Alternative methods for genetic transformation of Pseudozyma antarctica, a basidiomycetous yeast-like fungus.
Electroporation and Agrobacterium tumefaciens-mediated transformation (ATMT) were adapted and optimized for genetic transformation of the basidiomycetous yeast-like fungus Pseudozyma antarctica as alternatives to the cumbersome PEG/CaCl(2)-mediated transformation of protoplasts. Electroporation yielded 100-200 transformants per mug of DNA per 10(8) cells after 3 days on selective medium. For its part, ATMT yielded 60-160 transformants per 10(6) input cfu after 5-10 days on a selective medium. Transformants obtained from both methods showed stable hygromycin resistance and strong expression of green fluorescent protein. Analysis of integration events revealed a limited number of predominantly tandem insertions in the genome of transformants, an improvement over PEG/CaCl(2)-mediated transformation. Both protocols relied on intact conidia of P. antarctica as starting material and thus eliminated the need for cell wall-degrading or weakening agents such as lytic enzymes or chemicals. Other advantages over protoplast transformation included higher yield of transformants and shorter recovery time of transformed colonies on selective medium.
Département de Phytologie, Centre de Recherche en Horticulture, Pavillon de l'Envirotron, Université Laval, Québec, Québec, Canada G1K 7P4.
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Marchand
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Fortier
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Neveu
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Bolduc
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Belzile
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Bélanger
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D001487:Basidiomycota / Q000235:genetics* / Q000502:physiology; D018274:Electroporation / Q000379:methods*; D013172:Spores, Fungal; D014170:Transformation, Genetic