1.1. Why marker-free transgenic plants?
Selectable marker genes (SMGs), such as antibiotic or herbicide resistance genes, are used in nearly every plant transformation protocol to efficiently distinguish transformed from non-transformed cells. However, once a transgenic event has been selected, marker genes are generally of no use. On the contrary, the continued presence of marker genes in transgenic plants may raise public and regulatory concerns and may have technological disadvantages.
The main perceived risk is horizontal gene transfer of antibiotic resistance genes to pathogenic organisms or the transfer of herbicide resistance genes to weeds. Regulatory agencies may thus advice or require the absence of certain marker genes in commercialized transgenic plants .
Fears concerning SMGs center around the presence of antibiotic resistance genes in transgenic crops or its products that might reduce the efficacy of a clinically important antibiotic. A lot of attention has been spent on risk assessment concerning the transfer of antibiotic resistance genes from genetically modified (GM) plants to soil- and plant-related micro-organisms by horizontal gene transfer. For example, the transformation of bacteria in the food chain where free DNA persists in some materials for weeks, and moreover, some bacteria develop natural/chemical competence to take up DNA from the environment. In addition, in the gastrointestinal tract of humans and farm animals, DNA may remain stable for some time, particularly in the colon. However, degradation already begins before the DNA or the material containing the DNA arrives at the critical sites for horizontal gene transfer, which are generally believed to be the lower part of the small intestine, caecum, and the colon. In the case that DNA can arrive to this part, it will be mostly fractionated in pieces smaller than a gene sequence. Thus, breakdown of DNA in the gut, combined with the breakdown of the DNA due to food processing, strongly reduces the risk of dissemination . Moreover, the antibiotic resistance genes that are commonly used as selectable marker genes in transgenic plants actually have a bacterial origin . Indeed bacteria have developed very sophisticated mechanisms to eliminate competitors and guarantee their own survival producing antibiotics and genes to confer resistance to these antibiotics. Thus, the contribution of horizontal transfer of antibiotic resistance genes between transgenic plants and microorganism is most likely insignificant compared to the existing exchange of such genes between bacteria [3-6].
On the other hand the escape of herbicide resistance genes to wild relatives is also a concern. Many crops are sexually compatible with wild and/or weedy relatives, then if the plants grow close one to another, crop-to-weed or crop-to-wild relative gene ﬂow could result (reviewed by [6, 7]. The success of the introgression of a transgene in a wild relative has many barriers. Firstly, both have to grow in close proximity; secondly, both have to be flowering in overlapping time frames; thirdly, the progeny must be sufficiently fertile to propagate; and fourthly, a selective pressure should be applied (herbicide) . There will only be a selective advantage for the wild relative if the herbicide is used in the habitat where the relative grows. For example, it is well known that cultivated rice is sexually compatible with perennial wild red rice (
In some specific cases, selectable marker genes are needed after selection, for example in propagation of lines with nuclear male sterility . However, generally SMGs are not needed after the selection of the transgene event. On the contrary, their presence may have some technological drawbacks. It has been reported that some genes (selectable markers included) may induce pleiotropic effects under certain conditions [14, 15]. In fact, a transcriptome analysis of three
2. Strategies to obtain marker free transgenic plants
2.1. Transformation without selection
The most straightforward method to obtain marker-free plants is to transform without any selectable marker gene. However, most of the transformation protocols described are inefficient and just few cells integrate the foreign DNA. Nonetheless, some groups have studied the feasibility to obtain transgenic plants omitting selection. De Buck et al.  failed to obtain any transgenic plants when
2.2. Co-transformation of a marker gene and the gene-of-interest followed by segregation and selection of marker free progeny plants
Many approaches have been reported to remove selectable marker genes since the transformation technology was developed in the 80s. 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 (reviewed in [3, 30-32]). This strategy is based on the fact that cells selected for the presence of the marker gene, often contain the non-selected gene of interest as well. The SMG and the gene of interest can be delivered by: (i) two different
2.3. Placing the selectable marker gene or the gene-of-interest on a transposable element
Transposable elements (e.g.
This system has some advantages associated with the relocation of the gene of interest. For example, it permits to study a large range of position effects thereby generating an extensive qualitative and quantitative variation in expression levels from a single transpositionally active transformant line . Moreover, relocation allows elimination through recombination in the progeny of all sequences co-integrated at the original integration site. Thus the integration pattern is simplified and the relocated transposon-borne transgene may be less susceptible to gene silencing than at the original integration .
On the other hand, this system has several drawbacks. First, the transposition efficiency is variable in different species. Second, the method is labor intensive and time consuming because it requires crossing transgenic plants and the selection of the progeny [53, 54]. The method shows low efficiency of marker gene elimination because of the tendency of transposable elements to reinsert in positions genetically linked to the original position. Other disadvantages of this system are the genomic instability of transgenic plants because of the continuous presence of heterologous transposons and the generation of mutations because of insertion and excision cycles. Transposition can induce genome rearrangements, including deletions, inverted duplications, inversions, and translocations . Additionally, this system cannot be used for sterile plants and vegetatively propagated species and is impractical for plants with a long life cycle.
2.4. Homologous recombination
Another method developed for marker gene removal takes advantage of the DNA repair machinery of plant cells. Indeed, efficient repair of double-strand breaks (DSBs) is important for survival of all organisms. DSBs can be repaired via homologous recombination (HR) or non-homologous end-joining (NHEJ) . The ratio of HR to NHEJ events increases if homologous sequences near the brake are available . During the repair process the gene can be converted or deleted . Orel et al.  showed that deletion-associated pathway was about five times more frequent than the pathway resulting in gene conversion. These findings were exploited by Zubko et al. , who placed the selectable marker genes between two directly repeated 352 bp
2.5. Removal of the selectable marker gene after the selection procedure via site-specific recombinases or zinc finger nucleases
Another system to remove selectable marker genes is based on site-specific recombinases and was first reported about 20 years ago [62, 63]. Microbial site-specific recombinases have the ability to cleave DNA at specific sites and ligate it to the cleaved DNA at a second target sequence. The excision of foreign DNA that is placed in between recognition sites in a direct repeat orientation has been used to eliminate unwanted transgenic material from the nuclear genome of plants (Fig. 3). The most used recombination systems are Cre
The site-specific recombination systems can be divided in two categories according to the position of the recombinase gene. In a first category of strategies, the recombinase gene and the selectable marker are on a different vector and the recombinase gene is delivered to the plant containing the SMG by re-transformation [62, 72, 73] or by sexual crosses [63, 74-77].
A main limitation of both systems is that they require a time-consuming and labor-intensive breeding step, and that they are only applicable to sexually reproducing species or some species where the retransformation is available. An alternative approach depends on the expression of the recombinase transiently . Marker-free plants were also obtained after infection of PPT resistant
Nevertheless, as all technologies also the site-specific recombination systems have some drawbacks.
In a second category of methods using site-specific recombination, the selectable marker and the recombinase genes are on the same vector between the recombination sites (Fig. 4). This system is often referred to as “auto-excision”  or self-excision . The auto-excision strategy is a versatile system that could be applied in every species and that shows flexibility in spatial and temporal control. The expression of the recombinase gene can be induced by either external or intrinsic signals resulting in auto-excision of both the recombinase and marker genes placed within the excision site boundaries after their function is no longer needed. The control of excision is enabled by the regulated promoter used to control the recombinase gene. This approach was described with heat-shock inducible promoter-recombinase expression cassettes in
The recombinase can also be driven by chemically regulated promoters, like the GST-II-27 promoter from maize which is induced by an herbicide antidote Safener, to control the
A more refined approach comprises self-excision controlled by an endogenous stimulus that is a part of the plant life cycle. For example, Mlynárová et al.  reported the use of a microspore-specific NTM19 promoter from tobacco to drive the expression of the
The auto-excision strategy is very flexible in timing enabling the excision to take place in late (e.g. flowering or seedling) or early (e.g. somatic embryos) developmental stages. In addition many of these approaches are applicable to vegetatively propagated plant species and and long life cycle plants like perennial trees.
An additional feature of recombination based systems is the capability to resolve complex insertion sites containing multiple tandem insertions of the T-DNA down to more simple or even single copy structures. In wheat, maize and Arabidopsis it was demonstrated that complex integration patterns can be resolved by Cre-mediated recombination, thereby generating single copy transformants [87, 113, 114].
Additionally, the apparent disadvantage of the remaining presence of one
A number of novel recombinase systems have been identified that also show the ability to excise DNA in eukaryotic cells [90, 122-126]. So far, only ParA  and ΦC31  have been effectively used in plant.
An alternative to the site-specific recombination system would be to construct restriction endonucleases that recognize specific T-DNA sequences. Zinc finger nucleases (ZFNs) could for example be used to eliminate selectable marker genes or other unnecessary DNA sequences from the plant genome. ZFNs are artificial restriction enzymes that consist of a synthetic C2H2 zinc finger DNA-binding domain fused to the DNA cleavage domain of the restriction enzyme
However, many ZFNs have been reported to be toxic [131-133] presumably as a result of the creation of non-target DSBs . Thus, the strategy to address this problem would be the regulation of ZFN expression by the use of inducible promoters or the use of transient expression systems like the plant virus systems mentioned above. Another approach could be the redesigning of the
2.6. Removal of transplastome marker gene
In the last decade plastid genome (plastome or ptDNA) has become a popular target for engineering, as this has several advantages like potentially high level protein expression, maternal inheritance and non-dissemination of transgenes through pollen, high transgene copy number per cell and no detected gene silencing . However, selectable marker genes are unnecessary once transplastomic plant has been obtained. Moreover high levels of marker gene expression can cause metabolic problems. Additionally, for selection only spectinomycin and streptomycin (
2.6.1. Homology based SMG excision via directly repeated sequences
This approach is based on the efficient native homologous recombination apparatus of the plastid. This system relies on the presence of directly repeated identical sequences of plastid DNA. Then, any sequence between them could be excised [136, 137]. The first indication of this phenomenon was observed in the unicellular alga,
In the second version  marker-free tobacco plants were generated by the use of a vector that harboured an
2.6.2. Excision by phage site-specific recombinases
Site-specific recombinases have also been used to produce marker-free transplastomic plants. This approach exploits a two-step protocol. Step one is the production of transplastomic plants, which carry a SMG ﬂanked by two directly oriented recombinase target sites (Fig. 7). Afterward, marker-free plants could be obtained when the recombinase activity is introduced by nuclear transformation of a gene encoding a plastid-targeted recombinase[141, 142].
As an alternative, the ΦC31 phage site-specific integrase (Int) that mediates recombination between bacterial (
2.6.3. Transient cointegration of the marker gene
Based on the mechanism of integration of the foreign DNA in the plastid genome, Klaus et al.  designed a system to excise the SMG. Indeed, two homologous recombination events (Left and Rigth) are needed for DNA integration. However, considering that cointegrate formation is a common phenomenon that takes place in bacterial plasmid recombination, the authors assumed that in the chloroplast a transformation vector first forms a cointegrate following recombination between a single region of homology in the transformation vector and the plastome (Fig. 8A). Cointegrates are naturally unstable due to the presence of direct repeats in these molecules. Subsequent homology recombination events (between duplicated sequences) lead either to stable integration of both the GOI and SMG gene or to loss of the integrated vector, yielding a wild-type plastome (Fig. 8A) . In this work the authors used a vector where the marker gene (
The co-transformation-segregation method in plastid transformation technology is based on the same principle that has been applied in nuclear transformation. Indeed, the SMG and the gene of interest are inserted in two different plasmids and introduced into two locations (Fig. 9A) of the same plastid by biolistic transformation to generate heteroplastomic cells with both or either of the genes (Fig. 9B) [137, 146]. After segregation, a marker-free transplastomic plant could be obtained (Fig. 9C). The approach was developed to obtain antibiotic resistance gene-free plants with resistance to herbicides (glyphosate or phosphinothricin) due to the impossibility to obtain such plants directly. Indeed, transplastomic plants cannot be obtained directly by selection with herbicides after transformation with the resistance genes (
3. Marker free transgenic plants with agronomically useful genes
The various methods to obtain marker-free transgenic plants have proven their utility and are increasingly being deployed to obtain crop plants with agronomically useful genes. One of the crops that have received more attention is rice. Indeed some papers have described the production of marker-free transgenic plants with different genes of interest. Applying the co-transformation / segregation strategy with the use of ‘double right border’ twin T-DNA vectors Lu et al.  obtained marker-free transgenic rice plants harboring a
Sripriya et al.  generated marker-free transgenic plants with improved resistance to sheath blight. A single
On the other hand, Sengupta et al.  have exploited the Cre/
The first commercially available marker-free transgenic plant that was obtained through this system was developed by the company Renessen. They generated the transgenic corn line LY038, from which the
In another report, the production of marker-free transgenic soybean [
The expression of a chitinase gene,