A Comparative Study of Dormex® and Biostimulant Effects on Dormancy Release, Productivity, and Quality in ‘Royal Tioga®’ Sweet Cherry Trees (Prunus avium L.)

Abstract

Dormancy breakers are widely employed in regions with an insufficient chill accumulation to enhance floral bud break in sweet cherry production. Dormex (HC) has not been authorized in Europe and many other countries due to its detrimental effects on human health (carcinogenic) and the environment. This situation has increased the demand for alternative chemicals to HC. The current study was conducted in an orchard of 5-year-old ‘Royal Tioga^®’ sweet cherry trees (Prunus avium L.) grafted on MaxMa 14 rootstocks in the Karayusuflu Village (at an altitude of 50 m), Adana, a Mediterranean city in Türkiye. Levante (LV) and HC were applied approximately 30 days before the anticipated dormancy break of the buds. Chill accumulation was quantified monthly during the 2023–2024 winter season using the chilling hours below 7.2 °C (467 h) and chill units (280 CUs). This study evaluated the effects of dormancy breakers on hormonal changes in floral buds, focusing specifically on gibberellic acid (GA₃) and abscisic acid (ABA). Also, their impact on fruit set and quality parameters was assessed. The findings revealed that the LV and HC applications reduced the ABA content in flower buds during the dormancy period compared to the control. LV significantly improved bud break rates (56.8%) and fruit set (85.2%) while positively influencing the fruit quality parameters and yield. No significant differences between the LV and HC treatments were observed regarding average fruit weight (8.8–8.4 g) and yield values (3.7–3.5 kg tree⁻¹). These results suggest LV as an alternative to HC for early-season cherry production in warm climates.

1. Introduction

Türkiye is the leading producer of sweet cherries (Prunus avium L.), with an annual production of 656,041 tons. The country also plays a significant role in cherry exports. In 2022, the world cherry production amounted to 2,765,827 tons [1]. Sweet cherry is native to the South Caucasus, the Caspian Sea, and Northern Anatolia. In Türkiye, it is primarily cultivated in the Marmara, Aegean, Black Sea, and Mediterranean regions. These areas’ peak cherry production season occurs in June and July, aligning with the world cherry production season [2]. However, sweet cherry production is relatively low in April, May, and August compared to the primary growing season. There has been a notable increase in the cultivation of early cherry cultivars, particularly in the Çukurova region, which offers significant advantages for domestic consumption and export in Türkiye. Global warming and climate change have affected fruit production worldwide in recent years. These changes have adversely affected the fruit yield and quality parameters, particularly weight and color. For the commercial cultivation of many fruit tree species in low-chill regions, it is crucial to implement measures to break endo-dormancy during a mild winter [3]. Many fruit cultivars and species require chill accumulation to terminate endo-dormancy [4].

Recently, climate change has negatively impacted deciduous fruit crops, leading to significant alterations in the phenological phases. These include a prolonged growing season and insufficient chilling accumulation during winter, adversely affecting fruit growth and production. During bud dormancy, abscisic acid (ABA) and gibberellic acid (GA₃) act antagonistically to regulate dormancy maintenance and enhancement, respectively [5]. In warmer regions, fruit production is achievable only by using low-chill cultivars and implementing effective cultural practices, such as evaporative cooling, dormancy avoidance, and dormancy-breaking agents, mainly hydrogen cyanamide (HC). HC inhibits catalase activity and activates specific peroxidases [6]. The effective temperatures for the chilling requirements have been reported to range between 0 °C and 7 °C. Temperatures below 0 °C or above 7 °C are ineffective for chilling accumulation [7]. However, other studies suggest the optimal temperature range is between 2.5 °C and 9.1 °C [7,8]. A failure to fulfill the chilling requirements completely can lead to irregular flowering, reduced bud breaks, bud drop, bare shoots, anomalous flower development, growth disorders, and weak crop performance in many fruit trees [9]. Chemicals have been widely used to stimulate bud dormancy release in fruit trees in low-chill areas. Common chemicals include HC, potassium nitrate (KNO₃), and mineral oil, which exhibit an interdependent effect on bud breaks and influence the chemical composition of many fruit trees. Dormancy-breaking treatments effectively alter the levels of hormones, polyamines, and nitrogen in buds. HC, in particular, has been extensively used as a dormancy-breaking agent, with its effectiveness dependent on its interactions with plant hormones and amino acids [10,11]. Research on genetic and physiological functions has highlighted the pivotal role of hormones in dormancy regulation, particularly the involvement of ABA and GA₃ pathways in dormancy progression [12]. Although HC is widely used to accelerate dormancy release in grapevines and deciduous fruit species, its precise mechanism of action remains unclear [13]. While HC has been successfully employed to break endo-dormancy, it is one of the most commonly used dormancy-breaking agents (DBAs). HC is frequently applied in warm regions to release endo-dormancy and promote early blooming, especially in sweet cherry cultivation. Comparative studies have indicated that HC is more effective than other DBAs, such as KNO₃, mineral oil, calcium nitrate, and thiourea, particularly in regions with inadequate chill accumulation [14]. HC is a widely recognized commercial product and is considered the most effective DBA [10,15]. HC requires national authorization to be sold and is currently not authorized in Europe [16] but is regularly used in other countries such as the USA, India, and New Zealand, as well as South American countries [17]. Consequently, there is a pressing need to explore alternative chemicals with similar efficacy to HC.

The minimum nitrogen content in flower buds has been observed during dormancy, whereas maximum levels are recorded just before bud break [18]. Additionally, research has indicated that changes in amino acid composition are linked to flower bud break [2]. The effects of endogenous plant hormones on dormancy mechanisms have been extensively studied [8,19,20,21,22]. These studies have revealed that endogenous hormone concentrations, particularly ABA and GA₃, fluctuate during dormancy and significantly influence all dormancy stages [23]. To accurately determine the effects of growth regulators on dormancy release, selecting cultivars with higher chilling requirements is crucial, thereby allowing for an assessment of the impact of insufficient chilling. Cultivars with low chilling requirements should be avoided in such studies. Cherry cultivation in subtropical regions offers an opportunity to harvest cherries earlier, thus entering the early European cherry market. Based on this knowledge, the present study aims to investigate the dormancy-breaking effects of LV compared to HC and control treatments on ‘Royal Tioga^®’ sweet cherry trees, a cultivar struggling to break dormancy in warmer years.

2. Materials and Methods

2.1. Plant Material

The ‘Royal Tioga^®’ sweet cherry variety is self-fertile, characterized by low chilling requirements (350–500 h) and an early ripening period [24,25]. Five-year-old trees of the ‘Royal Tioga^®’ sweet cherry variety, grafted onto MaxMa 14 rootstock, served as the plant material for this study. These trees were in an orchard in Karayusuflu Village, Adana (40°20′02.19″ N latitude, 36°28′30.11″ E longitude, 50 m altitude). The orchard layout followed an east–west planting direction, with a 4 × 2.5 m spacing between the rows and within the rows. Ten trees were left as a buffer between the applications and the control. The trees were pruned using the Kym Green Bush (KGB) system. Standard orchard management practices, including regular irrigation and fertilization, were implemented. A drip irrigation system integrated with fertigation was employed throughout this study, maintaining soil moisture near field capacity from March to September. The system included two 16 mm diameter lateral pipes with in-line pressure-compensated emitters for each tree row. The emitters were spaced 50 cm apart, with a discharge rate of 4 L h⁻¹.

2.2. Experimental Design

A randomized block design model with three blocks was utilized in this study. Within each block, ten trees were selected for each recruitment of LV, and HC was applied to enhance dormancy breaking. The composition and formulation of these chemicals were as follows: LV 10% (v/v) (Nitrogen 11.1%, Calcium 4.1%, Lipids, Glycolipids, Oligosaccharides, and Polysaccharides) (Zoberbac Agrocompany S.L., Barcelona, Spain) and HC 4% (v/v) (Zhengzhou Delong Chemical Co., Ltd., Zhenzhou, China) [24]. The same amount of tap water was applied to the trees used as controls. Treatments were performed 30 days before the estimated bud break [13,23,26]. For ‘Royal Tioga^®’ sweet cherry trees, the appropriate application timing was determined to be approximately 30 days before the bud burst stage of the control trees (March 3) [8,25,27]. The randomly selected trees were labeled and treated on 2 February 2024. Foliar spray applications (4 L per tree, ten trees per treatment) were conducted. All treatments (HC, LV, control) were applied on 2 February using a 20 L portable sprayer. Triton B (0.05%) (Sigma Aldrich, Darmstadt, Germany) was used as a wetting agent in all solution preparations.

2.3. Phenological Observations

The number of flower buds was determined through counting. Bud break dates were recorded for each tree across all treatments when 50% of the flower buds displayed a green tip in the orchard. The final fruit set was calculated as the number of fruits per one hundred flower buds [28].

2.4. Chilling Accumulation of the Experimental Area

Experiments should be conducted during periods of insufficient chill accumulation to accurately assess the effects of the substances used to break dormancy. Thus, determining the chill accumulation in the experimental area is crucial. The most common methods for calculating the chill accumulation include the chilling hours and chill unit (Utah) models [29,30]. Trees must fulfill their chilling requirements to exit dormancy. Research has shown that the chill unit method suits studies in subtropical regions. This method employs effective temperature units, with the most effective temperature range between 2.5 °C and 9.1 °C and each hour is evaluated as a unit. The effects of other temperature ranges (1.5–2.4 °C and 9.2–12.4 °C) are calculated as 0.5 units, (<1.4 °C and 12.5–15.9 °C) as 0 units, and (>16 °C) as negative effects [31,32].

2.5. Fruit Analysis

A fruit analysis was conducted in triplicate, with each replicate consisting of 30 randomly selected fruits. A precision balance (Axis, ATA520 series, Gdańsk, Poland) with a precision of 0.1 g was utilized to measure fruit weight (g). Fruit dimensions (width, length, and height) (mm) were measured with a digital caliper (Digital ABS AOS Caliper, Mitutoyo, Kawasaki, Japan).

Total soluble solids (TSS, °Brix) were evaluated using a digital handheld refractometer (ATC-1, Atago, Tokyo, Japan). Fruit firmness (N) was assessed with a penetrometer (Landtek, FHT-1122, Digital Fruit Firmness Tester, Guangzhou, China). Fruit peel samples were measured with a 7.9 mm indentation (tip diameter) after cutting 2 cm from two opposite sides of the equatorial region with a fruit peeler.

Titratable acidity (TA, %) was determined using an automatic titration device (877 Titrino Plus, Metrohm, Herisau, Switzerland). This process involved titration with 0.1 mol L⁻¹ NaOH until a pH of 8.1 was reached. TA (%) was expressed as grams of citric acid per 100 mL. A Minolta 300 Chroma Meter (Minolta Camera Co., Ltd., Osaka, Japan) was used to assess the color parameters of the cherries. Fruit color was determined in terms of L, a*, b*, Chrome (C), and Hue (h°). L indicates a change in the brightness of the color (L; 0 black, 100 white), a* indicates a color change from green to red (positive values are red, negative values are green), and b* indicates a color change from yellow to blue (positive values are yellow, negative values are blue) [8].

Flower bud specimens were collected from each tree in every treatment group to analyze the internal hormone composition, specifically focusing on gibberellic acid (GA₃) and abscisic acid (ABA) levels. The bud samples were obtained randomly and immediately transferred to the laboratory in a container filled with liquid nitrogen. Approximately 0.5 g of flower buds were harvested every 15 days, starting in late November (after leaf fall) and continuing until the dormant period on February 28 (bud burst stage, green tip). After collection, the samples were flash-frozen in liquid nitrogen and stored at −70 °C for a subsequent hormone analysis.

Determining the plant growth regulators (PGRs) in the buds followed a previously established method, as outlined by [33]. Before injection, the extracts were filtered using a 0.45 μm pore size filter. The analysis was conducted using an Agilent 1100 HPLC system (Agilent Technologies, Palo Alto, CA, USA) controlled by ChemStation software running on a Windows NT platform.

The chromatographic separation was performed using a Luna reversed-phase C-18 column from Phenomenex (Torrance, CA, USA), measuring 4.6 mm × 250 mm with a 5 μm particle size. The mobile phase consisted of the following solvents: Solvent A, a mixture of water and formic acid (99:1, v/v), and Solvent B, a mixture of acetonitrile and Solvent A (60:40, v/v). The plant hormones were eluted under a flow rate of 0.5 mL min⁻¹ and a temperature of 25 °C. The elution process involved both isocratic and gradient conditions. From 0 to 10 min, the isocratic conditions with 0% Solvent B were maintained. The gradient conditions progressed as follows: 0% to 5% for Solvent B over 30 min, 5% to 15% for Solvent B in 18 min, 15% to 25% for Solvent B within 14 min, and 25% to 50% for Solvent B across 31 min. Finally, the proportion of Solvent B increased from 50% to 100% within 3 min, after which the column underwent washing and reconditioning.

The compounds were identified and assigned by comparing their UV spectra and retention times with authentic standards, with further confirmation via an LC-MS/MS analysis. Accordingly, an Agilent 6430 LC-MS/MS spectrometer with an electrospray ionization source was employed. The multiple reaction monitoring (MRM) method was employed for data acquisition, targeting specific mass transitions during the predetermined retention times [33]. For GA₃, the monitored MRM transitions were 345 → 239 and 345 → 301, while for ABA, they were 263 → 153 and 263 → 219 (Figure 1).

2.6. Biochemical Parameters

2.6.1. Total Phenolic Content

The Folin–Ciocalteu method [34] was used with minor modifications to determine the total phenolic content. Accordingly, 1 mL of the sample was combined with 9 mL of an 80% methanol solution. The mixture was centrifuged at 5500 rpm for 10 min (Elektro Mag, M 4808 PR Centrifuge Refrigerated-Full Swing-out Rotor w/Brushless Motor w/RCF Display, Türkiye, rotor diameter 17.5 cm). Subsequently, 50 µL of the supernatant was mixed with 250 µL of the Folin–Ciocalteu reagent. To this, 750 µL of a 20% (w/v) sodium carbonate solution was added, and the mixture was incubated at room temperature (21 °C) for 2 h. The optical density was then measured at 760 nm using a blank as the reference. A UV/VIS spectrophotometer (Thermo Fisher Scientific, Multiskan GO, FI-01620 Vantaa, Finland) was employed for the analysis. A freshly prepared calibration curve using the known gallic acid (GA) standard concentrations was used for quantification. The results were expressed as milligrams of gallic acid equivalents (mg GAE) per 100 g of sweet cherry dry weight (DW).

2.6.2. Total Anthocyanins

The pH differential method, as described by [35] with minor modifications, was utilized to determine the total anthocyanin content (TAC). Sweet cherries were homogenized in 10 mL of methanol containing 1% HCl for two minutes. The mixture was allowed to stand overnight and filtered through Whatman No. 2 filter paper (Sigma Aldrich, Germany). Two separate solutions were prepared for the analysis: one with a pH 1.0 potassium chloride buffer (1.86 g of KCl in 1 L of distilled water) and the other with a pH 4.5 sodium acetate buffer. Two extracts were prepared (54.43 g of CH₃CO₂Na.3H₂O in 1 L of distilled water), and two additional (CH₃CO₂Na.3H₂O) solutions were prepared. During the pH adjustment process, when the pH of a solution is elevated, acidic pH-reducing agents such as HCl are utilized to lower the pH to the desired level. Conversely, when the pH of a solution is low, basic pH-increasing agents such as NaOH are employed to raise the pH to the desired level. After a 15 min incubation period at room temperature, the absorbance of the extracts was measured using a Thermo Fisher Scientific Multiskan GO spectrophotometer (FI-01620 Vantaa, Finland) at wavelengths of 510 nm and 700 nm. The anthocyanin concentration in the triplicate-extracted samples was expressed as milligrams of cyanidin 3-glucoside equivalent (CGE) per liter, calculated using the following equation:

Total anthocyanin (mg L⁻¹) = (A × MW × DF × 1000)/∊ × L

where

• A = the Absorbance difference = (A₅₁₀ − A₇₀₀) pH _1.0 − (A₅₁₀ − A₇₀₀) pH _4.5;

• MW = the Molecular weight of cyanidin 3-glucoside = 445.2;

• DF = the Dilution Factor;

• ∊ = the Molar absorptivity of cyanidin 3-glucoside = 29,600;

• L = the Path length (usually 1 cm).

2.6.3. Determination of Antioxidant Capacity

Two distinct techniques, employing the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and Ferric Reducing Antioxidant Power (FRAP) assays, were used to assess antioxidant activity. The procedure commenced with weighing 1 g of each variety and adding 80% methanol. The samples were centrifuged at 10,000 rpm for 20 min at 4 °C. The resulting supernatant was promptly subjected to the assays following the protocols below. To ensure the reliability of the results, each extract was prepared in triplicate.

2.6.4. DPPH Assay

The radical scavenging activity and DPPH inhibition were evaluated using the method described by [36], with minor modifications. A 0.06 µM ethanolic DPPH solution was freshly prepared. Subsequently, 1950 µL of the DPPH solution was combined with 50 µL of the sample. The resulting mixture was shaken for 1 min and incubated in darkness at room temperature for 30 min. Absorbance was measured at 515 nm, with a blank reagent as a reference. The percentages of DPPH inhibition and radical scavenging activity were determined using the following equations:

DPPH % Inhibition = (Control Abs − (Sample Abs − Blank Abs)/Control Abs) × 100DPPH % Radical scavenging activity = (Control Abs − Sample Abs)/Control Abs) × 100

2.6.5. Determination of Sugar Content

An HPLC analysis was used to evaluate the concentrations of glucose, fructose, sucrose, and total sugar in the homogenized sweet cherry samples, following an established protocol [37]. Frozen juice samples were thawed at 25 °C before analysis, and a 1:4 ratio of juice to ultrapure water (Millipore Corp., Bedford, MA, USA) was prepared. The mixture underwent sonication at room temperature for 15 min in an ultrasonic bath and was centrifuged at 5500 rpm for 15 min. Before the HPLC analysis, the solution was filtered through Whatman nylon syringe filters (0.45 µm, 13 mm diameter).

Sugar content was determined in triplicate using a Shimadzu Prominence LC-20A HPLC system (Kyoto, Japan) equipped with a Refractive Index Detector (RID) and a Coregel-87C column (300 mm × 7.8 mm i.d., 5 µm). Separation was performed at 70 °C with a flow rate of 0.6 mL min⁻¹, employing isocratic ultrapure water as the eluent. Individual sugar concentrations were quantified using the corresponding standards and expressed as a percentage of dry weight (DW). The calibration curves generated from reference standards were utilized for content determination.

2.6.6. Organic Acids

The organic acids in the sweet cherries were determined using a high-performance liquid chromatography (HPLC) analysis, following the previously described methods [38]. For organic acid extraction, 1 mL of the sample was mixed with 4 mL of 3% metaphosphoric acid. The mixture was placed in an ultrasonic water bath at 80 °C for 15 min, sonicated, and centrifuged at 5500 rpm for 15 min (Elektro Mag, M 4808 PR Centrifuge Refrigerated-Full Swing-out Rotor w/Brushless Motor w/RCF Display, Rotor diameter Türkiye). The supernatant was filtered using Whatman nylon syringe filters (0.45 μm pore size, 13 mm diameter). The resulting filtrate was transferred to HPLC vials for analysis.

The organic acid extract was analyzed using an HPLC apparatus (Shimadzu LC 20AVP, Kyoto, Japan) equipped with a UV detector (Shimadzu SPD 20A VP) and an 87 H column (5 μm particle size, 300 mm × 7.8 mm, Transgenomic). The column temperature was maintained at 40 °C, with an injection volume of 20 μL. Detection was conducted at 210 nm and 242 nm wavelengths, with a flow rate of 0.8 mL min⁻¹. The mobile phase consisted of 0.05 mM of sulfuric acid.

The identification and quantification of organic acids were based on the retention times and spectral data, compared against standard compounds. Quantification was conducted using standard calibration curves for each identified acid. The malic, succinic, and citric acid results were calculated and expressed as a percentage of dry weight (DW). The L-ascorbic acid results were expressed as mg per 100 g.

2.7. Statistical Analyses

Each treatment consisted of three replicates, with each replicate comprising ten trees. One-way analysis of variance (ANOVA) was performed to evaluate the statistical differences in the spray treatments for each independent mean using the ‘aov’ function in R. Tukey’s post hoc test (p-value ≤ 0.05) was employed to identify significant differences among the treatments using the ‘Tukey HSD’ function. The results are reported as the mean ± standard deviation. Visualizations were generated with the R packages (version 4.4.2), ‘ggplot2’ (v3.4.4), ‘corrplot’ (v0.92), ‘FactoMineR’ (v2.9), and ‘factoextra’ (v1.0.7).

3. Results and Discussion

Chilling accumulations were calculated using the hours below 7.2 °C and chill units (CUs). During the 2022–2023 winter period, chilling accumulation was calculated as 460 h and 202 CUs, while in the 2023–2024 period, it was detected as 467 h and 280 CUs (Table 1). The chilling accumulation in the experimental area during these periods was insufficient to release the ‘Royal Tioga^®’ sweet cherry trees from dormancy. The differences in chilling accumulation between the years were consistent with the findings reported by [39], who studied the dormancy mechanism of sweet cherry cultivars.

The phenological results from the 2023 to 2024 period indicated no significant differences between the LV and HC applications regarding the dormancy-breaking date (28 February) and rate (56.8% and 56.6%, respectively), as well as the fruit set date (24 April) and rate (85.2% and 84.0%, respectively). However, significant differences were observed between the treatments and the control trees in the flower bud burst dates (3 March) and rates (53.3%), as well as the fruit set dates (4 May) and rates (53.8%) (Table 2). These findings demonstrate that the LV and HC applications significantly reduced the dormancy period and increased the dormant bud and fruit set ratios compared to the control trees (Table 2). The LV (56.8%) and HC (56.6%) applications provided slightly better results than the control (53.3%) in terms of the dormancy breaking rate. Similarly, the highest fruit set values were obtained with LV (85.2%) and HC (84.0%), while the lowest value was recorded for the control trees (53.8%) (Table 2).

The chemical treatments positively affected both bud break and fruit set in warmer regions. Previous studies by [11,40,41] reported that HC treatment was more effective in breaking dormancy in deciduous fruit trees compared to KNO₃ and mineral oil + HC treatments. In the present study, LV provided results similar to those of HC. Similar findings were reported for apples in [42]. Additionally, ref. [43] found that HC combined with 1% mineral oil was the most effective dormancy-breaking treatment for Golden Delicious apple trees. HC was also identified as the most effective treatment for breaking dormancy in the ‘Anna’ apple variety [10]. Furthermore, HC and an emulsified vegetable oil compound (EVOC, Waiken^®) were reported as the most effective treatments for breaking dormancy in the Lapins sweet cherry variety [26].

The chilling accumulation was considered insufficient during winter (2022–2023 and 2023–2024). No dormancy-breaking agents were applied during the first year (2022–2023). Consequently, no yield was obtained from the cherry trees. During the second winter period (2023–2024), dormancy-breaking agents (HC and LV) were applied to enhance yield and quality. The effects of these treatments on yield were compared. The lowest average yield, 0.5 kg tree⁻¹, was observed in the control group. In contrast, the highest yields were obtained with the LV (3.7 kg tree⁻¹) and HC (3.5 kg tree⁻¹) treatments. However, no statistically significant differences in yield were detected between the LV and HC applications. Despite this, the positive effects of the dormancy-breaking agents were evident during the low-chilling winter period, as a significant increase in yield was observed.

No statistically significant differences were found between the treatments and the control group regarding fruit weight, width, length, height, stone weight, stem height, or fruit juice pH levels. Fruit weight (g) emerged as the most critical parameter, with LV (8.8 g), HC (8.4 g), and the control (8.3 g) showing some variation. The pH values of fruit juice ranged from 3.2 to 3.3. The one-way ANOVA analysis revealed differences in the total soluble solids (TSSs) and total acidity (TA). The highest TSS was observed in the LV treatment group (16.1%), followed by the control group (15.9%). The lowest TSS content was recorded for the HC-treated fruits (15.1%). Regarding total acidity, LV treatment (4.6%) yielded the highest value, while the control (4.1%) and HC (4.3%) exhibited lower values. Fruit peel color showed similar results across treatments, ranging between 29.6° and 29.8° hue. In contrast, fruit firmness displayed significant differences, with the highest value recorded in the HC treatment group (41.5 N), followed by the LV (39.3 N) and the control groups (35.9 N). All pomological observations, including fruit weight, TSS, TA, and fruit elasticity, were in line with the previous research on cherry fruit quality parameters [13,26,44]. No statistically significant differences among the treatments were observed in relation to color parameters (measured as L, a*, b*, h°, and C) (Table 3).

Two representative phytohormones in plant samples, GA₃ and ABA, were analyzed in floral buds using the LC-MS/MS method. The electrospray ionization source was operated in negative ion mode, and quantitative measurements were performed in multiple reaction monitoring (MRM) mode. The MRM transitions monitored were 345 > 239 and 301 for GA₃, and 263 > 153 and 219 for ABA (Figure 1).

The LV and HC treatments reduced the ABA content in the flower buds of the ‘Royal Tioga^®’ sweet cherry trees compared to the control trees treated with tap water during the dormancy period. During the 2023–2024 winter season, the chilling accumulation in the experimental area was insufficient for the ‘Royal Tioga^®’ sweet cherries, resulting in no yield from the trees. Previous research has demonstrated that HC applications in sweet cherries promote uniform and frequent dormant flower bud bursts, improving fruit set and yield under low-chill conditions [45,46,47,48,49,50]. However, HC risks human health and the environment [51], highlighting the necessity of identifying reliable and eco-friendly alternatives for fruit production in low-chill regions.

The maximum ABA concentrations were detected at the beginning of the endo-dormancy period (1 October 2023) in the floral buds of both the treated and control trees. The ABA concentrations ranged from 6.91 to 0.76 μg g⁻¹ DW. The lowest ABA contents were observed in the LV and HC treatments on February 27, measuring 0.76 and 0.95 μg g⁻¹ DW, respectively, during the 2023–2024 dormancy period. The dormancy release in the floral buds was associated with a GA₃/ABA ratio higher than one [52]. Certain dormancy-breaking agents (DBAs), such as HC, mineral oil, and KNO₃, influence dormancy release by reducing the ABA concentrations and increasing the GA₃ levels in floral buds [53]. In this study, a negative correlation was observed between the GA₃ and ABA concentrations in the floral buds during dormancy (Figure 2). In terms of GA₃ concentrations, the LV treatment resulted in the highest levels across all the sampling dates, ranging from 0.36 to 1.68 μg g⁻¹ DW. The GA₃/ABA ratio was detected as 1 μg g⁻¹ DW, with dormancy breaking occurring only in these buds. The minimum GA₃ content was recorded on 1 December in the dormant flower buds of the control trees (0.24 μg g⁻¹ DW) (Figure 2). The highest GA₃/ABA ratio during the dormancy period was observed on 28 February in the flower buds treated with LV, measuring 2.17 μg g⁻¹ DW. The flower buds of all the trees exited dormancy on 28 February, coinciding with a sufficient chilling accumulation. On 27 February, the maximum GA₃/ABA ratios from the LV and HC treatments were 2.17 and 1.77 μg g⁻¹ DW, respectively, while the control had the lowest value at 0.75 μg g⁻¹ DW (Figure 2). Over the past decades, numerous studies have explored dormancy mechanisms [13,54,55,56,57], identifying ABA and GA₃ as key endogenous regulators acting antagonistically in warm regions [58,59].

3.1. Biochemical Parameters

Limited reports exist on the effects of DBAs on fruit quality parameters, including sugar, organic acid, total phenolic, total antioxidant, and total anthocyanin contents. In this study, the two chemicals used exhibited distinct impacts on these quality parameters, as summarized in Table 4.

3.2. Total Phenolic, Total Antioxidant, and Anthocyanin Contents

The observed increase in the total phenolic and antioxidant contents in the fruits from the trees treated with the dormancy-breaking agents can likely be attributed to an earlier budburst and flowering in these trees, resulting in an extended cultivation period. However, no statistically significant differences were observed in the total anthocyanin content between the treatment and control groups.

3.3. Determination of Sugar Content and Organic Acids

The present study identified four sugar components (sucrose, glucose, xylose, and fructose) in the ‘Royal Tioga^®’ cherries. The primary sugar component of sweet cherries is glucose, followed by fructose, sucrose, and xylose. In the sweet cherries treated with HC and LV, the sucrose content ranged from 802 to 807.35 mg 100 g⁻¹, the glucose content from 3980.7 to 4419.3 mg 100 g⁻¹, the fructose content from 3458.3 to 3851 mg 100 g⁻¹, and the xylose content ranged from 111.8 to 155 mg 100 g⁻¹ (Table 4). Five organic acids (oxalic, citric, tartaric, d-malic, and succinic acid) were analyzed in the ‘Royal Tioga^®’ cherries. Sweet cherries’ primary organic acid component is d-malic acid, followed by tartaric, citric, succinic, and oxalic acids. With the HC and LV treatments, the oxalic acid content ranged from 10.1 to 18.2 mg 100 g⁻¹, the citric acid content varied from 116.6 to 129.4 mg 100 g⁻¹, the tartaric acid content ranged from 524.3 to 565 mg 100 g⁻¹, the succinic acid content varied from 184 to 199 mg 100 g⁻¹, and the d-malic acid content ranged from 408 to 667.3 mg 100 g⁻¹ (Table 4). The HC and LV treatments boosted the sugar components (sucrose, glucose, xylose, and fructose) and organic acid components (oxalic, citric, tartaric, d-malic, and succinic acid) in the ‘Royal Tioga^®’ cherries. Researchers reported that HC stimulates the concentration of TSS and total sugars in grapevines [60]. The higher levels of sugar and organic acid components observed in the trees treated with LV and HC were likely due to an earlier budburst and flowering, which extended the cultivation period. Moreover, the practical effects of the HC and LV sprays on the physical and chemical quality of ‘Royal Tioga^®’ cherries can be attributed to the stimulatory action of these treatments in promoting bud sprouting and enhancing growth, ensuring an adequate leaf area (and thus photosynthetic capacity) through an earlier and more uniform bud break. The increased availability of photosynthates, resulting from a larger leaf area early in the season, optimizes cell division during fruit development and enhances the accumulation of sugars and organic acids [13,54,55]. Furthermore, the studies by [60,61,62] support these findings regarding various fruit quality characteristics.

3.4. Principal Component Analysis (PCA) and Pearson Correlation Analysis

A principal component analysis (PCA) was utilized to reduce the dimensionality of the large dataset. Two principal components (PCA1 and PCA2) accounted for most of the variability observed among the dormancy-breaking treatments (Figure 3). The vectors represent the original variables, with their lengths indicating the strength of each variable’s contribution to the principal components. PCA1 explained 43.9% of the morphological variation in the trees treated with dormancy-breaking agents. PCA2 accounted for 22.1% of the variation. The PCA results revealed that the first three principal components (PCs) explained a significant portion of the total variance. The eigenvalue of the first principal component (Dim.1) was 11.8, accounting for 43.9% of the variance. The second principal component (Dim.2) had an eigenvalue of 5.9, contributing an additional 22.1%, and resulting in a cumulative variance of 66.0%. The third principal component (Dim.3) had an eigenvalue of 3.9, explaining 14.5% of the variance, and resulting in a cumulative variance of 80.5%. These results indicate that the first three PCs captured the majority of the variability in the dataset, making them sufficient for downstream analyses. The PCA identified the distinct contributions of the variables to the first three principal components. Dimension 1 (PC1) was characterized by high loadings for sucrose, glucose, fructose, total sugar, citric acid, tartaric acid, and total phenolic content, primarily representing the variability associated with sugar content, organic acids, and phenolic compounds. Dimension 2 (PC2) was influenced by high oxalic acid and xylose loadings, capturing the variability related to organic acid composition and the sugars associated with cell wall polysaccharides. Dimension 3 (PC3) was associated with high loadings for the colorimetric variables a*, b*, and chroma, reflecting the differences in the color attributes of the samples, including redness, yellowness, and color intensity. Together, these dimensions explain the major sources of variation in the dataset, providing valuable insights into the biochemical and physical traits that drive sample differentiation. Consequently, the most effective descriptors were those associated with PCA1 and PCA2. The angles between the vectors indicated the correlations between variables; smaller angles represented positive correlations, as observed for sucrose, fructose, glucose, citric acid, oxalic acid, total phenol content, firmness, and L (Figure 3). Conversely, larger angles (approximately 180°) reflected negative correlations, particularly between D-malic acid and anthocyanin content (Figure 3). The samples were represented by distinct symbols and colors: blue for the control, yellow for the HC treatment, and red for the LV treatment (Figure 3). The clusters exhibited unique characteristics, underscoring the key relationships and distinctions within the dataset. The HC and Levante treatments were separated in the biplot, indicating that each group displayed distinct traits.

4. Conclusions

The primary findings of this research are summarized below based on the recorded data. The foliar application of LV and HC significantly accelerated the flower bud break in ‘Royal Tioga^®’ sweet cherry trees compared to the control group (sprayed with tap water).

The early bud break induced by LV was identified as one of its significant effects. LV, which contains organic materials, demonstrated greater efficiency in breaking dormancy than the control group. Furthermore, LV and HC gave similar results in terms of yield (3.7 kg tree⁻¹–3.5 kg tree⁻¹).

This study identified LV as the most effective chemical for dormancy breaking in ‘Royal Tiago^®’ sweet cherries. HC followed LV in terms of effectiveness, reducing the abscisic acid (ABA) levels while increasing the gibberellic acid (GA₃) levels in flower buds during dormancy. Furthermore, LV application resulted in an earlier bud break and improved fruit set and quality in ‘Royal Tiago^®’’ sweet cherries compared to the control group. These outcomes suggest that LV achieved superior results relative to the control regarding yield and quality parameters, particularly in the years with insufficient chilling. LV is recommended for other deciduous fruit cultivars in regions with warm climates, where insufficient chilling accumulation necessitates dormancy-breaking interventions. LV use mitigates the environmental and health risks associated with HC applications.

The LV and HC treatments showed almost the same values in terms of variables such as sucrose, tartaric acid, succinic acid, and total phenol content, whereas they varied in terms of glucose, xylose, fructose, oxalic acid, citric acid, D-malic acid, and total antioxidant content in ‘Royal Tioga^®’ sweet cherry cultivar.

This study also assessed the cost-effectiveness of HC and LV applications, finding that the costs per hectare and the yields obtained were comparable. The costs of the applications per hectare were determined as EUR 470 for HC and EUR 369 for LV. Consequently, LV represents a valuable alternative for cherry producers in countries where HC is prohibited due to its adverse effects on human health and the environment.

5. Patents

This section is not mandatory but may be added if patents result from the work reported in this manuscript.

Author Contributions

Conceptualization, A.B. and Z.K.; Methodology, B.İ. and S.Ç.; Validation, Ş.H.A. and C.K.; Formal analysis, N.E.K., Ö.F.B., A.E.G. and Ş.H.A.; Investigation, B.İ. and S.Ç.; Resources, B.İ., S.Ç., A.E.G. and A.B.; Data curation, N.E.K., Ö.F.B., A.B. and Ş.H.A.; Writing—original draft, B.İ., N.E.K., Ö.F.B., C.K. and Z.K.; Writing—review and editing, C.K. and Z.K.; Project administration, B.İ. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

The original contributions presented in this study are included in the article; further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Figures and Tables

Figure 1. LC-ESI-MS/MS MRM chromatograms of GA3 and ABA.

Figure 2. The effects of HC, LV, and control treatments on ABA, GA3, and GA3/ABA concentrations in the flower buds of ‘Royal Tioga®’ sweet cherry trees during the dormancy period (2023–2024). Different letters indicate statistical significance (p [less than] 0.05), and ‘n.s.’ denotes no statistically significant differences.

Figure 3. Principal component analysis of morphological and biochemical parameters of the ‘Royal Tiago®’ sweet cherry variety treated with HC, LV, and the control (95% confidence level).

Table 1

Chilling accumulations of the experimental area in the 2022–2023 and 2023–2024 winter periods.

Months	2023(2022–2023 Period)		2024(2023–2024 Period)
	Hours Below7.2 °C	Chill Unit(CU)	Hours Below7.2 °C	Chill Unit(CU)
November	68	1	19	6
December	51	77	122	24
January	167	69	157	128
February	105	38	113	98
March	69	17	56	24
Total	460	202	467	280

Table 2

The effects of chemicals on bud breaks, fruit set rates, and harvesting dates in ‘Royal Tioga^®’ sweet cherry trees (2024).

Treatments	Bud Burst (Green Tip Stage)		Fruit Set	Harvesting
	Date	Rate (%)	Rate (%)	Date
HC	28 February	56.6 ± 1.7 ns	84.0 ± 1.2 a	24 April
LV	28 February	56.8 ± 0.4 ns	85.2 ± 0.7 a	24 April
Control	3 March	53.3 ± 0.8 ns	53.8 ± 1.7 b	4 May

Each column represents mean ± standard deviation. Different letters indicate statistically significant differences at p < 0.05, and ‘n.s.’ denotes no statistically significant differences.

Table 3

The effects of dormancy-breaking treatments on fruit dimensions and quality parameters of the ‘Royal Tioga^®’ sweet cherry cultivar.

Treatments	Control	HC	LV
Fruit weight (g)	8.3 + 1.3 n.s	8.4 + 0.3 n.s	8.8 + 0.6 n.s
Fruit width (mm)	19.9 + 1.2 n.s	20.7 + 0.3 n.s	21.0 + 0.4 n.s
Fruit length (mm)	26.1 + 1.7 n.s	27.5 + 0.2 n.s	27.6 + 0.6 n.s
Fruit height (mm)	21.6 + 1.4 n.s	23.0 + 0.3 n.s	23.3 + 0.5 n.s
Stone weight (g)	0.4 + 0.1 n.s	0.4 + 0.0 n.s	0.4 + 0.0 n.s
Stem height (mm)	22.5 + 1.6 n.s	25.1 + 0.9 n.s	22.5 + 3.4 n.s
TA %	4.1 + 0.2 b	4.3 + 0.4 ab	4.6 + 0.4 a
pH	3.2 + 0.0 n.s	3.3 + 0.1 n.s	3.3 + 0.1 n.s
TSS%	15.9 + 0.1 n.s	15.1 + 0.4 n.s	16.1 + 0.9 n.s
Firmness (N)	35.9 + 2.4 b	41.5 + 2 a	39.3 + 1.3 ab
L	23.8 ± 1.4 b	29.0 ± 1.7 a	27.6 ± 1.7 ab
a*	22.9 ± 3.1 n.s	23.8 ± 3.4 n.s	22.7 ± 3.0 n.s
b*	9.0 ± 0.9 n.s	9.4 ± 2 n.s	9.0 ± 1.4 n.s
C	24.6 ± 3.1 n.s	25.6 ± 4.4 n.s	24.4 ± 3.3 n.s
h°	21.4 ± 1.6 n.s	21.5 ± 0.9 n.s	21.5 ± 0.7 n.s

Each column represents mean ± standard deviation. Different letters indicate statistically significant differences at p < 0.05, and ‘n.s.’ denotes no statistically significant differences.

Table 4

The effects of HC and LV on the sugar components (mg 100 g⁻¹), organic acid components (mg 100 g⁻¹), total antioxidant activities, total phenol content, and total anthocyanin content of the ‘Royal Tioga^®’ sweet cherry cultivar.

Treatments	Control	HC	LV
Sucrose	426.4 + 4.1 b	802 + 35.6 a	807.43 + 8 a
Glucose	3539 + 11 c	3980.7 + 8.0 b	4419.3 + 8 a
Xylose	116 + 5.4 b	111.8 + 9 b	155 + 4 a
Fructose	3103.7 + 9 c	3458.3 + 5.7 b	3851 + 10 a
Oxalic acid	12 + 1.8 b	10.1 + 1.9 b	18.2 + 1 a
Citric acid	99.33 + 2.0 c	129.4 + 3.5 a	116.6 + 1.19 b
Tartaric acid	423.67 + 21 b	524.3 + 22.5 a	565 + 3.5 a
D-malic acid	335.33 + 23 c	667.3 + 3.1 a	408 + 23.5 b
Succinic acid	159 + 8 b	184 + 11 a	199 + 4 a
Total Phenol (mg 100 g−1GAE)	40.32 + 5.1 b	62.38 + 4.1 a	61.3 + 8.4 a
DPPH (%)	65.18 + 3.6 ab	70 + 2.42 a	62.54 + 3.6 b
Anthocyanin (mg L−1)	6.17 + 0.1 n.s	6.72 + 0.45 n.s	6.2 + 0.1 n.s

Each column represents mean ± standard deviation. Different letters indicate statistically significant differences at p < 0.05, and ‘n.s.’ denotes no statistically significant differences.

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