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Perspective

Recent Progress in Genetic Transformation and Gene Editing Technology in Cucurbit Crops

by
Jing Feng
1,†,
Naonao Wang
2,†,
Yang Li
1,
Huihui Wang
1,
Wenna Zhang
2,
Huasen Wang
1 and
Sen Chai
1,*
1
Engineering Laboratory of Genetic Improvement of Horticultural Crops of Shandong Province, College of Horticulture, Qingdao Agricultural University, Qingdao 266109, China
2
Beijing Key Laboratory of Growth and Developmental Regulation for Protected Vegetable Crops, China Agricultural University, Beijing 100193, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2023, 13(3), 755; https://doi.org/10.3390/agronomy13030755
Submission received: 15 February 2023 / Revised: 2 March 2023 / Accepted: 2 March 2023 / Published: 5 March 2023
(This article belongs to the Special Issue Progress in Horticultural Crops - from Genotype to Phenotype)
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Abstract

:
Cucurbits (Cucurbitaceae) include major horticultural crops with high nutritional and economic value that also serve as model plants for studying plant development and crop improvement. Conventional breeding methods have made important contributions to the production of cucurbit crops but have led to a breeding bottleneck because of the narrow genetic bases and low variation rates of these crops. With the development of molecular techniques, innovations in germplasm development through transgenesis and gene editing have led to breakthroughs in horticultural crop breeding. Although the development of genetic transformation and gene editing techniques for cucurbit crops has lagged behind that for other major crops, great progress has been made in recent years. Here, we summarize recent advances in improving the genetic transformation efficiency of cucurbit crops, including the screening of germplasm and the application of physical treatments, morphogenic genes, and selection markers. In addition, we review the application of gene editing technology to cucurbit crops, including CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated nuclease 9)-mediated gene knockout and base editing. This work provides a reference for improving genetic transformation efficiency and gene editing technology for cucurbit crops.

1. Introduction

Cucurbits (Cucurbitaceae) are a major group of horticultural crops that are cultivated worldwide, including numerous species with extremely high economic and nutritional value. Cucurbit crops such as melon (Cucumis melo), pumpkin (Cucurbita moschata Duchesne), watermelon (Citrullus lanatus), and cucumber (Cucumis sativus) are very popular among consumers. Cucurbit plants are also used as model materials for studying plant development and quality improvement because of their variable shapes and abundance of flavor compounds [1]. In the past few decades, conventional genetic breeding technologies (cross breeding and mutation breeding) have played important roles in breeding selection of cucurbit varieties with high yields and quality. However, the low genetic diversity and variation rates in these crops limit the breeding of complex genetic characters and hamper innovation. With the development of modern biotechnology techniques, innovation via transgenesis and gene editing has become an important focus in the development of horticultural crops and has given rise to new breeding approaches such as “de novo domestication”, “rapid breeding”, “haploid breeding”, and “breaking of genetic linkage” [2,3]. Notably, the recent development of transgenic and gene editing techniques for multiple cucurbit species has greatly facilitated the study of the molecular mechanisms underlying gene function in these crops and should provide new methods for cultivating cucurbit crops with economically desirable traits.
Genetic transformation is a supporting technology for genetic engineering and includes the cloning, delivery, integration, and expression of target genes and the regeneration, screening, and identification of transgene-positive plants. Vector delivery is the first step in transformation, via methods such as protoplast transfection, Agrobacterium (Agrobacterium tumefaciens)-mediated transformation, and particle bombardment. Agrobacterium-mediated transformation is currently the most widely used genetic transformation method because of its high efficiency. Agrobacterium-mediated transformation involves the stable integration of T-DNA from a Ti plasmid containing the target gene into plant chromosomal DNA via infection. The transformation process relies on the efficiency of the plant regeneration system. Much progress has been made in the in vitro culture of cucurbit crops via common methods such as organ culture, somatic embryogenesis, anther culture, and protoplast culture [4,5,6,7]. The most common transformation method for cucurbit crops uses cotyledons as explants for direct or indirect plant regeneration; indirect regeneration is mainly used for watermelon and direct regeneration for cucumber and melons. Trulson et al. established the first system for Agrobacterium-mediated transformation of cucurbit crops using hypocotyls as explants to achieve the stable expression of the Neomycin Phosphotransferase II (NPTII) gene in cucumber [8]. In 1990, Fang et al. successfully transferred the NPTII gene into melons [9]. This was followed by the successful transformation of cucurbit crops such as watermelon, followed by pumpkin, opening the door to the genetic transformation of numerous cucurbit crops.
In recent years, the maturity of transgenic technology enables wide application of gene editing technology in many plants. In gene editing, an engineered endonuclease identifies specific DNA sequences or guide RNAs via its DNA-binding domain (DBD) and cuts the target DNA sequence precisely and efficiently, thus editing specific DNA sequences [10]. Researchers have developed many specific gene editing systems, including a class of nucleases that identify their targets via protein-DNA interactions, e.g., mega nucleases (MNs), zinc finger nucleases (ZFNs), and transcription-activator-like effector nucleases (TALENs) [11,12]. The other class of nucleases includes CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) and CRISPR/Cpf1 (a class 2/type V CRISPR RNA-guided endonuclease), which identify their targets through RNA–DNA base pairing [13]. CRISPR technology is regarded as the optimal method for acquiring genome edited crops given its low cost, high flexibility, and high reliability [14,15,16]. Gene editing technologies have been applied to cucurbit crops relatively recently, particularly CRISPR, which has been successfully used in cucumber, watermelon, melon, and pumpkin.
Although transformation systems have been established for some cucurbit crops, these systems suffer from low genetic transformation efficiency and poorly developed technology, and there are still gaps between these systems and those for other major crops. Researchers have tried to improve the genetic transformation efficiency of cucurbit crops in various ways and have optimized the genetic transformation steps to improve the transformation efficiency of multiple plant varieties. In addition, researchers have achieved gene knockout and base editing in cucurbit crops using CRISPR technology, and the genes responsible for many important agronomic traits have been explored. Here, we summarize the methods used to improve the genetic transformation efficiency of cucurbit crops and the application of gene editing technology to them.

2. Methods to Improve the Genetic Transformation Efficiency of Cucurbit Crops

Agrobacterium-mediated transformation using cotyledons as explants is currently the major technique adopted for many cucurbit crops. The efficiency of genetic transformation is influenced by many factors, including the regeneration efficiency of the plant, infection method, Agrobacterium strain, screening method, and use of exogenous phytohormones (Figure 1). Researchers have recently optimized the transformation process based on previous work, including genotype screening, Agrobacterium infection methods, application of morphogenic genes, and diversification of screening markers, thus improving the transformation efficiency (Figure 1) [17,18,19,20,21,22,23,24,25,26,27,28,29,30,31].
Seed germination: The sterilized seeds were sown on germination medium and cultured for 24–72 h until the cotyledons swelled and the hypocotyls extended to about 5–7 mm (different plant standards). Explant preparation: The cotyledons of the seeds were divided into two parts along the gap, and 2/3 of the adaxial end was taken as the explants. Physical treatments such as sonication, micro-brush, and vacuum infiltration were applied to assist the infection according to the experimental requirements. Coculture: The transformation vector with morphogenic genes was transformed into Agrobacteria and cocultured with explants for 3–4 days. Shoot regeneration: Explants after coculture were placed on the corresponding induction medium to induce shoots. Screening: Transformed positive shoots were identified using different screening markers. Root regeneration: Rooting was induced on positive shoots. Plant: Positive plants were obtained.

2.1. Germplasm Influences Transformation Efficiency

Differences in the genotypes of the target germplasm lead to significant differences in regeneration efficiency, representing an important factor influencing genetic transformation efficiency (Table 1). Identifying suitable germplasm is a direct method for improving the efficiency of genetic transformation. Germplasm screening has often been employed in watermelon, cucumber, and melon, less so in pumpkins, and rarely in other cucurbit crops. Different watermelon germplasms show significant differences in transformation efficiency. The transformation efficiency reached 8–17% in watermelon varieties such as Arka manik, Sugar baby, Arka muthu, IIHR-14, Feeling, and YL but only approximately 1–2% in China baby and Quality [17,18,19,20]. In general, the genetic transformation efficiency of cucumber ranges from 1% to 23%. The genetic transformation efficiency significantly differs among varieties, e.g., up to 23% in Jinyan no.7 but only approximately 1% in Eunchim and 404 [21,22]. The varieties Cu2, Poinsett 76, and Xintaimici are widely used by researchers and show stable genetic transformation efficiencies of 5–20% under different conditions [23,24,25,26]. Genetic transformation has been reported in more than 20 varieties of melon, such as Kirkagaç 637, CM-15, Charentais, and Silver Light, but their transformation efficiency is less than 7% in most cases. Fewer melon varieties with high transformation efficiency are currently available [27,32,33,34,35]. Finally, although it is difficult to transform pumpkins, successful transformation was recently achieved. Using JingXinZhen No. 4, Xin et al. achieved a transformation efficiency of up to 3.56% [22].

2.2. Auxiliary Physical Treatments Significantly Enhance Agrobacterium Infection

Various strains of Agrobacterium, an efficient natural carrier of transgenes, are widely used for the genetic transformation of plants because of their easy transformation and low cost. The most commonly used Agrobacterium strains for the genetic transformation of cucurbit crops include EHA105, GV3101, and LBA4404; different strains show somewhat different transformation efficiencies in various germplasms [26,27,28].
In addition to the effect of strain type, the key to successful transformation lies in the transmission process during infection. For cucurbit crops, the most commonly used explants are leaves, whose regenerable cells are located at the U-shaped cut end that is produced when generating explants. However, it is difficult for Agrobacteria to reach this region under routine infection conditions [43]. To achieve effective Agrobacterium infection, various methods are used, such as sonication, micro-brush (KITA, Nanotek Brush) treatment, and vacuum infiltration to promote the penetration of Agrobacteria into cells deep in the explant. Sonication generates many minor wounds on explants to facilitate the entry of Agrobacteria and improve the genetic transformation efficiency. Sonication improved the transformation efficiency of watermelon from the original rate of 8% to 17% [19]. Similarly, slightly scratching cucumber explants to allow better access by Agrobacteria significantly increased the entry of the transgene, as revealed by β-glucuronidase (GUS) staining [30]. Vacuum infiltration is another effective way to increase the probability of Agrobacterium entry. This method was first applied in the genetic transformation of soybean (Glycine max), Arabidopsis (Arabidopsis thaliana), and wheat (Triticum aestivum) and has also been successfully utilized in cucurbit crops [52]. A variety of cucumber germplasms were successfully transformed using vacuum infiltration. The “optimal infiltration intensity” strategy, which integrates multiple auxiliary methods, was recently proposed. The researchers used sonication and micro-brush techniques to wound cotyledonary node explants and created vacuum conditions with a sterile syringe, significantly improving the transformation efficiency of cucumber, melon, and pumpkin [22].

2.3. Morphogenic Genes Significantly Increase Transformation Efficiency

The genetic transformation efficiency of cucurbit crops can be improved by introducing some auxiliary physical methods. However, exploring and optimizing genetic transformation conditions requires a lot of time and effort, and the same genetic transformation system cannot be universally applied to different genotypes. Many genes capable of promoting or reprogramming cell fate can improve the genetic transformation efficiency of explants. These genes are known as morphogenic genes, including the wounding-associated Wound-Induced Dedifferentiation 1–4 (WIND1–4); epigenetic modification genes, including Polycomb Repressive Complex1/2 (PRC1/2) and Arabidopsis Trithorax4 (ATX4); growth regulatory genes, including Indole3-Acetic Acids (IAAs), YUCCAS (YUCs), and Arabidopsis Response Regulator7/15 (ARR7/15); and developmental regulatory genes, including Wuschel (WUS), Shoot Meristemless (STM), and Wuschel Related Homeobox5/11 (WOX5/11) [53,54,55]. Among morphogenic genes, the WUS-STM and Growth-Regulating Factor and GRF-Interacting Factor (GRF-GIF) complexes are commonly used to improve genetic transformation efficiency. For example, co-expressing WUS and STM greatly improved the in vitro transformation efficiency of monocotyledonous plants such as rice (Oryza sativa), sorghum (Sorghum bicolor), and some maize (Zea mays) inbred lines [56]. Co-expressing WUS and Isopentenyl Transferase (IPT) induced meristem growth and budding in Nicotiana benthamiana seedlings [57]. In addition, exogenously expressing AtGRF5 promoted budding and improved the genetic transformation efficiency of soybean, oilseed rape (Brassica napus), and sunflower (Helianthus annuus) [58]. Expressing fusion protein of TaGRF4 and OsGIF1 improved the regeneration speed and efficiency of wheat and rice and expanded the scope of varieties that could be used for genetic transformation [59].
Expressing morphogenic genes can improve the genetic transformation efficiency of watermelon. Among these genes, AtGRF5, TaGRF4-OsGIF1, ZmWUS, ZmWUS–ZmBBM, ZmWUS–IPT, and AtGRF5 showed the best effects, increasing the genetic transformation efficiency of watermelon cultivar WWl50 approximately 40-fold to 24.73% [28]. In another study, overexpressing the endogenous genes ClGRF4–ClGIF1 achieved a transformation efficiency of up to 47.02% in watermelon cultivar TC, representing an approximately 9-fold increase compared with the control (with a transformation efficiency of 5.23%). Mutating the miR396 target site in ClGRF4 further increased the transformation efficiency to 67.27% and had significant effects in eight other watermelon varieties, such as YL, M08, 148, and 97103 [29]. The role of morphogenic genes in increasing transformation efficiency has been verified in watermelon, for which indirect regeneration is mainly used, but whether these genes can be used for other cucurbit crops such as cucumber and melon (for which direct regeneration is mainly used) remains to be studied.

2.4. Diversification of the Screening Markers Used in Genetic Transformation

Introducing screening marker genes during transformation can facilitate the identification of positive transgenic events and can help determine whether the transformation was successful. Three major categories of screening markers are currently used in the genetic transformation of cucurbit crops. The first category is resistance genes, e.g., NPTII, Hygromycin phosphotransferase (HPT), Phosphinothricin N-acetyltransferase (BAR), and Acetolactate Synthase (ALS). The buds of explants harboring a resistance gene can grow normally under selection, whereas buds lacking the resistance gene cannot grow. The second category is reporter genes, e.g., Enhanced Green Fluorescent Protein (eGFP), Discosoma striata red Fluorescent Protein (DsRed), luciferase (LUC), and β-glucuronidase (GUS). Under specific physical/chemical conditions, buds harboring a reporter gene exhibit a different appearance from buds that have not been transformed. The third category is metabolism-related genes—e.g., Phosphomannose isomerase (pmi) and maize R, C1, and B transcription factor genes—which allow transformed regenerated buds to grow under specific nutritional conditions [31,60].
Among the resistance genes, NPTII has been used as a selection marker, but its effectiveness differs significantly between germplasms. Cotyledon explants of cucumber variety CMCC are extremely sensitive to hygromycin B, with good screening effects even at low concentrations [30]. However, escape is likely to occur when resistance genes are used; that is, buds free of any resistance gene can sometimes grow under selection [27]. In contrast, the use of reporter genes results in no escapes. Under specific physical/chemical conditions, reporter genes such as eGFP, DsRed, LUC, and GUS enable transgene-positive buds to exhibit a color different from that of non-transformed buds, thus achieving effective screening and avoiding the disadvantage of escapes. However, reporter genes are not perfect. They may be interfered with by the plants themselves, making it difficult to identify transgenic material under specific conditions. Moreover, high levels of reporter gene expression can damage the plant, leading to abnormal phenotypes. In addition to resistance genes and reporter genes, researchers have also developed metabolism-related genes, which allow transgene-positive buds to grow under specific nutritional conditions. For example, expression of the pmi gene enables the conversion of mannose-6-phosphate into fructose-6-phosphate, allowing regenerated buds to grow normally using mannose as a carbon source. Using the mannose selection system in cucumber, effective transgene-positive bud screening was achieved using a medium containing 10 g/L mannose and 10 g/L saccharose, leading to reduced damage and escape compared with the use of reporter genes and resistance genes [61].

2.5. Other Factors

Genetic transformation involves multiple factors and multiple steps. Therefore, various issues are likely to occur during transformation, which differ somewhat in different species. For example, cucumber is rich in endogenous ethylene, but the accumulation of ethylene is unfavorable for cell growth and differentiation and in severe cases leads to the aging of cells. AgNO3 can be added to the medium during transformation to inhibit ethylene production and promote cell differentiation and regeneration. However, Ag+ is a heavy metal that is somewhat toxic to plants, and the long-term use of AgNO3 can lead to plant deformity [43].
Browning, vitrification, and Agrobacterium contamination are likely to occur during melon transformation. Browning occurs when cells experience metabolic changes upon the activation of polyphenol oxidase in explants. This process can occur because of the germplasm selected, physiological conditions, culture conditions, and damage [62]. Antioxidants such as vitamin C, sodium thiosulfate, and cysteine can be added during melon transformation to inhibit browning. Vitrification is a condition in which the stems and leaves of explants are water-soaked and fragile. Vitrified seedlings tend to grow slowly and show weak differentiation ability. Vitrification may be related to phytohormone levels, culture conditions, and relative humidity. Vitrification of seedlings during transformation can be reduced by increasing the concentrations of agar and saccharose in the medium as appropriate, strengthening ventilation and air exchange, and controlling relative humidity levels. Finally, since Agrobacterium mediation is generally required for transformation, Agrobacterium contamination/overgrowth may occur during tissue culture. For Agrobacterium contamination, cefotaxime, plant preservative mixture (PPM), and Timentin can be added to the medium to inhibit the reproduction of the Agrobacteria and improve the survival rates of plants [43].

3. Application of Gene Editing Technology in Cucurbit Crops

Gene editing technology is an important strategy for gene function studies and molecular breeding. A gene editing system, which is introduced into a plant through genetic transformation, uses engineered endonucleases to edit the specific nucleotide sequences of the plant genome precisely and efficiently. CRISPR technology has become a mainstream gene editing technology used for cucurbit crops thanks to its efficiency, precision, and easy operation. After gene editing, the carrier fragments can be eliminated from the plants through hybridization [63]. CRISPR/Cas9-based knockout and base editing of genes have been successfully achieved in various cucurbit crops (Figure 2 and Table 2).

3.1. CRISPR/Cas9-Based Gene Knockout

CRISPR/Cas9 is a microbial adaptive immune mechanism that achieves precise gene editing based on the RNA-guided Cas9 nuclease within the Type II prokaryotic CRISPR/Cas system [87]. The present CRISPR-Cas9 systems are modifications of bacterial CRISPR/Cas9, comprising modified Cas9 endonuclease and a single guide (sg)RNA [88]. Under the guidance of a specific sgRNA, Cas9 specifically identifies the protospacer adjacent motif (PAM, NGG) in its target sequence and produces blunt-ended double-strand breaks approximately 3 bp upstream of the PAM to trigger the HDR or NHEJ pathway, thus achieving precise editing of plant genes [89].
In 2016, CRISPR/Cas9 was successfully applied to cucurbit crops for the first time. The researchers used CRISPR/Cas9 to mutate the DNA sequence at the target site of eukaryotic translation initiation factor 4E (eIF4E) at its N’ and C’ termini in cucumber. Two homozygous eIF4E knockout mutants exhibited immunity to Cucumber vein yellowing virus (Ipomovirus) infection and resistance to the potyviruses Zucchini yellow mosaic virus and Papaya ring spot mosaic virus-W [42]. Subsequently, Hu et al. used the cucumber U6 promoter to drive sgRNA to knock out WIP Domain Protein 1 (CsWIP1) using CRISPR/Cas9 technology and successfully created a gynoecious cucumber line [43]. Thereafter, Liu et al. explored the role of Sporocyteless (CsSPL) in ovule development and reproduction [73]. Since then, CRISPR/Cas9 has been widely used in cucumber. Researchers have successfully used CRISPR/Cas9 in breakthrough studies on the functions of genes for sex, fruit length, tendril formation, and stress resistance in cucumber [22,30,75,76,77,78,79,80,81].
In addition to the successful application of CRISPR/Cas9 in cucumber, gene knockout using CRISPR/Cas9 has also been reported in watermelon. Tian et al. knocked out the phytoene desaturase (ClPDS) gene in watermelon and successfully obtained albino regenerated buds, demonstrating the feasibility of applying CRISPR/Cas9 technology to watermelon [37]. In 2019, researchers used CRISPR/Cas9 to knock out the ClWIP1 gene and obtained a gynoecious line to explore the molecular mechanism that determines the sex of watermelon [66]. Researchers also used CRISPR/Cas9 to successfully knock out genes (e.g., a key component of the cohesin complex in meiosis (ClREC8), caffeic acid O-methyltransferase (ClCOMT1), β-glucosidase (ClBG1), NAC transcription factor68 (ClNAC68), vacuolar sugar transporter (ClVST1), alkaline alpha-galactosidase (ClAGA2), Citrullus lanatusSugars Will Eventually Be Exported Transporter 3 (ClSWEET3), Tonoplast Sugar Transporter (ClTST2), Citrullus lanatus Abnormal Tapetum 1 (ClATM1), and nuclear transcription factor Y subunit B-9-like (ClLEC1)) and achieved good results, increasing seed fertility, stress resistance, seed size, and sugar transport in watermelon [29,64,65,66,68,69,70,71]
Compared with that in cucumber and watermelon, the application of gene editing technology in other cucurbit crops is lagging. For melons, researchers successfully used CRISPR/Cas9 to knock out the CmPDS gene in 2019 and obtained albino regenerated buds [35]. Researchers subsequently used CRISPR/Cas9 to knock out eIF4E, ERECTA (CmER), and the NAC transcription factor gene Nonripening (CmNAC-NOR) and revealed their functions in virus resistance, plant architecture, and fruit ripening, respectively [22,82,83,84]. In pumpkin, researchers used Agrobacterium rhizogenes as an infection tool to develop an efficient CRISPR/Cas9-based root transformation system to analyze stem–root communication in cucurbit crops and successfully knocked out high-affinity K + transporter 1 (CmoHKT1;1) and Plasma membrane intrinsic proteins (CmoPIP1–4) [85,86]. Besides the root transformation system, Xin et al. obtained a knockout mutant of CmoER and achieved stable gene editing in pumpkin [22]. This team recently used CRISPR/Cas9 technology to achieve domain B knockout of CmoYABBY1, thus strengthening the translation of this gene and inhibiting stem growth proportionally in a dose-dependent manner, creating a pumpkin plant with a dominant bushy trait (Figure 2) [1].

3.2. Precise Editing via Cytosine Base Editors

In addition to the gene knockout achieved in cucurbit crops, precise single-nucleotide editing of genes in watermelon has been achieved using cytosine base editors (CBEs). CBEs consist of a Cas9 nickase (nCas9) with a D10A mutation, which deactivates RuvC (one of the two Cas9 nuclease domains), fused with a cytidine deaminase and a uracil DNA glycosylase (UDG) inhibitor (UGI). CBEs induce a C:G > T:A base change. Tian et al. used a novel Cas9 variant fused with CBE3 to achieve a single-nucleotide conversion from C to T in the Pro190 (CCG) codon of the ALS gene of watermelon without merging the DNA templates of the donor, thereby creating a non-GM herbicide-resistant watermelon variety [38]. It is worth noting that the first successful application of CRISPR/Cas9 technology in watermelon was also achieved by the same team [37].

4. Discussion and Future Prospects

In this review, we systematically summarized recent innovations and applications of genetic transformation and gene editing technology in cucurbit crops. Significant improvements have been made, but there are still many outstanding issues in the genetic transformation of cucurbit crops.

4.1. Establish Genotype-Free Transformation Systems

Genotypes play a pivotal role in transformation. Extensive studies have been performed on cucurbit crops, and the germplasms examined have varied widely among research teams. In contrast to other species, no widely used germplasm is currently available for the stable transformation of cucurbit crops. A uniform genotype would help researchers explore the functions of genes and analyze their genetic relationships. Therefore, testing the genetic transformation efficiency of the existing germplasms and identifying the most suitable receptor germplasms could help improve the genetic transformation efficiency of cucurbit crops. Certainly, for future molecular breeding, striving to establish a transformation system that is not limited by genotype is our ultimate goal, e.g., establishment of a genotype-free genetic transformation system using morphogenic genes. The use of morphogenic genes can significantly improve genetic transformation efficiency, as verified in multiple plant varieties [28,29]. However, the strong expression of morphogenic genes can affect the growth and development of plants, making it crucial to eliminate the abnormal phenotypes of regenerated seedlings when morphogenic genes are used. Various studies have used inducible 35s-MdBBM-GR vectors or the heat-induced Cre/loxP gene editing system during specific periods to reduce the damage caused by morphogenic genes [90,91]. In the future, innovative ways to solve this problem should continue to be explored.

4.2. Further Development of Gene Editing Technologies in Cucurbit Crops

The development prospects of gene editing technology are very broad. For example, the CRISPR-Cas9 system in tissue-specific or induced promoter control can help with spatial or temporal genomic alterations [92,93]. CRISPR-Cas9 also enables rapid functional identification of genes by generating gRNA libraries. As a result, CRISPR-Cas9 has become an efficient, simple, and fast tool for growing agriculturally improved crops that can both add better traits and remove undesirable traits [94]. Certainly, one of the best applications of the CRISPR-Cas9 system is the use of label-free “cisgene” plants with improved agronomic traits. These plants will eventually be immune to current transgene regulation because CRISPR-associated RNA-guided nucleases (RGENs) or ribonucleoproteins (RNPs) induce genomic mutations without any foreign DNA [95]. However, the development of gene editing technology in cucurbit crops is lagging, with successful results (including gene knockout) achieved only through CRISPR/Cas9 and base editing [37,38]. Efficient editing systems, such as “precise editing” via prime editing and gene editing systems optimized for the characteristics of cucurbit crops, remain to be further explored.
In conclusion, some progress has been made in the genetic transformation and gene editing of cucurbit crops, but many issues remain to be resolved.

Author Contributions

Conceptualization, J.F., N.W. and S.C.; formal analysis, J.F. and N.W.; investigation, J.F. and N.W.; resources, J.F., N.W. and H.W. (Huihui Wang); data curation, Y.L.; writing—original draft preparation, J.F., N.W. and S.C.; writing—review and editing, W.Z., N.W. and S.C.; visualization, J.F. and N.W.; supervision, H.W. (Huasen Wang) and S.C.; project administration, S.C.; funding acquisition, S.C. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (32102404 to S.C.), This work was also supported by the Natural Science Foundation of Shandong Province (ZR2020QC157 to S.C.).

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Efficient methods for the genetic transformation of cucurbit crops.
Figure 1. Efficient methods for the genetic transformation of cucurbit crops.
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Figure 2. Illustration of editing genes in cucurbit crops.
Figure 2. Illustration of editing genes in cucurbit crops.
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Table
Table 1. Details of some successful reports on Agrobacterium-mediated transformation of cucurbit crops.
Table 1. Details of some successful reports on Agrobacterium-mediated transformation of cucurbit crops.
SpeciesGermplasmStabilityAgrobacterium StrainsTransformation
Efficiency (%)		T1Reference
WatermelonFeeling, China baby, QualityKanamycinLBA44048.5–10%, 1–2.1%, 0–1.3% (PCR)Reported[18]
WatermelonFeeling, China baby, QualityKanamycinLBA44047%, 2.22%, 1.25% (PCR and Southern blot)NR[17]
Watermelonthe female parent line of Zhengkang No. 6Kanamycin/cefotaximeLBA44043 plantlets (PCR and Southern blot)Reported[36]
WatermelonPI179878Hygromycin BEHA1051.67% (shoots)NR[37]
WatermelonZG94Bialaphos resistanceEHA10523% (PCR)Reported[38]
WatermelonArka manik, Sugar baby, Arka muthu, IIHR-14BASTAEHA10517.33%, 12.33%, 16%, 14.66% (GUS, PCR, Southern hybridization)NR[19]
WatermelonTCNREHA1056.5% (GFP, PCR)NR[29]
WatermelonYLGlufosinate-ammoniumEHA10512.5% (PCR)NR[20]
CucumberEunchimPhospinotricinEHA1011.7%
(Northern blot)		NR[21]
CucumberPoinsett 76PhosphinothricinEHA105 LBA440421.0%, 8.5% (shoots)Reported[24]
CucumberEunsungParomomycinEHA1014.01%
(Southern blot)		Reported[39]
CucumberXintaimiciKanamycinLBA44044.8%
(PCR, Southern blot, Western blot)		NR[40]
CucumberShinhokusei 1KanamycinEHA10511.9 ± 3.5% (plants)Reported[41]
CucumberIlanKanamycinEHA1053 plants (PCR)Reported[42]
CucumberCu2GFPEHA1051.32% (plantlets)Reported[43]
CucumberXintaimiciKanamycinGV31018.10% (PCR)NR[26]
CucumberCCMCGUS/Hygromycin BEHA1050.24% (PCR)NR[30]
CucumberCu2, Xintaimici, 404, Eu1GFPEHA1055.18%, 2.20%, 1.97%, 2.46% (GFP)Reported[22]
MelonSilver LightKanamycinLBA4404Unspecified
(2 transgenic lines)		Reported[44]
MelonHetao Transformation via the pollen-tube pathway4.3% (PCR)Reported[45]
MelonVédrantisKanamycinGV22600.35–3.0% (PCR and GUS histochemical analysis)NR[46]
MelonHybrid line M01–3 Ovary-injection transformation2.7% (PCR)Reported[47]
MelonCantaloupe F39 Honeydew 150Kanamycin/GUSEHA1050.3%, 0.5%
(PCR, GUS and Southern blot)		NR[48]
MelonGeumnodajieunchunKanamycin/geneticinLBA44042.9%, 7.1% (PCR)NR[49]
MelonSilver LightKanamycinLBA440411 transgenic lines
(PCR and
Southern blot)		Reported[50]
MelonSilver LightKanamycinLBA44040.8% (PCR)Reported[33]
MelonCM-23KanamycinEHA1054% (PCR, Southern hybridization)NR[51]
MelonM-15KanamycinEHA10513% (PCR)Reported[32]
MelonCM-15KanamycinEHA1054 plants (PCR)Reported[34]
MelonCharentais, Galia,
Piel de sapo, Blanco		KanamycinEHA1051.3%, 1.6%, 3.8%, 2.5% (plants)Reported[27]
MelonCharentaisHygromycin BEHA1055.68% (shoots)NR[35]
Melonm1GFPEHA1053.95% (GFP)Reported[22]
PumpkinJingXinZhen No. 4GFPEHA1053.56% (GFP)Reported[22]
Table
Table 2. Details of editing genes of cucurbit crops.
Table 2. Details of editing genes of cucurbit crops.
SpeciesGeneIDGene Function or PhenotypeReferences
WatermelonClALSCla019277Herbicide resistance[38]
WatermelonClPDSCla97C07G142100Albino plants[28]
WatermelonClPDSCla010898Albino plants[37]
WatermelonClBG1Cla97C08G153160Regulation of seed size and seed germination[64]
WatermelonClNAC68Cla97C03G059250Regulation of fruit sugar content and seed development[65]
WatermelonClLEC1Cla97C10G188900Reproductive development[29]
WatermelonClWIP1Cla008537Sex determination[66]
WatermelonClVST1Cla97C02G031010Vacuolar sugar transporter[67]
WatermelonClAGA2,
ClSWEET3,
ClTST2		Cla97C04G070460,
Cla97C01G000640,
Cla97C02G036390		Carbohydrate partitioning[68]
WatermelonClREC8Cla97C07G132920Seedless watermelon[69]
WatermelonClCOMT1Cla97C10G188660Tolerance to abiotic stresses[70]
WatermelonClATM1Cla010576Male sterility[71]
CucumberCsALCCsa2G356640.1Pollen tube emergence[72]
CucumbereIF4EXM_004147349Antivirus[42]
CucumberCsWIP1, CsVFB1,
CsMLO8, CsGAD1		Csa4M290830, Csa4M641640,
Csa5M623470, Csa5M348050		Gynoecious inbred lines[43]
CucumberCsSPLCsa3M850670Male and female fertility and ovule development[73]
CucumberCsSF1, CsACF2Csa2G174140, Csa1G580750Cucurbit-specific RING-type E3 ligase,
Rate-limiting enzyme for ethylene biosynthesis		[74]
CucumberCsSF2Csa2G337260Fruit elongation[75]
CucumberCsTEN, CsACO1Csa5P644520.1, Csa6G160180Identity and mobility of tendrils[76]
CucumberCsHEC2Csa2G285890Fruit wart[77]
CucumberCsACS1, CsMYBCsaV3_6G044400, CsaV3_6G044410Female floral development[78]
CucumberCsHEC1, CsOVATE,
CsYUC4		Csa4G639900, Csa4G038760,
Csa2G379350		Fruit neck length[79]
CucumberCsGCN5Csa6G527060Gene transcription and plant development[30]
CucumberCsERCsaV3_4G036080Compact plant architecture[22]
CucumberCsGLDH, CsMIOXCsa4M236360.1, Csa2M000640Ascorbic acid biosynthesis[80]
CucumberCsAKT1CsaV3_1G029650Salt tolerance[81]
MelonCmPDSMELO3C017772.2Albino plants[35]
MelonCmNAC-NORMELO3C016540.2Fruit ripening[82]
MelonCmNAC-NORMELO3C016540Fruit coloring and ripening[83]
MeloneIF4EMELO3C002698.2Antivirus[84]
PumpkinCmoER1, CmoER2CmoCh09G003660, CmoCh01G017570Compact plant architecture[22]
PumpkinCmoPIP1-4CmoCh04G011950Salt stress[85]
PumpkinCmoHKT1;1CmoCh10G003830High-affinity K+ transporter1[86]
PumpkinCmoYABBY1CmoCh15G012090Architecture regulation[1]
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MDPI and ACS Style

Feng, J.; Wang, N.; Li, Y.; Wang, H.; Zhang, W.; Wang, H.; Chai, S. Recent Progress in Genetic Transformation and Gene Editing Technology in Cucurbit Crops. Agronomy 2023, 13, 755. https://doi.org/10.3390/agronomy13030755

AMA Style

Feng J, Wang N, Li Y, Wang H, Zhang W, Wang H, Chai S. Recent Progress in Genetic Transformation and Gene Editing Technology in Cucurbit Crops. Agronomy. 2023; 13(3):755. https://doi.org/10.3390/agronomy13030755

Chicago/Turabian Style

Feng, Jing, Naonao Wang, Yang Li, Huihui Wang, Wenna Zhang, Huasen Wang, and Sen Chai. 2023. "Recent Progress in Genetic Transformation and Gene Editing Technology in Cucurbit Crops" Agronomy 13, no. 3: 755. https://doi.org/10.3390/agronomy13030755

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