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  • 2000-2004  (19)
  • 1955-1959  (12)
  • 1930-1934
  • 1890-1899
  • Agrobacterium
  • gene transfer
  • protoplasts
  • 1
    Electronic Resource
    Electronic Resource
    Agrobacterium mediated transformation of chickpea (Cicer arietinum L.) embryo axes (2000)
    Springer
    Plant cell reports 19 (2000), S. 235-240 
    Details
    ISSN: 1432-203X
    Keywords: Key words Chickpea ; Transformation ; Agrobacterium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  Embryo axes of four accessions of chickpea (Cicer arietinum L.) were treated with Agrobacterium tumefaciens strains C58C1/GV2260 carrying the plasmid p35SGUSINT and EHA101 harbouring the plasmid pIBGUS. In both vectors the GUS gene is interrupted by an intron. After inoculation shoot formation was promoted on MS medium containing 0.5 mg/l BAP under a selection pressure of 100 mg/l kanamycin or 10 mg/l phosphinothricin, depending on the construct used for transformation. Expression of the chimeric GUS gene was confirmed by histochemical localization of GUS activity in regenerated shoots. Resistant shoots were grafted onto 5-day-old dark-grown seedlings, and mature plants could be recovered. T-DNA integration was confirmed by Southern analysis by random selection of putative transformants. The analysis of 4 plantlets of the T1 progeny revealed that none of them was GUS-positive, whereas the presence of the nptII gene could be detected by polymerase chain reaction.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002990050005
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  • 2
    Electronic Resource
    Electronic Resource
    An Agrobacterium tumefaciens transformation protocol effective on a variety of cottonwood hybrids (genus Populus) (2000)
    Springer
    Plant cell reports 19 (2000), S. 315-320 
    Details
    ISSN: 1432-203X
    Keywords: Key words Poplar ; Populus ; Aspen ; Cottonwood ; Transformation ; Agrobacterium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  We describe a protocol for Agrobacterium tumefaciens-mediated transformation of hybrid cottonwoods (Populus sections Tacamahaca Spach. and Aigeiros Duby). The protocol has allowed routine transformation of several economically important cottonwood hybrids (Populus trichocarpa Torr. & Gray×P. deltoides Bartr. ex. Marsh. and P. deltoides×P. nigra L.) that were previously difficult to transform. The procedure was applied to 11 different hybrid cottonwood genotypes and one P. deltoides genotype using kanamycin as the selection agent. Additional experiments showed a very strong interaction between auxin preculture and the effectiveness of various cytokinins for induction of shoot organogenesis. The data also demonstrated the superiority of Agrobacterium strain EHA105 over C58 and LBA4404 for T-DNA transfer based on transient assays with a reporter gene.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002990050019
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  • 3
    Electronic Resource
    Electronic Resource
    Agrobacterium tumefaciens-mediated transformation and transgenic-plant regeneration of onion (Allium cepa L.) (2000)
    Springer
    Plant cell reports 19 (2000), S. 376-381 
    Details
    ISSN: 1432-203X
    Keywords: Key words Allium cepa ; Transformation ; Regeneration ; gfp gene ; Agrobacterium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  An Agrobacterium tumefaciens-mediated transformation method has been developed for onions (Allium cepa L.) using immature embryos as the explant source. Transgenic plants were recovered from the open-pollinated onion cultivar Canterbury Longkeeper at a maximum transformation frequency from immature embryos of 2.7%. The method takes between 3–5 months from explant to primary regenerant entering the glasshouse. Multiple-shoot formation from primary transgenic material made possible the clonal multiplication of transformants. The binary vector used carried the nptII antibiotic resistance gene and the m-gfp5-ER reporter gene. Transgenic cultures were initially screened for their ability to fluoresce and to grow in the presence of geneticin (5–25 mg/l). The transgenic nature of individual plants was confirmed by Southern blot analysis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002990050743
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  • 4
    Electronic Resource
    Electronic Resource
    Effect of exogenous calcium on Agrobacterium tumefaciens-mediated gene transfer in Hevea brasiliensis (rubber tree) friable calli (2000)
    Springer
    Plant cell reports 19 (2000), S. 851-855 
    Details
    ISSN: 1432-203X
    Keywords: Key words Rubber tree ; Agrobacterium tumefaciens ; calcium ; gene transfer
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The influence of CaCl2 was investigated on Agrobacterium tumefaciens-mediated gene transfer in Hevea brasiliensis friable calli which are usually proliferated on maintenance medium (MM) containing 9 mM CaCl2.Five A. tumefaciens strains (C58pMP90, C58pGV2260, AGL1, LBA4404 and EHA 105) and two binary vectors (pGIN and pCAMBIA2301) were tested and the strain EHA105pC2301 was selected to conduct further experiments. The calli were precultured on MM containing a range of CaCl2 concentrations, then inoculated with Agrobacterium suspension. Transfer of friable calli from MM containing 9 mM CaCl2 to calcium-free medium significantly enhanced the transient β-glucuronidase activity. Interestingly, the use of calcium-free Agrobacterium resuspension medium to inoculate friable calli again dramatically increased the transformation efficiency. Induction of Agrobacterium's virulence with acetosyringone remained an important factor to stimulate transformation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002990000208
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  • 5
    Electronic Resource
    Electronic Resource
    Spacer Length Dependence on the Efficiency of Dimeric Anionic Peptides in Gene Transfer by Glycosylated Polylysine/Plasmid Complexes (2000)
    Springer
    Bioscience reports 20 (2000), S. 383-398 
    Details
    ISSN: 1573-4935
    Keywords: fusogenic peptides ; HA2 influenza virus hemagglutinin ; polylysine ; transfection ; gene transfer
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Amphiphilic anionic peptides have been used to enhance the efficiency of transfection by helping plasmids to escape from endosomes to the cytosol. It has been shown that efficiency of an eicosamers containing five glutamyl residues (E5), can be considerably enhanced either by transforming it into a dimer or by adding a tripeptide WYG in a C-terminal position (E5WYG). The dimerization of the peptide E5WYG leads to a more efficient tool when the dimerization device includes the tripeptide WYG unit and a longer spacer arm made of Gly-βAla-βAla residues, but to a 10-fold less efficient tool when the dimerization device includes a shorter spacer, a glycyl residue. Both dimers are taken up by the cells to a similar extent. Both dimers seem to be surrounded similarly as far as the environmental pH is concerned. In contrast, we found a correlation between the propensity of the peptides to adopt a helical structure at neutral pH and the gene transfer efficiency.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1010382001654
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  • 6
    Electronic Resource
    Electronic Resource
    Efficient Gene Delivery Using Anionic Liposome-Complexed Polyplexes (LPDII) (2000)
    Springer
    Bioscience reports 20 (2000), S. 419-432 
    Details
    ISSN: 1573-4935
    Keywords: liposomes ; gene therapy ; gene transfer ; nonviral vectors ; polylysine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Synthetic gene transfer vectors based on polyplexes complexed to anionic liposomes (LPDII vectors) were characterized for their transfection efficiency in cultured mammalian cells. The effects of polycation to DNA ratio, lipid to DNA ratio, choice of polycation and lipid composition were systematically evaluated in human oral carcinoma KB cells, using a luciferase reporter gene. For LPDII formulations containing poly-L-lysine and dioeoylphosphatidylethanolamine/cholesteryl hemisuccinate (DOPE/CHEMS) anionic liposomes, at a constant lipid to DNA ratio, an increase in the polycation/DNA (N/P) ratio resulted in an increase in transfection activity. Meanwhile, the optimal lipid to DNA ratio for efficient gene delivery was influenced by the N/P ratio used, and was increased at higher N/P ratios. For the DNA condensing agent, poly-L-lysine could be replaced by polyethylenimine (PEI) as the DNA condensing agent in the formulations. For the lipidic components, CHEMS could be replaced by other anioniclipids including oleic acid, dicetylphosphate and phosphatidylserine, but DOPE, a fusogenic helper lipid, could not be replaced by dioleolyphosphatidylcholine. LPDII formulation showed significantly less cytotoxicity compared to the commonly used cationic lipsomes or PEI mediated transfection and several cell lines were transfected with high efficiency. LPDII vectors avoid the use of toxic cationic lipids and may have potential application in gene therapy.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1010338219401
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  • 7
    Electronic Resource
    Electronic Resource
    Agrobacterium tumefaciens-mediated transformation of the aromatic shrub Lavandula latifolia (2000)
    Springer
    Molecular breeding 6 (2000), S. 539-552 
    Details
    ISSN: 1572-9788
    Keywords: Agrobacterium ; inheritance analysis ; Lavandula latifolia ; lavender ; organogenesis ; transgenic plants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Transgenic plants of the aromatic shrub Lavandula latifolia (Lamiaceae) were produced using Agrobacterium tumefaciens-mediated gene transfer. Leaf and hypocotyl explants from 35–40-day old lavender seedlings were inoculated with the EHA105 strain carrying the nptII gene, as selectable marker, and the reporter gusA gene with an intron. Some of the factors influencing T-DNA transfer to L. latifolia explants were assessed. Optimal transformation rates (6.0 ± 1.6% in three different experiments) were obtained when leaf explants precultured for 1 day on regeneration medium were subcultured on selection medium after a 24 h co-cultivation with Agrobacterium. Evidence for stable integration was obtained by GUS assay, PCR and Southern hybridisation. More than 250 transgenic plants were obtained from 37 independent transformation events. Twenty-four transgenic plants from 7 of those events were successfully established in soil. β-glucuronidase activity and kanamycin resistance assays in greenhouse-grown plants from two independent transgenic lines confirmed the stable expression of both gusA and nptII genes two years after the initial transformation. Evidence from PCR data, GUS assays and regeneration in the presence of kanamycin demonstrated a 1:15 Mendelian segregation of both transgenes among seedlings of the T1 progeny of two plants from one transgenic L. latifolia line.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1011332904024
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  • 8
    Electronic Resource
    Electronic Resource
    Study of the factors influencing Agrobacterium-mediated transformation of pea (Pisum sativum L.) (2000)
    Springer
    Molecular breeding 6 (2000), S. 185-194 
    Details
    ISSN: 1572-9788
    Keywords: Agrobacterium ; transformation ; pea ; Pisum sativum L. ; PCR analysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Factors influencing the efficiency of Agrobacterium-mediated transformation of pea were tested using highly efficient, direct regeneration system. The virulence of three Agrobacterium strains (octopine LBA 4404, nopaline C58C1 and succinamopine, hypervirulent EHA 105) clearly varied giving 1 transgenic plant per 100 explants for LBA 4404, 2.2 for C58C1 and 8.2 for EHA 105. To test the efficacy of selection agents we used the hypervirulent EHA 105 strain carrying pGPTV binary vector with one of four different selection genes: nptII, hpt, dhfr or bar. The mean number of transgenic, kanamycin-resistant plants for two cultivars tested was 4.2 per 100 explants and was slightly higher than the number of phosphinothricin-resistant plants (3.6 plants per 100 explants). The proportion of transgenics among kanamycin-selected plants was also higher than among phosphinothricin-resistant plants (35% and 28% respectively). There was no regeneration on hygromycin or methotrexate media (transformation with hpt and dhfr genes). Acetosyringone had no apparent influence on efficiency of transformation with hypervirulent EHA 105 strain, however it did affect the rate of transformation when moderately virulent C58C1 was used. Recovery of transgenic plants was enhanced after application of 5-azacytidine. The presence of integrated T-DNA was checked by PCR and confirmed by Southern hybridization. T-DNA was stably transmitted to the next generation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1009679908948
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  • 9
    Electronic Resource
    Electronic Resource
    Insulin-like Growth Factor-I cDNA Gene Transfer in vitro and in vivo (2000)
    Springer
    Biochemical genetics 38 (2000), S. 41-55 
    Details
    ISSN: 1573-4927
    Keywords: gene transfer ; liposome ; IGF-I ; wound healing ; rat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Our hypothesis is that gene transfer of an IGF-I CMV-cDNA with cholesterol containing cationic liposomes is an efficient tool for transient transfection of growth factors in vitro and in vivo. In vitro, we transiently cotransfected IGF-I cDNA with a CMV construct and a Lac Z CHKβCHK-galactosidase cDNA/CMV construct using cholesterol containing cationic liposomes and measured CHKβCHK-galactosidase and IGF-I mRNA and protein. In vivo, we subcutaneously injected 3-month-old male Sprague–Dawley rats with IGF-I cDNA and CHKβCHK-galactosidase cDNA into rat skin. After IGF-I and CHKβCHK-galactosidase were cotransfected into PC12 cells, Northern blot analysis showed that the peak time of IGF-I expression was 2 days for mRNA and 5 days for protein. In vivo, a cDNA/liposome ratio of 1:2 was most effective. IGF-I protein expression in IGF-I-transfected skin resulted in significant transfection from day 5 to day 7. In situ determination of CHKβCHK-galactosidase activity confirmed that transfections resulted in a restricted expression area.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1001880220039
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  • 10
    Electronic Resource
    Electronic Resource
    A Cationic Lipid Emulsion/DNA Complex as a Physically Stable and Serum-Resistant Gene Delivery System (2000)
    Springer
    Pharmaceutical research 17 (2000), S. 314-320 
    Details
    ISSN: 1573-904X
    Keywords: gene transfer ; lipid emulsions ; poly(ethylene glycol) ; transfection ; cationic lipids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. To develop a non-viral gene delivery system in the form ofan oil-in-water (o/w) lipid emulsionMethod. Cationic lipid emulsions were formulated with soybean oil,1,2-dioleoyl-sn-glycero-3-trimethylammonium-propane (DOTAP) as acationic emulsifier and other co-emulsifiers. The physicalcharacteristics of the lipid emulsion and the emulsion/DNA complex weredetermined. The in vitro transfection efficiency of the emulsion/DNAcomplex was determined in the presence of up to 90% serum. Results. The average droplet size and zeta potential of emulsions wereca. 180 nm and ca. +50 mV, respectively. Among the emulsions, astable formulation was selected to form a complex with a plasmidDNA encoding chloramphenicol acetyltransferase. By increasing theratio of emulsion to DNA, zeta-potential of the emulsion/DNA complexincreased monotonously from negative to positive without any changesin the complex size. The complex was stable against DNase I digestionand an anionic poly-l-aspartic acid (PLAA). The complex deliveredDNA into the cells successfully, and the transfection efficiency wasnot affected by complex formation time from 20 min to 2 h. Moreimportantly, the cationic lipid emulsion facilitated the transfer of DNAin the presence of up to 90% serum. Conclusions. The cationic lipid emulsion/DNA complex has physicalstability and serum resistant properties for gene transfer.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1007553106681
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  • 11
    Electronic Resource
    Electronic Resource
    pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation (2000)
    Springer
    Plant molecular biology 42 (2000), S. 819-832 
    Details
    ISSN: 1573-5028
    Keywords: Agrobacterium ; binary vectors ; plant transformation ; reporter genes ; selectable marker genes ; Ti vector
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Binary Ti vectors are the plasmid vectors of choice in Agrobacterium-mediated plant transformation protocols. The pGreen series of binary Ti vectors are configured for ease-of-use and to meet the demands of a wide range of transformation procedures for many plant species. This plasmid system allows any arrangement of selectable marker and reporter gene at the right and left T-DNA borders without compromising the choice of restriction sites for cloning, since the pGreen cloning sites are based on the well-known pBluescript general vector plasmids. Its size and copy number in Escherichia coli offers increased efficiencies in routine in vitro recombination procedures. pGreen can replicate in Agrobacterium only if another plasmid, pSoup, is co-resident in the same strain. pSoup provides replication functions in trans for pGreen. The removal of RepA and Mob functions has enabled the size of pGreen to be kept to a minimum. Versions of pGreen have been used to transform several plant species with the same efficiencies as other binary Ti vectors. Information on the pGreen plasmid system is supplemented by an Internet site (http://www.pgreen.ac.uk) through which comprehensive information, protocols, order forms and lists of different pGreen marker gene permutations can be found.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1006496308160
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  • 12
    Electronic Resource
    Electronic Resource
    Programmed cell death in cell cultures (2000)
    Springer
    Plant molecular biology 44 (2000), S. 359-368 
    Details
    ISSN: 1573-5028
    Keywords: Arabidopsis ; PCD induction ; programmed cell death ; protoplasts ; suspension cell cultures
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In plants most instances of programmed cell death (PCD) occur in a number of related, or neighbouring, cells in specific tissues. However, recent research with plant cell cultures has demonstrated that PCD can be induced in single cells. The uniformity, accessibility and reduced complexity of cell cultures make them ideal research tools to investigate the regulation of PCD in plants. PCD has now been induced in cell cultures from a wide range of species including many of the so-called model species. We will discuss the establishment of cell cultures, the fractionation of single cells and isolation of protoplasts, and consider the characteristic features of PCD in cultured cells. We will review the wide range of methods to induce cell death in cell cultures ranging from abiotic stress, absence of survival signals, manipulation of signal pathway intermediates, through the induction of defence-related PCD and developmentally induced cell death.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1026500810877
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  • 13
    Electronic Resource
    Electronic Resource
    Regulation of Sodium/Iodide Symporter (2000)
    Springer
    Reviews in endocrine & metabolic disorders 1 (2000), S. 205-215 
    Details
    ISSN: 1573-2606
    Keywords: Na+/I- symporter ; expression ; promoter ; gene transfer ; radioiodide therapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1010083132071
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  • 14
    Electronic Resource
    Electronic Resource
    Occurrence and diversity of plasmids in populations of streptomycetes in soil (2000)
    Springer
    Antonie van Leeuwenhoek 78 (2000), S. 323-329 
    Details
    ISSN: 1572-9699
    Keywords: gene transfer ; plasmids ; Streptomyces
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Studies were made of naturally occurring plasmids hosted in Streptomyces strains isolated from two different terrestrial ecosystems: an agricultural field and a protected forest area. Six out of the 147 screened isolates contained plasmids. The strains containing these plasmids were all isolated from the agricultural soil. Plasmids were not found among the strains isolated from the forest area. Cross hybridization of the six newly isolated plasmids revealed very high similarities between four of them. However, no similarities were found between the six newly isolated plasmids and well studied streptomycete plasmids such as pIJ101 and SCP2*. The host strains of the four similar plasmids belonged to three different species S. anulatus, S. rochei and S. diastaticus. This implies a possible conjugative transfer of these plasmids within the streptomycete population in the agricultural area. The reason for the absence of streptomycete plasmids from the populations derived from the forest area is discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1010229215567
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  • 15
    Electronic Resource
    Electronic Resource
    Plant regeneration from protoplasts of a commercial Asian long-grain javanica rice (2000)
    Springer
    Plant cell, tissue and organ culture 60 (2000), S. 79-82 
    Details
    ISSN: 1573-5044
    Keywords: cell suspensions ; Lolium multiflorum L. ; membrane filter-nurse culture ; Oryza sativa L. ; protoplasts ; two-step shoot regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A reproducible plant regeneration system has been developed for protoplasts from embryogenic cell suspension cultures of the commercial Asian long-grain javanica rice, Oryza sativa cv. Azucena. Protoplasts were isolated routinely from cell suspensions with yields of 5.5–12.0 × 106 g-1 fresh weight. A membrane filter nurse-culture method was adopted and was essential to support sustained mitotic division of protoplast-derived cells, leading to cell colony formation. The protoplast plating efficiency was higher when suspension cells of Lolium multiflorum, rather than those of the japonica rice O. sativa L. cv. Taipei 309, were employed as nurse cells. A two-step shoot regeneration procedure, in which protoplast-derived calli were cultured initially on medium semi-solidified with 1% (w/v) agarose followed by culture on medium containing 0.4% (w/v) agarose, induced plant regeneration from protoplast-derived calli. Fifteen percent of protoplast-derived tissues regenerated shoots; tissues not subjected to this treatment failed to develop shoots.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1006408109595
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  • 16
    Electronic Resource
    Electronic Resource
    Expression of maize γ-zein and β-zein genes in transgenic Nicotiana tabacum and Lotus corniculatus (2000)
    Springer
    Plant cell, tissue and organ culture 62 (2000), S. 141-151 
    Details
    ISSN: 1573-5044
    Keywords: Agrobacterium ; forage quality ; KDEL ; recombinant proteins ; sulphur-rich amino acids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Accumulation of zeins, the endosperm storage proteins of maize, in a heterologous plant expression system was attempted. Plants of Nicotiana tabacum and Lotus corniculatus were transformed by Agrobacterium with binary vectors harbouring genes that code for γ-zein and β-zein, two zeins rich in sulphur amino acids. Adding the ER retention signal KDEL to the C-terminal domain modified the zein polypeptides. Significant levels of γ-zein:KDEL and β-zein:KDEL were detected in primary transformants of tobacco. Moreover, the two zeins colocalized in leaf protein bodies of γ-/β-zein:KDEL plants derived from a cross between two primary transformants. Coexpression of γ-zein:KDEL and β-zein:KDEL could be a useful strategy to obtain genotypes of forage legumes which are able to accumulate sulphur amino acids to high levels. As a first step, L. corniculatus plants expressing γ-zein:KDEL in the leaves were obtained.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1026735106843
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  • 17
    Electronic Resource
    Electronic Resource
    Plant regeneration from embryogenic cell suspension-derived protoplasts of rubber (2000)
    Springer
    Plant cell, tissue and organ culture 61 (2000), S. 81-85 
    Details
    ISSN: 1573-5044
    Keywords: cell suspensions ; embryogenesis ; Hevea brasiliensis ; plant regeneration ; protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Embryogenic cell suspensions of rubber derived from immature inflorescences and inner integuments of immature fruits released 3.1 ± 0.2 × 107 protoplasts g-1 f. wt. (mean ± s.e.m, n = 10) and 3.2 ± 0.2 × 107 protoplasts g-1 f. wt., with mean viabilities of 83 ± 2% and 77 ± 8%, respectively. Sustained mitotic division was observed only when protoplasts were cultured in KPR liquid medium on nitrocellulose membranes overlying the same semi-solid medium containing Lolium multiflorum nurse cells. Protoplast-derived cell colonies were produced within 2 months of culture. Protoplast-derived cell colonies proliferated, upon subculture to MS-based regeneration medium, with 40% of the protoplast-derived calli developing somatic embryos. The latter germinated into plants on the same medium after 3 months of culture.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1006494404224
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  • 18
    Electronic Resource
    Electronic Resource
    Liposomes Containing Interferon-Gamma as Adjuvant in Tumor Cell Vaccines (2000)
    Springer
    Pharmaceutical research 17 (2000), S. 42-48 
    Details
    ISSN: 1573-904X
    Keywords: liposomes ; cancer vaccines ; cytokines ; immunotherapy ; interferon gamma ; gene transfer
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. Liposomal systems may be useful as a cytokine supplementin tumor cell vaccines by providing a cytokine reservoir at the antigenpresentation site. Here, we examined the effect of liposomeincorporation of mIFNγ on its potency as adjuvant in an established tumor cellvaccination protocol in the murine B16 melanoma model. Adjuvanticityof the mIFNγ-liposomes was compared to that achieved bymIFNγ-gene transfection of the B16 tumor cells. Furthermore, we studiedwhether liposomal incorporation of mIFNγ indeed increases theresidence time of the cytokine at the vaccination site. Methods. C57B1/6 mice were immunized with i) irradiated IFNγ-genetransfected B16 melanoma cells or ii) irradiated wild type B16 cellssupplemented with (liposomal) mIFNγ, followed by a challenge withviable B16 cells. The residence time of the (liposomal) cytokine at thesubcutaneous (s.c.) vaccination site was monitored using radiolabeledmIFNγ and liposomes. Results. Immunization with irradiated tumor cells admixed withliposomal mIFNγ generated comparable protection against B16 challenge asimmunization with mIFNγ-gene modified tumor cells. Irradiated tumorcells admixed with soluble mIFNγ did not generate any protectiveresponses. Radiolabeling studies indicated that free mIFNγ rapidlycleared from the s.c. injection site. Association of [125I]-mIFNγwith liposomes increased the local residence time substantially: liposomalassociation of mIFNγ resulted in a prolonged local residence time ofthe cytokine as reflected by a 4-fold increase of the area under thecurve. The amount of released cytokine in the optimal dose rangecorresponds to the amount released by the gene-transfected cells. Moderate but significant CTL-activity against B16 cells was found for miceimmunized with irradiated cells supplemented with mIFNγ-liposomescompared to untreated control animals. Conclusions. Prolonged presence of mIFNγ at the site of antigenpresentation is crucial for the generation of systemic immune responsesin the B16 melanoma model. These studies show that liposomalencapsulation of cytokines is an attractive strategy for paracrine cytokinedelivery in tumor vaccine development.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1007514424253
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  • 19
    Electronic Resource
    Electronic Resource
    Expression of murine adenosine deaminase (ADA) in transgenic maize (2000)
    Springer
    Transgenic research 9 (2000), S. 1-9 
    Details
    ISSN: 1573-9368
    Keywords: gene transfer ; selectable marker ; adenosine analogues ; transgenic maize ; adenosine deaminase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A murine adenosine deaminase (ADA) gene, driven by the maize ubi-1 promoter and intron region, was transformed into embryogenic maize callus, along with a bar and gusA gene-containing plasmid, using microparticle bombardment. Selection in the presence of either the herbicide Basta® or the adenosine analogue 2′-deoxyadenosine resulted in transgenic cultures that expressed GUS and accumulated a 41kD protein that immunoprecipated with an ADA-specific polyclonal antibody. ADA enzyme activity was observed in extracts from transgenic callus as well as regenerated plants and progeny. Cultures expressing ADA grew in the presence of 200mg/l 2′-deoxyadenosine, a concentration which completely inhibited the growth of non-transgenic cultures. ADA activity appeared to segregate in progeny of regenerated plants as a single, dominant Mendelian trait. These results suggest that ADA, in combination with adenosine analogue selection, represents a potentially viable selectable marker system for transgenic maize production.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008972101370
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  • 20
    Electronic Resource
    Electronic Resource
    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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  • 21
    Electronic Resource
    Electronic Resource
    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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  • 22
    Electronic Resource
    Electronic Resource
    Shoot apical meristems as a target for gene transfer by microballistics (1955)
    Springer
    Euphytica 85 (1955), S. 45-51 
    Details
    ISSN: 1573-5060
    Keywords: meristem ; shoot apex ; ballistic microtargeting ; gene transfer ; wheat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary The classical approach of gene transfer to a given plant species delivers the foreign gene to transformable cells and then puts the effort into generating plants. This approach is very difficult in many important crop plants, including cereals, and the results of regeneration are very genotype-dependent. In contrast, we use regenerable cells and try to transform them. Shoot apical meristems provide a tissue which regenerates in situ a fertile plant for most given genotypes or species. Transformation of meristem cells may lead to transgenic sectors in chimeras. These sectors may contribute to the gametes and, thus, to transgenic offspring, which then should be homohistonts and not sectorial chimeras like their parents. Our model plant for these studies is wheat. Microtargeting is a ballistic approach which is particularly suitable for the controlled delivery of microprojectiles to meristem cells in situ (Sautter et al., 1991). We summarize in this paper our experience with ballistic microtargeting of transgenes to wheat shoot apical meristem cells in situ.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00023929
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  • 23
    Electronic Resource
    Electronic Resource
    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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  • 24
    Electronic Resource
    Electronic Resource
    Transgenic barley by particle bombardment. Inheritance of the transferred gene and characteristics of transgenic barley plants (1955)
    Springer
    Euphytica 85 (1955), S. 81-88 
    Details
    ISSN: 1573-5060
    Keywords: gene transfer ; Hordeum vulgare ; neomycin phosphotransferase II ; particle bombardment ; transgenic barley
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Transgenic barley plants (Hordeum vulgare L. cv. Kymppi) were obtained by particle bombardment of various tissues. Immature embryos and microspore-derived cultures were bombarded with gold particles coated with plasmid DNA carrying the gene coding for neomycin phosphotransferase II (NPTII), together with plasmid DNA containing the gene for β-glucuronidase (GUS). Bombarded immature embryos were grown to plants without selection and NPTII activity was screened in small plantlets. One plant proved to be transgenic (T0). This chimeric plant passed the transferred nptII gene to its T1 progeny. The presence of the nptII gene was demonstrated by the PCR technique and enzyme activity was analyzed by an NPTII gel assay. Four T0 spikes and 15 T1 offspring were transgenic. The integration and inheritance was confirmed by Southern blot hybridization. Transgenic T2 and T3 plants were produced by isolating embryos from green grains of transgenic T1 and T2 plants, respectively and growing them to plants. After selfing, the ratio of transgenic to non-transgenic T2 offspring was shown to follow the rule of Mendelian inheritance. The general performance of transgenic plants was normal and no reduction in fertility was observed. Microspore-derived cultures were bombarded one and four weeks after microspore isolation. After bombardment, cultures were grown either with or without antibiotic selection (geneticin R or kanamycin). When cultures were grown without selection and regenerated plants were transferred to kanamycin selection in rooting phase, one out of a total of about 1500 plants survived. This plant both carried and expressed the transferred nptII gene. The integration was confirmed by Southern blot hybridization. This plant was not fertile.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00023933
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  • 25
    Electronic Resource
    Electronic Resource
    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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  • 26
    Electronic Resource
    Electronic Resource
    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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  • 27
    Electronic Resource
    Electronic Resource
    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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  • 28
    Electronic Resource
    Electronic Resource
    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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  • 29
    Electronic Resource
    Electronic Resource
    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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  • 30
    Electronic Resource
    Electronic Resource
    The sulphur-rich Brazil nut 2S albumin is specifically formed in transgenic seeds of the grain legume Vicia narbonensis (1955)
    Springer
    Euphytica 85 (1955), S. 181-192 
    Details
    ISSN: 1573-5060
    Keywords: Vicia narbonensis ; gene transfer ; gene expression ; seeds ; 2S albumin ; methionine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Epicotyl explants were co-cultivated with Agrobacterium tumefaciens EHA101 to transfer a chimeric 2S albumin gene construct carried in the binary Ti plasmid vectors pGSGLUC1 or pGA472 into the grain legume Vicia narbonensis. This gene encoding the sulphur-rich Brazil nut albumin was under the control of either the CaMV 35S promoter which permits gene expression in all organs, or the Vicia faba legumin B4 promoter which elicits seed-specific gene expression. After callus formation and selection for kanamycin resistance, somatic embryos were induced which, in the case of transformation with the vector pGSGLUC1, were screened for GUS activity. Embryos that produced GUS were in addition analysed for 2S albumin formation. Selected transgenic embryos were cloned by multiple shoot regeneration. Rooted and fertile plants were obtained by grafting transgenic shoots on the appropriate seedlings. R1 and R2 generations were raised and analysed for GUS as well as 2S albumin gene expression. Expression of the 35S promoter/2S albumin gene fusion took place in all organs of the transgenic plants including the cotyledons of seeds, whereas seed-specific gene expression was found in transformants with the legumin promoter/2S albumin gene fusion. The 2S albumin accumulated in the 2S protein fraction of transgenic seeds and its primary translation product was processed into the 9 and 3 kDa polypeptide chains. The foreign protein was localised in the protein bodies of the grain legume. Analysis of the R2 plants indicated Mendelian inheritance of the 2S albumin gene. In homozygous V. narbonensis plants the amounts of 2S albumin were twice that present in the corresponding heterozygous plants. Whereas only low level formation of the foreign protein was achieved if the gene was under the control of the 35S promoter, approximately 3.0% of the soluble seed protein was 2S albumin if seed-specific gene expression was directed by the legumin B4 promoter. Some of these transformants exhibited a three-fold increase in the methionine content of the salt-soluble protein fraction extracted from seeds.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00023947
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  • 31
    Electronic Resource
    Electronic Resource
    Strategies for variety-independent genetic transformation of important cereals, legumes and woody species utilizing particle bombardment (1955)
    Springer
    Euphytica 85 (1955), S. 13-27 
    Details
    ISSN: 1573-5060
    Keywords: gene transfer ; crop species ; particle bombardment ; transgenic plants ; cereals ; legumes ; woody plants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary The limiting component in the creation of transgenic crops has been the lack of effective means to introduce foreign genes into elite germplasm. However, the development of novel direct DNA transfer methodology, by-passing limitations imposed by Agrobacterium-host specificity and cell culture constraints, has allowed the engineering of almost all major crops, including formerly recalcitrant cereals, legumes and woody species. The creation of transgenic rice, wheat, maize, barley, oat, soybean, phaseolus, peanut, poplar, spruce, cotton and others, in an efficient and in some cases, variety-independent fashion, is a significant step towards the routine application of recombinant DNA methodology to the improvement of most important agronomic crops. In this review we will focus on key elements and advantages of particle bombardment technology in order to evaluate its impact on the accelerated commercialization of products based on agricultural biotechnology and its utility in studying basic plant developmental processes and function through transgenesis. Fundamental differences between conventional gene transfer methods, utilizing Agrobacterium vectors or protoplast/suspension cultures, and particle bombardment will be discussed in depth.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00023926
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