ISHS Acta Horticulturae 689: VII International Symposium on Grapevine Physiology and Biotechnology
MOLECULAR CHARACTERIZATION OF TRANSGENIC GRAPEVINE PLANTS |
| Authors: | I. Gribaudo, G. Gambino, S. Leopold, M. Laimer |
| Keywords: | Vitis vinifera, ´Nebbiolo´, seedless table grape, somatic embryogenesis, GFLV, methylation, pathogen-derived resistance |
| Abstract:
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 8 independent transformation events, while the 22 ‘7-3/2E1’ lines were likely derived from 6 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. | |
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