Somatic Embryogenesis in Vitis for Genome Editing: Optimization of Protocols for Recalcitrant Genotypes

Abstract

New Plant Breeding Techniques (NPBTs) protocols have been developed to produce new grape varieties with improved quantitative and qualitative characteristics. Reliable transformation protocols for grapes are based on the generation/induction of embryogenic callus cells that are then transformed. Varieties such as Italia have proven to be very recalcitrant to regeneration via somatic embryogenesis. In this work, the development of a protocol for improved production of embryogenic calluses is described. Two sterilization protocols were tested: (a) a lower active chlorine concentration for a longer time (LS); and (b) a higher chlorine concentration for a shorter time (HS), in combination with the absence or presence of citric acid in the growing substrate in the first growth media. The embryogenic calluses formation in Chardonnay, a cv. with a high embryogenic response, was significantly higher in presence of citric acid in the initial growing substrate regardless of the sterilization protocol. In Aglianico, a cv. with a lower embryogenic response, no significant differences were observed. Instead, in a recalcitrant cv. as Italia, we obtained a 13-fold increase in embryogenic calluses formation performing sterilization of flowers with the HS protocol compared to LS.

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

“…The results determined that all the explants were able to regenerate somatic embryos to a different extent according to the induction medium chosen. Our study demonstrated that full or half-strength MS basal salts worked best for Glera as there was no calli formation on the PIV medium, in contrast with findings from recent similar research performed on the same genotype, where the induction of somatic embryogenesis in various local Italian varieties was obtained using PIV medium (Gambino et al 2007;Forleo et al 2021). Embryogenic calli formation was nil in twelve of the fifteen media tested, and there is no absolute determining factor to explain the induction of embryogenic calli in Glera as the calli formation was somewhat random (embryogenic calli formed on media E, G, and H); the common factor in these three media was the presence of BAP and 2,4-D, in which, the concentration of auxin was double that of cytokinins.…”

“…Our results determined that Glera flower explants showed higher embryogenic potential when cultured on the MS-optimized media, compared to NN-based media. Recent findings from the study by Forleo et al (2021) performed on the same genotype, reported whole flowers as the best explant for somatic embryogenesis induction when initiated on PIV medium. While the optimized composition of the induction media assayed in this study led to individuate pistils as the most responsive starting explant on culture media G and H, enhancing the embryogenic potential of this cultivar.…”

“…The embryogenic competence of the recalcitrant cultivar Glera has also been fostered in this research, hence increasing embryogenic efficiency based on the scientific literature on the same genotype, this was achieved by initiating different explants on culture media having multiple basal salts and PGRs combinations (Forleo et al 2021). The results determined that all the explants were able to regenerate somatic embryos to a different extent according to the induction medium chosen.…”

“…Similarly, somatic embryogenesis induction in Ancellotta and Lambrusco Salamino has not been previously reported in scientific literature, although the in vitro propagation of these two cultivars has been successfully attempted by Gatti et al (2017). While for the cultivar Glera, known for 'Prosecco' wine, the regeneration via somatic embryogenesis has been described recently by only one study (Forleo et al 2021). Thus, the additional results obtained in our research will enhance future attempts in genetic engineering approaches of this important Italian cultivar, well-known for its recalcitrance to both in vitro regeneration and genetic transformation.…”

Phenotypic variation and epigenetic insight into tissue culture berry crops

“…There are few available detailed reports on SE in blueberries ( Ghosh et al., 2017 ; Ghosh et al., 2018 ). However, studies are available on SE in grapes ( Nakajima and Matsuta, 2003 ; Dhekney et al, 2016 ; Forleo et al., 2021 ), strawberry ( Biswas et al., 2009 ), Arbutus unedo (strawberry tree; Martins et al, 2022 ) and L. barbarum ( Verma et al., 2022 ) on semisolid media. Reports on SE in berry crops are scarce in a bioreactor system.…”