Preharvest Prohexadione-Ca Treatment Improves Fruit Set and Mechanical Properties in Cv. ‘Tip Top’ Sweet Cherries

Abstract

Sweet cherry (Prunus avium L.) cultivation is rapidly expanding in Northern Italy, where excessive vegetative vigor often limits fruit set and quality. This study aimed to evaluate the effects of Prohexadione-calcium (Pro-Ca) on the vegetative growth, productivity, and fruit quality of cv. ‘Tip Top’ sweet cherries grafted onto Gisela 6 and MaxMa 14 rootstocks. The growth regulator was applied twice between the flower bud and petal fall stages. Pro-Ca significantly reduced vigor and increased the fruit setting by 10%, resulting in an yield average of +3 kg per plant. Also preharvest treatment increased average cherry size compared with the control, particularly in plants grafted onto Gisela 6. Moreover, Pro-Ca-treated fruits exhibited a +20% red overcolor extension of the skin, improved skin firmness (+12%), and led to higher nutraceutical properties. In conclusion, Pro-Ca improved plant yield and fruit quality in ‘Tip Top’ sweet cherry, likely through the combined effects on hormonal balance, assimilate allocation, and canopy light distribution, supporting its potential as a valuable growth regulator in high-density sweet cherry orchards.

1. Introduction

Sweet cherry (Prunus avium L.) cultivation has intensified in Piedmont lowlands (Northwestern Italy), where planted hectares have increased by nearly 50% over the past decade [1]. This development has been driven by the need to replace less profitable crops such as peach [2] or those affected by severe phytosanitary issues like kiwifruit [3]. Cherry has therefore gained importance thanks to its favorable market value [4] and harvest timing, which does not overlap with that of other traditional crops of the region.

However, the changing spring climate in Northern Italy [5,6], characterized by heavy rainfall, hailstorms, and occasional frost events, poses challenges for cherry production. To mitigate some of these risks, modern orchards are typically equipped with anti-rain and anti-hail nets and are established at high planting densities using dwarfing rootstocks. Nevertheless, these conditions can also influence fruit quality, sometimes reducing firmness and cuticle strength [7,8]. In addition, the high vigor of P. avium and the dense planting system make pruning management particularly challenging. Maintaining an optimal vegetative/reproductive balance and adequate light penetration is difficult with the adoption of dwarfing rootstocks [9,10].

Among the recently introduced cultivars, the bicolored sweet cherry ‘Tip Top’ has gained commercial interest due to its appealing appearance and superior sugar concentration [11]. Still, this cultivar exhibits strong vigor, which can reduce fruit set [12] and result in insufficient skin pigmentation, leading to inadequate overcolor extension for the market. The use of dwarfing/semi-dwarfing rootstocks alone does not always ensure a proper balance between vegetative and reproductive growth, which is essential to achieving a high yield and appropriate fruit quality [13]. Moreover, its high vigor limits the adoption of high-density training systems, thereby reducing orchard profitability [14].

Several plant growth regulators (PGRs) may help manage excessive vigor [15,16], although their efficacy strongly depends on the timing of application and environmental conditions [17,18]. In regions with frequent spring rains, like Piedmont, PGRs application can be challenging. In this context, Prohexadione-calcium (Pro-Ca) represents a promising alternative due to its short absorption time (approximately 8 h) [19] and its ability to inhibit the biosynthesis of biologically active gibberellins (GA), thereby reducing vegetative growth [20,21]. While Pro-Ca has been widely studied in pome fruits [22,23,24,25,26,27] and strawberries [28,29,30], limited information is available for the sweet cherry, particularly under temperate and humid climatic conditions.

Therefore, the aim of this study was to evaluate the effects of Pro-Ca on the vigor and productivity of cv. ‘Tip Top’ sweet cherry grafted onto two dwarfing and semi-dwarfing rootstocks (Gisela 6 and MaxMa 14). In addition, the study also investigated the key quality attributes of sweet cherries, including firmness, color, and chemical composition, to clarify the contrasting results [31,32] reported in the literature regarding the influence of Pro-Ca on sweet cherry quality. Moreover, by addressing both physiological and fruit quality aspects, this study provides insights into the potential use of Pro-Ca for improving yield efficiency and fruit quality under high-density cherry orchard systems in Northern Italy, reducing the need for intensive agronomic interventions such as summer shoot thinning or heavy winter pruning, which often lead to a loss of flower buds and compromise the following season yield.

2. Materials and Methods

2.1. Plant Material and Experimental Design

The experiment was conducted during the 2025 growing season in two even-aged sweet cherry (Prunus avium L.) commercial orchards located in Dronero, Piedmont, Italy. The test involved the ‘TipTop’ variety, a bicolored cherry, grafted onto two rootstocks: Gisela 6 (G6) and Maxma 14 (M14). G6 rootstock is a semi-dwarf rootstock that induces moderate vigor (approximately 70–80% of that of trees of the P. avium seedling), early bearing, and high productivity, while M14 rootstock confers medium vigor (around 60–70% of the P. avium seedling), with good soil adaptability and regular yield performance. Both orchards were managed under protective netting to prevent damage from rain and hail.

The experiment design consisted of six randomized blocks per orchard. Each block included 5 trees, resulting in a total of 30 trees per orchard (6 blocks × 5 trees). Within each orchard, three blocks (15 trees) received a foliar PGR treatment (Pro-Ca), while the remaining three served as untreated controls (Ctrl). Overall, four experimental treatments were established: G6 Ctrl, G6 Pro-Ca, M14 Ctrl, and M14 Pro-Ca. Every block was randomly selected from inner orchard positions, avoiding border trees to reduce edge effects.

The foliar treatment was integrated into standard company management practices. The adopted PGR was the REGALIS^® (BASF SE, Ludwigshafen, Germany), composed of 10 g of Pro-Ca and 90 g of co-formulants per 100 g of product. Two applications were carried out at a rate of 1.8 g L⁻¹ and 1.5 L ha⁻¹ (Table 1), and harvesting was performed in June, coinciding with the local commercial maturity stage, defined by a soluble solids content >18 °Brix in the G6 Ctrl fruit. Throughout the growing season, measurements were taken for fruit set rate, tree vigor, and yield. After harvest, fruit samples were analyzed for quality attributes.

Table 1. Pre-harvest foliar treatment on cv. ‘TipTop’ sweet cherries during 2025 season.

Phenology Stage	Operation	Date
		G6	M14
Petals Fall	REGALIS® 1.5 L/ha	22 April	22 April
Fruit Sets	REGALIS® 1.5 L/ha	13 May	13 May
Commercial Ripeness	Harvest	20 June	26 June

2.2. Agronomic Measurement

Fruit set rate was monitored from full bloom until harvest. In each block, two representative branches were selected: one positioned in the upper and one in the lower canopy, on opposite canopy exposures, ensuring that the collected data represented the overall canopy variability. The number of flowers in each flower cluster was counted at full bloom, and just before harvest, the number of fruits per cluster was recorded. Since the number of flower clusters per branch was variable (due to biological differences among samples), all clusters present on each branch were counted to obtain the total number of flowers. The fruit set per branch was calculated as follows:

$$\mathrm{Fruit} \mathrm{set} \mathrm{rate}={\displaystyle \frac{N_{\mathrm{fruits}}}{N_{\mathrm{flowers}}}}\times 100$$

where $N_{\mathrm{flowers}}$ is the total number of flowers in all clusters of the branch and $N_{\mathrm{fruits}}$ is the total number of fruits in the same clusters. For each treatment, six branches (two per block × three blocks) were analyzed.

2.2.1. Plant Vigor

Plant vigor was measured as shoot elongation at monthly intervals following the second Pro-Ca application. One representative shoot per tree was selected and measured with a meter at 1, 2, 3, and 4 months post-treatment. To ensure representative sampling, shoots were chosen from different canopy positions (upper and lower parts, inner and outer zones, i.e., sun-exposed and shaded sides) within each block. Measurements ceased after the fourth month, corresponding to the sprout agostation.

2.2.2. Plant Yield

Yield was recorded for each tree at harvest. The weight of marketable fruit and waste (including molded, cracked, deformed, or undersized fruit) was recorded separately. Unmarketable yield (%) was calculated as:

$$\mathrm{Unmarketable} \mathrm{yield} (\mathrm{\%})={\displaystyle \frac{W_{\mathrm{waste}}}{W_{\mathrm{marketable}}+W_{\mathrm{waste}}}}\times 100$$

where $W_{waste}$ is the weight of the waste of each plant and $W_{marketable}$ is the weight of the marketable portion.

2.3. Fruit Quality Characteristics

Representative fruit samples (approximately 5 kg per block) were collected from each experimental block and transported to the DISAFA laboratory, University of Turin (Grugliasco, TO, Italy), for quality assessment. Fruits were inspected to ensure uniformity in health and absence of defects.

2.3.1. Fruit Size

Fruit diameter was measured with a caliper, with size classes defined as <Ø26 mm, Ø28 mm, Ø30 mm, and >Ø30 mm. Measurements were conducted on a sample of 10 kg per treatment.

2.3.2. Skin Color

Fruits were evaluated for their overcolor development, quantified as the percentage of red pigmentation relative to the total surface area of the fruit. Visual assessments were performed on 15 representative fruits per block for each treatment.

Moreover, skin color was also evaluated with a CR-400 colorimeter (Minolta Co., Ltd., Osaka, Japan) under consistent illumination conditions with a C illuminant and 2° observation angle. Measurements were conducted on 30 fruits per block and were expressed in the CIE LAB color space (Figure 1). Results for L* (lightness) and h° (hue angle) were recorded for both overcolor and ground color. The L* component determines the brightness of the color and has a range of 0 for black to 100 for white, whereas the hue refers to the dominant wavelength of a color, measured in degrees (0–360°) around a color wheel, where 0° represents red, 120° green, and 240° blue [33].

Figure 1. CIELAB coordinates in the color space [34].

2.3.3. Mechanical Properties

Fruit firmness was evaluated using a Texture Analyzer TA-XT2i (Stable Micro Systems Ltd., Godalming, Surrey, UK). Two mechanical tests were performed: a puncture test (PT), assessing the epidermis rupture force and therefore related to skin thickness, and a deformation/compression test (CT), assessing the flesh firmness.

The PT was carried out according to Silva et al. [35], with minor modifications. Each fruit was punctured in the equatorial zone with a 3 mm Ø cylindrical probe (P/3), penetrating 5 mm at a speed of 1 mm s⁻¹, using a 30 N load cell. The CT was performed according to Pinto De Andrade et al. [36] with some modifications. Cherries were compressed in the equatorial zone using a 75 mm Ø flat probe (P/75), applying 5% of strain at a speed of 1 mm s⁻¹. Both PT and CT were performed on different sets of fruits to avoid any structural interference between the two measurements. Only intact fruits, free from defects and with peduncles attached, were used for testing. Before analysis, samples were cooled to 6 °C to ensure a uniform surface temperature. 15 fruits per block were analyzed for each test, and firmness was expressed as the maximum force (N) recorded on the force–time curve.

2.3.4. Total Soluble Solids (TSS)

The analysis was performed on a clear juice extracted from 15 fruits per sample and centrifuged at 1500× g for 10 min (AVANTIM J-25, Beckman Instruments Inc., Villepinte, France). TSS was evaluated using a PR-32 digital refractometer (Atago Co., Ltd., Tokyo, Japan), and measurements were assessed in triplicate for each block, with results expressed in °Brix.

2.3.5. Titratable Acidity (TA) and pH

TA was determined by titrating 10 mL of clear cherry juice with 0.1 N NaOH to pH 8.1 using a Titralab AT1000 automatic titrator (Hach Lange SAS, L’Huisserie, France). Analysis was performed in triplicate for each, with results expressed as meq NaOH L⁻¹.

2.3.6. Nutraceutical Composition

Prior to analyzing the bioactive compounds, the nutraceutical components were first extracted. According to Šavikin et al. [37] 4 g of cherries were homogenized with 10 mL of extraction solvent (MeOH:H₂O, 20:1, v/v, acidified with HCl, 37%) using an UltraTurrax T18 homogenizer (IKA-Werke GmbH & Co. KG, Staufen im Breisgau, Germany) for 1 min. The homogenates were then sonicated at 50 Hz in a water bath at 50 °C for 20 min and centrifuged at 1000× g for 15 min. The resulting supernatants were collected and stored at −26 °C until further analysis. Three extracts per block (nine per treatment) were prepared.

TPC (Total Phenol Content) was determined using the Folin–Ciocalteu (FC) method, with gallic acid as the standard [38]. In brief, 40 μL of fruit extract was diluted with 160 μL of extraction solvent, and afterwards 1 mL of 0.1 N FC and 800 μL of 0.7 M Na₂CO₃ were added. The mixtures were dark-incubated for 5 min in a 50 °C water bath. Absorbance was measured spectrophotometrically (U-5100, Hitachi High-Tech Corporation, Tokyo, Japan) at 760 nm, and results are expressed as mg gallic acid equivalents (GAE) per 100 g fresh weight (mg GAE/100 g f.w.).

Antioxidant capacity (AOx) was assessed by the Ferric-Reducing Antioxidant Power (FRAP) assay [39]. In summary, 30 µL cherry extract and 90 µL of H₂0 were added to 900 µL of FRAP reagent. The FRAP reagent was prepared by mixing 25 mL of CH₃COONa buffer (pH 4.5), 2.5 mL of TPTZ (0.0156 g in 5 mL of 40 mM HCl), and 2.5 mL of FeCl₂ (0.135 g in 25 mL H₂O). Reduction of Fe³⁺-TPTZ to Fe²⁺ was measured at 595 nm after a 5 min reaction in a water bath at 37 °C. Results were expressed as mmol Fe²⁺ kg⁻¹ fresh weight (mmol Fe²⁺/kg f.w.).

2.4. Statistical Analysis

Data were analyzed using RStudio (v. 2025.090+387; RStudio PBC, Boston, MA, USA). A one-way analysis of variance (ANOVA) was performed to compare the effects of the four treatment combinations implemented in the field (G6 Ctrl, G6 Pro-Ca, M14 Ctrl, and M14 Pro-Ca) on agronomic performance and fruit quality, considering treatment as a fixed factor and block as a random factor. For plant vigor, a repeated-measured two-way ANOVA was conducted using treatment and month as fixed factors (treatment × month) and block as random. This model allowed testing of the time-dependent response to Pro-Ca while accounting for block-to-block variability.

Post hoc mean comparisons were performed using Tukey’s test, with statistical significance set at p ≤ 0.05.

3. Results

3.1. Pro-Ca Increases Yield and Inhibits Shoot Growth in Sweet Cherry Trees

Agronomic results highlighted how the fruit set is affected by Pro-Ca treatment (Figure 2). G6 Pro-Ca plants exhibited a significantly higher fruit set rate compared to all other treatments. In contrast, G6 Ctrl, M14 Ctrl, and M14 Pro-Ca displayed statistically similar and lower values. In the case of the M14 rootstock, no significant differences were observed between treated and untreated trees. Likewise, in the absence of Pro-Ca, the choice of rootstock did not result in significant differences in fruit set rate.

Figure 2. Fruit set rate of cv. ‘TipTop’ sweet cherries affected by Pro-Ca (Prohexadione Calcium) treatment compared to untreated control (Ctrl) grafted onto the rootstocks ‘Gisela 6’ (G6) and ‘Maxma 14’ (M14). Results are the mean (N = 6) ± SE; groups sharing the same letters are not statistically different, by a one-way ANOVA. Statistical significance at p < 0.001 by the Tukey LSD test.

The annual shoot elongation, an indicator of plant vigor, is reported in Figure 3. Both G6 Ctrl and M14 Ctrl plants exhibited greater vigor compared with their Pro-Ca-treated counterparts; still, in M14, the difference was not statistically significant. Among all treatments, G6 Pro-Ca showed the lowest vegetative growth, with an average shoot elongation of only 33 cm four months after treatment. Furthermore, G6 rootstock displayed a rapid vegetative growth during the first months after treatment, which then leveled off in the last months, when no significant differences over time were observed. Notably, G6 Pro-Ca showed a marked reduction in shoot elongation mainly in the first month after treatment and between the second and third month after treatment, whereas G6 Ctrl maintained significantly higher growth rates throughout this period. Regarding M14, both treatments showed a rather linear growth trend over the growing season, with no significant differences in shoot elongation observed between Pro-Ca and Ctrl at any sampling timepoint.

Figure 3. Plant vigor of cv. ‘TipTop’ sweet cherries affected by Pro-Ca (Prohexadione Calcium) treatment compared to untreated control (Ctrl) grafted onto the rootstocks ‘Gisela 6’ (G6) and ‘Maxma 14’ (M14). Results are the mean (N = 15) ± SE; groups sharing the same letters are not statistically different, by a two-way ANOVA (treatment × month). Statistical significance at p < 0.01 by the Tukey LSD test.

Regarding yield performance, G6 Pro-Ca resulted in the greatest plant production, with an average yield of 6.7 kg, which was statistically the highest (Figure 4A). Conversely, other treatments showed statistically lower and comparable yields. M14 Pro-Ca exhibited a slightly higher yield than M14 Ctrl, although this difference was not statistically significant. Similarly, in the absence of Pro-Ca, no significant differences were observed between the two rootstocks (G6 Ctrl and M14 Ctrl).

Figure 4. Yield performances of cv. ‘TipTop’ sweet cherries affected by Pro-Ca (Prohexadione Calcium) treatment compared to untreated control (Ctrl) grafted onto the rootstocks ‘Gisela 6’ (G6) and ‘Maxma 14’ (M14). (A): Plant Marketable yield (N = 15). (B): Unmarketable yield (N = 15). Results are the mean ± SE; groups sharing the same letters are not statistically different, by a one-way ANOVA. Statistical significance at p < 0.001 by the Tukey LSD test.

As for the proportion of unmarketable fruit (Figure 4B), a clear distinction between rootstocks was observed. The G6 rootstock showed the lowest waste percentage, with no significant differences between G6 Ctrl and G6 Pro-Ca. In contrast, the M14 rootstock exhibited a significantly higher waste percentage, with M14 Pro-Ca showing the highest unmarketable fraction among all treatments. Overall, G6 Pro-Ca proved to be the most effective treatment, combining the highest fruit yield per tree with the lowest waste percentage (<2.5%)

3.2. Pro-Ca Improves Mechanical Properties, Pigmentation, and Nutraceutical Compounds

Regarding fruit size distribution, the majority of cherries across all treatments fell within the 30 mm caliber class (Figure 5). This trend was more pronounced in the M14 samples compared with G6. Both G6 Pro-Ca and M14 Pro-Ca showed an increased proportion of fruits > 30 mm compared with their respective controls (G6 Ctrl and M14 Ctrl), while the proportion of Ø28 mm fruits decreased correspondingly. Fruits in the <26 mm class represented a small fraction of the total sample. The G6 Pro-Ca treatment had the highest proportion of these fruits, although this accounted for only 2% of the total sample and was therefore considered negligible.

Figure 5. Quantification (%) of cv. ‘TipTop’ sweet cherries in each caliber category (<26–28–30–>30 mm) affected by Pro-Ca (Prohexadione Calcium) treatment compared to untreated control (Ctrl) grafted onto the rootstocks ‘Gisela 6’ (G6) and ‘Maxma 14’ (M14).

Sweet cherries’ mechanical properties are reported in Table 2. PT, which reflects the epidermal rupture force and thus provides information about skin thickness, revealed that G6 Pro-Ca had the highest firmness values, indicating a thicker skin. Conversely, G6 Ctrl and M14 Ctrl exhibited significantly lower PT amounts. Overall, Pro-Ca-treated samples showed higher epidermal firmness than their untreated counterparts for both rootstocks. Conversely, no significant differences were observed between G6 Ctrl and M14 Ctrl.

Table 2. Mechanical properties of cv. ‘TipTop’ sweet cherries affected by Pro-Ca (Prohexadione Calcium) treatment compared to untreated control (Ctrl) grafted onto the rootstocks ‘Gisela 6’ (G6) and ‘Maxma 14’ (M14). Results are the mean (N = 45) ± SE of PT (puncture test) and CT (compression test). Groups sharing the same letters are not statistically different, by a one-way ANOVA. Significance levels: p < 0.01 (**), p ≥ 0.05 (ns, not significant).

Treatment	PT				CT
	(N)				(N)
G6 Ctrl	2.99	±	0.10	b	4.80	±	0.19
G6 Pro-Ca	3.35	±	0.09	a	4.38	±	0.18
M14 Ctrl	2.98	±	0.09	b	4.57	±	0.17
M14 Pro-Ca	3.20	±	0.09	ab	4.63	±	0.17
LSD (p ≤ 0.05)	**				ns

Regarding CT, representative of flesh firmness, no significant differences were detected among treatments. These results suggest that Pro-Ca mainly affected the cherry epidermis rather than the flesh texture, with a more pronounced effect observed in the G6 rootstock.

Regarding skin color, the results showed that G6 Pro-Ca and M14 Pro-Ca exhibited a significantly greater overcolor coverage compared with the controls (Table 3). Among all treatments, M14 Ctrl showed the lowest overcolor extension. Overall, within each rootstock, Pro-Ca-treated samples developed a wider red overcolor area than their respective controls.

Table 3. Fruit pigmentation of cv. ‘TipTop’ sweet cherries affected by Pro-Ca (Prohexadione Calcium) treatment compared to untreated control (Ctrl) grafted onto the rootstocks ‘Gisela 6’ (G6) and ‘Maxma 14’ (M14). Results are the mean (N = 90) ± SE of overcolor extension (%), overcolor L* (brightness), overcolor h° (hue angle), ground color L* (brightness), and ground color h° (hue angle). Groups sharing the same letters are not statistically different, by a one-way ANOVA. Significance levels: (***).

Treatment	Overcolor												Ground Color
	(%)				L*				h°				L*				h°
G6 Ctrl	51.6	±	2.1	b	55.7	±	0.8	a	47.7	±	1.4	a	66.2	±	0.5	a	85.1	±	1.8	a
G6 Pro-Ca	68.2	±	2.7	a	48.4	±	0.7	b	31.7	±	2.4	bc	64.4	±	0.9	b	82.7	±	1.9	ab
M14 Ctrl	41.8	±	2.5	c	50.3	±	1.4	b	34.8	±	1.6	b	64.5	±	1.2	b	79.2	±	2.2	b
M14 Pro-Ca	66.6	±	1.9	a	45.6	±	0.9	c	27.5	±	1.9	c	61.1	±	0.7	c	73.4	±	1.3	c
LSD (p ≤ 0.05)	***				***				***				***				***

Analysis of the overcolor colorimetric parameters revealed that M14 Pro-Ca and G6 Pro-Ca cherries exhibited a deeper red pigmentation compared with the other treatments, while G6 Ctrl showed overcolor hues shifted toward orange tones. Concerning lightness (L*), G6 Ctrl displayed the highest L* values for the overcolor, whereas M14 Pro-Ca had the lowest, indicating a darker and more intense coloration.

Regarding the ground color, G6 Ctrl exhibited the highest hue values, corresponding to yellowish tones and higher lightness, while M14 Pro-Ca showed the lowest hue values, corresponding to orange-red shades and significantly lower lightness. These results indicate that Pro-Ca application enhanced color development, leading to darker and more intensely pigmented fruits (Table 3).

Analysis of the chemical parameters of the cherries revealed that G6 Pro-Ca and M14 Pro-Ca samples exhibited significantly higher TSS concentrations compared to the controls, while the G6 Ctrl showed the statistically lowest value (Table 4). No significant differences were observed among treatments for pH. Regarding TA, the G6 Ctrl and M14 Ctrl showed significantly higher acidity values, whereas the Pro-Ca-treated trees displayed the lowest TA values.

Table 4. Chemical attributes of cv. ‘TipTop’ sweet cherries affected by Pro-Ca (Prohexadione Calcium) treatment compared to untreated control (Ctrl) grafted onto the rootstocks ‘Gisela 6’ (G6) and ‘Maxma 14’ (M14). Results are the mean (N = 9) ± SE of TSS (Total Soluble Solids), pH, TA (Titratable Acidity), TPC (Total Phenol Content), and AOx (antioxidant capacity). Groups sharing the same letters are not statistically different, by a one-way ANOVA. Significance level: p < 0.01 (**), p < 0.001 (***), p ≥ 0.05 (ns, not significant).

Treatment	TSS				pH			TA				TPC				AOx
	(°Brix)							(meqNaOH/l)				(mgGAE/100 g f.w.)				(mmolFe2+/kg f.w.)
G6 Ctrl	18.5	±	0.3	c	4.53	±	0.12	106.0	±	1.8	a	99.18	±	1.40	b	8.09	±	0.09	b
G6 Pro-Ca	20.8	±	0.1	a	4.39	±	0.15	98.4	±	1.3	b	137.29	±	1.61	a	12.14	±	0.40	a
M14 Ctrl	20.1	±	0.1	b	4.51	±	0.23	104.1	±	1.2	a	97.73	±	1.46	b	8.63	±	0.16	b
M14 Pro-Ca	20.8	±	0.2	a	4.38	±	0.19	96.8	±	2.4	b	135.64	±	1.83	a	11.83	±	0.27	a
LSD (p ≤ 0.05)	***				ns			**				***				***

Regarding the cherries’ nutraceutical composition, Pro-Ca treatment significantly enhanced both TPC and AOx, with G6 Pro-Ca and M14 Pro-Ca showing the statistically highest concentrations. In contrast, the G6 Ctrl and M14 Ctrl recorded the lowest values, without significant differences between those treatments.

4. Discussion

The application of Pro-Ca resulted in an increased fruit set, reduced vigor, and consequently higher plant yield. This effect can be attributed to the growth-retarding action of REGALIS^®, which inhibits the biosynthesis of biologically active gibberellins (GA₁ and GA₄), decreasing vegetative growth [40]. The reduction in shoot elongation may have promoted a better balance between the vegetative and reproductive phases, enhancing fruit set and overall yield [41]. These results are consistent with those reported by Lal et al. [42], who observed decreased vigor and increased productivity in several fruit crops after Pro-Ca application, as a consequence of an improved allocation of assimilates and metabolic energy toward developing fruits [43].

GA reduction may also have altered the hormonal balance within the abscission zone, modifying the IAA/GA ratio, and potentially lowering sensitivity to ethylene [44,45]. This could have contributed to a lower post-bloom fruit drop, as Pro-Ca may reduce the activity of enzymes involved in ethylene biosynthesis [46]. Although some studies suggest that Pro-Ca may inhibit ethylene biosynthesis in addition to GA₁ [47], no direct measurements of ACC oxidase activity have been reported in fruit crops so far. Therefore, the effect of Pro-Ca on ethylene production remains hypothetical; thus, further studies are needed to clarify how Pro-Ca regulates hormonal balance within the tree, including analysis of ACC oxidase activity following Pro-Ca treatments. In addition, the reduced vigor may have influenced the flow of nutrients and carbohydrates [48,49], which also affects abscission competence [50]. Since GA induces vegetative growth at the expense of reproductive sinks [51], reducing its levels may have redirected assimilates toward developing fruits, as demonstrated in strawberries, sweet cherries, and apples [24,28,31], thereby increasing fruit retention.

These effects were particularly evident in trees grafted on G6 rootstock; however, in plants grafted on M14, Pro-Ca did not significantly affect setting or yield. A temporary reduction in vigor was observed two and three months after treatment, but no significant differences were detected by the end of the growing season. In Ctrl trees, no significant yield differences were observed between the two rootstocks, consistent with findings that reported how G6 and M14 mainly differ in vigor rather than reproductive performance [52].

The decreased response to Pro-Ca in M14 may be due to its inherently higher vigor, which could have prevented a sufficient alteration of the IAA/GA ratio within the abscission zone [53]. Moreover, M14 may modulate the plant hormonal balance differently, maintaining a distinct IAA/GA/ABA ratio or producing higher levels of growth-promoting hormones [54,55]. As a result, although Pro-Ca reduces the pool of active GA, this modification might not be enough to significantly affect fruit set and vegetative growth in M14 grafting conditions.

A different pattern was observed for the unmarketable yield. A higher proportion of unmarketable fruits was recorded in trees grafted onto M14 rootstock, particularly those treated with Pro-Ca. This effect may be related to physiological changes induced by Pro-Ca, possibly affecting fruit firmness and elasticity, which in turn can influence susceptibility to cracking under certain environmental conditions. Several studies [56,57] have shown that rootstocks modulate fruit weight, firmness, and cuticle and epidermal strength, and these traits are often correlated with cracking incidence and other visual defects [58]. For example, comparative trials on Gisela, MaxMa, and other rootstocks reported significant differences in fruit firmness and cracking susceptibility. Thus, the greater proportion of unmarketable fruits on M14 observed, especially following Pro-Ca application, could result from a combination of rootstock and treatment effects on fruit development. Future work should quantify defect types separately (cracking vs. undersize vs. deformation) to test this hypothesis directly. However, this trend does not fully agree with the mechanical properties measured, since cuticle thickness values did not reflect the higher proportion of unmarketable fruits. Nevertheless, cuticle thickness mainly describes the structural aspect of the fruit skin rather than its elasticity. It is therefore possible that Pro-Ca treatment on M14 rootstock may have negatively affected the epidermal elasticity, increasing the susceptibility to cracking despite the apparently thicker skin. Previous studies have shown that fruit cracking is more closely related to skin elasticity and cuticle microcracks than to cuticle thickness itself [59].

The reduction in vegetative sinks during the fruit growth phase may have also contributed to the higher proportion of fruits with Ø ≥ 30 mm observed in G6 Pro-Ca and M14 Pro-Ca. This effect could have allowed a more efficient distribution of assimilates among developing fruits [60], promoting their enlargement and improving market value, as cherry prices are largely determined by fruit size [61]. Moreover, several studies have reported that Pro-Ca enhances photosynthetic efficiency and delays leaf senescence [29,60]. This may have compensated for the increased fruit load, ensuring sufficient assimilate supply during fruit development. Finally, the reduction in biologically active GA, involved in fruit enlargement [51], might have induced a relative increase in IAA or cytokinin activity within the fruit, stimulating cell division and expansion, and contributing to larger fruit size [17].

Pro-Ca treatments also affected mechanical properties, color development, and chemical characteristics. The observed differences in texture suggest that Pro-Ca mainly affected the skin (PT) rather than the flesh (CT) [35]. These findings may indicate that the higher penetration force measured in G6 Pro-Ca and M14 Pro-Ca reflects a thicker or denser skin, which may also confer improved resistance to mechanical damage [59,62]. Similar findings were reported in numerous studies [21,23,31] where an increase in overall fruit firmness was observed following Pro-Ca application, while flesh firmness often remained unchanged. The enhanced skin strength could be associated with modifications in the epidermal layers, possibly related to improved light penetration and carbohydrate distribution under reduced vegetative vigor. Indeed, Cares et al. [31] reported that Pro-Ca significantly reduced leaf area and shoot length, improving canopy light distribution, known to influence cuticle development and epidermal rigidity [10]. Conversely, Ağlar et al. [32] found no significant effects of Pro-Ca on flesh firmness, supporting the hypothesis that most textural changes occur at the fruit surface rather than the flesh. However, further investigation into the impact of reduced vegetative vigor on assimilate partitioning is needed to better explain the mechanisms behind improved fruit quality and size.

The same factors may also have influenced fruit coloration, sugar accumulation, and nutraceutical properties. Increased canopy light penetration promoted the development of a red overcolor and a more orange-toned ground color [63], slightly reducing fruit brightness in G6 Pro-Ca and M14 Pro-Ca. This condition also favored greater sugar accumulation, as reflected by the higher TSS/TA ratio in treated fruits, while control fruits remained more acidic. Several studies [64,65,66,67] report positive correlations between red skin overcolor and light exposure. Moreover, Berli et al. [68] showed that ABA, together with enhanced solar ultraviolets, not only increased TPC and skin pigmentation but also enhanced sugar content. Sunlight induces the expression of key genes involved in phenolic and anthocyanin biosynthesis, regulated by ABA signaling [7,8,69]. Therefore, the reduced canopy density and consequent improved light exposure of Pro-Ca-treated trees enhanced ABA accumulation and signaling, promoting anthocyanin synthesis and resulting in the more intensely colored, polyphenol-rich, and antioxidant cherries observed in G6 Pro-Ca and M14 Pro-Ca treatments.

5. Conclusions

The present study evaluated the effects of Pro-Ca application on vigorous sweet cherry trees cv. ‘TipTop’ during the early reproductive phase. Results indicate that Pro-Ca modulated the vegetative/reproductive balance, leading to higher yield, larger and firmer fruits, and improved color, taste, and antioxidant capacity. These outcomes likely resulted from a combination of hormonal regulation and more efficient assimilate allocation. In conclusion, Pro-Ca has been demonstrated to be a promising PGR for high-density sweet cherry orchards, not only for its efficient vigor control, but also for its potential to enhance fruit quality, ultimately leading to improved orchard profitability.