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Abstract Background This study aimed to produce Odontoglossum ringspot virus (ORSV)-free Cymbidium orchid ‘New True’ plants from ORSV-infected mother plants by culturing their meristems and successively repeating subcultures of protocorm-like bodies (PLBs) derived from the meristems. Results Initially, ORSV was confirmed as the causative agent of viral symptoms in orchid leaves via reverse transcription-polymerase chain reaction (RT-PCR) analysis. Meristems from infected plants were cultured to generate PLBs, which in sequence were repeatedly subcultured up to four times. RT-PCR and quantitative RT-PCR analyses revealed that while ORSV was undetectable in shoots derived from the first subculture, complete elimination of the virus required at least a second subculture. Genetic analysis using inter-simple sequence repeat markers indicated no somaclonal variation between regenerated plants and the mother plant, suggesting that genetic consistency was maintained. Conclusion Overall, our findings demonstrate that subculturing PLBs for a second time is ideal for producing genetically stable, ORSV-free Cymbidium orchids, thus offering a practical means of generating genetically stable, virus-free plants and enhancing plant health and quality in the orchid industry.

Abstract The study aimed to edit ethylene (ET) biosynthesis genes [1-aminocyclopropane-1-carboxylic acid (ACC) synthetase 1 (ACS1) and ACC oxidase 1 (ACO1)] in carnation using the CRISPR/Cas9 ribonucleoprotein (RNP) complex system. Initially, the conserved regions of the target genes (ACS1 and ACO1) were validated for the generation of different single guide RNAs (sgRNAs), followed by the use of an in vitro cleavage assay to confirm the ability of the sgRNAs to cleave the target genes specifically. The in vitro cleavage assay revealed that the sgRNAs were highly effective in cleaving their respective target regions. The complex of sgRNA: Cas9 was directly delivered into the carnation protoplast, and the target genes in the protoplast were deep-sequenced. The results revealed that the sgRNAs were applicable for editing the ET biosynthesis genes, as the mutation frequency ranged from 8.8 to 10.8% for ACO1 and 0.2–58.5% for ACS1. When sequencing the target genes in the callus derived from the protoplasts transformed with sgRNA: Cas9, different indel patterns (+ 1, − 1, and − 8 bp) in ACO1 and (− 1, + 1, and + 11) in ACS1 were identified. This study highlighted the potential application of CRISPR/Cas9 RNP complex system in facilitating precise gene editing for ET biosynthesis in carnation.

Floral senescence or petal abscission that determine cut flower quality and longevity are primarily caused by the regulation of endogenous ethylene and perception of endogenous and exogenous ethylene in floral organs (such as the petals and gynoecium), in ethylene-sensitive species. Several attempts have been made to prolong flower quality and longevity using silver ions and other chemicals that inhibit ethylene production and perception in floral organs. Among the available compounds, 1-methylcyclopropene (1-MCP) has been increasingly used as ethylene-action/perception inhibitor because of its ability to inhibit ethylene action/perception and it is environmentally safe. Although 1-MCP has been used in several cut flower crops for about 25 years, there has been no review yet that critically emphasizes the benefits or difficulties of using this compound for the prolongation of cut flower quality and longevity. Here, we outline the role of 1-MCP in inhibiting ethylene action/perception and petal senescence in cut flowers through the blocking of receptor binding sites, and we discuss how various factors affecting efficacy of 1-MCP (such as concentration, treatment time and temperature, genotype, and flower stage) are involved in the achievement of flower longevity. Moreover, we highlight the advantages of applying nonvolatile and liquid 1-MCP formulation types, as opposed to using the conventional 1-MCP treatment (powder formulation type). We expect that this review will provide useful information for the future utilization of 1-MCP for the maintenance of cut flower longevity.

Abstract Background This study investigated the effects of ethylene release compounds (ethephon), ethylene-action inhibitors (silver thiosulfate: STS), and nitric oxide donor (sodium nitroprusside: SNP) on stem bending of snapdragon flowers. Moreover, the effects of plant growth supplements [6-benzyladenine (BA), gibberellic acid 3 (GA3), and calcium chloride (CaCl2)] on the stem bending were also extensively investigated. Results Ethephon completely prevented stem bending until 9 days after treatment (9 DAT). STS exhibited the highest bending rate, while SNP did not significantly affect the bending compared to the controls. The bending results were associated with the results of stem curvature, relative shoot elongation, ethylene production, and lignin content, that are involved in the stem bending mechanism. This was proven by the expression analysis of genes involved in ethylene and lignin biosynthetic pathways. The addition of plant growth supplements slightly or significantly delayed stem bending in the treatments (control, SNP, and STS) and significantly reduced petal senescence in ethephon at 9 DAT. Conclusion These results show the preventive role of ethephon in the stem bending of cut snapdragon. Moreover, the combination of ethephon with supplements also provided information that could guide the development of strategies to delay stem bending in other cut flowers that undergo serious bending during a short vase life.

Ethylene plays a critical signaling role in the abiotic stress tolerance mechanism. However, the role of ethylene in regulating abiotic stress tolerance in petunia has not been well-investigated, and the underlying molecular mechanism by which ethylene regulates abiotic stress tolerance is still unknown. Therefore, we examined the involvement of ethylene in salt and drought stress tolerance of petunia using the petunia wild type cv. “Merage Rose” and the ethylene biosynthesis genes (PhACO1 and PhACO3)-edited mutants (phaco1 and phaco3). Here, we discovered that editing PhACO1 and PhACO3 reduced ethylene production in the mutants, and mutants were more sensitive to salt and drought stress than the wild type (WT). This was proven by the better outcomes of plant growth and physiological parameters and ion homeostasis in WT over the mutants. Molecular analysis revealed that the expression levels of the genes associated with antioxidant, proline synthesis, ABA synthesis and signaling, and ethylene signaling differed significantly between the WT and mutants, indicating the role of ethylene in the transcriptional regulation of the genes associated with abiotic stress tolerance. This study highlights the involvement of ethylene in abiotic stress adaptation and provides a physiological and molecular understanding of the role of ethylene in abiotic stress response in petunia. Furthermore, the finding alerts researchers to consider the negative effects of ethylene reduction on abiotic stress tolerance when editing the ethylene biosynthesis genes to improve the postharvest quality of horticultural crops.

Abiotic stress induces the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) in plants, which consequently enhances ethylene production and inhibits plant growth. The bacterial ACC deaminase enzyme encoded by the acdS gene reduces stress-induced ethylene production and improves plant growth in response to stress. In this study, overexpression of acdS in Petunia hybrida (‘Mirage Rose’) significantly reduced expression of the ethylene biosynthesis gene ACC oxidase 1 (ACO1) and ethylene production relative to those in wild type (WT) under various abiotic stresses (cold, drought, and salt). The higher reduction of stress-induced ethylene in the transgenic plants, which was due to the overexpression of acdS, led to a greater tolerance to the stresses compared to that in the WT plants. The greater stress tolerances were proven based on better plant growth and physiological performance, which were linked to stress tolerance. Moreover, expression analysis of the genes involved in stress tolerance also supported the increased tolerance of transgenics relative to that with the WT. These results suggest the possibility that acdS is overexpressed in ornamental plants, particularly in bedding plants normally growing outside the environment, to overcome the deleterious effect of ethylene on plant growth under different abiotic stresses. The development of stress-tolerant plants will be helpful to advance the floricultural industry.

Abstract Background Past research has shown that virus-induced phytoene desaturase (PDS) gene silencing via agroinjection in the attached and detached fruit of tomato plants results in a pale-yellow fruit phenotype. Although the PDS gene is often used as a marker for gene silencing in tomatoes, little is known about the role of PDS in fruit ripening. In this study, we investigated whether the pepper PDS gene silenced endogenous PDS genes in the fruit of two tomato cultivars, Dotaerang Plus and Legend Summer. Results We found that the pepper PDS gene successfully silenced endogenous PDS in tomato fruit at a silencing frequency of 100% for both cultivars. A pale-yellow silenced area was observed over virtually the entire surface of individual fruit due to the transcriptional reduction in phytoene desaturase (PDS), zeta-carotene (ZDS), prolycopene isomerase (CrtlSO), and beta-carotene hydroxylase (CrtR-b2), which are the carotenoid biosynthesis genes responsible for the red coloration in tomatoes. PDS silencing also affected the expression levels of the fruit-ripening genes Tomato AGAMOUS-LIKE1 (TAGL1), RIPENING INHIBITOR (RIN), pectin esterase gene (PE), lipoxygenase (LOX), FRUITFULL1/FRUITFUL2 (FUL1/FUL2), and the ethylene biosynthesis and response genes 1-aminocyclopropane-1-carboxylate oxidase 1 and 3 (ACO1 and ACO3) and ethylene-responsive genes (E4 and E8). Conclusion These results suggest that PDS is a positive regulator of ripening in tomato fruit, which must be considered when using it as a marker for virus-induced gene silencing (VIGS) experiments in order to avoid fruit-ripening side effects.

Abstract Background As strawberries are susceptible to somaclonal variation when propagated by tissue culture techniques, it is challenging to obtain the true-to-type plants necessary for continuous production of fruits of stable quality. Therefore, we aimed to develop an in vitro propagation method for the production of true-to-type plants of five different strawberry cultivars from meristems cultured in media containing different concentrations of kinetin (Kn). Results For all the cultivars, shoot induction was successful only in the meristems cultured in the medium without Kn and the medium containing 0.5 mg L−1 Kn. The shoots obtained from explants cultured in media supplemented with 0.5 mg L−1 Kn exhibited better plant growth parameters than those cultured in media without Kn and were genetically stable when compared with conventionally propagated plants for all the cultivars. Vegetative and sexual characters and fruit quality attributes observed in the plants derived from meristems cultured on 0.5 mg L−1 Kn and the conventionally propagated plants were not significantly different when grown for three continuous growing seasons under greenhouse conditions. Conclusion The culture of meristems in the medium containing 0.5 mg L−1 Kn is suitable for the efficient propagation of true-to-type plants of different strawberry cultivars and continuous production of fruits with stable quality. Hence, we expect that the method presented in this study will be helpful for the commercial production of true-to-type plants generated in vitro for other strawberry cultivars.

This study investigated whether ethylene is involved in the stem-bending mechanism of three different snapdragon cultivars ‘Asrit Red’, ‘Asrit Yellow’, and ‘Merryred Pink’, by treating their cut stems with an ethylene-releasing compound (ethephon), an ethylene-action inhibitor [silver thiosulfate (STS)], and distilled water (as the control). Ethephon completely prevented stem bending in all cultivars, whereas STS exhibited a higher bending rate compared with the control. The bending rates were influenced by several factors, such as the degree of stem curvature, relative shoot elongation, ethylene production, and lignin content, indicating their involvement in the stem-bending mechanism of the cultivars. The analysis of the expression of genes involved in the ethylene and lignin biosynthetic pathways also supported the importance of lignin and ethylene in the stem-bending mechanism. Taken together, as ethephon completely prevented stem bending of the three snapdragon cultivars, this study suggested that ethylene acts as a negative regulator of the stem-bending mechanism of snapdragon cultivars, and the information will be valuable for the prevention of stem bending in other commercially important ornamental flowers.

The efficacy of 1-methylcyclopropene (1-MCP) in maintenance of apple fruit quality can differ depending on apple cultivar, ethylene content at harvest, 1-MCP concentration, the interval between harvest and 1-MCP application, and the number of applications. In this study, we investigated whether the 1-MCP concentration and its application frequency differently affected fruit quality attributes of the two new apple cultivars ‘Arisoo’ and ‘Picnic’. The fruits were treated with 1-MCP (as single or double treatments) at the rate of 0 μL L−1 (control), 0.5 μL L−1, 1 μL L−1, 0.5 + 0.5 μL L−1, and 1 + 1 μL L−1 for 18 h, and they were stored at 0 °C up to six months. Comparatively, the fruit qualities of 1-MCP treated fruits were higher than that of the control during cold storage, with a higher suppression of internal ethylene content and a slower reduction of titratable acidity in 1-MCP treatments than the controls throughout the cold storage in both cultivars, regardless of the concentration and number of applications. Reduction of fruit firmness and soluble solid contents were maintained by single application of 0.5 μL L−1 1-MCP in ‘Arisoo’ apple, while double application of 0.5 + 0.5 μL L−1 was needed in ‘Picnic’ cultivar. The effective concentration for weight-loss reduction was found to be the single application of 1 μL L−1 1-MCP in both ‘Arisoo’ and ‘Picnic’. Conclusively, 1-MCP as single treatment at the rate of 0.5 μL L−1 could be sufficient in inhibiting ethylene action and maintaining fruit quality losses during cold storage, particularly in ‘Arisoo’. However, double application of 1-MCP might be necessary for some quality attributes in ‘Picnic’ apple.

We examined the effects of different sucrose concentrations (3%, 5%, and 7%) on anthocyanin accumulation and plant growth in wild type (WT) and transgenic (T2) torenia cultivar “Kauai Rose” overexpressing the anthocyanin regulatory transcription factors B-Peru + mPAP1 or RsMYB1. Sucrose increased anthocyanin production in both WT and transgenic plants, with higher anthocyanin production in transgenic plants compared to WT plants. Higher sucrose concentrations increased production of anthocyanin in transgenic and WT plants, with increased anthocyanin production associated with increased expression of anthocyanin biosynthesis genes. Higher sucrose concentrations reduced growth of WT and transgenic plants. Our results indicate that sucrose enhances anthocyanin production in torenia by regulating anthocyanin biosynthesis genes.

In this study, whether the addition of antifreeze protein (AFP) to a cryopreservative solution (plant vitrification solution 2 (PVS2)) is more effective in reducing freezing injuries in Hosta capitata than PVS2 alone at different cold exposure times (6, 24, and 48 h) is investigated. The upregulation of C-repeat binding factor 1 (CBF1) and dehydrin 1 (DHN1) in response to low temperature was observed in shoots. Shoots treated with distilled water (dH2O) strongly triggered gene expression 6 h after cold exposure, which was higher than those expressed in PVS2 and PVS2+AFP. However, 24 h after cold exposure, gene expressions detected in dH2O and PVS2 treatments were similar and higher than PVS2 + AFP. The expression was highest in PVS2+AFP when the exposure time was extended to 48 h. Similarly, nitric reductase activities 1 and 2 (Nia1 and Nia2) genes, which are responsible for nitric oxide production, were also upregulated in low-temperature-treated shoots, as observed for CBF1 and DHN1 expression patterns during cold exposure periods. Based on the gene expression patterns, shoots treated with PVS2+AFP were more likely to resist cold stress, which was also associated with the higher cryopreservation efficiency of PVS2+AFP compared to PVS2 alone. This finding suggests that the improvement of cryopreservation efficiency by AFP could be due to the transcriptional regulation of CBF1, DHN1, Nia1, and Nia2, which might reduce freezing injuries during cryopreservation. Thus, AFP could be potentially used as a cryoprotectant in the cryopreservation of rare and commercially important plant germplasm.

The RsMYB1 transcription factor (TF) controls the regulation of anthocyanin in radishes (Raphanus sativus), and its overexpression in tobacco and petunias strongly enhances anthocyanin production. However, there are no data on the involvement of RsMYB1 in the mechanisms underlying abiotic stress tolerance, despite strong sequence similarity with other MYBs that confer such tolerance. In this study, we used the anthocyanin-enriched transgenic petunia lines PM6 and PM2, which overexpress RsMYB1. The tolerance of these lines to heavy metal stress was investigated by examining several physiological and biochemical factors, and the transcript levels of genes related to metal detoxification and antioxidant activity were quantified. Under normal conditions (control conditions), transgenic petunia plants (T2-PM6 and T2-PM2) expressing RsMYB1, as well as wild-type (WT) plants, were able to thrive by producing well-developed broad leaves and regular roots. In contrast, a reduction in plant growth was observed when these plants were exposed to heavy metals (CuSO4, ZnSO4, MnSO4, or K2Cr2O7). However, T2-PM6 and T2-PM2 were found to be more stress tolerant than the WT plants, as indicated by superior results in all analyzed parameters. In addition, RsMYB1 overexpression enhanced the expression of genes related to metal detoxification [glutathione S-transferase (GST) and phytochelatin synthase (PCS)] and antioxidant activity [superoxide dismutase (SOD), catalase (CAT), and peroxidase (POX)]. These results suggest that enhanced expression levels of the above genes can improve metal detoxification activities and antioxidant activity, which are the main components of defense mechanism included in abiotic stress tolerance of petunia. Our findings demonstrate that RsMYB1 has potential as a dual-function gene that can have an impact on the improvement of anthocyanin production and heavy metal stress tolerance in horticultural crops.