Comparison of regeneration capacity and Agrobacterium-mediated cell transformation efficiency of different cultivars and rootstocks of Vitis spp. via organogenesis.

The success of in vitro plant regeneration and the competence of genetic transformation greatly depends on the genotype of the species of interest. In previous work, we developed a method for the efficient Agrobacterium-mediated genetic transformation via organogenesis of V. vinifera cultivar Thompson Seedless, by using meristematic bulk (MB) as starting tissue. In this study, we applied this method for the regeneration and transformation of MBs obtained from the Italian cultivar Ciliegiolo and two of the commonly used Vitis rootstocks, 110 Richter and Kober 5BB, in comparison with Thompson Seedless. The A. tumefaciens strain EHA105, harbouring pK7WG2 binary vector, was used for the transformation trials, which allowed selection through the enhanced-green fluorescent protein (eGFP) and the neomycin phosphotransferase (nptII) gene. Putative transformed tissues and/or shoots were identified by either a screening based on the eGFP expression alone or its use in combination with kanamycin in the medium. MBs obtained from Thompson Seedless showed the highest regeneration and transformation cell competence, which subsequently allowed the recovery of stably transformed plants. Ciliegiolo, 110 Richter, and Kober 5BB, produced actively growing transgenic calli showing eGFP fluorescence, more consistently on selective media, but had no regenerative competence

From induction to embryo proliferation: improved somatic embryogenesis protocol in grapevine for Italian cultivars and hybrid Vitis rootstocks

Somatic embryogenesis is the most common regeneration method for the application of new genomic techniques like cisgenesis/intragenesis, genome editing, and RNAi. However, some local important genotypes show recalcitrance to this morphogenetic strategy, which represents an obstacle for the application of genetic engineering techniques. Whole flowers, stamens, and pistils of three different Italian Vitis vinifera L. cultivars (Ancellotta, Glera, and Lambrusco Salamino), and four hybrid rootstocks (110 Richter, 17.37, SO4, Star 50) have been tested in several culture media with changing basal salts (NN and MS), different combinations of growth regulators (BAP, 2,4-D, NOA, PIC, and NAA), and gelling agents, to initiate somatic embryogenesis. The formation of embryogenic calli was observed mainly from whole flowers cultured on PIV medium (NN salts, B5 vitamins, 3 g L-1 gelrite, 60 g L-1 sucrose, 8.9 mu M BAP, and 4.5 mu M 2,4-D), and stamens on MS1 medium (MS salts and vitamins, 7 g L-1 plant agar, 20 g L-1 sucrose, 4.5 mu M BAP, and 5 mu M 2,4-D), in the cv. Ancellotta, Lambrusco Salamino, and all the rootstocks, except for Star 50, which showed the best embryogenetic response from pistils placed on MS1. In a recalcitrant cv. as Glera, pistils placed on MS medium supplemented with 1 mu M BAP, 5 mu M 2,4-D, and gelrite as gelling agent, showed the highest percentage of embryogenesis. In addition, a two-step protocol was efficiently optimized for further induction of secondary embryo production for the above-listed grapevine genotypes, which guaranteed the long-term maintenance of embryogenic cultures from clusters or single somatic embryos.Key message Different types of explants, induction media combinations and concentrations influence the efficiency of regeneration via somatic embryogenesis in recalcitrant Vitis cultivars and rootstocks

Biotechnological approaches: Gene overexpression, gene silencing, and genome editing to control fungal and oomycete diseases in grapevine

Downy mildew, powdery mildew, and grey mold are some of the phytopathological diseases causing economic losses in agricultural crops, including grapevine, worldwide. In the current scenario of increasing global warming, in which the massive use of agrochemicals should be limited, the management of fungal disease has become a challenge. The knowledge acquired on candidate resistant (R) genes having an active role in plant defense mechanisms has allowed numerous breeding programs to integrate these traits into selected cultivars, even though with some limits in the conservation of the proper qualitative characteristics of the original clones. Given their gene-specific mode of action, biotechnological techniques come to the aid of breeders, allowing them to generate simple and fast modifications in the host, without introducing other undesired genes. The availability of efficient gene transfer procedures in grapevine genotypes provide valid tools that support the application of new breeding techniques (NBTs). The expertise built up over the years has allowed the optimization of these techniques to overexpress genes that directly or indirectly limit fungal and oomycetes pathogens growth or silence plant susceptibility genes. Furthermore, the downregulation of pathogen genes which act as virulence effectors by exploiting the RNA interference mechanism, represents another biotechnological tool that increases plant defense. In this review, we summarize the most recent biotechnological strategies optimized and applied on Vitis species, aimed at reducing their susceptibility to the most harmful fungal and oomycetes diseases. The best strategy for combating pathogenic organisms is to exploit a holistic approach that fully integrates all these available tools

The bottlenecks in obtaining an efficient transformation protocol for RNAi-based sharka resistant peach

Stone fruits, especially peach (Prunus persica), are among the most important tree species grown in the Mediterranean basin subjected to viral infections, in particular caused by Plum Pox Virus (PPV), the etiologic agent of Sharka disease, which leads to significant agronomic and economic losses. At the moment, there are no means of direct struggle against such infection but only means of prevention, which are often not effective and associated to environmental sustainability issues and costs for farmers. For these reasons, many programs of genomic studies and traditional breeding are aimed at understanding the mechanisms of resistance. A possible integrative strategy to classical breeding techniques is represented by the use of genetic engineering technology to obtain transgenic plants of cultivars and rootstocks bearing resistance genes. Prunus, in particular peach, is considered one of the most recalcitrant species for what concern in vitro regeneration and transformation, especially when the starting plant material originates from mature tissues. The study here described has the main objective of transferring and adapt the protocol previously developed in grape (Mezzetti et al., 2002) to the in vitro regeneration, via organogenesis, and Agrobacterium-mediated transformation of the peach rootstock GF677 (Prunus persica x Prunus amygdalus). The ultimate purpose is the introduction of an anti PPV RNAi construct, named ihp35S-PPV194

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

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

IMPLEMENTATION OF A REGENERATION AND TRANSFORMATION PROTOCOL FOR VITIS VINIFERA VARIETIES AND ROOTSTOCK TO INDUCE GENE SILENCING AGAINST GFLV-GLRaV VIRUS

Grapevine cultivation is penalized by pathological problems with significant impact on production, quality and related costs. This species is affected by numerous viral diseases, such as \u201cfanleaf (GFLV)\u201d and \u201cleaf roll (GLRaV)\u201d diseases, which are the most diffused in Europe. The application of rigorous certification criteria is the only strategy available to control the diffusion of viruses. 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. Post Transcriptional Gene Silencing (PTGS) has emerged as alternative tool to induce resistance to virus in several plant species, even by using rootstocks as a source of RNAi controlling plant virus infection. For the application of this technology in grapevine it is really important to have efficient regeneration and transformation protocols for the most important cultivars and rootstocks. For this aim, the regeneration and transformation protocol via organogenesis (Mezzetti et al., 2002) was optimized for different grapevine cultivars (Vermentino, Albana, Pignoletto, SanGiovese), in comparison with the efficient table grape cultivar Thompson Seedless, and rootstocks (1103 Paulsen, 110 Richter and Kober 5BB). The meristematic bulks created for each clone were used as explants for Agrobacterium-mediated genetic transformation protocols with a gene construct that express the e-GFP as marker gene. 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