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Genetic transformation of Phytophthora ramorum with the jellyfish GFP geneAuthor(s): G. Calmin; M. Riedel; L. Belbahri; S. Wagner; S. Werres; F. Lefort
Source: Gen. Tech. Rep. PSW-GTR-221. Albany, CA: U.S. Department of Agriculture, Forest Service, Pacific Southwest Research Station: 270-272.
Publication Series: General Technical Report (GTR)
Station: Pacific Southwest Research Station
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DescriptionThe important quarantine organism Phytophthora ramorum has been dramatically increasing its host range in the past years and most of the studies concerning P. ramorum focus on these issues. Very little is known about the latency period. For sampling and analyzing potentially infected plant material, detailed information on the latency of the disease is of high importance. In order to provide such a tool for the infection process follow-up, we established a reliable method for the stable genetic transformation of P. ramorum isolates. The transferred genes were the marker gene nptII for resistance to geniticin and the target gene GFP. The first and most important step in this protocol was to develop a stable system to produce protoplasts from P. ramorum tissue. The next step was the transformation of protoplasts with plasmids containing marker gene and target gene, following an improved polyethylene glycol (PEG)-mediated protocol of protoplasts transformation. After transformation, protoplasts were cultivated on a selective medium and allowed to regenerate mycelium. The selected transformants were checked for integration and expression of transferred genes by PCR amplification and the use of anti-GFP antibodies. About forty-three different transformed isolates were produced and then tested for GFP fluorescence, GFP expression and GFP gene integration. They were further tested for their infection potential on Rhododendron plants.
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CitationCalmin, G.; Riedel, M.; Belbahri, L.; Wagner, S.; Werres, S.; Lefort, F. 2009. Genetic transformation of Phytophthora ramorum with the jellyfish GFP gene. In: Goheen, E.M.; Frankel, S.J., tech. coords. Proceedings of the fourth meeting of the International Union of Forest Research Organizations (IUFRO) Working Party S07.02.09: Phytophthoras in forests and natural ecosystems. Gen. Tech. Rep. PSW-GTR-221. Albany, CA: U.S. Department of Agriculture, Forest Service, Pacific Southwest Research Station: 270-272.
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