Chemical-induced autoexcision of selectable markers in elite tomato plants transformed with a gene conferring resistance to lepidopteran insects

Marker-free transgenic tomato plants harboring a synthetic Bacillus thuringiensis endotoxin gene, cryIAc, were obtained by using a chemically regulated, Cre/loxP-mediated site-specific DNA recombination system, in which the selectable marker neomycin phosphotransferase gene flanked by two directly oriented loxP sites was located between the cauliflower mosaic virus 35S promoter and a promoterless cryIAc. Upon induction by 2 μM β-estradiol, sequences encoding the selectable marker and cre sandwiched by two loxP sites were excised from the tomato genome, leading to activation of the downstream endotoxin gene cryIAc with high expression levels as shown by Northern blot and ELISA assay (250–790 ng g⁻¹ fresh wt) in T₁ generation. For transgenic line with single transgenic loci, 15% of T₁ progenies were revealed marker-free. This autoexcision strategy provides an effective approach to eliminate a selectable marker gene from transgenic tomato, thus expediting the public acceptance of genetically modified crop.     

Development of a simple and efficient system for excising selectable markers in Arabidopsis using a minimal promoter::Cre fusion construct

The development of rapid and efficient strategies to generate selectable marker-free transgenic plants could help increase the consumer acceptance of genetically modified (GM) plants. To produce marker-free transgenic plants without conditional treatment or the genetic crossing of offspring, we have developed a rapid and convenient DNA excision method mediated by the Cre/loxP recombination system under the control of a −46 minimal CaMV 35S promoter. The results of a transient expression assay showed that −46 minimal promoter::Cre recombinase (−46::Cre) can cause the loxP-specific excision of a selectable marker, thereby connecting the 35S promoter and β-glucuronidase (GUS) reporter gene. Analysis of stable transgenic Arabidopsis plants indicated a positive correlation between loxP-specific DNA excision and GUS expression. PCR and DNA gel-blot analysis further revealed that nine of the 10 tested T₁ transgenic lines carried both excised and nonexcised constructs in their genomes. In the subsequent T₂ generation plants, over 30% of the individuals for each line were marker-free plants harboring the excised construct only. These results demonstrate that the −46::Cre fusion construct can be efficiently and easily utilized for producing marker-free transgenic plants.     

Excision of a selectable marker in transgenic rice (Oryza sativa L.) using a chemically regulated Cre/loxP system

Removal of a selectable marker gene from genetically modified (GM) crops alleviates the risk of its release into the environment and hastens the public acceptance of GM crops. Here we report the production of marker-free transgenic rice by using a chemically regulated, Cre/loxP-mediated site-specific DNA recombination in a single transformation. Among 86 independent transgenic lines, ten were found to be marker-free in the T₀ generation and an additional 17 lines segregated marker-free transgenic plants in the T₁ generation. Molecular and genetic analyses indicated that the DNA recombination and excision in transgenic rice were precise and the marker-free recombinant T-DNA was stable and heritable.The first two authors contributed equally to the work     

Excision of a selective marker in transgenic rice using a novel Cre/<Emphasis Type="Italic">loxP</Emphasis> system controlled by a floral specific promoter

Based on the Cre/loxP system, we have developed a novel marker-free system mediating a direct auto-excision of loxP-flanked marker genes from T₁ transgenic rice without any treatment or further offspring crossing. To achieve this, the floral-specific promoter OsMADS45 was isolated from rice and the expression patterns of OsMADS45 promoter was characterised by using the pOs45:GUS transgenic plants. Furthermore, the binary vector with Cre recombinase under the control of OsMADS45 promoter was constructed and introduced into rice by Agrobacterium-mediated transformation and transgenic rice plants were generated. Southern blot analysis showed that auto-excision of the selective markers occurred in some T₁ progeny of the transgenic plants, suggesting that a high auto-excision frequency can be achieved with our Cre/loxP system. This auto-excision strategy provides an efficient way of removing the selectable marker gene from transgenic rice. Xianquan Bai and Qiuyun Wang contributed equally to the work.     

Salicylic-Acid-Induced Self-excision of the Marker Gene <Emphasis Type="Italic">npt</Emphasis>II from Transgenic Tomato Using the Cre–<Emphasis Type="Italic">loxP</Emphasis> System

The presence of antibiotic-resistant genes in genetically engineered crops together with the target gene has generated a number of environmental and consumer concerns. In order to alleviate public concerns over the safety of food derived from transgenic crops, marker gene elimination is desirable. Marker-free transgenic tomato plants were obtained by using a salicylic-acid-regulated Cre–loxP-mediated site-specific DNA recombination system in which the selectable marker neomycin phosphotransferase nptII and cre genes were flanked by two directly oriented loxP sites. Upon induction by salicylic acid, the cre gene produced a recombinase that eliminated sequences encoding nptII and cre genes, sandwiched by two loxP sites from the tomato genome. Regenerant plants with the Cre–loxP system were obtained by selection on kanamycin media and polymerase chain reaction (PCR) screening. Transgenic plants were screened for excision by PCR using nptII, cre, and PR-1a promoter primers following treatment with salicylic acid. The footprint of the excision was determined by sequencing the T-DNA borders after a perfect recombination event. The excision efficiency was 38.7%. A new plant transformation vector, pBLNSC (Genbank accession number EU327497), was developed, containing six cloning sites and the self-excision system. This provided an effective approach to eliminate the selectable marker gene from transgenic tomato, thus expediting public acceptance of genetically modified tomato.     

Enhancement of stress tolerance in transgenic tobacco plants constitutively expressing AtIpk2β, an inositol polyphosphate 6-/3-kinase from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Inositol phosphates (IPs) and their turnover products have been implicated to play important roles in stress signaling in eukaryotic cells. In higher plants genes encoding inositol polyphosphate kinases have been identified previously, but their physiological functions have not been fully resolved. Here we expressed Arabidopsis inositol polyphosphate 6-/3-kinase (AtIpk2β) in two heterologous systems, i.e. the yeast Saccharomyces cerevisiae and in tobacco (Nicotiana tabacum), and tested the effect on abiotic stress tolerance. Expression of AtIpk2β rescued the salt-, osmotic- and temperature-sensitive growth defects of a yeast mutant strain (arg82Δ) that lacks inositol polyphosphate multikinase activity encoded by the ARG82/IPK2 gene. Transgenic tobacco plants constitutively expressing AtIpk2β under the control of the Cauliflower Mosaic Virus 35S promoter were generated and found to exhibit improved tolerance to diverse abiotic stresses when compared to wild type plants. Expression patterns of various stress responsive genes were enhanced, and the activities of anti-oxidative enzymes were elevated in transgenic plants, suggesting a possible involvement of AtIpk2β in plant stress responses. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A Cre/loxP-mediated self-activating gene excision system to produce marker gene free transgenic soybean plants

Marker-gene-free transgenic soybean plants were produced by isolating a developmentally regulated embryo-specific gene promoter, app1, from Arabidopsis and developing a self-activating gene excision system using the P1 bacteriophage Cre/loxP recombination system. To accomplish this, the Cre recombinase gene was placed under control of the app1 promoter and, together with a selectable marker gene (hygromycin phosphotransferase), were cloned between two loxP recombination sites. This entire sequence was then placed between a constitutive promoter and a coding region for either β-glucuronidase (Gus) or glyphosate acetyltransferase (Gat). Gene excision would remove the entire sequence between the two loxP sites and bring the coding region to the constitutive promoter for expression. Using this system marker gene excision occurred in over 30% of the stable transgenic events as indicated by the activation of the gus reporter gene or the gat gene in separate experiments. Transgenic plants with 1 or 2 copies of a functional excision-activated gat transgene and without any marker gene were obtained in T0 or T1 generation. This demonstrates the feasibility of using developmentally controlled promoters to mediate marker excision in soybean.     

Inducible Excision of Selectable Marker Gene from Transgenic Plants by the Cre/<Emphasis Type="Italic">lox</Emphasis> Site-specific Recombination System

In a plant transformation process, it is necessary to use marker genes that allow the selection of regenerated transgenic plants. However, selectable marker genes are generally superfluous once an intact transgenic plant has been established. Furthermore, they may cause regulatory difficulties for approving transgenic crop release and commercialization. We constructed a binary expression vector with the Cre/lox system with a view to eliminating a marker gene from transgenic plants conveniently. In the vector, recombinase gene cre under the control of heat shock promoter and selectable marker gene nptII under the control of CaMV35S promoter were placed between two lox P sites in direct orientation, while the gene of interest was inserted outside of the lox P sites. By using this vector, both cre and nptII genes were eliminated from most of the regenerated plants of primary transformed tobacco through heat shock treatment, while the gene of interest was retained and stably inherited. This autoexcision strategy, mediated by the Cre/lox system and subjected to heat shock treatment to eliminate a selectable marker gene, is easy to adopt and provides a promising approach to generate marker-free transgenic plants.     

Generation of marker free salt tolerant transgenic plants of Arabidopsis thaliana using the gly I gene and cre gene under inducible promoters

Despite the advances in transgenesis, transformation technologies still rely on the introduction of a selectable marker gene to identify cells and tissues that have integrated the gene of interest in their genome. The continuous presence of the marker genes in the transgenics is often controversial as it can potentially have multiple undesirable impacts. The present study employed the self-excising Cre-loxP system to generate marker-free Arabidopsis thaliana expressing the agronomically important glyoxalase I (glyI) gene from Brassica juncea to confer salt stress tolerance. A binary vector was constructed wherein the salt-inducible rd29A promoter was used to drive the expression of the glyI gene and the transformants of A. thaliana were recovered using kanamycin resistance as the selectable marker. The neomycin phosphotransferase II (nptII) gene was flanked by the loxP sites followed by the introduction of a heat-inducible Cre-recombinase in between the loxP sites. The kanamycin-resistant transgenic lines of A. thaliana using this vector showed an ability to withstand stress imposed by 150 mM NaCl. The exposure of the T2 transgenic lines to a mild heat shock (37°C) resulted in the recovery of salt-tolerant, kanamycin-sensitive T3 progeny. Molecular analyses of the T3 transgenic lines following the heat shock treatment confirmed the excision of the nptII gene and the completion of their life cycle in the presence of 150 mM NaCl-induced stress.     

A passage through in vitro culture leads to efficient production of marker-free transgenic plants in <Emphasis Type="Italic">Brassica juncea</Emphasis> using the Cre–<Emphasis Type="Italic">loxP</Emphasis> system

The Cre–loxP site-specific recombination system was deployed for removal of marker genes from Brassica juncea (Indian mustard). Excision frequencies, monitored by removal of nptII or gfp genes in F₁ plants of crosses between LOX and CRE lines, were high in quiescent, differentiated somatic tissues but extremely poor in the meristematic regions (and consequently the germinal cells) thus preventing identification and selection of marker-free transgenic events which are devoid of both the marker gene as well as the cre gene, in F₂ progeny. We show that a passage through in vitro culture of F₁ leaf explants allows efficient development of marker-free transgenics in the F₂ generation addressing current limitations associated with efficient use of the Cre/loxP technology for marker gene removal. N. Arumugam and Vibha Gupta have contributed equally to this work.     

Expression of stilbene synthase gene in transgenic tomato using salicylic acid-inducible Cre/loxP recombination system with self-excision of selectable marker

A plant transformation vector, pCLKSCLA25 (EU327498), was developed to contain eight cloning sites and the inducible self-excision system which provided an effective approach to eliminate the selectable marker gene(s) from transgenic plants. Upon induction by salicylic acid, the cre gene produced a recombinase that eliminated sequences encoding the selectable marker neomycin phosphotransferase and cre itself. The excision efficiency was 41% in transgenic tomato regenarants. The stilbene synthase gene (vst1) from Vitis vinifera L. was cloned into pCLKSCLA25. The expression of vst1 gene contributed to the accumulation of trans-reveratrol from 3.4 to 8.7 μg/g fresh wt in different marker-free transgenic tomato lines. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Removal of the Selectable Marker Gene from Transgenic Tobacco Plants by Expression of Cre Recombinase from a Tobacco Mosaic Virus Vector through Agroinfection

Selection markers are often indispensable during the process of plant transformation, but dispensable once transgenic plants have been established. The Cre/lox site-specific recombination system has been employed to eliminate selectable marker genes from transgenic plants. Here we describe the use of a movement function-improved Tobacco Mosaic Virus (TMV) vector, m30B, to express Cre recombinase for elimination of the selectable marker gene nptII from transgenic tobacco plants. The transgenic tobacco plants were produced by Agrobacterium-mediated transformation with a specially designed binary vector pGNG which contained in its T-DNA region a sequence complex of 35S promoter-lox-the gfp coding sequence-rbcS terminator-Nos promoter-nptII-Nos terminator-lox-the gus coding region-Nos terminator. The expression of the recombinant viral vector m30B:Cre in plant cells was achieved by placing the viral vector under the control of the 35S promoter and through agroinoculation. After co-cultivating the pGNG-leaf discs with agro35S-m30B:Cre followed by shoot regeneration without any selection, plants devoid of the lox-flanked sequences including nptII were obtained with an efficiency of about 34% as revealed by histochemical GUS assay of the regenerants. Three of 11 GUS expressing regenerants, derived from two independent transgenic lines containing single copy of the pGNG T-DNA, proved to be free of the lox-flanked sequences by Southern blot analysis. Excision of the lox-flanked sequences in the three plants could be attributed to transient expression of Cre from the viral vector at the early stage of co-cultivation, since the cre sequence could not be detected in the viral RNA molecules accumulated in the plants, nor in their genomic DNA. The parental marker-free genotype was inherited in their selfed progeny, and all of the progeny were virus-free, apparently because TMV is not seed-transmissible. Therefore, expression of Cre from a TMV-based vector could be used to eliminate selectable marker genes from transgenic tobacco plants without sexual crossing and segregation, and this strategy could be extended to other TMV-infected plant species and applicable to other compatible virus–host plant systems.     

Co-transformation of gene expression cassettes via particle bombardment to generate safe transgenic plant without any unwanted DNA

The presence of resistant selectable marker genes and other added DNAs such as the vector backbone sequence in transgenic plant might be an unpredictable hazard to the ecosystem as well as to human health, which have affected the safe assessment of transgenic plants seriously. Using minimal gene expression cassette (containing the promoter, coding region, and terminator) without vector backbone sequence for particle bombardment is the new trend of plant genetic transformation. In the present paper, we co-transformed the selectable marker bar gene cassette and non-selected cecropinB gene cassette into rice (Oryza sativa L.) by particle bombardment, then eliminated the selectable marker bar gene in R₁ generation applying the hereditary segregation strategy and attained two safe transgenic plants only harboring cecropinB gene cassettes without any superfluous DNA. This is the fist report indicating that the combination of minimal gene cassettes transformation with the co-transformation and segregation strategy can generate selectable marker-free transgenic plants, which will promote the advancement in plant genetic engineering greatly.     

Systems for the removal of a selection marker and their combination with a positive marker

Many systems have been developed for the removal of a selection marker in order to generate marker-free transgenic plants. These systems consist of (1) a site-specific recombination system (Cre/lox) or a phage-attachment region (attP) to remove the selectable marker gene and (2) a transposable element system (Ac) or a co-transformation system to segregate the gene of interest from the selectable marker gene. Overall, the process is more time-consuming than conventional transformation methods because two rounds of transformation - two steps of regeneration or sexual crossings - are required to obtain the desired transgenic plants. Recently, removal systems combined with a positive marker, denoted as MAT vectors, have been developed to save time and effort by generating marker-free transgenic plants through a single-step transformation. We summarize here the transformation procedures using these systems and discuss their feasibility for practical use.     

Marker-Free Transgenic Chrysanthemum Obtained by <Emphasis Type="Italic">Agrobacterium</Emphasis>-Mediated Transformation with Twin T-DNA Binary Vectors

In this study, a superbinary vector was constructed to evaluate the potential of a twin T-DNA system for generating selectable marker-free transgenic chrysanthemum plants. The first T-DNA of the pCAMBIA 1300 vector contained the hygromycin phosphotransferase (hpt) selectable marker gene, while the second T-DNA carried the β-glucuronidase gene (uidA) and featuring the gene of interest. The two T-DNA regions were placed adjacent to each other with no intervening region. This vector was then used to transform transversal thin cell layers (1–2 mm thick) of internodal stem segments of chrysanthemum via Agrobacterium-mediated transfer. Putative transgenic plants were obtained and analyzed for presence and integration of the transgene using polymerase chain reaction amplification and Southern blotting. The primary cotransformation frequency was calculated at 38.4%. A total of 17 hpt-resistant/gus-positive T0 plants were evaluated for segregation in the next generation (T1), and among those approximately 15.7% carried the transgene. Overall, the two T-DNA system appeared to be a useful approach to generate marker-free transgenic chrysanthemum plants, thereby eliminating public concerns regarding proliferation of antibiotic and herbicide resistance genes into the environment.     

Chemical-regulated, site-specific DNA excision in transgenic plants

We have developed a chemical-inducible, site-specific DNA excision system in transgenic Arabidopsis plants mediated by the Cre/loxP DNA recombination system. Expression of the Cre recombinase was tightly controlled by an estrogen receptor-based fusion transactivator XVE. Upon induction by beta-estradiol, sequences encoding the selectable marker, Cre, and XVE sandwiched by two loxP sites were excised from the Arabidopsis genome, leading to activation of the downstream GFP (green fluorescent protein) reporter gene. Genetic and molecular analyses indicated that the system is tightly controlled, showing high-efficiency inducible DNA excision in all 19 transgenic events tested with either single or multiple T-DNA insertions. The system provides a highly reliable method to generate marker-free transgenic plants after transformation through either organogenesis or somatic embryogenesis.     

Reactivation of an integrated disabled viral vector using a Cre-loxP recombination system in Arabidopsis thaliana

Summary We developed an inactivated DNA replicon of Turnip Mosaic Virus (TuMV), which was reactivated by a recombination event based on the Cre-loxP system. Viral replication was prevented by the insertion of a translation terminator sequence flanked by two loxP sites at the junction of the P1–HCPro-coding genes. In vitro recombination was tested with purified Cre, which excised the floxed sequence from the TuMV DNA, leaving a single loxP site in the reactivated viral genome, and restored the open reading frame of the replicon. Arabidopsis thaliana plants were made transgenic for the inactivated TuMV replicon. Removal of the translation terminator sequence was achieved by the controlled expression of Cre. Delivery of the Cre recombinase to the transgenic plants was obtained by three methods: agroinfiltration, PVX-based production, or transgenic chemical-inducible expression. In each case, reactivation of TuMV replication was observed.     

Directed excision of a transgene from the plant genome

Summary The effectiveness of loxP-Cre directed excision of a transgene was examined using phenotypic and molecular analyses. Two methods of combining the elements of this system, re-transformation and cross pollination, were found to produce different degrees of excision in the resulting plants. Two linked traits, -glucuronidase (GUS) and a gene encoding sulfonylurea-resistant acetolactate synthase (ALS^r), were integrated into the genome of tobacco and Arabidopsis. The ALS^r gene, bounded by loxP sites, was used as the selectable marker for transformation. The directed loss of the ALS^T gene through Cre-mediated excision was demonstrated by the loss of resistance to sulfonylurea herbicides and by Southern blot analysis. The -glucuronidase gene remained active. The excision efficiency varied in F₁ progeny of different lox and Cre parents and was correlated with the Cre parent. Many of the lox × Cre F₁ progeny were chimeric and some F₂ progeny retained resistance to sulfonylureas. Re-transformation of lox/ALS/lox/GUS tobacco plants with cre led to much higher efficiency of excision. Lines of tobacco transformants carrying the GUS gene but producing only sulfonylurea-sensitive progeny were obtained using both approaches for introducing cre. Similarly, Arabidopsis lines with GUS activity but no sulfonylurea resistance were generated using cross pollinations.     

Tissue-specifically regulated site-specific excision of selectable marker genes in bivalent insecticidal, genetically-modified rice

Marker-free, genetically-modified rice was created by the tissue-specifically regulated Cre/loxP system, in which the Cre recombinase gene and hygromycin phosphotransferase gene (hpt) were flanked by two directly oriented loxP sites. Cre expression was activated by the tissue-specific promoter OsMADS45 in flower or napin in seed, resulting in simultaneous excision of the recombinase and marker genes. Segregation of T₁ progeny was performed to select recombined plants. The excision was confirmed by PCR, Southern blot and sequence analyses indicating that efficiency varied from 10 to 53 % for OsMADS45 and from 12 to 36 % for napin. The expression of cry1Ac and vip3A was detected by RT-PCR analysis in marker-free transgenic rice. These results suggested that our tissue-specifically regulated Cre/loxP system could auto-excise marker genes from transgenic rice and alleviate public concerns about the security of GM crops.     

Ovary-drip transformation: a simple method for directly generating vector- and marker-free transgenic maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) with a linear <Emphasis Type="Italic">GFP</Emphasis> cassette transformation

The presence of selectable marker genes and vector backbone sequences has affected the safe assessment of transgenic plants. In this study, the ovary-drip method for directly generating vector- and selectable marker-free transgenic plants was described, by which maize was transformed with a linear GFP cassette (Ubi-GFP-nos). The key features of this method center on the complete removal of the styles and the subsequent application of a DNA solution directly to the ovaries. The movement of the exogenous DNA was monitored using fluorescein isothiocyanate-labeled DNA, which showed that the time taken by the exogenous DNA to enter the ovaries was shortened compared to that of the pollen-tube pathway. This led to an improved transformation frequency of 3.38% compared to 0.86% for the pollen-tube pathway as determined by PCR analysis. The use of 0.05% surfactant Silwet L-77 + 5% sucrose as a transformation solution further increased the transformation frequency to 6.47%. Southern blot analysis showed that the transgenic plants had low transgene copy number and simple integration pattern. Green fluorescence was observed in roots and immature embryos of transgenic plants by fluorescence microscopy. Progeny analysis showed that GFP insertions were inherited in T₁ generation. The ovary-drip method would become a favorable choice for directly generating vector- and marker-free transgenic maize expressing functional genes of agronomic interest.