Melon (Cucumis melo L.) is a widely consumed fruit across the globe. While conventional breeding has contributed significantly to improving the quality and productivity of melon cultivars, there remains an urgent need for rapid and diversified genetic enhancements. Genome editing is regarded as a promising tool to meet these demands. However, most current genome editing protocols rely on genetic transformation and cell culture, yet melon presents inherent challenges due to its low transformation efficiency, often resulting in the generation of false-positive regenerated plants (escapes) (Shirazi Parsa et al., 2023). Moreover, melon is susceptible to ploidy alterations during cell culture, which can adversely affect morphology of regenerated plants (Ezura et al., 1992; Ayub et al., 1996; Liu et al., 2022; Nonaka et al., 2023). Consequently, the development of an efficient and reliable genome editing system for melon genetic improvement is imperative.

Previous studies have demonstrated a transgene-free genome editing method known as in planta particle bombardment-ribonucleoprotein (iPB-RNP), which has been successfully applied in wheat, barley, and soybean (Kumagai et al., 2022; Kuwabara et al., 2024; Tezuka et al., 2024). This technique involves the direct delivery of CRISPR/Cas9 ribonucleoproteins into the shoot apical meristem (SAM), targeting subepidermal L2 cells that serve as potential germline cells (Goldberg et al., 1993). As the iPB-RNP method circumvents the need for cell culture and plant regeneration, it offers considerable potential for application in recalcitrant crop species, including melon.

In the present study, an efficient genome editing platform for melon was established using the iPB-RNP method. CRISPR/Cas9 RNPs successfully induced targeted genome editing in E₀ plants (the first generation of genome-edited individuals), with a subset of the edited alleles transmitted to the next-generation. Targeting an ethylene biosynthesis gene using this platform enabled the generation of melon with extended shelf-life, wherein fruit ripening can be modulated by exogenous ethylene application.

To assess the feasibility of genome editing in melon via iPB-RNP, the efficiency of gold particle delivery into meristematic cells was first evaluated. Following 20 h of seed imbibition, one cotyledon was removed from germinated seeds (cv. Harukei-3) to expose the shoot apical meristem (SAM), and the remaining part of each seed was placed on an agar plate with the exposed SAM facing upward (Figure 1A). Gold particles coated with a GFP-expressing vector were then bombarded onto the SAMs (Figure 1A). After 16 h in darkness, GFP fluorescence was observed on the SAM surface in nearly all bombarded seeds, in contrast to untreated controls (Figure 1A; Supplementary Figure S1), consistent with previous observations in wheat and soybean (Hamada et al., 2017; Kuwabara et al., 2024). These results confirmed that the preparation of SAMs and the particle bombardment conditions were suitable for iPB application in melon.

To implement the iPB-RNP method in melon, CmGAD1, a glutamate decarboxylase gene (You et al., 2024), was selected as the initial genome editing target. Pre-assembled SpCas9 ribonucleoproteins (RNPs) targeting CmGAD1 (Supplementary Figure S2) were coated onto gold particles and delivered into the SAMs of germinated seeds. Plants regenerated from the bombarded seeds were analyzed after 4–5 weeks, and their sixth leaf was subjected to cleaved amplified polymorphic sequences (CAPS) analysis to detect mutations (Figure 1B). Seven of the 153 plants initially bombarded exhibited undigested bands in the CAPS assay (Figure 1C). Subsequent sequencing of these undigested bands confirmed the presence of insertion or deletion mutations involving single base pairs (Figure 1D). The editing efficiency in the primary (E₀) generation was 4.58% (7/153) (Figure 1G). As the E₀ mutants were chimeric, containing a mixture of edited and non-edited cells, seeds were collected from these plants and the progeny (E₁ generation) was analyzed to confirm stable inheritance of mutations. CAPS analysis detected mutant CmGAD1 alleles in two of the seven E₀ progenitor plants (Figures 1E,G). Among these, plants #2–13_2, #2–13_3, #2–16_5, and #2–16_6 were identified as homozygous mutants for CmGAD1 (Figure 1E), and sequencing confirmed that the E₁ mutations were consistent with those observed in the E₀ generation (Figures 1D,F). The overall editing efficiency of iPB-RNP for CmGAD1 was 1.31% (2/153 bombarded SAMs) (Figure 1G), which is comparable to editing efficiencies previously reported in wheat, barley, and soybean (Kumagai et al., 2022; Kuwabara et al., 2024; Tezuka et al., 2024).

Establishment of the iPB-RNP method in melon enabled the generation of mutants with an extended shelf-life, a highly desirable trait for reducing postharvest losses and expanding marketability. Because the gaseous phytohormone ethylene plays a central role in fruit ripening, CmACO1, a key gene involved in the final step of ethylene biosynthesis in melon, was selected as the target (Nonaka et al., 2023). A guide RNA was designed and synthesized to target CmACO1 (Figure 2A). Assembled Cas9 RNPs were delivered into SAMs via iPB-RNP, and resulting mutants were screened using CAPS analysis, as previously described. In total, 16 E₀ plants carried mutations, and heritable mutations were detected in three plants (#24–7, #30–11, and #30–16) (Figures 2B–E). Ultimately, three genome-edited lines were obtained from 227 bombarded SAMs, corresponding to a 1.32% editing efficiency (Figure 2F). CAPS analysis in the E₁ generation indicated that the #24–7_1 plant was a homozygous mutant, while the #30–11 and #30–16 plants yielded only heterozygous progeny (Figure 2D).

To investigate the phenotypic consequences of CmACO1 disruption, fruit from the wild-type (WT) and the cmaco1 homozygous mutant (#24–7) were compared. The #24–7 line carried a two-base deletion within the CmACO1 coding region, indicating a likely loss-of-function mutation (Figures 2E,G). Fruit size and shape in the cmaco1 mutant were similar to those of WT, and no significant difference in fresh fruit weight was observed between the two genotypes (Figure 2H). Eleven days after harvest (11 DAH), the epicarp of WT fruit had turned from green to cream-yellow, indicative of ripening, whereas the cmaco1 mutant fruit remained green (Figure 2H). To confirm whether the delayed ripening phenotype was attributable to reduced ethylene production, ethylene emission was measured post-harvest. WT fruit exhibited a sharp ethylene peak at 8 DAH, whereas cmaco1 fruit maintained significantly lower ethylene levels up to 16 DAH (Figure 2I). In a previous study, a cmaco1 mutant generated via Agrobacterium-mediated genome editing showed reduced fruit size and flattened shape, attributed to tetraploidy induced by somaclonal variation (Nonaka et al., 2023). In contrast, the present findings demonstrate that the iPB-RNP approach effectively avoids cell culture-associated complications, enabling the production of melons with extended shelf-life while maintaining normal morphology and growth characteristics.

It is commercially important for delayed-ripening melons to retain the ability to respond to exogenous ethylene to initiate ripening. To determine whether genome-edited cmaco1 fruits are capable of ripening under exogenous ethylene exposure, a short-term ethylene treatment was conducted. At 7 days after harvest (DAH), cmaco1 fruits were treated with a high concentration of ethylene (400 ppm) for 24 h (Figure 3A). A short-term treatment was selected to avoid the complications associated with continuous ethylene exposure, which requires precise control of ethylene concentration and periodic ventilation to prevent oxygen depletion (Guis et al., 1997; Nishiyama et al., 2007). Three days following the ethylene treatment (11 DAH), a slightly softened flesh texture was observed in the cross-section of the cmaco1 fruit, indicating that ripening had progressed (Figure 3B). In contrast, cmaco1 fruit not treated with ethylene retained a firm flesh texture even at 11 DAH (Figure 3B). Because flesh firmness and juice content are key indicators of fruit ripening (Chen et al., 2023), these parameters were measured in cmaco1 fruits before and after ethylene treatment. In wild-type (WT) fruits, a significant decrease in flesh firmness and an increase in extractable juice content were observed at 11 DAH compared to 5 DAH (Figure 3C). At 5 DAH, cmaco1 fruits displayed similar levels of firmness and juice content as WT, suggesting minimal ripening activity at this stage. However, at 11 DAH, cmaco1 fruits exhibited markedly firmer flesh and lower juice content than WT (Figure 3C). Upon ethylene treatment, these values in cmaco1 fruits shifted to levels comparable to those of fully ripened WT fruit at 11 DAH (Figure 3C). Previous studies reported that continuous ethylene exposure (50 ppm for 4 or 7 days) reversed the delayed-ripening phenotype of CmACO1 antisense melons (Ayub et al., 1996; Guis et al., 1997). The present data demonstrate that a short, high-concentration ethylene treatment can effectively induce ripening in the cmaco1 knockout mutant.

The effect of exogenous ethylene on the expression of fruit softening-related genes, Polygalacturonase1 (CmPG1) and Polygalacturonase2 (CmPG2), was analyzed in mesocarp tissue. Quantitative PCR (qPCR) analysis revealed that in WT fruit, CmPG1 and CmPG2 expression increased during ripening and declined by 11 DAH, when the fruit was fully softened (Figures 3B,D). In the cmaco1 mutant, CmPG1 expression was significantly reduced but was restored to WT levels following ethylene treatment (Figure 3D), indicating that CmPG1 expression is regulated by ethylene during ripening. Similarly, CmPG2 expression was also reduced in the cmaco1 mutant and restored by exogenous ethylene. Transient expression of CmPG1 and CmPG2 during ripening has been previously reported using gel-blot analysis (Hadfield et al., 1998; Nishiyama et al., 2007). The present qPCR analysis further revealed that CmPG1 expression was approximately twice as high as CmPG2 and that both genes were upregulated during ripening. These results suggest that ethylene-induced upregulation of CmPG genes contributes to flesh softening in ethylene-treated cmaco1 fruits.

To evaluate the expression of CmACO family genes (CmACO1–CmACO5) in mesocarp tissue, it was found that CmACO1 exhibited the highest and predominant expression in WT during fruit ripening (Supplementary Figure S3), consistent with the conclusion that postharvest ethylene production in melon is primarily mediated by CmACO1 (Nonaka et al., 2023). In cmaco1 mutants, only basal levels of CmACO2–CmACO5 transcripts were detected, both with and without ethylene treatment, and these levels were substantially lower than CmACO1 expression in WT (Supplementary Figure S3). These findings suggest that fruit ripening in cmaco1 is triggered by exogenous ethylene rather than endogenous ethylene synthesized via alternative CmACO genes. Expression of CmACO1 in cmaco1 mutants was slightly lower than that in WT at 2 DAH and dramatically decreased after 8 DAH (Figure 3D). Given that the cmaco1 mutant harbors a premature stop codon within the CmACO1 gene (Figure 2G), it is likely that aberrant CmACO1 transcripts are degraded via nonsense-mediated mRNA decay (Cai et al., 2018) once transcript levels reach a certain threshold.

In conclusion, a non-culture, DNA-free genome editing technique was successfully developed for melon using the iPB-RNP platform. The efficiency of producing genome-edited E₁ plants from bombarded SAMs was 1.31% for CmGAD1 and 1.32% for CmACO1 (Figures 1G, 2F), comparable to efficiencies reported for wheat, barley, and soybean (Kumagai et al., 2022; Kuwabara et al., 2024; Tezuka et al., 2024). As the method eliminates the need for cell culture, it is expected to overcome limitations inherent to traditional transformation approaches, such as genotype dependency and somaclonal variation (Nuñez-Palenius et al., 2008). The successful generation of an extended shelf-life melon line in this study highlights the broad potential of this technique for application in commercial melon breeding and across diverse species within the Cucurbitaceae family.