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Chapter 2
Strategies for Generating Marker-Free Transgenic Plants
Borys Chong-Pérez and Geert Angenon
Additional information is available at the end of the chapter
http://dx.doi.org/10.5772/55573
1. Introduction
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 [1].
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 gastrointes‐
tinal 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 [2]. Moreover, the antibiotic resistance
genes that are commonly used as selectable marker genes in transgenic plants actually have a
© 2013 Chong-Pérez and Angenon; licensee InTech. This is an open access article distributed under the terms
of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
18 Genetic Engineering
bacterial origin [3]. 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 overlap‐
ping time frames; thirdly, the progeny must be sufficiently fertile to propagate; and fourthly,
a selective pressure should be applied (herbicide) [8]. 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 (Oryza
rufipogon Griff.), considered a harmful weed. It grows in many of the same regions, often has
overlapping flowering times, and thus is a prime candidate for gene flow with cultivated rice.
Indeed, Chen et al. [9] showed that the gene flow rate was 0.01% under natural conditions.
This and other studies showed the risk of the transfer of transgene(s) to the wild relative or
weeds. Thus precautions should be taken into account to prevent gene flow and introgression.
A possible way consists in containing transgenic pollen by growing barrier crops in adjacent
areas or by alternating transgenic cultivars carrying different herbicide resistance genes [10].
Other strategies consists in the creation of biological containment, to limit the transfer of pollen
to plants in the surrounding area, e.g. by engineering male sterility or by delaying and/or
decreasing flowering [11, 12]. Alternatively, complete removal of the marker gene should
alleviate concerns regarding effects on human health and the environment.
In some specific cases, selectable marker genes are needed after selection, for example in
propagation of lines with nuclear male sterility [13]. 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 Arabidopsis transgenic lines containing pCAMBIA3300 vector (35S-bar-35S)
showed that they differ from their WT counterparts by expression of 7, 18 and 32 genes
respectively. However, only four genes were found to be significantly different in all three
lines compared with the wild type plant in glufosinate untreated plants [14]. Thereafter, 81
genes were found to be differentially expressed in the presence of glufosinate in transgenic
plants, in contrast to the 3762 differentially expressed genes in WT plants. From these 81 genes
29 were specific to transgenic plants [14]. These results suggested to the authors that glufosi‐
nate or a metabolic derivative of glufosinate activates unique detoxification pathways to offset
any effects on plant growth and development. Nevertheless, in the above mentioned work, no
indication or study of the position effect and/or effect of transgene regulatory sequences was
reported. Indeed the regulatory sequences (promoters and terminators) can influence the
activity of some genes in the same T-DNA or even endogenous genes that are close to the
Strategies for Generating Marker-Free Transgenic Plants 19
http://dx.doi.org/10.5772/55573
insertion site [16-18]. Furthermore, in systems where the number of efficient SMGs is limited,
the re-transformation with the same SMG is precluded by its presence. This is problematic as
most transformation protocols are indeed based on one or a few selectable marker genes only.
Miki and McHugh [3] reported that more than 90 % of the scientific publications that use
transgenic plants were based on three selection systems: the antibiotics kanamycin or hy‐
gromycin and the herbicide phosphinothricin. These outcomes provide an extra motivation to
remove SMGs and other unnecessary sequences as soon as possible after selection of transgenic
plants.
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 ineffi‐
cient 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. [19] failed to obtain any
transgenic plants when Arabidopsis roots were transformed via A. tumefaciens and shoots
regenerated on non-selective media. However, in tobacco protoplast transformation, these
authors obtained a total transformation frequency of 18%. Transformation protocols have
important influence on these and other results. For example, in a study where Arabidopsis was
transformed by the floral dip protocol and seedlings were grown on non-selective media,
transgenic plants could be obtained with an efficiency of 3.5% [20]. In citrus, 35 plants out of
620 analyzed were transgenic in the absence of selection [21]. The main objective of the
experiments mentioned until here was not to obtain marker-free plants, but they showed the
possibility or not to do so. Other experiments have as a goal to obtain marker free transgenic
plants. For example, in wheat transformation via micro-projectile bombardment, 23 out of 191
regenerated plants in non-selective media were transgenic (12%) [22]. Also in potato and
cassava transformation via A. tumefaciens, without selection pressure, resulted in transformed
shoots at an efficiency of 1–5% of the harvested shoots [23]. In this case the presence of chimeric
plants was less than 2% of transgenic plants. Other authors that mention the possibility to
obtain chimeric plants were Doshi et al.[24], which obtained a transformation frequency of
0.93% and 1.55% in triticale and wheat, respectively, without selection. These authors suggest
circumventing the chimera problem with two embryogenesis cycles, where the plantlets can
be regenerated from secondary embryos formed from transformed sectors within a primary
somatic embryo. A report of a non-selection approach for tobacco transformation showed a
transformation efficiency of 2.2-2.8% for the most effective binary vector; the authors found
that the number of chimeric plants was 28-56%, which is expected taking into account the
regeneration system applied [25]. In another interesting report the direct production of marker-
free citrus plants under non-selective conditions was assessed [26]. In two genotypes evalu‐
ated, only one produced transgenic plants with an efficiency of 1.7%. Remarkably, the
expression of the gene of interest (sgfp) was very low in transgenic plants. This phenomenon
has been reported before in citrus [21], Arabidopsis thaliana [20, 27] and white pine (Pinus
strobus L.) [28] transformed with A. tumefaciens and regenerated without selection. These
results are in line with the fact that T-DNA integrates randomly with respect to particular DNA
sequences in the genome, and that target sites include transcriptionally ‘silent’ regions, like
telomeres [29]. Cells with insertion events in such silent regions likely do not survive when
selection is applied.
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 transfor‐
mation 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 Agrobacterium strains each containing a binary plasmid carrying a single
T-DNA region (Fig. 1A) [33-37]; (ii) a single Agrobacterium strain, either containing one plasmid
with two separate T-DNAs (Fig. 1C) [33, 36, 38-41] or (iii) containing two separate plasmids
each containing a T-DNA (Fig. 1B) [42,43]. Alternatively, co-transformation can be achieved
by particle co-bombardment (Fig. 1D) [44, 45]. The co-transformation strategy is limited
because co-integration of both T-DNAs at the same genomic locus is frequently observed
leading to linkage between the marker and the transgene, which makes their segregation
impossible. This phenomenon has even more frequently been observed with particle bom‐
bardment-mediated transformation. Moreover, these methods cannot be applied to sterile
plants and vegetatively propagated species, and are not practical in plants with a long life cycle
such as trees [46]. This approach requires the generation of many transformants (to find
unlinked marker genes and genes-of-interest) and further crossing steps (to remove the marker
gene) making it a labor intensive work.
2.3. Placing the selectable marker gene or the gene-of-interest on a transposable element
Transposable elements (e.g. Ac/Ds from maize) can mediate repositioning of genetic material
in the plant genome. The Ac/Ds transposable element system has been used for relocation and
elimination of a selectable marker in tomato [47, 48] and rice [49]. Transposable elements can
be excised from the genome after the expression of the transposase; they can either re-insert
or not (Fig. 2). Taking into account these options, two approaches can be followed. In the first
one, if one counts on re-insertion of the transposon, the gene of interest (GOI) is placed on the
transposable element. Thus, the GOI will be excised and can be reinserted in a locus that is not
linked to the locus in which the selectable marker gene is located; they can be segregated in
the next generation [47, 49]. In a second approach, one relies on the fact that the transposon
will not be re-inserted [50]. An example of such a system is the one described by Ebinuma et
al. [51], in which the ipt selectable marker gene was inserted in an Ac derivative. However,
marker-free transgenic plants were obtained only with a very low efficiency (5%) as a result
of a high rate of re-insertion.
http://dx.doi.org/10.5772/55573
Figure 1. Co-transformation / segregation strategy to obtain marker-free transgenic plants. The SMG and the gene of
interest (GOI) are introduced on separate T-DNAs present in two different Agrobacterium strains (A), on separate vec‐
tors in the same Agrobacterium strain (B), or on the same vector (C); the two genes can also be delivered by a direct
gene transfer method such as particle bombardment (D). If the GOI and the SMG are integrated at unlinked positions,
progeny plants with only the GOI can be obtained after sexual propagation. LB: T-DNA left border, RB: T-DNA right
border, Prom: promoter; Term: terminator
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 [49]. 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 [52].
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 trans‐
posable 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 [55]. 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) [56]. The ratio of HR to NHEJ events increases if
homologous sequences near the brake are available [57]. During the repair process the gene
can be converted or deleted [58]. Orel et al. [56] 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. [59], who placed the selectable marker genes between two
directly repeated 352 bp attP regions of bacteriophage λ. This sequence is rich in A+T nucleo‐
tides that is supposed to have a stimulatory effect on recombination [60]. Moreover, these
elements were situated adjacent to a copy of the transformation booster sequence (TBS) from
Petunia hybrida, which was shown to increase both HR and NHEJ in Petunia, Nicotiana and
maize [61]. After selection on antibiotic (kanamycin) containing media, tobacco callus was
Figure 2. Transposon-mediated repositioning of the SMG. The SMG is cloned as part of a modified transposable ele‐
ment, e.g. the maize transposable element Ac, and linked to the gene of interest (GOI). Transposition may result in re-
insertion of the modified element with the SMG (A); if the re-insertion occurs in an unlinked position, marker-free
progeny may be obtained after crossing. Alternatively, no re-insertion occurs after excision of the modified transposa‐
ble element (B), also resulting in the loss of the SMG.
http://dx.doi.org/10.5772/55573
placed on antibiotic-free media to allow for the loss of SMG by homologous recombination.
Thereafter, plants were regenerated from callus and selection of marker free plants was based
on sensitivity to the antibiotic. Two clones showed sensitivity to the antibiotic and formed
green and white shoots. From these clones the authors regenerated 23 marker free plants.
However, from these marker-free plants, 20 lost the gene of interest that was outside of the
attP sites, probably because the NHEJ mechanism [59]. This protocol should produce marker-
free plants faster than do procedures involving re-transformation or cross-pollination, and also
avoid potential problems related with expression of recombinases (discussed below). None‐
theless, the method has some major disadvantages, like low efficiency, deletions of non-target
genes, the recombination cannot be controlled and many transgenic events can be lost during
the selection process. The mechanistic basis of the phenomenon is not yet understood and it
is not yet known how the system could be applied in other crops.
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/lox from bacteriophage
P1 [64, 65], FLP/FRT from Saccharomyces cerevisiae [66, 67] and R/RS from Zygosaccharomyces
rouxii [68]. These systems are belonging to the tyrosine recombinase family [69, 70]. After the
reaction, a recombination site (lox, FRT or RS) is remaining in the genome and it could
potentially serve as a site for integrative recombination. However, re-insertion of the elimina‐
tion fragment has not been detected [53, 71], probably because excision is an intramolecular
event, whereas integration needs interaction between unlinked sites; and second, the excised
circle cannot replicate autonomously and is probably rapidly lost in vivo [30].
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 [78]. Marker-free plants were also obtained after
infection of PPT resistant Nicotiana benthamiana and Arabidopsis thaliana leaves with a modified
plant virus carrying the cre gene (PVX-Cre) [79-81]; as well as in kanamycin resistant tobacco
with a TMV-Cre [82]. This method can be applied to vegetatively propagated and long life
cycle plants, but the lack of virus-based transformation system in these species is a drawback
that should be improved in the future.
Figure 3. Removal of selectable marker genes through site specific recombinases. The SMG is flanked by directly re‐
peated recombinase recognition sites, most often the lox, FRT or RS sites (black triangles; sequence of the sites is
shown in the upper right corner). The presence of the cognate recombinase enzyme, Cre, FLP or R respectively, directs
excision of the SMG. Re-insertion of the SMG occurs with low frequency if at all.
Nevertheless, as all technologies also the site-specific recombination systems have some
drawbacks. In vitro studies suggest that Cre can catalyze recombination between certain
naturally occurring “pseudo-lox sites” that can be highly divergent from the lox consensus
sequence [83]. It was also shown that constitutive expression of cre can lead in animal cells to
growth-inhibitory and genotoxic effects as a result of the endonuclease activity of Cre [84, 85].
This toxic effect was also investigated in cre expressing transgenic plants where a correlation
was found between aberrant phenotypes and constitutive cre expression [86]. Data regarding
the presence of cryptic FRT or RS sites or the infidelity of FLP- or R-recombinase activities in
higher eukaryotes do not appear to be available [30]. These findings suggest it may be useful
to limit cre expression both temporally and spatially, by placing it under the control of
regulated promoters.
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” [87] or self-excision [88]. 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
http://dx.doi.org/10.5772/55573
recombinase gene. This approach was described with heat-shock inducible promoter-recom‐
binase expression cassettes in Arabidopsis [89, 90], tobacco [91, 92], potato [93], maize [94],
Chinese white poplar (Populus tomentosa Carr.) [95], hybrid aspen (Populus tremula L. × P.
tremuloides Michx.) [96] and rice [97, 98]. In the latter experiment, the selectable marker gene
and cre gene were co-bombarded, but probably the efficiency could be higher if both were on
the same vector [98]. We have recently obtained transgenic banana plants devoid of the marker
gene using a Cre-lox auto-excision strategy induced by heat shock [99].
Figure 4. Site-specific recombinase based auto-excision systems. The site specific recombinase gene (REC) is under
control of an inducible promoter (indProm) and is placed together with the SMG between directly repeated recombi‐
nase recognition sites (black triangles). Induction of recombinase gene expression leads to excision of the SMG and
the recombinase gene.
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 R/RS
system in tobacco [100] and aspen [46, 101], with β-estradiol trans-induction of Cre/lox in
Arabidopsis [102], rice [103], and tomato [104] and with a dexamethasone-glucocorticoid
receptor ligand binding domain activated R/RS system in strawberry [105] and potato [106].
In the latter cases, the authors used a combined positive–negative selection scheme to obtain
marker- and recombinase-free genotypes [105, 106].
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. [107] reported the use of a
microspore-specific NTM19 promoter from tobacco to drive the expression of the cre gene.
Thus the excision of the marker gene is taking place during the microsporogenesis where the
efficiency was close to 100% in tobacco seeds. An improvement of this system was reported
by Luo et al. [108] and was called ‘GM-gene-deletor’. In this, the excision target unit is flanked
with two different fused target sites (lox-FRT), as an alternative to the use of one recombination
site (either lox or FRT) at each side. Activation of either recombinase (Cre or FLP) by a pollen
or pollen- and seed-specific promoter PAB5, gave up to 100% excision efficiency of lox-FRT
fusion-bounded transgenes in some transformation events, leaving residual LB and RB
elements flanking a lox-FRT site, in both pollen and/or seed. The use of germline-specific
promoters derived from the Arabidopsis APETALA1 and SOLO DANCERS genes, and com‐
bined with a positive-negative selection strategy, allowed Verweire et al. [87] to produce
completely marker- and recombinase-free Arabidopsis plants. Similarly, the expression of Cre
driven by the rice floral specific OsMADS45 gene promoter, excised the nptII gene flanked by
lox recombination sites in T1 rice generation [109]. In another approach, Li et al. [110] took
advantage of the somatic embryogenesis developmental stage required in soybean transfor‐
mation. In this report, the activation of cre gene was driven by the Arabidopsis app1 embryo-
specific gene promoter and successfully directed the production of marker- and recombinase-
free soybean; in 13% of the events complete excision was noted, whereas 31% yielded chimeras
and in 56% of the events the excision failed [110]. Excision systems have also been developed
based on seed inducible promoters. Indeed, the cruciferin C promoter from Arabidopsis was
used to control the expression of cre gene in tobacco seeds [88] but the excision efficiency was
low (10.2%). Additionally, in a similar strategy Brassica napus and tobacco marker free plants
were obtained when cre gene was driven by a seed-specific-napin promoter form Brassica
napus [111, 112]. In B. napus the efficiency ranged from 13 to 81% and in tobacco from 55 to
100% [111, 112].
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 lox site after the
excision is, in some cases, an important advantage. Indeed, the marker-free transgenic line
containing one lox site can be used as a target line for gene stacking. It has been reported that
the Cre/lox system can be used to introduce DNA via site-specific integration [115-120]. This
has three major advantages. First, the stacked trait is integrated in the genome in a genomic
locus giving predictable transgene expression. Second, the trait is introduced in a locus which
is already approved by the regulating authorities. Third, by stacking the traits in this way they
are linked to one another facilitating breeding programs [121].
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 [90] and ΦC31 [126] have been
effectively used in plant.
http://dx.doi.org/10.5772/55573
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 FokI [127, 128]. ZFNs are capable of inducing targeted double strand breaks[129]. Until
now, a unique approach for ZFN-mediated transgene deletion was reported by Petolino et al.
[130]. These authors crossed ZFN-overexpressing plants with target transgenic plants, which
were engineered to carry a GUS expression cassette that was flanked by recognition sites for
the ZFN. Both types of transgenic plants were homozygous for the transgene. The higher
frequency of GUS negative hybrid plants was 35 % for one particular cross. PCR and sequenc‐
ing analyses confirmed that the GUS cassette had indeed been removed.
However, many ZFNs have been reported to be toxic [131-133] presumably as a result of the
creation of non-target DSBs [134]. 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 FokI cleavage domain to create obligate heterodimers [134].
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 [135]. 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 (aadA) or kanamycin (nptII or kan and aphA-6) genes have been used. Then,
four strategies to produce marker-free transplastomic plant have been developed: (i) homol‐
ogy-based excision via directly repeated sequences, (ii) excision by phage site-specific recom‐
binanses, (iii) transient cointegration of the marker gene, and (iv) co-transformation-
segregation approach.
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, Chlamydomonas reinhardtii where
homologous recombination between two direct repeats allowed marker removal under non-
selective growth conditions [136]. Later experiments demonstrate marker excision in tobacco
chloroplasts after transformation with a construct carrying three transgenes (uidA, aadA and
bar genes) [135]. In the transformation vector, the authors placed two of the three genes under
the same promoter (Prrn promoter of the rRNA operon, and all the genes with the same
transcription terminator (TpsbA) (Fig. 5). Initial heteroplastomic clones were obtained by
selection for spectinomycin and streptomycin resistance conferred by aadA. Thereafter,

herbicide-resistant and -sensitive derivatives were identified in the absence of antibiotic
selection [135]. Finally the recombination between repeated sequences rendered two types of
stable marker-free plants: recombination R1 via the Prrn repeat produced herbicide resistant
clones and by recombination R2 via TpsbA, Gus expressing clones were obtained (Fig. 5).
Neither type of marker-free transgenic plants has repeated sequences. As herbicide resistance
genes could not be used to directly select plastid transformants [138], this strategy is very useful
to obtain marker-free highly herbicide resistant plants. Actually there are two versions of this
strategy. The first one allowed visual tracking of the SMG excision by creation of a pigment-
deficient zone due to the loss of a plastid photosynthetic gene rbcL [139]. The authors placed
the uidA gene under control of the PatpB promoter. The recombination between this sequence
with the native PatpB, which is located closed to the rbcL gene, allowed the deletion of a large
DNA segment that comprises the uidA-aadA-rbcL genes. The cells lacking rbcL could be visually
identified by their pale green color; these cells also lack the uidA and aadA genes. This approach
could facilitate advanced studies that require the isolation of double mutants in distant plastid
genes and the replacement of the deleted locus with site-directed mutant alleles and is not
easily achieved using other methods [139].
Figure 5. Homology-based marker gene excision via directly repeated sequences [135]. The repeats were the promot‐
ers (Prrn) and transcription terminators (TpsbA). Recombination via the Prrn promoter or TpsbA repeats yielded the
two stable marker-free ptDNA carrying only the uidA (recombination R2) or only the bar (recombination R1) gene. No
sequence is repeated in the final product. uidA: reporter gene encoding β-glucuronidase; aadA: spectinomycin resist‐
ance marker gene; bar: herbicide resistance gene.
In the second version [140] marker-free tobacco plants were generated by the use of a vector
that harboured an aadA gene disrupting the herbicide resistance gene hppd (4-hydroxyphenyl‐
pyruvate dioxygenase from Pseudomonas fluorescens (HPPD) enzyme that confers resistance to
sulcotrione and isoxaflutole). Initially, antibiotic-resistant clones were obtained. Marker-free
herbicide-resistant plants were identified after excision of the aadA marker gene by homolo‐
gous recombination within the overlapping region (403 bp) of the 5’ and 3’ halves of the
herbicide resistance gene. Excision of aadA led to reconstitution of a complete herbicide
resistance gene and expression of the HPPD (Fig. 6).
http://dx.doi.org/10.5772/55573
Figure 6. Homology-based marker gene excision via directly repeated sequences [140]. Integration of transgenes into
the wild-type (WT) tobacco plastid genome (A) after transformation with the designed vector, giving a transformed
plastome (B). After the recombination between the two P1 repeats, a marker-free plastome was obtained (C). HP1: 5’
fragment of the 4-hydroxyphenylpyruvate dioxygenase gene (hppd) coding region; LHRR and RHRR, left and right ho‐
mologous recombination regions; P1, repeat segment overlapping the 5’ and 3’ fragments; P1PD, 3’ fragment of the
hppd coding region.
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). After‐
ward, 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].
Figure 7. Marker gene excision from the plastid genome by Cre or Int site-specific recombinases [137]. A site-specific
recombinase gene (cre/int) introduced into the nucleus by transformation, pollination or transient Agroinfiltration,
encodes a plastid-targeted recombinase that excises selectable marker gene (SMG) from TP1-ptDNA after import into
plastids. Excision of the marker gene by phage recombinases via the target sites (black triangles) yields marker-free
TP2-ptDNA carrying only the gene of interest (GOI) and one recombinase recognition sequence [141-143].
Cre/lox was the first site-specific recombination system used to excise the SMG from the plastid
genome [141, 142]. In these works Cre activity was introduced by nuclear transformation and
marker-free transplastomic plants were obtained in tissue culture. However, these plants still
contain cre and nptII genes in their genome that had to be segregated away in the seed progeny
[141, 142]. Another way to introduce Cre activity is by pollination, in which apparently non-
specific Cre-induced re-arrangements between homologous ptDNA sequences were absent or
occurred significantly less often than in directly transformed plants [141]. On the other hand,
Lutz et al. [144] took into account the fact that not every T-DNA delivery results in stable
integration and expressed Cre transiently from T-DNA introduced by Agroinfiltration. As a
result in this experiment approximately 10% of the regenerated plants did not carry either a
plastid selectable marker or a nuclear cre gene. Nevertheless, Cre-mediated marker excision
can cause the deletion of ptDNA sequences by recombination via directly repeated non-lox
sequences that result in mutation of target plant [141, 142].
As an alternative, the ΦC31 phage site-specific integrase (Int) that mediates recombination
between bacterial (attB) and phage (attP) attachment sites was tested to excise the SMG [143].
The authors tested marker gene excision in a two-step process. In the first step, tobacco
chloroplast were transformed with a vector that contains the SMG (aadA gene) flanked with
directly oriented non-identical phage attP (215 bp) and bacterial attB (54 bp) recombination
sites, which are recognised by Int recombinase. The bar gene was used as gene of interest and
it was placed outside of the excision cassette. Spectinomycin-resistant clones were obtained
http://dx.doi.org/10.5772/55573
and these were stable in the absence of Int. In the second step, a plastid-targeted Int was
introduced by Agrobacterium-mediated nuclear transformation that directed efficient marker
gene excision. No fortuitous sequences appear to be present in the plastid genome that would
be recognized by the Int [143]. In the homology-mediated marker excision the frequency of
deletion is proportional to the length of the repeat. As the lox sequences are short (34 bp in
length), the probability to cause loss of the marker gene in the absence of Cre is not completely
absent. However the attB and attP sequences are not homologous, therefore plastid genomes
carrying att-flanked marker genes are predicted to be more stable than those with marker genes
flanked by identical lox sequences. The absence of homology between the attB and attP sites
and the absence of pseudo-att sites in ptDNA would make Int a preferred alternative to Cre
for plastid marker excision [137].
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.
[145] 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) [145]. In this work the authors used
a vector where the marker gene (aphA-6) was located outside of the recombination region. This
strategy allowed the selection for a cointegrate structure that forms by recombination via only
one of the targeting sequences (Fig. 8B). When selection for kanamycin resistance was
withdrawn, the second recombination event can take place and the marker gene is lost.
2.6.4. Co-transformation-segregation
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 impossi‐
bility to obtain such plants directly. Indeed, transplastomic plants cannot be obtained directly
by selection with herbicides after transformation with the resistance genes (CP4 or bar) because
cells harboring only a few copies of the transgene die [147, 148]. Nonetheless, when these genes
were co-transformed with a plasmid carrying the spectinomycin resistance (aadA) gene and
most ptDNA copies carry the genes, the cells and regenerated plants showed resistance to high
levels of the herbicides [147, 148].
Figure 8. Transient cointegration of the marker gene to obtain marker-free transplastomic plants [145]. Cointegrate
formation and subsequent recombination events with conventional and alternative plastid transformation vectors. (A)
Standard plastid transformation using a vector in which the SMG is cloned between the homologous flanks. Recombi‐
nation via a single flank (left or right) results in cointegration of the vector; subsequent loop-out recombination events
between direct repeats lead either to a stably transformed plastome containing the sequence of interest and marker
or wild-type plastome. (B) Plastid transformation using a vector in which the selection marker is cloned outside of the
homologous flanks. Again recombination via either left or right flanks results in cointegration of the vector; however,
following additional recombination events only the GOI is stably integrated and the marker gene is lost.
http://dx.doi.org/10.5772/55573
Figure 9. The cotransformation-segregation method to remove selectable marker genes from transplastomic plants
[149]. (A) Transformation of the plastid genome (ptDNA) with two vectors. Vector 1 containing the selectable marker
gene (SMG) and Vector 2 the gen of interest (GOI). (B) Transplastomic clones are identified by selection for antibiotic
resistance. The heteroplastomic cell carries wild-type ptDNA (wt), TP1-ptDNA obtained by transformation with Vector
1; TP2-ptDNA transformed with Vector 2; and TP3-ptDNA transformed with both vectors. (C) Replication and segrega‐
tion of ptDNA on non-selective medium eventually yields homoplastomic cells with TP1-ptDNA, TP2-ptDNA and TP3-
ptDNA. Desired marker-free transplastomic plants carry TP2-ptDNA and lack the antibiotic resistance marker (adapted
from [137]).
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. [39] obtained marker-free transgenic rice plants harboring a Rice ragged stunt virus
(RRSV) derived synthetic resistance gene. Another group obtained transgenic rice devoid of
selectable marker genes that produce high levels of carotenoids using the same strategy but
with two binary vectors in one Agrobacterium strain [150]. One of the vectors contained in the
T-DNA the phytoene synthase (psy) and phytoene desaturase (crtI) expression cassettes
whereas the other vector contained the hph, nptII and gus genes. Marker-free rice plants, with
improved resistance to Magnaporthe grisea were obtained by the expression of the rice pistil-
predominant chitinase gene using a vector system with two T-DNAs [151].
Sripriya et al. [152] generated marker-free transgenic plants with improved resistance to sheath
blight. A single A. tumefaciens strain harbored a cointegrate vector with the hph and gus genes and
a binary vector with the rice chitinase (chi1) gene. The elimination of SMG was accomplished by
segregation in T1 progeny. Some of the lines showed an enhanced resistance to Rhizoctonia solani.
Thereafter, the same group sequentially retransformed one of the chi1 lines with the tobacco
osmotin ap24 gene by co-transformation using an Agrobacterium strain harboring a single-copy
cointegrate vector pGV2260::pSSJ1 (hph and gus) and a multi-copy binary vector pBin19ΔnptII-
ap24 in the same cell [153]. They obtained one line in the T1 progeny where the SMG was absent
and chi1 and ap24 genes were integrated. Homozygous plants with both genes were obtained and
some of those showed enhanced resistance to R. solani. Selectable marker-free rice plants
expressing the Bacillus thuringiensis synthetic cry1B gene were obtained by transposon-mediat‐
ed repositioning of the GOI [49]. The Cry1B expression cassette was flanked by the inverted
terminal repeats of the maize Ac transposon that permit the repositioning of this cassette in the
rice genome. Preliminary bioassays suggested that the T-DNA free relocation events exhibit a
level of resistance to a major rice insect pest, Chilo suppressalis.
On the other hand, Sengupta et al. [154] have exploited the Cre/lox site-specific recombination
system to produce selectable marker-free transgenic rice plants with improved resistance to green
leafhoppers (Nephotettix virescens) and brown planthopper (Nilaparvata lugens). In this work two
independent vectors were used, one having the ASAL (Allium sativum leaf agglutinin) gene and
the hpt gene flanked by lox sites, and the other with the cre and bar genes. Cre activity was intro‐
duced by crossing single copy T0 plants and marker excision was detected in T1 hybrids. T2 progeny
showed the segregation of the cre-bar T-DNA and improved insect resistance.
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 nptII selectable marker gene, originally present between tandemly
oriented lox sites, was removed through introduction of the cre gene by a sexual cross [121].
For the market, this corn line has the name MaveraTM High Value Corn with Lysine, and was
developed for the feed industry. This line was obtained from a biolistic transformation event
http://dx.doi.org/10.5772/55573
where a cordapA gene encoding a seed-specifically expressed lysine insensitive dihydrodipi‐

colinate synthase enzyme. This approach was also applied to obtain marker-free salt tolerant
maize plants by the expression AtNHX1, a Na+ /H+ antiporter gene from Arabidopsis, but using
the FLP/FRT system [155].
In another report, the production of marker-free transgenic soybean [Glycine max (L.) Merr.]
is described which produces γ-linolenic acid and stearidonic acid that are important for
pharmaceutical and nutraceutical industries [41]. The authors applied the co-transformation /
segregation strategy, using a vector with two T-DNAs: the first harbored a cDNA of the Borago
officinalis L. Δ6 desaturase gene driven by the embryo- specific β-conglycinin promoter,
whereas the second T-DNA contained the selectable marker gene bar. In this work ~7% of the
transgenic lines were marker-free.
The expression of a chitinase gene, ChiC, on an ipt-type MAT (isopentenyl transferase-type

multi-auto-transformation) vector, allowed the production of marker-free disease-resistant
transgenic potato plants [156]. Based on transformation without selectable marker gene Stiller
et al. [157] obtained two potato lines with increased levels of the semi-essential amino acid
cysteine by expression of the serine acetyltransferase encoding cysE gene. This system was also
used by Ahmad et al. [158] to produce marker-free potato plants with enhanced tolerance to
oxidative stress by the expression of the superoxide dismutase and ascorbate peroxidase genes.
The same strategy was used in Chinese cabbage (Brassica campestris ssp. pekinensis (Lour)
Olsson) to produce Turnip mosaic virus (TuMV) resistant marker-free transgenic plants [159].
This approach was also applied in melon (Cucumis melo), where ripening was delayed by the
introduction of marker-free and vector-free antisense 1-aminocyclopropane-1-carboxylic acid
oxidase (ACC oxidase) construct [160].
Author details
Borys Chong-Pérez1 and Geert Angenon2*
*Address all correspondence to: [email protected]
1 Instituto de Biotecnología de las Plantas, Universidad Central “Marta Abreu” de Las Villas,
Santa Clara, Cuba
2 Laboratory of Plant Genetics, Vrije Universiteit Brussel, Brussels, Belgium
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Strategies for Generating Marker-Free Transgenic Plants

Borys Chong-Pérez and Geert Angenon

Additional information is available at the end of the chapter

1.1. Why marker-free transgenic plants?

2. Strategies to obtain marker free transgenic plants

2.1. Transformation without selection

2.2. Co-transformation of a marker gene and the gene-of-interest followed by segregation

continuous presence of heterologous transposons and the generation of mutations because of

2.4. Homologous recombination

A more refined approach comprises self-excision controlled by an endogenous stimulus that

An additional feature of recombination based systems is the capability to resolve complex

An alternative to the site-specific recombination system would be to construct restriction

2.6. Removal of transplastome marker gene

2.6.1. Homology based SMG excision via directly repeated sequences

selection for spectinomycin and streptomycin resistance conferred by aadA. Thereafter,

2.6.2. Excision by phage site-specific recombinases

2.6.3. Transient cointegration of the marker gene

The co-transformation-segregation method in plastid transformation technology is based on

3. Marker free transgenic plants with agronomically useful genes

where a cordapA gene encoding a seed-specifically expressed lysine insensitive dihydrodipi‐

The expression of a chitinase gene, ChiC, on an ipt-type MAT (isopentenyl transferase-type

Borys Chong-Pérez1 and Geert Angenon2*

*Address all correspondence to: [email protected]

2 Laboratory of Plant Genetics, Vrije Universiteit Brussel, Brussels, Belgium

[6] Ramessar K, Peremarti A, Gómez-Galera S, Naqvi S, Moralejo M, Muñoz P, Capell T,

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