African Journal of
Microbiology Research

  • Abbreviation: Afr. J. Microbiol. Res.
  • Language: English
  • ISSN: 1996-0808
  • DOI: 10.5897/AJMR
  • Start Year: 2007
  • Published Articles: 5211

Full Length Research Paper

Optimization of factors affecting protoplast preparation and transformation of smoke-tree wilt fungus Verticillium dahliae

Shuxiao Xiao, Yingjiao Sun, Chengming Tian, Yonglin Wang*
The Key Laboratory for Silviculture and Conservation of Ministry of Education, College of Forestry, Beijing Forestry University, Beijing 100083, China.
Email: [email protected]

  •  Accepted: 17 May 2013
  •  Published: 28 May 2013


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


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