Abstract
The factors influencing transfer of an intron — containing β-glucuronidase gene to apple leaf explants were studied during early steps of an Agrobacterium tumefaciens-mediated transformation procedure. The gene transfer process was evaluated by counting the number of β-glucuronidase expressing leaf zones immediately after cocultivation, as well as by counting the number of β-glucuronidase expressing calli developing on the explants after 6 weeks of postcultivation in the presence of 50 mg/l kanamycin. Of three different tested disarmed A. tumefaciens strains, EHA101(pEHA101) was the most effective for apple transformation. Cocultivation of leaf explants with A. tumefaciens on a medium with a high cytokinin level was more conducive to gene transfer than cocultivation on media with high auxin concentrations. Precultivation of leaf explants, prior to cocultivation, slightly increased the number of β-glucuronidase expressing zones measured immediately after cocultivation, but it drastically decreased the number of transformed calli appearing on the explants 6 weeks after infection. Other factors examined were: Agrobacterium cell density during infection, bacterial growth phase, nature of the carbon source, explant age, and explant genotype.
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Abbreviations
- 2,4-D:
-
2,4-dichlorophenoxyacetic acid
- CaMV35S:
-
35S RNA of cauliflower mosaic virus
- EDTA:
-
ethylenediaminetetraacetate
- FeNaEDTA:
-
ethylenediaminetetraacetate ferric-sodium salt
- GusA:
-
β-glucuronidase
- gusA :
-
ß-glucuronidase gene of Escherichia coli
- gusA-intron:
-
ß-glucuronidase gene containing an intron in the coding region
- IBA:
-
indole butyric acid
- 2iP:
-
N6-2-isopentenyl adenine
- NAA:
-
naphthaleneacetic acid
- nptII :
-
neomycinphosphotransferase II gene
- X-Gluc:
-
5-bromo-4-chloro-3-indolyl ß-D-glucuronide
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Communicated by M. R. Davey
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De Bondt, A., Eggermont, K., Druart, P. et al. Agrobacterium-mediated transformation of apple (Malus x domestica Borkh.): an assessment of factors affecting gene transfer efficiency during early transformation steps. Plant Cell Reports 13, 587–593 (1994). https://doi.org/10.1007/BF00234517
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DOI: https://doi.org/10.1007/BF00234517