Methods to produce marker‐free transgenic plants

Abstract

Selectable marker genes (SMGs) have been extraordinarily useful in enabling plant transformation because of the low efficiency of transgene integration. The most used SMGs encode proteins resistant to antibiotics or herbicides and use negative selection, i.e., by killing nontransgenic tissue. However, there are perceived risks in wide-scale deployment of SMG-transgenic plants, and therefore research has recently been performed to develop marker-free systems. In this review, transformation using markers not based on antibiotic or herbicide resistance genes, as well as different systems of marker gene deletion, are discussed.

Nonhomologous End Joining-Mediated Gene Replacement in Plant Cells

“…Transgene removal from transgenic plants is an important technology for molecular breeding and for gene containment (Moon et al, 2010), and it is frequently achieved using novel vectors and site-specific recombination systems (Darbani et al, 2007). Transgene deletion by ZFNs was recently reported by Petolino et al (2010), who flanked a transgene with the ZFN CCR5 (Perez et al, 2008).…”

Genetic transformation of apple (Malus x domestica) without use of a selectable marker gene

“…Horizontal gene transfer from plants to environmental and medically significant bacteria or from plant products consumed as food to intestinal microorganisms or to human cells is generally considered to be of extremely low frequency. However, the inherent risks have not been totally addressed, and there remains both regulatory and public concern in many countries (Darbani et al 2006 T679 T206 T306 T162 T468 T622 T850 T870 T1292 N Ag T670 T658 Fig. 4 Comparative RT-PCR for attacin E, GUS, nptII, EF-1α, and VirG in leaves of acclimated plants from M.26 transgenic lines obtained by markerless DNA transformation technology (T206 to T1292) and conventional transformation using nptII selection (T639 to T679) expressing the attacinE and gus genes under the control of the pin2 promoter and 35S promoter, respectively.…”

“…However, there is no evidence that the transgenic markers presently in use pose a health risk to humans or domestic animals. Nevertheless, some researchers and regulators have concluded that, although the transformation risk of plant-transmitted antibiotic resistance genes to pathogenic bacteria is very small, the use of markers conferring resistance to clinically relevant antibiotics should be phased out as suitable alternative technologies become available in plant biotechnology (Darbani et al 2006). Public concerns about the issue of the environmental safety of genetically modified plants have led to a demand for technologies allowing the production of transgenic plants without selectable (especially antibiotic resistance) markers.…”

Advances in genetic engineering of marine algae

“…The selection marker removal may significantly reduce the public acceptance of genetically modified plants (Miki and McHugh, 2004). Several marker elimination methods in higher plants have been developed during these years including co-transformation, which is usually applied in Agrobacterium-mediated transformation (Sripriya et al, 2008) and various site-specific recombination methods that eliminate the selection marker by deleting or inverting the marker gene with the help of an enzyme recombinase (Cotsaftis et al, 2002;Darbani et al, 2007, Kopertekh et al, 2004, Ow, 2002. The marine algal genetic transformation will certainly meet these biosafety issues (more discussion can be found in Section 6) when cultivating transgenic microalgae in an open pond of a coastal area or transgenic macroalgal sporophytes in the open sea.…”