CRISPR/Cas9 Targeted Editing of Genes Associated With Fungal Susceptibility in Vitis vinifera L. cv. Thompson Seedless Using Geminivirus-Derived Replicons

The woody nature of grapevine (Vitis vinifera L.) has hindered the development of efficient gene editing strategies to improve this species. The lack of highly efficient gene transfer techniques, which, furthermore, are applied in multicellular explants such as somatic embryos, are additional technical handicaps to gene editing in the vine. The inclusion of geminivirus-based replicons in regular T-DNA vectors can enhance the expression of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) elements, thus enabling the use of these multicellular explants as starting materials. In this study, we used Bean yellow dwarf virus (BeYDV)-derived replicon vectors to express the key components of CRISPR/Cas9 system in vivo and evaluate their editing capability in individuals derived from Agrobacterium-mediated gene transfer experiments of ‘Thompson Seedless’ somatic embryos. Preliminary assays using a BeYDV-derived vector for green fluorescent protein reporter gene expression demonstrated marker visualization in embryos for up to 33 days post-infiltration. A universal BeYDV-based vector (pGMV-U) was assembled to produce all CRISPR/Cas9 components with up to four independent guide RNA (gRNA) expression cassettes. With a focus on fungal tolerance, we used gRNA pairs to address considerably large deletions of putative grape susceptibility genes, including AUXIN INDUCED IN ROOT CULTURE 12 (VviAIR12), SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTER 4 (VviSWEET4), LESION INITIATION 2 (VviLIN2), and DIMERIZATION PARTNER-E2F-LIKE 1 (VviDEL1). The editing functionality of gRNA pairs in pGMV-U was evaluated by grapevine leaf agroinfiltration assays, thus enabling longer-term embryo transformations. These experiments allowed for the establishment of greenhouse individuals exhibiting a double-cut edited status for all targeted genes under different allele-editing conditions. After approximately 18 months, the edited grapevine plants were preliminary evaluated regarding its resistance to Erysiphe necator and Botrytis cinerea. Assays have shown that a transgene-free VviDEL1 double-cut edited line exhibits over 90% reduction in symptoms triggered by powdery mildew infection. These results point to the use of geminivirus-based replicons for gene editing in grapevine and other relevant fruit species.

Pre-Anthesis Cytokinin Applications Increase Table Grape Berry Firmness by Modulating Cell Wall Polysaccharides

The use of plant growth regulators (PGRs) is widespread in commercial table grape vineyards. The synthetic cytokinin CPPU is a PGR that is extensively used to obtain higher quality grapes. However, the effect of CPPU on berry firmness is not clear. The current study investigated the effects of pre-anthesis applications (BBCH15 and BBCH55 stages) of CPPU on ‘Thompson Seedless’ berry firmness at harvest through a combination of cytological, morphological, and biochemical analyses. Ovaries in CPPU-treated plants presented morphological changes related to cell division and cell wall modification at the anthesis stage (BBCH65). Moreover, immunofluorescence analysis with monoclonal antibodies 2F4 and LM15 against pectin and xyloglucan demonstrated that CPPU treatment resulted in cell wall modifications at anthesis. These early changes have major repercussions regarding the hemicellulose and pectin cell wall composition of mature fruits, and are associated with increased calcium content and a higher berry firmness at harvest.

Elevated carbon dioxide and temperature effects on rooting behaviour of grape cuttings

An experiment was laid out to study the impact of eCO2 (550ppm), eT (+3ºC) and their interaction (eCO2+eT) on rooting behaviour of cuttings of three grape varieties- Thompson Seedless, Bangalore Blue, and Dogridge in FATE and OTC facilities. Observations were recorded at 50 and 80 days after planting (DAP) and root growth data was recorded and analysed using WinRHIZO root scanner and its software. Analysis revealed that, among the selected grape varieties, Thompson Seedless cuttings has shown highest number of roots, root volume and dry biomass under eCO2 and eCO2+ eT conditions, while total root length and root length density were highest with Bangalore Blue. Under eT condition, Bangalore Blue showed highest number of roots, total root length and root length density, while root volume and dry biomass was highest with Thompson Seedless. The per se values of root parameters under all conditions and their response to eCO2 was lowest with Dogridge. Though eT condition reduced all the root parameters, their performance improved under eCO2+ eT indicating the presence of higher concentration of CO2 reduced the ill effects of high temperature. Overall, eCO2 and eCO2+eT conditions improved root parameters of grape varieties, while eT reduced them as compared to their performance under ambient condition and varietal variation is significant.

Grapevine Variety and Number of Canes Affect Dry-on-Vine (DOV) Raisin Production on an Overhead Arbor Trellis

Making raisins (dried grapes) is a laborious process and grape growers around the world are increasingly interested in adopting mechanized methods, especially dry-on-vine (DOV). The DOV method entails severing canes bearing mature fruit, causing them to DOV, after which they may be harvested by machine. The overhead arbor is an important DOV raisin trellis system, but basic agronomic performance of raisin varieties on that system are scant. Therefore, a three-year experiment was conducted to compare the performance of several raisin grape varieties (Vitis vinifera L.), on an overhead arbor trellis, for production of DOV raisins. Grapevine variety, ‘DOVine’, ‘Fiesta’, ‘Selma Pete’, or ‘Thompson Seedless’, was the main-plot factor, and number of canes, six or eight, was the sub-plot. ‘Fiesta’ produced the highest yields, averaging about 12.24 Mg/ha raisins. However, ‘Fiesta’ generally had the lowest soluble solids and among the lowest raisin grades. The later ripening of ‘Fiesta’ fruit delayed harvest pruning to initiate fruit drying compared to the other varieties. Therefore, its raisins did not dry sufficiently to meet industry standards. ‘Selma Pete’, ‘DOVine’, and ‘Thompson Seedless’ had similar yields, generally about 20% less than ‘Fiesta’, but ‘Selma Pete’ produced fruit that were the largest in size, with the highest soluble solids, and that produced raisins of the highest grades, compared to the other varieties. Vines pruned to eight canes produced higher yields without sacrificing raisin quality. In conclusion, ‘Fiesta’ was notable for its high productivity, and ‘Selma Pete’ for its earliness and high quality, and both varieties had sufficient capacity to carry eight 15-node canes.