Establishment of an Agrobacterium turnefaciens-mediated transformation system for Tilletia foetida

Tilletia foetida causes wheat common smut disease with severe loss of yield production and seed quality. In this study, a low-cost, rapid, and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for T. foetida mutagenesis was constructed: Transformants were screened with hygromycin B at 100 mu g/ml, cefotaxime sodium concentrations with 200 mu g/ml, Acetosyringone (AS) concentration at 200 mu mol/l, 1 x 10(6) T. foetida hypha cells/ml, co-cultivation at 22 degrees C with 24 h and culture was incubated at 16 degrees C up to day 7. Fourteen transformants were randomly selected and confirmed using the specific primers to amplify the fragment of hygromycin phosphotransferase gene. At the same time, PCR analysis was performed to detect Agrobacterium ttunefaciens Vir gene to eliminate false positives. The transformants were cultivated up to 8 generations on hygromycine B-containing complete medium (CM) and confirmed by PCR. The results indicated that 80% of T. foetida transformants were hygromycine B resistant. In conclusion, our analyses identified an efficient T-DNA insertion system for T. foetida and the results will be useful for further understanding the pathogenic mechanism via generation of the insertional mutants.