Orizalin-induced polyploidy in Capsicum annuum L.: stress-responsive proteins and breeding potential

Poliploidia induzida por orizalina em Capsicum annuum L.: proteínas responsivas ao estresse e potencial para o melhoramento

Abstract

Artificial polyploidization using β-tubulin depolymerizing agents like oryzalin represents a promising strategy for increasing genetic diversity in plant breeding programs. This study investigates the efficacy of oryzalin in inducing polyploidy in ornamental Capsicum annuum L., focusing on morphological, anatomical, cytogenetic, and molecular changes that support plant survival under toxic stress. Seeds from three genotypes (UFPB-001, UFPB-004, and Floribela) were subjected to varying oryzalin concentrations and exposure durations. While higher concentrations reduced survival rates, no lethal dose was observed, indicating lower phytotoxicity compared to colchicine. Molecular docking simulations revealed interactions between oryzalin and stress-related proteins 5YJS and 1SM4, suggesting the activation of defense mechanisms. Cytogenetic analysis confirmed polyploid induction in three plants, with rates of 25.0%, 33.3%, and 50.0% in UFPB-001, Floribela, and UFPB-004, respectively. Polyploid plants exhibited larger, thicker leaves, altered stomatal density, and increased chloroplasts per guard cell. These findings establish oryzalin as a potent polyploidy inducer, with potential applications in Capsicum breeding programs.

Keywords:
polyploidy; oryzalin; phytotoxicity; stress response; ornamental

Resumo

A poliploidização artificial utilizando agentes despolimerizantes de β-tubulina, como a orizalina, representa uma estratégia promissora para aumentar a diversidade genética em programas de melhoramento de plantas. Este estudo investiga a eficácia da orizalina na indução de poliploidia em genótipos ornamentais de Capsicum annuum L., com foco em mudanças morfológicas, anatômicas, citogenéticas e moleculares que suportam a sobrevivência das plantas sob estresse tóxico. Sementes de três genótipos (UFPB-001, UFPB-004 e Floribela) foram submetidas a diferentes concentrações de orizalina e tempos de exposição. Embora concentrações mais altas tenham reduzido as taxas de sobrevivência, nenhuma dose letal foi observada, indicando menor fitotoxicidade em comparação com a colchicina. Simulações de encaixe molecular revelaram interações significativas entre a orizalina e as proteínas 5YJS e 1SM4 relacionadas a mitigação do estresse, sugerindo a ativação de mecanismos de defesa. A análise citogenética confirmou a indução de poliploidia em três plantas, com taxas de 25,0%, 33,3% e 50,0% em UFPB-001, Floribela e UFPB-004, respectivamente. As plantas poliploides exibiram folhas maiores e mais espessas, densidade estomática alterada e aumento no número de cloroplastos por célula guarda. Esses resultados estabelecem a orizalina como um indutor eficaz de poliploidia, com potenciais aplicações em programas de melhoramento de Capsicum.

Palavras-chave:
poliploidia; orizalina; fitotoxidade; resposta ao estresse; ornamental

1. Introduction

Capsicum annuum L., one of the 38 species in the Capsicum genus (GRIN, 2024), is economically significant due to its diverse applications in food, industry, pharmaceuticals, and ornamentals. As a diploid species with a chromosome number of 2n = 2x = 24 (Dagnoko et al., 2013; Sousa et al., 2015), it is a prime candidate for breeding programs aimed at enhancing ornamental traits. The development of novel and visually appealing phenotypes is crucial for ornamental plant markets, where consumer preferences drive the demand for unique genetic varieties (Ascough et al., 2008; Lima et al., 2023; Pessoa et al., 2024).

Polyploidy has long played a central role in plant evolution, increasing genetic diversity and enabling species to adapt to diverse ecosystems. Artificial induction of polyploidy using chemical agents has emerged as a powerful tool for generating novel genotypes and phenotypes, providing desirable traits such as enhanced morphological characteristics, stress tolerance, and adaptability. These features can significantly benefit producers by increasing productivity and market value through the distinctive attributes of polyploid plants compared to their diploid counterparts (Chambhare and Nikam, 2021; Tossi et al., 2022; Yan et al., 2022).

Despite its utility, artificial polyploid induction commonly relies on β-tubulin depolymerizing agents that are highly toxic to the environment, human health, and plant tissues. Colchicine remains the most widely used agent for inducing polyploidy in plants, but its extreme toxicity has raised concerns about the sustainability of this approach (Tammu et al., 2021; Kulkarni and Borse, 2010).

To address these concerns, there is a growing need to develop protocols using less toxic and cheapers alternatives, such as oryzalin (Chemical Safety, 2024). Oryzalin has already shown promise in inducing polyploids in related species, such as eggplant (Solanum melongena) via leaf explants (Çeğil and Çürük, 2019) and Capsicum frutescens via seeds (Pliankong et al., 2017). However, the mechanisms that enable survival and adaptation in seeds exposed to oryzalin remain poorly understood, particularly in C. annuum.

This study aimed to evaluate the efficacy of oryzalin as a polyploid-inducing agent in ornamental pepper. We investigated the morphological and anatomical changes resulting from polyploidy and explored the molecular defense mechanisms activated in response to oryzalin-induced toxicity. By examining the molecular interactions between oryzalin and stress-response proteins, we seek to advance the understanding of polyploid induction and provide new opportunities for improving the genetic diversity and breeding potential of C. annuum.

2. Material and Methods

2.1. Plant material

Seeds from three genotypes of ornamental pepper plants (C. annuum) were used in this study. The accessions UFPB-001 and UFPB-004 belong to the Federal University of Paraíba (UFPB) Pepper Germplasm Bank, while the commercial cultivar Floribela was obtained from Feltrin®. The experiment was conducted in the Plant Biotechnology sector at UFPB, located in Areia, Paraíba, Brazil.

2.2. Experimental design

To evaluate the induction of polyploids in pepper plants using orizalin as an inducing agent, an experiment was conducted in a 3x5x4 triple factorial scheme with a completely randomized design, which combined three genotypes, five antimitotic concentrations, and four exposure times. Twenty-five seeds of each genotype were immersed in 2.0 mL Eppendorf tubes containing 1,000 µL of different antimitotic concentrations (0.0, 0.05, 0.1, 0.2, and 0.4%) and subjected to different exposure times (24, 48, 72, and 96 hours). Each treatment consisted of five replicates (5 seeds), totaling 60 treatments and 300 observations. After exposure, the seeds were sown in 200-cell polystyrene trays containing Plantmax HT® substrate. At 40 days after sowing (DAS), the seedlings were transplanted into 1L pots filled with Plantmax HT® substrate and labeled according to the treatments.

The plants were watered daily, and a nutrient solution was applied weekly according to the recommendations of Furlani et al. (1999), with the following composition in g/1,000L: 1,000g of calcium nitrate; 1,250g of potassium nitrate; 250g of MKP; 500g of magnesium sulphate; 1.5g of boric acid; 25g of chelatec AZ; 25g of ultraferro; 110g of potassium chloride; and 150g of potassium sulphate. Each plant received 100 mL of the nutrient solution per application. Pest and disease control was carried out weekly as a preventative measure throughout the crop cycle using the following products: Actara 250 WG®, Pirate®, and Kumulus® DF.

2.3. Treatment survival rate

Surviving seedlings were counted on the twenty-first day after sowing (DAS). The seedling survival rate (SSR) was calculated using the following formula (Equation 1) (Rodriguez, 1996):

$SSR\left(\text{\%}\right)=\left.\left(\frac{\text{total surviving seedlings}}{totalseedsplanted}\right.\right)x100$ (1)

2.4. Molecular interaction of orizalin with proteins

To better understand how plants survive when exposed to concentrations of orizalin at the selected exposure times, we investigated the molecular interaction between this phytotoxic compound and proteins that mediate stress responses in C. annuum. The molecular docking technique was employed to analyze the interaction of proteins involved in the stress response caused by exposure to orizalin, a compound used as a polyploidy inducer that is phytotoxic to plant cells and tissues. This technique helps elucidate how seeds exposed to concentrations of orizalin survive, grow, and develop, considering that the compound interferes with cell division by inhibiting spindle fiber formation, thereby inducing polyploidy.

The protein structures (PDB IDs: 5YJS and 1SM4), together with their respective ligands, were obtained from the Protein Data Bank (PDB). The PDB serves as a repository for three-dimensional structural information of proteins. Each entry includes atomic coordinates, polymer sequences, and metadata (Berman et al., 2000). Protein inhibitors and water molecules were removed using DISCOVERY STUDIO 2021 CLIENT software.

The ligand, orizalin, was modeled in 3D using ACD/ChemSketch software, while its 2D model was obtained from ChemSpider (Oryzalin ChemSpider ID: 27326). The docking simulation followed the "flexible ligand-rigid protein" approach using Autodock VINA within PyRx software (Trott and Olson, 2010; Eberhardt et al., 2021). After docking, ligand conformations were analyzed using Discovery Studio software. The grid dimensions and center coordinates for docking were defined as detailed in Table 1.

Table 1
Binding site coordinates and grid dimensions for different proteins.

Proteins	Center_x	Center_y	Center_z	Size_x	Size_y	Size_z
5YJS	-41.926	7.219	-20.655	126	126	126
1SM4	37.782	104.661	90.509	126	126	126

The interaction energy between ligands and amino acids was calculated using Discovery Studio software. Components included Van der Waals forces, electrostatic bonds, and hydrogen bonds (Santos et al., 2024).

2.5. Morphological analysis

The plants were analyzed morphologically according to the Capsicum descriptors provided by the International Plant Genetic Resources Institute (IPGRI). Variables included plant height (cm), stem diameter (cm), leaf length (cm), leaf width (cm), leaf shape index (leaf length/width), corolla diameter (mm), and flower shape index (petal length/width). Measurements were made using a graduated ruler and a digital caliper.

2.6. Anatomical analysis

Stomatal density, stomatal size, and the number of chloroplasts per guard cell were analyzed. Transparent nail polish was applied to the abaxial epidermis to capture stomatal impressions. Chloroplast counts were conducted using paradermal sections of leaf epidermis mounted on slides for microscopic observation.

2.7. Cytogenetic analysis

Root tips from plants with morphoanatomical changes were processed and stained with DAPI for cytogenetic analysis following standard protocols (Guerra and Souza, 2002).

2.8. Statistical analysis

The data were analyzed using R Studio® (Rstudio Team, 2020) with the "ExpDes.pt" package. ANOVA, the Scott-Knott mean test and regression were performed at a 5% probability level (Ferreira et al., 2018). Graphs and tables were generated using Excel 2021® and SigmaPlot 10.0®.

3. Results

The analysis of variance revealed significant differences in the seedling survival rate based on genotype (G), concentration (C), and exposure time (T), as well as their interactions (Table 2). Specifically, genotype × concentration (G × C) and concentration × exposure time (C × T) interactions were significant at the 1% probability level (p < 0.01), whereas genotype × exposure time (G × T) and G × C × T interactions were not statistically significant (p > 0.05). The coefficient of variation was 21.94%, indicating moderate variability in survival rates.

Table 2
Analysis of variance for seedling survival rates in three Capsicum annuum genotypes treated with oryzalin.

Source of Variation	Degrees Freedom	Survival Rate
Genotype (G)	2	1.06**
Concentration (C)	4	1.28**
Exposure time (T)	3	0.15**
G x C	8	0.12**
G x T	6	0.01ns
C x T	12	0.11**
G x C x T	24	0.02ns
Residue	240	0.04
Total	299	-

• Legend: Significant at 1% probability level, ns: Not significant (p > 0.05).

Significant isolated effects were found for genotype (G), concentration (C), and exposure time (T) on seedling survival rates. Interaction analyses revealed that higher concentrations resulted in reduced survival, with over 80% lethality observed at 0.2% and 0.4% concentrations for the UFPB accessions. In contrast, the Floribela cultivar displayed greater tolerance, with survival rates exceeding 60% for all concentrations except 0.4%. The regression models showed strong predictive capability, with R² values above 80% for all genotypes (Figure 1).

Figure 1
Survival rates of three C. annuum genotypes treated with varying oryzalin concentrations.

Prolonged exposure (beyond 48 hours) further decreased survival rates, particularly from the 0.1% dose upwards (Figure 2). Regression analysis confirmed that increasing both concentration and exposure time reduced seedling survival.

Figure 2
Survival rates of C. annuum seedlings exposed to different concentrations of oryzalin for various durations.

The molecular docking analysis revealed interaction energies of -8.514 and -9.515 kcal/mol for the 5YJS and 1SM4 proteins, respectively, indicating moderate-to-high affinity between oryzalin and C. annuum stress response proteins. Hydrogen and Pi-alkyl bonds stabilized the complexes, particularly through residues like Asn and Arg (5YJS) and Tyr and Ser (1SM4) (Figure 3). This stabilization suggests these proteins partially mitigate oryzalin toxicity, supporting its effectiveness in inducing polyploidy.

Figure 3
2D (A, C) and 3D (B, D) molecular interactions of oryzalin with C. annuum proteins 5YJS and 1SM4.

Cytogenetic analyses revealed that 0.1% oryzalin for 48–72 hours induced tetraploids in UFPB-001 (Figure 4C, D) and UFPB-004 (Figure 4G, H), while 0.2% for 48 hours induced a triploid in Floribela (Figure 4K, L). Controls remained diploid (24 chromosomes), whereas polyploids displayed 36 (triploid) or 48 chromosomes (tetraploid). Oryzalin concentrations of 0.1%–0.2% for 48–72 hours are recommended for polyploidy induction.

Figure 4
Chromosome analysis of diploid, triploid, and tetraploid C. annuum plants induced by oryzalin. Legend: UFPB-001 (A-B: Diploid and C-D: Tetraploid), UFPB-004 (E-F: Diploid and G-H: Tetraploid), Floribela (I-J: Diploid and K-L: Triploid).

Polyploid induction rates varied between 25%–50%, as shown in Table 3. Most treatments did not produce polyploids, but successful treatments offer valuable insights for breeding programs.

Table 3
Summary of polyploid induction rates in Capsicum annuum genotypes treated with oryzalin.

Genotype	Concentration × Time	Ploidy Level	Induction Rate (%)
UFPB-001	0.1% × 48 h	Tetraploid	25
UFPB-004	0.1% × 72 h	Tetraploid	50
Floribela	0.2% × 72 h	Triploid	33.3

Morphological analyses revealed that polyploids generally had larger leaves (Table 4). These plants showed greater leaf length and width compared to the diploids, except for the tetraploid derived from the UFPB-001 plant, which showed leaf width similar to the corresponding diploid (Table 4). No significant differences were observed in the leaf shape index (leaf length/width) between diploids and polyploids (p < 0.05).

Table 4
Leaf traits in diploid and polyploid C. annuum.

Genotypes	2n	Leaf length (cm)	Leaf width (cm)	Leaf index
Control
UFPB-001	2x = 24	4.46 c ± 0.60	1.73 b ± 0.30	2.59 ± 0.15
UFPB-004	2x = 24	4.01 c ± 0.62	1.71 b ± 0.17	2.34 ± 0.16
FLORIBELA	2x = 24	4.78 c ± 0.91	2.03 b ± 0.34	2.45 ± 0.81
Oryzalin
UFPB-001	4x = 48	6.65 b ± 0.56	2.42 b ± 0.38	2.78 ± 0.33
0.10% (48h)
UFPB-004	4x = 48	8.37 a ± 1.23	3.15 a ± 0.87	2.76 ± 0.51
0.10% (72h)
FLORIBELA	3x = 36	8.63 a ± 1.68	3.38 a ± 0.39	2.56 ± 0.44
0.20% (48h)

• Legend: Different letters in the column indicate statistically significant differences (p < 0.05).

Polyploids exhibited greater plant height and stem diameter than diploids, except for the tetraploid UFPB-001, which was similar in height to its diploid control (Figure 5). Stem diameter consistently increased with ploidy levels. Flower corolla diameter increased significantly in the tetraploid UFPB-004 compared to its diploid counterpart but decreased in other polyploids. The flower shape index did not differ significantly between diploid and triploid plants, although Floribela showed larger flowers across ploidy levels.

Figure 5
Morphological traits (height, stem diameter, and flower dimensions) of diploid and polyploid Capsicum annuum plants. Legend: Different letters in the bars indicate statistically significant differences (p < 0.05).

Polyploid leaves were significantly thicker than those of diploids (Figure 6). Tetraploid leaves, particularly in UFPB-001, displayed a leathery appearance. Floribela’s leaves, naturally thinner than UFPB accessions in diploids, increased in thickness at the triploid level, maintaining a delicate structure but with visibly enhanced dimensions. Anatomical analysis showed that stomatal density decreased while stomatal area increased with higher ploidy levels (Table 5). Tetraploids exhibited larger stomata compared to triploids. Figure 7A-H highlight these differences visually.

Figure 6
Comparative analysis of leaf thickness between diploids and polyploids (A) of Capsicum annuum. Photomicrograph of the thickness of diploid (B) and tetraploid (C) leaves of C. annuum. Legend: Different letters in the bars indicate statistically significant differences (p < 0.05).

Table 5
Stomatal parameters in diploid and polyploid C. annuum.

Genotypes	2n	Stomatal density (stomata/mm)	Stomatal area (µm2)	Stomatal length (µm)	Stomatal width (µm)
Control
UFPB-001	2x = 24	101.10 a ± 11.03	1030.51 c ± 58.21	43.08 c ± 1.59	30.75 b ± 1.33
UFPB-004	2x = 24	84.72 b ± 7.12	852.96 e ± 11.79	41.09 c ± 1.70	28.86 b ± 1.55
FLORIBELA	2x = 24	111.07 a ± 8.38	957.69 d ± 23.02	43.53 c ± 0.92	29.60 b ± 0.56
Oryzalin
UFPB-001	4x = 48	91.13 b ± 6.15	1277.14 a ± 46.55	50.77 a ± 1.85	33.18 a ± 1.68
0.10% (48h)
UFPB-004	4x = 48	42.01 c ± 9.41	1244.76 a ± 31.86	51.19 a ± 1.45	32.40 a ± 1.24
0.10% (72h)
FLORIBELA	3x = 36	116.05 a ± 10.24	1120.71 b ± 20.43	45.96 b ± 2.02	31.52 a ± 1.05
0.20% (48h)

• Legend: Different letters in the column indicate statistically significant differences (p < 0.05).

Figure 7
Stomatal characteristics and chloroplast counts in diploid and polyploid Capsicum annuum plants. Legend: UFPB-001 (A: Diploid and B: Tetraploid), UFPB-004 (C-G: Diploid; D-H: Tetraploid), Floribela (E: Diploid and F: Triploid).

Polyploids showed larger stomatal sizes and reduced density compared to diploids, except for the triploid Floribela, where stomatal density remained high despite an increase in stomatal size. Tetraploids exhibited approximately a 20% increase in stomatal size (length and width), aligning with the gygas effect. Chloroplast count per guard cell doubled in polyploids, from 9 in diploids to 18 in polyploids.

4. Discussion

The use of polyploidy-inducing agents is a valuable technique for accelerating the development of improved cultivars by expanding existing genetic variability. This expansion facilitates the selection of superior genotypes. However, such agents can occasionally cause phytotoxic effects on plant cells, tissues, and organs (Iannicelli et al., 2020; Touchell et al., 2020). Identifying the ideal combination of antimitotic agent, exposure time, and genotype survival mechanisms enhances the efficiency of artificial polyploidization, enabling faster selection of target species or genotypes (Niazian and Nalousi, 2020).

Colchicine is widely used as an antimitotic agent to induce polyploidy in Solanaceae species such as Solanum lycopersicum (Cola et al., 2014), Solanum melongena (Basay et al., 2011), Solanum tuberosum (Swaminathan, 1951), and Capsicum annuum (Selvakumar et al., 2022). However, its high toxicity to the environment, humans, and animals has led researchers to explore safer, cheaper and effective alternatives, such as oryzalin (Mir et al., 2021). Acting as a depolymerizing agent, oryzalin prevents β-tubulin formation throughout the cell cycle, making it a potent inducer of polyploidy. Its attributes have been studied to enhance plant diversity and address challenges like climate change and soil pollution (Touchell et al., 2020).

Compared to colchicine, oryzalin has lower toxicity, higher affinity for plant tubulins, and greater efficacy at lower doses (Touchell et al., 2020). These features make it an economically viable option for inducing polyploids (Ahmadi and Ebrahimzadeh, 2020). Molecular docking studies further demonstrate oryzalin's affinity for stress-response proteins in C. annuum, aiding in understanding plant survival under phytotoxic conditions (Ravindranath et al., 2015; Guterres and Im, 2020).

The binding energy between oryzalin and proteins such as 5YJS and 1SM4 (>-8.5 kcal/mol) suggests a strong affinity, indicating their role in mitigating phytotoxic effects in Capsicum annuum. Hydrogen bonding and pi-alkyl interactions stabilize these complexes, ensuring robust and selective responses to chemical stress (Stenström et al., 2021; Moon, 2024). These interactions are comparable to the SlMAPK1:Albaflavenone complex in Solanum lycopersicum, which enhances tolerance to water stress (Alfagham et al., 2024).

The combination of oryzalin concentration (0.1–0.2%), exposure time (48–72 hours), and genotype yielded tetraploids and one triploid in ornamental Capsicum annuum genotypes, demonstrating the feasibility of inducing polyploidy via seeds. This aligns with studies on Solanum commersonii (Tomé et al., 2016) and Musa acuminata (Costa et al., 2011), which showed higher efficacy and lower phytotoxicity of oryzalin compared to colchicine.

Stomatal size is a reliable polyploidy indicator in plants (Yan et al., 2022). Unlike findings in C. annuum treated with colchicine, which showed no significant stomatal differences (Tammu et al., 2021), this study confirmed stomatal size as a polyploidy marker in oryzalin-treated C. annuum. The observed increase in stomatal area and decrease in stomatal density align with results in Physalis spp. (Azeez et al., 2019).

Leaf thickness and chloroplast count per guard cell are additional indicators of polyploidy in species such as Kaempferia rotunda (Soonthornkalump et al., 2017), Musa sp. (Madail et al., 2022), and Dendrobium cariniferum (Zhang and Gao, 2021). These characteristics were validated in this study, as polyploids showed significant increases in both traits compared to diploids (p < 0.01).

These results reinforce oryzalin as a safer and more effective alternative to colchicine for producing polyploids in C. annuum. While Tammu et al. (2020) and Külahlioğlu (2017) reported limited success with colchicine in inducing polyploids in C. annuum and Solanum melongena, respectively, oryzalin demonstrated superior effectiveness. Future studies should consider genotype-specific responses and optimize concentration and exposure time for seed-based protocols.

Since the early 20th century, plant breeders have capitalized on the superiority of polyploids in various species, especially ornamentals (Sattler et al., 2016). Efforts to increase variability through polyploidization have contributed to the development of traits like drought tolerance in C. annuum (Maryam and Aminu, 2022), heat tolerance in Asparagus officinalis (Chen et al., 2020), and nematode resistance in Ipomoea batatas (Obata et al., 2022). The morphological and anatomical variability observed here supports the breeding program for ornamental peppers at the Federal University of Paraíba, enhancing its genetic base for further improvement.

5. Conclusions

Seed exposure to oryzalin concentrations of 0.1-0.2% for 48-72 h successfully induced polyploidy in Capsicum annuum, producing triploid and tetraploid plants with distinct phenotypic characteristics. Further studies will be conducted to evaluate potential variations in induction efficiency within these concentration and exposure time ranges. Moreover, Morphological and anatomical analyses confirmed that stomatal size, the number of chloroplasts per guard cell, and leaf thickness are reliable indicators for identifying polyploids.

Furthermore, the 5YJS and 1SM4 proteins play a critical role in mediating the response to chemical stress, activating defense mechanisms that enhance plant survival under oryzalin exposure. The polyploid genotypes developed in this study exhibit valuable characteristics and hold significant potential for incorporation into the Federal University of Paraíba's pepper breeding program, supporting the development of improved ornamental varieties.

Acknowledgements

This study was supported by the Graduate Program in Agronomy at the Federal University of Paraíba. We are grateful to the program, its faculty members, and all collaborators involved in this research. The authors also acknowledge the financial support provided by the Coordination for the Improvement of Higher Education Personnel (CAPES) - Brazil, under Funding Code 001.

Data Availability Statement

The entire data set that supports the results of this study was published in the article itself.

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Edited by

• Editor:
  Jairo Lizandro Schmitt

Publication Dates

• Publication in this collection
  29 Sept 2025
• Date of issue
  2025

History

• Received
  06 Mar 2025
• Accepted
  11 June 2025