Molecular characterization of transgenic grapevine plants

In an attempt to obtain grapevine plants resistant to the Grapevine Fanleaf Virus (GFLV), the GFLV coat protein (CP) gene was inserted into the Vitis vinifera cultivars ‘Nebbiolo’ and a seedless table grape ‘7-3/2E1’. Embryogenic calli were obtained from immature anthers and ovaries. The binary vectors used for transformation carried the full-length GFLV CP gene with an introduced start codon (pGA-CP+). The protocol adopted for selection and regeneration of transgenic embryos relied on prolonged culture on kanamycin (100 mg L-1) containing media. Forty lines of putatively transformed ‘Nebbiolo’ grapes and 22 lines of ‘7 -3/2E1’ were obtained, each derived from single somatic embryos. The transgenic status of all lines was assessed by PCR and Southern blot analysis. The number of T-DNA copies inserted in the genome ranged from 1 to 4. Digestion of genomic DNA with different restriction enzymes (HindIII and EcoRI) showed that several lines shared the same hybridization patterns. The 40 ‘Nebbiolo’ lines derived from 6 independent transformation events, while the 22 ‘7-3/2E1’ lines were likely derived from 10 independent transformation events. Southern and PCR data indicated that two groups of lines probably contained incomplete copies of T-DNA. No evidence of methylation of the transgenes at cytosine residues was found by Southern analysis of Nebbiolo DNA digested with methylation-sensitive restriction enzymes. The RT-PCR and Northern analyses showed the presence of the specific mRNA in all lines except for those that did not contain an intact T-DNA copy. Expression of GFLV CP gene was detected by DAS-ELISA: some ‘Nebbiolo’ lines accumulated the coat protein at a low level while in others the protein was not detectable by ELISA test..