency observed in medicinal plants are inefficient after applied transformation procedures. To date there have been attempts of genetic transformation by using A. tumefaciens of medicinal plants belonging to the families: Apocynaceae, Araceae, Araliaceae, Asphodelaceae, Asteraceae, Begoniaceae, Crassulaceae, Fabaceae, Lamiaceae, Linaceae, Papaveraceae, Plantaginaceae, Scrophulariaceae and Solanaceae.

Differential roles of glucosinolates and camalexin at different stages of Agrobacterium-mediated transformation.

PubMed

Shih, Po-Yuan; Chou, Shu-Jen; Müller, Caroline; Halkier, Barbara Ann; Deeken, Rosalia; Lai, Erh-Min

2018-03-02

Agrobacterium tumefaciens is the causal agent of crown gall disease in a wide range of plants via a unique interkingdom DNA transfer from bacterial cells into the plant genome. Agrobacterium tumefaciens is capable of transferring its T-DNA into different plant parts at different developmental stages for transient and stable transformation. However, the plant genes and mechanisms involved in these transformation processes are not well understood. We used Arabidopsis thaliana Col-0 seedlings to reveal the gene expression profiles at early time points during Agrobacterium infection. Common and differentially expressed genes were found in shoots and roots. A gene ontology analysis showed that the glucosinolate (GS) biosynthesis pathway was an enriched common response. Strikingly, several genes involved in indole glucosinolate (iGS) modification and the camalexin biosynthesis pathway were up-regulated, whereas genes in aliphatic glucosinolate (aGS) biosynthesis were generally down-regulated, on Agrobacterium infection. Thus, we evaluated the impacts of GSs and camalexin during different stages of Agrobacterium-mediated transformation combining Arabidopsis mutant studies, metabolite profiling and exogenous applications of various GS hydrolysis products or camalexin. The results suggest that the iGS hydrolysis pathway plays an inhibitory role on transformation efficiency in Arabidopsis seedlings at the early infection stage. Later in the Agrobacterium infection process, the accumulation of camalexin is a key factor inhibiting tumour development on Arabidopsis inflorescence stalks. In conclusion, this study reveals the differential roles of GSs and camalexin at different stages of Agrobacterium-mediated transformation and provides new insights into crown gall disease control and improvement of plant transformation. © 2018 THE AUTHORS. MOLECULAR PLANT PATHOLOGY PUBLISHED BY BRITISH SOCIETY FOR PLANT PATHOLOGY AND JOHN WILEY & SONS LTD.

Agrobacterium-mediated transformation of protocorm-like bodies in Cymbidium.

PubMed

Chin, Dong Poh; Mishiba, Kei-ichiro; Mii, Masahiro

2007-06-01

Genetically transformed plants of Cymbidium were regenerated after cocultivating protocorm-like bodies (PLB) with Agrobacterium tumefaciens strain EHA101 (pIG121Hm) that harbored genes for beta-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase II (nptII). PLB of three genotypes maintained in liquid new Dogashima medium (NDM), were subjected to transformation experiments. The PLB inoculated with Agrobacterium produced secondary PLB, 4 weeks after transfer onto 2.5 g L(-1) gellan gum-solidified NDM containing 10 g L(-1) sucrose, 20 mg L(-1) hygromycin and 40 mg L(-1) meropenem. Transformation efficiency was affected by genotype and the presence of acetosyringone during cocultivation. The highest transformation efficiency was obtained when PLB from the genotype L4 were infected and cocultivated with Agrobacterium on medium containing 100 muM acetosyringone. Transformation of the hygromycin-resistant plantlets regenerated from different sites of inoculated PLB was confirmed by histochemical GUS assay, PCR analysis and Southern blot hybridization.

Efficient Agrobacterium-mediated transformation of the liverwort Marchantia polymorpha using regenerating thalli.

PubMed

Kubota, Akane; Ishizaki, Kimitsune; Hosaka, Masashi; Kohchi, Takayuki

2013-01-01

The thallus, the gametophyte body of the liverwort Marchantia polymorpha, develops clonal progenies called gemmae that are useful in the isolation and propagation of isogenic plants. Developmental timing is critical to Agrobacterium-mediated transformation, and high transformation efficiency has been achieved only with sporelings. Here we report an Agrobacterium-mediated transformation system for M. polymorpha using regenerating thalli. Thallus regeneration was induced by cutting the mature thallus across the apical-basal axis and incubating the basal portion of the thallus for 3 d. Regenerating thalli were infected with Agrobacterium carrying binary vector that contained a selection marker, the hygromycin phosphotransferase gene, and hygromycin-resistant transformants were obtained with an efficiency of over 60%. Southern blot analysis verified random integration of 1 to 4 copies of the T-DNA into the M. polymorpha genome. This Agrobacterium-mediated transformation system for M. polymorpha should provide opportunities to perform genetic transformation without preparing spores and to generate a sufficient number of transformants with isogenic background.

N-METHYL GROUPS IN BACTERIAL LIPIDS

PubMed Central

Goldfine, Howard; Ellis, Martha E.

1964-01-01

Goldfine, Howard (Harvard Medical School, Boston, Mass.), and Martha E. Ellis. N-methyl groups in bacterial lipids. J. Bacteriol. 87:8–15. 1964.—The ability of bacteria to synthesize lecithin was examined by measuring the incorporation of the methyl group of methionine into the water-soluble moieties obtained on acid hydrolysis of bacterial lipids. Of 21 species examined, mostly of the order Eubacteriales, only 2, Agrobacterium radiobacter and A. rhizogenes, incorporated the methyl group of methionine into lipid-bound choline. Evidence was also obtained for the formation of lipid-bound N-methylethanolamine and N,N′-dimethylethanolamine in these two organisms. Two other species, Clostridium butyricum and Proteus vulgaris, incorporated the methyl group of methionine into lipid-bound N-methylethanolamine, but did not appear to be able to further methylate these lipids to form lecithin. The results of this study lend further strength to the generalization that bacteria, with the exception of the genus Agrobacterium, are unable to synthesize lecithin. PMID:14102879

Agrobacterium and Tumor Induction: A Model System.

ERIC Educational Resources Information Center

Lennox, John E.

1980-01-01

The author offers laboratory procedures for experiments using the bacterium, Agrobacterium tumefaciens, which causes crown gall disease in a large number of plants. Three different approaches to growing a culture are given. (SA)

Osmoregulation in Agrobacterium tumefaciens: accumulation of a novel disaccharide is controlled by osmotic strength and glycine betaine.

PubMed Central

Smith, L T; Smith, G M; Madkour, M A

1990-01-01

We have investigated the mechanism of osmotic stress adaptation (osmoregulation) in Agrobacterium tumefaciens biotype I (salt-tolerant) and biotype II (salt-sensitive) strains. Using natural-abundance 13C nuclear magnetic resonance spectroscopy, we identified all organic solutes that accumulated to significant levels in osmotically stressed cultures. When stressed, biotype I strains (C58, NT1, and A348) accumulated glutamate and a novel disaccharide, beta-fructofuranosyl-alpha-mannopyranoside, commonly known as mannosucrose. In the salt-sensitive biotype II strain K84, glutamate was observed but mannosucrose was not. We speculate that mannosucrose confers the extra osmotic tolerance observed in the biotype I strains. In addition to identifying the osmoregulated solutes that this species synthesizes, we investigated the ability of A. tumefaciens to utilize the powerful osmotic stress protectant glycine betaine when it is supplied in the medium. Results from growth experiments, nuclear magnetic resonance spectroscopy, and a 14C labeling experiment demonstrated that in the absence of osmotic stress, glycine betaine was metabolized, while in stressed cultures, glycine betaine accumulated intracellularly and conferred enhanced osmotic stress tolerance. Furthermore, when glycine betaine was taken up in stressed cells, its accumulation caused the intracellular concentration of mannosucrose to drop significantly. The possible role of osmoregulation of A. tumefaciens in the transformation of plants is discussed. PMID:2254260

Agrobacterium-medicated transformation of mature Prunus serotina (black cherry) and regeneration of trangenic shoots

Treesearch

Xiaomei Liu; Paula Pijut

2010-01-01

A protocol for Agrobacterium-mediated transformation was developed for in vitro leaf explants of an elite, mature Prunus serotina tree. Agrobacterium tumefaciens strain EHA105 harboring an RNAi plasmid with the black cherry AGAMOUS (AG) gene was used. Bacteria were induced...

Agrobacterium-mediated genetic transformation of pineapple (Ananas comosus L., Merr.).

PubMed

Mhatre, Minal

2013-01-01

Pineapple (Ananas comosus L., Merr.) is a commercially important crop, grown in the tropical and subtropical regions. However, the crop is faced with postharvest damage and poor varietal and nutritional improvement. Being a vegetatively propagated crop, conventional breeding programs take longer time for genetic improvement, which may not necessarily successfully develop an improved cultivar. Hence, the genetic modification of pineapple is an alternative handy approach to improve pineapple. We have established an Agrobacterium-mediated transformation system using leaf bases from in vitro-grown pineapple plants. Being a monocot, acetosyringone is added to the culture medium for overnight growth of Agrobacterium and transformation to transfer a gene of interest MSI99 soybean ferritin. Leaf bases isolated from in vitro shoot cultures are treated with Agrobacterium suspension at two dilutions, 10× and 20×, for 30 min. Explants are subsequently blot dried and cultured on gelrite solidified hormone-free Pin1 medium for 2 days (cocultivation). Periodic transfer is first done to the regeneration medium (Pin1) containing cefotaxime for the suppression of Agrobacterium growth. The transformants are selected by culturing on Pin1 medium containing cefotaxime and kanamycin. Multiple shoots, regenerated in leaf bases, are further multiplied and individually rooted in the liquid RM medium amended with antibiotics to recover plants. Putative transformants are analyzed for transgene integration and expression using standard molecular biological methods of PCR, RT-PCR, and genomic Southern.

Nopaline-type Ti plasmid of Agrobacterium encodes a VirF-like functional F-box protein.

PubMed

Lacroix, Benoît; Citovsky, Vitaly

2015-11-20

During Agrobacterium-mediated genetic transformation of plants, several bacterial virulence (Vir) proteins are translocated into the host cell to facilitate infection. One of the most important of such translocated factors is VirF, an F-box protein produced by octopine strains of Agrobacterium, which presumably facilitates proteasomal uncoating of the invading T-DNA from its associated proteins. The presence of VirF also is thought to be involved in differences in host specificity between octopine and nopaline strains of Agrobacterium, with the current dogma being that no functional VirF is encoded by nopaline strains. Here, we show that a protein with homology to octopine VirF is encoded by the Ti plasmid of the nopaline C58 strain of Agrobacterium. This protein, C58VirF, possesses the hallmarks of functional F-box proteins: it contains an active F-box domain and specifically interacts, via its F-box domain, with SKP1-like (ASK) protein components of the plant ubiquitin/proteasome system. Thus, our data suggest that nopaline strains of Agrobacterium have evolved to encode a functional F-box protein VirF.

77 FR 40880 - Agrobacterium radiobacter; Registration Review Proposed Decision; Notice of Availability

Federal Register 2010, 2011, 2012, 2013, 2014

2012-07-11

... perform its intended function without unreasonable adverse effects on human health or the environment... and other knowledge, including its effects on human health and the environment. DATES: Comments must... range of stakeholders including environmental, human health, farm worker, and agricultural advocates...

A Fruiting Body Tissue Method for Efficient Agrobacterium-Mediated Transformation of Agaricus bisporus

PubMed Central

Chen, Xi; Stone, Michelle; Schlagnhaufer, Carl; Romaine, C. Peter

2000-01-01

We describe a modified Agrobacterium-mediated method for the efficient transformation of Agaricus bisporus. Salient features of this procedure include cocultivation of Agrobacterium and fruiting body gill tissue and use of a vector with a homologous promoter. This method offers new prospects for the genetic manipulation of this commercially important mushroom species. PMID:11010906

The solid solution K3.84Ni0.78Fe3.19(PO4)5

PubMed Central

Strutynska, Nataliia Yu.; Ogorodnyk, Ivan V.; Livitska, Oksana V.; Baumer, Vyacheslav N.; Slobodyanik, Nikolay S.

2014-01-01

The title compound, tetra­potassium tetra­[nickel(II)/iron(III)] penta­kis­(orthophosphate), K3.84Ni0.78Fe3.19(PO4)5, has been obtained from a flux. The structure is isotypic with that of K4MgFe3(PO4)5. The three-dimensional framework is built up from (Ni/Fe)O5 trigonal bipyramids with a mixed Fe:Ni occupancy of 0.799 (8):0.196 (10) and isolated PO4 tetra­hedra, one of which is on a general position and one of which has -4.. site symmetry. Two K+ cations are statistically occupied and are distributed over two positions in hexa­gonally shaped channels that run parallel to [001]. One K+ cation [occupancy 0.73 (3)] is surrounded by nine O atoms, while the other K+ cation [occupancy 0.23 (3)] is surrounded by eight O atoms. PMID:25161510

High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda)

NASA Technical Reports Server (NTRS)

Wenck, A. R.; Quinn, M.; Whetten, R. W.; Pullman, G.; Sederoff, R.; Brown, C. S. (Principal Investigator)

1999-01-01

Agrobacterium-mediated gene transfer is the method of choice for many plant biotechnology laboratories; however, large-scale use of this organism in conifer transformation has been limited by difficult propagation of explant material, selection efficiencies and low transformation frequency. We have analyzed co-cultivation conditions and different disarmed strains of Agrobacterium to improve transformation. Additional copies of virulence genes were added to three common disarmed strains. These extra virulence genes included either a constitutively active virG or extra copies of virG and virB, both from pTiBo542. In experiments with Norway spruce, we increased transformation efficiencies 1000-fold from initial experiments where little or no transient expression was detected. Over 100 transformed lines expressing the marker gene beta-glucuronidase (GUS) were generated from rapidly dividing embryogenic suspension-cultured cells co-cultivated with Agrobacterium. GUS activity was used to monitor transient expression and to further test lines selected on kanamycin-containing medium. In loblolly pine, transient expression increased 10-fold utilizing modified Agrobacterium strains. Agrobacterium-mediated gene transfer is a useful technique for large-scale generation of transgenic Norway spruce and may prove useful for other conifer species.

Enhancing the biocatalytic manufacture of the key intermediate of atorvastatin by focused directed evolution of halohydrin dehalogenase.

PubMed

Luo, Yu; Chen, Yangzi; Ma, Hongmin; Tian, ZhenHua; Zhang, Yeqi; Zhang, Jian

2017-02-06

Halohydrin dehalogenases (HHDHs) are biocatalytically interesting enzymes due to their ability to form C-C, C-N, C-O, and C-S bonds. One of most important application of HHDH was the protein engineering of HheC (halohydrin dehalogenase from Agrobacterium radiobacter AD1) for the industrial manufacturing of ethyl (R)-4-cyano-3-hydroxybutanoate (HN), a key chiral synthon of a cholesterol-lowering drug of atorvastatin. During our development of an alternative, more efficient and economic route for chemo-enzymatic preparation of the intermediate of atorvastatin, we found that the HheC2360 previously reported for HN manufacture, had insufficient activity for the cyanolysis production of tert-butyl (3 R,5 S)-6-cyano-3,5-dihydroxyhexanoate (A7). Herein, we present the focused directed evolution of HheC2360 with higher activity and enhanced biocatalytic performance using active site mutagenesis. Through docking of the product, A7, into the crystal structure of HheC2360, 6 residues was selected for combined active sites testing (CASTing). After library screening, the variant V84G/W86F was identified to have a 15- fold increase in activity. Time course analysis of the cyanolysis reaction catalyzed by this variant, showed 2- fold increase in space time productivity compared with HheC2360. These results demonstrate the applicability of the variant V84G/W86F as a biocatalyst for the efficient and practical production of atorvastatin intermediate.

Female reproductive tissues are the primary target of Agrobacterium-mediated transformation by the Arabidopsis floral-dip method.

PubMed

Desfeux, C; Clough, S J; Bent, A F

2000-07-01

The floral-dip method for Agrobacterium-mediated transformation of Arabidopsis allows efficient plant transformation without need for tissue culture. To facilitate use with other plant species, we investigated the mechanisms that underlie this method. In manual outcrossing experiments, application of Agrobacterium tumefaciens to pollen donor plants did not produce any transformed progeny, whereas application of Agrobacterium to pollen recipient plants yielded transformants at a rate of 0.48%. Agrobacterium strains with T-DNA carrying gusA (encoding beta-glucuronidase [GUS]) under the control of 35S, LAT52, or ACT11 promoters revealed delivery of GUS activity to developing ovules, whereas no GUS staining of pollen or pollen tubes was observed. Transformants derived from the same seed pod contained independent T-DNA integration events. In Arabidopsis flowers, the gynoecium develops as an open, vase-like structure that fuses to form closed locules roughly 3 d prior to anthesis. In correlation with this fact, we found that the timing of Agrobacterium infection was critical. Transformants were obtained and GUS staining of ovules and embryo sacs was observed only if the Agrobacterium were applied 5 d or more prior to anthesis. A 6-fold higher rate of transformation was obtained with a CRABS-CLAW mutant that maintains an open gynoecium. Our results suggest that ovules are the site of productive transformation in the floral-dip method, and further suggest that Agrobacterium must be delivered to the interior of the developing gynoecium prior to locule closure if efficient transformation is to be achieved.

Global Analysis of Differentially Expressed Genes and Proteins in the Wheat Callus Infected by Agrobacterium tumefaciens

PubMed Central

Zhou, Xiaohong; Wang, Ke; Lv, Dongwen; Wu, Chengjun; Li, Jiarui; Zhao, Pei; Lin, Zhishan; Du, Lipu; Yan, Yueming; Ye, Xingguo

2013-01-01

Agrobacterium-mediated plant transformation is an extremely complex and evolved process involving genetic determinants of both the bacteria and the host plant cells. However, the mechanism of the determinants remains obscure, especially in some cereal crops such as wheat, which is recalcitrant for Agrobacterium-mediated transformation. In this study, differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were analyzed in wheat callus cells co-cultured with Agrobacterium by using RNA sequencing (RNA-seq) and two-dimensional electrophoresis (2-DE) in conjunction with mass spectrometry (MS). A set of 4,889 DEGs and 90 DEPs were identified, respectively. Most of them are related to metabolism, chromatin assembly or disassembly and immune defense. After comparative analysis, 24 of the 90 DEPs were detected in RNA-seq and proteomics datasets simultaneously. In addition, real-time RT-PCR experiments were performed to check the differential expression of the 24 genes, and the results were consistent with the RNA-seq data. According to gene ontology (GO) analysis, we found that a big part of these differentially expressed genes were related to the process of stress or immunity response. Several putative determinants and candidate effectors responsive to Agrobacterium mediated transformation of wheat cells were discussed. We speculate that some of these genes are possibly related to Agrobacterium infection. Our results will help to understand the interaction between Agrobacterium and host cells, and may facilitate developing efficient transformation strategies in cereal crops. PMID:24278131

Agrobacterium-mediated transformation of Mexican lime (Citrus aurantifolia Swingle) using optimized systems for epicotyls and cotelydons

USDA-ARS?s Scientific Manuscript database

Epicotyl and internodal stem segments provide the predominantly used explants for regeneration of transgenic citrus plants following co-cultivation with Agrobacterium. Previous reports using epicotyls segments from Mexican lime have shown low affinity for Agrobacterium tumefaciens infection which re...

X-ray structure of imidazolonepropionase from Agrobacterium tumefaciens at 1.87 Å resolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tyagi, Rajiv; Kumaran, Desigan; Burley, Stephen K.

2010-01-12

Histidine degradation in Agrobacterium tumefaciens involves four enzymes, including histidase (EC 4.3.1.3), urocanase (EC 4.2.1.49), imidazolonepropionase (EC 3.5.2.7), and N-formylglutamate amidohydrolase (EC 3.5.3.8). The third enzyme of the pathway, imidazolone-propionase, a 45.6 kDa protein, catalyzes conversion of imidazolone-5-propanoate to N-forminio-L-glutamate. Initial studies of the role of imidazolonepropionase in histidine degradation were published in 1953. Subsequent publications have been limited to enzyme kinetics, crystallization, and a recently reported structure determination. The imidazolonepropionases are members of metallodepenent-hydrolases (or amidohydroase) superfamily, which includs ureases, adenosine deaminases, phosphotriesterases, dihydroorotases, allantoinases, hydantoinases, adenine and cytosine deaminases, imidazolonepropionases, aryldial-kylphosphatases, chlorohydrolases, and formylmethanofuran dehydroases. Proteins belonging tomore » this large group share a common three-dimensional structural motif (an eightfold {alpha}/{beta} or TIM barrel) with similar active sites. Most superfamily members also share a conserved metal binding site, involving four histidine residues and one aspartic acid. Imidazolonepropionase is one of the targets selected for X-ray crystallpgrahpic structure determination by the New York Structural GenomiX Research Consortium (NYSGXRC) Target ID: 9252b to correlate the structure function relationship of poorly studied by important enzyme. Here they report the crystal structure of imidazolonepropionase from Agrobacterium tumefaciens determined at 1.87 {angstrom} resolution.« less

Female Reproductive Tissues Are the Primary Target of Agrobacterium-Mediated Transformation by the Arabidopsis Floral-Dip Method1

PubMed Central

Desfeux, Christine; Clough, Steven J.; Bent, Andrew F.

2000-01-01

The floral-dip method for Agrobacterium-mediated transformation of Arabidopsis allows efficient plant transformation without need for tissue culture. To facilitate use with other plant species, we investigated the mechanisms that underlie this method. In manual outcrossing experiments, application of Agrobacterium tumefaciens to pollen donor plants did not produce any transformed progeny, whereas application of Agrobacterium to pollen recipient plants yielded transformants at a rate of 0.48%. Agrobacterium strains with T-DNA carrying gusA (encoding β-glucuronidase [GUS]) under the control of 35S, LAT52, or ACT11 promoters revealed delivery of GUS activity to developing ovules, whereas no GUS staining of pollen or pollen tubes was observed. Transformants derived from the same seed pod contained independent T-DNA integration events. In Arabidopsis flowers, the gynoecium develops as an open, vase-like structure that fuses to form closed locules roughly 3 d prior to anthesis. In correlation with this fact, we found that the timing of Agrobacterium infection was critical. Transformants were obtained and GUS staining of ovules and embryo sacs was observed only if the Agrobacterium were applied 5 d or more prior to anthesis. A 6-fold higher rate of transformation was obtained with a CRABS-CLAW mutant that maintains an open gynoecium. Our results suggest that ovules are the site of productive transformation in the floral-dip method, and further suggest that Agrobacterium must be delivered to the interior of the developing gynoecium prior to locule closure if efficient transformation is to be achieved. PMID:10889238

Agrobacterium- and Biolistic-Mediated Transformation of Maize B104 Inbred.

PubMed

Raji, Jennifer A; Frame, Bronwyn; Little, Daniel; Santoso, Tri Joko; Wang, Kan

2018-01-01

Genetic transformation of maize inbred genotypes remains non-routine for many laboratories due to variations in cell competency to induce embryogenic callus, as well as the cell's ability to receive and incorporate transgenes into the genome. This chapter describes two transformation protocols using Agrobacterium- and biolistic-mediated methods for gene delivery. Immature zygotic embryos of maize inbred B104, excised from ears harvested 10-14 days post pollination, are used as starting explant material. Disarmed Agrobacterium strains harboring standard binary vectors and the biolistic gun system Bio-Rad PDS-1000/He are used as gene delivery systems. The herbicide resistant bar gene and selection agent bialaphos are used for identifying putative transgenic type I callus events. Using the step-by-step protocols described here, average transformation frequencies (number of bialaphos resistant T 0 callus events per 100 explants infected or bombarded) of 4% and 8% can be achieved using the Agrobacterium- and biolistic-mediated methods, respectively. An estimated duration of 16-21 weeks is needed using either protocol from the start of transformation experiments to obtaining putative transgenic plantlets with established roots. In addition to laboratory in vitro procedures, detailed greenhouse protocols for producing immature ears as transformation starting material and caring for transgenic plants for seed production are also described.

Agrobacterium-mediated transformation of finger millet (Eleusine coracana (L.) Gaertn.) using shoot apex explants.

PubMed

Ceasar, S Antony; Ignacimuthu, S

2011-09-01

A new Agrobacterium-mediated transformation system was developed for finger millet using shoot apex explants. The Agrobacterium strain LBA4404 harboring binary vector pCAMBIA1301, which contained hygromycin phosphotransferase (hptII) as selectable marker gene and β-glucuronidase (GUS) as reporter gene, was used for optimization of transformation conditions. Two finger millet genotypes, GPU 45 and CO 14, were used in this study. The optimal conditions for the Agrobacterium-mediated transformation of finger millet were found to be the co-cultivation of explants obtained on the 16th day after callus induction (DACI), exposure of explants for 30 min to agrobacterial inoculum and 3 days of co-cultivation on filter paper placed on medium supplemented with 100 μM acetosyringone (AS). Addition of 100 μM L: -cysteine in the selection medium enhanced the frequency of transformation and transgenic plant recovery. Both finger millet genotypes were transformed by Agrobacterium. A frequency of 19% transient expression with 3.8% stable transformation was achieved in genotype GPU 45 using optimal conditions. Five stably transformed plants were fully characterized by Southern blot analysis. A segregation analysis was also performed in four R(1) progenies, which showed normal Mendelian pattern of transgene segregation. The inheritance of transgenes in R(1) progenies was also confirmed by Southern blot analysis. This is the first report on Agrobacterium-mediated transformation of finger millet. This study underpins the introduction of numerous agronomically important genes into the genome of finger millet in the future.

Agrobacterium-mediated genetic transformation of Fraxinus americana hypocotyls

Treesearch

Kaitlin J. Palla; Paula M. Pijut

2015-01-01

An Agrobacterium tumefaciens-mediated genetic transformation system was successfully developed for white ash (Fraxinus americana) using hypocotyls as the initial explants. Hypocotyls isolated from mature embryos germinated on Murashige and Skoog (MS) medium supplemented with 22.2 µM 6-benzyladenine (BA) and 0.5 µM...

Sonication, Vacuum Infiltration and Thiol Compounds Enhance the Agrobacterium-Mediated Transformation Frequency of Withania somnifera (L.) Dunal

PubMed Central

Sivanandhan, Ganeshan; Kapil Dev, Gnajothi; Theboral, Jeevaraj; Selvaraj, Natesan; Ganapathi, Andy; Manickavasagam, Markandan

2015-01-01

In the present study, we have established a stable transformation protocol via Agrobacterium tumafacines for the pharmaceutically important Withania somnifera. Six day-old nodal explants were used for 3 day co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring the vector pCAMIBA2301. Among the different injury treatments, sonication, vacuum infiltration and their combination treatments tested, a vacuum infiltration for 10 min followed by sonication for 10 sec with A. tumefaciens led to a higher transient GUS expression (84% explants expressing GUS at regenerating sites). In order to improve gene integration, thiol compounds were added to co-cultivation medium. A combined treatment of L-Cys at 100 mg/l, STS at 125 mg/l, DTT at 75 mg/l resulted in a higher GUS expression (90%) in the nodal explants. After 3 days of co-cultivation, the explants were subjected to three selection cycles with increasing concentrations of kanamycin [100 to 115 mg/l]. The integration and expression of gusA gene in T0 and T1 transgenic plants were confirmed by polymerase chain reaction (PCR), and Southern blott analysis. These transformed plants (T0 and T1) were fertile and morphologically normal. From the present investigation, we have achieved a higher transformation efficiency of (10%). Withanolides (withanolide A, withanolide B, withanone and withaferin A) contents of transformed plants (T0 and T1) were marginally higher than control plants. PMID:25927703

An improved Agrobacterium-mediated transformation of recalcitrant indica rice (Oryza sativa L.) cultivars.

PubMed

Shri, Manju; Rai, Arti; Verma, Pankaj Kumar; Misra, Prashant; Dubey, Sonali; Kumar, Smita; Verma, Sikha; Gautam, Neelam; Tripathi, Rudra Deo; Trivedi, Prabodh Kumar; Chakrabarty, Debasis

2013-04-01

Agrobacterium-mediated transformation of indica rice varieties has been quite difficult as these are recalcitrant to in vitro responses. In the present study, we established a high-efficiency Agrobacterium tumefaciens-mediated transformation system of rice (Oryza sativa L. ssp. indica) cv. IR-64, Lalat, and IET-4786. Agrobacterium strain EHA-101 harboring binary vector pIG121-Hm, containing a gene encoding for β-glucuronidase (GUS) and hygromycin resistance, was used in the transformation experiments. Manipulation of different concentrations of acetosyringone, days of co-culture period, bacterial suspension of different optical densities (ODs), and the concentrations of L-cysteine in liquid followed by solid co-culture medium was done for establishing the protocol. Among the different co-culture periods, 5 days of co-culture with bacterial cells (OD600 nm = 0.5-0.8) promoted the highest frequency of transformation (83.04 %) in medium containing L-cysteine (400 mg l(-1)). Putative transformed plants were analyzed for the presence of a transgene through genomic PCR and GUS histochemical analyses. Our results also suggest that different cultural conditions and the addition of L-cysteine in the co-culture medium improve the Agrobacterium-mediated transformation frequencies from an average of 12.82 % to 33.33 % in different indica rice cultivars.

Endophytic occupation of root nodules and roots of Melilotus dentatus by Agrobacterium tumefaciens.

PubMed

Wang, Ling Ling; Wang, En Tao; Liu, Jie; Li, Ying; Chen, Wen Xin

2006-10-01

Agrobacterium strains have been frequently isolated from the root nodules of different legumes. Various possible mechanisms have been proposed to explain the existence of these bacteria in nodules, but there is no sufficient experimental evidence to support the estimations. In this work, we proved that the Agrobacterium strain CCBAU 81181, which was originally isolated from the root nodules of Onobrychis viciaefolia, and a symbiotic strain of Sinorhizobium meliloti CCBAU 10062 could coinhabit the root nodules of Melilotus dentatus. Analyses were performed by using a fluorescence marker, reisolation of bacteria from nodules, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole cellular proteins, and polymerase chain reaction amplification of symbiotic genes. The inoculation of A. tumefaciens CCBAU 81181 did not affect the growth and nodulation of plants. CCBAU 81181 and 24 other Agrobacterium strains isolated from nodules were incapable of nodulating on their original or alternative host and 22 strains of these strains were endophytes in the roots and stems of their hosts. Also, the tumor-inducing A. tumefaciens strains IAM 13129(T) and C58 were found capable of entering the roots of Glycyrrhiza pallidiflora, but did not cause pathogenic symptoms. With these results, we conclude that A. tumefaciens strains could be endophytic bacteria in the roots, stems, and root nodules. This finding partially explains why Agrobacterium strains were frequently isolated from the surface-sterilized nodules.

Horizontal gene transfer from Agrobacterium to plants.

PubMed

Matveeva, Tatiana V; Lutova, Ludmila A

2014-01-01

Most genetic engineering of plants uses Agrobacterium mediated transformation to introduce novel gene content. In nature, insertion of T-DNA in the plant genome and its subsequent transfer via sexual reproduction has been shown in several species in the genera Nicotiana and Linaria. In these natural examples of horizontal gene transfer from Agrobacterium to plants, the T-DNA donor is assumed to be a mikimopine strain of A. rhizogenes. A sequence homologous to the T-DNA of the Ri plasmid of Agrobacterium rhizogenes was found in the genome of untransformed Nicotiana glauca about 30 years ago, and was named "cellular T-DNA" (cT-DNA). It represents an imperfect inverted repeat and contains homologs of several T-DNA oncogenes (NgrolB, NgrolC, NgORF13, NgORF14) and an opine synthesis gene (Ngmis). A similar cT-DNA has also been found in other species of the genus Nicotiana. These presumably ancient homologs of T-DNA genes are still expressed, indicating that they may play a role in the evolution of these plants. Recently T-DNA has been detected and characterized in Linaria vulgaris and L. dalmatica. In Linaria vulgaris the cT-DNA is present in two copies and organized as a tandem imperfect direct repeat, containing LvORF2, LvORF3, LvORF8, LvrolA, LvrolB, LvrolC, LvORF13, LvORF14, and the Lvmis genes. All L. vulgaris and L. dalmatica plants screened contained the same T-DNA oncogenes and the mis gene. Evidence suggests that there were several independent T-DNA integration events into the genomes of these plant genera. We speculate that ancient plants transformed by A. rhizogenes might have acquired a selective advantage in competition with the parental species. Thus, the events of T-DNA insertion in the plant genome might have affected their evolution, resulting in the creation of new plant species. In this review we focus on the structure and functions of cT-DNA in Linaria and Nicotiana and discuss their possible evolutionary role.

Impact of biological amendments on Agrobacterium tumefaciens soil survival

USDA-ARS?s Scientific Manuscript database

Paradox, the primary walnut rootstock used in California, is susceptible to Agrobacterium tumefaciens, which causes crown gall. While A. tumefaciens is susceptible to commonly used fumigants such as methyl bromide (MeBr) and Telone-C35 (1,3-dichloropropene and chloropicrin), these fumigants also sig...

Use of Ti plasmid DNA probes for determining tumorigenicity of agrobacterium strains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burr, T.J.; Norelli, J.L.; Katz, B.H.

1990-06-01

Probes consisting of T-DNA genes from the Ti plasmid of Agrobacterium tumefaciens were used for determining tumorigenicity of strains. Two {sup 32}P-labeled probes hybridized with 28 of 28 tumorigenic strains of the pathogen but not with 20 of 22 nontumorigenic strains. One probe, pTHE17, consists of all but the far left portion of the T-DNA of strain C58. Probe SmaI7 consists of SmaI fragment 7 of pTiC58, including onc genes 1, 4, and 6a and most of 2. Another probe, pAL4044, consisting of the vir region of strain Ach-5, hybridized with several nontumorigenic as well as tumorigenic strains. Colony hybridizationsmore » were done with 28 tumorigenic and 22 nontumorigenic Agrobacterium strains. About 10{sup 6} CFU of the different tumorigenic strains were detectable with this method. Southern analyses confirmed the presence or absence of Ti plasmids in strains for which tumorigenicity was questioned. Colony hybridization with the T-DNA probes provides a rapid and sensitive means for determining the tumorigenic nature of Agrobacterium strains.« less

Agrobacterium-mediated transformation of the haploid liverwort Marchantia polymorpha L., an emerging model for plant biology.

PubMed

Ishizaki, Kimitsune; Chiyoda, Shota; Yamato, Katsuyuki T; Kohchi, Takayuki

2008-07-01

Agrobacterium-mediated transformation has not been practical in pteridophytes, bryophytes and algae to date, although it is commonly used in model plants including Arabidopsis and rice. Here we present a rapid Agrobacterium-mediated transformation system for the haploid liverwort Marchantia polymorpha L. using immature thalli developed from spores. Hundreds of hygromycin-resistant plants per sporangium were obtained by co-cultivation of immature thalli with Agrobacterium carrying the binary vector that contains a reporter, the beta-glucuronidase (GUS) gene with an intron, and a selection marker, the hygromycin phosphotransferase (hpt) gene. In this system, individual gemmae, which arise asexually from single initial cells, were analyzed as isogenic transformants. GUS activity staining showed that all hygromycin-resistant plants examined expressed the GUS transgene in planta. DNA analyses verified random integration of 1-5 copies of the intact T-DNA between the right and the left borders into the M. polymorpha genome. The efficient and rapid Agrobacterium-mediated transformation of M. polymorpha should provide molecular techniques to facilitate comparative genomics, taking advantage of this unique model plant that retains many features of the common ancestor of land plants.

Detection of Oil Palm Root Penetration by Agrobacterium-Mediated Transformed Ganoderma boninense, Expressing Green Fluorescent Protein.

PubMed

Govender, Nisha; Wong, Mui-Yun

2017-04-01

A highly efficient and reproducible Agrobacterium-mediated transformation protocol for Ganoderma boninense was developed to facilitate observation of the early stage infection of basal stem rot (BSR). The method was proven amenable to different explants (basidiospore, protoplast, and mycelium) of G. boninense. The transformation efficiency was highest (62%) under a treatment combination of protoplast explant and Agrobacterium strain LBA4404, with successful expression of an hyg marker gene and gus-gfp fusion gene under the control of heterologous p416 glyceraldehyde 3-phosphate dehydrogenase promoter. Optimal transformation conditions included a 1:100 Agrobacterium/explant ratio, induction of Agrobacterium virulence genes in the presence of 250 μm acetosyringone, co-cultivation at 22°C for 2 days on nitrocellulose membrane overlaid on an induction medium, and regeneration of transformants on potato glucose agar prepared with 0.6 M sucrose and 20 mM phosphate buffer. Evaluated transformants were able to infect root tissues of oil palm plantlets with needle-like microhyphae during the penetration event. The availability of this model pathogen system for BSR may lead to a better understanding of the pathogenicity factors associated with G. boninense penetration into oil palm roots.

Agrobacterium-mediated transformation of Fraxinus pennsylvanica hypocotyls and plant regeneration

Treesearch

Ningxia Du; Paula M. Pijut

2009-01-01

A genetic transformation protocol for green ash (Fraxinus pennsylvanica) hypocotyl explants was developed. Green ash hypocotyls were transformed using Agrobacterium tumefaciens strain EHA105 harboring binary vector pq35GR containing the neomycin phosphotransferase (nptII) and β-glucuronidase (GUS) fusion...

Component identification of electron transport chains in curdlan-producing Agrobacterium sp. ATCC 31749 and its genome-specific prediction using comparative genome and phylogenetic trees analysis.

PubMed

Zhang, Hongtao; Setubal, Joao Carlos; Zhan, Xiaobei; Zheng, Zhiyong; Yu, Lijun; Wu, Jianrong; Chen, Dingqiang

2011-06-01

Agrobacterium sp. ATCC 31749 (formerly named Alcaligenes faecalis var. myxogenes) is a non-pathogenic aerobic soil bacterium used in large scale biotechnological production of curdlan. However, little is known about its genomic information. DNA partial sequence of electron transport chains (ETCs) protein genes were obtained in order to understand the components of ETC and genomic-specificity in Agrobacterium sp. ATCC 31749. Degenerate primers were designed according to ETC conserved sequences in other reported species. DNA partial sequences of ETC genes in Agrobacterium sp. ATCC 31749 were cloned by the PCR method using degenerate primers. Based on comparative genomic analysis, nine electron transport elements were ascertained, including NADH ubiquinone oxidoreductase, succinate dehydrogenase complex II, complex III, cytochrome c, ubiquinone biosynthesis protein ubiB, cytochrome d terminal oxidase, cytochrome bo terminal oxidase, cytochrome cbb (3)-type terminal oxidase and cytochrome caa (3)-type terminal oxidase. Similarity and phylogenetic analyses of these genes revealed that among fully sequenced Agrobacterium species, Agrobacterium sp. ATCC 31749 is closest to Agrobacterium tumefaciens C58. Based on these results a comprehensive ETC model for Agrobacterium sp. ATCC 31749 is proposed.

Agrobacterium-mediated virus-induced gene silencing assay in cotton.

PubMed

Gao, Xiquan; Britt, Robert C; Shan, Libo; He, Ping

2011-08-20

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation(1). To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions. Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation(2,3). Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies(3,4). As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation. In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development(6), and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves(7), providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

PubMed Central

Gao, Xiquan; Britt Jr., Robert C.; Shan, Libo; He, Ping

2011-01-01

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions. Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation. In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino

Factors enhancing Agrobacterium tumefaciens-mediated gene transfer in peanut (Arachis hypogaea L.)

NASA Technical Reports Server (NTRS)

Egnin, M.; Mora, A.; Prakash, C. S.; Mortley, D. G. (Principal Investigator)

1998-01-01

Parameters enhancing Agrobacterium-mediated transfer of foreign genes to peanut (Arachis hypogaea L.) cells were investigated. An intron-containing beta-glucuronidase uidA (gusA) gene under the transcriptional control of CaMV 35S promoter served as a reporter. Transformation frequency was evaluated by scoring the number of sectors expressing GUS activity on leaf and epicotyl explants. The 'Valencia Select' market type cv. New Mexico was more amenable to Agrobacterium transformation than the 'runner' market type cultivars tested (Florunner, Georgia Runner, Sunrunner, or South Runner). The disarmed Agrobacterium tumefaciens strain EHA101 was superior in facilitating the transfer of uidA gene to peanut cells compared to the disarmed strain C58. Rinsing of explants in half-strength Murashige-Skoog (MS) media prior to infection by Agrobacterium significantly increased the transformation efficiency. The use of cocultivation media containing high auxin [1.0 or 2.5 mg/l (4.53 micromolar or 11.31 micromolar) 2,4-D] and low cytokinin [0.25 or 0.5 mg/l (1.0 micromolar or 2.0 micromolar) BA] promoted higher transformation than either hormone-free or thidiazuron-containing medium. The polarity of the epicotyl during cocultivation was important; explants incubated in an inverted (vertically) manner followed by a vertically upright position resulted in improved transformation and shoot regeneration frequencies. Preculture of explants in MS basal medium or with 2.5 mg thidiazuron per l prior to infection drastically decreased the number of transformed zones. The optimized protocol was used to obtain transient transformation frequencies ranging from 12% to 36% for leaf explants, 15% to 42% for epicotyls. Initial evidence of transformation was obtained by polymerase chain reaction and subsequently confirmed by Southern analysis of regenerated plants.

A new QRT-PCR assay designed for the differentiation between elements provided from Agrobacterium sp. in GMOs plant events and natural Agrobacterium sp. bacteria.

PubMed

Nabi, Nesrine; Chaouachi, Maher; Zellama, Mohamed Salem; Ben Hafsa, Ahmed; Mrabet, Besma; Saïd, Khaled; Fathia, Harzallah Skhiri

2016-04-01

The question asked in the present work was how to differentiate between contamination of field samples with and GM plants contained sequences provided from this bacterium in order to avoid false positives in the frame of the detection and the quantification of GMO. For this, new set of primers and corresponding TaqMan Minor Groove Binder (MGB) probes were designed to target Agrobacterium sp. using the tumor-morphology-shooty gene (TMS1). Final standard curves were calculated for each pathogen by plotting the threshold cycle value against the bacterial number (log (colony forming units) per milliliter) via linear regression. The method designed was highly specific and sensitive, with a detection limit of 10CFU/ml. No significant cross-reaction was observed. Results from this study showed that TaqMan real-time PCR, is potentially an effective method for the rapid and reliable quantification of Agrobacterium sp. in samples containing GMO or non GMO samples. Copyright © 2015 Elsevier Ltd. All rights reserved.

Susceptibility of Juglans Species andInterspecific Hybrids to Agrobacterium tumefaciens

Treesearch

James R. McKenna; Lynn Epstein

2003-01-01

Crown gall, caused by the common soil-borne bacterium Agrobacterium tumefaciens, can be an economic problem in walnut nurseries and production orchards in Caliiornia. The principal rootstocks used for commercial walnut production in California are the native Northern California black walnut, Juglans hindsii, and "Paradox,...

Agrobacterium tumefaciens-mediated transformation of oleaginous yeast Lipomyces species

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dai, Ziyu; Deng, Shuang; Culley, David E.

Background: Because of interest in the production of renewable bio-hydrocarbon fuels, various living organisms have been explored for their potential use in producing fuels and chemicals. The oil-producing (oleaginous) yeast Lipomyces starkeyi is the subject of active research regarding the production of lipids using a wide variety of carbon and nutrient sources. The genome of L. starkeyi has been published, which opens the door to production strain improvements using the tools of synthetic biology and metabolic engineering. However, using these tools for strain improvement requires the establishment of effective and reliable transformation methods with suitable selectable markers (antibiotic resistance ormore » auxotrophic marker genes) and the necessary genetic elements (promoters and terminators) for expression of introduced genes. Chemical-based methods have been published, but suffer from low efficiency or the requirement for targeting to rRNA loci. To address these problems, Agrobacterium-mediated transformation was investigated as an alternative method for L. starkeyi and other Lipomyces species. Results: In this study, Agrobacterium-mediated transformation was demonstrated to be effective in the transformation of both L. starkeyi and other Lipomyces species and that the introduced DNA can be reliably integrated into the chromosomes of these species. The gene deletion of Ku70 and Pex10 was also demonstrated in L. starkeyi. In addition to the bacterial antibiotic selection marker gene hygromycin B phosphotransferase, the bacterial -glucuronidase reporter gene under the control of L. starkeyi translation elongation factor 1 promoter was also stably expressed in seven different Lipomyces species. Conclusion: The results from this study clearly demonstrate that Agrobacterium-mediated transformation is a reliable genetic tool for gene deletion and integration and expression of heterologous genes in L. starkeyi and other Lipomyces species.« less

Agrobacterium tumefaciens-mediated transformation of oleaginous yeast Lipomyces species.

PubMed

Dai, Ziyu; Deng, Shuang; Culley, David E; Bruno, Kenneth S; Magnuson, Jon K

2017-08-01

Interest in using renewable sources of carbon, especially lignocellulosic biomass, for the production of hydrocarbon fuels and chemicals has fueled interest in exploring various organisms capable of producing hydrocarbon biofuels and chemicals or their precursors. The oleaginous (oil-producing) yeast Lipomyces starkeyi is the subject of active research regarding the production of triacylglycerides as hydrocarbon fuel precursors using a variety of carbohydrate and nutrient sources. The genome of L. starkeyi has been published, which opens the door to production strain improvements through the development and use of the tools of synthetic biology for this oleaginous species. The first step in establishment of synthetic biology tools for an organism is the development of effective and reliable transformation methods with suitable selectable marker genes and demonstration of the utility of the genetic elements needed for expression of introduced genes or deletion of endogenous genes. Chemical-based methods of transformation have been published but suffer from low efficiency. To address these problems, Agrobacterium-mediated transformation was investigated as an alternative method for L. starkeyi and other Lipomyces species. In this study, Agrobacterium-mediated transformation was demonstrated to be effective in the transformation of both L. starkeyi and other Lipomyces species. The deletion of the peroxisomal biogenesis factor 10 gene was also demonstrated in L. starkeyi. In addition to the bacterial antibiotic selection marker gene hygromycin B phosphotransferase, the bacterial β-glucuronidase reporter gene under the control of L. starkeyi translation elongation factor 1α promoter was also stably expressed in six different Lipomyces species. The results from this study demonstrate that Agrobacterium-mediated transformation is a reliable and effective genetic tool for homologous recombination and expression of heterologous genes in L. starkeyi and other Lipomyces

Assessment of factors affecting Agrobacterium-mediated genetic transformation of the unicellular green alga, Chlorella vulgaris.

PubMed

Cha, Thye San; Yee, Willy; Aziz, Ahmad

2012-04-01

The successful establishment of an Agrobacterium-mediated transformation method and optimisation of six critical parameters known to influence the efficacy of Agrobacterium T-DNA transfer in the unicellular microalga Chlorella vulgaris (UMT-M1) are reported. Agrobacterium tumefaciens strain LBA4404 harbouring the binary vector pCAMBIA1304 containing the gfp:gusA fusion reporter and a hygromycin phosphotransferase (hpt) selectable marker driven by the CaMV35S promoter were used for transformation. Transformation frequency was assessed by monitoring transient β-glucuronidase (GUS) expression 2 days post-infection. It was found that co-cultivation temperature at 24°C, co-cultivation medium at pH 5.5, 3 days of co-cultivation, 150 μM acetosyringone, Agrobacterium density of 1.0 units (OD(600)) and 2 days of pre-culture were optimum variables which produced the highest number of GUS-positive cells (8.8-20.1%) when each of these parameters was optimised individually. Transformation conducted with the combination of all optimal parameters above produced 25.0% of GUS-positive cells, which was almost a threefold increase from 8.9% obtained from un-optimised parameters. Evidence of transformation was further confirmed in 30% of 30 randomly-selected hygromycin B (20 mg L(-1)) resistant colonies by polymerase chain reaction (PCR) using gfp:gusA and hpt-specific primers. The developed transformation method is expected to facilitate the genetic improvement of this commercially-important microalga.

Application of succulent plant leaves for Agrobacterium infiltration-mediated protein production

USDA-ARS?s Scientific Manuscript database

Infiltration of tobacco leaves with a suspension of Agrobacterium tumefaciens harboring a binary plant expression plasmid provides a convenient method for laboratory scale protein production. When expressing plant cell wall degrading enzymes in the widely used tobacco (Nicotiana benthamiana), diffic...

Efficient and genotype-independent Agrobacterium--mediated tomato transformation.

PubMed

Park, Sung Hun; Morris, Jay L; Park, Jung Eun; Hirschi, Kendal D; Smith, Roberta H

2003-10-01

An efficient method to transform five cultivars of tomato (Lycopersicon esculentum), Micro-Tom, Red Cherry, Rubion, Piedmont, and E6203 is reported. A comparison was made of leaf, cotyledon, and hypocotyl explants on 7 different regeneration media without Agrobacterium tumefaciens cocultivation and on 11 different media with cocultivation. Although all cultivars and explants formed callus and regenerated on the initial 7 media, cocultivation with A. tumefaciens significantly reduced the callus induction and regeneration. From these experiments, a transformation methodology using either hypocotyls or cotyledons cultured for one day on BA 1 mgL-1, NAA 0.1 mgL-1 and 3 days cocultivation with the Agrobacterium on this same medium followed by a transfer to a medium with zeatin 2 mgL-1 and IAA 0.1 mgL-1 for 4-6 weeks resulted in a greater than 20% transformation frequency for all five cultivars tested. In this transformation method, no feeder layers of tobacco, petunia or tomato suspension cultures were used, and the subculture media was minimal. Stable integration and transmission of the transgene in T1 generation plants were confirmed by Southern blot analysis. This procedure represents a simple, efficient and general means of transforming tomato.

Agrobacterium tumefaciens Promotes Tumor Induction by Modulating Pathogen Defense in Arabidopsis thaliana[W

PubMed Central

Lee, Chil-Woo; Efetova, Marina; Engelmann, Julia C; Kramell, Robert; Wasternack, Claus; Ludwig-Müller, Jutta; Hedrich, Rainer; Deeken, Rosalia

2009-01-01

Agrobacterium tumefaciens causes crown gall disease by transferring and integrating bacterial DNA (T-DNA) into the plant genome. To examine the physiological changes and adaptations during Agrobacterium-induced tumor development, we compared the profiles of salicylic acid (SA), ethylene (ET), jasmonic acid (JA), and auxin (indole-3-acetic acid [IAA]) with changes in the Arabidopsis thaliana transcriptome. Our data indicate that host responses were much stronger toward the oncogenic strain C58 than to the disarmed strain GV3101 and that auxin acts as a key modulator of the Arabidopsis–Agrobacterium interaction. At initiation of infection, elevated levels of IAA and ET were associated with the induction of host genes involved in IAA, but not ET signaling. After T-DNA integration, SA as well as IAA and ET accumulated, but JA did not. This did not correlate with SA-controlled pathogenesis-related gene expression in the host, although high SA levels in mutant plants prevented tumor development, while low levels promoted it. Our data are consistent with a scenario in which ET and later on SA control virulence of agrobacteria, whereas ET and auxin stimulate neovascularization during tumor formation. We suggest that crosstalk among IAA, ET, and SA balances pathogen defense launched by the host and tumor growth initiated by agrobacteria. PMID:19794116

Nuclear Data Sheets A = 84

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abriola, D.; Sonzogni, A.; Bostan,M. Erturk,S.

The evaluated spectroscopic data are presented for 12 known nuclides of mass 84 (Ga, Ge, As, Se, Br, Kr, Rb, Sr, Y, Zr, Nb, Mo). Except for the stable nuclides {sup 84}Sr and {sup 84}Kr, extensive new data are available for all the other nuclides since the 1997 evaluation by J.K. Tuli (1997Tu02) of A = 84 nuclides. Many precise Penning-trap mass measurements since AME-2003 for A = 84 nuclides (2009Re03,2008Ha23,2008We10,2007Ke09,2006Ka48,2006De36,2006Ri15) have resulted in improved Q values and separation energies. However, many deficiencies still remain. Some examples are given below. Excited-state data for {sup 84}Ga and {sup 84}As are nonexistent,more » and those for {sup 84}Ge are scarce. The radioactive decay schemes of {sup 84}Ga, {sup 84}Ge, {sup 84}Se, {sup 84}Y (39.5 min), {sup 84}Y (4.6 s), {sup 84}Zr and {sup 84}Nb suffer from incompleteness and that for {sup 84}Mo decay is not known at all. The energy ordering of the two activities (39.5 min and and 4.6 s) of {sup 84}Y is not well established, although, high-spin with tentative spin-parity of (6+) is adopted here as the ground state of {sup 84}Y based on weak arguments. From a conference report published in 2000, it is clear that extensive experiments were done to investigate decays of {sup 84}Zr and {sup 84}Y, but details of these studies never appeared in literature and none were made available to the evaluators when requested from original authors. This evaluation was carried out as part of ENSDF workshop for Nuclear Structure and Decay Data Evaluators, organized and hosted by the 'Horia Hulubei' National Institute for Physics and Nuclear Engineering, Bucharest, Romania during March 30, 2009 - April 3, 2009. Names of the evaluators principally responsible for evaluation of individual nuclides are given under the respective Adopted data sets.« less

Mutants of Agrobacterium tumefaciens with elevated vir gene expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pazour, G.J.; Ta, C.N.; Das, A.

1991-08-15

Expression of Agrobacterium tumefaciens virulence (vir) genes requires virA, virG, and a plant-derived inducing compound such as acetosyringone. To identify the critical functional domains of virA and virG, a mutational approach was used. Agrobacterium A136 harboring plasmid pGP159, which contains virA, virG, and a reporter virB:lacZ gene fusion, was mutagenized with UV light or nitrosoguanidine. Survivors that formed blue colonies on a plate containing 5-bromo-4-chloro-3-indolyl beta-D-galactoside were isolated and analyzed. Quantification of beta-galactosidase activity in liquid assays identified nine mutant strains. By plasmid reconstruction and other procedures, all mutations mapped to the virA locus. These mutations caused an 11- tomore » 560-fold increase in the vegetative level of virB:lacZ reporter gene expression. DNA sequence analysis showed that the mutations are located in four regions of VirA: transmembrane domain one, the active site, a glycine-rich region with homology to ATP-binding sites, and a region at the C terminus that has homology to the N terminus of VirG.« less

Agrobacterium-assisted selenium nanoparticles: molecular aspect of antifungal activity

NASA Astrophysics Data System (ADS)

Kumar, Anil; Bera, Smritilekha; Singh, Man; Mondal, Dhananjoy

2018-03-01

Selenium nanoparticles (SeNPs) were synthesized through the bioreduction of sodium selenite (Na2SeO3) using gram-negative agrobacterium (AGBT) species. Subsequently, their physicochemical properties (pH, viscosity and surface tension) and medicinal activities as anti-dermatophyte against soil keratinophilic fungi at the molecular level were assessed. UV-visible and FTIR spectroscopic data of the biologically synthesized SeNPs were then recorded for confirming the presence of native biological materials adhered to nanoparticles, which are inherently required to enhance the stability and solubility through inhibition of the nanoparticle’s natural aggregation and agglomeration. The λ max value between 290-300 nm in the absorption spectra of the biogenic materials in different concentrations of the Na2SeO3 corroborated the presence of SeNPs in the solution. The interaction of SeNPs in solution state was further studied through the determination of pH, viscosity and surface tension values of agrobacterium-derived SeNPs in different solvents. The pH value of SeNPs dispersed in water is reported as above 7.0 and the average viscosity, and surface tensions of the SeNPs are appeared as near to the water. The particle size distribution was further determined by DLS and the highest % of particle size of the synthesized SeNPs is found in between 200-300 nm. The anti-dermatophyte activity and molecular interaction with fungi DNA molecules were assessed providing the highest anti-dermatophyte activity at 0.1 M concentration and it is observed that the quantities and qualities of fungi DNA were affected by SeNPs. Considering all the outcomes of the studies together, our findings suggest that agrobacterium-mediated synthesis of SeNPs is dependent on bacterial metabolisms but not on the concentration of Na2SeO3 and are promising selenium-derived species with potential application in the prevention of fungal infection through denaturation of fungi DNA.

Agrobacterium-mediated transformation of Easter lily (Lilium longiflorum cv. Nellie White)

USDA-ARS?s Scientific Manuscript database

Conditions were optimized for transient transformation of Lilium longiflorum cv. Nellie White using Agrobacterium tumefaciens. Bulb scale and basal meristem explants were inoculated with A. tumefaciens strain AGL1 containing the binary vector pCAMBIA 2301 which has the uidA gene that codes for ß-gl...

A Pyranose-2-Phosphate Motif Is Responsible for Both Antibiotic Import and Quorum-Sensing Regulation in Agrobacterium tumefaciens

PubMed Central

El Sahili, Abbas; Li, Si-Zhe; Lang, Julien; Virus, Cornelia; Planamente, Sara; Ahmar, Mohammed; Guimaraes, Beatriz G.; Aumont-Nicaise, Magali; Vigouroux, Armelle; Soulère, Laurent; Reader, John; Queneau, Yves; Faure, Denis; Moréra, Solange

2015-01-01

Periplasmic binding proteins (PBPs) in association with ABC transporters select and import a wide variety of ligands into bacterial cytoplasm. They can also take up toxic molecules, as observed in the case of the phytopathogen Agrobacterium tumefaciens strain C58. This organism contains a PBP called AccA that mediates the import of the antibiotic agrocin 84, as well as the opine agrocinopine A that acts as both a nutrient and a signalling molecule for the dissemination of virulence genes through quorum-sensing. Here, we characterized the binding mode of AccA using purified agrocin 84 and synthetic agrocinopine A by X-ray crystallography at very high resolution and performed affinity measurements. Structural and affinity analyses revealed that AccA recognizes an uncommon and specific motif, a pyranose-2-phosphate moiety which is present in both imported molecules via the L-arabinopyranose moiety in agrocinopine A and the D-glucopyranose moiety in agrocin 84. We hypothesized that AccA is a gateway allowing the import of any compound possessing a pyranose-2-phosphate motif at one end. This was structurally and functionally confirmed by experiments using four synthetic compounds: agrocinopine 3’-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate. By combining affinity measurements and in vivo assays, we demonstrated that both L-arabinose-2-phosphate and D-glucose-2-phosphate, which are the AccF mediated degradation products of agrocinopine A and agrocin 84 respectively, interact with the master transcriptional regulator AccR and activate the quorum-sensing signal synthesis and Ti plasmid transfer in A. tumefaciens C58. Our findings shed light on the role of agrocinopine and antibiotic agrocin 84 on quorum-sensing regulation in A. tumefaciens and reveal how the PBP AccA acts as vehicle for the importation of both molecules by means of a key-recognition motif. It also opens future possibilities for the rational design of

A Pyranose-2-Phosphate Motif Is Responsible for Both Antibiotic Import and Quorum-Sensing Regulation in Agrobacterium tumefaciens.

PubMed

El Sahili, Abbas; Li, Si-Zhe; Lang, Julien; Virus, Cornelia; Planamente, Sara; Ahmar, Mohammed; Guimaraes, Beatriz G; Aumont-Nicaise, Magali; Vigouroux, Armelle; Soulère, Laurent; Reader, John; Queneau, Yves; Faure, Denis; Moréra, Solange

2015-08-01

Periplasmic binding proteins (PBPs) in association with ABC transporters select and import a wide variety of ligands into bacterial cytoplasm. They can also take up toxic molecules, as observed in the case of the phytopathogen Agrobacterium tumefaciens strain C58. This organism contains a PBP called AccA that mediates the import of the antibiotic agrocin 84, as well as the opine agrocinopine A that acts as both a nutrient and a signalling molecule for the dissemination of virulence genes through quorum-sensing. Here, we characterized the binding mode of AccA using purified agrocin 84 and synthetic agrocinopine A by X-ray crystallography at very high resolution and performed affinity measurements. Structural and affinity analyses revealed that AccA recognizes an uncommon and specific motif, a pyranose-2-phosphate moiety which is present in both imported molecules via the L-arabinopyranose moiety in agrocinopine A and the D-glucopyranose moiety in agrocin 84. We hypothesized that AccA is a gateway allowing the import of any compound possessing a pyranose-2-phosphate motif at one end. This was structurally and functionally confirmed by experiments using four synthetic compounds: agrocinopine 3'-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate. By combining affinity measurements and in vivo assays, we demonstrated that both L-arabinose-2-phosphate and D-glucose-2-phosphate, which are the AccF mediated degradation products of agrocinopine A and agrocin 84 respectively, interact with the master transcriptional regulator AccR and activate the quorum-sensing signal synthesis and Ti plasmid transfer in A. tumefaciens C58. Our findings shed light on the role of agrocinopine and antibiotic agrocin 84 on quorum-sensing regulation in A. tumefaciens and reveal how the PBP AccA acts as vehicle for the importation of both molecules by means of a key-recognition motif. It also opens future possibilities for the rational design of

Development of an Agrobacterium-Mediated Stable Transformation Method for the Sensitive Plant Mimosa pudica

PubMed Central

Mano, Hiroaki; Fujii, Tomomi; Sumikawa, Naomi; Hiwatashi, Yuji; Hasebe, Mitsuyasu

2014-01-01

The sensitive plant Mimosa pudica has long attracted the interest of researchers due to its spectacular leaf movements in response to touch or other external stimuli. Although various aspects of this seismonastic movement have been elucidated by histological, physiological, biochemical, and behavioral approaches, the lack of reverse genetic tools has hampered the investigation of molecular mechanisms involved in these processes. To overcome this obstacle, we developed an efficient genetic transformation method for M. pudica mediated by Agrobacterium tumefaciens (Agrobacterium). We found that the cotyledonary node explant is suitable for Agrobacterium-mediated transformation because of its high frequency of shoot formation, which was most efficiently induced on medium containing 0.5 µg/ml of a synthetic cytokinin, 6-benzylaminopurine (BAP). Transformation efficiency of cotyledonary node cells was improved from almost 0 to 30.8 positive signals arising from the intron-sGFP reporter gene by using Agrobacterium carrying a super-binary vector pSB111 and stabilizing the pH of the co-cultivation medium with 2-(N-morpholino)ethanesulfonic acid (MES) buffer. Furthermore, treatment of the explants with the detergent Silwet L-77 prior to co-cultivation led to a two-fold increase in the number of transformed shoot buds. Rooting of the regenerated shoots was efficiently induced by cultivation on irrigated vermiculite. The entire procedure for generating transgenic plants achieved a transformation frequency of 18.8%, which is comparable to frequencies obtained for other recalcitrant legumes, such as soybean (Glycine max) and pea (Pisum sativum). The transgene was stably integrated into the host genome and was inherited across generations, without affecting the seismonastic or nyctinastic movements of the plants. This transformation method thus provides an effective genetic tool for studying genes involved in M. pudica movements. PMID:24533121

Development of an Agrobacterium-mediated stable transformation method for the sensitive plant Mimosa pudica.

PubMed

Mano, Hiroaki; Fujii, Tomomi; Sumikawa, Naomi; Hiwatashi, Yuji; Hasebe, Mitsuyasu

2014-01-01

The sensitive plant Mimosa pudica has long attracted the interest of researchers due to its spectacular leaf movements in response to touch or other external stimuli. Although various aspects of this seismonastic movement have been elucidated by histological, physiological, biochemical, and behavioral approaches, the lack of reverse genetic tools has hampered the investigation of molecular mechanisms involved in these processes. To overcome this obstacle, we developed an efficient genetic transformation method for M. pudica mediated by Agrobacterium tumefaciens (Agrobacterium). We found that the cotyledonary node explant is suitable for Agrobacterium-mediated transformation because of its high frequency of shoot formation, which was most efficiently induced on medium containing 0.5 µg/ml of a synthetic cytokinin, 6-benzylaminopurine (BAP). Transformation efficiency of cotyledonary node cells was improved from almost 0 to 30.8 positive signals arising from the intron-sGFP reporter gene by using Agrobacterium carrying a super-binary vector pSB111 and stabilizing the pH of the co-cultivation medium with 2-(N-morpholino)ethanesulfonic acid (MES) buffer. Furthermore, treatment of the explants with the detergent Silwet L-77 prior to co-cultivation led to a two-fold increase in the number of transformed shoot buds. Rooting of the regenerated shoots was efficiently induced by cultivation on irrigated vermiculite. The entire procedure for generating transgenic plants achieved a transformation frequency of 18.8%, which is comparable to frequencies obtained for other recalcitrant legumes, such as soybean (Glycine max) and pea (Pisum sativum). The transgene was stably integrated into the host genome and was inherited across generations, without affecting the seismonastic or nyctinastic movements of the plants. This transformation method thus provides an effective genetic tool for studying genes involved in M. pudica movements.

Biolistic- and Agrobacterium-mediated transformation protocols for wheat.

PubMed

Tamás-Nyitrai, Cecília; Jones, Huw D; Tamás, László

2012-01-01

After rice, wheat is considered to be the most important world food crop, and the demand for high-quality wheat flour is increasing. Although there are no GM varieties currently grown, wheat is an important target for biotechnology, and we anticipate that GM wheat will be commercially available in 10-15 years. In this chapter, we summarize the main features and challenges of wheat transformation and then describe detailed protocols for the production of transgenic wheat plants both by biolistic and Agrobacterium-mediated DNA-delivery. Although these methods are used mainly for bread wheat (Triticum aestivum L.), they can also be successfully applied, with slight modifications, to tetraploid durum wheat (T. turgidum L. var. durum). The appropriate size and developmental stage of explants (immature embryo-derived scutella), the conditions to produce embryogenic callus tissues, and the methods to regenerate transgenic plants under increasing selection pressure are provided in the protocol. To illustrate the application of herbicide selection system, we have chosen to describe the use of the plasmid pAHC25 for biolistic transformation, while for Agrobacterium-mediated transformation the binary vector pAL156 (incorporating both the bar gene and the uidA gene) has been chosen. Beside the step-by-step methodology for obtaining stably transformed and normal fertile plants, procedures for screening and testing transgenic wheat plants are also discussed.

Regulation of hydantoin-hydrolyzing enzyme expression in Agrobacterium tumefaciens strain RU-AE01.

PubMed

Jiwaji, Meesbah; Dorrington, Rosemary Ann

2009-10-01

Optically pure D-: amino acids, like D-: hydroxyphenylglycine, are used in the semi-synthetic production of pharmaceuticals. They are synthesized industrially via the biocatalytic hydrolysis of p-hydroxyphenylhydantoin using enzymes derived from Agrobacterium tumefaciens strains. The reaction proceeds via a three-step pathway: (a) the ring-opening cleavage of the hydantoin ring by a D-: hydantoinase (encoded by hyuH), (b) conversion of the resultant D-: N-carbamylamino acid to the corresponding amino acid by a D-: N-carbamoylase (encoded by hyuC), and (c) chemical or enzymatic racemization of the un-reacted hydantoin substrate. While the structure and biochemical properties of these enzymes are well understood, little is known about their origin, their function, and their regulation in the native host. We investigated the mechanisms involved in the regulation of expression of the hydantoinase and N-carbamoylase enzyme activity in A. tumefaciens strain RU-AE01. We present evidence for a complex regulatory network that responds to the growth status of the cells, the presence of inducer, and nitrogen catabolite repression. Deletion analysis and site-directed mutagenesis were used to identify regulatory elements involved in transcriptional regulation of hyuH and hyuC expression. Finally, a comparison between the hyu gene clusters in several Agrobacterium strains provides insight into the function of D-: selective hydantoin-hydrolyzing enzyme systems in Agrobacterium species.

Nuclear Data Sheets for A = 84

NASA Astrophysics Data System (ADS)

Abriola, Daniel; Bostan, Melih; Erturk, Sefa; Fadil, Manssour; Galan, Monica; Juutinen, Sakari; Kibédi, Tibor; Kondev, Filip; Luca, Aurelian; Negret, Alexandru; Nica, Ninel; Pfeiffer, Bernd; Singh, Balraj; Sonzogni, Alejandro; Timar, Janos; Tuli, Jagdish; Venkova, Tsanka; Zuber, Kazimierz

2009-11-01

The evaluated spectroscopic data are presented for 12 known nuclides of mass 84 (Ga, Ge, As, Se, Br, Kr, Rb, Sr, Y, Zr, Nb, Mo). Except for the stable nuclides 84Sr and 84Kr, extensive new data are available for all the other nuclides since the 1997 evaluation by J.K. Tuli (1997Tu02) of A = 84 nuclides. Many precise Penning-trap mass measurements since AME-2003 for A = 84 nuclides (2009Re03,2008Ha23,2008We10,2007Ke09,2006Ka48,2006De36,2006Ri15) have resulted in improved Q values and separation energies. However, many deficiencies still remain. Some examples are given below. Excited-state data for 84Ga and 84As are nonexistent, and those for 84Ge are scarce. The radioactive decay schemes of 84Ga, 84Ge, 84Se, 84Y (39.5 min), 84Y (4.6 s), 84Zr and 84Nb suffer from incompleteness and that for 84Mo decay is not known at all. The energy ordering of the two activities (39.5 min and and 4.6 s) of 84Y is not well established, although, high-spin with tentative spin-parity of (6+) is adopted here as the ground state of 84Y based on weak arguments. From a conference report published in 2000, it is clear that extensive experiments were done to investigate decays of 84Zr and 84Y, but details of these studies never appeared in literature and none were made available to the evaluators when requested from original authors. This evaluation was carried out as part of ENSDF workshop for Nuclear Structure and Decay Data Evaluators, organized and hosted by the "Horia Hulubei" National Institute for Physics and Nuclear Engineering, Bucharest, Romania during March 30, 2009 - April 3, 2009. Names of the evaluators principally responsible for evaluation of individual nuclides are given under the respective Adopted data sets.

Nuclear data sheets for A=84.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abriola, D.; Bostan, M.; Erturk, S.

The evaluated spectroscopic data are presented for 12 known nuclides of mass 84 (Ga, Ge, As, Se, Br, Kr, Rb, Sr, Y, Zr, Nb, Mo). Except for the stable nuclides {sup 84}Sr and {sup 84}Kr, extensive new data are available for all the other nuclides since the 1997 evaluation by J.K. Tuli (1997Tu02) of A = 84 nuclides. Many precise Penning-trap mass measurements since AME-2003 for A = 84 nuclides (2009Re03,2008Ha23,2008We10,2007Ke09,2006Ka48,2006De36,2006Ri15) have resulted in improved Q values and separation energies. However, many deficiencies still remain. Some examples are given below. Excited-state data for {sup 84}Ga and {sup 84}As are nonexistent,more » and those for {sup 84}Ge are scarce. The radioactive decay schemes of {sup 84}Ga, {sup 84}Ge, {sup 84}Se, {sup 84}Y (39.5 min), {sup 84}Y (4.6 s), {sup 84}Zr and {sup 84}Nb suffer from incompleteness and that for {sup 84}Mo decay is not known at all. The energy ordering of the two activities (39.5 min and 4.6 s) of {sup 84}Y is not well established, although, high-spin with tentative spin-parity of (6+) is adopted here as the ground state of {sup 84}Y based on weak arguments. From a conference report published in 2000, it is clear that extensive experiments were done to investigate decays of {sup 84}Zr and {sup 84}Y, but details of these studies never appeared in literature and none were made available to the evaluators when requested from original authors. This evaluation was carried out as part of ENSDF workshop for Nuclear Structure and Decay Data Evaluators, organized and hosted by the 'Horia Hulubei' National Institute for Physics and Nuclear Engineering, Bucharest, Romania during March 30, 2009 - April 3, 2009. Names of the evaluators principally responsible for evaluation of individual nuclides are given under the respective Adopted data sets.« less

Agrobacterium tumefaciens-mediated transformation of Mucor circinelloides.

PubMed

Nyilasi, I; Acs, K; Papp, T; Nagy, E; Vágvölgyi, C

2005-01-01

The Agrobacterium tumefaciens-mediated transformation of the zygomycetous fungus Mucor circinelloides is described. A method was also developed for the hygromycin B-based selection of Mucor transformants. Transformation with the hygromycin B phosphotransferase gene of Escherichia coli controlled by the heterologous Aspergillus nidulans trpC promoter resulted in hygromycin B-resistant clones. The presence of the hygromycin resistance gene in the genome of the transformants was verified by polymerase chain reaction and Southern hybridization: the latter analyses revealed integrations in the host genome at different sites in different transformants. The stability of transformants remained questionable during the latter analyses.

24 CFR 84.84 - Procurement standards.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 24 Housing and Urban Development 1 2010-04-01 2010-04-01 false Procurement standards. 84.84... EDUCATION, HOSPITALS, AND OTHER NON-PROFIT ORGANIZATIONS Use of Lump Sum Grants § 84.84 Procurement standards. (a) Purpose of procurement standards. Paragraphs (b) through (i) of this section set forth...

[Agrobacterium rubi strains from blueberry plants are highly diverse].

PubMed

Abrahamovich, Eliana; López, Ana C; Alippi, Adriana M

2014-01-01

The diversity of a collection of Agrobacterium rubi strains isolated from blueberries from different regions of Argentina was studied by conventional microbiological tests and molecular techniques. Results from biochemical and physiological reactions, as well as from rep-PCR and RFLP analysis of PCR-amplified 23S rDNA showed high phenotypic and genotypic intraspecific variation. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España. All rights reserved.

Feedback regulation of an Agrobacterium catalase gene katA involved in Agrobacterium-plant interaction.

PubMed

Xu, X Q; Li, L P; Pan, S Q

2001-11-01

Catalases are known to detoxify H2O2, a major component of oxidative stress imposed on a cell. An Agrobacterium tumefaciens catalase encoded by a chromosomal gene katA has been implicated as an important virulence factor as it is involved in detoxification of H2O2 released during Agrobacterium-plant interaction. In this paper, we report a feedback regulation pathway that controls the expression of katA in A. tumefaciens cells. We observed that katA could be induced by plant tissue sections and by acidic pH on a minimal medium, which resembles the plant environment that the bacteria encounter during the course of infection. This represents a new regulatory factor for catalase induction in bacteria. More importantly, a feedback regulation was observed when the katA-gfp expression was studied in different genetic backgrounds. We found that introduction of a wild-type katA gene encoding a functional catalase into A. tumefaciens cells could repress the katA-gfp expression over 60-fold. The katA gene could be induced by H2O2 and the encoded catalase could detoxify H2O2. In addition, the katA-gfp expression of one bacterial cell could be repressed by other surrounding catalase-proficient bacterial cells. Furthermore, mutation at katA caused a 10-fold increase of the intracellular H2O2 concentration in the bacteria grown on an acidic pH medium. These results suggest that the endogenous H2O2 generated during A. tumefaciens cell growth could serve as the intracellular and intercellular inducer for the katA gene expression and that the acidic pH could pose an oxidative stress on the bacteria. Surprisingly, one mutated KatA protein, exhibiting no significant catalase activity as a result of the alteration of two important residues at the putative active site, could partially repress the katA-gfp expression. The feedback regulation of the katA gene by both catalase activity and KatA protein could presumably maintain an appropriated level of catalase activity and H2O2 inside A

Cytoskeletal dynamics in interphase, mitosis and cytokinesis analysed through Agrobacterium-mediated transient transformation of tobacco BY-2 cells.

PubMed

Buschmann, H; Green, P; Sambade, A; Doonan, J H; Lloyd, C W

2011-04-01

Transient transformation with Agrobacterium is a widespread tool allowing rapid expression analyses in plants. However, the available methods generate expression in interphase and do not allow the routine analysis of dividing cells. Here, we present a transient transformation method (termed 'TAMBY2') to enable cell biological studies in interphase and cell division. Agrobacterium-mediated transient gene expression in tobacco BY-2 was analysed by Western blotting and quantitative fluorescence microscopy. Time-lapse microscopy of cytoskeletal markers was employed to monitor cell division. Double-labelling in interphase and mitosis enabled localization studies. We found that the transient transformation efficiency was highest when BY-2/Agrobacterium co-cultivation was performed on solid medium. Transformants produced in this way divided at high frequency. We demonstrated the utility of the method by defining the behaviour of a previously uncharacterized microtubule motor, KinG, throughout the cell cycle. Our analyses demonstrated that TAMBY2 provides a flexible tool for the transient transformation of BY-2 with Agrobacterium. Fluorescence double-labelling showed that KinG localizes to microtubules and to F-actin. In interphase, KinG accumulates on microtubule lagging ends, suggesting a minus-end-directed function in vivo. Time-lapse studies of cell division showed that GFP-KinG strongly labels preprophase band and phragmoplast, but not the metaphase spindle. © 2010 The Authors. New Phytologist © 2010 New Phytologist Trust.

Nuclear Data Sheets for A = 84

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abriola, Daniel; Bostan, Melih; Erturk, Sefa

The evaluated spectroscopic data are presented for 12 known nuclides of mass 84 (Ga, Ge, As, Se, Br, Kr, Rb, Sr, Y, Zr, Nb, Mo). Except for the stable nuclides {sup 84}Sr and {sup 84}Kr, extensive new data are available for all the other nuclides since the 1997 evaluation by J.K. Tuli (1997Tu02) of A = 84 nuclides. Many precise Penning-trap mass measurements since AME-2003 for A = 84 nuclides (2009Re03,2008Ha23,2008We10,2007Ke09,2006Ka48,2006De36,2006Ri15) have resulted in improved Q values and separation energies. However, many deficiencies still remain. Some examples are given below. Excited-state data for {sup 84}Ga and {sup 84}As are nonexistent,more » and those for {sup 84}Ge are scarce. The radioactive decay schemes of {sup 84}Ga, {sup 84}Ge, {sup 84}Se, {sup 84}Y (39.5 min), {sup 84}Y (4.6 s), {sup 84}Zr and {sup 84}Nb suffer from incompleteness and that for {sup 84}Mo decay is not known at all. The energy ordering of the two activities (39.5 min and and 4.6 s) of {sup 84}Y is not well established, although, high-spin with tentative spin-parity of (6+) is adopted here as the ground state of {sup 84}Y based on weak arguments. From a conference report published in 2000, it is clear that extensive experiments were done to investigate decays of {sup 84}Zr and {sup 84}Y, but details of these studies never appeared in literature and none were made available to the evaluators when requested from original authors. This evaluation was carried out as part of ENSDF workshop for Nuclear Structure and Decay Data Evaluators, organized and hosted by the 'Horia Hulubei' National Institute for Physics and Nuclear Engineering, Bucharest, Romania during March 30, 2009 - April 3, 2009. Names of the evaluators principally responsible for evaluation of individual nuclides are given under the respective Adopted data sets.« less

Application of succulent plant leaves for Agrobacterium infiltration-mediated protein production.

PubMed

Jones, Richard W

2016-01-01

When expressing plant cell wall degrading enzymes in the widely used tobacco (Nicotiana benthamiana) after Agrobacterium infiltration, difficulties arise due to the thin leaf structure. Thick leaved succulents, Kalanchoe blossfeldiana and Hylotelephium telephium, were tested as alternatives. A xyloglucanase, as well as a xyloglucanase inhibitor protein was successfully produced. Published by Elsevier B.V.

Metabolic engineering of Agrobacterium sp. strain ATCC 31749 for production of an α-Gal epitope

PubMed Central

2010-01-01

Background Oligosaccharides containing a terminal Gal-α1,3-Gal moiety are collectively known as α-Gal epitopes. α-Gal epitopes are integral components of several medical treatments under development, including flu and HIV vaccines as well as cancer treatments. The difficulty associated with synthesizing the α-Gal epitope hinders the development and application of these treatments due to the limited availability and high cost of the α-Gal epitope. This work illustrates the development of a whole-cell biocatalyst for synthesizing the α-Gal epitope, Gal-α1,3-Lac. Results Agrobacterium sp. ATCC 31749 was engineered to produce Gal-α1,3-Lac by the introduction of a UDP-galactose 4'-epimerase:α1,3-galactosyltransferase fusion enzyme. The engineered Agrobacterium synthesized 0.4 g/L of the α-Gal epitope. Additional metabolic engineering efforts addressed the factors limiting α-Gal epitope production, namely the availability of the two substrates, lactose and UDP-glucose. Through expression of a lactose permease, the intracellular lactose concentration increased by 60 to 110%, subsequently leading to an improvement in Gal-α1,3-Lac production. Knockout of the curdlan synthase gene increased UDP-glucose availability by eliminating the consumption of UDP-glucose for synthesis of the curdlan polysaccharide. With these additional engineering efforts, the final engineered strain synthesized approximately 1 g/L of Gal-α1,3-Lac. Conclusions The Agrobacterium biocatalyst developed in this work synthesizes gram-scale quantities of α-Gal epitope and does not require expensive cofactors or permeabilization, making it a useful biocatalyst for industrial production of the α-Gal epitope. Furthermore, the engineered Agrobacterium, with increased lactose uptake and improved UDP-glucose availability, is a promising host for the production of other medically-relevant oligosaccharides. PMID:20067629

Agrobacterium rhizogenes-mediated transformation: root cultures as a source of alkaloids.

PubMed

Sevón, Nina; Oksman-Caldentey, Kirsi-Marja

2002-10-01

Hairy roots, transformed with Agrobacterium rhizogenes, have been found to be suitable for the production of secondary metabolites because of their stable and high productivity in hormone-free culture conditions. A number of plant species including many medicinal plants have been successfully transformed with Agrobacterium rhizogenes. Transformed root cultures have also been found to be a potential source of high-value pharmaceuticals. In this article the most important alkaloids produced by hairy roots are summarised. Several different methods have been used to increase the alkaloid accumulation in hairy root cultures. The selection of high productive root lines based on somaclonal variation offers an interesting option to enhance the productivity. Elicitors and modification of culture conditions have been shown to increase the growth and the alkaloid production in some cases. Genetic engineering is a modern tool to regulate the secondary metabolism also in hairy roots. However, our knowledge on biosynthesis of many alkaloids is still poor. Only a limited number of enzymes and their respective genes which regulate the biosynthetic pathways are fully characterised.

Crown gall transformation of tobacco callus cells by cocultivation with Agrobacterium tumefaciens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muller, A.; Manzara, T.; Lurquin, P.F.

1984-09-17

Incubation of cells from squashed tobacco callus tissue with virulent Agrobacterium tumefaciens leads to the production of cells displaying a crown gall phenotype. In vitro crown gall transformation of dicotyledonous plant cells has been demonstrated after cocultivation of cell-wall regenerating mesophyll protoplasts with Agrobacterium tumefaciens cells. In addition, it has been shown that protoplasts freshly isolated from suspension cultures, when treated with A. tumefaciens spheroplasts and a fusogen, also generated cells displaying a typical crown gall phenotype, i.e., phytohormone-independent growth and opine synthesis. Subsequently, both techniques were used to transfer and express foreign genes in plant cells via A. tumefaciensmore » T-DNA integration. For practical purposes, it would be advantageous to be able to perform crown gall transformation of plant cells in tissue culture. The authors report here for the first time the production of Nicotiana tabacum crown gall cells after cocultivation of callus tissue with A. tumefaciens A136 cells. 11 references, 1 figure, 1 table.« less

Agrobacterium-mediated transient MaFT expression in mulberry (Morus alba L.) leaves.

PubMed

Wu, Su-Li; Yang, Xiao-Bing; Liu, Li-Qun; Jiang, Tao; Wu, Hai; Su, Chao; Qian, Yong-Hua; Jiao, Feng

2015-01-01

To optimize Agrobacterium-mediated transient transformation assay in mulberry (Morus alba L.), various infiltration methods, Agrobacterium tumefaciens (A. tumefaciens) strains, and bacterial concentrations were tested in mulberry seedlings. Compared with LBA4404, GV3101 harboring pBE2133 plasmids presented stronger GUS signals at 3 days post infiltration using syringe. Recombinant plasmids pBE2133:GFP and pBE2133:GFP:MaFT were successfully constructed. Transient expression of MaFT:GFP protein was found in leaves, petiole (cross section), and shoot apical meristem (SAM) of mulberry according to the GFP signal. Moreover, MaFT:GFP mRNA was also detected in leaves and SAM via RT-PCR and qRT-PCR. An efficient transient transformation system could be achieved in mulberry seedlings by syringe using A. tumefaciens GV3101 at the OD600 of 0.5. The movement of MaFT expression from leaves to SAM might trigger the precocious flowering of mulberry.

Agrobacterium tumefaciens-mediated transformation of Phellodendron amurense Rupr. using mature-seed explants.

PubMed

Yang, Jingli; Zhao, Bo; Kim, Yeon Bok; Zhou, Chenguang; Li, Chunyan; Chen, Yunlin; Zhang, Haizhen; Li, Cheng Hao

2013-01-01

An efficient transformation protocol was developed for Agrobacterium-mediated transformation of Phellodendron amurense Rupr. for using explants from mature seeds. The binary vector pCAMBIA1303, which contained hygromycin phosphotransferase (hptII) as a selectable marker gene and β-glucuronidase (GUS) as a reporter gene, was used for transformation studies. Different factors that affect survival of transformed buds, namely Agrobacterium infection method, bacterial strain, pre-culture duration, acetosyringone concentration, co-culture duration, and co-culture temperature were examined and optimized for transformation efficiency on the basis of GUS staining of hygromycin-resistant buds. Polymerase chain reaction (PCR), Southern blot and reverse transcription PCR confirmed the presence of the GUS gene. A transformation frequency of 13.1 % was achieved under optimized conditions for transformation (A. tumefaciens strain EHA105, 4 days co-cultivation at 4 °C, and infection of the pre-cultured mature-seed explants for 2 days). This is the first report of a successful genetic transformation protocol for P. amurense.

Persistence of Agrobacterium tumefaciens in transformed conifers.

PubMed

Charity, Julia A; Klimaszewska, Krystyna

2005-01-01

Previous studies have shown that the widely used plant transformation vector Agrobacterium tumefaciens can persist in genetically engineered plants in vitro and in transgenic greenhouse-grown plants, despite the use of counter-selective antibiotics. However, little is known regarding Agrobacterium persistence in tree species. To understand the kinetics of A. tumefaciens decline and persistence in transformation experiments, we assayed for the presence of A. tumefaciens in spruce and pine embryogenic tissue for up to 10 weeks post-transformation. The A. tumefaciens populations declined rapidly in the first five days post-cocultivation but generally declined more slowly in pine, relative to spruce. No bacteria were detected in spruce embryogenic tissue beyond four weeks after cocultivation, however in pine there were -100 colony forming units per g tissue at 10 weeks post-cocultivation. We present evidence that the detection limit for PCR using virD2 primers to detect A. tumefaciens in a background of pine needle DNA was approximately 10(9)-10(10) A. tumefaciens cells per g of tissue. We also assayed for A. tumefaciens in transgenic pine and spruce embryogenic tissue and from needles, branches, stems and roots of transformed plants, up to four years post-inoculation. Occasionally A. tumefaciens was detected in embryogenic tissue up to 12 months post-inoculation. A. tumefaciens was never detected in cultured embryogenic tissue more than twelve months after inoculation, nor in developing somatic embryos or germinating plantlets, nor any of the parts of greenhouse-grown plants. From these data we conclude that if A. tumefaciens persists in transgenic conifers, it does so beneath our ability to detect it.

Mapping of the Interaction Between Agrobacterium tumefaciens and Vanda Kasem's Delight Orchid Protocorm-Like Bodies.

PubMed

Gnasekaran, Pavallekoodi; Subramaniam, Sreeramanan

2015-09-01

Physical contact between A. tumefaciens and the target plant cell walls is essential to transfer and integrate the transgene to introduce a novel trait. Chemotaxis response and attachment of Agrobacterium towards Vanda Kasem's Delight (VKD) protocorm-like bodies (PLBs) were studied to analyse the interaction between Agrobacterium and PLB during the transformation event. The study shows that initially A. tumefaciens reversibly attached to PLB surface via polar and lateral mode of adherence followed by the irreversible attachment which involved the production of cellulosic fibril by A. tumefaciens. Cellulosic fibril allows formation of biofilm at the tip of trichome. Contrarily, attachment mutant Escherichia coli strain DH5α was significantly deficient in the attachment process. Spectrophotometric GUS assay showed the mean value of attachment by A. tumefaciens was 8.72 % compared to the negative control E. coli strain DH5α that produced 0.16 %. A. tumefaciens swarmed with sharper and brighter edge when severe wounding was applied to the PLBs producing the highest swarming ratio of 1.46 demonstrating the positive effect of the plant exudates on bacterial movement. The study shows that VKD's PLBs are the suitable explants for Agrobacterium-mediated transformation since the bacteria expressed higher competency rate.

Development of efficient plant regeneration and transformation system for impatiens using Agrobacterium tumefaciens and multiple bud cultures as explants.

PubMed

Dan, Yinghui; Baxter, Aaron; Zhang, Song; Pantazis, Christopher J; Veilleux, Richard E

2010-08-09

Impatiens (Impatiens walleriana) is a top selling floriculture crop. The potential for genetic transformation of Impatiens to introduce novel flower colors or virus resistance has been limited by its general recalcitrance to tissue culture and transformation manipulations. We have established a regeneration and transformation system for Impatiens that provides new alternatives to genetic improvement of this crop. In a first step towards the development of transgenic INSV-resistant Impatiens, we developed an efficient plant regeneration system using hypocotyl segments containing cotyledonary nodes as explants. With this regeneration system, 80% of explants produced an average of 32.3 elongated shoots per initial explant plated, with up to 167 elongated shoots produced per explant. Rooting efficiency was high, and 100% of shoots produced roots within 12 days under optimal conditions, allowing plant regeneration within approximately 8 weeks. Using this regeneration system, we developed an efficient Agrobacterium-mediated Impatiens transformation method using in vitro multiple bud cultures as explants and a binary plasmid (pHB2892) bearing gfp and nptII genes. Transgenic Impatiens plants, with a frequency up to 58.9%, were obtained within 12 to 16 weeks from inoculation to transfer of transgenic plants to soil. Transgenic plants were confirmed by Southern blot, phenotypic assays and T1 segregation analysis. Transgene expression was observed in leaves, stems, roots, flowers, and fruit. The transgenic plants were fertile and phenotypically normal. We report the development of a simple and efficient Agrobacterium-mediated transformation system for Impatiens. To the best of our knowledge, there have been no reports of Agrobacterium-mediated transformation of Impatiens with experimental evidence of stable integration of T-DNA and of Agrobacterium-mediated transformation method for plants using in vitro maintained multiple bud cultures as explants. This transformation system

Agrobacterium-mediated genetic transformation of yam (Dioscorea rotundata): an important tool for functional study of genes and crop improvement

PubMed Central

Nyaboga, Evans; Tripathi, Jaindra N.; Manoharan, Rajesh; Tripathi, Leena

2014-01-01

Although genetic transformation of clonally propagated crops has been widely studied as a tool for crop improvement and as a vital part of the development of functional genomics resources, there has been no report of any existing Agrobacterium-mediated transformation of yam (Dioscorea spp.) with evidence of stable integration of T-DNA. Yam is an important crop in the tropics and subtropics providing food security and income to over 300 million people. However, yam production remains constrained by increasing levels of field and storage pests and diseases. A major constraint to the development of biotechnological approaches for yam improvement has been the lack of an efficient and robust transformation and regeneration system. In this study, we developed an Agrobacterium-mediated transformation of Dioscorea rotundata using axillary buds as explants. Two cultivars of D. rotundata were transformed using Agrobacterium tumefaciens harboring the binary vectors containing selectable marker and reporter genes. After selection with appropriate concentrations of antibiotic, shoots were developed on shoot induction and elongation medium. The elongated antibiotic-resistant shoots were subsequently rooted on medium supplemented with selection agent. Successful transformation was confirmed by polymerase chain reaction, Southern blot analysis, and reporter genes assay. Expression of gusA gene in transgenic plants was also verified by reverse transcription polymerase chain reaction analysis. Transformation efficiency varied from 9.4 to 18.2% depending on the cultivars, selectable marker genes, and the Agrobacterium strain used for transformation. It took 3–4 months from Agro-infection to regeneration of complete transgenic plant. Here we report an efficient, fast and reproducible protocol for Agrobacterium-mediated transformation of D. rotundata using axillary buds as explants, which provides a useful platform for future genetic engineering studies in this economically important

DETECTION AND IMPLICATIONS OF EARLY AGROBACTERIUM TUMEFACIENS INFECTION OF PARADOX SEEDS AND SEEDLINGS

USDA-ARS?s Scientific Manuscript database

Paradox (Juglans hindsii x J. regia), the dominant rootstock used in California, USA walnut production, has many desirable horticultural characteristics, but is highly susceptible to crown gall. Crown gall, caused by the soil-borne bacterium Agrobacterium tumefaciens, can not be consistently control...

Increased Mechanical Properties Through the Addition of Zr to GRCop-84

NASA Technical Reports Server (NTRS)

Ellis, David L.; Lerch, Bradley A.

2011-01-01

GRCop-84 (Cu-8 at.% Cr-4 at.% Nb) has shown exceptional mechanical properties above 932 F (773 K). However, its properties below 932 F (773 K) are inferior to precipitation strengthened alloys such as Cu-Cr, Cu-Zr and Cu-Cr-Zr when they are in the fully aged, hard-drawn condition. It has been noted that the addition of small amounts of Zr, typically 0.1 wt.% to 0.5 wt.%, can greatly enhance the mechanical properties of copper-based alloys. Limited testing was conducted upon GRCop-84 with an addition of 0.4 wt.% Zr to determine its tensile, creep and low cycle fatigue (LCF) properties. Very large increases in strength (up to 68%) and ductility (up to 123%) were observed at both room temperature and 932 F (773 K). Creep properties at 932 F (773 K) demonstrated more than an order of magnitude decrease in the creep rate relative to unmodified GRCop-84 with a corresponding order of magnitude increase in creep life. Limited LCF testing showed that the modified alloy had a comparable LCF life at room temperature, but it was capable of sustaining a much higher load. While more testing and composition optimization are required, the addition of Zr to GRCop-84 has shown clear benefits to mechanical properties.

45 CFR 84.56-84.60 - [Reserved

Code of Federal Regulations, 2012 CFR

2012-10-01

... 45 Public Welfare 1 2012-10-01 2012-10-01 false [Reserved] 84.56-84.60 Section 84.56-84.60 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION NONDISCRIMINATION ON THE BASIS OF HANDICAP IN PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Health, Welfare, and Social...

45 CFR 84.56-84.60 - [Reserved

Code of Federal Regulations, 2011 CFR

2011-10-01

... 45 Public Welfare 1 2011-10-01 2011-10-01 false [Reserved] 84.56-84.60 Section 84.56-84.60 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION NONDISCRIMINATION ON THE BASIS OF HANDICAP IN PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Health, Welfare, and Social...

45 CFR 84.56-84.60 - [Reserved

Code of Federal Regulations, 2013 CFR

2013-10-01

... 45 Public Welfare 1 2013-10-01 2013-10-01 false [Reserved] 84.56-84.60 Section 84.56-84.60 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION NONDISCRIMINATION ON THE BASIS OF HANDICAP IN PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Health, Welfare, and Social...

45 CFR 84.56-84.60 - [Reserved

Code of Federal Regulations, 2014 CFR

2014-10-01

... 45 Public Welfare 1 2014-10-01 2014-10-01 false [Reserved] 84.56-84.60 Section 84.56-84.60 Public Welfare Department of Health and Human Services GENERAL ADMINISTRATION NONDISCRIMINATION ON THE BASIS OF HANDICAP IN PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Health, Welfare, and Social...

45 CFR 84.56-84.60 - [Reserved

Code of Federal Regulations, 2010 CFR

2010-10-01

... 45 Public Welfare 1 2010-10-01 2010-10-01 false [Reserved] 84.56-84.60 Section 84.56-84.60 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION NONDISCRIMINATION ON THE BASIS OF HANDICAP IN PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Health, Welfare, and Social...

Agrobacterium tumefasciens-mediated transformation of the aquatic fungus Blastocladiella emersonii.

PubMed

Vieira, André L G; Camilo, César M

2011-08-01

Agrobacterium tumefaciens is widely used for plant DNA transformation and more recently, has also been used to transform yeast, filamentous fungi and even human cells. Using this technique, we developed the first transformation protocol for the saprobic aquatic fungus Blastocladiella emersonii, a Blastocladiomycete localized at the base of fungal phylogenetic tree, which has been shown as a promising and interesting model of study of cellular function and differentiation. We constructed binary T-DNA vectors containing hygromycin phosphotransferase (hph) or enhanced green fluorescent protein (egfp) genes, under the control of Aspergillus nidulans trpC promoter and terminator sequences. 24 h of co-cultivation in induction medium (IM) agar plates, followed by transfer to PYG-agar plates containing cefotaxim to kill Agrobacterium tumefsciens and hygromycin to select transformants, resulted in growth and sporulation of resistant transformants. Genomic DNA from the pool o resistant zoospores were shown to contain T-DNA insertion as evidenced by PCR amplification of hph gene. Using a similar protocol we could also evidence the expression of enhanced green fluorescent protein (EGFP) in zoospores derived from transformed cells. This protocol can also open new perspectives for other non-transformable closely related fungi, like the Chytridiomycete class. Copyright © 2011 Elsevier Inc. All rights reserved.

Novel primers for detection of genetically diverse virulent Agrobacterium tumefaciens bv1 strains

USDA-ARS?s Scientific Manuscript database

Novel primers were developed to amplify a 243 bp fragment of an intergenic region between gene5 and tms2 on the T-DNA of Agrobacterium tumefaciens. These primers exhibit 100% positive correlation with strain virulence, 100% negative correlation with avirulence and did not generate extraneous bands,...

Agrobacterium-Mediated Transformation of Bread and Durum Wheat Using Freshly Isolated Immature Embryos

NASA Astrophysics Data System (ADS)

Huixia, Wu; Angela, Doherty; Jones, Huw D.

Agrobacterium-mediated transformation of wheat is becoming a viable alternative to the more established biolistic protocols. It offers advantages in terms of simple, low-copy-number integrations and can be applied with similar efficiencies to specific durum wheat and spring and winter bread wheat types varieties.

Factors influencing Agrobacterium-mediated embryogenic callus transformation of Valencia sweet orange (Citrus sinensis) containing the pTA29-barnase gene.

PubMed

Li, D D; Shi, W; Deng, X X

2003-12-01

Valencia sweet orange (Citrus sinensis (L.) Osbeck) calluses were used as explants to develop a new transformation system for citrus mediated by Agrobacterium tumefaciens. Factors affecting Agrobacterium-mediated transformation efficiency included mode of pre-cultivation, temperature of cocultivation and presence of acetosyringone (AS). The highest transformation efficiency was obtained with a 4-day pre-cultivation period in liquid medium. Transformation efficiency was higher when cocultivation was performed for 3 days at 19 degrees C than at 23 or 28 degrees C. Almost no resistant callus was obtained if the cocultivation medium lacked AS. The transformation procedure yielded transgenic Valencia plants containing the pTA29-barnase gene, as verified by PCR amplification and confirmed by Southern blotting. Because male sterility is a common factor leading to seedlessness in citrus cultivars with parthenocarpic characteristics, production of seedless citrus genotypes by Agrobacterium-mediated genetic transformation is a promising alternative to conventional breeding methods.

Agrobacterium-mediated transformation in Alpinia galanga (Linn.) Willd. for enhanced acetoxychavicol acetate production.

PubMed

Rao, Kiranmayee; Chodisetti, Bhuvaneswari; Mangamoori, Lakshmi Narasu; Giri, Archana

2012-09-01

Agrobacterium-mediated transformations ensure elevated amounts of secondary metabolite accumulation with genetic and biosynthetic stability. In the present study, Alpinia galanga rich in bioactive compounds was genetically transformed using different strains of Agrobacterium rhizogenes viz. LBA 9402, A(4), 532, 2364 and PRTGus. Even though a higher growth rate was obtained with the LBA 9402 strain, maximum acetoxychavicol acetate accumulation (ACA) was seen in the PRTGus transformant. PRTGus root line has shown 10.1 fold higher ACA content in comparison to the control roots. The lowest ACA production was shown by the A(4) transformant (4.9 fold). The quantification of ACA in the transformed roots was carried out by using HPLC, which was found to be in the order of PRTGus > LBA 9402 > 2364 > 532 > A(4). The fast growth rate of hairy roots, genetic stability and their ability to synthesize more than one metabolite offer a promising system for the production of valuable secondary metabolites.

Morphogenetic and chemical stability of long-term maintained Agrobacterium-mediated transgenic Catharanthus roseus plants.

PubMed

Verma, Priyanka; Sharma, Abhishek; Khan, Shamshad Ahmad; Mathur, Ajay Kumar; Shanker, Karuna

2015-01-01

Transgenic Catharanthus roseus plants (transgenic Dhawal [DT] and transgenic Nirmal [NT]) obtained from the Agrobacterium tumefaciens and Agrobacterium rhizognenes-mediated transformations, respectively, have been maintained in vitro for 5 years. Plants were studied at regular intervals for various parameters such as plant height, leaf size, multiplication rate, alkaloid profile and presence of marker genes. DT plant gradually lost the GUS gene expression and it was not detected in the fifth year while NT plant demonstrated the presence of genes rolA, rolB and rolC even in the fifth year, indicating the more stable nature of Ri transgene. Vindoline content in the DT was two times more than in non-transformed control plants. Alkaloid and tryptophan profiles were almost constant during the 5 years. The cluster analysis revealed that the DT plant is more close to the control Nirmal plant followed by NT plant.

Agrobacterium-mediated genetic transformation and plant regeneration of the hardwood tree species Fraxinus profunda

Treesearch

Micah E. Stevens; Paula M. Pijut

2014-01-01

Using mature hypocotyls as the initial explants, an Agrobacterium tumefaciens-mediated genetic transformation system was successfully developed for pumpkin ash (Fraxinus profunda). This transformation protocol is an invaluable tool to combat the highly aggressive, non-native emerald ash borer (EAB), which has the potential to...

Development of 72/84kV Dry Air Insulated Dead Tank Type VCB

NASA Astrophysics Data System (ADS)

Saito, Hitoshi; Nagatake, Kazuhiro; Komatsu, Hideki; Takeshita, Yukihiro; Matsui, Yoshihiko; Katsumata, Kiyohito; Sakaki, Masayuki

As a circuit breaker for over 84kV, SF6 gas circuit breaker (GCB) has been used for a long time, in virtue of its excellent characteristics as arc extinction and insulating medium. Although, SF6 gas has very high global warming potential (GWP) of 23,900, and it was designated to regulation object in COP3 in Kyoto in 1997. A lot of efforts have been done to reduce the amount of SF6 gas usage and emission from conventional equipments. On the other hand, SF6 gas free equipment has been researching and one strong candidate is air-insulated type switchgears with vacuum interrupters. In last few years, air-insulated switchgears, which include GIS, Cubicle type GIS (C-GIS) and dead tank type VCB, have been developed in succession. So far, we have already more than three years operation record for the air-insulated dead tank type VCB, and over 100 units is in-service in power systems. Recently, VCB technology, that is essential for SF6 gas-free equipments, has been advanced in the field of high-voltage, large current interruption and environment-conscious design. In this paper, the advanced dead tank type VCB and its technology is descried.

Transformation of lettuce (Lactuca sativa) mediated by Agrobacterium tumefaciens.

PubMed

Michelmore, R; Marsh, E; Seely, S; Landry, B

1987-12-01

Lactuca sativa can be routinely transformed using Ti plasmids of Agrobacterium tumefaciens containing a chimeric kanamycin resistance gene (NOS.NPTII.NOS). Critical experimental variables were plant genotype, bacterial concentration, presence of a nurse culture and timing of transfers between tissue culture media. Transformation was confirmed by the ability to callus and root in the presence of kanamycin, nopaline production, and by hybridization in Southern blots. Transformation has been achieved with several Ti vectors. Several hundred transformed plants have been regenerated. Kanamycin resistance was inherited monogenically. Homozygotes can be selected by growing R2 seedlings on media containing G418.

Agrobacterium rhizogenes-induced cotton hairy root culture as an alternative tool for cotton functional genomics

USDA-ARS?s Scientific Manuscript database

Although well-accepted as the ultimate method for cotton functional genomics, Agrobacterium tumefaciens-mediated cotton transformation is not widely used for functional analyses of cotton genes and their promoters since regeneration of cotton in tissue culture is lengthy and labor intensive. In cer...

Effect of selection agents to Chrysanthemum (Chrysanthemum morifolium) callus growth after Agrobacterium-mediated genetic transformation

NASA Astrophysics Data System (ADS)

Sjahril, R.; Jamaluddin, I.; Nadir, M.; Asman; Dungga, N. E.

2018-05-01

Genetic transformation mediated by Agrobacterium tumefaciens requires an efficient selection method for successful progress of transformation. This study aims to determine the concentration and kind of antibiotics and selection agents used during transformation to formulate standard protocol of chrysanthemum in the process of propagating disease resistant Chrysanthemum mediated by Agrobacterium tumefaciens EHA105 (pEKB-WD). The experiments were performed by planting chrysanthemum explants leaf cutting (5 mm diameter on NAA medium 2 mg L-1 BAP 2 mg L-1) with addition of Kanamycin: 25, 50, 100, 150 and 200 (mg L-1); Hygromycin: 5, 10, 25, 50 and 75 (mg L-1); Paromomycin: 10, 25, 50, 75 and 100 (mg L-1). Experiment was arranged in a Completely Randomized Design (CRD). Each treatment was repeated five times thus 75 bottles of culture were used; each bottle consists of 5 pieces of leaf cuttings, resulted in total of 375 pieces. The results showed that selection agent had a critical value for Hygromycin 25 mg L-1 and Kanamycin 100 mg L-1 which can make explant experienced necrosis better than Paromomycin. Paromomycin at 100 mg L-1 was only able to kill explant’s periphery. Remained callus stayed fresh more than 50% so that when used as the selection agent could produce more escape cell. The optimum transformation with concentration of 10% Agrobacterium (vol/vol) with 30 minutes co-cultivation can produce more efficient transformed callus. Considering the high price of Hygromycin, it was best to use Kanamycin as selective agents.

42 CFR 84.84 - Hand-operated valves; minimum requirements.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 1 2011-10-01 2011-10-01 false Hand-operated valves; minimum requirements. 84.84 Section 84.84 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Self...

42 CFR 84.84 - Hand-operated valves; minimum requirements.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 1 2010-10-01 2010-10-01 false Hand-operated valves; minimum requirements. 84.84 Section 84.84 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Self...

42 CFR 84.84 - Hand-operated valves; minimum requirements.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 1 2013-10-01 2013-10-01 false Hand-operated valves; minimum requirements. 84.84 Section 84.84 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Self...

42 CFR 84.84 - Hand-operated valves; minimum requirements.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 1 2014-10-01 2014-10-01 false Hand-operated valves; minimum requirements. 84.84 Section 84.84 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Self...

42 CFR 84.84 - Hand-operated valves; minimum requirements.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 1 2012-10-01 2012-10-01 false Hand-operated valves; minimum requirements. 84.84 Section 84.84 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Self...

Efficient and rapid Agrobacterium-mediated genetic transformation of durum wheat (Triticum turgidum L. var. durum) using additional virulence genes.

PubMed

Wu, Huixia; Doherty, Angela; Jones, Huw D

2008-06-01

Genetic transformation of wheat, using biolistics or Agrobacterium, underpins a range of specific research methods for identifying genes and studying their function in planta. Transgenic approaches to study and modify traits in durum wheat have lagged behind those for bread wheat. Here we report the use of Agrobacterium strain AGL1, with additional vir genes housed in a helper plasmid, to transform and regenerate the durum wheat variety Ofanto. The use of the basic pSoup helper plasmid with no additional vir genes failed to generate transformants, whereas the presence of either virG542 or the 15 kb Komari fragment containing virB, virC and virG542 produced transformation efficiencies of between 0.6 and 9.7%. Of the 42 transgenic plants made, all but one (which set very few seeds) appeared morphologically normal and produced between 100 and 300 viable seeds. The transgene copy number and the segregation ratios were found to be very similar to those previously reported for bread wheat. We believe that this is the first report describing successful genetic transformation of tetraploid durum wheat (Triticum turgidum L. var. durum) mediated by Agrobacterium tumefaciens using immature embryos as the explant.

Genotype-independent and enhanced in planta Agrobacterium tumefaciens-mediated genetic transformation of peanut [Arachis hypogaea (L.)].

PubMed

Karthik, Sivabalan; Pavan, Gadamchetty; Sathish, Selvam; Siva, Ramamoorthy; Kumar, Periyasamy Suresh; Manickavasagam, Markandan

2018-04-01

Agrobacterium infection and regeneration of the putatively transformed plant from the explant remains arduous for some crop species like peanut. Henceforth, a competent and reproducible in planta genetic transformation protocol is established for peanut cv. CO7 by standardizing various factors such as pre-culture duration, acetosyringone concentration, duration of co-cultivation, sonication and vacuum infiltration. In the present investigation, Agrobacterium tumefaciens strain EHA105 harboring the binary vector pCAMBIA1301- bar was used for transformation. The two-stage selection was carried out using 4 and 250 mg l -1 BASTA ® to completely eliminate the chimeric and non-transformed plants. The transgene integration into plant genome was evaluated by GUS histochemical assay, polymerase chain reaction (PCR), and Southern blot hybridization. Among the various combinations and concentrations analyzed, highest transformation efficiency was obtained when the 2-day pre-cultured explants were subjected to sonication for 6 min and vacuum infiltrated for 3 min in Agrobacterium suspension, and co-cultivated on MS medium supplemented with 150 µM acetosyringone for 3 days. The fidelity of the standardized in planta transformation method was assessed in five peanut cultivars and all the cultivars responded positively with a transformation efficiency ranging from minimum 31.3% (with cv. CO6) to maximum 38.6% (with cv. TMV7). The in planta transformation method optimized in this study could be beneficial to develop superior peanut cultivars with desirable genetic traits.

DC-Analyzer-facilitated combinatorial strategy for rapid directed evolution of functional enzymes with multiple mutagenesis sites.

PubMed

Wang, Xiong; Zheng, Kai; Zheng, Huayu; Nie, Hongli; Yang, Zujun; Tang, Lixia

2014-12-20

Iterative saturation mutagenesis (ISM) has been shown to be a powerful method for directed evolution. In this study, the approach was modified (termed M-ISM) by combining the single-site saturation mutagenesis method with a DC-Analyzer-facilitated combinatorial strategy, aiming to evolve novel biocatalysts efficiently in the case where multiple sites are targeted simultaneously. Initially, all target sites were explored individually by constructing single-site saturation mutagenesis libraries. Next, the top two to four variants in each library were selected and combined using the DC-Analyzer-facilitated combinatorial strategy. In addition to site-saturation mutagenesis, iterative saturation mutagenesis also needed to be performed. The advantages of M-ISM over ISM were that the screening effort is greatly reduced, and the entire M-ISM procedure was less time-consuming. The M-ISM strategy was successfully applied to the randomization of halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) when five interesting sites were targeted simultaneously. After screening 900 clones in total, six positive mutants were obtained. These mutants exhibited 4.0- to 9.3-fold higher k(cat) values than did the wild-type HheC toward 1,3-dichloro-2-propanol. However, with the ISM strategy, the best hit showed a 5.9-fold higher k(cat) value toward 1,3-DCP than the wild-type HheC, which was obtained after screening 4000 clones from four rounds of mutagenesis. Therefore, M-ISM could serve as a simple and efficient version of ISM for the randomization of target genes with multiple positions of interest.

Progress of cereal transformation technology mediated by Agrobacterium tumefaciens.

PubMed

Hiei, Yukoh; Ishida, Yuji; Komari, Toshihiko

2014-01-01

Monocotyledonous plants were believed to be not transformable by the soil bacterium Agrobacterium tumefaciens until two decades ago, although convenient protocols for infection of leaf disks and subsequent regeneration of transgenic plants had been well established in a number of dicotyledonous species by then. This belief was reinforced by the fact that monocotyledons are mostly outside the host range of crown gall disease caused by the bacterium and by the failures in trials in monocotyledons to mimic the transformation protocols for dicotyledons. However, a key reason for the failure could have been the lack of active cell divisions at the wound sites in monocotyledons. The complexity and narrow optimal windows of critical factors, such as genotypes of plants, conditions of the plants from which explants are prepared, tissue culture methods and culture media, pre-treatments of explants, strains of A. tumefaciens, inducers of virulence genes, transformation vectors, selection marker genes and selective agents, kept technical hurdles high. Eventually it was demonstrated that rice and maize could be transformed by co-cultivating cells of callus cultures or immature embryos, which are actively dividing or about to divide, with A. tumefaciens. Subsequently, these initial difficulties were resolved one by one by many research groups, and the major cereals are now transformed quite efficiently. As many as 15 independent transgenic events may be regenerated from a single piece of immature embryo of rice. Maize transformation protocols are well established, and almost all transgenic events deregulated for commercialization after 2003 were generated by Agrobacterium-mediated transformation. Wheat, barley, and sorghum are also among those plants that can be efficiently transformed by A. tumefaciens.

Progress of cereal transformation technology mediated by Agrobacterium tumefaciens

PubMed Central

Hiei, Yukoh; Ishida, Yuji; Komari, Toshihiko

2014-01-01

Monocotyledonous plants were believed to be not transformable by the soil bacterium Agrobacterium tumefaciens until two decades ago, although convenient protocols for infection of leaf disks and subsequent regeneration of transgenic plants had been well established in a number of dicotyledonous species by then. This belief was reinforced by the fact that monocotyledons are mostly outside the host range of crown gall disease caused by the bacterium and by the failures in trials in monocotyledons to mimic the transformation protocols for dicotyledons. However, a key reason for the failure could have been the lack of active cell divisions at the wound sites in monocotyledons. The complexity and narrow optimal windows of critical factors, such as genotypes of plants, conditions of the plants from which explants are prepared, tissue culture methods and culture media, pre-treatments of explants, strains of A. tumefaciens, inducers of virulence genes, transformation vectors, selection marker genes and selective agents, kept technical hurdles high. Eventually it was demonstrated that rice and maize could be transformed by co-cultivating cells of callus cultures or immature embryos, which are actively dividing or about to divide, with A. tumefaciens. Subsequently, these initial difficulties were resolved one by one by many research groups, and the major cereals are now transformed quite efficiently. As many as 15 independent transgenic events may be regenerated from a single piece of immature embryo of rice. Maize transformation protocols are well established, and almost all transgenic events deregulated for commercialization after 2003 were generated by Agrobacterium-mediated transformation. Wheat, barley, and sorghum are also among those plants that can be efficiently transformed by A. tumefaciens. PMID:25426132

Agrobacterium tumefaciens mutants affected in attachment to plant cells.

PubMed Central

Douglas, C J; Halperin, W; Nester, E W

1982-01-01

An analysis of Agrobacterium tumefaciens mutants with Tn5 insertions in chromosomal DNA showed that the chromosome of A. tumefaciens codes for a specific ability of this bacterium to attach to plant cells. This ability is associated with tumorigenesis by A. tumefaciens, the ability of avirulent A. tumefaciens to inhibit tumorigenesis, and the ability to adsorb certain phages. A second class of chromosomal mutations affects tumorigenesis without altering the ability to attach to plant cells. The attachment of A. tumefaciens to plant cells was assayed by mixing radiolabeled bacteria with suspensions of tobacco tissue culture cells or freshly isolated Zinnia leaf mesophyll cells. Under the conditions of this assay, an avirulent Ti plasmid-cured strain attached to the same extent as the same strain containing pTiB6806. Six of eight avirulent mutants with Tn5 insertions in chromosomal DNA showed defective attachment, whereas two retained wild-type attachment ability. In contrast to the strains showing wild-type attachment, the attachment-defective mutants failed to inhibit tumorigenesis when inoculated onto Jerusalem artichoke slices before inoculation of a virulent strain and also showed a loss of sensitivity to two Agrobacterium phages. The loss of phage sensitivity appeared to be due to a loss of ability to adsorb the phages. Staining with Calcofluor indicated that the mutants retained the ability to synthesize cellulose fibrils, which have been implicated in the attachment process. Southern filter hybridizations demonstrated that each mutant contained a single Tn5 insertion, and genetic linkage between the Tn5 insertion in one mutant and the attachment phenotype has also been demonstrated. Images PMID:6292165

Agrobacterium-mediated transformation of two Serbian potato cultivars (Solanum tuberosum L. cv. Dragacevka and cv. Jelica)

USDA-ARS?s Scientific Manuscript database

An efficient protocol for Agrobacterium-mediated transformation of Serbian potato cultivars Dragacevka and Jelica, enabling the introduction of oryzacystatin genes OCI and OCII, was established. Starting with leaf explants a two-stage transformation protocol combining procedures of Webb and Wenzler...

An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize.

PubMed

Char, Si Nian; Neelakandan, Anjanasree K; Nahampun, Hartinio; Frame, Bronwyn; Main, Marcy; Spalding, Martin H; Becraft, Philip W; Meyers, Blake C; Walbot, Virginia; Wang, Kan; Yang, Bing

2017-02-01

CRISPR/Cas9 is a powerful genome editing tool in many organisms, including a number of monocots and dicots. Although the design and application of CRISPR/Cas9 is simpler compared to other nuclease-based genome editing tools, optimization requires the consideration of the DNA delivery and tissue regeneration methods for a particular species to achieve accuracy and efficiency. Here, we describe a public sector system, ISU Maize CRISPR, utilizing Agrobacterium-delivered CRISPR/Cas9 for high-frequency targeted mutagenesis in maize. This system consists of an Escherichia coli cloning vector and an Agrobacterium binary vector. It can be used to clone up to four guide RNAs for single or multiplex gene targeting. We evaluated this system for its mutagenesis frequency and heritability using four maize genes in two duplicated pairs: Argonaute 18 (ZmAgo18a and ZmAgo18b) and dihydroflavonol 4-reductase or anthocyaninless genes (a1 and a4). T 0 transgenic events carrying mono- or diallelic mutations of one locus and various combinations of allelic mutations of two loci occurred at rates over 70% mutants per transgenic events in both Hi-II and B104 genotypes. Through genetic segregation, null segregants carrying only the desired mutant alleles without the CRISPR transgene could be generated in T 1 progeny. Inheritance of an active CRISPR/Cas9 transgene leads to additional target-specific mutations in subsequent generations. Duplex infection of immature embryos by mixing two individual Agrobacterium strains harbouring different Cas9/gRNA modules can be performed for improved cost efficiency. Together, the findings demonstrate that the ISU Maize CRISPR platform is an effective and robust tool to targeted mutagenesis in maize. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Quorum Sensing and Quorum Quenching in Agrobacterium: A "Go/No Go System"?

PubMed

Dessaux, Yves; Faure, Denis

2018-04-16

The pathogen Agrobacterium induces gall formation on a wide range of dicotyledonous plants. In this bacteria, most pathogenicity determinants are borne on the tumour inducing (Ti) plasmid. The conjugative transfer of this plasmid between agrobacteria is regulated by quorum sensing (QS). However, processes involved in the disturbance of QS also occur in this bacteria under the molecular form of a protein, TraM, inhibiting the sensing of the QS signals, and two lactonases BlcC (AttM) and AiiB that degrade the acylhomoserine lactone (AHL) QS signal. In the model Agrobacterium fabrum strain C58, several data, once integrated, strongly suggest that the QS regulation may not be reacting only to cell concentration. Rather, these QS elements in association with the quorum quenching (QQ) activities may constitute an integrated and complex “go/no go system” that finely controls the biologically costly transfer of the Ti plasmid in response to multiple environmental cues. This decision mechanism permits the bacteria to sense whether it is in a gall or not, in a living or decaying tumor, in stressed plant tissues, etc. In this scheme, the role of the lactonases selected and maintained in the course of Ti plasmid and agrobacterial evolution appears to be pivotal.

High-pressure synthesis, crystal structure, and magnetic properties of KSbO3-type 5d oxides K0.84OsO3 and Bi2.93Os3O11

NASA Astrophysics Data System (ADS)

Yuan, Yahua; Feng, Hai L.; Shi, Youguo; Tsujimoto, Yoshihiro; Belik, Alexei A.; Matsushita, Yoshitaka; Arai, Masao; He, Jianfeng; Tanaka, Masahiko; Yamaura, Kazunari

2014-12-01

5d Solid-state oxides K0.84OsO3 (Os5.16+; 5d 2.84) and Bi2.93Os3O11 (Os4.40+; 5d 3.60) were synthesized under high-pressure and high-temperature conditions (6 GPa and 1500-1700 °C). Their crystal structures were determined by synchrotron x-ray diffraction and their 5d electronic properties and tunnel-like structure motifs were investigated. A KSbO3-type structure with a space group of Im-3 and Pn-3 was determined for K0.84OsO3 and Bi2.93Os3O11, respectively. The magnetic and electronic transport properties of the polycrystalline compounds were compared with those obtained theoretically. It was revealed that the 5d tunnel-like structures are paramagnetic with metallic charge conduction at temperatures above 2 K. This was similar to what was observed for structurally relevant 5d oxides, including Bi3Re3O11 (Re4.33+; 5d 2.66) and Ba2Ir3O9 (Ir4.66+; 5d 4.33). The absence of long-range magnetic order seems to be common among 5d KSbO3-like oxides, regardless of the number of 5d electrons (between 2.6 and 4.3 per 5d atom).

Genetic transformation of Fusarium oxysporum f.sp. gladioli with Agrobacterium to study pathogenesis in Gladiolus

USDA-ARS?s Scientific Manuscript database

Fusarium rot caused by Fusarium oxysporum f.sp. gladioli (Fog) is one of the most serious diseases of Gladiolus, both in the field and in stored bulbs. In order to study the pathogenesis of this fungus, we have transformed Fog with Agrobacterium tumefaciens binary vectors containing the hygromycin B...

Linear Chromosome-generating System of Agrobacterium tumefaciens C58: Protelomerase Generates and Protects Hairpin Ends

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Wai Mun; DaGloria, Jeanne; Fox, Heather

2012-09-05

Agrobacterium tumefaciens C58, the pathogenic bacteria that causes crown gall disease in plants, harbors one circular and one linear chromosome and two circular plasmids. The telomeres of its unusual linear chromosome are covalently closed hairpins. The circular and linear chromosomes co-segregate and are stably maintained in the organism. We have determined the sequence of the two ends of the linear chromosome thus completing the previously published genome sequence of A. tumefaciens C58. We found that the telomeres carry nearly identical 25-bp sequences at the hairpin ends that are related by dyad symmetry. We further showed that its Atu2523 gene encodesmore » a protelomerase (resolvase) and that the purified enzyme can generate the linear chromosomal closed hairpin ends in a sequence-specific manner. Agrobacterium protelomerase, whose presence is apparently limited to biovar 1 strains, acts via a cleavage-and-religation mechanism by making a pair of transient staggered nicks invariably at 6-bp spacing as the reaction intermediate. The enzyme can be significantly shortened at both the N and C termini and still maintain its enzymatic activity. Although the full-length enzyme can uniquely bind to its product telomeres, the N-terminal truncations cannot. The target site can also be shortened from the native 50-bp inverted repeat to 26 bp; thus, the Agrobacterium hairpin-generating system represents the most compact activity of all hairpin linear chromosome- and plasmid-generating systems to date. The biochemical analyses of the protelomerase reactions further revealed that the tip of the hairpin telomere may be unusually polymorphically capable of accommodating any nucleotide.« less

Agrobacterium-derived cytokinin influences plastid morphology and starch accumulation in Nicotiana benthamiana during transient assays

PubMed Central

2014-01-01

Background Agrobacterium tumefaciens-based transient assays have become a common tool for answering questions related to protein localization and gene expression in a cellular context. The use of these assays assumes that the transiently transformed cells are observed under relatively authentic physiological conditions and maintain ‘normal’ sub-cellular behaviour. Although this premise is widely accepted, the question of whether cellular organization and organelle morphology is altered in Agrobacterium-infiltrated cells has not been examined in detail. The first indications of an altered sub-cellular environment came from our observation that a common laboratory strain, GV3101(pMP90), caused a drastic increase in stromule frequency. Stromules, or ‘stroma-filled-tubules’ emanate from the surface of plastids and are sensitive to a variety of biotic and abiotic stresses. Starting from this observation, the goal of our experiments was to further characterize the changes to the cell resulting from short-term bacterial infestation, and to identify the factor responsible for eliciting these changes. Results Using a protocol typical of transient assays we evaluated the impact of GV3101(pMP90) infiltration on chloroplast behaviour and morphology in Nicotiana benthamiana. Our experiments confirmed that GV3101(pMP90) consistently induces stromules and alters plastid position relative to the nucleus. These effects were found to be the result of strain-dependant secretion of cytokinin and its accumulation in the plant tissue. Bacterial production of the hormone was found to be dependant on the presence of a trans-zeatin synthase gene (tzs) located on the Ti plasmid of GV3101(pMP90). Bacteria-derived cytokinins were also correlated with changes to both soluble sugar level and starch accumulation. Conclusion Although we have chosen to focus on how transient Agrobacterium infestation alters plastid based parameters, these changes to the morphology and position of a single

Highly Efficient Agrobacterium-Mediated Transformation of Wheat Via In Planta Inoculation

NASA Astrophysics Data System (ADS)

Risacher, Thierry; Craze, Melanie; Bowden, Sarah; Paul, Wyatt; Barsby, Tina

This chapter details a reproducible method for the transformation of spring wheat using Agrobacterium tumefaciens via the direct inoculation of bacteria into immature seeds in planta as described in patent WO 00/63398(1. Transformation efficiencies from 1 to 30% have been obtained and average efficiencies of at least 5% are routinely achieved. Regenerated plants are phenotypically normal with 30-50% of transformation events carrying introduced genes at single insertion sites, a higher rate than is typically reported for transgenic plants produced using biolistic transformation methods.

Properties of the Only Thorium Fullerene, Th@C84, Uncovered.

PubMed

Kaminský, Jakub; Vícha, Jan; Bouř, Petr; Straka, Michal

2017-04-27

Only a single thorium fullerene, Th@C 84 , has been reported to date (Akiyama, K.; et al. J. Nucl. Radiochem. Sci. 2002, 3, 151-154). Although the system was characterized by UV-vis and XANES (X-ray absorption near edge structure) spectra, its structure and properties remain unknown. In this work we used the density functional calculations to identify molecular and electronic structure of the Th@C 84 . Series of molecular structures satisfying the ThC 84 stoichiometric formula were studied comprising 24 IPR and 110 non-IPR Th@C 84 isomers as well as 9 ThC 2 @C 82 IPR isomers. The lowest energy structure is Th@C 84 -C s (10) with the singlet ground state. Its predicted electronic absorption spectra are in agreement with the experimentally observed ones. The bonding between the cage and Th was characterized as polar covalent with Th in formal oxidation state IV. The NMR chemical shifts of Th@C 84 -C s (10) were predicted to guide the future experimental efforts in identification of this compound.

Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants

NASA Technical Reports Server (NTRS)

Cardoza, V.; Stewart, C. N.

2003-01-01

An efficient protocol for the production of transgenic Brassica napus cv. Westar plants was developed by optimizing two important parameters: preconditioning time and co-cultivation time. Agrobacterium tumefaciens-mediated transformation was performed using hypocotyls as explant tissue. Two variants of a green fluorescent protein (GFP)-encoding gene--mGFP5-ER and eGFP--both under the constitutive expression of the cauliflower mosaic virus 35S promoter, were used for the experiments. Optimizing the preconditioning time to 72 h and co-cultivation time with Agrobacterium to 48 h provided the increase in the transformation efficiency from a baseline of 4% to 25%. With mGFP5-ER, the transformation rate was 17% and with eGFP it was 25%. Transgenic shoots were selected on 200 mg/l kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 10 g/l sucrose and 0.5 mg/l indole butyric acid in the presence of kanamycin.

Genetic Transformation of Metroxylon sagu (Rottb.) Cultures via Agrobacterium-Mediated and Particle Bombardment

PubMed Central

Ibrahim, Evra Raunie

2014-01-01

Sago palm (Metroxylon sagu) is a perennial plant native to Southeast Asia and exploited mainly for the starch content in its trunk. Genetic improvement of sago palm is extremely slow when compared to other annual starch crops. Urgent attention is needed to improve the sago palm planting material and can be achieved through nonconventional methods. We have previously developed a tissue culture method for sago palm, which is used to provide the planting materials and to develop a genetic transformation procedure. Here, we report the genetic transformation of sago embryonic callus derived from suspension culture using Agrobacterium tumefaciens and gene gun systems. The transformed embryoids cells were selected against Basta (concentration 10 to 30 mg/L). Evidence of foreign genes integration and function of the bar and gus genes were verified via gene specific PCR amplification, gus staining, and dot blot analysis. This study showed that the embryogenic callus was the most suitable material for transformation as compared to the fine callus, embryoid stage, and initiated shoots. The gene gun transformation showed higher transformation efficiency than the ones transformed using Agrobacterium when targets were bombarded once or twice using 280 psi of helium pressure at 6 to 8 cm distance. PMID:25295258

Effect of Medium Supplements on Agrobacterium rhizogenes Mediated Hairy Root Induction from the Callus Tissues of Camellia sinensis var. sinensis

PubMed Central

Rana, Mohammad M.; Han, Zhuo-Xiao; Song, Da-Peng; Liu, Guo-Feng; Li, Da-Xiang; Wan, Xiao-Chun; Karthikeyan, Alagarsamy; Wei, Shu

2016-01-01

Tea (Camellia sinensis L.) is recalcitrant to Agrobacterium-mediated genetic transformation largely due to the bactericidal effects of tea polyphenols and phenolics oxidation induced by necrosis of explant tissue over the process of transformation. In this study, different antioxidants/adsorbents were added as supplements to the co-cultivation and post co-cultivation media to overcome these problems for the transformation improvement. Tea-cotyledon-derived calli were used as explants and Agrobacterium rhizognes strain ATCC 15834 was used as a mediator. Results showed that Agrobacterium growth, virulence (vir) gene expression and browning of explant tissue were greatly influenced by different supplements. Murashige and Skoog (MS) basal salts medium supplemented with 30 g·L−1 sucrose, 0.1 g·L−1 l-glutamine and 5 g·L−1 polyvinylpolypyrrolidone (PVPP) as co-cultivation and post co-cultivation media could maintain these parameters better that ultimately led to significant improvement of hairy root generation efficiency compared to that in the control (MS + 30 g·L−1 sucrose). Additionally, the reporter genes β-glucuronidase (gusA) and cyan fluorescent protein (cfp) were also stably expressed in the transgenic hairy roots. Our study would be helpful in establishing a feasible approach for tea biological studies and genetic improvement of tea varieties. PMID:27428960

Effect of Medium Supplements on Agrobacterium rhizogenes Mediated Hairy Root Induction from the Callus Tissues of Camellia sinensis var. sinensis.

PubMed

Rana, Mohammad M; Han, Zhuo-Xiao; Song, Da-Peng; Liu, Guo-Feng; Li, Da-Xiang; Wan, Xiao-Chun; Karthikeyan, Alagarsamy; Wei, Shu

2016-07-15

Tea (Camellia sinensis L.) is recalcitrant to Agrobacterium-mediated genetic transformation largely due to the bactericidal effects of tea polyphenols and phenolics oxidation induced by necrosis of explant tissue over the process of transformation. In this study, different antioxidants/adsorbents were added as supplements to the co-cultivation and post co-cultivation media to overcome these problems for the transformation improvement. Tea-cotyledon-derived calli were used as explants and Agrobacterium rhizognes strain ATCC 15834 was used as a mediator. Results showed that Agrobacterium growth, virulence (vir) gene expression and browning of explant tissue were greatly influenced by different supplements. Murashige and Skoog (MS) basal salts medium supplemented with 30 g·L(-1) sucrose, 0.1 g·L(-1) l-glutamine and 5 g·L(-1) polyvinylpolypyrrolidone (PVPP) as co-cultivation and post co-cultivation media could maintain these parameters better that ultimately led to significant improvement of hairy root generation efficiency compared to that in the control (MS + 30 g·L(-1) sucrose). Additionally, the reporter genes β-glucuronidase (gusA) and cyan fluorescent protein (cfp) were also stably expressed in the transgenic hairy roots. Our study would be helpful in establishing a feasible approach for tea biological studies and genetic improvement of tea varieties.

Characterization of an Agrobacterium tumefaciens d-Psicose 3-Epimerase That Converts d-Fructose to d-Psicose

PubMed Central

Kim, Hye-Jung; Hyun, Eun-Kyung; Kim, Yeong-Su; Lee, Yong-Joo; Oh, Deok-Kun

2006-01-01

The noncharacterized gene previously proposed as the d-tagatose 3-epimerase gene from Agrobacterium tumefaciens was cloned and expressed in Escherichia coli. The expressed enzyme was purified by three-step chromatography with a final specific activity of 8.89 U/mg. The molecular mass of the purified protein was estimated to be 132 kDa of four identical subunits. Mn2+ significantly increased the epimerization rate from d-fructose to d-psicose. The enzyme exhibited maximal activity at 50°C and pH 8.0 with Mn2+. The turnover number (kcat) and catalytic efficiency (kcat/Km) of the enzyme for d-psicose were markedly higher than those for d-tagatose, suggesting that the enzyme is not d-tagatose 3-epimerase but d-psicose 3-epimerase. The equilibrium ratio between d-psicose and d-fructose was 32:68 at 30°C. d-Psicose was produced at 230 g/liter from 700-g/liter d-fructose at 50°C after 100 min, corresponding to a conversion yield of 32.9%. PMID:16461638

The genome of cultivated sweet potato contains Agrobacterium T-DNAs with expressed genes: An example of a naturally transgenic food crop

PubMed Central

Kyndt, Tina; Quispe, Dora; Zhai, Hong; Jarret, Robert; Ghislain, Marc; Liu, Qingchang; Gheysen, Godelieve

2015-01-01

Agrobacterium rhizogenes and Agrobacterium tumefaciens are plant pathogenic bacteria capable of transferring DNA fragments [transfer DNA (T-DNA)] bearing functional genes into the host plant genome. This naturally occurring mechanism has been adapted by plant biotechnologists to develop genetically modified crops that today are grown on more than 10% of the world’s arable land, although their use can result in considerable controversy. While assembling small interfering RNAs, or siRNAs, of sweet potato plants for metagenomic analysis, sequences homologous to T-DNA sequences from Agrobacterium spp. were discovered. Simple and quantitative PCR, Southern blotting, genome walking, and bacterial artificial chromosome library screening and sequencing unambiguously demonstrated that two different T-DNA regions (IbT-DNA1 and IbT-DNA2) are present in the cultivated sweet potato (Ipomoea batatas [L.] Lam.) genome and that these foreign genes are expressed at detectable levels in different tissues of the sweet potato plant. IbT-DNA1 was found to contain four open reading frames (ORFs) homologous to the tryptophan-2-monooxygenase (iaaM), indole-3-acetamide hydrolase (iaaH), C-protein (C-prot), and agrocinopine synthase (Acs) genes of Agrobacterium spp. IbT-DNA1 was detected in all 291 cultigens examined, but not in close wild relatives. IbT-DNA2 contained at least five ORFs with significant homology to the ORF14, ORF17n, rooting locus (Rol)B/RolC, ORF13, and ORF18/ORF17n genes of A. rhizogenes. IbT-DNA2 was detected in 45 of 217 genotypes that included both cultivated and wild species. Our finding, that sweet potato is naturally transgenic while being a widely and traditionally consumed food crop, could affect the current consumer distrust of the safety of transgenic food crops. PMID:25902487

Characterization of Cold Sprayed CuCrAl-Coated and Uncoated GRCop-84 Substrates for Space Launch Vehicles

NASA Technical Reports Server (NTRS)

Raj, S. V.; Karthikeyan, J.; Lerch, B. A.; Barrett, C.; Garlich, R.

2007-01-01

A newly developed Cu-23(wt.%)Cr-5%Al (CuCrAl) alloy is currently being considered as a protective coating for GRCop-84 (Cu-8(at.%)Cr-4%Nb). The coating was deposited on GRCop-84 substrates by the cold spray deposition technique. Cyclic oxidation tests conducted in air on both coated and uncoated substrates between 773 and 1073 K revealed that the coating remained intact and protected the substrate up to 1073 K. No significant weight loss of the coated specimens were observed at 773 and 873 K even after a cumulative cyclic time of 500 h. In contrast, the uncoated substrate lost as much as 80% of its original weight under similar test conditions. Low cycle fatigue tests revealed that the fatigue lives of thinly coated GRCop-84 specimens were similar to the uncoated specimens within the limits of experimental scatter. It is concluded that the cold sprayed CuCrAl coating is suitable for protecting GRCop-84 substrates.

An efficient Agrobacterium-mediated transformation method for the edible mushroom Hypsizygus marmoreus.

PubMed

Zhang, Jin jing; Shi, Liang; Chen, Hui; Sun, Yun qi; Zhao, Ming wen; Ren, Ang; Chen, Ming jie; Wang, Hong; Feng, Zhi yong

2014-01-01

Hypsizygus marmoreus is one of the major edible mushrooms in East Asia. As no efficient transformation method, the molecular and genetics studies were hindered. The glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of H. marmoreus was isolated and its promoter was used to drive the hygromycin B phosphotransferase (HPH) and enhanced green fluorescent protein (EGFP) in H. marmoreus. Agrobacterium tumefaciens-mediated transformation (ATMT) was successfully applied in H. marmoreus. The transformation parameters were optimized, and it was found that co-cultivation of bacteria with protoplast at a ratio of 1000:1 at a temperature of 26 °C in medium containing 0.3 mM acetosyringone resulted in the highest transformation efficiency for Agrobacterium strain. Besides, three plasmids, each carrying a different promoter (from H. marmoreus, Ganoderma lucidum and Lentinula edodes) driving the expression of an antibiotic resistance marker, were also tested. The construct carrying the H. marmoreus gpd promoter produced more transformants than other constructs. Our analysis showed that over 85% of the transformants tested remained mitotically stable even after five successive rounds of subculturing. Putative transformants were analyzed for the presence of hph gene by PCR and Southern blot. Meanwhile, the expression of EGFP in H. marmoreus transformants was detected by fluorescence imaging. This ATMT system increases the transformation efficiency of H. marmoreus and may represent a useful tool for molecular genetic studies in this mushroom species. Copyright © 2014 Elsevier GmbH. All rights reserved.

Historical account on gaining insights on the mechanism of crown gall tumorigenesis induced by Agrobacterium tumefaciens

PubMed Central

Kado, Clarence I.

2014-01-01

The plant tumor disease known as crown gall was not called by that name until more recent times. Galls on plants were described by Malpighi (1679) who believed that these extraordinary growth are spontaneously produced. Agrobacterium was first isolated from tumors in 1897 by Fridiano Cavara in Napoli, Italy. After this bacterium was recognized to be the cause of crown gall disease, questions were raised on the mechanism by which it caused tumors on a variety of plants. Numerous very detailed studies led to the identification of Agrobacterium tumefaciens as the causal bacterium that cleverly transferred a genetic principle to plant host cells and integrated it into their chromosomes. Such studies have led to a variety of sophisticated mechanisms used by this organism to aid in its survival against competing microorganisms. Knowledge gained from these fundamental discoveries has opened many avenues for researchers to examine their primary organisms of study for similar mechanisms of pathogenesis in both plants and animals. These discoveries also advanced the genetic engineering of domesticated plants for improved food and fiber. PMID:25147542

Early events in Agrobacterium-mediated genetic transformation of citrus explants.

PubMed

Peña, Leandro; Pérez, Rosa M; Cervera, Magdalena; Juárez, José A; Navarro, Luis

2004-07-01

Genetic transformation of plants relies on two independent but concurrent processes: integration of foreign DNA into plant cells and regeneration of whole plants from these transformed cells. Cell competence for regeneration and for transformation does not always fall into the same cell type/developmental stage, and this is one of the main causes of the so-called recalcitrance for transformation of certain plant species. In this study, a detailed examination of the first steps of morphogenesis from citrus explants after co-cultivation with Agrobacterium tumefaciens was performed, and an investigation into which cells and tissues are competent for regeneration and transformation was carried out. Moreover, the role of phytohormones in the co-cultivation medium as possible enhancers of gene transfer was also studied. A highly responsive citrus genotype and well-established culture conditions were used to perform a histological analysis of morphogenesis and cell competence for transformation after co-cultivation of citrus epicotyl segments with A. tumefaciens. In addition, the role of phytohormones as transformation enhancers was investigated by flow cytometry. It is demonstrated that cells competent for transformation are located in the newly formed callus growing from the cambial ring. Conditions conducive to further development of this callus, such as treatment of explants in a medium rich in auxins, resulted in a more pronounced formation of cambial callus and a slower shoot regeneration process, both in Agrobacterium-inoculated and non-inoculated explants. Furthermore, co- cultivation in a medium rich in auxins caused a significant increase in the rate of actively dividing cells in S-phase, the stage in which cells are more prone to integrate foreign DNA. Use of proper co-cultivation medium and conditions led to a higher number of stably transformed cells and to an increase in the final number of regenerated transgenic plants.

Use of Agrobacterium rhizogenes strain 18r12v and paromomycin selection for transformation of Brachypodium distachyon and Brachypodium sylvaticum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Collier, Ray; Bragg, Jennifer; Hernandez, Bryan T.

In this study, the genetic transformation of monocot grasses is a resource intensive process, the quality and efficiency of which is dependent in part upon the method of DNA introduction, as well as the ability to effectively separate transformed from wildtype tissue. Agrobacterium-mediated transformation of Brachypodium has relied mainly on Agrobacterium tumefaciens strain AGL1. Currently the antibiotic hygromycin B has been the selective agent of choice for robust identification of transgenic calli in Brachypodium distachyon and Brachypodium sylvaticum but few other chemicals have been shown to work as well for selection of transgenic Brachypodium cells in tissue culture. This studymore » demonstrates that Agrobacterium rhizogenes strain 18r12v and paromomycin selection can be successfully used for the efficient generation of transgenic B. distachyon and B. sylvaticurn. Additionally we observed that the transformation rates were similar to or higher than those obtained with A. turnefaciens strain AGL1 and hygromycin selection. The A. rhizogenes strain 18r12v harboring the pARS1 binary vector and paromomycin selection is an effective means of generating transgenic Brachypodium plants. This novel approach will facilitate the transgenic complementation of T-DNA knockout mutants of B. distachyon which were created using hygromycin selection, as well as aid the implementation of more complex genome manipulation strategies which require multiple rounds of transformation.« less

Use of Agrobacterium rhizogenes strain 18r12v and paromomycin selection for transformation of Brachypodium distachyon and Brachypodium sylvaticum

DOE PAGES

Collier, Ray; Bragg, Jennifer; Hernandez, Bryan T.; ...

2016-05-24

In this study, the genetic transformation of monocot grasses is a resource intensive process, the quality and efficiency of which is dependent in part upon the method of DNA introduction, as well as the ability to effectively separate transformed from wildtype tissue. Agrobacterium-mediated transformation of Brachypodium has relied mainly on Agrobacterium tumefaciens strain AGL1. Currently the antibiotic hygromycin B has been the selective agent of choice for robust identification of transgenic calli in Brachypodium distachyon and Brachypodium sylvaticum but few other chemicals have been shown to work as well for selection of transgenic Brachypodium cells in tissue culture. This studymore » demonstrates that Agrobacterium rhizogenes strain 18r12v and paromomycin selection can be successfully used for the efficient generation of transgenic B. distachyon and B. sylvaticurn. Additionally we observed that the transformation rates were similar to or higher than those obtained with A. turnefaciens strain AGL1 and hygromycin selection. The A. rhizogenes strain 18r12v harboring the pARS1 binary vector and paromomycin selection is an effective means of generating transgenic Brachypodium plants. This novel approach will facilitate the transgenic complementation of T-DNA knockout mutants of B. distachyon which were created using hygromycin selection, as well as aid the implementation of more complex genome manipulation strategies which require multiple rounds of transformation.« less

Improved Agrobacterium-mediated transformation and high efficiency of root formation from hypocotyl meristem of spring Brassica napus 'Precocity' cultivar.

PubMed

Liu, X X; Lang, S R; Su, L Q; Liu, X; Wang, X F

2015-12-14

Rape seed (Brassica napus L.) is one of the most important oil seed crops in the world. Genetic manipulation of rapeseed requires a suitable tissue culture system and an efficient method for plant regeneration, as well as an efficient transformation procedure. However, development of transgenic B. napus has been problematic, and current studies are limited to cultivated varieties. In this study, we report a protocol for regeneration of transgenic rape after Agrobacterium-mediated transformation of hypocotyls from the spring B. napus 'Precocity' cultivar. We analyzed the effects of plant growth regulators in the medium on regeneration. Additionally, factors affecting the transformation efficiency, including seedling age, Agrobacterium concentration, infection time, and co-cultivation time, were assessed by monitoring GUS expression. Results from these experiments revealed that transformation was optimized when the meristematic parts of the hypocotyls were taken from 8 day-old seedlings, cultured on Murashinge and Skoog basal media containing 0.1 mg/L 1-naphthaleneacetic acid and 2.5 mg/L 6-benzylaminopurine, and incubated in Agrobacterium suspension (OD600 = 0.5) for 3 to 5 min, followed by 2 days of co-cultivation. Integration of T-DNA into the plant genome was confirmed by polymerase chain reaction (PCR), b-glucuronidase histochemical staining, and quantitative real-time PCR. The protocols developed for regeneration, transformation, and rooting described in this study could help to accelerate the development of transgenic spring rape varieties with novel features.

Effect of pre-plant soil fumigants on Agrobacterium tumefaciens, pythiaceous species, and subsequent soil recolonization by A. tumefaciens

USDA-ARS?s Scientific Manuscript database

Paradox (Juglans hindsii x J. regia), the dominant rootstock used in the California walnut industry, is susceptible to crown gall, caused by Agrobacterium tumefaciens. In practice, soil fumigation has been a common preplant management strategy for crown gall, but even an industry standard, methyl b...

Iodate and nitrate transformation by Agrobacterium/Rhizobium related strain DVZ35 isolated from contaminated Hanford groundwater.

PubMed

Lee, Brady D; Ellis, Joshua T; Dodwell, Alex; Eisenhauer, Emalee E R; Saunders, Danielle L; Lee, M Hope

2018-05-15

Nitrate and radioiodine ( 129 I) contamination is widespread in groundwater underneath the Central Plateau of the Hanford Site. 129 I, a byproduct of nuclear fission, is of concern due to a 15.7 million year half-life, and toxicity. The Hanford 200 West Area contains plumes covering 4.3 km 2 with average 129 I concentrations of 3.5 pCi/L. Iodate accounts for 70.6% of the iodine present and organo-iodine and iodide make up 25.8% and 3.6%, respectively. Nitrate plumes encompassing the 129 I plumes have a surface area of 16 km 2 averaging 130 mg/L. A nitrate and iodate reducing bacterium closely related to Agrobacterium, strain DVZ35, was isolated from sediment incubated in a 129 I plume. Iodate removal efficiency was 36.3% in transition cultures, and 47.8% in anaerobic cultures. Nitrate (10 mM) was also reduced in the microcosm. When nitrate was spiked into the microcosms, iodate removal efficiency was 84.0% and 69.2% in transition and anaerobic cultures, respectively. Iodate reduction was lacking when nitrate was absent from the growth medium. These data indicate there is simultaneous reduction of nitrate and iodate by DVZ35, and iodate is reduced to iodide. Results provide the scientific basis for combined nitrogen and iodine cycling throughout the Hanford Site. Copyright © 2018. Published by Elsevier B.V.

Re-examination of cellular cyclic beta-1,2-glucans of Rhizobiaceae: distribution of ring sizes and degrees of glycerol-1-phosphate substitution.

PubMed

Zevenhuizen, L P; van Veldhuizen, A; Fokkens, R H

1990-04-01

Gel-filtration and thin layer chromatography of low molecular weight carbohydrates from culture filtrates of Agrobacterium radiobacter, Isolate II, have shown, that next to the neutral beta-1,2-glucan fraction a major acidic fraction was present which was found to be glycerophosphorylated cyclic beta-1,2-glucans. Re-examination of cyclic beta-1,2-glucan preparations which had been obtained by extraction of Rhizobium cells with hot phenol-water also showed these acidic modified beta-1,2-glucans to be present. Cyclic beta-1,2-glucans from R. leguminosarum (9 strains) and of R. phaseoli (1 strain) had ring size distribution with degrees of polymerisation (DPs) of 19 and 20 as major ring sizes of which a minor part was glycerophosphorylated; beta-1,2-glucans of R. trifolii (3 strains) had ring sizes with DPs measuring 19-22 as prominent components which were largely unsubstituted, and R. meliloti (7 strains) had beta-1,2-glucans with ring size distributions extending to still higher DPs of 19-25 of which the major part appeared to be glycerophosphorylated.

Reduction in pathogen populations at grapevine wound sites is associated with the mechanism underlying the biological control of crown gall by rhizobium vitis strain ARK-1.

PubMed

Kawaguchi, Akira

2014-09-17

A nonpathogenic strain of Rhizobium (=Agrobacterium) vitis, ARK-1, limited the development of grapevine crown gall. A co-inoculation with ARK-1 and the tumorigenic strain VAT07-1 at a 1:1 cell ratio resulted in a higher population of ARK-1 than VAT07-1 in shoots without tumors, but a significantly lower population of ARK-1 than VAT07-1 in grapevine shoots with tumors. ARK-1 began to significantly suppress the VAT07-1 population 2 d after the inoculation. This result indicated that ARK-1 reduced the pathogen population at the wound site through biological control. Although ARK-1 produced a zone of inhibition against other tumorigenic Rhizobium spp. in in vitro assays, antibiosis depended on the culture medium. ARK-1 did not inhibit the growth of tumorigenic R. radiobacter strain AtC1 in the antibiosis assay, but suppressed the AtC1-induced formation of tumors on grapevine shoots, suggesting that antibiosis by ARK-1 may not be the main mechanism responsible for biological control.

Improved catalytic properties of halohydrin dehalogenase by modification of the halide-binding site.

PubMed

Tang, Lixia; Torres Pazmiño, Daniel E; Fraaije, Marco W; de Jong, René M; Dijkstra, Bauke W; Janssen, Dick B

2005-05-03

Halohydrin dehalogenase (HheC) from Agrobacterium radiobacter AD1 catalyzes the dehalogenation of vicinal haloalcohols by an intramolecular substitution reaction, resulting in the formation of the corresponding epoxide, a halide ion, and a proton. Halide release is rate-limiting during the catalytic cycle of the conversion of (R)-p-nitro-2-bromo-1-phenylethanol by the enzyme. The recent elucidation of the X-ray structure of HheC showed that hydrogen bonds between the OH group of Tyr187 and between the Odelta1 atom of Asn176 and Nepsilon1 atom of Trp249 could play a role in stabilizing the conformation of the halide-binding site. The possibility that these hydrogen bonds are important for halide binding and release was studied using site-directed mutagenesis. Steady-state kinetic studies revealed that mutant Y187F, which has lost both hydrogen bonds, has a higher catalytic activity (k(cat)) with two of the three tested substrates compared to the wild-type enzyme. Mutant W249F also shows an enhanced k(cat) value with these two substrates, as well as a remarkable increase in enantiopreference for (R)-p-nitro-2-bromo-1-phenylethanol. In case of a mutation at position 176 (N176A and N176D), a 1000-fold lower catalytic efficiency (k(cat)/K(m)) was obtained, which is mainly due to an increase of the K(m) value of the enzyme. Pre-steady-state kinetic studies showed that a burst of product formation precedes the steady state, indicating that halide release is still rate-limiting for mutants Y187F and W249F. Stopped-flow fluorescence experiments revealed that the rate of halide release is 5.6-fold higher for the Y187F mutant than for the wild-type enzyme and even higher for the W249F enzyme. Taken together, these results show that the disruption of two hydrogen bonds around the halide-binding site increases the rate of halide release and can enhance the overall catalytic activity of HheC.

Evaluations and modifications of semi-selective media for improved isolation of Agrobacterium tumefaciens biovar 1 from cultivated walnut

USDA-ARS?s Scientific Manuscript database

Agrobacterium tumefaciens, the causal agent of crown gall of walnut, is an aerobic, Gram negative bacterium belonging to the family Rhizobiaceae. Like many in this group, A. tumefaciens is a common inhabitant of soil and plant host tissue. Isolation from these complex environments is difficult even ...

Chemical modification of L-glutamine to alpha-amino glutarimide on autoclaving facilitates Agrobacterium infection of host and non-host plants: A new use of a known compound

PubMed Central

2011-01-01

Background Accidental autoclaving of L-glutamine was found to facilitate the Agrobacterium infection of a non host plant like tea in an earlier study. In the present communication, we elucidate the structural changes in L-glutamine due to autoclaving and also confirm the role of heat transformed L-glutamine in Agrobacterium mediated genetic transformation of host/non host plants. Results When autoclaved at 121°C and 15 psi for 20 or 40 min, L-glutamine was structurally modified into 5-oxo proline and 3-amino glutarimide (α-amino glutarimide), respectively. Of the two autoclaved products, only α-amino glutarimide facilitated Agrobacterium infection of a number of resistant to susceptible plants. However, the compound did not have any vir gene inducing property. Conclusions We report a one pot autoclave process for the synthesis of 5-oxo proline and α-amino glutarimide from L-glutamine. Xenobiotic detoxifying property of α-amino glutarimide is also proposed. PMID:21624145

Cross sections for the reactions e+e-→K+K-π+π-, K+K-π0π0, and K+K-K+K- measured using initial-state radiation events

NASA Astrophysics Data System (ADS)

Lees, J. P.; Poireau, V.; Prencipe, E.; Tisserand, V.; Garra Tico, J.; Grauges, E.; Martinelli, M.; Milanes, D. A.; Palano, A.; Pappagallo, M.; Eigen, G.; Stugu, B.; Sun, L.; Brown, D. N.; Kerth, L. T.; Kolomensky, Yu. G.; Lynch, G.; Koch, H.; Schroeder, T.; Asgeirsson, D. J.; Hearty, C.; Mattison, T. S.; McKenna, J. A.; Khan, A.; Blinov, V. E.; Buzykaev, A. R.; Druzhinin, V. P.; Golubev, V. B.; Kravchenko, E. A.; Onuchin, A. P.; Serednyakov, S. I.; Skovpen, Yu. I.; Solodov, E. P.; Todyshev, K. Yu.; Yushkov, A. N.; Bondioli, M.; Curry, S.; Kirkby, D.; Lankford, A. J.; Mandelkern, M.; Stoker, D. P.; Atmacan, H.; Gary, J. W.; Liu, F.; Long, O.; Vitug, G. M.; Campagnari, C.; Hong, T. M.; Kovalskyi, D.; Richman, J. D.; West, C. A.; Eisner, A. M.; Kroseberg, J.; Lockman, W. S.; Martinez, A. J.; Schalk, T.; Schumm, B. A.; Seiden, A.; Cheng, C. H.; Doll, D. A.; Echenard, B.; Flood, K. T.; Hitlin, D. G.; Ongmongkolkul, P.; Porter, F. C.; Rakitin, A. Y.; Andreassen, R.; Dubrovin, M. S.; Meadows, B. T.; Sokoloff, M. D.; Bloom, P. C.; Ford, W. T.; Gaz, A.; Nagel, M.; Nauenberg, U.; Smith, J. G.; Wagner, S. R.; Ayad, R.; Toki, W. H.; Spaan, B.; Kobel, M. J.; Schubert, K. R.; Schwierz, R.; Bernard, D.; Verderi, M.; Clark, P. J.; Playfer, S.; Watson, J. E.; Bettoni, D.; Bozzi, C.; Calabrese, R.; Cibinetto, G.; Fioravanti, E.; Garzia, I.; Luppi, E.; Munerato, M.; Negrini, M.; Piemontese, L.; Baldini-Ferroli, R.; Calcaterra, A.; de Sangro, R.; Finocchiaro, G.; Nicolaci, M.; Pacetti, S.; Patteri, P.; Peruzzi, I. M.; Piccolo, M.; Rama, M.; Zallo, A.; Contri, R.; Guido, E.; Lo Vetere, M.; Monge, M. R.; Passaggio, S.; Patrignani, C.; Robutti, E.; Bhuyan, B.; Prasad, V.; Lee, C. L.; Morii, M.; Edwards, A. J.; Adametz, A.; Marks, J.; Uwer, U.; Bernlochner, F. U.; Ebert, M.; Lacker, H. M.; Lueck, T.; Dauncey, P. D.; Tibbetts, M.; Behera, P. K.; Mallik, U.; Chen, C.; Cochran, J.; Crawley, H. B.; Meyer, W. T.; Prell, S.; Rosenberg, E. I.; Rubin, A. E.; Gritsan, A. V.; Guo, Z. J.; Arnaud, N.; Davier, M.; Derkach, D.; Grosdidier, G.; Le Diberder, F.; Lutz, A. M.; Malaescu, B.; Roudeau, P.; Schune, M. H.; Stocchi, A.; Wormser, G.; Lange, D. J.; Wright, D. M.; Bingham, I.; Chavez, C. A.; Coleman, J. P.; Fry, J. R.; Gabathuler, E.; Hutchcroft, D. E.; Payne, D. J.; Touramanis, C.; Bevan, A. J.; Di Lodovico, F.; Sacco, R.; Sigamani, M.; Cowan, G.; Paramesvaran, S.; Brown, D. N.; Davis, C. L.; Denig, A. G.; Fritsch, M.; Gradl, W.; Hafner, A.; Alwyn, K. E.; Bailey, D.; Barlow, R. J.; Jackson, G.; Lafferty, G. D.; Cenci, R.; Hamilton, B.; Jawahery, A.; Roberts, D. A.; Simi, G.; Dallapiccola, C.; Salvati, E.; Cowan, R.; Dujmic, D.; Sciolla, G.; Lindemann, D.; Patel, P. M.; Robertson, S. H.; Schram, M.; Biassoni, P.; Lazzaro, A.; Lombardo, V.; Palombo, F.; Stracka, S.; Cremaldi, L.; Godang, R.; Kroeger, R.; Sonnek, P.; Summers, D. J.; Nguyen, X.; Taras, P.; De Nardo, G.; Monorchio, D.; Onorato, G.; Sciacca, C.; Raven, G.; Snoek, H. L.; Jessop, C. P.; Knoepfel, K. J.; LoSecco, J. M.; Wang, W. F.; Honscheid, K.; Kass, R.; Brau, J.; Frey, R.; Sinev, N. B.; Strom, D.; Torrence, E.; Feltresi, E.; Gagliardi, N.; Margoni, M.; Morandin, M.; Posocco, M.; Rotondo, M.; Simonetto, F.; Stroili, R.; Ben-Haim, E.; Bomben, M.; Bonneaud, G. R.; Briand, H.; Calderini, G.; Chauveau, J.; Hamon, O.; Leruste, Ph.; Marchiori, G.; Ocariz, J.; Sitt, S.; Biasini, M.; Manoni, E.; Rossi, A.; Angelini, C.; Batignani, G.; Bettarini, S.; Carpinelli, M.; Casarosa, G.; Cervelli, A.; Forti, F.; Giorgi, M. A.; Lusiani, A.; Neri, N.; Oberhof, B.; Paoloni, E.; Perez, A.; Rizzo, G.; Walsh, J. J.; Lopes Pegna, D.; Lu, C.; Olsen, J.; Smith, A. J. S.; Telnov, A. V.; Anulli, F.; Cavoto, G.; Faccini, R.; Ferrarotto, F.; Ferroni, F.; Gaspero, M.; Li Gioi, L.; Mazzoni, M. A.; Piredda, G.; Bünger, C.; Hartmann, T.; Leddig, T.; Schröder, H.; Waldi, R.; Adye, T.; Olaiya, E. O.; Wilson, F. F.; Emery, S.; Hamel de Monchenault, G.; Vasseur, G.; Yèche, Ch.; Aston, D.; Bard, D. J.; Bartoldus, R.; Benitez, J. F.; Cartaro, C.; Convery, M. R.; Dorfan, J.; Dubois-Felsmann, G. P.; Dunwoodie, W.; Field, R. C.; Franco Sevilla, M.; Fulsom, B. G.; Gabareen, A. M.; Graham, M. T.; Grenier, P.; Hast, C.; Innes, W. R.; Kelsey, M. H.; Kim, H.; Kim, P.; Kocian, M. L.; Leith, D. W. G. S.; Lewis, P.; Li, S.; Lindquist, B.; Luitz, S.; Luth, V.; Lynch, H. L.; MacFarlane, D. B.; Muller, D. R.; Neal, H.; Nelson, S.; Ofte, I.; Perl, M.; Pulliam, T.; Ratcliff, B. N.; Roodman, A.; Salnikov, A. A.; Santoro, V.; Schindler, R. H.; Snyder, A.; Su, D.; Sullivan, M. K.; Va'vra, J.; Wagner, A. P.; Weaver, M.; Wisniewski, W. J.; Wittgen, M.; Wright, D. H.; Wulsin, H. W.; Yarritu, A. K.; Young, C. C.; Ziegler, V.; Park, W.; Purohit, M. V.; White, R. M.; Wilson, J. R.; Randle-Conde, A.; Sekula, S. J.; Bellis, M.; Burchat, P. R.; Miyashita, T. S.; Alam, M. S.; Ernst, J. A.; Gorodeisky, R.; Guttman, N.; Peimer, D. R.; Soffer, A.; Lund, P.; Spanier, S. M.; Eckmann, R.; Ritchie, J. L.; Ruland, A. M.; Schilling, C. J.; Schwitters, R. F.; Wray, B. C.; Izen, J. M.; Lou, X. C.; Bianchi, F.; Gamba, D.; Lanceri, L.; Vitale, L.; Lopez-March, N.; Martinez-Vidal, F.; Oyanguren, A.; Ahmed, H.; Albert, J.; Banerjee, Sw.; Choi, H. H. F.; King, G. J.; Kowalewski, R.; Lewczuk, M. J.; Lindsay, C.; Nugent, I. M.; Roney, J. M.; Sobie, R. J.; Gershon, T. J.; Harrison, P. F.; Latham, T. E.; Puccio, E. M. T.; Band, H. R.; Dasu, S.; Pan, Y.; Prepost, R.; Vuosalo, C. O.; Wu, S. L.

2012-07-01

We study the processes e+e-→K+K-π+π-γ, K+K-π0π0γ, and K+K-K+K-γ, where the photon is radiated from the initial state. About 84 000, 8000, and 4200 fully reconstructed events, respectively, are selected from 454fb-1 of BABAR data. The invariant mass of the hadronic final state defines the e+e- center-of-mass energy, so that the K+K-π+π-γ data can be compared with direct measurements of the e+e-→K+K-π+π- reaction. No direct measurements exist for the e+e-→K+K-π0π0 or e+e-→K+K-K+K- reactions, and we present an update of our previous result based on a data sample that is twice as large. Studying the structure of these events, we find contributions from a number of intermediate states and extract their cross sections. In particular, we perform a more detailed study of the e+e-→ϕ(1020)ππγ reaction and confirm the presence of the Y(2175) resonance in the ϕ(1020)f0(980) and K+K-f0(980) modes. In the charmonium region, we observe the J/ψ in all three final states and in several intermediate states, as well as the ψ(2S) in some modes, and measure the corresponding products of branching fraction and electron width.

Establishment of an efficient virus-induced gene silencing (VIGS) assay in Arabidopsis by Agrobacterium-mediated rubbing infection.

PubMed

Manhães, Ana Marcia E de A; de Oliveira, Marcos V V; Shan, Libo

2015-01-01

Several VIGS protocols have been established for high-throughput functional genomic screens as it bypasses the time-consuming and laborious process of generation of transgenic plants. The silencing efficiency in this approach is largely hindered by a technically demanding step in which the first pair of newly emerged true leaves at the 2-week-old stage are infiltrated with a needleless syringe. To further optimize VIGS efficiency and achieve rapid inoculation for a large-scale functional genomic study, here we describe a protocol of an efficient VIGS assay in Arabidopsis using Agrobacterium-mediated rubbing infection. The Agrobacterium inoculation is performed by simply rubbing the leaves with Filter Agent Celite(®) 545. The highly efficient and uniform silencing effect was indicated by the development of a visibly albino phenotype due to silencing of the Cloroplastos alterados 1 (CLA1) gene in the newly emerged leaves. In addition, the albino phenotype could be observed in stems and flowers, indicating its potential application for gene functional studies in the late vegetative development and flowering stages.

Optimization of factors influencing microinjection method for Agrobacterium tumefaciens-mediated transformation of tomato.

PubMed

Vinoth, S; Gurusaravanan, P; Jayabalan, N

2013-02-01

A simple and efficient protocol for Agrobacterium-mediated genetic transformation of tomato was developed using combination of non-tissue culture and micropropagation systems. Initially, ESAM region of 1-day-old germinated tomato seeds were microinjected for one to five times with Agrobacterium inoculums (OD(600) = 0.2-1.0). The germinated seeds were cocultivated in the MS medium fortified with (0-200 mM) acetosyringone and minimal concentrations of (0-20 mg L(-1)) kanamycin, and the antibiotic concentration was doubled during the second round of selection. Bacterial concentration of OD(600) = 0.6 served as an optimal concentration for infection and the transformation efficiency was significantly higher of about 46.28 %. In another set of experiment, an improved and stable regeneration system was adapted for the explants from the selection medium. Four-day-old double cotyledonary nodal explants were excised from the microinjected seedlings and cultured onto the MS medium supplemented with 1.5 mg L(-1) thidiazuron, 1.5 mg L(-1) indole-3-butyric acid, 30 mg L(-1) kanamycin, and 0-1.5 mg L(-1) adenine sulphate. Maximum of 9 out of 13 micropropagated shoots were shown positive to GUS assay. By this technique, the transformation efficiency was increased from 46.28 to 65.90 %. Thus, this paper reports the successful protocol for the mass production of transformants using microinjection and micropropagation techniques.

Disassembly of synthetic Agrobacterium T-DNA–protein complexes via the host SCFVBF ubiquitin–ligase complex pathway

PubMed Central

Zaltsman, Adi; Lacroix, Benoît; Gafni, Yedidya; Citovsky, Vitaly

2013-01-01

One the most intriguing, yet least studied, aspects of the bacterium–host plant interaction is the role of the host ubiquitin/proteasome system (UPS) in the infection process. Increasing evidence indicates that pathogenic bacteria subvert the host UPS to facilitate infection. Although both mammalian and plant bacterial pathogens are known to use the host UPS, the first prokaryotic F-box protein, an essential component of UPS, was identified in Agrobacterium. During its infection, which culminates in genetic modification of the host cell, Agrobacterium transfers its T-DNA—as a complex (T-complex) with the bacterial VirE2 and host VIP1 proteins—into the host cell nucleus. There the T-DNA is uncoated from its protein components before undergoing integration into the host genome. It has been suggested that the host UPS mediates this uncoating process, but there is no evidence indicating that this activity can unmask the T-DNA molecule. Here we provide support for the idea that the plant UPS uncoats synthetic T-complexes via the Skp1/Cullin/F-box protein VBF pathway and exposes the T-DNA molecule to external enzymatic activity. PMID:23248273

Development of a rapid and simple Agrobacterium tumefaciens mediated transformation system for the fungal pathogen Heterobasidion annosum

Treesearch

Nicklas Samils; Malin Elfstrand; Daniel L. Lindner Czederpiltz; Jan Fahleson; Ake Olson; Christina Dixelius; Jan Stenlid

2006-01-01

Heterobasidion annosum causes root and butt-rot in trees and is the most serious forest pathogen in the northern hemisphere. We developed a rapid and simple Agrobacterium-mediated method of gene delivery into H. annosum to be used in functional studies of candidate genes and for visualization of mycelial interactions. Heterobasidion annosum TC 32-1 was cocultivated at...

Efficient Agrobacterium-Mediated Transformation of Hybrid Poplar Populus davidiana Dode × Populus bollena Lauche

PubMed Central

Han, Xue; Ma, Shurong; Kong, Xianghui; Takano, Tetsuo; Liu, Shenkui

2013-01-01

Poplar is a model organism for high in vitro regeneration in woody plants. We have chosen a hybrid poplar Populus davidiana Dode × Populus bollena Lauche. By optimizing the Murashige and Skoog medium with (0.3 mg/L) 6-benzylaminopurine and (0.08 mg/L) naphthaleneacetic acid, we have achieved the highest frequency (90%) for shoot regeneration from poplar leaves. It was also important to improve the transformation efficiency of poplar for genetic breeding and other applications. In this study, we found a significant improvement of the transformation frequency by controlling the leaf age. Transformation efficiency was enhanced by optimizing the Agrobacterium concentration (OD600 = 0.8–1.0) and an infection time (20–30 min). According to transmission electron microscopy observations, there were more Agrobacterium invasions in the 30-day-old leaf explants than in 60-day-old and 90-day-old explants. Using the green fluorescent protein (GFP) marker, the expression of MD–GFP fusion proteins in the leaf, shoot, and root of hybrid poplar P. davidiana Dode × P. bollena Lauche was visualized for confirmation of transgene integration. Southern and Northern blot analysis also showed the integration of T-DNA into the genome and gene expression of transgenic plants. Our results suggest that younger leaves had higher transformation efficiency (~30%) than older leaves (10%). PMID:23354481

Influence of volatile organic compounds emitted by Pseudomonas and Serratia strains on Agrobacterium tumefaciens biofilms.

PubMed

Plyuta, Vladimir; Lipasova, Valentina; Popova, Alexandra; Koksharova, Olga; Kuznetsov, Alexander; Szegedi, Erno; Chernin, Leonid; Khmel, Inessa

2016-07-01

The ability to form biofilms plays an important role in bacteria-host interactions, including plant pathogenicity. In this work, we investigated the action of volatile organic compounds (VOCs) produced by rhizospheric strains of Pseudomonas chlororaphis 449, Pseudomonas fluorescens B-4117, Serratia plymuthica IC1270, as well as Serratia proteamaculans strain 94, isolated from spoiled meat, on biofilms formation by three strains of Agrobacterium tumefaciens which are causative agents of crown-gall disease in a wide range of plants. In dual culture assays, the pool of volatiles emitted by the tested Pseudomonas and Serratia strains suppressed the formation of biofilms of A. tumefaciens strains grown on polycarbonate membrane filters and killed Agrobacterium cells in mature biofilms. The individual VOCs produced by the tested Pseudomonas strains, that is, ketones (2-nonanone, 2-heptanone, 2-undecanone), and dimethyl disulfide (DMDS) produced by Serratia strains, were shown to kill A. tumefaciens cells in mature biofilms and suppress their formation. The data obtained in this study suggest an additional potential of some ketones and DMDS as protectors of plants against A. tumefaciens strains, whose virulence is associated with the formation of biofilms on the infected plants. © 2016 APMIS. Published by John Wiley & Sons Ltd.

Purification, crystallization and preliminary crystallographic study of an IDS-epimerase from Agrobacterium tumefaciens BY6

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bäuerle, Bettina; Sandalova, Tatyana; Schneider, Gunter

2006-08-01

This is the first report of the crystallization of an IDS-epimerase from A. tumefaciens BY6 and its l-selenomethionine derivative. The initial degradation of all stereoisomers of the complexing agent iminodisuccinate (IDS) is enabled by an epimerase in the bacterial strain Agrobacterium tumefaciens BY6. This protein was produced in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method. Crystals of IDS-epimerase were obtained under several conditions. The best diffracting crystals were grown in 22% PEG 3350, 0.2 M (NH{sub 4}){sub 2}SO{sub 4} and 0.1 M bis-Tris propane pH 7.2 at 293 K. These crystals belong to the monoclinic space groupmore » P2{sub 1}, with unit-cell parameters a = 55.4, b = 104.2, c = 78.6 Å, β = 103.3°, and diffracted to 1.7 Å resolution. They contain two protein molecules per asymmetric unit. In order to solve the structure using the MAD phasing method, crystals of the l-selenomethionine-substituted epimerase were grown in the presence of 20% PEG 3350, 0.2 M Na{sub 2}SO{sub 4} and 0.1 M bis-Tris propane pH 8.5.« less

Gene disruption in Trichoderma atroviride via Agrobacterium-mediated transformation.

PubMed

Zeilinger, Susanne

2004-02-01

A modified Agrobacterium-mediated transformation method for the efficient disruption of two genes encoding signaling compounds of the mycoparasite Trichoderma atroviride is described, using the hph gene of Escherichia coli as selection marker. The transformation vectors contained about 1 kb of 5' and 3' non-coding regions from the tmk1 (encoding a MAP kinase) or tga3 (encoding an alpha-subunit of a heterotrimeric G protein) target loci flanking a selection marker. Transformation of fungal conidia and selection on hygromycin-containing media applying an overlay-based procedure, which overcomes the lack of formation of distinct single colonies by the fungus, led to stable clones for both disruption constructs. Southern and PCR analyses proved gene disruption by single-copy homologous integration with a frequency of approximately 60% for both genes; and the loss of tmk1 and tga3 transcript formation in the disruptants was demonstrated by RT-PCR.

Coordinated Regulation of Species-Specific Hydroxycinnamic Acid Degradation and Siderophore Biosynthesis Pathways in Agrobacterium fabrum

PubMed Central

Baude, Jessica; Vial, Ludovic; Villard, Camille; Campillo, Tony; Lavire, Céline; Nesme, Xavier

2016-01-01

ABSTRACT The rhizosphere-inhabiting species Agrobacterium fabrum (genomospecies G8 of the Agrobacterium tumefaciens species complex) is known to degrade hydroxycinnamic acids (HCAs), especially ferulic acid and p-coumaric acid, via the novel A. fabrum HCA degradation pathway. Gene expression profiles of A. fabrum strain C58 were investigated in the presence of HCAs, using a C58 whole-genome oligoarray. Both ferulic acid and p-coumaric acid caused variations in the expression of more than 10% of the C58 genes. Genes of the A. fabrum HCA degradation pathway, together with the genes involved in iron acquisition, were among the most highly induced in the presence of HCAs. Two operons coding for the biosynthesis of a particular siderophore, as well as genes of the A. fabrum HCA degradation pathway, have been described as being specific to the species. We demonstrate here their coordinated expression, emphasizing the interdependence between the iron concentration in the growth medium and the rate at which ferulic acid is degraded by cells. The coordinated expression of these functions may be advantageous in HCA-rich but iron-starved environments in which microorganisms have to compete for both iron and carbon sources, such as in plant roots. The present results confirm that there is cooperation between the A. fabrum-specific genes, defining a particular ecological niche. IMPORTANCE We previously identified seven genomic regions in Agrobacterium fabrum that were specifically present in all of the members of this species only. Here we demonstrated that two of these regions, encoding the hydroxycinnamic acid degradation pathway and the iron acquisition pathway, were regulated in a coordinated manner. The coexpression of these functions may be advantageous in hydroxycinnamic acid-rich but iron-starved environments in which microorganisms have to compete for both iron and carbon sources, such as in plant roots. These data support the view that bacterial genomic species

Use of Agrobacterium rhizogenes strain 18r12v and paromomycin selection for transformation of Brachypodium distachyon and Brachypodium sylvaticum

USDA-ARS?s Scientific Manuscript database

The genetic transformation of monocot grasses is a resource intensive process, the quality and efficiency of which is dependent in part upon the method of DNA introduction, as well as the ability to effectively separate transformed from wildtype tissue. Agrobacterium-mediated transformation of Brac...

Influences of Agrobacterium rhizogenes strains, plant genotypes, and tissue types on the induction of transgenic hairy roots in Vitis species

USDA-ARS?s Scientific Manuscript database

Agrobacterium rhizogenes-mediated induction of transgenic hairy roots was previously demonstrated in Vitis vinifera L. and a few other Vitis species. In this study, 13 Vitis species, including V. aestivalis, V. afghanistan, V. champinii, V. doaniana, V. flexuosa, V. labrusca, V. nesbittiana, V. pal...

Exploration of new perspectives and limitations in Agrobacterium mediated gene transfer technology. Progress report, [June 1, 1992-- May 31, 1994

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marton, L.

1994-12-31

This report describes progress aimed at constructing gene-transfer technology for Nicotiana plumbaginifolia. Most actual effort as described herein has so far been directed at exploring new perspectives and limitations in Agrobacterium mediated gene transfer. Accomplishments are described using a core homologous gene targeting vector.

Genomic Species Are Ecological Species as Revealed by Comparative Genomics in Agrobacterium tumefaciens

PubMed Central

Lassalle, Florent; Campillo, Tony; Vial, Ludovic; Baude, Jessica; Costechareyre, Denis; Chapulliot, David; Shams, Malek; Abrouk, Danis; Lavire, Céline; Oger-Desfeux, Christine; Hommais, Florence; Guéguen, Laurent; Daubin, Vincent; Muller, Daniel; Nesme, Xavier

2011-01-01

The definition of bacterial species is based on genomic similarities, giving rise to the operational concept of genomic species, but the reasons of the occurrence of differentiated genomic species remain largely unknown. We used the Agrobacterium tumefaciens species complex and particularly the genomic species presently called genomovar G8, which includes the sequenced strain C58, to test the hypothesis of genomic species having specific ecological adaptations possibly involved in the speciation process. We analyzed the gene repertoire specific to G8 to identify potential adaptive genes. By hybridizing 25 strains of A. tumefaciens on DNA microarrays spanning the C58 genome, we highlighted the presence and absence of genes homologous to C58 in the taxon. We found 196 genes specific to genomovar G8 that were mostly clustered into seven genomic islands on the C58 genome—one on the circular chromosome and six on the linear chromosome—suggesting higher plasticity and a major adaptive role of the latter. Clusters encoded putative functional units, four of which had been verified experimentally. The combination of G8-specific functions defines a hypothetical species primary niche for G8 related to commensal interaction with a host plant. This supports that the G8 ancestor was able to exploit a new ecological niche, maybe initiating ecological isolation and thus speciation. Searching genomic data for synapomorphic traits is a powerful way to describe bacterial species. This procedure allowed us to find such phenotypic traits specific to genomovar G8 and thus propose a Latin binomial, Agrobacterium fabrum, for this bona fide genomic species. PMID:21795751

Crystallization and preliminary X-ray analysis of an exotype alginate lyase Atu3025 from Agrobacterium tumefaciens strain C58, a member of polysaccharide lyase family 15

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ochiai, Akihito; Yamasaki, Masayuki; Mikami, Bunzo

2006-05-01

The crystallization and preliminary X-ray characterization of a family PL-15 exotype alginate lyase are presented. Almost all alginate lyases depolymerize alginate in an endolytical fashion via a β-elimination reaction. The alginate lyase Atu3025 from Agrobacterium tumefaciens strain C58, consisting of 776 amino-acid residues, is a novel exotype alginate lyase classified into polysaccharide lyase family 15. The enzyme was crystallized at 293 K by sitting-drop vapour diffusion with polyethylene glycol 4000 as a precipitant. Preliminary X-ray analysis showed that the Atu3025 crystal belonged to space group P2{sub 1} and diffracted to 2.8 Å resolution, with unit-cell parameters a = 107.7, bmore » = 108.3, c = 149.5 Å, β = 91.5°.« less

Agrobacterium-mediated transformation of maize (Zea mays) with Cre-lox site specific recombination cassettes in BIBAC vectors.

PubMed

Vega, Juan M; Yu, Weichang; Han, Fangpu; Kato, Akio; Peters, Eric M; Zhang, Zhanyuan J; Birchler, James A

2008-04-01

The Cre/loxP site-specific recombination system has been applied in various plant species including maize (Zea mays) for marker gene removal, gene targeting, and functional genomics. A BIBAC vector system was adapted for maize transformation with a large fragment of genetic material including a herbicide resistance marker gene, a 30 kb yeast genomic fragment as a marker for fluorescence in situ hybridization (FISH), and a 35S-lox-cre recombination cassette. Seventy-five transgenic lines were generated from Agrobacterium-mediated transformation of a maize Hi II line with multiple B chromosomes. Eighty-four inserts have been localized among all 10 A chromosome pairs by FISH using the yeast DNA probe together with a karyotyping cocktail. No inserts were found on the B chromosomes; thus a bias against the B chromosomes by the Agrobacterium-mediated transformation was revealed. The expression of a cre gene was confirmed in 68 of the 75 transgenic lines by a reporter construct for cre/lox mediated recombination. The placement of the cre/lox site-specific recombination system in many locations in the maize genome will be valuable materials for gene targeting and chromosome engineering.

Characterization of a putative periplasmic transport system for octopine accumulation encoded by Agrobacterium tumefaciens Ti plasmid pTiA6.

PubMed Central

Valdivia, R H; Wang, L; Winans, S C

1991-01-01

Neoplastic crown gall tumors incited by Agrobacterium tumefaciens release novel amino acid or sugar derivatives known as opines, whose synthesis is directed by genes transferred to plant cells. Agrobacterium cells can transport and catabolize these compounds as sources of carbon and nitrogen. This article describes a region of the pTiA6 plasmid which is required for catabolism of the opine octopine and whose transcription is induced by octopine. This region of the plasmid contains four open reading frames, occQ, occM, occP, and occJ, which show homology to the family of so-called shock-sensitive permeases. TnphoA mutagenesis demonstrated that the OccJ and OccM proteins lie fully or partly in the periplasmic space. The OccJ protein was identified by electrophoresis and found to be fully localized in the periplasmic space. When these proteins were expressed in Escherichia coli, radiolabeled octopine became cell-associated. Images FIG. 6 PMID:1655707

Occurrence of enzymes involved in biosynthesis of indole-3-acetic acid from indole-3-acetonitrile in plant-associated bacteria, Agrobacterium and Rhizobium.

PubMed Central

Kobayashi, M; Suzuki, T; Fujita, T; Masuda, M; Shimizu, S

1995-01-01

The occurrence of a hitherto unknown pathway involving the action of two enzymes, a nitrile hydratase and an amidase for the biosynthesis of indole-3-acetic acid was discovered in phytopathogenic bacteria Agrobacterium tumefaciens and in leguminous bacteria Rhizobium. The nitrile hydratase acting on indole-3-acetonitrile was purified to homogeneity through only two steps from the cell-free extract of A. tumefaciens. The molecular mass of the purified enzyme estimated by HPLC was about 102 kDa, and the enzyme consisted of four subunits identical in molecular mass. The enzyme exhibited a broad absorption spectrum in the visible range with absorption maxima at 408 nm and 705 nm, and it contained cobalt and iron. The enzyme stoichiometrically catalyzed the hydration of indole-3-acetonitrile into indole-3-acetamide with a specific activity of 13.7 mol per min per mg and a Km of 7.9 microM. Images Fig. 1 PMID:11607511

Cyclic Oxidation Behavior of Cold Sprayed CuCrAl-Coated and Uncoated GRCop-84 Substrates for Space Launch Vehicles

NASA Technical Reports Server (NTRS)

Raj, S. V.; Barrett, C.; Karthikeyan, J.; Garlick, R.

2006-01-01

A newly developed Cu-23 (wt %) Cr-5%Al (CuCrAl) alloy shown to resist hydridation and oxidation in an as-cast form is currently being considered as a protective coating for GRCop-84, which is an advanced copper alloy containing 8 (at.%) Cr and 4 (at.%) Nb. The coating was deposited on GRCop-84 substrates by the cold spray deposition technique. Cyclic oxidation tests conducted in air on both coated and uncoated substrates between 773 and 1073 K revealed that the coating remained intact and protected the substrate up to 1073 K. No significant weight loss of the coated specimens were observed at 773 and 873 K even after a cumulative cyclic time of 500 h. About a 10 percent weight loss observed at 973 and 1073 K was attributed to the excessive oxidation of the uncoated sides. In contrast, the uncoated substrate lost as much as 80 percent of its original weight under similar test conditions. It is concluded that the cold sprayed CuCrAl coating is suitable for protecting GRCop-84 substrates.

Transport properties of ultrathin BaFe1.84Co0.16As2 superconducting nanowires

NASA Astrophysics Data System (ADS)

Yuan, Pusheng; Xu, Zhongtang; Li, Chen; Quan, Baogang; Li, Junjie; Gu, Changzhi; Ma, Yanwei

2018-07-01

Superconducting nanowire single-photon detectors (SNSPDs) have an absolute advantage over other types of single-photon detectors, except for the low operating temperature. Therefore, much effort has been devoted to finding high-temperature superconducting materials that are suitable for preparing SNSPDs. Copper-based and MgB2 ultrathin superconducting nanowires have already been reported. However, the transport properties of iron-based ultrathin superconducting nanowires have not been studied. In this work, a 10 nm thick × 200 nm wide × 30 μm long high-quality superconducting nanowire was fabricated from ultrathin BaFe1.84Co0.16As2 films by a lift-off process. The precursor BaFe1.84Co0.16As2 film with a thickness of 10 nm and root-mean-square roughness of 1 nm was grown on CaF2 substrates by pulsed laser deposition. The nanowire shows a high superconducting critical temperature {T}{{c}}{{zero}} = 20 K with a narrow transition width of ΔT = 2.5 K and exhibits a high critical current density J c of 1.8 × 107 A cm-2 at 10 K. These results of ultrathin BaFe1.84Co0.16As2 nanowire will attract interest in electronic applications, including SNSPDs.

A new high-frequency Agrobacterium-mediated transformation technique for Sesamum indicum L. using de-embryonated cotyledon as explant.

PubMed

Chowdhury, Supriyo; Basu, Arpita; Kundu, Surekha

2014-09-01

In spite of the economic importance of sesame (Sesamum indicum L.) and the recent availability of its genome sequence, a high-frequency transformation protocol is still not available. The only two existing Agrobacterium-mediated transformation protocols that are available have poor transformation efficiencies of less than 2%. In the present study, we report a high-frequency, simple, and reproducible transformation protocol for sesame. Transformation was done using de-embryonated cotyledons via somatic embryogenic stages. All the critical parameters of transformation, like incubation period of explants in pre-regeneration medium prior to infection by Agrobacterium tumefaciens, cocultivation period, concentrations of acetosyringone in cocultivation medium, kanamycin concentration, and concentration of plant hormones, including 6-benzylaminopurine, have been optimized. This protocol is superior to the two existing protocols in its high regeneration and transformation efficiencies. The transformed sesame lines have been tested by PCR, RT-PCR for neomycin phosphotransferase II gene expression, and β-glucuronidase (GUS) assay. The regeneration frequency and transformation efficiency are 57.33 and 42.66%, respectively. T0 and T1 generation transgenic plants were analyzed, and several T1 plants homozygous for the transgenes were obtained.

A rapid, highly efficient and economical method of Agrobacterium-mediated in planta transient transformation in living onion epidermis.

PubMed

Xu, Kedong; Huang, Xiaohui; Wu, Manman; Wang, Yan; Chang, Yunxia; Liu, Kun; Zhang, Ju; Zhang, Yi; Zhang, Fuli; Yi, Liming; Li, Tingting; Wang, Ruiyue; Tan, Guangxuan; Li, Chengwei

2014-01-01

Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale.

A Rapid, Highly Efficient and Economical Method of Agrobacterium-Mediated In planta Transient Transformation in Living Onion Epidermis

PubMed Central

Xu, Kedong; Huang, Xiaohui; Wu, Manman; Wang, Yan; Chang, Yunxia; Liu, Kun; Zhang, Ju; Zhang, Yi; Zhang, Fuli; Yi, Liming; Li, Tingting; Wang, Ruiyue; Tan, Guangxuan; Li, Chengwei

2014-01-01

Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale. PMID:24416168

Lifestyle of the biotroph Agrobacterium tumefaciens in the ecological niche constructed on its host plant.

PubMed

González-Mula, Almudena; Lang, Julien; Grandclément, Catherine; Naquin, Delphine; Ahmar, Mohammed; Soulère, Laurent; Queneau, Yves; Dessaux, Yves; Faure, Denis

2018-07-01

Agrobacterium tumefaciens constructs an ecological niche in its host plant by transferring the T-DNA from its Ti plasmid into the host genome and by diverting the host metabolism. We combined transcriptomics and genetics for understanding the A. tumefaciens lifestyle when it colonizes Arabidopsis thaliana tumors. Transcriptomics highlighted: a transition from a motile to sessile behavior that mobilizes some master regulators (Hfq, CtrA, DivK and PleD); a remodeling of some cell surface components (O-antigen, succinoglucan, curdlan, att genes, putative fasciclin) and functions associated with plant defense (Ef-Tu and flagellin pathogen-associated molecular pattern-response and glycerol-3-phosphate and nitric oxide signaling); and an exploitation of a wide variety of host resources, including opines, amino acids, sugars, organic acids, phosphate, phosphorylated compounds, and iron. In addition, construction of transgenic A. thaliana lines expressing a lactonase enzyme showed that Ti plasmid transfer could escape host-mediated quorum-quenching. Finally, construction of knock-out mutants in A. tumefaciens showed that expression of some At plasmid genes seemed more costly than the selective advantage they would have conferred in tumor colonization. We provide the first overview of A. tumefaciens lifestyle in a plant tumor and reveal novel signaling and trophic interplays for investigating host-pathogen interactions. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

SurR9C84A protects and recovers human cardiomyocytes from hypoxia induced apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ashok, Ajay; Department of Pathology, Case Western Reserve University, 2103 Cornell Rd. WRB 5128, Cleveland, OH 44106-7288; Kanwar, Jagat Rakesh

Survivin, as an anti-apoptotic protein and a cell cycle regulator, is recently gaining importance for its regenerative potential in salvaging injured hypoxic cells of vital organs such as heart. Different strategies are being employed to upregulate survivin expression in dying hypoxic cardiomyocytes. We investigated the cardioprotective potential of a cell permeable survivin mutant protein SurR9C84A, for the management of hypoxia mediated cardiomyocyte apoptosis, in a novel and clinically relevant model employing primary human cardiomyocytes (HCM). The aim of this research work was to study the efficacy and mechanism of SurR9C84A facilitated cardioprotection and regeneration in hypoxic HCM. To mimic hypoxicmore » microenvironment in vitro, well characterized HCM were treated with 100 µm (48 h) cobalt chloride to induce hypoxia. Hypoxia induced (HI) HCM were further treated with SurR9C84A (1 µg/mL) in order to analyse its cardioprotective efficacy. Confocal microscopy showed rapid internalization of SurR9C84A and scanning electron microscopy revealed the reinstatement of cytoskeleton projections in HI HCM. SurR9C84A treatment increased cell viability, reduced cell death via, apoptosis (Annexin-V assay), and downregulated free cardiac troponin T and MMP-9 expression. SurR9C84A also upregulated the expression of proliferation markers (PCNA and Ki-67) and downregulated mitochondrial depolarization and ROS levels thereby, impeding cell death. Human Apoptosis Array further revealed that SurR9C84A downregulated expression of pro-apoptotic markers and augmented expression of HSPs and HTRA2/Omi. SurR9C84A treatment led to enhanced levels of survivin, VEGF, PI3K and pAkt. SurR9C84A proved non-toxic to normoxic HCM, as validated through unaltered cell proliferation and other marker levels. Its pre-treatment exhibited lesser susceptibility to hypoxia/damage. SurR9C84A holds a promising clinical potential for human cardiomyocyte survival and proliferation following hypoxic

Exploration of new perspectives and limitations in Agrobacterium-mediated gene transfer technology. Final report, June 1, 1992--May 31, 1995

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marton, L.

1996-02-01

Genetic manipulation of plants often involves the introduction of homologous or partly homologous genes. Ectropic introduction of homologous sequences into plant genomes may trigger epigenetic changes, making expression of the genes unpredictable. The main project objective was to examine the feasibility of using Agrobacterium-mediated gene transfer for homologous gene targeting in plants.

[Transgenic wheat (Triticum aestivum L.) with increased resistance to the storage pest obtained by Agrobacterium tumefaciens--mediated].

PubMed

Bi, Rui-Ming; Jia, Hai-Yan; Feng, De-Shun; Wang, Hong-Gang

2006-05-01

The transgenic wheat of improved resistance to the storage pest was production. We have introduced the cowpea trypsin inhibitor gene (CpTI) into cultured embryonic callus cells of immature embryos of wheat elite line by Agrobacterium-mediated method. Independent plantlets were obtained from the kanamycin-resistant calli after screening. PCR and real time PCR analysis, PCR-Southern and Southern blot hybridization indicated that there were 3 transgenic plants viz. transformed- I, II and III (T- I, T-II and T-III). The transformation frequencies were obviously affected by Agrobacterium concentration, the infection duration and transformation treatment. The segregations of CpTI in the transgenic wheat progenies were not easily to be elucidated, and some transgenic wheat lines (T- I and T-III) showed Mendelian segregations. The determinations of insect resistance to the stored grain insect of wheat viz. the grain moth (Sitotroga cerealella Olivier) indicated that the 3 transgenic wheat progeny seeds moth-resistance was improved significantly. The seed moth-eaten ratio of T- I, T-II, T-III and nontransformed control was 19.8%, 21.9%, 32.9% and 58.3% respectively. 3 transgenic wheat T1 PCR-positive plants revealed that the 3 transgenic lines had excellent agronomic traits. They supplied good germplasm resource of insect-resistance for wheat genetic improvement.

Isolation of plasma membrane fractions from the intestinal epithelial model T84.

PubMed

Kaoutzani, P; Parkos, C A; Delp-Archer, C; Madara, J L

1993-05-01

The human intestinal epithelial cell line T84 is widely used as a model for studies of Cl- secretion and crypt cell biology. We report a fractionation approach that permits separation of purified apical and basolateral T84 plasma membrane domains. T84 cellular membranes were isolated by nitrogen cavitation and differential centrifugation from monolayers grown on permeable supports. Membranes were then fractionated by isopycnic sucrose density gradient sedimentation, and fractions were assessed, using enzymatic and Western blot techniques, for apical (alkaline phosphatase) and basolateral (Na(+)-K(+)-ATPase) plasma membrane markers and for cytosolic, lysosomal, Golgi, and mitochondrial markers. Buffer conditions were defined that permitted separation of enriched apical and basolateral markers. The validity of the selected markers for the apical and basolateral domains was verified by selective apical and basolateral surface labeling studies using trace iodinated wheat germ agglutinin or biotinylation. This approach allows for separation of apical and basolateral plasma membranes of T84 cells for biochemical analyses and should thus be of broad utility in studies of this model polarized and transporting epithelium.

VIP1 and Its Homologs Are Not Required for Agrobacterium-Mediated Transformation, but Play a Role in Botrytis and Salt Stress Responses

PubMed Central

Lapham, Rachelle; Lee, Lan-Ying; Tsugama, Daisuke; Lee, Sanghun; Mengiste, Tesfaye; Gelvin, Stanton B.

2018-01-01

The bZIP transcription factor VIP1 interacts with the Agrobacterium virulence protein VirE2, but the role of VIP1 in Agrobacterium-mediated transformation remains controversial. Previously tested vip1-1 mutant plants produce a truncated protein containing the crucial bZIP DNA-binding domain. We generated the CRISPR/Cas mutant vip1-2 that lacks this domain. The transformation susceptibility of vip1-2 and wild-type plants is similar. Because of potential functional redundancy among VIP1 homologs, we tested transgenic lines expressing VIP1 fused to a SRDX repression domain. All VIP1-SRDX transgenic lines showed wild-type levels of transformation, indicating that neither VIP1 nor its homologs are required for Agrobacterium-mediated transformation. Because VIP1 is involved in innate immune response signaling, we tested the susceptibility of vip1 mutant and VIP1-SRDX plants to Pseudomonas syringae and Botrytis cinerea. vip1 mutant and VIP1-SRDX plants show increased susceptibility to B. cinerea but not to P. syringae infection, suggesting a role for VIP1 in B. cinerea, but not in P. syringae, defense signaling. B. cinerea susceptibility is dependent on abscisic acid (ABA) which is also important for abiotic stress responses. The germination of vip1 mutant and VIP1-SRDX seeds is sensitive to exogenous ABA, suggesting a role for VIP1 in response to ABA. vip1 mutant and VIP1-SRDX plants show increased tolerance to growth in salt, indicating a role for VIP1 in response to salt stress. PMID:29946325

VPS GRCop-84 Liner Development Efforts

NASA Technical Reports Server (NTRS)

Elam, Sandra K.; Holmes, Richard; McKechnie, Tim; Hickman, Robert; Pickens, Tim

2003-01-01

For the past several years, NASA's Marshall Space Flight Center (MSFC) has been working with Plasma Processes, Inc. (PPI) to fabricate combustion chamber liners using the Vacuum Plasma Spray (VPS) process. Multiple liners of a variety of shapes and sizes have been created. Each liner has been fabricated with GRCop-84 (a copper alloy with chromium and niobium) and a functional gradient coating (FGC) on the hot wall. While the VPS process offers versatility and a reduced fabrication schedule, the material system created with VPS allows the liners to operate at higher temperatures, with maximum blanch resistance and improved cycle life. A subscal unit (5K lbf thrust class) is being cycle tested in a LOX/Hydrogen thrust chamber assembly at MSFC. To date, over 75 hot-fire tests have been accumulated on this article. Tests include conditions normally detrimental to conventional materials, yet the VPS GRCop-84 liner has yet to show any signs of degradation. A larger chamber (15K lbf thrust class) has also been fabricated and is being prepared for hot-fire testing at MSFC near the end of 2003. Linear liners have been successfully created to further demonstrate the versatility of the process. Finally, scale up issues for the VPS process are being tackled with efforts to fabricate a full size, engine class liner. Specifically, a liner for the SSME's Main Combustion Chamber (MCC) has recently been attempted. The SSME size was chosen for convenience, since its design was readily available and its size was sufficient to tackle specific issues. Efforts to fabricate these large liners have already provided valuable lessons for using this process for engine programs. The material quality for these large units is being evaluated with destructive analysis and these results will be available by the end of 2003.

Structural Analysis of ADP-Glucose Pyrophosphorylase From the Bacterium Agrobacterium Tumefaciens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cupp-Vickery, J.R.; Igarashi, R.Y.; Perez, M.

2009-05-14

ADP-glucose pyrophosphorylase (ADPGlc PPase) catalyzes the conversion of glucose 1-phosphate and ATP to ADP-glucose and pyrophosphate. As a key step in glucan synthesis, the ADPGlc PPases are highly regulated by allosteric activators and inhibitors in accord with the carbon metabolism pathways of the organism. Crystals of Agrobacterium tumefaciens ADPGlc PPase were obtained using lithium sulfate as a precipitant. A complete anomalous selenomethionyl derivative X-ray diffraction data set was collected with unit cell dimensions a = 85.38 {angstrom}, b = 93.79 {angstrom}, and c = 140.29 {angstrom} ({alpha} = {beta} = {gamma} = 90{sup o}) and space group I{sub 222}. Themore » A. tumefaciens ADPGlc PPase model was refined to 2.1 {angstrom} with an R{sub factor} = 22% and R{sub free} = 26.6%. The model consists of two domains: an N-terminal {alpha}{beta}{alpha} sandwich and a C-terminal parallel {beta}-helix. ATP and glucose 1-phosphate were successfully modeled in the proposed active site, and site-directed mutagenesis of conserved glycines in this region (G20, G21, and G23) resulted in substantial loss of activity. The interface between the N- and the C-terminal domains harbors a strong sulfate-binding site, and kinetic studies revealed that sulfate is a competitive inhibitor for the allosteric activator fructose 6-phosphate. These results suggest that the interface between the N- and C-terminal domains binds the allosteric regulator, and fructose 6-phosphate was modeled into this region. The A. tumefaciens ADPGlc PPase/fructose 6-phosphate structural model along with sequence alignment analysis was used to design mutagenesis experiments to expand the activator specificity to include fructose 1,6-bisphosphate. The H379R and H379K enzymes were found to be activated by fructose 1,6-bisphosphate.« less

Investigations Concerning Cavitation and Frost Fatigue in Clonal 84K Poplar Using High-Resolution Cavitron Measurements1[OPEN

PubMed Central

Feng, Feng; Ding, Fei; Tyree, Melvin T.

2015-01-01

Both drought and freezing-thawing of stems induce a loss of hydraulic conductivity (percentage loss of conductivity [PLC]) in woody plants. Drought-induced PLC is often accompanied by physical damage to pit membranes, causing a shift in vulnerability curves (cavitation fatigue). Hence, if cavitated stems are flushed to remove embolisms, the next vulnerability curve is different (shifted to lower tensions). The 84K poplar (Populus alba × Populus glandulosa) clone has small vessels that should be immune from frost-induced PLC, but results demonstrated that freezing-thawing in combination with tension synergistically increased PLC. Frost fatigue has already been defined, which is similar to cavitation fatigue but induced by freezing. Frost fatigue caused a transition from a single to a dual Weibull curve, but drought-fatigued stems had single Weibull curves shifted to lower tensions. Studying the combined impact of tension plus freezing on fatigue provided evidence that the mechanism of frost fatigue may be the extra water tension induced by freezing or thawing while spinning stems in a centrifuge rather than direct ice damage. A hypothesis is advanced that tension is enhanced as ice crystals grow or melt during the freeze or thaw event, respectively, causing a nearly identical fatigue event to that induced by drought. PMID:25786827

Incidence of Agrobacterium tumefaciens biovar 1 in and on ‘Paradox’ (Juglans hindsii x Juglans regia) walnut seed collected from commercial nurseries

USDA-ARS?s Scientific Manuscript database

The walnut rootstock Paradox (Juglans hindsii (Jeps) Rehder x J. regia L.) is susceptible to Agrobacterium tumefaciens (7) which often results in a high incidence of crown gall in nursery or walnut production orchards. Though A. tumefaciens is susceptible to the commonly used preplant soil fumigant...

Draft Genome Sequence of Agrobacterium sp. Strain UHFBA-218, Isolated from Rhizosphere Soil of Crown Gall-Infected Cherry Rootstock Colt

PubMed Central

Dua, Ankita; Sangwan, Naseer; Kaur, Jasvinder; Saxena, Anjali; Kohli, Puneet; Gupta, A. K.

2013-01-01

We report here the draft genome sequence of the alphaproteobacterium Agrobacterium sp. strain UHFBA-218, which was isolated from rhizosphere soil of crown gall-infected cherry rootstock Colt. The draft genome of strain UHFBA-218 consists of 112 contigs (5,425,303 bp) and 5,063 coding sequences with a G+C content of 59.8%. PMID:23723402

42 CFR 84.256 - Quality control requirements.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 1 2013-10-01 2013-10-01 false Quality control requirements. 84.256 Section 84.256... § 84.256 Quality control requirements. (a) In addition to the construction and performance requirements specified in §§ 84.251, 84.252, 84.253, 84.254, and 84.255, the quality control requirements in paragraphs...

42 CFR 84.256 - Quality control requirements.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 1 2012-10-01 2012-10-01 false Quality control requirements. 84.256 Section 84.256... § 84.256 Quality control requirements. (a) In addition to the construction and performance requirements specified in §§ 84.251, 84.252, 84.253, 84.254, and 84.255, the quality control requirements in paragraphs...

42 CFR 84.256 - Quality control requirements.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 1 2010-10-01 2010-10-01 false Quality control requirements. 84.256 Section 84.256... § 84.256 Quality control requirements. (a) In addition to the construction and performance requirements specified in §§ 84.251, 84.252, 84.253, 84.254, and 84.255, the quality control requirements in paragraphs...

42 CFR 84.256 - Quality control requirements.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 1 2011-10-01 2011-10-01 false Quality control requirements. 84.256 Section 84.256... § 84.256 Quality control requirements. (a) In addition to the construction and performance requirements specified in §§ 84.251, 84.252, 84.253, 84.254, and 84.255, the quality control requirements in paragraphs...

42 CFR 84.256 - Quality control requirements.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 1 2014-10-01 2014-10-01 false Quality control requirements. 84.256 Section 84.256... § 84.256 Quality control requirements. (a) In addition to the construction and performance requirements specified in §§ 84.251, 84.252, 84.253, 84.254, and 84.255, the quality control requirements in paragraphs...

40 CFR 406.84 - [Reserved

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 28 2010-07-01 2010-07-01 true [Reserved] 406.84 Section 406.84 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS GRAIN MILLS POINT SOURCE CATEGORY Hot Cereal Subcategory § 406.84 [Reserved] ...

40 CFR 406.84 - [Reserved

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 30 2013-07-01 2012-07-01 true [Reserved] 406.84 Section 406.84 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS GRAIN MILLS POINT SOURCE CATEGORY Hot Cereal Subcategory § 406.84 [Reserved] ...

40 CFR 406.84 - [Reserved

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 30 2012-07-01 2012-07-01 false [Reserved] 406.84 Section 406.84 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS GRAIN MILLS POINT SOURCE CATEGORY Hot Cereal Subcategory § 406.84 [Reserved] ...

40 CFR 406.84 - [Reserved

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 29 2011-07-01 2009-07-01 true [Reserved] 406.84 Section 406.84 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS GRAIN MILLS POINT SOURCE CATEGORY Hot Cereal Subcategory § 406.84 [Reserved] ...

40 CFR 406.84 - [Reserved

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 29 2014-07-01 2012-07-01 true [Reserved] 406.84 Section 406.84 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS GRAIN MILLS POINT SOURCE CATEGORY Hot Cereal Subcategory § 406.84 [Reserved] ...

40 CFR 428.84 - [Reserved

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 31 2013-07-01 2013-07-01 false [Reserved] 428.84 Section 428.84 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS (CONTINUED) RUBBER MANUFACTURING POINT SOURCE CATEGORY Wet Digestion Reclaimed Rubber Subcategory § 428.84...

40 CFR 428.84 - [Reserved

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 29 2010-07-01 2010-07-01 false [Reserved] 428.84 Section 428.84 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS RUBBER MANUFACTURING POINT SOURCE CATEGORY Wet Digestion Reclaimed Rubber Subcategory § 428.84 [Reserved] ...

40 CFR 428.84 - [Reserved

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 30 2014-07-01 2014-07-01 false [Reserved] 428.84 Section 428.84 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS (CONTINUED) RUBBER MANUFACTURING POINT SOURCE CATEGORY Wet Digestion Reclaimed Rubber Subcategory § 428.84...

40 CFR 428.84 - [Reserved

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 30 2011-07-01 2011-07-01 false [Reserved] 428.84 Section 428.84 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS RUBBER MANUFACTURING POINT SOURCE CATEGORY Wet Digestion Reclaimed Rubber Subcategory § 428.84 [Reserved] ...

40 CFR 428.84 - [Reserved

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 31 2012-07-01 2012-07-01 false [Reserved] 428.84 Section 428.84 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS (CONTINUED) RUBBER MANUFACTURING POINT SOURCE CATEGORY Wet Digestion Reclaimed Rubber Subcategory § 428.84...

34 CFR 84.301 - [Reserved

Code of Federal Regulations, 2010 CFR

2010-07-01

... 34 Education 1 2010-07-01 2010-07-01 false [Reserved] 84.301 Section 84.301 Education Office of the Secretary, Department of Education GOVERNMENTWIDE REQUIREMENTS FOR DRUG-FREE WORKPLACE (FINANCIAL ASSISTANCE) Requirements for Recipients Who Are Individuals § 84.301 [Reserved] ...

34 CFR 84.660 - Recipient.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 34 Education 1 2010-07-01 2010-07-01 false Recipient. 84.660 Section 84.660 Education Office of the Secretary, Department of Education GOVERNMENTWIDE REQUIREMENTS FOR DRUG-FREE WORKPLACE (FINANCIAL ASSISTANCE) Definitions § 84.660 Recipient. Recipient means any individual, corporation, partnership...

Agrobacterium uses a unique ligand-binding mode for trapping opines and acquiring a competitive advantage in the niche construction on plant host.

PubMed

Lang, Julien; Vigouroux, Armelle; Planamente, Sara; El Sahili, Abbas; Blin, Pauline; Aumont-Nicaise, Magali; Dessaux, Yves; Moréra, Solange; Faure, Denis

2014-10-01

By modifying the nuclear genome of its host, the plant pathogen Agrobacterium tumefaciens induces the development of plant tumours in which it proliferates. The transformed plant tissues accumulate uncommon low molecular weight compounds called opines that are growth substrates for A. tumefaciens. In the pathogen-induced niche (the plant tumour), a selective advantage conferred by opine assimilation has been hypothesized, but not experimentally demonstrated. Here, using genetics and structural biology, we deciphered how the pathogen is able to bind opines and use them to efficiently compete in the plant tumour. We report high resolution X-ray structures of the periplasmic binding protein (PBP) NocT unliganded and liganded with the opine nopaline (a condensation product of arginine and α-ketoglurate) and its lactam derivative pyronopaline. NocT exhibited an affinity for pyronopaline (K(D) of 0.6 µM) greater than that for nopaline (KD of 3.7 µM). Although the binding-mode of the arginine part of nopaline/pyronopaline in NocT resembled that of arginine in other PBPs, affinity measurement by two different techniques showed that NocT did not bind arginine. In contrast, NocT presented specific residues such as M117 to stabilize the bound opines. NocT relatives that exhibit the nopaline/pyronopaline-binding mode were only found in genomes of the genus Agrobacterium. Transcriptomics and reverse genetics revealed that A. tumefaciens uses the same pathway for assimilating nopaline and pyronopaline. Fitness measurements showed that NocT is required for a competitive colonization of the plant tumour by A. tumefaciens. Moreover, even though the Ti-plasmid conjugal transfer was not regulated by nopaline, the competitive advantage gained by the nopaline-assimilating Ti-plasmid donors led to a preferential horizontal propagation of this Ti-plasmid amongst the agrobacteria colonizing the plant-tumour niche. This work provided structural and genetic evidences to support the niche

Crystal Structure of AGR_C_4470p from Agrobacterium tumefaciens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vorobiev,S.; Neely, H.; Seetharaman, J.

2007-01-01

We report here the crystal structure at 2.0 {angstrom} resolution of the AGR{_}C{_}4470p protein from the Gram-negative bacterium Agrobacterium tumefaciens. The protein is a tightly associated dimer, each subunit of which bears strong structural homology with the two domains of the heme utilization protein ChuS from Escherichia coli and HemS from Yersinia enterocolitica. Remarkably, the organization of the AGR{_}C{_}4470p dimer is the same as that of the two domains in ChuS and HemS, providing structural evidence that these two proteins evolved by gene duplication. However, the binding site for heme, while conserved in HemS and ChuS, is not conserved inmore » AGR{_}C{_}4470p, suggesting that it probably has a different function. This is supported by the presence of two homologs of AGR{_}C{_}4470p in E. coli, in addition to the ChuS protein.« less

Natural Transformation of Pseudomonas fluorescens and Agrobacterium tumefaciens in Soil

PubMed Central

Demanèche, Sandrine; Kay, Elisabeth; Gourbière, François; Simonet, Pascal

2001-01-01

Little information is available concerning the occurrence of natural transformation of bacteria in soil, the frequency of such events, and the actual role of this process on bacterial evolution. This is because few bacteria are known to possess the genes required to develop competence and because the tested bacteria are unable to reach this physiological state in situ. In this study we found that two soil bacteria, Agrobacterium tumefaciens and Pseudomonas fluorescens, can undergo transformation in soil microcosms without any specific physical or chemical treatment. Moreover, P. fluorescens produced transformants in both sterile and nonsterile soil microcosms but failed to do so in the various in vitro conditions we tested. A. tumefaciens could be transformed in vitro and in sterile soil samples. These results indicate that the number of transformable bacteria could be higher than previously thought and that these bacteria could find the conditions necessary for uptake of extracellular DNA in soil. PMID:11375171

40 CFR 427.84 - [Reserved

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 29 2010-07-01 2010-07-01 false [Reserved] 427.84 Section 427.84 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS ASBESTOS MANUFACTURING POINT SOURCE CATEGORY Coating or Finishing of Asbestos Textiles Subcategory § 427.84...

34 CFR 84.670 - Suspension.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 34 Education 1 2010-07-01 2010-07-01 false Suspension. 84.670 Section 84.670 Education Office of the Secretary, Department of Education GOVERNMENTWIDE REQUIREMENTS FOR DRUG-FREE WORKPLACE (FINANCIAL ASSISTANCE) Definitions § 84.670 Suspension. Suspension means an action taken by a Federal agency that...

24 CFR 84.43 - Competition.

Code of Federal Regulations, 2010 CFR

2010-04-01

... NON-PROFIT ORGANIZATIONS Post-Award Requirements Procurement Standards § 84.43 Competition. All... 24 Housing and Urban Development 1 2010-04-01 2010-04-01 false Competition. 84.43 Section 84.43... free competition. The recipient shall be alert to organizational conflicts of interest as well as...

45 CFR 84.2 - Application.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 45 Public Welfare 1 2012-10-01 2012-10-01 false Application. 84.2 Section 84.2 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION NONDISCRIMINATION ON THE BASIS OF HANDICAP IN PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE General Provisions § 84.2 Application. This...

24 CFR 84.34 - Equipment.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 24 Housing and Urban Development 1 2010-04-01 2010-04-01 false Equipment. 84.34 Section 84.34 Housing and Urban Development Office of the Secretary, Department of Housing and Urban Development UNIFORM... NON-PROFIT ORGANIZATIONS Post-Award Requirements Property Standards § 84.34 Equipment. (a) Title to...

34 CFR 84.640 - Employee.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 34 Education 1 2010-07-01 2010-07-01 false Employee. 84.640 Section 84.640 Education Office of the Secretary, Department of Education GOVERNMENTWIDE REQUIREMENTS FOR DRUG-FREE WORKPLACE (FINANCIAL ASSISTANCE) Definitions § 84.640 Employee. (a) Employee means the employee of a recipient directly engaged in the...

34 CFR 84.630 - Debarment.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 34 Education 1 2010-07-01 2010-07-01 false Debarment. 84.630 Section 84.630 Education Office of the Secretary, Department of Education GOVERNMENTWIDE REQUIREMENTS FOR DRUG-FREE WORKPLACE (FINANCIAL ASSISTANCE) Definitions § 84.630 Debarment. Debarment means an action taken by a Federal agency to prohibit a...

34 CFR 84.655 - Individual.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 34 Education 1 2010-07-01 2010-07-01 false Individual. 84.655 Section 84.655 Education Office of the Secretary, Department of Education GOVERNMENTWIDE REQUIREMENTS FOR DRUG-FREE WORKPLACE (FINANCIAL ASSISTANCE) Definitions § 84.655 Individual. Individual means a natural person. (Authority: E.O.s 12549 and...

34 CFR 84.615 - Conviction.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 34 Education 1 2010-07-01 2010-07-01 false Conviction. 84.615 Section 84.615 Education Office of the Secretary, Department of Education GOVERNMENTWIDE REQUIREMENTS FOR DRUG-FREE WORKPLACE (FINANCIAL ASSISTANCE) Definitions § 84.615 Conviction. Conviction means a finding of guilt (including a plea of nolo...

Renilla luciferase-based quantitation of Potato virus A infection initiated with Agrobacterium infiltration of N. benthamiana leaves.

PubMed

Eskelin, K; Suntio, T; Hyvärinen, S; Hafren, A; Mäkinen, K

2010-03-01

A quantitation method based on the sensitive detection of Renilla luciferase (Rluc) activity was developed and optimized for Potato virus A (PVA; genus Potyviridae) gene expression. This system is based on infections initiated by Agrobacterium infiltration and subsequent detection of the translation of PVA::Rluc RNA, which is enhanced by viral replication, first within the cells infected initially and later by translation and replication within new cells after spread of the virus. Firefly luciferase (Fluc) was used as an internal control to normalize the Rluc activity. An approximately 10-fold difference in the Rluc/Fluc activity ratio between a movement-deficient and a replication-deficient mutant was observed starting from 48h post Agrobacterium infiltration (h.p.i.). The Rluc activity derived from wild type (wt) PVA increased significantly between 48 and 72h.p.i. and the Rluc/Fluc activity deviated clearly from that of the mutant viruses. Quantitation of the Rluc and Fluc mRNAs by semi-quantitative RT-PCR indicated that increases and decreases in the Renillareniformis luciferase (rluc) mRNA levels coincided with changes in Rluc activity. However, a subtle increase in the mRNA level led to pronounced changes in Rluc activity. PVA CP accumulation was quantitated by enzyme-linked immunosorbent assay. The increase in Rluc activity correlated closely with virus accumulation. Copyright (c) 2009 Elsevier B.V. All rights reserved.

Hairy root cultures of butterfly pea (Clitoria ternatea L.): Agrobacterium × plant factors influencing transformation.

PubMed

Swain, S S; Sahu, L; Pal, A; Barik, D P; Pradhan, C; Chand, P K

2012-02-01

Transformed rhizoclones were developed from Agrobacterium-treated explants of the medicinally important twinning legume Clitoria ternatea L. Several key factors influencing transformation events were optimized. A4T was the most infectious among the strains employed. Internode segments were more responsive than leaves, outdoor-grown explants preferred to those from in vitro cultures. High frequency transformation, resulting in up to 85.8% rhizogenesis, was attained using pre-pricked internodal explants for immersion (10 min) in Agrobacterium rhizogenes suspension grown overnight with acetosyringone (100 μM) to an OD(660) ≅ 0.6, diluted to a density of 10(9) cells ml(-1), followed by 5-day co-cultivation. Roots were individually cultured in MS0 supplemented with the bacteriostatic antibiotic cefotaxime (500 μg ml(-1)). Rhizoclones were renewed through successive subcultures in MS0 under diffused illumination. The T ( L )-DNA rolB and rolC ORF were detected in rhizoclones through PCR amplification. The T ( R )-DNA gene encoding mannopine synthase (man2) was revealed by positive amplification and opine gene expression substantiated by agropine and mannopine biosynthesis in all selected transformed rhizoclones. The implication of such findings is discussed on the context of utilization of such genetically transformed root cultures towards sustainable production of medicinally useful phytocompounds, besides providing a means for plant conservation.

Plant Cell Division Analyzed by Transient Agrobacterium-Mediated Transformation of Tobacco BY-2 Cells.

PubMed

Buschmann, Henrik

2016-01-01

The continuing analysis of plant cell division will require additional protein localization studies. This is greatly aided by GFP-technology, but plant transformation and the maintenance of transgenic lines can present a significant technical bottleneck. In this chapter I describe a method for the Agrobacterium-mediated genetic transformation of tobacco BY-2 cells. The method allows for the microscopic analysis of fluorescence-tagged proteins in dividing cells in within 2 days after starting a coculture. This transient transformation procedure requires only standard laboratory equipment. It is hoped that this rapid method would aid researchers conducting live-cell localization studies in plant mitosis and cytokinesis.

24 CFR 81.84 - Hearings.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 24 Housing and Urban Development 1 2011-04-01 2011-04-01 false Hearings. 81.84 Section 81.84... LOAN MORTGAGE CORPORATION (FREDDIE MAC) Procedures for Actions and Review of Actions § 81.84 Hearings. (a) Applicability. The hearing procedures in this section apply to hearings on the record to review...

24 CFR 81.84 - Hearings.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 24 Housing and Urban Development 1 2013-04-01 2013-04-01 false Hearings. 81.84 Section 81.84... LOAN MORTGAGE CORPORATION (FREDDIE MAC) Procedures for Actions and Review of Actions § 81.84 Hearings. (a) Applicability. The hearing procedures in this section apply to hearings on the record to review...

24 CFR 81.84 - Hearings.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 24 Housing and Urban Development 1 2014-04-01 2014-04-01 false Hearings. 81.84 Section 81.84... LOAN MORTGAGE CORPORATION (FREDDIE MAC) Procedures for Actions and Review of Actions § 81.84 Hearings. (a) Applicability. The hearing procedures in this section apply to hearings on the record to review...

42 CFR 84.258 - Fees.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 1 2010-10-01 2010-10-01 false Fees. 84.258 Section 84.258 Public Health PUBLIC... RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Special Use Respirators § 84.258 Fees. The following fees shall be charged for the examination, inspection, and testing of complete assemblies and...

1 CFR 8.4 - Indexes.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 1 General Provisions 1 2011-01-01 2011-01-01 false Indexes. 8.4 Section 8.4 General Provisions ADMINISTRATIVE COMMITTEE OF THE FEDERAL REGISTER SPECIAL EDITIONS OF THE FEDERAL REGISTER CODE OF FEDERAL REGULATIONS § 8.4 Indexes. A subject index to the entire Code shall be annually revised and separately...

1 CFR 8.4 - Indexes.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 1 General Provisions 1 2010-01-01 2010-01-01 false Indexes. 8.4 Section 8.4 General Provisions ADMINISTRATIVE COMMITTEE OF THE FEDERAL REGISTER SPECIAL EDITIONS OF THE FEDERAL REGISTER CODE OF FEDERAL REGULATIONS § 8.4 Indexes. A subject index to the entire Code shall be annually revised and separately...

45 CFR 1356.84 - Sampling.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 45 Public Welfare 4 2011-10-01 2011-10-01 false Sampling. 1356.84 Section 1356.84 Public Welfare....84 Sampling. (a) The State agency may collect and report the information required in section 1356.83(e) of this part on a sample of the baseline population consistent with the sampling requirements...

45 CFR 1356.84 - Sampling.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 45 Public Welfare 4 2010-10-01 2010-10-01 false Sampling. 1356.84 Section 1356.84 Public Welfare....84 Sampling. (a) The State agency may collect and report the information required in section 1356.83(e) of this part on a sample of the baseline population consistent with the sampling requirements...

11 CFR 8.4 - Contents.

Code of Federal Regulations, 2010 CFR

2009-01-01

... 11 Federal Elections 1 2009-01-01 2009-01-01 false Contents. 8.4 Section 8.4 Federal Elections FEDERAL ELECTION COMMISSION NATIONAL VOTER REGISTRATION ACT (42 U.S.C. 1973gg-1 et seq.) National Mail Voter Registration Form § 8.4 Contents. (a) Information about the applicant. The application shall...

11 CFR 8.4 - Contents.

Code of Federal Regulations, 2010 CFR

2008-01-01

... 11 Federal Elections 1 2008-01-01 2008-01-01 false Contents. 8.4 Section 8.4 Federal Elections FEDERAL ELECTION COMMISSION NATIONAL VOTER REGISTRATION ACT (42 U.S.C. 1973gg-1 et seq.) National Mail Voter Registration Form § 8.4 Contents. (a) Information about the applicant. The application shall...

1 CFR 8.4 - Indexes.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 1 General Provisions 1 2012-01-01 2012-01-01 false Indexes. 8.4 Section 8.4 General Provisions ADMINISTRATIVE COMMITTEE OF THE FEDERAL REGISTER SPECIAL EDITIONS OF THE FEDERAL REGISTER CODE OF FEDERAL REGULATIONS § 8.4 Indexes. A subject index to the entire Code shall be annually revised and separately...

1 CFR 8.4 - Indexes.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 1 General Provisions 1 2014-01-01 2012-01-01 true Indexes. 8.4 Section 8.4 General Provisions ADMINISTRATIVE COMMITTEE OF THE FEDERAL REGISTER SPECIAL EDITIONS OF THE FEDERAL REGISTER CODE OF FEDERAL REGULATIONS § 8.4 Indexes. A subject index to the entire Code shall be annually revised and separately...

1 CFR 8.4 - Indexes.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 1 General Provisions 1 2013-01-01 2012-01-01 true Indexes. 8.4 Section 8.4 General Provisions ADMINISTRATIVE COMMITTEE OF THE FEDERAL REGISTER SPECIAL EDITIONS OF THE FEDERAL REGISTER CODE OF FEDERAL REGULATIONS § 8.4 Indexes. A subject index to the entire Code shall be annually revised and separately...

34 CFR 84.665 - State.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 34 Education 1 2010-07-01 2010-07-01 false State. 84.665 Section 84.665 Education Office of the Secretary, Department of Education GOVERNMENTWIDE REQUIREMENTS FOR DRUG-FREE WORKPLACE (FINANCIAL ASSISTANCE) Definitions § 84.665 State. State means any of the States of the United States, the District of Columbia, the...

40 CFR 82.84 - Requirements.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 17 2010-07-01 2010-07-01 false Requirements. 82.84 Section 82.84... STRATOSPHERIC OZONE Federal Procurement § 82.84 Requirements. (a) No later than October 24, 1994, each... requirements and policies of title VI of the Clean Air Act, 42 U.S.C. 7671-7671g. Each such regulation shall...

40 CFR 82.84 - Requirements.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 18 2014-07-01 2014-07-01 false Requirements. 82.84 Section 82.84... STRATOSPHERIC OZONE Federal Procurement § 82.84 Requirements. (a) No later than October 24, 1994, each... requirements and policies of title VI of the Clean Air Act, 42 U.S.C. 7671-7671g. Each such regulation shall...

40 CFR 82.84 - Requirements.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 18 2013-07-01 2013-07-01 false Requirements. 82.84 Section 82.84... STRATOSPHERIC OZONE Federal Procurement § 82.84 Requirements. (a) No later than October 24, 1994, each... requirements and policies of title VI of the Clean Air Act, 42 U.S.C. 7671-7671g. Each such regulation shall...

40 CFR 82.84 - Requirements.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 17 2011-07-01 2011-07-01 false Requirements. 82.84 Section 82.84... STRATOSPHERIC OZONE Federal Procurement § 82.84 Requirements. (a) No later than October 24, 1994, each... requirements and policies of title VI of the Clean Air Act, 42 U.S.C. 7671-7671g. Each such regulation shall...

40 CFR 82.84 - Requirements.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 18 2012-07-01 2012-07-01 false Requirements. 82.84 Section 82.84... STRATOSPHERIC OZONE Federal Procurement § 82.84 Requirements. (a) No later than October 24, 1994, each... requirements and policies of title VI of the Clean Air Act, 42 U.S.C. 7671-7671g. Each such regulation shall...

21 CFR 814.84 - Reports.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Reports. 814.84 Section 814.84 Food and Drugs FOOD... APPROVAL OF MEDICAL DEVICES Postapproval Requirements § 814.84 Reports. (a) The holder of an approved PMA... otherwise, any periodic report shall: (1) Identify changes described in § 814.39(a) and changes required to...

21 CFR 814.84 - Reports.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Reports. 814.84 Section 814.84 Food and Drugs FOOD... APPROVAL OF MEDICAL DEVICES Postapproval Requirements § 814.84 Reports. (a) The holder of an approved PMA... otherwise, any periodic report shall: (1) Identify changes described in § 814.39(a) and changes required to...

Effects of plant density on recombinant hemagglutinin yields in an Agrobacterium-mediated transient gene expression system using Nicotiana benthamiana plants.

PubMed

Fujiuchi, Naomichi; Matsuda, Ryo; Matoba, Nobuyuki; Fujiwara, Kazuhiro

2017-08-01

Agrobacterium-mediated transient expression systems enable plants to rapidly produce a wide range of recombinant proteins. To achieve economically feasible upstream production and downstream processing, it is beneficial to obtain high levels of two yield-related quantities of upstream production: recombinant protein content per fresh mass of harvested biomass (g gFM -1 ) and recombinant protein productivity per unit area-time (g m -2 /month). Here, we report that the density of Nicotiana benthamiana plants during upstream production had significant impacts on the yield-related quantities of recombinant hemagglutinin (HA). The two quantities were smaller at a high plant density of 400 plants m -2 than at a low plant density of 100 plants m -2 . The smaller quantities at the high plant density were attributed to: (i) a lower HA content in young leaves, which usually have high HA accumulation potentials; (ii) a lower biomass allocation to the young leaves; and (iii) a high area-time requirement for plants. Thus, plant density is a key factor for improving upstream production in Agrobacterium-mediated transient expression systems. Biotechnol. Bioeng. 2017;114: 1762-1770. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Description of recovery method used for curdlan produced by Agrobacterium sp. IFO 13140 and its relation to the morphology and physicochemical and technological properties of the polysaccharide

PubMed Central

Mangolim, Camila Sampaio; da Silva, Thamara Thaiane; Fenelon, Vanderson Carvalho; Koga, Luciana Numata; Ferreira, Sabrina Barbosa de Souza; Bruschi, Marcos Luciano; Matioli, Graciette

2017-01-01

Curdlan is a linear polysaccharide considered a dietary fiber and with gelation properties. This study evaluated the structure, morphology and the physicochemical and technological properties of curdlan produced by Agrobacterium sp. IFO 13140 recovered by pre-gelation and precipitation methods. Commercial curdlan submitted or otherwise to the pre-gelation process was also evaluated. The data obtained from structural analysis revealed a similarity between the curdlan produced by Agrobacterium sp. IFO 13140 (recovered by both methods) and the commercial curdlans. The results showed that the curdlans evaluated differed significantly in terms of dispersibility and gelation, and only the pre-gelled ones had significant potential for food application, because this method influence on the size of the particles and in the presence of NaCl. In terms of technological properties, the curdlan produced by Agrobacterium sp. IFO 13140 (pre-gelation method) had a greater water and oil holding capacity (64% and 98% greater, respectively) and a greater thickening capacity than the pre-gelled commercial curdlan. The pre-gelled commercial curdlan displayed a greater gelling capacity at 95°C than the others. When applied to food, only the pre-gelled curdlans improved the texture parameters of yogurts and reduced syneresis. The curdlan gels, which are rigid and stable in structure, demonstrated potential for improving the texture of food products, with potential industrial use. PMID:28245244

Description of recovery method used for curdlan produced by Agrobacterium sp. IFO 13140 and its relation to the morphology and physicochemical and technological properties of the polysaccharide.

PubMed

Mangolim, Camila Sampaio; Silva, Thamara Thaiane da; Fenelon, Vanderson Carvalho; Koga, Luciana Numata; Ferreira, Sabrina Barbosa de Souza; Bruschi, Marcos Luciano; Matioli, Graciette

2017-01-01

Curdlan is a linear polysaccharide considered a dietary fiber and with gelation properties. This study evaluated the structure, morphology and the physicochemical and technological properties of curdlan produced by Agrobacterium sp. IFO 13140 recovered by pre-gelation and precipitation methods. Commercial curdlan submitted or otherwise to the pre-gelation process was also evaluated. The data obtained from structural analysis revealed a similarity between the curdlan produced by Agrobacterium sp. IFO 13140 (recovered by both methods) and the commercial curdlans. The results showed that the curdlans evaluated differed significantly in terms of dispersibility and gelation, and only the pre-gelled ones had significant potential for food application, because this method influence on the size of the particles and in the presence of NaCl. In terms of technological properties, the curdlan produced by Agrobacterium sp. IFO 13140 (pre-gelation method) had a greater water and oil holding capacity (64% and 98% greater, respectively) and a greater thickening capacity than the pre-gelled commercial curdlan. The pre-gelled commercial curdlan displayed a greater gelling capacity at 95°C than the others. When applied to food, only the pre-gelled curdlans improved the texture parameters of yogurts and reduced syneresis. The curdlan gels, which are rigid and stable in structure, demonstrated potential for improving the texture of food products, with potential industrial use.

The hypothetical protein Atu4866 from Agrobacterium tumefaciens adopts a streptavidin-like fold

PubMed Central

Ai, Xuanjun; Semesi, Anthony; Yee, Adelinda; Arrowsmith, Cheryl H.; Choy, Wing-Yiu; Li, Shawn S.C.

2008-01-01

Atu4866 is a 79-residue conserved hypothetical protein of unknown function from Agrobacterium tumefaciens. Protein sequence alignments show that it shares ≥60% sequence identity with 20 other hypothetical proteins of bacterial origin. However, the structures and functions of these proteins remain unknown so far. To gain insight into the function of this family of proteins, we have determined the structure of Atu4866 as a target of a structural genomics project using solution NMR spectroscopy. Our results reveal that Atu4866 adopts a streptavidin-like fold featuring a β-barrel/sandwich formed by eight antiparallel β-strands. Further structural analysis identified a continuous patch of conserved residues on the surface of Atu4866 that may constitute a potential ligand-binding site. PMID:18042676

Generation of Marker- and/or Backbone-Free Transgenic Wheat Plants via Agrobacterium-Mediated Transformation.

PubMed

Wang, Gen-Ping; Yu, Xiu-Dao; Sun, Yong-Wei; Jones, Huw D; Xia, Lan-Qin

2016-01-01

Horizontal transfer of antibiotic resistance genes to animals and vertical transfer of herbicide resistance genes to the weedy relatives are perceived as major biosafety concerns in genetically modified (GM) crops. In this study, five novel vectors which used gusA and bar as a reporter gene and a selection marker gene, respectively, were constructed based on the pCLEAN dual binary vector system. Among these vectors, 1G7B and 5G7B carried two T-DNAs located on two respective plasmids with 5G7B possessing an additional virGwt gene. 5LBTG154 and 5TGTB154 carried two T-DNAs in the target plasmid with either one or double right borders, and 5BTG154 carried the selectable marker gene on the backbone outside of the T-DNA left border in the target plasmid. In addition, 5BTG154, 5LBTG154, and 5TGTB154 used pAL154 as a helper plasmid which contains Komari fragment to facilitate transformation. These five dual binary vector combinations were transformed into Agrobacterium strain AGL1 and used to transform durum wheat cv Stewart 63. Evaluation of the co-transformation efficiencies, the frequencies of marker-free transgenic plants, and integration of backbone sequences in the obtained transgenic lines indicated that two vectors (5G7B and 5TGTB154) were more efficient in generating marker-free transgenic wheat plants with no or minimal integration of backbone sequences in the wheat genome. The vector series developed in this study for generation of marker- and/or backbone-free transgenic wheat plants via Agrobacterium -mediated transformation will be useful to facilitate the creation of "clean" GM wheat containing only the foreign genes of agronomic importance.

Better GGA and meta-GGA Functionals: VT84, meta-VMT, meta-VT84

NASA Astrophysics Data System (ADS)

Vela, Alberto; Martin Del Campo, J.; Gazquez, J. L.; Trickey, S. B.

2011-03-01

The goal of fast DFT calculations on large families of highly complicated systems (e.g. large clusters, biomolecules) implicitly conflicts with the heavy emphasis of recent years on inclusion of exact exchange. In response we have worked on improving non-empirical GGA X functionals. Here we report extension of our VMT GGA functional (J. Chem. Phys. 130 244103 (2009)) to satisfy a relevant asymptotic constraint, yielding the VT{84} X functional. With the PBE C functional, VT{84} gives about 10% improvement over VMT in energetics on the G3 223 molecule set. At the meta-GGA level of complexity, we have both meta-VMT and meta-{84}. The former is about 10% better on the G3 set than the TPSS meta-GGA, while meta-VT{84} gives roughly 10% further improvement over meta-VMT. Details of these assessments, including improvements in chemical shifts, will be presented. SBT acknowledges US DOE Grant DE-SC0002139.

The roles of plant phenolics in defence and communication during Agrobacterium and Rhizobium infection.

PubMed

Bhattacharya, Amita; Sood, Priyanka; Citovsky, Vitaly

2010-09-01

Phenolics are aromatic benzene ring compounds with one or more hydroxyl groups produced by plants mainly for protection against stress. The functions of phenolic compounds in plant physiology and interactions with biotic and abiotic environments are difficult to overestimate. Phenolics play important roles in plant development, particularly in lignin and pigment biosynthesis. They also provide structural integrity and scaffolding support to plants. Importantly, phenolic phytoalexins, secreted by wounded or otherwise perturbed plants, repel or kill many microorganisms, and some pathogens can counteract or nullify these defences or even subvert them to their own advantage. In this review, we discuss the roles of phenolics in the interactions of plants with Agrobacterium and Rhizobium.

Thermodynamic properties of scapolites at temperatures ranging from 10 K to 1000 K

USGS Publications Warehouse

Komada, N.; Moecher, D.P.; Westrum, E.F.; Hemingway, B.S.; Zolotov, M. Yu; Semenov, Y.V.; Khodakovsky, I.L.

1996-01-01

The heat capacities of five mineral samples from the scapolite solid-solution series, Na4Al3Si9O24Cl (marialite) to Ca4Al6Si6O24CO3 (meionite), were measured by the adiabatic method from T=8 K to T=350 K and by the differential scanning calorimetry (d.s.c.) method from T = 300 K to T = 1000 K. The meionite (Me) content in per cent {Me=100 Ca*/(Ca* + Na*)} (where the asterisk indicates that possible substituents are included) and molar heat capacity (Cp,m/R) at T=298.15 K for each sample is: Me28, 82.07; Me44, 82.09; Me55, 83.95; Me69, 85.80; Me88, 84.54. The standard molar entropies, {S,om(298.15 K)-Som(0 K)} R-1 (R=8.31451 J??K-1??mol-1), at T=298.15 K for the respective compositions are: 85.05??0.26, 83.78??0.50, 85.22??0.24, 85.76??0.21, and 84.17??0.59. The calculated standard molar entropies (as above) at T=298.15 K for the end-members marialite and meionite, and for an intermediate composition (mizzonite=Me75) are 84.85, 83.94 and 86.15, respectively. Values of the coefficients in the equation Cp,m/R = a + bT+ cT2 + dT-1/2 + eT-2 (valid from T = 300 K to T =1000 K) are: (Mex, a, b/K, c/K2, d/K-1/2, e/K-2 Me88), 315.580, -0.0795676, 1.52825.10-5, -3954.83, 1808460; Me69, 261.285, -0.0415017, 8.73053.10-7, -3028.28, 1083666; Me55, 232.236, -0.0352222, 6.49875.10-6, 2505.99, 601750; Me44, 276.696, -0.0756614, 2.39722.10-5, -3210.40, 1044363; Me28, 149.917, 0.0229399, -1.23180.10-5, 1208.87, -318470. Smoothed thermodynamic functions for the five samples are also presented. The enthalpies of solution for five natural scapolites were measured in 2PbO??B2O3 melts at T= 973 K by Calvet-type calorimetry. The values of ??solHom/R??K are: Me11, 32.14??0.7; Me28, 32.34??0.4; Me44, 33.66??0.8; Me69, 35.29??0.8; Me88, 32.87??0.3. The calculated enthalpies of formation for stoichiometric scapolites ??fHom/103??R??K at T= 298.15 K are: Me0, -1467.4??1.3; Me11, -1491.2??1.2; Me28, -1527.6??0.9; Me44, -1564.1??1.1; Me55, -1587.4??1.1; Me69, -1619.7??1.1; Me75, -1633.1??1.1; Me

Characterization of curdlan produced by Agrobacterium sp. IFO 13140 cells immobilized in a loofa sponge matrix, and application of this biopolymer in the development of functional yogurt.

PubMed

Ortiz Martinez, Camila; Pereira Ruiz, Suelen; Carvalho Fenelon, Vanderson; Rodrigues de Morais, Gutierrez; Luciano Baesso, Mauro; Matioli, Graciette

2016-05-01

Agrobacterium sp. IFO 13140 cells were immobilized on a loofa sponge and used to produce curdlan over five successive cycles. The interaction between microbial cells and the loofa sponge as well as the produced curdlan were characterized by Fourier transform infrared-attenuated total reflectance (FTIR-ATR) spectrometry. The purity of the curdlan was also evaluated. The storage stability of the immobilized cells was assessed and the produced curdlan was used in a functional yogurt formulation. The average curdlan production by immobilized cells was 17.84 g L(-1) . The presence of the microorganism in the sponge was confirmed and did not cause alterations in the matrix, and the chemical structure of the curdlan was the same as that of commercial curdlan. The purity of both was similar. The immobilized cells remained active after 300 days of storage at -18 °C. The use of the produced curdlan in a functional yogurt resulted in a product with lower syneresis. A large number of cells physically adhered to the surface of loofa sponge fibers, and its use as an immobilization matrix to produce curdlan was effective. The use of the produced curdlan in yogurt allowed the development of a more stable product. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

Genetic transformation of tobacco NT1 cells with Agrobacterium tumefaciens.

PubMed

Mayo, Kristin J; Gonzales, Barbara J; Mason, Hugh S

2006-01-01

This protocol is used to produce stably transformed tobacco (Nicotiana tabacum) NT1 cell lines, using Agrobacterium tumefaciens-mediated DNA delivery of a binary vector containing a gene encoding hepatitis B surface antigen and a gene encoding the kanamycin selection marker. The NT1 cultures, at the appropriate stage of growth, are inoculated with A. tumefaciens containing the binary vector. A 3-day cocultivation period follows, after which the cultures are rinsed and placed on solid selective medium. Transformed colonies ('calli') appear in approximately 4 weeks; they are subcultured until adequate material is obtained for analysis of antigen production. 'Elite' lines are selected based on antigen expression and growth characteristics. The time required for the procedure from preparation of the plant cell materials to callus development is approximately 5 weeks. Growth of selected calli to sufficient quantities for antigen screening may require 4-6 weeks beyond the initial selection. Creation of the plasmid constructs, transformation of the A. tumefaciens line, and ELISA and Bradford assays to assess protein production require additional time.

Beyond Agrobacterium-Mediated Transformation: Horizontal Gene Transfer from Bacteria to Eukaryotes.

PubMed

Lacroix, Benoît; Citovsky, Vitaly

2018-03-03

Besides the massive gene transfer from organelles to the nuclear genomes, which occurred during the early evolution of eukaryote lineages, the importance of horizontal gene transfer (HGT) in eukaryotes remains controversial. Yet, increasing amounts of genomic data reveal many cases of bacterium-to-eukaryote HGT that likely represent a significant force in adaptive evolution of eukaryotic species. However, DNA transfer involved in genetic transformation of plants by Agrobacterium species has traditionally been considered as the unique example of natural DNA transfer and integration into eukaryotic genomes. Recent discoveries indicate that the repertoire of donor bacterial species and of recipient eukaryotic hosts potentially are much wider than previously thought, including donor bacterial species, such as plant symbiotic nitrogen-fixing bacteria (e.g., Rhizobium etli) and animal bacterial pathogens (e.g., Bartonella henselae, Helicobacter pylori), and recipient species from virtually all eukaryotic clades. Here, we review the molecular pathways and potential mechanisms of these trans-kingdom HGT events and discuss their utilization in biotechnology and research.

Purification, gene cloning, and characterization of γ-butyrobetainyl CoA synthetase from Agrobacterium sp. 525a.

PubMed

Fujimitsu, Hiroshi; Matsumoto, Akira; Takubo, Sayaka; Fukui, Akiko; Okada, Kazuma; Mohamed Ahmed, Isam A; Arima, Jiro; Mori, Nobuhiro

2016-08-01

The report is the first of purification, overproduction, and characterization of a unique γ-butyrobetainyl CoA synthetase from soil-isolated Agrobacterium sp. 525a. The primary structure of the enzyme shares 70-95% identity with those of ATP-dependent microbial acyl-CoA synthetases of the Rhizobiaceae family. As distinctive characteristics of the enzyme of this study, ADP was released in the catalytic reaction process, whereas many acyl CoA synthetases are annotated as an AMP-forming enzyme. The apparent Km values for γ-butyrobetaine, CoA, and ATP were, respectively, 0.69, 0.02, and 0.24 mM.

45 CFR 84.21 - Discrimination prohibited.

Code of Federal Regulations, 2011 CFR

2011-10-01

... BASIS OF HANDICAP IN PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Accessibility § 84.21 Discrimination prohibited. No qualified handicapped person shall, because a recipient's facilities are... 45 Public Welfare 1 2011-10-01 2011-10-01 false Discrimination prohibited. 84.21 Section 84.21...

34 CFR 84.610 - Controlled substance.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 34 Education 1 2010-07-01 2010-07-01 false Controlled substance. 84.610 Section 84.610 Education Office of the Secretary, Department of Education GOVERNMENTWIDE REQUIREMENTS FOR DRUG-FREE WORKPLACE (FINANCIAL ASSISTANCE) Definitions § 84.610 Controlled substance. Controlled substance means a controlled...

45 CFR 84.5 - Assurances required.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 45 Public Welfare 1 2012-10-01 2012-10-01 false Assurances required. 84.5 Section 84.5 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION NONDISCRIMINATION ON THE BASIS OF HANDICAP IN PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE General Provisions § 84.5...

45 CFR 84.4 - Discrimination prohibited.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 45 Public Welfare 1 2013-10-01 2013-10-01 false Discrimination prohibited. 84.4 Section 84.4... § 84.4 Discrimination prohibited. (a) General. No qualified handicapped person shall, on the basis of... discrimination under any program or activity which receives Federal financial assistance. (b) Discriminatory...

45 CFR 84.4 - Discrimination prohibited.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 45 Public Welfare 1 2011-10-01 2011-10-01 false Discrimination prohibited. 84.4 Section 84.4... § 84.4 Discrimination prohibited. (a) General. No qualified handicapped person shall, on the basis of... discrimination under any program or activity which receives Federal financial assistance. (b) Discriminatory...

45 CFR 84.4 - Discrimination prohibited.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 45 Public Welfare 1 2010-10-01 2010-10-01 false Discrimination prohibited. 84.4 Section 84.4... § 84.4 Discrimination prohibited. (a) General. No qualified handicapped person shall, on the basis of... discrimination under any program or activity which receives Federal financial assistance. (b) Discriminatory...

45 CFR 84.4 - Discrimination prohibited.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 45 Public Welfare 1 2014-10-01 2014-10-01 false Discrimination prohibited. 84.4 Section 84.4... § 84.4 Discrimination prohibited. (a) General. No qualified handicapped person shall, on the basis of... discrimination under any program or activity which receives Federal financial assistance. (b) Discriminatory...

20 CFR 631.84 - Allowable projects.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 20 Employees' Benefits 3 2010-04-01 2010-04-01 false Allowable projects. 631.84 Section 631.84... THE JOB TRAINING PARTNERSHIP ACT Disaster Relief Employment Assistance § 631.84 Allowable projects...) Shall be used exclusively to provide employment on projects that provide food, clothing, shelter and...

18 CFR 401.84 - Hearing procedure.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 18 Conservation of Power and Water Resources 2 2011-04-01 2011-04-01 false Hearing procedure. 401.84 Section 401.84 Conservation of Power and Water Resources DELAWARE RIVER BASIN COMMISSION ADMINISTRATIVE MANUAL RULES OF PRACTICE AND PROCEDURE Administrative and Other Hearings § 401.84 Hearing...

20 CFR 631.84 - Allowable projects.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 20 Employees' Benefits 3 2011-04-01 2011-04-01 false Allowable projects. 631.84 Section 631.84... THE JOB TRAINING PARTNERSHIP ACT Disaster Relief Employment Assistance § 631.84 Allowable projects...) Shall be used exclusively to provide employment on projects that provide food, clothing, shelter and...

20 CFR 631.84 - Allowable projects.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 20 Employees' Benefits 3 2012-04-01 2012-04-01 false Allowable projects. 631.84 Section 631.84... THE JOB TRAINING PARTNERSHIP ACT Disaster Relief Employment Assistance § 631.84 Allowable projects...) Shall be used exclusively to provide employment on projects that provide food, clothing, shelter and...

Development of an Efficient Agrobacterium-Mediated Transformation System and Production of Herbicide-Resistant Transgenic Plants in Garlic (Allium sativum L.)

PubMed Central

Ahn, Yul-Kyun; Yoon, Moo-Kyoung; Jeon, Jong-Seong

2013-01-01

The genetic improvement of garlic plants (Allium sativum L.) with agronomical beneficial traits is rarely achieved due to the lack of an applicable transformation system. Here, we developed an efficient Agrobacterium-mediated transformation procedure with Danyang, an elite Korean garlic cultivar. Examination of sGFP (synthetic green fluorescence protein) expression revealed that treatment with 2-(N-morpholino) ethanesulfonic acid (MES), L-cysteine and/or dithiothreitol (DTT) gives the highest efficiency in transient gene transfer during Agrobacterium co-cultivation with calli derived from the roots of in vitro plantlets. To increase stable transformation efficiency, a two-step selection was employed on the basis of hygromycin resistance and sGFP expression. Of the hygromycin-resistant calli initially produced, only sGFP-expressing calli were subcultured for selection of transgenic calli. Transgenic plantlets produced from these calli were grown to maturity. The transformation efficiency increased up to 10.6% via our optimized procedure. DNA and RNA gel-blot analysis indicated that transgenic garlic plants stably integrated and expressed the phosphinothricin acetyltransferase (PAT) gene. A herbicide spraying assay demonstrated that transgenic plants of garlic conferred herbicide resistance, whilst non-transgenic plants and weeds died. These results indicate that our transformation system can be efficiently utilized to produce transgenic garlic plants with agronomic benefits. PMID:23832764

Development of an efficient Agrobacterium-mediated transformation system and production of herbicide-resistant transgenic plants in garlic (Allium sativum L.).

PubMed

Ahn, Yul-Kyun; Yoon, Moo-Kyoung; Jeon, Jong-Seong

2013-08-01

The genetic improvement of garlic plants (Allium sativum L.) with agronomical beneficial traits is rarely achieved due to the lack of an applicable transformation system. Here, we developed an efficient Agrobacterium-mediated transformation procedure with Danyang, an elite Korean garlic cultivar. Examination of sGFP (synthetic green fluorescence protein) expression revealed that treatment with 2-(N-morpholino) ethanesulfonic acid (MES), L-cysteine and/or dithiothreitol (DTT) gives the highest efficiency in transient gene transfer during Agrobacterium co-cultivation with calli derived from the roots of in vitro plantlets. To increase stable transformation efficiency, a two-step selection was employed on the basis of hygromycin resistance and sGFP expression. Of the hygromycin-resistant calli initially produced, only sGFP-expressing calli were subcultured for selection of transgenic calli. Transgenic plantlets produced from these calli were grown to maturity. The transformation efficiency increased up to 10.6% via our optimized procedure. DNA and RNA gel-blot analysis indicated that transgenic garlic plants stably integrated and expressed the phosphinothricin acetyltransferase (PAT) gene. A herbicide spraying assay demonstrated that transgenic plants of garlic conferred herbicide resistance, whilst nontransgenic plants and weeds died. These results indicate that our transformation system can be efficiently utilized to produce transgenic garlic plants with agronomic benefits.

24 CFR 84.32 - Real property.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 24 Housing and Urban Development 1 2012-04-01 2012-04-01 false Real property. 84.32 Section 84.32... NON-PROFIT ORGANIZATIONS Post-Award Requirements Property Standards § 84.32 Real property. HUD prescribes the following requirements for recipients concerning the use and disposition of real property...

24 CFR 84.32 - Real property.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 24 Housing and Urban Development 1 2010-04-01 2010-04-01 false Real property. 84.32 Section 84.32... NON-PROFIT ORGANIZATIONS Post-Award Requirements Property Standards § 84.32 Real property. HUD prescribes the following requirements for recipients concerning the use and disposition of real property...

24 CFR 84.32 - Real property.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 24 Housing and Urban Development 1 2011-04-01 2011-04-01 false Real property. 84.32 Section 84.32... NON-PROFIT ORGANIZATIONS Post-Award Requirements Property Standards § 84.32 Real property. HUD prescribes the following requirements for recipients concerning the use and disposition of real property...

24 CFR 84.32 - Real property.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 24 Housing and Urban Development 1 2013-04-01 2013-04-01 false Real property. 84.32 Section 84.32... NON-PROFIT ORGANIZATIONS Post-Award Requirements Property Standards § 84.32 Real property. HUD prescribes the following requirements for recipients concerning the use and disposition of real property...

24 CFR 84.32 - Real property.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 24 Housing and Urban Development 1 2014-04-01 2014-04-01 false Real property. 84.32 Section 84.32... NON-PROFIT ORGANIZATIONS Post-Award Requirements Property Standards § 84.32 Real property. HUD prescribes the following requirements for recipients concerning the use and disposition of real property...

32 CFR 776.84 - Ethics investigation.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 32 National Defense 5 2011-07-01 2011-07-01 false Ethics investigation. 776.84 Section 776.84... Complaint Processing Procedures § 776.84 Ethics investigation. (a) Whenever an ethics investigation is... ethics investigation: (1) To request a hearing before the investigating officer (IO); (2) To inspect all...

32 CFR 776.84 - Ethics investigation.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 32 National Defense 5 2010-07-01 2010-07-01 false Ethics investigation. 776.84 Section 776.84... Complaint Processing Procedures § 776.84 Ethics investigation. (a) Whenever an ethics investigation is... ethics investigation: (1) To request a hearing before the investigating officer (IO); (2) To inspect all...

32 CFR 776.84 - Ethics investigation.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 32 National Defense 5 2012-07-01 2012-07-01 false Ethics investigation. 776.84 Section 776.84... Complaint Processing Procedures § 776.84 Ethics investigation. (a) Whenever an ethics investigation is... ethics investigation: (1) To request a hearing before the investigating officer (IO); (2) To inspect all...

32 CFR 776.84 - Ethics investigation.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 32 National Defense 5 2014-07-01 2014-07-01 false Ethics investigation. 776.84 Section 776.84... Complaint Processing Procedures § 776.84 Ethics investigation. (a) Whenever an ethics investigation is... ethics investigation: (1) To request a hearing before the investigating officer (IO); (2) To inspect all...

32 CFR 776.84 - Ethics investigation.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 32 National Defense 5 2013-07-01 2013-07-01 false Ethics investigation. 776.84 Section 776.84... Complaint Processing Procedures § 776.84 Ethics investigation. (a) Whenever an ethics investigation is... ethics investigation: (1) To request a hearing before the investigating officer (IO); (2) To inspect all...

42 CFR 84.10 - Application procedures.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 1 2010-10-01 2010-10-01 false Application procedures. 84.10 Section 84.10 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Application for Approval § 84.10...

42 CFR 84.10 - Application procedures.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 1 2012-10-01 2012-10-01 false Application procedures. 84.10 Section 84.10 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Application for Approval § 84.10...

42 CFR 84.10 - Application procedures.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 1 2011-10-01 2011-10-01 false Application procedures. 84.10 Section 84.10 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Application for Approval § 84.10...

42 CFR 84.10 - Application procedures.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 1 2014-10-01 2014-10-01 false Application procedures. 84.10 Section 84.10 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Application for Approval § 84.10...

42 CFR 84.10 - Application procedures.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 1 2013-10-01 2013-10-01 false Application procedures. 84.10 Section 84.10 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Application for Approval § 84.10...

10 CFR 61.84 - Criminal penalties.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 10 Energy 2 2012-01-01 2012-01-01 false Criminal penalties. 61.84 Section 61.84 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) LICENSING REQUIREMENTS FOR LAND DISPOSAL OF RADIOACTIVE WASTE Records, Reports, Tests, and Inspections § 61.84 Criminal penalties. (a) Section 223 of the Atomic Energy Act of...

10 CFR 61.84 - Criminal penalties.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 10 Energy 2 2014-01-01 2014-01-01 false Criminal penalties. 61.84 Section 61.84 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) LICENSING REQUIREMENTS FOR LAND DISPOSAL OF RADIOACTIVE WASTE Records, Reports, Tests, and Inspections § 61.84 Criminal penalties. (a) Section 223 of the Atomic Energy Act of...

10 CFR 61.84 - Criminal penalties.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 10 Energy 2 2011-01-01 2011-01-01 false Criminal penalties. 61.84 Section 61.84 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) LICENSING REQUIREMENTS FOR LAND DISPOSAL OF RADIOACTIVE WASTE Records, Reports, Tests, and Inspections § 61.84 Criminal penalties. (a) Section 223 of the Atomic Energy Act of...

24 CFR 84.83 - Property standards.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 24 Housing and Urban Development 1 2010-04-01 2010-04-01 false Property standards. 84.83 Section 84.83 Housing and Urban Development Office of the Secretary, Department of Housing and Urban... EDUCATION, HOSPITALS, AND OTHER NON-PROFIT ORGANIZATIONS Use of Lump Sum Grants § 84.83 Property standards...

14 CFR 399.84 - Price advertising.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 14 Aeronautics and Space 4 2012-01-01 2012-01-01 false Price advertising. 399.84 Section 399.84... STATEMENTS STATEMENTS OF GENERAL POLICY Policies Relating to Enforcement § 399.84 Price advertising. The Board considers any advertising or solicitation by a direct air carrier, indirect air carrier, or an...

14 CFR 399.84 - Price advertising.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Price advertising. 399.84 Section 399.84... STATEMENTS STATEMENTS OF GENERAL POLICY Policies Relating to Enforcement § 399.84 Price advertising. The Board considers any advertising or solicitation by a direct air carrier, indirect air carrier, or an...

14 CFR 399.84 - Price advertising.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 14 Aeronautics and Space 4 2011-01-01 2011-01-01 false Price advertising. 399.84 Section 399.84... STATEMENTS STATEMENTS OF GENERAL POLICY Policies Relating to Enforcement § 399.84 Price advertising. The Board considers any advertising or solicitation by a direct air carrier, indirect air carrier, or an...

45 CFR 84.11 - Discrimination prohibited.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 45 Public Welfare 1 2013-10-01 2013-10-01 false Discrimination prohibited. 84.11 Section 84.11... § 84.11 Discrimination prohibited. (a) General. (1) No qualified handicapped person shall, on the basis of handicap, be subjected to discrimination in employment under any program or activity to which this...

45 CFR 84.11 - Discrimination prohibited.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 45 Public Welfare 1 2014-10-01 2014-10-01 false Discrimination prohibited. 84.11 Section 84.11... § 84.11 Discrimination prohibited. (a) General. (1) No qualified handicapped person shall, on the basis of handicap, be subjected to discrimination in employment under any program or activity to which this...

45 CFR 84.11 - Discrimination prohibited.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 45 Public Welfare 1 2011-10-01 2011-10-01 false Discrimination prohibited. 84.11 Section 84.11... § 84.11 Discrimination prohibited. (a) General. (1) No qualified handicapped person shall, on the basis of handicap, be subjected to discrimination in employment under any program or activity to which this...

34 CFR 84.620 - Cooperative agreement.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 34 Education 1 2013-07-01 2013-07-01 false Cooperative agreement. 84.620 Section 84.620 Education... (FINANCIAL ASSISTANCE) Definitions § 84.620 Cooperative agreement. Cooperative agreement means an award of... term does not include cooperative research and development agreements as defined in 15 U.S.C. 3710a...

34 CFR 84.620 - Cooperative agreement.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 34 Education 1 2014-07-01 2014-07-01 false Cooperative agreement. 84.620 Section 84.620 Education... (FINANCIAL ASSISTANCE) Definitions § 84.620 Cooperative agreement. Cooperative agreement means an award of... term does not include cooperative research and development agreements as defined in 15 U.S.C. 3710a...

34 CFR 84.620 - Cooperative agreement.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 34 Education 1 2010-07-01 2010-07-01 false Cooperative agreement. 84.620 Section 84.620 Education... (FINANCIAL ASSISTANCE) Definitions § 84.620 Cooperative agreement. Cooperative agreement means an award of... term does not include cooperative research and development agreements as defined in 15 U.S.C. 3710a...

45 CFR 84.39 - Private education.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 45 Public Welfare 1 2011-10-01 2011-10-01 false Private education. 84.39 Section 84.39 Public... Secondary Education § 84.39 Private education. (a) A recipient that provides private elementary or secondary education may not, on the basis of handicap, exclude a qualified handicapped person if the person can, with...

45 CFR 84.39 - Private education.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 45 Public Welfare 1 2014-10-01 2014-10-01 false Private education. 84.39 Section 84.39 Public... Secondary Education § 84.39 Private education. (a) A recipient that provides private elementary or secondary education may not, on the basis of handicap, exclude a qualified handicapped person if the person can, with...

45 CFR 84.39 - Private education.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 45 Public Welfare 1 2012-10-01 2012-10-01 false Private education. 84.39 Section 84.39 Public... Secondary Education § 84.39 Private education. (a) A recipient that provides private elementary or secondary education may not, on the basis of handicap, exclude a qualified handicapped person if the person can, with...

45 CFR 84.39 - Private education.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 45 Public Welfare 1 2013-10-01 2013-10-01 false Private education. 84.39 Section 84.39 Public... Secondary Education § 84.39 Private education. (a) A recipient that provides private elementary or secondary education may not, on the basis of handicap, exclude a qualified handicapped person if the person can, with...

45 CFR 84.39 - Private education.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 45 Public Welfare 1 2010-10-01 2010-10-01 false Private education. 84.39 Section 84.39 Public... Secondary Education § 84.39 Private education. (a) A recipient that provides private elementary or secondary education may not, on the basis of handicap, exclude a qualified handicapped person if the person can, with...

24 CFR 35.84 - Effective dates.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 24 Housing and Urban Development 1 2011-04-01 2011-04-01 false Effective dates. 35.84 Section 35.84 Housing and Urban Development Office of the Secretary, Department of Housing and Urban Development... Paint and/or Lead-Based Paint Hazards Upon Sale or Lease of Residential Property § 35.84 Effective dates...

33 CFR 84.23 - Maneuvering light.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Maneuvering light. 84.23 Section... RULES ANNEX I: POSITIONING AND TECHNICAL DETAILS OF LIGHTS AND SHAPES § 84.23 Maneuvering light. Notwithstanding the provisions of § 84.03(f), the maneuvering light described in Rule 34(b) shall be placed...

33 CFR 84.23 - Maneuvering light.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 33 Navigation and Navigable Waters 1 2011-07-01 2011-07-01 false Maneuvering light. 84.23 Section... RULES ANNEX I: POSITIONING AND TECHNICAL DETAILS OF LIGHTS AND SHAPES § 84.23 Maneuvering light. Notwithstanding the provisions of § 84.03(f), the maneuvering light described in Rule 34(b) shall be placed...

33 CFR 84.23 - Maneuvering light.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 33 Navigation and Navigable Waters 1 2013-07-01 2013-07-01 false Maneuvering light. 84.23 Section... RULES ANNEX I: POSITIONING AND TECHNICAL DETAILS OF LIGHTS AND SHAPES § 84.23 Maneuvering light. Notwithstanding the provisions of § 84.03(f), the maneuvering light described in Rule 34(b) shall be placed...

33 CFR 84.23 - Maneuvering light.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 33 Navigation and Navigable Waters 1 2014-07-01 2014-07-01 false Maneuvering light. 84.23 Section... RULES ANNEX I: POSITIONING AND TECHNICAL DETAILS OF LIGHTS AND SHAPES § 84.23 Maneuvering light. Notwithstanding the provisions of § 84.03(f), the maneuvering light described in Rule 34(b) shall be placed...

33 CFR 84.23 - Maneuvering light.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 33 Navigation and Navigable Waters 1 2012-07-01 2012-07-01 false Maneuvering light. 84.23 Section... RULES ANNEX I: POSITIONING AND TECHNICAL DETAILS OF LIGHTS AND SHAPES § 84.23 Maneuvering light. Notwithstanding the provisions of § 84.03(f), the maneuvering light described in Rule 34(b) shall be placed...

A T-DNA gene required for agropine biosynthesis by transformed plants is functionally and evolutionarily related to a Ti plasmid gene required for catabolism of agropine by Agrobacterium strains.

PubMed Central

Hong, S B; Hwang, I; Dessaux, Y; Guyon, P; Kim, K S; Farrand, S K

1997-01-01

The mechanisms that ensure that Ti plasmid T-DNA genes encoding proteins involved in the biosynthesis of opines in crown gall tumors are always matched by Ti plasmid genes conferring the ability to catabolize that set of opines on the inducing Agrobacterium strains are unknown. The pathway for the biosynthesis of the opine agropine is thought to require an enzyme, mannopine cyclase, coded for by the ags gene located in the T(R) region of octopine-type Ti plasmids. Extracts prepared from agropine-type tumors contained an activity that cyclized mannopine to agropine. Tumor cells containing a T region in which ags was mutated lacked this activity and did not contain agropine. Expression of ags from the lac promoter conferred mannopine-lactonizing activity on Escherichia coli. Agrobacterium tumefaciens strains harboring an octopine-type Ti plasmid exhibit a similar activity which is not coded for by ags. Analysis of the DNA sequence of the gene encoding this activity, called agcA, showed it to be about 60% identical to T-DNA ags genes. Relatedness decreased abruptly in the 5' and 3' untranslated regions of the genes. ags is preceded by a promoter that functions only in the plant. Expression analysis showed that agcA also is preceded by its own promoter, which is active in the bacterium. Translation of agcA yielded a protein of about 45 kDa, consistent with the size predicted from the DNA sequence. Antibodies raised against the agcA product cross-reacted with the anabolic enzyme. These results indicate that the agropine system arose by a duplication of a progenitor gene, one copy of which became associated with the T-DNA and the other copy of which remained associated with the bacterium. PMID:9244272

40 CFR 427.84 - [Reserved

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 30 2014-07-01 2014-07-01 false [Reserved] 427.84 Section 427.84 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS (CONTINUED) ASBESTOS MANUFACTURING POINT SOURCE CATEGORY Coating or Finishing of Asbestos Textiles...

40 CFR 427.84 - [Reserved

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 31 2012-07-01 2012-07-01 false [Reserved] 427.84 Section 427.84 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS (CONTINUED) ASBESTOS MANUFACTURING POINT SOURCE CATEGORY Coating or Finishing of Asbestos Textiles...

40 CFR 427.84 - [Reserved

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 31 2013-07-01 2013-07-01 false [Reserved] 427.84 Section 427.84 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS (CONTINUED) ASBESTOS MANUFACTURING POINT SOURCE CATEGORY Coating or Finishing of Asbestos Textiles...

7 CFR 983.84 - Derogation.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 8 2010-01-01 2010-01-01 false Derogation. 983.84 Section 983.84 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE PISTACHIOS GROWN IN CALIFORNIA...

Ultralow noise performance of an 8.4-GHz maser-feedhorn system

NASA Technical Reports Server (NTRS)

Johnson, D. L.; Petty, S. M.; Kovatch, J. J.; Glass, G. W.

1990-01-01

A total system noise temperature of 6.6 K was demonstrated with an 8.4-GHz traveling wave maser and feedhorn operating in a cryogenic environment. Both the maser and feedhorn were inserted in the helium cryostat, with the maser operating in the 1.6-K liquid bath and the feedhorn cooled in the helium gas, with a temperature gradient along the horn ranging from the liquid bath temperature at its lower end to room temperature at its top. The ruby maser exhibited 43 dB of gain with a bandwidth of 76 MHz(-3 dB) centered at 8400 MHz. Discussions of the maser, cooled feedhorn, and cryostat designs are presented along with a discussion of the noise temperature measurements.

7 CFR 955.84 - Derogation.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 8 2014-01-01 2014-01-01 false Derogation. 955.84 Section 955.84 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS AND ORDERS; FRUITS, VEGETABLES, NUTS), DEPARTMENT OF AGRICULTURE VIDALIA ONIONS GROWN IN GEORGIA...

7 CFR 955.84 - Derogation.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 8 2011-01-01 2011-01-01 false Derogation. 955.84 Section 955.84 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE VIDALIA ONIONS GROWN IN GEORGIA...

7 CFR 955.84 - Derogation.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 8 2012-01-01 2012-01-01 false Derogation. 955.84 Section 955.84 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE VIDALIA ONIONS GROWN IN GEORGIA...

7 CFR 955.84 - Derogation.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 8 2013-01-01 2013-01-01 false Derogation. 955.84 Section 955.84 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS AND ORDERS; FRUITS, VEGETABLES, NUTS), DEPARTMENT OF AGRICULTURE VIDALIA ONIONS GROWN IN GEORGIA...

7 CFR 955.84 - Derogation.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 8 2010-01-01 2010-01-01 false Derogation. 955.84 Section 955.84 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE VIDALIA ONIONS GROWN IN GEORGIA...

Production of human interferon alfa 2b in plants of Nicotiana excelsior by Agrobacterium-mediated transient expression.

PubMed

Sindarovska, Y R; Gerasymenko, I M; Sheludko, Y V; Olevinskaya, Z M; Spivak, N Y; Kuchuk, N V

2010-01-01

Human interferon alpha2b gene was transiently expressed in Nicotiana excelsior plants. Fusion with N. plumbaginifolia calreticulin signal peptide for improved apoplast targeting and carrying out the expression under optimized conditions resulted in maximal interferon activity of 3.2 x 10(3) IU/g fresh weight (FW) with an average of 2.1 +/- 0.8 x 10(3) IU/g FW. It proves that N. excelsior is a suitable host for Agrobacterium-mediated transient expression of genes encoding physiologically active human proteins. The transient expression conditions optimized for GFP marker protein were confirmed to be preferable for hIFN alpha2b.

9 CFR 381.84 - Airsacculitis.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 9 Animals and Animal Products 2 2010-01-01 2010-01-01 false Airsacculitis. 381.84 Section 381.84 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE AGENCY... CERTIFICATION POULTRY PRODUCTS INSPECTION REGULATIONS Post Mortem Inspection; Disposition of Carcasses and Parts...

32 CFR 644.84 - Counteroffers.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 32 National Defense 4 2011-07-01 2011-07-01 false Counteroffers. 644.84 Section 644.84 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY (CONTINUED) REAL PROPERTY REAL ESTATE... Department of Justice; travel costs of all Government personnel and consultants participating in trial...

42 CFR 84.1101 - Definitions.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 1 2014-10-01 2014-10-01 false Definitions. 84.1101 Section 84.1101 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Dust, Fume, and Mist; Pesticide...

42 CFR 84.1101 - Definitions.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 1 2013-10-01 2013-10-01 false Definitions. 84.1101 Section 84.1101 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Dust, Fume, and Mist; Pesticide...

42 CFR 84.1101 - Definitions.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 1 2011-10-01 2011-10-01 false Definitions. 84.1101 Section 84.1101 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Dust, Fume, and Mist; Pesticide...

42 CFR 84.1101 - Definitions.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 1 2010-10-01 2010-10-01 false Definitions. 84.1101 Section 84.1101 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Dust, Fume, and Mist; Pesticide...

42 CFR 84.1101 - Definitions.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 1 2012-10-01 2012-10-01 false Definitions. 84.1101 Section 84.1101 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Dust, Fume, and Mist; Pesticide...

A phage display-selected peptide inhibitor of Agrobacterium vitis polygalacturonase.

PubMed

Warren, Jeremy G; Kasun, George W; Leonard, Takara; Kirkpatrick, Bruce C

2016-05-01

Agrobacterium vitis, the causal agent of crown gall of grapevine, is a threat to viticulture worldwide. A major virulence factor of this pathogen is polygalacturonase, an enzyme that degrades pectin components of the xylem cell wall. A single gene encodes for the polygalacturonase gene. Disruption of the polygalacturonase gene results in a mutant that is less pathogenic and produces significantly fewer root lesions on grapevines. Thus, the identification of peptides or proteins that could inhibit the activity of polygalacturonase could be part of a strategy for the protection of plants against this pathogen. A phage-displayed combinatorial peptide library was used to isolate peptides with a high binding affinity to A. vitis polygalacturonase. These peptides showed sequence similarity to regions of Oryza sativa (EMS66324, Japonica) and Triticum urartu (NP_001054402, wild wheat) polygalacturonase-inhibiting proteins (PGIPs). Furthermore, these panning experiments identified a peptide, SVTIHHLGGGS, which was able to reduce A. vitis polygalacturonase activity by 35% in vitro. Truncation studies showed that the IHHL motif alone is sufficient to inhibit A. vitis polygalacturonase activity. © 2015 BSPP AND JOHN WILEY & SONS LTD.

Multipole induced splitting of metal-cage vibrations in crystalline endohedral D2d-M2@C84 dimetallofullerenes.

PubMed

Krause, M; Popov, V N; Inakuma, M; Tagmatarchis, N; Shinohara, H; Georgi, P; Dunsch, L; Kuzmany, H

2004-01-22

Metal-carbon cage vibrations of crystalline endohedral D2d-M2@C84 (M=Sc,Y,Dy) dimetallofullerenes were analyzed by temperature dependent Raman scattering and a dynamical force field model. Three groups of metal-carbon cage modes were found at energies of 35-200 cm(-1) and assigned to metal-cage stretching and deformation vibrations. They exhibit a textbook example for the splitting of molecular vibrations in a crystal field. Induced dipole-dipole and quadrupole-quadrupole interactions account quantitatively for the observed mode splitting. Based on the metal-cage vibrational structure it is demonstrated that D2d-Y2@C84 dimetallofullerene retains a monoclinic crystal structure up to 550 K and undergoes a transition from a disordered to an ordered orientational state at a temperature of approximately 150 K.

Improved production of transgenic Dioscorea zingiberensis (Dioscoreaceae) by Agrobacterium tumefaciens-mediated transformation.

PubMed

Shi, L; Fan, J Q; Hu, C G; Luo, J; Yao, J L

2012-02-03

The establishment of high-efficiency Agrobacterium-mediated transformation techniques could improve the production of Dioscorea zingiberensis, a medicinal species with a high diosgenin content. We co-cultivated embryogenic calli induced from mature seeds with A. tumefaciens strain EHA105. A binary vector, pCAMBIA1381, which contains the gfp and hpt genes under the control of the ubiquitin promoter and the CaMV 35S promoter, respectively, was used for transformation. Pre-culture, basic medium, acetosyringone, and bacterial density were evaluated to establish the most efficient protocol. The optimal conditions consisted of MS medium without CaCl(2) for pre- and co-cultivation, three days for pre-culture, addition of 200 μM AS, and an OD(600) of 0.5. The transgenic plants grown under selection were confirmed by PCR analysis and Southern blot analysis. This protocol produced transgenic D. zingiberensis plants in seven months, with a transformation efficiency of 6%.

32 CFR 552.84 - Purpose.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 32 National Defense 3 2010-07-01 2010-07-01 true Purpose. 552.84 Section 552.84 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY MILITARY RESERVATIONS AND NATIONAL CEMETERIES... training areas. Uninterrupted military use is vital to maintain and improve the combat readiness of the US...

24 CFR 84.22 - Payment.

Code of Federal Regulations, 2011 CFR

2011-04-01

... NON-PROFIT ORGANIZATIONS Post-Award Requirements Financial and Program Management § 84.22 Payment. (a... management systems that meet the standards for fund control and accountability as established in § 84.21. Cash advances to a recipient organization shall be limited to the minimum amounts needed and be timed...

24 CFR 84.22 - Payment.

Code of Federal Regulations, 2010 CFR

2010-04-01

... NON-PROFIT ORGANIZATIONS Post-Award Requirements Financial and Program Management § 84.22 Payment. (a... management systems that meet the standards for fund control and accountability as established in § 84.21. Cash advances to a recipient organization shall be limited to the minimum amounts needed and be timed...

45 CFR 84.1 - Purpose.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 45 Public Welfare 1 2012-10-01 2012-10-01 false Purpose. 84.1 Section 84.1 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION NONDISCRIMINATION ON THE BASIS OF HANDICAP IN... eliminate discrimination on the basis of handicap in any program or activity receiving Federal financial...

9 CFR 381.84 - Airsacculitis.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 2 2011-01-01 2011-01-01 false Airsacculitis. 381.84 Section 381.84 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE AGENCY... carcasses may be passed for food after complete removal and condemnation of all affected tissues including...

9 CFR 381.84 - Airsacculitis.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 2 2013-01-01 2013-01-01 false Airsacculitis. 381.84 Section 381.84 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE AGENCY... carcasses may be passed for food after complete removal and condemnation of all affected tissues including...

9 CFR 381.84 - Airsacculitis.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 2 2014-01-01 2014-01-01 false Airsacculitis. 381.84 Section 381.84 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE AGENCY... carcasses may be passed for food after complete removal and condemnation of all affected tissues including...

9 CFR 381.84 - Airsacculitis.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 2 2012-01-01 2012-01-01 false Airsacculitis. 381.84 Section 381.84 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE AGENCY... carcasses may be passed for food after complete removal and condemnation of all affected tissues including...

Proline antagonizes GABA-induced quenching of quorum-sensing in Agrobacterium tumefaciens

PubMed Central

Haudecoeur, E.; Planamente, S.; Cirou, A.; Tannières, M.; Shelp, B. J.; Moréra, S.; Faure, D.

2009-01-01

Plants accumulate free L-proline (Pro) in response to abiotic stresses (drought and salinity) and presence of bacterial pathogens, including the tumor-inducing bacterium Agrobacterium tumefaciens. However, the function of Pro accumulation in host-pathogen interaction is still unclear. Here, we demonstrated that Pro antagonizes plant GABA-defense in the A. tumefaciens C58-induced tumor by interfering with the import of GABA and consequently the GABA-induced degradation of the bacterial quorum-sensing signal, 3-oxo-octanoylhomoserine lactone. We identified a bacterial receptor Atu2422, which is implicated in the uptake of GABA and Pro, suggesting that Pro acts as a natural antagonist of GABA-signaling. The Atu2422 amino acid sequence contains a Venus flytrap domain that is required for trapping GABA in human GABAB receptors. A constructed atu2422 mutant was more virulent than the wild type bacterium; moreover, transgenic plants with a low level of Pro exhibited less severe tumor symptoms than did their wild-type parents, revealing a crucial role for Venus flytrap GABA-receptor and relative abundance of GABA and Pro in host-pathogen interaction. PMID:19706545

Mutational analysis of the transcriptional activator VirG of Agrobacterium tumefaciens.

PubMed Central

Scheeren-Groot, E P; Rodenburg, K W; den Dulk-Ras, A; Turk, S C; Hooykaas, P J

1994-01-01

To find VirG proteins with altered properties, the virG gene was mutagenized. Random chemical mutagenesis of single-stranded DNA containing the Agrobacterium tumefaciens virG gene led with high frequency to the inactivation of the gene. Sequence analysis showed that 29% of the mutants contained a virG gene with one single-base-pair substitution somewhere in the open reading frame. Thirty-nine different mutations that rendered the VirG protein inactive were mapped. Besides these inactive mutants, two mutants in which the vir genes were active even in the absence of acetosyringone were found on indicator plates. A VirG protein with an N54D substitution turned out to be able to induce a virB-lacZ reporter gene to a high level even in the absence of the inducer acetosyringone. A VirG protein with an I77V substitution exhibited almost no induction in the absence of acetosyringone but showed a maximum induction level already at low concentrations of acetosyringone. Images PMID:7961391

76 FR 1412 - Investing in Innovation Fund; Catalog of Federal Domestic Assistance (CFDA) Numbers: 84.396A, 84...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-01-10

... DEPARTMENT OF EDUCATION [Docket ID ED-2011-OII-0001] Investing in Innovation Fund; Catalog of Federal Domestic Assistance (CFDA) Numbers: 84.396A, 84.396B and 84.396C AGENCY: Office of Innovation and... selection criteria. SUMMARY: The Assistant Deputy Secretary for Innovation and Improvement proposes to amend...

34 CFR 84.650 - Grant.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 34 Education 1 2010-07-01 2010-07-01 false Grant. 84.650 Section 84.650 Education Office of the Secretary, Department of Education GOVERNMENTWIDE REQUIREMENTS FOR DRUG-FREE WORKPLACE (FINANCIAL ASSISTANCE... value to the recipient to carry out a public purpose of support or stimulation authorized by a law of...

50 CFR 217.84 - Mitigation.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 50 Wildlife and Fisheries 10 2014-10-01 2014-10-01 false Mitigation. 217.84 Section 217.84 Wildlife and Fisheries NATIONAL MARINE FISHERIES SERVICE, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION...-monitoring zone, the animals is moving away from the area, and the area is clear of marine mammals for at...

50 CFR 217.84 - Mitigation.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 50 Wildlife and Fisheries 10 2013-10-01 2013-10-01 false Mitigation. 217.84 Section 217.84 Wildlife and Fisheries NATIONAL MARINE FISHERIES SERVICE, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION...-monitoring zone, the animals is moving away from the area, and the area is clear of marine mammals for at...

50 CFR 217.84 - Mitigation.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 50 Wildlife and Fisheries 10 2012-10-01 2012-10-01 false Mitigation. 217.84 Section 217.84 Wildlife and Fisheries NATIONAL MARINE FISHERIES SERVICE, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION...-monitoring zone, the animals is moving away from the area, and the area is clear of marine mammals for at...

Tensile Properties of GRCop-84

NASA Technical Reports Server (NTRS)

Ellis, David L.; Loewenthal, William S.; Yun, Hee-Man

2012-01-01

This is a chapter in the final report on GRCop-84 for the Reusable Launch Vehicle (RLV) Second Generation/Project Constellation Program. It contains information on the tensile properties of GRCop-84. GRCop-84 (Cu-8 at.% Cr-4 at.% Nb) was produced by extrusion and Hot Isostatic Pressing (HIPing). Some of the extrusions were rolled to plate and sheet while other extrusions were drawn into tubing. The material was further subjected to various heat treatments corresponding to annealing, anticipated typical brazing conditions, an end-of-life condition and various elevated temperature exposures to attempt to improve creep resistance. As anticipated, cold work increased strength while decreasing ductility. Annealing at 600 C (1112 F) and higher temperatures was effective. An exposure for 100 h at 500 C (932 F) resulted in an increase in strength rather than the anticipated decrease. High temperature simulated-braze cycles and thermal exposures lowered the strength of GRCop-84, but the deceases were small compared to precipitation strengthened copper alloys. It was observed that the excess Cr could form large precipitates that lower the reduction in area though it appears a minimum amount is required. Overall, GRCop-84 exhibits good stability of its tensile properties, which makes it an excellent candidate for rocket engine liners and many other high temperature applications.

45 CFR 84.61 - Procedures.

Code of Federal Regulations, 2013 CFR

2013-10-01

... provisions applicable to title VI of the Civil Rights Act of 1964 apply to this part. These procedures are... 45 Public Welfare 1 2013-10-01 2013-10-01 false Procedures. 84.61 Section 84.61 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION NONDISCRIMINATION ON THE BASIS OF HANDICAP IN...

45 CFR 84.61 - Procedures.

Code of Federal Regulations, 2011 CFR

2011-10-01

... provisions applicable to title VI of the Civil Rights Act of 1964 apply to this part. These procedures are... 45 Public Welfare 1 2011-10-01 2011-10-01 false Procedures. 84.61 Section 84.61 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION NONDISCRIMINATION ON THE BASIS OF HANDICAP IN...

45 CFR 84.61 - Procedures.

Code of Federal Regulations, 2012 CFR

2012-10-01

... provisions applicable to title VI of the Civil Rights Act of 1964 apply to this part. These procedures are... 45 Public Welfare 1 2012-10-01 2012-10-01 false Procedures. 84.61 Section 84.61 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION NONDISCRIMINATION ON THE BASIS OF HANDICAP IN...

45 CFR 84.61 - Procedures.

Code of Federal Regulations, 2014 CFR

2014-10-01

... provisions applicable to title VI of the Civil Rights Act of 1964 apply to this part. These procedures are... 45 Public Welfare 1 2014-10-01 2014-10-01 false Procedures. 84.61 Section 84.61 Public Welfare Department of Health and Human Services GENERAL ADMINISTRATION NONDISCRIMINATION ON THE BASIS OF HANDICAP IN...

10 CFR 72.84 - Violations.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 10 Energy 2 2010-01-01 2010-01-01 false Violations. 72.84 Section 72.84 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) LICENSING REQUIREMENTS FOR THE INDEPENDENT STORAGE OF SPENT NUCLEAR FUEL, HIGH-LEVEL... violation of the provisions of— (1) The Atomic Energy Act of 1954, as amended; (2) Title II of the Energy...

45 CFR 84.8 - Notice.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 45 Public Welfare 1 2012-10-01 2012-10-01 false Notice. 84.8 Section 84.8 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION NONDISCRIMINATION ON THE BASIS OF HANDICAP IN... recipient that it does not discriminate on the basis of handicap in violation of section 504 and this part...

PopZ identifies the new pole, and PodJ identifies the old pole during polar growth in Agrobacterium tumefaciens

PubMed Central

Grangeon, Romain; Zupan, John R.; Anderson-Furgeson, James; Zambryski, Patricia C.

2015-01-01

Agrobacterium tumefaciens elongates by addition of peptidoglycan (PG) only at the pole created by cell division, the growth pole, whereas the opposite pole, the old pole, is inactive for PG synthesis. How Agrobacterium assigns and maintains pole asymmetry is not understood. Here, we investigated whether polar growth is correlated with novel pole-specific localization of proteins implicated in a variety of growth and cell division pathways. The cell cycle of A. tumefaciens was monitored by time-lapse and superresolution microscopy to image the localization of A. tumefaciens homologs of proteins involved in cell division, PG synthesis and pole identity. FtsZ and FtsA accumulate at the growth pole during elongation, and improved imaging reveals FtsZ disappears from the growth pole and accumulates at the midcell before FtsA. The L,D-transpeptidase Atu0845 was detected mainly at the growth pole. A. tumefaciens specific pole-organizing protein (Pop) PopZAt and polar organelle development (Pod) protein PodJAt exhibited dynamic yet distinct behavior. PopZAt was found exclusively at the growing pole and quickly switches to the new growth poles of both siblings immediately after septation. PodJAt is initially at the old pole but then also accumulates at the growth pole as the cell cycle progresses suggesting that PodJAt may mediate the transition of the growth pole to an old pole. Thus, PopZAt is a marker for growth pole identity, whereas PodJAt identifies the old pole. PMID:26324921

Assessment of bioavailability of soil-sorbed atrazine.

PubMed

Park, Jeong-Hun; Feng, Yucheng; Ji, Pingsheng; Voice, Thomas C; Boyd, Stephen A

2003-06-01

Bioavailability of pesticides sorbed to soils is an important determinant of their environmental fate and impact. Mineralization of sorbed atrazine was studied in soil and clay slurries, and a desorption-biodegradation-mineralization (DBM) model was developed to quantitatively evaluate the bioavailability of sorbed atrazine. Three atrazine-degrading bacteria that utilized atrazine as a sole N source (Pseudomonas sp. strain ADP, Agrobacterium radiobacter strain J14a, and Ralstonia sp. strain M91-3) were used in the bioavailability assays. Assays involved establishing sorption equilibrium in sterile soil slurries, inoculating the system with organisms, and measuring the CO(2) production over time. Sorption and desorption isotherm analyses were performed to evaluate distribution coefficients and desorption parameters, which consisted of three desorption site fractions and desorption rate coefficients. Atrazine sorption isotherms were linear for mineral and organic soils but displayed some nonlinearity for K-saturated montmorillonite. The desorption profiles were well described by the three-site desorption model. In many instances, the mineralization of atrazine was accurately predicted by the DBM model, which accounts for the extents and rates of sorption/desorption processes and assumes biodegradation of liquid-phase, but not sorbed, atrazine. However, for the Houghton muck soil, which manifested the highest sorbed atrazine concentrations, enhanced mineralization rates, i.e., greater than those expected on the basis of aqueous-phase atrazine concentration, were observed. Even the assumption of instantaneous desorption could not account for the elevated rates. A plausible explanation for enhanced bioavailability is that bacteria access the localized regions where atrazine is sorbed and that the concentrations found support higher mineralization rates than predicted on the basis of aqueous-phase concentrations. Characteristics of high sorbed-phase concentration

Assessment of Bioavailability of Soil-Sorbed Atrazine

PubMed Central

Park, Jeong-Hun; Feng, Yucheng; Ji, Pingsheng; Voice, Thomas C.; Boyd, Stephen A.

2003-01-01

Bioavailability of pesticides sorbed to soils is an important determinant of their environmental fate and impact. Mineralization of sorbed atrazine was studied in soil and clay slurries, and a desorption-biodegradation-mineralization (DBM) model was developed to quantitatively evaluate the bioavailability of sorbed atrazine. Three atrazine-degrading bacteria that utilized atrazine as a sole N source (Pseudomonas sp. strain ADP, Agrobacterium radiobacter strain J14a, and Ralstonia sp. strain M91-3) were used in the bioavailability assays. Assays involved establishing sorption equilibrium in sterile soil slurries, inoculating the system with organisms, and measuring the CO2 production over time. Sorption and desorption isotherm analyses were performed to evaluate distribution coefficients and desorption parameters, which consisted of three desorption site fractions and desorption rate coefficients. Atrazine sorption isotherms were linear for mineral and organic soils but displayed some nonlinearity for K-saturated montmorillonite. The desorption profiles were well described by the three-site desorption model. In many instances, the mineralization of atrazine was accurately predicted by the DBM model, which accounts for the extents and rates of sorption/desorption processes and assumes biodegradation of liquid-phase, but not sorbed, atrazine. However, for the Houghton muck soil, which manifested the highest sorbed atrazine concentrations, enhanced mineralization rates, i.e., greater than those expected on the basis of aqueous-phase atrazine concentration, were observed. Even the assumption of instantaneous desorption could not account for the elevated rates. A plausible explanation for enhanced bioavailability is that bacteria access the localized regions where atrazine is sorbed and that the concentrations found support higher mineralization rates than predicted on the basis of aqueous-phase concentrations. Characteristics of high sorbed-phase concentration

Thermophysical Properties of GRCop-84

NASA Technical Reports Server (NTRS)

Ellis, David L.; Keller, Dennis J.; Nathal, Michael (Technical Monitor)

2000-01-01

The thermophysical properties and electrical resistivity of GRCop-84 (Cu - 8 at.% Cr-4 at.% Nb) were measured from cryogenic temperatures to near its melting point. The data were analyzed using weighted regression to determine the properties as a function of temperature and assign appropriate confidence intervals. The results showed that the thermal expansion of GRCop-84 was significantly lower than NARloy-Z (Cu-3 wt. % Ag-0.5 wt. % Zr), the currently used thrust cell liner material. The lower thermal expansion is expected to translate into lower thermally induced stresses and increases in thrust cell liner lives between 2X and 41X over NARloy-Z. The somewhat lower thermal conductivity of GRCop-84 can be offset by redesigning the liners to utilize its much greater mechanical properties. Optimized designs are not expected to suffer from the lower thermal conductivity. Electrical resistivity data, while not central to the primary application, show that GRCop-84 has potential for applications where a combination of good electrical conductivity and strength is required.

Sequence and transcriptional analysis of the genes responsible for curdlan biosynthesis in Agrobacterium sp. ATCC 31749 under simulated dissolved oxygen gradients conditions.

PubMed

Zhang, Hong-Tao; Zhan, Xiao-Bei; Zheng, Zhi-Yong; Wu, Jian-Rong; Yu, Xiao-Bin; Jiang, Yun; Lin, Chi-Chung

2011-07-01

Expression at the mRNA level of ten selected genes in Agrobacterium sp. ATCC 31749 under various dissolved oxygen (DO) levels during curdlan fermentation related to electron transfer chain (ETC), tricarboxylic acid (TCA) cycle, peptidoglycan/lipopolysaccharide biosynthesis, and uridine diphosphate (UDP)-glucose biosynthesis were determined by qRT-PCR. Experiments were performed at DO levels of 30%, 50%, and 75%, as well as under low-oxygen conditions. The effect of high cell density on transcriptional response of the above genes under low oxygen was also studied. Besides cytochrome d (cyd A), the transcription levels of all the other genes were increased at higher DO and reached maximum at 50% DO. Under 75% DO, the transcriptional levels of all the genes were repressed. In addition, transcription levels of icd, sdh, cyo A, and fix N genes did not exhibit significant fluctuation with high cell density culture under low oxygen. These results suggested a mechanism for DO regulation of curdlan synthesis through regulation of transcriptional levels of ETCs, TCA, and UDP-glucose synthesis genes during curdlan fermentation. To our knowledge, this is the first report that DO concentration apparently regulates curdlan biosynthesis in Agrobacterium sp. ATCC 31749 providing essential lead for the optimization of the fermentation at the industrial scale.

Agrobacterium tumefaciens-mediated transformation of Narcissus tazzeta var. chinensis.

PubMed

Lu, Gang; Zou, Qingcheng; Guo, Deping; Zhuang, Xiaoying; Yu, Xiaolin; Xiang, Xun; Cao, Jiashu

2007-09-01

Phytoene synthase (PSY), as a key regulatory enzyme for carotene biosynthesis, plays an important role in regulating color formation in many species. In the present study, a protocol was developed for the transformation of Narcissus tazzeta var chinensis using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pCAMBIA1301 plasmid which contained an antisense phytoene synthase gene, a reporter beta-glucuronidase gene and a selectable marker hygromycin phosphotransferase gene. Effects of some factors on efficiency of transformation and regeneration were examined. Preculture of the explants for 6 days before inoculation enhanced the transient GUS expression. The addition of acetosyringone (AS) at 100 micromol l(-1) for inoculation and a period of 3 days co-cultivation yielded efficient transient GUS expression. Transformants were obtained through selection on MS medium containing 5 mg l(-1) 6-benzylaminopurine (BA), 0.1 mg l(-1)alpha-naphthalene acetic acid (NAA) and 40 mg l(-1) hygromycin. The transformation frequency was 1.24% based on PCR analysis of gus gene. One or two copies of transgene were demonstrated in different transformations by Southern blotting analyses. Northern blotting results confirmed that the transcription of the endogenous psy gene in transgenic plants was inhibited or silenced. The method reported here provides new opportunities for improvement of quality traits of Narcissus tazzeta via genetic transformation.

7 CFR 1956.84 - Approval or rejection.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 14 2010-01-01 2009-01-01 true Approval or rejection. 1956.84 Section 1956.84 Agriculture Regulations of the Department of Agriculture (Continued) RURAL HOUSING SERVICE, RURAL BUSINESS... Housing § 1956.84 Approval or rejection. (a)-(d) [Reserved] (e) Appeal rights. A debtor whose debt...

7 CFR 982.84 - Duration of immunities.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 8 2013-01-01 2013-01-01 false Duration of immunities. 982.84 Section 982.84... WASHINGTON Order Regulating Handling Miscellaneous Provisions § 982.84 Duration of immunities. The benefits, privileges, and immunities conferred upon any person by virtue of this subpart shall cease upon the...

7 CFR 982.84 - Duration of immunities.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 8 2014-01-01 2014-01-01 false Duration of immunities. 982.84 Section 982.84... WASHINGTON Order Regulating Handling Miscellaneous Provisions § 982.84 Duration of immunities. The benefits, privileges, and immunities conferred upon any person by virtue of this subpart shall cease upon the...

7 CFR 982.84 - Duration of immunities.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 8 2011-01-01 2011-01-01 false Duration of immunities. 982.84 Section 982.84... WASHINGTON Order Regulating Handling Miscellaneous Provisions § 982.84 Duration of immunities. The benefits, privileges, and immunities conferred upon any person by virtue of this subpart shall cease upon the...

7 CFR 982.84 - Duration of immunities.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 8 2010-01-01 2010-01-01 false Duration of immunities. 982.84 Section 982.84... WASHINGTON Order Regulating Handling Miscellaneous Provisions § 982.84 Duration of immunities. The benefits, privileges, and immunities conferred upon any person by virtue of this subpart shall cease upon the...

7 CFR 982.84 - Duration of immunities.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 8 2012-01-01 2012-01-01 false Duration of immunities. 982.84 Section 982.84... WASHINGTON Order Regulating Handling Miscellaneous Provisions § 982.84 Duration of immunities. The benefits, privileges, and immunities conferred upon any person by virtue of this subpart shall cease upon the...

34 CFR 84.625 - Criminal drug statute.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 34 Education 1 2011-07-01 2011-07-01 false Criminal drug statute. 84.625 Section 84.625 Education Office of the Secretary, Department of Education GOVERNMENTWIDE REQUIREMENTS FOR DRUG-FREE WORKPLACE (FINANCIAL ASSISTANCE) Definitions § 84.625 Criminal drug statute. Criminal drug statute means a Federal or...

34 CFR 84.635 - Drug-free workplace.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 34 Education 1 2013-07-01 2013-07-01 false Drug-free workplace. 84.635 Section 84.635 Education Office of the Secretary, Department of Education GOVERNMENTWIDE REQUIREMENTS FOR DRUG-FREE WORKPLACE (FINANCIAL ASSISTANCE) Definitions § 84.635 Drug-free workplace. Drug-free workplace means a site for the...

34 CFR 84.635 - Drug-free workplace.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 34 Education 1 2011-07-01 2011-07-01 false Drug-free workplace. 84.635 Section 84.635 Education Office of the Secretary, Department of Education GOVERNMENTWIDE REQUIREMENTS FOR DRUG-FREE WORKPLACE (FINANCIAL ASSISTANCE) Definitions § 84.635 Drug-free workplace. Drug-free workplace means a site for the...

34 CFR 84.625 - Criminal drug statute.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 34 Education 1 2014-07-01 2014-07-01 false Criminal drug statute. 84.625 Section 84.625 Education Office of the Secretary, Department of Education GOVERNMENTWIDE REQUIREMENTS FOR DRUG-FREE WORKPLACE (FINANCIAL ASSISTANCE) Definitions § 84.625 Criminal drug statute. Criminal drug statute means a Federal or...

34 CFR 84.625 - Criminal drug statute.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 34 Education 1 2013-07-01 2013-07-01 false Criminal drug statute. 84.625 Section 84.625 Education Office of the Secretary, Department of Education GOVERNMENTWIDE REQUIREMENTS FOR DRUG-FREE WORKPLACE (FINANCIAL ASSISTANCE) Definitions § 84.625 Criminal drug statute. Criminal drug statute means a Federal or...

34 CFR 84.635 - Drug-free workplace.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 34 Education 1 2014-07-01 2014-07-01 false Drug-free workplace. 84.635 Section 84.635 Education Office of the Secretary, Department of Education GOVERNMENTWIDE REQUIREMENTS FOR DRUG-FREE WORKPLACE (FINANCIAL ASSISTANCE) Definitions § 84.635 Drug-free workplace. Drug-free workplace means a site for the...

42 CFR 84.11 - Contents of application.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 1 2014-10-01 2014-10-01 false Contents of application. 84.11 Section 84.11 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Application for Approval § 84.11...

42 CFR 84.11 - Contents of application.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 1 2011-10-01 2011-10-01 false Contents of application. 84.11 Section 84.11 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Application for Approval § 84.11...

42 CFR 84.32 - Notice of disapproval.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 1 2013-10-01 2013-10-01 false Notice of disapproval. 84.32 Section 84.32 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Approval and Disapproval § 84.32...

42 CFR 84.11 - Contents of application.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 1 2013-10-01 2013-10-01 false Contents of application. 84.11 Section 84.11 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Application for Approval § 84.11...

42 CFR 84.32 - Notice of disapproval.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 1 2011-10-01 2011-10-01 false Notice of disapproval. 84.32 Section 84.32 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Approval and Disapproval § 84.32...

42 CFR 84.32 - Notice of disapproval.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 1 2014-10-01 2014-10-01 false Notice of disapproval. 84.32 Section 84.32 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Approval and Disapproval § 84.32...

42 CFR 84.32 - Notice of disapproval.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 1 2012-10-01 2012-10-01 false Notice of disapproval. 84.32 Section 84.32 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Approval and Disapproval § 84.32...

42 CFR 84.11 - Contents of application.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 1 2010-10-01 2010-10-01 false Contents of application. 84.11 Section 84.11 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Application for Approval § 84.11...

42 CFR 84.11 - Contents of application.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 1 2012-10-01 2012-10-01 false Contents of application. 84.11 Section 84.11 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Application for Approval § 84.11...

42 CFR 84.32 - Notice of disapproval.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 1 2010-10-01 2010-10-01 false Notice of disapproval. 84.32 Section 84.32 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Approval and Disapproval § 84.32...

46 CFR 151.50-84 - Sulfur dioxide.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 5 2014-10-01 2014-10-01 false Sulfur dioxide. 151.50-84 Section 151.50-84 Shipping... BULK LIQUID HAZARDOUS MATERIAL CARGOES Special Requirements § 151.50-84 Sulfur dioxide. (a) Sulfur... respiratory protective device that protects the wearer against sulfur dioxide vapors and provides respiratory...

46 CFR 151.50-84 - Sulfur dioxide.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 5 2012-10-01 2012-10-01 false Sulfur dioxide. 151.50-84 Section 151.50-84 Shipping... BULK LIQUID HAZARDOUS MATERIAL CARGOES Special Requirements § 151.50-84 Sulfur dioxide. (a) Sulfur... respiratory protective device that protects the wearer against sulfur dioxide vapors and provides respiratory...

46 CFR 151.50-84 - Sulfur dioxide.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 5 2013-10-01 2013-10-01 false Sulfur dioxide. 151.50-84 Section 151.50-84 Shipping... BULK LIQUID HAZARDOUS MATERIAL CARGOES Special Requirements § 151.50-84 Sulfur dioxide. (a) Sulfur... respiratory protective device that protects the wearer against sulfur dioxide vapors and provides respiratory...

46 CFR 151.50-84 - Sulfur dioxide.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 5 2011-10-01 2011-10-01 false Sulfur dioxide. 151.50-84 Section 151.50-84 Shipping... BULK LIQUID HAZARDOUS MATERIAL CARGOES Special Requirements § 151.50-84 Sulfur dioxide. (a) Sulfur... respiratory protective device that protects the wearer against sulfur dioxide vapors and provides respiratory...

34 CFR 84.635 - Drug-free workplace.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 34 Education 1 2010-07-01 2010-07-01 false Drug-free workplace. 84.635 Section 84.635 Education Office of the Secretary, Department of Education GOVERNMENTWIDE REQUIREMENTS FOR DRUG-FREE WORKPLACE (FINANCIAL ASSISTANCE) Definitions § 84.635 Drug-free workplace. Drug-free workplace means a site for the...

34 CFR 84.625 - Criminal drug statute.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 34 Education 1 2010-07-01 2010-07-01 false Criminal drug statute. 84.625 Section 84.625 Education Office of the Secretary, Department of Education GOVERNMENTWIDE REQUIREMENTS FOR DRUG-FREE WORKPLACE (FINANCIAL ASSISTANCE) Definitions § 84.625 Criminal drug statute. Criminal drug statute means a Federal or...

41 CFR 60-741.84 - Effective date.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 41 Public Contracts and Property Management 1 2011-07-01 2009-07-01 true Effective date. 60-741.84 Section 60-741.84 Public Contracts and Property Management Other Provisions Relating to Public Contracts... WITH DISABILITIES Ancillary Matters § 60-741.84 Effective date. This part shall become effective August...

12 CFR 34.84 - Future bank expansion.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 12 Banks and Banking 1 2011-01-01 2011-01-01 false Future bank expansion. 34.84 Section 34.84 Banks and Banking COMPTROLLER OF THE CURRENCY, DEPARTMENT OF THE TREASURY REAL ESTATE LENDING AND APPRAISALS Other Real Estate Owned § 34.84 Future bank expansion. A national bank normally should use real...

40 CFR 97.84 - Opt-in process.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 20 2010-07-01 2010-07-01 false Opt-in process. 97.84 Section 97.84 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) FEDERAL NOX BUDGET TRADING PROGRAM AND CAIR NOX AND SO2 TRADING PROGRAMS Individual Unit Opt-ins. § 97.84 Opt-in...

34 CFR 5.84 - Consideration on review.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 34 Education 1 2010-07-01 2010-07-01 false Consideration on review. 5.84 Section 5.84 Education Office of the Secretary, Department of Education AVAILABILITY OF INFORMATION TO THE PUBLIC PURSUANT TO PUB. L. 90-23 (Eff. until 7-14-10) Administrative Review § 5.84 Consideration on review. Review shall...

14 CFR 33.84 - Engine overtorque test.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Engine overtorque test. 33.84 Section 33.84... STANDARDS: AIRCRAFT ENGINES Block Tests; Turbine Aircraft Engines § 33.84 Engine overtorque test. (a) If approval of a maximum engine overtorque is sought for an engine incorporating a free power turbine...

Hairy Root Transformation Using Agrobacterium rhizogenes as a Tool for Exploring Cell Type-Specific Gene Expression and Function Using Tomato as a Model1[W][OPEN

PubMed Central

Ron, Mily; Kajala, Kaisa; Pauluzzi, Germain; Wang, Dongxue; Reynoso, Mauricio A.; Zumstein, Kristina; Garcha, Jasmine; Winte, Sonja; Masson, Helen; Inagaki, Soichi; Federici, Fernán; Sinha, Neelima; Deal, Roger B.; Bailey-Serres, Julia; Brady, Siobhan M.

2014-01-01

Agrobacterium rhizogenes (or Rhizobium rhizogenes) is able to transform plant genomes and induce the production of hairy roots. We describe the use of A. rhizogenes in tomato (Solanum spp.) to rapidly assess gene expression and function. Gene expression of reporters is indistinguishable in plants transformed by Agrobacterium tumefaciens as compared with A. rhizogenes. A root cell type- and tissue-specific promoter resource has been generated for domesticated and wild tomato (Solanum lycopersicum and Solanum pennellii, respectively) using these approaches. Imaging of tomato roots using A. rhizogenes coupled with laser scanning confocal microscopy is facilitated by the use of a membrane-tagged protein fused to a red fluorescent protein marker present in binary vectors. Tomato-optimized isolation of nuclei tagged in specific cell types and translating ribosome affinity purification binary vectors were generated and used to monitor associated messenger RNA abundance or chromatin modification. Finally, transcriptional reporters, translational reporters, and clustered regularly interspaced short palindromic repeats-associated nuclease9 genome editing demonstrate that SHORT-ROOT and SCARECROW gene function is conserved between Arabidopsis (Arabidopsis thaliana) and tomato. PMID:24868032

STS-84 Crew Portrait

NASA Technical Reports Server (NTRS)

1997-01-01

The crew assigned to the STS-84 mission included (seated front left to right) Jerry M Linenger, mission specialist; Charles J. Precourt, commander; and C. Michael Foale, mission specialist. On the back row (left to right) are Jean-Francois Clervoy (ESA), mission specialist; Eileen M. Collins, pilot; Edward T. Lu, mission specialist; Elena V. Kondakova (RSA), mission specialist; and Carlos I. Noriega, mission specialist. Launched aboard the Space Shuttle Atlantis on May 15, 1997 at 4:07:48 am (EDT), the STS-84 mission served as the sixth U.S. Space Shuttle-Russian Space Station Mir docking.

42 CFR 84.310 - Post-approval testing.

Code of Federal Regulations, 2014 CFR

2014-10-01

... Respirators § 84.310 Post-approval testing. (a) NIOSH will periodically test the capacity and performance of units of approved CCERs. (b) NIOSH may test units that are new and/or units that have been deployed in... 42 Public Health 1 2014-10-01 2014-10-01 false Post-approval testing. 84.310 Section 84.310 Public...

42 CFR 84.310 - Post-approval testing.

Code of Federal Regulations, 2013 CFR

2013-10-01

... Respirators § 84.310 Post-approval testing. (a) NIOSH will periodically test the capacity and performance of units of approved CCERs. (b) NIOSH may test units that are new and/or units that have been deployed in... 42 Public Health 1 2013-10-01 2013-10-01 false Post-approval testing. 84.310 Section 84.310 Public...

42 CFR 84.310 - Post-approval testing.

Code of Federal Regulations, 2012 CFR

2012-10-01

... Respirators § 84.310 Post-approval testing. (a) NIOSH will periodically test the capacity and performance of units of approved CCERs. (b) NIOSH may test units that are new and/or units that have been deployed in... 42 Public Health 1 2012-10-01 2012-10-01 false Post-approval testing. 84.310 Section 84.310 Public...

42 CFR 84.88 - Breathing bag test.

Code of Federal Regulations, 2014 CFR

2014-10-01

... Apparatus § 84.88 Breathing bag test. (a) Breathing bags will be tested in an air atmosphere saturated with... Institute upon request. (d) The air within the bag(s) shall not contain more than 100 parts per million of... 42 Public Health 1 2014-10-01 2014-10-01 false Breathing bag test. 84.88 Section 84.88 Public...

Agrobacterium rhizogenes-mediated DNA transfer to Aesculus hippocastanum L. and the regeneration of transformed plants.

PubMed

Zdravković-Korać, S; Muhovski, Y; Druart, P; Calić, D; Radojević, L

2004-04-01

Hairy roots were induced from androgenic embryos of horse chestnut (Aesculus hippocastanum L.) by infection with Agrobacterium rhizogenes strain A4GUS. Single roots were selected according to their morphology in the absence of antibiotic or herbicide resistance markers. Seventy-one putative transformed hairy root lines from independent transformation events were established. Regeneration was induced in MS liquid medium supplemented with 30 microM 6-benzylaminopurine (BA), and the regenerants were multiplied on MS solid medium containing 10 microM BA. Following elongation on MS medium supplemented with 1 microM BA and 500 mg/l polyvinylpyrrolidone, the shoots were subjected to a root-inducing treatment. Stable integration of TL-DNA within the horse chestnut genome was confirmed by Southern hybridization. The copy number of transgenes was estimated to be from two to four.

Hfq Influences Multiple Transport Systems and Virulence in the Plant Pathogen Agrobacterium tumefaciens

PubMed Central

Wilms, Ina; Möller, Philip; Stock, Anna-Maria; Gurski, Rosemarie; Lai, Erh-Min

2012-01-01

The Hfq protein mediates gene regulation by small RNAs (sRNAs) in about 50% of all bacteria. Depending on the species, phenotypic defects of an hfq mutant range from mild to severe. Here, we document that the purified Hfq protein of the plant pathogen and natural genetic engineer Agrobacterium tumefaciens binds to the previously described sRNA AbcR1 and its target mRNA atu2422, which codes for the substrate binding protein of an ABC transporter taking up proline and γ-aminobutyric acid (GABA). Several other ABC transporter components were overproduced in an hfq mutant compared to their levels in the parental strain, suggesting that Hfq plays a major role in controlling the uptake systems and metabolic versatility of A. tumefaciens. The hfq mutant showed delayed growth, altered cell morphology, and reduced motility. Although the DNA-transferring type IV secretion system was produced, tumor formation by the mutant strain was attenuated, demonstrating an important contribution of Hfq to plant transformation by A. tumefaciens. PMID:22821981

Bioassays of quorum sensing compounds using Agrobacterium tumefaciens and Chromobacterium violaceum.

PubMed

Chu, Weihua; Vattem, Dhiraj A; Maitin, Vatsala; Barnes, Mary B; McLean, Robert J C

2011-01-01

In most bacteria, a global level of regulation exists involving intercellular communication via the production and response to cell density-dependent signal molecules. This cell density-dependent regulation has been termed quorum sensing (QS). QS is a global regulator, which has been associated with a number of important features in bacteria including virulence regulation and biofilm formation. Consequently, there is considerable interest in understanding, detecting, and inhibiting QS. Acyl homoserine lactones (acyl HSLs) are used as extracellular QS signals by a variety of Gram-negative bacteria. Chromobacterium violaceum, a Gram-negative bacterium commonly found in soil and water, produces the characteristic purple pigment violacein, the production of which is regulated by acyl HSL-mediated QS. Based on this readily observed pigmentation phenotype, C. violaceum strains can be used to detect various aspects of acyl HSL-mediated QS activity. In another commonly used bioassay organism, Agrobacterium tumefaciens, QS can be detected by the use of a reporter gene such as lacZ. Here, we describe several commonly used approaches incorporating C. violaceum and A. tumefaciens that can be used to detect acyl HSLs and QS inhibition.

7 CFR 993.84 - Personal liability.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 8 2011-01-01 2011-01-01 false Personal liability. 993.84 Section 993.84 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE DRIED PRUNES PRODUCED IN CALIFORNIA...

7 CFR 993.84 - Personal liability.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 8 2012-01-01 2012-01-01 false Personal liability. 993.84 Section 993.84 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE DRIED PRUNES PRODUCED IN CALIFORNIA...

7 CFR 993.84 - Personal liability.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 8 2014-01-01 2014-01-01 false Personal liability. 993.84 Section 993.84 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS AND ORDERS; FRUITS, VEGETABLES, NUTS), DEPARTMENT OF AGRICULTURE DRIED PRUNES PRODUCED IN CALIFORNIA...

7 CFR 993.84 - Personal liability.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 8 2013-01-01 2013-01-01 false Personal liability. 993.84 Section 993.84 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS AND ORDERS; FRUITS, VEGETABLES, NUTS), DEPARTMENT OF AGRICULTURE DRIED PRUNES PRODUCED IN CALIFORNIA...

Characterization of three Agrobacterium tumefaciens avirulent mutants with chromosomal mutations that affect induction of vir genes.

PubMed

Metts, J; West, J; Doares, S H; Matthysse, A G

1991-02-01

Three Agrobacterium tumefaciens mutants with chromosomal mutations that affect bacterial virulence were isolated by transposon mutagenesis. Two of the mutants were avirulent on all hosts tested. The third mutant, Ivr-211, was a host range mutant which was avirulent on Bryophyllum diagremontiana, Nicotiana tabacum, N. debneyi, N. glauca, and Daucus carota but was virulent on Zinnia elegans and Lycopersicon esculentum (tomato). That the mutant phenotype was due to the transposon insertion was determined by cloning the DNA containing the transposon insertion and using the cloned DNA to replace the wild-type DNA in the parent bacterial strain by marker exchange. The transposon insertions in the three mutants mapped at three widely separated locations on the bacterial chromosome. The effects of the mutations on various steps in tumor formation were examined. All three mutants showed no alteration in binding to carrot cells. However, none of the mutants showed any induction of vir genes by acetosyringone under conditions in which the parent strain showed vir gene induction. When the mutant bacteria were examined for changes in surface components, it was found that all three of the mutants showed a similar alteration in lipopolysaccharide (LPS). LPS from the mutants was larger in size and more heavily saccharide substituted than LPS from the parent strain. Two of the mutants showed no detectable alteration in outer membrane and periplasmic space proteins. The third mutant, Ivr-225, was missing a 79-kDa surface peptide. The reason(s) for the failure of vir gene induction in these mutants and its relationship, if any, to the observed alteration in LPS are unknown.

Heat capacity and entropy at the temperatures 5 K to 720 K and thermal expansion from the temperatures 298 K to 573 K of synthetic enargite (Cu3AsS4)

USGS Publications Warehouse

Seal, R.R.; Robie, R.A.; Hemingway, B.S.; Evans, H.T.

1996-01-01

The heat capacity of synthetic Cu3AsS4 (enargite) was measured by quasi-adiabatic calorimetry from the temperatures 5 K to 355 K and by differential scanning calorimetry from T = 339 K to T = 720 K. Heat-capacity anomalies were observed at T = (58.5 ?? 0.5) K (??trsHom = 1.4??R??K; ??trsSom = 0.02??R) and at T = (66.5 ?? 0.5) K (??trsHom = 4.6??R??K; ??trsSom = 0.08??R), where R = 8.31451 J??K-1??mol-1. The causes of the anomalies are unknown. At T = 298.15 K, Cop,m and Som(T) are (190.4 ?? 0.2) J??K-1??mol-1 and (257.6 ?? 0.6) J??K-1??mol-1, respectively. The superambient heat capacities are described from T = 298.15 K to T = 944 K by the least-squares regression equation: Cop,m/(J??K-1??mol-1) = (196.7 ?? 1.2) + (0.0499 ?? 0.0016)??(T/K) -(1918 000 ?? 84 000)??(T/K)-2. The thermal expansion of synthetic enargite was measured from T = 298.15 K to T = 573 K by powder X-ray diffraction. The thermal expansion of the unit-cell volume (Z = 2) is described from T = 298.15 K to T = 573 K by the least-squares equation: V/pm3 = 106??(288.2 ?? 0.2) + 104??(1.49 ?? 0.04)??(T/K). ?? 1996 Academic Press Limited.

42 CFR 84.89 - Weight requirement.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 1 2012-10-01 2012-10-01 false Weight requirement. 84.89 Section 84.89 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Self-Contained Breathing...

42 CFR 84.89 - Weight requirement.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 1 2013-10-01 2013-10-01 false Weight requirement. 84.89 Section 84.89 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Self-Contained Breathing...

42 CFR 84.89 - Weight requirement.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 1 2011-10-01 2011-10-01 false Weight requirement. 84.89 Section 84.89 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Self-Contained Breathing...

42 CFR 84.89 - Weight requirement.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 1 2010-10-01 2010-10-01 false Weight requirement. 84.89 Section 84.89 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Self-Contained Breathing...

45 CFR 84.37 - Nonacademic services.

Code of Federal Regulations, 2014 CFR

2014-10-01

... athletics, transportation, health services, recreational activities, special interest groups or clubs... 45 Public Welfare 1 2014-10-01 2014-10-01 false Nonacademic services. 84.37 Section 84.37 Public Welfare Department of Health and Human Services GENERAL ADMINISTRATION NONDISCRIMINATION ON THE BASIS OF...

45 CFR 84.37 - Nonacademic services.

Code of Federal Regulations, 2012 CFR

2012-10-01

... athletics, transportation, health services, recreational activities, special interest groups or clubs... 45 Public Welfare 1 2012-10-01 2012-10-01 false Nonacademic services. 84.37 Section 84.37 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION NONDISCRIMINATION ON THE BASIS OF...

45 CFR 84.37 - Nonacademic services.

Code of Federal Regulations, 2013 CFR

2013-10-01

... athletics, transportation, health services, recreational activities, special interest groups or clubs... 45 Public Welfare 1 2013-10-01 2013-10-01 false Nonacademic services. 84.37 Section 84.37 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION NONDISCRIMINATION ON THE BASIS OF...

36 CFR 9.84 - Application requirements.

Code of Federal Regulations, 2010 CFR

2010-07-01

... environmentally sound manner utilizing the least impacting technology suitable for the purposes of the project... MINERALS MANAGEMENT Alaska Mineral Resource Assessment Program § 9.84 Application requirements. (a) By... Director an application pursuant to § 9.84(b) for proposed AMRAP projects and activities discussed and...

36 CFR 9.84 - Application requirements.

Code of Federal Regulations, 2013 CFR

2013-07-01

... environmentally sound manner utilizing the least impacting technology suitable for the purposes of the project... MINERALS MANAGEMENT Alaska Mineral Resource Assessment Program § 9.84 Application requirements. (a) By... Director an application pursuant to § 9.84(b) for proposed AMRAP projects and activities discussed and...

36 CFR 9.84 - Application requirements.

Code of Federal Regulations, 2011 CFR

2011-07-01

... environmentally sound manner utilizing the least impacting technology suitable for the purposes of the project... MINERALS MANAGEMENT Alaska Mineral Resource Assessment Program § 9.84 Application requirements. (a) By... Director an application pursuant to § 9.84(b) for proposed AMRAP projects and activities discussed and...

36 CFR 9.84 - Application requirements.

Code of Federal Regulations, 2012 CFR

2012-07-01

... environmentally sound manner utilizing the least impacting technology suitable for the purposes of the project... MINERALS MANAGEMENT Alaska Mineral Resource Assessment Program § 9.84 Application requirements. (a) By... Director an application pursuant to § 9.84(b) for proposed AMRAP projects and activities discussed and...

36 CFR 9.84 - Application requirements.

Code of Federal Regulations, 2014 CFR

2014-07-01

... environmentally sound manner utilizing the least impacting technology suitable for the purposes of the project... MINERALS MANAGEMENT Alaska Mineral Resource Assessment Program § 9.84 Application requirements. (a) By... Director an application pursuant to § 9.84(b) for proposed AMRAP projects and activities discussed and...

7 CFR 993.84 - Personal liability.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 8 2010-01-01 2010-01-01 false Personal liability. 993.84 Section 993.84 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... responsible, either individually of jointly with others, in any way whatsoever, to any person, for errors in...

42 CFR 84.1130 - Respirators; description.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 1 2010-10-01 2010-10-01 false Respirators; description. 84.1130 Section 84.1130 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Dust, Fume, and Mist...

42 CFR 84.1130 - Respirators; description.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 1 2014-10-01 2014-10-01 false Respirators; description. 84.1130 Section 84.1130 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Dust, Fume, and Mist...

42 CFR 84.1130 - Respirators; description.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 1 2011-10-01 2011-10-01 false Respirators; description. 84.1130 Section 84.1130 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Dust, Fume, and Mist...

42 CFR 84.1130 - Respirators; description.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 1 2013-10-01 2013-10-01 false Respirators; description. 84.1130 Section 84.1130 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Dust, Fume, and Mist...

42 CFR 84.1130 - Respirators; description.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 1 2012-10-01 2012-10-01 false Respirators; description. 84.1130 Section 84.1130 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Dust, Fume, and Mist...

45 CFR 84.21 - Discrimination prohibited.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 45 Public Welfare 1 2012-10-01 2012-10-01 false Discrimination prohibited. 84.21 Section 84.21... Discrimination prohibited. No qualified handicapped person shall, because a recipient's facilities are... in, or otherwise be subjected to discrimination under any program or activity to which this part...

45 CFR 84.21 - Discrimination prohibited.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 45 Public Welfare 1 2014-10-01 2014-10-01 false Discrimination prohibited. 84.21 Section 84.21... Discrimination prohibited. No qualified handicapped person shall, because a recipient's facilities are... in, or otherwise be subjected to discrimination under any program or activity to which this part...

15 CFR 8.4 - Discrimination prohibited.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 15 Commerce and Foreign Trade 1 2011-01-01 2011-01-01 false Discrimination prohibited. 8.4 Section... General Provisions; Prohibitions: Nondiscrimination Clause; Applicability to Programs § 8.4 Discrimination... discrimination under, any program to which this part applies. (b) Specific discriminatory acts prohibited. (1) A...

45 CFR 84.21 - Discrimination prohibited.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 45 Public Welfare 1 2013-10-01 2013-10-01 false Discrimination prohibited. 84.21 Section 84.21... Discrimination prohibited. No qualified handicapped person shall, because a recipient's facilities are... in, or otherwise be subjected to discrimination under any program or activity to which this part...

15 CFR 8.4 - Discrimination prohibited.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 15 Commerce and Foreign Trade 1 2013-01-01 2013-01-01 false Discrimination prohibited. 8.4 Section... General Provisions; Prohibitions: Nondiscrimination Clause; Applicability to Programs § 8.4 Discrimination... discrimination under, any program to which this part applies. (b) Specific discriminatory acts prohibited. (1) A...

45 CFR 84.21 - Discrimination prohibited.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 45 Public Welfare 1 2010-10-01 2010-10-01 false Discrimination prohibited. 84.21 Section 84.21... Discrimination prohibited. No qualified handicapped person shall, because a recipient's facilities are... in, or otherwise be subjected to discrimination under any program or activity to which this part...

15 CFR 8.4 - Discrimination prohibited.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 15 Commerce and Foreign Trade 1 2012-01-01 2012-01-01 false Discrimination prohibited. 8.4 Section... General Provisions; Prohibitions: Nondiscrimination Clause; Applicability to Programs § 8.4 Discrimination... discrimination under, any program to which this part applies. (b) Specific discriminatory acts prohibited. (1) A...

15 CFR 8.4 - Discrimination prohibited.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 15 Commerce and Foreign Trade 1 2014-01-01 2014-01-01 false Discrimination prohibited. 8.4 Section... General Provisions; Prohibitions: Nondiscrimination Clause; Applicability to Programs § 8.4 Discrimination... discrimination under, any program to which this part applies. (b) Specific discriminatory acts prohibited. (1) A...

45 CFR 84.47 - Nonacademic services.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 45 Public Welfare 1 2012-10-01 2012-10-01 false Nonacademic services. 84.47 Section 84.47 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION NONDISCRIMINATION ON THE BASIS OF... Nonacademic services. (a) Physical education and athletics. (1) In providing physical education courses and...

45 CFR 84.47 - Nonacademic services.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 45 Public Welfare 1 2013-10-01 2013-10-01 false Nonacademic services. 84.47 Section 84.47 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION NONDISCRIMINATION ON THE BASIS OF... Nonacademic services. (a) Physical education and athletics. (1) In providing physical education courses and...

45 CFR 84.47 - Nonacademic services.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 45 Public Welfare 1 2011-10-01 2011-10-01 false Nonacademic services. 84.47 Section 84.47 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION NONDISCRIMINATION ON THE BASIS OF... Nonacademic services. (a) Physical education and athletics. (1) In providing physical education courses and...

45 CFR 84.47 - Nonacademic services.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 45 Public Welfare 1 2014-10-01 2014-10-01 false Nonacademic services. 84.47 Section 84.47 Public Welfare Department of Health and Human Services GENERAL ADMINISTRATION NONDISCRIMINATION ON THE BASIS OF... Nonacademic services. (a) Physical education and athletics. (1) In providing physical education courses and...

Effect of manganese doping on remnant polarization and leakage current in (K0.44,Na0.52,Li0.04)(Nb0.84,Ta0.10,Sb0.06)O3 epitaxial thin films on SrTiO3

NASA Astrophysics Data System (ADS)

Abazari, M.; Akdoǧan, E. K.; Safari, A.

2008-05-01

Single phase, epitaxial, ⟨001⟩ oriented, undoped and 1mol% Mn-doped (K0.44,Na0.52,Li0.04)(Nb0.84,Ta0.10,Sb0.06)O3 thin films of 400nm thickness were synthesized on SrRuO3 coated SrTiO3. Such films exhibit well saturated hysteresis loops and have a spontaneous polarization (Ps) of 10μC /cm2, which is a 150% higher over the Ps of the undoped composition. The coercive field of 1mol% Mn doped films is 13kV/cm. Mn-doping results in three orders of magnitude decrease in leakage current above 50kV/cm electric field, which we attribute to the suppression of intrinsic p-type conductivity of undoped films by Mn donors.

7 CFR 984.84 - Personal liability.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 8 2010-01-01 2010-01-01 false Personal liability. 984.84 Section 984.84 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... with others, in any way whatsoever, to any handler or any person for errors in judgment, mistakes, or...

24 CFR 8.4 - Discrimination prohibited.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 24 Housing and Urban Development 1 2011-04-01 2011-04-01 false Discrimination prohibited. 8.4... DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT General Provisions § 8.4 Discrimination prohibited. (a) No... in, be denied the benefits of, or otherwise be subjected to discrimination under any program or...

24 CFR 8.4 - Discrimination prohibited.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 24 Housing and Urban Development 1 2010-04-01 2010-04-01 false Discrimination prohibited. 8.4... DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT General Provisions § 8.4 Discrimination prohibited. (a) No... in, be denied the benefits of, or otherwise be subjected to discrimination under any program or...

24 CFR 8.4 - Discrimination prohibited.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 24 Housing and Urban Development 1 2012-04-01 2012-04-01 false Discrimination prohibited. 8.4... DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT General Provisions § 8.4 Discrimination prohibited. (a) No... in, be denied the benefits of, or otherwise be subjected to discrimination under any program or...

24 CFR 8.4 - Discrimination prohibited.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 24 Housing and Urban Development 1 2013-04-01 2013-04-01 false Discrimination prohibited. 8.4... DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT General Provisions § 8.4 Discrimination prohibited. (a) No... in, be denied the benefits of, or otherwise be subjected to discrimination under any program or...

24 CFR 8.4 - Discrimination prohibited.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 24 Housing and Urban Development 1 2014-04-01 2014-04-01 false Discrimination prohibited. 8.4... DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT General Provisions § 8.4 Discrimination prohibited. (a) No... in, be denied the benefits of, or otherwise be subjected to discrimination under any program or...

Registration of 'UFCP 84-1047' Sugarcane

USDA-ARS?s Scientific Manuscript database

UFCP 84-1047 (Reg. No.; PI xxxx) was released by the United States Department of Agriculture-Agricultural Research Services (USDA-ARS), Canal Point (CP), Florida, and the University of Florida (UF) for its potential use in cellulosic ethanol production. UFCP 84-1047 is a high fiber sugarcane (Saccha...

31 CFR 103.84 - Withdrawing requests.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 31 Money and Finance: Treasury 1 2010-07-01 2010-07-01 false Withdrawing requests. 103.84 Section 103.84 Money and Finance: Treasury Regulations Relating to Money and Finance FINANCIAL RECORDKEEPING... requests. A person may withdraw a request for an administrative ruling at any time before the ruling has...

42 CFR 84.308 - Additional testing.

Code of Federal Regulations, 2013 CFR

2013-10-01

... §§ 84.304 through 84.307. These units will be evaluated for fire and explosion hazards using the tests....S.C. 552(a) and 1 CFR Part 51. All approved material is available for inspection at NIOSH, National Personal Protection Technology Laboratory (NPPTL), Bruceton Research Center, 626 Cochrans Mill Road...

42 CFR 84.308 - Additional testing.

Code of Federal Regulations, 2014 CFR

2014-10-01

... §§ 84.304 through 84.307. These units will be evaluated for fire and explosion hazards using the tests....S.C. 552(a) and 1 CFR Part 51. All approved material is available for inspection at NIOSH, National Personal Protection Technology Laboratory (NPPTL), Bruceton Research Center, 626 Cochrans Mill Road...

42 CFR 84.308 - Additional testing.

Code of Federal Regulations, 2012 CFR

2012-10-01

... §§ 84.304 through 84.307. These units will be evaluated for fire and explosion hazards using the tests....S.C. 552(a) and 1 CFR Part 51. All approved material is available for inspection at NIOSH, National Personal Protection Technology Laboratory (NPPTL), Bruceton Research Center, 626 Cochrans Mill Road...

45 CFR 84.44 - Academic adjustments.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 45 Public Welfare 1 2013-10-01 2013-10-01 false Academic adjustments. 84.44 Section 84.44 Public... Academic adjustments. (a) Academic requirements. A recipient to which this subpart applies shall make such modifications to its academic requirements as are necessary to ensure that such requirements do not discriminate...

45 CFR 84.44 - Academic adjustments.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 45 Public Welfare 1 2014-10-01 2014-10-01 false Academic adjustments. 84.44 Section 84.44 Public... Academic adjustments. (a) Academic requirements. A recipient to which this subpart applies shall make such modifications to its academic requirements as are necessary to ensure that such requirements do not discriminate...

45 CFR 84.44 - Academic adjustments.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 45 Public Welfare 1 2012-10-01 2012-10-01 false Academic adjustments. 84.44 Section 84.44 Public... Academic adjustments. (a) Academic requirements. A recipient to which this subpart applies shall make such modifications to its academic requirements as are necessary to ensure that such requirements do not discriminate...

45 CFR 84.23 - New construction.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 45 Public Welfare 1 2012-10-01 2012-10-01 false New construction. 84.23 Section 84.23 Public... construction. (a) Design and construction. Each facility or part of a facility constructed by, on behalf of, or... of the facility is readily accessible to and usable by handicapped persons, if the construction was...

45 CFR 84.23 - New construction.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 45 Public Welfare 1 2013-10-01 2013-10-01 false New construction. 84.23 Section 84.23 Public... construction. (a) Design and construction. Each facility or part of a facility constructed by, on behalf of, or... of the facility is readily accessible to and usable by handicapped persons, if the construction was...

45 CFR 84.13 - Employment criteria.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 45 Public Welfare 1 2013-10-01 2013-10-01 false Employment criteria. 84.13 Section 84.13 Public... administered to an applicant or employee who has a handicap that impairs sensory, manual, or speaking skills... sensory, manual, or speaking skills (except where those skills are the factors that the test purports to...

45 CFR 84.13 - Employment criteria.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 45 Public Welfare 1 2014-10-01 2014-10-01 false Employment criteria. 84.13 Section 84.13 Public... administered to an applicant or employee who has a handicap that impairs sensory, manual, or speaking skills... sensory, manual, or speaking skills (except where those skills are the factors that the test purports to...

45 CFR 84.13 - Employment criteria.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 45 Public Welfare 1 2011-10-01 2011-10-01 false Employment criteria. 84.13 Section 84.13 Public... administered to an applicant or employee who has a handicap that impairs sensory, manual, or speaking skills... sensory, manual, or speaking skills (except where those skills are the factors that the test purports to...

45 CFR 84.44 - Academic adjustments.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 45 Public Welfare 1 2010-10-01 2010-10-01 false Academic adjustments. 84.44 Section 84.44 Public..., manual, or speaking skills as will best ensure that the results of the evaluation represents the student... speaking skills (except where such skills are the factors that the test purports to measure). (d) Auxiliary...

45 CFR 84.13 - Employment criteria.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 45 Public Welfare 1 2010-10-01 2010-10-01 false Employment criteria. 84.13 Section 84.13 Public... administered to an applicant or employee who has a handicap that impairs sensory, manual, or speaking skills... sensory, manual, or speaking skills (except where those skills are the factors that the test purports to...

45 CFR 84.44 - Academic adjustments.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 45 Public Welfare 1 2011-10-01 2011-10-01 false Academic adjustments. 84.44 Section 84.44 Public..., manual, or speaking skills as will best ensure that the results of the evaluation represents the student... speaking skills (except where such skills are the factors that the test purports to measure). (d) Auxiliary...

Highly efficient Agrobacterium-mediated transformation of embryogenic cell suspensions of Musa acuminata cv. Mas (AA) via a liquid co-cultivation system.

PubMed

Huang, Xia; Huang, Xue-Lin; Xiao, Wang; Zhao, Jie-Tang; Dai, Xue-Mei; Chen, Yun-Feng; Li, Xiao-Ju

2007-10-01

A high efficient protocol of Agrobacterium-mediated transformation of Musa acuminata cv. Mas (AA), a major banana variety of the South East Asia region, was developed in this study. Male-flower-derived embryogenic cell suspensions (ECS) were co-cultivated in liquid medium with Agrobacterium strain EHA105 harboring a binary vector pCAMBIA2301 carrying nptII and gusA gene in the T-DNA. Depending upon conditions and duration of co-cultivation in liquid medium, 0-490 transgenic plants per 0.5 ml packed cell volume (PCV) of ECS were obtained. The optimum duration of inoculation was 2 h, and the highest transformation frequency was achieved when infected ECS were co-cultivated in liquid medium first for 12 h at 40 rpm and then for 156 h at 100 rpm on a rotary shaker. Co-cultivation for a shorter duration (72 h) or shaking constantly at 100 rpm at the same duration gave 1.6 and 1.8 folds lower transformation efficiency, respectively. No transgenic plants were obtained in parallel experiments carried on semi-solid media. Histochemical GUS assay and molecular analysis in several tissues of the transgenic plants demonstrated that foreign genes were stably integrated into the banana genome. Compared to semi-solid co-cultivation transformation in other banana species, it is remarkable that liquid co-cultivation was much more efficient for transformation of the Mas cultivar, and was at least 1 month faster for regenerating transgenic plants.

7 CFR 1779.84 - Additional loans or advances.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 12 2010-01-01 2010-01-01 false Additional loans or advances. 1779.84 Section 1779.84 Agriculture Regulations of the Department of Agriculture (Continued) RURAL UTILITIES SERVICE, DEPARTMENT OF AGRICULTURE (CONTINUED) WATER AND WASTE DISPOSAL PROGRAMS GUARANTEED LOANS § 1779.84 Additional loans or...

34 CFR 84.645 - Federal agency or agency.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 34 Education 1 2010-07-01 2010-07-01 false Federal agency or agency. 84.645 Section 84.645 Education Office of the Secretary, Department of Education GOVERNMENTWIDE REQUIREMENTS FOR DRUG-FREE WORKPLACE (FINANCIAL ASSISTANCE) Definitions § 84.645 Federal agency or agency. Federal agency or agency...

KEY COMPARISON: CCQM-K32 key comparison and P84 pilot study: Amount of silicon oxide as a thickness of SiO2 on Si

NASA Astrophysics Data System (ADS)

Seah, M. P.

2008-01-01

CCQM-K32 and P84 were conducted following the pilot study P-38 to demonstrate and document the capability of interested National Metrology Institutes to measure the amount of silicon oxide on silicon wafers expressed as a thickness of SiO2 for nominal thicknesses in the range 1.5 nm to 8 nm. 'Amount of substance' may be expressed in many ways and here the measurand is the thickness of the silicon oxide layer on each of a total of 9 samples of nominal thicknesses in the range 1.5 to 8 nm on (100) and (111) Si substrates, expressed as the thickness of SiO2. This report presents the results from K32 and P84. It includes the data received for the measured values and their associated uncertainties, at 95% confidence, for the 9 samples prior to the deadline for receipt of data. The materials are grown by thermal oxidation in very clean furnaces designed for high quality gate oxides on Si wafers in European and US facilities at the same time as those for the pilot study, P-38. Separate samples were provided to each institute in special containers limiting the carbonaceous contamination to below about 0.3 nm. The 9 samples included 5 samples of ultra-thin SiO2 on (100) orientated wafers of Si and 4 samples of ultra-thin SiO2 on (111) orientated wafers of Si. The measurements from the 11 participating laboratories were conducted using ellipsometry, neutron reflectivity (NR), x-ray photoelectron spectroscopy (XPS) or x-ray reflectivity measurements (XRR), guided by the protocol developed in the pilot study P-38 and reproduced in the Appendix. The measurements are given in tables 2 and 3. A very small correction is then made for the different samples that each laboratory received as in table 4. Where appropriate, method offset values deduced from the pilot study P-38 are given in table 5 leading to comparative data in tables 6 and 7. Values for the key comparison reference values (KCRVs) and their associated uncertainties are made from the weighted means and the expanded

24 CFR 84.24 - Program income.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 24 Housing and Urban Development 1 2010-04-01 2010-04-01 false Program income. 84.24 Section 84.24... income. (a) HUD shall apply the standards set forth in this section in requiring recipient organizations to account for program income related to projects financed in whole or in part with Federal funds. (b...

19 CFR 122.84 - Intermediate airport.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 19 Customs Duties 1 2011-04-01 2011-04-01 false Intermediate airport. 122.84 Section 122.84... Intermediate airport. (a) Application. The provisions of this section apply at any U.S. airport to which an... aircraft arrives at the next airport, the aircraft commander or agent shall make entry by filing the: (1...

19 CFR 122.84 - Intermediate airport.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 19 Customs Duties 1 2010-04-01 2010-04-01 false Intermediate airport. 122.84 Section 122.84... Intermediate airport. (a) Application. The provisions of this section apply at any U.S. airport to which an... aircraft arrives at the next airport, the aircraft commander or agent shall make entry by filing the: (1...

19 CFR 122.84 - Intermediate airport.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 19 Customs Duties 1 2012-04-01 2012-04-01 false Intermediate airport. 122.84 Section 122.84... Intermediate airport. (a) Application. The provisions of this section apply at any U.S. airport to which an... aircraft arrives at the next airport, the aircraft commander or agent shall make entry by filing the: (1...

19 CFR 122.84 - Intermediate airport.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 19 Customs Duties 1 2014-04-01 2014-04-01 false Intermediate airport. 122.84 Section 122.84... Intermediate airport. (a) Application. The provisions of this section apply at any U.S. airport to which an... aircraft arrives at the next airport, the aircraft commander or agent shall make entry by filing the: (1...

19 CFR 122.84 - Intermediate airport.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 19 Customs Duties 1 2013-04-01 2013-04-01 false Intermediate airport. 122.84 Section 122.84... Intermediate airport. (a) Application. The provisions of this section apply at any U.S. airport to which an... aircraft arrives at the next airport, the aircraft commander or agent shall make entry by filing the: (1...

42 CFR 84.41 - Quality control plans; contents.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 1 2014-10-01 2014-10-01 false Quality control plans; contents. 84.41 Section 84... AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Quality Control § 84.41 Quality control plans; contents. (a) Each quality control plan shall contain provisions for the...

42 CFR 84.41 - Quality control plans; contents.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 1 2012-10-01 2012-10-01 false Quality control plans; contents. 84.41 Section 84... AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Quality Control § 84.41 Quality control plans; contents. (a) Each quality control plan shall contain provisions for the...

42 CFR 84.41 - Quality control plans; contents.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 1 2013-10-01 2013-10-01 false Quality control plans; contents. 84.41 Section 84... AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Quality Control § 84.41 Quality control plans; contents. (a) Each quality control plan shall contain provisions for the...

42 CFR 84.41 - Quality control plans; contents.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 1 2010-10-01 2010-10-01 false Quality control plans; contents. 84.41 Section 84... AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Quality Control § 84.41 Quality control plans; contents. (a) Each quality control plan shall contain provisions for the...

42 CFR 84.41 - Quality control plans; contents.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 1 2011-10-01 2011-10-01 false Quality control plans; contents. 84.41 Section 84... AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Quality Control § 84.41 Quality control plans; contents. (a) Each quality control plan shall contain provisions for the...

42 CFR 84.111 - Gas masks; required components.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 1 2013-10-01 2013-10-01 false Gas masks; required components. 84.111 Section 84.111 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Gas Masks § 84...

42 CFR 84.111 - Gas masks; required components.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 1 2012-10-01 2012-10-01 false Gas masks; required components. 84.111 Section 84.111 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Gas Masks § 84...

42 CFR 84.123 - Exhalation valve leakage test.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 1 2014-10-01 2014-10-01 false Exhalation valve leakage test. 84.123 Section 84.123 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Gas Masks § 84...

42 CFR 84.111 - Gas masks; required components.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 1 2011-10-01 2011-10-01 false Gas masks; required components. 84.111 Section 84.111 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Gas Masks § 84...

42 CFR 84.111 - Gas masks; required components.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 1 2010-10-01 2010-10-01 false Gas masks; required components. 84.111 Section 84.111 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Gas Masks § 84...

42 CFR 84.123 - Exhalation valve leakage test.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 1 2013-10-01 2013-10-01 false Exhalation valve leakage test. 84.123 Section 84.123 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Gas Masks § 84...

42 CFR 84.123 - Exhalation valve leakage test.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 1 2010-10-01 2010-10-01 false Exhalation valve leakage test. 84.123 Section 84.123 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Gas Masks § 84...

42 CFR 84.111 - Gas masks; required components.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 1 2014-10-01 2014-10-01 false Gas masks; required components. 84.111 Section 84.111 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Gas Masks § 84...

42 CFR 84.123 - Exhalation valve leakage test.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 1 2011-10-01 2011-10-01 false Exhalation valve leakage test. 84.123 Section 84.123 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Gas Masks § 84...

42 CFR 84.123 - Exhalation valve leakage test.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 1 2012-10-01 2012-10-01 false Exhalation valve leakage test. 84.123 Section 84.123 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES OCCUPATIONAL SAFETY AND HEALTH RESEARCH AND RELATED ACTIVITIES APPROVAL OF RESPIRATORY PROTECTIVE DEVICES Gas Masks § 84...

Agrobacterium tumefaciens-mediated transformation of the entomopathogenic fungus Nomuraea rileyi.

PubMed

Shao, Changwen; Yin, Youping; Qi, Zhaoran; Li, Ren; Song, Zhangyong; Li, Yan; Wang, Zhongkang

2015-10-01

An Agrobacterium-mediated genetic transformation system for the entomopathogenic fungus Nomuraea rileyi was established. Three binary T-DNA vectors, pPZP-Hph, pPZP-Hph-RNAi and pPZP-Hph-DsRed2, were constructed. The trpc promoter from Aspergillus nidulans was used as the cis-regulatory element to drive the expression of hygromycin phosphotransferase (hph) gene and DsRed2, which conferred the hygromycin B (Hyg B) resistance and red fluorescence visualization, respectively. The blastospores and conidia were used as the recipients. The blastospores' transformation efficiency reached ∼20-40 transformants per 10(6) blastospores, whereas the conidia were not transformed. Based on an analysis of five generations of subcultures, PCR and Southern blotting assays, the Ptrpc-hph cassette had integrated into the genomes of all transformants, which contained single copy of the hph gene and showed mitotic stability. Abundant altered morphologic phenotypes in colonies, blastospores and hyphae formations were observed in the arbitrary insertional mutants of N. rileyi, which made it possible to study the relationships between the functions and the interrupted genes over the whole genome. The transformation protocol will promote the functional characterization of genes, and the construction of genetically engineered strains of this important entomopathogenic fungus, and potentially of other similar fungal pathogens. Copyright © 2015 Elsevier Inc. All rights reserved.

40 CFR 86.602-84 - Definitions.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 19 2014-07-01 2014-07-01 false Definitions. 86.602-84 Section 86.602... New Light-Duty Vehicles, Light-Duty Trucks, and Heavy-Duty Vehicles § 86.602-84 Definitions. (a) The definitions in this section apply to this subpart. (b) As used in this subpart, all terms not defined herein...

40 CFR 86.602-84 - Definitions.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 20 2013-07-01 2013-07-01 false Definitions. 86.602-84 Section 86.602... Auditing of New Light-Duty Vehicles, Light-Duty Trucks, and Heavy-Duty Vehicles § 86.602-84 Definitions. (a) The definitions in this section apply to this subpart. (b) As used in this subpart, all terms not...

40 CFR 86.602-84 - Definitions.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 19 2010-07-01 2010-07-01 false Definitions. 86.602-84 Section 86.602... Auditing of New Light-Duty Vehicles, Light-Duty Trucks, and Heavy-Duty Vehicles § 86.602-84 Definitions. (a) The definitions in this section apply to this subpart. (b) As used in this subpart, all terms not...

40 CFR 86.602-84 - Definitions.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 20 2012-07-01 2012-07-01 false Definitions. 86.602-84 Section 86.602... Auditing of New Light-Duty Vehicles, Light-Duty Trucks, and Heavy-Duty Vehicles § 86.602-84 Definitions. (a) The definitions in this section apply to this subpart. (b) As used in this subpart, all terms not...

40 CFR 86.602-84 - Definitions.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 19 2011-07-01 2011-07-01 false Definitions. 86.602-84 Section 86.602... Auditing of New Light-Duty Vehicles, Light-Duty Trucks, and Heavy-Duty Vehicles § 86.602-84 Definitions. (a) The definitions in this section apply to this subpart. (b) As used in this subpart, all terms not...

45 CFR 84.54 - Education of institutionalized persons.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 45 Public Welfare 1 2011-10-01 2011-10-01 false Education of institutionalized persons. 84.54..., Welfare, and Social Services § 84.54 Education of institutionalized persons. A recipient to which this... its program or activity is provided an appropriate education, as defined in § 84.33(b). Nothing in...

45 CFR 84.54 - Education of institutionalized persons.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 45 Public Welfare 1 2014-10-01 2014-10-01 false Education of institutionalized persons. 84.54..., Welfare, and Social Services § 84.54 Education of institutionalized persons. A recipient to which this... its program or activity is provided an appropriate education, as defined in § 84.33(b). Nothing in...

45 CFR 84.54 - Education of institutionalized persons.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 45 Public Welfare 1 2012-10-01 2012-10-01 false Education of institutionalized persons. 84.54..., Welfare, and Social Services § 84.54 Education of institutionalized persons. A recipient to which this... its program or activity is provided an appropriate education, as defined in § 84.33(b). Nothing in...

45 CFR 84.54 - Education of institutionalized persons.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 45 Public Welfare 1 2013-10-01 2013-10-01 false Education of institutionalized persons. 84.54..., Welfare, and Social Services § 84.54 Education of institutionalized persons. A recipient to which this... its program or activity is provided an appropriate education, as defined in § 84.33(b). Nothing in...

45 CFR 84.51 - Application of this subpart.

Code of Federal Regulations, 2014 CFR

2014-10-01

... Social Services § 84.51 Application of this subpart. Subpart F applies to health, welfare, and other... 45 Public Welfare 1 2014-10-01 2014-10-01 false Application of this subpart. 84.51 Section 84.51 Public Welfare Department of Health and Human Services GENERAL ADMINISTRATION NONDISCRIMINATION ON THE...

45 CFR 84.51 - Application of this subpart.

Code of Federal Regulations, 2012 CFR

2012-10-01

... Social Services § 84.51 Application of this subpart. Subpart F applies to health, welfare, and other... 45 Public Welfare 1 2012-10-01 2012-10-01 false Application of this subpart. 84.51 Section 84.51 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION NONDISCRIMINATION ON THE...

45 CFR 84.51 - Application of this subpart.

Code of Federal Regulations, 2011 CFR

2011-10-01

... Social Services § 84.51 Application of this subpart. Subpart F applies to health, welfare, and other... 45 Public Welfare 1 2011-10-01 2011-10-01 false Application of this subpart. 84.51 Section 84.51 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION NONDISCRIMINATION ON THE...

45 CFR 84.51 - Application of this subpart.

Code of Federal Regulations, 2013 CFR

2013-10-01

... Social Services § 84.51 Application of this subpart. Subpart F applies to health, welfare, and other... 45 Public Welfare 1 2013-10-01 2013-10-01 false Application of this subpart. 84.51 Section 84.51 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION NONDISCRIMINATION ON THE...

45 CFR 84.51 - Application of this subpart.

Code of Federal Regulations, 2010 CFR

2010-10-01

... Social Services § 84.51 Application of this subpart. Subpart F applies to health, welfare, and other... 45 Public Welfare 1 2010-10-01 2010-10-01 false Application of this subpart. 84.51 Section 84.51 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION NONDISCRIMINATION ON THE...