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  • 1
    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
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  • 2
    ISSN: 1573-5028
    Keywords: cotton fiber ; transgenics ; particle bombardment ; fiber properties ; cDNA and genes ; nucleotide sequence
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Two genes, each corresponding to fiber mRNA E6, were isolated from cotton cultivars Coker 312 (Gossypium hirsutum L.) and Sea Island (G. barbadense L.). E6 is one of the predominant fiber-specific mRNAs present during early fiber development. The distinguishing feature of the nucleotide-derived E6 protein is the presence of a motif where a dimer, Ser-Gly, is repeated several times. Two of the Sea Island genes contained a pentameric motif, Ser-Gly, while one of the Coker genes had one and the other had four motifs each. cDNA clones containing one or five Ser-Gly motifs were also identified. Thus, it appears that the strict conservation of this motif may not be critical to E6 protein function. Sequence characterizations of the genes and cDNAs showed that multiple members of the E6 family are transcribed in fiber and may result in proteins 238 to 246 amino acids long. The 3′ ends of the genes and cDNAs showed considerable heterology among them. Transgenic plants containing antisense genes were generated to decipher E6 function. Transgenic cotton with reduced E6 protein levels in the range of 60 to 98% were identified. However, no discernible phenotypic changes in fiber development or properties were apparent. This result leads to the conclusion that E6 is not critical to the normal development or structural integrity of cotton fibers.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1573-9368
    Keywords: cultured liverwort cells ; hygromycin phosphotransferase (HPT) ; Marchantia polymorpha ; particle bombardment ; stable transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Suspension-cultured cells (A-18 line) of the liverwortMarchanta polymorpha were bombarded by a pneumatic particle gun with plasmid pCH harbouring the hygromycin phosphotransferase (HPT) gene (hpt) under the control of the cauliflower mosaic virus (CaMV) 35 S promoter and the nopaline synthase polyadenylation region. Nine weeks after bombardments, 128 hygromycin-resistant calluses were obtained from an approximate total of 7×106 cells. Ten cell lines chosen randomly were analysed further. Southern blot analysis showed that all of the ten lines contain thehpt gene in the genome, demonstrating that these lines are transformants. An HPT enzyme activity assay confirmed the expression of the gene in all of the transformant lines.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1572-9788
    Keywords: bar gene ; glufosinate ; herbicide tolerance ; particle bombardment ; Oryza sativa L.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The commerical cultivars ‘Gulfmont’, ‘IR72’ and ‘Koshihikari’ were genetically engineered using electric discharge particle bombardment to express the bar gene which confers resistance to the broad-range herbicide glufosinate. Southern and northern blot analyses of transgenics material revealed stable integration and expression of introduced transgenes in the lines evaluated. In a few plants, silencing of the uidA marker gene was detected at the transcriptional level. Field studies were conducted in 1993 and 1994 at the Rice Research Station near Crowley, LA. This report summarizes results from the first two years of field trial for transgenic Gulfmont and Koshihikari. Transgenic cultivar IR72 was tested in 1995 and preliminary results are similar to those reported for transgenic Gulfmont. All 11 independently derived transgenic lines produced fertile, normal looking seed at maturity. Significant differences were observed in the absence of the herbicide between parental cultivars and transgenic Gulfmont-and Koshihikari-derived lines for days to 50% heading (20% of transgenic lines), plant height (13%), and grain yield (7%). Foliar application of glufosinate had little or no effect on agronomic performance of all transgenic Gulfmont and IR72 lines, while herbicide applications affected grain, yield and plant height of some transgenic Koshhikari. Non-transgenic plants of all three cultivars at the 4-leaf stage were killed within 7 days after 1.12 or 2.24 kg/ha glufosinate applications. Significant differences among certain transgenic lines were observed for agronomic traits after herbicide applications. These results demonstrate that the bar gene was effective in conferring field-level resistance to glufosinate in rice. Variation among transgenic lines required traditional breeding selection procedures to identify superior agronomic types with high levels of herbicide resistance and showed the necessity to generate several independent transgenic lines of each cultivar.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1572-9788
    Keywords: cassava ; genetic modification ; luciferase ; particle bombardment
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Cassava embroids derived from friable embryogenic callus of the genotype TMS60444 were bombarded with DNA of the constructs pJIT100 or pJIT64. Both constructs contain the non-invasive reporter gene luciferase from firefly driven by the CaMV 35S promoter. The influence of several particle gun machine parameters and pretreatment of plant material on transient luciferase activity were studied to determine the most essential conditions for stable transformation. Two weeks after bombardment pieces of friable calli with luciferase activity were selected. In total, 67 independent selected calli with luciferase activity (spots), derived from five different experiments, were further cultured either in liquid or on solid medium. Per plate or flask one spot was cultured. In subsequent selection rounds all spots of one individual plate or flask were cultured as one individual group. In this way different transformation events were separated and multiplied. Eight weeks after bombardment 34 cultures still contained luciferase activity. The mean number of luciferase spots per culture had increased from 1 to 4.6 spots in liquid and to 2.5 spots on solid medium. After two more months of subsequent culture and luciferase selection presence of the construct in these cultures was confirmed at the molecular level using the polymerase chain reaction assay and Southern analysis. Friable embryos derived from four transformation events were cultured for maturation. Between 3% and 21% of the mature embryos of the different transformation events were luciferase-positive. After multiplication of the luciferase-positive mature embryos by secondary somatic embryogenesis they were germinated. The plantlets analysed contained one to several copies of the inserted DNA. The method presented enables the transformation of this particular cassava genotype, thus allowing the genetic improvement of this important tropical crop by transgenesis.
    Type of Medium: Electronic Resource
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