Abstract

Verticillium dahliae is a causal agent of vascular wilt smoke-tree Cotinus coggygrial. The genetic transformation of V. dahliae has been carried out by Agrobacterium and polyethylene glycol-mediated transformation. Due to technical simplicity and efficiency, PEG-mediated transformation is the most commonly used method for genetic transformation in filamentous fungi. However, polyethylene glycol (PEG)-mediated transformation is still deficient in V. dahliae. In this study, we studied the main factors affecting the release of protoplasts from mycelia of V. dahliae such as lytic enzymes, enzymolysis buffers and incubation time. The optimal combination for protoplast preparation from smoke-tree wilt fungus V. dahliae can release 2.8 × 10⁶ protoplasts/ml from fresh mycelia of smoke-tree wilt fungus. The majority of spheroplast contained nuclei and well regenerated. After PEG mediated transformation, green fluorescent protein (GFP) expressing V. dahliae strains were obtained. Therefore, this study provides a protocol suitable for protoplast preparation and transformation of smoke-tree wilt fungusV. dahliae.

Key words: Verticillium dahliae, smoke-tree wilt, protoplast preparation, polyethylene glycol (PEG) transformation

Abbreviation

PEG, Polyethylene glycol; ATMT, Agrobacterium tumefaciens-mediated transformation; GFP, green fluorescent protein; PDA, potato dextrose agar DAPI, 4’, 6-diamidino-2-phenylindole.