Effect of Plant Growth Regulators on Germination and Seedling Growth of Passiflora alata and Passiflora edulis

Abstract

The yellow (Passiflora edulis) and sweet (Passiflora alata) passion fruit plants hold significant economic importance in tropical fruit cultivation, valued not only for the quality of their fruit but also for their medicinal properties. Conventional propagation through seeds faces challenges due to irregular and slow germination, affecting the time required for seedling formation and the viability and uniformity of plantations. The use of plant growth regulators has been explored as a strategy to overcome these barriers, improving both the rate and uniformity of seed germination. This study aimed to evaluate the influence of seed imbibition with plant growth regulators on the germination and subsequent growth of yellow and sweet passion fruit seedlings. Gibberellic acid (GA₃) and GA₄₊₇ combined with 6-benzyladenine (GA₄₊₇+6BA) were applied in five different concentrations (0, 250, 500, 750, and 1000 mg L⁻¹ a.i.). The experiments were conducted in both laboratory and greenhouse conditions, following a completely randomized design with a 2 × 5 factorial scheme. The varieties tested were ‘BRS Mel do Cerrado’ for sweet passion fruit and ‘IAC-275’ for yellow passion fruit. Quantitative parameters, such as germination percentage, germination speed index, fresh and dry biomass of roots and shoots, and enzymatic activity, were assessed. The results indicated that GA₃, at higher concentrations, significantly enhanced both germination percentage and speed index in both Passiflora alata and Passiflora edulis when compared to the control. Additionally, seedlings treated with GA₃ showed a marked increase in shoot and root biomass, particularly at concentrations of 500 and 750 mg L⁻¹. Enzymatic assays revealed heightened catalase and peroxidase activities in treated seedlings, indicating improved stress tolerance. In contrast, the GA₄₊₇+6BA treatment showed less pronounced effects on seedling growth. Overall, GA₃ was more effective in enhancing germination and seedling growth in both species, suggesting its potential application in improving the propagation of passion fruit.

1. Introduction

The cultivation of passion fruit, in its yellow (Passiflora edulis Sims f. flavicarpa Deg.) and sweet (Passiflora alata Curtis) varieties, is economically significant, primarily due to the high market value of its fruits both nationally and internationally, which is attributed to their organoleptic and nutritional properties. In 2022, Brazilian passion fruit production reached 697,859 thousand tons over a cultivated area of 45,602 thousand hectares [1]. This production volume accounts for more than 70% of the world’s passion fruit production, establishing Brazil as the largest producer and consumer of passion fruit globally.

Despite this prominence, the productive efficiency of these species is often compromised by agronomic challenges, especially during the seed germination phase, which is crucial for the successful establishment of the crop. This phase is often characterized by inconsistent germination and seed dormancy, phenomena that can delay or even prevent uniform seedling development [2].

Seed germination is a critical stage in the life cycle of Passiflora species, as it directly impacts the success of seedling establishment and, consequently, the overall efficiency of commercial cultivation. Given the growing demand for passion fruit, particularly in Brazil, which is the world’s largest producer, achieving uniform and rapid germination is essential for large-scale propagation. However, Passiflora species often exhibit irregular and slow germination rates due to physiological dormancy and variability in seed quality. These challenges can lead to delayed seedling establishment and hinder the uniformity of plantations, affecting both fruit yield and quality. Therefore, optimizing seed germination through the application of plant growth regulators presents a valuable strategy to overcome these barriers and enhance the propagation process, ensuring a more consistent supply of healthy seedlings for commercial production.

In this context, the physiological management of seeds using plant growth regulators such as gibberellic acid (GA₃) and GA₄₊₇ combined with 6-benzyladenine (GA₄₊₇+6BA) emerges as a viable strategy to optimize seedling formation, establishment, and initial plant development [3]. GA₃ is a widely recognized plant growth regulator known for its ability to break seed dormancy and accelerate germination by promoting the hydrolysis of reserves essential for embryo growth. During germination, GA₃ plays a crucial role in protein and RNA synthesis, facilitating cell expansion and contributing to seedling development [4]. Furthermore, GA₃ has been associated with increased plant height, improved photosynthesis, and the advancement of the crop life cycle.

On the other hand, cytokinins are recognized for their ability to induce cell division. During the germination phase, this hormone modulates gene expression, regulates protein functions, and adjusts membrane permeability, as well as the levels of other plant hormones, such as gibberellins [4]. Cytokinins are responsible for rapid responses in the germination process, aiding in radicle development and contributing to the absorption of gibberellins by plant tissue [4]. The use of GA₄₊₇+6BA is often associated with increased plant biomass, the development of more robust roots, and overall improvement in seedling quality [5].

The combination of GA₃ and GA₄₊₇+6BA can further optimize the germination process and seedling growth of passion fruit and Rangpur lime plants [3]. The application of these plant growth regulators can stimulate both root and shoot development, promoting faster and more robust seedling establishment in the field [3,6]. This integrated approach is essential for overcoming the challenges faced in passion fruit cultivation, ensuring more uniform plant development from the earliest stages and, consequently, promoting higher productivity at the end of the cycle.

Given the economic importance of passion fruit and the challenges faced in its production, particularly with regard to obtaining vigorous seedlings, the present study aimed to investigate the effects of GA₃ and GA₄₊₇+6BA application on the germination, growth, and development of yellow and sweet passion fruit seedlings.

2. Materials and Methods

2.1. Location of the Experimental Area, Seed Acquisition, Experimental Design, and Treatments

The experiments were conducted at the Department of Plant Production of the Faculty of Agronomic Sciences, São Paulo State University “Júlio de Mesquita Filho” (UNESP), located at the Botucatu Campus, in the State of São Paulo, Brazil. The physiological quality of yellow passion fruit (P. edulis) and sweet passion fruit (P. alata) seeds was evaluated after manual extraction from fully ripe fruits, washing in running water until complete removal of the aril, and being shade-dried.

For both experiments, a completely randomized design with a 2 × 5 factorial arrangement was used, testing the effects of the plant growth regulators GA₃ and GA₄₊₇+6Benzyladenine (GA₄₊₇+6BA) at concentrations of 0, 250, 500, 750, and 1000 mg L⁻¹ a.i. The gibberellin source used consisted of 4% GA₃ and 96% inert ingredients. Yet, the GA₄₊₇+6BA mixture was composed of gibberellins GA₄₊₇ (1.8%) and the cytokinin 6-benzyladenine (1.8%).

In the laboratory tests for germination evaluation, four replicates of 50 seeds each were distributed in “gerbox” containers with moistened blotting paper, maintained in a germination chamber with alternating temperature and light cycles (16 h of light at 30 °C and 8 h of darkness at 20 °C), following the guidelines of [7]. Germination was confirmed by the emergence of at least 2 mm of the radicle [8].

In a greenhouse environment, seeds subjected to the treatments were sown in individual cells of polystyrene trays containing 72 units, filled with a commercial substrate that includes sphagnum peat moss, vermiculite, limestone, and agricultural gypsum (pH 5.0; electrical conductivity of 0.7 mS cm⁻¹; density of 101 kg m⁻³; water retention capacity of 55%). After 60 days, the seedlings were transplanted into pots containing a mixture of soil and organic compounds in a 3:1 ratio, enriched with limestone (2 kg m⁻³), single superphosphate (1.5 kg m⁻³), and potassium chloride (0.5 kg m⁻³).

2.2. Seed Imbibition Process and Application of Treatments

The dynamics of water absorption were studied using four replications of 25 seeds, which were immersed in distilled water with an aeration system. The seeds were periodically removed, superficially dried, and weighed on a high-precision balance. Subsequently, the seed moisture content was determined by the oven-drying method at 105 °C [7]. Afterward, the seeds were subjected to imbibition in solutions containing plant growth regulators with adequate aeration.

2.3. Evaluations

2.3.1. Germination Analysis

The variables analyzed included:

Germination percentage: determined at the end of the tests using the ratio G = (N/A) × 100, where G is the germination percentage, N is the number of germinated seeds, and A is the total number of seeds tested [7,9].

Germination speed index (GSI): obtained by the daily sum of germinated seeds, divided by the number of days until the first germination [10].

Length of shoots and roots and fresh and dry mass of seedlings: measured with precision, with seedlings dried to determine the dry mass in an oven at 80 °C until a constant weight was reached.

Percentage of abnormal seedlings: calculated as PA = (N/A) × 100, where PA represents the proportion of abnormal seedlings, N is the number of abnormal seedlings, and A is the total number of seeds in the sample.

2.3.2. Physiological Analysis

In the greenhouse, analyses of the fresh and dry mass of shoots and roots were conducted on the seedlings using a ruler graduated in millimeters and a balance with a precision of 0.00001 g. For this, after determining the fresh masses, leaves and roots were dried in an air-circulating oven at 80 °C until a constant weight was reached.

Detailed biochemical analyses and antioxidant enzyme activity were also conducted to understand the plants’ capacity to mitigate oxidative stress and to assess the metabolic state and response of yellow and sweet passion fruit seedlings to treatments with plant growth regulators:

Quantification of total proteins: the determination of total protein concentration was performed using the colorimetric method by [11]. Leaf tissue samples were homogenized in an appropriate extraction buffer, and the protein content was evaluated by measuring the absorbance at 595 nm.

Peroxidase (POD): The activity of POD was determined spectrophotometrically by measuring the oxidation of specific substrates in the presence of hydrogen peroxide, according to the methodology adapted from [12]. The reaction temperature was maintained at 25 °C, and enzyme activity was expressed in units per milligram of protein, where one unit is defined as the amount of enzyme required to cause an increase in absorbance by 0.001 units per minute.

Superoxide dismutase (SOD): SOD activity, essential in the dismutation of superoxide into oxygen and hydrogen peroxide, was assessed using the technique described by [13]. The assay was conducted at 25 °C, and one unit of SOD activity was defined as the amount of enzyme required to inhibit the reduction of nitroblue tetrazolium by 50%.

Catalase (CAT): The activity of CAT was measured by the rate of hydrogen peroxide decomposition, as described by [14]. The reaction temperature was maintained at 25 °C, and enzyme activity was expressed in units per milligram of protein, where one unit is defined as the amount of enzyme that decomposes 1 μmol of H₂O₂ per minute.

Ascorbate peroxidase (APX): APX activity, involved in the detoxification of hydrogen peroxide in the ascorbate-glutathione cycle, was measured by the reduction of ascorbate monitored at 290 nm, according to [15]. The reaction temperature was maintained at 25 °C, and enzyme activity was expressed in units per milligram of protein, where one unit is defined as the amount of enzyme that oxidizes 1 μmol of ascorbate per minute.

2.4. Statistical Analyses

The results obtained were subjected to analysis of variance and Tukey’s test (p < 0.005%). To verify the clustering of responses, a multivariate analysis was conducted using the Statistical Analysis Software 4.0 (SAS) program, employing principal component analysis (PCA) to characterize the interactions between plant growth regulators and doses.

3. Results and Discussion

3.1. Yellow Passion Fruit (P. edulis)

3.1.1. Germination Seeds

A significant response of the combination of treatments on the analyzed characteristics was observed, showing a significant relationship (p < 0.01) between the types of plant growth regulators and the applied doses on all variables related to the germination of yellow passion fruit seeds. Treatment with gibberellic acid (GA₃) resulted in a notable reduction in the number of abnormal seedlings, especially at concentrations of 250 mg L⁻¹ and 1000 mg L⁻¹ (Figure 1a). On the other hand, the combination of GA₄₊₇+6Benzyladenine (GA₄₊₇+6BA) induced the formation of abnormal seedlings at all tested doses (Figure 1a).

Although it enhanced the shoot development of seedlings at higher concentrations (Figure 1d), 6BA did not proportionally stimulate root development (Figure 1e), resulting in unbalanced growth and, consequently, more abnormal seedlings (Figure 1a). Cytokinins are naturally more abundant in the apical meristems, with the root tip being the primary site of this hormone’s biosynthesis [16], suggesting that treatments with higher concentrations of 6BA may have caused antagonistic effects [3]. The results showed a clear effect of GA₄₊₇+6BA on increasing shoot length as the concentration increased, but they are inconclusive regarding the stimulation of root growth.

Higher doses of GA₃ (500, 750, and 1000 mg L⁻¹) were more effective in increasing the germination percentage and the length of both roots and shoots of the seedlings compared to GA₄₊₇+6BA (Figure 1d–f). This treatment also contributed to a significant increase in the fresh and dry mass of the seedlings (Figure 1b,c), indicating more vigorous and healthy growth. Seedlings with greater biomass are generally more resilient to environmental stresses, such as drought, pests, and diseases.

The different doses of GA₃ showed a consistent effect on the germination of yellow passion fruit seeds (Figure 1f). Gibberellic acid plays a crucial role in germination by stimulating the production of enzymes that break dormancy and activate metabolic processes essential for germination. This includes promoting the synthesis of proteins, carbohydrates, and other compounds vital for the plant’s initial development, as well as regulating the hydrolysis of energy reserves through the induction of new α-amylase, the enzyme responsible for starch hydrolysis [4]. The efficacy of gibberellic acid in increasing the germination rate is corroborated by previous studies, such as those by [3,17,18], although the responses may vary depending on the species and doses used.

GA₃ proved to be more effective than GA₄₊₇+6BA in accelerating the germination of yellow passion fruit seeds, improving the Germination Speed Index (GSI) at all tested concentrations (Figure 2). The application of GA₃ during seed imbibition significantly increased water absorption and the activation of metabolic processes necessary for germination. In all cases, GA₃ resulted in a strong physiological response, leading to faster germination compared to the GA₄₊₇+6BA treatment. These results are likely related to the fact that gibberellins control the stages of activation of the embryo’s vegetative growth, the weakening of the endosperm layer surrounding the embryo, and the mobilization of energy reserves in the storage tissues [19].

3.1.2. Growth and Development of Seedlings

When analyzing the characteristics of yellow passion fruit seedlings, it was found that the dry mass of the shoots was influenced by GA₃ and GA₄₊₇+6BA, especially at concentrations of 750 and 1000 mg L⁻¹ (Figure 3d). This effect can be explained by GA₃’s ability to induce more vigorous cell growth, resulting in an increase in dry mass due to the greater production of organic matter without a corresponding increase in cellular water content, which would influence fresh mass.

Additionally, GA₃ may have promoted the synthesis of structural components such as cellulose and lignin, which are substantial for plant dry mass but do not directly affect the water content or other components related to fresh mass [20]. Thus, the effect of GA₃ on the dry mass of yellow passion fruit seedlings may be primarily associated with the stimulation of cell growth and increased production of organic matter without significantly impacting the cellular water content.

Although GA₃ and GA₄₊₇+6BA are recognized as regulators of plant growth and development, they did not significantly influence the fresh mass of the shoots or roots nor the dry mass of the roots of yellow passion fruit seedlings (Figure 3a,b). This lack of influence can be attributed to the specific response of each plant species to hormonal treatments. Additionally, the regulators primarily acted on the germination process without exhibiting prolonged residual effects that could promote biomass accumulation in the subsequent seedlings. It is possible that yellow passion fruit has limited sensitivity to GA₃ and GA₄₊₇+6BA in terms of biomass accumulation in shoots and roots, potentially due to the doses used or the mode of application of the regulators, which were applied during seed imbibition and may have been consumed during the germination process and initial biomass accumulation by the seedlings [18].

3.1.3. Protein Levels and Oxidative Enzymes of Seedlings

The application of GA₃ at a concentration of 500 mg L⁻¹ proved to be more effective in significantly increasing the synthesis and accumulation of proteins in yellow passion fruit seedlings (Figure 4a). GA₃ is known for its crucial role in plant growth and development, influencing various physiological processes, including protein synthesis. Proteins are essential in plant responses to biotic and abiotic stresses, aiding in the maintenance of cellular integrity and metabolic functioning. Therefore, the increase in protein levels in the seedlings may enhance their stress response capacity, resulting in superior field performance and overall greater plant resistance.

The plant growth regulators GA₃ and GA₄₊₇+6BA, at different concentrations, demonstrated distinct effects on the activities of antioxidant enzymes in P. edulis seedlings. Both substances reduced catalase (CAT) activity (Figure 4d) while increasing superoxide dismutase (SOD) activity (Figure 4b), with the action of GA₄₊₇+6BA at a concentration of 250 mg L⁻¹ being particularly notable. This alteration can be explained by the ability of the regulators to decrease the production of reactive oxygen species (ROS) or increase the effectiveness of other antioxidant enzymes, such as SOD, leading to a reduced need for CAT to neutralize hydrogen peroxide, a common byproduct in oxidative processes [21].

ROS are known to alter normal cellular metabolism through oxidative damage to lipids, proteins, and nucleic acids, and they can also damage vital cellular components, such as photosystem II and lipid membranes [22]. Therefore, the selection of plant growth regulators and their doses should be guided to minimize oxidative stress, avoiding excessive energy consumption required for the activation of antioxidant enzymes, which could compromise the performance and vigor of the seedlings.

Furthermore, it was observed that GA₃ tends to result in lower enzymatic activities, as indicated by various measurements (Figure 4), suggesting that reduced levels of these enzymes may indicate delays in the seedling senescence process. This effect is particularly relevant for the development of fruit seedlings, as delaying senescence can result in a prolonged and potentially more productive vegetative period [3], which is extremely beneficial for the cultivation of yellow passion fruit.

3.1.4. Principal Component Analysis (PCA) of P. edulis

The principal component analysis (PCA) indicates that the higher concentrations of plant growth regulators, such as D750 and D1000, have a significant impact on the growth and antioxidant responses of P. edulis seedlings (Figure 5). The seedlings treated with these higher concentrations are distinctly positioned relative to the control (D0), suggesting a notable alteration in the physiological characteristics of these seedlings. These results underscore the importance of the dosage of plant growth regulators in the development of yellow passion fruit seedlings (Figure 5).

The PCA indicates the relationship between the physiological and growth variables of the seedlings and the two main components of the PCA. It is observed that variables such as fresh and dry shoot mass (MFPA, MSPA) and root biomass (MFR, MSR) are strongly associated with the first principal component. This suggests that the plant growth regulators exert a strong influence on the growth of the seedlings, both in terms of shoot and root biomass.

The plant growth regulators, especially at higher doses, demonstrate a substantial impact not only on vegetative growth but also on the antioxidant responses of the seedlings. Antioxidant variables such as CAT (catalase) and SOD (superoxide dismutase) are positively correlated with growth variables (Figure 5). This may indicate that while growth is stimulated, the plants also mobilize antioxidant defense mechanisms to combat the potential stress induced by the rapid increase in biomass.

The action of plant growth regulators, as demonstrated, suggests that they not only promote growth through mechanisms such as cell division and elongation but also adjust the plant’s physiology to deal with the challenges associated with accelerated growth. This increase in antioxidant activity is crucial, as it protects cells from potential damage caused by reactive oxygen species, which are often a byproduct of accelerated metabolism.

Therefore, the use of plant growth regulators, especially in high concentrations, can be an effective strategy to optimize the production of yellow passion fruit seedlings, leveraging both the growth and the physiological resilience of the seedlings and better preparing them for transplanting and subsequent adaptation to the field environment.

3.2. Sweet Passion Fruit (P. alata)

3.2.1. Germination Seeds

Comparing the effect of GA₄₊₇+6BA and GA₃ on the germination results of P. alata seeds, it was observed that GA₄₊₇+6BA resulted in 100% abnormal seedlings, regardless of the concentration applied. On the other hand, treatment with GA₃, except at the concentration of 1000 mg L⁻¹, resulted in an insignificant occurrence of abnormal seedlings, with approximately 8% of the seedlings affected in this specific case alone (Figure 6a). The effectiveness of GA₃ as a promoter in germination and seedling production results in the significant formation of normal plants, optimizing the morphological quality of the plants and highlighting its potential as a beneficial plant regulator in the initial development of sweet passion fruit.

Although it contributes to root growth in other species [19], the concentrations of GA₄₊₇+6BA tested in the present study resulted in compromised root development during the germination process of sweet passion fruit (Figure 6e). This limitation contrasts with the stimulating effect observed in the shoot part of the plant. This difference may be attributed to specific conditions, such as variability in the sensitivity of P. alata seedlings to cytokinin.

On the other hand, GA₃ has proven effective in promoting root growth and increasing the dry mass of seedlings (Figure 6c,e), reinforcing its well-known ability to stimulate cell expansion and overall seedling growth [3,16]. In addition, GA₃ plays a crucial role in overcoming dormancy by inducing enzymes that weaken the endosperm [3,23,24], thereby contributing to the development of more vigorous and robust seedlings.

Additionally, seeds treated with GA₄₊₇+6BA exhibited a high Germination Speed Index (GSI) at a concentration of 250 mg L⁻¹ (Figure 7), which can be attributed to the potential of these plant growth regulators to rapidly stimulate embryonic cell division and accelerate germination. However, it was observed that increasing the concentrations of GA₄₊₇+6BA resulted in a reduction in GSI, indicating that the efficacy of the germination stimulus is concentration-dependent.

The higher germination speed is closely related to the faster development of the plant, which is extremely important for the formation of sweet passion fruit seedlings in a shorter time. It is noteworthy that both gibberellins and cytokinins play crucial roles in the synthesis of enzymes that degrade the reserves stored in the endosperm, forming simple sugars, amino acids, and nucleic acids, which are absorbed and transported to the growth regions of the embryo, stimulating cell elongation. They also play a role in breaking seed dormancy in various species, including those of the Passifloraceae family, underscoring the utility of these regulators in improving seed germination parameters [19,25] and Citrus limonia [3].

Gibberellic acid showed a significant increase in the Germination Speed Index (GSI) when applied at a concentration of 750 mg L⁻¹ (Figure 7), reinforcing that this hormone has a stimulating effect on germination processes [3], especially in seeds that are not in a state of dormancy [26]. Research conducted by [17] demonstrated that the highest GSI values for sweet passion fruit seeds were achieved with the application of 500 mg L⁻¹ of GA₃. Additionally, Refs. [27,28] identified that 150 and 300 mg L⁻¹ of GA₃, respectively, were effective doses for moistening the substrate with sweet passion fruit seeds, and [3] observed that the application of GA₃ improves the GSI of rangpur lime (Citrus limonia), highlighting that the methodology of applying the regulator to the seed is a determining factor in choosing the appropriate dose.

3.2.2. Growth and Development of Seedlings

GA₃ positively contributed to the length and fresh mass of the roots of sweet passion fruit seedlings, with the best results obtained at higher concentrations (500, 750, and 1000 mg L⁻¹) (Figure 8b,c). Good root development is essential for increasing the capacity to absorb water and nutrients from the soil, contributing to the initial adaptation of seedlings to the field, promoting more effective establishment, and, subsequently, more vigorous plant development.

Both GA₃ and the mixture of GA₄₊₇+6BA did not show significant effects on the dry mass of the shoots and roots of sweet passion fruit seedlings (Figure 8d). This lack of response may be attributed to the variable sensitivity of this species’ seeds to these plant growth regulators, particularly in terms of dry biomass accumulation. It is not uncommon for different plant species to exhibit distinct responses to the same plant regulator stimuli, which can also depend on the doses and application methods used.

3.2.3. Protein Levels and Oxidative Enzymes of Seedlings

However, at higher doses (500, 750, and 1000 mg L⁻¹), both treatments were able to increase the protein content in sweet passion fruit seedlings (Figure 9a and Figure 10). Higher protein content in seedlings can confer increased resistance to biotic and abiotic stresses, contributing to the maintenance of cellular integrity and metabolic functionality [29]. Thus, seedlings with high protein content not only exhibit a better stress response capacity but also tend to perform better when transplanted to the field.

In the context of antioxidant enzymes, GA₃ was shown to reduce the activity of peroxidase (POD), superoxide dismutase (SOD), and ascorbate peroxidase (APX), indicating decreased activity in POD at 500 mg L⁻¹ (Figure 9c), SOD at 500 and 1000 mg L⁻¹ (Figure 9b), and APX at 750 mg L⁻¹ (Figure 9e and Figure 10). This suggests that specific doses of the regulator can differentially influence antioxidant enzymes, impacting plant defense mechanisms. The reduction in the activity of these antioxidant enzymes may be a consequence of the lower production of reactive oxygen species (ROS), as proposed by [21], resulting in a reduced need for antioxidant enzyme activity due to the modulatory action of plant growth regulators on plant oxidative stress.

The application of GA₄₊₇+6BA at concentrations of 250 mg L⁻¹ and 750 mg L⁻¹ during seed imbibition resulted in a reduction of catalase (CAT) activity in P. alata seedlings (Figure 9d). This phenomenon illustrates that enzymatic activity is influenced by the specific interaction between plant growth regulators and the applied doses. Each enzyme has specific binding sites for regulator molecules, which can significantly alter the metabolic activity of plants [23,24]. The reduction in the activity of antioxidant enzymes such as CAT is often associated with lower levels of oxidative stress, which is beneficial during the seedling stage, making them more vigorous and capable of adapting to adverse edaphoclimatic conditions.

On the other hand, doses of 500 mg L⁻¹, 750 mg L⁻¹, and 1000 mg L⁻¹ of gibberellic acid (GA₃) were effective not only in increasing the protein content and fresh biomass of the seedlings but also in reducing antioxidant activity. GA₃ is known to decrease the production of reactive oxygen species (ROS), which can cause oxidative damage to essential cellular components such as lipids, proteins, and nucleic acids and directly damage critical structures such as photosystem II and lipid membranes [3,21,22]. Therefore, the choice of plant growth regulators and appropriate doses is crucial to minimize oxidative stress, avoiding excessive energy expenditure on the activation of antioxidant enzymes, which could result in performance losses in the seedlings.

Thus, the use of GA₃ in the imbibition of P. alata seeds emerges as a promising strategy, facilitating not only the robust development of seedlings but also enhancing their ability to withstand adverse conditions without the risk of elevated oxidative stress.

3.2.4. Principal Component Analysis (PCA) of P. alata

Principal component analysis (PCA) suggests that the use of GA₃, especially at higher doses, has a substantial impact on the growth of P. alata seedlings, significantly increasing the biomass of both the aerial parts and the roots. This is evident from the positive correlation between growth variables and the first principal component. On the other hand, GA₄₊₇+6BA appears to have a less pronounced influence on these growth characteristics (Figure 10).

Figure 10. Principal component analysis (PCA) of sweet passion fruit (P. alata) seedlings from seeds imbibed in GA₃ and GA₄₊₇+6BA.

Figure 10. Principal component analysis (PCA) of sweet passion fruit (P. alata) seedlings from seeds imbibed in GA₃ and GA₄₊₇+6BA.

The variation in enzymatic responses indicates that plant growth regulators may distinctly affect the mechanisms of antioxidant defense in seedlings, which is crucial for protection against oxidative stress. Understanding these interactions helps optimize the use of plant growth regulators to enhance seedling growth and health, ideally adapting them for transplantation and cultivation under field conditions.

Therefore, this PCA provides valuable insights for researchers, producers, and nursery operators on how different hormonal treatments can be used to maximize the development and quality of sweet passion fruit seedlings. GA₃, especially at higher doses, appears to promote a substantial increase in both shoot and root dry and fresh mass. Physiologically, GA₃ is known to stimulate cell expansion through the promotion of nucleic acid and protein synthesis, which is essential for cell growth. It may also play a role in mobilizing nutrient reserves within the seed, accelerating initial growth after germination [4]. Different doses and types of plant growth regulators not only affect growth but also the biochemical and physiological response of seedlings. The lower activity of antioxidant enzymes at some specific doses may indicate that these concentrations are optimized to reduce metabolic stress in plants, while other concentrations may induce different adaptive responses. GA₃ reduces the activity of antioxidant enzymes at certain doses, which can be interpreted as a reduction in oxidative stress. Physiologically, this suggests that GA₃ can modulate the balance of reactive oxygen species (ROS), potentially reducing the oxidative cell damage that normally occurs during intense metabolic processes associated with rapid growth [4].

GA₃ can maximize seedling growth while minimizing metabolic stress, transforming them into more resilient and adaptable plants. This is crucial for the success of seedlings when transplanted to the field, where they have to deal with environmental variations and stresses such as drought and diseases. Seedlings with well-developed root systems, for example, can absorb water and nutrients more efficiently, which is fundamental for the successful establishment of plants in new environments, such as those obtained with high doses of GA₃ (Figure 8c).

4. Conclusions

The treatment of Passiflora alata and Passiflora edulis seeds with GA3 significantly accelerates the germination process, increasing both the germination percentage and the germination speed index. Additionally, seedlings treated with GA3 exhibited enhanced root and shoot biomass accumulation, as well as improved enzymatic activity (particularly catalase and peroxidase), which are indicators of better stress tolerance. These results suggest that GA3 not only promotes faster seedling emergence but also contributes to the production of healthier, more vigorous seedlings that are better equipped to withstand environmental stresses, making it a promising tool for improving passion fruit propagation.