Abstract
Abstract Objective: The transformation system for the asparagus stem blight pathogen Phomopsis asparagi (Sacc.) Bubak has not yet been reported. In the present study, we intend to achieve and optimize the genetic transformation of P. asparagi. This study aims at establishing the foundation for understanding the pathogenic mechanism of P. asparagi, which will be of great theoretical and practical significance. Methods: The Agrobacterium tumefaciens-mediated transformation (ATMT) system for P. asparagi was constructed at three aspects, i.e. condition optimization, insertion verification and transformant stability. Results: The optimal conditions for this ATMT system were as follows: 8 h of pre-induction, 48 h of co-culture, 200 μmol/L AS in ISM, co-culture at 25-28°C and pH 5.6-5.8 of ISM at the co-culture phase. The PCR result of the hph gene revealed that an expected band of about 500 bp was amplified from all the 10 transformants selected at random, and the PCR result of the Vir gene revealed that an expected band of about 730 bp was amplified from the four strains of A. tumefacien as positive controls, whereas no corresponding DNA band could be amplified from the 10 transformants. The results of the two PCR amplifications clearly showed that T-DNA was indeed inserted into the genome of target isolate FJ2. The stability result revealed that transformants still displayed high resistance to hygromycin B and could grow normally after subculture for five generations. Conclusion: A stable and efficient ATMT transformation system for P. asparagus was constructed systematically, in which a high transformation rate was achieved. For improving this system, the trasformation conditions were optimized through gradient experiments, T-DNA insertion was verified through dual PCR and the insertion segment containing hph gene in the transformant was proved hereditary stable through subculture. This system layed a foundation for the research on pathogenic mechanism and pathogenicity-related genes of P. asparagi.
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