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Elimination of selection markers from transgenic plants
139
Elimination of selection markers from transgenic plants Barbara Hohn*, Avraham A Levy† and Holger Puchta‡ Selection markers, which were necessary for the isolation of transgenic plants, are no longer required in mature plants, especially when they are grown in fields. Regimes to achieve their efficient elimination, mostly through site-specific recombination or transposition, are being developed. Addresses *Friedrich Miescher Institute, Maulbeerstrasse 66, 4058 Basel, Switzerland; e-mail: [email protected] † Plant Sciences Department, The Weizmann Institute of Sciences, Rehovot, 76100 Israel; e-mail: [email protected] ‡ AG DNA Rekombination, IPK, Corrensstrasse 3, D-06466 Gatersleben, Germany; e-mail: [email protected] Current Opinion in Biotechnology 2001, 12:139–143 0958-1669/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved.
Introduction The development of transgenic plants requires the use of selectable marker genes, because the efficiency of plant transformation is less than optimal for many important plant species. The maintenance of resistance genes in transgenic plants causes no concerns for laboratory (or greenhouse) experiments. However, their persistence in the field is, depending on one’s point of view, unnecessary, undesirable or unacceptable. These arguments are sufficient to warrant strong efforts to develop strategies for the efficient elimination of marker genes after selection; ‘The moor has fulfilled his duty, the moor can leave’ [1]. Moreover, for conducting further rounds of transformation it is advantageous to have freed the plants from the resistance genes that are no longer necessary, as the list of selectable genes that can be used for any given plant species is not very long. In addition, it may be wise to remove the promoter used to drive the selectable gene, in order to reduce the chance of (transcriptional) gene silencing of the desired transgene linked to the same promoter [2••]. These issues were recognized some time ago and possibilities for the elimination of resistance marker genes have been suggested.
Positive selection of transgenic plants Parallel to, and in combination with, marker elimination, a new set of markers are being developed that are called positive selection markers [3]. The principle of this system is that nontransformed cells are not killed, as in the procedures using antibiotic or herbicide resistance genes, but transformed cells experience a metabolic or developmental advantage. This leads to an increased efficiency of regeneration of transformed plants. Of added value is the expected nontoxicity of the selective chemicals compared with antibiotics and herbicides. In the article by Joersbo and Okkels [3], the β-glucuronidase gene from Escherichia coli was used as a selectable gene and a glucuronide derivative
of the cytokinin benzyladenine was used as the selective agent. This compound requires the activity of β-glucuronidase to activate a cytokinin. The efficiency of transformation was reported to be about twofold higher than with kanamycin; however, transformants were not further characterized. The xylose isomerase gene of Thermoanaerobacterium thermosulfurogenes has also been employed for positive selection: only transformed cells can live on a diet consisting of D-xylose as the sole carbon source. In some, but not all, of the tested species, a higher fraction of transformed cells could be recovered [4]. So far, the best-established system is that which utilizes the phosphomannose isomerase gene (pmi) of E. coli as the selectable gene and mannose as the selective agent [5]. After uptake, mannose is phosphorylated by a hexokinase to mannose-6-phosphate, which accumulates and causes drastic growth inhibition due to the lack of phosphomannose isomerase activity in plants. The transformation of sugar beet with Agrobacterium tumefaciens was found to yield transgenic shoots, even at low levels of expression of the selective gene. Frequencies of transformation were one order of magnitude higher than those obtained with kanamycin as the selective agent. This system has been successfully adapted to Agrobacterium-mediated transformation of maize [6•]. Immature embryos surviving selection were recovered with a frequency of up to 30% and they developed into normal fertile plants with confirmed integration of the transgene. Genes encoding enzymes in the hormone pathway originating from Agrobacterium have also been successfully used for the selection of transformed plants, although in all reported cases the presence or the activity of the respective gene had to be eliminated or turned down. This was necessary to avoid the detrimental effects of hormone overdoses on plant development. In addition, the phenotype, not being cell autonomous, would otherwise lead to chimeric plants. Ebinuma et al. [7] used the isopentenyl transferase (ipt) gene from the T-DNA of Agrobacterium coupled to the constitutive 35S promoter of cauliflower mosaic virus (CaMV) for tobacco transformation. Growth-retarded transformants appeared with occasional normal sideshoots in which the ipt part of the transgene had removed itself due to the activity of an Ac transposon. Higher frequencies of excision of the selectable transgene were established by replacing the transposon by a site-specific recombinase and its respective target sequences flanking the selectable gene ([8••]; see below). The ipt gene also provided the basis for the development of an inducible plant transformation system. The use of a dexamethasone-inducible promoter driving the ipt gene led to the recovery of lettuce and tobacco transformants under inducing conditions [9•].
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Figure 1 (a)
Site-specific recombinase Marker
Transgene
Transgene Marker Transposase
Marker
Transgene
Transgene
Marker
(b) Transposase Marker
Transgene
Marker
Transgene Current Opinion in Biotechnology
Genes that conferred auxin-independent growth onto tobacco transformants were obtained from Agrobacterium rhizogenes [10]. The bacterially derived rol genes are responsible for the proliferation of hairy roots by increasing auxin sensitivity. Transgenic plants arising from this transformation procedure also displayed abnormal phenotypes, such as wrinkled leaves, shortened internodes and reduced apical dominance, again necessitating eviction of the selectable marker. These regimes using plant hormone genes will have to be adapted individually to different plant species by adjustment of plant-internal hormone levels. Improvements might result from varying the promoters used to allow the development of specific organs. The recovery of marker-free plants without the necessity of sexual crossings is certainly an advantage. In addition, some of the selectable genes mentioned may in the future be exchanged for native plant genes, thus eliminating the use of foreign genes as selective markers altogether.
Elimination of marker genes by cotransformation One way to separate selectable marker transgenes from the transgene of interest is to separate them at the stage of transformation. The method of choice for transformation is to use Agrobacterium-mediated processes, as these are more likely to lead to separate integration events than, for instance, particle gun mediated gene delivery methods. Depending on the plant species, the bacterial strains and vectors, and the transformation procedure used, a fraction of transformants will carry the two transgenes linked or not linked [11–13]. An improvement in the cotransformation procedure was introduced by Komari et al. [14] using binary plasmids containing two T-DNAs. Cotransformation frequencies using these ‘superbinary’ vectors were reported to be as high as 47%, with a high proportion of both tobacco and rice transformants carrying unlinked transgenes. Of course this procedure requires fertile plants for genetic separation of the two transgene loci.
Schemes for marker gene elimination. (a) Enzymes acting at specific sites that flank the marker gene to be evicted lead to efficient marker gene elimination. Reinsertion of the marker gene at ectopic positions can be screened for or selected against (see text). (b) The transgene by itself is mobile; the activation of transposase allows the relocation of the desired transgene to new chromosomal positions. Genetic crosses and/or segregation will dissociate the two transgenes. In this case, the presence of the marker gene can also be counter-selected. Sequences required for transposition are represented by black triangles, direct repeats of targets for sitespecific recombinases by black arrows, transgenic DNA by thick black lines and plant DNA by thin solid or dashed back lines.
Elimination of marker genes by site-specific recombinases A general scheme for marker excision using site-specific recombinases is presented in the upper part of Figure 1a. Marker and transgene, originally linked on one transgenic unit, are separated by the activity of an enzyme, the specific recognition sites of which flank the marker to be excised. In pioneering work a decade ago, Dale and Ow [15] used the Cre recombinase of the E. coli bacteriophage P1 to remove a selectable marker gene flanked by lox target sites from a transgenic locus in transformed tobacco. Upon removal of the Cre-encoding locus by segregation, plants were recovered that had incorporated only the desired transgene. In addition, Arabidopsis plants that were free of a selectable marker gene were also recovered using the same site-specific recombination system [16]. Other single-chain recombinases were also found to be useful for the removal of marker genes: the site-specific recombination system of the Streptomyces bacteriophage φC31 (D Ow and R Calendar, personal communication), the FLP/FRT system of the 2 µ plasmid of Saccharomyces cerevisiae [17,18] and the R-RS system of the pSR1 plasmid of Zygosaccharomyces rouxii [19,20]. The common feature in these systems is the generation of transgenic plants that contain two directly oriented recognition sites for the respective recombinase flanking the sequence to be excised. Upon expression of the single-chain recombinase the recombination reaction is initiated, resulting in marker-free transgenic plants. In order to speed up this process, marker gene constructs and tissue culture conditions are being developed that allow marker gene elimination soon after transformation. In one approach, the selectable marker was located adjacent to a counterselectable marker, whereby both markers resided inside the ‘elimination cassette’ [21•]. This approach is especially fast if the recombinase is introduced into the plant cells only transiently (see below).
Elimination of selection markers from transgenic plants Hohn, Levy and Puchta
Alternatively, the expression cassette of the recombinase can be placed inside the elimination cassette; using this strategy, marker-free plants carrying a single copy of the transgene could be recovered even in the absence of counter-selection [8••,20,22•]. It remains to be tested whether a combination of these tricks — namely, using an elimination cassette that contains genes for selection and recombination as well as counter-selection — would further improve the efficiency and speed of marker elimination. In cases in which complex integration patterns of transgenes have to be resolved, the use of site-specific recombination can be of added value: concomitant with the conversion to a single transgene unit, the adjacent resistance marker could be eliminated in transgenic wheat [23••].
Elimination of marker genes by transposases Transposable elements can be harnessed to allow the production of marker-free transgenic plants owing to three key properties: elements from maize such as Ac/Ds were shown to transpose to both linked and unlinked sites in all the heterologous plant hosts analyzed [24]; transposable elements can be engineered in such a way that the transposase can be expressed from a nonmobile construct, whereas the mobile unit only has to contain terminal sequences that allow its transposition; and many excision events are not associated with reinsertion, possibly because of the loss of the element [25•]. On the basis of these properties, transposable elements can be employed for the dissociation of marker and desirable gene in two ways. In the first (Figure 1a, lower part), the marker gene is placed on the mobile element, which is lost after transposition [26]. Marker-free transgenic tobacco and aspen plants have been generated at low frequencies by inserting the selectable ipt gene (see above) into the transposable element Ac [7]. The second possibility for the transposoninduced dissociation of marker gene and desired gene consists of the relocation of the desired gene away from the original transgene locus (Figure 1b). The feasibility of this approach was demonstrated in tomato [27]. The advantage of this system is not only in unlinking the marker gene, but also in creating ‘clean’ insertions of the desired transgene. In addition, a series of plants with different transgene loci can be obtained from one original transformant, which is especially important if recalcitrant plants have to be transformed. This repositioning allows the expression of the transgene at different genomic positions and consequently at different levels.
Recombinases and transposases can be introduced into plants as DNA, RNA or protein Site-specific recombinases and transposases can be expressed from a transgenic locus that is introduced into the plant carrying the marker and desired transgene by a further round of transformation or by crossing. In several of the above mentioned experiments, the transient expression of enzyme genes was also used. Agrobacterium-mediated transfer of T-DNA leads to an early wave of gene expression most
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likely from unintegrated T-DNA molecules [28]. The obvious advantage for marker elimination lies in marker excision in the absence of an integrated version of the enzyme gene. In one study, this approach was combined with a negatively selectable marker to select for events of marker loss [23••]. This technology is especially useful for the generation of transgenic crops that are vegetatively propagated. A transposase mRNA, produced in vitro and introduced into protoplasts, was also documented to activate a transgenic Ds element [29]. In a most interesting recent report, the Crerecombinase was introduced into plants from Agrobacterium that was manipulated to contain Cre-recombinase VirE2 or VirF fusion proteins [30••]. These experiments document that these two virulence proteins are indeed transported into plant cells; however, in the context of this review they suggest that enzymes needed for marker excision may be introduced by Agrobacterium tumefaciens.
Intrachromosomal recombination for marker gene elimination?
In an attempt to use the bacteriophage λ integration/excision system for marker elimination, the unexpected excision of an NPTII gene from tobacco in the absence of any enzyme activity was reported ([31•]; discussed in [32]). The anticipated target for the λ enzymes was a pair of 352 base pair attachment regions of λ. From two of the 11 transgenic calli that contained a resistance gene between these elements, shoots grew up that contained white kanamycinsensitive sectors. Three out of 23 of these shoots proved to have lost the resistance cassette by homologous recombination, and the majority had also lost adjacent transgene sequences. This experiment thus documents a surprisingly high incidence of intrachromosomal homologous recombination, several orders higher than previously reported [33]. It is important to clarify whether the λ-attachment region is intrinsically recombinogenic, whether by chance the two transgenic loci are recombinational hotspots, and whether the employed transformation booster sequence or the special culture conditions influenced the recombination behaviour of these two transgenic lines.
Conclusions Despite the recent advances in the transformation of plants [34], technologies for the efficient generation of transgenic plants in the absence of any selection were optimized only for tobacco [35]. Hope remains that other plants will become less recalcitrant to transformation by improving culture conditions and transformation devices. Meanwhile, the marker improvement and marker elimination systems described in this review will hopefully become standard for crop improvement and the acceptance of improved crops.
Update Integration of foreign genes into the plastid genome has special advantages [36•]: gene containment may be enhanced because in many crop plants plastids are inherited from the maternal parent preventing spread of transgenes;
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homologous recombination in chloroplasts allows precise gene targeting into a small genome; and high levels of gene expression can be achieved. However, because of the high copy number and the prokaryotic expression signals of the selection markers it may be especially advisable to remove them after the generation of transplastomic plants. The efficient homologous recombination system of chloroplasts was exploited to remove genes conferring herbicide resistance and coding for a screenable marker from transplastomic tobacco plants [37••].
Acknowledgements We acknowledge the communication of unpublished information from H Ebinuma, D Ow, R Calendar and P Hooykaas. E Bucher kindly provided the figure and I Kovalchuk critically reviewed the manuscript. Special thanks go to D Ow for contributing with expertise, and to W Hörr-Szalay for information on [1]. BH acknowledges financial support from the Novartis Research Foundation.
11. Depicker A, Herman L, Jacobs S, Schell J, van Montagu M: Frequencies of simultaneous transformation with different T-DNAs and their relevance to the Agrobacterium plant cell interaction. Mol Gen Genet 1985, 201:477-484. 12. De Block M, Debrouwer D: Two T-DNAs co-transformed into Brassica napus by a double Agrobacterium infection are mainly integrated at the same locus. Theor Appl Genet 1991, 82:257-263. 13. McKnight TD, Lillis MT, Simpson RB: Segregation of genes transferred to one plant cell from two separate Agrobacterium strains. Plant Mol Biol 1987, 8:439-445. 14. Komari T, Hiei Y, Saito Y, Murai N, Kumashiro T: Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J 1996, 10:165-174. 15. Dale EC, Ow DW: Gene transfer with subsequent removal of the selection gene from the host genome. Proc Natl Acad Sci USA 1991, 88:10558-10562. 16. Russell SH, Hoopes JL, Odell JT: Directed excision of a transgene from the plant genome. Mol Gen Genet 1992, 234:49-59. 17.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Schiller F: ‘Der Mohr hat seine Schuldigkeit getan; der Mohr kann gehen’ (translation ‘The moor has fulfilled his duty; the moor can leave’). In ‘Die Verschwörung des Fiesco’ first performance 1784.
2. Matzke and Matzke (eds): Special issue on plant gene silencing. •• Plant Mol Biol 2000, 43:121-418. In this issue of Plant Molecular Biology, devoted to plant gene silencing, various silencing phenomena are described, silencing mechanisms discussed and practical considerations are presented. 3.
Joersbo M, Okkels FT: A novel principle for selection of transgenic plant cells: positive selection. Plant Cell Reports 1996, 16:219-221.
4.
Haldrup A, Petersen SG, Okkels FT: The xylose isomerase gene from Thermoanaerobacterium thermosulfurogenes allows effective selection of transgenic plant cells using D-xylose as the selection agent. Plant Mol Biol 1998, 37:287-296.
Kilby NJ, Davies GJ, Snaith MR: FLP recombinase in transgenic plants: constitutive activity in stably transformed tobacco and generation of marked cell clones in Arabidopsis. Plant J 1995, 8:637-652.
18. Lyznik LA, Rao KV, Hodges TK: FLP-mediated recombination of FRT sites in the maize genome. Nucleic Acids Res 1996, 24:3784-3789. 19. Onouchi H, Nishihama R, Kudo M, Machida Y, Machida C: Visualization of site-specific recombination catalyzed by a recombinase from Zygosaccharomyces rouxii in Arabidopsis thaliana. Mol Gen Genet 1995, 247:653-660. 20. Sugita K, Kasahara T, Matsunaga E, Ebinuma H: A transformation vector for the production of marker-free transgenic plants containing a single copy transgene at high frequency. Plant J 2000, 5:461-469. 21. Gleave AP, Mitra DS, Mudge SR, Morris BA: Selectable marker-free • transgenic plants without sexual crossing: transient expression of cre recombinase and use of a conditional lethal dominant gene. Plant Mol Biol 1999, 40:223-235. The ‘elimination-cassette’ of this vector system contains a selectable, as well as a counter-selectable gene, which together are flanked by lox sites. 5-Fluorouracil-resistant plants have lost the selectable gene as well as the counter-selectable cytosine deaminase (cod A) gene.
6. •
22. Zuo J, Nui Q-W, Geir Møller S, Chua N-H: Chemical-regulated, site• specific DNA excision in transgenic plants. Nat Biotechnol 2001, 19:157-161. Plants were transformed with a T-DNA which contained, adjacent to the desired transgene and between lox sites, a selectable marker gene and a Cre open reading frame under the control of an inducible promoter. Upon induction of the promoter (in this case by β-estradiol) the marker gene including the inducible system can efficiently be removed at any time of choice.
7.
23. Srivastava V, Anderson OD, Ow DW: Single-copy transgenic wheat •• generated through the resolution of complex integration patterns. Proc Natl Acad Sci USA 1999, 96:1117-11121. Multiple transgene loci in wheat plants were resolved using a site-specific recombinase. Concomitant with this conversion was the removal of the marker gene used for transformant selections.
5.
Joersbo M, Donaldson I, Kreiberg J, Petersen SG, Brundstedt J, Okkels FT: Analysis of mannose selection used for transformation of sugar beet. Mol Breed 1998, 4:111-117.
Negrotto D, Jolley M, Beer S, Wenck AR, Hansen G: The use of phosphomannose isomerase as a selectable marker to recover transgenic maize plants (Zea mays L.) via Agrobacterium transformation. Plant Cell Reports 2000, 19:798-803. The positively selectable marker phosphomannose isomerase, introduced in [5], was employed for the recovery of transgenic maize plants in high yield; 30% of treated immature embryos gave rise to fertile transgenic plants. Ebinuma H, Sugita K, Matsunaga E, Yamakado M: Selection of marker-free transgenic plants using the isopentenyl transferase gene. Proc Natl Acad Sci USA 1997, 94:2117-2121.
8. ••
Sugita K, Matsunaga E, Ebinuma H: Effective selection system for generating marker-free transgenic plants independent of sexual crossing. Plant Cell Reports 1999, 18:941-947. A vector system is described (the MAT vector system) in which the selectable isopentenyltransferase gene is removed from morphologically abnormal transgenic shoots by site-specific recombination. 9. •
Kunkel T, Niu Q-W, Chan Y-S, Chua N-H: Inducible isopentenyl transferase as a high efficiency marker for plant transformation. Nat Biotechnol 1999, 17:916-919. Expression of isopentenyltransferase in a dexamethasone-inducible manner allows the selection of transgenic plants. 10. Ebinuma H, Sugita K, Matunaga E, Endo S, Kasahara E: Selection of marker-free transgenic plants using the oncogenes (IPT, ROL A, B, C) of Agrobacterium as selectable markers. In Molecular Biology of Woody Plants. Edited by Jarn SM, Minocha SC. Netherlands: Kluwer Academic Publishers; 2000:24-46.
24. Kunze R: The maize transposable element Activator (Ac). In Current Topics in Microbiology and Immunology, Transposable Elements. vol 24. Edited by Saedler H, Gierl A. Springer-Verlag; 1996:162-187. 25. Gorbunova V, Levy AA: Analysis of extrachromosomal Ac/Ds • transposable elements. Genetics 2000, 155:349-359. Analysis of extrachromosomal Ac/Ds excision products suggests that these species are not true transposition intermediates and that, therefore, excision does not necessarily lead to reintegration of transposable elements. 26. Yoder JI, Goldsbrough AP: Transformation systems for generating marker-free transgenic plants. Biotechnology 1994, 12:263-267. 27.
Goldsbrough AP, Lastrella CN, Yoder JI: Transposition mediated re-positioning and subsequent elimination of marker genes from transgenic tomato. Biotechnology 1993, 11:1286-1292.
28. Janssen B-J, Gardner RC: Localized transient expression of Gus in leaf discs cocultivated with Agrobacterium. Plant Mol Biol 1989, 14:61-72.
Elimination of selection markers from transgenic plants Hohn, Levy and Puchta
29. Lebel EG, Masson J, Bogucki A, Paszkowski J: Transposable elements as plant transformation vectors for long stretches of foreign DNA. Theor Appl Genet 1995, 91:899-906. 30. Vergunst AC, Schrammeijer B, den Dulk-Ras A, de Vlaam CMT, •• Regensburg-Tuink JG, Hooykaas PJ: VirB/D4-dependent protein translocation from Agrobacterium into plant cells. Science 2000, 290:979-982. Transport of VirE2 and VirF Cre fusion proteins from Agrobacterium tumefaciens to plants was monitored by a Cre-mediated recombination event resulting in a selectable plant phenotype. 31. Zubco E, Scutt C, Meyer P: Intrachromosomal recombination • between attP regions as a tool to remove selectable marker genes from tobacco transgenes. Nat Biotechnol 2000, 18:442-445. Following the transfer of a vector containing a resistance gene flanked by two 352 base pair attachment regions of bacteriophage λ, somatic tissue could be isolated at a remarkably high frequency in which sequences internal (and partially external) to the attachment sites were spontaneously deleted. 32. Puchta H: Removing selectable marker genes: taking the shortcut. Trends Plant Sci 2000, 5:273-274.
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33. Puchta H, Hohn B: From centiMorgans to base pairs: homologous recombination in plants. Trends Plant Sci 1996,1:340-348. 34. Hansen G, Wright MS: Recent advances in the transformation of plants. Trends Plant Sci 1999, 4:226-231. 35. Shillito RD, Saul MW, Paszkowski J, Müller M, Potrykus I: High efficiency direct gene transfer to plants. Biotechnology 1985, 3:1099-1103. 36. Heifetz PB: Genetic engineering of the chloroplast. Biochimie • 2000, 82:655-666. An extensive review is presented on the potential of using the prokaryotically derived genome, present at high copy numbers in plant cells, for accepting and expressing introduced genes. 37. Jamtham S, Day A: Removal of antibiotic resistance genes from •• transgenic tobacco plastids. Nat Biotech 2000, 18:1172-1176. The expression of transgenic sequences incorporated into plastid genomes offers a range of possibilities and advantages [36•]. Homologous recombination, the prevailing mode of integration of foreign sequences in the chloroplast genome, has been efficiently used to remove marker genes used for selection of transformants.
Elimination of selection markers from transgenic plants Barbara Hohn*, Avraham A Levy† and Holger Puchta‡ Selection markers, which were necessary for the isolation of transgenic plants, are no longer required in mature plants, especially when they are grown in fields. Regimes to achieve their efficient elimination, mostly through site-specific recombination or transposition, are being developed. Addresses *Friedrich Miescher Institute, Maulbeerstrasse 66, 4058 Basel, Switzerland; e-mail: [email protected] † Plant Sciences Department, The Weizmann Institute of Sciences, Rehovot, 76100 Israel; e-mail: [email protected] ‡ AG DNA Rekombination, IPK, Corrensstrasse 3, D-06466 Gatersleben, Germany; e-mail: [email protected] Current Opinion in Biotechnology 2001, 12:139–143 0958-1669/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved.
Introduction The development of transgenic plants requires the use of selectable marker genes, because the efficiency of plant transformation is less than optimal for many important plant species. The maintenance of resistance genes in transgenic plants causes no concerns for laboratory (or greenhouse) experiments. However, their persistence in the field is, depending on one’s point of view, unnecessary, undesirable or unacceptable. These arguments are sufficient to warrant strong efforts to develop strategies for the efficient elimination of marker genes after selection; ‘The moor has fulfilled his duty, the moor can leave’ [1]. Moreover, for conducting further rounds of transformation it is advantageous to have freed the plants from the resistance genes that are no longer necessary, as the list of selectable genes that can be used for any given plant species is not very long. In addition, it may be wise to remove the promoter used to drive the selectable gene, in order to reduce the chance of (transcriptional) gene silencing of the desired transgene linked to the same promoter [2••]. These issues were recognized some time ago and possibilities for the elimination of resistance marker genes have been suggested.
Positive selection of transgenic plants Parallel to, and in combination with, marker elimination, a new set of markers are being developed that are called positive selection markers [3]. The principle of this system is that nontransformed cells are not killed, as in the procedures using antibiotic or herbicide resistance genes, but transformed cells experience a metabolic or developmental advantage. This leads to an increased efficiency of regeneration of transformed plants. Of added value is the expected nontoxicity of the selective chemicals compared with antibiotics and herbicides. In the article by Joersbo and Okkels [3], the β-glucuronidase gene from Escherichia coli was used as a selectable gene and a glucuronide derivative
of the cytokinin benzyladenine was used as the selective agent. This compound requires the activity of β-glucuronidase to activate a cytokinin. The efficiency of transformation was reported to be about twofold higher than with kanamycin; however, transformants were not further characterized. The xylose isomerase gene of Thermoanaerobacterium thermosulfurogenes has also been employed for positive selection: only transformed cells can live on a diet consisting of D-xylose as the sole carbon source. In some, but not all, of the tested species, a higher fraction of transformed cells could be recovered [4]. So far, the best-established system is that which utilizes the phosphomannose isomerase gene (pmi) of E. coli as the selectable gene and mannose as the selective agent [5]. After uptake, mannose is phosphorylated by a hexokinase to mannose-6-phosphate, which accumulates and causes drastic growth inhibition due to the lack of phosphomannose isomerase activity in plants. The transformation of sugar beet with Agrobacterium tumefaciens was found to yield transgenic shoots, even at low levels of expression of the selective gene. Frequencies of transformation were one order of magnitude higher than those obtained with kanamycin as the selective agent. This system has been successfully adapted to Agrobacterium-mediated transformation of maize [6•]. Immature embryos surviving selection were recovered with a frequency of up to 30% and they developed into normal fertile plants with confirmed integration of the transgene. Genes encoding enzymes in the hormone pathway originating from Agrobacterium have also been successfully used for the selection of transformed plants, although in all reported cases the presence or the activity of the respective gene had to be eliminated or turned down. This was necessary to avoid the detrimental effects of hormone overdoses on plant development. In addition, the phenotype, not being cell autonomous, would otherwise lead to chimeric plants. Ebinuma et al. [7] used the isopentenyl transferase (ipt) gene from the T-DNA of Agrobacterium coupled to the constitutive 35S promoter of cauliflower mosaic virus (CaMV) for tobacco transformation. Growth-retarded transformants appeared with occasional normal sideshoots in which the ipt part of the transgene had removed itself due to the activity of an Ac transposon. Higher frequencies of excision of the selectable transgene were established by replacing the transposon by a site-specific recombinase and its respective target sequences flanking the selectable gene ([8••]; see below). The ipt gene also provided the basis for the development of an inducible plant transformation system. The use of a dexamethasone-inducible promoter driving the ipt gene led to the recovery of lettuce and tobacco transformants under inducing conditions [9•].
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Figure 1 (a)
Site-specific recombinase Marker
Transgene
Transgene Marker Transposase
Marker
Transgene
Transgene
Marker
(b) Transposase Marker
Transgene
Marker
Transgene Current Opinion in Biotechnology
Genes that conferred auxin-independent growth onto tobacco transformants were obtained from Agrobacterium rhizogenes [10]. The bacterially derived rol genes are responsible for the proliferation of hairy roots by increasing auxin sensitivity. Transgenic plants arising from this transformation procedure also displayed abnormal phenotypes, such as wrinkled leaves, shortened internodes and reduced apical dominance, again necessitating eviction of the selectable marker. These regimes using plant hormone genes will have to be adapted individually to different plant species by adjustment of plant-internal hormone levels. Improvements might result from varying the promoters used to allow the development of specific organs. The recovery of marker-free plants without the necessity of sexual crossings is certainly an advantage. In addition, some of the selectable genes mentioned may in the future be exchanged for native plant genes, thus eliminating the use of foreign genes as selective markers altogether.
Elimination of marker genes by cotransformation One way to separate selectable marker transgenes from the transgene of interest is to separate them at the stage of transformation. The method of choice for transformation is to use Agrobacterium-mediated processes, as these are more likely to lead to separate integration events than, for instance, particle gun mediated gene delivery methods. Depending on the plant species, the bacterial strains and vectors, and the transformation procedure used, a fraction of transformants will carry the two transgenes linked or not linked [11–13]. An improvement in the cotransformation procedure was introduced by Komari et al. [14] using binary plasmids containing two T-DNAs. Cotransformation frequencies using these ‘superbinary’ vectors were reported to be as high as 47%, with a high proportion of both tobacco and rice transformants carrying unlinked transgenes. Of course this procedure requires fertile plants for genetic separation of the two transgene loci.
Schemes for marker gene elimination. (a) Enzymes acting at specific sites that flank the marker gene to be evicted lead to efficient marker gene elimination. Reinsertion of the marker gene at ectopic positions can be screened for or selected against (see text). (b) The transgene by itself is mobile; the activation of transposase allows the relocation of the desired transgene to new chromosomal positions. Genetic crosses and/or segregation will dissociate the two transgenes. In this case, the presence of the marker gene can also be counter-selected. Sequences required for transposition are represented by black triangles, direct repeats of targets for sitespecific recombinases by black arrows, transgenic DNA by thick black lines and plant DNA by thin solid or dashed back lines.
Elimination of marker genes by site-specific recombinases A general scheme for marker excision using site-specific recombinases is presented in the upper part of Figure 1a. Marker and transgene, originally linked on one transgenic unit, are separated by the activity of an enzyme, the specific recognition sites of which flank the marker to be excised. In pioneering work a decade ago, Dale and Ow [15] used the Cre recombinase of the E. coli bacteriophage P1 to remove a selectable marker gene flanked by lox target sites from a transgenic locus in transformed tobacco. Upon removal of the Cre-encoding locus by segregation, plants were recovered that had incorporated only the desired transgene. In addition, Arabidopsis plants that were free of a selectable marker gene were also recovered using the same site-specific recombination system [16]. Other single-chain recombinases were also found to be useful for the removal of marker genes: the site-specific recombination system of the Streptomyces bacteriophage φC31 (D Ow and R Calendar, personal communication), the FLP/FRT system of the 2 µ plasmid of Saccharomyces cerevisiae [17,18] and the R-RS system of the pSR1 plasmid of Zygosaccharomyces rouxii [19,20]. The common feature in these systems is the generation of transgenic plants that contain two directly oriented recognition sites for the respective recombinase flanking the sequence to be excised. Upon expression of the single-chain recombinase the recombination reaction is initiated, resulting in marker-free transgenic plants. In order to speed up this process, marker gene constructs and tissue culture conditions are being developed that allow marker gene elimination soon after transformation. In one approach, the selectable marker was located adjacent to a counterselectable marker, whereby both markers resided inside the ‘elimination cassette’ [21•]. This approach is especially fast if the recombinase is introduced into the plant cells only transiently (see below).
Elimination of selection markers from transgenic plants Hohn, Levy and Puchta
Alternatively, the expression cassette of the recombinase can be placed inside the elimination cassette; using this strategy, marker-free plants carrying a single copy of the transgene could be recovered even in the absence of counter-selection [8••,20,22•]. It remains to be tested whether a combination of these tricks — namely, using an elimination cassette that contains genes for selection and recombination as well as counter-selection — would further improve the efficiency and speed of marker elimination. In cases in which complex integration patterns of transgenes have to be resolved, the use of site-specific recombination can be of added value: concomitant with the conversion to a single transgene unit, the adjacent resistance marker could be eliminated in transgenic wheat [23••].
Elimination of marker genes by transposases Transposable elements can be harnessed to allow the production of marker-free transgenic plants owing to three key properties: elements from maize such as Ac/Ds were shown to transpose to both linked and unlinked sites in all the heterologous plant hosts analyzed [24]; transposable elements can be engineered in such a way that the transposase can be expressed from a nonmobile construct, whereas the mobile unit only has to contain terminal sequences that allow its transposition; and many excision events are not associated with reinsertion, possibly because of the loss of the element [25•]. On the basis of these properties, transposable elements can be employed for the dissociation of marker and desirable gene in two ways. In the first (Figure 1a, lower part), the marker gene is placed on the mobile element, which is lost after transposition [26]. Marker-free transgenic tobacco and aspen plants have been generated at low frequencies by inserting the selectable ipt gene (see above) into the transposable element Ac [7]. The second possibility for the transposoninduced dissociation of marker gene and desired gene consists of the relocation of the desired gene away from the original transgene locus (Figure 1b). The feasibility of this approach was demonstrated in tomato [27]. The advantage of this system is not only in unlinking the marker gene, but also in creating ‘clean’ insertions of the desired transgene. In addition, a series of plants with different transgene loci can be obtained from one original transformant, which is especially important if recalcitrant plants have to be transformed. This repositioning allows the expression of the transgene at different genomic positions and consequently at different levels.
Recombinases and transposases can be introduced into plants as DNA, RNA or protein Site-specific recombinases and transposases can be expressed from a transgenic locus that is introduced into the plant carrying the marker and desired transgene by a further round of transformation or by crossing. In several of the above mentioned experiments, the transient expression of enzyme genes was also used. Agrobacterium-mediated transfer of T-DNA leads to an early wave of gene expression most
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likely from unintegrated T-DNA molecules [28]. The obvious advantage for marker elimination lies in marker excision in the absence of an integrated version of the enzyme gene. In one study, this approach was combined with a negatively selectable marker to select for events of marker loss [23••]. This technology is especially useful for the generation of transgenic crops that are vegetatively propagated. A transposase mRNA, produced in vitro and introduced into protoplasts, was also documented to activate a transgenic Ds element [29]. In a most interesting recent report, the Crerecombinase was introduced into plants from Agrobacterium that was manipulated to contain Cre-recombinase VirE2 or VirF fusion proteins [30••]. These experiments document that these two virulence proteins are indeed transported into plant cells; however, in the context of this review they suggest that enzymes needed for marker excision may be introduced by Agrobacterium tumefaciens.
Intrachromosomal recombination for marker gene elimination?
In an attempt to use the bacteriophage λ integration/excision system for marker elimination, the unexpected excision of an NPTII gene from tobacco in the absence of any enzyme activity was reported ([31•]; discussed in [32]). The anticipated target for the λ enzymes was a pair of 352 base pair attachment regions of λ. From two of the 11 transgenic calli that contained a resistance gene between these elements, shoots grew up that contained white kanamycinsensitive sectors. Three out of 23 of these shoots proved to have lost the resistance cassette by homologous recombination, and the majority had also lost adjacent transgene sequences. This experiment thus documents a surprisingly high incidence of intrachromosomal homologous recombination, several orders higher than previously reported [33]. It is important to clarify whether the λ-attachment region is intrinsically recombinogenic, whether by chance the two transgenic loci are recombinational hotspots, and whether the employed transformation booster sequence or the special culture conditions influenced the recombination behaviour of these two transgenic lines.
Conclusions Despite the recent advances in the transformation of plants [34], technologies for the efficient generation of transgenic plants in the absence of any selection were optimized only for tobacco [35]. Hope remains that other plants will become less recalcitrant to transformation by improving culture conditions and transformation devices. Meanwhile, the marker improvement and marker elimination systems described in this review will hopefully become standard for crop improvement and the acceptance of improved crops.
Update Integration of foreign genes into the plastid genome has special advantages [36•]: gene containment may be enhanced because in many crop plants plastids are inherited from the maternal parent preventing spread of transgenes;
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homologous recombination in chloroplasts allows precise gene targeting into a small genome; and high levels of gene expression can be achieved. However, because of the high copy number and the prokaryotic expression signals of the selection markers it may be especially advisable to remove them after the generation of transplastomic plants. The efficient homologous recombination system of chloroplasts was exploited to remove genes conferring herbicide resistance and coding for a screenable marker from transplastomic tobacco plants [37••].
Acknowledgements We acknowledge the communication of unpublished information from H Ebinuma, D Ow, R Calendar and P Hooykaas. E Bucher kindly provided the figure and I Kovalchuk critically reviewed the manuscript. Special thanks go to D Ow for contributing with expertise, and to W Hörr-Szalay for information on [1]. BH acknowledges financial support from the Novartis Research Foundation.
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