A rapid and simple method for isolation of Neurospora crassa homokaryons using microconidia

Current approaches for obtaining homokaryons of asexually growing N. crassa rely on serial passages of macroconidia, which are usually multinuclear (Davis and de Serres 1970 Methods Enzymol. 17A:79- 143). This method is time consuming and labor intensive. We have devised a simple method for purifying viable uninucleate microconidia from conidiating strains of N. crassa grown on Westergaard and Mitchell synthetic crossing medium (SC) supplemented with iodoacetate (IAA). Rossier, Oulevey and Turian (1973 Arch. Mikrobiol. 91:345-353) showed that standing liquid cultures containing 1x SC and 1 mM IAA produced microconidia. We have modified their method to reliably obtain microconidia from 150 mm slants of solid agar medium (0.1 x SC/0.5% sucrose/2% agar/1 mM IAA). We have purified these microconidia free of macroconidia and mycelia using Millipore Durapore Millex 5 µm filters. We are using this technique for the one step purification of homokaryons following DNA-mediated transformation. These methods may be generally useful for studies with heterokaryons.