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Four main methods have been suggested to create marker gene-free transformed plants. The methods are: 1. Co-Transformation 2. Site-Specific Recombination 3. Marker Gene Elimination by Transposase 4. Marker Gene Elimination by Intrachromosomal Recombination.
Method # 1. Co-Transformation:
Co-transformation strategies are popularly employed for the production of transformed plants free from marker gene(s). In this novel approach, gene of interest and marker genes are placed on two separate T-DNA in single plasmids. Both marker gene and transgene are inserted at loci, which should recombine at reasonably high frequencies.
Therefore, the gene of interest can be segregated from the selectable marker gene in the next generation (Fig. 14.22). Using this approach marker from plants was developed in rice, tobacco and rape seed plants. The frequency in which marker gene and transgene segregate is determined by the location of the transgene and marker gene in the plant genome.
Method # 2. Site-Specific Recombination:
In the second approach, marker genes are flanked with direct repeats of recognition sites for a site-specific recombinase. The enzyme recombinase excise, marker gene from plant genome by enzyme mediated site-specific recombination.
The bacteriophage p1 cre/lox recombination system is one which consist to 38 Ka product of the cregene in codes are recombination and to asymmetric 34 bp lox sites, composed of two 13 bp invert repeats will 8 bp core regions.
This core region is asymmetric and provides direction to the site. The cre/lox system has been used to direct site-specific integration of incoming plasmids at the lox sites previously placed in to genome by direct gene transfer or Agrobacterium mediated transfer.
Cre-lox system facilitates accurate insertion of single copy DNA into genomic targets (Fig. 14.23). The cloned marker gene between two lox site is eliminated after transformation. This particular approach was utilised in the production of marker free transgenic tobacco and Arabidopsis.
Method # 3. Marker Gene Elimination by Transposase:
Separation of marker gene from target gene can be accomplished by employing transposable elements. This is generally achievable in two ways. In one of the ways, the marker gene is inserted on mobile element, which is lost after transposition.
For example, marker-free transgenic tobacco have been produced by inserting the selectable ipt gene into transposable element AC, while in another approach make the transgene by itself mobile and the activation of transposase allows the identification of the target gene to a new chromosomal position.
The genetic crosses and segregation will dissociate to target gene and marker gene. Marker free tobacco plants have now been successfully produced by this approach (Fig. 14.24).
Method # 4. Marker Gene Elimination by Intrachromosomal Recombination:
Another approach to eliminate marker gene, a new strategy was developed in which DNA deletion based on intrachromosomal homologous recombination (ICR) between two homologous sequences. For example, marker gene npt II of negative selectable marker tms 2 gene was flanked by two 352 bp attachment p (attp) regions of phage λ in a binary vector (pattp- ICR).
This construct was introduced into tobacco and resistant calli were selected with kanamycin. Out of 11 calli, 2 produced in a mixture of green and white shoots, after subjected for shoot formation. Then results show that there is a loss of npt II gene.
