Strategy to produce marker-free transgenic plants
Author: Lalbahadur Singh


To produce transgenic plants, selection systems are used that lead to the selective growth of transformed cells. Genes encoding for resistance to specific antibiotics or herbicides have been found to be effective for selection and provide a means for rapidly identifying transformed cells, tissues, and regenerated shoots. Antibiotics and herbicides kill cells by a variety of mechanisms and resistance genes have been widely used in transgenic plant production.

However, because SMGs are integrated into the plant genome, there are concerns about widespread occurrence of transgenes in novel ecosystems ( e.g., antibiotic resistance in crops and their agro ecosystems). Horizontal gene transfer from plants to environmental or medically related bacteria, or from plant products consumed as food to intestinal microorganisms or human cells, are generally considered to be not likely, but the inherent risks have not been totally addressed, and therefore there remain both regulatory and public concerns in many places in the world. So therefore, Following are some of the strategy/ method for generation of marker free transgenic plants are;

Co-transformation

Co-transformation is a method for production of marker free transformants based on Agrobacterium- or biolistics mediated transformation in which a SMG and gene of interest are on separate constructs. Three approaches are used for co-transformation: introduction of two T-DNAs, in separate Agrobacterium strains or biolistics introduction of two plasmids in the same tissue; (ii) introduction of two T-DNAs carried by different replicons within the same Agrobacterium strain; and (iii) introduction of two T-DNAs located on the same replicon within an Agrobacterium. In all of these variants, SMGs can subsequently be removed from the plant genome during segregation and recombination that occurs during sexual reproduction by selecting on the transgene of interest and not the SMG in progeny.

Using plant DNA (P-DNA)

Recent studies have shown that plants have T-DNA border-like sequences in rice and Arabidopsis and these might be used in transformation. Because this so called plant DNA (P-DNA) lacks any open reading frames and contains a high A/T content, it is likely the footprint of ancient Agrobacterium-mediated natural transformation events via horizontal gene transfer. It has been demonstrated that plant-derived P-DNA fragments can be used to replace the universally employed Agrobacterium T-DNA for transformation. In addition, co transformation of the inserted desired transgene into P-DNA and SMG-containing T-DNA is capable of producing marker-free and backbone-free transgenic plants.

Negative selection

This strategy is based on the incorporation of a negative selection step. The use of a negative SMG next to a positive SMG in the same construct is a powerful method to create marker gene-free transgenic plants. In this method, transformed offspring are selected for the absence of negative SMG under the selection pressure of a negative marker gene and the presence of the desired transgene. This negative selection method allows researchers to decrease their search for selectable marker-free transgenic plants without having to resort to copious molecular analyses, i.e., thousands of PCR analysis.

Site-specific recombination-mediated marker deletion

Recombination is a universal phenomenon that can occur at any place between two homologous DNA molecules. There are three well-described site-specific recombination systems that might be useful for the production of marker-free transgenic plants: Cre/loxP system from bacteriophage P1, where the Cre enzyme recognizes its specific target sites, FLP/ FRT recombination system from Saccharomyces cerevisiae, where the FLP recombinase acts on the FRT sites and R/RS recombination system from Zygosaccharomyces rouxii, where R and RS are the recombinase and recombination site, respectively.

In these systems, elimination of SMG would require recombinase expression in transgenic plants. Alternatively, a transgenic plant of interest can be crossed with a plant that expresses a recombinase gene. After segregation, marker-free transgenic progeny plants can be identified.

Transposon-based marker elimination

Use of transposable elements for marker gene removal involves several steps: (i) insertion of the marker gene onto a transposon, a segment of DNA that hops around in the plant’s genome; (ii) co-transformation with gene of interest; and (iii) segregation of the marker gene.

Intrachromosomal recombination system

As above, recombination sites are engineered into the plant, but no recombinase is expressed. The attachment site from phage origin is denoted POP’ (P for phage) or attP, and the attachment site from bacterial origin is denoted BOB’ (B for bacteria) or attB. Intrachromosomal recombination in plants is obtained by insertion of SMG between two direct repeats of attP that

facilitates spontaneous excision. Base composition of the attP site sequence is A + T rich, which is conjectured to play a recombination-stimulating role.

Removal of chloroplast marker genes

Mitochondria and chloroplasts have independent genomes in plants that have been the target (especially chloroplasts) of genetic transformation. Chloroplast transformation vectors are designed with homologous flanking sequences on either side of the transgene. In addition, chloroplast engineering overcomes the challenges of gene silencing, position effects, and multi-step engineering of multiple genes, which are current limitations of nuclear transformation. Homologous recombination (the use of identical sequences for example in promoters and terminators between genes) and site-specific recombination (for example Cre/lox recombination- based systems) or transient expression of recombinase are all potentially suitable for producing marker- free engineered chloroplast of plants.

Use of markers not based on antibiotic or herbicide resistance

Recently, an Escherichia coli-derived phosphomannose isomerase (PMI) was used to convert mannose-6-phosphate to fructose-6-phosphate for positive selectable marker in plant transformation. Only transformed cells are capable of utilizing mannose as a carbon source. Another marker, xylose isomerase (xylA) gene of Streptomyces rubiginosus can be used as the selectable marker and xylose as the selective agent. The enzyme from S. rubiginosus catalyses the isomerization of D-xylose to D-xylulose. The non-transformed plant cells cannot utilize the D-xylose as a sole carbon source, but xylA transformed cells with are capable of growing on xylose.

References

1. Darbani, B., Eimanifar, A., Stewart, C. N., Jr. Camargo, W. N. (2007). Methods to produce marker-free transgenic plants. Biotechnol. J., 2, 83-90.

About Author / Additional Info:
Ph.D. scholar, biotechnology, Indian agricultural research institute (IARI), new delhi. Currently I am working in the area of miRNAs in pulse crop.