Clean vector technology for marker-free transgenic fruit crops

Marker-free transgenic crops confer several advantages over transgenic crops equipped with selection genes coding e.g. for antibiotic resistance. Firstly, the European Union has prepared a guidance document for risk assessment of GM-crops to be introduced in the environment (E.U. Joint Working Group on Novel Foods and GMO’s, 2003). In this document based on compliance to consumer demands the EU encourages to “avoid or minimise the inclusion of superfluous transgenes or sequences”. EU thus promotes the use of clean vector systems. Secondly, the number of selection genes allowing the preferential growth of transformed cells and tissues is limited. Often a gene transfer protocol for a specific crop or even a cultivar depends on the use of one specific selectable marker gene. Hence, stacking of genes within the same transgenic line is difficult once a selectable marker gene has been introduced. If these marker genes can be removed, the subsequent introduction of the next gene-of-interest is greatly facilitated. At Plant Research International a system has been developed for specific elimination of any introduced DNA/gene sequences using site-specific recombination combined with selection for successful removal using a negative selection system. Completely marker-free transgenic plants have been obtained using a model vector, both in an efficient transformation system (strawberry) as well as in a non-efficient transformation system (apple). Frequencies were more than adequate. Presently a versatile vector set providing a choice of several selectable markers and carrying a multiple cloning site for receiving cassettes of the gene-of-interest is available for application in, amongst others, fruit crops