Elaboration of a Reliable Strategy Based on Real-Time PCR To Characterize Genetically Modified Plantlets and To Evaluate the Efficiency of a Marker Gene Removal in Grape (Vitis spp.)

We have developed an effective strategy based on real-time PCR assay for the molecular characterization of genetically modified grape and to quantify the efficiency of a marker gene removal. This research has been implemented in Vitis vinifera cv. Brachetto plantlets where exogenes were inserted during cocultures of embryogenic calli with Agrobacterium tumefaciens carrying the chemically inducible site-specific cre/loxP pX6 vector where the expression of the cre recombinase is regulated by 17-beta-estradiol. The neomycin phosphotransferase gene (nptll) for the kanamycin resistance trait was inserted as part of the gene transfer protocol, and this exogene was employed as a case study for carrying out our research. The 9-cis-epoxycarotenoid dioxygenase (nced2) and chalcone isomerase (chi) genes coding for two enzymes, involved respectively in abscisic acid and flavonoid biosynthesis, proved to be valuable reference endogenes for real-time PCR assays. Two types of duplo-target plasmids were exploited for building the standard curves: in one type (p-nptll/nced2) the nptll sequence is linked to the nced2 sequence; in the other (p-nptll/chi) it is linked to the chi. These calibrators were intended to simulate an ideal genetically modified plant carrying a homozygous single-copy exogene insertion. The repeatability test confirmed the suitability of both plasmid calibrators. Foreign gene stability can be monitored during long-term plant preservation, and the method proved to be suitable for quantifying the efficiency of nptll gene removal induced by 17-beta-estradiol.