Jing Gao1, Yanbo Li, Xiwang Ke, Zhensheng Kang, Lili Huang. 1. National Key Laboratory of Crops Stress Biology for Dryland and College of Plant Protection, Northwest A&F University, Yangling 712100, China. gaojingshai@yahoo.cn
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-GFP-GUS carrying hph gene was used and Valsa mali var. mali 03-8 isolate was used as the host strain. RESULT: At 50 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 x 10(7) 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.
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-GFP-GUS carrying hph gene was used and Valsa mali var. mali 03-8 isolate was used as the host strain. RESULT: At 50 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 x 10(7) 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.