Possibility of genome editing for melon breeding.

Genome editing technologies are promising for conventional mutagenesis breeding, which takes a long time to remove unnecessary mutations through backcrossing and create new lines because they directly modify the target genes of elite strains. In particular, this technology has advantages for traits caused by the loss of function. Many efforts have been made to utilize this technique to introduce valuable features into crops, including maize, soybeans, and tomatoes. Several genome-edited crops have already been commercialized in the US and Japan. Melons are an important vegetable crop worldwide, produced and used in various areas. Therefore, many breeding efforts have been made to improve its fruit quality, resistance to plant diseases, and stress tolerance. Quantitative trait loci (QTL) analysis was performed, and various genes related to important traits were identified. Recently, several studies have shown that the CRISPR/Cas9 system can be applied to melons, resulting in its possible utilization as a breeding technique. Focusing on two productivity-related traits, disease resistance, and fruit quality, this review introduces the progress in genetics, examples of melon breeding through genome editing, improvements required for breeding applications, and the possibilities of genome editing in melon breeding.

Efficient transient protein expression in tomato cultivars and wild species using agroinfiltration-mediated high expression system.

KEY MESSAGE: The new transient protein expression system using the pBYR2HS vector is applicable to several tomato cultivars and wild species with high level of protein expression. Innovation and improvement of effective tools for transient protein expression in plant cells is critical for the development of plant biotechnology. We have created the new transient protein expression system using the pBYR2HS vector that led to about 4 mg/g fresh weight of protein expression in Nicotiana benthamiana. In this study, we validated the adaptability of this transient protein expression system by agroinfiltration to leaves and fruits of several tomato cultivars and wild species. Although the GFP protein was transiently expressed in the leaves and fruits of all tomato cultivars and wild species, we observed species-specific differences in protein expression. In particular, GFP protein expression was higher in the leaves and fruits of Micro-Tom, Solanum pimpinellifolium (0043) and S. pimpinellifolium (0049-w1) than in those of cultivars and wild species. Furthermore, Agrobacterium with GABA transaminase enhanced transient expression in tomato fruits of Micro-Tom. Taken together with these results, our system is applicable to several tomato cultivars and species as well as a model tomato, even though characteristics are often different among tomato cultivars or species. Thus, the system is an effective, simple, and valuable tool to achieve rapid transgene expression to examine gene function in tomato plant cells.

Melonet-DB, a Grand RNA-Seq Gene Expression Atlas in Melon (Cucumis melo L.).

Melon (Cucumis melo L.) is an important Cucurbitaceae crop produced worldwide, exhibiting wide genetic variations and comprising both climacteric and non-climacteric fruit types. The muskmelon cultivar "'Earl's favorite Harukei-3 (Harukei-3)"' known for its sweetness and rich aroma is used for breeding of high-grade muskmelon in Japan. We conducted RNA sequencing (RNA-seq) transcriptome studies in 30 different tissues of the 'Harukei-3' melon. These included root, stems, leaves, flowers, regenerating callus and ovaries, in addition to the flesh and peel sampled at seven stages of fruit development. The expression patterns of 20,752 genes were determined with fragments per kilobase of transcript per million fragments sequenced (FPKM) >1 in at least one tissue. Principal component analysis distinguished 30 melon tissues based on the global gene expression profile and, further, the weighted gene correlation network analysis classified melon genes into 45 distinct coexpression groups. Some coexpression groups exhibited tissue-specific gene expression. Furthermore, we developed and published web application tools designated "'Gene expression map viewer"' and "'Coexpression viewer"' on our website Melonet-DB (http://melonet-db.agbi.tsukuba.ac.jp/) to promote functional genomics research in melon. By using both tools, we analyzed melon homologs of tomato fruit ripening regulators such as E8, RIPENING-INHIBITOR (RIN) and NON-RIPENING (NOR). The "'Coexpression viewer"' clearly distinguished fruit ripening-associated melon RIN/NOR/CNR homologs from those expressed in other tissues. In addition, several other MADS-box, NAM/ATAF/CUC (NAC) and homeobox transcription factor genes were identified as fruit ripening-associated genes. Our tools provide useful information for research not only on melon but also on other fleshy fruit plants.

Comparative effects of ethylene inhibitors on Agrobacterium-mediated transformation of drought-tolerant wild watermelon.

Ethylene (C2H4), a phytohormone that is produced in response to both abiotic and biotic stresses, is an important factor influencing the efficiency of Agrobacterium-mediated transformation. In this study, effects of various ethylene inhibitors on the efficiency of Agrobacterium-mediated genetic transformation in drought-tolerant wild watermelon was comparatively examined. Consequently, in comparison to the application of chemical inhibitors such as AgNO3 and aminoethoxyvinylglycine (AVG), lower ethylene level was observed when the infecting Agrobacterium contained a gene for 1-aminocyclopropane-carboxylic acid (ACC) deaminase (acdS), which cleaves ethylene precursor ACC into α-ketobutyrate and ammonia. GUS histochemical and spectrophotometric enzyme assays showed that acdS was more effective in enhancing gene transfer than the chemical ethylene inhibitors. Efficiency of transgenic shoots formation was higher in acdS- and AVG-treated explants. These observations demonstrated that controlling the ethylene level during co-cultivation and shoot formation, particularly using the acdS-harboring Agrobacterium, is advantageous for enhancing the transformation efficiency in this plant.

Jasmonate-Responsive ERF Transcription Factors Regulate Steroidal Glycoalkaloid Biosynthesis in Tomato.

Steroidal glycoalkaloids (SGAs) are cholesterol-derived specialized metabolites produced in species of the Solanaceae. Here, we report that a group of jasmonate-responsive transcription factors of the ETHYLENE RESPONSE FACTOR (ERF) family (JREs) are close homologs of alkaloid regulators in Cathranthus roseus and tobacco, and regulate production of SGAs in tomato. In transgenic tomato, overexpression and dominant suppression of JRE genes caused drastic changes in SGA accumulation and in the expression of genes for metabolic enzymes involved in the multistep pathway leading to SGA biosynthesis, including the upstream mevalonate pathway. Transactivation and DNA-protein binding assays demonstrate that JRE4 activates the transcription of SGA biosynthetic genes by binding to GCC box-like elements in their promoters. These JRE-binding elements occur at significantly higher frequencies in proximal promoter regions of the genes regulated by JRE genes, supporting the conclusion that JREs mediate transcriptional co-ordination of a series of metabolic genes involved in SGA biosynthesis.

A Universal Positive-Negative Selection System for Gene Targeting in Plants Combining an Antibiotic Resistance Gene and Its Antisense RNA.

Gene targeting (GT) is a useful technology for accurate genome engineering in plants. A reproducible approach based on a positive-negative selection system using hygromycin resistance and the diphtheria toxin A subunit gene as positive and negative selection markers, respectively, is now available. However, to date, this selection system has been applied exclusively in rice (Oryza sativa). To establish a universally applicable positive-negative GT system in plants, we designed a selection system using a combination of neomycin phosphotransferaseII (nptII) and an antisense nptII construct. The concomitant transcription of both sense and antisense nptII suppresses significantly the level of expression of the sense nptII gene, and transgenic calli and plants become sensitive to the antibiotic geneticin. In addition, we were able to utilize the sense nptII gene as a positive selection marker and the antisense nptII construct as a negative selection marker for knockout of the endogenous rice genes Waxy and 33-kD globulin through GT, although negative selection with this system is relatively less efficient compared with diphtheria toxin A subunit. The approach developed here, with some additional improvements, could be applied as a universal selection system for the enrichment of GT cells in several plant species.

A novel rice cytochrome P450 gene, CYP72A31, confers tolerance to acetolactate synthase-inhibiting herbicides in rice and Arabidopsis.

Target-site and non-target-site herbicide tolerance are caused by the prevention of herbicide binding to the target enzyme and the reduction to a nonlethal dose of herbicide reaching the target enzyme, respectively. There is little information on the molecular mechanisms involved in non-target-site herbicide tolerance, although it poses the greater threat in the evolution of herbicide-resistant weeds and could potentially be useful for the production of herbicide-tolerant crops because it is often involved in tolerance to multiherbicides. Bispyribac sodium (BS) is an herbicide that inhibits the activity of acetolactate synthase. Rice (Oryza sativa) of the indica variety show BS tolerance, while japonica rice varieties are BS sensitive. Map-based cloning and complementation tests revealed that a novel cytochrome P450 monooxygenase, CYP72A31, is involved in BS tolerance. Interestingly, BS tolerance was correlated with CYP72A31 messenger RNA levels in transgenic plants of rice and Arabidopsis (Arabidopsis thaliana). Moreover, Arabidopsis overexpressing CYP72A31 showed tolerance to bensulfuron-methyl (BSM), which belongs to a different class of acetolactate synthase-inhibiting herbicides, suggesting that CYP72A31 can metabolize BS and BSM to a compound with reduced phytotoxicity. On the other hand, we showed that the cytochrome P450 monooxygenase CYP81A6, which has been reported to confer BSM tolerance, is barely involved, if at all, in BS tolerance, suggesting that the CYP72A31 enzyme has different herbicide specificities compared with CYP81A6. Thus, the CYP72A31 gene is a potentially useful genetic resource in the fields of weed control, herbicide development, and molecular breeding in a broad range of crop species.

WIND1-based acquisition of regeneration competency in Arabidopsis and rapeseed.

Callus formation and de novo organogenesis often occur in the wounded tissues of plants. Although this regenerative capacity of plant cells has been utilized for many years, molecular basis for the wound-induced acquisition of regeneration competency is yet to be elucidated. Here we find that wounding treatment is essential for shoot regeneration from roots in the conventional tissue culture of Arabidopsis thaliana. Furthermore, we show that an AP2/ERF transcription factor WOUND INDUCED DEDIFFERENTIATION1 (WIND1) plays a pivotal role for the acquisition of regeneration competency in the culture system. Ectopic expression of WIND1 can bypass both wounding and auxin pre-treatment and increase de novo shoot regeneration from root explants cultured on shoot-regeneration promoting media. In Brassica napus, activation of Arabidopsis WIND1 also greatly enhances de novo shoot regeneration, further corroborating the role of WIND1 in conferring cellular regenerative capacity. Our data also show that sequential activation of WIND1 and an embryonic regulator LEAFY COTYLEDON2 enhances generation of embryonic callus, suggesting that combining WIND1 with other transcription factors promote efficient and organ-specific regeneration. Our findings in the model plant and crop plant point to a possible way to efficiently induce callus formation and regeneration by utilizing transcription factors as a molecular switch.

Simultaneous determination of N(G)-monomethyl-L-arginine, N(G),N(G)-dimethyl-L-arginine, N(G),N(G')-dimethyl-L-arginine, and L-arginine using monolithic silica disk-packed spin columns and a monolithic silica column.

We have developed and validated a high-performance liquid chromatography method that uses monolithic silica disk-packed spin columns and a monolithic silica column for the simultaneous determination of N(G)-monomethyl-L-arginine, N(G),N(G)-dimethyl-L-arginine, and N(G),N(G')-dimethyl-L-arginine in human plasma. For solid-phase extraction, our method employs a centrifugal spin column packed with monolithic silica bonded to propyl benzenesulfonic acid as a cation exchanger. After pretreatment, the methylated arginines are converted to fluorescent derivatives with 4-fluoro-7-nitro-2,1,3-benzoxadiazole, and then the derivatives are separated on a monolithic silica column. L-arginine concentration was also determined in diluted samples. Standard calibration curves revealed that the assay was linear in the concentration range 0.2-1.0 µM for methylated arginines and 40-200 µM for L-arginine. Linear regression of the calibration curve yielded equations with correlation coefficients of 0.999 (r(2)). The sensitivity was satisfactory, with a limit of detection ranging from 3.75 to 9.0 fmol for all four compounds. The RSDs were 4.3-4.8% (intraday) and 3.0-6.8% (interday). When this method was applied to samples from six healthy donors, the detected concentrations of N(G)-monomethyl-L-arginine, N(G),N(G)-dimethyl-L-arginine, N(G),N(G')-dimethyl-L-arginine and L-arginine were 0.05 ± 0.01, 0.41 ± 0.07, 0.59 ± 0.11, and 83.8 ± 30.43 µM (n = 6), respectively.

Targeted modification of CmACO1 by CRISPR/Cas9 extends the shelf-life of Cucumis melo var. reticulatus melon.

The gaseous plant hormone ethylene is a regulator of fruit shelf-life, one of the essential traits in fruits. Extending fruit shelf-life reduces food loss, thereby expected to contribute to food security. The enzyme 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) is the final step of the ethylene production pathway. Its suppression via antisense technology has been demonstrated to extend the shelf-life of melon, apple, and papaya. Genome editing technology is an innovative technique for plant breeding. Because the genome editing technology would not leave the exogenous genes in the final crop products, the crops via genome editing can be considered non-genetically modified yields; compared to conventional breeding, such as mutation breeding, the breeding term would be expected to be relatively short. These points include the advantage of this technique in utilization for commercial applications. We attempted to extend the shelf-life of the Japanese luxury melon (Cucumis melo var. reticulatus, 'Harukei-3') via modification of the ethylene synthesis pathway with the genome editing technology, CRISPR/Cas9 system. The Melonet-DB (https://melonet-db.dna.affrc.go.jp/ap/top) showed that the melon genome had the five CmACOs and the gene CmACO1 predominantly expressed in harvested fruits. From this information, CmACO1 was expected to be a key gene for shelf-life in melons. Based on this information, the CmACO1 was selected as the target of the CRISPR/Cas9 system and introduced the mutation. The final product of this melon did not have any exogenous genes. The mutation was inherited for at least two generations. In the T2 generation, the fruit phenotypes 14 days after harvest were as follows: ethylene production was reduced to one-tenth that of the wild type, pericarp colour remained green, and higher fruit firmness. Early fermentation of the fresh fruit was observed in the wild-type fruit but not in the mutant. These results show that CmACO1 knockout via CRISPR/Cas9 extended the melon's shelf-life. Moreover, our results suggest that genome editing technology would reduce food loss and contribute to food security.

Sequential monitoring of transgene expression following Agrobacterium-mediated transformation of rice.

Although Agrobacterium-mediated transformation technology is now used widely in rice, many varieties of indica-type rice are still recalcitrant to Agrobacterium-mediated transformation. It was reported recently that T-DNA integration into the rice genome could be the limiting step in this method. Here, we attempted to establish an efficient sequential monitoring system for stable transformation events by visualizing stable transgene expression using a non-destructive and highly sensitive visible marker. Our results demonstrate that click beetle luciferase (ELuc) is an excellent marker allowing the observation of transformed cells in rice callus, exhibiting a sensitivity >30-fold higher than that of firefly luciferase. Since we have previously shown that green fluorescent protein (GFP) is a useful visual marker with which to follow transient and/or stable expression of transgenes in rice, we constructed an enhancer trap vector using both the gfbsd2 (GFP fused to the N-terminus of blasticidin S deaminase) and eluc genes. In this vector, the eluc gene is under the control of the Cauliflower mosaic virus 35S minimal promoter, while the gfbsd2 gene is under the control of the full-length rice elongation factor gene promoter. Observation of transformed callus under a dissecting microscope demonstrated that the level of ELuc luminescence reflected exclusively stable transgene expression, and that both transient and stable expression could be monitored by the level of GFP fluorescence. Moreover, we show that our system enables sequential quantification of transgene expression via differential measurement of ELuc luminescence and GFP fluorescence.

Suppression of Ku70/80 or Lig4 leads to decreased stable transformation and enhanced homologous recombination in rice.

Evidence for the involvement of the nonhomologous end joining (NHEJ) pathway in Agrobacterium-mediated transferred DNA (T-DNA) integration into the genome of the model plant Arabidopsis remains inconclusive. Having established a rapid and highly efficient Agrobacterium-mediated transformation system in rice (Oryza sativa) using scutellum-derived calli, we examined here the involvement of the NHEJ pathway in Agrobacterium-mediated stable transformation in rice. Rice calli from OsKu70, OsKu80 and OsLig4 knockdown (KD) plants were infected with Agrobacterium harboring a sensitive emerald luciferase (LUC) reporter construct to evaluate stable expression and a green fluorescent protein (GFP) construct to monitor transient expression of T-DNA. Transient expression was not suppressed, but stable expression was reduced significantly, in KD plants. Furthermore, KD-Ku70 and KD-Lig4 calli exhibited an increase in the frequency of homologous recombination (HR) compared with control calli. In addition, suppression of OsKu70, OsKu80 and OsLig4 induced the expression of HR-related genes on treatment with DNA-damaging agents. Our findings suggest strongly that NHEJ is involved in Agrobacterium-mediated stable transformation in rice, and that there is a competitive and complementary relationship between the NHEJ and HR pathways for DNA double-strand break repair in rice.

Modification of tomato breeding traits and plant hormone signaling by target-AID, the genome-editing system inducing efficient nucleotide substitution.

Target activation-induced cytidine deaminase (Target-AID), a novel CRISPR/Cas9-based genome-editing tool, confers the base-editing capability on the Cas9 genome-editing system. It involves the fusion of cytidine deaminase (CDA), which catalyzes cytidine (C) to uridine (U) substitutions, to the mutated nickase-type nCas9 or deactivated-type dCas9. To confirm and extend the applicability of the Target-AID genome-editing system in tomatoes (Solanum lycopersicum L.), we transformed the model tomato cultivar "Micro-Tom" and commercial tomato cultivars using this system by targeting SlDELLA, which encodes a negative regulator of the plant phytohormone gibberellic acid (GA) signaling pathway. We confirmed that the nucleotide substitutions were induced by the Target-AID system, and we isolated mutants showing high GA sensitivity in both "Micro-Tom" and the commercial cultivars. Moreover, by successfully applying this system to ETHYLENE RECEPTOR 1 (SlETR1) with single sgRNA targeting, double sgRNA targeting, as well as dual-targeting of both SlETR1 and SlETR2 with a single sgRNA, we demonstrated that the Target-AID genome-editing system is a promising tool for molecular breeding in tomato crops. This study highlights an important aspect of the scientific and agricultural potential of the combinatorial use of the Target-AID and other base-editing systems.

The accumulation of recombinant miraculin is independent of fruit size in tomato.

The taste-modifying protein miraculin (MIR) has received increasing interest as a new low-calorie sweetener. In our previous study using the tomato variety 'Micro-Tom,' it was shown that in transgenic tomatoes in which MIR was expressed by using the cauliflower mosaic virus 35S promoter (p35S) and a heat shock protein terminator (tHSP) cassette (p35S-MIR-tHSP), higher levels of miraculin accumulated than when MIR was driven by the nopaline synthase terminator (tNOS) cassette (p35S-MIR-tNOS). 'Micro-Tom' is a dwarf tomato used for research and shows a low yield. To achieve high productivity of MIR, it is essential to improve the MIR accumulation potential by using high-yielding cultivars. In this study, we evaluate whether the high MIR accumulation trait mediated by the tHSP appears even when fruit size increases. A line in which the p35S-MIR-tHSP cassette was introduced into a high-yielding variety was bred by backcrossing. The line homozygous for MIR showed higher accumulation of MIR than the heterozygous line. Despite large differences in fruit size, the MIR level in the backcross line was similar to that in the p35S-MIR-tHSP line (background 'Micro-Tom'). It was approximately 3.1 times and 4.0 times higher than those in miracle fruits and the p35S-MIR-tNOS tomato line 5B ('Moneymaker' background, which exhibits the highest miraculin productivity achieved thus far), respectively. These results demonstrate that the high MIR accumulation trait mediated by the tHSP appears even when fruit size is increased.

Genome editing in PDS genes of tomatoes by non-selection method and of Nicotiana benthamiana by one single guide RNA to edit two orthologs.

The CRISPR/Cas9 system is widely used for targeted mutagenesis in many organisms including plants. For application of this system, tissue culture methods need to be established. In this study, detailed methods for introduction of mutations in tomato and Nicotiana benthamiana plants using the CRISPR/Cas9 system are described. The methods include tissue culture protocols for tomato and N. benthamiana. We also demonstrate the methodology to generate Cas9-free genome edited tomato plants and use of one single guide RNA (sgRNA) to edit two orthologs in N. benthamiana. The examples of editing the PHYTOENE DESATURASE (PDS) genes in these plants are also provided. The Cas9-free tomato line was obtained when tomato plants were cultured on a non-selective medium after transformation with the CRISPR/Cas9 system. Two orthologs of PDS in N. benthamiana were mutated using a sgRNA, because these orthologs contain the same nucleotide sequences with PAM motif. These mutations were inherited to the next generation. The mutations in the PDS genes resulted in an albino phenotype in tomato and N. benthamiana plants. These results demonstrate that the non-selective method is one of the ways to obtain Cas9-free genome editing in tomato plants and that the two orthologs can be edited by one sgRNA in N. benthamiana.

Comparative genomics of muskmelon reveals a potential role for retrotransposons in the modification of gene expression.

Melon exhibits substantial natural variation especially in fruit ripening physiology, including both climacteric (ethylene-producing) and non-climacteric types. However, genomic mechanisms underlying such variation are not yet fully understood. Here, we report an Oxford Nanopore-based high-grade genome reference in the semi-climacteric cultivar Harukei-3 (378 Mb + 33,829 protein-coding genes), with an update of tissue-wide RNA-seq atlas in the Melonet-DB database. Comparison between Harukei-3 and DHL92, the first published melon genome, enabled identification of 24,758 one-to-one orthologue gene pairs, whereas others were candidates of copy number variation or presence/absence polymorphisms (PAPs). Further comparison based on 10 melon genome assemblies identified genome-wide PAPs of 415 retrotransposon Gag-like sequences. Of these, 160 showed fruit ripening-inducible expression, with 59.4% of the neighboring genes showing similar expression patterns (r > 0.8). Our results suggest that retrotransposons contributed to the modification of gene expression during diversification of melon genomes, and may affect fruit ripening-inducible gene expression.

Super-Agrobacterium ver. 4: Improving the Transformation Frequencies and Genetic Engineering Possibilities for Crop Plants.

Agrobacterium tumefaciens has been utilized for both transient and stable transformations of plants. These transformation methods have been used in fields such as breeding GM crops, protein production in plant cells, and the functional analysis of genes. However, some plants have significantly lower transient gene transfer and stable transformation rates, creating a technical barrier that needs to be resolved. In this study, Super-Agrobacterium was updated to ver. 4 by introducing both the ACC deaminase (acdS) and GABA transaminase (gabT) genes, whose resultant enzymes degrade ACC, the ethylene precursor, and GABA, respectively. A. tumefaciens strain GV2260, which is similar to other major strains (EHA105, GV3101, LBA4404, and MP90), was used in this study. The abilities of the Super-Agrobacterium ver. 4 were evaluated in Erianthus ravennae, Solanum lycopersicum "Micro-Tom," Nicotiana benthamiana, and S. torvum. Super-Agrobacterium ver. 4 showed the highest T-DNA transfer (transient transformation) frequencies in E. ravennae and S. lycopersicum, but not in N. benthamiana and S. torvum. In tomato, Super-Agrobacterium ver. 4 increased the stable transformation rate by 3.6-fold compared to the original GV2260 strain. Super-Agrobacterium ver. 4 enables reduction of the amount of time and labor required for transformations by approximately 72%, and is therefore a more effective and powerful tool for plant genetic engineering and functional analysis, than the previously developed strains. As our system has a plasmid containing the acdS and gabT genes, it could be used in combination with other major strains such as EHA105, EHA101, LBA4404, MP90, and AGL1. Super-Agrobacterium ver. 4, could thus possibly be a breakthrough application for improving basic plant science research methods.

Author Correction: Efficient increase of ɣ-aminobutyric acid (GABA) content in tomato fruits by targeted mutagenesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

1-Aminocyclopropane-1-carboxylate deaminase enhances Agrobacterium tumefaciens-mediated gene transfer into plant cells.

Agrobacterium-mediated gene transfer is widely used for plant molecular genetics, and efficient techniques are required. Recent studies show that ethylene inhibits the gene transfer. To suppress ethylene evolution, we introduced 1-aminocyclopropane-1-carboxylate (ACC) deaminase into Agrobacterium tumefaciens. The ACC deaminase enhanced A. tumefaciens-mediated gene transfer into plants.

Ethylene production in plants during transformation suppresses vir gene expression in Agrobacterium tumefaciens.

Ethylene evolution from plants inhibits Agrobacterium-mediated genetic transformation, but the mechanism is little understood. In this study, the possible role of ethylene in Agrobacterium-mediated genetic transformation was clarified. It was tested whether or not plant ethylene sensitivity affected genetic transformation; the sensitivity might regulate bacterial growth during co-cultivation and vir gene expression in Agrobacterium tumefaciens. For these experiments, melon (Cucumis melo) was used, in which ethylene sensitivity was controlled by chemicals, and Arabidopsis ethylene-insensitive mutants. Agrobacterium-mediated genetic transformation was inhibited in ethylene-sensing melon, whereas, in Arabidopsis ethylene-insensitive mutant, it was enhanced. However, the ethylene sensitivity did not affect bacterial growth. vir gene expression was inhibited by application of plant exudate from ethylene-sensitive plants. The inhibitory effect of the ethylene sensitivity on genetic transformation relieved the activation of vir gene expression in A. tumefaciens with vir gene inducer molecule (acetosyringone, AS) or A. tumefaciens mutant strain which has constitutive vir gene expression. These results indicate that ethylene evolution from a plant inoculated with A. tumefaciens inhibited vir gene expression in A. tumefaciens through the ethylene signal transduction in the plant, and, as a result, Agrobacterium-mediated genetic transformation was inhibited.