Restriction enzyme-mediated insertional mutagenesis: an efficient method of Rosellinia necatrix transformation

Rosellinia necatrix: causing root rot disease is a very destructive pathogen of woody plants and is responsible for yield losses to a large number of fruit trees. The genetic analysis of this pathogen has not been picked up because of difficulty in generating mutations in Rosellinia necatrix for many reasons. A number of methods have been proposed for inducing mutations in Rosellinia necatrix but none of them proved worth because of very low transformation efficiencies. Here, we propose an efficient method for Rosellinia necatrix protoplast production, where protoplasts in the tune of 10⁷ per ml can be easily generated. We also propose a restriction enzyme-mediated integration (REMI)-based methods for efficient transformation of Rosellinia necatrix. In the present study, an approximate of 800 transformants was obtained from 5 μg of linearized plasmid. Out of 47 single spored transformants analyzed, only 33 showed hygromycin gene amplification using PCR and only 19 transformants showed single gene integration in southern hybridization, which accounted for single gene integration percentage of 42%, highest amongst all the previous reports on Rosellinia necatrix transformations. Some of the transformants studied for pathogenicity phenotype also showed a marked reduction in pathogenicity. Thus, in the present investigation, 42% single gene integrations among the transformed colonies can be considered as excellent transformation efficiency.