A Cre/loxP-mediated self-activating gene excision system to produce marker gene free transgenic soybean plants

Abstract

Marker-gene-free transgenic soybean plants were produced by isolating a developmentally regulated embryo-specific gene promoter, app1, from Arabidopsis and developing a self-activating gene excision system using the P1 bacteriophage Cre/loxP recombination system. To accomplish this, the Cre recombinase gene was placed under control of the app1 promoter and, together with a selectable marker gene (hygromycin phosphotransferase), were cloned between two loxP recombination sites. This entire sequence was then placed between a constitutive promoter and a coding region for either beta-glucuronidase (Gus) or glyphosate acetyltransferase (Gat). Gene excision would remove the entire sequence between the two loxP sites and bring the coding region to the constitutive promoter for expression. Using this system marker gene excision occurred in over 30% of the stable transgenic events as indicated by the activation of the gus reporter gene or the gat gene in separate experiments. Transgenic plants with 1 or 2 copies of a functional excision-activated gat transgene and without any marker gene were obtained in T0 or T1 generation. This demonstrates the feasibility of using developmentally controlled promoters to mediate marker excision in soybean.

Generation of marker free salt tolerant transgenic plants of Arabidopsis thaliana using the gly I gene and cre gene under inducible promoters

“…However, no transgene of agronomic importance was introduced in the transgenic plants. Recently an embryo specific promoter driving the cre gene has been used for generating marker excision in soybean (Li et al 2007). Other workers (Zuo et al 2001;Sreekala et al 2005;Zhang et al 2003) have used chemically induced autoexcision of selectable markers.…”

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

“…Individual transformed events were selected on hygromycin, and embryos from each event were matured as described previously (Cahoon et al, 1999;Li et al, 2007).…”

Elimination of Transgenic Sequences in Plants by Cre Gene Expression

“…Third, the excisional recombination method was combined with trait genes, illustrating the development from laboratory experiments to practical utilization; this tendency can mainly be observed for the marker gene elimination technology. Among commercial traits combined with the Cre-lox system are modification of protein composition (Ow, 2007), tolerance to environmental stress (Zhang et al, 2009) as well as herbicide (Lutz et al, 2006;Li et al, 2007) and insect (Chakraborti et al, 2008;Zhang et al, 2006) resistance. The first marker-free commercial maize event LY038, which received the US regulatory approval in 2006, provides higher lysine content (Ow, 2007).…”