Abstract
Worldwide grapevine cultivation is based on the use of elite cultivars, in many cases strictly linked to local important wine brands. Most ofVitis viniferacultivars have high susceptibility to fungal and viral diseases therefore, new breeding techniques (e.g. Cisgenesis, RNAi and gene editing) offer the possibility to introduce new clones of the main cultivars with increased diseases resistance, in order to reduce environmental impact and improve quality in the intensive wine grape industry. This study is finalized to develop efficientin vitroregeneration and transformation protocols to extend the application of these technologies in wine grape cultivars and rootstocks. With this aim,in vitroregeneration protocols based on the production of meristematic bulks (Mezzetti et al., 2002) were optimized for different grapevine cultivars (Glera, Vermentino, Sangiovese, Thompson Seedless) and rootstocks (1103 Paulsen, and 110 Richter). The meristematic bulks were then used as explants forAgrobacteriummediated genetic transformation protocols, by comparing the use of NPTII and e-GFP as marker genes. Results confirmed the efficiency of meristematic bulks as the regenerating tissue to produce new modified plants in almost all the above genotypes. The highest regeneration efficiency in some genotypes allowed the selection of stable modified lines/calli with only the use of e-GFP marker gene. This protocol can be applied in the use of MYB marker gene for the production of cisgenic lines. Genotypes having the highest regeneration and transformation efficiency were also used for transformation experiments using a hairpin gene construct designed to silence the RNA-dependent RNA polymerase (RpRd) of the GFLV and GLRaV3, which would induce multiple virus resistances, and the Dicer-like protein 1 (Bc-DCL1) and Bc-DCL2 to controlB. cinereainfection.
Highlights
Grapes (Vitis spp.) are among the world’s most important fruit crops for their diverse uses
Traditional breeding techniques are limited in that there is reduced genetic resources and an increase in variability that is unacceptable for the preservation of traditional grapevine clones
The sequencing of V.vinifera genome has allowed the identification of gene sequences useful for controlling several important agronomical characteristics in grapevine, which has been mostly validated by Agrobacterium-mediated transformation [3,4,5]
Summary
Grapes (Vitis spp.) are among the world’s most important fruit crops for their diverse uses. The application of New Breeding Techniques (NBTs), such as cisgenesis, RNAi and gene editing, can be a useful alternative to classical breeding methods to introduce or modify important agronomical traits in grapevine clones [1, 2]. The main bottleneck in the application of biotechnological tools for grapevine genetic improvement is the development of efficient and reproducible in vitro regeneration and transformation protocols, which often need to be optimized for the individual genotypes being studied [5]. In vitro grapevine shoots regeneration was obtained via organogenesis, which represents a good alternative regeneration method to somatic embryogenesis, avoiding some technical problems related to embryogenetic cultures induction and maintenance [9,10,11,12]
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