Among the New Plant Breeding Techniques (NPBTs), the CRISPR/Cas9 system represents a useful tool for target gene editing, improving the traits of the plants rapidly. This technology allows targeting one or more sequences simultaneously, as well as introducing new genetic variations by homology-directed recombination. However, the technology of CRISPR/Cas9 remains a challenge for some polyploid woody species, since all the different alleles for which the mutation is required must be simultaneously targeted. In this work we describe improved protocols adapting the CRISPR/Cas9 system to highbush blueberry (Vaccinium corymbosum L.), using Agrobacterium-mediated transformation. As a proof of concept, we targeted the gene encoding for phytoene desaturase, whose mutation disrupts chlorophyll biosynthesis allowing for the visual assessment of knockout efficiency. Leaf explants of in vitro-cultured blueberry cv. Berkeley has been transformed with a CRISPR/Cas9 construct containing two guide RNAs (gRNA1 and gRNA2) targeting two conserved gene regions of pds and subsequently maintained on a selection medium enriched with kanamycin. After 4 weeks in culture on the selection medium, the kanamycin-resistant lines were isolated, and the genotyping of these lines through Sanger sequencing revealed successful gene editing. Some of mutant shoot lines included albino phenotypes, even if the editing efficiencies were quite low for both gRNAs, ranging between 2.1 and 9.6% for gRNA1 and 3.0 and 23.8 for gRNA2. Here we showed a very effective adventitious shoot regeneration protocol for the commercial cultivar of highbush blueberry “Berkeley”, and a further improvement in the use of CRISPR/Cas9 system in Vaccinium corymbosum L., opening the way to the breeding mediated by biotechnological approaches.

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.

Polyploid induction is of utmost importance in horticultural plants for the development of new varieties with desirable morphological and physiological traits. Polyploidy may occur naturally due to the formation of unreduced gametes or can be artificially induced by doubling the number of chromosomes in somatic cells. In this experiment, a protocol for in vitro polyploid induction of highbush blueberry (Vaccinium corymbosum L.) leaf tissues was studied by using different concentrations of colchicine and oryzalin. Oryzalin was found to be highly toxic to this species, while the adventitious shoot organogenesis media enriched with 25 and 250 µM colchicine was able to induce polyploidization, with significant differences among the treatments used. Higher concentrations of both antimitotic agents led to the browning and death of the leaf tissues. The polyploids obtained showed several morphological differences when compared with the diploid shoots. Flow cytometry analysis was used to confirm the ploidy level of the regenerated shoots, demonstrating that a total of 15 tetraploids and 34 mixoploids were obtained. The stomatal sizes (length and width) of the tetraploids were larger than those of the diploids, but a reduced stomatal density was observed as compared to the controls. These shoots will be acclimatized and grown until they reach the reproductive phase in order to test their potential appeal as new varieties or their use for breeding and genetic improvement.

Castanea sativa is one of the main multipurpose tree species valued for its timber and nuts. This species is susceptible to two major diseases, ink disease and chestnut blight, caused by Phytophthora spp. and Cryphonectria parasitica, respectively. The loss-of-function mutations of genes required for the onset of pathogenesis, referred to as plant susceptibility (S) genes, are one mechanism of plant resistance against pathogens. On the basis of sequence homology, functional domain identification, and phylogenetic analyses, we report for the first time on the identification of S-genes (mlo1, dmr6, dnd1, and pmr4) in the Castanea genus. The expression dynamics of S-genes were assessed in C. sativa and C. crenata plants inoculated with P. cinnamomi and C. parasitica. Our results highlighted the upregulation of pmr4 and dmr6 in response to pathogen infection. Pmr4 was strongly expressed at early infection phases of both pathogens in C. sativa, whereas in C. crenata, no significant upregulation was observed. The infection of P. cinnamomi led to a higher increase in the transcript level of dmr6 in C. sativa compared to C. crenata-infected samples. For a better understanding of plant responses, the transcript levels of defense genes gluB and chi3 were also analyzed.

Climate change is deeply impacting the food chain production, lowering quality and yield. In this context, the international scientific community has dedicated many efforts to enhancing resilience and sustainability in agriculture. Italy is among the main European producers of several fruit trees; therefore, national research centers and universities undertook several initiatives to maintain the specificity of the ‘Made in Italy’ label. Despite their importance, fruit crops are suffering from difficulties associated with the conventional breeding approaches, especially in terms of financial commitment, land resources availability, and long generation times. The ‘new genomic techniques’ (NGTs), renamed in Italy as ‘technologies for assisted evolution’ (TEAs), reduce the time required to obtain genetically improved cultivars while precisely targeting specific DNA sequences. This review aims to illustrate the role of the Italian scientific community in the use of NGTs, with a specific focus on Citrus, grapevine, apple, pear, chestnut, strawberry, peach, and kiwifruit. For each crop, the key genes and traits on which the scientific community is working, as well as the technological improvements and advancements on the regeneration of local varieties, are presented. Lastly, a focus is placed on the legal aspects in the European and in Italian contexts.

The European hazelnut (Corylus avellana L.; 2n = 2x = 22) is a worldwide economically important tree nut that is cross-pollinated due to sporophytic incompatibility. Therefore, any individual plant is highly heterozygous. Cultivars are clonally propagated using mound layering, rooted suckers, and micropropagation. In recent years, the interest in this crop has increased, due to a growing demand related to the recognized health benefits of nut consumption. C. avellana cv “Tonda Gentile delle Langhe” (“TGdL”) is well-known for its high kernel quality, and the premium price paid for this cultivar is an economic benefit for producers in northern Italy. Assembly of a high-quality genome is a difficult task in many plant species because of the high level of heterozygosity. We assembled a chromosome-level genome sequence of “TGdL” with a two-step approach. First, 10X Genomics Chromium Technology was used to create a high-quality sequence, which was then assembled into scaffolds with cv “Tombul” genome as the reference. Eleven pseudomolecules were obtained, corresponding to 11 chromosomes. A total of 11,046 scaffolds remained unplaced, representing 11% of the genome (46,504,161 bp). Gene prediction, performed with Maker-P software, identified 27,791 genes (AED ≤0.4 and 92% of BUSCO completeness), whose function was analyzed with BlastP and InterProScan software. To characterize “TGdL” specific genetic mechanisms, Orthofinder was used to detect orthologs between hazelnut and closely related species. The “TGdL” genome sequence is expected to be a powerful tool to understand hazelnut genetics and allow detection of markers/genes for important traits to be used in targeted breeding programs.