Plant Biotechnology Journal
(2004)
2
, pp. 233–240
doi: 10.1111/j.1467-7652.2004.00067.x
© 2004 Blackwell Publishing Ltd
233
Blackwell Publishing, Ltd.
Effective production of marker-free transgenic strawberry
plants using inducible site-specific recombination and a
bifunctional selectable marker gene
Jan G. Schaart*, Frans A. Krens, Koen T. B. Pelgrom, Odette Mendes and Gerard J. A. Rouwendal
Plant Research International, Wageningen University and Research Centre, PO Box 16, 6700 AA Wageningen, The Netherlands
Summary
Public concerns about the issue of the environmental safety of genetically modified plants
have led to a demand for technologies allowing the production of transgenic plants without
selectable (antibiotic resistance) markers. We describe the development of an effective
transformation system for generating such marker-free transgenic plants, without the
need for repeated transformation or sexual crossing. This system combines an inducible
site-specific recombinase for the precise elimination of undesired, introduced DNA
sequences with a bifunctional selectable marker gene used for the initial positive selection
of transgenic tissue and subsequent negative selection for fully marker-free plants. The
described system can be generally applied to existing transformation protocols, and was
tested in strawberry using a model vector in which site-specific recombination leads to a
functional combination of a cauliflower mosaic virus 35S promoter and a GUS encoding
sequence, thereby enabling the histochemical monitoring of recombination events. Fully
marker-free transgenic strawberry plants were obtained following two different selection/
regeneration strategies.
Received 6 October 2003;
revised 5 December 2003;
accepted 9 December 2003.
*
Correspondence
(fax +31 317 418094;
e-mail jan.schaart@wur.nl)
Keywords:
marker-free transgenic
plants, negative selection, site-specific
recombination, strawberry.
Introduction
To date, two different a