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Kornelia Smalla - One of the best experts on this subject based on the ideXlab platform.
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transformation of acinetobacter sp strain bd413 pfg4δnptii with Transgenic Plant dna in soil microcosms and effects of kanamycin on selection of transformants
Applied and Environmental Microbiology, 2000Co-Authors: Kaare Magne Nielsen, J D Van Elsas, Kornelia SmallaAbstract:Bacterial antibiotic resistance markers are the most fre-quently inserted genes in Transgenic Plants. However, the re-sistance genes do not encode desirable traits in commerciallyused Plant varieties. Of the 15 different resistance genes incor-porated into Plants (17, 30), several encode resistance to clin-ically used antibiotics. Since Plant DNA has been shown topersist in soil over extended periods of time (8, 25, 38, 39),concerns that these transgenes may spread horizontally to bac-teria have been raised (17, 18, 22, 29). Sequence comparisonsof genes isolated from wild Plants and bacteria have indicatedthat horizontal gene transfer has occurred naturally betweenthem (13, 32). Moreover, whole-genome analyses of bacteriasuggest horizontal transfer of genetic material to be commonand a major force in bacterial evolution (14, 40).One mechanism of gene transfer that allows uptake of ge-netic material from diverged species in bacteria is naturaltransformation, which facilitates uptake of naked DNA incompetent bacteria (15). Based on this mechanism, severallaboratory studies have been conducted to elucidate the po-tential for Plant-harbored resistance determinants to be takenup by naturally occurring bacterial recipients (2, 3, 21, 28).These studies have, however, not been able to demonstrateuptake of such determinants, nor have studies of bacteria ob-tained from soil samples from field trials with Transgenic Plants(8, 25). Detection of horizontal transfer in these studies reliedupon the uptake of expressed and selectable genes in thebacterial recipients grown under optimized conditions or apositive DNA hybridization signal or PCR amplification ofPlant transgenes in the bacterial fraction of soil. However,direct analyses of DNA from soil samples often fail to dem-onstrate integration of Plant transgenes into bacterial ge-nomes. Transfer of smaller DNA fragments or nonexpressedor nonselected genes would rarely be detected in these studies.Recently, uptake of Transgenic Plant-harbored DNA frag-ments by bacteria based on restoration of a partially deleted(10- or 317-bp internal deletion) bacterial kanamycin (KM)resistance gene (
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monitoring field releases of genetically modified sugar beets for persistence of Transgenic Plant dna and horizontal gene transfer
FEMS Microbiology Ecology, 1999Co-Authors: Frank Gebhard, Kornelia SmallaAbstract:Field releases of Transgenic rizomania-resistant sugar beet (Beta vulgaris) Plants were accompanied by a study of the persistence of DNA from Transgenic sugar beet litter in soil and of horizontal gene transfer of Plant DNA to bacteria. The Transgenic sugar beets contained the marker genes nptII and bar under the control of the bidirectional TR1/2 promoter conferring kanamycin (Km) and glufosinate ammonium resistance to the Plant. Primer systems targeting the construct allowed the specific and sensitive detection of the Transgenic DNA in soil. Soil samples were analyzed by cultivation of bacteria on nonselective and Km-selective media to determine the proportion of Km-resistant bacteria and to monitor the culturable fraction for incorporation of Transgenic Plant DNA. To detect the presence of Transgenic DNA independently from cultivation, total soil DNA was extracted and amplified by PCR with three different primer sets specific for the Transgenic DNA. Long-term persistence of Transgenic DNA could be shown under field conditions (up to 2 years) and also in soil microcosms with introduced Transgenic Plant DNA. No construct-specific sequences were detected by dot blot hybridizations of bacterial isolates. The experimental limitations of detecting horizontal gene transfer from Plants to bacteria under field conditions are discussed.
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transformation of acinetobacter sp strain bd413 by Transgenic sugar beet dna
Applied and Environmental Microbiology, 1998Co-Authors: Frank Gebhard, Kornelia SmallaAbstract:The ability of Acinetobacter sp. strain BD413(pFG4ΔnptII) to take up and integrate Transgenic Plant DNA based on homologous recombination was studied under optimized laboratory conditions. Restoration of nptII, resulting in kanamycin-resistant transformants, was observed with plasmid DNA, Plant DNA, and homogenates carrying the gene nptII. Molecular analysis showed that some transformants not only restored the 317-bp deletion but also obtained additional DNA.
Frank Gebhard - One of the best experts on this subject based on the ideXlab platform.
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monitoring field releases of genetically modified sugar beets for persistence of Transgenic Plant dna and horizontal gene transfer
FEMS Microbiology Ecology, 1999Co-Authors: Frank Gebhard, Kornelia SmallaAbstract:Field releases of Transgenic rizomania-resistant sugar beet (Beta vulgaris) Plants were accompanied by a study of the persistence of DNA from Transgenic sugar beet litter in soil and of horizontal gene transfer of Plant DNA to bacteria. The Transgenic sugar beets contained the marker genes nptII and bar under the control of the bidirectional TR1/2 promoter conferring kanamycin (Km) and glufosinate ammonium resistance to the Plant. Primer systems targeting the construct allowed the specific and sensitive detection of the Transgenic DNA in soil. Soil samples were analyzed by cultivation of bacteria on nonselective and Km-selective media to determine the proportion of Km-resistant bacteria and to monitor the culturable fraction for incorporation of Transgenic Plant DNA. To detect the presence of Transgenic DNA independently from cultivation, total soil DNA was extracted and amplified by PCR with three different primer sets specific for the Transgenic DNA. Long-term persistence of Transgenic DNA could be shown under field conditions (up to 2 years) and also in soil microcosms with introduced Transgenic Plant DNA. No construct-specific sequences were detected by dot blot hybridizations of bacterial isolates. The experimental limitations of detecting horizontal gene transfer from Plants to bacteria under field conditions are discussed.
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transformation of acinetobacter sp strain bd413 by Transgenic sugar beet dna
Applied and Environmental Microbiology, 1998Co-Authors: Frank Gebhard, Kornelia SmallaAbstract:The ability of Acinetobacter sp. strain BD413(pFG4ΔnptII) to take up and integrate Transgenic Plant DNA based on homologous recombination was studied under optimized laboratory conditions. Restoration of nptII, resulting in kanamycin-resistant transformants, was observed with plasmid DNA, Plant DNA, and homogenates carrying the gene nptII. Molecular analysis showed that some transformants not only restored the 317-bp deletion but also obtained additional DNA.
Ramon J. Seidler - One of the best experts on this subject based on the ideXlab platform.
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Quantification of Transgenic Plant marker gene persistence in the field
Molecular Ecology, 2003Co-Authors: Franco Widmer, Katherine K. Donegan, Ramon J. Seidler, Gary ReedAbstract:Methods were developed to monitor persistence of genomic DNA in decaying Plants in the field. As a model, we used recombinant neomycin phosphotransferase II (rNPT-II) marker genes present in genetically engineered Plants. Polymerase chain reaction (PCR) primers were designed, complementary to 20-bp sequences of the nopaline synthase promoter in a Transgenic tobacco and the cauliflower mosaic virus 35S promoter in a Transgenic potato. The PCR reverse primer was complementary to a 20-bp sequence of the N-terminal NPT-II coding region. The PCR protocol allowed for quantification of as few as 10 rNPT-II genes per reaction. We analysed rNPT-II marker gene amounts in samples obtained from two field experiments performed at different locations in Oregon. In Transgenic tobacco leaves, buried at 10 cm depth in a field plot in Corvallis, marker DNA amount dropped to 0.36% during the first 14 days and was detectable for 77 days at a final level of 0.06% of the initial amount. Monitoring of residual potato Plant litter, from the soil surface of a test field in Hermiston, was performed for 137 days. After 84 days marker gene amounts dropped to 2.74% (leaf and stem) and 0.50% (tuber) of the initially detected amount. At the final sample date 1.98% (leaf and stem) and 0.19% (tuber) were detectable. These results represent the first quantitative analysis of Plant DNA stability under field conditions and indicate that a proportion of the Plant genomic DNA may persist in the field for several months.
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decomposition of genetically engineered tobacco under field conditions persistence of the proteinase inhibitor i product and effects on soil microbial respiration and protozoa nematode and microarthropod populations
Journal of Applied Ecology, 1997Co-Authors: Katherine K. Donegan, Ramon J. Seidler, V J Fieland, D L Schaller, C J Palm, L M Ganio, D M Cardwell, Yosef SteinbergerAbstract:To evaluate the potential effects of genetically engineered (Transgenic) Plants on soil ecosystems, litterbags containing leaves of non-engineered (parental) and Transgenic tobacco Plants were buried in field plots. The Transgenic tobacco Plants were genetically engineered to constitutively produce proteinase inhibitor I, a protein with insecticidal activity. The litterbag contents and surrounding soil, as well as soil from control plots without litterbags, were sampled over a 5-month period at 2- to 4-week intervals and assayed for proteinase inhibitor concentration, litter decomposition rates, carbon and nitrogen content, microbial respiration rates and population levels of nematodes, protozoa and microarthropods. The proteinase inhibitor concentration in the Transgenic Plant litter after 57 days was 0.05% of the sample day 0-value and was not detectable on subsequent sample days. Although the carbon content of the Transgenic Plant litter was comparable to that of the parental Plant litter on sample day 0, it became significantly lower over the course of the experiment. Nematode populations in the soil surrounding the Transgenic Plant litterbags were greater than those in the soil surrounding parental Plant litterbags and had a different trophic group composition, including a significantly higher ratio of fungal feeding nematodes to bacterial feeding nematodes on sample day 57. In contrast, Collembola populations in the soil surrounding the Transgenic Plant litterbags were significantly lower than in the soil surrounding parental Plant litterbags. Our results demonstrated that under field conditions proteinase inhibitor remained immunologically active in buried Transgenic Plant litter for at least 57 days and that decomposing parental and Transgenic Plant litter differed in quality (carbon content) and in the response of exposed soil organisms (Collembola and nematodes).
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sensitive detection of Transgenic Plant marker gene persistence in soil microcosms
Molecular Ecology, 1996Co-Authors: Franco Widmer, Ramon J. Seidler, Lidia S WatrudAbstract:Genetic engineering offers the opportunity to generate Plants with useful new traits conferred by genes originating from a variety of organisms. The objectives of this study were to establish methods for investigating persistence of recombinant Plant marker DNA after introduction into soil and to collect data from controlled laboratory test systems. As a model system, we studied the stability of DNA encoding recombinant neomycin phosphotransferase II (rNPT-II), a neomycin/kanamycin resistance marker, used in Plant genetic engineering. The recombinant nature of the target (i.e. fusion of nopaline synthase promoter and NPT-II coding region) allowed us to design a rNPT-II-specific PCR primer pair. DNA preparation and quantitative PCR protocols were established. Effects of temperature and moisture, on DNA persistence in soil were determined in two laboratory test systems. In the first system, purified plasmid DNA was added to soil and incubated under controlled conditions. Up to 0.08% of the rNPT-II target sequences were detectable after 40 days. In the second system, fresh leaf tissue of Transgenic tobacco was ground, added to soil, and incubated under controlled conditions. After 120 days, up to 0.14% of leaf tissue-derived genomic rNPT-II sequences were detectable. Under most experimental conditions, leaf tissue-derived and plasmid DNA were initially degraded at a high rate. A small proportion of the added DNA resisted degradation and was detectable for several months. We hypothesize that this DNA may have been adsorbed to soil particles and was protected from complete degradation.
Kokichi Hinata - One of the best experts on this subject based on the ideXlab platform.
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Transgenic Plant production from leaf discs of Moricandia arvensis using Agrobacterium tumefaciens
Plant Cell Reports, 1996Co-Authors: Hamid Rashid, Kinya Toriyama, Kokichi HinataAbstract:A high frequency shoot regeneration (80%) was developed from callus of leaf discs and stem internodes of Moricandia arvensis. Leaf discs were shown to be a preferable starting material for transformation experiments. Agrobacterium tumefaciens strain GV3101/pMP90 used in this study contained a binary vector with genes for kanamycin resistance, hygromycin resistance and β-glucuronidase (GUS). Maximum transformation efficiency (10.3%) was achieved by using kanamycin at the rate of 200 mg/l as a selection agent. Presence of tobacco suspension culture during co-cultivation and a pre-selection period of seven days after co-cultivation was essential for successful transformation. Transgenic Plants grew to maturity and exhibited flowering in a glasshouse. GUS activity was evident in all parts of leaf and the presence of GUS gene in Plant gemone was confirmed by PCR analysis.
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Transgenic Plant production mediated by agrobacterium in indica rice
Plant Cell Reports, 1996Co-Authors: Hamid Rashid, Kinya Toriyama, Shuuji Yokoi, Kokichi HinataAbstract:A reproducible system has been developed for the production of Transgenic Plants in indica rice using Agrobacterium-mediated gene transfer. Three-week-old scutella calli served as an excellent starting material. These were infected with an Agrobacterium tumefaciens strain EHA101 carrying a plasmid pIG121Hm containing genes for β-glucuronidase (GUS) and hygromycin resistnace (HygR). Hygromycin (50 mg/l) was used as a selectable agent. Inclusion of acetosyringone (50μM) in the Agrobacterium suspension and co-culture media proved to be indispensable for successful transformation. Transformation efficiency of Basmati 370 was 22% which was as high as reported in japonica rice and dicots. A large number of morphologically normal, fertile Transgenic Plants were obtained. Integration of foreign genes into the genome of Transgenic Plants was confirmed by Southern blot analysis. GUS and HygR genes were inherited and expressed in R1 progeny. Mendelian segregation was observed in some R1 progeny.
Kaare Magne Nielsen - One of the best experts on this subject based on the ideXlab platform.
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transformation of acinetobacter sp strain bd413 pfg4δnptii with Transgenic Plant dna in soil microcosms and effects of kanamycin on selection of transformants
Applied and Environmental Microbiology, 2000Co-Authors: Kaare Magne Nielsen, J D Van Elsas, Kornelia SmallaAbstract:Bacterial antibiotic resistance markers are the most fre-quently inserted genes in Transgenic Plants. However, the re-sistance genes do not encode desirable traits in commerciallyused Plant varieties. Of the 15 different resistance genes incor-porated into Plants (17, 30), several encode resistance to clin-ically used antibiotics. Since Plant DNA has been shown topersist in soil over extended periods of time (8, 25, 38, 39),concerns that these transgenes may spread horizontally to bac-teria have been raised (17, 18, 22, 29). Sequence comparisonsof genes isolated from wild Plants and bacteria have indicatedthat horizontal gene transfer has occurred naturally betweenthem (13, 32). Moreover, whole-genome analyses of bacteriasuggest horizontal transfer of genetic material to be commonand a major force in bacterial evolution (14, 40).One mechanism of gene transfer that allows uptake of ge-netic material from diverged species in bacteria is naturaltransformation, which facilitates uptake of naked DNA incompetent bacteria (15). Based on this mechanism, severallaboratory studies have been conducted to elucidate the po-tential for Plant-harbored resistance determinants to be takenup by naturally occurring bacterial recipients (2, 3, 21, 28).These studies have, however, not been able to demonstrateuptake of such determinants, nor have studies of bacteria ob-tained from soil samples from field trials with Transgenic Plants(8, 25). Detection of horizontal transfer in these studies reliedupon the uptake of expressed and selectable genes in thebacterial recipients grown under optimized conditions or apositive DNA hybridization signal or PCR amplification ofPlant transgenes in the bacterial fraction of soil. However,direct analyses of DNA from soil samples often fail to dem-onstrate integration of Plant transgenes into bacterial ge-nomes. Transfer of smaller DNA fragments or nonexpressedor nonselected genes would rarely be detected in these studies.Recently, uptake of Transgenic Plant-harbored DNA frag-ments by bacteria based on restoration of a partially deleted(10- or 317-bp internal deletion) bacterial kanamycin (KM)resistance gene (
