Overcoming breeding barriers with genome editing in autopolyploid crops

Abstract

Autopolyploid crops play a central role in global agriculture, yet their complex genomes pose significant barriers to genetic improvement. High allelic diversity, extensive redundancy, and polysomic inheritance impede both conventional breeding and the implementation of modern biotechnological tools. Genome editing offers a powerful alternative by enabling precise, multi-allelic modification of traits associated with yield, quality, and stress resilience. However, progress across autopolyploid crops remains uneven due to low transformation and regeneration efficiencies, limited genomic resources, and challenges in achieving complete allele disruption. This review focuses on recent advances in genome editing across four economically important autopolyploid crops—potato (Solanum tuberosum), alfalfa (Medicago sativa), sugarcane (Saccharum spp.), and blueberry (Vaccinium corymbosum). We highlight the diversity of traits targeted through CRISPR/Cas systems, including reporter and selectable marker validation, tuber and forage quality, biomass composition, stress tolerance, flowering modulation, and plant regeneration. We also describe technical constraints affecting genome editing in autopolyploids including genotype-dependent recalcitrance, low transformation and editing efficiency, multiallelic targeting and chimerism, outlining emerging solutions such as multiplexed designs, endogenous promoters, morphogenic regulators and virus-based approaches, among others. Together, these developments provide a path toward efficient and heritable genome editing in complex polyploid genomes, setting the stage for next-generation precision breeding in crops vital to food, forage, and bioenergy security.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08862-7.

Background

Global climate projections and crop production models predict notable declines in yield in the coming decades, raising concerns for food and nutritional security [1, 2]. While conventional breeding, including selective breeding and hybridization, has contributed significantly to crop improvement, modern biotechnological tools, including transgenesis and genome editing, offer additional opportunities to address these challenges and accelerate genetic gains [3, 4]. These approaches have been widely applied in diploid species, yet their applications in polyploid crops continue to face substantial barriers that require tailored and more efficient solutions.

Polyploidy is defined as a whole genome duplication (WGD), resulting in more than two sets of chromosomes, which can be derived from hybridization, unreduced gametes or synthetic generation [5]. Although polyploidy occurs in animals and unicellular organisms, this phenomenon is prevalent in plants [6]. Polyploidy offers various advantages compared to their diploid counterparts; as a result of the extra gene copies, polyploids present a buffer effect against deleterious alleles, contributing to the transition to self-pollination, an increase in genetic diversity and gene neofunctionalization, promoting their adaptation to harsher environments [5–8].

Polyploids can be classified based on the nature of the WGD into allopolyploids and autopolyploids. Allopolyploids result from interspecific hybridization, whereas autopolyploids arise from genomic duplications within a single species [9]. Allopolyploids exhibit preferential chromosomal pairing, often originating from well-documented interspecific hybridization, facilitating breeding and genomic-based approaches. In contrast, breeding strategies in autopolyploid crop species can be more difficult due to their polysomic inheritance, mostly as a consequence of a non-preferential pairing during meiosis that can result in abnormal segregation patterns [6, 9]. Likewise, performing genome-based approaches in autopolyploid crops, such as genome assembly, annotation, or genome editing, is more challenging due to high gene redundancy and highly similar homologous chromosomes [10].

Autopolyploid crops, namely potatoes (Solanum tuberosum), alfalfa (Medicago sativa), sugarcane (Saccharum spp.) and blueberries (Vaccinium corymbosum), are cultivated worldwide, with annual productions ranging from 50 to over 2,000 million tons [11]. These crops serve as essential sources of carbohydrates, forage, biofuel, and beneficial health-related compounds for human and animal consumption, as well as for industrial applications. Traditional breeding efforts have primarily focused on improving superior agronomic and quality-related traits, increasing yields, and enhancing resistance to biotic and abiotic stresses. Yet, breeding for these autopolyploid crops is constrained by high heterozygosity, extensive allelic diversity, and frequent chromosomal rearrangements [12–15]. Additionally, hybridization can promote inbreeding depression, limit genetic gains, and reduce fitness due to the accumulation of deleterious mutations as observed in potatoes and alfalfa [12, 13, 16, 17].

In this context, genome editing has emerged as a reliable breeding method with transformative potential in sustainable agriculture [18, 19]. While traditional breeding can disrupt favorable allelic combinations and introduce thousands to millions of random mutations, carefully designed genome editing techniques can precisely target single or multiple DNA locations [20, 21]. Notably, genome editing offers an efficient alternative for accelerating trait improvement in autopolyploid crops that would otherwise take multiple breeding cycles under traditional breeding methods. Although significant progress has been made in genome editing applications for model crop species [22, 23], advancements in autopolyploid crops have lagged due to their complex genomes, lower transformation and regeneration efficiencies, and, in some cases, limited genomic resources [24, 25].

This review summarizes recent advancements in genome editing across four major autopolyploid crops that are essential to both small- and large-scale agricultural systems worldwide. Although genome editing has been applied to a broad range of agronomic and biological traits, progress varies substantially across species: potato accounts for most published studies, followed by alfalfa, and sugarcane, whereas blueberry remains largely underexplored, with only a few proof-of-concept reports. This review also highlights the application of genome editing strategies for improving traits of agronomic and biological significance for these crops. It addresses key challenges that limit editing efficiency and discusses emerging strategies and technologies that may facilitate more efficient genome editing in these complex species. Together, these insights support the development of next-generation precision breeding approaches for autopolyploid species of global importance.

Overview of genome editing systems

Genome editing relies on programmable nucleases to introduce double-strand breaks in DNA, which are then repaired by either non-homologous end joining or homology-directed repair, with the latter allowing precise sequence replacement [26, 27]. Early genome editing tools, including Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs), combined engineered DNA-binding proteins with nucleases to target specific sites in the genome [28, 29]. While ZFNs and TALENs were effective, their complexity and high cost limited their widespread use. The emergence of the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein (CRISPR/Cas) system, first reported in plants in 2013 [30], revolutionized genome editing by replacing protein-based DNA recognition with a programmable RNA-guided mechanism. Its simplicity, efficiency, and ability to target multiple loci quickly made CRISPR/Cas the dominant genome-editing platform. In the widely used CRISPR/Cas9 platform, genome editing relies on two components: the Cas9 nuclease and a single guide RNA (sgRNA), which fuses the CRISPR RNA (crRNA) and trans‑activating crRNA (tracrRNA) into a single transcript [31]. The sgRNA–Cas9 complex guides Cas9 to a complementary genomic site with a 20-nucleotide sequence, requiring a 5′-NGG protospacer adjacent motif (PAM) for target recognition, where Cas9 then introduces a double-strand break [30]. The discovery of diverse Cas orthologs, including Cas12 and Cas13, has greatly expanded its capabilities, enabling alternative protospacer adjacent motif (PAM) recognition, RNA targeting, and multiplexed editing across diverse plant species [32–34].

More recent advancements allow precise modifications without generating double-strand breaks (DSBs). Among them, Base Editors (BEs) convert single nucleotides (C→T or A→G) using a modified Cas-deaminase fusions [35] and Prime Editors (PEs) introduce targeted substitutions, insertions, or deletions via a Cas9 nickase–reverse transcriptase fusion [33, 36]. Recently, Virus-Induced Genome Editing (VIGE) has allowed the delivery of CRISPR components transiently through viral vectors such as Potato virus X, enabling DNA-free, systemic editing without tissue culture [37, 38]. Together, these approaches have expanded the genome editing toolbox, offering versatile strategies for functional genomics and precision breeding in crops where conventional methods face limitations.

Precision breeding applications in autopolyploid crops through genome editing

Genome editing has been successfully applied in autopolyploid crops despite the inherent complexities imposed by multiple chromosome sets. Genome editing pipelines across the four autopolyploid species show that potato has the most advanced system, supported by well-established regeneration protocols from leaves, protoplasts, and stem tissues (Table 1). This flexibility enables multiple transformation methods, including PEG-mediated protoplast transfection, Agrobacterium, biolistic delivery, and vacuum infiltration, and the routine use of multiple editing platforms such as Ribonucleoproteins (RNPs), TALENs, CRISPR/Cas9, and CBE. Alfalfa also has a well-established pipeline, with several responsive explants including leaves, cotyledons, germinated seedlings, petioles, and efficient Agrobacterium-based transformation; additional enhancements such as ultrasonic-assisted delivery have been reported to further improve editing outcomes. In contrast, sugarcane relies almost exclusively on embryogenic callus for regeneration; although Agrobacterium and biolistic transformation are feasible, the recovery of uniformly edited plants is hindered by high chimerism and the extreme genomic complexity of the crop. Blueberry remains restricted to regeneration from leaf explants and axillary buds, and its Agrobacterium- mediated transformation remains inefficient and strongly genotype-dependent.

Table 1.

Summary of the status of the genome editing pipeline in the autopolyploid crops reported in this study

Crop	Regeneration (Explants)	Transformation (Delivery methods)	Genome Editing (System Used)
Potato	Well established (leaf discs, protoplasts, stem internodes)	Well established (PEG-mediated protoplast transfection, Agrobacterium, biolistic, vacuum infiltration)	Well established (RNP delivery, TALENs, CRISPR/Cas9, CBE)
Alfalfa	Well established (leaf, cotyledon, germinated seedlings, petiole, leaflet/foliole)	Well established (Agrobacterium and Rhizobium)	Well established (CRISPR/Cas9, CBE)
Sugarcane	Well established (embryogenic callus)	Established with limitations (Agrobacterium and biolistic transformation)	Established with limitations (TALENs, CRISPR/Cas9)
Blueberry	Established (leaf and axillary buds)	Not well established (Agrobacterium transformation)	Emerging/Not well established (CRISPR/Cas9)

Overall, advances in CRISPR/Cas systems, base editing, and transcriptional regulation have expanded opportunities for marker validation, improve productivity, nutritional quality, food safety, sustainability, and resilience in these species (Table 2, Supplementary Tables 1, and Fig. 1). While the advancements are relative for each of the crops, some traits are common across species, while others are more species-specific.

Table 2.

Genome editing implementation for technical validation and trait improvement in autopolyploid crops

Crop	Breeding Goal/Technical Objective	Gene Targets	Genome Editing Method	Outcome/Impact	References
Potato	Validate editing reagents and allele targeting	StPDS, StF3H, StALS, StGWD1, StDMR6-1	CRISPR/Cas9*, CBEµ	Pigment-altered phenotypes; herbicide-resistant lines; demonstration of locus-dependent editing efficiency	[44–50]
	Food safety	StVInv, StAS1	CRISPR/Cas9, TALENs ß	Reduced acrylamide formation	[51–55]
	Amylose-free starch	StGBSSI, StSBE (1, 2, 3)	CRISPR/Cas9, CBE	Amylose-free starch	[45, 56–62]
	Toxicity reduction	StSSR2, St16DOX	CRISPR/Cas9, TALEN	Glycoalkaloid reduction	[63–65]
	Enzymatic browning reduction	StPPO2	CRISPR/Cas9	Reduced enzymatic browning	[66]
	Stress resilience	StMYB44, StAOX	CRISPR/Cas9	Enhanced stress tolerance with improved photosynthesis and phosphate intake	[67, 68]
	Pathogen resistance	StCERK1, StNPR3, StCCoAOMT, StSR4, StERF3, StNRL1, StDMR6-1, StMLO, StDND1, StHDS, StTTM2, StCHL1	CRISPR/Cas9	Enhanced pathogen resistance	[69–76]
	Virus resistance	StCOILIN, StEIF4E1, Viral factors	CRISPR/Cas9, CRISPR/Cas13a	Enhanced resistance to virus	[77–80]
Alfalfa	Validate editing reagents	MsPDS, MsPALM1, MsALS, MsNOD26, MsBFT, MsTFL1A, MsTFL1B, MsTFL1C, RH1, RUBY, GUS, MsNOD26	CRISPR/Cas9	Albino mutants, improved leaf architecture, herbicide resistance, anthocyanin accumulation, and betalain biosynthesis	[40, 42], 81– [83]
	Flowering modulation	MsFTa1, MsNP1, MSAD_264347	CRISPR/Cas9	Late flowering and induced male sterility	[84–87]
	Increased biomass	MsSPL8	CRISPR/Cas9	Improved yield and stress resistance	[88]
	Lignin reduction	MsC3H	CRISPR/Cas9	Enhanced forage quality and improved digestibility	[89]
	Nutrient efficiency	MsPHO2	CRISPR/Cas9	Improved phosphate uptake	[90]
	Plant regeneration	MsPLT1-2, MsWOX5, MsBBM, MsWUS, MsREV, MsWIND1	CRISPR-a¢	Enhanced regeneration efficiency	[91]
Sugarcane	Validate editing reagents; evaluate HDR† precision	SoMgCh, SoALS	CRISPR/Cas9, CRISPRi§	Light-green phenotypes and herbicide-resistant lines	[92–95]
	Lignin reduction	SoCOMT, SoLIM	CRISPR/Cas9, TALENs	Reduced lignin content	[96–98]
	Improve canopy architecture	SoLG1	CRISPR/Cas9	Enhanced photosynthesis	[99]
Blueberry	Validate editing reagents	VcPDS, GUS	CRISPR/Cas9, CRISPR/Cas12a	Albino mutants and vector modulation	[100, 101]
	Flowering modulation	VcCEN	CRISPR/Cas9	Early flowering	[102, 103]

^†HDR homology-directed repair, *CRISPR/Cas9 Clustered Regularly Interspaced ShortPalindromic Repeats/CRISPR-associated protein 9 genome-editing system, ^μCBE Cytidine Base Editor, ^ßTALENs Transcription Activator-LikeEffector Nucleases, ^¢CRISPR-a CRISPR activation system, ^§CRISPRi CRISPR interference system

Fig. 1.

Genome editing applications in model autopolyploid crops. a Targeted traits, across selected autopolyploid species, include those producing changes in plant color or morphology, increasing biomass and early or late flowering, improving tuber quality, enhancing plant resistance against plant pathogens and improved abiotic tolerance, including phosphorus uptake and light exposure. Others, such as plant regeneration-related traits, are not included in this figure. b Three main delivery methods have been leveraged to target relevant traits, primarily through Agrobacterium-mediated transformation, biolistic and PEG-mediated transformation using protoplasts

Shared genome editing applications across crop species

A key common step in establishing genome-editing platforms in autopolyploid crops has been the implementation of a visual marker-based screening system to efficiently evaluate transformation and editing outcomes. For this, targeted mutagenesis of the PHYTOENE DESATURASE (PDS), FLAVANONE 3-HYDROXYLASE (F3H), and MAGNESIUM CHELATASE (MgCh) have produced easily scorable pigment phenotypes, while targeting ACETOLACTATE SYNTHASE (ALS) conferred herbicide resistance. Transgenic reporters, including β-GLUCURONIDASE (GUS), further enabled systematic evaluation of promoter activity and nuclease performance (Table 2, Supplementary Table 1).

These marker-based platforms have been essential for testing editing components, validating both biological (Agrobacterium-based) and non-biological (biolistics, PEG-mediated DNA/RNP uptake) delivery systems (Fig. 1b), and improving allele-targeting efficiency in autopolyploid crops. Alfalfa provides one of the strongest examples: multiplex CRISPR/Cas9 constructs combined with reporter lines have generated reliable, high-throughput screening systems that form the foundation of robust genome-editing toolkits [39–42]. Comparable refinements in potato and sugarcane show that the strategic use of visual and selectable markers accelerates the development of efficient workflows, even in species with high ploidy and complex genome architecture. In blueberry, similar strategies have been implemented, although overall transformation and editing efficiencies remain comparatively low [43]. Continued improvements in editing systems, sgRNA expression, and delivery methods will be essential to increaseediting success in recalcitrant crops.

Crop-specific genome editing applications

Genome editing displays varying degrees of technical maturity across autopolyploid crops, from extensively characterized systems in potato to emerging advances in alfalfa, sugarcane, and proof-of-concept studies in blueberry, highlighting its versatility for trait improvement. Despite substantial differences in the development and implementation of genome editing strategies among these species, current work demonstrates that genome editing can effectively target both simple and complex traits. The following sections summarize progress in the four selected autopolyploid crops, emphasizing technical feasibility, biological insights gained through targeted edits, and the translational potential of these tools for future precision breeding applications.

Potato (Solanum tuberosum)

Genome editing in potato has evolved rapidly, reflecting economic importance and corresponding significant research investment in this crop. Editing efforts have focused on validation tools, quality improvement, and stress resilience, revealing both technical feasibility and translational potential in this complex autotetraploid species (Table 2, Supplementary Table 1, Fig. 1).

Tuber quality and public health-related safety traits have been extensively modified through CRISPR/Cas9, CBE, and dMac3-Cas9 (an improved system incorporating dMac3 as a translational enhancer for Cas9) [61]. Targeting GRANULE-BOUND STARCH SYNTHASE (StGBSS) and STARCH-BRANCHING ENZYMES (StSBE1–3) enabled modulation of amylose-to-amylopectin ratios, producing both waxy and high-amylose starches suitable for industrial processing and health-promoting applications [45, 57–62, 104]. Similarly, extended shelf life and reduction of tuber toxicity have been achieved by knockout of POLYPHENOL OXIDASE (StPPO2), reducing enzymatic browning and enhancing postharvest quality and storage stability [66], while disruption of STEROL SIDE CHAIN REDUCTASE2 (StSSR2) and OXOGLUTARATE-DEPENDENT DIOXYGENASE (St16DOX) decreased toxic glycoalkaloid accumulation [63–65]. Similarly, edits of VACUOLAR INVERTASE (StVInv) and ASPARAGINE SYNTHETASE (StAS1) mitigated cold-induced sweetening and acrylamide formation during frying, improving food safety and product value [51–55]. Notably, StVInv knockout lines are advancing toward registration as non-GMO cultivars, exemplifying translational success [105]. Although these results have been fundamental in accelerating tuber quality improvement, only one cultivar (StPPO knockout) has been approved, but it has not yet been commercialized (Gene Editing Tracker: https://crispr-gene-editing-regs-tracker.geneticliteracyproject.org).

Resistance to plant stresses is one of the most extensively explored genome-editing applications in potato. Among these, resistance to biotic stresses has been studied by targeting genes involved in plant defense responses and host negative regulators. For oomycete and fungal pathogens such as Phytophthora infestans and Alternaria solani, knockout of the pattern-recognition receptor CHITIN ELICITOR RECEPTOR KINASE 1 (StCERK1) confirmed the signaling role of this gene in defense responses [70]. Edits in susceptibility (S) genes including DOWNY MILDEW RESISTANT 6 − 1 (StDMR6-1), DEFENSE NO DEATH 1 (StDND1), and SIGNAL RESPONSE 4 (StSR4) enhanced salicylic acid-mediated defense responses [72, 75], while modifications in ETHYLENE RESPONSE FACTOR 3 (StERF3) and CRYPTOCHROME-INTERACTING bHLH1/HBI1-like (StCHL) modulated other hormone signaling pathways [73, 75]. Targeting NPH3/RPT2-LIKE1 (StNRL1) elucidated effector signaling, whereas and knock-ins in CAFFEOYL-COA O-METHYLTRANSFERASE (StCCoAOMT) improved cell wall integrity while reducing pathogen biomass [71, 74].

For bacterial pathogens, targeting of StCERK1 and StDMR6-1 elucidated interactions with Ralstonia solanacearum and Streptomyces scabies, while mutations of NON-EXPRESSOR OF PATHOGENESIS-RELATED 3 (StNPR3) provided resistance to Candidatus Liberibacter solanacearum (Zebra Chip) [69, 70, 76]. For viral diseases, edits of EUKARYOTIC TRANSLATION INITIATION FACTOR 4E (eIF4E), Cajal body regulators, and CRISPR/Cas13d–mediated RNA targeting of coat proteins provided effective resistance to Potato virus Y (PVY) and other viruses as well [77–80].

Abiotic stress tolerance studies in autotetraploid potato have elucidated regulatory pathways as a foundation for improving stress resilience. Overexpression of the transcription factor StMYB44 reduced phosphate accumulation, while knockout had negligible effects, emphasizing its role in Pi homeostasis [106, 107]. CRISPR-mediated disruption of PLANT ALTERNATIVE OXIDASE (StAOX) increased reactive oxygen species (ROS) levels, lipid peroxidation, and altered photosynthetic efficiency, unveiling its importance in oxidative stress regulation under nutrient deficiency and high-light conditions [68]. These findings highlight the use of genome editing to dissect complex stress-related mechanisms.

Potato stands as the best-characterized autopolyploid crop for genome editing, covering multiple traits from tuber quality to pathogen resistance due to its status as a staple crop. The largest impact that genome editing will have on the commercialization of potatoes is the development of transgene-free plants through methods like RNP or transient expression, integrating new breading techniques into potato breeding programs and reducing regulatory controls and consumer acceptance concerns.

Alfalfa (Medicago sativa)

Although less thoroughly implemented than potato, genome editing in alfalfa has made noticeable advancement towards the development of an enhanced edited forage crop. However, as a highly heterozygous and autopolyploid forage crop, alfalfa continues to offer challenges in the implementation of genome editing, but potential remains for improving forage quality, biomass, and stress resilience. Editing efforts have evolved across flowering modulation, nutrient efficiency, and regeneration (Table 2, Supplementary Table 1, Fig. 1).

Targeted modifications of flowering regulators have demonstrated clear potential for improving yield and facilitating hybrid breeding. Knockouts of MSAD_264347 and FLOWERING LOCUS T homolog (MsFTa1) delayed flowering while increasing crude protein, mineral content, and reducing lignin [86, 87], whereas mutation of NO POLLEN 1 (MsNP1) induced male sterility without compromising vegetative vigor, supporting controlled hybrid seed production [84].

Besides reproductive traits, edits in COUMARATE 3-HYDROXYLASE (MsC3H) reduced lignin and improved digestibility [89], and modifications in SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 8 (MsSPL8) enhanced drought tolerance and increased biomass production [88, 108]. For nutrient use efficiency, multi-allelic disruption of PHOSPHATE2 (MsPHO2) via CRISPR/Cas9 and TALENs resulted in phosphate hyper-accumulation and demonstrated promise for improving metabolic efficiency under nutrient-limiting conditions [109]. Together, these edits illustrate how targeted manipulation of flowering time, lignin biosynthesis, and stress-response pathways can potentially bolster productivity in environments with water scarcity or low phosphorus availability.

Regeneration efficiency remains a central limitation for editing in alfalfa, yet a recent advancement provides promising solutions. Activation of PLETHORA1 (MsPLT1) using the ER-Tag system, which couples the β-estradiol-inducible XVE switch with CRISPR-SunTag activation, significantly improved callus formation and shoot regeneration in recalcitrant genotypes [91]. This approach offers a promising tool to increase transformation and genome editing efficiencies in autopolyploid crops and enables broader implementation in agronomically important but recalcitrant cultivars with valuable genetic backgrounds.

Although the number of genome editing studies in alfalfa remains lower than in potato, existing research demonstrates consistent improvements in flowering control, stress tolerance, nutrient uptake, and regeneration capacity. Collectively, these advances represent essential tools for enabling effective genome editing in alfalfa and support its continued development as a high-value forage crop under increasingly variable environmental conditions.

Sugarcane (Saccharum spp.)

Sugarcane is an economically important yet genetically complex crop because of its extremely high ploidy and large, heterozygous genome. Consequently, genome editing efforts have primarily centered on proof-of-concept applications aimed at validating editing systems (describe earlier) and enhancing biomass composition (Table 2, Supplementary Table 1, Fig. 1).

Improving biomass quality has been a central objective for bioenergy optimization. Targeted knockouts of CAFFEIC ACID O-METHYLTRANSFERASE (SoCOMT) and LIM-domain transcription factor–related genes (SoLIM) using TALENs and CRISPR/Cas9 have successfully reduced lignin content by 19.5–51.4% without compromising yield [96–98]. Similarly, modification of LIGULELESS1 (SoLG1) altered leaf architecture toward intermediate upright forms, increasing light interception and biomass accumulation under field conditions [99].

Although genome editing in sugarcane remains technically demanding due to recalcitrant transformation and regeneration, and high allele diversity, current studies demonstrate both system robustness and meaningful agronomic impact. Reduced lignin content, and increased biomass accumulation collectively highlight the growing feasibility of implementing genome editing to support sustainable bioenergy and feed production in this highly polyploid crop.

Blueberry (Vaccinium corymbosum)

Genome editing research in blueberry remains limited but has generated significant proof-of-concept outcomes, specifically in regeneration and flowering regulation. The woody nature and long juvenile period of the species make it a challenging yet promising candidate for future improvement (Table 2, Supplementary Table 1, Fig. 1).

In flowering regulation, targeting the negative regulator CENTRORADIALIS (VcCEN) shortened the juvenile phase. Although initial experiments did not achieve early flowering, succeeding work effectively induced it, implying that genome editing can accelerate reproductive transitions and shorten breeding cycles [102, 103]. While still in early-stage development, genome editing in blueberry reveals technical feasibility for both validation and trait modification. Continued progress will rely on advancing transformation and regeneration systems, laying out the groundwork to accelerate precision breeding of high-value cultivars.

Technical and biological barriers to efficient genome editing in autopolyploid crops

Genome editing in autopolyploid crops presents more complex challenges than those of diploid and even allopolyploid species, including the requirement for multiallelic targeting, the occurrence of mosaicism or chimerism, genotype-dependent recalcitrance to transformation, and inherently low editing efficiencies (Fig. 2). These constraints have limited the effective implementation of genome editing technologies, especially in species such as sugarcane and blueberry. This reduced efficiency is likely a consequence of the complex interaction of technical and biological factors that influence both transformation success and the recovery of edited mutants. Understanding these bottlenecks is essential to develop more reliable genome editing strategies in these crops. The following sections examine these constraints across the selected autopolyploid crops, outlining how they shape editing efficiency and limit the recovery of fully edited plants.

Fig. 2.

Challenges and prospective solutions for genome editing in autopolyploid crops. Autopolyploid crops face multiple obstacles that limit the efficiency and precision of genome editing. Key challenges include pronounced genotype-dependent variation in regeneration responses, low transformation and editing efficiencies, the requirement for simultaneous multiallelic disruption to obtain detectable phenotypes and the frequent occurrence of chimerism during callus-based regeneration. Current and emerging strategies offer promising solutions. In planta transformation approaches could support the generation of uniform, heritable edited lines in recalcitrant cultivars or species. Additionally, using native regulatory elements can improve expression of editing components, and advanced transformation system, including delivery of developmental regulators (DRs), ternary vectors incorporating additional vir genes, and type III secretion systems (T3SS), to enhance T-DNA transfer and promote efficient shoot regeneration. Multiplexed sgRNA designs increase the probability of achieving tetra-allelic knockouts. Improved Cas activity, through translational enhancers or Cas–exonuclease fusions (e.g., Cas9–TREX2), can boost editing frequencies and broaden mutational outcomes. Protoplast-based transformation enables single-cell editing and regeneration, reducing mosaicism, while co-editing strategies, such as dual delivery of base editors targeting the Acetolactate synthase gene alongside CRISPR constructs for genes of interest, could facilitate selection of uniformly edited cells. Together, these innovations expand the technical toolbox for overcoming the intrinsic complexity of autopolyploid genomes and improving editing success across highly recalcitrant species. Green–yellow sugarcane plants represent mosaic knockouts of MAGNESIUM CHELATASE (MgCh). Albino blueberry and potato plants/branch represent knockouts of PHYTOENE DESATURASE (PDS)

Genotype-dependent recalcitrance in transformation

Even among autopolyploid crops, blueberry remain particularly challenging in genome editing due to its exceptionally low transformation and regeneration efficiencies. Agrobacterium tumefaciens–mediated transformation often yields only a small number of transgenic shoots, and these efficiencies improve only marginally when CRISPR/Cas9 is incorporated [110, 111]. Some blueberry cultivars are also highly sensitive to antibiotic-based selection: commonly used agents such as kanamycin and hygromycin frequently either fail to suppress non-transgenic escapes or severely inhibit regeneration even at low concentrations [100, 112, 113].

Another barrier is the pronounced genotype dependence of both transformation and regeneration. Multiple studies demonstrate that each blueberry cultivar requires unique hormone combinations and culture conditions to induce organogenesis [110, 111, 114, 115]. Even within the same cultivar, regeneration responses differ markedly across research groups, highlighting strong genotype-by-protocol interactions and complicating the development of broadly applicable transformation systems.

Blueberry is well known for its high phenolic content not only in fruits but also in leaves [116]. These abundant phenolic compounds can oxidize rapidly upon wounding, and such oxidation is known to inhibit cell division, interfere with Agrobacterium infection, and reduce explant viability, as reported in several plant species [117]. Similar constraints have been documented in polyploid monocots such as bamboo and banana, where polyphenol-induced tissue necrosis severely limited transformation efficiency. In those systems, the incorporation of antioxidants, surfactants (e.g., Tween-20), and inhibitors of hypersensitive responses into the infection medium effectively reduced browning and markedly improved Agrobacterium-mediated transformation [118, 119]. Although not explicitly reported for blueberry transformation, the presence of high phenolic levels likely contributes to its recalcitrance to both transformation and regeneration, and the targeted use of such additives may represent a practical strategy to enhance transformation efficiency in this crop.

Multiallelic editing challenges

Gene editing in autopolyploid crops remains particularly challenging due to their extensive genetic redundancy, which requires the simultaneous disruption of multiple homologous alleles to generate discernible phenotypes. Although multiallelic edits have been reported for easily scorable marker genes such as PDS in alfalfa and blueberry using a single sgRNA, most agronomically relevant or complex traits typically require multiple sgRNAs to achieve edits in at least one allele [39, 52, 90, 98, 101]. This highlights the limited likelihood of obtaining complete or tetra-allelic mutations when relying on a single sgRNA.

One major limitation is the availability of high-quality, haplotype-resolved genome assemblies, which restrict precise allele identification and hinder efficient screening of multiallelic edits. This challenge is especially pronounced in blueberry and sugarcane, where high allelic diversity complicates detection of low-frequency and mosaic edits. Most studies in these species to date have used Sanger sequencing of colony-derived or bulk PCR amplicons [43, 52, 55, 73, 92, 93, 100], methods insufficient for detecting rare or mixed genotypes. In contrast, high-throughput amplicon sequencing and third-generation sequencing have enabled accurate identification of tetra-allelic variants, even with single sgRNA [39].

Interestingly, some studies in potato and alfalfa have reported tetra-allelic edits through high-resolution melting (HRM), high-resolution fragment analysis (HRFA), or Sanger sequencing [46, 48, 56, 78, 120] using a sgRNA. These discrepancies likely reflect differences in transformation systems, such as PEG-mediated protoplast transfection, which enables uniform single-cell editing, or the use of alternative Agrobacterium strains (e.g., C58pMP90 instead of GV3101), which may alter T-DNA delivery efficiencies in potato. Similarly, these species have high quality genome assemblies for allele identification, improving precise identification of sgRNA and detection of editions. Moreover, the strategies used to detect multiallelic events can strongly influence the likelihood of identifying fully edited genotypes. In both alfalfa and sugarcane, multiallelic editing was assessed primarily on the activity of individual sgRNAs, which limited the recovery of complete allelic knockouts and overlooked the potential combinatorial effects of multiplex strategies. As a result, only a subset of alleles, such as three disrupted stMsSPL8 copies in alfalfa, were identified [88]. These cases highlight that relying on single-guide-based screening underestimates true editing outcomes.

Together, these findings highlight the need for multiplex guide design, optimized delivery systems, and high-resolution genotyping methods to reliably achieve full allelic disruption in autopolyploid crops [89]. As more phased genome assemblies and high-throughput analytical tools become available, precise multiallelic editing will become increasingly feasible across these complex species.

Chimerism as a major bottleneck in genome editing

Although chimerism or mosaicism could be a challenge across autopolyploid crops, mainly when explants have multiple somatic edited cells, its impact is particularly severe in sugarcane due to its extremely high ploidy level (8–12×) and the presence of more than ten homologous alleles per locus (Fig. 2). Despite transformation efficiencies of ~ 20–30%, regenerated plants commonly exhibit extensive chimerism [93]. For example, CRISPR/Cas9 mutagenesis of SoMgCh co-edited 49 of 59 alleles (> 80% molecular efficiency) but produced uniform phenotypes in only 3 of 30 lines (20% phenotypic efficiency). This discrepancy reflects the nature of biolistic transformation into highly heterogeneous embryogenic callus. Since sugarcane is largely incompatible with Agrobacterium, biolistic delivery introduces CRISPR reagents into multiple founder cells, producing regenerants composed of edited and unedited sections. Polyploid redundancy further masks phenotypes when unedited homeologs compensate; even tri-allelic ALS knockouts confer only partial herbicide resistance [92]. Furthermore, heat-shock treatments were attempted to reduce chimerism, but produced no significant results [93].

Additional factors contributing to low editing efficiency across autopolyploids

Reported editing efficiencies vary widely across autopolyploid crops and depend on the delivery method, cassette design, and target gene. Among available platforms, Agrobacterium-mediated transformation generally achieves modest efficiencies (e.g., 8% tetra-allelic StSBE3; [62]). Biolistic transformation shows broader but inconsistent efficiencies, particularly in sugarcane, where it remains the main option due to the recalcitrance to Agrobacterium-mediated systems and dependence on embryogenic tissues in this crop [93, 94]. In contrast, PEG-mediated protoplast transfection can achieve up to 72% efficiency, though its use is largely restricted to potato because of the laborious regeneration process, and lack of standardized protoplast isolation and regeneration protocols in the other crops. Collectively, the delivery system represents a key bottleneck, influencing not only editing frequency but also the ability to regenerate viable plants.

The cassette design also plays a crucial role in determining editing efficiency. Cas9 remains the most widely used nuclease, yet codon optimization alone has not yielded consistent improvements in polyploids such as sugarcane [84]. In contrast, using endogenous promoters has substantially enhanced editing performance, reaching up to 96.9% in alfalfa [84], while similar strategies in blueberry achieved slightly lower efficiencies compared to heterologous systems [43]. Conversely, Cas12a has shown limited success in autopolyploids, likely due to its temperature sensitivity and context-dependent activity, with one report in blueberry showing no detectable edits [100]. These outcomes highlight that efficient expression of editing components must consider both host-specific regulatory elements and the structural properties of the nuclease used.

Finally, the target gene itself can profoundly affect observed editing efficiency. Genes with redundant or essential functions often require full allelic disruption to express a visible phenotype, which is difficult to achieve in autopolyploids. For example, single functional alleles of MsPDS in alfalfa and StGBSS1 in potato preserved wild-type phenotypes, while tetra-allelic mutants resulted in severe dwarfism, albinism, or loss of amylose [39, 45]. In contrast, partial mutations in StNPR3, StF3H, and StVInv were sufficient to confer reduced disease severity, altered pigmentation, or decreased reducing sugars accumulation [47, 54, 69]. In some cases, full knockouts are lethal, as reported for StHDS in potato [75], underscoring how gene function and pathway context can limit recoverable mutants. Thus, both the biological role of the target and the extent of allelic disruption directly shape the perceived efficiency of genome editing in autopolyploid systems.

Strategies to overcome barriers to genome editing in autopolyploid crops

Despite significant advances in CRISPR-based technologies, editing efficiencies in autopolyploid crops remain considerably lower than in diploid species [61, 78]. However, recent innovations in regeneration systems, delivery technologies, multiplex editing, nuclease optimization and regulatory element engineering offer promising avenues to improve editing precision and heritable allele disruption [62, 89]. The following sections synthesize the major strategies that collectively enhance genome editing in autopolyploid crops (Fig. 2).

Improving regeneration and transformation to enhance editing efficiencies

Transformation and regeneration remain major limitations for genome editing in autopolyploid crops like sugarcane and blueberry, where tissue culture responses, T-DNA delivery, and strong genotype effects constrain editing efficiency. At the regeneration stage, enhancing developmental competence is critical. Manipulating auxin–cytokinin pathways according to cultivar-specific profiles [121, 122] and overexpressing PLETHORA, WUSCHEL, or BABY BOOM can increase regeneration 5–10 times in perennial crops [123, 124]. Additional regulatory layers, including microRNAs such as miR156-mediated dedifferentiation [125], epigenetic regulation such as Polycomb Repressive Complex 1/2 (PRC1/2) inhibitors [126], and CLV3-like peptides [127], further improve regeneration capacity. Specifically, for blueberry, integrating CRISPRa activation of VcPLT1/VcPLT5, miR156 induction, and hormone-optimized temporary immersion bioreactors [128] provides a promising framework for overcoming recalcitrance.

Improvements in Agrobacterium-mediated systems, such as hypervirulent strains, ternary-helper vectors, auxotrophic backgrounds, type three secretion systems (T3SS) and VirG overexpression, have increased T-DNA transfer and reduced overgrowth in recalcitrant tissues [129–132]. Alternative biological platforms offer additional benefits. Rhizobium rhizogenes enables rapid, tissue-culture-independent genome editing and sgRNA validation, achieving > 90% transformation and editing rates in potato and alfalfa [41, 65, 133]. Recent in planta strategies, including de novo meristem induction [134], virus-induced genome editing [135, 136], and WOUND INDUCED DEDIFFERENTIATION 1 (WIND1)-driven regeneration [137] further reduced chimerism by minimizing callus phases.

Similarly, emerging physical and chemical delivery systems also show promise. Silicon carbide (SiC) whiskers can co-deliver plasmids and CRISPR RNPs [138], while cell-penetrating peptides (CPPs) mediate macromolecule transport across plant cell walls [139–141]. CPP structure optimization may enable tissue-culture-free editing in highly recalcitrant autopolyploids [142, 143]. Collectively, these innovations expand the transformation toolbox for complex genomes, offering effective routes to increase heritable, multiallelic editing efficiency in autopolyploid crops such as blueberry, alfalfa, and sugarcane.

Multiplex genome editing to enable multi-allelic disruption

In autopolyploid crops, effective trait modification requires the simultaneous disruption of all homologous alleles at a given locus. Multiplex CRISPR designs using three to six sgRNAs per gene generally outperform single-guide systems by increasing the probability of editing every allele and by promoting larger deletions that ensure gene knockout [48, 56, 61]. In alfalfa, for example, a single sgRNA targeting COUMARATE 3-HYDROXYLASE (MsC3H) failed to induce mutations, whereas combining three additional sgRNAs successfully generated indels across multiple alleles, leading to tetra-allelic mutants [89]. Similarly, multiplex polycistronic systems have achieved editing efficiencies of up to 85.7% in alfalfa and 88% in potato [48, 49, 90]. Thus, multiplexing enhances editing efficiency and phenotypic penetrance along with a high-throughput detection system, making it a preferred approach for recalcitrant polyploids such as blueberry and sugarcane, where single guides rarely achieve complete allele disruption.

Leveraging endogenous promoters to improve editing efficiency

Endogenous regulatory elements often outperform heterologous promoters in perennial and polyploid species. In alfalfa, an elective screen of Medicago-derived U6 promoters paired with visual reporters identified strong Pol III promoters that, when incorporated into a tRNA-sgRNA polycistronic architecture, enhanced multiplex editing efficiency [41]. Similar promoter optimization has boosted editing in other perennials; for example, grapevine lines expressing sgRNAs from native U3/U6 promoters and Cas9 from an endogenous ubiquitin promoter showed markedly higher edit rates than constructs with heterologous Arabidopsis elements [144].

Although early blueberry studies suggested benefits to native promoters [43], stable transformations did not show marked improvements relative to heterologous promoters [102], likely due to limitations of early genome assemblies [145]. With more complete Vaccinium assemblies, pangenomes and transcriptomic resources available [146], systematic mining of native U3/U6 and ubiquitin/actine or other strong promoters is now feasible and expected to improve editing not only in blueberry but also in sugarcane and potato.

Optimizing sgRNA design for autopolyploid genomes

The efficiency and specificity of CRISPR-based genome editing is also dependent on the design of the sgRNA [147, 148]. A well-designed sgRNA not only maximizes the frequency of on-target cleavage but also minimizes unintended edits [149]. While factors such as GC content and secondary structure influence Cas-sgRNA binding affinity and cleavage activity, positional attributes of the target site, whether located within the 5′ or 3′ regions, exons, introns, or untranslated regions (UTRs) can affect accessibility and repair outcomes [150–153].

While there is no strong evidence that designing the sgRNA by targeting at the 5’ region of the gene improves editing efficiency, targeting of the GLUCAN WATER DIKINASE 1 (StGWD1) gene in potato exhibited higher editing efficiency rates than those directed at the 3’ region [50]. However, in the same study, no such positional difference was found in StDMR6-1. This suggests that the local chromatin context, transcriptional activity, and DNA accessibility near promoter-proximal regions may enhance Cas9 recruitment and cleavage efficiency in certain loci. However, positional effects appear to be gene-specific, as comparable efficiencies were not consistently observed across all targets.

Targeting untranslated regions has emerged as a promising strategy for improving gene expression while avoiding deleterious coding mutations. In sugarcane, transcriptional repression of the SoMgCh gene for chlorophyll reduction using a catalytically inactive Cas9 (dCas9) directed at the 5′ UTR achieved up to 74% editing efficiency, substantially outperforming conventional exon-targeted Cas9 approaches [93, 94]. This underscores the importance of considering regulatory regions, such as promoters and UTRs. Moreover, editing of conserved 5′ UTR motifs may offer improved specificity and cross-allelic uniformity in polyploid genomes. In cases where the functional relevance of an intronic region is already established, targeted editing can be effective. For example, editing an enhancer in the intronic region of the VInv gene in potato, whose role in cold-induced sweetening is well characterized, successfully reduced this trait. Such approaches, however, remain highly case-specific and applicable only when prior evidence confirms intronic involvement in gene function [52].

Enhancing editing efficiency through Cas modulation and exonuclease fusions

Enhancing Cas expression is an important strategy to improve gene-editing efficiency, particularly in autopolyploid crops where achieving multi-allelic mutations is inherently challenging. Mutation frequencies are influenced not only by the choice of Cas nuclease and sgRNA design but also by the strength of their regulatory elements and by post-transcriptional and translational mechanisms that govern nuclease stability and activity. Inefficient Cas expression may result in mosaicism or partial editing across alleles, thereby limiting the recovery of complete knockouts in polyploid genomes.

A first attempt in improving Cas9 translation efficiency was the introduction of the dMac3 translational enhancer, a 161-nucleotide sequence derived from the 5′ untranslated region (UTR) of Oryza sativa Mac3 mRNA. When incorporated into binary vectors for CRISPR/Cas9 editing in potato, the dMac3 element significantly boosted editing frequencies and increased the proportion of tetra-allelic mutants for StGBSSI and StSBE3 [61, 62]. Complementary work in Arabidopsis has further demonstrated that post-transcriptional regulation can enhance Cas9 stability. Hung et al., (2025) engineered a construct combining the 3′ UTR of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) with a minimal binding site for BRUNO-LIKE 1 (BRN1) tethered to RIBOSOMAL PROTEIN S6 (RPS6), a known regulator of translation efficiency [154]. These modifications enhanced Cas9 protein synthesis, prevented RNA interference–mediated silencing, and improved editing rates.

Beyond expression strengthening, Cas–exonuclease fusions have emerged as a promising route to increase mutagenesis efficiency. Fusions of Cas nucleases with T5 exonuclease (T5exo) or TREX2 (the human 3′ repair exonuclease) promote larger deletions and increase editing frequency in both Cas9- and Cas12-based systems [155, 156]. Notably, Cas9–TREX2 fusions have been shown to improve editing efficiency in allopolyploids like wheat [156]. Although direct evidence for reduced off-target activity remains limited, these fusions may enhance mutational specificity by biasing DNA repair toward error-prone pathways.

Collectively, these studies highlight the importance of optimizing Cas expression and nuclease architecture to achieve uniform, high efficiency editing in complex autopolyploid genomes. Combining translational enhancers, post-transcriptional regulatory elements, endogenous promoters, and engineered nuclease fusions offers a powerful framework to improve multiallelic editing, mitigate mosaicism, and overcome the low transformation and regeneration efficiencies characteristic of polyploid crops.

Reducing mosaicism and chimerism

Mosaicism represents a persistent obstacle to genome editing in autopolyploid crops, largely arising from the asynchronous differentiation of somatic cells during callus-based regeneration. Because multiple meristematic lineages simultaneously contribute to shoot formation, edited and unedited sectors frequently coexist within a single plant, reducing both phenotypic uniformity and heritable transmission of edits [157]. Several emerging strategies aim to minimize this cellular heterogeneity by shifting regeneration toward single-cell or clonally uniform origins.

One promising approach involves the use of Rhizobium rhizogenes-mediated hairy root systems, described above, which generates adventitious shoots from individual transformed cells and therefore substantially reduce chimerism in citrus [158]. Protoplast-based editing provides an alternative route to clonal uniformity, as it enables direct delivery of multiplex CRISPR RNPs into individual cells prior to regeneration and has been successfully applied in several systems [159]. Additional advances in selective culturing strategies have further improved the enrichment of fully edited cell populations, by suppressing escape events and maintaining the dominance of edited genotypes across regeneration cycles [160–162].

One such approach is co-editing, which most often uses herbicide resistance trait conferred by SNPs in the ALS gene, which is targeted alongside with the trait of interest. Since both edits are induced within the same early editing window, cells expressing the selectable phenotype are highly enriched for associated edits at the target gene of interest. Regenerating shoots exclusively from these edited cells minimizes the contribution of unedited or partially edited cells that typically give rise to chimeric cells, besides providing a possibility to achieve transgene-free edits [163–165]. Virus-induced genome editing (VIGE) systems offer yet another avenue for reducing mosaicism by enabling systemic, homogeneous expression of sgRNAs throughout developing tissues, thereby increasing the probability that edits occur early enough to be transmitted throughout the plant [166]. Together, these methodologies illustrate a shift toward regeneration systems that either bypass or minimize callus phases, ultimately enabling the production of non-chimeric, heritable edits, a requirement for genome editing in complex polyploid crops.

Minimizing off-target mutations

Although off-target effects pose a lesser barrier than transformation or mosaicism, they are relevant for future cultivar release. One of the reasons for increased off-target activity is attributed to the prevalence and expression of the editing components, Cas9 and the sgRNA for longer periods than required in the cells [167, 168]. This can be reduced by using RNPs to deliver the CRISPR components, as observed predominantly in mammalian cells [169, 170] and allohexaploid wheat [168]. Liang et al., (2015) showed that using RNPs instead of plasmids reduced off-target mutations by 28-fold, while also improving the on-target mutation by 1.6-fold for the Overlapping Transcript 3 (OT3-18) gene in mammalian cells [171]. This improvement likely results from the short lifespan of RNPs, which act quickly after transfection and degrade within a day, limiting Cas9 exposure and unwanted edits [172].

Concluding remarks and future trends

Genome editing is rapidly transforming the improvement of autopolyploid crops, offering new opportunities to overcome long-standing barriers in breeding species such as potato, alfalfa, sugarcane, and blueberry. Despite their agricultural and economic relevance, these crops remain challenging to engineer due to high ploidy levels, difficulty of haplotype resolving, extensive heterozygosity, allelic diversity, and recalcitrance to transformation. However, notable progress has been made in targeted modification of traits related to biomass accumulation, lignin content, starch metabolism, flowering control, biotic and abiotic stress tolerance, and quality attributes. These achievements demonstrate that precise genome editing is feasible in complex genomes, although routine deployment in breeding pipelines still requires substantial technical innovation.

High-quality, haplotype-resolved genome assemblies remain foundational for accurate target selection and reliable edit identification in autopolyploids. The development of long-read sequencing technologies, single-cell genomics [173], advanced phasing methods [174], and emerging pan-genomic resources is accelerating the generation of chromosome-scale references capable of distinguishing homoeologous loci. Such resources support improved sgRNA design, accurate prediction of off-target sites, and allele-specific editing, as shown in alfalfa where a chromosome-level genome facilitated tetra-allelic PDS editing [39]. Integration of new CRISPR approaches including base editing, prime editing, and epigenome editing [175], will further expand the precision and types of genetic variation accessible in autopolyploid crops.

Achieving heritable and phenotypically scorable edits requires strategies explicitly tailored to polyploid allele dosage. Multiplexed sgRNA systems have consistently improved multi-allelic editing, enabling robust phenotypic outcomes through large deletions or tetra-allelic knockouts. Promoter optimization also plays a critical role: endogenous U6/U3 promoters and species-specific regulatory elements enhance sgRNA expression and editing activity, as demonstrated in alfalfa and potato. These innovations underscore the importance of engineering expression systems that are specifically designed at the cellular context of each crop.

Emerging applications of artificial intelligence and machine learning are likely to reshape genome editing in autopolyploids by enabling data-driven sgRNA design, improved off-target prediction, nuclease selection, and optimization of delivery systems. AI-assisted frameworks also hold promise for risk assessment, biosafety monitoring, and regulatory compliance, supporting responsible integration of editing technologies into agricultural practice.

Although autopolyploids present challenges such as polysomic inheritance, self-incompatibility, and genotype-dependent regeneration, their genomic redundancy also enables unique opportunities to generate graded allelic series and tune gene dosage [75]. Progress in targeting conserved regions, developing allele-specific editing, and improving multi-allelic screening pipelines [108] will continue to enhance the efficiency with which elite edited individuals are identified. Complementary strategies, such as editing performed in diploid progenitors followed by introgression into polyploid backgrounds, may offer additional routes to accelerate genetic improvement.

Finally, regulatory policies for gene-edited crops are evolving globally, with several countries classifying transgene-free edited lines as non-GMO [176]. This trend has enabled the approval of low-lignin alfalfa in Canada and non-browning potato in Argentina (Gene Editing Tracker: https://crispr-gene-editing-regs-tracker.geneticliteracyproject.org). Given this regulatory landscape, DNA-free editing technologies, including RNP delivery, viral vectors, and regeneration-free Cut-Dip-Budding approaches [177], will be particularly important for clonally propagated autopolyploids, where removal of foreign DNA through segregation is not feasible. However, integrating T-DNA-containing plants into conventional breeding schemes offers an alternative approach, as segregating progeny can eliminate foreign DNA. This strategy can bypass regulatory constraints, although it extends the timeline for releasing new cultivars.

Together, the convergence of high-resolution genomic resources, improved editing architectures, optimized delivery platforms, and advanced computational tools will enable the next generation of efficient, predictable, and publicly acceptable genome editing applications in autopolyploid crops. These developments hold significant promise for delivering cultivars with improved productivity, resilience, and quality to meet future agricultural and environmental demands.

Abbreviations

16DOX

2-Oxoglutarate-dependent dioxigenase

ALS

Acetolactate synthase

AOX

Plant alternative oxidase

AS

Asparagine synthetase

BE

Base editors

C3H

Coumarate 3-hydroxylase

CBE

Cytidine Base Editor

CCoAOMT

Caffeoyl-COA O-methyltransferase

CHL

Cryptochrome-interacting bHLH1/HBI1-like

CPP

Cell-Penetrating Peptide

CEN

Centroradialis

CERK1

Chitin elicitor receptor kinase1

COMT

Caffeic acid o-methyltransferase

CRISPR

Clustered regularly interspaced short palindromic repeats

CRISPRa

CRISPR activation

CRISPRi

CRISPR interference

DMR6-1

Downy mildew resistant 6 − 1

DNA

Deoxyribonucleic Acid

DND1

Defense, no death 1

DR

Developmental Regulator

DSBs

Double-strand breaks

eIF4E

Eukaryotic translation initiation factor 4e

ERF3

Ethylene response factor 3

F3H

Flavanone 3-hydroxylase

FT

flowering locus T

GBSS

Granule-bound starch synthase

GUS

β-glucuronidase

GWD1

Glucan water dikinase 1

HDR

Homology-Directed Repair

HDS

Hydroxy-2-methyl-2-(e)-butenyl 4-diphosphate synthase

HRM

high-resolution melting

HRFA

high-resolution fragment analysis

Pi

Inorganic Phosphate

InDels

Insertions and Deletions

LG1

Liguleless1

MgCh

Magnesium chelatase

NP1

No pollen 1

NPR3

Non-expressor of pathogenesis-related3

non-GMOs

Non-Genetically Modified Organisms

NRL1

NPH3/RPT2-LIKE1

OT3

Overlapping Transcript 3

PALM1

PALMATE-LIKE PENTAFOLIATA1

PAM

protospacer adjacent motif

PE

Prime editors

PHO2

Phosphate2

PDS

Phytoene desaturase

PLT

Plethora

PEG

Polyethylene Glycol

PPO2

Polyphenol oxidase2

PRC1/2

Polycomb Repressive Complex

PVY

Potato Virus Y

ROS

Reactive Oxygen Species

RH1

Red heart1

RNA

Ribonucleic Acid

RNP

Ribonucleoprotein

SBEs

Starch-branching enzymes

sgRNAs

Single guide RNAs

SiC

Silicon Carbide

SNP

Single Nucleotide Polymorphism

SR1

Signal response 1

SSR2

Sterol side chain reductase2

TALENs

Transcription Activator-Like Effector Nucleases

TFL1

Terminal flower1

TREX2

Three prime repair exonuclease2

UTR

Untranslated Region

VIGE

Virus-Induced Genome Editing

VInv

Vacuolar invertase

WGD

Whole Genome Duplication

WIND1

Wound induced dedifferentiation 1

Authors’ contributions

PM and FER developed the idea and structured the manuscript. FER, LSB, GAGM, JHK, YK, FAP and TJ wrote, reviewed, and edited the manuscript. FER created the figures. HH and PM reviewed and edited the manuscript. All authors read and approved the manuscript.

Funding

This work was supported by royalties from the University of Florida Blueberry Breeding program. Authors GAGM and LSB, have received funding from the Ministerio de Ciencia y Tecnología de Colombia - MinCiencias (project ref. 9734-937-103718), carried out by Agrosavia.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.