Marker Gene Controversy in Transgenic Plants

Biosafety implications of selectable marker genes that are integrated into the transgenic plants are discussed. In the laboratory, selectable marker genes are used at two stages to distinguish transformed cells out of a large population of nontransformed cells: 1) initial assembly of gene cassettes is generally done in E. coli on easily manipulatable plasmid vectors that contain the selectable marker genes which often code for antibiotic inactivating enzymes, and 2) Then the gene cassettes are inserted into the plant genome by various transformation methods. For selection of transformed plant cells, antibiotic and herbicide resistance genes are widely used. Consequently, transgenic plants can end up with DNA sequences of selectable markers that are functional in E. coli and plants. The potential for horizontal gene transfer of selectable markers from transgenic plants to other organisms both in the environment and in the intestine of humans and animals is evaluated. Mechanisms and consequences of the transfer of marker genes from plants to other organisms is examined. Strategies to avoid marker genes in plants are discussed. It is possible to avoid the use of controversial selectable markers in the construction of transgenic plants.     

Introduction and expression of the octopine T-DNA oncogenes in tobacco plants and their progeny

The tumour-inducing T-DNA genes 1, 2 and 4 of the octopine Ti-plasmid pTiAch5 were cloned and introduced into tobacco cells by cocultivation or leaf disk transformation using pTi derived vectors. When a selectable marker was needed, we used a aminoglycoside phosphotransferase II (nos-APH(3′)II) chimeric gene conferring kanamycin resistance to plant cells. The expression of gene 4 in transformed tissue cultures precluded the regeneration of normal transformed plants. Normal transformed plants were obtained with the construction carrying genes 1 or 2. We report in vivo complementation of genes 1 and 2 after crosses of transformed plants. Strategies are described for the use of genes 1 and 2 as selection or screening markers in plant cells or regenerated plants.     

Genetic transformation in higher plants

Successful transformation of plant cells has been obtained utilizing vectors and DNA delivery methods derived from the plant pathogen, Agrobacterium tumefaciens. This soil bacterium is capable of transferring a DNA segment (T‐DNA), located between specific nucleotide border sequences, from its large tumor inducing (Ti) plasmid into the nuclear DNA of infected plant cells. The exploitation of the Agrobacterium/Ti plasmid system for plant cell transformation has been facilitated by (1) the construction of modified Agrobacterium strains in which the genes responsible for pathogenicity have been deleted; (2) the design of intermediate vectors containing selectable drug markers for introducing foreign genes into the Ti plasmid and subsequently into plant cells; and (3) the development of efficient in vitro methods for transforming plant cells and tissues with engineered Agrobacterium strains. These modifications have led to the development of a simple, efficient, and reproducible transformation system from which morphologically normal transformed plants can be readily regenerated. The foreign genes are stably maintained and expressed in the resulting plants and are inherited by progeny as typical Mendelian traits. The availability of transformation systems has already facilitated numerous studies on gene expression and regulation in plants and should eventually allow for the modification of various crop species in an agronomically significant manner. The needs and possibilities for the development of alternate vectors and transformation procedures will be discussed.     

Improved method for the transformation of Arabidopsis thaliana with chimeric dihydrofolate reductase constructs which confer methotrexate resistance

Summary A modified root explant transformation method has been developed that is effective in producing transgenic Arabidopsis thaliana plants which are methotrexate resistant due to the integration of T-DNA vectors containing a chimeric dihydrofolate reductase gene. Molecular analysis shows that transformed methotrexate resistant plants contain the expected T-DNA construct with the chimeric gene. This transformation method also works well with other plant selectable markers, including hygromycin phosphotransferase and neomycin phosphotransferase II.Abbreviations DHFR (dihydrofolate reductase) - HPT (hygromycin phosphotransferase) - NPTII (neomycin phosphotransferase) - CaMV (Cauliflower Mosaic Virus) - MES (2-[N-morpholino]ethanesulfonic acid) - BAP (N6-benzylaminopurine) - NAA (naphthaleneacetic acid) - 2,4-D (2,4 dichlorophenoxyacetic acid) - 2iP (6-(dimethylallylamino)-purine) - 2iPAde (6-(dimethylallylamino)-ade-nine) - IAA (indole-3-acetic acid) - IBA (indole-3-butyric acid)     

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.     

Alternative selectable markers for potato transformation using minimal T-DNA vectors

Alternative selection systems for plant transformation are especially valuable in clonal crops, such as potato (Solanum tuberosum L.), to pyramid transgenes into the same cultivar by successive transformation events. We have modified the pGPTV series of binary vectors to construct pMOA1 to pMOA5, resulting in a series of essentially identical binary vectors except for the presence of different selectable marker genes. These selectable marker genes are tightly inserted between the left and right T-DNA borders and confer resistance to kanamycin (nptII), hygromycin (hpt), methotrexate (dhfr), phosphinothricin (bar), or phleomycin (ble). The T-DNA of all the vectors is based on the minimal features necessary for plant transformation, with no extraneous DNA segments that may be unacceptable to regulatory authorities for general release of transgenic plants. A series of unique restriction sites exists between the right border and each selectable marker gene for subsequent insertion of useful genes. We have also developed improved culture procedures for potato transformation and used the pMOA1 to pMOA5 binary vectors to define stringent selection conditions for each marker gene. Combining these advances improved the frequency of recovering transformed potato plants while maintaining a low frequency of escapes. The relative efficiency of recovering transgenic potato lines with each selectable marker gene can be summarised as: kanamycin resistance>hygromycin resistance>phosphinothricin resistance>phleomycin resistance>methotrexate resistance.     

Expression of foreign genes in regenerated plants and in their progeny

Chimeric genes comprised of the nopaline synthase promoter and bacterial coding sequences specifying resistance to kanamycin, chloramphenicol or methotrexate, were inserted into the non-oncogenic Ti plasmid vector pGV3850 by recombination (through homologous pBR322 sequences present in the chimeric gene constructs and pGV3850). These co-integrates in Agrobacterium were used to infect single plant protoplasts of Nicotiana by co-cultivation. The resistance traits allowed the selection of transformed calli in tissue culture in the presence of the appropriate antibiotic. Furthermore, as a non-oncogenic Ti plasmid was used for the protoplast transformation, phenotypically normal and fertile plants could be regenerated from the resistant calli. We have shown that these fully differentiated plant tissues exhibit functional expression of resistance traits (Km^R and Cm^R). All plants carrying the chimeric genes developed normally, flowered, and set seeds. The inheritance of several of these resistance traits was analyzed and shown to be Mendelian. These results are model experiments to demonstrate that genes of interest can be systematically transferred to the genome of plants using non-oncogenic Ti plasmid derivatives; and that transformed plants are capable of normal growth and differentiation, thus providing a natural environment for the study of gene expression and development of plant cells.     

Advances in selectable marker genes for plant transformation

Plant transformation systems for creating transgenics require separate process for introducing cloned DNA into living plant cells. Identification or selection of those cells that have integrated DNA into appropriate plant genome is a vital step to regenerate fully developed plants from the transformed cells. Selectable marker genes are pivotal for the development of plant transformation technologies because marker genes allow researchers to identify or isolate the cells that are expressing the cloned DNA, to monitor and select the transformed progeny. As only a very small portion of cells are transformed in most experiments, the chances of recovering transgenic lines without selection are usually low. Since the selectable marker gene is expected to function in a range of cell types it is usually constructed as a chimeric gene using regulatory sequences that ensure constitutive expression throughout the plant. Advent of recombinant DNA technology and progress in plant molecular biology had led to a desire to introduce several genes into single transgenic plant line, necessitating the development of various types of selectable markers. This review article describes the developments made in the recent past on plant transformation systems using different selection methods adding a note on their importance as marker genes in transgenic crop plants.     

Chimeric genes as dominant selectable markers in plant cells

Opine synthases are enzymes produced in dicotyledonous plants as the result of a natural gene transfer phenomenon. Agrobacteria contain Ti plasmids that direct the transfer, stable integration and expression of a number of genes in plants, including the genes coding for octopine or nopaline synthase. This fact was used as the basis for the construction of a number of chimeric genes combining the 5' upstream promoter sequences and most of the untranslated leader sequence of the nopaline synthase (nos) gene with the coding sequence of two bacterial genes: the aminoglycoside phosphotransferase (APH(3')II) gene of Tn5 and the methotrexate-insensitive dihydrofolate reductase (DHFR Mtx^R) of the R67 plasmid. The APH(3')II enzyme inactivates a number of aminoglycoside antibiotics such as kanamycin, neomycin and G418. Kanamycin, G418 and methotrexate are very toxic to plants. The chimeric NOS-APH(3')II gene, when transferred to tobacco cells using the Ti plasmid as a gene vector, was expressed and conferred resistance to kanamycin to the plant cells. Kanamycin-resistant tobacco cells were shown to contain a typical APH(3')II phosphorylase activity. This chimeric gene can be used as a potent dominant selectable marker in plants. Similar results were also obtained with a NOS-DHFR Mtx^R gene. Our results demonstrate that foreign genes are not only transferred but are also functionally expressed when the appropriate constructions are made using promoters known to be active in plant cells.     

A highly efficient sulfadiazine selection system for the generation of transgenic plants and algae

The genetic transformation of plant cells is critically dependent on the availability of efficient selectable marker gene. Sulfonamides are herbicides that, by inhibiting the folic acid biosynthetic pathway, suppress the growth of untransformed cells. Sulfonamide resistance genes that were previously developed as selectable markers for plant transformation were based on the assumption that, in plants, the folic acid biosynthetic pathway resides in the chloroplast compartment. Consequently, the Sul resistance protein, a herbicide‐insensitive dihydropteroate synthase, was targeted to the chloroplast. Although these vectors produce transgenic plants, the transformation efficiencies are low compared to other markers. Here, we show that this inefficiency is due to the erroneous assumption that the folic acid pathway is located in chloroplasts. When the RbcS transit peptide was replaced by a transit peptide for protein import into mitochondria, the compartment where folic acid biosynthesis takes place in yeast, much higher resistance to sulfonamide and much higher transformation efficiencies are obtained, suggesting that current sul vectors are likely to function due to low‐level mistargeting of the resistance protein to mitochondria. We constructed a series of optimized transformation vectors and demonstrate that they produce transgenic events at very high frequency in both the seed plant tobacco and the green alga Chlamydomonas reinhardtii. Co‐transformation experiments in tobacco revealed that sul is even superior to nptII, the currently most efficient selectable marker gene, and thus provides an attractive marker for the high‐throughput genetic transformation of plants and algae.     

Utilization of the plant methionine sulfoxide reductase B genes as selectable markers in Arabidopsis and tomato transformation

Plant transformation is an important tool for basic research and agricultural biotechnology. In most cases, selection of putative transformants is based on antibiotic or herbicide resistance. Overexpression of plant genes that provide protection from abiotic or biotic stresses can result in a conferred phenotype that can be used as a means for selection. We have demonstrated herein that specific methionine sulfoxide reductase B (MsrB) genes that are overexpressed in transgenic plants may constitute a new selectable marker with concomitantly increased tolerance to methyl viologen (MV) treatment. Arabidopsis transformants overexpressing cytosolic MsrB7, MsrB8 or MsrB9 are viable and survive after MV selection. To establish whether these native plant origin genes serve as new non-antibiotic markers that can be applied to crop transformation, tomato cotyledons were used as transformation materials. MsrB7 transgenic tomato plants were successfully obtained by Agrobacterium-mediated transformation and selection on medium supplemented with MV. We suggest that specific MsrB genes that are overexpressed in transgenic plants may constitute a new selectable marker with increased tolerance to oxidative stress concomitant with MV treatment.     

An efficient and novel plant selectable marker based on organomercurial resistance

A selectable marker gene facilitates the detection of genetically modified plant cells during transformation experiments. So far, these marker genes are almost exclusively of two types, conferring either antibiotic resistance or herbicide tolerance. However, more selectable markers must be developed as additional transgenic traits continue to be incorporated into transgenic plants. Here, we used mercury resistance, conferred by the organomercurial lyase gene, as a selectable marker for transformation. The merB gene fromStreptococcus aureus was modified for plant expression and transferred to a hybrid poplar(Populus alba xPopulus glandulosa), using the stem segment-agrobacteria co-cultivation method. The transformed cells were selected on a callus-inducing medium containing as little as 1 μM methylmercury. Subsequent plant regeneration was done in the presence of methylmercury. Resistance to Hg was stably maintained in mature plants after two years of growth in the nursery. We suggest that this gene could serve as an excellent selectable marker for plant transformation.     

Transformation of Brassica napus and Brassica oleracea Using Agrobacterium tumefaciens and the Expression of the bar and neo Genes in the Transgenic Plants

An efficient and largely genotype-independent transformation method for Brassica napus and Brassica oleracea was established based on neo or bar as selectable marker genes. Hypocotyl explants of Brassica napus and Brassica oleracea cultivars were infected with Agrobacterium strains containing chimeric neo and bar genes. The use of AgNO₃ was a prerequisite for efficient shoot regeneration under selective conditions. Vitrification was avoided by decreasing the water potential of the medium, by decreasing the relative humidity in the tissue culture vessel, and by lowering the cytokinin concentration. In this way, rooted transformed shoots were obtained with a 30% efficiency in 9 to 12 weeks. Southern blottings and genetic analysis of S1-progeny showed that the transformants contained on average between one and three copies of the chimeric genes. A wide range of expression levels of the chimeric genes was observed among independent transformants. Up to 25% of the transformants showed no detectable phosphinotricin acetyltransferase or neomycin phosphotransferase II enzyme activities although Southern blottings demonstrated that these plants were indeed transformed.     

Chromosomal localization of foreign genes in Petunia hybrida

Summary A F1 hybrid of Petunia hybrida, heterozygous for at least one marker on each of the seven chromosomes, was transformed with a modified strain of Agrobacterium tumefaciens in which the phytohormone biosynthetic genes in the transferred DNA (T-DNA) were replaced with a NOS/NPTII/NOS chimeric gene and a wildtype nopaline synthase (NOS) gene. The chimeric gene, which confers kanamycin resistance, was used as selectable marker during the transformation process and the NOS gene was used as a scorable marker in the genetic studies. After plants had been regenerated from the transformed tissues, the transgenic plants that expressed both of these markers were backcrossed to the parental lines. The offspring were examined for the segregation of the NOS gene and the Petunia markers. Genetic mapping was thus accomplished in a single generation.By Southern hybridization analysis we confirmed the presence of the expected T-DNA fragments in the transformed plants. Four out of the six plants presented here, had just one monomeric T-DNA insertion. The sizes of the plant/T-DNA junction fragments suggest that the integration occurred in different sites of the Petunia genome. One transformant gave a more complicated hybridization pattern and possibly has two T-DNA inserts. Another transgenic plant was earlier reported (Fraley et al. 1985) to have two, possibly tandemly repeated T-DNAs.Data is presented on the genetic localization of the T-DNA inserts in six independently obtained transgenic plants. The T-DNA inserts in three plants were mapped to chromosome I. However, the distances between the NOS gene and the marker gene on this chromosome were significantly different. In another transgenic plant the NOS gene was coinherited with the marker on chromosome IV. Two other transgenic plants have the T-DNA insert on chromosome III. A three point cross enabled us to determine that both plants have the NOS gene distally located from the peroxidaseA (prxA) marker and both plants showed about 18% recombination. However, Southern hybridization analysis shows that the sizes of the plant/T-DNA junction fragments in these transgenic plants are different, thus suggesting that the integrations occurred in different sites.     

A set of novel Ti plasmid-derived vectors for the production of transgenic plants

Summary We describe in this paper the construction and use of a set of novel Ti plasmid-derived vectors that can be used to produce transgenic plants. These vectors are based on one of two strategies: 1) double recombination into the wild-type Ti plasmid of genetic information flanked by two T-DNA fragments on a wide-host range plasmid; 2) the binary vector strategy. The vector based on the double recombination principle contains a kanamycin resistance gene for use as a plant selectable marker, a polylinker for the insertion of foreign genes, and a nopaline synthase gene. The vector was constructed such that a disarmed T-DNA results from the double recombination event. The binary vector combines several advantageous features including an origin of replication that is stable in Agrobacterium in the absence of selection, six unique sites for insertion of foreign genes, an intact nopaline synthase gene, and a kanamycin resistance marker for selection of transformed plant cells. All of these vectors have been used to produce tobacco plants transformed with a variety of foreign genes.     

Genetic transformation of apple (<Emphasis Type="Italic">Malus</Emphasis> x <Emphasis Type="Italic">domestica</Emphasis>) without use of a selectable marker gene

Selectable marker genes are widely used for the efficient transformation of crop plants. In most cases, antibiotic or herbicide resistance marker genes are preferred because they tend to be most efficient. Due mainly to consumer and grower concerns, considerable effort is being put into developing strategies (site-specific recombination, homologous recombination, transposition, and cotransformation) to eliminate the marker gene from the nuclear or chloroplast genome after selection. For the commercialization of genetically transformed plants, use of a completely marker-free technology would be desirable, since there would be no involvement of antibiotic resistance genes or other marker genes with negative connotations for the public. With this goal in mind, a technique for apple transformation was developed without use of any selectable marker. Transformation of the apple genotype “M.26” with the constructs pPin2Att35SGUSintron and pPin2MpNPR1 was achieved. In different experiments, 22.0–25.4% of regenerants showed integration of the gene of interest. Southern analysis in some transformed lines confirmed the integration of one copy of the gene. Some of these transformed lines have been propagated and used to determine the uniformity of transformed tissues in the plantlets. The majority of the lines are uniformly transformed plants, although some lines are chimeric, as also occurs with the conventional transformation procedure using a selectable marker gene. A second genotype of apple, “Galaxy,” was also transformed with the same constructs, with a transformation efficiency of 13%.     

Agrobacterium tumefaciens-mediated transformation of bush monkey-flower (Mimulus aurantiacus Curtis) with a new reporter gene ZsGreen

A successful in vitro Agrobacterium-mediated transformation protocol was developed for Mimulus aurantiacus, a model species for ecological and evolutionary genetics and a promising ornamental plant. Three binary vectors were tested, each containing the hptII selectable marker gene and one of the reporter genes: gusA, EGFP or ZsGreen, all of them under CaMV 35S promoter. Genetic transformation was achieved through 4 days of co-cultivation of leaf, petiole and hypocotyl explants with Agrobacterium tumefaciens strain LBA 4404. Explants produced transformed callus tissue on solid modified Murashige and Skoog medium supplemented with 1 mg L^?1 6-benzylaminopurine, 0.5 mg L^?1 1-naphthaleneacetic acid, 30 g L^?1 sucrose and 20 or 50 mg L^?1 hygromycin B. All three reporter genes were expressed in callus tissue but the intensity of expression gradually decreased during further plant development. The new reporter gene ZsGreen proved suitable for plant transformation experiments since very intense and bright fluorescence was detected. Out of 1,760 co-cultured explants, 110 plants were regenerated and all of them were found to be PCR positive for the selection and/or reporter genes. Chemiluminescent Southern blot analysis revealed that 91 % of the regenerated plants (100 T₀ plants) contained T-DNA integrated in their genome. Transformation efficiency varied from 1.4 to 23.3 % for hypocotyl and petiole explants, respectively. Integration of some backbone sequences in plant genomes was confirmed in 75.3 % of T₀ plants. Using this protocol, stable transformants expressing selectable marker gene hptII and one of the reporter genes (gusA, ZsGreen or EGFP) were obtained in 4–5 months.     

A Modified MultiSite Gateway Cloning Strategy for Consolidation of Genes in Plants

The genome information is offering opportunities to manipulate genes, polygenic characters and multiple traits in plants. Although a number of approaches have been developed to manipulate traits in plants, technical hurdles make the process difficult. Gene cloning vectors that facilitate the fusion, overexpression or down regulation of genes in plant cells are being used with various degree of success. In this study, we modified gateway MultiSite cloning vectors and developed a hybrid cloning strategy which combines advantages of both traditional cloning and gateway recombination cloning. We developed Gateway entry (pGATE) vectors containing attL sites flanking multiple cloning sites and plant expression vector (pKM12GW) with specific recombination sites carrying different plant and bacterial selection markers. We constructed a plant expression vector carrying a reporter gene (GUS), two Bt cry genes in a predetermined pattern by a single round of LR recombination reaction after restriction endonuclease-mediated cloning of target genes into pGATE vectors. All the three transgenes were co-expressed in Arabidopsis as evidenced by gene expression, histochemical assay and insect bioassay. The pGATE vectors can be used as simple cloning vectors as there are rare restriction endonuclease sites inserted in the vector. The modified multisite vector system developed is ideal for stacking genes and pathway engineering in plants.     

Mercury-tolerant Transgenic Poplars Expressing Two Bacterial Mercury-metabolizing Genes

Mercury is one of the most toxic metals to various organisms, including humans. Genes involved in mercury metabolism have been cloned fromStaphylococcus aureus, and were modified here to be expressed in plants. Transgenic poplars containing both chimeric genes (p35S-merA andp35S-merB) were developed via two rounds of transformation usingnos-nptll andnos-hpt genes as selectable markers. Although expression levels varied among transgenic lines, tolerance to either ionic mercury or organic mercury matched well with the degree of expression revealed by northern hybridization. In culture, these trees were tolerant to 50 μM HgCl₂ and 2 μM CH₃HgCI. Variations in mercury tolerance among the transgenic lines indicates that vigorous selection is required to select the best clones for use in phytoremediation.     

Sulfonamide resistance gene for plant transformation

The sulfonamide resistance gene from plasmid R46 encodes for a mutated dihydropteroate synthase insensitive to inhibition by sulfonamides. Its coding sequence was fused to the pea ribulose bisphosphate carboxylase/oxygenase transit peptide sequence. Incubation of isolated chloroplasts with the fusion protein synthesised in vitro, showed that the bacterial enzyme was transported to the chloroplast stroma and processed into a mature form. Expression of the gene fusion in transgenic plants resulted in a high level of resistance to sulfonamides. Direct selection of transformed shoots on leaf explants was efficient using sulfonamides as sole selective agents. Transformed shoots rooted normally on sulfonamides at concentrations toxic for untransformed ones. Sulfonamide resistance was transmitted to the progeny of transformed plants as a single Mendelian dominant character. These results demonstrate that this chimeric gene can be used as an efficient and versatile selectable marker for plant transformation.