Site-Specific Integration of Transgenes in Soybean via Recombinase-Mediated DNA Cassette Exchange

Abstract

A targeting method to insert genes at a previously characterized genetic locus to make plant transformation and transgene expression predictable is highly desirable for plant biotechnology. We report the successful targeting of transgenes to predefined soybean (Glycine max) genome sites using the yeast FLP-FRT recombination system. First, a target DNA containing a pair of incompatible FRT sites flanking a selection gene was introduced in soybean by standard biolistic transformation. Transgenic events containing a single copy of the target were retransformed with a donor DNA, which contained the same pair of FRT sites flanking a different selection gene, and a FLP expression DNA. Precise DNA cassette exchange was achieved between the target and donor DNA via recombinase-mediated cassette exchange, so that the donor DNA was introduced at the locus previously occupied by the target DNA. The introduced donor genes expressed normally and segregated according to Mendelian laws.

An Improved Variant of Soybean Type 1 Diacylglycerol Acyltransferase Increases the Oil Content and Decreases the Soluble Carbohydrate Content of Soybeans

“…Recombination-mediated cassette exchange (Li et al, 2009(Li et al, , 2010b) was used to create transgenic soybean events that expressed the GmDGAT1b-MOD variant under the control of the promoter of the soybean b-conglycinin a-prime subunit gene (Glyma.10G246300) known to confer strong seed-preferred gene expression (Beachy et al, 1985). A total of four events were created that harbor a single-copy transgene at the same location in the soybean genome (Supplemental Fig.…”

Directed genome engineering for genome optimization

“…However, site-specific recombination in the plant genome has only been possible at a pre-engineered recombination site and did not allow precise engineering at will at any desired position in the plant genome. Successful targeting of transgenes to preengineered recombination sites was reported for crop plants such as rice (Srivastava and Ow, 2001, Srivastava et al, 2004, Chawla et al, 2006 and soybean (Li et al, 2009). Recently, considerable progress has been made in the engineering of zinc-finger recombinases and TALE recombinases.…”

Strategies for Improvement of Soybean Regeneration via Somatic Embryogenesis and Genetic Transformation

“…Several studies about soybean transformation via particle bombardment using embryogenic tissues have been published (Finer & McMullen, 1991;Finer et al, 1992;Stewart et al, 1996;Droste et al, 2002;Homrich et al, 2008;Schmidt et al, 2008;Wu et al, 2008;Li et al, 2009;Hernandez-Garcia et al, 2009;Xing et al, 2010).…”

“…Sato et al (1993) proved that somatic embryo proliferation occurred from single epidermal cells of existing somatic embryos. Using proliferative embryos as target tissue many studies succeed in regenerating completely transformed plants (Finer & McMullen, 1991;Finer et al, 1992;Stewart et al, 1996;Trick & Finer, 1998;Droste et al, 2002;Homrich et al, 2008;Schmidt et al, 2008;Wu et al, 2008;Li et al, 2009;Hernandez-Garcia et al, 2009;Xing et al, 2010;Wiebke-Strohm et al, 2011).…”

“…Even after more than two decades, the stable transformation of soybean cannot yet be considered as routine because it depends on the ability to bring together efficient transformation and regeneration techniques. Two methods have been successfuly used: particle bombardment Finer & McMullen, 1991;Christou & McCabe, 1992;Finer et al, 1992;Stewart et al, 1996;Aragão et al, 2000;Droste et al, 2002;Homrich et al, 2008;Wu et al, 2008;Li et al, 2009;Hernandez-Garcia et al, 2009;Xing et al, 2010;Viana et al, 2011) and Agrobacterium tumefaciens system Trick & Finer, 1998;Aragão et al, 2000;Yan et al, 2000;Ko et al, 2003Ko et al, , 2004Paz et al, 2006;Hong et al, 2007;Miklos et al, 2007;Liu et al, 2008;Wang & Xu, 2008;Wiebke-Strohm et al, 2011).…”