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An optimized Agrobacterium tumefaciens-mediated transformation protocol for Ceratocystis albifundus

Brenda Wingfield: Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria

<div>Despite the availability of genome sequences for the wattle pathogen, <em>Ceratocystis albifundus</em>, functional association of loci with specific biological traits remains lacking. This is mainly because an efficient transformation system is not available for the fungus. Here, we present an optimized <em>Agrobacterium tumefaciens</em>-mediated transformation protocol for genetic manipulation of this fungus. For the development of this protocol, <em>A. tumefaciens</em> strain AGL-1 and four binary T-DNA vectors (conferring hygromycin and geneticin resistance and expressing green fluorescent protein [GFP]) were used for transforming germinated conidia of three <em>C. albifundus</em> strains. Stable expression of GFP or the antibiotic resistance genes was confirmed through sequential sub-culturing of transformants on selective and non-selective media. Incorporation of these genes into the genome of the fungus was also confirmed using PCR, Sanger sequencing and Southern hybridization analysis. The range of experimental parameters optimised included: (i) the concentrations of hygromycin and geneticin required for inhibiting growth of the fungi, (ii) concentration of the fungal conidial suspension, (iii) bacterial cell concentration, and (iv) fungus-bacterium co-cultivation durations. To the best of our knowledge, this is the first report of a transformation protocol for <em>C. albifundus</em> and its availability will be invaluable for functional genetics studies in this important fungus.</div>