Gibberellic Acid and Zeatin Delay “Harton” Plantain (Musa paradisiaca) Ripening

Abstract

Plantain (Musa paradisiaca) is a climacteric fruit with high endogenous ethylene production, which accelerates ripening and limits shelf life, especially during transport and exportation, leading to significant losses for producers and distributors. This study evaluated the effect of gibberellic acid (GA₃) and zeatin (Zea) on delaying the ripening of Hartón plantains grown in Colombia. The goal was to assess whether these plant regulators could delay physicochemical changes under simulated cold chain conditions. A completely randomized design was used with nine treatments, plus a control, each with five replicates. Fruits were stored at 11 ± 2 °C and 75% relative humidity for 25 days. Pulp firmness, soluble solids, titratable acidity, pH, starch, chlorophyll, carotenoids, total polyphenols, and polyphenol oxidase activity were assessed. The combination of GA₃ + Zea was effective in preserving firmness, maintaining starch and chlorophyll content, and limiting increases in soluble solids and polyphenol oxidase activity associated with senescence. This delayed ripening did not affect structural integrity or caused oxidative stress. Combined application of GA₃ and Zea is a low-cost and effective strategy to extend the shelf life of plantains for export, benefiting the tropical agri-food chain. This approach offers a practical alternative for maintaining fruit quality without the need for costly preservation technologies.

1. Introduction

The plantain (Musa paradisiaca AAB, subgrup Plantain cv. ‘Hartón’) is an essential staple food in the diet of millions of people, especially in tropical and subtropical areas. Its nutritional richness, energy content, and culinary versatility make it an essential product for both local consumption and export, being one of the most affordable and widely consumed fruits in the world, with more than 100 billion bananas consumed annually worldwide [1]. After wheat, rice, and corn, the banana is the fourth-most important food crop in the world. Bananas contain approximately 400 milligrams of potassium per 100 g of fresh fruit, and their consumption helps regulate blood pressure and reduce the risk of heart disease by up to 27% [2,3]. In addition to their high potassium content, bananas are also a source of vitamin C and vitamin B6. Especially in rural areas of developing countries, bananas can provide up to 25% of a family’s daily calorie intake. Many African countries, such as Uganda, Rwanda, and Cameroon, consume more than 200 kg of bananas per capita, while Filipinos consume around 60 kg of bananas per year.

Additionally, the constant emergence of diseases, along with the increasing size of the global population, creates a demand for new vaccines that can address challenges that the conventional ones have not been able to overcome. Banana is a cheap and reliable fruit to use as an orally delivered vaccine [4]. A lecitin-like protein was identified by Covés-Datson et al. [5] exhibiting expansive activity against all influenza strains. The application of transgenic plants in the production of pharmaceuticals has led to a new approach: plant-based, orally delivered vaccines. Horie et al. [6] studied the effects of Banafine^® administration on influenza vaccine antibody titer in elderly patients (average age ∼80 years) receiving gastrostomy tube feeding with higher success. An edible vaccine against Hepatitis-B was developed by Maji using transgenic bananas.

In Latin American countries, the terms “banana” and “plantain” are often used interchangeably. Still, they refer to different fruits, although they belong to the same genus (Musa) but distinct species, such as Musa acuminata (banana) and Musa paradisiaca (plantain) [7,8]. The main difference between the two fruits is that bananas are usually eaten raw as fresh fruit. It is sweet, has a soft texture, and is considered ideal for snacks, desserts, and smoothies. In contrast, the plantain (also known as “plátano macho” in some countries) has a less sweet flavor and a firmer texture, thus being typically eaten cooked, either fried, boiled, or roasted. It is a staple ingredient in many Latin American cuisines, especially in savory dishes. In terms of starch, bananas have a higher content of natural sugars, making them sweeter to the palate. Plantains, on the other hand, are richer in starch and less sweet when green, although they become somewhat sweeter when ripe. This difference influences both their flavor and their culinary uses. Finally, the banana is generally smaller and curved, with thinner, easier-to-peel skin. The plantain is usually larger, straighter, and has a thicker skin, requiring more effort to peel, especially when green [9].

Regarding 2023 world banana production report [10], Uganda is the largest world producer (10,440,849 tons), followed by Congo (4,887,511 tons), Ghana (4,819,199 tons), Cameroon (4,660,387 tons), Philippines (3,113,584 tons), Colombia (2,478,699 tons), Côte d’Ivoire (2,113,309 tons), Sri Lanka (1,529,919 tons), Myanmar (1,281,598 tons), the Dominican Republic (1,151,333 tons), and Rwanda (903,786 tons) complete the 11 largest banana producers in the world. In this sense, Colombia, despite ranking sixth worldwide, is the leader in banana production among all Latin American countries [10]. In 2023, Colombia produced approximately 4,844,783 tons of plantain, harvested from a planted area of 486,876 hectares, with a yield of 10.6 tons per hectare [11]. In that same year, Colombia exported 87,851 tons of plantain and imported approximately 13,475 tons [11]. From 2019 to 2023, the total area planted with plantain increased by approximately 9%, from 447,913 hectares in 2019 to 486,876 hectares in 2023. Consequently, total production increased by 18.3%—from 4,094,459 tons to 4,844,783 tons. The plantain production chain is strategically crucial for countries such as Colombia, Ecuador, Peru, and other Central American countries, where it represents a significant source of rural income and employment. Despite technical developments in production, post-harvest handling remains a vulnerable link.

This research arises from the problem of the accelerated ripening process of Hartón plantains during cold storage and transportation, which prevents them from reaching export destinations in optimal condition. This phenomenon is linked to the intensive production of ethylene [12]. Ethylene production in plantains increases significantly with temperature, from 0.1–2 µL kg⁻¹ h⁻¹ at 13 °C to 0.3–10 µL kg⁻¹ h⁻¹ at 20 °C [13]. Furthermore, the respiration rate also increases with temperature, which can further accelerate ripening if the temperature is not adequately controlled [14]. To mitigate these effects, it is essential to control storage and transportation conditions, such as temperature and ambient ethylene concentration. Therefore, post-harvest handling strategies should consider techniques that attenuate or delay these processes, including storage under specific conditions, the use of modified atmospheres, and the application of ethylene inhibitors [15]. Post-harvest handling therefore becomes a critical link, especially when considering the high loss rates recorded due to premature ripening in the absence of adequate control [16].

In general, cytokinins and gibberellins are involved in the primary regulation of ripening in climacteric fruits, such as bananas and plantains, and perform antagonistic or modulatory functions concerning ethylene, the primary regulator of ripening in these fruits. Cytokinins act mainly by delaying fruit ripening, inhibiting the synthesis and/or action of ethylene [17]. The primary mechanism involves the transcriptional regulation of genes for key enzymes in ethylene biosynthesis, such as ACC synthase (ACS) and ACC oxidase (ACO), as well as the reduction of 1-aminocyclopropane-1-carboxylic acid (ACC) production, an immediate precursor of ethylene [18]. Cytokinins also interfere with ethylene transduction pathways, thereby reducing the expression of ethylene-responsive genes, including EIN3 and ERFs (ethylene response factors). Consequently, even when ethylene is present, its signaling is reduced.

Furthermore, cytokinins can induce antioxidant enzymes, which protect cells from the oxidative stress associated with maturation [19]. Gibberellins, especially gibberellic acid (GA₃), have a cytokinin-like effect, inhibiting or delaying the action of ethylene. The main mechanisms include reducing the expression of ACS and ACO. Gibberellins also interfere with tissue sensitivity to ethylene, delaying the physiological processes it triggers, such as softening and color change. However, gibberellins promote cell elongation and the maintenance of juvenile metabolic activity (“juvenile effect”), thereby delaying the signs of ethylene-induced senescence [20]. Therefore, both cytokinins and gibberellins can be used as biotechnological or agronomic tools to extend the post-harvest life of bananas/plantains [17,21,22].

This study hypothesizes that the combined application of gibberellic acid (GA₃) and zeatin (Zea) extends the post-harvest shelf life of plantains by delaying key physiological ripening markers, such as pigment degradation, starch conversion, and enzymatic browning, through the regulation of plant growth regulators on ethylene-induced processes at the tissue level. Our primary goal was to evaluate the effects of gibberellin and zeatin on delaying plantain fruit ripening and assessing the physicochemical components of plantain fruit quality.

2. Materials and Methods

2.1. Experimental Design

Fruit samples were collected from the Edith Villace farm, Turbo city, Antioquia, Colombia (8°4′12″ N, 76°39′47″ W; 12 m.a.s.l.). The region is characterized by an Af climate (tropical rainforest climate), with air temperatures ranging from 26 °C to 32 °C and an average annual rainfall of 2427 mm. A 20-foot container, with dimensions of 6.1 m × 2.4 m × 2.6 m (length, width, height), 38 m³ simulated refrigerated container within intermodal transport refrigerated intermodal container with temperature calibrated to 8.8 ± 2.5 °C in accordance with Lukasse et al. [23] was provided by Uniban Corporation, Medellin, Antioquia, Colombia, where the fruit chamber was individually prepared. The air humidity and temperature inside the container were measured every 5 min using an automatic temperature and air humidity datalogger (Akrom Eclectronic Devices, São Leopoldo, RS, Brazil) during the storage of the fruits.

The harvest and post-harvest of the plantain bunches were carried out according to the quality protocols established by the regional marketers (length, thickness, and physical appearance of the fruit). For each treatment, four bunches were selected from the plants located in the usable plot, on which the measurements planned for the study were performed.

To evaluate the influence of gibberellin and cytokinin on fruit ripening delay, 10 treatments were designed (

Table 1. Evaluated plant growth regulator (PGR) treatments in the ripening control of “Hartón” plantains.

Control	0	T0
Gibberellic acid (GA3; μM)	250	T1
	275	T2
	300	T3
Zeatin (Zea; μM)	390	T4
	430	T5
	480	T6
GA3 (250 μM) + Zea (390 μM)		T7
GA3 (275 μM) + Zea (430 μM)		T8
GA3 (300 μM) + Zea (480 μM)		T9

), composed of three gibberellin (GA₃) or zeatin (Zea) concentrations, combined or not and five storage times (0, 10, 15, 20, and 25 days after harvest); all with five biological repetitions, each experimental unit consisted of 20 plantains, making a total of 50 experimental units.

All Hartón plantain fruits were uniform in size and primary ripeness, with a uniform harvesting time for all experimental units. For each treatment, a 30 cm³ PGRs solution, supplemented with surfactant (Surfare, Germinare S.A. de C.V., San Nicolás de los Garza, Nuevo León, Mexico), was applied to the fruit via nebulization to facilitate adherence. In the control treatment, only distilled water and surfactant were applied. After that, the remaining solution on the fruits was dried with absorbent paper, and then all the fruits were returned to their shipping boxes.

2.2. Chlorophyll and Carotenoids in the Epicarp (Fruit Peel)

In each evaluation period, both chlorophyll and carotenoids were measured. For the extraction of pigments from the fruit peel, five fragments ~1 cm² were used. These peel fragments were collected at equivalent distances to peduncle, center, and apex, corresponding to 25, 50, and 75% of the fruit length, external curvature, and peduncle and center, internal curvature as recommended by Velásquez et al. [24]. The peel fragments were homogenized with 2 mL of cold methanol [25]. The samples were centrifuged at 15,000× g at 4 °C, and the upper portion was used to measure the absorbance at wavelengths of 470 nm, 652 nm, and 665 nm. A fourth wavelength of 720 nm was used to exclude interferences, serving as a blank test as proposed by Wellburn [26].

2.3. Fruit Peel Color

For all plantain samples, the chromatic parameters a* (chromaticity on the green (-) to red (+) axis), b* (chromaticity on the blue (-) to yellow (+) axis) (chroma), L* (luminosity or clarity), saturation (C) and tone or tint (H) will be determined in a Color Flex EZ45 colorimeter (ASTM E308 and a standard illuminant D65—HunterLab^® brand, Reston, VA, USA), with an observation angle of 90°. After reading, the colorimeter was calibrated using a standard black ceramic plate (X = 13.80, Y = 19.23, Z = 13.88) and a standard white plate (X = 80.09, Y = 84.99, Z = 87.67). In each experimental unit, five plantains were sampled. L, a, and b values were taken from 5 portions of each fruit: next to the peduncle, the portion furthest from the peduncle, and three measurements from the intermediate portion of the fruit. In colorimetry studies applied to fruits, converting L*, a*, and b* coordinates to the hue angle (h°) is a standard procedure that allows for a more objective quantification of color tone during the ripening process. The hue angle is calculated using the inverse-ratio arctangent trigonometric function adjusted for quadrants, according to Scalisi et al. [27]:

$$h^o=atan2\left(b^{*}, a^{*}\right) \times {\displaystyle \frac{180}{\pi }}$$

This method considers the sign of both variables and returns the angle in radians. This result must be converted to degrees and, if negative, adjusted to the range of 0° to 360° using MOD [27]. The hue angle allows for the interpretation of color: values close to 0° indicate red, 90° indicate yellow, 180° indicate green, and 270° indicate blue. During fruit ripening, it is common to observe a transition in h° from higher values (green) to lower values (red-yellow), making this index a valuable tool for objectively assessing color evolution (Figure 1).

2.4. The Total Soluble Solids (°Brix), pH, and Titrable Acidity

For total soluble solids, fruit juice was extracted at each of five stages of ripeness, and values were measured using a digital refractometer (Pal-1 Atago refractometer, Ribeirão Preto, SP, Brazil). The pH was evaluated on a digital pH meter (SevenExcellence pH meter S400-Std, Mettler Toledo, Barueri, SP, Brazil. Titratable acidity was determined according to the AOAC method 942.15 [28]. One gram of plantain pulp was homogenized with 5 mL of distilled water and filtered to remove solid particles. Then, 1 mL of the extract was transferred to an Erlenmeyer flask, where a few drops of the phenolphthalein indicator were added. The solution was titrated with 0.1 M sodium hydroxide (NaOH) until the endpoint was reached, indicated by the appearance of a persistent pink color for at least 30 s. The consumed volume of the base was used to calculate the acidity, expressed as a percentage of equivalent citric acid, using the appropriate conversion factor, as described in detail by Benavides Arévalo et al. [29].

2.5. The Fruit Firmness

Fruit firmness was assessed on fresh plantains in all ripening stages using a portable penetrometer (Fruit Hardness Tester, mod. PTR-300, Instruterm, São Paulo, SP, Brazil) with a 2 mm pressure point. The lever was gently lowered over the trapped fruits so that the pressure point hit each sample at a distance corresponding to the stem, center, and apex, equivalent to 25, 50, and 75% of the external curvature length, obtaining a final average as suggested by Velásquez et al. [24]. This average represents the peel penetration capacity, which increases as the fruit ripens. The pressure at which the penetrometer was used to break the peel was measured in kg cm⁻² using a methodology similar to that described for seeds [30].

2.6. Starch and Soluble Sugars

To analyze the starch, a portion of 500 mg of plantain pulp was processed according to AOC 996.11 [31] in the presence of 50 mM MOPS buffer (pH 7.5), 5 mM calcium chloride, and 0.02% sodium azide. The enzymes α-amylase (3000 U mL⁻¹) and amyloglucosidase (200 U mL⁻¹) were used to hydrolyze starch to glucose. One unit (U) of α-amylase activity or amyloglucosidase is the amount of enzyme required to release 1 μmole of p-nitrophenol. The final product (glucose) was measured spectrophotometrically at 510 nm.

To analyze the soluble sugars, 500 mg of plantain pulp was carefully ground in liquid nitrogen and stored at −20 °C until use. For the extraction of soluble sugars, samples were solubilized in 50% (v/v) ethanol for 90 min. The samples were centrifuged at 15,000× g at 4 °C. The supernatant was used to measure the soluble sugars as described in detail in Lozano-Isla et al. [32].

2.7. Total Polyphenols and Polyphenol Oxidase Activity

For total polyphenols measurements, the Folin–Ciocalteu method was used [33]. The samples were prepared by homogenizing 10 g of plantain pulp in 100 mL of 80% cold methanol for 24 h at 4 °C. The extract was then filtered, and a 1 mL aliquot was taken, which was mixed with 5 mL of the Folin–Ciocalteu reagent diluted 1:10 with distilled water and 4 mL of 7.5% sodium carbonate. The mixture was incubated for 30 min at room temperature, and the absorbance was measured at 765 nm using a UV-Vis spectrophotometer. The results will be expressed as milligrams of gallic acid equivalents per 100 g of sample (mg GAE 100 g⁻¹ fruit pulp), as previously reported in Musa spp. [34].

To measure the activity of polyphenol oxidase (PPO), five grams of fruit pulp were homogenized in 60 mL of 0.1 M sodium phosphate buffer and 0.1 M potassium phosphate, pH 7.0, with 3% Tween 80. After filtering the homogenate through a cotton cloth, the filtrate was centrifuged at 10,000× g at 4 °C for 20 min, and the supernatant was used as the primary PPO solution. For the analysis of PPO, the solution was diluted 10 to 250 times with 0.01 M sodium phosphate. The activity of PPO was determined using the colorimetric method as described by Fujita et al. [35]. One unit (U) of PPO activity was defined as the amount of enzyme required to metabolize catechol, producing ortho-benzoquinone (molar extinction coefficient − ε of 3.6 × 10³ M⁻¹·cm⁻¹) measured in one minute at 420 nm (1 cm of light path) [36].

2.8. Statistical Analysis

All experiments were arranged in a completely randomized design with five repetitions. All data were analyzed using R statistical software, version 4.4.2, with an analysis of variance (ANOVA) to assess differences between treatments. Means were compared using the Student–Newman–Keuls test at a significance level of p < 0.05. All graphs were generated with SigmaPlot for Windows v. 11.0 (Systat Software, Inc., San Jose, CA, USA).

Figure 2 illustrates a diagram summarizing all the procedures used in this study.

3. Results and Discussion

3.1. Climatological Measurements Inside the Container

All climatic measurements, i.e., temperature and relative humidity in the microenvironment inside the refrigerated container were sampled from 11 March 2024 to 4 April (Figure 3). The graph displays data corresponding to the calibrated temperature, the actual temperature supplied by the system, and relative humidity values, which allow for an evaluation of the stability of environmental control and the accuracy of the sensors used in the measurement.

The calibrated temperature, represented by the calibrated temperature curve, remained constant throughout the entire observation period. In contrast, the temperature exhibited slight fluctuations around the reference value, which is expected in active thermal regulation systems and indicates the adequate performance of the air conditioning mechanism. Since the temperature measured by the sensors presented a slight discrepancy as compared to the supplied temperature, it was necessary to calculate the root mean square error (RMSE), which reached a value of 0.901 °C, while the mean absolute error (MAE) was 0.517 °C. These results reflect an average deviation of less than 1 °C between the measured and calibrated temperatures, indicating high accuracy of the measurement system. The low magnitude of these indicators confirms the reliability of the thermal sensors used, considering the inherent variations in heat exchange processes and the instruments’ sensitivity.

Furthermore, it is essential to highlight that this experiment was conducted in a microenvironment where the container was opened for sampling at each evaluation (every 5 days). This situation does not represent the real-life exportation scenario, in which the container remains completely sealed from departure until arrival at the destination. These periodic openings introduced unwanted thermal changes, disrupting the internal equilibrium and, most likely, artificially increasing measurement errors. Despite these unfavorable conditions, the system managed to maintain a temperature difference of less than 1 °C, which reinforces its ability to maintain a stable temperature inside the container. In the context of plantain transport—a fruit susceptible to thermal variations—this ability to maintain temperatures close to the set point even under human intervention suggests that, under real-life export conditions, product quality would not be compromised.

This scenario is highly relevant in the context of fruit and vegetable transportation, where thermal stability is crucial for maintaining quality and extending product shelf life. Guo et al. [37] showed that even the implementation of advanced cold storage systems can compromise temperature control accuracy. In their study, the integration of LSTM neural networks achieved an RMSE of less than 0.105 °C, highlighting the need for precision in these systems to meet international standards. Considering that a RMSE of 0.105 °C was achieved only with the use of neural networks, our RMSE of 0.901 °C is excellent.

The relative humidity recorded during the experiment ranged from 80% to 96%, which is consistent with the recommended ranges for sensitive products, such as tropical fruits. Kader [38] stated that a relative humidity ranging from 90% to 95% is ideal for minimizing water loss, preventing wilting, and reducing the risk of accelerated decomposition, especially in export products such as bananas. Recently, Zuo et al. [39] confirmed that maintaining high humidity levels during cold storage improves resistance to physiological damage and keeps the visual and nutritional quality of fruits and vegetables. The storage of ‘Niagara Rosada’ table grapes demonstrated that a humidity range of 90–95% not only minimized mass loss but also the occurrence of post-harvest diseases [40]. This type of evidence supports the reliability of the monitoring system described in the present study, thereby reinforcing its applicability in commercial contexts, where environmental control is crucial and margins of error are minimal.

3.2. Chlorophyll and Carotenoids in the Fruit Peel

This section examines the effects of GA₃, zeatin, and their combinations on pigment changes during plantain ripening. Plantain fruits treated with isolated GA₃ have a lesser impact on the degradation of chlorophyll than fruits treated with isolated Zea. However, combined treatments showed an intermediate chlorophyll degradation, especially at higher concentrations.

The chlorophyll “a” levels decrease across all treatments, reflecting the degradation process of this pigment during plantain ripening (Figure 4). Initially, the chlorophyll levels were uniform across all treatments. However, over the past 25 days, a marked reduction was observed, particularly in treatments with gibberellic acid (GA₃). Treatment with 250 μM GA₃ showed a 75% decrease of chlorophyll “a”, while 300 μM GA₃ decreased this variable by ~80%, indicating an accelerated ripening effect. The evolution of chlorophyll “a” in the control treatment was one of the least effective at preserving the pigment. From 0 to 25 days after harvest (DAH), the chlorophyll “a” decreased sharply, nearly 90%, reflecting accelerated ripening in the PGR-free treatment. Similarly, treatments with zeatin applied alone, particularly at concentrations of 390 μM and 430 μM, failed to delay the chlorophyll degradation. In these cases, chlorophyll “a” decreased 70% to 80%, demonstrating that zeatin alone was not sufficient to maintain color stability and was not efficient in delaying plantain ripening. These results suggest that neither the application of zeatin alone nor the complete absence of treatment can inhibit the catabolic chlorophyll mechanisms during fruit ripening. In contrast, the combined treatments of GA₃ + Zeatin (as 250 μM GA₃ plus 390 μM Zea) maintained higher levels of chlorophyll “a” up to day 15, with a more moderate decrease of approximately 55% by day 25. The most effective treatment in preserving chlorophyll “a” in plantain fruit was GA₃ without combination with zeatin; particularly 250 μM GA₃, which showed a chlorophyll “a” reduction of about 35.6%, in contrast to PGR-free, which showed a 56.2% reduction after 25 DAH. Other treatments, also effective, included 275 μM GA₃ (−41.5%) and 300 μM GA₃ (−34%). These results indicate that zeatin, either alone (mean reduction of 36.2%) or in combination with GA₃ (mean decrease of 52.8%), does not have an essential role in protecting the integrity of chlorophyll “a” against the catabolic processes induced by plantain ripening.

Regarding chlorophyll “b”, a similar trend to chlorophyll “a” was observed, although with an even more pronounced degradation rate. Fruits treated with GA₃ at 250, 275, and 300 μM showed a decrease of 29.3%, 48.3%, and 48.4%, respectively. In contrast, the PGR-free showed an 83.5% decrease in chlorophyll “b” after 25 DAH (Figure 4B). These data are consistent with an intensified ripening effect. Treatments with zeatin, regardless of concentration, resulted in a gradual reduction of approximately 75–80% with similar results from the control, indicating no effective role in delaying the degradation of chlorophyll “b”. However, the combinations of GA₃ and Zea showed more effectiveness in preserving the chlorophyll “b” than isolated Zea were 275 μM GA₃ plus 430 μM Zea, and 300 μM GA₃ plus 480 μM Zea showed a reduction of 48.3% and 51.5%, respectively, in chlorophyll “b” after 25 days of ripening (Figure 4B).

The results of the present study are consistent with previous research highlighting the effectiveness of GA₃ on climacteric fruits. For example, Osman and Abu-Goukh [41] demonstrated that immersing bananas in a 100 ppm GA₃ solution, together with polyethylene films, delayed ripening and maintained fruit quality during storage. Huang and He [17] described that the exogenous application of cytokinins, such as forchlorfenuron (CPPU), regulated cytokinin oxidase activity and antioxidant activity, which contributes to maintaining chlorophyll pigment during banana fruit ripening. On the other hand, treatments with zeatin showed a more gradual reduction in chlorophyll “b”, with losses ranging from 50% to 60%, suggesting a retarding effect on pigment degradation. Huang and He [17] demonstrated that the exogenous application of cytokinins regulates cytokinin oxidase activity and antioxidant activity, thereby maintaining chlorophyll levels during banana ripening. Li et al. [42] reported that GA₃ retarded the senescence of shoots, and the degradation of proteins and Chl by reducing the chlorophyllase and Mg-dechelation activity, key enzymes in chlorophyll catabolism.

Total carotenoids showed a general upward trend during ripening. Treatments with GA₃ at any concentration stimulated a sharp increase in carotenoids, with estimated increases of around 20–43% after 25 DAH. Zeatin alone showed a moderate increase, ranging from 54% to 70%, suggesting that isolated Zea has a role in carotenoid synthesis. However, Zeatin (at 430–480 μM) combined with GA₃ (275–300 μM) promoted the lowest carotenoid increase, such as 29.6% and 30.5%, respectively (Figure 4C). It is important to note that no-PCR promotes an increase in carotenoids by 2.33-fold. These results showed that low GA₃ concentration or its combination with higher Zea (275 μM GA₃ plus 430 μM Zea or 300 μM GA₃ plus 480 μM Zea), suggesting a negative modulation of carotenoid synthesis when both regulators are present. These data were consistent with those presented in Figure 5, since these fruits remained greener than the previous ones.

These results indicate that carotenoids serve as positive markers of the ripening process, especially when stimulated by GA₃, but the presence of zeatin can regulate their intensity. These results are consistent with those of Lima et al. [43], which described that the application of cytokinins and gibberellins did not significantly affect chlorophyll degradation or carotenoid synthesis in banana fruit peel. However, Aremu et al. [22] showed that the combination of 200 mg L⁻¹ GA₃ + 200 mg·L⁻¹ kinetin had a minor inhibitory effect on overall fruit maturation, though not specifically on peel pigmentation or biochemical ripening markers. Notwithstanding, Ghimire et al. [21] showed that GA₃ and kinetin played a key role in delaying the maturation of banana fruits by inhibiting chlorophyll degradation and reducing carotenoid accumulation, thereby retarding peel color change and the progression of ripening. According to Ghimire et al. [21], GA₃ at 300 ppm showed the most pronounced inhibitory effect on banana ripening, resulting in the lowest peel color rating.

3.3. Fruit Peel Color Measurement

This study utilizes the hue angle as an objective color metric to track pigment changes in plantain peel during ripening. As chlorophyll declines and carotenoids rise, Hue values drop—shifting from green to yellow. Figure 5 shows that combined GA₃ and zeatin treatments slow this shift, while untreated or low-zeatin fruits ripen faster, confirming Hue angle as a reliable ripeness indicator. In this figure, Hue angles [44] are used as an objective chromatic indicator of peel color changes. Hue value is particularly valuable in fruit ripening studies, since it allows the visual perception of color—associated with relative pigment content—to be quantifiably transformed into a measurable physical parameter. Thus, as chlorophyll degrades (a trend observed in Figure 4) and carotenoids accumulate, the green color fades, giving way to yellow and orange hues, as reflected in the progressive shift in Hue value (Figure 5). This approach not only provides visual validation of the previous pigment data but also allows the differential effects of each treatment to be clearly distinguished. For example, treatments such as GA₃ in combination with zeatin (300 μm GA₃ plus 480 μm Zea) preserved chlorophylls and moderately promoted carotenoids—showed higher Hue values, consistent with slower ripening.

In contrast, less effective treatments, such as PGR-free, isolated GA₃, or Zea, exhibited rapid chlorophyll loss and greater carotenoid accumulation, showing significantly lower Hue values, which reflected a greater yellow color intensity. The data in Figure 5 reinforces the transformation of peel colors during ripening in an integrated manner, making color measurement a robust and essential tool for studying fruit ripeness. This pattern has been corroborated by other studies [45] that observed a significant decrease in Hue values during storage of 1-MCP-treated bananas, indicating slower ripening compared to untreated fruits. Likewise, Ringer and Blanke [46] developed a banana ripening index based on non-invasive measurements of color and chlorophyll, demonstrating that Hue values consistently decrease from approximately 98° at early stages to 82° at advanced stages of ripening. These findings support the usefulness of Hue degrees as a robust tool for monitoring banana fruit ripeness.

3.4. pH, Total Soluble Solids (°Brix), and Titrable Acidity

The assessment of physicochemical parameters such as pH, soluble solids (°Brix), and titratable acidity is essential for monitoring ripening in climacteric fruits like plantain. These indicators reflect key metabolic changes, including starch breakdown, sugar accumulation, and acid neutralization. They are widely used to evaluate the effectiveness of plant growth regulator (PGR) treatments in managing fruit maturation.

The pH assessment in plantain fruits subjected to different PGR treatments reveals clear patterns that correlate with the fruit’s ripening stage (Figure 6A). The pH showed a tendency to increase as the fruit ripens, due to the progressive neutralization of organic acids by endogenous metabolic processes [47], recently confirmed by Batista-Silva et al. [48]. In this context, the PGR-free treatment showed a higher pH value, reaching significantly high levels, which reflects advanced ripening without hormonal intervention. Similar results were observed in response to zeatin, regardless of concentration (Figure 6), in which the pH also increased markedly, exceeding the values observed in several combined treatments. In contrast, treatments with GA₃, especially in combination with zeatin—such as 275 μM GA₃ plus 430 μM Zea and 300 μM GA₃ plus 480 μM Zea—showed the lowest pH values at the end of the testing period. The pH increase in these treatments was, respectively, 7% and 13.6% in contrast to a 36.1% increase in PGR-free, indicating that the combination of GA₃ and Zea was effective in reducing the ripening process. The highest increase in pH was verified in those treatments with isolated Zea, which showed a pH rise of 46.8% (Zea 390 μM), 49% (Zea 430 μM), and 53.4% (Zea 480 μM), indicating their inefficiency to retard plantain fruit ripeness.

These treatments delayed the neutralization of organic acids, maintaining an acid profile more characteristic of unripe fruits. Recent studies have shown that the application of GA₃ and cytokinins, such as zeatin, slows down the ripening process, maintaining lower pH levels over prolonged storage periods. For example, Ghimire et al. [21] described that treatment with GA₃ at 300 ppm resulted in a final pH of 4.8 in bananas stored at room temperature as compared to a higher pH in untreated fruits, indicating slower ripening and better preservation of the fruit’s natural acidity. Likewise, Zomo et al. [49] reported that the combination of 150 ppm GA₃ with storage at 15 °C in ‘Sabri’ bananas allowed for maintaining a lower pH and higher titratable acidity compared to fruits stored at higher temperatures or PGR-free, evidencing slower ripening. These findings support the strategy of using PGR in combination with reduced storage temperatures to preserve the quality and extend the shelf life of bananas destined for distant markets.

Analysis of soluble solids content (°Brix) provides an accurate assessment of fruit sweetness, which is directly associated with the starch hydrolysis process during ripening. As the fruit begins its ripening process, sugars are formed through starch hydrolysis, increasing the °Brix value. Figure 6B shows that the PGR-free treatment had the highest °Brix values (24 ± 0.3—an increase of 243%), indicating rapid starch conversion and uncontrolled ripening. Similarly, treatments with isolated GA₃ at 250 μM (+157%), 275 μM (+150%), and 300 μM (+212%) showed a similar pattern. In contrast, zeatin at 390 μM (+39%), 430 μM (+45%), and 480 μM (+67%) showed the lowest °Brix values, remaining close to each other and reflecting a partial blockage or delay in the enzymatic activity associated with starch degradation. The combination of GA₃ and Zea recorded the intermediate values (~132%) (Figure 6B).

The titratable acidity is another key parameter in evaluating ripening in climacteric fruits, such as plantains, as it reflects the concentration of organic acids responsible for the fruit’s flavor and stability. This phenomenon has been documented in studies such as Campuzano et al. [50], which reported a significant reduction in titratable acidity in Cavendish bananas as ripening progressed. Similarly, Giraldo et al. [51] observed a decrease in titratable acidity in Dominico-Hartón bananas during the ripening process, which was correlated with increased Brix levels and decreased pH. Additionally, Ghimire et al. [21] demonstrated that bananas treated with GA₃ at 300 ppm exhibited a lower final °Brix and higher acidity, indicating an effective ripening delay. According to Zomo et al. [49], ‘Sabri’ banana fruits stored at 15 °C combined with GA₃ preserved titratable acidity and avoided sudden increases in soluble sugars, thus prolonging the fruit’s shelf life. Batista-Silva et al. [48] investigated the molecular mechanisms involved, highlighting that the regulation of enzymes associated with the conversion of starch into sugars is closely linked to hormonal signals and environmental conditions, confirming that adequate modulation of these factors can successfully interfere with ripening. In our study, the lowest titratable acidity was verified in treatments with isolated GA₃ at 250 μM (+17%), 275 μM (+8%), and 300 μM (+19%). Those treatments showed a decrease in titratable acidity in about 25.7%, 26.5%, and 27.4% in comparison to PGR-free value (+69%). Together, these data reinforce the effectiveness of GA₃ in preserving not only acidity and firmness but also limiting sugar accumulation, a crucial aspect in the export logistics of climacteric fruits such as plantains as reported in the literature.

3.5. Fruit Firmness

Fruit firmness is a key physical indicator of ripening in plantains, reflecting the structural integrity of peel and pulp tissues. As ripening progresses, enzymatic activity—particularly from polygalacturonases and cellulases—leads to cell wall degradation, resulting in tissue softening [52]. Measuring the force required to penetrate the fruit provides a reliable, objective method to evaluate the effectiveness of plant growth regulator (PGR) treatments in delaying this softening process.

Plantain fruit firmness was measured by the force required to penetrate both the peel and the pulp (Figure 7). This physical parameter is a direct indicator of fruit ripeness. Green fruits have dense, compact tissues that are resistant to mechanical deformation. At the same time, the progression towards ripening involves the progressive softening of said tissues, associated with enzymatic degradation processes of the cell wall [53].

The PGR-free fruits showed the lowest penetration force (7.7 ± 0.8 kg cm⁻²; −86.3%) values in both peel and pulp. A similar trend was observed in treatments with isolated zeatin, especially at concentrations of 430 μM L⁻¹ (9.4 ± 0.5 kg cm⁻²) and 480 μM L⁻¹ (7.5 ± 0.2 kg cm⁻²) which also showed significant reductions of 81.9% and 85.5% in the pulp firmness, respectively, indicating that Zea application alone does not effectively preserve the structural integrity of the fruit. In contrast, the GA₃ at 250 μM (−60.8%), 275 μM (−36.8%), and 300 μM (−39.3%) exhibited the highest penetration forces at 25 DAH. These penetration forces are 2.8-, 4.8-, and 5.4-fold higher than PGR-free at 25 DAH.

In this process, polygalacturonases are activated, which hydrolyze the α-1,4-galacturon bonds in the pectin chain, leading to pectin solubilization and loss of cohesion between cells, and resulting in a softer fruit texture [54]. This process has been widely studied among various fruit species. For example, in strawberries, the suppression of the FaPG1 gene using gene silencing techniques has been shown to maintain fruit firmness, highlighting the central role of PGs in softening [55].

On the other hand, cellulases catalyze the degradation of cellulose, a structural polysaccharide of the cell wall, contributing to the loss of fruit firmness. In studies with tomatoes, it was shown that cellulase activity increases during fruit ripening, correlating with the decrease in fruit firmness [56]. In summary, peel and pulp firmness as an objective mechanical measure of ripeness confirms that combined applications of GA₃ and zeatin are significantly more effective in delaying structural softening of the fruit.

3.6. Starch and Soluble Sugars

Starch degradation and sugar accumulation are central metabolic processes during plantain ripening, directly linked to enzymatic activity and hormonal regulation. Measuring these parameters provides insight into how plant growth regulators (PGRs), such as gibberellins and cytokinins, influence the rate of carbohydrate metabolism. This analysis is crucial for evaluating strategies designed to delay ripening and extend post-harvest shelf life, particularly in export-oriented production systems [57].

The data presented in Figure 8A show that the PGR-free exhibited a slow reduction in starch content when compared to GA₃ (300 μM) + Zea (480 μM) from 0 to 20 DAH. However, after 25 DAH, the treatments 250 μM GA₃ + 390 μM Zea, 275 μM GA₃ + 430 μM Zea, and 300 μM GA₃ + 480 μM Zea showed, respectively, 2.9%, 2.9%, and 4.1% more starch than PGR-free—all values being statistically distinct. These values could be interpreted as a stabilization of starch hydrolysis, catalyzed mainly by enzymes such as amylases and glucosidases, with few effects of PGR on starch synthesis or metabolization. Distinct results were described by Khattak et al. [58,59], who described that starch content decreases from 25% in the pre-climacteric stage to around 1% in the climacteric stage. Rossetto et al. [60] observed that bananas treated with GA₃ significantly delayed starch degradation, maintaining higher levels of this polysaccharide during storage. This effect is attributed to the inhibition of the expression of genes related to the synthesis of hydrolytic enzymes. In ‘Nanica’ bananas, the combination of cytokinins and GA₃ not only delayed ripening but also preserved firmness and reduced acidity loss, evidenced by lower pH values compared to untreated fruits [43]. These findings suggest that co-application of GA₃ and cytokinins can modulate the enzymatic activity responsible for starch hydrolysis, thus preserving fruit quality during storage and transport, which is especially relevant for the export of bananas to distant markets.

On the other hand, the accumulation of total sugars is a direct result of starch degradation and is closely related to the progression of ripening. The data of Figure 8B show that PGR-free fruits experienced a marked increase in total sugar levels, with an estimated increase of more than 400% at 25 DAH compared to the initial content, evidencing an advanced ripening process. Treatments with isolated 275 μM GA₃, 430 μM Zea, or a combination with 275 μM GA₃ plus 430 μM Zea showed 8.2%, which was 8.7% less sugar concentration compared PGR-free at 25 DAH, confirming its limited effectiveness in slowing carbohydrate mobilization. Contrasting these values, combined treatments, particularly 300 μM GA₃ plus 480 μM Zea, showed an increase of around 380% in sugars, while 275 μM GA₃ plus 430 μM Zea showed an increase of nearly 370%. The worst treatment was isolated Zea at 390 μM, which showed a 420% increase in sugars compared to initial values. These findings support the hypothesis that any PGR had a significant effect on sugar accumulation during ripening in plantain fruits. In cherry fruits, the application of 20 mg L⁻¹ GA₃ increased fruit firmness at harvest, decreased the rate of fruit softening, and delayed fruit maturity 5–8 days [60]. Wei et al. [61,62] described that several persimmon genotypes treated with 300 mg L⁻¹ GA₃ delayed fruit maturation, resulting in the inhibition of the consumption of fructose and glucose. This phenomenon primarily results from the disruption of the balance between the biosynthesis and degradation of starch, a critical situation for the post-harvest handling of fruits destined for export, which require maintaining the green state for a more extended period [57,63,64,65]. According to Cordenunsi-Lysenko et al. [63], the use of PGR can alter the expression of genes associated with carbohydrate metabolism, resulting in a delay in starch conversion. Soares et al. [66] also documented that the starch content in different plantain varieties can remain elevated until later stages, depending on external intervention and the activated or suppressed enzyme profile. This action is closely linked to the control of hydrolytic enzyme activity, which determines the rate of starch decomposition.

Finally, it is essential to note that this physiological modulation has been supported by molecular and proteomic approaches [61]. Dong et al. [67] identified, through comparative analyses, that plantain varieties subjected to different hormonal conditions differentially express proteins associated with carbon metabolism, such as starch hydrolases and sugar transporters. Furthermore, fruit ripening is the result of a complex hormonal regulatory network, in which ethylene plays a central role, but whose action can be modulated by gibberellins and cytokinins that act as partial suppressors of catalytic genes [68].

3.7. Polyphenols and Polyphenol Oxidase Activity

Polyphenols are an essential biochemical indicator associated with the fruit’s antioxidant capacity, resistance to oxidative stress, and tissue stability during ripening. Polyphenols contribute to antioxidant defense and tissue preservation, while PPO is linked to enzymatic browning and degradation of these compounds [34,69,70]. Our results showed that treatments with zeatin, applied in isolation, notably Zea 430 μM L⁻¹ and Zea 480 μM L⁻¹, resulted in a lower reduction in phenolic content, at 7.1% and 17.4%, respectively. Similarly, 300 μM GA₃ plus 480 μM Zea promoted a decrease of 20.6% (Figure 9A), indicating a more balanced modulation of phenolic metabolism. These treatments, although they did not fully preserve polyphenols, were more effective in delaying their degradation compared to single hormonal treatments. An inverse pattern was verified in isolated GA₃, which promoted a higher decrease of up to 34% in comparison to PGR-free. The worst treatment was 250 μM GA₃ plus 390 μM Zea with a polyphenols reduction of 44%.

Polyphenol oxidase (PPO) is a key enzyme involved in enzymatic browning and the degradation of phenolic compounds, factors that directly affect the visual, sensory, and nutritional quality of the fruit [70]. This study observed that PPO activity increases significantly throughout ripening, as expected, given the progressive breakdown of cell membranes and the release of phenolic substrates. In our study, the isolated GA₃ showed the worst results, with lower inhibition of PPO activity with 6.5% (250 μM), 11.2% (275 μM), and 23.3% (300 μM). Similarly, lower concentration of isolated Zea (390 μM) or combined with 250 μM GA₃ showed reduction in PPO activity by 13.2% and 0.3%, respectively, indicating that GA₃ isolated or combined with a lower concentration of Zea is unable to block the activity of PPO. The inverse situation was verified under Zea at 430–480 μM, where PPO activity was inhibited by 39.2% and 31.6%, respectively. Higher GA₃ (300 μM) combined with higher Zea (480 μM) also showed a good inhibition of PPO activity (28.9%). Thus, the combined treatments offer an intermediate alternative, and the isolated treatments represent a clear risk of accelerating oxidative senescence.

The results described in this study are in line with those reported by Fujita et al. [35], who highlight the role of ethylene and other PGRs in PPO activation and polyphenol degradation. Majaliwa et al. [69] also stated that the stability of phenolic composts ranged more between cultivars than ripening stages, indicating the importance of fruit genetics in the response to ripening. However, the decrease in phenolic content during ripening is inversely correlated with antioxidant activity and directly correlated with PPO activity, confirming that precise PGRs management is essential to delay oxidative catabolism [34]. On the other hand, simple PGRs treatments such as the isolated application of GA₃ or Zeatin tend to maintain PPO activity and polyphenol degradation. Ha et al. [34] observed increases of up to 80% in PPO activity in banana (Musa sapientum) fruits, which aligns with the drastic decrease in phenols. At the same time, Galeazzi [71] identified PPO as a key catalyst for phenolic oxidation on “nanica” banana (Musa cavendishii). Likewise, Ketsa and Wisutiamonkul [72] highlighted that the decrease in dopamine in the banana peel is associated with enzymatic browning and the high affinity of PPO for said substrate. This observation emphasizes the need to modulate ripening to avoid premature loss of antioxidant compounds, especially in the outer layers of the fruit, which are more exposed to the action of PPO.

In contrast, combined treatments of GA₃ and Zeatin appear to offer a more conservative and efficient strategy, as proposed by Wang et al. [73]. These authors identified that banana cultivars rich in gallocatechin are less susceptible to oxidation, suggesting that phenolic conservation can be achieved with less aggressive treatments. In this regard, Ha et al. [34] support this perspective by showing that phenolics such as catechins, ferulic acid, and quercetin not only act as antioxidants but also directly inhibit PPO by forming complexes with its active copper site. Therefore, as shown in the present study, combined treatments do not completely eliminate PPO activity, but they do regulate it, maintaining an adequate proportion of protective compounds in plant tissues.

Finally, the physiological control of ripening through balanced hormonal strategies not only delays visual and chemical deterioration of the fruit but also preserves its biochemical functionality during prolonged storage, an essential aspect for export markets. Our results demonstrate that combined treatments with GA₃ and zeatin achieved moderate activation of PPO (30–40%) and less loss of polyphenols as compared to isolated PGRs applications. These findings are consistent with those reported by Shujita et al. [35], who highlighted that the reduction in polyphenols compromises the fruit’s natural defense against pathogens. This interpretation, which shows that fruits in the early stages of ripening retain higher levels of phenolic compounds and present a greater antioxidant capacity, is also negatively correlated with PPO activity [34]. On the other hand, Majaliwa et al. [69] confirmed that bananas (Musa spp.) with higher gallocatechin content are more resistant to oxidative degradation, indicating that treatment selection should also consider varietal genetics. Therefore, it is established that a combined PGRs supplementation, such as the joint use of GA₃ and zeatin, does not seek to eliminate ripening processes, but rather to carefully modulate them to preserve the biochemical integrity of the fruit without compromising its natural physiological evolution.

The canonical analysis provided indirect and vital information regarding plantain ripening (Figure 10). The first canonical analysis (Figure 10A) reveals that higher peel color values were associated with higher chlorophyll “b” values (r = 1.1019; Supplementary Table S1), a fact easily demonstrated in Figure 5, where green fruits, with more chlorophyll, had higher peel color values. On the contrary, higher peel color values were negatively correlated with pH (r = −0.3614; Supplementary Table S1). It is essential to note that chlorophyllases are enzymes that convert chlorophylls into chlorophyllide, hydrolyzing the phytol side chain and signaling this protein for degradation [74]. According to Suzuki et al. [75], chlorophyllase activity increased with increasing pH, up to pH 7, after which it decreased. Additionally, a Mg-dechelating activity of Chenopodium album [75,76] was described as having its best activity at pH 7.5 and completely inactivated at a pH higher than 8.2. Thus, it can be concluded that high pH decreases chlorophyllase activity, a fact that preserves the color of fruits with higher peel color values (Figure 10C). This hypothesis is corroborated in the present study by the positive correlation between peel color and total soluble solids (r = 0.8423; Supplementary Table S1), since this variable decreased strongly with increasing pH (r = −0.7864; Supplementary Table S1). Although this study has shown that increasing pH strongly favors carotenoid synthesis (r = 0.7891; Supplementary Table S1), the effect of pH on carotenoid synthesis does not appear to be a consensus in the literature. Iwata-Reuyl et al. [77], showed that phytoene synthase, an enzyme that catalyzes the condensation of two molecules of geranylgeranyl diphosphate to generate prephytoene diphosphate (PPPP) and the subsequent rearrangement of the cyclopropylcarbinyl intermediate to phytoene (beginning of carotenoid synthesis), had an optimal pH in the range of 8.2. Thus, carotenoid synthesis increases with increasing pH, as shown in Figure 10C. However, Chen et al. [78], showed that the activity of isopentenyl diphosphate isomerase (IDI) from Saccharomyces cerevisiae has its maximum activity at pH 7.5 and is completely inactivated at a pH level above 9. The degradation of chlorophyll “b” is also strongly contributed to by the increase in titratable acidity (r = −2.0871; Figure 10B; Supplementary Table S1), reinforcing the hypotheses mentioned above. pH also positively affects the synthesis of polyphenols (r = 0.8293; Supplementary Table S1), since the enzyme phenylalanine ammonia lyase (PAL), which catalyzes the non-oxidative degradation of L-phenylalanine to trans cinnamic acid plus a free ammonium ion and participates in the phenylpropanoid pathway which is used to channel carbon from primary to secondary metabolism, is strongly activated with increasing pH up to pH 7.6, when it begins to present slow activation values at strongly alkaline pH. On the other hand, the reduction in polyphenols, normally linked to the activity of the PPO enzyme, is strongly activated at pH below 7 and undergoes strong inactivation at higher pH [74,75]. These biochemical data, even if not measured directly in this study, show how de-greening, carotenoid synthesis and polyphenol synthesis are intrinsically linked to pH, a factor of extreme relevance, since the ripening of bananas is stimulated by more alkaline or less acidic pH. Therefore, it can be indirectly concluded that pH control during the ripening of plantain fruits should be avoided, which presents yet another challenge for producers, especially plantain exporters worldwide.

Future research should then focus on unraveling the molecular mechanisms behind the observed physiological responses, particularly through transcriptomic and proteomic profiling of ripening-related pathways. It will also be critical to assess varietal responsiveness to different PGRs, refine application dosages, and validate this protocol in real-world export settings where container openings are minimized. Moreover, environmental sustainability and cost-effectiveness analyses are necessary to assess the large-scale applicability. Extending this strategy to other climacteric fruits could provide a versatile alternative to conventional preservation technologies and enhance resilience in tropical agri-food supply chains.

4. Conclusions

This study demonstrated that plant growth regulators had a significant impact on the ripening of Hartón plantains during cold storage. GA₃ alone accelerated pigment degradation, sugar accumulation, and polyphenol loss, while zeatin alone had a limited impact. Conversely, combined GA₃ and zeatin preserved chlorophyll, starch, and polyphenols, reduced pH and PPO activity, and maintained firmness longer. Then, among treatments, 300 μM GA₃ with 480 μM zeatin was the most effective, offering modulation of ethylene action, delaying starch hydrolysis and pigment degradation, and mitigating oxidative stress—key factors in extending shelf life and preserving post-harvest quality. Canonical correlation analysis also indicated that pH plays a central role in modulating ripening-related parameters, including pigment stability and polyphenol metabolism, supporting its value as a secondary physiological marker. The findings support the use of GA₃ + Zea as a practical, low-cost strategy for maintaining export-quality plantains without compromising fruit integrity.

Our results demonstrate that the effects of plant growth regulators (PGRs) on the post-harvest physiology of Hartón plantains were not stable throughout the storage period. However, PGRs showed a positive effect on delaying post-harvest ripening of Hartón plantains, demonstrating that GA₃ + Zea combinations, especially 275 μM GA₃ + 430 μM Zea, were the best options for delaying fruit ripening. However, this effect was significantly dependent on the ripening stage, highlighting a strong interaction between treatment and evaluation time. This was particularly evident in physiological parameters such as pigment retention (chlorophyll a and b), carotenoid accumulation, acidity, firmness, sugar metabolism, and oxidative activity, all of which showed statistically significant treatment × time interaction effects. These interactions indicate that the impact of PGRs is modulated by ripening progression, highlighting the importance of temporal dynamics in evaluating post-harvest treatments, but also strongly demonstrating that isolated PGRs have a milder effect than their combinations. The full statistical validation of these interaction effects is presented in Supplementary Table S2, and the impact of the different PGRs, regardless of evaluation time, is presented in Supplementary Table S3, which complements the raw data previously provided in Supplementary Table S4. Collectively, our data support the strategic use of RCPs not only to delay ripening but also to adapt fruit quality management at different storage stages.