Effect of G - 418 on growth of os - 1 protoplastsa

The formation of stable protoplasts from os-1 (NM233t) (FGSC #4493 or 4494) mycelial cultures involves the germinaUse of partially purified Polyoxin B tion of macroconidia in Vogel's medium N containing 10% sorbose and 200-400 μg/ml purified Polyoxin B at 37° C (Selitrenfor forming protoplasts. nikoff et al., 1981 Exper. Mycol. 5: 155-161). Polyoxin B is purified from Polyoxin AL Wettable Powder (Kaken Chemical Co.) by Norite and Dowex chromatography. Although not overly difficult, complete purification is time consuming. As an alternative, partially purified (40%) polyoxin was used and found to be as effective in promoting protoplasts as was purified (b 70%) polyoxin. Simply, AL Wettable Powder is made to 10% in water (100 mls), the insoluble material filtered using Whatman #l paper and the dark brown filtrate adsorbed to a granular Norite column, washed with H20, and eluted with 60% acetone. The acetone was removed and the brown powder dissolved in H20, filtered, sterilized, and stored frozen at -20° C until used (up to six months without detectable loss of activity). A granular Norite column is extremely fast flowing so that the entire partial purification procedure requires less than a day. If the Norite column step is omitted, i.e., if crude polyoxin is used, then protoplast formation is inhibited. (Supported by National Science Foundation award PCM 8112212.) Department of Anatomy, University of Colorado Health Sciences Center, Denver, CO 80262.