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

Abstract

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 µM BAP, and 4.5 µM 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 µM BAP, and 5 µM 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 µM BAP, 5 µM 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.

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

“…However, similar experiments on other woody plants are scarce, probably due to the high level of heterozygosity and the need to cultivate clones identical to those typical of the selected crop variety ( Capriotti et al., 2020 ). For this reason, SEs, obtained following the newly established protocols on Ancellotta, Lambrusco Salamino, and Thompson Seedless grapevine cultivars ( Capriotti et al., 2022 ), were sectioned by separating cotyledons from hypocotyls, and used as starting explants in this study to optimize regeneration and Agrobacterium -mediated transformation protocols via organogenesis. Despite the regeneration from cotyledons and hypocotyls of mature SEs having been attempted in previous studies for other grapevine genotypes ( Vilaplana and Mullins, 1989 ; Martinelli et al., 2001b ), this is the first report in which both type of explants were efficiently employed to induce high regeneration.…”

“…Well-developed somatic embryos of the Vitis vinifera cultivars Ancellotta, Lambrusco Salamino, and Thompson Seedless were used as source of starting explants ( Figure 1A ). These explants derive from the development of embryogenic calli induced from whole flowers, stamens, and pistils of the respective Vitis vinifera cultivars, following the protocols optimized by Capriotti et al., 2022 . In sterile conditions, mature somatic embryos (SE) at the cotyledonary stage were sliced, separating hypocotyls from cotyledons, and discarding the primary radicle ( Figure 1A ).…”

“…Hence, most genetic transformations in grapevine have been performed on embryogenic calli or somatic embryos as starting explants induced from the most disparate somatic tissues ( Dhekney et al., 2019 ; Zhang et al., 2021 ; Nuzzo et al., 2022 ). There is no unique developmental pathway to obtain somatic embryos, indeed, it has been demonstrated that somatic embryo formation can occur directly or indirectly from a starting explant, and the entire process can be initiated from differentiated somatic cells or pericycle-like stem cells ( Fehér, 2019 ; Capriotti et al., 2022 ). Recent publications highlighted the importance to have well-established somatic embryogenesis regeneration protocols for the application of “new genomic techniques” (NGTs) on additional cultivars and rootstocks of Italian and international interest ( Forleo et al., 2021 ; Capriotti et al., 2022 ; Nerva et al., 2023 ).…”

Biotechnologies and Strategies for Grapevine Improvement

“…The acquisition of embryogenic competence is related to different patterns of gene expression involving internal cell reprogramming leading to a reversion of the differentiation state [128,155,156]. Somatic embryos may be obtained through the development of an embryogenic callus (indirect embryogenesis) followed by the emergence of pro-embryogenic masses (PEMs), from which new somatic embryos are formed [157], or may occur directly from the explants without the callus developmental phase.…”

The Role of Italy in the Use of Advanced Plant Genomic Techniques on Fruit Trees: State of the Art and Future Perspectives

“…For example, this issue is quite relevant in grapevine, where the most diffuse tissue type for transformation events and the only one available for totipotent protoplasts isolation and transfection is the embryogenic callus [ 63 , 64 ]. In grapevine, it can be obtained from floral tissues (e.g., anthers and ovaries) but the competency in producing embryogenic callus is genotype-specific, and many varieties are still recalcitrant [ 65 ].…”