The genetic improvement of grapevine through second-generation sustainable biotechnologies represents a powerful tool to face current challenges of modern viticulture, such as cultivar adaptation to ongoing climate change or reduction in the pesticide use.
These innovative technologies can indeed allow the development of novel genotypes through the introduction of target genome modifications with high precision and efficiency; a strategy particularly useful for the genetic improvement of those varieties and clones of high economic value and resulting from long breeding selection processes.
The somatic embryogenesis process (i.e. the initiation of embryos from plant somatic tissues), used as in vitro regeneration system that exploits the production of embryogenic callus, is the most suited tool in grapevine for supporting dedicated genetic improvement programs based on traditional and second generation (i.e. cis-genesis and genome editing) biotechnological approaches.
Since 1997, a dedicated platform for the production and maintenance of grapevine embryogenic callus has been developed at the IPSP-CNR Functional Genomics and Ecophysiology laboratory. The starting point is the in vitro culture of thousands of floral explants collected every year in the field from different Vitis vinifera cultivars (Chardonnay, Brachetto, Sangiovese, Cabernet Sauvignon, Nebbiolo, Syrah, Müller-Thurgau, Grenache and Riesling renano) and rootstock genotypes (110R, 1103P and Vitis rupestris) (Gribaudo et al., 2004; Gambino et al. 2007; Gambino et al., 2011; Gribaudo et al., 2017).
Besides other applications (e.g. regeneration of virus-free plants, germplasm preservation and collection), the embryogenic callus is used by the IPSP-CNR research staff as the basis for producing genetically transformed grapevines via Agrobacterium approach (see related functional genomics studies: Gambino et al., 2005; Gambino et al., 2009; Perrone et al., 2012; Vitali et al., 2016) and cis-genic lines. Embryogenic calli are also processed for isolation of protoplasts, which are following subjected to transfection with CRISPR/Cas9 vectors and finally in vitro cultured to regenerate DNA-free edited vines (EditGrape IPSP-CNR research project). In the last years, the development of an optimized grapevine transformation protocol significantly improved the effectiveness of the transformation platform, leading to regenerate considerable numbers of transformed plants within 5 months from the embryogenic callus/Agrobacterium co-culture.
The possibility to regenerate grapevines from somatic embryos (somaclones) has also the great advantage to allow the exploitation of potential somaclonal variability, referred to as the genetic variability associated with gene mutations or epigenetic marks randomly occurring during the somatic embryogenesis process. That is undoubtedly instrumental for genetic improvement purposes, for instance for the selection of stress-resistant genotypes (GrapeFit IPSP-CNR research project). In the frame of this context, the research team has recently developed explant regeneration protocols addressed to prime somaclonal genetic variability through the application of in vitro selective pressure agents on the culture media. The final objective is to orient the establishment of genetic variability events that can facilitate the selection of plant lines displaying increased tolerance to drought stress and fungal diseases, such as downy and powdery mildews (EditGrape IPSP-CNR research project).
- Gribaudo I, Gambino G, Vallania R (2004). Somatic Embryogenesis from Grapevine Anthers: The Optimal Developmental Stage for Collecting Explants. American Journal of Enology and Viticulture, 55:4
- Gambino G, Gribaudo I, Leopold S, Schartl A, Laimer M (2005). Molecular characterization of grapevine plants transformed with GFLV resistance genes: I. Plant Cell Reports 24(11): 655-662.
- Gambino G, Ruffa P, Vallania R. Gribaudo I (2007). Somatic embryogenesis from whole flowers, anthers and ovaries of grapevine (Vitis spp.). Plant Cell Tissue and Organ Culture 90: 79-83
- Gambino G, Chitarra W, Maghuly F, Laimer M, Boccacci P, Torello Marinoni D, Gribaudo I (2009). Characterization of T-DNA insertions in transgenic grapevines obtained by Agrobacterium-mediated transformation. Molecular Breeding 24(3): 305-320
- Gambino G, Minuto M, Boccacci P, Perrone I, Vallania R, Gribaudo I (2011). Characterization of expression dynamics of WOX homeodomain transcription factors during somatic embryogenesis in Vitis vinifera. Journal of Experimental Botany, 62: 1089–1101
- Perrone I, Gambino G, Chitarra W, Vitali M, Pagliarani C, Riccomagno N, Balestrini R, Kaldenhoff R, Uehlein N, Gribaudo I, Schubert A, Lovisolo C. (2012). The Grapevine Root-Specific Aquaporin VvPIP2;4N Controls Root Hydraulic Conductance and Leaf Gas Exchange Under Well-Watered Conditions but Not Under Water Stress. Plant Physiology, 160:965-77
- Vitali M, Cochard H, Gambino G, Ponomarenko A, Perrone I, Lovisolo C (2016). VvPIP2;4N aquaporin involvement in controlling leaf hydraulic capacitance and resistance in grapevine. Physiologia Plantarum 158: 284–296
- Gribaudo I, Gambino G, Boccacci P, Perrone I, Cuozzo D (2017). A multi-year study on the regenerative potential of several Vitis genotypes. Acta Horticulturae, 1155, 45-50