( somewhat lower than the frequency ...)Transformation of Filamentous Fungi by Microprojectile Bombardment

somewhat lower than the frequency observed by chemical transformation of protoplasts (20 4 transformants/g DNA, Herzog et al ., 1996). However, only about 30% of the transformants obtained from regenerated protoplasts were stable transformants (for further details see Herzog et al ., 1996). On the other hand, among Biolistic transformants, 6070% were mitotically stable. Therefore, the frequency with which stable A. nidulans transformants were obtained was similar for both techniques (4 transformants/g DNA for Biolistic transformation, 6 transformants/g DNA for chemical transformation). The reason for this difference may be because fungal protoplasts are typically enucleate to multinucleate, while conidia of A. nidulans are uninucleate (Bennett & Klich, 1992). Consequently, in a protoplast, only one of several nuclei might be transformed, thus resulting in a lower proportion of stable transformants compared to Biolistic transformation of Aspergillus conidia. A more detailed analysis of this phenomenon is given by Lorito et al . (1993) who made similar observations during their study of T. harzianum and G. virens.

Analysis of genomic DNA by Southern blot hybridization showed that Biolistic transformants were indistinguishable from transformants isolated previously following PEG-mediated transformation (Ballance et al ., 1983). Furthermore, microprojectile bombardment often resulted in the delivery of several copies of the pRG-1 plasmid per conidium. Tandem repeat integration of the whole plasmid with varying copy number (Figure 3, lanes 79) as well as integrations at multiple sites had taken place (Figure 3, lanes 5 and 6). Such integration is thought to result from a single cross-over event (type I or type
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