Development Of A Highly Efficient "Gene-Deletor" System And Its Potential Applications To Address The Concern Over The Safety Of GM Plants

IntroductionPlants produce large amounts of pollen that can disperse over long distances via insects and wind. Gene transfer through pollination routinely occurs within the major world crops, such as wheat, rice, maize, soybean, barley, and cotton, and also between these crops and their wild relatives (Chevre et al. 1997). To date, more than 52 million hectares of transgenic crops have been grown worldwide. In the case of canola, one of the first GM crops, Rieger et al. (2002) have demonstrated that cross-pollination between commercial canola fields can occur over considerable distances. In addition, pollen of some GM crops might cause adverse effects on non-target organisms (Wisniewski et al. 2002). The claims that Bt corn pollen killed larval monarch butterfly (Losey et al. 1999) and that transgenic maize genes had introgressed to wildtype maize in remote areas of Mexico (Quist and Chapela, 2001) have raised storms of controversy about GM plants (Shelton and Sears, 2001; Wisniewski et al. 2002). In recent years, several technologies have been developed to address the pollen-mediated "transgene pollution" problem but none has proved broadly applicable in field conditions (Daniell, 2002). Inserting transgenes into the chloroplast genome appears to be most promising but pollen of some angiosperm and gymnosperm species such as alfalfa, tobacco, pea, rice and conifer contains chloroplasts and transmits chloroplast genes in these species (Cummins, 1998). Also, it has been recently shown that DNA can be transferred from the chloroplast and integrated into the nucleus at a frequency of one in approximately 16,000 tobacco pollen grains (Huang et al. 2003). Furthermore, because of technical difficulties, chloroplast transformation has been achieved only in a very small number of species so far (Bock, 2001).Site-specific recombination systems such as Cre/loxP of phage P1, R/RS of Zygosaccharomyces rouxii and 7LP/FRT of Saccharmyces cerevisiae, function through interactions of a recombinase with its specific recognition sites (Craig, 1988). Depending on the orientation of the recombinase recognition sites, intramolecular (excision) or intermolecular (integration) recombination can occur. Intramolecular recombination between directly oriented sites results in excision of the intervening DNA between the two sites and leaves one site behind. Although strategies for removing marker genes (e.g., antibiotic resistance genes) or short spacer sequences from the host plant genome usingsite-specific recombinases have been developed (Hare and Chua, 2002; Luo et al. 2000; Ow, 2002), the possibility of creating non-transgenic organs or plants from transgenic plants has not been addressed. Keenan and Stemmer (2002) have recently proposed to use a site-specific recombinase to remove transgenes from specific tissues in crop plants to address safety concerns regarding GM plants but the proposed technology has not been demonstrated experimentally. One of major technical challenges is that efficiencies of the existing site-specific recombination systems are generally low in higher plants (Hare and Chua, 2002; Ow, 2002). Low efficiency of transgene deletion can make the proposed technology little useful for large-scale crop production because even if a small fraction of pollen grains, seeds or fruits remains transgenic, the total number of pollen grains, seeds or fruits that contain transgenes can be enormously large. A chemical inducible excision of a transformation marker gene recently described by Zuo et al. (2001) appears to be highly efficient in a laboratory setting but uniform application and effective penetration of the chemical to target tissues under field conditions can be technically difficult.Recently, Lauth et al. (2002) reported that the use of loxP on one side and FRT on the other side as the flanking sites for a Cre or FLP expression vector resulted in significantly higher rates of recombination. To develop a recombinase-based system for excising transgenes from the pollen genome, a fused recognition site, inc...