High efficiency transformation of Fusarium solani f. sp. cucurbitae race 2 (mating population V).
A cosmid vector, suitable for library construction and DNA transformation in filamentous fungi, has been constructed and a reliable and highly efficient PEG-mediated DNA transformation system for F. solani f. sp. cucurbitae, based on resistance to hygromycin B, has been developed for use with this vector. This transformation system yielded 10(4) transformants per micrograms of DNA when using 10(7) protoplasts. Factors important in achieving high efficiency included: the maintenance of an osmoticum in all transformation steps, PEG 4000 concentration, and the ratio of transforming vector DNA to protoplasts. Approximately 60% of transformants stably integrated vector DNA. Molecular analysis revealed multiple copies of the plasmid integrated into the genome at one or more sites. The frequency of transformation achieved will facilitate the isolation of genes from this fungus by complementation.
Crowhurst
R N
RN
DSIR Plant Protection, Auckland, New Zealand.
Rees-George
J
J
Rikkerink
E H
EH
Templeton
M D
MD
D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D003360:Cosmids / Q000235:genetics*; D004352:Drug Resistance, Microbial / Q000235:genetics; D005670:Fusarium / Q000187:drug effects / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D006921:Hygromycin B / Q000494:pharmacology; D008969:Molecular Sequence Data; D011092:Polyethylene Glycols / Q000494:pharmacology; D013012:Sorbitol / Q000494:pharmacology; D014162:Transfection; D014170:Transformation, Genetic / Q000235:genetics*
D011092:Polyethylene Glycols; D006921:Hygromycin B; D013012:Sorbitol