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Transformation using positive selection, e.g. on kanamycin (nptII)
Removal of marker by chemical induction of Recombinase R activity
Selection for marker-free plants using negative selection (cod A) on 5-FC
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Figure 1. Selection scheme for producing marker-free transgenic plants using pMF1. A similar selection scheme can be followed using pMF2 or pMF3 vectors, when positive selection on either hygromycin or phosphinothricin, respectively, is preferred.
The marker removal system (schemes in Figures 1 & 2) fits easily in existing Agrobacterium -mediated transformation protocols. After positive selection of transgenic tissue or plants, chemical induction of Recombinase R activity will result in elimination of DNA-sequences that are flanked by intact recombination sites (Rs). The Recombinase R protein is inactivated by the presence of the Ligand Binding Domain (LBD) of the glucocorticoid receptor. Activation of the Recombinase R protein activity is achieved by an overnight incubation of plant tissue in a 10 µM dexamethasone solution. Subsequent (negative) selection on 5-FC should prevent development of plant tissue still containing the cod A-gene, thereby preventing the occurrence of chimeras due to incomplete DNA excision.
Figure 2. Schematic representation depicting the T-DNA of pMF1 before and after Recombinase R-mediated removal of the DNA-sequences that are flanked by the recombination sites (Rs). Black arrows indicate primer locations for PCR-check for confirmation of the recombination event.