In order to construct the TDNA insertional mutagenesis transformation system for rice sheath blight pathogen Rhizoctonia solani AG1 ⅠA, the virulent isolate GD118 of this pathogen was selected as the initial isolate for transformation. The conditions for the transformation of GD118 were optimized in 5 aspects, i.e. preinduction time, coculture time, acetosyringone concentration at coculture stage, coculture temperature and pH values of solid induction medium (SIM) at coculture phase. Finally, an Agrobacterium tumefaciensmediated transformation (ATMT) system for R. solani AG1 ⅠA was established successfully. The optimal conditions for this ATMT system are as follows: the concentration of hygromycin B at 30 μg/mL for transformant screening, 8 h of preinduction time, 20 h of coculture time, 200 μmol/L of acetosyringone in SIM at coculture stage, coculture temperature at 25℃ and pH 5.6 to 5.8 of SIM at coculture phase. The transformants still showed high resistance to hygromycin B after 5 generations’ subcultures. Ten transformants were randomly picked out for PCR verification using the specific primers designed from the hph gene, and the results revealed that an expected band of 500 bp was amplified from all of the 10 transformants. Meantime, PCR amplification for these 10 transformants was carried out using specific primers designed from the Vir gene of A. tumefaciens, with 4 strains of A. tumefaciens as positive controls for eliminating the falsepositive caused by A. tumefaciens′s contamination, and the results showed that an expected band of 730 bp could be amplified from the 4 strains of A. tumefaciens, whereas no corresponding DNA band could be amplified from the 10 transformants. The results of above two PCR amplifications clearly showed that TDNA was indeed inserted into the targeted isolate GD118.
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