Dong Yan-Hong, Wang Shu-Tong
College of Plant Protection, Hebei Agricultural University, Baoding, China.
Methods Mol Biol. 2022;2391:63-73. doi: 10.1007/978-1-0716-1795-3_6.
Agrobacterium tumefaciens-mediated transformation (ATMT) is becoming a popular effective system as an insertional mutagenesis tool in filamentous fungi. An efficient Agrobacterium tumefaciens-mediated transformation approach was developed for the plant pathogenic fungus, F. oxysporum, the causal agent of Apple replant disease (ARD) in China. Four parameters were selected to optimize efficiencies of transformation. A. tumefaciens concentration, conidial concentration of F. oxysporum, and co-culture temperature and time have a significant influence on all parameters. Transformants emit green fluorescence under fluorescence microscopy. The integration of a mitotically stable hygromycin resistance gene (hph) in the genome is confirmed by PCR. The transformation efficiency can reach up to 300 transformants per 10 conidia under optimal conditions. This ATMT method is an efficient tool for insertional mutagenesis of F. oxysporum.
根癌农杆菌介导的转化（ATMT）作为丝状真菌插入诱变工具正成为一种流行的有效系统。针对中国苹果再植病（ARD）的病原菌——植物病原真菌尖孢镰刀菌，开发了一种高效的根癌农杆菌介导的转化方法。选择了四个参数来优化转化效率。根癌农杆菌浓度、尖孢镰刀菌分生孢子浓度、共培养温度和时间对所有参数都有显著影响。转化体在荧光显微镜下发出绿色荧光。通过PCR确认有丝分裂稳定的潮霉素抗性基因（hph）整合到基因组中。在最佳条件下，转化效率可达每10个分生孢子300个转化体。这种ATMT方法是尖孢镰刀菌插入诱变的有效工具。
