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Transformation of Filamentous Fungi by Microprojectile Bombardment

. When at least 107 conidia were plated, colonies with a prototrophic phenotype were found, indicating that the pyrG89 mutation of the pyrimidine auxotrophic A. nidulans strain was successfully complemented by transformation with pRG-1 plasmid DNA (Figure 1). The first transformants were visible 2436 hours after bombardment. After 48 hours, transformants were counted and transferred onto fresh selective plates for further study.

Conidial density was found to be a crucial parameter in optimizating transformation frequency (Figure 2A). In subsequent experiments, 108 conidia were spread on each plate. Further increasing the spore density, overincubation of plates, or prolonged storage of conidial suspensions prior to the transformation experiment resulted in the occurrence of background colonies. Transformants were found under a wide range of helium pressures (8001,600 psi, Figure 2B) with a reduced transformation frequency at 1,600 psi, possibly due to increased cell death. No transformants were produced by bombardment at 400 psi. No significant difference in transformation frequency was observed when M5 (mean diameter 0.4 m), M10 (0.7m) and M17 (1.1 m) tungsten particles were compared (data not shown). Particles made of material other than tungsten were not tested. The addition of an osmotic stabilizer to the media (1 M sorbitol) did not increase the transformation efficiency. This result is consistent with the results of others who reported that fungal spores or mycelia were transformed in the absence of an osmoticum (see references in Table 1).

Microprojectile bombardment resulted in an overall transformation frequency (6 0.5 transformants/g DNA) that was
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