Transformation of enhanced green fluorescent protein gene in Pythium oligandrum mediated by Agrobacterium tumefaciens

Abstract:

[Objective] Pythium oligandrum is an environmental friendly mycoparasitic biocontrol fungus that can antagonize several plant pathogens and promote plant growth. We constructed Agrobacterium tumefaciens-mediated transformation of P. oligandrum.[Methods] Three A. tumefaciens strains:EH105, AGL-1 and LBA4404 were compared to find out the strain which showed the best genetic transformation efficiency within P. oligandrum. The factors of transformation system were screened and optimized to establish the genetic transformation system within P. oligandrum mediated by Agrobacterium tumefaciens.[Results] EHA105 strain showed the best genetic transform efficiency within P. oligandrum among three tested strains, followed by AGL-1 strain, and LBA4404 strain performed poorly. Then, the optimized genetic transformation system was as follows:A. tumefaciens EHA105 (OD₆₀₀=0.6) suspension was mixed with P. oligandrum (10⁶-10⁷ spores per milliliter) suspension in a proportion of 1-10:1, then incubated in dark conditions for 72 h between 25 and 26℃ in the presence of induction medium (pH 5.0) containing acetosyringone at 300 μmol/L. Afterwards, antibiotic was added in to eliminate A. tumefaciens before 24 hours reviving cultivation. In the end, the transformants were selected by medium containing 200 mg/L of Basta and 250 mg/L of cefotaxime sodium on PDA. In this work, egfp was successfully transformed into P. oligandrum with the efficiency of 130 transformants per 10⁶ spores.[Conclusion] We developed an Agrobacterium tumefaciens-mediated genetic transformation system for P. oligandrum for the first time to provide powerful tool for the study of P. oligandrum molecular breeding and biological control mechanisms.

Key words:Pythium oligandrum;biocontrol fungus;Agrobacterium tumefaciens;genetic transformation;enhanced green fluorescent protein gene