Less is more: strategies to remove marker genes from transgenic plants

Abstract

Selectable marker genes (SMGs) and selection agents are useful tools in the production of transgenic plants by selecting transformed cells from a matrix consisting of mostly untransformed cells. Most SMGs express protein products that confer antibiotic- or herbicide resistance traits, and typically reside in the end product of genetically-modified (GM) plants. The presence of these genes in GM plants, and subsequently in food, feed and the environment, are of concern and subject to special government regulation in many countries. The presence of SMGs in GM plants might also, in some cases, result in a metabolic burden for the host plants. Their use also prevents the re-use of the same SMG when a second transformation scheme is needed to be performed on the transgenic host. In recent years, several strategies have been developed to remove SMGs from GM products while retaining the transgenes of interest. This review describes the existing strategies for SMG removal, including the implementation of site specific recombination systems, TALENs and ZFNs. This review discusses the advantages and disadvantages of existing SMG-removal strategies and explores possible future research directions for SMG removal including emerging technologies for increased precision for genome modification.

Efficient Generation of Marker-Free Transgenic Rice Plants Using an Improved Transposon-Mediated Transgene Reintegration Strategy

“…For the cotransformation, site-specific recombination, and other T-DNA-based strategies for marker gene removal (Ebinuma et al, 2001;Wang et al, 2011;Kapusi et al, 2013;Yau and Stewart, 2013), the complex transgene integration patterns such as multiple T-DNA loci, tandem-linked T-DNA copies, T-DNA truncations, and cotransferred non-T-DNA vector backbone sequence in transgenic plants (Kim et al, 2003;Rai et al, 2007) may complicate the molecular analysis of transgene integration and affect the stability of transgene expression. In this study, we validated that the maize Ac/Ds transposable element can be routinely used to generate marker-free transgenic plants in rice.…”

“…However, the retention of such marker genes in transgenic plants is undesirable because of their possible toxicity or allergenicity to humans and their unpredictable hazards to the environment (Miki and McHugh, 2004;Ramessar et al, 2007;Yau and Stewart, 2013). Therefore, the production of marker-free transgenic crops is crucial to avoid these risks and promote their commercial deployment.…”

“…Several strategies have been developed to remove selectable markers from transgenic plants while retaining the gene of interest (GOI; Ebinuma et al, 2001;Cotsaftis et al, 2002;Wang et al, 2011;Kapusi et al, 2013;Yau and Stewart, 2013;Oliva et al, 2014). Transposition is an advantageous strategy for marker gene removal, because it allows intact transgene insertion with defined boundaries and requires only a few primary transformants (Cotsaftis et al, 2002).…”

7. Themenbereich grüne Gentechnologie: Pflanzenzüchtung und Agrarwirtschaft

“…48 Neben der Verwendung alternativer Markergene kommt auch der Erzeugung markerfreier transgener Pflanzen eine steigende Bedeutung zu. Hierbei wird das verwendete Selektionsmarker-Gen durch unterschiedliche Mechanismen nach erfolgter Transformation wieder aus dem Genom entfernt (Khan et al, 2011;Yau/Stewart, 2013 …”

Marker-free transgenic rice lines with a defensin gene are potentially active against phytopathogenic fungus Sarocladium oryzae

“…Many approaches have been reported to remove selectable marker genes since the transformation technology was developed. One of the earliest methods was based on co-transformation of a transgene and a selectable marker delivered by two separate DNA molecules and thereafter, segregation of both in the progeny (Yau and Stewart, 2013).…”