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Transformation of Filamentous Fungi by Microprojectile Bombardment

h addition the suspension was vigorously vortexed and incubated on ice for 10 min. The DNA-coated tungsten particles were then pelleted in a microfuge for 10 seconds. After removing the supernatant, the tungsten particles were washed with ethanol, resuspended in 30 l ethanol, and 8 l loaded onto each of three macrocarrier discs for bombardment in the PDS-1000/He instrument. Target distance (6 cm), vacuum (28 in Hg) and other parameters were identical to conditions optimized for nuclear transformation of S. cerevisiae cells (Heiser, 1992). The bombardment pressure was varied from 400 to 1,600 psi. Bombarded plates were incubated at 37 C for 48 hours. Control plates were bombarded with tungsten particles which were prepared as described above but not coated with plasmid.

Formation and transformation of protoplasts
PEG-mediated transformation of protoplasts was performed as described by Herzog et al ., 1996.

DNA isolations and Southern blot analysis
DNA was isolated from transformed and non-transformed mycelia by the procedure of Raeder and Broda (1985). Samples of DNA were digested with PstI which cuts pRG-1 at a single site (Waring et al ., 1989). Southern blot hybridizations were carried out as outlined by Sambrook et al . (1989). Plasmid DNA probes were radioactively labeled using the Random Primed DNA Labeling Kit from United States Biochemical (Cleveland, Ohio).

Results and Discussion
In initial experiments using M10 tungsten particles, a helium pressure of 1,200 psi, and other bombardment parameters were used as optimized for nuclear transformation of S. cerevisiae (Heiser, 1992). The spore density was varied from 106108 conidia per plate
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