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-ofinterest is greatly facilitated. At Plant Research International a system has been developed for specific elimination of any introduced DNA/gene sequences using sitespecific 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-ofinterest is available for application in, amongst others, fruit crops. BACKGROUND Genetically modified (GM) food products are looked upon with a certain degree of mistrust by the majority of European consumers. Almost 50% of consumers think that GM food is less safe for human consumption than non-GM food, 33% are not sure. Only 20% of the consumers know that there is no scientific basis to these doubts on food safety. While on the one hand players in the food production chain, such as breeding companies, growers/farmers and biotechnology firms, are convinced of the technical and economic benefits (Graff and Newcombe, 2003), the consumers on the other hand perceive risks to human health, to the environment and to biodiversity. In the USA and other countries around the globe, e.g. Argentina, Canada, China and India, GM crops are increasingly implemented in agriculture, however, the European input in research and development of GM crops has been reduced since 1998. This de-facto moratorium on the introduction of new GM crops has been in force since 1999. As a consequence of this, 51% of the European Small and Medium Enterprises (SMEs) at present do not use genetic engineering approaches, most of them indicated that there has been a change in their strategy in the last few years abandoning GM related R&D projects (Menrad and Menrad, 2003). In July 2003, the ban on import of GM food products was lifted by the European Union (EU), with the proviso that all products containing more than 0.9% of EUapproved GM material have to be labeled. Also, the European Parliament has formulated guidelines based on their desire to provide European consumers with a choice to either accept or avoid the consumption of any GM or GM-derived food products. For this, completely separate production and processing chains will have to be set up. In practice, XIth Eucarpia Symp. on Fruit Breed. & Genetics Eds. F. Laurens and K. Evans Acta Hort. 663, ISHS 2004 432 this might turn out to be too expensive for the industry, presenting yet another barrier to the implementation of GM food in Europe. Another consequence of this EU policy is the negative impact it has on imports of food from the USA and the reluctance of developing countries to grow GM crops out of fear of reducing their export possibilities to the EU. In order to change the perception of EU consumers it might not be advisable to force GM crops and food upon them by enforcing official WTO rules and by pressure from the USA. Instead, consumers should regain confidence and trust in their scientists and government organizations. This can only be achieved by communicating clearly the benefits of particular GM crops and by highlighting the relevance of these crops to the individual consumer. Consumers should be able to relate to the goals of the modification and to the way these goals are achieved. If applicable, it should be explained that alternatives do not really exist or are less beneficial than the GM approach. In parallel to this, technical solutions to some of the concerns that consumers expressed are sought by science and can be applied. In a ‘Guidance Document for the risk assessment of GM plants and derived food and feed’ by the Joint Working Group on Novel Foods and GMOs prepared for the EU, recommendations include the encouragement of notifiers to develop GM crops in which only DNA essential to the desired modification is introduced, e.g. clean vector technology. Overall, three principle ways are identified: avoid or minimize the inclusion of superfluous transgenes or sequences avoid or minimize superfluous expression of the transgene avoid or minimize the dispersal of transgenes in the environment. Plant Research International has added to this list the preferential use of gene sequences or promoters which are speciesor at least plant-derived. This, combined with PRIs own clean vector system and transparent communication on PRI arguments why and when to use GM technology, will hopefully contribute to a broader public acceptance of genetic modification of plants. CLEAN VECTOR TECHNOLOGY Clean vector technology aims to produce GM plants with only the gene-of-interest as the newly introduced gene function, without any superfluous gene sequences. Primarily, the goal is to avoid the use or the continued presence of antibiotic resistance genes as selectable markers. Four approaches to achieve this can be followed. A. No Selectable Marker Here, GM plants are produced by Agrobacterium inoculation followed by regeneration of shoots without the use of a selectable agent. This will lead to a (great) number of plants, the majority of which are non-transgenic. However, depending on the regeneration and gene transfer frequencies, some plantlets will be transgenic and they will have to be identified, e.g. by a dedicated PCR screening on DNA of several sets of pooled plants. A prerequisite is a regeneration/transformation protocol of high efficiency. So far, this method is limited to model species and a low number of specific crop cultivars, e.g. in potato. B. Cotransformation In this system the selectable marker gene is physically separated from the gene-ofinterest. This can be on different T-DNAs residing on the same or on separate binary vector(s). The separate binary vectors can be present in the same or in separate Agrobacterium strains. The two T-DNAs should become integrated in two genetically unlinked loci. After selection for the GM plants by growth on antibiotic or herbicide containing media subsequent segregation after sexual crossing of resistant regenerants should result in GM plants equipped only with the gene-of-interest. A prerequisite here is that the crop can be sexually propagated without losing too many traits or cultivar identity and this within a reasonable time frame. For vegetatively propagated crops or crops with a very long sexual cycle, such as tulip or apple, this approach is less feasible.