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19 December 2025

Advancements in Genetic Transformation of Grapevine (Vitis spp.)

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1
Institute of Forestry and Pomology, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100093, China
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Beijing Engineering Research Center for Deciduous Fruit Trees, Beijing 100093, China
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Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (North China), Ministry of Agriculture and Rural Affairs, Beijing 100093, China
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Authors to whom correspondence should be addressed.
Horticulturae2026, 12(1), 7;https://doi.org/10.3390/horticulturae12010007 
(registering DOI)
This article belongs to the Section Viticulture
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Abstract

Traditional methods for grapevine (Vitis spp.) breeding are marked by lengthy breeding cycles with usually low efficiency, rendering them inadequate for the demands of the rapidly evolving grapevine industry. While grapevine genetic transformation holds significant potential for improvement, its application is hampered by bottlenecks in efficiency, speed, and genotype dependence. In this context, this review systematically examines the factors influencing and challenges associated with key steps in grapevine genetic transformation—specifically, gene delivery and plant regeneration. It posits that the development and application of marker genes, the exploration and utilization of developmental regulators, and the establishment of novel genetic transformation systems are effective strategies to overcome current limitations. In this paper, we present a foundation and methodological guidance for creating efficient and stable genetic transformation systems for grapevine, with significant theoretical and practical implications.

1. Introduction

Grapevines possess significant economic, social, ecological, and cultural value, ranking as one of the world’s major fruit crops [1]. In recent years, the grapevine industry has encountered severe challenges due to the increasing frequency of extreme weather events like droughts and floods [2], ongoing soil degradation [3], escalating pest and disease pressures [4], and rising consumer demands for superior fruit quality [5]. Therefore, grapevine breeding programs aim to develop new cultivars that combine multiple resistance traits with a range of desirable fruit quality characteristics. Traditional methods for grapevine breeding, characterized by long cycles, high labor demands, and substantial randomness, are insufficient to meet the rapidly changing needs of the grapevine industry. However, the ongoing development and application of molecular biological technologies have significantly accelerated the grapevine breeding process [6]. Advances in plant genetic engineering techniques, in particular, have enabled the precise modification of grapevine varietal traits. These techniques allow for the enhancement or modification of existing traits, facilitating the combination of multiple desirable quality and resistance characteristics, which greatly speeds up grapevine germplasm innovation and the development of new cultivars [7].
Integrated multi-omics analysis enables the precise identification of key genes underlying target traits and elucidates related molecular mechanisms, while genetic transformation provides essential functional validation and supports varietal improvement. The release of the first grapevine genome sequence in 2007 established a foundation for precision breeding in this species [8]. Taking grapevine downy mildew (caused by Plasmopara viticola, a major grapevine disease) as an example, proteomic analysis has been used to identify resistance genotypes [9] and clarify the combined resistance mechanisms of Rpv1 and Rpv3 [10], multi-omics approaches have facilitated the development of molecular markers [11], metabolomics has uncovered early disease detection markers [12], and proteomics has further revealed molecular interaction networks between grapevine and the pathogen [13,14]. Genetic transformation plays a critical role in this context; for instance, transient expression of RxLR_PVITv1008311 in resistant V. riparia and susceptible V. vinifera leaves validated its role in pathogen recognition [11], while heterologous overexpression of VpPR10.2 and VvPR10.3 in tobacco (Nicotiana tabacum) conferred disease resistance, supporting their importance in grapevine response to downy mildew [13]. If these candidate genes could be stably introduced through genetic transformation or precisely edited to generate whole transgenic plants, the results of multi-omics analyses would be further substantiated [15,16].
Genome editing mediated by the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system (CRISPR/Cas9) represents an efficient and promising approach for crop improvement, having been successfully applied in various plant species [17,18,19,20,21,22]. In 2016, the CRISPR/Cas9 system was first employed to edit the L-idonate dehydrogenase gene (IdnDH) in ‘Chardonnay’, resulting in the generation of edited grapevine plants [23]. Subsequently, the Vitis vinifera phytoene desaturase gene (VvPDS) in ‘Neo Muscat’ was also edited, further confirming the efficacy of this system in grapevine [24]. Since then, CRISPR/Cas9 has been extensively utilized in functional genomics studies in grapevine, including: editing the VvCCD8 gene in the rootstock ‘41B’ to investigate its role in shoot branching [25]; targeting the VvWRKY52 [26], VvMLO3 [27], VvPR4b [16], and VvbZIP36 [28] genes in ‘Thompson Seedless’ to study their effects on noble rot resistance, powdery mildew resistance, downy mildew resistance, and anthocyanin accumulation, respectively; editing the VviTAS4b and VviMYBA7 genes in the rootstock ‘101-14’ to examine their resistance to Pierce’s disease and grapevine red blotch virus [29];targeting the VviPLATZ1 gene in ‘Microvine’ to explore its function in female flower morphology [30]; editing the VvEPFL9-1 gene in ‘Sugraone’ to analyze its influence on stomatal density [31].
The CRISPR/Cas9 system has undergone continuous optimization and development. For instance, the use of a maize-codon optimized Cas9 (zCas9i) achieved up to 100% biallelic mutation in ‘Chardonnay’ [32]. Additionally, CRISPR/dCas9 (deactivated Cas9)-based transcriptional activation has been successfully applied to modulate endogenous gene expression via VP64 and TV activators in grapevine [33]. Beyond CRISPR/Cas9, the CRISPR/LbCas12a (also known as CRISPR/Cpf1) system has also demonstrated efficient genome editing in grapevine [34].
The introduction of exogenous DNA during the editing process remains a major limitation for the commercial application of gene-edited crops [35]. Although self-pollination or crossing can segregate out transgenes to obtain DNA-free progeny, this approach often leads to unintended genetic changes and extends the breeding timeline [36]. To circumvent transgene integration, Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) complexes were delivered directly into GFP-overexpressing ‘Thompson Seedless’ suspension cells, followed by protoplast regeneration, yielding DNA-free edited plants [37]. However, this method has so far been validated only with a reporter GFP gene, and its applicability to other endogenous genes requires further investigation.
Genetic transformation and genome editing share many procedural similarities yet differ fundamentally in their objectives [38]. Both rely largely on the same core steps—gene delivery and plant regeneration [37]. However, their goals are distinct: genetic transformation primarily aims to introduce and express foreign genes, whereas genome editing focuses on precise modification of endogenous sequences, ideally without leaving any exogenous DNA behind [39]. The two approaches are complementary; genetic transformation often serves as the vehicle to deliver editing components, and the efficacy of genome editing depends heavily on transformation efficiency. In this sense, genome editing represents an advanced application built upon the foundation of genetic transformation.
Although genetic transformation in grapevines was first achieved in the 1990s [40], current transformation systems still face challenges such as low efficiency, lengthy cycles, and genotype dependence.
Acquiring stable transgenic grapevine plants through genetic transformation is often challenging and time-consuming. In contrast, transient transformation systems effectively address these limitations. Transient transformation of grapevine tissues, such as leaves [41], fruits [42,43,44], callus [45], and protoplasts [46], offers an efficient method for functional gene validation and exploring the molecular mechanisms behind physiological and biochemical processes. Additionally, heterologous expression of grapevine genes in model plants like Arabidopsis (Arabidopsis thaliana) [47], tobacco [48,49], and tomato (Solanum lycopersicum) [50] can achieve similar objectives. However, neither transient nor heterologous expression systems can directly accomplish genetic improvement in grapevine cultivars.
Genetic transformation is currently the sole method for achieving stable transgenic grapevine plants [51]. This process involves incorporating target genes into the grapevine genome through either direct or indirect techniques, ensuring their stable expression in recipient materials or entire plants. The transformation process consists of two main steps: introducing target genes into recipient materials and regenerating whole plants from the successfully transformed tissues [52].
This review offers a comprehensive overview of the current state of grapevine genetic transformation, emphasizing two key aspects: target gene delivery and plant regeneration. Microprojectile bombardment and pollen-tube pathway transformation are the primary direct gene transfer methods for grapevines, whereas Agrobacterium-mediated transformation is the leading indirect approach [51]. In terms of regeneration, organogenesis and somatic embryogenesis are the two pathways for grapevine explants, with somatic embryogenesis being the preferred method [53,54]. To tackle existing challenges such as low efficiency, lengthy processes, and genotype-specific limitations, three strategic solutions are proposed: developing and applying highly efficient marker genes; using developmental regulators (DRs) to boost transformation efficiency and overcome genotype barriers; and establishing new genetic transformation systems. The objective of this review is to provide a theoretical foundation for establishing stable and efficient genetic transformation systems across a broader range of grapevine cultivars.

2. Target Gene Delivery

Methods for delivering target genes into recipient materials, based on the requirement for biological mediators, can be broadly categorized into two types: direct gene transfer and indirect gene transfer [52].

2.1. Direct Gene Transfer

Direct gene transfer methods bypass biological mediators by using physical or chemical techniques to insert target genes directly into recipient materials. Key methods include microprojectile bombardment, pollen-tube pathway transformation, and polyethylene glycol (PEG)-mediated transformation, with microprojectile bombardment being the most prominent (Table 1).
Table 1. Utilization of direct gene transfer methods in grapevine genetic transformation.
Microprojectile bombardment utilizes a cold shock wave to deliver microparticles coated with target genes into plant cells, enabling transgene expression [64]. The primary advantage of this method lies in its capacity to deliver diverse biological materials into various recipient tissues. In the early stages of grapevine genetic transformation research, a limited number of attempts were made. In 1993, Hébert et al. were the first to apply this method to the genetic transformation of the grapevine cultivar ‘Chancellor’ [55]. Subsequently, the same procedure was used for ‘Chardonnay’, ‘Merlot’, and ‘Albariño’ (Table 1). The technique can also be combined with Agrobacterium-mediated transformation. For instance, in 1995, Scorza et al. performed two rounds of bombardment on somatic embryos followed by Agrobacterium infection [60]. Besides high costs, other limitations—such as low transformation efficiency, high chimerism rates, regeneration difficulties, and unpredictable transgene expression due to multi-copy random insertion events—all of which have contributed to a stagnation in its application within grapevine research. In recent years, interdisciplinary advances have led to the development of the flow guiding barrel (FGB), which increases transformation efficiency by 2 to 30 fold and reduces the occurrence of multi copy transgene insertion events [65]. This innovation holds significant potential for application in genetically recalcitrant grapevine cultivars.
The pollen-tube pathway method utilizes pollen tubes to deliver target gene solutions into the ovary, enabling transformation of zygotic embryos or early embryonic cells, which subsequently develop into transgenic seeds [66]. This approach is often combined with Agrobacterium-mediated transformation and has been studied by only a very limited number of research groups in grapevine. By immersing inflorescences in an Agrobacterium infection solution, Yang [61] and Zhou [62] successfully introduced the rolC and iaaM genes, respectively, into the ‘Manicure Finger’ cultivar in 2008 and 2009. A similar attempt was made in 2011 on the cultivar ‘Wink’ [63]. The key advantages of this method lie in its use of the plant reproductive system as a natural vector, its independence from tissue culture, and its operational simplicity, which allows high-throughput implementation. However, grapevines have a relatively short flowering period and are susceptible to cleistogamy. Furthermore, the applicability of this technique in grapevine requires further validation by more research teams. In recent years, advances in nanomaterials have enabled the successful application of nanoparticle-mediated pollen-tube pathway transformation in various plant species [67], revealing new potential for genetic transformation in grapevines.
The PEG-mediated transient transformation system for grapevine was established in 2015 [46], yet its application remained limited due to challenges in protoplast regeneration. It was not until 2022 [37] that complete gene-edited plants were successfully obtained through this approach. Further development is required to advance the utilization of this method in grapevine genetic transformation.
Microparticle bombardment, pollen-tube pathway, and PEG-mediated transformation all exhibit inherent limitations, resulting in their minimal application in grapevine genetic transformation.

2.2. Indirect Gene Transfer

In grapevine genetic transformation research, indirect transformation methods are generally preferred over direct gene transfer techniques [68]. Utilizing the ability of Agrobacterium to transfer and integrate T-DNA into the plant genome, target genes can be introduced into grapevine recipients and expressed [69]. Agrobacterium-mediated transformation stands out due to its high efficiency, simplicity of operation, genetic stability, and ability to transfer large DNA fragments [52,70].
Several key factors significantly impact the efficiency of Agrobacterium-mediated transformation in grapevine. These include recipient material, Agrobacterium strain, infection solution concentration, infection duration, co-culture period, acetosyringone (AS) concentration, selective agents and concentration (Table 2).
The selection of recipient materials should be the primary consideration in grapevine genetic transformation. Tissues with vigorous division and differentiation capabilities, such as somatic embryos (SE), embryogenic callus (EC), pro-embryonic masses (PEM), and meristematic bulks (MB), are commonly used as recipient materials for genetic transformation (Table 2). The influence of recipient materials on transformation is largely mediated through their impact on regeneration. Detailed discussions on grapevine regeneration will be presented in subsequent sections.
In selecting specific parameters for Agrobacterium strain, A. tumefaciens EHA105, GV3101, and LBA4404 are predominantly used as the vector for grapevine genetic transformation. Zhao infected petioles of Vitis amurensis with EHA105, GV3101, and LBA4404 at OD600 = 0.5, achieving transformation efficiencies of 25.31%, 29.91%, and 3.14%, respectively, with EHA105 and GV3101 significantly outperforming LBA4404 [71].
Infection solution concentration, typically measured by optical density at 600 nm (OD600), plays a crucial role in determining transformation efficiency. An OD600 range of 0.2–1.0 is optimal for grapevine genetic transformation (Table 2). Low Agrobacterium concentrations during infection can significantly boost transformation success rates [72]. For example, in ‘Syrah’ callus transformation experiments, a bacterial concentration of OD600 = 0.2 improved transformation and regeneration outcomes, whereas higher concentrations caused callus browning and necrosis [73]. However, an OD600 range of 0.8–1.0 is also frequently used. This is viable because thoroughly rinsing the explants with 200–500 mg/L of cefotaxime (Cef), carbenicillin (Carb), or timentin (Tm) in water or liquid medium after infection effectively suppresses Agrobacterium overgrowth and minimizes its harmful effects [53,74].
Table 2. Application of Agrobacterium-mediated transformation for recovering transgenic grapevine plants.
Table 2. Application of Agrobacterium-mediated transformation for recovering transgenic grapevine plants.
CultivarRecipient MaterialAgrobacterium StrainInfection Solution ConcentrationInfection DurationCo-Culture PeriodAS ConcentrationSelective Agents and ConcentrationTarge GeneRegeneration PathwayReference
Aligote, Podarok MagarachaSEEHA105OD600 = 0.272 h\\Kanamycin (Kan) 50 mg/Lm-GFP5-er, NPTIIsomatic embryogenesis[54]
Cabernet Sauvignonimmature zygotic embryosEHA105OD600 = 0.620 min\\\VvBBM, GFP, NPTII\[75]
Podarok MagarachaleavesEHA105OD600 = 0.8\72 h\Kan 50 mg/LGFP, NPTIIorganogenesis[53]
Chardonnayimmature zygotic embryosEHA105OD600 = 0.620 min\200 μMKan 30 mg/LeGFP, NPTII\[76]
Thompson SeedlesscallusGV3101OD600 = 0.810 min\\Kan 75 mg/LVyUSPA3, RNAi-VyUSPA3, GFP, NPTIIorganogenesis[77]
Red GlobeECGV3101\10 min48 h100 μMphosphinothricin (PPT) 150 µL/mLVaERD15, Barsomatic embryogenesis[78]
Thompson SeedlesscallusGV3101\10 min48 h200 μM\VaSAP15, GFPorganogenesis[79]
Shine MuscatECLBA44041 × 108 cfu/mL15 min96 h100 μMKan 25 mg/LsGFP, NPTIIsomatic embryogenesis[80]
Thompson SeedlessMBEHA105OD600 = 0.5–1.015 min48 h100 μMKan 70 mg/LeGFP, NPTIIorganogenesis[81]
Thompson SeedlessSEAgrobacteriumOD600 = 0.67–10 min48–72 h\Kan 100 mg/LVvMybA1, NPTIIsomatic embryogenesis[82]
Thompson SeedlessPEMGV3101OD600 = 0.4–0.68 min48 h\Kan 75 mg/LVpPR10.1, GFP, NPTIIsomatic embryogenesis[83]
Thompson SeedlessPEMEHA1051 × 108 cfu/mL\48 h100 μMKan 100 mg/LVaTLP, NPTIIsomatic embryogenesis[84]
Chardonnay, Thompson Seedless, Red Globe, Cabernet Sauvignon, St. George, 101-14MgtMBEHA105OD600 = 0.5–1.015 min48 h100 μMKan 100 mg/L, hygromycin (Hyg) 1–2.5 mg/LGFP, NPTII, HPTIIorganogenesis[85]
Thompson SeedlessMBGV3101OD600 = 0.4–0.68 min48 h\Kan 75 mg/LVqDUF642, GFP, NPTII, HPTIIsomatic embryogenesis[86]
Thompson SeedlessleavesLBA4404\\48 h\bialophos 3.0 mg/LP5CS, Barorganogenesis[87]
Thompson Seedless, Bronx SeedlessSEEHA105\8–10 min72 h\Kan 100 mg/LVvMybA1, GUS, NPTIIsomatic embryogenesis[88]
Thompson SeedlessPEMGV3101OD600 = 0.4–0.68 min48 h\Kan 75 mg/LVqSTS6, NPTIIsomatic embryogenesis[89]
Dornfelder, RieslingSELBA4404OD550 = 1.220 min48 h100 μMKan 100 mg/LGUS, NPTIIsomatic embryogenesis[90]
ChardonnayPEMGV3101OD600 = 0.3–0.410 min72 h\Hyg 10 mg/LVpSTSgDNA2, HPTIIsomatic embryogenesis[91]
Thompson SeedlessEC, PEM, SEGV3101OD600 = 0.4–0.68 min48 h\Kan 75 mg/LVpPUB23, NPTIIsomatic embryogenesis[92]
Ramsey, Gloire, St. George, Cabernet Franc, Cabernet Sauvignon, Chardonnay, Merlot, Orange Muscat, Pinot Noir, Sauvignon Blanc, Shiraz, Superior Seedless, Thompson Seedless, Zinfandel, Conquistador, Freedom, Harmony, Richter 110, Seyval BlancSEEHA105OD600 = 0.8–1.010 min72 h\\eGFP, NPTIIsomatic embryogenesis[93]
Crimson Seedless, SugraoneECEHA105OD600 = 0.06, 0.210 min48 h\Kan 20 mg/L, 50 mg/LsGFP, NPTIIsomatic embryogenesis[94]
Merlot, Shiraz, Thompson Seedless, Seyval BlancSEEHA105OD600 = 0.8–1.010 min72 h\Kan 100 mg/LeGFP, GUS, NPTIIsomatic embryogenesis[95]
Arich DresséSELBA4404OD600 = 1.030 min48 h200 μMPPT 2.5 mg/LGUS, Barsomatic embryogenesis[96]
Thompson SeedlessECGV3101OD600 = 0.4–0.610 min72 h200 μMHyg 12 mg/LSTS, GUS, mGFP, HPTIIsomatic embryogenesis[97]
Alachua, CarlosSEEHA105OD600 = 0.8–1.08 min72 h\Kan 100 mg/LGFP, NPTIIsomatic embryogenesis[98]
Thompson Seedlessshoot apical meristemsEHA105OD600 = 0.810 min48 h\Kan 16 mg/LeGFP, NPTIIorganogenesis[99]
Red GlobeECEHA105OD630 = 0.610 min48 h100 μMparomomycin (Prm) 25 mg/LGUS, NPTIIsomatic embryogenesis[100]
Chardonnay, Thompson SeedlessECEHA1011 × 109 cfu/mL5 min48 h20 μMKan 100 mg/LPGIP, GFP, GUS, NPTIIsomatic embryogenesis[101]
Portan, ShirazECEHA105, A4OD550 = 0.410 min48 h100 μMKan 150 mg/Lm-GFP5-er, NPTIIsomatic embryogenesis[102]
Cabernet Sauvignon, Shiraz, Chardonnay, Riesling, Sauvignon Blanc, Chenin Blanc, Muscat Gordo BlancoECEHA101, EHA105OD550 = 0.37 min48 h100 μMKan 100 mg/Lm-GFP5-er, GUS, NPTIIsomatic embryogenesis[103]
SultanaECEHA101, EHA105\7 min48 h\Kan 100 mg/LGUS, NPTIIsomatic embryogenesis[104]
KoshusangjakuECA13\10 min72 h20 μMKan 50 mg/LGUS, NPTIIsomatic embryogenesis[105]
The duration of infection also needs optimization within a specific range (7–20 min) (Table 2). If the duration is too short, it compromises transformation efficiency, while excessive exposure can cause tissue damage and bacterial contamination, thereby reducing the regenerative potential of the recipient materials. When immature zygotic embryos of ‘Cabernet Sauvignon’ were treated with an EHA105 infection suspension at OD600 = 0.6, the highest transformation efficiency reached 12.38% at an infection duration of 20 min. When the infection time was extended to 30 min, widespread mortality of the immature zygotic embryos occurred, and no positive transgenic plants were detected [76].
The co-culture period refers to the time between the completion of infection and the start of selection, usually lasting 48 or 72 h and typically conducted in the dark (Table 2). During this phase, no selective agents harmful to grapevine cells are added to the medium. Although not strictly required, a well-timed co-culture period aids in the recovery and proliferation of recipient materials. During the optimization of genetic transformation conditions for Vitis amurensis, it was found that the transformation efficiency exhibited an initial increase followed by a decline with prolonged co-culture duration. The highest efficiency reached 24.14% after 2 days of co-culture, while efficiencies corresponding to 1, 3, and 4 days of co-culture were 1.10%, 10.83%, and 9.69%, respectively [71].
As a phenolic compound, AS initiates T-DNA transfer and enhances transformation efficiency [106]. The optimal concentration of AS is typically 100 or 200 μM, which effectively increases the efficiency of grapevine genetic transformation (Table 2). However, elevated concentrations can inhibit cellular growth. Studies have shown that the transformation efficiency of grape immature zygotic embryos was 0.00%, 1.19%, 9.53%, and 4.76% when AS concentrations were 0, 100, 200, and 300 μM, respectively [76].
Following infection, antibiotics or herbicides are routinely used for selecting and initially identifying acquired explant materials. The type and concentration of these selective agents are crucial for determining screening efficiency. Kan is a commonly used selective agent in grapevine genetic transformation, with an optimal concentration range of 50–100 mg/L (Table 2). However, genotypic differences require concentration adjustments, as seen in ‘Shine Muscat,’ which needs just 15 mg/L for optimal results [80]. Gradually increasing Hyg concentrations (3, 6, 9, 12 mg/L) also yields effective selection [97]. Additionally, glyphosate and Prm are sometimes added to culture media as alternative selective agents [91,96]. Furthermore, antibiotics can also be employed to address the issue of chimeras. Capriotti et al. successfully obtained fully fluorescent shoots in the ‘Ancellotta’ cultivar by transferring chimeric tissues to a medium containing higher concentrations of the selective agent (increasing Kan from 146 μM to 208.6 μM) [107].
Establishing an efficient grapevine genetic transformation system requires careful optimization of multiple factors, including recipient material, Agrobacterium strain, infection solution concentration, infection duration, co-culture period, AS concentration, as well as selective agents and their concentrations. It should be noted that the optimal infection conditions for one genotype or explant type are not necessarily applicable to other genotypes or explant types [53,54].

3. Grapevine Regeneration

Regeneration is the biological process by which plant tissues or organs repair and replace themselves after injury or stress [108]. Establishing an efficient regeneration system is essential for genetic transformation in grapevine, involving two main pathways: organogenesis and somatic embryogenesis.

3.1. Organogenesis in Grapevine

The organogenesis pathway involves the formation of adventitious shoots either directly from explants or indirectly through callus intermediates, eventually developing into complete plants [108]. In grapevine organogenesis, explants such as shoot apical meristems [85,99], leaf blades [53], petioles [53], cotyledons [107], and hypocotyls [107] are commonly used. Regeneration conditions and efficiency vary significantly across different genotypes, and explant types. In a comparison of organogenesis capacity across 22 genotypes, significant variation was observed when cultured on media supplemented with thidiazuron (TDZ) and indole-3-butyric acid (IBA), ranging from a minimum of 1.7% (‘Magarach no. TT2’) to a maximum of 100.0 ± 7.2% (‘Podarok Magaracha’) [53]. Capriotti et al. compared the organ regeneration ability in ‘Thompson Seedless’ using MB, hypocotyls (derived from somatic embryos), and cotyledons (derived from somatic embryos). The regeneration rates of transgenic positive shoots were 1%, 7%, and 26%, respectively, showing significant differences [107]. The organogenesis pathway in grapevine offers the advantage of short regeneration cycles, with adventitious shoots typically appearing within weeks [53]. Using leaf blades or petioles as explants in ‘Kober 5BB’ and ‘Podarok Magaracha’, well-developed shoots were obtained after 11 weeks (including 2 weeks in dark culture, 4 weeks in light culture, and 5 weeks in dark culture), with regeneration efficiencies ranging from 5.6% to 6.2% and 15.8% to 16.1%, respectively [53]. However, this method often encounters chimerism issues in genetic transformation applications, as the shoots originate from multiple cell lineages [109]. When using cotyledons of ‘Ancellotta’ for organogenesis, only one positive shoot was obtained, which exhibited a chimeric phenotype [107]. To mitigate chimerism, antibiotics or herbicides are added to the culture medium for selection and suppression, although this process often further reduces regeneration efficiency. The study found that the callus formation capacity of Vitis amurensis petioles is inversely proportional to the concentration of Kan. In the absence of Kan, nearly all petioles (96.86%) formed callus, whereas no callus formation occurred when the Kan concentration reached 30 mg/L [71]. Moreover, compared to the somatic embryogenesis pathway, the organogenesis approach is more severely genotype-dependent, which significantly limits its broader application in grapevine genetic transformation [53,54].

3.2. Somatic Embryogenesis in Grapevine

The somatic embryogenesis pathway is now the favored method for grapevine genetic transformation systems [51]. In grapevines, materials such as floral organs, stem segments, petioles, leaves, immature zygotic embryos, and hypocotyls can induce SE [68]. Among the various tissues, floral organs exhibit the highest efficiency in somatic embryogenesis. When using filaments of ‘Shine Muscat’ to induce somatic embryos, the somatic embryo induction rate reached a remarkably high level of 44.9%. Furthermore, 7 positive EC were subcultured for the regeneration of positive plants, successfully yielding a total of 49 plants, with each EC producing between 2 and 16 plants [80]. The somatic embryogenesis regeneration process, from explants to plants, takes approximately 12 months, with the induction of EC alone requiring about 7 months [54]. The brief flowering period of grapevines, combined with the lengthy cycle and low efficiency of somatic embryogenesis induction, makes the long-term preservation of SE especially important. Subculturing EC in liquid MS medium supplemented with 2% sucrose, 2.0 mg/L zeatin (Zea), and 0.1 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D) enables the maintenance of embryogenic potential for up to two years while yielding abundant material [54]. By repeatedly cycling SE through dedifferentiation to form EC and redifferentiation into new SE, this iterative method facilitates mass production for genetic transformation and long-term preservation of SE. Zhou tested the re-induction rates at five different stages of SE and found that the torpedo and mid-cotyledonary stages exhibited the highest performance, approximately 80% [92]. However, it requires considerable human and material resources. Additionally, with extended subculturing, the proliferative and differentiation capacities of callus gradually diminish, while the risk of somaclonal variation significantly rises [110].
Somatic embryogenesis and organogenesis, the two principal regeneration pathways in grapevine, exhibit substantial differences in their procedural frameworks and temporal cycles. Somatic embryogenesis requires initial induction of somatic embryos, followed by progression through defined embryonic developmental stages, with the complete cycle taking approximately 12 months [54]. In contrast, organogenesis involves direct shoot regeneration from explants or indirect regeneration via callus intermediates, with shoot induction typically requiring about 11 weeks [53]. Somatic embryogenesis often necessitates different culture media at various developmental stages, whereas organogenesis generally requires fewer media changes. For example, the somatic embryogenesis pathway in ‘Podarok Magaracha’ employs 6 distinct media, while its organogenesis pathway uses only 2 [53,54]. However, because somatic embryos develop from single cells and organs regenerate from tissue aggregates, organogenesis is more prone to chimera formation. Moreover, the somatic embryogenesis pathway allows for the maintenance of large quantities of somatic embryos, bypassing the need for repeated initial induction and enabling their recurrent utilization [54,92].

3.3. Factors Influencing Grapevine Regeneration

An efficient regeneration system is crucial for the success of genetic transformation protocols [52,111]. The grapevine regeneration process is shaped by several critical factors, including explant selection, hormone regimens, organic supplements, and the occurrence of abnormal plantlets.
The selection of explants constitutes the foremost consideration in establishing an efficient grapevine regeneration system [70]. Regenerative capacity is an inherent genotypic trait in plants [53]. Maletich et al. successively attempted to establish organogenesis protocols for 22 genotypes and somatic embryogenesis protocols for 10 genotypes, with successful regeneration achieved only in ‘Podarok Magaracha’, ‘Aligote’, ‘Malbec’, ‘Carménère’, and the rootstock ‘Kober 5BB’, ‘SO4’ [53,54] The complex genetic background and varying regenerative capacities among explants have led to a research bias in grapevine genetic transformation, with a predominant focus on a few cultivars (Table 2).
The regulation of plant regeneration is governed by a complex hormonal network, in which auxins and cytokinins play pivotal roles [52,112]. Different hormones of the same type exhibit distinct induction effects. For example, a comparative study using somatic embryos from three grape cultivars (‘Malbec’, ‘Aligote’, and ‘Podarok Magaracha’) evaluated the effects of different auxins (Indole-3-acetic acid (IAA), IBA, 2-Naphthoxyacetic acid (NOA), 1-Naphthaleneacetic acid (NAA), and 2,4-D) on embryo germination. IAA demonstrated the highest efficiency, with germination rates of 3.4%, 4.2%, and 2.2%, respectively, whereas IBA and NOA showed inhibitory effects [54]. The impact of varying hormone concentrations on regeneration has long been a focal point in grapevine regeneration research [53,54,75,76,107]. However, a universally applicable hormone combination has yet to be developed, which may be attributed to the complex genetic background of grapevines.
In addition to conventional phytohormones, low concentrations of melatonin (MT) (0.6 mg/L) have been shown to reduce the induction time for cotyledonary embryo development and secondary embryogenesis in ‘Chardonnay’ seeds [113]. The addition of amino acids, such as glutamate, also promotes somatic embryogenesis [114]. Research indicates that picloram is preferred over 2,4-D because of its milder dedifferentiation capacity and enhanced proliferation of embryogenic callus, with optimal induction occurring at a concentration of 1.0 mg/L [115].
Tissue culture is a crucial step in plant regeneration, yet grapevine tissue cultures often produce abnormal plantlets, which exacerbates the already low regeneration efficiency [116]. Ji et al. attempted to recycle abnormal embryos and found that the fused cotyledon-type embryos were capable of inducing somatic embryos within two months, achieving an efficiency of 72.22% [116]. The formation of aberrant embryos is linked to several factors, such as medium composition, subculture frequency and duration, and environmental conditions. Notably, the chemical components of the culture medium, especially exogenous hormones and growth regulators, are key factors influencing normal somatic embryo development [117]. When leaves or petioles were used for organogenesis, shoots obtained on MS medium supplemented with TDZ and BA were mostly deformed or vitrified, whereas normal shoots were observed on IM medium supplemented with BA and IBA [53].

4. The Future of Genetic Transformation in Grapevine

4.1. Development and Application of Marker Genes

Marker genes play important roles in genetic transformation and are categorized into selectable marker genes and reporter genes [52]. During vector construction, selectable marker genes are used for the screening and identification of positive bacterial strains, ensuring both the correctness and efficiency of the vector assembly. After the delivery of the target gene, selectable markers enable rapid elimination of non-positive individuals, thereby preventing unnecessary effort [53,82,107]. Moreover, selectable marker genes can help address the issue of chimerism that may arise during genetic transformation [107]. Reporter genes serve functions such as dynamic monitoring, quantitative analysis, and visual tracking in genetic transformation; their easily detectable expression products indirectly reflect the expression activity, regulatory mechanisms, or cellular events of the target gene [52].
In grapevine genetic transformation, the primary marker genes include NPTII, HPTII, and Bar, which confer resistance to Kan, Hyg, and PPT, respectively, as shown in Table 2.
The development of reporter genes can be divided into three stages: the first stage represented by the GUS gene; the second stage represented by the GFP gene; and the third stage represented by the RUBY gene.
The GUS gene is commonly used as a reporter during the early stages of grapevine genetic transformation (Table 2). Using 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc) as a substrate allows for histochemical staining, which turns positive tissues blue, facilitating visual detection both macroscopically and microscopically. Tissues subjected to GUS histochemical staining lose viability due to the destructive nature of the assay [118].
GFP and its variants are the most widely used reporter genes in grapevine genetic transformation (Table 2). Detection with GFP reporter systems is straightforward, relying on green fluorescence emission under ultraviolet illumination to identify positive tissues, thereby reducing losses associated with destructive detection methods. However, the green fluorescence signal is sometimes detectable only in juvenile tissues but absent in mature tissues, which may lead to false-negative results.
The RUBY gene, as a recently developed reporter system, enables betalain biosynthesis to produce visually discernible red pigmentation, thereby significantly reducing detection complexity and facilitating chimera identification [119,120,121,122,123]. However, its application in grapevine genetic transformation requires careful discrimination from endogenous anthocyanin accumulation that generates similar red phenotypes. Overexpression of the grapevine endogenous gene VvMybA1 induces anthocyanin accumulation, generating conspicuous color differentiation [88]. Since this system involves no introduction of exogenous genetic material, it avoids potential risks associated with transgene insertion, making it a promising candidate for developing grape-optimized reporter genes.
The development and application of visual marker genes have significantly alleviated the workload for transgenic material identification, shortened detection timelines, and consequently accelerated progress in grapevine genetic transformation research.

4.2. Exploration and Utilization of DRs

Research has shown that specific genes actively facilitate plant regeneration during genetic transformation. Targeted regulation of these genes allows for the development of stable, efficient, and genotype-independent transformation systems [124].
GROWTH-REGULATING FACTORS (GRFs) are a highly conserved family of plant transcription factors that typically form functional transcriptional complexes with GIF cofactors, playing crucial roles in growth, development, and regeneration [1]. Studies have shown that the expression of homologous or heterologous GRF genes promotes regeneration in sugar beet [125], soybean [125], sunflower [125], watermelon [126,127], and rubber tree [128]. Although the grapevine genome contains 10 GRF genes and 4 GIF genes [49], existing cases mainly demonstrate their regenerative roles in other species: in citrus, heterologous expression of the VvGRF–GIF chimera that was resistant to miR396 (rVvGRF–GIF) increased regeneration efficiency by 7.4-fold [129]; in diploid Fragaria vesca Hawaii 4, overexpression of VvGRF-GIF1 significantly enhanced regeneration efficiency [130]; in tobacco, overexpression of rVvGRF8-GIF2 yielded the highest regeneration efficiency at 83.43%, 3.1 times that of the control [49]. Regrettably, to date, no reports have demonstrated the application of these genes in promoting grapevine regeneration.
BABY BOOM (BBM), a member of the AP2/ERF transcription factor family, plays a critical role in inducing somatic embryogenesis [131] and promoting plant regeneration in various species, including sweet pepper [132], maize [133,134], and apple [135]. In immature zygotic embryos of ‘Cabernet Sauvignon’, overexpression of VvBBM significantly improved germination rates and enhanced overall growth and development of transformed plants under non-tissue culture conditions [75]. VvBBM holds the potential to overcome genotype- and tissue culture-dependent limitations. However, there is a lack of additional application cases, particularly in somatic embryogenesis or organogenesis.
WUSCHEL (WUS)-related homeobox (WOX) genes play an important role in early embryogenesis. Previous studies have demonstrated their ability to promote regeneration in wheat [136], maize [137], apple [122], and kiwifruit [122]. The grapevine genome contains 12 VvWOX genes, and expression dynamic analysis in ‘Chardonnay’ embryogenic tissues revealed that VvWOX2 and VvWOX9 are the primary WOX genes expressed during somatic embryogenesis [138]. Enhancing the expression of VvWOX2 and VvWOX9 may improve grape regeneration efficiency; however, this hypothesis currently lacks empirical validation.
In addition, genes such as WIND [139], REF [140], IPT [141], and LEC [142] genes have also been shown to promote regeneration. When applying DRs to enhance genetic transformation, multiple genes are sometimes combined to achieve higher regeneration rates [133,143].
While developmental regulators hold promise for improving regeneration, their associated drawbacks cannot be overlooked, including pleiotropic effects, abnormal development, and reduced fertility [144]. In apple, overexpression of BBM resulted in abnormal phenotypes such as compact plant architecture and curled leaves in some lines [135]. Although overexpression of GRF5 in soybean promoted transgenic shoot formation, it simultaneously inhibited adventitious root development, thereby affecting the regeneration of complete plants [125]. Wheat plants overexpressing the GRF4-GIF1 chimera exhibited a 23.9% reduction in grain number per spike [129].
Combinatorial expression of DRs may help mitigate the drawbacks associated with single-gene approaches. Furthermore, optimizing promoters to achieve tissue-specific or inducible expression can avoid the adverse effects of constitutive expression [122,134]. Altruistic transformation strategies, such as co-inoculation with two Agrobacterium strains carrying morphogenic genes and target trait genes separately, have successfully yielded normal transgenic plants containing only the trait of interest in poplar [145].
The application of DRs represents a powerful approach to address regeneration recalcitrance, improve regeneration efficiency, overcome genotype dependency, and ultimately enhance genetic transformation efficiency in grapevine. However, it is regrettable that despite existing research on related genes in grapevine, practical cases demonstrating their effectiveness in promoting grapevine regeneration—whether using heterologous or homologous genes—remain scarce.

4.3. Establishment of Novel Genetic Transformation Systems in Grapevine

Transient transformation offers significant advantages in grapevine gene research due to its high efficiency and rapidity [41,146]. Beyond the commonly used Agrobacterium-mediated transient transformation, a PEG-mediated grapevine protoplast transient gene expression system has been developed. This system provides high gene delivery efficiency (60–70%), a short experimental cycle (results can be detected 20 h post-transfection), and operational simplicity, making it an efficient platform for functional validation of genes in grapevine [46].
DNA-free editing has remained a central objective in genome editing [36]. In grapevine, this goal has been achieved by delivering ribonucleoprotein (RNP) complexes into protoplasts via PEG-mediated transfection, thereby enabling editing without introducing exogenous DNA [37,147]. Additionally, advances in nanomaterial science have opened new avenues for DNA-free genome editing. For instance, carbon nanotube (CNT)-mediated delivery technology has been demonstrated as an effective method for introducing genetic materials into plants [148,149].
Despite established genetic transformation systems for grapevine, these processes are still notoriously time-consuming, inefficient, and poorly reproducible, often limited to a few specific cultivars. Thus, developing novel transformation systems is crucial. In recent years, many innovative genetic transformation strategies have emerged [150].
For species capable of shoot regeneration from roots, target genes can be introduced into hairy roots, with subsequent shoot regeneration from these roots producing complete transgenic plants [111,121,151,152]. However, grapevine lacks the natural ability to regenerate shoots from roots, and there are no successful reports of complete plant regeneration from root tissues under in vitro conditions. The significant improvement in complete plant regeneration efficiency from transgenic positive roots through overexpression of developmental regulators in apple and kiwifruit offers valuable inspiration for grapevine research [122].
For species with strong regenerative capacity, in planta transformation introduces target genes into wounded sites, utilizing the plant’s innate regeneration potential for highly efficient shoot regeneration [121]. These approaches still require validation in grapevine. The in planta genome editing in citrus (IPGEC) system enables effective genome editing and shoot regeneration without the need for tissue culture by using Agrobacterium to transiently co-express three modular components in citrus seedling stems: gene editing elements, shoot induction/regeneration factors, and T-DNA delivery enhancers [123]. This method is highly relevant for enhancing grapevine genetic transformation techniques.
Zhang et al. developed a transformation system using immature zygotic embryos of ‘Chardonnay’ [76]. This approach significantly shortens the operational cycle to about two months compared to traditional grapevine genetic transformation protocols, achieving a 42.85% transformation efficiency without tissue culture [75].
Innovative methodologies are urgently needed in grapevine genetic transformation to accelerate genomic research and biomolecular technology development, thereby advancing genetic improvement in grapevine.

5. Conclusions

Genetic transformation serves as a powerful tool for validating gene function, elucidating molecular mechanisms, and achieving varietal improvement in grapevine [73,77,78,79,146,153,154,155]. However, current transformation systems are predominantly established on only a few cultivars and suffer from low efficiency [53,54,74,75,76]. The associated regeneration systems are often optimized for a limited number of genotypes, lacking broad applicability [53,54,81,107,156].
DRs can promote plant regeneration and have been successfully applied in multiple species, particularly in woody plants with relatively low regenerative capacity, such as rubber tree [128], apple [122,135], and kiwifruit [122]. Although studies on related genes in grapevine exist [49,75,138], successful cases of using developmental regulators to enhance grapevine regeneration remain scarce [75]. The application of developmental regulators in grapevine holds promise for improving genetic transformation efficiency and overcoming the challenges associated with recalcitrant cultivars.
Novel genetic transformation systems are being continuously developed, such as IPGEC system [123], Fast-TrACC system [143], and Cut-dip-budding (CDB) system [151]. These systems bypass the tedious and time-consuming tissue culture procedures, often offering simpler and more efficient alternatives, thereby providing valuable references for establishing new grapevine transformation platforms. Furthermore, the development of transient RNP delivery systems has shown initial success [37,147]. The commercial application of gene-edited plants heavily relies on the advancement of such systems.
Additionally, integrating single-cell or multi-omics data to decipher the mechanisms of grapevine regeneration and to identify key regulatory factors aims to inform and optimize targeted regeneration strategies [157].

Author Contributions

Conceptualization, W.L., X.W. and A.Y.; methodology, W.L., J.R. and Z.L.; validation, W.L., X.W. and H.W.; formal analysis, W.L., X.W. and H.W.; investigation, W.L., X.W. and H.W.; resources, W.L. and J.R.; data curation, W.L. and A.Y.; writing—original draft preparation, W.L.; writing—review and editing, W.L., Z.L. and L.S.; visualization, W.L. and J.R.; supervision, L.S.; project administration, Z.L. and L.S.; funding acquisition, L.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by BAAFS Funding for the Development of Distinguished Scholars (JKZX202402), Beijng Academy of Agriculture and Forestry Sciences Innovation Capability Construction Special Project (KJCX20251002), and National Natural Science Foundation of China (grant number 32472677).

Data Availability Statement

No new data were created or analyzed in this study. Data sharing is not applicable.

Conflicts of Interest

The authors declare no conflicts of interest.

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