Transformation of Phomopsis perniciosa Mediated by Agrobacterium tumefaciens

We developed an Agrobacterium-mediated transformation system for P.perniciosa by using the α-conidia of strain LXS230101 as transformation recipients,A.tumefacien strain EHA105 carring plasmid pBIG3C harboring the hygromycin B phosphotransferase gene(hph).Successful transformation of P.perniciosa was performord and the highest effiency reached on 686 transformants per 1×106 spores.The optimal transformation conditions were that 1×106 spores per milliliter of P.perniciosa α-conidia suspension were co-cultured with Agrobacterium cells at 25 ℃ for 72 h,in the presence of Co-culture medium containing acetosyringone(AS) at 200 μmol/mL.The transformants were verified by PCR amplification and by Southern blot analysis with the hph primers and probe,respectively.The results showed that all the detected transformants could be amplified the target bands and the T-DNA was inserted into the genome of P.perniciosa.In addition,the transformants were stable when grown on PDA medium without hygromycin for five times.