pMF-vectors for marker-free technology
pMF is a new optimized plant transformation vector produced by Plant Research International, Wageningen for generation of genetically modified plants which are free from undesired DNA-sequences.
|
GM stawbberry shoots after transformation with pRCNG (model vector, Schaart et al. 2004).
Left: GM shoot prior to Dex treatment; right: GM shoot after marker-excision. |
Use of the pMF vectors for plant transformation enables effective removal of undesired DNA sequences, like antibiotic resistance genes, from the obtained transgenic plants or plant tissues. Removal of selectable marker genes may be desirable for the production of genetically modified crops with increased consumer acceptance. In addition, marker removal enables stacking of transgenes by recurrent transformations
Principle
The pMF vector provides an inducible site-specific recombination system for removal of undesired DNA sequences. A negative selection step using the cod A (cytosine deaminase) gene, ensures the ultimate production of completely marker-free plants (ref. Schaart et al. (2004) Plant Biotechnology J. 2: 223-240). Cod A is a conditionally lethal dominant gene encoding an enzyme that converts non-toxic 5-fluorocytosine (5-FC) to cytotoxic 5-fluorouracil (5-FU).
|
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
|
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.
Patents
A patent application for our marker-free system is pending (US2004185567/EP1264891/WO02097102). Granted in NZ.
The Recombinase-LBD part falls under patent EP0632054, lapsed in SE, PT, DK, MC, LU, GR and NL.