Isolation of protoplasts from <i>Aspergillus nidulans</i> conidiospores

Abstract

Protoplasts were prepared from conidiospores of Aspergillus nidulans. The mononucleated conidia gave protoplasts of a uniform size, approximately 5-micron diameter, depending on the strain and the stabilizing medium used. Conidia were preincubated with 2-deoxy-D-glucose in a minimal medium at 37 degrees C for 3 h. The swollen conidia were collected, resuspended in a buffer containing 0.4 M (NH4)2SO4 as stabilizer, and incubated with Oerskovia lytic enzymes at 30 degrees C for 3 or 4 h. Approximately 80% of the conidia were converted into protoplasts. The protoplasts were separated from cell wall fragments and intact conidia by centrifugation over 30% sucrose. This isolation procedure gives a suspension of mononucleated or binucleated protoplasts suitable for recombination experiments and other studies for which a homogenous protoplast suspension is required. The procedure was also successful for Aspergillus niger.