ISSN:
1432-203X
Keywords:
Abbreviations: BAP, benzylaminopurine; CTAB, hexadecyltrimethylammoniumbromide; Na2EDTA, ethylenediamine-tetra-acetate di-sodium salt; FeNaEDTA, ethylenediamine-tetra-acetate, ferric-sodium salt; GA3, gibberellic acid 3; GusA, β-glucuronidase; gusA, β-glucuronidase gene of Escherichia coli; IAA, indole acetic acid; IBA, indole butyric acid; 2iP, N6-2-isopentenyl adenine; NAA, naphthalene acetic acid; nptII, neomycinphosphotransferase II gene; 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 β-D-glucuronide; Zea, trans-Zeatin
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
Notes:
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 combing 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 β-glucuronidase assays and Souther analysis. The transformation procedure has also been successfully applied to several other apple cultivars.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/s002990050072
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