Site-specific excisional recombination strategies for elimination of undesirable transgenes from crop plants

Abstract

A major limitation of crop biotechnology and breeding is the lack of efficient molecular technologies for precise engineering of target genomic loci. While transformation procedures have become routine for a growing number of plant species, the random introduction of complex transgenenic DNA into the plant genome by current methods generates unpredictable effects on both transgene and homologous native gene expression. The risk of transgene transfer into related plant species and consumers is another concern associated with the conventional transformation technologies. Various approaches to avoid or eliminate undesirable transgenes, most notably selectable marker genes used in plant transformation, have recently been developed. These approaches include cotransformation with two independent T-DNAs or plasmid DNAs followed by their subsequent segregation, transposon-mediated DNA elimination, and most recently, attempts to replace bacterial T-DNA borders and selectable marker genes with functional equivalents of plant origin. The use of site-specific recombination to remove undesired DNA from the plant genome and concomitantly, via excision-mediated DNA rearrangement, switch-activate by choice transgenes of agronomical, food or feed quality traits provides a versatile "transgene maintenance and control" strategy that can significantly contribute to the transfer of transgenic laboratory developments into farming practice. This review focuses on recent reports demonstrating the elimination of undesirable transgenes (essentially selectable marker and recombinase genes) from the plant genome and concomitant activation of a silent transgene (e.g., a reporter gene) mediated by different site-specific recombinases driven by constitutive or chemically, environmentally or developmentally regulated promoters. These reports indicate major progress in excision strategies which extends application of the technology from annual, sexually propagated plants towards perennial, woody and vegetatively propagated plants. Current trends and future prospects for optimization of excision-activation machinery and its practical implementation for the generation of transgenic plants and plant products free of undesired genes are discussed.

Generating Marker-Free Transgenic Wheat Using Minimal Gene Cassette and Cold-Inducible Cre/Lox System

“…Cre/lox auto-excision system was successfully applied for eliminating the undesirable parts of the transformation vectors as the selection marker gene, in many species (Gidoni et al 2008;Tuteja et al 2012). For the induction of cre, in auto-excision strategy, many types of promoters have already been tested with different efficiency (Chakraborti et al 2008;ChongPerez et al 2012ChongPerez et al , 2013Russell et al 1992).…”

“…It is no question therefore, how to introduce foreign genes into the host genome. However precise incorporation and controlled expression still present a challenge, because the most efficient methods allow random introduction of transgenes into the plant genome generating unpredictable effects on both transgene and native gene expression (Gidoni et al 2008). Beside to the appropriate gene expression, the risk of utilization of transgenic plant would be decreased by controlling gene integration and transcription and thus the public concerns would be appeased.…”

“…An additional method is the site-specific recombination which proved to be the most efficient way of marker gene elimination because of its relative simplicity. The natural function of these systems is the control of the precise excision or integration of defined DNA units into the host genome (Gidoni et al 2008;Nandy and Srivastava 2012). The phage mediated recombination was successfully used in plant biotechnology (Kempe et al 2010;Kittiwongwattana et al 2007).…”

“…The limited expression of the recombinase gene can be achieved by using tissue specific, developmentally regulated or inducible promoters in self-excising cre-recombinase vectors (Gidoni et al 2008). Numerous transformation systems, based on inducible promoters, were constructed, and used successfully for transgene elimination experiments in many species (Chong-Perez et al 2012;Liu et al 2005;Zuo et al 2001).…”

Next generation biotechnology: how sophisticated constructs lead to further insights and new approaches towards biotechnology’s demands

“…In the last twenty years there have been several successful attempts of marker gene excision in a variety of plants (Russell et al 1992, Dale & Ow 1992, Kerbach et al 2005, Darbani et al 2006, Gidoni et al 2008. While the first attempts of marker excision were carried out with constitutive promoters, the last five years have brought a shift to a finer regulated on demand activation of the marker excision.…”