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  • Articles: DFG German National Licenses  (94)
  • 1995-1999  (86)
  • 1955-1959  (8)
  • 1920-1924
  • Agrobacterium
  • protoplasts
  • 1
    Electronic Resource
    Electronic Resource
    A Modular Vector for Agrobacterium Mediated Transformation of Wheat (1999)
    Springer
    Plant molecular biology reporter 17 (1999), S. 323-331 
    Details
    ISSN: 1572-9818
    Keywords: Agrobacterium ; modular vector ; transformation ; wheat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Wheat (cv Chinese Spring) tissues were transformed using Agrobacterium tumefasciens and a new plasmid modular vector, pMVTBP. We constructed pMVTBP with unique restriction sites connecting (1) the CaMV 35S promoter, (2) a Kozak sequence, (3) the FLAG epitope, (4) the (His)6 epitope, (5) a coding region (for wheat TATA Binding Protein, wTBP) and (6) the CaMV 35S 3′UTR. This vector thus allows easy exchange of different regulatory or coding sequences. Explants of either germinating mature seeds, or immature embryos, were induced to callus for up to two weeks, treated with virulence-induced bacteria for one hour, then regenerated into plantlets. Transient expression of a GUS reporter gene, assayed at about one week, occurred in 10–12% of calluses. Expression of the FLAG-tagged wTBP was also detected, by immunostaining. Stable expression, by selective growth on geneticin, and by GUS expression at about six weeks, occurred in 1–2% of calluses, quite comparable to that achieved by other methods.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1007686408369
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    Paper (German National Licenses)
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  • 2
    Electronic Resource
    Electronic Resource
    Transgenic almond (Prunus dulcis Mill.) plants obtained by Agrobacterium-mediated transformation of leaf explants (1999)
    Springer
    Plant cell reports 18 (1999), S. 387-393 
    Details
    ISSN: 1432-203X
    Keywords: Key words Almond ; Prunus ; Transformation ; Agrobacterium ; Adventitious regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Almond (Prunus dulcis Mill.) leaves were transformed with the marker genes gusA (β-glucuronidase) and nptII (neomycin phosphotransferase II) via Agrobacterium-mediated transformation. Bacterial strains and preculture of explants affected efficiency of gene transfer evaluated by transient expression assays. Following transformation, shoots were induced from primary explants on medium without kanamycin and exposed to selection 20 days after cocultivation. From 1419 original leaves, four shoots (A, B, C and D) were obtained that showed amplification of the predicted DNA fragments by polymerase chain reaction (PCR). After micropropagation of these shoots, only those cloned from shoot D gave consistently positive results in histochemical GUS detection and PCR amplification. Southern blot hybridisation confirmed stable transgene integration in clone D, which was also negative in PCR amplification of an Agrobacterium gene. Additional molecular analysis suggested that the remaining three shoots (A, B and C) were chimeric.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002990050591
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  • 3
    Electronic Resource
    Electronic Resource
    Green-fluorescent protein facilitates rapid in vivo detection of genetically transformed plant cells (1999)
    Springer
    Plant cell reports 18 (1999), S. 707-714 
    Details
    ISSN: 1432-203X
    Keywords: Key words Green-fluorescent protein ; Transformation ; Particle bombardment ; Agrobacterium ; Sugarcane
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Early detection of plant transformation events is necessary for the rapid establishment and optimization of plant transformation protocols. We have assessed modified versions of the green fluorescent protein (GFP) from Aequorea victoria as early reporters of plant transformation using a dissecting fluorescence microscope with appropriate filters. Gfp-expressing cells from four different plant species (sugarcane, maize, lettuce, and tobacco) were readily distinguished, following either Agrobacterium-mediated or particle bombardment-mediated transformation. The identification of gfp-expressing sugarcane cells allowed for the elimination of a high proportion of non-expressing explants and also enabled visual selection of dividing transgenic cells, an early step in the generation of transgenic organisms. The recovery of transgenic cell clusters was streamlined by the ability to visualize gfp-expressing tissues in vitro.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002990050647
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  • 4
    Electronic Resource
    Electronic Resource
    Stable genetic transformation of white pine (Pinus strobus L.) after cocultivation of embryogenic tissues with Agrobacterium tumefaciens (1999)
    Springer
    Molecular breeding 5 (1999), S. 429-440 
    Details
    ISSN: 1572-9788
    Keywords: transgenic trees ; scaffold attachment region ; Agrobacterium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract A genetic transformation procedure for white pine has been developed after cocultivation of embryogenic tissues with Agrobacterium tumefaciens. This efficient transformation procedure led to an average of four independent transformed lines per gram of cocultivated embryogenic tissue and up to 50 transformed lines can be obtained in a routine experiment. Constructs bearing the uidA gene or the green fluorescent protein (GFP) gene were introduced and β-glucuronidase (GUS) activity was followed over time. The expression of the uidA gene was lowest with a 35S-gus-intron construct and was 20-fold higher with a 35S-35S-AMVgus::nptII construct. The addition of scaffold attachment region (SAR) sequences surrounding the gus::nptII fusion did not significantly enhance the GUS activity. Transformed mature somatic embryos have been germinated and plantlets are presently being acclimatized.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1009683605841
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  • 5
    Electronic Resource
    Electronic Resource
    Analysis of ribosomal RNA genes in somatic hybrids between wild and cultivated Solanum species (1999)
    Springer
    Molecular breeding 5 (1999), S. 11-20 
    Details
    ISSN: 1572-9788
    Keywords: plant breeding ; protoplasts ; somatic fusion ; ribosomal RNA genes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Ribosomal RNA genes were exploited as markers to identify somatic hybrids between Solanum tuberosum cv. Brodick and wild diploid Solanum species, S. megistacrolobum, S. sanctae-rosae and S. sparsipilum and DNA methylation as a possible regulatory factor in gene expression was investigated. Specific restriction enzyme/probe combinations revealed useful polymorphisms in the conserved coding and variable intergenic spacer regions of the ribosomal RNA genes. Some intermediate ribosomal RNA gene profiles indicate hybridity whereas others were characteristic of S. tuberosum cv. Brodick. This evidence is suggestive of somatic exchange/re-arrangement between the NOR region of S. sanctae-rosae and S. tuberosum cv. Brodick. Ribosomal RNA gene copy number analysis of the somatic hybrids did not reveal hexaploid values suggesting that these products are not symmetric hybrids derived from the parental diploid and tetraploid plants. The results indicate site-specific methylation of ribosomal RNA gene sequences for the parental plants; while some somatic hybrids display a reduction, others show an increase. The significance of the findings for somatic cell genetics and plant breeding studies is discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1009618311294
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  • 6
    Electronic Resource
    Electronic Resource
    Production and testing of plants regenerated from protoplasts of photoperiod sensitive genic male sterile rice (Oryza sativa L.) (1999)
    Springer
    Euphytica 105 (1999), S. 167-172 
    Details
    ISSN: 1573-5060
    Keywords: rice ; Oryza sativa L. ; photoperiod sensitive genic male sterility (PGMS) ; protoplasts ; flow cytometry ; tetraploid
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Plants were regenerated from protoplasts isolated from embryonic suspension cultures of N5047S, a photoperiod sensitive genic male sterile (PGMS) Japonica rice line. Flow cytometric analyses of nuclear DNA content identified some tetraploid regenerates whose agronomic traits could be distinguished from diploid regenerates. Pollen and female fertility of diploid protoplast-derived clones grown under different light and temperature conditions was compared. A promising PGMS protoplast clone, ZAU11S, was developed from these clones. Its male sterility was confirmed as a photoperiod × temperature interaction type.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1003442100236
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  • 7
    Electronic Resource
    Electronic Resource
    Positive-negative selection and T-DNA stability in Arabidopsis transformation (1999)
    Springer
    Plant molecular biology 39 (1999), S. 83-93 
    Details
    ISSN: 1573-5028
    Keywords: Arabidopsis ; Agrobacterium ; T-DNA ; CodA ; positive-negative selection ; recombination
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have analysed the application of positive-negative selection for the selection of homologous recombination interactions between the chromosome and a T-DNA molecule after transformation of plant cells. Two different genomic loci in a cell suspension of Arabidopsis thaliana were chosen to study gene targeting events. One was the chalcone synthase (CHS) gene present as a single copy and the second an hemizygous chromosomally inserted T-DNA containing the hpt gene, conferring resistance to hygromycin, flanked by CHS sequences. The target lines were transformed with replacement-type T-DNA vectors which contained a positive selectable marker flanked by the regions of the CHS gene and a negative selectable marker to counter-select random insertions. As negative marker we used the Escherichia coli codA gene encoding cytosine deaminase, conferring upon the cells sensitivity to 5-flourocytosine (5-FC). Doubly selected transformants represent 1–4% of the primary transformed cells. Targeting events were not found at the chalcone synthase locus nor at the artificial hpt locus in a total of 4379 doubly selected calli, corresponding to at least 109 475 individual primary transformants. We show by PCR and Southern analysis that the 5-FC resistance in the majority of these cells is associated with substantial deletions of the T-DNA molecule from the right-border end.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1006192225464
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  • 8
    Electronic Resource
    Electronic Resource
    Characterization of a pine multigene family containing elicitor- responsive stilbene synthase genes (1999)
    Springer
    Plant molecular biology 39 (1999), S. 221-229 
    Details
    ISSN: 1573-5028
    Keywords: heartwood constituent ; phytoalexin ; pinosylvin ; Pinus sylvestris ; protoplasts ; stilbene synthase ; transient expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Young pine seedlings respond to environmental stress by induced synthesis of pinosylvin, a stilbene phytoalexin. Heartwood of pine trees is characterized by a high content of pinosylvin. The formation of pinosylvin from cinnamoyl-CoA and three molecules malonyl-CoA catalysed by pinosylvin synthase is typical of the genus Pinus. Its enzyme activity not detectable in unstressed seedlings is substantially increased upon application of stimuli like UV-light or infection with the phytopathogenic fungus Botrytis cinerea. A genomic DNA library was screened with pinosylvin synthase cDNA pSP-54 as a probe. Ten clones were isolated and grouped into five subclasses according to the size of their introns. After subcloning into plasmid T7T3, four different members of the five gene subclasses were characterized by sequencing. Emphasis was put on isolating various promoters and analyzing and comparing their responsiveness. The amino acid sequences deduced from genes PST-1, PST-2, PST-3 and PST-5 shared an overall identity of more than 95%. In gene PST-5, the putative translation start site ATG was replaced by CTG. While promoter regions near the TATAA box were almost identical PST-1, PST-2 and PST-3, further upstream sequences differed substantially. Differences in promoter strength were analysed both in transgenic tobacco plants and by transient expression in tobacco protoplasts. Constructs used contained the bacterial β-glucuronidase under the control of the promoters of pine genes PST-1, PST-2 and PST-3. Upon treatment with UV light or fungal elicitor, the promoter of PST-1 showed highest responsiveness and led to tissue-specific expression in vascular bundles. The data suggest that in pine the gene product of PST-1 is responsible for both the stress response in seedlings and pinosylvin formation in the heartwood.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1006163030646
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  • 9
    Electronic Resource
    Electronic Resource
    Plant regeneration from protoplasts derived from cell suspension of adventive somatic embryos in sugarcane (Saccharum spp. hybrid cv. CoL-54 and cv. CP-43/33) (1999)
    Springer
    Plant cell, tissue and organ culture 56 (1999), S. 155-162 
    Details
    ISSN: 1573-5044
    Keywords: adventive somatic embryo ; cell culture ; protoplasts ; regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Adventive somatic embryos were initiated from the cut edges of juvenile leaf explants of two cultivars of sugarcane (Saccharum spp. hybrid cv. CoL-54 and cv. CP-43/33). This response was achieved using MS medium containing 9 μmol (2 mg l-1) 2,4-D and 500 mg l-1 CH under either continuous or 16-h photoperiod. Regeneration from somatic embryos was achieved under either continuous or 16-h photoperiod on MS basal medium in 5–6 weeks. Using adventive somatic embryos of 20–25 days of age as an explant source, homogeneous cell suspension cultures were initiated in both AA and MS media supplemented with 2 mg l-1 2,4-D and 500 mg l-1 CH. Protoplasts were isolated from homogeneous cell suspension cultures, an average yield being 2.5×107 ml-1 for both the cultivars. The best division efficiency (1.5 and 0.80%) and microcalluses for cv. CoL-54 and cv. CP-43/33, respectively were achieved using modified KPR medium under dark conditions in 6–8 weeks. Microcalluses were proliferated and plant regeneration was achieved from protocalluses.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1006296320725
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  • 10
    Electronic Resource
    Electronic Resource
    Sequence and functional analysis of the left-hand part of the T-region from the nopaline-type Ti plasmid, pTiC58 (1999)
    Springer
    Plant molecular biology 41 (1999), S. 765-776 
    Details
    ISSN: 1573-5028
    Keywords: Agrobacterium ; Ti plasmid ; oncogenes ; e gene ; 3′ gene
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The Agrobacterium tumefaciens nopaline strain C58 transfers a large, 29 kb T-DNA into plant cells during infection. Part of this DNA (the `common DNA') is also found on the T-DNA of octopine strains, the remaining DNA is nopaline strain-specific. Up to now, only parts of the C58 T-DNA and related T37 T-DNA have been sequenced. We have sequenced the remainder of the nopaline-specific T-DNA (containing genes a to d) and acs to iaaM. Gene c codes for a new unknown T-DNA protein. Gene a is homologous to the agrocinopine synthase gene. Genes b, c′, d and e are part of a larger family: they are related to the T-DNA genes 5, rolB, lso and 3′. Genes 5, rolB and lso induce or modify plant growth and have been called T-DNA oncogenes. Our studies show that gene 3′ (located on the TR-DNA of octopine strains) is also oncogenic. Although the b–e T-DNA fragment from C58 and its individual genes lack growth-inducing activity, an a-acs deletion mutant was distinctly less virulent on Kalanchoe daigremontiana and showed reduced shoot formation on Kalanchoe tubiflora. Shoot formation could be restored by genes c and c′ in co-infection experiments. Contrary to an earlier report, a C58 e gene deletion mutant was fully virulent on all plants tested.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1006370207379
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  • 11
    Electronic Resource
    Electronic Resource
    Evidence of Migration and Endophytic Presence of Agrobacterium tumefaciens in Rose Plants (1999)
    Springer
    European journal of plant pathology 105 (1999), S. 39-50 
    Details
    ISSN: 1573-8469
    Keywords: Agrobacterium ; crown gall ; systemic infection ; rose ; endophyte
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Agrobacterium tumefaciens was isolated from stem tumors of several rose cultivars showing that the bacterium is the causal agent of aerial galls in rose plants. No differences were observed in the characteristics of the Agrobacterium isolates from crown or aerial galls. Stem inoculation of ten rose cultivars showed that all of them were susceptible to A. tumefaciens but differences in the size of the resulting tumors were observed. The movement of A. tumefaciens in rose plants was demonstrated using two wild type strains and two antibiotic resistant mutants. Three months after inoculation, the inoculated strains were recovered in the roots, crown and below and above the inoculation site but low numbers of pathogenic Agrobacterium cells were isolated. New tumors appeared in 5% of the noninoculated wounds. A. tumefaciens was isolated from the stem at different distances from the tumor in naturally infected plants. In symptomless commercial plants, the isolation from the roots, crown and at different stem levels demonstrated the existence of systemic and latent infections in rose. Direct isolation using a nonselective and selective media with or without a previous enrichment step were efficient methods for isolating tumorigenic Agrobacterium from the different parts of rose plants.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008660500107
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  • 12
    Electronic Resource
    Electronic Resource
    Origin and evolution of plasmids (1998)
    Springer
    Antonie van Leeuwenhoek 73 (1998), S. 117-126 
    Details
    ISSN: 1572-9699
    Keywords: riboplasmids ; encapsidation ; pseudovirions ; selfish plasmids ; replicons ; ribozyme ; Agrobacterium ; Rhizobium ; grapevines ; L-tartrate ; lignin ; methoxyphenols ; satellite viruses ; opines ; crown gall ; T-DNA ; origin of replication
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Studies on the origin and evolution of plasmids may provide valuable insights on the promiscuous nature of DNA. The first examples of the selfish nature of nucleic acids are exemplified by primordial oligoribonucleotides which evolved into primitive replicons. The propagation of these molecules were likely patterned after the current viral RNA ribozymes, which have been recently shown to possess RNA synthesizing and template mediated polymerizing capabilities in the absence of proteins. The parasitic nature of nucleic acids is depicted by satellite nucleic acid molecules associated with viruses. The satellites of adenovirus and tobacco ringspot virus serve as established examples: they contain no open reading frames. Comparative analysis of the replication origins of virions and plasmids show them to be conserved, originating from the simplest autocatalytic replicon to highly complex and evolved plasmids, replicating by a rolling circle mechanism. The eventual association of proteins with nucleic acids provided added efficiency and protective advantages for molecular perpetuation. The promiscuous and selfish nature of plasmids is demonstrated by their ability to genetically engineer their host so that the host cell is best able to cope and survive in hostile environments. Survival of the host ensures survival of the plasmid. Sequestering of genes by plasmids occurs when the environmental conditions negatively affect the host. The sequestering mechanism is fundamental and forms the outreach mechanisms to generate and propagate macromolecules of increasing size when necessary for survival. The level of sophistication of plasmids increases with the addition of new genes such as those that allow the host to occupy a specific environment normally inhospitable to the host cell. The vast range of plasmid types which have obtained genes interchangeably reflect the levels of sophistication achieved by these macromolecules. The Ti plasmid in Agrobacterium tumefaciens and the pSym and accessory plasmids in Rhizobium illustrate the level of complexity attained by replicons.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1000652513822
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  • 13
    Electronic Resource
    Electronic Resource
    Transformation and regeneration of Lobelia erinus using Agrobacterium tumefaciens (1998)
    Springer
    Plant cell reports 18 (1998), S. 308-311 
    Details
    ISSN: 1432-203X
    Keywords: Key words Campanulaceae ; Lobelia erinus ; Transformation ; Agrobacterium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A transformation/regeneration system was developed for the common garden Lobelia (Lobelia erinus). Using an Agrobacterium-based protocol, over 40 transformants have been generated with four different binary vectors. The explant source was hypocotyl-root sections from axenically grown seedlings. Stable transformation was demonstrated by Southern hybridization analysis, β-glucuronidase staining, and transmission of the T-DNA to progeny. This extends the ever-widening range of transformable plant species to the Campanulaceae and will allow molecular studies of development and physiology in this easily cultured and popular garden plant.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002990050577
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  • 14
    Electronic Resource
    Electronic Resource
    Factors affecting soybean cotyledonary node transformation (1998)
    Springer
    Plant cell reports 18 (1998), S. 180-186 
    Details
    ISSN: 1432-203X
    Keywords: Key words Soybean ; SAAT (sonication assisted Agrobacterium-mediated transformation) ; Agrobacterium ; Transformation ; KYRT1
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cotyledonary node transformation efficiency was evaluated using a sonication assisted Agrobacterium-mediated transformation (SAAT) protocol, three dissimilar A. tumefaciens strains, and explants derived from 28 diverse cultivars and/or genotypes of soybean [Glycine max (L.) Merr.]. The explants were evaluated at 10 and 45 days after co-cultivation for transformation with a binary vector containing both a GUS-intron gene and an NPTII selectable marker. The best overall strain of A. tumefaciens was determined to be KYRT1 based on stable GUS transformation of soybean cotyledonary node explants measured at the terminal 45 day evaluation point. SAAT did not increase stable transformation at 45 days post co-cultivation. SAAT was determined to significantly decrease shoot proliferation of some genotypes, but it is unclear what effect this may have on the recovery of transformed shoots. Significant differences were also detected between genotypes for transformation and shoot proliferation frequency.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002990050553
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  • 15
    Electronic Resource
    Electronic Resource
    Transgenic peppermint (Mentha×piperita L.) plants obtained by cocultivation with Agrobacterium tumefaciens (1998)
    Springer
    Plant cell reports 17 (1998), S. 165-171 
    Details
    ISSN: 1432-203X
    Keywords: Key words Peppermint ; Transformation ; Agrobacterium ; GUS ; Transgenic
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The first transgenic peppermint (Mentha×piperita L. cultivar Black Mitcham) plants have been obtained by Agrobacterium-mediated transformation by cocultivation with morphogenically responsive leaf explants. Basal leaf explants with petioles, from leaves closest to the apex of in-vitro-culture-maintained shoots (5 cm), exhibited optimal shoot organogenetic responsiveness on medium supplemented with thidiazuron (8.4 µm). Shoot formation occurred at sites of excision on the leaf blade and petiole either directly from cells of the explant or via a primary callus. Analyses of transient GUS activity data indicated that DNA delivery by microprojectile bombardment was more effective than Agrobacterium infection. However, no transgenic plants were obtained from over 22,000 leaf explants after particle bombardment. Cocultivation of leaf explants with Agrobacterium strain EHA 105 and kanamycin selection produced transgenic plants. Greater transient and stable -glucuronidase (GUS) activities were detected in explants or propagules transformed with the construct where gusA was driven by the pBISN1 promoter rather than a CaMV 35S promoter. Eight plants were subsequently regenerated and verified as transgenic based on detection of the nptII transgene by PCR and Southern blot analyses. The Southern analyses indicated that the plants were derived from eight unique transformation events. All transgenic plants appeared morphologically normal. Analyses of GUS activities in leaves sampled from different portions of these transgenic plants, 10 months after transfer to the greenhouse, indicated that six out of the eight original regenerants were uniformly transformed, i.e., did not exhibit chimeric sectors.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002990050372
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  • 16
    Electronic Resource
    Electronic Resource
    Agrobacterium tumefaciens-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants (1998)
    Springer
    Plant cell reports 17 (1998), S. 675-680 
    Details
    ISSN: 1432-203X
    Keywords: Key words Genetic transformation ; Agrobacterium ; Eucalyptus ; Regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract An efficient system for Agrobacterium-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants was developed. Transformation was accomplished by cocultivation of hypocotyl segments with Agrobacterium tumefaciens containing a binary Ti-plasmid vector harboring chimeric neomycin phosphotransferase and β-glucuronidase (GUS) genes. A modified Gamborg's B5 medium used in this study was effective for both callus induction and regeneration of transgenic shoots. This medium could also effectively maintain the organogenic capability of callus for more than a year. Culturing transgenic shoots in Murashige and Skoog medium supplemented with 0.1 mg ⋅ l–1 benzylaminopurine prior to root induction in rooting medium markedly increased the rootability of shoots that were recalcitrant to rooting. Histochemical assay revealed the expression of the GUS gene in leaf, stem, and root tissues of transgenic plants. Insertion of the GUS gene in the nuclear genome of transgenic plants was verified by genomic Southern hybridization analysis, further confirming the integration and expression of T-DNA in these plants.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002990050464
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  • 17
    Electronic Resource
    Electronic Resource
    Activity of the CaMV 35S promoter in various parts of transgenic early flowering birch clones (1998)
    Springer
    Plant cell reports 18 (1998), S. 243-248 
    Details
    ISSN: 1432-203X
    Keywords: Key wordsBetula pendula ; Transformation ; Agrobacterium ; Gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Early flowering together with small size would be useful for various biotechnical or genetic studies on trees. We report here the selection and micropropagation of early flowering birch (Betula pendula) clones (BPM1–12) obtained from seeds of birches bred elsewhere for early flowering. Under conditions that accelerate flowering (a high CO2 level, strong and continuous illumination), the first male inflorescences emerged in 3–5 months, the trees then being 20–80 cm high. Transgenic lines (CaMV 35S-GUS INT) were produced through Agrobacterium-mediated gene transfer from BPM2, BPM5 and JR1/4 (a normally flowering birch). β-Glucuronidase (GUS) activities in the different lines, assayed 1–1.5 years after transformation, varied greatly. During further in vitro culture for 10 months, the activities decreased to 0.3–7% of the original values. GUS activities were detected in all organs studied, including the developing male inflorescences; the highest activity was in the roots.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002990050564
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  • 18
    Electronic Resource
    Electronic Resource
    Transformation of arctic bramble (Rubus arcticus L.) by Agrobacterium tumefaciens (1998)
    Springer
    Plant cell reports 17 (1998), S. 822-826 
    Details
    ISSN: 1432-203X
    Keywords: Key words Rosaceae ; β-Glucuronidase ; Regeneration ; Agrobacterium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Genetic transformation of arctic bramble (Rubus arcticus L.) was achieved utilizing a Ti-plasmid vector system of Agrobacterium tumefaciens. Internodal stem segments were inoculated with Agrobacterium strain EHA101 carrying a T-DNA with the CaMV 35 S promoter-gus-int marker gene from which β-glucuronidase (GUS) is expressed only in plants. Regenerants were produced on Murashige and Skoog medium. Growth of Agrobacterium was inhibited with cefotaxime. Kanamycin was used as the selective agent for the transformants. Regenerants were assayed by histochemical GUS staining, and by Southern analysis using a gus-int probe. Transgenic arctic bramble plants containing gus-int and expressing GUS were recovered. Expression has been stable for 3 years in micropropagation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002990050491
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  • 19
    Electronic Resource
    Electronic Resource
    CaMV 35S promoter directs β-glucuronidase expression in the laticiferous system of transgenic Hevea brasiliensis (rubber tree) (1998)
    Springer
    Plant cell reports 17 (1998), S. 621-625 
    Details
    ISSN: 1432-203X
    Keywords: Key wordsHevea brasiliensis ; Agrobacterium ; CaMV 35S ; β-Glucuronidase ; Latex
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Hevea brasiliensis anther calli were genetically transformed using Agrobacterium GV2260 (p35SGUSINT) that harboured the β-glucuronidase (gus) and neomycin phosphotransferase (nptII) genes. β-Glucuronidase protein (GUS) was expressed in the leaves of kanamycin-resistant plants that were regnerated, and the presence of the gene was confirmed by Southern analysis. GUS was also observed to be expressed in the latex and more importantly in the serum fraction. Transverse sections of the leaf petiole from a transformed plant revealed GUS expression to be especially enhanced in the phloem and laticifers. GUS expression was subsequently detected in every one of 194 plants representing three successive vegetative cycles propagated from the original transformant. Transgenic Hevea could thus facilitate the continual production of foreign proteins expressed in the latex.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002990050454
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  • 20
    Electronic Resource
    Electronic Resource
    Genetic transformation and regeneration of mature tissues of woody fruit plants bypassing the juvenile stage (1998)
    Springer
    Transgenic research 7 (1998), S. 51-59 
    Details
    ISSN: 1573-9368
    Keywords: sweet orange ; Citrus ; woody ; transformation ; Agrobacterium ; mature
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Regeneration and transformation systems from mature plant material of woody fruit species have to be achieved as a necessary requirement for the introduction of useful genes into specific cultivars and the rapid evaluation of resulting horticultural traits. We report here, for the first time, a procedure for genetic transformation and regeneration of mature tissues of woody plants that overcomes the long juvenile periods and high heterozygosity that are characteristic of most of these species. An improved regeneration frequency from mature explants was obtained by invigoration of the plant material through grafting of mature buds on juvenile seedlings. Co-cultivation of the explants in feederplates after inoculation with Agrobacterium tumefaciens resulted in enhanced transformation frequencies. Furthermore, in vitro shoot-tip grafting of the regenerated mature shoots on seedling rootstocks provided a rapid and efficient system for plant production. Citrus is the most extensivel y grown fruit crop worldwide and sweet orange (Citrus sinensis L. Osbeck) accounts for approximately 70% of the Citrus total production. Mature transgenic sweet orange plants have been obtained, which flowered and bore fruit in 14 months
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008855922283
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  • 21
    Electronic Resource
    Electronic Resource
    An efficient protocol for sugarcane (Saccharum spp. L.) transformation mediated by Agrobacterium tumefaciens (1998)
    Springer
    Transgenic research 7 (1998), S. 213-222 
    Details
    ISSN: 1573-9368
    Keywords: Saccharum ; Agrobacterium ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract This is the first successful report of the recovery of morphologically normal transgenic sugarcane plants from co-cultivation of calluses with Agrobacterium tumefaciens. Transformation frequencies (total of transgenic plants/number of cell clusters) were between 9.4 × 10−3 and 1.15 × 10−2. In our experiments, both LBA4404 (pTOK233) and EHA101 (pMTCA3IG), carrying a super-binary vector or supervirulent strain, respectively, were successful for sugarcane transformation. We found that three main factors: (1) the use of young regenerable calluses as target explants; (2) induction and/or improvement of the A. tumefaciens virulence system with sugarcane cell cultures and (3) pre-induction of organogenesis or somatic-embryogenesis-like sexual embryos, seem to be crucial in order to increase the cells competence for T-DNA transfer process. Patterns generated by Southern hybridization confirmed that T-DNAs were randomly integrated into sugarcane genome without th e persistence of A. tumefaciens in the transgenic plants
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008845114531
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  • 22
    Electronic Resource
    Electronic Resource
    Agrobacterium tumefaciens-mediated transformation of cauliflower, Brassica oleracea var. botrytis (1998)
    Springer
    Molecular breeding 4 (1998), S. 531-541 
    Details
    ISSN: 1572-9788
    Keywords: Agrobacterium ; Brassica oleracea ; cauliflower ; regeneration ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract We have developed an efficient and simpler method for genetic transformation and regeneration of cauliflower, Brassica oleracea var. botrytis plants. Explants from 4-day old seedlings were inoculated and cocultivated with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector with the neomycin phosphotransferase-II gene under the regulatory control of nopaline synthase promoter and terminator sequences, permitting transformed shoots to be selected on kanamycin containing medium. After three months rooted transformed plantlets were successfully transferred and grown under glasshouse conditions. Higher numbers of transformed plants were obtained from cotyledon than hypocotyl explants, presumably indicating cotyledons of cauliflower are more amenable to genetic transformation. Integration and expression of the introduced transgene were analysed by DNA gel blot and PCR analysis and NPT-II expression assay. Factors influencing transformation efficiency include explant age, concentration of bacterium used for infection, duration of infection and cocultivation with Agrobacterium. Transgenic plants of three commercial genotypes of cauliflower were produced using this method. We also show that introduction of antisense Bcp1 (pollen-specific gene) linked to a pollen-specific promoter (Lat52) resulted in the expected sterility of 50% pollen carrying this transgenic construct.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1009658614579
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  • 23
    Electronic Resource
    Electronic Resource
    Investigation of Agrobacterium-mediated transformation of apple using green fluorescent protein: high transient expression and low stable transformation suggest that factors other than T-DNA transfer are rate-limiting (1998)
    Springer
    Plant molecular biology 37 (1998), S. 549-559 
    Details
    ISSN: 1573-5028
    Keywords: Agrobacterium ; apple ; GFP ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract To investigate early events of Agrobacterium-mediated transformation of apple cultivars, a synthetic green fluorescent protein gene (SGFP) was used as a highly sensitive, vital reporter gene. Leaf explants from four apple cultivars (‘Delicious’, ‘Golden Delicious’, ‘Royal Gala’ and ‘Greensleeves’) were infected with Agrobacterium EHA101 harboring plasmid pDM96.0501. Fluorescence microscopy indicated that SGFP expression was first detected 48 h after infection and quantitative analysis revealed a high T-DNA transfer rate. Plant cells with stably incorporated T-DNA exhibited cell division and developed transgenic calli, followed by formation of transgenic shoots at low frequencies. The detection of SGFP expression with an epifluorescence stereomicroscope confirmed the effectiveness of SGFP as a reporter gene for detection of very early transformation events and for screening of putative transformants. The efficiency of the transformation and regeneration process decreased ca. 10000-fold from Agrobacterium infection to transgenic shoot regeneration, suggesting that factors other than Agrobacterium interaction and T-DNA transfer are rate-limiting steps in Agrobacterium-mediated transformation of apple.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1006041313209
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  • 24
    Electronic Resource
    Electronic Resource
    The use of visual marker genes as cell-specific reporters of Agrobacterium-mediated T-DNA delivery to wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.). (1998)
    Springer
    Euphytica 99 (1998), S. 17-25 
    Details
    ISSN: 1573-5060
    Keywords: Agrobacterium ; barley ; C1/Lc ; GFP ; GUS ; wheat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Transfer of T-DNA from Agrobacterium tumefaciens and A. rhizogenes to cells of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) is demonstrated following the inoculation of immature embryos and immature embryo-derived callus. Agrobacterium T-DNA vectors containing the C1/Lc anthocyanin-biosynthesis regulatory genes, the gusA gene or a synthetic green fluorescent protein gene (sgfp-S65T) were constructed from original binary vectors. The visual T-DNA markers were used as cell-autonomous reporters of early Agrobacterium-mediated transformation events in the wheat and barley cells. This localization of the transformed cells revealed a non-random distribution throughout each embryo and callus piece.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1018303102488
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  • 25
    Electronic Resource
    Electronic Resource
    Metabolite control of Sorghum C4 phosphoenolpyruvate carboxylase catalytic activity and phosphorylation state (1998)
    Springer
    Photosynthesis research 58 (1998), S. 153-162 
    Details
    ISSN: 1573-5079
    Keywords: C4 photosynthesis ; enzyme kinetics ; protein phosphorylation ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Kinetic analyses were performed on the nonphosphorylated and in vitro phosphorylated forms of recombinant Sorghum C4 phospho enolpyruvate carboxylase (C4 PEPC). The native enzyme was purified by immunoaffinity chromatography and its integrity demonstrated by Western blot analyses using anti N- and C-terminus antibodies. At suboptimal pH (7.1 to 7.3) and PEP concentration (2.5 mM), phosphorylation, positive metabolite effectors e.g., glucose-6-phosphate, glycine and dihydroxyacetone-phosphate, or an increase in pH strongly activated the enzyme and lowered the inhibitory effect of L-malate. C4 PEPC phosphorylation strengthened the effect of the positive effectors thereby decreasing further the enzyme's sensitivity to this inhibitor. L-malate also decreased the phosphorylation rate of C4 PEPC, a process antagonized by positive metabolite effectors. This was shown both in vitro, in a reconstituted phosphorylation assay containing the catalytic subunit of a cAMP-dependent protein kinase or the Sorghum leaf PEPC-PK and in situ, during induction of C4 PEPC phosphorylation in mesophyll cell protoplasts.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1006164209129
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  • 26
    Electronic Resource
    Electronic Resource
    Cre/lox-mediated site-specific integration of Agrobacterium T-DNA in Arabidopsis thaliana by transient expression of cre (1998)
    Springer
    Plant molecular biology 38 (1998), S. 393-406 
    Details
    ISSN: 1573-5028
    Keywords: Agrobacterium ; Arabidopsis ; Cre/lox ; recombination ; site-specific integration ; T-DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The Cre/lox system was used to obtain targeted integration of an Agrobacterium T-DNA at a lox site in the genome of Arabidopsis thaliana. Site-specific recombinants, and not random events, were preferentially selected by activation of a silent lox-neomycin phosphotransferase (nptII) target gene. To analyse the effectiveness of Agrobacterium-mediated transfer we used T-DNA vectors harbouring a single lox sequence (this vector had to circularize at the T-DNA left- and right-border sequences prior to site-specific integration) or two lox sequences (this vector allowed circularization at the lox sequences within the T-DNA either prior to or after random integration, followed by targeting of the circularized vector), respectively. Furthermore, to control the reversibility of the integration reaction, Cre recombinase was provided transiently by using a cotransformation approach. One precise stable integrant was found amongst the recombinant calli obtained after transformation with a double-lox T-DNA vector. The results indicate that Agrobacterium-mediated transformation can be used as a tool to obtain site-specific integration.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1006024500008
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  • 27
    Electronic Resource
    Electronic Resource
    ABA synthesis in protoplasts of different origin in response to osmotic stress (1998)
    Springer
    Plant growth regulation 25 (1998), S. 135-141 
    Details
    ISSN: 1573-5087
    Keywords: ABA synthesis ; Amaranthus ; fluridone ; osmotic stress ; protoplasts ; rose petal
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract All the protoplasts analysed in this study whatever the original plant material were able to accumulate ABA under osmotic stress. The time course of ABA accumulation strongly differed according to the plant material. In both rose petal or Amaranthus leaf protoplasts, the increase in ABA level was significant but transient. Protoplasts prepared from Amaranthus cell suspensions behaved differently, showing a late and durable accumulation of ABA. Similar patterns of changes in ABA accumulation were observed in the original plant material under osmotic stress. A pretreatment of plant material by fluridone induced a strong inhibition of ABA accumulation whatever the origin of protoplasts was. This result suggests that ABA could be synthesised via the carotenoid pathway in the absence of the cell wall.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1006085327980
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  • 28
    Electronic Resource
    Electronic Resource
    Transgenic Populus tremula: a step-by-step protocol for its Agrobacterium-mediated transformation (1997)
    Springer
    Plant molecular biology reporter 15 (1997), S. 219-235 
    Details
    ISSN: 1572-9818
    Keywords: Agrobacterium ; Populus ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In recent years, Populus species have acquired an important place in basic and applied research of woody plants. The practical role of Populus species in world forestry and their importance to research as a woody-plant model have led to increasing interest in tissue-culture and molecular techniques, as well as the development of transformation procedures for this genus. A simple technical procedure is described here step-by-step, for the first time, as a routine method for transforming Populus tremula using a disarmed Agrobacterium tumefaciens hypervirulent strain. The procedure begins with the inoculation of stem explants with bacterial suspension, followed by a short period of co-cultivation on a highly regenerative medium. Transformed shoots are selected on regeneration medium containing antibiotics and the presence of the inserted target genes is checked using a rapid and efficient PCR test. Selected shoots are transferred to a rooting medium, under the same selection pressure, and propagated via stem cuttings. Selected plants can be hardened and transferred to the green-house within 4 months of inoculation. The method has proven efficient for several gene constructs, selection on Kan or Hyg, and three different Agrobacterium strains.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1007484917759
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  • 29
    Electronic Resource
    Electronic Resource
    Role of the host cell cycle in theAgrobacterium-mediated genetic transformation ofPetunia: Evidence of an S-phase control mechanism for T-DNA transfer (1997)
    Springer
    Planta 201 (1997), S. 160-172 
    Details
    ISSN: 1432-2048
    Keywords: Agrobacterium ; Leaf mesophyll cells ; Petunia ; Transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Chimeric β-glucuronidase (GUS) gene expression in an efficientAgrobacterium-mediated transformation system utilising mesophyll cells ofPetunia hybrida synchronized with cell cycle phase-specific inhibitors (mimosine and colchicine) was used to show the absolute requirement of S-phase for transfer and/or integration of the transferred DNA (T-DNA). Flow-cytometric analysis of nuclear DNA content and immunohistological detection of bromodeoxyuridine (BrdUrd) incorporation showed that, prior to phytohormone treatment, most (98%) mesophyll cells were at GO-Gl-phase (quiescent phase) and no cell division was occurring. After 48 h and 72 h of phytohormone treatment, there was a rapid increase in S-G2-M-phase populations (〉 75%) and a concomitant decrease (down to 24%) in G0–-G1-phase cells. Assays of GUS showed that maximum transformation (〉 95% of explants) also occurred after this period. Our data showed that mimosine and colchicine blocked the mesophyll cells at late Gl-phase and M-phase, respectively. No transformation (= GUS expression) was observed in phytohormone-treated cells inhibited in late G1 by mimosine. However, after removal of mimosine, 82% of the explants were transformed, indicating the non-toxic and reversible effect of the inhibitor. On the other hand, a relatively high transformation frequency (65% of explants) was observed after blocking the cell cycle at M-phase with colchicine. However, only transient, but no stable, gene expression (= kanamycin-resistant callus formation) was observed in colchicine-treated M-phase-arrested cells. Similarly, endoreduplication of nuclear DNA, which occurred during the 48 h of phytohormone treatment in some mesophyll cells and cells located along the minor veins in the leaf explants, resulted in transient GUS expression only. These observations indicate a direct correlation between endoreduplication and transient GUS gene expression. Obviously, for stable GUS gene expression, cell division and proliferation are required, indicating that both DNA duplication (S-phase) and cell division (M-phase) are strongly related to stable transformation. We propose that the present system should facilitate further dissection of the process of T-DNA integration in the host genome and therefore should aid in developing new strategies for transformation of recalcitrant plants.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01007700
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  • 30
    Electronic Resource
    Electronic Resource
    Expression and inheritance of foreign genes in transgenic peanut plants generated byAgrobacterium-mediated transformation (1997)
    Springer
    Plant cell reports 16 (1997), S. 541-544 
    Details
    ISSN: 1432-203X
    Keywords: Transgenic peanut ; Agrobacterium ; Transformation ; Transgene expression ; Transgene inheritance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract To evaluate and characterize the stability of traits transferred viaAgrobacterium transformation, foreign gene expression must be examined in sexually derived progeny. The objective of this study was to analyze three transgenic peanut plants, 1-10, 12-1, and 17-1, for the inheritance and expression of their foreign genes. Segregation ratios for the introduced genes in T2 plants gave either 100% or 3:1 expression of the β-glucuronidase (GUS) gene, demonstrating recovery of both homozygous and heterozygous T1 plants. Fluorometric GUS assay in T1 and T2 generations of all three plants showed that the GUS gene was stably expressed in the progeny. DNA analyses showed 100% concordance between the presence of the foreign gene and enzyme activity. Our results demonstrate that transgenes in peanut introduced byAgrobacterium can be inherited in a Mendelian manner.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01142320
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  • 31
    Electronic Resource
    Electronic Resource
    Isolation of new 6-methylnicotinic-acid-degrading bacteria, one of which catalyses the regioselective hydroxylation of nicotinic acid at position C2 (1997)
    Springer
    Archives of microbiology 168 (1997), S. 355-361 
    Details
    ISSN: 1432-072X
    Keywords: Key words 6-Methylnicotinic acid ; 2-Hydroxy-6-methylnicotinic acid ; Nicotinic acid ; 2-Hydroxynicotinic acid ; Ralstonia ; Burkholderia ; Paenibacillus ; Agrobacterium ; Rhizobium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract 2-Hydroxynicotinic acid is an important building block for herbicides and pharmaceuticals. Enrichment strategies to increase the chances of finding microorganisms capable of hydroxylating at the C2 position and to avoid the degradation of nicotinic acid via the usual intermediate, 6-hydroxynicotinic acid, were used. Three bacterial strains (Mena 23/3–3c, Mena 25/4–1, and Mena 25/ 4–3) were isolated from enrichment cultures with 6-methylnicotinic acid as the sole source of carbon and energy. Partial characterization of these strains indicated that they represent new bacterial species. All three strains completely degraded 6-methylnicotinic acid, and evidence is presented that the first step in the degradation pathway of strain Mena 23/3–3c is hydroxylation at the C2 position. Resting cells of this strain grown on 6-methylnicotinic acid also hydroxylated nicotinic acid at the C2 position, but did not further degrade the product. Strain Mena 23/ 3–3c showed the highest degree of 16S rRNA sequence similarity to members of the genera Ralstonia and Burkholderia.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002030050509
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  • 32
    Electronic Resource
    Electronic Resource
    Characterization of RNA-mediated resistance to tomato spotted wilt virus in transgenic tobacco plants expressing NSM gene sequences (1997)
    Springer
    Plant molecular biology 33 (1997), S. 235-243 
    Details
    ISSN: 1573-5028
    Keywords: movement protein ; protoplasts ; resistance ; tomato spotted wilt virus (TSWV) ; tospovirus ; transgenic plants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Transgenic Nicotiana tabacum plants expressing RNA sequences of the tomato spotted wilt virus NSM gene, which encodes the putative viral movement protein, were found to be highly resistant to infection with the virus. Expression of untranslatable as well as anti-sense RNA of the NSM gene resulted in resistance levels as high as those in plants expressing translatable RNA sequences. For all three types of transgenic plants resistance levels of up to 100% were reached in the S2 progeny. These results indicate that the resistance mediated by the NSM gene is accomplished by expression of transcripts rather than protein in transgenic plants, similar to previously observed N gene-mediated resistance. Protoplast inoculations revealed that resistant plants expressing NSM are, in contrast to N transgenic resistant plants, not resistant at the cellular level. This suggests the RNA-mediated resistance mechanism against TSWV targets viral mRNAs rather than the viral genome.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1005729808191
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  • 33
    Electronic Resource
    Electronic Resource
    Excision of the maize transposable element Ac in periclinal chimeric leaves of 35S-Ac-rolC transgenic aspen-Populus (1997)
    Springer
    Plant molecular biology 33 (1997), S. 1097-1103 
    Details
    ISSN: 1573-5028
    Keywords: Ac ; Agrobacterium ; periclinal chimera ; rolC ; transgenic Populus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The transposable element Ac from maize, in combination with the phenotypic selectable marker rolC, was employed in transformation experiments of a hybrid aspen clone. A number of transgenic clones exhibited light-green sectors on green leaves. In vitro regeneration from leaves showing a high number of light-green spots resulted in R2 plants, which also showed light-green sectored leaves. However, only one out of 385 regenerated plants obtained showed green leaves. Both PCR and northern analysis indicated Ac excision and restoration of rolC expression. In Southern blot analysis of this green plant additional bands were observed as compared to the original R1 plant. The occurrence of these bands and a suggested Ac excision in the non-green L1-epidermal layer leading to periclinal chimerism of this plant is discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1005788706864
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  • 34
    Electronic Resource
    Electronic Resource
    Tapetum-specific expression of theOsg6B promoter-β-glucuronidase gene in transgenic rice (1997)
    Springer
    Plant cell reports 16 (1997), S. 363-367 
    Details
    ISSN: 1432-203X
    Keywords: Agrobacterium ; Tapetum-specific promoter ; Transformation ; Rice
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The promoter of an anther tapetum-specific gene,Osg6B, was fused to aβ-glucuronidase (GUS) gene and introduced into rice byAgrobacterium-mediated gene transfer. Fluorometric and histochemical GUS assay showed that GUS was expressed exclusively within the tapetum of anthers from the uninucleate microspore stage (7 days before anthesis) to the tricellular pollen stage (3 days before anthesis). This is the first demonstration of an anther-specific promoter directing tapetum-specific expression in rice.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01146774
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  • 35
    Electronic Resource
    Electronic Resource
    Expression and inheritance of foreign genes in transgenic peanut plants generated by Agrobacterium-mediated transformation (1997)
    Springer
    Plant cell reports 16 (1997), S. 541-544 
    Details
    ISSN: 1432-203X
    Keywords: Key words Transgenic peanut ; Agrobacterium ; Transformation ; Transgene expression ; Transgene inheritance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract To evaluate and characterize the stability of traits transferred via Agrobacterium transformation, foreign gene expression must be examined in sexually derived progeny. The objective of this study was to analyze three transgenic peanut plants, 1-10, 12-1, and 17-1, for the inheritance and expression of their foreign genes. Segregation ratios for the introduced genes in T2 plants gave either 100% or 3:1 expression of the β-glucuronidase (GUS) gene, demonstrating recovery of both homozygous and heterozygous T1 plants. Fluorometric GUS assay in T1 and T2 generations of all three plants showed that the GUS gene was stably expressed in the progeny. DNA analyses showed 100% concordance between the presence of the foreign gene and enzyme activity. Our results demonstrate that transgenes in peanut introduced by Agrobacterium can be inherited in a Mendelian manner.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01142320
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  • 36
    Electronic Resource
    Electronic Resource
    Genetic transformation of Populus genotypes with different chimaeric gene constructs: transformation efficiency and molecular analysis (1997)
    Springer
    Transgenic research 6 (1997), S. 111-121 
    Details
    ISSN: 1573-9368
    Keywords: Ac ; Agrobacterium ; aspen ; Populus ; transformation ; transposition
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Aspen (Populus tremula) and hybrid aspen (P. tremula × P. tremuloides) were transformed with different gene constructs using two types of promoter. The aim was to determine the influence of the reporter gene rolC, controlled by promoters of viral or plant origin, on genetic and morphologic expression of different transgenic aspen clones. An improved transformation method using leaf discs was developed, by which putative transgenic plantlets were regenerated at high efficiencies (up to 34%) on kanamycin-containing medium. Transgenic aspen carrying the rolC gene from Agrobacterium rhizogenes under control of the cauliflower-35S-promoter are reduced in size with smaller leaves, whereas aspen transgenic for the same rolC gene, but under control of the light inducible rbcS promoter from potato, are only slightly reduced in size compared to untransformed controls. However, all clones carrying 35S-rolC and rbcS-rolC genes revealed light-green colouration of leaves when compared to untransformed aspen. Owing to this special feature, constructs were used in which expression of the rolC gene was inhibited by insertion of a transposable element, Ac, from maize. Transgenic aspen transformed with the 35S-Ac-rolC and rbcS-Ac-rolC genes were morphologically similar to untransformed aspen, but out of 54 independently regenerated 35S-Ac-rolC transgenic aspen clones, 30 clones showed light-green/dark green variegated leaves. In contrast, out of 19 independently transformed rbcS-Ac-rolC aspen clones, only two clones revealed light-green/dark green variegated leaves. The role of bacterial strains in transformation, and molecular genetics of transgenic aspen plants (including the function of the transposable element, Ac, in the aspen genome) are discussed
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1018421620040
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  • 37
    Electronic Resource
    Electronic Resource
    Induction of callose formation is a sensitive marker for genotypic aluminium sensitivity in maize (1997)
    Springer
    Plant and soil 192 (1997), S. 23-30 
    Details
    ISSN: 1573-5036
    Keywords: aluminium ; callose ; protoplasts ; resistance ; toxicity ; Zea mays
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The screening of 37 Zea mays L. cultivars in nutrient solution using root elongation (24 h) as a parameter showed large genotypic differences in Al resistance among the genetic material evaluated. Callose concentrations in root tips were closely and positively related to Al-induced inhibition of root elongation. Therefore, Al-induced callose formation in root tips appears to be an excellent indicator of Al injury and can be used as a selection criteria for Al sensitivity. In contrast, aluminium concentrations in root tips were not related to Al-induced inhibition of root elongation, nor to Al-induced callose formation. Callose formation was also induced by short-term A1 treatment in root tip protoplasts, and the response of protoplasts clearly reflected the cultivar-specific response to Al of intact roots. This indicates that in maize, Al sensitivity is expressed on the protoplast level.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1004204120863
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  • 38
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    Electronic Resource
    Cell suspension, isolation and culture of protoplasts of Allium cepa (1997)
    Springer
    Plant cell, tissue and organ culture 51 (1997), S. 43-47 
    Details
    ISSN: 1573-5044
    Keywords: Allium cepa ; onion ; organogenesis ; protoplasts ; suspension culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Tissue culture techniques involving cell suspension, protoplast fusion and culture in the genus Allium are different as Allium is recalcitrant due to the biological peculiarity of the genus. A procedure is described for the establishment of a regenerable suspension culture and for the isolation and culture of protoplasts of Allium cepa.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1005886732424
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  • 39
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    Electronic Resource
    Plant regeneration from protoplasts isolated from primary calli using mature embryos of two Brazilian rice cultivars (1997)
    Springer
    Euphytica 94 (1997), S. 01-05 
    Details
    ISSN: 1573-5060
    Keywords: Oryza sativa ; japonica rice cultivar ; mature embryo ; primary calli ; protoplasts ; green plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract A plant regeneration system from rice protoplasts using calli derived from mature embryos was established for the two Brazilian modern rice cultivars IAC-201 and IAC-165. After 30 to 40 days of in vitro culture it was possible to obtain on average 6 million protoplasts per gram of callus. Microscopic selection of embryogenic calli was a key step for protoplast isolation. The production of embryogenic calli increased when L-proline and casein hydrolysate were used in the callus induction medium. The Oc or IR52 nurse cell lines were essential for protoplast division. Different regeneration media were studied and 139 plants were regenerated which set seed. Some of the regenerated plants showed morphological variation such as the presence of awns in spite of the short time of the in vitro culture.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1002962724080
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  • 40
    Electronic Resource
    Electronic Resource
    Photosynthesis response to triacontanol correlates with increased dynamics of mesophyll protoplast and chloroplast membranes (1997)
    Springer
    Plant growth regulation 21 (1997), S. 145-152 
    Details
    ISSN: 1573-5087
    Keywords: chloroplasts ; membrane dynamics ; Pisum ; photosymthesis ; protoplasts ; triacontanol
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The effects of a long chain aliphatic alcohol 1-triacontanol (TRIA) on the photosynthesis and membrane properties of mesophyll protoplasts and chloroplasts isolated from pea leaves were studied. In vitro treatments of isolated protoplasts caused a large enhancement (166% ) of the CO2-fixation rate after 60 min of TRIA (10-6 M) application as compared to the control. An enhanced photosynthetic response was observed in in vitro treated leaf pieces. Application of octacosanol (OCTA) under the same experimental conditions did not result in any stimulating effects. In vivo treatments of pea seedlings also resulted in a significant increase of the net CO2 uptake to 109% and 119% in 10-8 M and 10-6 M TRIA-treated plants respectively. It was demonstrated that the incubation of both protoplasts and chloroplasts with TRIA resulted in a rise of the excimer/monomer (IE/IM ) ratio of pyrene (Py) fluorescence, thus indicating remarkable fluidization and/or disordering of the lipid matrix of their membranes. This effect depended on the incubation time and became evident at very low concentrations of TRIA (0.3 μM). The increase of membrane fluidity was accompanied by TRIA-induced alterations in the dielectric environment in the membrane regions where Py molecules are situated. The results are discussed in terms of specific concentration dependent TRIA-induced alterations of the dynamic properties of protoplast and chloroplast membranes and their possible involvement in the initiation of the integral physiological response to exogenous application of TRIA.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1005790121111
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  • 41
    Electronic Resource
    Electronic Resource
    Agrobacterium rhizogenes and A. tumefaciens co-transformation to obtain grapevine hairy roots producing the coat protein of grapevine chrome mosaic nepovirus (1997)
    Springer
    Plant cell, tissue and organ culture 49 (1997), S. 53-62 
    Details
    ISSN: 1573-5044
    Keywords: Agrobacterium ; coat protein ; grapevine ; hairy root ; nepovirus ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Hairy root cultures of grapevine were obtained from plantlets co-inoculated by virulent Agrobacterium rhizogenes strains and disarmed A. tumefaciens strains harbouring the binary vectors pKHG4 and pKVHG 2+. These plasmids contain the nptII, hpt and gus genes and differ for the presence of the gene encoding for the grapevine chrome mosaic virus coat protein. For the cultivar ‘Gravesac’, 72% of the excised root tips initiated hairy root cultures on growth regulator-free media. According to the nature of the strains used in co-inoculation, co-transformation frequencies of the hairy root clones ranged from 4 to 16%. Co-transformed roots showed resistance to kanamycin and hygromycin but responses varied from clone to clone. Fluorometric GUS expression and GCMV coat protein production showed a large variability among hairy root clones co-transformed by pKHVG2+. Though the presence of gus, nptII and GCMV coat protein genes was checked by polymerase chain reaction and Southern blotting, it was difficult to establish a clear relationship between expression of the different transgenes. The regeneration of plants was not achieved, but the possibility to graft in vitro transgenic roots to non transformed shoot systems could permit rapid testing of the resistance induced by nepovirus coat protein in roots of cultivars that are recalcitrant to A. tumefaciens-mediated transformation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1005854212592
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  • 42
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    Electronic Resource
    Isolation and identification of N2-fixing microorganisms from the rhizosphere of Capparis spinosa (L.) (1997)
    Springer
    Plant and soil 197 (1997), S. 19-23 
    Details
    ISSN: 1573-5036
    Keywords: Agrobacterium ; Capparis spinosa ; Comamonas ; N2 fixation ; Pseudomonas ; rhizosphere ; Sphingobacterium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Four bacterial strains, Pseudomonas stutzeri var. mendocina, Comamonas sp., Agrobacterium tumefaciens biovar. 2 and Sphingobacterium sp., isolated from the rhizosphere of wild-grown caper (Capparis spinosa L.) plants were able to fix N2 as shown by their growth in nitrogen-free medium and by the acetylene reduction test. P. stutzeri var. mendocina and Comamonas sp. contained DNA homologous to the Klebsiella pneumoniae M5a1 nifHDK genes. No hybridization was found with total DNA from either A. tumefaciens biovar. 2 or Sphingobacterium sp. using nifHDK probes from either K. pneumoniae or Rhizobium meliloti.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1004211909641
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  • 43
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    Electronic Resource
    An improved method for direct gene transfer and subsequent regeneration ofArabidopsis thaliana Landsbergerecta and two marker lines (1997)
    Springer
    Plant cell, tissue and organ culture 47 (1997), S. 111-118 
    Details
    ISSN: 1573-5044
    Keywords: PEG-mediated transformation ; protoplasts ; transgene inheritance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A protocol for PEG-mediated transformation of protoplasts is described forA. thaliana ecotype Landsbergerecta and two marker lines derived from it, M4 and M10. The optimal transformation conditions were: 14 µg plasmid DNA and 28 µg carrier DNA per 6 x 105 protoplasts in 15% (w/v) PEG solution. Based on the hygromycin resistance conferred by the transgene, relative transformation frequencies of 2.5–3.2% and absolute transformation frequencies of 1–2 x 10−4, were obtained. Shoot regeneration frequencies of 40–60% were achieved, and fertile transgenic plants of the three tested lines were obtained. Southern blot hybridizations demonstrated multi-copy integration patterns in most cases. Hygromycin resistance segregation patterns of 3:1 and 15:1 were found, as well as unexpected segregation patterns, suggesting that modifications in gene expression took place and that these can progressively occur over successive generations.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF02318946
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  • 44
    Electronic Resource
    Electronic Resource
    Plant regeneration in sweet potato (Ipomoea batatas L., Convolvulaceae) (1997)
    Springer
    Euphytica 96 (1997), S. 143-152 
    Details
    ISSN: 1573-5060
    Keywords: Sweet potato ; Ipomoea batatas ; plant regeneration ; somatic embryogenesis ; protoplasts ; genetic variation ; somaclonal variation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The application of new techniques for improvement of sweet potato crops, particularly including the exploitation of somaclonal variation, gene transfer by genetic transformation and somatic hybridization, requires the control of plant regeneration from tissue cultures. Shoots can easily be regenerated from explants of stems, petioles, leaves and roots, while callus cultures do not produce any shoots. The potential of somatic embryogenesis and plant regeneration via embryogenesis was evaluated for 10 cultivars of sweet potato. Protocols for plant regeneration from cultured protoplasts have also been developed. Since mesophyll was resistant to enzyme digestion, fragments of stems and petioles, callus and cell suspensions were used as source of protoplasts of sweet potato. Series of transfers of protoplast-derived calluses, particularly those which had been obtained from in vitro plants, to media containing a high level of zeatin resulted in successful formation of shoots in only two sweet potato cultivars. In addition, the embryogenic potential was irreversibly lost through protoplast culture, since protoplasts isolated from embryogenic cell suspensions developed into non-embryogenic callus. Consequently, an alternative protocol is being successfully developed to improve plant regeneration from cultured protoplasts of sweet potato, involving first root formation from which shoots can then be regenerated. Preliminary evaluation in field conditions in Gabon revealed that plants regenerated from cultured protoplasts exhibited a great genetic variability in their growth and tuber formation in particular.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1002997319342
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  • 45
    Electronic Resource
    Electronic Resource
    Maintenance of steroid 11-hydroxylation activity in immobilized Cunninghamella elegans protoplasts (1997)
    Springer
    World journal of microbiology and biotechnology 13 (1997), S. 469-473 
    Details
    ISSN: 1573-0972
    Keywords: 2-Deoxy-d-glucose ; hydroxylation ; immobilization ; polyoxin ; protoplasts ; steroids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1018540620234
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  • 46
    Electronic Resource
    Electronic Resource
    The effect of polyamines on the development of sugar beet protoplasts (1997)
    Springer
    Biologia plantarum 39 (1997), S. 561-567 
    Details
    ISSN: 1573-8264
    Keywords: Beta vulgaris ; plating efficiency ; protoplasts ; putrescine ; spermidine ; spermine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The influence of the exogenous polyamines: putrescine, spermidine and spermine, on the frequency of protoplast divisions for 2 genotypes of sugar beet (Beta vulgaris L.) was analyzed. Protoplasts were cultured by the agarose disk method on Saunders and Doley medium supplemented with either hormones or polyamines, or hormones combined with polyamines. The latter supplement led to a statistically significant increase in plating efficiency. The improvement in division index was caused mainly by spermine.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1000926714622
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  • 47
    Electronic Resource
    Electronic Resource
    SAAT: sonication-assisted Agrobacterium-mediated transformation (1997)
    Springer
    Transgenic research 6 (1997), S. 329-336 
    Details
    ISSN: 1573-9368
    Keywords: Agrobacterium ; SAAT ; sonication ; transformation ; wounding
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Plant transformation via Agrobacterium can be limited by both host specificity and the inability of Agrobacterium to reach the proper cells in the target tissue. Described here is a new and efficient Agrobacterium-based transformation technology that overcomes these barriers and enhances DNA transfer in such diverse plant groups as dicots, monocots, and gymnosperms. This new technology, called sonication-assisted Agrobacterium-mediated transformation (SAAT), involves subjecting the plant tissue to brief periods of ultrasound in the presence of Agrobacterium. Scanning electron and light microscopy reveal that SAAT treatment produces small and uniform fissures and channels throughout the tissue allowing the Agrobacterium easy access to internal plant tissues. Unlike other transformation methods, this system has the potential to transform meristematic tissue buried under several cell layers. SAAT increases transient transformation efficiency in several different plant tissues including leaf tissue, immature cotyledons, somatic and zygotic embryos, roots, stems, shoot apices, embryogenic suspension cells and whole seedlings. A 100- to 1400-fold increase in transient β- glucuronid ase expression has been demonstrated in various tissues of soybean, Ohio buckeye, cowpea, white spruce, wheat and maize. Stable transformation of both soybean and Ohio buckeye has been obtained using SAAT of embryogenic suspension culture tissues. For soybean, SAAT treatment was necessary to obtain stable transformation with this tissue
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1018470930944
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  • 48
    Electronic Resource
    Electronic Resource
    Matrix attachment regions (MARs) enhance transformation frequency and transgene expression in poplar (1997)
    Springer
    Transgenic research 6 (1997), S. 415-420 
    Details
    ISSN: 1573-9368
    Keywords: GUS ; matrix attachment regions ; Populus ; transformation ; transgene expression ; Agrobacterium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We tested the value of a matrix attachment region (MAR) fragment derived from a tobacco gene for increasing the frequency of Agrobacterium-mediated transformation. A binary vector that carried a GUS reporter gene containing an intron and an nptII gene was modified to contain flanking MAR elements within the T-DNA borders. Vectors containing or lacking MARs were then used to transform tobacco, a readily transformabl e poplar clone (Populus tremula × P. alba), and a recalcitrant poplar clone (Populus trichocarpa × P. deltoides). MARs increased GUS gene expression approximately 10-fold in the two hybrid poplar clones and twofold in tobacco one month after cocultivation with Agrobacterium; MARs also increased the frequency of kanamycin-resistant poplar shoots recovered
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1018439518649
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  • 49
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    Electronic Resource
    Activation of the Ty1-copia group retrotransposons of potato (Solatium tuberosum) during protoplast isolation (1996)
    Springer
    Plant cell reports 15 (1996), S. 949-953 
    Details
    ISSN: 1432-203X
    Keywords: Ty1-copia group retrotransposons ; potato ; protoplasts ; expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The potato (Solanum tuberosum) genome contains a highly heterogeneous population of Ty1-copia group retrotransposons. Here we identify the first such transposable element known to be transcribed in this species. The elements are transcriptionally activated during protoplast isolation. The majority of the activated Ty1-copia sequences are similar to elements which are transcriptionally induced under the same conditions in tobacco (Nicotiana tabacum). We also show that a previously identified potato element M166, which has no known equivalent in tobacco is also transcribed under these conditions. It appears that the control of transcription of this particular Ty1-copia group retrotransposons has been broadly conserved between these two species.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00231594
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  • 50
    Electronic Resource
    Electronic Resource
    Plant regeneration from protoplasts of Japanese lawngrass (1996)
    Springer
    Plant cell reports 15 (1996), S. 737-741 
    Details
    ISSN: 1432-203X
    Keywords: Zoysia japonica ; embryogenic callus ; protoplasts ; plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Embryogenic callus of Japanese lawngrass (Zoysia japonica Steud.) was induced from sterile mature seeds on LS medium with 5 mg / l of 2,4-D. Embryogenic callus selected visually under microscope was proliferated in liquid N6 medium with amino acids (N6-AA medium). Protoplasts were isolated from suspension cells by the treatment of enzyme mixture containing pectolyase Y-23 and cultured in K8p medium with 2 mg / l of 2,4-D at the density of 106 / ml. Plants were regenerated by transferring the protoplasts derived callus to MS medium and incubating at 28 °C under light for two months. Plantlets were successfully transplanted in the soil.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00232218
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  • 51
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    Electronic Resource
    Transgenic plant production mediated by Agrobacterium in Indica rice (1996)
    Springer
    Plant cell reports 15 (1996), S. 727-730 
    Details
    ISSN: 1432-203X
    Keywords: Acetosyringone ; Agrobacterium ; Indica rice ; Oryza sativa L. ; Transgenic plants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary 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.
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    URL: http://dx.doi.org/10.1007/BF00232216
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  • 52
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    Electronic Resource
    Transient expression of uidA constructs in in vitro onion (Allium cepa L.) cultures following particle bombardment and Agrobacterium-mediated DNA delivery (1996)
    Springer
    Plant cell reports 15 (1996), S. 958-962 
    Details
    ISSN: 1432-203X
    Keywords: Transformation ; particle bombardment ; Agrobacterium ; Allium cepa.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Particle bombardment and Agrobacterium-mediated DNA delivery into immature embryos and microbulbs were used to investigate the expression of the uidA gene in in vitro onion cultures. Both methods were successful in delivering DNA and subsequent uidA expression was observed. Optimal transient β-glucuronidase activity was observed in immature embryos that had been pre-cultured for three days and bombarded at a distance of 3 cm from the stopping plate, under 25 in Hg vacuum, using 900–1300 psi rupture discs. The CaMV35S-uidA gene construct gave five fold higher transient β-glucuronidase activity than the uidA gene construct regulated by any of four other promoters initially chosen for high experession in monocotyledonous tissues.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00231596
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  • 53
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    Electronic Resource
    Production of an extracellular polysaccharide byAgrobacterium sp DS3 NRRL B-14297 isolated from soil (1996)
    Springer
    Journal of industrial microbiology and biotechnology 16 (1996), S. 129-133 
    Details
    ISSN: 1476-5535
    Keywords: extracellular polysaccharide ; Agrobacterium ; viscous polysaccharide
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A bacterium isolated from soil and identified asAgrobacterium sp produced a water-soluble extracellular polysaccharide capable of producing highly viscous solutions. Gas chromatographic analysis revealed a sugar composition of glucose, galactose and mannose in the molar ratio of 7.5∶2.4∶1, together with 3.7% (w/w) pyruvic acid. Methylation analyses showed the presence of (1→3)-, (1→4)- and (1→6)-linked glucose, (1→3)- and (1→4, 1→6)-linked galactose and a small portion of (1→3)-linked mannose residues. Succinic acid was not present. The molecular weight of the polysaccharide was estimated by light scattering to be 2×106 Da. The viscosity of solutions containing the polysaccharide remained constant from pH 3 to 11, and decreased by 50% when heated from 5 to 55°C. Maximum yield of the polysaccharide, 20 g L−1, was reached in 48 h at 30°C incubation.
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    URL: http://dx.doi.org/10.1007/BF01570073
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  • 54
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    Electronic Resource
    Agrobacterium tumefaciens-mediated transformation of japonica and indica rice varieties (1996)
    Springer
    Planta 199 (1996), S. 612-617 
    Details
    ISSN: 1432-2048
    Keywords: Agrobacterium ; indica rice ; Inheritance ; Japonica rice ; Oryza ; Transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Genetic transformation of rice (Oryza sativa L.) mediated by Agrobacterium ttumefaciens has been confirmed for japonica varieties and extended to include the more recalcitrant indica varieties. Immature embryos were inoculated with either A. tumefaciens At656 (pCNL56) or LBA4404 (pTOK233). Experimental conditions were developed initially for immature embryos treated with strain At656, based upon both transient and stable β-glucuromdase (GUS) activities. However, plant regeneration following selection on G418 (pCNL56 contained the nptII gene) did not occur. Using the same basic protocol, but inoculating immature embryos of rice with LBA4404 (pTOK233), resulted in efficient (about 27%) production of transgenic plants of the japonica variety, Radon, and an acceptable efficiency (from 1–5%) for the indica varieties IR72 and TCS10. Transformation was based upon resistance to hygromycin (pTOK233 contains the hpt gene), the presence of GUS activity (from the gusA gene), Southern blots for detection of the integrated gusA gene, and transmission of GUS activity to progeny in a Mendelian 3:1 segregation ratio. Southern blots indicated two to three copies of the gene integrated in most transformants. Transgenic plants of both the japonica and indica varieties were self-fertile and comparable in this respect to seed-grown plants. Key factors facilitating the transformation of rice by Agrobacterium tumefaciens appeared to be the use of embryos as the expiant, the use of hygromycin as the selection agent (which does not interfere with rice regeneration), the presence of extra copies of certain vir genes on the binary vector of pTOK233, and maintaining high concentrations of acetosyringone for inducing the vir genes during co-cultivation of embryos with Agrobacterium.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00195194
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  • 55
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    Electronic Resource
    Plant regeneration from protoplasts isolated from callus ofGentiana crassicaulis (1996)
    Springer
    Plant cell reports 16 (1996), S. 88-91 
    Details
    ISSN: 1432-203X
    Keywords: Gentiana crassicaulis Duthie ex Burk ; protoplasts ; callus ; plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Fast growing calli induced from hypocotyl segments ofGentiana crassicaulis were used for preparation of protoplasts. High yields of viable protoplasts were produced in an enzyme solution containing 1–2% cellulase, I% pecfinase, and 0.5% Hemicellulase. Protoplasts were cultured in KM8P medium containing 1 mg/l 2,4-D, 0.5 mg/l 6BA, 500 mg/l LH, 0.5 M glucose and 0.1 M mannitol by the solid-liquid dual layer culture method. First division occurred within 4–5 days of culture at a frequency of 17.8%. Sustained divisions led to callus formation. Periodically diluting the cultures with freshly prepared liquid medium containing 1% glucose was critical for colony formation. Protocolonies about 2 mm in size were transferred onto MS medium supplemented with 3 mg/l ZT, 2 mg/l 6BA, 1 mg/l GA3, 1 mg/l NAA and 6% sucrose to obtain embryogenic calli. Plantlets were regenerated via somatic embryogenesis at high frequency on hormone-free MS Medium.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01275457
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  • 56
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    Electronic Resource
    Sequence and characterisation of a ribosomal RNA operon from Agrobacterium vitis (1996)
    Springer
    Molecular genetics and genomics 251 (1996), S. 99-107 
    Details
    ISSN: 1617-4623
    Keywords: Key words Ribosomal genes ; Agrobacterium ; Evolution ; 23S rRNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  One of the four ribosomal RNA operons (rrnA) from the Agrobacterium vitis vitopine strain S4 was sequenced. rrnA is most closely related to the rrn operons of Bradyrhizobium japonicum and Rhodobacter sphaeroides and carries an fMet-tRNA gene downstream of its 5S gene, as in the case of R. sphaeroides. The 16S rRNA sequence of S4 differs from the A. vitis K309 type strain sequence by only one nucleotide, in spite of the fact that S4 and K309 have very different Ti plasmids. The predicted secondary structure of the S4 23S rRNA shows several features that are specific for the alpha proteobacteria, and an unusual branched structure in the universal B8 stem. The 3′ ends of the three other rrn copies of S4 were also cloned and sequenced. Sequence comparison delimits the 3′ ends of the four repeats and defines two groups: rrnA/rrnB and rrnC/rrnD.
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    URL: http://dx.doi.org/10.1007/BF02174350
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  • 57
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    Transformation ofBrassica oleracea L.: a critical review (1996)
    Springer
    Molecular breeding 2 (1996), S. 185-210 
    Details
    ISSN: 1572-9788
    Keywords: Brassica oleracea ; Agrobacterium ; transformation ; direct gene transfer ; regeneration ; virulence ; flowering ; rol genes ; transgene expression ; transgene inactivation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Brassica oleracea is a highly polymorphic species encompassing a wide range of important vegetable and fodder crops. Gene transfer into cultivated forms of this species requires reproducible and efficient methods for genetic transformation and plant regeneration. In this review, we have collated the research experience on transformation ofB. oleracea to highlight the problems encountered. Most research effort has been directed at developingAgrobacterium-mediated transformation methods with relatively little emphasis to date on direct gene transfer techniques. Common procedures for the transformation ofB. oleracea have not emerged, due to the inherent variability between and amongst genotypes. Future progress would be facilitated by the use of genetically fixed material, such as double-haploid or inbred lines, to reduce variation of response within genotypes and would avoid the need for cultivar-specific transformation protocols if responsive lines amenable to crossing with cultivated forms could be identified. The principal difficulties relate to combining efficient plant regeneration with gene transfer. Methods that enhance bacterial virulence and increase the proportion of cells susceptible to transformation and competent for regeneration are discussed. Inefficient selection is a major cause of poor transformation frequencies inB. oleracea and has resulted in the regeneration of chimeric plants uponAgrobacterium tumefaciens-mediated transformation. Promising results have been obtained withAgrobacterium rhizogenes-mediated transformation but the impact of therol genes on flowering of primary transformants has not yet been fully assessed. Strategies to reduce the deleterious effects of therol genes on flowering are discussed. Few agronomically useful characters have been introduced, the majority of research having been confined to the introduction of marker and reporter genes; possible candidate genes are discussed.
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    URL: http://dx.doi.org/10.1007/BF00564197
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    Sequence and characterisation of a ribosomal RNA operon fromAgrobacterium vitis (1996)
    Springer
    Molecular genetics and genomics 251 (1996), S. 99-107 
    Details
    ISSN: 1617-4623
    Keywords: Ribosomal genes ; Agrobacterium ; Evolution ; 23S rRNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract One of the four ribosomal RNA operons (rrnA) from theAgrobacterium vitis vitopine strain S4 was sequenced.rrnA is most closely related to therrn operons ofBradyrhizobium japonicum andRhodobacter sphaeroides and carries an fMet-tRNA gene downstream of its 5S gene, as in the case ofR. sphaeroides. The 16S rRNA sequence of S4 differs from theA. vitis K309 type strain sequence by only one nucleotide, in spite of the fact that S4 and K309 have very different Ti plasmids. The predicted secondary structure of the S4 23S rRNA shows several features that are specific for the alpha proteobacteria, and an unusual branched structure in the universal B8 stem. The 3′ ends of the three otherrrn copies of S4 were also cloned and sequenced. Sequence comparison delimits the 3′ ends of the four repeats and defines two groups:rrnA/rrnB andrrnC/rrnD.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF02174350
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  • 59
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    Corresponding 16S rRNA gene segments in Rhizobiaceae and Aeromonas yield discordant phylogenies (1996)
    Springer
    Plant and soil 186 (1996), S. 69-74 
    Details
    ISSN: 1573-5036
    Keywords: Agrobacterium ; population genetics ; Rhizobium ; systematics ; taxonomy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Previous evidence has indicated that the 16S rRNA genes in certain species of Aeromonas may have a history of lateral transfer and recombination. A comparative analysis of patterns of 16S nucleotide sequence polymorphism among species of Rhizobium and Agrobacterium was conducted to determine if there is similar evidence for chimeric 16S genes in members of the Rhizobiaceae. Results from phylogenetic analyses and comparison of patterns of nucleotide sequence polymorphism in portions of rhizobial 16S genes revealed the same type of segment-dependent polymorphic site partitioning that was previously reported for Aeromonas. These results support the hypothesis that certain 16S segments in rhizobia may have a history of lateral transfer and recombination.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00035057
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  • 60
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    Identification of Agrobacterium and Rhizobium species based on cellular fatty acid composition (1996)
    Springer
    Plant and soil 184 (1996), S. 143-158 
    Details
    ISSN: 1573-5036
    Keywords: Agrobacterium ; FAME ; fatty acid analysis ; rapid identification ; Rhizobium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The increasing number of phylogenetically defined species in the genera Agrobacterium, Rhizobium and Sinorhizobium suggests a need for a rapid identification method which will distinguish between these species. We have examined 65 strains of Agrobacterium representing: A. tumefacies, (34); A. rhizogenes, (16) A. vitis, (10) A. rubi (2) and some unclassified strains, and 150 strains of Rhizobium and Sinorhizobium representing: R. etli (21); R. galegae (20); R. huakuii (17); R. leguminosarum (20); R. loti (16); R. topici (18); S. fredii (19); and S. meliloti (20). Fatty acid methyl esters (FAME) were obtained from each strain, as previously described, and analysed by gas-chromatography using the MIDI Hewlett-Packard Microbial Identification System (MIS). Fatty acid profiles were recorded, characteristic fatty acids noted and the overall similarities between fatty acid profiles for each species calculated. Relationships between species were also derived from the fatty acid data by principal component analysis. This showed overlapping clusters for strains of R. leguminosarum and R. etli, R. topici and A. rhizogenes and S. fredii and S. meliloti within one supercluster. Strains of A. tumefaciens, A rubi, A. vitis and R. galegae formed a second supercluster while R. loti and R. huakuii strains formed a third cluster well separated from all the other strains. The fatty acid profiles were used to correctly identify at least 94% of the strains representing each species in the collection except R. etli. R. etli strains (23.8%) were misidentified as R. leguminosarum. This was attributed to the high similarity (44.7%) between R. etli and R. leguminosarum. It is concluded that whole cell fatty acid analysis should form part of the polyphasic description of new species of root nodule bacteria, with the proviso that growth conditions and analytical methods be carefully standardized. It is suggested that FAME-MIS system and the database we have compiled provide a basis for future development.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00029284
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    A single cell model system to study hormone signal transduction (1996)
    Springer
    Plant growth regulation 18 (1996), S. 149-154 
    Details
    ISSN: 1573-5087
    Keywords: cell culture ; auxins ; cytokinins ; confocal laser scanning microscope ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The model system presented here is based on immobilised single cells, derived directly from tobacco mesophyll protoplasts. It allows the adequate steering of cell populations towards expansion, cell cycling or cell resting. Using this approach cells always have the same predictable response to auxins and cytokinins whatever their actual physiological status. This model system opens new ways to study cellular parameters governing these hormone responses, some of which have been explored so far; a) the cytokinin response can equally well be induced by endogenous as by exogenous cytokinins; b) at least two intracellular components, microtubuli and the ER, adapt their architecture to the hormone-induced status of the cell; c) addition of NAA to the cells does not induce a change in the cytoplasmic pH.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00028500
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    Production and regeneration of protoplasts from the mycorrhizal fungus Suillus granulatus (1996)
    Springer
    World journal of microbiology and biotechnology 12 (1996), S. 625-628 
    Details
    ISSN: 1573-0972
    Keywords: Mycorrhizae ; protoplasts ; Suillus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Experiments were performed with the mycorrhizal fungus Suillus granulatus to define the parameters for production and regeneration of protoplasts. Protoplasts were released at frequencies between 1 and 3×107/ml from mycelium 3 to 7 days old. The best osmotic stabilizer for protoplast release was MgSO4 (0.7 m). To optimize protoplast release and regeneration an enzyme (Novozym 234) concentration 1.7 mg/ml was chosen, with a digestion time of 1 to 2 h. Regenerated colonies formed mycorrhizae within 60 days after inoculation in Pinus caribaea var. hondurensis seedlings.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00327726
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    Oil palm (Elaeis guineensis) protoplasts: isolation, culture and microcallus formation (1996)
    Springer
    Plant cell, tissue and organ culture 46 (1996), S. 35-41 
    Details
    ISSN: 1573-5044
    Keywords: Elaeis guineensis ; oil palm ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Procedures are deseribed for the efficient isolation of protoplasts from a variety of oil palm (Elaeis guineensis Jacq.) tissues. Various factors including donor source, composition of enzyme mixture and culture medium affected the yield and viability of the protoplasts Polyembryogenic cultures of oil palm were the most suitable starting material in terms of yield, viability and metabolic competence. Pectolyase Y-23 in association with cellulase and hemicellulase was required for the efficient release of protoplasts from the oil palm tissues. Limited cell division to form microcallus was observed at very low frequency (〈0.01%) when glutathione and catalase were incorporated in the culture medium.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00039694
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    Intraspecific somatic and sexual hybridisation between dihaploid lines of Solanum tuberosum L.: evaluation of morphological traits and resistance to late blight Phytophthora infestans (Mont.) de Bary in the foliage (1996)
    Springer
    Euphytica 90 (1996), S. 209-216 
    Details
    ISSN: 1573-5060
    Keywords: protoplasts ; potato ; somatic hybrids ; sexual hybrids ; late blight ; Phytophthora infestans
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Intraspecific tetraploid somatic and sexual hybrid plants have been resynthesised following protoplast fusion and by sexual crosses between two dihaploid potato (Solanum tuberosum) lines each possessing complementary agronomic traits. The dihaploid PDH 40 possesses good tuber shape and yield but has foliage susceptibility to late blight (Phytophthora infestans). On the other hand, the dihaploid PDH 727 possesses resistance to blight in the foliage but has a low yield of small and irregular shaped tubers. Since it was only possible to use a partial selection strategy based on culture media to facilitate recovery of somatic hybrid plants-further morphological and esterase isozyme based characterisations were performed to identify somatic hybrid plants from amongst the non-hybrid plant material. When the blight resistance of both the intraspecific somatic and sexual hybrid plants was assessed there was no significant difference in the mean resistance value and it was intermediate between those of their parents. However, the range of resistance was much wider among the sexual hybrids than among the plants derived from somatic fusion. An assessment of tuber yield between tetraploid sexual and somatic hybrids showed no significant difference and it was higher than that of either parent value. The implication of these results in the context of potato genetics and breeding is discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00023860
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    Elementary auxin response chains at the plasma membrane involve external abp1 and multiple electrogenic ion transport proteins (1996)
    Springer
    Plant growth regulation 18 (1996), S. 23-28 
    Details
    ISSN: 1573-5087
    Keywords: auxin receptor ; ion channels ; auxin-binding protein ; protoplasts ; electrophysiology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Studies of membrane electrical responses of isolated protoplasts to auxin have demonstrated the existence of elementary response chains to auxin at the plasma membrane, presently defined only by their uttermost ends. At one side, as demonstrated by several lines of evidence, the auxin perception unit involves proteins homologous to ZmER-abp1 (abp1), the most abundant auxin-binding protein from maize coleoptiles. At the other side, multiple ion transport proteins appear as targets of the auxin signal; the proton pump ATPase, an anion channel and potassium channels. We investigated early electrical responses to auxin at the plasma membrane of tobacco protoplasts. The work presented here will initially focus on abp1 and its functional role at the membrane. The C-terminus abp1 peptide (Pz151–163) was recently reported to modulate K+ currents at the plasma membrane of intact guard cells from broad bean [23] and induce plasma membrane hyperpolarisation of tobacco mesophyll protoplasts. These results further demonstrate that proteins involved in plasma membrane responses to auxin are related to maize abp1, and provide clues as to the region of the protein possibly involved in the interaction of abp1 with the plasma membrane. Secondly, this report concentrates on one of the targets of auxin, a voltage-dependent and ATP-regulated anion channel that we characterised on protoplasts from tobacco cell suspensions. This anion channel was specifically modulated by auxin, as already observed for the anion channel of guard cells [14]. Further work will be needed to assess if this auxin modulation involves a direct interaction between the hormone and the anion channel protein(s), or follows from the activation of a perception chain including abp1 homologues.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00028484
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    Agrobacterium-mediated transformation of Javanica rice (1996)
    Springer
    Molecular breeding 2 (1996), S. 267-276 
    Details
    ISSN: 1572-9788
    Keywords: Agrobacterium ; Javanica rice ; regeneration ; rice ; TAIL-PCR ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Difficulties frequently encountered using direct DNA transfer methods for transformation of Javanica varieties of rice (Oryza sativa L.) have limited the application of biotechnology to these varieties. We now reportAgrobacterium-mediated transformation of Javanica cultivars Gulfmont and Jefferson that are, respectively, widely used or about to enter commercial cultivation in the southern USA. Vigorous, phenotypically normal, fertile plants expressing both the selectable marker and the gene of interest were obtained. Southern analysis showed that only one or two copies of the T-DNA insert were present. Sequence analysis of right border fragments of one line confirmed that insertion was into a coding region of rice nuclear DNA. This analysis also revealed the presence of relatively short regions of permuted T-DNA border sequences, similar to those found afterAgrobacterium-mediated transformation of dicots. Progeny analysis of lines bearing two copies showed co-segregation, indicating that they were located relatively closely on the same chromosome. The introduced genes were transmitted to the R1 and R2 generations in a Mendelian fashion, confirming the suitability of this approach for biotechnological improvement of elite rice cultivars.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00564204
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    Efficient plant regeneration from hairy root-derived protoplasts of Hyoscyamus muticus (1995)
    Springer
    Plant cell reports 14 (1995), S. 738-742 
    Details
    ISSN: 1432-203X
    Keywords: Hyoscyamus muticus ; plant regeneration ; protoplasts ; transformed roots
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Successful plant regeneration was achieved for the first time from hairy root-derived protoplasts of Hyoscyamus muticus. High yields (7 × 106 / g fresh weight) of protoplasts were isolated directly from the transformed roots of Hyoscyamus muticus using an enzyme mixture comprising 1 % macerozyme and 2 % cellulase in an osmoticum consisting of 0.2 M CaCl2 and 0.6 M mannitol. Protoplasts were first cultured in liquid NT/PRO I medium and further on semi-solid NT/PRO II agar medium. The procedure permits highly efficient formation of colonies. The plating efficiency varied from 1–9 %. The small individual colonies regenerated easily into shoots and roots at frequencies of 18 % and 70 %, respectively. The time required for the development of small plantlets from protoplasts was 8–11 weeks. The regenerated plants contained rolB from Ri-T-DNA and exhibited an altered phenotype compared to the control plants.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00232659
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    Expression of Agrobacterium rhizogenes auxin biosynthesis genes in transgenic tobacco plants (1995)
    Springer
    Plant molecular biology 28 (1995), S. 123-136 
    Details
    ISSN: 1573-5028
    Keywords: Agrobacterium rhizogenes ; auxin biosynthesis genes ; plant cell division ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Plant oncogenes aux1 and aux2 carried by the TR-DNA of Agrobacterium rhizogenes strain A4 encode two enzymes involved in the auxin biosynthesis pathway in transformed plant cells. The short divergent promoter region between the two aux-coding sequences contains the main regulatory elements. This region was fused to the uidA reporter gene and introduced into Nicotiana tabacum in order to investigate the regulation and the tissue specificity of these genes. Neither wound nor hormone induction could be detected on transgenic leaf discs. However, phytohormone concentration and auxin/cytokinin balance controlled the expression of the chimaeric genes in transgenic protoplasts. The expression was localised in apical meristems, root tip meristems, lateral root primordia, in cells derived from transgenic protoplasts and in transgenic calli. Histological analysis showed that the expression was located in cells reactivated by in vitro culture. Experiments using cell-cycle inhibitors such as hydroxyurea or aphidicolin on transgenic protoplast cultures highly decreased the β-glucuronidase activity of the chimaeric genes. These results as well as the histological approach suggest a correlation between expression of the aux1 and aux2 genes and cell division.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00042044
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    A novel wound-inducible extensin gene is expressed early in newly isolated protoplasts of Nicotiana sylvestris (1995)
    Springer
    Plant molecular biology 29 (1995), S. 279-292 
    Details
    ISSN: 1573-5028
    Keywords: Agrobacterium tumefaciens ; extensin ; hydroxyproline-rich cell wall protein ; Nicotiana sylvestris ; protoplasts ; wounding
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A cDNA clone (6PExt 1.2) encoding a novel extensin was isolated from a cDNA library made from 6 h old mesophyll protoplasts of Nicotiana sylvestris. The screening was performed with a heterologous probe from carrot. The encoded polypeptide showed features characteristic of hydroxyproline-rich glycoproteins such as Ser-(Pro)4 repeats and a high content in Tyr and Lys residues. The presence of four Tyr-X-Tyr-Lys motifs suggests the possibility for intramolecular isodityrosine cross-links whereas three Val-Tyr-Lys motifs may participate in intermolecular cross-links. The analysis of genomic DNA gel blots using both the N. sylvestris and the carrot clones as probes showed that the 6PExt 1.2 gene belongs to a complex multigene family encoding extensin and extensin-related polypeptides in N. sylvestris as well as in related Nicotianeae including a laboratory hybrid. This was confirmed by the analysis of RNA gel blots: a set of mRNAs ranging in size from 0.3 kb to 3.5 kb was found by the carrot extensin probe. The 6PExt 1.2 probe found a 1.2 kb mRNA in protoplasts and in wounded tissues as well as a 0.9 kb mRNA which seemed to be stem-specific. The gene encoding 6PExt 1.2 was induced by wounding in protoplasts, in leaf strips and after Agrobacterium tumefaciens infection of stems.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00043652
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    5′-Regulatory region of Agrobacterium tumefaciens T-DNA gene 6b directs organ-specific, wound-inducible and auxin-inducible expression in transgenic tobacco (1995)
    Springer
    Plant molecular biology 29 (1995), S. 1299-1304 
    Details
    ISSN: 1573-5028
    Keywords: Agrobacterium ; gene 6b ; phytohormonal regulation of expression ; T-DNA promoter
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The regulatory activity of a 826 bp DNA fragment located upstream of the pTiBo542 TL-DNA gene 6b coding region was analysed in transgenic tobacco, using β-glucuronidase (gus) as a reporter gene. The region was shown to drive organ-specific, wound- and auxin-inducible expression of the reporter, the effect being dependent on the type and concentration of auxin.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00020470
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    The three-dimensional structure of vascular tissues in Agrobacterium tumefaciens-induced crown galls and in the host stems of Ricinus communis L. (1995)
    Springer
    Planta 196 (1995), S. 597-605 
    Details
    ISSN: 1432-2048
    Keywords: Agrobacterium ; Crown gall ; Fiber ; Phloem anastomoses ; Phloem and xylem differentiation ; Phytohormones (auxin, cytokinin, ethylene, gibberellin) ; Ray differentiation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The three-dimensional pattern of phloem and xylem in 10-d-to two-month-old tumors induced by Agrobacterium tumefaciens (C58) and in adjacent Ricinus communis L. stem tissues was studied in thick sections by clearing with lactic acid and by staining with lacmoid. The crown galls contained two types of vascular strands: treelike branched bundles, which developed towards the tumor surface in fast-growing regions, and globular bundles in the slowly developing parts. Both types of vascular bundles contained xylem and phloem and were continuous with the vascular system of the host plant. The tumor bundles were interconnected by a dense net of phloem anastomoses, consisting of sieve tubes but no vessels. These vascular patterns reflect the apparent synthesis sites, concentration gradients and flow pathways of the plant hormones additionally produced in the tumors upon expression of the T-DNA-encoded genes. The A. tumefaciens-induced crown gall affected vascular differentiation in the host stem. In the basipetal direction, the tumor induced more xylem differentiation directly below it, where the crown-gall bundles joined the vascular system of the host. In the centripetal direction, the crown gall caused the development of pathologic xylem characterized by narrow vessels, giant rays and absence of fibers. On the other hand, most probably as a consequence of its gibberellic acid content, the host plant stimulated a local differentiation of regenerative phloem and xylem fibers with unique ramifications, only at the base of the tumor. However, fibers were absent from the main body of the crown gall. The study shows that A. tumefaciens-induced crown galls are characterized by a sophisticated network of vascular tissues in the tumor and are accompanied by a perturbated vessel system in the host. The hormonal mechanisms controlling vascular differentiation in the tumor and neighboring host tissues are discussed. In addition, the “gall constriction hypothesis” is proposed for explaining the mechanism which gives priority in water supply to the growing gall over the host shoot.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00203661
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    The effect of auxin on cytokinin levels and metabolism in transgenic tobacco tissue expressing an ipt gene (1995)
    Springer
    Planta 196 (1995), S. 84-94 
    Details
    ISSN: 1432-2048
    Keywords: Agrobacterium ; Auxin ; Cytokinin metabolism ; Cytokinin oxidase ; ipt gene ; Nicotiana (cytokinin)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The ipt gene from the T-DNA of Agrobacterium tumefaciens was transferred to tobacco (Nicotiana tabacum L.) in order to study the control which auxin appears to exert over levels of cytokinin generated by expression of this gene. The transgenic tissues contained elevated levels of cytokinins, exhibited cytokinin and auxin autonomy and grew as shooty calli on hormone-free media. Addition of 1-naphthylacetic acid to this culture medium reduced the total level of cytokinins by 84% while 6-benzylaminopurine elevated the cytokinin level when added to media containing auxin. The cytokinins in the transgenic tissue were labelled with 3H and auxin was found to promote conversion of zeatin-type cytokinins to 3H-labelled adenine derivatives. When the very rapid metabolism of exogenous [3H]zeatin riboside was suppressed by a phenylurea derivative, a noncompetitive inhibitor of cytokinin oxidase, auxin promoted metabolism to adenine-type compounds. Since these results indicated that auxin promoted cytokinin oxidase activity in the transformed tissue, this enzyme was purified from the tobacco tissue cultures. Auxin did not increase the level of the enzyme per unit tissue protein, but did enhance the activity of the enzyme in vitro and promoted the activity of both glycosylated and non-glycosylated forms. This enhancement could contribute to the decrease in cytokinin level induced by auxin. Studies of cytokinin biosynthesis in the transgenic tissues indicated that trans-hydroxylation of isopentenyladenine-type cytokinins to yield zeatin-type cytokinins occurred principally at the nucleotide level.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00193221
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    Regeneration of whole plants from protoplasts isolated from tissue-cultured shoot primordia of garlic (Allium sativum L.) (1995)
    Springer
    Plant cell reports 15 (1995), S. 17-21 
    Details
    ISSN: 1432-203X
    Keywords: garlic ; Allium sativum L. ; shoot primordia ; protoplasts ; plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Protoplasts derived from tissue-cultured shoot primordia of garlic (Allium sativum L.) initiated successive cell divisions within 4 days and formed small individual calli (0.2mm in diameter) after 5 weeks of culture on Gamborg's B5 medium supplemented with 0.1% casein hydrolysate, 1mg/1 1-naphthaleneacetic acid and 1mg/1 6-benzylaminopurine. Plating efficiency was roughly 5% at the density of 1x104 protoplasts/ml of medium. Adventitious buds developed from the calli during subsequent subculture on Gamborg's B5 medium supplemented with 40mg/l adenine and 10% coconut milk. When transferred to the same medium without supplements, these buds grew into shoots and rooted. The regenerated garlic plantlets were successfully transferred to the greenhouse and grew into whole plants.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01690245
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    Variability in the response toPseudomonas solanacearum of transgenic lines of potato carrying a cecropin gene analogue (1995)
    Springer
    Potato research 38 (1995), S. 371-378 
    Details
    ISSN: 1871-4528
    Keywords: bacterial wilt ; resistance ; transformation ; Agrobacterium ; S. tuberosum L. ; transgenic plant
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Transgenic potato plants of cv. Désirée carrying an antibacterial gene, coding for a cecropin lytic peptide analogue, were inoculated with a virulent strain ofPseudomonas solanacearum under controlled conditions. The disease index scored during three repeated infection trials indicated an increased variability in plant response among the transgenic lines which gave either a more susceptible or a more resistant response to the pathogen when compared with untransformed Désirée. Immunity toP. solanacearum was not observed, but it was possible to select a group of transgenic lines that showed resistance levels and disease development curves comparable to the field resistant cv. Cruza 148.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF02357742
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    Rapid micropreps and minipreps of Ti plasmids and binary vectors fromAgrobacterium tumefaciens (1995)
    Springer
    Transgenic research 4 (1995), S. 349-351 
    Details
    ISSN: 1573-9368
    Keywords: Agrobacterium ; Ti plasmid ; binary vector ; minipreparation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A simple and versatile procedure has been developed for the isolation of both large helper/Ti plasmids and binary vectors fromAgrobacterium tumefaciens. Using a slightly modified alkaline lysis protocol, intact plasmid can be recovered from cultures grown in standard micro-centrifuge tubes or culture tubes in sufficient yield and purity to allow for restriction analysis on ethidium bromide stained gels of the 〉200 kb Ti plasmid DNA. Contamination by chromosomal DNA is minimal and there is thus no need for isopycnic gradient purification. This same procedure can be combined with a high temperature treatment (37°C) and antibiotic selection to generate preparations containing binary vector DNA that are virtually free of interfering Ti plasmid DNA. Restriction patterns produced from these binary vector DNA preparations are unambiguous and therefore preliminary screening by Southern hybridization can be eliminated.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01972532
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    Cell competence forAgrobacterium-mediated DNA transfer inPisum sativum L. (1995)
    Springer
    Transgenic research 4 (1995), S. 184-191 
    Details
    ISSN: 1573-9368
    Keywords: competence ; transient ; transformation ; Agrobacterium ; grain legume ; histochemical GUS-detection
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Distribution and properties of pea (Pisum sativum L.) cells, competent forAgrobacterium-mediated transformation were analysed byin situ histochemical detection of GUS (β-glucuronidase) activity, 4 d after inoculation with engineeredAgrobacterium tumefaciens. The vector system consisted of the hypervirulent disarmed strain EHA101 and the binary plasmid pIBGUS, carrying an intron-containing, 35S-promotor drivengusA (oruidA) gene and two selectable marker genes. Cells competent for transformation were mainly restricted to the dedifferentiating cells neighbouring the vascular system of cotyledon and epicotyl explants. A standardized assay was developed, allowing determination and quantification of factors influencing number and distribution of competent cells. In etiolated seedlings, competence for transformation decreased with the distance of the epicotyl explant from the shoot apex and was specifically induced by the exogenous application of auxins. Transient expression ofgusA afterAgrobacterium-mediated DNA transfer was dramatically reduced upon application of cell-cycle and DNA replication inhibitors aphidicolin, colchicine and nalidixic acid. GUS expression after direct DNA transfer of double-stranded plasmid DNA (via PEG into protoplasts or via particle bombardment of epicotyl segments) was independent of cell-division/DNA replication. A GUS-positive mutant of EHA101 was constructed to allowin situ analysis of attaching bacteria within the plant tissue. Attachment and invasion was inhibited by well-developed cuticula but was restored after chloroform treatment of the tissue surface. Moreover, no correlation was found between distribution of attaching bacteria and the pattern of transformation-competent cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01968783
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  • 77
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    Ti plasmid-encoded octopine and nopaline catabolism in Agrobacterium: specificities of the LysR-type regulators OccR and NocR, and protein-induced DNA bending (1995)
    Springer
    Molecular genetics and genomics 249 (1995), S. 102-110 
    Details
    ISSN: 1617-4623
    Keywords: Agrobacterium ; Ti plasmid ; Activator ; Opines ; LysR family
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The occ and noc regions in octopine and nopaline Ti plasmids, respectively, are responsible for the catabolism of octopine and nopaline in Agrobacterium. The functions are activated in the presence of the opines by OccR and NocR, two related regulatory proteins, and the promoters contain common sequence motifs. We have investigated heterologous interactions between the regulators and the promoters. Previous experiments using all possible heterologous combinations of opines, regulators, and promoters in vivo had demonstrated that only the combination of nopalme, NocR, and the occ promoter led to limited promoter activation. We now show that OccR and NocR bind to the heterologous promoters in vitro and in vivo. The weak or non-existent promoter activation actually observed could be explained by the assumption that OccR and NocR use different activation mechanisms; we investigated protein-induced DNA bending because of reports that the two regulators differ in this respect. Analysis with a bending vector showed that both OccR and NocR induced a DNA bend that is relaxed in the presence of the respective opine. The data suggest that subtle differences in regulator/promoter interactions are responsible for the inactivity of the heterologous combinations. Investigations with a chimeric NocR/OccR protein indicated that it induced a DNA bend in both promoters. No opine-induced relaxation was detectable with the hybrid, and the inducible promoter was not activated. These findings suggest that bend relaxation may be an integral part of promoter activation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00290241
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  • 78
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    Analysis of viability and cell types of macroalgal protoplasts using flow cytometry (1995)
    Springer
    Journal of applied phycology 7 (1995), S. 413-420 
    Details
    ISSN: 1573-5176
    Keywords: protoplasts ; flow cytometry ; seaweed ; biotechnology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The ability to rapidly distinguish viable sub-populations of cells within populations of macroalgal protoplast isolations was demonstrated using flow cytometry. Viable protoplasts from Ulva sp. and Porphyra perforata J. Ag. were distinguished from non-viable protoplasts based on differential fluorescein accumulation. The identities of cortical and epidermal protoplasts from Macrocystis pyrifera (L.) C. Ag. were inferred based on light-scattering and chlorophyll a autofluorescence. Three cell types could be distinguished among protoplasts released from thalli of P. perforata based on chlorophyll a and phycoerythrin autofluorescence. Mixed protoplast populations of Ulva sp. and P. perforata were also discernable based on relative chlorophyll a and phycoerythrin autofluorescence. The ability to screen heterogenous protoplast populations rapidly, combined with the cell sorting capabilities of many flow cytometers, should prove valuable for seaweed biotechnology.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00003799
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  • 79
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    New plant promoter and enhancer testing vectors (1995)
    Springer
    Molecular breeding 1 (1995), S. 419-423 
    Details
    ISSN: 1572-9788
    Keywords: binary vectors ; gus ; T-DNA ; Agrobacterium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract We describe here a set of binary vectors suitable forAgrobacterium-mediated gene transfer and specially designed for studying plant promoters. These vectors are based on the use of thegus reporter gene, contain multiple unique restriction sites upstream of thegus gene, and minimal promoters for testing the effect of enhancers or activator elements. In addition, an intron-containinggus (uidA) gene was introduced into one of these vectors in order to examine reporter gene activity in tissues whereAgrobacterium contamination may be a problem or in transient expression assays.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01248419
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  • 80
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    Parameters influencing transient and stable transformation of barley (Hordeum vulgare L.) protoplasts (1995)
    Springer
    Plant cell, tissue and organ culture 41 (1995), S. 125-138 
    Details
    ISSN: 1573-5044
    Keywords: Hordeum vulgare ; plasmid uptake ; polyethylene glycol (PEG) ; protoplasts ; transient gene expression ; stable transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cat gene expression has been investigated following PEG-mediated plasmid uptake into barley protoplasts. The uptake conditions optimised for transient expression were employed for stable transformation. Transformed protoplast-derived calli of the cvs. Dissa and Igri, were selected on medium containing G418 at 40 μg ml−1 or kanamycin sulphate at 250 μg ml−1. Absolute transformation frequencies of 28.9×10−5 and 21.3×10−5 were recorded for Dissa with kanamycin sulphate and G418 selection, respectively. The frequency for Igri was 11.5×10−5 with G418 selection. Antibiotic resistant protoplast-derived colonies expressed NPTII activity; Southern hybridisation confirmed integration of the nptII gene into barley genomic DNA.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00051581
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  • 81
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    Plasma membrane of younger and outer cells is the primary specific site for aluminium toxicity in roots (1995)
    Springer
    Plant and soil 171 (1995), S. 105-112 
    Details
    ISSN: 1573-5036
    Keywords: Al tolerance ; 1 ; 3-β-glucan ; K leakage ; plasma membrane ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Experiments were carried out to identify the primary site for aluminium (Al) toxicity in roots. Al accumulated in large amounts in the younger and outer cells in roots of pea and was retarded when the ionic strength of the Al solution was high. Cell destruction was extensive in the regions with high Al accumulation. The accumulation of Al in, and potassium (K) leakage from, the root tip were in the order pea〉maize〉rice, the same order as their sensitivity to Al. The protoplasts from the root tip portion of pea incubated with Al showed a wrinkled and uneven surface. The protoplasts progressively shrank and eventually collapsed. Viability decreased in this process. In the control protoplasts of maize, β-glucan formation was uniform on the spherical surfaces, whereas it was spotty in the Al-treated protoplasts; the cell wall material of the latter contained partly 1, 3-β-glucan which is known to be synthesised by 1, 3-β-glucan synthase embedded in the plasma membrane. These results suggest that the specific site for Al toxicity is the plasma membrane of younger and outer cells in roots and that Al tolerance depends largely on the integrity of the plasma membrane.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00009571
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  • 82
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    Plant regeneration from explant and protoplast derived calluses of Medicago littoralis (1995)
    Springer
    Plant cell, tissue and organ culture 41 (1995), S. 41-48 
    Details
    ISSN: 1573-5044
    Keywords: callus ; Medicago littoralis ; plant regeneration ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Plant regeneration from explant and protoplast derived callus has been achieved in Medicago littoralis cv. Harbinger 1886, an annual legume resistant to the fungus Pseudopeziza medicaginis. Callus was induced from different tissue explants and the fastest growth rate was observed for hypocotyls in B5 medium with 2 mg l−1 2,4-dichlorophenoxyacetic acid and 0.5 mg l−1 N6-benzyladenine. Protoplasts were isolated from cotyledons and leaves of sterile plants and from callus; the first two kinds of protoplasts showed a plating efficiency of 5.6% and 5%, respectively, when embedded in agarose. Plant regeneration occurred on media containing % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9qq-f0-yqaqVeLsFr0-vr% 0-vr0db8meaabaqaciGacaGaaeqabaWaaeaaeaaakeaacaqGobWaaW% baaSqabeaacaqG2aaaaOGaaeOVfiaabs5adaahaaWcbeqaaiaaikda% aaGccaqG+waaaa!3F97!\[{\text{N}}^{\text{6}} {\text{\Delta }}^2 {\text{}}\]isopentenyl-adenine combined with indole-3-acetic acid or 1,2-benzisoxazole-3-acetic acid, and on media with N6-benzyladenine plus α-naphtaleneacetic acid; a cytokinin/auxin ratio higher than 1 induced embryos while a ratio around 1 stimulated shoot formation. Embryo development and rooting of shoots were performed in RL medium without growth regulators.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00124085
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  • 83
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    The growth form of the inducing microorganism and chitin addition affect mycolytic enzyme production byTrichoderma harzianum (1995)
    Springer
    Journal of industrial microbiology and biotechnology 15 (1995), S. 397-400 
    Details
    ISSN: 1476-5535
    Keywords: mycolytic enzymes ; Trichoderma harzianum ; protoplasts ; Rhizopus nigricans ; induction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The effect of the growth form of the inducing microorganism on specificTrichoderma harzianum mycolytic enzyme production was studied. The pelleted form ofRhizopus nigricans gave a better product concerning protoplast formation ability. The maximum yield of protoplasts from the target fungusCochliobolus lunatus was 1×108 ml−1. Analysis of individual specific enzyme activities inTrichoderma mycolytic enzyme preparations confirms the importance of high chitinase and low protease activity for high protoplast yields. Supplementation of the production medium with chitin increased the chitinase activity in theTrichoderma exoenzyme mixture.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01569963
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  • 84
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    Tissue culture response in various wild and cultivated Solanum germplasm accessions for exploitation in potato breeding (1995)
    Springer
    Plant cell, tissue and organ culture 41 (1995), S. 151-158 
    Details
    ISSN: 1573-5044
    Keywords: potato ; protoplasts ; regeneration ; Solanum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The response to different in vitro methods for use in potato breeding has been evaluated in 11 genotypes of 5 Solanum species, S. etuberosum, S. lycopersicoides, S. maglia, S. rickii, and S. tuberosum. Callus induction and growth, and shoot regeneration were strongly influenced by the genotype, explant source, and medium utilized. Furthermore, considerable differences among the 11 genotypes were found both in plating efficiency and shoot regeneration from protoplast culture. Some interesting correlations were found between different tissue culture responses, suggesting linkage and/or pleiotropic effect of genes. The potential application to potato breeding of the in vitro techniques analyzed is discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00051584
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  • 85
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    Efficient transformation and regeneration of carnation cultivars usingAgrobacterium (1995)
    Springer
    Molecular breeding 1 (1995), S. 283-293 
    Details
    ISSN: 1572-9788
    Keywords: Agrobacterium ; carnation ; Dianthus caryophyllus ; regeneration ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract We have developed an efficient method for transformation and regeneration of plants from carnation,Dianthus caryophyllus L. Whole leaves fromin vitro shoot cultures were mixed withAgrobacterium, cocultivated for 5 days and then plated on 2 µg/l chlorsulfuron (CS). Regenerated shoots and shoot clusters were divided into smaller sections and plated on 3 µg/l CS for selection to produce fully transformed shoots. Geneticin (G418) and kanamycin used were not as effective selective agents as CS. All regenerated shoots were vitrified. These were normalized, rooted and transferred to the greenhouse. 100% of regenerated plants were transformed based on rooting assay, GUS assay, PCR and Southern analysis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF02277428
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  • 86
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    Crop improvement through tissue culture (1995)
    Springer
    World journal of microbiology and biotechnology 11 (1995), S. 409-415 
    Details
    ISSN: 1573-0972
    Keywords: Breeding ; embryo culture ; haploids ; micropropagation ; protoplasts ; synthetic seed ; transformation ; wide hybridization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Plant tissue culture comprises a set of in vitro techniques, methods and strategies that are part of the group of technologies called plant biotechnology. Tissue culture has been exploited to create genetic variability from which crop plants can be improved, to improve the state of health of the planted material and to increase the number of desirable germplasms available to the plant breeder. Tissue-culture protocols are available for most crop species, although continued optimization is still required for many crops, especially cereals and woody plants. Tissueculture techniques, in combination with molecular techniques, have been successfully used to incorporate specific traits through gene transfer. In vitro techniques for the culture of protoplasts, anthers, microspores, ovules and embryos have been used to create new genetic variation in the breeding lines, often via haploid production. Cell culture has also produced somaclonal and gametoclonal variants with crop-improvement potential. The culture of single cells and meristems can be effectively used to eradicate pathogens from planting material and thereby dramatically improve the yield of established cultivars. Large-scale micropropagation laboratories are providing millions of plants for the commercial ornamental market and the agricultural, clonally-propagated crop market. With selected laboratory material typically taking one or two decades to reach the commercial market through plant breeding, this technology can be expected to have an ever increasing impact on crop improvement as we approach the new millenium.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00364616
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  • 87
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    Protoplast technology for the breeding of top-fruit trees (Prunus, Pyrus, Malus, Rubus) and woody ornamentals (1955)
    Springer
    Euphytica 85 (1955), S. 287-294 
    Details
    ISSN: 1573-5060
    Keywords: protoplasts ; protoclonal variation ; somatic hybridization ; top-fruit trees ; woody ornamentals
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Until recently, temperate fruit trees and woody ornamentals have been regarded as recalcitrant to biotechnological breeding approaches based on protoplasts. This however should no longer be the case, as procedures are now available, not only for the regeneration of complete plants from protoplasts of various tissues of such species, but also for the exploitation of protoplast technology for their genetic manipulation. This paper will examine the recent advances and state of the art in this domain, with particular attention to the use of protoplast technology as a novel tool in the breeding of rosaceous top-fruit tree species and woody ornamentals. Problems and their solutions within the context of regenerating plants from isolated protoplasts of stone (Prunus spp.), pome (Pyrus spp., Malus spp.) and small (Rubus spp.) fruits, and of several shrubby ornamental genotypes (Lonicera spp., Weigela spp., Forsythia spp., Cotoneaster spp.) will be addressed. Interspecific (Prunus spinosa + Prunus cerasifera) and intergeneric (Forsythia spp. + Syringa spp.) somatic hybridization within this group of species, as well as the use of protoplasts for host/pathogen interaction studies (Pyrus/Erwinia amylovora) will also be discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00023958
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  • 88
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    Transformation of lily by Agrobacterium (1955)
    Springer
    Euphytica 85 (1955), S. 97-100 
    Details
    ISSN: 1573-5060
    Keywords: Agrobacterium ; transformation ; lily ; β-glucuronidase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Lily cv. Harmony was inoculated with several Agrobacterium strains to study its susceptibility to Agrobacterium infection and transformation. Tumorous tissue formation on inoculated stem internodes of sterile-grown plantlets, as well as expression of a β-glucuronidase marker gene interrupted by an intron in cells of inoculated stem nodes, indicate that the monocotyledon Lilium is a host for Agrobacterium.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00023935
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  • 89
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    A study of different (CaMV 35S and mas) promoter activities and risk assessment of field use in transgenic rapeseed plants (1955)
    Springer
    Euphytica 85 (1955), S. 411-416 
    Details
    ISSN: 1573-5060
    Keywords: Agrobacterium ; Brassica napus ; CaMV 35S promoter ; mas promoter ; gene expression ; risk assessment
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Gene fusions between the β-glucuronidase (GUS) reporter gene and the promoters of the cauliflower mosaic virus 35S RNA transcript (CaMV 35S) and the mannopine synthase (mas) genes were introduced into rapeseed varieties via Agrobacterium-mediated transformation. Fluorometric assay of β-glucuronidase activity indicated different expression patterns for the two promoters. In seedlings, the CaMV 35S promoter had maximum activity in the primary roots, while the mas promoter was most active in the cotyledons. Etiolated seedlings cultured in the dark showed reduced activity of the mas promoter. Before vernalization at the rosette stage, both promoters were more active in older plant parts than in younger ones. At this stage the highest activity was recorded in cotyledons. After the plants had bolted reduced promoter function was detected in the upper parts of the transformed plants. Both promoters were found to be functional in the majority of the studied organs of transgenic rapeseed plants, but the promoter activity varied considerably between the organs at different developmental stages. The ability of pollen to transfer the introduced genes to other varieties and related species (e.g. Brassica napus and Diplotaxus muralis) by cross-pollination was studied in greenhouse experiments, and field trials were carried out to estimate the distance for biologically-relevant gene dispersal. In artificial crossing, the introduced marker gene was transferable into other varieties of Brassica napus. In field trials, at a distance of 1 metre from the source of transgenic plants, the frequency of an outcrossing event was relatively high (10-3). Resistant individuals were found at 16 and 32 metres from the transgenic pollen donors, but the frequency of an outcrossing event dropped to 10-5.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00023974
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  • 90
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    Gene specificity is maintained in transient expression assays with protoplasts derived from different tissues of barley (1955)
    Springer
    Euphytica 85 (1955), S. 203-207 
    Details
    ISSN: 1573-5060
    Keywords: barley ; electroporation ; PEG-mediated DNA uptake ; promoter analysis ; protoplasts ; transient expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary In some cereal species that are still recalcitrant to stable transformation and regeneration, transient expression in isolated protoplasts is a useful tool for the study of gene expression and regulation. We have successfully applied these techniques to barley protoplasts derived from developing endosperm, aleurone, leaves and roots in order to characterize functionally cis-acting motives in two gene promoters, corresponding to trypsin inhibitor BTI-CMe and to sucrose synthase Ss1. Gene specificity is maintained in transient expression assays with protoplasts isolated from these different barley tissues and the pattern of expression parallels the mRNA levels observed for the corresponding genes in the same tissues.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00023949
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  • 91
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    The phenotypic characterisation of R2 generation transgenic rice plants under field and glasshouse conditions (1955)
    Springer
    Euphytica 85 (1955), S. 395-401 
    Details
    ISSN: 1573-5060
    Keywords: Oryza sativa L. cv. Taipei 309 ; rice ; protoplasts ; direct DNA uptake ; kanamycin-resistant transgenic plants ; field trial ; glasshouse trial ; neomycin phosphotransferase II (npt II) gene ; gene expression and inheritance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary The phenotypes of seed progeny (R2 generation) of Oryza sativa L. cv. Taipei 309, which carried the neomycin phosphotransferase II (npt II) gene, were compared with those of non-transformed, protoplast-derived plants of the same generation and non-transformed, seed-derived plants under field and glasshouse conditions. Under both conditions the transgenic plants were generally smaller, took longer to flower and had reduced fertility. Significant differences were observed between individuals within the group of transgenic plants. The npt II gene was present in most of the transgenic plants, but NPT II activity was only detected in a minority of individuals.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00023972
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  • 92
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    Studies of the mechanism of transgene integration into plant protoplasts: improvement of the transformation rate (1955)
    Springer
    Euphytica 85 (1955), S. 53-61 
    Details
    ISSN: 1573-5060
    Keywords: bleomycin ; direct gene transfer ; expression ; irradiation ; petunia ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary The production of transgenic plants by means of direct gene transfer to protoplasts is now a widely-used technique. The biological mechanisms underlying the transformation are still poorly understood, but many investigations have attempted to shed light on some components of this process. Varying the experimental conditions has in some cases led to better transformation rates, but further improvements of the protocols are possible. Such improvements will require a better understanding of how the alien DNA enters the cells, becomes integrated into the chromosomes and is treated as a part of the plant genome. Irradiation with sublethal doses of X-rays or UV-light has been shown to increase the transformation frequency, while certain drugs have been shown to act in a similar manner. The effects of these and other factors are discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00023930
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  • 93
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    An assessment of morphogenic and transformation efficiency in a range of varieties of potato (Solanum tuberosum L.) (1955)
    Springer
    Euphytica 85 (1955), S. 101-108 
    Details
    ISSN: 1573-5060
    Keywords: Agrobacterium ; plant regeneration ; potato ; Solanum tuberosum ; tissue culture ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary To provide a truly genotype-independent transformation system, it is necessary to be able to transform a wide range of potato genotypes. The ability to regenerate shoots in vitro was determined for 34 potato varieties using tuber disc explants. Following a culture regime used extensively in previous studies with the variety Desiree, half of the varieties could be regenerated from tuber discs and half could not. From a sample of varieties that could be regenerated from tuber discs, all but one variety gave transgenic plants. Twelve varieties were evaluated for the capacity to regenerate shoots from leaf and internode explants excised from in vitro grown plants. All of the varieties tested regenerated adventitious shoots. Leaf and internode explants from 5 varieties were subsequently used for transformation, and transgenic plants were produced from two potato varieties that did not give transgenic plants from tuber disc explants. Some varieties could not be transformed by either method, and will require modification of the in vitro regeneration and transformation system to be successful.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00023936
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  • 94
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    Some methodological aspects of apple transformation by Agrobacterium (1955)
    Springer
    Euphytica 85 (1955), S. 131-134 
    Details
    ISSN: 1573-5060
    Keywords: apple ; transformation ; Agrobacterium ; preculture ; azacytidine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Leaf explants of apple cvs Gala and Golden Delicious were infected with the Agrobacterium tumefaciens strain AGL0(pMOG410). The effects of a 2 d preculture of the explants before infection and the addition of 5-azacytidine to the selection medium were studied. The percentages of GUS-positive explants after 5 w did not significantly alter due to these treatments. One of the ‘Gala’ shoots, which was removed from a leaf explant cultured for 8 w on selection medium, proved to be GUS-positive and will be analyzed further. In general, however, it should be concluded that regeneration of transgenic shoots directly from leaf tissue was not very effective.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00023941
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