Biotechnological Advances for Enhancing European Chestnut Resistance to Pests, Diseases, and Climate Change

Abstract

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.

1. Introduction

The genus Castanea (Fagaceae family) comprises 13 species distributed across forest ecosystems of the northern hemisphere. Among them, Castanea sativa Mill. (European chestnut), C. dentata (Marsh.) Borkh. (American chestnut), C. crenata Siebold & Zucc. (Japanese chestnut) and C. mollissima Blume (Chinese chestnut) are the most widely studied, particularly in relation to disease resistance and biotechnology. In Europe, the native C. sativa holds significant ecological and economic value. Palynological studies indicate that it was widespread during the Tertiary period and retreated to southern refugia during glaciations, followed by a rapid, human-mediated expansion during the Roman Empire [1]. Today, C. sativa is distributed across central and southern Europe and Asia Minor [2] (Figure 1). Its durable wood is used in furniture, fencing, and construction [3], while its edible nuts, which are rich in complex carbohydrates, fiber, vitamins, and antioxidants [4], remain central to rural economies and agroforestry systems. The European chestnut also retains deep cultural significance and was historically described as the “bread of the forests”, supporting ancient communities and Roman legions [5]. It is tied to southern Europe’s local traditions and seasonal festivals and continues to symbolize resilience, sustainability, and forest-based heritage.

Over the past century, two diseases have severely affected the European chestnut, with ink disease being the most destructive [6]. First reported in Spain and Portugal in the 19th century, ink disease is now widespread across many European countries (Figure 1). It is primarily caused by Phytophthora cinnamomi Rands, which is prevalent in Spain, Portugal, and France, and by P. cambivora (Petri) Buisman, which is more common in Italy and Greece [7]. These oomycetes infect the roots, disrupting water and nutrient transport and leading to dieback and tree death [8]. They spread rapidly in moist soils, and their impact is expected to increase with climate change [8,9]. The distribution of P. cinnamomi is strongly limited by low temperatures [10,11,12,13], and some historic records from cold areas [14] (included in Figure 1) may reflect temporary introductions rather than established populations. The other major disease affecting the European chestnut is chestnut blight caused by Cryphonectria parasitica (Murr.) Barr. [15]. It was first reported in 1938 near Genoa (Italy) and has since spread throughout much of Europe (Figure 1). The fungus kills bark tissues, girdling branches or the trunk, leading to the death of the affected organ [6]. Although the tree may partially survive, both wood quality and fruit production are severely impacted.

The chestnut gall wasp Dryocosmus kuriphilus Yasumatsu has emerged as an additional threat to chestnut cultivation and is now established in major chestnut-growing regions worldwide, including Italy, France, Spain, and Portugal (Figure 1). Persistent infestations significantly alter the branch architecture and reduce leaf area and bud formation, causing a decline in wood and flower production [16,17,18] that leads to a substantial reduction in nut yield. Asian chestnut species have coevolved resistance to ink disease and chestnut blight, as the pathogens are native to Asia [3], although they remain susceptible to the gall wasp [19]. In contrast, the American chestnut, which is now functionally extinct due to C. parasitica, is highly vulnerable to all three threats.

Biotechnology has been instrumental in efforts to manage these threats. In Europe, most work has focused on ink disease, with classical breeding programs producing interspecific hybrids resistant to P. cinnamomi, which are widely used in orchards [20,21,22]. Chestnut blight management has relied heavily on hypovirulence, which has proven effective in many European regions [23]. However, recent blight outbreaks in Spain and northern Portugal [24,25] have renewed concerns and prompted efforts to find blight-resistant European chestnut genotypes and hybrids. In North America, where P. cinnamomi cannot spread into colder environments [10,12] and hypovirulence has been largely ineffective [26], efforts have mainly focused on fighting chestnut blight, providing insights relevant to European programs.

Figure 1. Map illustrating the geographic distribution of natural populations of Castanea sativa (European chestnut), C. dentata (American chestnut), C. mollissima (Chinese chestnut), and C. crenata (Japanese chestnut), along with the main biotic stresses affecting each species, including chestnut blight (Cryphonectria parasitica), ink disease (Phytophthora cinnamomi), and the chestnut gall wasp (Dryocosmus kuriphilus). The map was created using data from several sources [2,14,27,28,29,30,31] with Inkscape v1.4.2 [32]. The map background is available at https://commons.wikimedia.org/wiki/File:Simplified_blank_world_map_without_Antartica_(no_borders).svg (accessed on 21 October 2025).

Figure 1. Map illustrating the geographic distribution of natural populations of Castanea sativa (European chestnut), C. dentata (American chestnut), C. mollissima (Chinese chestnut), and C. crenata (Japanese chestnut), along with the main biotic stresses affecting each species, including chestnut blight (Cryphonectria parasitica), ink disease (Phytophthora cinnamomi), and the chestnut gall wasp (Dryocosmus kuriphilus). The map was created using data from several sources [2,14,27,28,29,30,31] with Inkscape v1.4.2 [32]. The map background is available at https://commons.wikimedia.org/wiki/File:Simplified_blank_world_map_without_Antartica_(no_borders).svg (accessed on 21 October 2025).

In contrast, progress toward gall wasp resistance has been slower, likely due to the pest’s later introduction into Europe, its initially underestimated ecological and economic impacts, and the early reliance on biological control for management. The parasitoid Torymus sinensis Kamijo remains the most effective strategy used in several countries, including Japan, Korea, Italy, and the United States [18,33,34,35]. However, the continued spread and economic losses highlight the need for improved Integrated Pest Management (IPM) programs and for identifying resistant or less susceptible genotypes. Natural resistance in chestnut species has been suggested [36] but has not yet been systematically studied.

Climate change further exacerbates these challenges. Rising temperatures, an increased drought frequency, irregular precipitation patterns, and shifts in pest and pathogen dynamics threaten C. sativa resilience, productivity, and distribution [37]. Biotechnology supports assisted migration strategies by producing genotypes adapted to future climate scenarios. This involves predictive modeling combined with biotechnological breeding. Climate change drives biotechnology toward developing chestnut genotypes resilient to abiotic stresses and emerging pests, while enhancing disease resistance under shifting pathogen dynamics.

To address these complex challenges, modern biotechnological tools are increasingly combined with classical breeding methods. These tools include the development of genetic and genomic resources, genetic improvement strategies, advanced in vitro propagation technologies, and field validation approaches for deploying improved plant materials. Together, these components create a pipeline to deliver disease- and climate-resilient genotypes while preserving genetic diversity and supporting sustainable chestnut forestry. Figure 2 illustrates this integrated framework, summarizing the main biotechnological domains discussed in this review that collectively contribute to the development of improved chestnuts.

2. Classical Biotechnology: Controlled Hybridization

While tolerance could theoretically be achieved through conventional breeding programs, such long-term efforts have rarely been implemented for long-rotation hardwood species due to the extended generation times. As noted by Savill et al. [38], projects spanning more than 40 years have historically been considered unattractive, a sentiment echoed by Vieitez et al. [39]. European chestnut, however, stands out as one of the few hardwoods in which classical genetic improvement programs, focused primarily on increasing tolerance to P. cinnamomi, have been actively pursued. Interspecific hybridization has long underpinned European chestnut breeding, particularly for increasing resistance to ink disease and chestnut blight, and for improving timber production, nut quality, and grafting compatibility [20,21,40,41,42]. Since the early 20th century, crosses between C. sativa and Asian species (C. crenata and C. mollissima) have served as the basis for introgressing desirable traits [43,44,45,46,47].

While C. crenata shows higher resistance to diseases, its poor climatic adaptation to early frost and drought conditions, undesirable phenotypic traits, and graft incompatibility with C. sativa cultivars have limited its direct use for nut or timber production [40,46,48]. Nonetheless, C. crenata has been the main source of ink disease resistance used in Europe, and several successful hybrid clones have been derived from multiple breeding programs in France, Spain, Portugal, and Italy. These hybrids were commercialized for nut and timber production, as well as for use as rootstocks [48,49,50,51]. In contrast, C. mollissima has been particularly valued as a donor of blight resistance and, to a lesser extent, for desirable nut traits such as easy peeling and a larger seed size. Although not directly adapted to European environments, its inclusion has complemented C. crenata, enabling the development of hybrids with combined tolerance to both major pathogens and improved nut quality [45,46]. Several clones from Spanish breeding programs (111-1, 7521, 2671, and 1483) are widely used as rootstocks due to their high tolerance to ink disease and high graft compatibility with fruit varieties [22]. In Portugal, more than fifty ink-disease-resistant genotypes were selected during the first decade of the 2000s [52,53]. Following these efforts, the genetic improvement program launched at the Instituto Nacional de Investigação Agrária e Veterinária (INIAV) in 2006 focused on resistance to ink disease, given its continued negative impact on national orchards. This initiative also aimed to modernize plant materials for new plantations, especially rootstocks, to better adapt to the current climate conditions since most of the varieties available then still originated from breeding programs of the previous century [49,54,55]. Seven hybrid genotypes developed through controlled crosses (C. sativa × C. crenata/mollissima) were selected using controlled inoculations, standardized symptom scales, mortality indices, and pathogenicity tests based on Koch’s postulates. The robustness of these tests was reinforced through inoculations on biological replicates of each genotype obtained by micropropagation. The results revealed marked differences among genotypes, with some exhibiting significantly attenuated symptoms, reduced root necrosis, limited lesion progression to the collar, and higher survival rates [56]. Three of these genotypes are now registered in the Portuguese National Register of Fruit Varieties, including SC1202 (Variety RIVERA), for which CPVO (Community Plant Variety Office) protection is under review. Parallel efforts in Spain by the public company TRAGSA (Orense, Spain) selected seven new ink-disease-resistant hybrids (C003, C004, C042, C053, P011, P042, and P043) registered in the Spanish National Catalogue of Basic Plant Materials [57].

Comparable breeding progress has been achieved in France and Italy, where Euro-Asian cultivars such as ‘Bouche de Bétizac’ and ‘Primato’, rootstocks such as ‘Maridonne’ and ‘Marlhac’, and rootstocks used directly as fruit producers like ‘Marigoule’ and ‘Maraval’, combine disease resistance with improved agronomic and adaptive traits [35,58,59], with some also showing resistance to the chestnut gall wasp [35,60,61]. Outside Europe, C. mollissima and C. crenata have contributed to backcross breeding programs aimed at restoring the American chestnut [62], while Japanese breeding has focused on gall wasp resistance and nut traits such as easy peeling [63].

Despite these advances, achieving the optimal adaptation of hybrids to European climate conditions remains challenging. Recently, increased drought and heat, acting synergistically with ink disease, have exacerbated damage in southern European chestnut stands [3,64]. Under these conditions, many C. sativa × C. crenata/mollissima hybrids show poor tolerance to abiotic stresses [65,66,67]. This has stimulated new initiatives in Spain to develop genotypes resilient to multiple stressors associated with climate change [68,69,70], leading to the selection of two C. sativa clones that combine resistance to P. cinnamomi with drought and heat tolerance [37]. These clones are currently undergoing varietal registration and represent promising parental material for future breeding programs (Figure 3).

Overall, a century of classical hybridization demonstrates the potential of interspecific breeding to deliver genetic solutions for major chestnut diseases. However, the growing interaction between biotic and abiotic stresses under climate change highlights the limitations of classical approaches alone. Future advances are expected to result from combining controlled hybridization with genome-assisted selection tools to accelerate the development of resilient C. sativa genotypes adapted to Mediterranean environments.

3. Modern Biotechnological Tools

Although classical breeding has generated valuable disease-tolerant hybrids, many show certain limitations, including a reduced tree size, less desirable growth form, smaller nuts, and, as mentioned above, low tolerance to Mediterranean climatic conditions. Such drawbacks limit their suitability for timber-oriented systems or high-quality nut production, highlighting the need to balance resistance with the preservation of agronomically important traits of C. sativa. Modern forest biotechnology offers promising tools to accelerate tree improvement by integrating molecular techniques into classic breeding programs [39,71,72]. These include marker-assisted selection, genotyping for individual tree identification, the discovery of genes or alleles linked to pathogen resistance, genetic engineering approaches, in vitro culture techniques, and cryopreservation for the development, propagation, and long-term conservation of improved genotypes [3,73,74] (Figure 2). In particular, genomic selection holds great potential to substantially shorten the time required for the genetic evaluation of desirable traits in trees, thereby increasing the efficiency and precision of breeding efforts [72,75].

3.1. Perspective for Marker-Assisted Selection (MAS)

Advancements in molecular breeding must be integrated into chestnut breeding programs to support the increasing demand for elite chestnut genotypes. Thus, the application of marker-assisted selection (MAS) will be instrumental in accelerating the introgression of disease resistance and other beneficial traits, such as environmental adaptability and agronomic performance. A foundational step was the development and deployment of molecular markers capable of identifying traits of interest and serving as tools for MAS programs. In C. sativa, genomic simple sequence repeats (gSSRs) were first developed approximately 20 years ago [76,77]. Since then, numerous studies have been conducted using these markers, and they have provided the basis for MAS programs.

3.1.1. Genetic Characterization of Cultivars as a Source for Selection

Early applications of gSSRs demonstrated a robust genotyping capacity, enabling accurate varietal discrimination [78,79,80,81]. Pereira-Lorenzo et al. [82] used 24 highly polymorphic SSR markers to evaluate 271 accessions corresponding to 118 European cultivars, establishing a comprehensive European reference database for the identification and characterization of chestnut varieties.

In the Iberian Peninsula and surrounding regions, diversification and conservation of chestnut cultivars have also been explored using molecular tools. Pereira-Lorenzo et al. [83] performed a large-scale SSR-based survey on 593 grafted chestnut trees from the Iberian Peninsula, the Azores, and the Canary Islands, including trees older than 300 years. They identified 356 distinct genotypes and reported a clonality rate of 33%, reflecting the widespread use of grafting. Despite clonal propagation, high genetic variability was maintained through hybridization and mutation, which emerged as the main driver of diversification. Ten cultivar groups were defined, exhibiting strong geographic structuring and evidence of long-distance dissemination to Atlantic islands, underscoring the role of human-mediated dispersal. The importance of molecular markers in distinguishing hybrids and detecting introgression from Asian species was highlighted by Pereira-Lorenzo et al. [84]. More recently, instant domestication has been highlighted to explain how traditional practices of selection and propagation rapidly shaped chestnut diversity [85]. The introduction of new sequencing techniques enabled the transformation of Single Nucleotide Polymorphisms (SNPs) into Kompetitive Allele-Specific PCR (KASP) markers, which have been successfully evaluated for varietal discrimination [86].

3.1.2. Adaptive Diversity as a Source for Selection

Information for MAS programs has also been obtained using functional markers such as Expressed Sequence Tag–Simple Sequence Repeats (EST-SSRs), which target expressed genes and therefore provide opportunities to directly associate genetic variation with adaptive traits. Their potential lies in supporting early selection for stress tolerance and disease resistance, complementing gSSR markers. Applications of EST-SSRs have included both the characterization of cultivated germplasm and wild populations to evaluate the genetic and adaptive potential of chestnut in Europe [79,87]. Their potential has been further demonstrated through studies that revealed associations with water stress responses and tolerance to P. cinnamomi, supporting their use in the identification of drought-tolerant and disease-resistant individuals within breeding populations [68,88]. In particular, several EST-SSR loci have been linked to genes involved in key physiological and molecular mechanisms governing plant responses to abiotic and biotic stresses, including signaling pathways mediated by abscisic acid, osmotic adjustment processes, antioxidant activity, and pathogen recognition mechanisms, suggesting that they can act as indirect indicators of stress resilience [68,89,90,91].

Population-level studies have further strengthened the case for functional markers. Studies using gSSRs revealed distinct gene pools across coppice and wild populations that are shaped by geography and environmental gradients [92,93]. More recent work based on EST-SSRs expanded this approach, identifying loci potentially involved in stress responses. Castellana et al. [94] used the same EST-SSR markers to study variations across European chestnut populations and reported a potential association between the FIR059 allele and climatic variables, suggesting a role in the abiotic stress adaptation. Similarly, Dorado et al. [69] used molecular markers associated with heat stress (VIT099 and POR016) to assess tolerance within and between populations, highlighting loci under positive selection. These markers stand out as promising candidates for the early selection of heat-tolerant C. sativa individuals.

Crucially, recent work indicates that responses to abiotic and biotic stresses are not independent and that breeding for combined stress resilience requires markers that reflect those interactions. Experimental warming and infection trials revealed that thermal regimes can modulate chestnut susceptibility to P. cinnamomi and alter secondary metabolite profiles associated with defense, indicating that some genotypes respond better to combined heat and pathogen exposure than to single stresses [8]. Complementary dual-transcriptome studies have resolved early host defense pathways and pathogen effectors, providing candidate genes whose expression is modified upon infection and that may also interact with abiotic stress signaling [95]. These genes are obvious targets for EST-SSR/SNP development aimed at detecting resilience to multiple stressors.

3.1.3. QTL Mapping and Integration into MAS Pipelines

The availability of multilocus marker sets (SSRs, SNPs, and double digest restriction site-associated DNA sequencing, ddRAD-seq) has also enabled the construction of high-density genetic linkage maps, which, when combined with functional annotations of quantitative trait locus (QTL) intervals, provide a molecular basis for MAS and a platform for future genomic selection efforts. The first genetic linkage map for C. sativa [96], based on Random Amplified Polymorphic DNA (RAPD), Inter-Simple Sequence Repeat (ISSR), and isozyme markers, was constructed using the two-way pseudo-testcross strategy. This map facilitated the identification of QTLs associated with adaptive traits such as growth and water use efficiency [97]. Comparative genetic and QTL mapping between Quercus robur L. and C. sativa identified homologous genomic regions, allowing putative candidate genes for bud burst to be inferred from the colocation of EST-derived markers and QTLs [98].

Further refinement of genetic maps has been achieved through bin mapping approaches, which cluster markers into cosegregating bins to improve map resolution and reduce redundancy, facilitating high-throughput genotyping and QTL discovery [99,100]. These genomic resources are now actively translated into MAS pipelines in C. sativa breeding programs. For example, breeders can pyramid resistance alleles for ink disease and gall wasp across generations using flanking SSR or SNP markers, while markers linked to heat tolerance allow the early selection of individuals better adapted to increased temperatures. These approaches have already accelerated cultivar development, demonstrating how genomics-guided MAS complements classical breeding.

3.2. Molecular and Genomic Approaches

Progress in chestnut improvement increasingly depends on high-resolution molecular data. The effectiveness of MAS ultimately relies on identifying the genes, pathways, and regulatory networks underlying resistance and adaptation. These resources support the discovery of actionable targets for future breeding and biotechnological interventions.

Extensive research aimed at understanding the genetic basis of resistance and susceptibility to chestnut’s main biotic stresses has accelerated in response to the urgent need to safeguard chestnut forests and orchard health. Advances in genetics, cell and molecular biology, bioinformatics, and complementary insights from histopathology and physiology have produced a robust foundation of knowledge, largely driven by high-throughput sequencing technologies. Over the past two decades, these efforts have generated extensive genomic resources that support more effective, timely disease management and breeding strategies.

Building on this foundation, the following section integrates and summarizes current findings from transcriptomic, proteomic, and metabolomic studies, QTL mapping, molecular marker development, and whole-genome sequencing. It provides a comprehensive overview of the molecular and genomic underpinnings of chestnut defense against P. cinnamomi, C. parasitica, and D. kuriphilus, with a particular focus on European chestnut and its hybrids. Key candidate genes and pathways are highlighted, along with the broader genetic architecture shaping chestnut responses to these biotic stresses.

3.2.1. Molecular Mechanisms of Castanea Defense Against Phytophthora cinnamomi

• Constitutive and inducible defense responses

Transcriptomics, gene expression profiling and hormone studies have revealed that resistance and tolerance to P. cinnamomi in C. sativa and its hybrids with Asian chestnuts involve a complex interplay of constitutive and inducible defense mechanisms, including gene expression regulation, biochemical barriers, and cellular responses [91,95,101,102].

Pre-formed, constitutive defenses represent a key component of plant immunity, acting as the first barrier against pathogen invasion. In resistant C. crenata, high basal expression of defense-related genes, including receptor-like kinases (RLKs) and antifungal proteins (Cast_Gnk2-like), may fortify the root environment and limit P. cinnamomi colonization. In contrast, susceptible C. sativa shows lower constitutive expression of those genes, facilitating early pathogen ingress [102]. Additionally, reduced levels of pattern recognition receptors (PRRs), such as BAK1 orthologs, may impair pathogen-associated molecular patterns (PAMPs) and delay immune responses [95]. Overall, weak constitutive defenses appear to underlie C. sativa susceptibility.

Time is a pivotal factor in Castanea spp. responses to P. cinnamomi. Resistant C. crenata rapidly activates cellular defenses within 0.5–2 h after inoculation [103], largely reflecting constitutive gene expression rather than induction, as indicated by high basal levels of resistance-related genes [102] and minimal transcriptional changes after infection [95]. In contrast, C. sativa shows an early but transient defense response, with gene expression declining at 48–72 h. During this period, P. cinnamomi shows higher expression of genes encoding elicitins/elicitin-like (oomycete PAMPs) and necrosis-inducing-like proteins (NLPs) in C. sativa than in C. crenata [95]. This temporal mismatch enables the pathogen to bypass initial plant defenses and deploy effectors that suppress host immunity, promoting disease progression. Sustained and timely defense responses thus emerge as critical for resistance, while their collapse in C. sativa underpins susceptibility.

Resistant C. crenata mounts a rapid and robust defense against P. cinnamomi characterized by the upregulation of pathogenesis-related (PR) and antifungal protein genes. In contrast, C. sativa displays delayed and reduced expression of these genes, correlating with severe symptoms and higher mortality [101,102]. Using digital PCR, Santos et al. [102] analyzed eight candidate genes across resistant, susceptible, and hybrid genotypes, revealing clear genotype-dependent patterns. Remarkably, Cast_Gnk2-like exhibited high constitutive expression and rapid induction in C. crenata following P. cinnamomi inoculation, whereas C. sativa showed low basal and induced levels. This positions Cast_Gnk2-like as a potential marker of resistance.

Cast_Gnk2-like shares homology with the ginkbilobin-2 (Gnk2) gene from Ginkgo biloba, which encodes a cysteine-rich repeat secreted protein with a DUF26 domain conferring antifungal activity [104]. Gnk2 has been linked to Programmed Cell Death (PCD) [105] induction and functions as a lectin with high affinity for D-mannose [106], a carbohydrate present in P. cinnamomi cell walls, potentially explaining its inhibitory effect on pathogen growth. Based on this result, Cast_Gnk2-like is hypothesized to exert a direct antifungal effect [107], prompting studies to validate its function in C. sativa and other Fagaceae species via overexpression in susceptible genotypes (see Section 3.4.1) [108,109,110].

Comparative transcriptomic studies show that resistant genotypes like C. crenata upregulate a wider spectrum of genes involved in pathogen perception, signal transduction, transcription factor activation, and the biosynthesis of defense-related metabolites [95,101]. In contrast, C. sativa exhibits a narrower set of differentially expressed genes (DEGs), and lower expression of key defense markers upon P. cinnamomi inoculation. Proteomic analyses reinforce these trends, revealing reduced abundances of proteins linked to salicylic acid (SA) signaling, reactive oxygen species (ROS) metabolism, and cell wall reinforcement in infected C. sativa tissues [111].

• Hormones involved in inducible defense responses

Plant hormones play a crucial role in modulating immune responses to pathogens. The induction of SA-mediated responses results in the activation of PR proteins, cell wall reinforcement, and hypersensitive response (HR), all of which are key for resistance to hemibiotrophic pathogens [112]. Moreover, the interplay between SA and other hormones such as jasmonic acid (JA), abscisic acid (ABA), and ethylene (ET) is critical for fine-tuning the immune response. ABA often antagonizes SA-mediated responses.

Recent studies using physiological and biochemical methods indicate that resistance to P. cinnamomi in C. sativa × C. crenata genotypes is associated with early and robust SA signaling in roots, the antagonism of ABA signaling, stable primary and secondary metabolism, and transient oxidative stress followed by recovery [91]. In contrast, susceptible C. sativa genotypes involve delayed and weak JA signaling, a lack of SA induction, high ABA accumulation in leaves, impaired carbohydrate and secondary metabolism, and fluctuating oxidative stress without a sufficient antioxidant response. Supporting this finding, gene expression analyses suggest that SA signaling, involving transcription factors such as Cast_WRKY31 and Myb-related protein 4 (Cast_Myb4), is activated more rapidly in resistant than in susceptible genotypes [102].

Expression profiling of susceptibility (S) genes during P. cinnamomi infection provided further evidence supporting the delayed activation of SA-mediated defenses in C. sativa [113]. Specifically, powdery mildew resistance 4 (pmr4, encoding a callose synthase) and downy mildew resistance 6 (dmr6, repressor of the SA pathway) are rapidly upregulated upon infection, correlating with the suppression of SA-dependent defenses and increased host susceptibility. In contrast, C. crenata shows no significant upregulation of these genes, supporting its resistance.

The C. crenata allene oxide synthase gene (CcAOS), a key component of the JA pathway, was identified from transcriptome data [101] due to its strong induction in inoculated C. crenata compared to C. sativa. A functional analysis confirmed its relevance in defending against P. cinnamomi: the overexpression of CcAOS in the susceptible Arabidopsis ecotype Ler-0 delayed pathogen progression and increased tolerance [114].

• Reinforcement of cell wall defenses

Cell wall reinforcement through phenolic compound accumulation is a key defense mechanism against P. cinnamomi. Transcriptomic data indicate that genes involved in lignin biosynthesis, structural proteins, and cell wall-modifying enzymes, including pectinesterase 2 (Cast_PE-2) and the TF Cast_Myb4, are expressed at lower levels in C. sativa than in C. crenata [95,102]. Resistant genotypes (C. crenata and C. sativa × C. crenata) rapidly upregulate these genes after pathogen detection, promoting cell wall thickening. Studies at the cellular level support this timing and reveal these temporal differences, with phenolic compound accumulation occurring at 30 min in C. crenata versus 72 h in C. sativa after inoculation [103].

Although C. sativa can accumulate callose around intracellular hyphae within 24 h after inoculation [103], this response, which is possibly mediated by the S gene pmr4 [113], is ineffective. Delayed and insufficient structural defenses permit extensive P. cinnamomi colonization and tissue necrosis, as evidenced by larger infection areas [103] and higher pathogen levels in C. sativa [113]. Moreover, the lack of sustained activation of enzymes responsible for the cell wall cross-linking further increases C. sativa’s vulnerability [95].

• ROS-mediated mechanisms and other phenolic compounds

Rapid ROS production is one of the hallmark responses of plants to pathogen attack, as ROS serve both as direct antimicrobial agents and as secondary signals that activate further defense responses. In resistant C. crenata, transcriptomic data suggest a pronounced ROS burst that may trigger HR, a rapid, localized form of pathogen-induced cell death that restricts pathogen spread [95,103]. In C. sativa, ROS generation may be transient, with limited expression of respiratory burst oxidase homolog protein B (RBOHB) and increased detoxification by antioxidant enzymes, thereby reducing the antimicrobial effects. The upregulation of negative regulators of PCD, such as BON1-associated protein 2-like (BAP2-like), further suppresses HR, enabling P. cinnamomi’s transition from the biotrophic to the necrotrophic phase. This weak and short-lived ROS response, coupled with the premature suppression of HR, likely contributes to the inability of C. sativa to restrict pathogen proliferation.

Dorado et al. [8] studied C. sativa defense mechanisms against P. cinnamomi under warming scenarios. Their findings indicate that resistance relies on both morphological traits (growth and root biomass) and the accumulation of phenolic compounds with antioxidant and antimicrobial properties, namely, quercetin 3-O-glucuronide, 3-feruloylquinic acid, gallic acid ethyl ester, and ellagic acid. Plants previously exposed to moderate warming exhibited greater resilience to the pathogen, whereas those cultivated under normal or heatwave conditions were more susceptible. Plants that survived the infection showed elevated levels of the four metabolites, highlighting their role in the adaptive responses to combined heat and pathogen stress.

• Ubiquitin-mediated regulation

The ubiquitin/26S proteasome system plays a pivotal role in regulating plant immunity by degrading proteins involved in hormone signaling and defenses [115]. It can also inhibit pathogen effectors by triggering PAMP- and ETI-mediated responses [115]. Transcriptomic analyses reveal that C. crenata substantially upregulates the 26S proteasome regulatory subunit 4 homolog A and several proteases after P. cinnamomi inoculation, unlike C. sativa [95]. The lack of such modulation in C. sativa may allow negative regulators to persist, weakening defense responses. Although the specific components in chestnut remain unclear, the evidence highlights the importance of post-translational modifications in fine-tuning the balance between resistance and susceptibility during P. cinnamomi infection.

• Marker development and QTL mapping

Leveraging available genomic resources, including transcriptomic data [101], Santos et al. [89] developed 43 novel EST-SSR markers from DEGs linked to host responses to infection. These markers exhibited high amplification success and interspecific transferability across four Castanea species. Their average expected heterozygosity (0.61) exceeded previous reports for chestnut EST-SSRs, reinforcing their value for genetic diversity and breeding studies.

A major milestone in C. sativa breeding programs was the development of the first interspecific genetic linkage map for C. sativa × C. crenata [116]. Built with 452 SSRs and SNPs and spanning 498.9 cM, this map enabled the identification of QTLs for ink disease resistance on linkage groups E and K, overlapping with QTLs from American × Chinese chestnut populations [117] and suggesting conserved defense mechanisms against P. cinnamomi. Notably, QTLs on group E colocalized with defense-related genes, including those putatively encoding PR proteins (NDR1/HIN1-like protein 3), phospholipid transporters, transcriptional regulators (RNA polymerase II-associated factor 1, PAF1 homolog), and epigenetic modulators (zinc-finger PHD-type) [116].

Genetic linkage maps and the identification of QTLs associated with ink disease resistance have revealed the polygenic nature of this trait in chestnut. These studies not only provided evidence of the genetic architecture underlying ink disease resistance in chestnut but also identified molecular markers with strong potential for MAS.

The identification of QTLs associated with ink disease would greatly benefit from genotyping-by-sequencing (GBS), which offers a cost-effective approach for generating high-density, genome-wide markers essential for accurate QTL mapping. By enabling the identification of genomic regions linked to phenotypic traits, GBS facilitates the discovery of markers for MAS, accelerating the introgression of favorable alleles into elite genotypes [118]. GBS is particularly valuable for perennial, outcrossing species such as C. sativa, where the large genome size, high heterozygosity, and long generation cycles hinder conventional breeding. Through high-resolution genetic mapping, GBS supports the development of improved chestnut varieties with enhanced disease resistance and adaptability.

Table 1. Biotechnological tools used to understand chestnut responses to Phytophthora cinnamomi, with a focus on Castanea sativa (susceptible) and Castanea crenata (resistant).

Methodology	Species	Main Findings	References
Comparative transcriptomics	C. sativa C. crenata	C. crenata upregulates genes involved in pathogen perception, signaling, transcription factors, defense metabolites and proteome regulation. C. sativa shows limited and transient gene expression.	[95,101]
Gene expression profiling	C. sativa C. crenata C. sativa × C. crenata	C. crenata shows high basal and induced expression of PR genes (e.g., RLKs and Cast_Gnk2-like), enabling early defense activation. C. sativa presents lower expression, allowing rapid pathogen colonization.	[95,102] in accordance with the results in [103]
Histopathology and cellular studies	C. sativa C. crenata	C. crenata responds more rapidly and efficiently than C. sativa; pathogen growth is restricted by the early activation of callose deposition, HR-like cell death, cell wall thickening, and the accumulation of phenolic-like compounds.	[103] in accordance with the results in [95,113]
Validation of gene function	C. sativa C. dentata Quercus ilex Quercus suber Arabidopsis thaliana	Cast_Gnk2-like is relevant in Castanea and Quercus defenses; CcAOS increases tolerance in A. thaliana Ler-0.	[108,109,110,114] corroborated by results in [102]
Proteomics	C. sativa	Upon infection, C. sativa downregulates proteins involved in SA signaling.	[111] in accordance with the results in [95,101]
Physiological and biochemical assays	C. sativa C. sativa × C. crenata	C. sativa × C. crenata shows early SA signaling, ABA antagonism, and oxidative stress recovery. C. sativa shows delayed JA signaling, high ABA levels, impaired metabolism, and a weak antioxidant response.	[91] in accordance with the results in [102]
Analysis of susceptibility gene expression	C. sativa C. crenata	C. sativa upregulates pmr4 and dmr6 early in the infection, putatively contributing to suppressing SA defenses; putative callose accumulation induced by pmr4 is not sufficient to restrict pathogen growth.	[113] in accordance with the results in [103]
Metabolite analysis	C. sativa	Moderate warming enhances C. sativa resilience to the pathogen. Surviving plants accumulate key phenolics (e.g., quercetin 3-O-glucuronide and ellagic acid), contributing to defenses.	[8]
Molecular marker development	C. sativa C. crenata	Forty-three EST-SSR markers were identified from DEGs associated with host responses to infection.	[89] supported by the results in [101]
Genetic mapping	C. sativa × C. crenata	Interspecific linkage mapping enabled the detection of QTLs for pathogen resistance on linkage groups E and K that colocalized with defense-related genes.	[116] supported by the results in [89]

ABA—abscisic acid; CcAOS—allene oxide synthase; DEGs—differentially expressed genes; dmr6—downy mildew-resistant 6; EST-SSR—Expressed Sequence Tag–Simple Sequence Repeats; HR—hypersensitive response; JA—jasmonic acid; pmr4—powdery mildew-resistant 4; PR—pathogenesis-related; QTL—quantitative trait locus; RLKs—receptor-like kinases; SA—salicylic acid.

summarizes the methods used to gain key insights into the responses of C. sativa and C. crenata to P. cinnamomi infection.

3.2.2. Molecular Mechanisms of Castanea Defense Against Cryphonectria parasitica

Chestnut blight poses a significant threat to chestnut species worldwide. While the American chestnut is highly susceptible, the European chestnut displays relatively lower susceptibility and, in some cases, tolerance. Among Asian species, the Chinese chestnut is considered more resistant to blight than the Japanese chestnut, which is attributed to a combination of rapid immune response activation, efficient pathogen recognition, and robust structural defenses [119].

• Castanea sativa: partial tolerance or susceptibility

European chestnut can be susceptible to C. parasitica, particularly to virulent strains that cause extensive necrosis and canker formation. Infection occurs mainly through wounds or bark fissures and is often facilitated by environmental stressors such as drought or mechanical damage. Once established, the pathogen forms mycelial fans that physically disrupt tissues and secrete hydrolytic enzymes (laccases, cellulases, and cutinases) to degrade host structures. It also produces phytotoxic metabolites (skyrin, rugulosin, diaportin, and nitrogen-containing compounds), causing rapid tissue necrosis, especially in apical shoots and leaves, with mortality observed within 8 days [120]. Host defenses, including cell wall lignification and wound periderm formation, are largely ineffective against fungal progression. Occasional recovery in certain genotypes or under favorable conditions reflects tolerance rather than resistance [121], allowing survival despite persistent infection and canker development.

The physiological and biochemical responses of C. sativa to C. parasitica include a reduction in photosynthetic pigments and increased activity of antioxidant enzymes such as ascorbate peroxidase (APX), guaiacol peroxidase (POD), and superoxide dismutase (SOD). Additionally, infected tissues accumulate stress markers like proline—an osmolyte that stabilizes proteins and scavenges ROS—and malondialdehyde, an indicator of lipid peroxidation under oxidative stress [122].

• Hypovirulent fungal strains in Europe carrying Cryphonectria hypovirus 1

Despite C. sativa susceptibility to C. parasitica, the presence of hypovirulent fungal strains and the use of biological control agents like Cryphonectria hypovirus 1 (CHV1) have proven effective at reducing disease severity in Europe [123]. These strains exhibit reduced growth, sporulation, and virulence enzyme activity (e.g., laccase), causing only superficial necrosis that may heal through callus formation, enabling the compartmentalization of the infection [120,124]. Inoculation trials with C. sativa × C. crenata hybrids revealed improved tolerance [125,126]. These studies confirmed that while the hybrids can exhibit enhanced tolerance and genotype-dependent recovery, C. sativa remains susceptible, confirming the lack of innate immunity.

Chitinases, key hydrolytic enzymes targeting chitin in the fungal cell wall, are important inducible defense proteins in plants [127]. In C. sativa, gene and protein expression studies showed the systemic induction of chitinases and β-1,3-glucanases in response to infection, with higher activity in trees inoculated with hypovirulent strains [128,129]. Three of four chitinases [130] purified from C. sativa inhibited hyphal growth in vitro, with hypovirulent strains being more susceptible than virulent strains, suggesting that reduced fungal growth may be related to vulnerability to host chitinases. Additionally, an endochitinase-like protein, Ch3, from C. sativa cotyledons showed antifungal activity [131], and its gene was isolated for further validation as a resistance gene [132]. These findings indicate that hypovirulence-associated viruses may enhance host recognition or defense responses.

Pavese et al. [113] performed gene expression profiling of C. sativa inoculated with C. parasitica. They reported that the upregulation of S genes such as pmr4 and dmr6 may trigger stress-related pathways that downregulate SA-mediated defenses, potentially facilitating pathogen progression. Additionally, PR proteins such as chitinases and glucanases showed strong induction upon fungal inoculation.

SA plays a central role in plant defenses, particularly in activating pathogen resistance genes [133]. Biochemical studies in C. sativa show SA accumulation after inoculation with both virulent and hypovirulent strains of C. parasitica, with higher levels observed in response to infections with hypovirulent strains, suggesting either an enhanced host response or suppression by virulent strains. Similar patterns in chitinase gene expression have been observed [129]. Transcriptomic data from C. dentata and C. mollissima (detailed ahead) confirm increased SA-related gene expression in canker tissue, supporting its role in defense across species [134]. SA signaling also involves the activation of defense genes, regulation of cell death, and antagonism/fine-tuning of JA-ET pathways, functioning as both a host defense mechanism and a potential target for pathogen manipulation [133,135].

Overall, C. sativa exhibits partial and often ineffective defenses against C. parasitica. Fungal suppression of host responses appears to underlie its limited resistance compared to Asian chestnut species. While hypovirulent strains provide some potential for disease control, the species remains susceptible without external measures or genetic improvement.

• Castanea mollissima: robust resistance

C. mollissima has co-evolved with chestnut blight in its native range, developing genetically encoded resistance mechanisms. It employs both constitutive and inducible defenses, such as a rapid wound response, cell wall lignification, and the activation of resistance genes upon infection. These traits are supported by genomic studies in the sequenced genome of the ‘Vanuxem’ cultivar, which have identified candidate resistance genes, supporting its use in restoration breeding programs in North America and Europe [136].

The reference genome of C. mollissima comprises over 36,000 gene models and extensive transcriptomic data, facilitating the identification of selection signatures and resistance loci distinguishing it from susceptible species such as C. dentata and C. sativa [136]. Moreover, high genetic diversity across natural populations, especially in the Qinling–Daba Mountains, highlights these regions as reservoirs of resistance genes [137].

The complete chloroplast genome of wild C. mollissima, comprising 131 genes linked to stress responses and metabolic regulation, has been sequenced. Phylogenetic analyses confirm its close relationship with other resistant Fagaceae species, reinforcing its value as a genetic donor in breeding programs [138].

• The role of the OxO gene in blight tolerance

The stark contrast between C. sativa and C. mollissima has driven interspecific hybridization efforts in Europe, aiming to introgress blight resistance traits from the latter into the former (as described in Section 2). However, phenotyping for blight tolerance in hybrids between C. sativa and Asian species has largely focused on ink disease tolerance rather than chestnut blight.

On the other hand, because blight is most severe in American chestnut, decades of efforts in the US have concentrated on breeding programs crossing C. dentata with C. mollissima [139]. These initiatives are far more focused than those targeting pathogens in Europe. Furthermore, genomic studies indicate that blight resistance is a multigenic, quantitative trait, supported by genetic mapping that identified resistance loci across all 12 chromosomes [62,140].

Simultaneously, the transformation of C. dentata with the wheat oxalate oxidase gene (OxO) represents a complementary strategy to the labor-intensive backcross breeding approach. Transgenic C. dentata lines expressing OxO have shown promising results by degrading oxalic acid secreted by C. parasitica, thereby reducing lesion severity [141]. These trees are undergoing regulatory review, with a draft review by the US Department of Agriculture Animal and Plant Health Inspection Service (USDA-APHIS) concluding that they are unlikely to pose a plant pest risk [142], while the Environmental Protection Agency (EPA) and Food and Drug Administration (FDA) still need to complete their reviews. Approval would mark a major milestone for transgenesis in ecological conservation. Unlike the US, European legislation imposes strict limitations on the cultivation of transgenic plants. Discussions are ongoing regarding the use and commercialization of plants produced through genome editing (see Section 4.5), which means that transgenics cannot be considered a viable option to accelerate chestnut breeding programs.

• Transcriptomic and genomic insights

Barakat et al. [143] used high-throughput pyrosequencing to compare transcriptomes of blight-susceptible American chestnut and blight-resistant Chinese chestnut following C. parasitica inoculation. RNA from healthy and infected stem tissues generated over a million reads and tens of thousands of unigenes per species. The study revealed numerous stress- and defense-related genes, including transcription factors (e.g., WRKY, zinc finger, and MYB), enzymes (e.g., cinnamoyl-CoA reductase and alpha-dioxygenase), and transporters that were more highly expressed in canker tissues.

C. mollissima exhibited a broader and more robust transcriptional response, consistent with its higher resistance, while functional annotation revealed similar overall gene function distributions between species, with subtle differences in categories such as transcription factor activity and the stress response.

Building on previous work, Barakat et al. [134] showed that chestnut genes exhibit greater similarity to those of woody plants rather than herbaceous species. A comparative transcriptome analysis identified hundreds of genes with differential expression between cankered and healthy stem tissues, many of which were linked to defense-related pathways, such as cell wall biosynthesis, ROS production, hormone signaling (SA, JA, ET, and ABA), HR, and PCD. While HR and PCD are generally associated with immunity against biotrophs, necrotrophic pathogens often exploit host cell death generated by HR to facilitate infection [144]. Moreover, molecular and genetic studies indicate that HR and PCD are not universally coupled to resistance, as their components can be activated independently, and resistance to necrotrophs may occur with minimum cell death [145].

Peroxidase activity, particularly that of Class II peroxidases, has been implicated in the host response to C. parasitica, with increased transcript levels observed in the canker tissue of C. mollissima [134]. These secreted plant enzymes, which are involved in one-electron oxidation and peroxide production, play roles in pathogen defenses, namely, in auxin metabolism, cell wall reinforcement, lignin and suberin synthesis, and the regulation of reactive oxygen and nitrogen species [127,146,147]. Four distinct peroxidases have been identified in Castanea stem and leaf tissues [148], though their enzymatic properties remain poorly characterized. The evidence is conflicting: Castanea pumila Mill., a blight-susceptible species, showed higher peroxidase transcript abundance than the resistant C. mollissima [149], and infection was found to reduce peroxidase activity in both C. dentata and C. mollissima, with a smaller decrease in the latter [148]. These findings suggest possible pathogen-mediated suppression of host peroxidase activity, but their precise roles in blight resistance remain unclear due to the lack of follow-up studies.

Barakat et al. [134,143] generated valuable genomic resources and identified candidate genes and networks linked to resistance to C. parasitica. While gene expression profiles in canker tissues were broadly similar between C. dentata and C. mollissima, notable differences emerged: C. dentata exhibited increased activation of housekeeping genes, whereas C. mollissima showed a stronger induction of PR transcripts. The authors hypothesized that blight tolerance may depend more on the rapidity of the host response than on the presence of specific defense-related genes.

Nie et al. [150] performed transcriptomic profiling of the resistant wild genotype ‘HBY-1’ at 0, 3, and 9 h after inoculation with C. parasitica using Illumina sequencing. The analysis revealed that 9 h post-infection is a critical point for defense activation. Two hundred eighty-three DEGs were identified and clustered into metabolism-related pathways (secondary metabolite and phenylpropanoid biosynthesis, and photosynthesis) and defense-related pathways (plant–pathogen interaction and MAPK signal transduction). These pathways were interconnected via phosphatidylinositol, phytohormone, and α-linolenic acid signaling. Notably, genes involved in JA biosynthesis were significantly upregulated, indicating early activation of the JA pathway. The study highlights that C. mollissima mounts a rapid and coordinated defense involving hormone signaling, pathogen recognition, and metabolic reprogramming.

Recently, Westbrook et al. [139] employed an integrative approach combining genomics, transcriptomics, and statistical modeling to accelerate the restoration of American chestnut with improved resistance to blight and ink disease. They identified candidate resistance and susceptibility genes, many colocalizing with known blight QTLs. New whole-genome sequencing of C. dentata (‘Ellis’) and C. mollissima (‘Mahogany’) supported genome-wide association studies (GWAS), revealing four potential resistance genes, including a chitinase, and 26 susceptibility candidates for targeted genome editing.

• Metabolomic insights

The metabolomic dynamics governing the interaction between C. parasitica and Castanea species remain largely unexplored. The review by Lovat and Donnelly [119] provides a comprehensive analysis of the mechanisms and metabolites involved in the interaction between chestnuts and C. parasitica.

Chestnut cell walls contain compounds related to lignin barriers and wound periderm that contribute to blight defense. Tannins, a diverse group of polyphenolic compounds including phenolic acids, flavonoids, and sugars, play key roles in defense, antioxidant activity, and structural integrity [151]. Their interaction with C. parasitica is complex; while tannase activity can increase fungal growth in tannin-rich media [152,153], species-specific tannin profiles appear to influence disease resistance. Asian chestnuts like C. mollissima contain higher levels of vescalagin and castalagin, which may inhibit fungal growth more effectively than hamamelitannin, which is prevalent in susceptible C. dentata and C. sativa [119]. Histological and hormonal studies suggest that esterase activity and JA signaling modulate tannin availability and the response to infection [154,155]. Tannins may also inhibit fungal enzymes such as polygalacturonase, underscoring their complex roles in chestnut blight pathogenesis [156]. Overall, tannins are pivotal in host–pathogen interactions, acting both as substrates and modulators of fungal virulence.

A synthesis of the strategies and key findings for understanding chestnut responses to C. parasitica is presented in

Table 2. Biotechnological tools used to understand chestnut resistance to Cryphonectria parasitica, with a focus on C. sativa (tolerant/susceptible), C. dentata (susceptible), and C. mollissima (resistant).

Methodology	Species/Genotypes	Results/Findings	References
Physiological and biochemical responses	C. sativa	Reduced levels of photosynthetic pigments; increased levels of APX, POD, and SOD; accumulation of proline and MDA.	[122]
Biological control through CHV1 hypovirus	C. sativa	Mitigation of disease severity via hypovirulent strains.	[123]
Chitinase and β-1,3-glucanase expression	C. sativa	Systemic induction; higher activity with hypovirulent strains; antifungal activity of Ch3 protein.	[128,129,130,131]
Susceptibility gene expression profiling	C. sativa	Upregulation of pmr4 and dmr6; suppression of SA-mediated responses.	[113]
SA accumulation studies (metabolite and transcriptome analysis)	C. sativa C. dentata C. mollissima	Higher SA levels with hypovirulent strains; SA-related gene expression in canker tissue.	[129,134]
Genomic and transcriptomic studies	C. mollissima ‘Vanuxem’	Identification of resistance genes; rapid wound response and cell wall lignification.	[136]
Chloroplast genome sequencing	Wild C. mollissima	A total of 131 genes are involved in stress responses and metabolic regulation.	[138]
Genetic mapping and GWAS	C. dentata × C. mollissima	Resistance loci were identified on all chromosomes; candidate resistance and susceptibility genes were identified.	[62,139,140]
Transgenic OxO expression	C. dentata	Oxalate oxidase degrades oxalic acid from the pathogen; field trials and regulatory review are ongoing.	[141,157]
Transcriptome comparison via pyrosequencing	C. dentata vs. C. mollissima	A stronger and faster defense response was observed in C. mollissima and involved TFs, enzymes (e.g., peroxidases), and PR proteins; expression of housekeeping genes in C. dentata.	[134,143]
Transcriptomic profiling	Wild C. mollissima ‘HBY-1’	C. mollissima shows a rapid and coordinated defense involving hormone signaling (relevance of JA), pathogen recognition, and metabolic reprogramming.	[150]
Tannin profiling (metabolite analysis)	C. sativa C. mollissima C. dentata	Prevalence of hamamelitannin in C. sativa and C. dentata. Higher vescalagin and castalagin levels in C. mollissima, resulting in the efficient inhibition of fungal enzymes. Esterase activity and JA signaling modulate tannin availability.	[119,154,155,156]

APX—ascorbate peroxidase; ch3—chitinase; CHV1—Cryphonectria hypovirus 1; DEGs—differentially expressed genes; dmr6—downy mildew-resistant 6; GWAS—genome-wide association study; JA—jasmonic acid; MDA—malondialdehyde; OxO—oxalate oxidase; TFs—transcription factors; ROS—reactive oxygen species; SA—salicylic acid; ET—ethylene; ABA—abscisic acid; HR—hypersensitive response; PCD—programmed cell death; PR—pathogenesis-related; pmr4—powdery mildew-resistant 4; POD—guaiacol peroxidase; SA—salicylic acid; SOD—superoxide dismutase.

.

3.2.3. Molecular Mechanisms of Castanea Defense Against Dryocosmus kuriphilus

Identifying resistant genotypes is essential to understand chestnut–pest interactions. Resistance to D. kuriphilus has been documented in the hybrid Bouche de Bétizac (C. sativa × C. crenata). Early studies reported no infestations for 3 years [158], and this resistance was later confirmed after nearly a decade of observations [35]. Although the wasp can still lay eggs in the buds, larvae fail to develop beyond the first instar, most likely due to HR triggered by the hybrid. Dini et al. [159] demonstrated this using diaminobenzidine (DAB) staining to detect H₂O₂ accumulation in vivo, which indicates the activity of stress-response glycoproteins germin and germin-like proteins (GLPs). GLPs, which have OxO activity, are associated with PCD and HR. Bouche de Bétizac buds exhibited strong DAB staining, whereas the susceptible cultivar Madonna (C. sativa) showed no staining, regardless of the infestation state. Furthermore, high expression of a putative GLP during early budburst in Bouche de Bétizac reinforced the presence of HR. This response was also proposed in preliminary transcriptomic studies; using a differential display analysis, Botta et al. [160] identified differentially expressed bands between Bouche de Bétizac and the susceptible cultivar Marrone (C. sativa), including sequences putatively encoding resistance-related proteins, mitogen-activated proteins, vesicle-associated membrane proteins, and 14-3-3 proteins.

The molecular basis of resistant and susceptible responses was further investigated by Acquadro et al. [161] using a transcriptomic analysis of buds from Bouche de Bétizac and Madonna. The two assemblies contained 34,081 and 30,605 unigenes, respectively. Bouche de Bétizac unigenes were functionally characterized, whereas the Madonna assembly was used mainly for RNA-seq data analysis. This work identified 1444 putative resistance gene analogs (RGAs) and approximately 1135 unigenes predicted as miRNA targets. Global transcriptome profiling revealed significantly enriched Gene Ontology terms, particularly the response to stimulus and developmental processes (e.g., post-embryonic development). Upregulated genes included approximately 60 genes predicted to encode leucine-rich repeat (LRR) proteins, along with several transcriptional regulators, such as 6 APETALA2/Ethylene (AP2/ERF) and 16 WRKY (e.g., WRKY33). A putative homolog of the RAV1 transcription factor, which is typically a negative regulator of growth, was also upregulated and suggested to participate in the developmental adaptation to gall-induced stimuli [161]. Additional upregulated genes included protein regulators (e.g., regulatory-associated protein of TOR 1b; RAPTOR1B), storage proteins (e.g., late embryogenesis abundant protein D29; LEA D29), and more than 100 genes associated with death and apoptosis processes, including those involved in HR [161], reinforcing earlier findings [159]. This study also produced valuable genomic resources, including the first reference unigene catalog for European chestnut and approximately 7k SSR and 335k SNP/INDEL markers.

Another breakthrough in understanding the genetic resistance of Bouche de Bétizac came from high-density mapping of interspecific hybrids. A large-effect QTL, Rdk1, was mapped to linkage group K, explaining 67–69% of the phenotypic variance [60]. Twenty-six candidate genes were located within this region, including metacaspase-1b and a receptor of the resistance to Peronospora parasitica 13 locus (RPP13) subfamily. Both genes are known to be involved in HR.

In addition to Bouche de Bétizac, Sartor et al. [35] identified six other resistant cultivars, including two C. sativa: the Italian cultivar ‘Pugnenga’ and the French cultivar ‘Savoye’. This is particularly significant, as it suggests the possibility of transmitting resistant traits within the species. More recently, resistance was also detected in natural C. sativa populations in Greece [61]. GWAS revealed a small region on pseudochromosome 3 (Chr3) associated with high resistance that contains 12 candidate genes, including members of the Cytochrome P450, UDP-glycosyltransferase, and Rac-like GTP-binding protein families. Twenty-one SNPs within this region were identified, representing promising markers for MAS in breeding programs.

Insights from other chestnut species also inform European chestnut research and breeding programs. Zhu et al. [162] suggested that the peroxidase pathway may contribute to resistance in a partially resistant Chinese chestnut variety and identified four transcription factors (CmbHLH130, CmWRKY31, CmNAC50, and CmPHL12) as potential regulators.

Although further research is needed to clarify the genetic basis of chestnut responses to gall wasp, current evidence consistently highlights oxidative stress signaling as a central mechanism. Several candidate resistance genes have been identified, providing a strong foundation for future functional studies. Moreover, the recent publication of a high-quality D. kuriphilus reference genome [163] provides new opportunities to investigate the pest’s molecular weapons and the associated host transcriptional responses during infestation. These advances will support a more comprehensive understanding of chestnut–gall wasp interactions and inform the development of IPM strategies beyond the current reliance on biological control with T. sinensis, including genetics-based methods [164] and next-generation approaches such as RNA interference (RNAi) [165].

Table 3. Biotechnological strategies used to understand European chestnut resistance to Dryocosmus kuriphilus.

Methodology	Species/Genotypes	Results/Findings	References
Biological control Torymus sinensis	Various (wild and cultivated)	Effective at reducing infestations, but the pest continues to spread	[18,33,34]
Phenotypic resistance screening	C. sativa C. crenata Euro Japanese hybrids	7 resistant cultivars were identified: C. sativa ‘Pugnenga’ & ‘Savoye’; C. crenata ‘Idae’; Hybrids ‘BB’, ‘Marlhac’, Maridonne’, and ‘Vignols’	[35,158]
Histochemistry and gene expression	‘BB’ (R) vs. ‘Madonna’ (C. sativa, S)	The detection of H2O2 accumulation and strong GLP expression in the R hybrid was linked to HR	[159]
RNA-seq transcriptome analysis	‘BB’ (R) vs. ‘Madonna’ (C. sativa, S)	1444 RGAs and 1135 miRNA targets; upregulation of LRRs, WRKYs, AP2/ERFs, RAV1, LEA D29, and RAPTOR1B; identification of HR-related genes	[161]
	C. mollissima ‘Shuhe Wuyingli’ (PR) vs. ‘HongLi’ (S)	The peroxidase pathway was implicated; 4 TFs were identified (CmbHLH130, CmWRKY31, CmNAC50, and CmPHL12)	[162]
Genomic resources development	C. sativa	Reference unigene catalog; ~7k SSRs and 335k SNP/INDELs were identified	[161]
QTL mapping	Interspecific hybrids ‘BB’ × ‘Madonna’	The Rdk1 locus explains 67–69% of resistance variance; candidate genes include metacaspase-1b and RPP13 receptor	[60]
GWAS	Greek C. sativa provenances (R)	Region on Chr3 with 12 candidate genes (cytochrome P450, UDP-GT, and Rac-like GTPases); 21 SNPs were identified	[61]
Genome sequencing	D. kuriphilus (pathogen)	High-quality reference genome published; enables host-pest interaction studies	[163]

BB—‘Bouche de Bétizac’; GLP—germin-like protein; GWAS—genome-wide association study; HR—hypersensitive response; INDEL—insertion deletion; PR—partially resistant; QTL—quantitative trait loci; R—resistant; RGAs—resistance gene analogs; S—susceptible; SNP—single nucleotide polymorphism; SSR—simple sequence repeat; TF—transcription factor.

provides an overview of the strategies used to understand chestnut resistance to the chestnut gall wasp.

3.2.4. Whole Genome Sequencing

Whole genome sequencing (WGS) has become a cornerstone in advancing molecular knowledge of defense mechanisms in chestnut species, particularly in response to pathogens, pests, and climate change. By providing comprehensive insights into the genetic architecture of chestnut trees, WGS enables the identification of resistance-associated genes and regulatory elements that govern responses to biotic and abiotic stressors.

Genome assemblies differing in completeness are now available for four chestnut species. For C. sativa, two assemblies have been published: one for the cultivar ‘Marrone di Chiusa Pesio’ using Oxford Nanopore and Illumina technologies [166] and another for the Anatolian cultivar ‘Sarı Aşılama’ [167]. The genome of C. mollissima has been sequenced for multiple cultivars, including ‘Vanuxem’, a donor of blight resistance in C. dentata restoration breeding [136]. For C. crenata, a chromosome-level genome assembly has revealed conserved chromosomal segments and a large repertoire of protein-coding genes, supporting its known resistance to diseases and pests [168]. The links to access C. dentata genome data are https://phytozome-next.jgi.doe.gov/info/Cdentata_v1_1 (accessed on 14 November 2025) and https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1148431 (accessed on 14 November 2025).

These genomic resources facilitate comparative analyses to identify candidate genes under selection and enable MAS for resistance traits. WGS also supports landscape genomic approaches, mapping adaptive genetic variation across environmental gradients and predicting genomic offset under future climate scenarios [169]. An example in progress is the restoration of C. dentata in the US, where genome-enabled breeding programs are incorporating adaptive diversity into backcross populations, ensuring that restored American chestnut trees are not only disease-resistant but also ecologically viable across diverse habitats [139]. This program results from a collaborative effort between non-profit research organizations, state research institutions, federal agencies, and universities fully dedicated to improving C. dentata. Unfortunately, a comparable coordinated initiative for C. sativa has not been feasible in Europe, despite multiple attempts to secure funding for integrated research among Spanish, Portuguese, Italian, and U.S. groups. Efforts to disseminate C. sativa research and attract investment from chestnut producers and related companies have been made, but so far have yielded very limited financial support.

Importantly, WGS provides the foundation for genome editing technologies, such as CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9), by enabling the precise identification of target genes and regulatory sequences. The availability of C. sativa reference genomes significantly increases the accuracy and efficiency of such editing methodologies (detailed in Section 3.4.2), allowing for targeted modifications that improve disease resistance, stress tolerance, and other agronomic traits. However, C. sativa reference genomes still require higher resolution to enable accurate comparisons of complete gene sequences between resistant and susceptible species. At the transcriptional level, coding sequences are highly similar, and many defense mechanisms are shared. Therefore, a focus on non-coding regions (e.g., promoters) is essential, as subtle differences in these sequences may influence gene expression. Identifying these cis-regulatory differences could provide targets for gene editing to modulate expression in C. sativa toward the patterns observed in resistant species.

In summary, WGS provides a foundational platform for integrating molecular genetics with ecological and evolutionary frameworks, accelerating both traditional breeding and cutting-edge genome editing approaches to develop resilient chestnut populations capable of withstanding current and emerging challenges.

3.3. Micropropagation Techniques

The ability to clonally propagate C. sativa and its hybrids is essential for preserving and multiplying wild, elite, and conventionally bred genotypes. Micropropagation in chestnut relies primarily on two approaches: axillary budding and somatic embryogenesis (SE). Axillary budding is currently the only technique suitable for large-scale commercial production and is used to generate uniform plant material for field trials, graft compatibility studies, and physiological or molecular assays. In contrast, SE, although not yet commercially deployable, offers major advantages for genetic improvement, cryopreservation, and as a regeneration platform for genetic transformation and genome editing.

3.3.1. Axillary Budding Micropropagation

The initial objective of micropropagation techniques in European chestnut was to develop efficient axillary budding systems for the clonal propagation of trees selected for tolerance to P. cinnamomi. This method was conceived as a complementary alternative to traditional ground-layering. European chestnut is a recalcitrant species, particularly with respect to rooting, a difficulty that persists even under in vitro conditions. To overcome these limitations, Spanish researchers initially optimized their methodology using juvenile plant material, successfully establishing a reliable micropropagation system [170]. This protocol was later adapted for mature plant material, which in some cases required rejuvenation techniques (e.g., etiolation or repeated grafting) to elicit a suitable in vitro response [171,172,173]. Since then, numerous studies and reviews have been published on the in vitro propagation of European chestnut and its hybrids via axillary bud micropropagation [5,174,175,176,177,178].

Axillary budding micropropagation has become a strategic tool for rapidly disseminating superior chestnut genotypes with enhanced P. cinnamomi tolerance, selected from European breeding programs. In Spain, a robust protocol capable of cloning any European chestnut genotype was validated and transferred to the private sector, enabling commercial-scale production [179] (Figure 4). For example, TRAGSA Company (Orense, Spain) has recently produced 40,000 plants in its first full-scale campaign to assess market capacity for increased volumes. Production in the ongoing campaign is expected to reach 50,000–60,000 plants, with plans to scale up to 100,000 hybrid chestnut plants in 2025. Meanwhile, CULTIGAR Company (A Coruña, Spain)has produced an average annual output of 58,000 plants in recent seasons.

In Portugal, micropropagation is central to producing homogeneous experimental material for physiological, molecular, and pathogenicity studies [56,95,102], as well as for multiplying rootstocks tolerant to P. cinnamomi [49] (return to Section 2 for details on registered new chestnut varieties). To support the transition from the laboratory to the field, a pilot facility was established in Marvão (a traditional chestnut-growing region in North Alentejo) to produce thousands of clonal plants with ensured uniformity, traceability, and phytosanitary quality. Field trials are currently evaluating new genotypes exposed to natural infections of P. cinnamomi for vegetative vigor, survival, productivity, and health. These results are crucial for assessing the commercial viability of new materials and informing technical recommendations to producers. In parallel, graft compatibility studies are ongoing to evaluate rootstock–scion interactions to ensure compatibility with traditional, commercially valued varieties. This integrated approach protects regional genetic heritage while introducing resilience into production systems.

Factors such as plant vigor, rooting uniformity, and acclimatization performance were significantly improved, marking a major step toward the large-scale, energy-efficient production of elite European chestnut genotypes. A recent advancement is the use of LED-based systems, which have emerged as powerful tools to further enhance chestnut micropropagation. Along this line, Marino et al. [180] demonstrated that specific LED spectra substantially improved both the multiplication and rooting phases, doubling the proliferation index and achieving 100% rooting success compared to conventional fluorescent lighting.

3.3.2. Somatic Embryogenesis

SE has been established in European chestnut and its hybrids, although current protocols remain unsuitable for commercial application. Nevertheless, SE has proven invaluable for developing cryopreservation, genetic transformation, and gene editing methodologies (Figure 5). The first well-documented report of true somatic embryo induction and subsequent plant regeneration from immature zygotic embryos of Castanea sativa × C. crenata hybrids was published by Vieitez et al. [181], and later studies confirmed SE in both hybrid (C. sativa × C. crenata) and pure C. sativa genotypes [182,183,184,185,186].

• Somatic Embryogenesis Induction

SE induction typically involves a two-step culture protocol: (1) an induction phase using high auxin concentrations and (2) an expression phase with low or no plant growth regulators (PGRs). Among auxins, 2,4-dichlorophenoxyacetic acid (2,4-D) is most commonly used and considered essential for initiating embryogenic cultures. It is often supplemented with low concentrations of cytokinins such as benzyladenine (BA), kinetin, or zeatin to support embryo development. Sezgin and Dumanoğlu [184] described an alternative induction system using indole-3-butyric acid (IBA) with thidiazuron (TDZ).

Induction efficiency strongly depends on the developmental stage of the zygotic embryo, which is linked to the timing of collection. In northwest Spain, embryos are most competent for SE when collected at 6 to 12 weeks post-anthesis (late July to early September). Both whole zygotic embryos and cotyledonary segments have been used as explants, with embryonic axes exhibiting twice the induction efficiency of cotyledonary pieces [182]. The induction percentages vary by the genotype of the mother tree and collection date, ranging from 2% in hybrids [181,186] to 11.1% in C. sativa [182,185].

Reliable SE induction from non-zygotic tissues remains challenging, hindering its effective use in the large-scale propagation of known genotypes. Successful induction from such explants has only been achieved in European chestnut by Corredoira et al. [182,187]. Somatic embryos were induced from the most apical leaves excised from proliferating shoot cultures using a three-step protocol: (1) culture on medium with 4 mg/L naphthaleneacetic acid (NAA) and 0.5 mg/L BA, (2) subculture onto medium with the concentrations of both PGRs reduced to 0.1 mg/L, and (3) transfer to a PGR-free expression medium. Unlike SE induction using zygotic embryos, 2,4-D and IBA were ineffective in these explants. Induction rates were considerably lower than those from zygotic embryos and did not exceed 1% [182]. However, adding 2 mg/L larch wood extract, a compound rich in arabinogalactan proteins (AGPs), substantially improved responses, increasing SE induction to 5.3%, the highest value reported for non-zygotic explants in European chestnut [188].

• Plant regeneration from somatic embryos

A major bottleneck in European chestnut SE, as in many other woody species, is efficient plant regeneration, namely, the simultaneous development of both shoot and root systems from somatic embryos. Several factors influence somatic embryo maturation and conversion into viable plantlets, including osmotic agents, pre-germination treatments, and PGRs [74]. The source and concentration of carbon are critical. Among the tested sugars, 3% maltose yielded the highest recovery rate (39%), including 6% full plantlet conversions and 33% of explants developing only shoots [187]. Increasing the agar concentration in the maturation medium to 1.1% also improved maturation and conversion (10–25%) in embryogenic lines derived from zygotic embryos [183]. ABA, which is commonly used to promote maturation in other embryogenic systems, proved largely ineffective in chestnut [186].

Direct transfer of somatic embryos from maturation to germination media often results in poor germination and abnormal plantlets, making pre-germination treatments such as cold storage, desiccation, or gibberellic acid (GA₃) application necessary to improve conversion [74]. Two months of cold storage increased conversion rates up to 38.9% in C. sativa lines [187] and 29–32% in hybrid material [186,189]. Fast desiccation for 2 h reduced moisture to approximately 57% and increased plant quality [190]. Similarly, Sezgin & Dumanoğlu [185] achieved 40% plant regeneration after cold storage followed by slow desiccation using a saturated salt solution.

During germination, somatic embryos are generally cultured on media supplemented with low cytokinin concentrations, alone or in combination with auxins. The combination of 0.1 mg/L BA with 0.1 mg/L NAA or IBA yielded the highest conversion rates [184,190]. Plantlet development was further improved by adding 200–438 mg/L glutamine or 150 µM Fe-Na-EDTA to the germination medium [186,190].

Despite substantial progress, SE in chestnut remains limited by strong genotype effects, low induction frequencies (particularly in non-zygotic tissues), and incomplete or asynchronous shoot and root development, restricting its use for large-scale propagation (Figure 5). Advancing SE requires a deeper understanding of the molecular mechanisms regulating embryogenesis. In Chinese chestnut, several marker genes are associated with SE induction [191], including Agamous-Like 11 (CmAGL11), which appears to regulate gibberellin, auxin, and ethylene pathways during SE [192]. These molecular insights can inform more efficient induction systems and accelerate chestnut genetic improvement and germplasm innovation.

3.4. Genetic Engineering Strategies

A fundamental premise for the successful production of transgenic plants is having an in vitro regeneration system capable of developing plants from cells, organs, or tissues susceptible to infection by Agrobacterium tumefaciens. Traditional genetic transformation and modern genome editing technologies have been employed in European chestnut for research and breeding using SE (Figure 5). Despite its limitations, SE remains the most effective regeneration system, providing higher transformation efficiency and reducing the occurrence of chimeric plants compared with other systems [74,182]. Remarkably, C. sativa stands out as one of the first forest tree species where gene editing has been successfully implemented, representing a significant advance in the genetic improvement of woody plants.

3.4.1. Agrobacterium-Mediated Genetic Transformation

The first genetic transformation of European chestnut was reported by Seabra and Pais [193] using Agrobacterium-mediated transformation of hypocotyl segments from in vitro-germinated seedlings. They used strain LBA 4404 carrying the binary vector p35SGUSINT, which included the neomycin phosphotransferase (nptII) gene for kanamycin (kan) resistance and the reporter gene uidA for β-glucuronidase (GUS) activity. Although transgenic shoots were regenerated, molecular analyses revealed that many were chimeric.

Stable genetic transformation was later achieved using somatic embryos as explants and the same marker genes [194,195]. Early experiments determined that the transformation efficiency was significantly influenced by Agrobacterium strain/plasmid combinations and the co-cultivation time [194]. The highest transformation efficiency (25%) was obtained with strain EHA105 harboring plasmid pUbiGUSINT after 4 days of co-cultivation.

Follow-up studies investigated the effects of acetosyringone, bacterial density, the somatic embryo genotype, and developmental stage [195,196]. Unexpectedly, acetosyringone reduced the transformation efficiency. All bacterial densities yielded kan-resistant embryos, with the highest efficiency (20.1%) obtained with Agrobacterium in the exponential growth phase (OD₆₀₀ ≈ 0.6), though the differences between densities were not statistically significant. Both the developmental stage and the genotype of somatic embryos strongly influenced transformation success. Globular and heart-shaped embryos, as well as embryo clumps, exhibited higher transformation frequencies (up to 30%) than cotyledonary-stage embryos, likely due to greater proliferation potential and proportion of actively embryogenic cells [197]. Genotype-dependent variation was also evident: among the seven lines tested, two showed relatively high transformation efficiencies (21.7% and 33.8%), while the others ranged from 1.7% to 10%.

Optimized transformation protocols enabled the introduction of candidate resistance genes identified in molecular studies (Section 3.2). Two PR genes—thaumatin-like protein (CsTL1) [198] and chitinase (CsCh3) [131]—were successfully integrated into three C. sativa somatic embryo genotypes [132,199]. These studies generated transgenic lines suitable for downstream functional validation under greenhouse and field conditions to assess the effectiveness of these genes against Phytophthora spp. and/or C. parasitica infection. Several lines carried low transgene copy numbers, and GFP-based transgene activity was detected across tissues, with no pleiotropic effects observed (Figure 5). CsTL1 was strongly overexpressed in some lines, reaching up to 13.5-fold higher expression than the non-transformed counterpart [199]. Using the same transformation system, twelve C. sativa lines carrying the Cast_Gnk2-like antifungal gene were generated. Transgene copy number and relative expression were analyzed in highly proliferative lines [107], and four were selected for P. cinnamomi tolerance assays. One transgenic line showed significantly improved tolerance compared to the non-transformed control, exhibiting reduced root necrosis and overall disease symptoms [200].

Adapted transformation and regeneration systems have also enabled Cast_Gnk2-like functional validation studies in other Fagaceae species susceptible to P. cinnamomi: C. dentata [107,110], Quercus ilex [108], and Q. suber [109]. Tolerance assays for C. dentata are ongoing, while oak plantlets overexpressing Cast_Gnk2-like appear to exhibit improved tolerance compared to non-transformed plantlets. Further supporting its role in conferring tolerance, the recombinant protein expressed in Escherichia coli showed activity against P. cinnamomi in pathogenicity tests, validating its potential as a protective agent and providing perspectives for bioactive compounds in integrated disease management [201].

Collectively, these studies highlight the value of traditional genetic transformation for functional validation, the elucidation of resistance mechanisms, and the development of innovative protection tools for Fagaceae species. Moreover, in those studies, the introduced gene belongs to the same species or a closely related family. Consequently, the resulting plants cannot be considered strictly transgenic; they may instead be classified as cisgenic or intragenic.

Transgenic plant regeneration was consistently low, irrespective of the genotype. These low conversion rates significantly limit the number of plantlets available for downstream analyses. However, some somatic embryos undergo partial germination, producing shoots without roots. These shoots can be leveraged through axillary shoot proliferation and subsequently induced to root, allowing for the propagation of an unlimited number of transgenic plantlets [202]. This method provides sufficient plant material for essential molecular validation and disease resistance assays. Although improving the direct conversion efficiency of somatic embryos into complete plantlets remains a key objective, axillary shoot culture is currently the only reliable and scalable strategy for multiplying transgenic chestnut lines for research and functional evaluation.

3.4.2. New Plant Breeding Techniques

New Plant Breeding Techniques (NPBTs) enable precise genome modifications without introducing foreign genes [203]. Among NPBTs, CRISPR/Cas9 has emerged as the most widely adopted system due to its versatility and ability to target multiple loci simultaneously [204].

An advance over standard CRISPR/Cas9 delivery is DNA-free genome editing using RNP complexes [205]. In conventional vector-based systems, Cas9 can remain active for a long time, increasing the risk of off-target mutations. In contrast, RNPs act only for a short time before being degraded by the cell, which limits nuclease exposure and prevents the stable integration of foreign DNA [203]. RNPs can be delivered through particle bombardment, protoplast electroporation, or polyethylene glycol (PEG)-mediated uptake, but their use, along with CRISPR/Cas9 in general, remains limited in woody species [206].

• CRISPR/Cas9 Genome Editing in Castanea sativa

The first successful CRISPR/Cas9 application in European chestnut targeted the phytoene desaturase (pds) gene, establishing a proof-of-concept for genome editing in this recalcitrant woody species [207]. Edited embryogenic lines regenerated into plantlets displaying the expected albino phenotype, and sequencing confirmed small insertions and deletions at the target site. This study demonstrated that CRISPR/Cas9 can be effectively applied in C. sativa using SE-derived tissues (Figure 5).

More recently, this system has been extended to genes of agronomic relevance. In particular, susceptibility genes are being targeted to improve tolerance to P. cinnamomi [113] and to develop chestnut genotypes with enhanced disease resistance, improved nut quality, and better stress tolerance.

• DNA-Free Genome Editing Using Ribonucleoproteins

Pavese et al. [208] developed the first protocol for DNA-free genome editing in European chestnut using pre-assembled Cas9–sgRNA RNP complexes. In this system, RNPs targeting pds were directly delivered into protoplasts (derived from somatic embryos), enabling transient Cas9 activity without stable integration of recombinant DNA (Figure 5). Successful editing of the target gene was confirmed by sequencing, demonstrating that RNP-mediated editing is feasible in C. sativa. Because RNPs are rapidly degraded by the cell, this method reduces the risk of off-target mutations and enables the generation of edited plants free of transgene sequences [203]. This could potentially ease public acceptance compared to traditional transgenics and be an advantage for regulatory approval, providing new opportunities for breeding programs.

The main limitation is still regeneration: producing whole plants from edited protoplasts is a major bottleneck for C. sativa and many other woody species [209]. Integrating RNP delivery with improved SE-based regeneration strategies is crucial to fully exploit DNA-free genome editing for the traits of interest.

3.5. Germplasm Conservation Through Cryopreservation

Cryopreservation offers a reliable strategy for the long-term conservation of chestnut germplasm by storing biological tissues in liquid nitrogen (LN) [210]. This approach is crucial for conserving genetic diversity, particularly in species with recalcitrant seeds that cannot be preserved through conventional extended storage, like chestnut species. Beyond safeguarding wild genetic resources, cryopreservation also enables the stable storage of genetically modified embryogenic lines while introduced or modified genes are functionally evaluated.

As a result of these investigations, cryopreservation of European chestnut can effectively complement traditional field genebanks and has already been successfully applied to zygotic embryos, somatic embryos, and in vitro axillary shoot tips [174] (Figure 5).

3.5.1. Cryopreservation of Zygotic Embryos

Corredoira et al. [211] developed a highly efficient protocol for zygotic embryos, identifying the moisture content as a critical factor influencing post-cryopreservation survival. The study involved isolating embryonic axes and subjecting them to various desiccation periods (up to 7 h) before immersion in LN. The highest survival (100%) and plant regeneration (63%) rates were obtained when the moisture content was approximately 20% after 5 h of desiccation. A comparable whole-plant regeneration rate (64%) was later reported by Gaidamashvili et al. [212] using an encapsulation–vitrification protocol with activated charcoal. However, this method is significantly more complex and labor-intensive than the desiccation process.

3.5.2. Cryopreservation of Somatic Embryos

Significant progress has been made in recent years in the cryopreservation of somatic embryos in woody species, including European chestnut [210]. A vitrification-based protocol combining a 3-day preculture on 0.3 M sucrose medium, Plant Vitrification Solution 2 (PVS2) treatment [213] for 60 min at 0 °C, and rapid immersion in LN enabled 68% recovery of globular and heart-stage embryo clumps. These embryos remained stable after 10 years of cryostorage (Corredoira, unpublished data), confirming the method’s long-term reliability. This approach has been successfully used for transgenic embryogenic lines carrying CsTL1 [199] and CsCh3 [132], with embryo recovery rates up to 92%, underscoring its value for the long-term preservation of genetically modified material. In contrast, desiccation-based cryopreservation resulted in only 33% of embryo recovery, highlighting the greater efficiency of vitrification-based techniques for cryopreserving chestnut embryogenic tissues [199,211]. Somatic plantlets derived from genetically modified cultures can be rigorously field-tested for multiple years while the cultures remain in cryostorage, reducing maintenance, contamination risks, and labor costs.

3.5.3. Cryopreservation of Shoot Tips

In vitro shoot tips have also been cryopreserved after optimizing several parameters [214]. The most effective protocol used 0.5–1.0 mm shoot tips cold-hardened for two weeks and precultured for two days in 0.2 M sucrose medium, followed by a two-step vitrification process (incubation in the loading solution for 20 min and then in modified PVS2 for 120 min) before immersion in LN. This method resulted in 38–54% shoot recovery across five chestnut clones (of both juvenile and mature origin), with successful plant regeneration in all cases. The same protocol enabled the establishment of the first European chestnut germplasm cryobank for P. cinnamomi-tolerant genotypes, a collaboration between the Spanish National Research Council and the public company TRAGSA [215]. Of the 46 tested genotypes, 43 survived cryopreservation, although only 63% maintained the shoot regeneration capacity.

These advances position cryopreservation as a promising yet still underutilized tool for the long-term conservation of European chestnut.

4. Conclusions and Future Perspectives

4.1. Climate Change and Chestnut Vulnerability

Climate change is impacting European chestnut populations by altering phenology, increasing susceptibility to drought and heat, and affecting growth, fruit quality, and survival. Warmer and wetter conditions also favor the spread of diseases and pests, increasing tree vulnerability. In this context, biotechnological tools such as in vitro culture, genetic transformation, omics-based approaches, gene editing (e.g., CRISPR/Cas9), and cryopreservation provide a powerful and complementary toolkit for modern breeding. These approaches enable the precise introduction of disease resistance, characterization of genetic diversity, conservation, and targeted enhancement of key agronomic traits, ultimately contributing to resilient, productive, and sustainable cultivars.

4.2. Propagation and Conservation Strategies

Micropropagation through axillary bud proliferation is a well-established technique in European chestnut and is widely applied at a commercial scale. Spanish companies such as TRAGSA and CULTIGAR currently produce thousands of plants annually, primarily hybrid genotypes, using this approach. Moreover, efforts to bridge laboratory findings with field deployment are exemplified by the Pilot Facility in Marvão, Portugal, which supports the practical validation of new genotypes under natural conditions. Somatic embryogenesis is considered a key tool for mass propagation and advanced breeding programs. However, the development of embryogenic systems from non-zygotic material, especially mature genotypes, will require considerable effort to optimize. Progress has relied on empirical testing of culture media, growth conditions, donor age, and explant type; future advances will depend on identifying novel small chemical promoters to enhance cellular reprogramming and regeneration [216]. Cryopreservation protocols for European chestnut, which were successfully developed for multiple explant types, offer significant potential for long-term germplasm conservation. Despite high efficiency, these methods are not routinely applied, and their practical implementation remains challenging. Establishing cryobanks should therefore be prioritized, especially by public institutions, to safeguard biodiversity under the pressures of climate change. This approach not only provides a long-term secure storage strategy but also reduces the vulnerability of germplasms to pests, diseases, and environmental hazards affecting field collections.

4.3. Functional Analysis and Genetic Improvement

Traditional Agrobacterium-mediated transformation has enabled genes to be introduced into embryogenic tissues to study disease resistance and stress tolerance. More recently, CRISPR/Cas9 gene editing offers precise modifications of endogenous genes without foreign DNA insertion, providing a socially acceptable alternative. Although still in early stages for chestnut, these tools hold promise for accelerating breeding and functional validation of candidate genes. Together, traditional transformation and genome editing form a comprehensive platform for European chestnut genetic improvement and research.

4.4. Omics and High-Throughput Phenotyping

Advances in genomics, transcriptomics, and metabolomics have deepened the understanding of European chestnut biology, enabling the identification of genes linked to disease resistance and the adaptation to abiotic stress. Integrating multiomics with high-resolution genomes and high-throughput phenotyping (HTP) will reveal defense mechanisms and guide improvement strategies. Besides the contribution to ongoing efforts in MAS for chestnut controlled and expedited breeding, the expanding repository of genetic and functional information creates new opportunities for applying gene editing tools with greater precision and efficiency by targeting multiple well-characterized genes. Consequently, genome editing approaches that combine resistance allele stacking in breeding strategies with the modification of susceptibility genes will accelerate the development of elite chestnut varieties adapted to the challenges of climate change, with enhanced biotic stress resilience, productivity, and sustainability.

New chestnut varieties that result from improvement programs and are resistant to pests and diseases need to be tested for susceptibility to climatic extremes (i.e., heat waves, prolonged droughts, and late frosts) to evaluate genotype resilience in the context of climate change projections. For HTP, UAV (unmanned aerial vehicle)-based multispectral imaging, automated classification, and root imaging techniques [e.g., X-ray computed tomography (CT), magnetic resonance imaging (MRI) and ground-penetrating radar (GPR)] combined with AI offer scalable, non-destructive alternatives. Coupling aerial and root HTP with genomic tools will accelerate the selection of genotypes combining pathogen/pest resistance and drought resilience, which is critical for climate adaptation.

4.5. Regulatory and Socio-Economic Considerations

European legislation imposes strict limits on the cultivation of transgenic plants. Discussions on cisgenic plants produced via genome editing are ongoing, and future EU regulations on New Genomic Techniques will determine the commercial viability of edited chestnuts [217]. DNA-free editing methods, such as RNP-mediated protoplast transfection, will be essential for compliance. Socio-economic factors, including propagation efficiency, production costs, and monitoring, will influence adoption. Complementary strategies such as dsRNA-based protection and microbial consortia may further enhance orchard resilience.

The convergence of biotechnology, omics, and advanced phenotyping positions European chestnut breeding at the forefront of innovation. Coordinated efforts in research, regulation and technology transfer will be key to developing resilient, productive and sustainable chestnut orchards adapted to climate change and emerging threats.