Development of Genetic Transformation system of Valsa mali of apple mediated by PEG

Abstract:

Abstract: ［Objective］The genetic transformation of Valsa mali var． mali was developed by PEG-mediated protoplasts transformation．［Method］It was transformed by PEG-induced fusion of protoplasts． The plasmid pBIG2RHPH2-GFPGUS carrying hph gene was used and Valsa mali var． mali 03-8 isolate was used as the host strain．［Result］At50 mg/mL driselase + 10 mg/mL lysing enzymes concentration，the mycelium of Valsa mali var． mali cultured in YEPD medium for 48 h was hydrolyzed in 10 mL enzymes liquid /0. 5 g wet mycelium for 2 h． The protoplast yield was 4×107 CFU /mg． The transformation efficiency was 44 per g DNA． Analysis of the transformants by PCR and Southern blotting showed that the selectable marker gene hph was integrated effectively into the genome of Valsa mali var． mali．After 5 subculturing on PDA，87. 5% transformants could grow． This stability test of transformants suggested that the foreign gene hph was stable in heredity．［Conclusion］This transformation system is a valuable and important tool for the further study of the pathogenic gene of Valsa mali．

Key words:Keywords: protoplast，transformant，hph gene，transformation efficiency，stability test