ISHS Acta Horticulturae 689: VII International Symposium on Grapevine Physiology and Biotechnology
INDUCTION OF SILENCING IN TRANSGENIC GRAPEVINES (VITIS SP.) |
| Authors: | G.M. Reustle, R. Ebel, P. Winterhagen, T. Manthey, C. Dubois, A. Bassler, M. Sinn, P. Cobanov, T. Wetzel, G. Krczal, R. Jardak-Jamoussi, A. Ghorbel |
| Keywords: | nepovirus, virus resistance, movement protein, inverted repeat, somatic embryogenesis |
| DOI: | 10.17660/ActaHortic.2005.689.64 |
| Abstract:
Grapevine Fanleaf degeneration, caused by a group of nepoviruses, is a major viral disease in viticulture world-wide. Due to a lack of natural genetic resources for viral resistance, suitable for breeding programs, a transgenic approach was chosen to develop rootstocks and cultivars resistant against the most relevant agents of the disease in Germany and Tunisia. Highly conserved sequences of Grapevine Fanleaf Virus (GFLV), Arabis Mosaic Virus (ArMV) and Raspberry Ringspot Virus (RpRSV), were combined with defective interfering (DI)-sequences from Tomato Bushy Stunt Virus (TBSV) and/or used to clone direct or inverted repeat constructs. This strategy allows the induction of sequence specific RNA-silencing by the expression of aberrant and/or double-stranded RNAs (dsRNA), resulting in resistance against viruses with homologues sequences. For proof of concept the constructs were genetically transferred into tobacco (N. benthamiana) by Agrobacterium-mediated transformation. Challenge inoculation with the relevant viruses yielded transgenic lines showing immunity, recovery, retarded infection and susceptibility. Northern analysis using a virus-derived sequence as probe, could detect transgene specific small interfering RNA (siRNA) in the resistant lines. To genetically engineer grapevine, embryogenic tissue of rootstocks (‘SO4’, ‘125AA’, ‘5C’, ‘Binova’, ‘5BB’) and the Tunisian cultivar ‘Arich dressé’ were induced from anther and ovule cultures and used for Agrobacterium (LBA 4404) mediated transformation. After 16 to 20 weeks of selection on Phosphinothricin (PPT) containing media, newly generated somatic embryos were harvested and cultivated on PPT free media for regeneration. Repeated propagation of putative transgenic lines by one-node cuttings and PCR analysis from the obtained plants yielded non-chimeric transgenic grapevines. Southern analysis showed different copy number in the transgenic lines (1 to 5 copies). Transgene specific siRNAs were detected by Northern analysis in some transgenic grapevine lines. | |
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