Study on physiological characteristics of Passiflora edulis Sims with different ploidy under drought stress

Abstract

Passiflora edulis Sims, an economically important fruit crop known as the “king of juices,” is severely affected by drought, which significantly constrains its growth and yield. To investigate whether polyploidy enhances drought resistance, we compared the physiological responses of diploid and triploid tissue-cultured seedlings of the P. edulis ‘Mantianxing’ cultivar subjected to Polyethylene Glycol 6000 (PEG-6000) for 7 days. Physiological parameters were analyzed in mixed samples of mid-shoot segments (mixed stem and leaf segments or leaves) from three biological replicates, with the data subjected to one-way ANOVA and Duncan’s test (p < 0.05). The experimental results confirmed that: (1) The optimal rooting medium for sterile seedlings of diploid and triploid is 0.1 mg/l NAA + 0.2 g AC + 1/2 MS. (2) Under drought stress, particularly at 40% and 50% PEG-6000 concentrations, triploids exhibited significantly higher activities of peroxidase (POD) and catalase (CAT), higher proline (Pro) and soluble sugar contents, and lower malondialdehyde (MDA) content than diploids. Superoxide dismutase (SOD) activity did not differ significantly by ploidy. Under severe drought (50% PEG-6000), triploids maintained higher soluble sugar but lower soluble protein content. These results demonstrate that triploid ‘Mantianxing’ possesses superior drought adaptability, mediated by enhanced osmotic adjustment and a more robust antioxidant system, providing a physiological basis for polyploid breeding in P. edulis.

Introduction

Passiflora edulis Sims, a member of the genus Passiflora in the Passifloraceae family, is a perennial evergreen vine and a plant grown for beverage production [1], known as the “King of Drinks” [2]. It has widespread use in fresh food [3], medicinal applications [4–6], and as an ornamental plant [7–11]. Native to north-central South America, it is primarily distributed across Central and South America, as well as Australia, Africa, and Asia [12]. In China, its primary distribution is in the southwest, south-central, and southern regions, with Yunnan having the highest number of species [13]. Currently, large-scale cultivation in China is limited to purple passion fruit, yellow passion fruit, their hybrids, and three other strains [14]. And ‘Mantianxing’ is a purple-fruited passionfruit variety; the fruit is larger and has a purple-red rind, with a surface covered in dark spots. The branches and vines are green, so the name ‘Mantianxing’ has strong processing and utilization value [15].

Abiotic stresses, particularly drought, salinity, heat, and cold, are hampering crop productivity worldwide. Furthermore, severe or sustained abiotic stresses can disrupt cellular homeostasis and even kill entire plants, thus fully compromising crop yield and productivity [16–18]. Due to global warming, drought events have become increasingly common in many regions worldwide [19]. Drought can notably affect plant growth and development [20]. However, water is an essential factor for plants, playing a direct role in plant metabolism, transpiration, photosynthesis, and other physiological processes [21]. Additionally, its content generally accounts for 60–90% of the plant’s weight [22]. Water scarcity caused by drought can directly affect various physiological and biochemical activities in crops, such as increasing oxidative damage by elevating the levels of osmotic regulators like soluble sugars [23], soluble proteins [24], proline [25], and others, along with regulating the activity of antioxidant enzymes [26], which may even lead to reduced crop yields or crop death [27].

Polyethylene glycol (PEG) is a classic osmotic agent used to simulate drought stress [28]. Reducing the external water potential creates an osmotic gradient that induces water loss in plant tissues, consequently triggering water stress responses [29]. Critically, PEG molecules cannot be absorbed by plant cells, thus avoiding unintended physiological interference. Owing to these characteristics, PEG has become a reproducible tool for evaluating drought tolerance at the seedling stage [30]. Numerous studies have confirmed the reliability of this method for screening drought-tolerant germplasm [31–33]. The controlled environment of an in vitro system serves as a simplified but powerful analytical tool [34]. While it cannot fully replicate the complexity of field conditions, this very limitation is what enables the system to yield unique insights. This controlled setting allows us to eliminate external interference and focus on dissecting the core physiological and biochemical pathways triggered by drought stress itself in plants.

It has been found that polyploid plants often exhibit excellent resistance to adversity, such as low-temperature [35–37], drought [38–40], salt stress [41–45], and other adverse conditions, indicating a stronger ability to survive. Therefore, induced polyploidy is frequently used to improve plant resistance. There are many reports also confirming the excellent drought resistance of polyploids, such as in the drought stress test on diploid Pellicle Melon and its homologous tetraploid, it was found that the tetraploid P. melon has an obvious drought resistance advantage over the diploid, no matter in the growth indexes, the water status of the leaves, or the content of chlorophyll, soluble sugars, and proline. The cultivation of tetraploid P. melon is a practical method for drought-resistant breeding of P. melon [46]. In the determination of xylem hydraulic conductivity of the Chamerion angustifolium, it was also found that tetraploid C. angustifolium has higher xylem hydraulic conductivity than diploid, which proves that when drought occurs, tetraploid C. angustifolium will have a more substantial water transportation capacity, and thus faster adaptation to drought and stronger drought resistance [47]. Research on drought stress in diploid and tetraploid Brachypodium distachyon revealed that different ploidy levels of Brachypodium distachyon exhibited distinct photosynthetic characteristics. The tetraploid Brachypodium distachyon exhibited higher photosynthetic rates and greater stomatal conductance, attributed to its increased heterozygosity and genetic variability. Consequently, the tetraploid Brachypodium distachyon possessed more plasticity and environmental adaptability than its diploid counterparts [48]. The ultimate goal of artificial selection and breeding new varieties is to obtain crops with excellent growth traits, ecological adaptability, and superior stress resistance [49, 50]. It is understood that P. edulis is a kind of vine with high adaptability. Still, its water demand is very high, especially during the reproductive period, which is sensitive to water deficit. Sufficient water is necessary to support twigging and budding in the early stages of fertility, and a lack of water during the flowering and fruiting stages will directly impact fruit quality and yield [51].

In recent years, substantial research has been conducted on P. edulis; however, reports on its drought tolerance are sporadic. Based on this, this study used two different ploidy levels of P. edulis ‘Mantianxing’ as the starting material. To ensure a consistent genetic background for a valid ploidy comparison, both diploid and triploid plants were derived from the same genotype via tissue culture: the triploids were regenerated from endosperm culture of a selected mother plant. In contrast, the diploids were propagated from stem segments of the same plant. The ploidy of all triploid plants was confirmed by flow cytometry (using diploid plants as an internal reference), as reported in our previous work [52]. By selecting the optimal hormone combination for rooting diploids and triploids, we can produce consistent, stable experimental materials for subsequent drought-stress testing, ensuring the accuracy of the drought challenge. We hypothesize that triploid ‘Mantianxing’ will show superior drought tolerance to diploids under PEG-6000 induction, mediated by higher activities of SOD, POD, CAT, and lower MDA content (antioxidant defense), as well as increased soluble sugar, soluble protein, and proline (osmotic adjustment). To test this, we analyzed changes in these physiological indices and their correlations with drought tolerance. This research provides theoretical and data support for breeding drought-tolerant polyploid ‘Mantianxing’ varieties, promoting further study and industrial development of P. edulis.

Materials and methods

Plant material

P. edulis ‘Mantianxing’ seeds were sourced from the Institute of Tropical Crop Science in Yunnan Province. A diploid material with verified ploidy (2n = 2x = 18) was obtained by means of sterile embryo germination, and the endosperm culture technique was applied to successfully obtain triploid aseptic seedlings (2n = 3x = 27) of ‘Mantianxing’ [53].

Screening of aseptic seedling rooting media

Triploid sterile seedlings grown in the medium were cut about 1 cm from the top of the plant in an ultra-clean bench, then placed in 1/2 MS (with 30 g/L sucrose and 4.3 g/L agar) solid medium supplemented with different hormones. As shown in Table 1, a total of nine treatments were set up, comprising 0.1, 0.5, 0.75, 1, and 1.5 mg/L IBA and 0.1, 0.3, and 0.5 mg/L NAA, all supplemented with 0.2 g/L activated charcoal (abbreviated as AC) in the medium—the 1/2 MS medium without hormones served as the control. Seven sterile seedlings per treatment, with three replicates, were grown in the culture room at 25 °C ± 2 °C, under a 12-hour light cycle and a light intensity of 1000–1500 lx. Rooting was observed after 30 days of culture.

Table 1.

Screening formulations for rooting medium of triploid ‘Mantianxing’

Treatment	Medium formulation
1	0.2g AC + 1/2MS
2	0.1 mg/L IBA + 0.2g AC + 1/2 MS
3	0.5 mg/L IBA + 0.2g AC + 1/2 MS
4	0.75 mg/L IBA + 0.2g AC + 1/2 MS
5	1.0 mg/L IBA + 0.2g AC + 1/2 MS
6	1.5 mg/L IBA + 0.2g AC + 1/2 MS
7	0.1 mg/L NAA + 0.2g AC + 1/2 MS
8	0.3 mg/L NAA + 0.2g AC + 1/2 MS
9	0.5 mg/L NAA + 0.2g AC + 1/2 MS

The most suitable formula for rooting of triploid aseptic seedlings screened by the group and the most suitable rooting medium formula for diploid aseptic seedlings screened by the group in the previous period (0.5 mg/L IBA + 0.2 g AC + 1/2 MS) were compared to contrast the most suitable medium for the two ploidy levels, which will lay the foundation for the subsequent experiments. The sterile seedlings were cut to a height of 1 cm and transferred to the two media. Ten sterile seedlings of each ploidy were set up in three replicates, and the temperature of the culture room was 25 ± 2 ℃, light duration was set to 12 h/d, light intensity to 1000–1500 lx, and rooting was monitored after 30 days of culture.

PEG − 6000 simulated rough stress experiment

1–2 cm sterile seedlings of the triploid and diploid P. edulis ‘Mantianxing’ were selected and placed in rooting medium (0.1 mg/L NAA + 0.2 g AC + 1/2 MS), which was screened over the two-month pre-screening period to ensure the establishment of a robust root system and achieve uniform plantlet size, which is critical for consistent stress response. Then, the seedlings were placed in a liquid medium containing different concentrations of PEG-6000, followed by a 7-day stress incubation period. After 7 days, the antioxidant enzyme activities of the two ploidy levels were determined. The results are summarized in the following table. To prepare hormone-free MS liquid medium, 30 g of sugar was added per liter of medium, and no agar was included. After the MS liquid medium was prepared, polyethylene glycol 6000 (PEG-6000) was added at 0%, 20%, 30%, 40%, and 50% (w/v). After the configuration was completed, the seedlings were divided into 300 mL culture bottles, and rooted seedlings of similar size were selected, with 2 plants per bottle. Each treatment consisted of five such bottles (ten seedlings) and was conducted with three independent biological replicates, resulting in a total of 30 plantlets per treatment. For the blank control, medium without PEG was employed. The materials were then transferred to the group culture room for treatment at 25 ± 2 ℃, with a light duration of 12 h per day, a light intensity of 1000–1500 lx, and a drought stress treatment lasting 7 days.

Determination of physiological and biochemical indices

After 7 days of stress treatment, sample collection and index determination were performed. To analyze the related physiological indices of diploid and triploid ‘Mantianxing’, plants with uniform growth potential were selected, and samples were collected as follows: For the determination of soluble sugar and soluble protein contents, the middle stem and leaf segments were collected and mixed; for the determination of drought-resistant enzyme activities CAT, POD, SOD, MDA and Pro content, 4–6 upper-middle leaves were collected from each plant. Three replicates were set for each ploidy, i.e., three seedlings were selected, and the corresponding sample (mixed stem and leaf segments or leaves) from each seedling was regarded as one independent replicate. The specific sampling method is visually illustrated in Fig. 1. All indices were determined using commercial kits from Suzhou Grace Biotechnology Co., Ltd (Suzhou, China, www.geruisi-bio.com, accessed on 26 August 2025).

Fig. 1.

Schematic diagram of sampling method for P. edulis ‘Mantianxing’ seedlings with different ploidy levels under drought stress

Soluble protein content was determined using the Coomassie Brilliant Blue G-250 method with a commercial kit (Cat. No.: G0417F). Briefly, 0.3 g of fresh mixed stem and leaf segments was weighed and ground into a homogenate with 1 mL of extraction buffer. The homogenate was centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was collected as the crude sample extract and stored on ice for later use. The crude extract was diluted to an appropriate multiple with distilled water. A total of 160 µL of the diluted sample was added to an EP tube, followed by 800 µL of kit-supplied Reagent 1. After mixing thoroughly, the mixture was incubated at room temperature for 10 min. A blank control tube was set up simultaneously (160 µL of distilled water + 800 µL of Reagent 1, treated under the same conditions). All reaction solutions were transferred to 1 mL glass cuvettes, and the absorbance was measured at 600 nm using a visible spectrophotometer (Shanghai Precision Instrument Co., Ltd., model V-5800). The ΔA value was calculated as A_sample − A_blank, and the soluble protein content was computed by substituting ΔA into the standard curve equation (y = 5.8086x + 0.0322) and the formula provided in the kit.

MDA content was determined using the thiobarbituric acid (TBA) method with a commercial kit (Cat. No.: G0109W48). 0.1 g of fresh leaves was weighed and ground into a homogenate with 1 mL of kit-supplied extraction buffer on ice. The homogenate was centrifuged at 12,000 rpm for 10 min at 4℃, and the supernatant was collected and kept on ice for subsequent analysis. A total of 200 µL of supernatant was mixed with 300 µL of the kit working solution, and the mixture was incubated in a water bath at 90 ~ 95℃ for 30 min. After being taken out and cooled on ice, the mixture was centrifuged at 12,000 rpm for 10 min at 25℃. Then 200 µL of supernatant was added to a 96-well plate, and the absorbance was measured at 532 nm and 600 nm (ΔA = A₅₃₂−A₆₀₀) using an ELISA Reader (Beijing Perlong New Technology Co., Ltd., Model DNM-9602). MDA content was calculated according to the formula provided in the kit.

Soluble sugar (Cat. No.: G0501W48), Pro (Cat. No.: G0111W48) contents, and CAT (Cat. No.: G0105W48), POD (Cat. No.: G0107W48), SOD (Cat. No.: G0101W48) activities were determined using commercial kits. Some 0.1 g fresh plant samples were weighed and put in a mortar. For soluble sugar extraction, 0.8 mL of 80% ethanol was added; for the other indices (Pro, CAT, POD, SOD), 1 mL of the kit-supplied extraction buffer was used. The samples were then homogenized on ice. After centrifugation at 4℃ × 12,000 rpm for 10 min, the supernatant was collected and placed on ice for measurement. For soluble sugar content determination, 25 µL of supernatant was mixed with 75 µL of distilled water, 30 µL of the kit working solution, and 250 µL of concentrated sulfuric acid. The mixture was heated in a boiling water bath for 10 min, and 200 µL of it was transferred to a 96-well plate after cooling. For PRO content determination, 150 µL of supernatant was mixed with 150 µL of glacial acetic acid and 300 µL of kit Reagent 1, heated in a 95℃ water bath for 30 min, and 200 µL of the mixture was transferred to a 96-well plate after cooling. For CAT, POD, and SOD activity determination, appropriate volumes of supernatant were mixed with corresponding kit-supplied reagents in proportion. All indicators were measured for absorbance using an ELISA Reader (Beijing Perlong New Technology Co., Ltd., DNM-9602) at 620 nm (soluble sugar), 520 nm (Pro), 470 nm (POD), 450 nm (SOD), and 510 nm (CAT), respectively. After calculating the ΔA values, the contents or enzyme activities were computed according to the formulas provided in each kit.

Data processing and analysis

Data processing was conducted in Excel (2021), while one-way and two-way analysis of variance (ANOVA) were performed using SPSS 20.0, prior to performing the normality of all experimental data was tested using the Shapiro-Wilk test, and the homogeneity of variances was verified via Levene’s test. Multiple comparisons were performed using Duncan’s method, and all data were expressed as mean ± standard error. Diagrams were plotted using Origin (2021) and Adobe Illustrator (2021).

Results and analysis

The impact of different hormones on rooting in triploid ‘Mantianxing.’

After triploid ‘Mantianxing’ was cultured with various hormone types and concentrations for 30 days, the rooting rate, main root length, number of lateral roots, and lateral root length were measured. These data helped identify the most suitable rooting formulations for group-cultivated triploid ‘Mantianxing’ seedlings. As shown in Table 2, the triploid ‘Mantianxing’ exhibited the highest rooting rate in the medium containing 0.1 mg/L NAA + 0.2 g AC + 1/2 MS. Given that this medium, with its highest rooting rate, was optimal for generating more rooted seedlings, it was selected as the most suitable formulation.

Table 2.

Impacts of various hormones on rooting in triploid P. edulis ‘Mantianxing’

Treatment		Number of rooted plants (plants)	Number of main roots (roots)	Length of main roots (cm)	Number of lateral roots (roots)	Length of lateral roots (cm)	Average seedling height (cm)	Rooting rate (%)
1	0.33 ± 0.58b		0.33 ± 0.58d	0.60 ± 1.04d	0.67 ± 1.15c	0.08 ± 0.14a	1.56 ± 0.23 cd	4.67 ± 0.08b
2	2.67 ± 2.31ab		0.33 ± 2.89 cd	3.29 ± 2.85c	5.67 ± 5.13bc	0.33 ± 0.31a	1.17 ± 1.02d	38.00 ± 0.33ab
3	4.00 ± 1.00a		13.00 ± 4.36ab	3.48 ± 0.25c	6.00 ± 1.00bc	0.29 ± 0.04a	3.07 ± 0.25a	57.00 ± 0.14a
4	4.67 ± 2.08a		6.67 ± 2.08bcd	4.43 ± 0.5c	16.67 ± 10.69b	1.05 ± 0.15b	2.00 ± 0.17bc	66.67 ± 0.33a
5	3.33 ± 2.31ab		11.00 ± 7.81abc	0.34 ± 0.07d	4.00 ± 3.46bc	0.42 ± 0.08a	2.33 ± 0.37b	48.00 ± 0.30ab
6	3.00 ± 1.00ab		6.00 ± 2.65bcd	3.28 ± 0.25c	8.33 ± 2.52bc	0.87 ± 0.40b	2.22 ± 0.13bc	43.00 ± 0.14ab
7	6.00 ± 1.73a		19.67 ± 7.09a	6.57 ± 0.40b	16.00 ± 6.08bc	0.82 ± 0.16b	3.27 ± 0.21a	85.67 ± 0.25a
8	5.00 ± 2.65a		12.33 ± 6.81ab	12.83 ± 1.61a	37.00 ± 19.31a	2.80 ± 0.30a	2.32 ± 0.10b	71.33 ± 0.38a
9	4.67 ± 1.15a		7.00 ± 1.00bcd	3.26 ± 0.19c	10.00 ± 2.00bc	0.92 ± 0.11b	1.74 ± 0.05bcd	66.67 ± 0.17a

Note: Data in the table are mean ± SD, n = 3; Significant differences within the same column were determined by one-way ANOVA followed by Duncan’s multiple range test (p < 0.05) and are indicated by different lowercase letters. The same as below

Rooting of diploid and triploid ‘Mantianxing’ under optimum medium

According to Table 3, the medium with the highest rooting rate for triploid ‘Mantianxing’ was identified and compared with the medium with the highest rooting rate for diploid ‘Mantianxing’ from the previous stage. The most suitable medium for rooting both ploidy levels of ‘Mantianxing’ was selected—this is the rooting formula for ‘Mantianxing’. As shown in Table 3, treatments AD and AT involved rooting diploid and triploid ‘Mantianxing’ in a formulation (0.1 mg/L NAA + 0.2 g AC + 1/2 MS) that was most suitable for the triploid ‘Mantianxing’. Treatments BD and BT involved rooting diploid and triploid ‘Mantianxing’ in the optimal diploid ‘Mantianxing’ formulation (0.5 mg/L IBA + 0.2 g AC + 1/2 MS) for 30 days. From the rooting rate results, it is clear that the best triploid ‘Mantianxing’ rooting medium identified at this stage is most suitable for rooting both diploid and triploid ‘Mantianxing’. This medium was used to root cultures in subsequent experiments.

Table 3.

Effect of two rooting medium formulations on rooting in diploid and triploid P. edulis ‘Mantianxing’

Treatment	Number of rooted plants (plants)	Number of main roots (roots)	Length of main roots (cm)	Number of lateral roots (roots)	Length of lateral roots (cm)	Average seedling height (cm)	Rooting rate (%)
AD	6.67 ± 1.15a	15.33 ± 3.06a	12.17 ± 4.37a	52.33 ± 12.01a	1.03 ± 0.21a	3.47 ± 1.04a	66.67 ± 0.12a
AT	8.33 ± 0.58a	25.33 ± 8.39a	6.10 ± 1.49a	57.33 ± 8.08a	1.03 ± 0.64a	3.27 ± 0.50a	83.33 ± 0.06a
BD	5.33 ± 2.08a	10.67 ± 4.51a	5.94 ± 0.68a	16.33 ± 7.51a	1.56 ± 0.33a	2.47 ± 0.40a	53.33 ± 0.21a
BT	7.33 ± 0.58a	17.33 ± 1.53a	6.46 ± 0.83a	35.33 ± 3.51a	0.43 ± 0.09a	3.03 ± 0.15a	73.33 ± 0.06a

Note: Treatment codes correspond to combinations of ploidy and formulation: “D” stands for diploid, “T” for triploid; “A” refers to the formulation (0.1 mg/L NAA + 0.2 g AC + 1/2 MS), and “B” to the formulation (0.5 mg/L IBA + 0.2 g AC + 1/2 MS). For instance, “AD” indicates diploids cultured in formulation A, with other codes following this logic. Data in the table are mean ± SD. Within the same formulation, different letters denote significant differences between treatments at the p < 0.05 level for both ploidies of ‘Mantianxing’

Variations in soluble sugar and soluble protein levels under drought stress

Under varying PEG-6000 concentrations, soluble sugar levels in both ploidies of ‘Mantianxing’ increased but with differing trends, as shown in Fig. 2. The increase in the amplitude for diploid P. edulis was greater than for the triploid. When the PEG-6000 concentrations were 20%, 30%, and 40%, the soluble sugar contents of diploid P. edulis were higher, reaching 0.26, 0.29, and 0.26 mg/g, respectively, which were 18.18%, 31.82%, and 13.04% higher than those of triploid (0.22, 0.22, and 0.23 mg/g). Conversely, at 0% and 50% PEG-6000, the triploid ‘Mantianxing’ exhibited higher levels (0.17 and 0.25 mg/g) than the diploid (0.09 and 0.16 mg/g), corresponding to increases of 88.89% and 36%. Severe drought stress (50% PEG-6000) caused a significant reduction in diploid soluble sugar content, whereas the triploid showed a non-significant increase.

Fig. 2.

Changes in soluble sugar content of diploid and triploid ‘Mantianxing’ under PEG-6000 treatment. Data are presented as mean ± SE (n = 3). Different capital letters indicate that the differences between the two ploidy ‘Mantianxing’ at each concentration are significant at the p < 0.05 level, and other lowercase letters indicate that the discrepancies between different concentrations of ploidy ‘Mantianxing’ are also crucial at the p < 0.05 level, the same as below

Both ploidy levels of ‘Mantianxing’ showed increased soluble protein content under different concentrations of PEG-6000 treatment. However, the trend of increase differed: as shown in Fig. 3, with rising stress concentration, the soluble protein content in diploid ‘Mantianxing’ increased significantly, while the triploid ‘Mantianxing’ showed a non-significant trend, although it still risen from 0% to 50% (18.68, 19.07, 19.40, 20.42, 21.11 mg/g), with slight fluctuations. Starting from 20% PEG-6000 concentration, the soluble protein levels of diploid ‘Mantianxing’ were significantly higher than those of the triploid across all treatments. It indicates that under drought stress, the diploid ‘Mantianxing’ primarily maintains cellular osmotic balance and protects against damage by increasing soluble protein content. Triploids respond differently to stress than diploids due to genetic changes that may result from ploidy alteration, leading to a different mode of plant regulation.

Fig. 3.

Changes in soluble protein content of diploid and triploid ‘Mantianxing’ under PEG-6000 treatment

Changes in enzyme activity content under drought stress

Under PEG-6000-induced drought stress, SOD activity in both ploidies of ‘Mantianxing’ exhibited a trend of first increasing and then decreasing, as shown in Fig. 4. The triploid ‘Mantianxing’ showed the highest SOD activity at 20% PEG-6000, which was 24.34% higher than the control (CK). After this point, SOD activity began to decline. Similarly, the diploid ‘Mantianxing’ also peaked at 20% PEG-6000, with a 22.41% increase compared to CK, followed by a decrease. This suggests that a plant’s capacity to acclimate to adverse conditions cannot persist indefinitely. When stress becomes too intense, the plant’s regulatory capacity decreases, resulting in reduced SOD enzyme activity.

Fig. 4.

Changes in diploid and triploid ‘Mantianxing’ SOD activity content under PEG-6000 treatment

Overall, under simulated drought stress with PEG-6000, triploid ‘Mantianxing’ consistently exhibited higher SOD activity than the diploid, with both ploidies showing a similar trend in SOD activity. However, there was no significant difference in SOD enzyme activity between the two ploidy levels of ‘Mantianxing’ across treatments. This reflects that changes in ploidy had little effect on the SOD enzyme activity of the triploid.

As shown in Fig. 5, the POD enzyme activities of both ploidy ‘Mantianxing’ under PEG-6000 drought stress treatment displayed a continuous upward trend. The POD enzyme activity of triploid ‘Mantianxing’ at 50% stress concentration was 2.29 times higher than that of diploid and 6.63 times higher than CK. The POD enzyme activity in diploid ‘Mantianxing’ reached 50%, which was 4.33 times that of CK. In all five treatments, the POD enzyme activity of triploid ‘Mantianxing’ was consistently higher than that of the diploid, with a greater increase. When the drought level reached 20%, the POD enzyme activity content of triploid ‘Mantianxing’ was significantly higher than that of the diploid. Elevated gene dosage resulted in notably greater POD activity in triploid plants compared to diploids following drought stress, suggesting that the triploid’s capacity to scavenge hydrogen peroxide strengthened as drought severity increased.

Fig. 5.

Changes in POD activity content of diploid and triploid ‘Mantianxing’ under PEG-6000 treatment

With increasing drought severity, the change in CAT enzyme activity in the two-ploidy ‘Mantianxing’ displayed a pattern similar to that of SOD enzyme activity, with both initially increasing and then decreasing. As indicated in Fig. 6, the CAT enzyme activities of both ploidy levels of ‘Mantianxing’ reached their peak values of 191.93 µmol/min/g FW and 372.76 µmol/min/g FW when the concentration of PEG-6000 was 20%. The CAT enzyme activities of triploids of ‘Mantianxing’ were 1.94 times higher than those of diploids and 2.31 times higher than those of CK at this concentration. At this concentration, the CAT activity of triploid ‘Mantianxing’ was 1.94-fold higher than that of the diploid and 2.31-fold higher than the CK group. When the concentration of PEG-6000 exceeded 20%, the CAT enzyme activity of both ploidies of ‘Mantianxing’ began to decline, indicating that the two ploidies shared similar characteristics. The CAT enzyme activity began to lose its regulatory ability at a drought stress concentration of 20%. The CAT content also decreased, aligning with the change in SOD content. Additionally, the CAT enzyme activity in triploid ‘Mantianxing’ under drought stress showed an additive benefit after gene doubling.

Fig. 6.

Changes in the CAT activity content of diploid and triploid ‘Mantianxing’ under PEG-6000 treatment

Changes in malondialdehyde content under drought stress

MDA, a terminal product of membrane lipid peroxidation, is closely linked to the degree of plant cell damage: greater adversity-induced injury generally leads to higher MDA accumulation. As depicted in Fig. 7, under PEG-6000-induced drought stress, MDA levels in both ploidies of ‘Mantianxing’ rose steadily. Notably, diploid ‘Mantianxing’ consistently had higher MDA levels than the triploid, implying that triploid P. edulis ‘Mantianxing’ suffered less cellular damage than the diploid under drought stress.

Fig. 7.

Changes in MDA content of diploid and triploid ‘Mantianxing’ under PEG-6000 treatment

Changes in proline content under drought stress

Proline serves as a key osmotic regulator in plants; under drought conditions, plants typically modulate proline levels to reduce drought-induced harm. As shown in Fig. 8, the diploid ‘Mantianxing’ proline content increased, then decreased, as drought stress intensified. The plant raises its proline levels to maintain balance in response to environmental stress. However, the proline content of diploid ‘Mantianxing’ peaked at 30% of the PEG-6000 concentration and then declined, possibly due to severe drought conditions that the plant itself cannot regulate.

Fig. 8.

Changes of Pro content in diploid and triploid ‘Mantianxing’ under PEG-6000 treatment

The proline content in triploid ‘Mantianxing’ rose as drought stress intensified, but it began to decline at the 40% stress level, with the decline non-significant. In the absence of PEG-6000, the proline content of diploid ‘Mantianxing’ was higher than that of the triploid. After drought stress treatment, the proline content of triploid ‘Mantianxing’ increased rapidly, except at 20% and 30% PEG-6000 levels. The proline content of triploid ‘Mantianxing’ increased rapidly after drought stress and was significantly higher than that of diploid under most drought conditions, except at 20% and 30% PEG-6000.

It is commonly accepted that plants with stronger stress resistance synthesize greater amounts of proline under adverse conditions, which helps lower water potential and thereby preserve osmotic equilibrium. The triploid ‘Mantianxing’ apparently produces higher levels of proline when subjected to severe drought stress, and the increased ploidy gives it a more drought-resistant advantage.

An exploratory Pearson correlation analysis was conducted on pooled data from all treatments (n = 18, comprising 6 treatments × 3 independent biological replicates), with each data point derived from an independently harvested plant sample to ensure statistical independence. The physiological indicators of P. edulis show both conserved and ploidy-specific correlation patterns. Conserved patterns include the positive correlations among soluble protein, soluble sugar, POD, MDA, and Pro, as well as the negative correlation between SOD and POD, which are significant (p ≤ 0.05) in both triploid and diploid, potentially reflecting fundamental physiological regulatory mechanisms. Ploidy-specific variations, such as the stronger positive association between soluble sugars and POD in triploids and differences in the intensity of proline-related correlations, indicate that triploidy may alter the coordination among physiological processes, potentially influencing metabolic efficiency or stress responses compared to diploid P. edulis.

The correlation analysis heatmap in Fig. 9 shows that diploid and triploid P. edulis exhibit broadly similar correlation patterns across multiple indicators. Soluble proteins and sugars are positively associated with POD, whereas MDA exhibits a significant positive association with soluble sugars. SOD is significantly negatively correlated with POD and is negatively related to soluble proteins. Ploidy-specific differences, such as the stronger positive correlation between soluble sugars and POD in triploids and the varying strength of Pro correlations, suggest that triploids may alter the coordination of physiological processes. Compared to diploid P. edulis, this could potentially affect metabolic efficiency or stress responses.

Fig. 9.

Correlation analysis of diploid and triploid indicators of P. edulis. The size of the circle represents the absolute value of the Pearson correlation coefficient. * means p ≤ 0.05. Analysis was performed on pooled data from all treatments (n = 18). This is an exploratory analysis without multiple-testing correction; results are presented as preliminary associative patterns

Discussion

Rooting culture of purple passion fruit ‘Mantianxing.’

Rooting of aseptic seedlings often uses adventitious shoots or stem segments for rooting, thus the rooting of aseptic seedlings is adventitious primarily, the root primordium is formed through the stimulation of exogenous hormones, and supplementing plant growth regulators in the medium may facilitate root formation [54], the plant growth regulators include cytokinins and schizophyllins [55], and the cytokinins mainly promote the formation of axillary buds in the cell division. They are mostly added during proliferation. In plant rooting culture, growth factors are often used to promote rooting. NAA (naphthalene acetic acid) and IBA (indole butyric acid) are widely used growth regulators for inducing rooting in sterile seedlings. They can regulate stem growth rate, suppress lateral buds, and promote rooting, with this rooting-promoting effect being a key application [56–58].

Different plants exhibit varying sensitivities to growth-regulating hormones, necessitating the screening of different hormone types and formulations to achieve optimal rooting results [59]. Comparing the effects of two hormones (NAA and IBA) on the rooting of two ploidy varieties, the following results were observed: for ‘Mantianxing’, NAA exhibited a superior rooting effect compared to IBA. Under the same hormone formulation, the rooting impact of the triploid ‘Mantianxing’ was better than that of the diploid P. edulis.

Effect of drought stress on the two ploidy levels of P. edulis ‘Mantianxing.’

Although this study was conducted under PEG-simulated drought stress, it cannot fully replicate field drought conditions. This is because natural drought involves more complex, multifactorial processes, such as uneven water distribution, reduced soil aeration, temperature fluctuations, and alterations in nutrient availability and microbial dynamics [60]. Consequently, further research is necessary to evaluate the performance of these methods under actual field conditions. The experimental system using in vitro-derived plantlets and abrupt PEG exposure offers the key advantage of high experimental control, ensuring uniform seedling physiological status and precise quantification of osmotic stress responses by eliminating heterogeneous environmental interference. Yet this system has inherent limitations: in vitro plantlets, acclimated to a sterile, low-stress culture environment, may exhibit distinct physiological responses to PEG stress compared with soil-grown seedlings; meanwhile, abrupt PEG-induced acute osmotic shock differs from gradual natural soil drought, and PEG may exert specific physicochemical effects independent of osmotic stress [34, 61], these factors collectively limit the direct extrapolation of our results to field drought scenarios. Nonetheless, PEG remains a valuable tool for comparative assessment of drought tolerance at the seedling stages, as demonstrated in this study.

Under drought stress, soluble sugars and soluble proteins in plants, along with osmoregulatory substances such as proline, help maintain cellular osmotic pressure. The accumulation of these two substances is closely linked to the physiological processes underlying plant stress resistance [62, 63]. As drought stress intensified, the soluble sugar content in triploid ‘Mantianxing’ rose, though the fluctuation was slight, and the increase in soluble protein was also insignificant. The growth of soluble sugar content of diploid ‘Mantianxing’ increased firstly and then decreased, which may be the result of the plant’s drought environment in which the Leaf starch hydrolysis was enhanced, which led to the accumulation of sugar [64–66], with the aggravation of drought stress, the plant osmotic system suffered damage, and the accumulation of soluble sugar began to decline. Still, during severe drought, the soluble sugar content in triploid ‘Mantianxing’ remained stable, without a decline. The triploid plants had a stronger osmotic adjustment capacity and consequently showed reduced sensitivity to drought stress relative to diploids. The soluble protein levels in diploid ‘Mantianxing’ increased with the aggravation of drought stress, and the ability of diploid ‘Mantianxing’ to accumulate soluble proteins appeared to be stronger than that of the triploid when the plant was subjected to an adverse environment. Unlike the diploid’s strategy of active soluble protein accumulation, the soluble protein content in triploid ‘Mantianxing’ remained relatively stable under drought stress, which may correspond to a different drought adaptation mechanism.

Under normal circumstances, the ROS content in the plant body and the reactive oxygen species scavenging mechanism are in a state of dynamic equilibrium, when the plant is subjected to external stress [67–70], this equilibrium is broken, the ROS accumulates in substantial amounts, the extent of cellular lipid peroxidation becomes more severe^[44], and a large quantity of MDA is secreted. The greater the amount of MDA, the more severe the plant damage [71–76]. In the present study, we used PEG-6000 to simulate drought stress on sterile seedlings at two ploidy levels of ‘Mantianxing’. With the deepening of drought, the MDA content in the sterile seedlings continued to rise, and the MDA content of diploid ‘Mantianxing’ was always greater than that of the triploid in different treatment concentrations. The MDA content was always greater than that of the triploid, indicating that under drought stress, cellular damage in triploid passion fruit ‘Mantianxing’ was less severe than in diploids, with the former showing stronger drought resistance.

In this study, SOD and CAT activities showed a tendency to rise first and then decline as stress increased, whereas POD activity continued to grow. Both SOD and CAT indices reached their maximum values at 20% PEG-6000. Then they began to decline, indicating that ROS levels increased in the plant body under mild drought stress. Meanwhile, SOD, POD, and CAT rapidly accumulated in the plant, allowing it to remove free radicals and restore dynamic balance quickly. However, during severe drought, the accumulation of reactive oxygen species (ROS) in the plant body was excessive, leading to plasma membrane damage, a disorganized antioxidant enzyme system, and a decline in the activities of SOD and CAT. The plant could not maintain normal growth due to its regulatory issues. SOD, POD, and CAT enzyme activities of triploid ‘Mantianxing’ showed superiority over diploid under PEG-6000 simulated drought stress. This is similar to the results of studies on the physiological characteristics of Populus ussuriensis Kom. with different ploidy levels under salt stress.

Under salt stress, the SOD and POD levels of triploid and tetraploid pine trees are higher than those of diploid P. ussuriensis, and the antioxidant systems of polyploid pine trees are stronger than those of diploid P. ussuriensis [77]. Studies on diploid and tetraploid P. edulis subjected to low-temperature stress suggest that polyploid plants have advantages in antioxidant enzyme activity (such as SOD, POD, and CAT), which is consistent with the increased SOD, POD, and CAT activity observed in this study [78]. In addition to the antioxidant system, triploid ‘Mantianxing’ proline content was higher than that of diploids under drought stress. In general, in the face of adversity, plants accumulate a large amount of proline to maintain cellular water balance, stabilize the structure of biomolecules, and ensure normal plant growth [79]. In drought stress, the triploid ‘Mantianxing’ accumulated a large amount of proline in its body at high stress concentration. At the same time, diploid ‘Mantianxing’ decreased its proline content in its body at high stress concentration, suggesting that under severe drought, triploid ‘Mantianxing’ may have a stronger ability to tolerate drought.

In conclusion, this study provides the first systematic comparison of physiological responses between diploid and triploid P. edulis under a drought stress gradient. By integrating measurements of oxidative damage (MDA), key antioxidant enzymes (SOD, POD, CAT), and major osmoregulatory substances, and employing correlation analysis, we offer a holistic view beyond isolated trait comparisons.

These results collectively reveal a dynamic, stress-intensity-dependent strategy in triploids. Under mild to moderate drought, their superior antioxidant defense (evidenced by higher POD/CAT and lower MDA) appears dominant. Under severe drought, as enzymatic defenses decline, robust osmotic adjustment (maintaining soluble sugars and high proline) becomes critical, likely working in concert with non-enzymatic mechanisms to mitigate oxidative damage. This integrated strategy explains the mixed pattern (e.g., unchanged SOD, higher diploid soluble protein). It underscores that triploid drought tolerance stems from the flexible prioritization of different protective mechanisms across a stress gradient, rather than a uniform enhancement of all traits. Thus, our study highlights strategic flexibility, rather than across-the-board enhancement, as a core component of triploid drought tolerance. This refines the general understanding of polyploid advantage in stressful environments.

Conclusion

In the current study, the optimal rooting medium for diploid and triploid P. edulis ‘Mantianxing’ was determined to be 0.1 mg/L NAA + 0.2 g AC + 1/2 MS. This research assessed and elaborated on the variations in physiological and biochemical characteristics between the two ploidy types of P. edulis ‘Mantianxing’ under simulated drought stress. Findings revealed that triploid P. edulis maintained higher levels of soluble sugars, soluble proteins, and proline, which help regulate cellular osmotic pressure. Meanwhile, their MDA levels remained consistently lower than those in diploid plants. Furthermore, triploid P. edulis showed enhanced activity of antioxidant enzymes (SOD, POD, and CAT), which help eliminate excessive ROS, thereby exhibiting stronger overall adaptability to drought conditions. These results deepen the understanding of how polyploid P. edulis ‘Mantianxing’ responds to drought stress and shed light on the physiological and biochemical mechanisms underlying the performance of triploid plants under such stress. The study lays a foundation for germplasm innovation in P. edulis, enriches knowledge of polyploid resistance to adverse environments, and provides valuable theoretical references for polyploid breeding. This work holds significant implications for the development of related industries in arid and semi-arid areas.

Authors’ contributions

**Zhang Xiaoyue: ** Writing – original draft, Methodology, Investigation, Data Curation. **Zhu Yajing: ** Methodology, Investigation, Data Curation. **Cai Nianhui: ** Writing – review & editing, Supervision, Methodology. **Xu Yulan: ** Writing – review & editing, Supervision, Project administration, Methodology, Funding acquisition.

Funding

This work was supported by the Project of Science and Technology Talent and Platform Program of Yunnan Provincial Department of Science and Technology (grant no. 202605AF350003), the Yunnan Provincial First-Class Discipline Construction Fund for Forestry at Southwest Forestry University (grant no. 05000-524005), Yunnan Graduate Tutor Team Building Project (grant no. 2024-61), and Research and Innovation Fund Project for Postgraduate Students of Southwest Forestry University (grant no. YJS-KCJJ-2025-31).

Data availability

The data are available from the corresponding author.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.