Agrobacterium-mediated transformation of apple (Malus x domestica Borkh.): an assessment of factors affecting regeneration of transgenic plants |
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Authors: | An De Bondt Kristel Eggermont Iris Penninckx Inge Goderis Willem F Broekaert |
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Institution: | (1) F. A. Janssens Laboratorium voor Genetica, Katholieke Universiteit Leuven, Willem de Croylaan 42, B-3001 Heverlee, Belgium;(2) N. V. Jo Nicolai & Co, Gorsemdorp 51, B-3803 Saint-Truiden, Belgium |
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Abstract: | We have previously developed a protocol for efficient gene transfer and regeneration of transgenic calli following cocultivation of apple (cv. Jonagold) explants with Agrobacterium tumefaciens (De Bondt et al. 1994, Plant Cell Reports 13: 587–593). Now we report on the optimization of postcultivation conditions for efficient and reproducible regeneration of transgenic shoots from the apple cultivar Jonagold. Factors which were found to be essential for efficient shoot regeneration were the use of gelrite as a gelling agent and the use of the cytokinin-mimicing thidiazuron in the selective postcultivation medium. Improved transformation efficiencies were obtained by combining the hormones thidiazuron and zeatin and by using leaf explants from in vitro grown shoots not older than 4 weeks after multiplication. Attempts to use phosphinothricin acetyl transferase as a selectable marker were not successful. Using selection on kanamycin under optimal postcultivation conditions, about 2% of the leaf explants developed transgenic shoots or shoot clusters. The presence and expression of the transferred genes was verified by -glucuronidase assays and Southern analysis. The transformation procedure has also been succesfully applied to several other apple cultivars.Abbreviations BAP
benzylaminopurine
- CTAB
hexadecyltrimethylammoniumbromide
- Na2EDTA
ethylenediamine-tetra-acetate ferric-sodium salt
- FeNaEDTA
ethylenediamine-tetra-acetate ferric-sodium salt
- GA3
gibberellic acid 3
- GusA
-glucuronidase
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gusA
-glucuronidase gene of Escherichia coli
- IAA
indole acetic acid
- IBA
indole butyric acid
- 2iP
N6-2-isopentenyl adenine
- NAA
naphthalene acetic acid
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nptII
neomycinphosphotransferase II gene
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bar
phosphinothricin acetyl transferase gene
- PCR
polymerase chain reaction
- PPT
phosphinothricin
- STS
silver thiosulphate
- T-DNA
transferred DNA
- TDZ
thidiazuron
- X-Gluc
5-bromo-4-chloro-3-indolyl -D-glucuronide
- Zea
trans-Zeatin |
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