Transformation of Filamentous Fungi by Microprojectile Bombardment

The vast majority of transformation protocols for filamentous fungi are based on permeabilizing cell membranes with polyethylene glycol (PEG) or electroporation. Both methods typically require preparing protoplasts or osmotically sensitive cells prior to transformation (Herzog et al., 1996; Goldman et al., 1990; a detailed review of fungal transformation systems is given by Lemke and Peng, 1995). Over the last few years, microprojectile bombardment has become a powerful tool for transformation of intact cells, particularly for the transformation of fungal strains that do not yield sufficient numbers of regenerating protoplasts or species that cannot be grown in culture, such as obligate plant pathogens. Biolistic® transformation of Saccharomyces cerevisiae cells was optimized using the PDS-1000/He system (Heiser, 1992). In addition, several filamentous fungi have been transformed by means of microprojectile bombardment (Table 1). In this study, the PDS-1000/He system was used for stable nuclear transformation of an Aspergillus nidulans strain deficient in pyrimidine synthesis by selection for prototrophic transformants. The effect of conidial density, helium pressure, and size of microprojectiles on transformation frequency were investigated. Transformation frequency, mitotic stability of transformants, and Southern blot hybridization patterns were compared between conidia transformed by particle bombardment and protoplasts transformed by chemical (PEG-mediated) transformation. In addition, optimized conditions for microprojectile bombardment were compared with literature data on Biolistic transformation of other fungal species. Materials and Methods