Transient gene expression in electroporated protoplasts of Eucalyptus citriodora Hook |
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Authors: | G. Manders A. V. P. dos Santos F. B. d'Utra Vaz M. R. Davey J. B. Power |
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Affiliation: | (1) Plant Genetic Manipulation Group, Department of Life Science, University of Nottingham, NG7 2RD Nottingham, UK;(2) Universidade Federal de Alagoas, Rio Largo, 57100, Alagoas, Brazil |
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Abstract: | Protoplasts isolated from cotyledons of Eucalyptus citriodora were electroporated using a rectangular pulse, with plasmid carrying the cat gene. The levels of transient expression and protoplast viability were influenced by the voltage and pulse duration. At a field strength of 800 V cm-1 (1000 s), a protoplast viability of 57%, and 47% conversion of 14C-chloramphenicol to its acetylated forms, were obtained. Expression levels were improved by an increase in plasmid concentration (up to 60 g ml-1), and also by the addition of carrier DNA. Gene expression was further enhanced by the addition of 40% (w/v) PEG, in the presence of the carrier DNA, to the protoplasts after electroporation.Abbreviations BAP 6-benzylaminopurine - CAT chloramphenicol acetyltransferase - CPW 13M CPW salts medium with 13% (w/v) mannitol - DC direct current - FDA fluorescein diacetate - f. wt fresh weight - GUS -glucuronidase - K Kao (1977) - MES 2-N-morpholinoethane sulfonic acid - MS Murashige & Skoog (1962) - NAA -naphthaleneacetic acid - PVP-10 polyvinylpyrrolidone (Av MW 10,000) - TLC thin layer chromatography |
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Keywords: | chloramphenicol acetyltransferase electroporation Eucalyptus citriodora protoplasts transient gene expression |
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