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Transformation of Filamentous Fungi by Microprojectile Bombardment

s for microprojectile bombardment were compared with literature data on Biolistic transformation of other fungal species.


Materials and Methods
Strain and plasmid
Aspergillus nidulans strain A773 (pyrG89, wA3, pyroA4) was obtained from the Fungal Genetics Stock Center, Dept. of Microbiology, University of Kansas Medical Center, Kansas City, Kansas, and maintained on solid YG medium [0.5% yeast extract, 2% glucose, 2% agar, pH 6.3] supplemented with 0.12% uracil and 0.12% uridine. Plates were incubated for 68 days at 37 C at which time conidia were harvested in 0.9% sterile saline. The resuspended conidia were sonicated in an ultra sound sonicator for 1 min prior to being used for protoplast formation or for microprojectile bombardment.

Plasmid pRG-1, a 5 kb plasmid containing the Neurospora crassa pyr4 gene cloned into pUC9 was used for transformation experiments. This plasmid has been shown to complement the pyrG89 mutation in A. nidulans (Ballance et al ., 1983; Waring et al ., 1989).

Biolistic transformation
Intact conidia were spread onto 10 ml of solid YG medium (in a 100 x 15 mm petri dish), briefly air-dried under sterile conditions, and used for microprojectile bombardment within 13 hours. Each plate was bombarded twice. Tungsten particles (M5, M10, or M17 particles, Bio-Rad Laboratories, Hercules, California) were prepared and coated with plasmid DNA as described by Daniell (1993). The following were added in order to a 1.5 ml microcentrifuge tube: 25 l tungsten particle suspension (1.5 mg in 50% glycerol), 5 l plasmid DNA (0.5 g/l), 25 l 2.5 M CaCl2, and 5 l 1 M spermidine free base. After eac
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