Marker-free PLRV resistant potato mediated by Cre-loxP excision and RNAi.

An inverted repeat construct corresponding to a segment of the potato leaf roll virus coat protein gene was created under control of a constitutive promoter and transferred into a transformation vector with a heat inducible Cre-loxP system to excise the nptII antibiotic resistance marker gene. Fifty-eight transgenic events were evaluated for resistance to PLRV by greenhouse inoculations, which lead to the identification of 7 highly resistant events, of which 4 were extremely resistant. This resistance was also highly effective against accumulation in subsequent tuber generations from inoculated plants, which has not been reported before. Northern blot analysis showed correlation of PLRV specific siRNA accumulation with the level of PLRV resistance. Heat mediated excision of the nptII antibiotic resistance gene in PLRV resistant events was highly efficient in one event with full excision in 71 % of treated explants. On the other hand 8 out of 10 analyzed events showed truncated T-DNA insertions lacking one of the two loxP sites as determined by PCR and confirmed by sequencing flanking regions in 2 events, suggesting cryptic LB sites in the non-coding region between the nptII gene and the flanking loxP site. Accordingly, it is proposed to modify the Cre-loxP vector by reducing the 1 kb size of the region between nptII, loxP, and the LB.

Self-excision of the antibiotic resistance gene nptII using a heat inducible Cre-loxP system from transgenic potato.

Resistance to antibiotics mediated by selectable marker genes remains a powerful selection tool for transgenic event production. However, regulatory agencies and consumer concerns favor these to be eliminated from food crops. Several excision systems exist but none have been optimized or shown to be functional for clonally propagated crops. The excision of the nptII gene conferring resistance to kanamycin has been achieved here using a gene construct based on a heat-inducible cre gene producing a recombinase that eliminates cre and nptII genes flanked by two loxP sites. First-generation regenerants with the Cre-loxP system were obtained by selection on kanamycin media. Following a heat treatment, second generation regenerants were screened for excision by PCR using nptII, cre, and T-DNA borders primers. Excision efficiency appeared to be at 4.7% depending on the heat treatment. The footprint of the excision was shown by sequencing between T-DNA borders to correspond to a perfect recombination event. Selectable marker-free sprouts were also obtained from tubers of transgenic events when submitted to similar heat treatment at 4% frequency. Spontaneous excision was not observed out of 196 regenerants from untreated transgenic explants. Biosafety concerns are minimized because the expression of cre gene driven by the hsp70 promoter of Drosophila melanogaster was remarkably low even under heat activation and no functional loxP site were found in published Solanum sequence database. A new plant transformation vector pCIP54/55 was developed including a multiple cloning site and the self-excision system which should be a useful tool not only for marker genes in potato but for any gene or sequence removal in any plant.