Castanea sativa Mill. is a highly versatile species of great ecological, economic, and cultural importance throughout Europe. However, it faces serious threats such as ink disease and chestnut blight, caused respectively by Phytophthora cinnamomi (Pc) and Cryphonectria parasitica, whose severity has increased in recent years due to climate change. A promising gene editing strategy to confer pathogen tolerance involves inactivating susceptibility genes (S genes) in plants. Here, the susceptibility gene Cspmr4, which encodes a callose synthase, was knocked-out for the first time in a forest species using CRISPR/Cas9. To achieve this, somatic embryos from two embryogenic chestnut lines (CI-9 and CI-3) were transformed using Agrobacterium tumefaciens strain EHA105 carrying a CRISPR/Cas9 construct targeting two sites in Cspmr4. Only line CI-9 produced kanamycin resistant transgenic embryos, with an average transformation efficiency of 5.9%. To assess editing efficiency and identify mutation types, target sites were amplified using PCR, followed by Sanger sequencing and mutation analysis using TIDE and ICE tools. Editing was confirmed in five of seven embryogenic lines. All five lines have at least 97% editing efficiency in at least one gRNA. Edited somatic embryos showed the highest survival rates, exceeding 50% in three of the analyzed lines. In addition, regenerated plants from line CI-9-pmr4-1 exhibited a marked reduction in root necrosis, decreasing from 57% in wild-type explants to only 8% in the edited line.

Woody fruit crops represent a cornerstone of global agriculture, yet their genetic improvement is severely hindered by the persistent issue of in vitro recalcitrance. This phenomenon represents a significant bottleneck for large-scale micropropagation and the application of groundbreaking breeding technologies, thereby slowing the development of new, resilient cultivars. This review provides a comprehensive state-of-the-art overview of the physiological, hormonal, and molecular factors underlying regenerative responses in woody species, emphasizing the critical role of explant type, developmental stage, genetic, and epigenetic factors beyond morphogenic competence. We highlight how endogenous and exogenous phytohormone interactions, in conjunction with complex genetic and epigenetic signalling networks, modulate adventitious organogenesis and somatic embryogenesis, often resulting in unpredictable and genotype-dependent outcomes. Particular attention is dedicated to biotechnological strategies aimed at overcoming recalcitrance, including the use of morphogenic regulators, inducible gene expression systems to minimize pleiotropic effects, and site-specific recombination technologies for marker-free transformation. Examples from woody fruit crops highlight both the progress made and the limitations that still hinder the establishment of efficient and reproducible protocols. Finally, we discuss current knowledge gaps and open questions, including the need to elucidate the molecular mechanisms of recalcitrance, the potential of in planta and tissue culture-free transformation methods, and the future integration of Artificial Intelligence and Machine Learning tools to accelerate protocol optimization. Together, these insights outline the path toward developing genotype-independent and scalable transformation systems for recalcitrant woody fruit species.

New Plant Breeding Techniques (NPBTs) are emerging as innovative tools for plant breeding, with the CRISPR/Cas9 system being a valuable method for accelerating genetic improvement. Currently, the combination of the CRISPR/Cas9 system with protoplast technology offers a promising approach to producing transgene-free edited plants. In this study, we established the first protoplast isolation protocol in Corylus avellana L., cv ‘Tonda Gentile Trilobata’. Friable calli were obtained from in vitro-derived leaves cultured on a callus induction medium based on a modified Murashige and Skoog medium with half-strength macroelements, supplemented with 0.2 mg/l benzylaminopurine (BAP) and 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4D). For cell-wall digestion, the solution contained Cellulase Onozuka R-10 (2

This study investigates the genetic basis of fruit traits in European hazelnut ( Corylus avellana L.), with a focus on identifying the genetic determinants that influence nut and kernel traits as well as the genes associated with flowering time. The research aims to shorten hazelnut breeding cycles and understand the genetic interplay affecting these traits. Using a mapping population from 'Tonda Gentile delle Langhe' (TGdL) and 'Merveille de Bollwiller' (MB) cultivars (175 F1 progeny), extensive phenotyping and genotypic analysis were conducted. Quantitative trait loci (QTL) analysis identified 131 QTLs (52 major) associated with 12 nut and kernel traits were identified across the 11 LGs with 68 on the TGdL map and 63 on the MB map. Among them stable QTLs over the three years were detected on TGdL map (nine QTLs on five LGs) and MB map (13 QTLs on four LGs). The most interesting were on MB_04 and MB_05 LGs for fruit size traits, on TGdL_04 and TGdL_11 for fruit shape traits and on MB_02 for cluster traits. Through exploration of candidate genes within these regions a linkage between nut and kernel traits and flowering time were identified, along with candidate genes involved in key developmental processes such as cell proliferation and circadian clock regulation. Notably, genes affecting flowering time were also linked to fruit development traits, suggesting a genetic connection. Our findings highlight a significant genetic overlap between nut and kernel traits and flowering time, providing valuable genetic markers for use in hazelnut breeding programs. This research underlines the potential for using molecular breeding techniques to enhance trait selection efficiency, thereby supporting the development of new hazelnut varieties with improved quality and adaptability to changing environmental conditions. These insights are crucial for advancing sustainable agricultural practices and meeting the increasing global demand for hazelnuts.

The sweet chestnut (Castanea sativa Mill.) is one of the most widespread cultivated temperate trees in Europe, valued both for its edible nuts and high-quality timber. Due to its ecological, economic, and cultural importance, it has been the focus of extensive genetic studies. However, its mitochondrial genome has remained largely unexplored. Here, we present the first complete mitochondrial genome of C. sativa (cultivar ‘Marrone di Chiusa Pesio’), assembled using high-throughput sequencing and characterised through comparative analyses with closely related species. The final assembly consists of six contigs with a total length of 402,729 bp, comprising 35 protein-coding genes, 33 tRNA genes, and 3 rRNA genes. Compared to the congeneric C. mollissima, C. sativa shows a relatively low repeat content in terms of both number and length. Codon usage patterns were found to be highly similar among C. sativa, C. mollissima, and C. henryi. Additionally, homologous fragments between the plastid and mitochondrial genomes were identified, totaling 4,671 bp (1.16

Corylus avellana L. is a rare and endangered species in Kazakhstan, included in the national Red Book. The results of morphological and genetic characterization of the sole known natural population of C. avellana in the Western Kazakhstan region are presented in this study. Sixty wild accessions were evaluated based on tree and leaf morphological traits using standard descriptors in accordance with Bioversity International guidelines. Genetic diversity was assessed using ten nuclear simple sequence repeat (SSR) markers. A total of 120 alleles were detected across the nuclear loci, with the number of alleles per locus ranging from 9 to 16 and an average of 12. The mean effective number of alleles (Ne) per locus was 3.862. A high level of intraspecific polymorphism was observed, with an average observed heterozygosity (Ho) of 0.70. The population showed considerable genetic diversity, as highlighted by a mean Shannon’s diversity index of 1.526. STRUCTURE, PCoA, and phylogenetic analyses confirmed strong differentiation between the wild Kazakh population and the cultivated hazelnut germplasm. Due to the lack of viable seeds, in vitro conservation was initiated using vegetative shoots. A two-step disinfection protocol, involving Plant Preservative Mixture and mercuric chloride, significantly improved explant survival, enabling successful establishment of an aseptic in vitro collection. These findings highlight the urgent need for targeted conservation strategies and show the potential of biotechnological approaches for safeguarding Kazakhstan’s only natural C. avellana population.

Castanea sativa is one of the most appreciated species in Italy, due to timber and fruit quality; nuts are usually intended for fresh consumption, but also for the production of industrial foods. The demand for an efficient traceability system to identify Castanea species and cultivars along the supply chain, from the chestnut orchard to the processed product, is crucial to protect consumers and producers from frauds. Here we present a traceability protocol to identify the species of the Castanea genus and trace the cultivars of Castanea sativa, based on DNA markers (SSRs and SNPs) analyses. Microsatellites were applied to identify the cultivar in plant material (leaves, seed, and episperm) and processed food (creams, beverages, flour, and cookies). Although SSRs made it possible to trace cultivar starting from somatic tissues (e.g.: leaves), the presence of foreign paternal DNA from pollenizer plants in seed and processed food, interferes with the identification of the maternal one. To overcome this issue, we Illumina sequenced six C. sativa cultivars to highlight maternal-inherited markers. A set of SNP/INDEL markers in two chloroplastic regions were identified, able to effectively distinguish and trace C. sativa from C. mollissima and C. crenata. The High Resolution Melting (HRM) analysis was performed as a practical and efficient method for SNP/INDELvalidation and to develop a fast, reliable, and reproducible protocol for chestnut traceability in food.

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 106 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 NH4NO3 and KNO3 supplemented with 0.1 mg/L benzylaminopurine and 0.1 mg/L 2,4-dichlorophenoxyacetic acid.

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. 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

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 106 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 NH4NO3 and KNO3 supplemented with 0.1 mg/L benzylaminopurine and 0.1 mg/L 2,4-dichlorophenoxyacetic acid.

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 increasing interest in European hazelnut (Corylus avellana L.) cultivation registered in the last years has led to a significant increase in worldwide hazelnut growing areas, also involving regions characterized by a marginal presence of hazelnut orchards. Despite this increasement, world production still relies on the cultivation of few varieties, most of which are particularly suitable to the environment where they have been selected. Therefore, it is necessary to develop new cultivars with high environmental plasticity capable of providing constant and high-quality productions in the new environments and under the climatic change conditions of traditional growing areas. Over the years, many molecular markers for genetic breeding programs have been developed and omics sciences also provided further information about the genetics of this species. These data could be of support to the application of new plant breeding techniques (NPBTs), which would allow the development of cultivars with the desired characteristics in a shorter time than traditional techniques. However, the application of these methodologies is subordinated to the development of effective regeneration protocols which, to date, have been set up exclusively for seed-derived explants. A further aspect to be exploited is represented by the possibility of cultivating hazelnut cells and tissues in vitro to produce secondary metabolites of therapeutic interest. This review aims to consolidate the state of the art on biotechnologies and in vitro culture techniques applied on this species, also describing the various studies that over time allowed the identification of genomic regions that control traits of interest.

COPYRIGHT © 2023 Cristofori, Botta, Rovira, Molnar and Mehlenbacher. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. TYPE Editorial PUBLISHED 04 January 2023 DOI 10.3389/fpls.2022.1120595

Castanea sativa cv. ‘Garrone Rosso’ and ‘Marrone di Castel del Rio’ are two of the most prized varieties in Italy due to their valuable and healthy nuts used for fresh consumption and in the confectionery industry. Despite the growing demand for chestnuts, there are constraints regarding plant propagation that hamper the renewal and new planting of orchards in different areas. Castanea sativa is susceptible to diseases that have caused a reduction in its area of production. For this reason, in vitro culture represents a valuable technique for germplasm preservation and plant multiplication enabling production of a high number of plants for use in breeding programs. Here we present an in vitro micropropagation protocol for Italian Castanea sativa cv. ‘Marrone di Castel del Rio’ and cv. ‘Garrone Rosso’ to contribute to the preservation and enhancement of the Italian germplasm. Nodal explants were used as the starting material for in vitro establishment. The cv. ‘Marrone di Castel del Rio’ showed a high percentage of survival explants (92%) when subjected to long bleach exposure (25 min), in contrast to what was observed for the ‘Garrone Rosso’ cultivar. Ascorbic acid was found to be the best compound to counteract phenol exudation. The MS3B and DKW media supplied with 0.5 mg/L BAP were effective for in vitro establishment, while the DKW medium (0.1 mg/L BAP and 0.05 mg/L IBA) was preferable for the proliferation phase. A double-layer rooting methodology was used and 35% rooting was observed with 25 mg/L IBA rooting treatment.

The request for an efficient traceability system able to identify hazelnut cultivars along the entire processing chain is becoming a critical point for avoiding fraudulent practices and safeguarding the interests of growers, food processors and consumers. In this study, DNA was extracted from different hazelnut matrices, including plant material (leaf, kernel and kernel episperm), and processed foods (paste, grain, flour and different types of snacks containing hazelnuts). The efficiency of Simple Sequence Repeat (SSR) markers was tested to identify the hazelnut cultivar ‘Tonda Gentile’ in all the supply chain. The analysis at 10 SSR loci was able to verify the presence/absence of the alleles of a declared cultivar contained in these matrices. The SSR analysis of DNA from raw episperm offers the possibility of identifying the mother cultivar and is suggested as an effective way to discover frauds since DNA analysis can be performed on individual kernels. For food matrices containing hazelnuts, the presence of the mother cultivar’s DNA can be assessed based on the identification of its alleles in the sample, although the presence of multiple alleles from the pollenizers makes the interpretation of results more difficult.

Different climatic conditions are known to affect the synthesis of primary and secondary metabolites. Therefore, the phenolic contents in new growing areas could affect the quality and flavor of hazelnuts. The aim of this study was to determine the variability of the phenolic contents of the kernels in different commercial hazelnut cultivars depending on their growing area. Five cultivars ('Tonda Gentile delle Langhe', 'Merveille de Bollwiller', 'Pauetet', 'Tonda di Giffoni', and 'Barcelona' (syn. 'Fertile de Coutard')) grown in different European collection orchards were included in the study. High-performance liquid chromatography coupled with mass spectrometry was used to identify and quantify the phenolic compounds. Thirteen phenols were identified in the hazelnut kernels, including 7 flavanols, 2 hydroxybenzoic acids, 3 flavonols, and one dihydrochalcone. Catechin and procyanidin dimers were the main phenolic compounds found in the hazelnut kernels. The highest contents of catechin and total flavanols were determined in cultivars cultivated in Spain and northern Italy, and the lowest in Slovenia and France. Flavanols were the major phenolic groups independent of the place of cultivation, as they accounted for more than 50% of all phenolic compounds identified. The flavanols were followed by hydroxybenzoic acids, flavonols, and dihydrochalcones. Higher contents of flavanols and flavonols were found in kernels from areas characterized by higher natural irradiation, which stimulates their accumulation. The contents of hydroxybenzoic acids correlated with altitude, which stimulated phenolic acid synthesis. A negative correlation was observed between the dihydrochalcone content and annual rainfall, probably due to hydric stress.