Genetic transformation, electrophoretic karyotyping and isolation of insertional mutants in the tree pathogenic fungus Neonectria galligena
The ascomycete tree pathogen Neonectria galligena was transformed using several plasmid vectors containing the bacterial hygromycin phosphotransferase gene (hph) conferring resistance to hygromycin B. Protoplasts were produced by digesting germinating conidia in a Novozym 234 solution osmotically stabilized with NaCl. The protoplasts regenerated at ca. 70% on complete medium supplemented with either sucrose or NaCl. Up to 115 hygromycin B-resistant transformants per microgram of transforming DNA per 108 protoplasts were obtained. Southern analysis of genomic DNA from the transformants, separated by either standard agarose gel electrophoresis or by pulsed field gel electrophoresis, confirmed that hygromycin B resistance resulted from integration of the vector. Transformants expressing the green fluorescent protein were also obtained. A total of 209 transformants were then screened for aggressiveness and mycelial radial growth. Four mutants were identified including one mutant with reduced aggressiveness but normal growth in vitro, two mutants with reduced growth in vitro but normal aggressiveness, and one mutant in which both aggressiveness and in vitro growth were reduced.
Document Type: Research Article
Publication date: December 1, 2003