Methods to produce marker-free transgenic plants

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

Strategies for developing marker-free transgenic plants

The development of marker-free transgenic plants has responded to public concerns over the safety of biotechnology crops. It seems that continued work in this area will soon remove the question of unwanted marker genes from the debate concerning the public acceptability of transgenic crop plants. Selectable marker genes are co-introduced with genes of interest to identify those cells that have integrated the DNA into their genome. Despite the large number of different selection systems, marker genes that confer resistance to the antibiotics, hygromycin (hpt) and kanamycin (nptII) or herbicide phosphinothricin (bar), have been used in most transgenic research and crop development techniques. The techniques that remove marker gene are under development and will eventually facilitate more precise and subtle engineering of the plant genome, with widespread applications in both fundamental research and biotechnology. In addition to allaying public concerns, the absence of resistance genes in transgenic plants could reduce the costs of developing biotechnology crops and lessen the need for time-consuming safety evaluations, thereby speeding up the commercial production of biotechnology crops. Many research results and various techniques have been developed to produce marker-free transgenic plants. This review describes the strategies for eliminating selectable marker genes to generate marker-free transgenic plants, focusing on the three significant marker-free technologies, co-transformation, site-specific recombinase-mediated excision, and non-selected transformation.     

Development of selection marker-free transgenic potato plants with enhanced tolerance to oxidative stress

A binary vector devoid of a plant selection-marker gene (designated as pSSA-F) was constructed to overcome bio-safety concerns about genetically modified plants. This vector carried chloroplast-targeted superoxide dismutase (SOD) and ascorbate peroxidase (APX) genes under the control of an oxidative stress-inducible(SWPA2) promoter, and was utilized to transform potato (Solanum tuberosum L.). Integration of these foreign genes into transgenic plants was primarily performed via PCR with genomic DNA. Twelve marker-free transgenic lines were obtained by inoculating stem explants. The maximum transformation efficiency was 6.25% and averaged 2.2%. Successful integration of the SOD and APX genes rendered transgenic plants tolerant to methyl viologen-mediated oxidative stress at the leaf-disc and whole-plant levels. Our findings suggest that this technique for developing selection marker-free transgenic plants is feasible and can be employed with other crop species.     

The ways to produce biologically safe marker-free transgenic plants

The review considers the basic strategies used to produce biologically safe marker-free transgenic plants and analyzes their advantages and disadvantages. The systems of positive and negative selection as safer approaches for transformant identification are briefly described. The application of co-transformation, transposition, and site-specific recombination for production of marker-free plants is described. Special attention is paid to novel approaches to create marker-free plants initially containing no selective genes in their genomes.     

Engineering plants with increased levels of the antioxidant chlorogenic acid

The trend to view many foods not only as sustenance but also as medicine, so-called functional foods, is increasing. Phenolics are the most widespread dietary antioxidants, and among these, chlorogenic acid (CGA) accumulates to high levels in some crop plants. CGA acts as an antioxidant in plants and protects against degenerative, age-related diseases in animals when supplied in their diet. cDNA clones encoding the enzyme that synthesizes CGA, hydroxycinnamoyl-CoA quinate: hydroxycinnamoyl transferase (HQT), were characterized from tomato and tobacco. Gene silencing proved HQT to be the principal route for accumulation of CGA in solanaceous species. Overexpression of HQT in tomato caused plants to accumulate higher levels of CGA, with no side-effects on the levels of other soluble phenolics, and to show improved antioxidant capacity and resistance to infection by a bacterial pathogen. Tomatoes with elevated CGA levels could be used in foods with specific benefits for human health.     

Effective selection system for generating marker-free transgenic plants independent of sexual crossing

 In a previous report, a novel selection protocol termed "the MAT-vector system" for generating marker-free transgenic plants (MFTPs) was presented. The first stage of the system is visual selection of morphologically abnormal transgenic shoots, ipt-shooty, that have lost apical dominance and rooting ability. The second stage involves elimination of the ipt gene and the appearance of MFTPs free of ipt gene influence. The present report describes a practical MAT-vector in which removal of the ipt gene is efficiently mediated by the site-specific recombination system R/RS from Zygosaccharomyces rouxii, in place of the maize transposable element Ac, used previously. This improved MAT-vector produced MFTPs from 39% of moderate ipt-shooty and 70% of extreme ipt-shooty lines. These results are superior to the previous MAT-vector which produced MFTPs from only 5% of ipt-shooty lines. The present novel system also induced direct development of MFTPs from adventitious buds without production of ipt-shooty intermediates. The presence of β-glucuronidase (GUS) and neomycin phosphotransferase (NPTII) genes of interest, and the absence of the ipt gene were verified by a GUS histochemical assay, NPTII assay, and molecular analysis. Received: 19 June 1998 / Revision received: 4 December 1998 / Accepted: 18 December 1998     

Asexual production of selectable marker-free transgenic woody plants, vegetatively propagated species

We describe here a practical system for generating selectable marker-free transgenic woody plants independent of sexual crossing. We previously reported that the GST-MAT vector system could produce marker-free transgenic tobacco plants containing a single-copy transgene at high frequency. The GST-MAT vector system consists of a DNA excision cassette of the R/RS site-specific recombination system from Zygosaccharomyces rouxii into which the isopentenyltransferase gene from Agrobacterium tumefaciens has been inserted. In this study, we applied this new GST-MAT vector to hybrid aspen (Populus Sieboldii X Populus grandidentata), a model of vegetatively propagated plant species, to produce selectable marker-free transgenic woody plants. In the new GST-MAT vector, the chimeric ipt gene fused with a light-inducible rbcS promoter efficiently produced transgenic ipt-shooty with GUS activity from 38.0% of infected stems. Upon excision of the R and ipt genes between RS sites, regulated by the inducible promoter of the maize glutathione-S-transferase (GST-II-27) gene, 3 (21.4%) transgenic hybrid aspen plants with marker-free and normal phenotype were generated from 14 ipt-shooty lines within 2 months after cutting induction. These results suggest that the MAT-vector system might be useful for removing a selectable marker gene independent of sexual crossing in vegetatively propagated species.     

A novel double T-DNA system for producing stack and marker-free transgenic plants

This study aimed to develop a new vector system to remove selection genes and to introduce two or more genes of interest into plants in order to express them in a coordinated manner. A multigene expression vector was established based on pCamBIA2300 using a selectable marker gene (SMG)-free system based on the combination of the isocaudamer technique and double T-DNA. The vector DT7 containing seven target genes was constructed and introduced into tobacco using Agrobacterium-mediated transformation. Twenty-one of 27 positive transgenic plants contained both T-DNA regions. The co-transformation frequency was 77.8 %. The frequency of unlinked integration of two intact T-DNAs was 22.22 % (6/27). The frequency of removal of SMG from transgenic T1 plants was 19.10 %. These results suggest that this vector system was functional and effective for multigene expression and SMG-free transgenic plant cultivation. At least seven target genes can be co-expressed using this system. Overall, these findings provide a new and highly effective platform for multigene and marker-free transgenic plant production.     

Recent advances in development of marker-free transgenic plants: Regulation and biosafety concern

During the efficient genetic transformation of plants with the gene of interest, some selectable marker genes are also used in order to identify the transgenic plant cells or tissues. Usually, antibiotic- or herbicide-selective agents and their corresponding resistance genes are used to introduce economically valuable genes into crop plants. From the biosafety authority and consumer viewpoints, the presence of selectable marker genes in released transgenic crops may be transferred to weeds or pathogenic microorganisms in the gastrointestinal tract or soil, making them resistant to treatment with herbicides or antibiotics, respectively. Sexual crossing also raises the problem of transgene expression because redundancy of transgenes in the genome may trigger homology-dependent gene silencing. The future potential of transgenic technologies for crop improvement depends greatly on our abilities to engineer stable expression of multiple transgenic traits in a predictable fashion and to prevent the transfer of undesirable transgenic material to non-transgenic crops and related species. Therefore, it is now essential to develop an efficient marker-free transgenic system. These considerations underline the development of various approaches designed to facilitate timely elimination of transgenes when their function is no longer needed. Due to the limiting number of available selectable marker genes, in future the stacking of transgenes will be increasingly desirable. The production of marker-free transgenic plants is now a critical requisite for their commercial deployment and also for engineering multiple and complex trait. Here we describe the current technologies to eliminate the selectable marker genes (SMG) in order to develop marker-free transgenic plants and also discuss the regulation and biosafety concern of genetically modified (GM) crops.     

Construction of marker-free transplastomic plants

Because of its prokaryotic-type gene expression machinery, maternal inheritance and the opportunity to express proteins at a high level, the plastid genome (plastome or ptDNA) is an increasingly popular target for engineering. The ptDNA is present as up to 10,000 copies per cell, making selection for marker genes essential to obtain plants with uniformly transformed ptDNA. However, the marker gene is no longer desirable when homoplastomic plants are obtained. Marker-free transplastomic plants can now be obtained with four recently developed protocols: homology-based excision via directly repeated sequences, excision by phage site-specific recombinanses, transient cointegration of the marker gene, and the cotransformation-segregation approach. Marker excision technology will benefit applications in agriculture and in molecular farming.     

Development of transgenic rice plants expressing maize anthocyanin genes and increased blast resistance

The functional association of flavonoids with plant stress responses, though widely reported in the literature, remains to be documented in rice. Towards this end we chose a transgenic approach with well characterized regulatory and structural genes from maize involved in flavonoid biosynthesis. Activation of anthocyanin pathway in rice was investigated with the maize genes. Production of purple anthocyanin pigments were observed in transformed Tp309 (a japonica rice variety) calluses upon the introduction of the maize regulatory genes C1 (coloured-1), R (red) and the structural gene C2 (coloured-2, encoding chalcone synthase). In addition, stable transgenic plants carrying the maize C2 gene under the control of the maize Ubiquitin promoter were generated. A localized appearance of purple/red pigment in the leaf blade and leaf sheath of R₀ C2 transgenic seedlings was observed. Such a patchy pattern of the transgene expression appears to be conditioned by the genetic background of Tp309, which is homozygous for dominant color inhibitor gene(s) whose presence was unravelled by appropriate genetic crosses. Southern blot analysis of the transgenic plants demonstrated that c2 cDNA was integrated into the genome. Western blot analysis of these primary transgenics revealed the CHS protein while it was not detected in the control untransformed Tp3O9, suggesting that Tp309 might have a mutation at the corresponding C2 locus or that the expression of this gene is suppressed in Tp309. Further analysis of C2 transgenics revealed CHS protein only in three out of sixteen plants that were western-positive in the R₀ generation, suggesting gene silencing. Preliminary screening of these R₁ plants against the rice blast fungus Magnaporthe grisea revealed an increase in resistance.     

Evaluation of a morphological marker selection and excision system to generate marker-free transgenic cassava plants

The efficacy of the ipt-type Multi-Auto-Transformation (MAT) vector system to transform the extensively grown cassava cultivar “KU50” was evaluated. This system utilizes the isopentenyltransferase (ipt) gene as morphological marker for visual selection of transgenic lines. The extreme shooty phenotype (ESP) of transgenic lines is lost due to the removal of ipt gene mediated by the yeast Rint/RS system. As a result, phenotypically normal shoots, considered marker-free transgenic plants, could be obtained. When transforming KU50 cassava cultivar with two different ipt-type MAT vectors, transformation frequency at 19–21% was observed. Among the total number of ESP explants, 32–38% regained normal extended shoot phenotype and 88–96% of which were confirmed to represent the marker-free transgenic plants. This is the first demonstration of the efficacy of Rint/RS system in promoting excision of ipt marker gene in cassava specie, with the consequent rapid production of marker-free transgenic plants. The high efficiency of this system should facilitate pyramiding a number of transgenes by repeated transformation without having to undergo through laborious, expensive and time-consuming processes of sexual crossing and seed production. The generation of marker-free, thus environmentally safe, genetically modified cassava clones should also ease the public concerns regarding the use of transgenic cassava in both food and nonfood industries.     

Use of the gene of antimicrobial peptide cecropin P1 for producing marker-free transgenic plants

The marker-free transgenic tobacco plants carrying a synthetic gene encoding the antimicrobial peptide cecropin P1 (cecP1) under the control of the cauliflower mosaic virus 35S RNA promoter were produced. The binary vector pBM, free of any selective genes of resistance to antibiotics or herbicides intended for selecting transgenic plants, was used for transformation. The transformants were screened on a nonselective medium by detecting cecropin P1 in plant cells according to the antibacterial activity of plant extracts and enzyme immunoassay. According to the two used methods, 2% of the analyzed regenerants were transformants. The resulting marker-free plants displayed a considerably increased resistance to microbial phytopathogens—the bacterium Erwinia carotovora and fungus Sclerotinia sclerotiorum. Thus, the gene cecP1 can be concurrently used as a target gene and a screening marker. The utility of cecP1 as a selective gene for direct selection of transformed plants is discussed.     

Co-transformation with one Agrobacterium tumefaciens strain containing two binary plasmids as a method for producing marker-free transgenic plants

Co-transformation was investigated as a method that would allow the use of a selectable marker during plant regeneration followed by recovery of progeny which contain the desired gene(s) but lack a marker gene. Rapeseed (Brassica napus cv `212/86') and tobacco (Nicotiana tabacum cv `Xanthi NC') were co-cultivated with a single Agrobacterium tumefaciens strain containing two binary plasmids. Genes from both plasmids were expressed in approximately 50% of the primary transformants. Progeny expressing only one of the transgenes were observed in about 50% of the co-transformed lines, indicating that the genes were inserted at different loci. This single-strain co-transformation method allowed the use of a selectable marker during plant regeneration and subsequent recovery of marker-free progeny. Received: 23 December 1996 / Revision received: 23 September 1997 / Accepted: 11 October 1997     

Selectable marker-free transgenic orange plants recovered under non-selective conditions and through PCR analysis of all regenerants

Selectable marker (SM) genes have been considered necessary to achieve acceptable rates in the generation of transgenic plants. Genes encoding antibiotic or herbicide resistance are widely used for this purpose. In most cases, once transgenic plants have been regenerated, permanence of SM genes in the plant genome is no longer necessary, and it becomes a matter of public concern. Moreover, the removal of SM genes from transgenic plants could facilitate gene stacking through successive transformations, particularly when the availability of these markers is rather limited for most crop plants. In the genus Citrus, with highly heterozygotic species of long generation cycles, methods implying the segregation and removal of marker transgenes in the progeny are not feasible. Here, we have evaluated the direct production of SM-free citrus plants under non-selective conditions, using a “clean” binary vector carrying only the transgene of interest, and through the recovery of transformants by polymerase chain reaction (PCR) analysis of all regenerated shoots. The response of two different citrus genotypes, Carrizo citrange (intergeneric hybrid of C. sinensis L. Osb. X Poncirus trifoliata L. Raf.) and Pineapple sweet orange (C. sinensis L. Osb.), was evaluated. Our results indicate that, in this system, the competence between transgenic and non-transgenic cells is the main factor determining final transgenic regeneration frequencies. For Carrizo citrange, no transgenic plant could be recovered. For Pineapple sweet orange, marker-free transformation efficiency was 1.7%, paving the way for the viable production of orange transformants carrying only the transgene(s) of interest.     

An efficient method for the production of marker-free transgenic plants of peanut (Arachis hypogaea L.)

Recombinant genes conferring resistance to antibiotics or herbicides are widely used as selectable markers in plant transformation for selecting the primary transgenic events. However, these become redundant once the transgenic plants have been developed and identified. Although, there is no evidence that the selectable marker genes are unsafe for consumers and the environment, it would be desirable if the marker genes can be eliminated from the final transgenic events. The availability of efficient transformation methods can enable the possibility of developing transgenic events that are devoid of the marker gene/s upfront. Taking advantage of the high and consistent transformation potential of peanut, we report a technique for developing its transgenics without the use of any selectable marker gene. Marker-free binary vectors harboring either the phytoene synthase gene from maize (Zmpsy1) or the chitinase gene from rice (Rchit) were constructed and used for Agrobacterium tumefaciens-mediated transformation of peanut. The putative transgenic events growing in vitro were initially identified by PCR and further confirmed for gene integration and expression by dot blots assays, Southern blots, and RT-PCR where they showed a transformation frequency of over 75%. This system is simple, efficient, rapid, and does not require the complex segregation steps and analysis for selection of the transgenic events. This approach for generation of marker-free transgenic plants minimizes the risk of introducing unwanted genetic changes, allows stacking of multiple genes and can be applicable to other plant species that have high shoot regeneration efficiencies.