Chiang Mai Journal of Science

 Establishment of an efficient and stable genetic transformation system in cabbage Fusarium wilt pathogen (Fusarium oxysporum f. sp. conglutinans) is a key step for studying gene function and the specific phenotypes of mutants. The optimal conditions for protoplast preparation and generation are as follows: A mycelia growth time of 32 h in CM medium, Driselase as the cell wall-degrading enzyme, 1.5 ml of enzyme solution (20 mg/ml Driselase) to 1 gram of mycelia, a treatment time of 4 h at 28C, and 0.7 M NaCl buffer. With the above conditions, the pKNTG vector containing green fluorescent protein gene was transformed into the protoplasts of F. oxysporum strain A6 by polyethylene glycol (PEG), and 20 positive transformants were obtained and used for the pathogenicity test. The linearized pUCATPH plasmid containing the hygromycin resistance gene was also transformd into the protoplasts of A6 strain, and HindIII and EcoRI were used in this procedure. Finally, a library with 1506 transformants was constructed by the restriction enzyme mediated integration (REMI) method. Southern blot analysis was used to prove the feasibility of the transformatiom and the REMI transformation system of the Fusarium oxysporum f. sp. Conglutinans were established successfully.