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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.

CRISPR/Cas9 has emerged as the most important tool for genome engineering due to its simplicity, design flexibility, and high efficiency. This technology makes it possible to induce point mutations in one or some target sequences simultaneously, as well as to introduce new genetic variants by homology-directed recombination. However, this approach remains largely unexplored in forest species. In this study, we reported the first example of CRISPR/Cas9-mediated gene editing in Castanea genus. As a proof of concept, we targeted the gene encoding phytoene desaturase ( pds ), whose mutation disrupts chlorophyll biosynthesis allowing for the visual assessment of knockout efficiency. Globular and early torpedo-stage somatic embryos of Castanea sativa (European chestnut) were cocultured for 5 days with a CRISPR/Cas9 construct targeting two conserved gene regions of pds and subsequently cultured on a selection medium with kanamycin. After 8 weeks of subculture on selection medium, four kanamycin-resistant embryogenetic lines were isolated. Genotyping of these lines through target Sanger sequencing of amplicons revealed successful gene editing. Cotyledonary somatic embryos were maturated on maltose 3% and cold-stored at 4°C for 2 months. Subsequently, embryos were subjected to the germination process to produce albino plants. This study opens the way to the use of the CRISPR/Cas9 system in European chestnut for biotechnological applications

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.

The CRISPR/Cas9 ribonucleoprotein (RNP)-mediated technology represents a fascinating tool for modifying gene expression or mutagenesis as this system allows for obtaining transgene-free plants, avoiding exogenous DNA integration. Holm oak ( Quercus ilex ) has an important social, economic, and ecological role in the Mediterranean climate zones of Western Europe and North Africa and is severely affected by oak decline syndrome. Here we report the first example of the application of the CRISPR/Cas9-RNP technology in holm oak. Firstly, we evaluated the protoplast isolation from both in vitro leaves and proembryogenic masses. Proembryogenic masses represented the best material to get high protoplast yield (11 x 10 6 protoplasts/ml) and viability. Secondly, the protoplast transfection ability was evaluated through a vector expressing green fluorescence protein as marker gene of transfection, reaching a transfection percentage of 62% after 24 hours. CRISPR/Cas9 RNPs were successfully delivered into protoplasts resulting in 5.6% ± 0.5% editing efficiency at phytoene desaturase ( pds ) target genomic region. Protoplasts were then cultured in semisolid media and, after 45 days in culture, developed embryogenic calli were observed in a Murashige and Skoog media with half concentration of NH 4 NO 3 and KNO 3 supplemented with 0.1 mg/L benzylaminopurine and 0.1 mg/L 2,4-dichlorophenoxyacetic acid.

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.

Biotechnological tools have emerged as key alternatives for the protection, improvement, and sustainable use of forest species. This paper analyzes the main biotechnological strategies applied to the European chestnut, a species of significant ecological, economic, and cultural importance in many temperate regions. However, in recent decades, it has been seriously threatened by various factors, including devastating diseases such as chestnut blight and ink disease, as well as the impacts of climate change. First, classical and assisted breeding techniques are discussed, including controlled hybridization and the use of molecular markers to accelerate the selection of genotypes of interest. In the field of molecular biotechnology, studies related to the identification of key genes, the development of genetic markers (e.g., SSRs and SNPs), and the omics characterization of chestnut are reviewed. The use of micropropagation techniques for the clonal multiplication of elite individuals is also included. Furthermore, advances in genetic modifications are explored, highlighting the introduction of resistance genes through transgenic and cisgenic approaches, as well as emerging technologies such as CRISPR/Cas9. In the future, the integration of classical breeding with advanced genomics will enable the precise selection and accelerated development of European chestnut varieties, combining traditional trait improvement with genomic tools such as marker-assisted selection, genomic prediction, and gene editing to enhance disease resistance and climate resilience.

An increasing interest in the cultivation of (European) hazelnut ( Corylus avellana ) is driving a demand to breed cultivars adapted to non-conventional environments, particularly in the context of incipient climate change. Given that plant phenology is so strongly determined by genotype, a rational approach to support these breeding efforts will be to identify quantitative trait loci (QTLs) and the genes underlying the basis for adaptation. The present study was designed to map QTLs for phenology-related traits, such as the timing of both male and female flowering, dichogamy, and the period required for nuts to reach maturity. The analysis took advantage of an existing linkage map developed from a population of F 1 progeny bred from the cross “Tonda Gentile delle Langhe” × “Merveille de Bollwiller,” consisting in 11 LG. A total of 42 QTL-harboring regions were identified. Overall, 71 QTLs were detected, 49 on the TGdL map and 22 on the MB map; among these, 21 were classified as major; 13 were detected in at least two of the seasons (stable-major QTL). In detail, 20 QTLs were identified as contributing to the time of male flowering, 15 to time of female flowering, 25 to dichogamy, and 11 to time of nut maturity. LG02 was found to harbor 16 QTLs, while 15 QTLs mapped to LG10 and 14 to LG03. Many of the QTLs were clustered with one another. The major cluster was located on TGdL_02 and consisted of mainly major QTLs governing all the analyzed traits. A search of the key genomic regions revealed 22 candidate genes underlying the set of traits being investigated. Many of them have been described in the literature as involved in processes related to flowering, control of dormancy, budburst, the switch from vegetative to reproductive growth, or the morphogenesis of flowers and seeds.

Objectives The sweet chestnut Castanea sativa Mill. is the only native Castanea species in Europe, and it is a tree of high economic value that provides appreciated fruits and valuable wood. In this study, we assembled a high-quality nuclear genome of the ancient Italian chestnut variety ‘Marrone di Chiusa Pesio’ using a combination of Oxford Nanopore Technologies long reads, whole-genome and Omni-C Illumina short reads. Data description The genome was assembled into 238 scaffolds with an N50 size of 21.8 Mb and an N80 size of 7.1 Mb for a total assembled sequence of 750 Mb. The BUSCO assessment revealed that 98.6% of the genome matched the embryophyte dataset, highlighting good completeness of the genetic space. After chromosome-level scaffolding, 12 chromosomes with a total length of 715.8 and 713.0 Mb were constructed for haplotype 1 and haplotype 2, respectively. The repetitive elements represented 37.3% and 37.4% of the total assembled genome in haplotype 1 and haplotype 2, respectively. A total of 57,653 and 58,146 genes were predicted in the two haplotypes, and approximately 73% of the genes were functionally annotated using the EggNOG-mapper. The assembled genome will be a valuable resource and reference for future chestnut 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.

Qualitative and DNA analysis can be performed by taking a multidisciplinary approach to evaluate apple juices, the relevant values of which are a function of the origin, processing method and cultivar used. In detail, the aims of this study were to characterize apple juices through physiochemical analysis, sensory analysis and DNA analysis to evaluate the efficiency of simple sequence repeat (SSR) markers for cultivar identification. Six apple juices made with cv Golden Delicious, cv Granny Smith and a mix of these cultivars from an e-commerce platform (Samples A and B), DISAFA (Samples C and D) and a local farm (Piedmont, Italy) (Samples E and F) were considered. Apple juices A, B, E and F (clarified and pasteurized) can be considered as being of high quality, while Samples C and D were unclarified, unpasteurized and made with apples purchased from a local store. Considering the qualitative analysis, it was observed that the cultivar of apple affected the parameters assessed. In the case of total phenolic compounds, the highest values were observed for juices made only with cv Granny Smith, suggesting how this cultivar contributes to maintaining these nutraceutical compounds more than cv Golden Delicious. Regarding DNA analysis, a limited number of markers, i.e., 4 and 3, respectively, for the apple juices originating from e-commerce and a local farm could successfully produce reproducible amplified fragments. These results can be related to the different procedures used in processing apple juices of different origins.