Bacterial dihydrodipicolinate synthase and desensitized aspartate kinase: Two novel selectable markers for plant transformation. Guerineau, F. Sulfonamide resistance gene for plant transformation. Haughn, G. Transformation with a mutant Arabidopsis acetolactate synthase gene renders tobacco resistant to sulfonylurea herbicides.
Goddijn, O. A chimaeric tryptophan decarboxylase gene as a novel selectable marker in plant cells. Fuchs, R. Purification and characterization of microbially expressed neomycin phospho-transferase II NTPII protein and its equivalence to the plant expressed protein. Nap, J. Biosafety of kanamycin resistant plants: An overview. Transgenic Research 1 : — Redenbaugh, K. Safety assessment of genetically engineered fruits and vegetables: A case of the Flavr Savr tomato. Dale, E.
Gene transfer and subsequent removal of the selection gene from the host genome. USA 88 : — Transposition mediated repositioning and subsequent elimination of marker genes from transgenic tomato. Matzke, M. Reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants. Jorgensen, R. Silencing of plant genes by homologous transgenes.
AgBiotech News and Infor. Tanksley, S. Regulatory considerations: Genetically engineered plants: a summary of a workshop held at the Boyce Thompson Institute for Plant Research.
San Francisco: Center for Science Information. Depicker, A. Frequencies of simultaneous transformation with different T-DNAs and their relevance to the Agrobaclerium plant cell interaction. Two T-DNAs co-transformed into Brassica napus by a double Agrobacterium infection are mainly integrated at the same locus. McKnight, T. Segregation of genes transferred to one plant cell from two separate Agrobacterium strains. Cregg, J. Use of site-specific recombination to regenerate selectable markers.
Golic, K. The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome. Cell 59 : — O'Gorman, S. Recombinase-mediated gene activation and site-specific integration in mammalian cells. Lyznik, L. Activity of yeast FLP recombinase in maize and rice protoplasts. Acids Res. Intra- and intermolecular site-specific recombination in plant cells mediated by bacteriophage P1 recombinase. Gene 91 : 79— Odell, J. Site-directed recombination in the genome of transgenic tobacco.
Onouchi, H. Operation of an efficient site-specific recombination system of Zygosaccharomyces rouxii in tobacco cells. Maeser, S. The Gin recombinase of phage Mu can catalyze site-specific recombination in plant protoplasts. Russell, S. Directed excision of a transgene from the plant genome. Baker, B. USA 83 : — Yoder, J. Ac transposition in transgenic tomato plants.
Fedoroff, N. Maize transposable elements, p. In : Mobile DNA. Berg, D. Amercian Society for Microbiology, Washington D. Greenblatt, I. A chromosome replication pattern deduced from pericarp phenotypes resulting from movements of the transposable element, Modulator , in maize. Genetics : — Preferential transposition of the maize element Activator to linked chromosomal locations in tobacco. Plant Cell 2 : — Belzile, F. Consequently, the elongation of the polypeptide chain is inhibited and protein synthesis interrupted, causing the same symptoms described for the other aminoglycoside antibiotics Benveniste and Davies, The hpt gene has been extensively utilized, especially when the use the neo gene is not possible.
This is the case of several monocotyledonous species that show high levels of natural resistance to kanamycin Wilmink and Dons, ; Vasil, Hygromycin B is usually more toxic than kanamycin and kills sensitive cells more quickly. In this system, chloroplasts or plastids are transformed with genes for herbicide resistance as in the selection marker process because most target proteins for herbicides are compartmentalized within this organelle.
Chloroplast transformation can be accomplished by both the biolistic and polyethylene glycol-mediated strategies Daniell et al. Vectors that specifically address the integration of the herbicide-resistant marker gene in the chloroplasmatic genome such as the "tobacco vector " or the "universal chloroplast vector" should be preferentially used Daniell et al.
These vectors possess flanking sequences from highly conserved chloroplast genes to allow integration by homologous recombination. The advantages that chloroplast expression systems may give over routinely nuclear expression are Bogorad, :. The chloroplast genome expresses maternal inheritance in most crop plants;. In addition, other sub-cellular organelles such as mitochondria might also provide more favorable environments than the nuclear-cytoplasmic compartment for certain biochemical reactions or for high, predictable, uniform and stable transgene expression, not subject to gene silencing Bogorad, Antibiotic-resistant marker genes could be also used in this system, facilitating the extension of plastid transformation to non-green plastids such as in embryogenic cells of cereal crops Kavanagh et al.
Antibiotic resistance genes ARGs have been introduced into transgenic plant genomes. The ARGs are used under control of prokaryotic promoters to select bacteria in vector production for direct plant transformation.
In some cases, these prokaryotic ARGs are introduced in the vector along with the gene of interest. In addition, ARGs under the control of eukaryotic promoters are widely used as selection marker genes. Due to biosafety concerns, there have been complex evaluations to study the potential impacts of ARGs present in transgenic plants on human health and the environment.
Although there is no evidence of deleterious effects in the use of transgenic plants carrying ARGs, its removal is already stated as "good laboratory practice" by several regulatory committees U.
Food And Drug Administration, Thus it is recommended that those who are developing genetically modified foods for the market should be encouraged to phase out ARGs use Puchta, ; Ow, This recommendation could also be applied to the other selection marker genes, since they are not necessary once the transgenic plant is obtained.
Consequently, several strategies to remove the selection marker genes have been developed. Co-transformation: In this system, the transformation is achieved using two separate plasmid vectors: one containing the gene of interest and other the selective marker gene. In this way, the selective marker gene can be further eliminated by progeny segregation. Thus, the co-transformation system allows the use of a selective agent during plant regeneration and subsequent recovery of marker-free progeny, which contains only the gene of interest.
Other reports stated that, under certain conditions depending on the transformation vector, transformation methodology, strains of Agrobacterium , plant species etc. MAT multi-auto-transformation system: The ipt gene that codes for the enzyme isopentenyl phosphotransferase EC 2. The MAT system is primarily based on the visual selection of transgenic plants containing the ipt gene Ebinuma et al. In the presence of this gene, the transformed plant loses the apical dominance and ability to root.
The acquired abnormal phenotype was called extreme shooty phenotype ESP and is easy to detect visually. In a second step, the unsuitable ipt gene is removed from the transgenic plant through the transposition of the Ac transposable element from maize, which is transferred along with the selection marker gene. In this way, marker-free transgenic plants could be generated with the normal phenotype restored and containing only the gene of interest. Cytokinins stimulate organogenesis in several in vitro cultivated plants and are widely used to regenerate plants after the transformation event.
In the transposition process, only a few excised transposable Ac elements containing the marker gene disappear from transgenic cells because the Ac elements do not reinsert or because they reinsert into a sister chromatid that is further lost by somatic segregation. Therefore, the frequency of the Ac element elimination to recover marker-free transgenic plants is low, ranging from 0.
When the expression of the ipt gene is controlled by a dexamethasone-inducible system, the co-introduction of multiple genes, in addition to ipt , is more efficient and the recovery of marker-free transgenic plants is high Kunkel et al.
The MAT system is particularly valuable for plants with long generation cycles such as fruit and forest trees, providing a promising way to shorten breeding time. Once the transgenic plant has been obtained, the Ds element and the marker gene will be transferred to a new locus of the plant genome or eliminated when in the presence of the transposase. The gene of interest will be left in the first insertion locus Goldsbrough et al.
Transposase can be introduced in this plant as an additional element in the transformation vector by a second transformation or by sexual crossing. The advantage of this system is that the selection marker gene will be lost in some somatic tissues due to failure of the Ds element reintegration.
This makes the strategy suitable for removal of marker genes in vegetatively propagated plants Yoder and Goldsbrough, ; Pavingerova et al. In a similar way, systems to remove selection marker gene by intrachromosomal recombination between the bacteriophage l attachment attP regions Zubko et al.
Site-specific recombination system : This is a two-component system that requires an enzyme that acts in trans to catalyze the recombination between two short specific DNA sequences, which flank the selective marker gene to be eliminated.
This system has already been demonstrated to be efficient for yeast. However, in plants, the site-specific recombination rate is very low and the current knowledge of homologous recombination is still limited Mengiste and Paszkowski, ; Vergunst and Hooykaas, In this recombination system, the plant should be previously transformed with a selection marker gene cloned between two sequences of the lox gene, each with 34 bp repeats in direct orientation.
In a second stage, the Cre gene should be introduced in this plant by a second transformation, either by sexual crossing or by transient expression. Once the Cre gene is expressed, the Cre enzyme catalyses the recombination between the lox repeat sequences, thus eliminating the marker gene in the secondary transformants.
This recombination strategy can also be used to target the insertion of new genes in an already transformed plant containing the lox sites, or another known sequence, inserted at a "suitable" chromosomal position Gallego et al. Site-specific recombination can also be used to compare transgenic plant lines without the effect of the integration site position.
Acknowledgements: We would like to thank Dr. Manoel Teixeira Souza Jr. Abrir menu Brasil. Brazilian Journal of Plant Physiology. Abrir menu. Plant Physiol. W5 Norte, Markers genes for positive selection Some marker genes for positive selection enable the identification and selection of genetically modified cells without injury or death of the non-transformed cell population negative selection.
Marker genes for negative selection The first category of negative selection markers is the genes that express resistance to herbicides. Chloroplast transformation In this system, chloroplasts or plastids are transformed with genes for herbicide resistance as in the selection marker process because most target proteins for herbicides are compartmentalized within this organelle.
The advantages that chloroplast expression systems may give over routinely nuclear expression are Bogorad, : - The retention of the transgene flow especially marker genes in microspores is reduced. The chloroplast genome expresses maternal inheritance in most crop plants; - The expression level of the transgene is extremely high compared with the expression of the same gene integrated in the nuclear genome, due to the large copy number of chloroplast genomes 5, to 10, per cell; - The insertion in the genome is driven site-specific , which avoids position effects and facilitates comparative studies; - The quasi absence of gene silence in the inserted transgenes Daniell et al.
Strategies to recover marker-free transgenic plants Antibiotic resistance genes ARGs have been introduced into transgenic plant genomes. Merril] plants at high frequency. Benveniste R, Davies J Mechanisms of antibiotic resistance in bacteria. Nature Bogorad L Engineering chloroplasts: an alternative site for foreign genes, proteins, reactions and products. Trends Biotech. Bowen BA Markers for plant gene transfer. For example, you could not test a cell in tissue culture for the expression of a trait that influences grain protein content.
What if you extracted the DNA from each cell and searched for the gene? You would have to separate each cell, allow the cell to multiply into a pile of callus, and sample a portion of each callus pile and extract DNA. Then tests would have to be perforemed to identify the presence of the gene. This would be just as cumbersome as trying to grow each cell into a plant. Selecting out transgenic cells is done by co-transforming the cells with the transgene plus an additional gene called a selectable marker gene.
Selectable marker genes encode a trait that is easily detectable, even at the cellular level. The two most commonly used selectable marker genes encode the traits of herbicide and antibiotic resistance. To select out the transgenic cells, all of the cells are grown on media containing the herbicide or antibiotic.
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