Biotechnol. J. 2007, 2, 83–90 DOI 10.1002/biot.200600182 www.biotechnology-journal.

com

Review

Methods to produce marker-free transgenic plants

Behrooz Darbani1, Amin Eimanifar2, C. Neal Stewart, Jr.3 and William N. Camargo4
1Agriculture Biotechnology Research Institute for Northwest & West of Iran, Tabriz, Iran
2Iranian Artemia Reference Center, Urmia, Iran
3Department of Plant Sciences, University of Tennessee, Knoxville, TN, USA
4Fisheries and Illinois Aquaculture Center, Southern Illinois University at Carbondale, Carbondale, IL, USA

Selectable marker genes (SMGs) have been extraordinarily useful in enabling plant transformation
Received 13 September 2006
because of the low efficiency of transgene integration. The most used SMGs encode proteins re- Revised 16 October 2006
sistant to antibiotics or herbicides and use negative selection, i.e., by killing nontransgenic tissue. Accepted 30 October 2006
However, there are perceived risks in wide-scale deployment of SMG-transgenic plants, and there-
fore research has recently been performed to develop marker-free systems. In this review, trans-
formation using markers not based on antibiotic or herbicide resistance genes, as well as differ-
ent systems of marker gene deletion, are discussed.

Keywords: Biosafety · Marker genes · Negative selection · Selectable markers · Site-specific recombination · Transposons

1 Introduction (PAT), which converts PPT into the nontoxic acetylated

form [4]. The application of selection pressure in regener-
To produce transgenic plants, selection systems are used ation media, transformed cells with selectable marker
that lead to the selective growth of transformed cells. genes (SMGs) can survive, while non-transformed cells
Genes encoding for resistance to specific antibiotics or are killed. Screening approaches for the identification of
herbicides have been found to be particularly effective for transgenic lines among many more non-transgenic lines
selection and provide a means for rapidly identifying are time consuming, and have not been widely used in
transformed cells, tissues, and regenerated shoots. An- the absence of SMGs. In addition, in the absence of
tibiotics and herbicides kill cells by a variety of mecha- SMGs, the size of the regenerated plant events is reduced
nisms and resistance genes have been widely used in almost tenfold [6, 7].
transgenic plant production. For instance, kanamycin However, because SMGs are integrated into the plant
and related aminoglycoside antibiotics kill cells by inhibi- genome, there are concerns about widespread occur-
tion of protein translation [1, 2] and the bacterially derived rence of transgenes in novel ecosystems (e.g., antibiotic
nptII gene, encoding neomycin phosphotransferase, inac- resistance in crops and their agroecosystems). Horizontal
tivates these antibiotics by phosphorylation [3]. The her- gene transfer from plants to environmental or medically
bicide phosphinothricin (PPT), an analog of glutamine, is related bacteria, or from plant products consumed as food
toxic to plants by irreversibly inhibiting glutamine syn- to intestinal microorganisms or human cells, are general-
thetase, a key enzyme for ammonium assimilation and the ly considered to be not likely, but the inherent risks have
regulation of nitrogen assimilation in plants [4, 5]. The bar not been totally addressed, and therefore there remain
gene, cloned from the bacterium Streptomyces hygro- both regulatory and public concerns in many places in the
scopicus, encodes phosphinothricin acetyltransferase world. Transfer of plant DNA into microbial or mammalian
cells under normal conditions of dietary exposure [8]
would require all of the following events to occur: (i) re-
Correspondence: Professor C. Neal Stewart Jr., Department of Plant
moval of the relevant gene(s) from the plant genome,
Sciences, University of Tennessee, 252 Ellington Plant Sciences, Knoxville,
TN 37919, USA
probably as linear fragments; (ii) protection of the gene(s)
E-mail: nealstewart@utk.edu from nuclease degradation in the plant as well as animal
gastrointestinal tract; (iii) uptake of the gene(s) with di-
Abbreviation: SMG, selectable marker gene etary DNA; (iv) transformation of bacteria or competent

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mammalian cells; (v) insertion of the gene(s) into the host can subsequently be removed from the plant genome dur-
DNA by rare repair or recombination events into a tran- ing segregation and recombination that occurs during
scribable unit; and finally, (vi) continuous stabilization of sexual reproduction by selecting on the transgene of in-
the inserted gene, e.g., there would be low-to-no-cost to terest and not the SMG in progeny. Therefore, co-trans-
the host harboring the new gene in the absence of selec- formation methods cannot be used for vegetatively prop-
tion pressure. Numerous experiments have evaluated the agated plants [18]. These procedures not only require fer-
possible transfer of plant DNA into microbes and mam- tile plants, but are also very time consuming [19]. In sex-
malian cells. There are reports that bacteriophage and ually reproducing plants, selection schemes are required
plasmid DNA, when fed to mice at very high levels, can to select the stable integration of two T-DNAs, which can
later be detected in their cells [9], but no data exist to be segregated in subsequent progeny [20]. However, tight
demonstrate that plant DNA can be transferred into and linkage between co-integrated DNAs limits the efficiency
be stably maintained or expressed in mammalian cells [8]. of co-transformation. Indeed, integration of an SMG and
There are some experimental data indicating the transfer the transgene of interest on separate loci are required [21].
of plant DNA into bacteria under laboratory conditions, Moreover, it is not applicable to transgenic trees with long
but only if homologous recombination is facilitated [10, generation times [22] that require the use of transient se-
11]. However, there is no evidence that the transgenic lection [23]. The outcome of one co-transformation study
markers presently in use pose a health risk to humans or indicated that co-transfer of T-DNAs present in the same
domestic animals. Nevertheless, some researchers and “super-binary” plant transformation vector in one strain
regulators have concluded that, although the transforma- was considerably more efficient than transfer from two
tion risk of plant-transmitted antibiotic resistance genes different strains [24]. However, the overall advantages of
to pathogenic bacteria is quite small, the use of markers these methods remain unclear [16, 25, 26].
conferring resistance to clinically relevant antibiotics
should be phased out as suitable alternative technologies 2.1 Using plant DNA
become available in plant biotechnology [8, 12, 13].
In addition to the risk of horizontal gene transfer, there Recent studies have shown that plants have T-DNA bor-
is also a “vertical cross-species” transfer risk that could po- der-like sequences in rice and Arabidopsis [27, 28] and
tentially create enhanced weediness problems [14]. Pro- these might be used in transformation. Because this so-
duction of marker-free transgenic crops eliminates risk of called plant DNA (P-DNA) lacks any open reading frames
horizontal gene transfer and could mitigate vertical gene and contains a high A/T content, it is likely the footprint
transfer. We discuss two strategies to achieve these goal: of ancient Agrobacterium-mediated natural transforma-
one approach uses markers not based on antibiotic or her- tion events via horizontal gene transfer [29]. It has been
bicide resistance genes, and the second is to excise or seg- demonstrated that plant-derived P-DNA fragments can
regate marker genes from the host genome after regener- be used to replace the universally employed Agrobacteri-
ation of transgenic plants [15], which includes co-transfor- um T-DNA for transformation [23]. In addition, co-trans-
mation (i.e., separate transformation of marker and trans- formation of the inserted desired transgene into P-DNA
gene), site-specific recombinase-mediated marker and SMG-containing T-DNA is capable of producing
deletion (e.g., Cre/loxP, FLP/FRT and R/RS site-specific re- marker-free and backbone-free transgenic plants [23].
combination systems), transposon-based expelling sys- Moreover, insertion of mutated Agrobacterium virD2 gene
tems (e.g., Ac transposon), intrachromosomal recombina- into T-DNA served to impair the integration of transferred
tion based excision, and transformation by marker genes T-DNAs [30] and thus allowing a higher frequency for
not based on herbicide or antibiotic selection. marker-free P-DNA population compared with T-DNA
containing an SMG. Apparently, the mutated VirD2 pro-
tein has no effect on P-DNA integration. Inserting the
2 Co-transformation bacterial ipt cytokinin expression cassette into the back-
bone of P-DNA vector enabled an increase in the frequen-
Co-transformation is a method for production of marker- cy of backbone-free transgenic plant in the recovered
free transformants based on Agrobacterium- or biolistics- population [23].
mediated transformation in which a SMG and gene of in-
terest are on separate constructs. Three approaches are 2.2 Negative selection
used for co-transformation: (i-a) introduction of two T-
DNAs, in separate Agrobacterium strains or (i-b) biolistics An alternative and potentially more efficient strategy is
introduction of two plasmids in the same tissue; (ii) intro- based on the incorporation of a negative selection step.
duction of two T-DNAs carried by different replicons The use of a negative SMG next to a positive SMG in the
within the same Agrobacterium strain; and (iii) introduc- same construct is a powerful method to create marker
tion of two T-DNAs located on the same replicon within gene-free transgenic plants. In this method, transformed
an Agrobacterium [16, 17]. In all of these variants, SMGs offspring are selected for the absence of negative SMG

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under the selection pressure of a negative marker gene gene. After segregation, marker-free transgenic progeny
and the presence of the desired transgene. This negative plants can be identified. To eliminate the breeding step, a
selection method allows researchers to decrease their co-transformation based on transient expression of the
search for selectable marker-free transgenic plants with- site-specific recombination system in combination with a
out having to resort to copious molecular analyses, i.e., conditional lethal dominant gene, coda was proposed
thousands of PCRs [31]. Finally, the combination of using [40–42]. Furthermore, the characterization and use of in-
a mixture of mechanisms, transient selection, sequential ducible promoters, CLX vector system, and GST-MAT
transformation, negative marker genes, P-DNA and a mu- vector system (multi-auto-transformation) including
tated virD2 gene together should be capable of producing oncogenes for cell proliferation and regeneration of trans-
high frequency marker-free transgenic plants by co-trans- genic plants (see Fig. 2), to express of recombinase genes
formation methods. Recently, a novel marker gene has would be useful. After applying the induction agent, the
been characterized, dao1, encoding D-amino acid oxidase recombinase would be expressed with induced excision
that it can be used as for either positive or negative mark- of SMGs and all sequences between the two recombina-
er, depending on the substrate [32]. Therefore, it is possi- tion sites [43, 44]. Also, tissue-specific promoters for pro-
ble to apply the negative selection after a positive selec- ducing marker-free transformants could be useful for fine-
tion using one marker gene, dao1, via changing D-alanine tuning the excision patterns. Recently, plant Cre virus
or D-serine to D-isoleucine or D-valine for the substrates. vectors (TMV-Cre and PVX-Cre) for transient expression
of cre recombinase has been developed as an alternative
method for the production of marker-free transgenic N.
3 Site-specific recombination-mediated marker benthamiana plants. In this method, transgenic plants
deletion containing lox sites and the bar SMG are inoculated with
PVX-Cre and TMV-Cre recombinant viruses. PVX-Cre
Recombination is a universal phenomenon that can occur and TMV-Cre systemically infect leaves and allow regen-
at any place along two homologous DNA molecules. In eration without selection pressure. This strategy can be
temperate bacteriophages, there is a second type of re- applied to plant species that depend on organogenesis or
combination called site-specific recombination, which somatic embryogenesis for regeneration, particularly,
takes place only between defined excision sites in the soybean, potato and a number of woody plant species
phage and in the bacterial chromosome. Positions of the [45]. Also, a tightly controlled microspore-specific pro-
site-specific recombination in the bacterial and phage moter and a site-specific recombination system was re-
DNA are called the bacterial and phage attachment sites, cently employed in an efficient marker gene removal in to-
respectively. Each attachment site consists of three seg- bacco pollen [46].
ments. The central segment has the conserved nucleotide Another strategy was proposed employing two site-
sequence that sites the recombination event. A phage specific recombination systems: one for integrating the
protein, an integrase, catalyzes the site-specific recombi- DNA in a recombination site into the host genome at the
nation events, which lead to physical exchange of DNA. designated genomic target site, and a second for remov-
Excision requires the phage enzyme integrase plus an ad- ing sequences that are not needed after DNA transfer.
ditional phage protein called excisionase [33]. There are This strategy is based on the tandem use of the Cre/lox,
three well-described site-specific recombination systems FLP/FRT and R/RS inducible systems (Fig. 3). In this
that might be useful for the production of marker-free method it is feasible to achieve site-specific integrations
transgenic plants: Cre/loxP system from bacteriophage at an efficient rate with predictable transgene expression.
P1, where the Cre enzyme recognizes its specific target
sites [34, 35], FLP/FRT recombination system from Sac-
charomyces cerevisiae, where the FLP recombinase acts
on the FRT sites [36, 37] and R/RS recombination system
from Zygosaccharomyces rouxii, where R and RS are the
recombinase and recombination site, respectively [38].
Recognition sites for recombinases consist of palin-
dromes, which are flanked with 7–12-bp core sequences
[39]. Cleavage of the sites occurs at the borders between
the recombinase binding elements and the core sequence
(Fig. 1). In these systems, elimination of SMG would re-
quire recombinase expression in transgenic plants. The
recombinase gene cassette can be introduced into trans-
formed plants that contain the SMG between two recog-
nition sites. Alternatively, a transgenic plant of interest Figure 1. Cre/loxP recombination system (M: marker gene, and GOI: gene
can be crossed with a plant that expresses a recombinase of interest). See [34].

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Figure 2. (A) Structure of the CLX vector system. See [44]. Cre-int includes eight copies of the LexA operator sequence fused to the -46 CaMV35S promoter.
Sequence of Cre is interrupted by an intron. Excision is produced via the ‚-estradiol-induced site-specific DNA recombination. (B) MAT Vector System. See
[43]. Recombinase genes (R) promoting with GST promoter and hpt marker gene flanked by two directly oriented RS sites. XVE G10-90 and GOI are hybrid
transactivator constitutive promoter and gene of interest, respectively.

Figure 3. ‘Combined step’ strategy based on two site-specific recombination systems to remove excess DNA after site-specific integration. See [47]. (A) Use
of Cre/lox and inducible FLP/FRT system to integrate a circular DNA and deleting of excess DNA from the integration locus, respectively. Introduction of
circular DNA containing a lox75 (left arm mutant) site into cells containing a lox76 (right arm mutant) locus, site-specific integration of the gene of interest
(GOI), and formation of a double mutant lox (dmlox) site, which stabilizes the integration locus. (B) Use of R/RS system to integrate a linear T-DNA, deliv-
ered by Agrobacterium, into target RS sites, followed by use of the inducible Cre/loxP system to remove excess DNA from the integration locus. hpt, hy-
gromycin phosphotransferase gene; npt, neomycin phosphotransferase; P, promoter; Pi, inducible promoter (key in the box).

In this system single copy events at the designated target thousands of bases long. They code at least one protein,
site ranged from 40% to 60% [47]. which enables them to replicate. The most widely studied
transposon is the P element from the fruitfly (Drosophila
melanogaster) [49]. Transposable elements can also be
4 Transposon-based marker methods used to produce marker-free transgenic plants (Fig. 4).
Use of transposable elements for marker gene removal in-
In the 1940s, Barbara McClintock made an astonishing volves several steps: (i) insertion of the marker gene onto
discovery. She detected two factors of DNA transposition a transposon, a segment of DNA that “hops” around in the
in maize: a Ds (disassociation) element that was located plant’s genome; (ii) co-transformation with gene of inter-
at a chromosome break site and an unlinked genetic fac- est; and (iii) segregation of the marker gene.
tor (Ac) that was required to activate the breakage of A MAT vector system containing the ipt gene and Ac
chromosome 9. McClintock concluded that such an un- element has been designed so that when tobacco leaf seg-
stable phenotype resulted from the movement or transpo- ments were transformed and selected, subsequent exci-
sition of Ds [48]. These came to be known as transposons. sion of the modified Ac produced marker-free transgenic
Transposons are DNA sequences between hundreds to tobacco plants without sexual crosses or seed production

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tages are: (i) expression of a heterologous recombinase

and sexual reproduction are not necessary; (ii) there is a
one step selection procedure for transgenic calli (lengthy
propagation two-step time as above might increase the
risk of somaclonal variation); (iii) it utilizes a natural nu-
clear recombination systems present in plants; (iv) the fre-
quency of intrachromosomal recombination between two
homologous sequences in plants might be increased by
Figure 4. Ac transposon-based expelling of nuclear marker genes stimulation of repair systems; and (v) the efficiency of ho-
(M: marker gene, and GOI: gene of interest). See [50]. mologous recombination is directly correlated with the
size of the homologous regions [18, 51, 52].

[50]. However, there are several drawbacks of using a

transposable element system for marker gene removal: 6 Removal of chloroplast marker genes
(i) different species have variable rates of transposition ef-
ficiency; (ii) this method requires labor and time costs for Mitochondria and chloroplasts have independent
crossing transgenic plants and the selection of the proge- genomes in plants that have been the target (especially
ny [19, 51]; (iii) there is low efficiency of marker-free trans- chloroplasts) of genetic transformation. Biosafety might
genic plant generation, because of the tendency of trans- be facilitated by maternal inheritance, which is the case
posable elements to reinsert elsewhere in the genome; in most plant species, in which transgenes in plastids
(iv) imprecise excision; (v) generation of mutations be- would not be disseminated via pollen [58]. Chloroplast
cause of insertion and excision cycles; and (vi) genomic transformation vectors are designed with homologous
instability of transgenic plants because the continuous flanking sequences on either side of the transgene. In ad-
presence of heterologous transposons [18] decreases dition, chloroplast engineering overcomes the challenges
efficiency. of gene silencing, position effects, and multi-step engi-
neering of multiple genes, which are current limitations of
nuclear transformation [58–60]. Homologous recombina-
5 Intrachromosomal recombination system tion (the use of identical sequences for example in pro-
moters and terminators between genes) ([61], Fig. 5) and
A variant of site-specific recombination systems de- site-specific recombination (for example Cre/lox recombi-
scribed above employs an intrachromosomal recombina- nation-based systems) or transient expression of recom-
tion system. As above, recombination sites are engi- binase [62] are all potentially suitable for producing mark-
neered into the plant, but no recombinase is expressed. er-free engineered chloroplast of plants. Re-transforma-
The attachment site from phage origin is denoted POP’ (P tion using the same marker gene has been recently
for phage) or attP, and the attachment site from bacterial demonstrated, and provides first rigorous proof that de-
origin is denoted BOB’ (B for bacteria) or attB [33]. Intra- spite the high copy number of chloroplast genes, all
chromosomal recombination in plants [52] is obtained by copies of a marker gene can be removed by homologous
insertion of SMG between two direct repeats of attP that recombination [63]. Recently, a construct co-integration
facilitates spontaneous excision. Base composition of the system followed by a homologous recombination event
attP site sequence is A + T rich, which is conjectured to between single homologous regions in vector and plas-
play a recombination-stimulating role [53]. Possibly, the tome was developed (Fig. 6). After recombination, co-in-
formation of a recombination hot spot is caused via the in- tegrates are inherently unstable because of direct repeats.
duction of double-strand breaks (DSBs) [54], but may also Therefore, subsequent loop-out recombination events
reduce of the stability of transgene sequences later on. create either the stable integration of a transgene of in-
Thirty-three percent efficiency of marker gene by ISceI
expression, a site-specific homing endonuclease encoded
by a mitochondrial intron of Saccharomyces cerevisiae,
demonstrated that induced DSB-mediated recombination
by highly specific endonucleases could be a feasible al-
ternative to site-specific recombinases for marker elimi-
nation [55, 56]. In addition, the inclusion of a transforma-
tion booster sequence (TBS) from Petunia hybrida insert-
ed into the construct adjacent to the attachment se-
quences increased the frequency of intrachromosomal
recombination and illegitimate recombination events in Figure 5. Homologous recombination based removal chloroplast marker
Petunia, Nicotiana and maize [57]. The potential advan- gene (M: for marker gene and GOI: gene of interest). See [61].

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Most recently, a notable replacement of the bacterial

kanamycin-resistant nptII gene is an Arabidopsis thaliana
ATP binding cassette (ABC) transporter (Atwbc19) gene.
When overexpressed in transgenic tobacco, it yielded
roughly equivalent degrees of kanamycin resistance in
plants; however, because of cellular targeting to the tono-
plast, it is not expected to confer kanamycin resistance in
bacteria if horizontal gene flow were to occur [74]. Other
plant-based markers are plant counterparts of aspartate
kinase (AK), and dihydrodipicolinate synthase (DHPS)
genes for lysine inhibition [73].

8 Conclusions and perspectives

There are many compelling reasons to produce trans-

Figure 6. Co-integrated based marker excision method for generation of genic plants with as little foreign DNA as possible. Many
marker-free plastid transformants (M: marker gene, LF: left and RF: right
companies that produce transgenic crops are taking this
homologous fragments, GOI: gene of interest, and WT: wild type). See
[64].
minimalist approach now in their research and marketing
strategies, resulting in fewer regulatory and consumer-
based concerns for their products. Concurrently with this
terest or loss of the integrated vector, which then yields a implementing this corporate strategy, there has been a re-
wild-type plastome [64]. cent explosion of technologies that, when further devel-
oped, will allow the removal of DNA in plants almost as
easily as it is inserted today. In fact, we might expect
7 Markers not based on antibiotic or herbicide technological development to move at a similar pace to
resistance that of transformation technology in the 1980s and 1990s.
In a 10-year period, techniques were developed so that
Recently, researchers have described substitute marker nearly all row crops could be transformed with both bi-
genes of non-bacterial origin that could have inherently olistics and Agrobacterium. SMGs are the most obvious
increased biosafety. Of most interest are marker genes candidate sequences to be deleted from transgenic plants
from plants themselves [65]. One potential alternative since they largely serve no purpose after selection of
method to produce transformants without any antibiot- transformants, thus their removal will be beneficial to
ic/herbicide marker gene is so-called positive selection end-users. There is no doubt that transgene removal will
systems. Recently, an Escherichia coli-derived phospho- eventually be routine as some of the techniques reviewed
mannose isomerase (PMI) was used to convert mannose- here are perfected.
6-phosphate to fructose-6-phosphate for positive selec-
table marker in plant transformation. Only transformed
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Behrooz Darbani has a degree in plant Neal Stewart obtained his PhD in Plant
breeding and holds a MSc in plant bio- Physiology from the Virginia Polytech-
technology from the Tabriz University nic Institute and State University in
of Iran. He joined the genomics labora- 1993 (VA; USA). From 1993 to 1995 he
tory of the Agriculture Biotechnology was Postdoctoral Associate in Plant Ge-
Research Institute for Northwest & netics (Department of Crop and Soil
West Iran as a researcher in 2005. Sciences, The University of Georgia,
Presently, his research interests focuse GA; USA) and Assistant & Associate
on recombinant protein production in Professor of Biology, University of
prokaryotes as well as on molecular North Carolina (Greensboro, NC, USA)
markers and genetic engineering of plants. in 1995–2002. He joined the University of Tennessee (TN, USA) in
June 2002 assuming the Racheff Chair of Excellence in Plant Molecular
Genetics. His laboratory is housed in the new state of the art Plant
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