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.
Euscepes postfasciatus is one of the most important sweetpotato pests in the South Pacific, Caribbean basin, and some countries of Central and South America. Development of host resistance will greatly improve the effects of integrated pest management (IPM) for this pest. Ten transgenic clones of `Jewel' sweetpotato with cowpea trypsin inhibitors and snowdrop lectin, developed by Axis Agri. Genetics, Ltd., were assayed for weevil resistance using a no-choice bioassay. A replicated experiment was conducted in the screenhouse. Five storage roots from each clone were infested with five pairs of adults. Non-transformed `Jewel' was used as a check. Resistance was assessed 60 days after infestation by estimating the percentage of internal damage and the weevil population in the storage roots. A five-grade damage index was recorded. The experiment was repeated twice. Significant enhancement of resistance was found in the transgenic clones. Clone CTI-13 with cowpea trypsin inhibitor and clone PCG-7 with both cowpea trypsin inhibitor and snowdrop lectin demonstrated moderate resistance to E. postfasciatus, whereas the non-transformed `Jewel' was susceptible. This result shows that resistance to Euscepes postfasciatus can be achieved through genetic transformation.
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