Introduction

Cymbidium, a commercially important orchid which ranks sixth in global floriculture trade, commanded the highest market price among cut flowers at $3.60 per stem at the Royal Flora Holland in 2018 (An et al., 2015;De, 2022;Faust and Dole, 2021). Cymbidium is economically significant and widely cultivated in various countries (Shim et al., 2021). The National Institute of Horticultural and Herbal Science of the Rural Development Administration has successfully bred around 58 varieties of Cymbidium in Korea (RDA, 2022).

After flowers are cut from the maternal plant, transporting them with preservative solutions and sustaining postharvest quality ensure prolonged marketability (Thakur, 2020). Vase life of cut flowers is a key indicator of postharvest quality and is determined based on attributes such as diameter and length, flower opening, changes in fresh weight, stem or pedicel size, senescence pattern, petal color, total longevity, and foliage burning (De and Singh, 2019). The various senescence symptoms of cut Cymbidium, such as lip coloration, tip burn, and wilting, are caused by decreased water uptake as result of stem blockage due to microbial growth, air blockage, or physiological plugging (De and Singh, 2019;Kim et al., 2016). Dry storage increased wilting and reduced flower quality and consumer preference in importing countries, resulting in export decline (Roh et al., 2018). A preservative solution designed for transportation is employed to facilitate overseas export and ensure the quality of cut Cymbidium during long-term transport. However, the limited efficacy of conventional preservative solutions under diverse transport conditions and cultivar-specific responses underscores the imperative to develop innovative preservation strategies.

The xylem occlusion (i.e. blockage of xylem vessel by air and microorganism) is the major problem behind the deterioration of cut flowers (van Doorn, 1997). Bactericides play a crucial role in suppressing bacterial proliferation during prolonged transport and preventing vascular occlusion in cut flowers (Roh et al., 2018). As it has some special properties like it act as an antibacterial agent and can maximize the uptake of water (Sharma and Thakur, 2020). Sugars serve as essential components in flower food, supplying vital carbohydrates to cut stems and sustaining metabolic processes necessary for extending vase life (van Doorn, 1997). However, sucrose must be combined with antimicrobial compounds to deter microbial accumulation in the solutions. Numerous prior studies aimed to enhance vase life through diverse preservative solutions, typically incorporating both biocides and sugars. In a previous investigation, a treatment of 2% sucrose + 200 ppm 8-hydroxyquinoline sulfate (8-HQS) exhibited a vase life of 77.6 days, while 2% sucrose + 100 ppm Al₂ (SO₄) treatments resulted in a vase life of 77.4 days, surpassing the control in tap water (65 days) (De et al., 2015). In addition to widely used formulations, a variety of commercial floral preservatives are available as hydration, holding, and vase solutions, many of which have been proven effective and are well-documented in the literature (Fatima et al., 2022).

Nanobubbles (NB) are nanosized gas bubbles (generally over 200 nm, and less than 1000 nm in diameter) suspended in liquids, where their negatively charged surfaces promote rapid dissolution and facilitate efficient gas–liquid exchange (Atkinson et al., 2019;Takahashi, 2005). Their negatively charged surfaces enhance gas– liquid exchange and promote the generation of reactive oxygen species (ROS), contributing to their effectiveness in water treatment (Atkinson et al., 2019). In aquaculture, ozone nanobubble (O₃NB) has been used successfully to control bacterial populations and prevent disease outbreaks (Jhunkeaw et al., 2021). Nanobubble technology, especially oxygen nanobubbles (O₂NB), improves plant growth by increasing dissolved oxygen and enhancing nutrient uptake in agricultural systems (Ahmed et al., 2018;Noh et al., 2022).

Although O₂NB and O₃NB are rarely used in cut flower preservation, they effectively reduce bacterial counts in water with proven stability (Hu and Xia, 2018). However, limited studies have explored their potential in extending vase life and maintaining postharvest quality in cut flowers. The application of hydrogen NB in vase solutions for cut roses has been reported to delay wilting, reduce the generation of ROS, and suppress the activity of senescence-related enzymes (Li et al., 2021). In our previous studies, we also found that the use of oxygen and ozone nanobubbles as preservative solutions improved the postharvest quality of cut C. ‘Spring Pearl’ and C. ‘Lovely King’ (Chung et al., 2022;2024).

We hypothesized that combining oxygen and ozone nanobubbles with sucrose would extend the vase life of cut flowers by reducing bacterial growth and providing nutrients. Therefore, this study aimed to evaluate the efficacy of oxygen and ozone nanobubbles, both individually and in combination with sucrose, as preservative solutions for maintaining postharvest quality and extending vase life of cut C. ‘Lovely King’ during simulated transport conditions.

Materials and Methods

Plant materials

Cut stems of Cymbidium ‘Lovely King’, cultivated in pots with bark under greenhouse conditions, were harvested in Haepyeong farm (Gongju, Korea) on February 20, 2023. Flowers were harvested at a stage when over 75% of the florets had fully opened (De and Singh, 2019). The flowers were transported to the laboratory of Seoul Women’s University (Seoul, Korea) immediately for 3 h, and the stems were recut in tap water before treatment. The randomly selected flowers were uniform in size and color without defects.

Experimental environments

The flowers were treated with seven different preservative solutions in plastic tubes (one stem per tube), then packaged in plastic bags and placed in boxes to simulate a transportation environment. The boxes put in the chamber and the environments had a temperature of 5.2 ± 0.8°C and a relative humidity of 56.1 ± 8.4%. Seven days after the treatment in the chamber, all of the cut flower stems were individually placed in glass bottles filled with 500 mL tap water at room temperature in the laboratory, to simulate typical household conditions where flowers are generally placed in vases filled with tap water. The room environments had a temperature of 20.8 ± 0.1°C, relative humidity of 53.0 ± 0.1%, and light intensity of 29.6 ± 0.4 μmol·m^-2·s^-1 (cool white fluorescent tubes) in 12/12 h (light/dark) cycles.

Preservative solutions

Preservative solutions for transportation consisted of tap water (TW, control), sucrose 2% (Suc), Floralife (FL), oxygen nanobubble (O₂NB), O₂NB + suc 2% (O₂NBS), ozone nanobubble (O₃NB), O₃NB + suc 2% (O₃NBS). The concentration of 2% sucrose was selected based on a previous study (De et al., 2014). FL is an abbreviation for Floralife and generally denotes vase life extension products (such as Express Universal) manufactured by the company. Express Universal (Floralife®, Kent, OH, USA), a commercial preservative, was applied at a rate of 5 mL (one packet) per 500 mL of water for vase life extension, following the manufacturer’s instructions. The O₂NB and O₃NB were generated using a CR-P3W oxygen concentrator (Fawoo Nanotech Co., Ltd., Bucheon, Republic of Korea) and an nanobubble generator (SNT-03, Fawoo Nanotech Co. Ltd., Bucheon, Republic of Korea). In the initially prepared NB solutions, the ozone and dissolved oxygen (DO) concentrations of NB treatments were 0.0 and 12.3 mg･L^-1 (TW, control), 0.0 and 49.3 mg･L^-1 (O₂NB), 0.0 and 49.0 mg･L^-1 (O₂NBS), 6.0 and 55.3 mg･L^-1 (O₃NB), and 5.4 and 55.23 mg･L^-1 (O₃NBS), respectively. NBs (< 200 nm) with a concentration of approximately 500 million bubbles mL^-1 were produced, and most of the bubbles were less than 214.8 ± 101.8 nm in diameter (Fawoo Nanotech, 2023). The ozone is not separate because O₃NB was generated simultaneously with O₂NB. The DO and ozone concentrations were measured using a research-grade meter (HI 5421, HANNA Instruments, Woonsocket, RI, USA) and a portable color reader (Q-03-2, Shenzhen Sinsche Technology Co., Ltd., Guangdong, China), respectively.

Data collection and analysis

The following senescence symptoms were monitored, floret abscission, stem bending (bent neck; neck angle > 90°), and wilting. Vase life was defined as the point at which more than 50% of the florets exhibited senescence symptoms and lost ornamental value, or when the fresh weight decreased by more than 70% compared to day 0. This definition was established with reference to previous studies, in which vase life was defined as the period until more than 70% of the florets senesced and dropped or showed discoloration and loss of ornamental quality, or as the duration until the first floret abscission from the inflorescence (Biswas et al., 2022;Kim et al., 2016). Bacterial counts in the preservative solutions from individual water tubes were determined. The 1 mL of each solution was diluted with 0.75% saline, and 0.1 mL of the diluted samples was plated on tryptic soy agar and incubated at 37°C for 48 hours. The number of bacterial colonies was then counted visually. For anatomical observation, 0.5 mm segments were excised from the basal ends of cut C. ‘Lovely King’ stems subjected to seven different treatments, using a sterile blade. The samples were stained with diluted methylene blue on days 7 and 19. Vascular tissues and xylem occlusions at the stem ends were examined and photographed under 40× magnification using a BX61 optical microscope (Olympus, Tokyo, Japan). The relative fresh weight (RFW) of cut flowers was determined using the formula: RFW (%) = (W_t/W₀) × 100, where W_t represents the stem weight (g) on the given experimental day, and W₀ denotes the stem weight (g) on day 0. Daily water uptake was assessed by recording the weight loss of the vase solution in the absence of cut stems. It was calculated as: Water uptake = S_t−1 − S_t, where S_t and S_t−1 indicate the weight (g) of the vase solution on the current and previous day, respectively. Floral diameter was measured on the sixth flower from the base of the inflorescence, recording the maximum span using digital calipers. Statistical analyses were conducted using R software (version 4.3.0; R Core Team, Vienna, Austria). Data were analyzed via analysis of variance (ANOVA), and treatment means were compared using Duncan’s multiple range test at a significance level of P < 0.05. Graphical representations were created with Sigma Plot (version 10.0; Systat Software Inc., San Jose, CA, USA).

Results

Vase life

The O₂NB treatment extended the vase life of C. ‘Lovely King’ to 20.4 days, which was the longest among all treatments and significantly longer than that of O₂NBS (14.1 days) (Table 1). The vase life in the O₂NBS was not significantly different compared to that of the control (TW), which had a vase life of 17.3 days. Flowers treated with O₂NBS exhibited more severe senescence symptoms, such as wilting and discoloration, while those under O₂NB treatment remained fully open with minimal visual degradation (Fig. 1). Although the O₂NB treatment extended vase life by 2.9 days compared to TW, the difference was not statistically significant. Flowers treated with the commercial preservative FL had a vase life of 18.7 days, which was shorter than that of the O₂NB group. The O₃NB and O₃NBS treatments resulted in vase lives of 18.7 and 17.9 days, respectively.

Bacterial counts and microscopy observation on vascular

The bacterial counts in C. ‘Lovely King’ treated with O₂NB was 4.4 log ₁₀ CFU mL^-1, which was the lowest among all treatments and significantly lower than the control (Fig. 2). The bacterial counts in C. ‘Lovely King’ treated with O₃NB and O₃NBS were 4.6 and 4.9 log ₁₀ CFU mL^-1, respectively, both of which were also significantly lower than that of the TW (control) (5.9 log ₁₀ CFU mL^-1). The Suc treatment showed a bacterial counts of 5.6 log ₁₀ CFU mL^-1. In contrast, no bacterial counts was detected in the FL treatment. Optical microscopy observations showed that vascular tissues at the stem ends maintained relatively large and uniform size on day 7 (Fig. 3). Fewer dark-stained parts were observed at this point compared to day 19. As the vase life progressed, the vascular areas outlined in red became smaller, and dark occlusions were increasingly observed in the phloem by day 19.

Relative fresh weight and water uptake

The RFW of C. ‘Lovely King’ increased across all treatments until day 7, followed by a gradual decline over time (Fig. 4). According to one-way ANOVA followed by Duncan’s multiple range test (p < 0.05), the RFW values on day 7 were significantly higher in the FL (101.4%), O₃NB (101.3%), and O₂NB (101.1%) treatments compared to the tap water (control, 100.6%). The RFW of C. ‘Lovely King’ treated with O₂NBS and Suc reached 100.5% respectively, both lower than the control with no significant differences on day 7. The RFW treated with O₂NB treatment remained significantly higher than that in the O₂NBS treatment throughout the experiment (P < 0.05). The RFW treated with O₂NBS showed the earliest decline and exhibited the lowest RFW values from days 9 to 23, with significantly lower values than those of tap water (control) between days 9 and 15. Flowers treated with O₃NB maintained significantly higher RFW values than the control on days 7, 9, 11, 19, and 23 (P < 0.05), ranging from 98.2% to 81.0%. Moreover, the RFW values in the O₃NB treatment were consistently higher than those in the O₃NBS treatment, with statistically significant differences observed between days 9 and 19. The RFW of C. ‘Lovely King’ treated with FL was significantly higher than that of the control until day 9, and remained significantly higher than those of the O₂NBS and O₃NBS treatments until day 15. However, the RFW of flowers under FL and O₂NBS treatments fell below the 70% threshold, generally considered the minimum standard for vase life, in the later stages.

The water uptake of C. ‘Lovely King’ treated with O₂NB and O₃NB was initially measured at 12.0 g and 10.5 g, respectively, both statistically significantly greater than that of O₂NBS (6.9 g) (P < 0.05), although the differences from the control were not statistically significant (Fig. 5). The water uptake of C. ‘Lovely King’ treated with O₃NB was the highest among other treatments from days 9 to 15, ranging between 7.0 g and 12.0 g, and which were statistically significantly higher than that of the control. The water uptake of C. ‘Lovely King’ treated with O₂NB and O₃NB was higher than that of O₂NBS and O₃NBS throughout the experiment, with statistically significant differences observed especially between days 11 and 15 (P < 0.05). Conversely, the water uptake of C. ‘Lovely King’ treated with FL was the lowest among all treatments on days 13 (4.8 g), 19 (3.1 g), and 23 (2.3 g). The water uptake treated with O₂NBS was the lowest among treatments on days 9 (6.8 g), 11 (6.2 g), and 15 (5.1 g). The water uptake of C. ‘Lovely King’ treated with O₃NBS showed significantly reduced levels on days 11 to 15, yet exhibited a sharp increase and became the highest among treatments on days 19 and 23. Meanwhile, the Suc treatment consistently outperformed both O₂NBS and O₃NBS in terms of water uptake throughout the entire experiment.

Flower diameter

The flower diameter of C. ‘Lovely King’ treated with O₂NBS showed no statistically significant difference compared to the control on day 0, but was the highest among all treatments on all other days except day 11, showing statistically significant differences compared to the control (Fig. 6). It gradually increased from 71.4 mm to 91.2 mm by day 23. The flower diameter of C. ‘Lovely King’ treated with control was the lowest at 65.0 mm on day 0, which was the smallest among all treatments, and although it varied during the experiment, it remained consistently low. The flower diameter treated with O₂NB increased from 70.9 mm to a peak of 83.7 mm by day 11, followed by a gradual decline thereafter. In the Suc treatment, flower diameter remained relatively low until day 15. Throughout the vase period, the diameter fluctuated irregularly due to the characteristic petal movement of cut C. ‘Lovely King’, where petals tend to curl inward or outward as senescence progresses.

Discussion

This study demonstrated that various preservative solutions for transportation of O₂NB, O₃NB, sucrose (Suc), and Floralife (FL, commercial preservatives) affected the vase life and postharvest quality of cut Cymbidium ‘Lovely King’ flowers. The vase life of cut flowers is affected by postharvest water relations, carbohydrate status, the type of holding solution used, hormonal balance, and environmental conditions (Azizollah et al., 2021; Rabiza-Świder et al., 2020). The use of various preservative solutions and commercial preservatives containing a biocide and a sugar remains a common method (Rabiza-Świder et al., 2020). In this study, the O₂NB treatment significantly extended vase life compared to O₂NBS, with no significant difference from the control. Moreover, the O₂NB, O₃NB, and O₃NBS treatments significantly reduced the bacterial counts compared to the control, Suc, and O₂NBS (Fig. 2), indicating that nanobubble treatments have effective antimicrobial properties in preservative solution. Notably, O₃NBS further reduced bacterial levels compared to O₂NBS, which may be attributed to the stronger oxidizing capacity of ozone (Hayakumo et al., 2014). These findings imply that nanobubble-based solutions were effective in extending vase life by suppressing microbial growth, potentially reducing vascular blockage, and improving water uptake.

Sucrose supply as a nutritional source in preservative solutions contributes positively to the extension of vase life in cut flowers (van Doorn, 1997). However, it also promotes microbial proliferation in solutions without antimicrobials, leading to xylem occlusion and inhibiting water uptake (Pun and Ichimura, 2003). We hypothesized that the combination of NB and sucrose would increase the vase life of cut C. ‘Lovely King’ by reducing bacterial counts and providing additional nutrients, but the vase life of the O₂NBS treatment was shorter than that of O₂NB alone by 6.3 days (Table 1). This result was contradictory to the previous study that the solution containing 200 mg･L^-1 8-hydroxyquinoline sulfate (8-HQS) combined with 2% sucrose improved postharvest quality of cut snapdragon flowers (Asrar, 2012). This result may be attributed to the use of a concentration combination that was not optimal for C. ‘Lovely King’, which may have limited the efficacy of sucrose. Although 2% sucrose was selected based on previous studies reporting its effectiveness in extending the vase life of cut Cymbidium (De et al., 2014), the optimal concentration may differ by cultivar. In C. ‘Red Princess’, pulsing with 5% sucrose increased vase life to 56 days, followed by 8% sucrose (54.78 days), while in C. ‘Baltic Glaciers Mint Ice’, pulsing with 5% sucrose followed by 150 ppm 8-HQS most effectively increased the vase life of flowers (De and Singh, 2019). To clarify the role of sucrose under appropriate conditions, further studies examining a wider range of concentration combinations are required.

Water deficit is one of the primary factors in flower wilting, and maintaining cell turgor is essential for extending vase life (van Doorn, 1997). The biocide might be to inhibit the growth of microorganisms in the vessels and stimulate water absorption, as observed in previous studies helping to maintain the turgor of flowers (Halevy and Mayak, 1981;Jiang and Reid, 2012). Treatments with O₂NB and O₃NB, particularly O₃NB, maintained higher RFW values over time (Fig. 4). In contrast, sucrose-containing treatments such as O₂NBS and O₃NBS resulted in lower initial water uptake and faster RFW decline, likely due to microbial blockage of xylem vessels. In this study, a tendency toward increased flower diameter was observed in treatments with reduced water uptake and lower relative fresh weight, which is likely associated with petal expansion during the senescence process (Figs. 4–6). Such a phenomenon may reflect the late-stage morphological changes accompanying tissue senescence, rather than an indication of improved ornamental quality.

Furthermore, lower water loss is generally associated with extended vase life, as it helps maintain cell turgor and delays wilting. It has also been reported that increasing relative humidity (RH) reduces water loss from floral tissues, thereby affecting water uptake dynamics (Fanourakis et al., 2013). In this study, RH levels in the laboratory ranged from 43.5 to 56.4% (data not shown), and a decline in water uptake was observed between days 9 and 13 as RH increased (Fig. 5), which may have influenced the fresh weight and overall longevity of the cut flowers.

The sterilization mechanisms of O₂NB and O₃NB are derived from their applications in wastewater and drinking water treatment, owing to their capacity to produce highly reactive free radicals (Meegoda et al., 2018). When nanobubbles collapse, they release free radicals that break down organic carbon in wastewater, thereby functioning as an effective biocide (Ebina et al., 2013). The formulation and overall quality of the holding solution play crucial roles in determining the vase life of plants (De et al., 2015). In the present study, tap water was used as the control to simulate farm-level conditions where tap water is typically used; however, its chemical composition may vary depending on the region, potentially influencing the experimental results. Therefore, further research is warranted to evaluate the standardization of vase life assessments using deionized water and thoroughly cleaned and disinfected vases. Additionally, oxidative stress and carbohydrate depletion are key factors that contribute to the premature senescence of cut flowers, particularly in ethylene-insensitive species (Saeed et al., 2014). Previous investigations on cut Cymbidium have shown that the senescence process is linked to elevated H₂O₂ levels in pollinated flowers (Attri et al., 2008). The collapse of nanobubbles produces free radicals that can damage floral tissues and vascular structures, concurrently generating ROS, which play an essential role in the aging of cut flower petals (Agarwal et al., 2011). Furthermore, ROS are critically involved in mediating lipid peroxidation and the wilting of petals. Future research should focus on optimizing NB-sucrose concentration ratios, quantifying ROS dynamics in petal tissues, and validating these effects under commercialscale postharvest logistics conditions.