Artificial Polyploidization Enhances Morphological, Physiological, and Biological Characteristics in Melothria scabra Naudin

Abstract

Cucamelon (Meltric scabra Naudin, Cucurbitaceae) is a climbing, herbaceous perennial plant with various culinary and medicinal uses. The current study aimed to develop novel autopolyploid genotypes of M. scabra through in vitro polyploidization and assess their morphological and phytochemical characteristics. For polyploid induction, oryzalin was employed as an antimitotic agent, and it was applied at various concentrations (40, 60, and 80 μM) and durations (24 and 48 h). Flow cytometry analysis confirmed the successful induction of polyploids, with polyploidization efficiency ranging from 2.5% to 15%. From a total of 240 treated plants, a total of 20 autotetraploid plants were obtained. The obtained polyploid and control diploid genotypes were cultivated under greenhouse conditions. Further, the plants were transferred to field conditions, and the leaves, flowers, and fruits were harvested to be evaluated for the morphological, biochemical, and biological activity variations among the obtained genotypes. Morphological comparisons between diploid and autotetraploid plants revealed significant differences in flower characteristics, fruit attributes, and leaf morphology. Nutritional evaluation demonstrated enhancement of key parameters in the induced polyploids compared to the diploid plants, including glucose, fructose, carotenoid, polyphenol, and antioxidant contents, highlighting the potential impact of polyploidization on these traits. The results from this study highlight the potential of artificial chromosome doubling as an effective breeding strategy in M. scabra and related plant species.

1. Introduction

Cucamelon, also known as Mexican miniature watermelon (Melothria scabra Naudin), is a horticultural crop from the Cucurbitaceae family [1]. It is a diploid (2n = 2× = 24) plant species that originated from Mexico and Central America but has spread widely across other regions of the world [2]. Cucamelon is cultivated for its diminutive, edible fruit, which resembles a miniature striped watermelon (Citrullus lanatus). Morphologically, the fruit is oval with an average length of 2.4 cm and a width of 1.5 cm and is consumed either in its raw form or processed as pickles and vegetables [1,3]. The fruits of M. scabra contain alkaloids, tannins, flavonoids, terpenoids, and saponins [1]. Apart from these phytochemicals, cucamelons possess numerous ethnopharmacological properties. For example, the leaves of these plants have antidiabetic activity [4], it is used to reduce blood pressure in Sulawesi medicinal folklore, and the extract of cucamelon plant is also applied as a mosquito repellent in some parts of the world [1].

Although cucamelons have numerous uses, they have not benefited from systematic breeding attempts, unlike the major crops like wheat or rice. This neglect presents a compelling opportunity to explore the untapped potential of this crop. In this scenario, artificial chromosome doubling using antimitotic agents offers an exciting avenue. Artificial chromosome doubling or polyploidization is a fast and cost-effective approach in plant breeding for generating novel genotypes with superior traits in relatively less time [5,6,7,8]. Artificial polyploidy induction has been a great breeding strategy, particularly in horticultural crops [9], where it has successfully yielded genotypes with novel morphological, physiological, and phytochemical characteristics [10,11,12,13].

The most common antimitotic agent used to induce autopolyploid plants is colchicine due to its thermostability and effectivity [12,14]. However, due to its toxic nature and higher binding affinity to animal cell tubulin protein than to plant tubulin, other antimitotic agents like oryzalin are preferred [13,15]. In recent years, the application of oryzalin has gained popularity for chromosome duplication due to its higher success compared to the predominantly used colchicine, particularly within the Cucurbitaceae family [16,17,18,19]. For example, a previous study reported a comparably high tetraploid induction efficiency (up to 14%) in Cucumis melo var. Makuwa, leveraging the application of oryzalin in conjunction with amiprophos-methyl (APM), when compared to traditional colchicine treatment [20]. Similarly, another study noted a higher effectiveness of chromosome doubling in Cucumis sativus L. through the utilization of oryzalin in contrast to trifluralin and colchicine in their study [16]. These collective findings underscore the promising potential of oryzalin as a valuable alternative for efficient polyploid induction and its relevance to crop improvement and breeding programs.

Artificial polyploidization has never been carried out in M. scabra to date. However, several attempts towards polyploid induction in plants from the Cucurbitaceae family have been made. For instance, in Cucumis melo L., polyploids were successfully induced using oryzalin, where the obtained polyploid plants exhibited substantial morphological changes in seeds, leaves, and stomata compared with the diploid mother plants [18]. A similar study on the same species reported a higher total soluble solid content in tetraploid fruits than in diploid fruits [20]. In Cucumis sativus L., polyploids were produced using colchicine that displayed larger leaf size, flower diameter, stoma size, pollen grains, and more chloroplast numbers in guard cells [21]. In the same species, doubled haploid (DH) plants were obtained using colchicine, trifluralin, and oryzalin. The DH plants obtained exhibited larger leaves, flowers, and fruit sizes compared to their haploid relatives [16]. Polyploidy was also induced in Citrullus lanatus (Thunb.) Matsum and Nakai by colchicine treatment where the obtained polyploid plants had broader and thicker leaves of dark green color, and larger stomata, pollen grains, seeds, and fruits than their respective diploids [22,23]. Along with the morphological parameters, polyploidization could also influence the ratio of existing components in the phytochemical profile of the plants. For example, autotetraploidy in Cichorium intybus L. significantly influenced the concentrations of total phenolic compounds and chlorogenic acid in its leaves [24]. In Thymus vulgaris L., the essential oil yield and thymol content demonstrated notable increases [12], while the levels of macro- and micronutrients saw a rise in Callisia fragrans (Lindl.) Woodson [15].

Considering these developments in the Cucurbitaceae family, it could be hypothesized that artificial chromosome doubling could be an effective approach for obtaining genotypes with novel and desirable traits. Hence, the objective of this study was to obtain the autopolyploid plants in M. scabra Naudin by in vitro polyploidization using oryzalin as an antimitotic agent for the first time. This study also aimed to examine the influence of polyploidization events on the morphological, biochemical, and anatomical attributes in cucamelons. The findings obtained in this present study will serve as a foundation for future breeding attempts in this and related species. To the best of our knowledge, this was the first attempt at induced polyploidization in M. scabra.

2. Materials and Methods

2.1. Plant Material Acquisition and In Vitro Transfer

Plantlets of M. scabra were sourced from the plant collection at the Faculty of Tropical AgriSciences, Czech University of Life Sciences Prague, Czech Republic. These plants were maintained in a 5 × 5 cm plastic pot within a greenhouse environment. The potting mix comprised sand, soil, peat moss, and vermiculite in a 1:1:1:1 ratio. The greenhouse maintained an average temperature of 23 °C, with relative air humidity levels fluctuating between 70% and 80%. Nodal segments were harvested from these maintained plants and subjected to surface sterilization. Surface sterilization included washing the nodal segments under running tap water for 10 min, followed by 1 min 70% ethanol wash and 10 min treatment with a 1% NaCl solution containing two drops of Tween 20. Finally, the nodal segments were rinsed thrice before transferring onto a basic Murashige and Skoog (MS) medium [25] without phytohormones. The explants were grown at 25/20 ± 0.5 °C under 16 h light/8 h darkness photoperiod, controlled automatically. Illumination intensity was 3800 lux (51.3 µmol m⁻² s⁻¹) from cool white fluorescent lamps. The plants were subcultured every 30 days until a sufficient number of plants were achieved for polyploid induction.

2.2. Autopolyploid Induction

The polyploidy in M. scabra was induced using the nodal segments. The nodal segments were treated with 40, 60, and 80 μM of oryzalin for 24 and 48 h using the submersing method previously described [12]. The oryzalin solutions were prepared by dissolving an appropriate amount of oryzalin in a 1% DMSO (dimethyl sulfoxide) solution. A total of 40 nodal segments were treated in each concentration. After the treatment, the nodal segments were removed from the oryzalin solution, rinsed three times in sterilized distilled water, and cultivated individually in 100 mL Erlenmeyer flasks on the MS basal medium for the regeneration of the new shoots. Three months after multiplication, the ploidy level was determined by a flow cytometer (Partec GmbH, Münster, Germany).

2.3. Flow Cytometry Analysis

The flow cytometry analysis was performed according to a previous study with slight modifications [12]. Approximately 1 × 1 cm² of leaf tissue was chopped using a sharp razor blade in a Petri dish containing 1 mL of Otto I buffer (0.1 M C₆H₈O₇ in 0.5% Tween 20). Crude suspensions of the samples containing the isolated nuclei were subsequently filtered through a nylon mesh of 50 μm mesh size. The filtered supernatant was subjected to the dyeing step, where 1 mL of Otto II buffer (0.4 M Na₂HPO₄·12 H2O) containing fluorescent dye DAPI (2 μg/mL) was added to the filtered samples. All measurements to detect ploidy levels were executed using a Partec PAS flow cytometer (Partec GmbH, Münster, Germany), where at least 10,000 nuclei were recorded. Histograms of the relative DNA content were evaluated using the Flomax software package (Version 2.3). The stability of the ploidy level was retested after 10 months.

2.4. Transfer to Ex Vitro Condition

For further evaluation of the morphophysiological parameters, two new autopolyploid genotypes (31 and 52) with stable ploidy levels were chosen, which showed good development and a great visual difference under in vitro culture conditions compared to the diploid genotype. The plants were selected and transferred to the greenhouse conditions where the average temperature was 23 °C with a relative humidity of 60–70%. The plantlets with well-developed root systems were removed from MS medium and transferred to plastic pots (5 × 5 cm) containing sand:soil:peat moss:vermiculite (1:1:1:1; v/v) mixture. The plants were maintained for 7 days covered with transparent polythene sheet under high humidity, and then the humidity was gradually lowered. The percentage of ex vitro survival was evaluated after 4 weeks.

2.5. Quantitative and Morphological Evaluation

In both autotetraploid and diploid (control) plants, various characteristics were assessed in the fruits, including weight, width, length, shape, and color. Seed attributes such as weight (weight of a hundred seeds), shape, and germination rates were also evaluated. Flower analysis involved examining size, petal count, and color. Leaves were assessed for both shape and color. All data were collected during the flowering stage and fruit harvesting period.

2.6. Determination of Dry Matter, Ash, and Crude Protein Content among the Diploid and Tetraploid Fruits

Dry matter determination was performed based on two 5 g weights of each sample into pre-dried aluminum trays, which were then placed in an oven (Memmert, Germany) heated to 103 °C for 4 h. After drying, the trays were allowed to cool in a desiccator and then weighed. The nitrogen content was evaluated according to the Kjeldahl method ISO 5983-1:2005 [26] using a Kjeltec 2400 analyzer (FOSS, Hilleroed, Denmark). The crude protein content was calculated using a nitrogen-to-protein factor of 6.25. Ash contents were analyzed after mineralization of the fruits from all the genotypes at 550 °C in a muffle furnace (Nabertherm, Germany). The analyses of the parameters above were carried out in triplicate.

2.7. Determination of Vitamin C, Sugars, and Citric Acid among the Diploid and Tetraploid Fruits

Preparation of samples for determination of vitamin C was carried out according to a previously described protocol [27]. To determine glucose, fructose, and citric acid, one gram of lyophilized sample was weighed into the 10 mL volumetric flask and 0.3 mL of Carrez reagent II (300 g of ZnSO₄·7H₂O in 1 L of demineralized water) was added, followed by 0.3 mL of Carrez reagent I (150 g of K₄[Fe(CN)6]·3H₂O in 1 L of demineralized water). The prepared solution was topped up to the mark with demineralized water. Subsequently, the solution was centrifuged in a CompactStar CS 4 centrifuge (VWR, Leuven, Belgium) and the aqueous extract was filtered with a 0.45 µm PTFE filter (Agilent, Santa Clara, CA, USA).

A liquid chromatograph 1260 Infinity II (with vial sampler, pump, and column thermostat–MCT) (Agilent, Santa Clara, CA, USA) was used for the analysis. An Aminex HPX–87H Organic Acid Analysis Column 300 × 7.8 mm (Bio-Rad, Hercules, CA, USA) with RID detector (Agilent, Santa Clara, CA, USA) was used for sugars and a DAD WR detector (Agilent, Santa Clara, CA, USA) for citric acid analysis. A total of 20 µL of the sample was injected with a column flow rate of 0.6 mL.min⁻¹ with the heat of the thermostat 55 °C. A total of 0.005 M sulfuric acid solution was used as the mobile phase. A Poroshell 120 EC-C18 2.7 µm 3.0 × 150 mm column (Agilent, Santa Clara, CA, USA) and a DAD WR detector (Agilent, Santa Clara, CA, USA) were used for vitamin C analysis. For this analysis, 20 µL samples were injected and the column flow rate was 0.3 mL.min⁻¹ with the heat of the thermostat 25 °C. The mobile phase was composed of methanol and demineralized water, in a ratio of 40:760, and 0.2 µL of H₃PO₄ (Lachema, Brno, Czech Republic) was added to adjust the pH to 3. For each of the parameters, 3 biological and 3 technical replicates were used.

2.8. Total Antioxidant Activity Comparison among the Diploid and Tetraploid Fruits

A total of 1 g of the homogenized lyophilisate was weighed into 15 mL plastic centrifuge tubes and extracted with 10 mL of methanol overnight; the solubility of the substances was supported with an ultrasonic water bath for 10 min. The samples were subsequently centrifuged (5 °C, 5 min, 3186 rcf). The supernatant was further diluted with methanol (1:1; v/v). Subsequently, 100 μL of the sample was added to 1.9 mL of methanolic DPPH solution (adjusted to absorbance A = 0.6 at 515 nm), and the contents were thoroughly mixed and left for 20 min at room temperature. Subsequently, the absorbance of the solution was measured at 515 nm (Thermo Fisher Scientific, Waltham, MA, USA). The samples were prepared in three parallel replicates and the total antioxidant activity was expressed as the average value in Trolox equivalent (TE) in µg/g sample dry weight. Three biological and three technical replicates were used.

2.9. Total Phenolic Content among the Diploid and Tetraploid Fruits

The extract was prepared in the same way as for the determination of antioxidant activity. Next, 1 mL of extract was measured into 25 mL volumetric flasks, and 1 mL of Folin–Ciocalteu reagent and 3 mL of 20% aqueous sodium carbonate solution were added. The flasks were made up to 25 mL with demi-water and left for 2 h at room temperature. Subsequently, the absorbance at 765 nm was measured against a blank. The results were expressed as average values from three parallel repetitions in gallic acid equivalent (GAE) in µg/g dry weight of the sample. Three biological and three technical replicates were used.

2.10. Analysis of Carotenoids among the Diploid and Tetraploid Fruits

Briefly, the carotenoid-containing extract obtained from 0.5 g of lyophilized sample was subjected to alkaline hydrolysis with ethanolic potassium hydroxide at room temperature for 2 h. After liquid–liquid extraction of the hydrolysate with water and an ether:hexane mixture (1:1; v/v), the organic fraction was purified with water, concentrated, reconstituted with a suitable solvent, and analyzed by HPLC-DAD. The exact procedure for sample preparation, chromatographic separation, identification, and quantification of carotenoids is described in [28]. Three biological and three technical replicates were used.

2.11. Statistical Analysis

Quantitative measurements were evaluated utilizing the Kruskal–Wallis test, a non-parametric method suitable for comparing more than two independent groups. A post hoc multiple comparison test of the mean rank order was conducted to scrutinize differences between specific groups further. For all analyses, a significance level of p < 0.05 was set to establish statistical significance, indicating substantial differences between the groups under investigation. The STATISTICA software package (Version 13.3) was used for all the analyses.

3. Results

3.1. Effect of Oryzalin on the Survival and Polyploid Induction Rate

A total of 240 nodal segments were exposed to oryzalin across three concentrations and two time durations. The survival rate of explants was evaluated after 4 weeks. The survival rate varied from 20% to 70.5%, depending on the concentration and length of exposure of oryzalin to the explant. The explant survival rate was lower at a higher concentration and a longer exposure time (20%, at 80 µM and 40 h). On the other hand, at lower concentrations and shorter duration of exposure, the oryzalin was less toxic (Table 1). The surviving and regenerated plantlets were micropropagated, and after two months their ploidy levels were determined by flow cytometry analysis (FCM). A total of 20 autotetraploid and 15 mixoploid plants were detected (Table 1). Figure 1a,b show histograms generated by FCM depicting the relative DNA content among diploid and tetraploid plants. Polyploidization efficiency of oryzalin in M. scabra ranged between 2.5% and 15% (Table 1). The highest number of polyploids was obtained in the treatment where explants were treated with 80 µM oryzalin for 40 h (six tetraploids).

3.2. Morphological Comparisons between Diploid and Tetraploids of Melothria scabra

The plants were cultivated in greenhouse conditions. Diploid and polyploid plants had vegetation periods of 120 days and 155 days, respectively. The flowering time among the polyploid genotypes differed, with the first flowers in diploid plants observed after 50 days of cultivation and in polypoid plants after 57 (for genotype 31) and 60 (for genotype 52) days of cultivation. The flowers from diploid and tetraploid genotypes varied significantly (Table 2 and Figure 2). The flowers varied in the width of the entire flower. Polyploid plants had larger flowers (36% on average) with the same number of petals as diploid plants (Table 2). The development of fruits till maturity took, on average, 17 days in control and 20 and 25 days in genotypes 52 and 31, respectively, from the beginning of flowering. There was a significant difference in the length, width, and weight of the fruit (Table 3 and Figure 3). Fruits of polyploid plants (genotype 52) were on average 10% shorter than those of diploid plants, but all polyploid plants had wider (13–15%) fruit with significantly higher weight (14–23%). However, when the seeds obtained from the fruits were compared, control fruits had the longest seeds (3.1 cm), the widest seeds (1.7 cm), and the highest average weight of 100 seeds (375.25 mg). In contrast, both polyploid genotypes 52 and 31 exhibited shorter and narrower seeds with significantly lower average seed weights (Table 3 and Figure 4). Additionally, the induced polyploids failed to germinate, whereas the diploid seeds had a germination rate of 85%. Tetraploid induction also had a significant effect on morphological traits of the leaf (Figure 5). The leaf blade margins of polyploid plants were more dentate than those of diploid plants. Changes in the color of the leaves were also distinctly different. The leaves of genotype 31 had a light green coloration, and genotype 52 showed a dark green coloration compared to their diploid forms.

3.3. Nutritional Comparisons between Diploid and Autotetraploids of Melothria scabra

Ripe fruits were collected and analyzed immediately after harvesting within a few hours. All fruits were harvested and processed under the same conditions. In this comparative study, we assessed the polyploid genotypes (genotypes 31 and 52) and compared them to the control genotype for nutritional and physiological parameters. The findings revealed significant differences among the genotypes for several key parameters. The control genotype exhibited the highest dry matter content (10.30 g/100 g fresh weight (FW)), crude protein content (21.15 g/100 g DM), ash content (4.19 g/100 g DM) and vitamin C content (14.49 mg/kg DM), while genotypes 31 and 52 generally showed lower values in these categories. However, genotypes 31 and 52 had significantly higher glucose and fructose content than the control genotype (Table 4). Citric acid was significantly similar in the control (15.26 g/100 g DM) and in genotype 52 (13.50 g/100 g DM), whereas genotype 31 (11.35 g/100 g DM) exhibited lower values. These results highlight significant variations in nutritional and physiological parameters among the polyploid and the control genotypes, emphasizing the potential impact of polyploidization on these characteristics.

3.4. Polyphenol, Antioxidant, and Carotenoid Contents

The results revealed notable differences in these biochemical parameters among the control fruits and the fruits from the induced polyploid genotypes, genotype 31 and genotype 52. The control group exhibited a polyphenol content of 1183 μg GAE/g. Notably, both genotype 31 and genotype 52 displayed significantly higher polyphenol content, with values of 2055 μg GAE/g and 2377 μg GAE/g, respectively. Conversely, the antioxidant activity of all the variants did not differ, where the control group exhibited an antioxidant content of 1509 μg TE/g. Genotype 31 and genotype 52 had a comparable antioxidant content of 1467 μg TE/g and 1527 μg TE/g, respectively (Table 5). The carotenoid content in the fruits was further analyzed by measuring the concentrations of specific carotenoids, including lutein, zeaxanthin, α-carotene, and β-carotene. Notably, genotype 52 exhibited a substantial increase in lutein content, recording a value of 68.2 μg/g DW compared to the control (62.61 μg/g DW) and genotype 31 (44.63 μg/g DW). Additionally, genotype 52 also displayed a significantly higher zeaxanthin content (3.46 μg/g DW) compared to the control (2.89 μg/g DW) and genotype 31 (2.48 μg/g DW). However, no significant differences were observed in the levels of α-carotene and β-carotene among the three groups.

In this study, the polyploid genotypes (genotypes 31 and 52) were compared with the control genotype for the content of carotenoids. The control genotype had the highest lutein content (62.61 μg/g), which was significantly different from genotype 31 (44.63 μg/g) and genotype 52 (68.2 μg/g). Zeaxanthin content did not significantly differ among the genotypes, with values ranging from 2.48 to 3.46 μg/g. Genotype 31 exhibited the highest α-carotene content (0.87 μg/g), which was significantly different from the control genotype (0.72 μg/g) and genotype 52 (0.84 μg/g). The β-carotene content did not show significant differences among the genotypes, with values ranging from 18.34 to 21.93 μg/g.

4. Discussion

Polyploidization is a natural biological process that can result in novel genetic material, giving rise to unique characteristics distinct from those found in the progenitor forms. Typically, polyploid organisms demonstrate enhanced traits with increased resilience to both biotic and abiotic stresses compared to their diploid counterparts [17]. Artificially, polyploidization as a breeding tool has long been used for the improvement of numerous plant species [9,10,11,12,13,15,29]. It is a fast, cost-effective, and reliable approach for introducing novel genotypes with desirable traits [9]. Oryzalin was used as an antimitotic agent in this study as a polyploid induction agent in M. scabra. The efficiency of polyploidization achieved using oryzalin reached up to 15%. Similar to the current study, a polyploidization rate of up to 11.11% was obtained in oriental melon when oryzalin was used as an antimitotic agent [20].

Morphological parameters are often used as the first primary screening criteria for polyploids but are generally not completely reliable. Hence, chromosome counting is considered as the most direct and accurate method to identify polyploids [30]. However, this approach has several drawbacks, including that it is time-consuming and laborious. It often needs to be optimized for individual plant species, and often the counting of smaller chromosomes leads to inaccuracies [9,30,31]. In contrast, flowcytometry is a fast and reliable approach to identifying polyploid individuals. This technique eliminates the need for labor-intensive chromosome counting, providing a rapid and reliable assessment of ploidy levels [31]. In the current study, the flow cytometric analysis employed was effective in screening out the polyploids from the treated population. While chromosome counting is a valuable complementary method, its omission did not compromise the accuracy of polyploid identification through flow cytometry.

It was elucidated from the current study that artificial polyploidization in M. scabra triggers gigantism, where the polyploid plants displayed significantly larger leaves, flowers, and fruits. The phenomenon of gigantism caused by polyploidization has been frequently reported in plants from Cucurbitaceae and other families. For instance, in C. lanatus [22,23] and C. melo [18], induced tetraploid plants exhibited significantly larger flowers, leaves, seeds, and fruit size than diploid plants. However, a study by Bae et al. [17] was not in line with these findings for tetraploid plants of C. lanatus, and the induced tetraploid plants showed small, thick, and crumpled leaves. This discrepancy suggests that the effects of polyploidization on plant morphology may vary depending on the type of plant species employed. One crucial finding from the current study was observed in the seed characteristics. The induced polyploid plants exhibited a notable decrease in the average seed weight, suggesting inadequately filled endosperm. This could potentially explain the observed failure in germination, as the seeds might lack the required nutrient reserves for germination. Further molecular and physiological investigations could provide deeper insights into the underlying mechanisms. While polyploid genotypes with chaffy seeds or kernels could pose challenges in terms of seed germination and crop establishment, the polyploid minimelon fruits with chaffy seeds could have positive implications from a consumer perspective.

The primary agronomical gain from this plant is the striped fruit, which is sweet and resembles a watermelon. The effect of polyploidization on the fruit characteristics constitutes an essential aspect from the breeding point of view. Several key parameters, including dry weight, ash weight, wet weight, vitamin C, glucose, fructose, and citric acid content, were systematically examined in the current study. The fresh weight of the fruits from the polyploid genotype displayed significantly higher values (14–23%). A higher weight of fruits can directly translate into a higher yield of a fruit crop. While the wet weight of the fruits increased significantly, the dry weight and the ash weight of the polyploid fruit were about 22–25% less than in the control. This indicates that the polyploid fruits had higher water content. Noteworthily, the polyploidization positively influenced the glucose and fructose content of the fruits from both genotypes. Glucose content increased by 73% in genotype 31 and 142% in genotype 52 compared to the control variant. Similarly, the fructose content increased by 55% in genotype 31 and 71.32% in genotype 52 compared to the control variant. This is a significant finding, as increased sugar content can enhance the sweetness and palatability of the fruits, potentially making them more appealing to consumers. Similar results where induced polyploid genotypes demonstrated higher fruit weight and elevated sugar contents of the fruits have been previously reported [32,33,34]. These enhancements could be attributed to the changes in gene expression, involving both the upregulation and downregulation of processes related to biosynthesis, transport, reception of primary and secondary metabolites, and various enzymes [34,35]. Overall, these findings highlight the potential of synthetic polyploidization in improving desired fruit traits.

In this study comparing a control genotype with two induced polyploid genotypes, several notable differences in biological activity were observed. Specifically, polyphenols were present in higher amounts in the polyploid genotypes compared to the control. Polyphenols are known to have numerous health benefits, including protection against cardiovascular diseases, diabetes, insulin resistance, and certain cancers [36,37]. Polyphenols can also act as antioxidants to fight against the oxidative damage caused to the cells [37]. While the polyphenols were higher in the polyploids, the levels of antioxidant activity among the polyploid and diploid genotypes were not significantly different. Despite having higher polyphenols, the comparable antioxidant activity among polyploid genotypes and the control genotype could be attributed to the higher amount of vitamin C (13–21%) present in the control genotype compared to the fruits from the polyploid genotypes. Vitamin C is a well-known antioxidant found in plants, vegetables, and fruits [38]. Carotenoids are natural nutrients found in fruits and vegetables that possess numerous health benefits [39]. For example, a higher lutein intake can help to remedy conditions related to eyesight, like cataracts [40]. To the best of our knowledge, the current study assessed the carotenoids, namely lutein, zeaxanthin, α-carotene, and β-carotene, for the first time in M. scabra. Lutein and β-carotene were found to be present in major portions in the fruits of the control and polyploid genotypes. The polyploid genotypes displayed a very similar profile to that of the diploid for the assessed carotenoids except for zeaxanthin in genotype 52, which had significantly higher value, and lutein, where genotype 31 exhibited lower values. Overall, the results suggest that induced polyploidy, particularly in genotype 52, can significantly impact the biological activity of these genotypes, offering potential applications in agriculture and nutrition.

Another advantage of polyploidy is that it leads to novel genotypes resistant to various biotic and abiotic stresses [41]. There are studies that report that polyploid plants have a greater resistance to biotic and abiotic stress factors. For example, in apple (Malus x domestica Borkh), the resistance of autotetraploids to Venturia inaequalis [42], Alternaria alternata, and Colletotrichum gloeosporioides [43] infection is reported in comparison to their diploid forms. Similar results were presented for Anemone sylvestris, where the autotetraploids had a better response to Phytophthora plurivora infection [10]. In the case of M. scabra in the current study, the diploid (control) plants were observed to have a fungal infection of cucurbit powdery mildew (Sphaerotheca fuliginea and Erysiphe cichoracearum). On the other hand, the polyploid plants were only slightly attacked by these fungi, even though they were growing in proximity to the diploid plants. Considering these previous findings and preliminary observations from the current study, it would not be baseless to assume that polyploid plants of M. scabra could be more tolerant to biotic factors compared to their diploid forms. However, a systematic study to confirm this hypothesis needs to be carried out. The research on the effects of polyploidy on biotic and abiotic factors could help to obtain new genotypes of horticultural crops with greater resistance, and thus obtain high quantitative and qualitative yields.

5. Conclusions

In summary, our study successfully induced polyploidy in M. scabra through the application of oryzalin, resulting in substantial morphological and biochemical transformations. The induced autotetraploid plants exhibited distinct vegetative and reproductive characteristics, including enlarged flowers, increased fruit weight, and altered leaf morphology. Nutritional analysis unveiled variations in key parameters, including an elevation in sugar levels in polyploids, underscoring the potential of polyploidization to enhance nutritional content. Moreover, the generated polyploid genotypes demonstrated enhanced biological activities. These comprehensive findings provide valuable insights into the prospective applications of polyploidy for crop enhancement, emphasizing improved nutritional quality in cucamelons and related crops. Future studies should be aimed to assess these novel genotypes for their resilience to both abiotic and biotic stresses, further advancing our understanding and potential utilization of polyploidization in agricultural contexts.

Author Contributions

Conceptualization, E.F.-C. and R.B.; Formal analysis, E.F.-C.; Funding acquisition, E.F.-C.; Investigation, R.B., T.A.J., P.Š., L.P., Z.K., M.O. and L.K.; Methodology, E.F.-C., R.B., T.A.J., P.Š., L.P., Z.K., M.O. and L.K.; Project administration, E.F.-C.; Software, R.B.; Supervision, E.F.-C.; Writing—original draft, E.F.-C. and R.B.; Writing—review and editing, R.B., P.Š., L.P., M.O. and L.K. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

Data are contained within the article.

Acknowledgments

We express our sincere appreciation to Miroslav Klíma of the Division of Crop Genetics and Breeding at the Crop Research Institute, Prague, for his invaluable assistance in preparing the figures.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

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Figures and Tables

Figure 1. Histograms of flow cytometry analysis from M. scabra: (a) control plant (diploid) and (b) polyploid plants (tetraploid).

Figure 2. Morphological variation between diploid (a) and induced tetraploid (b) flowers of M. scabra cultivated in greenhouse conditions.

Figure 3. Morphological variation between diploid (2×) and induced tetraploid (genotypes 31 and 52) fruit of M. scabra.

Figure 4. Comparison of seed morphology among diploid and polyploid genotypes.

Figure 5. Morphological variation between diploid (2×) and induced tetraploid (4×) (genotypes 31 and 52) leaves of M. scabra.

Table 1

Effect of polyploidization on nodal segments of M. scabra Naudin using oryzalin.

Treatment	Oryzalin(µM)	Number of Treated Explants	Time of Treatment(h)	Survival Rate(%)	Number of TetraploidPlants	Number of Mixoploid Plants	Polyploidization Efficiency of Tetraploids(%)
T1	4040	40	24	70.5	3	3	7.5
T2		40	48	62.5	3	1	7.5
T3	6060	40	24	60.0	3	4	7.5
T4		40	48	50.0	4	3	10.0
T5	8080	40	24	37.5	1	2	2.5
T6		40	48	20.0	6	2	15.0

Table 2

Morphological evaluation of the flower from control and polyploid genotypes.

Variant	Flower Width(mm)	Receptacle Width(mm)	Flower Height(mm)	Number of Petals
Control	8.46 ± 1.74 a	2.28 ± 0.71 a	2.89 ± 0.57 a	5 ± 0 a
Genotype 31	11.18 ± 1.24 b	2.83 ± 0.35 a	3.05 ± 0.64 a	5 ± 0 a
Genotype 52	11.5 ± 1.29 b	2.69 ± 0.57 a	3.33 ± 0.45 a	5 ± 0 a

Different letters within the same column differ significantly. Data were tested by Kruskal–Wallis test with a p-value of 0.05.

Table 3

Morphological evaluation of the fruits and seeds from control and polyploid genotypes.

Variant	Fruit Length(mm)	Fruit Width(mm)	Fruit Weight(g)	Seed Length (mm)	Seed Width (mm)	Average Weight of 100 Seeds (mg)
Control	27.1 ± 1.63 a	14.9 ± 0.64 a	3.4 ± 0.45 a	3.1 ± 0.1 a	1.7 ± 0.04 a	375.2 ± 27.5 a
Genotype 31	27.7 ± 2.42 a	17.0 ± 0.97 b	4.2 ± 0.58 b	2.9 ± 0.1 ab	1.8 ± 0.09 a	83.3 ± 7.5 b
Genotype 52	24.5 ± 2.01 b	17.2 ± 0.91 b	3.9 ± 0.39 b	2.7 ± 0.1 b	1.5 ± 0.01 b	76.2 ± 5.8 c

Different letters within the same column differ significantly. Data were tested by Kruskal–Wallis test with a p-value of 0.05.

Table 4

Nutritional evaluation of the fruits from control and polyploid genotypes of M. scabra Naudin.

Variant	Dry Weight g/100 g FW	Crude Protein g/100 g DM	Ash g/100 g DM	Vitamin C mg/kg DM	Glucose mg/100 g DM	Fructose g/100 g DM	Citric Acid g/100 g DM
Control	10.30 ± 0.07 a	21.15 ± 0.88 a	4.19 ± 0.07 a	14.49 ± 0,71 a	109.67 ± 3.04 c	8.30 ± 0.18 c	15.26 ± 0.72 a
Genotype 31	7.98 ± 0.05 b	15.74 ± 0.60 b	3.25 ± 0.06 b	12.77 ± 0.66 b	189.28 ± 0.75 b	12.84 ± 0.01 b	11.35 ± 0.19 b
Genotype 52	7.73 ± 0.12 b	16.23 ± 0.62 b	3.16 ± 0.03 b	11.96 ± 0.20 b	265.65 ± 0.75 a	14.22 ± 0.01 a	13.50 ± 0.01 a

Different letters within the same column differ significantly. Data were tested by Kruskal–Wallis test with a p-value of 0.05. DM = dry matter, FW = fresh weight.

Table 5

Comparison of polyphenols, antioxidant activity, and carotenoids content in fruits of M. scabra Naudin and its induced polyploids.

Variant	Polyphenols(μg GAE/g DW)	Antioxidant Activity(μg TE/g DW)	Carotenoids (μg/g DW)
			Lutein	Zeaxanthin	α-Carotene	β-Carotene
Control	1183 ± 26.66 c	1509 ± 42.00 a	62.61 ± 5.60 a	2.89 ± 0.29 b	0.72 ± 0.24 a	18.46 ± 4.85 a
Genotype 31	2055 ± 14.04 b	1467 ± 49.35 a	44.63 ± 2.04 b	2.48 ± 0.04 b	0.87 ± 0.11 a	18.34 ± 2.86 a
Genotype 52	2377 ± 22.27 a	1527 ± 17.95 a	68.2 ± 9.63 a	3.46 ± 0.40 a	0.84 ± 0.25 a	21.93 ± 5.79 a

Different letters within the same column differ significantly. Data were tested by Kruskal–Wallis test with a p-value of 0.05.

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