A plant regeneration platform to apply new breeding techniques for improving disease resistance in grapevine rootstocks and cultivars

Abstract

Worldwide grapevine cultivation is based on the use of elite cultivars, in many cases strictly linked to local important wine brands. Most of Vitis 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 efficient in vitro regeneration and transformation protocols to extend the application of these technologies in wine grape cultivars and rootstocks. With this aim, in vitro regeneration 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 for Agrobacteriummediated 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 control B. cinerea infection.

New Perspectives in Grapevine (Vitis spp.) Breeding

“…Various explant sources, such as leaves [23] and anthers [24] have been studied under inducing conditions to explore the possibilities of obtaining somatic embryos. In the initial transformation studies, leaf tissues of different V. vinifera cultivars [25] and different rootstocks [26][27][28] were studied, resulting in non-regenerating transgenic callus. Somatic embryogenesis has been used by different researchers in micro propagation and genetic transformation studies of various woody perennial plant species.…”

Efficient protocol of de novo shoot organogenesis from somatic embryos for grapevine genetic transformation

“…In Vitis spp., although de novo shoot organogenesis has been successfully reported using different organs as starting explants (leaf segments, in vitro leaves, internodes, petioles, apical and lateral meristems), the number of reports describing the use of this regeneration process to obtain genetically transformed lines are limited ( Xie et al., 2016 ; Zhang et al., 2021 ). The highest transformation efficiencies (from 10% up to almost 50%) have been reported in more than one Vitis genotype, including the table grape cultivar Thompson Seedless, by using slices of a vegetative structure called meristematic bulk (MB), resulting from the mechanical treatment and repeated subcultures of meristematic shoot apices in media with increasing cytokinin concentrations ( Mezzetti et al., 2002 ; Xie et al., 2016 ; Sabbadini et al., 2019a ; Sabbadini et al., 2019b ). What these studies has in common is the strong dependence of the success of genetic transformation on the genotype used.…”

Improvement of technological methods of production and cultivation of grafted grape seedlings

“…Vegetative grape plants, both grafted and root seedlings, are in a vegetative state, intended for planting on a plantation (permanent place) without prior planting in nursery garden. Such seedlings are widely used to accelerate the propagation of new promising technical and table grape varieties [2,3,4].…”