Study On The Transformation Of Cryphonectria Parasitica With Agrobacterium Tumefaciens

Chestnut blight caused by Cryphonectria parasitica is the most devastating disease around the world. In this paper, T-DNA insertional transformation technique of strain 7-6 of C. parasitica mediated by Agrobacterium tumefaciens was founded. The T-DNA insertional transformant library was constructed and the quality of the library was evaluated.To optimize the effiency of transformation, spores from 20 dpi slant with consentration 10~8/mL was used and the tempreture to co-incubation was determined as 23℃. Two hundred and thirty-five C. parasitica T-DNA transformants were obtained using the transformation technique.To confirm the transformants obtained, PCR amplification was conducted using genomic DNA as template and primers specific to T-DNA sequence of pTFCM. There was no significant difference on colony extension between wild-type strain 7-6 and 10 randomly selected transformants. Six successive generations of the 10 transformants obtained on PDA grew well on PDA containing 35ug/mL hygromycin B which showed that the transformants were stable genetically.