Establishment Of Agrobacterium Tumefaciens-Mediated Transformation System For Penicillium Digitatum

China is the largest producer of Citrus in the word. Nevertheless, there are still numerous challenges being faced during the production. And the most critical issue is that the frequent outbreak of various diseases in the postharvest storing and transportation period leaded to huge losses in Citrus industry. Among the fungal diseases in Citrus, the green mold is one of the most serious diseases which is caused by Penicillium digitatum. Generally, the fungi invade the fruit from the wound after the citrus harvest, and then the green mold outbreaks resulting in decayed citrus.The technique of Agrobacterium tumefaciens-mediated transformation, increases our understanding of pathogenic mechanism of P.digitatum and helps us analyzing the function of pathogenic relative gene. It also provides theoretical evidence for the disease control. In this study, a number of parameters which affect the transformation frequency were been probed and the transformants were molecular analyzed. The results are presented below.Agrobacterium-mediated preliminary transformation system for penicillium digitatum were established. The result showed the optimal condition for transformation.The concentration of Penicillium digitatum conidia is1×106spore/ml, that of Agrobacterium cells is OD600of0.15-0.25. The time of co-cultivation was48h. The selective concentration of hygromycin b is40μg/ml.Mix the1×106spore/ml Penicillium digitatum conidia with equal volume1Agrobacteriumd cells which were diluted to OD600of0.15-0.25. Subsequently, plat the mixtures onto CO-IM medium with cellophane sheets evenly. After incubating at24℃for48h in the dark, remove the cellophane sheets into sterilized Petri dishes. And move cellophane sheets, which contained40μg/ml of hygromycin B and200μg/ml of cefotaxime sodium, covering with selective medium. Then incubat at24℃for4-5d. The addition of AS(200μmol/l)during the pre-culture and Agrobacterium co-cultivation period are required for transformation. Transformants emerging from the seleetive medium were grown in PDA supplemented with40jig/ml hygromycin B. Successively subculturing on PDA for five rounds of conidia, after that transformants were removed to selective medium containing hygromycin B(40μg/ml), incubating at25℃for5days. The transformant is mitotically stable if it grow. Molecular analyses the transformants. Extract genomic DNA of wild fungi Penicillium digitatum and transformants. The hph resistance gene in transformants genomic DNA has been amplified by PCR with primer hphl and hph2. The result showes that transformants cantianed the expected hph gene fragment. And the transformant T-DNA flanking genomic DNA was amplified by Tail-PCR.