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  • 1
    ISSN: 1432-203X
    Keywords: Key words:Forsythia, genetic transformation, regeneration, woody ornamentals. ; Abbreviations.BAP: 6‐benzyl-aminopurine; CaMV: Cauliflower Mosaic Virus; GUS and gus: β-glucuronidase; IAA: indole-3-acetic acid; IBA: indole-3-butyric acid; MS: Murashige and Skoog; NOS: nopaline synthase; NPT II and npt II: neomycin phosphotransferase II; PCR: polymerase chain reaction; SDS: sodium dodecyl sulphate; SSC: sodium chloride-sodium citrate; X-Gluc: 5-bromo-4-chloro-3-indolyl glucuronide.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Internode explants of in vitro plants of Forsythia×intermedia "Spring Glory" were transformed with the gus and npt II genes after inoculation with the A. tumefaciens strain EHA 101 harbouring the plasmid pFAJ3000. Shoot organogenesis took place from callused edges of explants. The first transformed buds were detected 4 to 6 weeks after transfer on regeneration medium, containing 25 mg/l kanamycin as selective agent. An averge of 1% of explants regenerated transgenic shoots. β-glucuronidase assays and culture on kanamycin-containing medium provided the first indication of integration and expression of introduced genes in transformants. Southern blot and polymerase chain reaction amplification analyses gave molecular confirmation of genetic transformation. Transgenic plants were acclimatized in the greenhouse. Enzymatic assays on several organs of mature plants still showed β-glucuronidase activity, thus confirming stable integration of T-DNA in the plant genome.
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  • 2
    ISSN: 1432-203X
    Keywords: Forsythia ; genetic transformation ; regeneration ; woody ornamentals
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Internode explants ofin vitro plants ofForsythia x intermedia “Spring Glory” were transformed with thegus andnpt II genes after inoculation with theA. tumefaciens strain EHA 101 harbouring the plasmid pFAJ3000. Shoot organogenesis took place from callused edges of explants. The first transformed buds were detected 4 to 6 weeks after transfer on regeneration medium, containing 25 mg/l kanamycin as selective agent. An average of 1% of explants regenerated transgenic shoots. β-glucuronidase assays and culture on kanamycin-containing medium provided the first indication of integration and expression of introduced genes in transformants. Southern blot and polymerase chain reaction amplification analyses gave molecular confirmation of genetic transformation. Transgenic plants were acclimatized in the greenhouse. Enzymatic assays on several organs of mature plants still showed β-glucuronidase activity, thus confirming stable integration of T-DNA in the plant genome.
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  • 3
    ISSN: 1573-5028
    Keywords: competitive PCR ; flavonoid pathway ; Forsythia ; gene expression ; transformation ; woody ornamentals
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The expression, during flower development, of the gene encoding the anthocyanin pathway key enzyme dihydroflavonol 4-reductase (DFR) was investigated in floral organs of Forsythia × intermedia cv. ‘Spring Glory’. Full-length DFR and partial chalcone synthase (CHS) cDNAs, the gene of interest and a flavonoid pathway control gene respectively, were obtained from petal RNA by reverse transcription PCR. Whereas for CHS northern blot analysis enabled the study of its expression pattern, competitive PCR assays were necessary to quantify DFR mRNA levels in wild-type plants and in petals of 2 transgenic clones containing a CaMV 35S promoter-driven DFR gene of Antirrhinum majus. Results indicated a peak of CHS and DFR transcript levels in petals at the very early stages of anthesis, and different expression patterns in anthers and sepals. In comparison to wild-type plants, transformants showed a more intense anthocyanin pigmentation of some vegetative organs, and a dramatic increase in DFR transcript concentration and enzymatic activity in petals. However, petals of transformed plants did not accumulate any anthocyanins. These results indicate that other genes and/or regulatory factors should be considered responsible for the lack of anthocyanin production in Forsythia petals.
    Type of Medium: Electronic Resource
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