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  • 1
    ISSN: 1573-5044
    Keywords: Agrobacterium tumefaciens ; Catharanthus roseus ; cytokinin ; isopentenyl-transferase ; stress response
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cytokinins play a critical role in several aspects of plant growth, metabolism and development. We previously reported that adding cytokinins to the culture medium of a suspension-cultured cell line of periwinkle increased the accumulation of indole alkaloids, and our aim was to compare the effect of exogenously-applied cytokinins with that of elevated levels of endogenous cytokinins on indole alkaloid production. We used an Agrobacterium tumefaciens strain yielding a plasmid with the isopentenyl transferase gene under control of its own promoter. Co-culture of suspension cells with the bacteria caused a severe stress response leading to cell necrosis. Therefore, we failed to transform this material but we succeeded in transforming periwinkle cotyledons. We verified that callus cultures generated from the isopentenyl transferase-transgenic cotyledons accumulated high cytokinin concentrations. Treating normal callus cultures (generated from untransformed cotyledons) with cytokinins enhanced their alkaloid production. By contrast, the enhanced concentration of endogenous cytokinins in transgenic calli did not increase indole alkaloid production, and thus did not mimic the effect of exogenously-applied cytokinins. Hypothesis to explain this discrepancy are discussed.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1573-5028
    Keywords: Catharanthus roseus ; cell biochemical differentiation ; cytokinin ; intracellular pathogenesis-related protein ; pollen allergen ; RT- and RACE-PCR
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cytokinin treatment of periwinkle callus cultures increased the accumulation of a protein, designated T1, in two-dimensional separated protein extracts. The first 30 NH2-terminal amino acids were determined by Edman degradation and showed significant sequence homology with intracellular pathogenesis-related (IPR) plant proteins and the Bet v 1 allergen family. The deduced amino acid sequence of cDNAs coding for T1, isolated by RT-PCR and 5′ RACE-PCR, exhibited an average sequence identity of 40% with both IPR and Bet v 1-related allergens. T1 and all related proteins contained a p-loop motif typically found in nucleotide-binding proteins as the most conserved sequence feature. Northern blot analysis showed that cytokinin treatment of periwinkle callus induced T1 transcripts, whereas addition of 2,4-dichlorophenoxyacetic acid inhibited this accumulation. Hybridization of genomic periwinkle DNA with the T1 cDNA suggested that the protein is encoded by a single-copy gene. Immunoblot studies with a panel of Bet v 1-specific antibodies and sera from Bet v 1 allergic individuals identified T1 as a protein that is immunologically distinct from the Bet v 1 allergen family and has no allergenic properties.
    Type of Medium: Electronic Resource
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