Generation of selectable marker-free transgenic rice using double right-border (DRB) binary vectors

Abstract

Currently employed transformation systems require selectable marker genes encoding antibiotic or herbicide resistance, along with the gene of interest (GOI), to select transformed cells from among a large population of untransformed cells. The continued presence of these selectable markers, especially in food crops such as rice (Oryza sativa L.), is of increasing public concern. Techniques based on DNA recombination and Agrobacterium-mediated co-transformation with two binary vectors in a single or two different Agrobacterium strains, or with super-binary vectors carrying two sets of T-DNA border sequences (twin T-DNA vectors), have been employed by researchers to produce selectable marker-free (SMF) transgenic progeny. We have developed a double right-border (DRB) binary vector carrying two copies of T-DNA right-border (RB) sequences flanking a selectable marker gene, followed by a GOI and one copy of the left border sequence. Two types of T-DNA inserts, one initiated from the first RB containing both the selectable gene and the GOI, and the other from the second RB containing only the GOI, were expected to be produced and integrated into the genome. In the subsequent generation, these inserts could segregate away from each other, allowing the selection of the progeny with only the GOI. We tested this vector using two selectable marker genes and successfully obtained progeny plants in which the second selectable marker gene segregated away from the first. Using the DRB binary vector system, we recovered SMF transgenic lines containing a rice ragged stunt virus (RRSV)-derived synthetic resistance gene in the rice cultivars Jarrah and Xiu Shui. Approximately 36–64% of the primary transformants of these cultivars yielded SMF progeny. Among SMF Jarrah transgenic progeny <50% of plants contained the RRSV transgene. Thus, we have developed an efficient vector for producing SMF plants that allows straightforward cloning of any GOIs in comparison with the published ‘twin T-DNA’ vectors.

A large-scale study of rice plants transformed with different T-DNAs provides new insights into locus composition and T-DNA linkage configurations

“…Previously, we showed that such an approach is prone to underestimate transgenic locus number and therefore to present a distorted view of T-DNA linkage groups in plants (Vain et al 2003). Recently, it has been shown that 30-60% of the plants transformed with different T-DNAs can contain several types of T-DNA inserts (Lu et al 2001;Miller et al 2002) with some inserts containing either linked or unlinked T-DNAs or a mixture of both. The further development of the multiple T-DNA strategy relies, therefore, on a better understanding of locus constitution and T-DNA linkage in populations of transgenic plants.…”

“…The multiple T-DNA approach was also the first to be implemented in cereals and is now used to produce markerfree transgenic rice Lu et al 2001), barley (Matthew et al 2001) and maize (Miller et al 2002) plants. Rice plants containing only a rice ragged stunt virus resistance transgene have been produced using the one vector/one strain version of this strategy (Lu et al 2001). The multiple T-DNA approach is likely to be one of the preferred type of "clean-gene" technology to produce transgenic cereal crops free of selectable marker genes in the near future.…”

“…However, important limitations still lie in the uncontrolled factors affecting the integration and behaviour of transgenes. Strategies to control transgene integration (Paszkowski et al 1988;Albert et al 1995;Terada et al 2002) and to limit unwanted or multiple integrated DNA sequences (Hanson et al 1999;Srivastava et al 1999;Lu et al 2001;Cotsaftis et al 2002) are central to the development of advanced plant transformation technologies. Among these, the production of marker-free transgenic plants is particularly important.…”

“…Most previous studies on T-DNA linkage, with few exceptions (Bhattacharyya et al 1994;Cluster et al 1996;Lu et al 2001;Matthew et al 2001), were based on the molecular analysis of primary transformants (Southern blot analysis of T 0 plants) combined with the segregation analysis of the transgene phenotype. Previously, we showed that such an approach is prone to underestimate transgenic locus number and therefore to present a distorted view of T-DNA linkage groups in plants (Vain et al 2003).…”

Cre/lox-mediated marker gene excision in transgenic maize (Zea mays L.) plants

“…A number of methods for marker gene removal from transgenic plants have been reported in the literature, including cotransformation of T-DNAs followed by segregation of the marker gene from the trait gene (Depicker et al 1985;de Frammond et al 1986;McKnight et al 1987;De Block and Debrouwer 1991;Komari et al 1996;De Neve et al 1997;Daley et al 1998;Xing et al 2000;Lu et al 2001;McCormac et al 2001), homologous recombination between direct repeats (Lichtenstein et al 1994;Zubko et al 2000) and sitespecific recombination. A number of site-specific recombinases of prokaryotic or yeast origin have been shown to function in transgenic plants for marker removal, including Cre/lox from bacteriophage P1 Ow 1990, phage Mu (Maeser andKahmann 1991).…”