First Report on Genome Editing via Ribonucleoprotein (RNP) in Castanea sativa Mill.

Abstract

Castanea sativa is an important tree nut species worldwide, highly appreciated for its multifunctional role, in particular for timber and nut production. Nowadays, new strategies are needed to achieve plant resilience to diseases, climate change, higher yields, and nutritional quality. Among the new plant breeding techniques (NPBTs), the CRISPR/Cas9 system represents a powerful tool to improve plant breeding in a short time and inexpensive way. In addition, the CRISPR/Cas9 construct can be delivered into the cells in the form of ribonucleoproteins (RNPs), avoiding the integration of exogenous DNA (GMO-free) through protoplast technology that represents an interesting material for gene editing thanks to the highly permeable membrane to DNA. In the present study, we developed the first protoplast isolation protocol starting from European chestnut somatic embryos. The enzyme solution optimized for cell wall digestion contained 1% cellulase Onozuka R-10 and 0.5% macerozyme R-10. After incubation for 4 h at 25 °C in dark conditions, a yield of 4,500,000 protoplasts/mL was obtained (91% viable). The transfection capacity was evaluated using the GFP marker gene, and the percentage of transfected protoplasts was 51%, 72 h after the transfection event. The direct delivery of the purified RNP was then performed targeting the phytoene desaturase gene. Results revealed the expected target modification by the CRISPR/Cas9 RNP and the efficient protoplast editing.

Accelerating wood domestication in forest trees through genome editing: Advances and prospects

“…The use of RNPs to achieve DNA-free genome editing has been demonstrated in a variety of plant species [51, 66,67], including a limited number of woody species, such as grapevine, rubber tree, apple, chestnut and pine [68][69][70][71][72]. However, no regeneration from gene-edited cells has been reported yet.…”

Advances in Quercus ilex L. breeding: the CRISPR/Cas9 technology via ribonucleoproteins

“…Among these, the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) system represents a real revolution in all areas of biotechnology, enabling the production of ideal cultivars free of negative or undesired genetic traits ( Pavese et al., 2021a ; Vaia et al., 2022 ). However, until now, the use of this technique in forest species remains limited ( Walawage et al., 2019 ; Wang et al., 2020 ; Poovaiah et al., 2021 ; Pavese et al., 2021a , 2022 ) due to their recalcitrance to in vitro regeneration and the low transformation efficiency ( Corredoira et al., 2019 ). This applies to holm oak, in which although somatic embryogenesis has been induced in zygotic embryos ( Mauri and Manzanera, 2003 ), flower tissues ( Blasco et al., 2013 ; Barra-Jimenez et al., 2014 ) and leaf and apex explants derived from in vitro cultures ( Martinez et al., 2017 ; 2020 ; 2021 ), the induction frequency is usually low and the success of the technique is highly genotype dependent.…”

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

“…The protoplast represents a valuable material for the transfection process due to its high permeability to exogenous DNA [ 142 ]. The first experiment conducted in chestnut reported the setup of a protoplast isolation protocol and a transfection event using the pds gene [ 143 ]. A high number of protoplasts/mL was obtained (4,500,000), 91% viable.…”