3Faculty of Agricultural Sciences , Payame Noor University, Tehran, Iran.
One of the important technical obstacles in the study of many filamentous fungi is the development of efficient transformation system. Transformation of filamentous fungi is difficult because they have a cell wall and for most frequently used approaches preparing of protoplast is necessary. Protoplast preparation is batch-dependent and often frustrating work. In this study, a modified method was introduced for gene transfer to the plant pathogenic fungus, Fusarium graminearum, the major causal agent of Fusarium Head Blight disease in small grains. This protocol was based on protoplast-PEG method. Age of mycelia, enzymes exposure time and mycelium/enzyme ratio were optimized for the purpose of protoplast preparation. The outcome showed that the best result for protoplast preparation was obtained when 1.5 × 105 spores were let germinate for 6 h then exposed to 10 ml of enzyme solution for 3 h. The effect of other parameters that might enhance transformation yields including PEG concentration, DNA quantity and number of protoplasts was also examined. The most efficient condition for transformation involved the use of 106 protoplasts, 20 µg DNA and 30% PEG (w/v). In the course of this study, a simple and appropriate modified protocol for transformation of F. graminearum was established. The method introduced is also more economical and faster than other current methods.