Reliability in Transformation of the Basidiomycete Coprinopsis cinerea

Order of Publishing in Issue: 
8
Volume :6
Issue :3
July, 2012
Page No: 
340-355
Authors: 
Bastian Dörnte and Ursula Kües*
Address: 
Büsgen-Institute, Division of Molecular Wood Biotechnology and Technical Mycology, University of Goettingen, Büsgenweg 2, 37077 Goettingen, Germany
Email-ID: 
ukuees@gwdg.de

Abstract

Transformation of the basidiomycete Coprinopsis cinerea makes use of unicellular haploid asexual spores called oidia. Protoplasts of oidia are generated by a cellulase/chitinase enzyme mix. Protoplasts and DNAs are incubated together in 25 mM Ca2+ and 5% PEG (polyethylene glycol) 4000 on ice and more PEG is added (23% final concentration) after a ‘heat shock’ step at RT (room temperature). Upon regeneration on selective media, transformation rates of several hundreds of clones might be obtained per 1 μg DNA and 107 protoplasts. Although the technique has been invented 25 years ago by Binninger et al. (1), there are reoccurring pitfalls in the method that can cause failure. Successful transformation needs a good amount of skilful knowhow about the fungus and the method. Here we present our experiences with C. cinerea transformations, call attention to potential flaws and to optimal handlings in fungal cultivation, harvesting, protoplasting, transformation, and subsequent regeneration of the fungus.

Keywords: 
Transformation, protoplasts, C. cinerea vectors, oidia, basidiomycete
AttachmentSize
Paper-8.pdf275.79 KB

 
To download full text articles and issues in PDF at no cost.All you have to do is fill out a brief registration form. It's easy and it's free. At any time, you may request removal of your contact information from our database.
Click here to Register | Click here if you have already registered

2018 ABAP Awards

Invitation for Nominations to 2019 ABAP Awards
The Association of Biotechnology and Pharmacy invites nominations for the following awards from the eligible talented people with proven track record and achievements on or before August 31, 2019 in the prescribed format.

DOWNLOAD CIRCULAR | APPLICATION FORM

AWARDS

User login