Toxicity of polyploidy-inducing agents in Capsicum spp. and their impacts on seedling emergence and morphological alterations

Toxicidade de agentes indutores de poliploidia em Capsicum spp. e seus impactos na emergência de plântulas e diversificação morfológica

ABSTRACT

Capsicum spp. are model taxa for studying evolutionary mechanisms due to their morphological diversity. This study investigates the phytotoxic effects of the polyploidy-inducing agents oryzalin and trifluralin on four Capsicum genotypes, elucidating their role in seedling emergence and morphological diversification. Seeds were exposed to five agents’ concentrations across four exposure durations at 25°C. Both agents reduced seedling emergence in genotypes (<37%) and emergence speed index in a concentration-dependent manner, with genotype-specific tolerance reflecting divergent evolutionary adaptations to mitotic disruption. Induced morphological alterations, such as thickened leaves and reduced internodes, mirror patterns observed in natural polyploids, suggesting that genomic instability caused by antimitotics may mimic evolutionary diversification processes in Capsicum. This genotypic variability provides insights for breeding, as genotypes with higher tolerance to specific antimitotics could facilitate controlled polyploid induction, aiming to create variability for developing superior cultivars with enhanced hybrid vigor, abiotic stress tolerance, or larger fruits. Unexpected survival at high trifluralin concentrations correlated with delayed mean emergence time, indicating potential selection of tolerant or polyploid subpopulations. These findings position antimitotic agents as tools to explore stress-induced genomic plasticity and its taxonomic implications in Capsicum, while paving the way for innovative strategies in breeding programs.

Keywords:
Solanaceae; phytotoxic; seedling survival; vigor; variability; breeding

RESUMO

Capsicum spp. são táxons modelo para estudos de mecanismos evolutivos devido à diversidade morfológica. Este estudo investiga os efeitos fitotóxicos dos agentes indutores de poliploidia, orizalina e trifluralina, em quatro genótipos de Capsicum, analisando seu impacto na emergência de plântulas e diversificação morfológica. Sementes foram expostas a cinco concentrações dos agentes em quatro durações a temperatura de 25^°C. Ambos os agentes reduziram a emergência (<37%) e o índice de velocidade de emergência, de modo dependente da concentração, com tolerância genótipo-específica refletindo adaptações evolutivas à disrupção mitótica. Alterações como folhas espessadas e entrenós reduzidos assemelham-se a padrões de poliploides naturais, sugerindo que a instabilidade genômica ocasionada por antimitóticos pode simular processos evolutivos de diversificação em Capsicum. A variabilidade genotípica oferece bases para o melhoramento, pois genótipos tolerantes a antimitóticos específicos permitem indução controlada de poliploidia, visando cultivares superiores com vigor híbrido, tolerância a estresses abióticos ou frutos maiores. Sobrevivência inesperada em altas concentrações de trifluralina correlacionou-se com atraso na emergência, indicando seleção de subpopulações tolerantes ou poliploides. Esses resultados destacam antimitóticos como ferramentas para explorar plasticidade genômica induzida por estresse e implicações taxonômicas em Capsicum, além de orientar estratégias inovadoras em programas de melhoramento.

Palavras-chave:
Solanaceae; fitotoxicidade; sobrevivência de plântulas; vigor; variabilidade; melhoramento genético

Capsicum species (Solanaceae), native to Central and South America, represent a plant group of significant taxonomic and evolutionary importance, with five domesticated species: C. annuum L., C. chinense Jacq., C. frutescens L., C. pubescens Ruiz and Pav., and C. baccatum L. (Cansian Júnior et al., 2021). Their remarkable phenotypic plasticity, expressed through morphological variations in fruits (color, shape, and dimensions), plant architecture (growth habit and branching patterns), and leaves (pigmentation and texture), reflects evolutionary adaptations to heterogeneous environmental pressures (Rego et al., 2022). This diversity, sustained by genomic mechanisms such as polyploidy, positions the genus as a model for studying evolutionary diversification (Zhang et al., 2025).

The enhancement of genetic diversity, a central goal of modern breeding (Xia et al., 2020; Tracy et al., 2020; Rodrigues et al., 2023), mirrors natural processes of genomic innovation. Chemical agents such as dinitroaniline herbicides (oryzalin and trifluralin), whose inhibition of microtubule polymerization during mitosis (Chen et al., 2021) mimics genotoxic stresses encountered in natural environments, have been used to induce artificial polyploidy in plants (Trojak-Goluch et al., 2021). This approach enables exploration of how induced genomic changes may catalyze adaptive traits, akin to those observed in wild polyploids.

The efficacy of these agents varies by genotype, concentration, and exposure duration (Eeckhaut et al., 2018), reflecting divergent evolutionary strategies for stress tolerance. Successful protocols in species such as Passiflora edulis (Rego et al., 2011) and Agastache foeniculum (Talebi et al., 2017) demonstrate the universality of polyploidy as an adaptive response in angiosperms. In Capsicum, although colchicine has been extensively tested in seeds of C. annuum and C. frutescens (Pal & Ramanujam, 1939; Tammu et al., 2021), the evolutionary potential of other chemical inducers remains underexplored, particularly their phytotoxic effects during early developmental stages.

Phytotoxicity in seed-based polyploidization protocols, manifested through reduced germination rates and delayed seedling emergence (Mo et al., 2020), provides insights into evolutionary trade-offs between genomic stability and stress adaptation. Studies such as Silva et al. (2019) reinforce that dynamic analysis of emergence can reveal critical thresholds between cellular damage and tolerance mechanisms, essential parameters for survival in extreme environments.

This study evaluates the effects of oryzalin and trifluralin on seedling emergence in Capsicum, investigating how genotypic variation in tolerance to these agents reflects underlying evolutionary mechanisms driving genus diversification. By examining genotype-concentration-exposure interactions, we aim to elucidate phenotypic variability arising from genomic stress and propose hypotheses for future testing.

MATERIAL AND METHODS

The study was conducted at the Biotechnology and Plant Breeding Laboratory of the Center for Agricultural Sciences (CCA), Federal University of Paraíba (UFPB), located in Areia, Paraíba State, Brazil (6°57'42"S, 35°41'43"W; 573 m altitude). To investigate evolutionary responses to genomic stress, four Capsicum genotypes were selected: two C. annuum accessions (UFPB-001 and UFPB-004) from the UFPB Germplasm Bank, representing natural genetic diversity, and two commercial cultivars (‘Floribela’ - C. annuum and ‘Biquinho’ - C. chinense), obtained from Feltrin^®. This selection encompasses intraspecific and interspecific variability, enabling comparisons of adaptive strategies across distinct genotypes.

Two independent experiments evaluated the effects of oryzalin and trifluralin, dinitroaniline herbicides that mimic natural genotoxic stress by inhibiting microtubule polymerization. The experimental design followed a 4×5×4 factorial arrangement (four genotypes, five concentrations [0.0%, 0.05%, 0.1%, 0.2%, 0.4%], four exposure durations [24, 48, 72, 96 hours]) at 25^°C, totaling 80 treatments with five replicates, with one seed per plot. The present study aimed to expand the concentration ranges and exposure durations reported in the literature for Solanaceae species traditionally treated with colchicine, a widely used but highly toxic antimitotic agent. This study sought to adapt the methodology from a sustainability perspective by employing antimitotic compounds with greater plant affinity, lower toxicity, and reduced cost, such as oryzalin and trifluralin. This approach aims to extend promising results previously reported in Capsicum species (Amanah et al., 2016; Tammu et al., 2021).

Seeds were immersed in 2 mL Eppendorf tubes containing 1 mL of treatment solution, simulating acute exposure to genotoxic agents. After each exposure period, seeds were surface-dried on sterile filter paper and sown in polystyrene trays filled with Plantmax^® substrate. Trays were irrigated manually once daily using 10 L watering cans to restore substrate field capacity, ensuring uniform moisture for seedling emergence. Emergence was monitored from 7 to 21 days after sowing (DAS). During this period, the trays containing the seeds were maintained in a greenhouse under the following environmental conditions: mean temperature of 25.4°C (maximum 29.9°C and minimum 21.7°C), mean relative humidity of 76%, and mean daily insolation of 6.6 h. The evaluated parameters included seedling emergence (SE, %) (ISTA, 2015), emergence speed index (ESI) (Maguire, 1962), and mean emergence time (MET) (Labouriau, 1983).

Seedling emergence (SE, in %) was estimated using the following equation (ISTA, 2015):

$SE=\left(\frac{n}{N}\right)x100$

where: n is the number of emerged seeds, and N is the total number of seeds.

Emergence speed index (ESI) was estimated according to Maguire (1962):

$ESI={\sum }_{i=1}^k\left(ni/ti\right)$

where: ni is the number of seeds emerged on each day of daily counting until the last count, andtiis the number of days after the start of the test on each count.

Mean emergence time (MET, in days) was estimated using the equation proposed by Labouriau (1983):

$AET={\sum }_{i=1}^{k}nixti/{\sum }_{i=1}^{k}ni$

where: ni is the number of seeds emerged per day (not the cumulative number, but the number corresponding to the i-th observation), and ti is the time from the start of the emergence test to the i-th observation.

All data were transformed using the square-root transformation (x+0.5)¹/² to improve normality and were subjected to factorial ANOVA using R (v. 4.2.1) in RStudio (v4.2.1) with the ExpDes.pt package, followed by Scott-Knott cluster analysis (p<0.05). Regression models (SigmaPlot 10.0, Excel 2021) identified dose-response relationships, while graphical outputs highlighted genotypic patterns in stress tolerance.

RESULTS AND DISCUSSION

General results

Trifluralin was markedly more phytotoxic than oryzalin, reducing seedling emergence to 15.68%, compared to 36.56% for oryzalin and 78.75% in controls (Figure 1). While both agents suppressed emergence across genotypes, the cultivars 'Floribela' (C. annuum) and 'Biquinho' (C. chinense) tolerated oryzalin better, especially at 0.1-0.2%. UFPB-004 showed the greatest sensitivity (≈90% reduction), followed by UFPB-001 (≈70%). Trifluralin effects were more uniform, with all genotypes strongly inhibited.

Polyploidy-inducing agents (PIAs), oryzalin and trifluralin, impair β-tubulin polymerization, disrupting mitosis and embryo development (Pereira et al., 2012; Taiz et al., 2017). The toxicity resulting from concentration and exposure duration may lead to embryonic cell or tissue death, severely affecting embryo growth resumption (Rego et al., 2011; Pereira et al., 2012; Lan et al., 2020; Niazian & Nalousi, 2020). Trifluralin’s stronger inhibition aligns with its higher binding affinity to plant tubulin (Hansen & Andersen, 1996). Tolerance in 'Floribela' and 'Biquinho' may reflect genotype-specific adaptive mechanisms (Zhang et al., 2020).

Oryzalin effects

Analysis of variance (Table 1) showed highly significant effects of genotype, concentration, and exposure time on emergence parameters, with concentration as the main factor. The genotype × concentration interaction was significant for seedling emergence (p<0.05) and emergence speed index (p<0.01). Prolonged exposure durations consistently reduced seedling emergence, with significant effects on SE (p<0.01), ESI (p<0.05), and mean emergence time (MET; p<0.01).

A clear concentration-dependent inhibition was observed (Table 2). 'Floribela' maintained >65% emergence at 0.2%, while other genotypes dropped to less than 35%. 'Biquinho' also showed greater survival than UFPB accessions at higher concentrations. These contrasts mirror findings in Vitis vinifera (Kara et al., 2021) and Limonium sinuatum (Mori et al., 2021), where antimitotic sensitivity varied with genotype-specific genomic.

Mo et al. (2020) reported that oryzalin significantly inhibited germination and emergence in Rhododendron fortunei at concentrations as low as observed in this study for Capsicum genotypes exposed beyond 24 hours. The accelerated emergence under high oryzalin concentrations and prolonged exposure likely reflects seed survival mechanisms, a pattern also noted in Centrosema virginianum (Battistin et al., 1993). Further studies are required to elucidate cellular and biochemical underpinnings of this response.

Figure 1
Comparative phytotoxic effects of oryzalin and trifluralin on seedling emergence across four Capsicum genotypes. Areia, UFPB, 2023.

Table 1
Analysis of variance for seedling emergence (SE), emergence speed index (ESI), and mean emergence time (MET) of four Capsicum genotypes exposed to oryzalin. Areia, UFPB, 2023.

Source of variation	DF	SE	ESI	MET
Genotype (G)	3	0.73**	0.10**	8.52**
Concentration (C)	4	1.57**	0.22**	17.34**
Exposure time (T)	3	0.31**	0.03*	5.73**
G x C	12	0.09*	0.02**	1.55ns
G x T	9	0.03ns	0.01ns	1.20ns
C x T	12	0.07ns	0.01ns	1.15ns
G x C x T	36	0.04ns	0.01ns	1.19ns
Residue	320	0.04	0.01	0.99
Total	399	-	-	-
CV (%)		21.94	12.30	62.32

• Significance levels: ^* p<0.05, ^** p<0.01, ^ns not significant (p>0.05).

Table 2
Seedling emergence (%) of four Capsicum genotypes under oryzalin concentrations. Areia, UFPB, 2023.

Genotypes	Oryzalin concentration (%)
	0.00	0.05	0.10	0.20	0.40
UFPB-001	75.00 a	60.00 a	25.00 b	10.00 c	5.00 a
UFPB-004	70.00 a	25.00 b	40.00 b	5.00 c	5.00 a
Floribela	95.00 a	90.00 a	70.00 a	65.00 a	10.00 a
Biquinho	75.00 a	75.00 a	45.00 b	35.00 b	20.00 a

• * Values within a column followed by different lowercase letters differ significantly (p<0.05).

Oryzalin treatments produced mutant seedlings with distinct morphological abnormalities (Figure 2). All genotypes produced visually mutant seedlings following oryzalin treatments, including UFPB-001 (three seedlings), UFPB-004 (one seedling), cv. Floribela (one seedling), and cv. Biquinho (one seedling). Affected seedlings showed reduced internodes and twisted leaves (Figure 2A), along with visibly thicker, leathery leaves displaying irregular shapes (Figure 2B). Developmental anomalies included leaves with bifurcated tips and abnormal venation patterns (Figure 2C), and in some specimens, leaves fused at the base showing a succulent-like morphology (Figure 2D).

Figure 2
Oryzalin-induced morphological abnormalities in Capsicum seedlings. A: UFPB-001 at 0.1% (48 h) Internode reduction and leaf twisting. B: UFPB-001 at 0.2% (24 h) Thickened, leathery leaves. C: UFPB-004 at 0.1% (72 h) Bifurcated leaf tips with abnormal venation. D: cv. Biquinho at 0.1% (96 h) Basal leaf fusion (scale bar = 1 cm). Areia, UFPB, 2023.

In addition to the described morphological alterations, mutant seedlings exhibited leaves with pronounced surface rugosity (Figure 2B) and irregular margin morphology, including serrations and undulations (Figure 2A, B and C). These textural abnormalities co-occurred with the previously noted thickening and fusion phenotypes.

Oryzalin-induced morphological anomalies, including reduced height, thickened and leathery twisted leaves, bifurcated tips, and fused organs (Figure 2), directly reflect impaired cell division and expansion, consistent with dinitroaniline-induced microtubule dysfunction (Zeng et al., 2019). Traits such as internode shortening are common in polyploid angiosperms, arising from novel genomic combinations catalyzed by chromosomal duplication, which drive evolutionary diversification (Soltis & Soltis, 2016).

Thicker, broader, twisted, and coriaceous leaves have been reported in Vanda limbata ‘Jawa’ (Dwiati et al., 2025) and in Citrullus lanatus (Bae et al., 2020) following exposure to oryzalin concentrations. However, to the best of our knowledge, the present study is the first to describe bifurcated leaf apices and leaf blades united by a single petiole. Dose-response regression confirmed genotype-specific patterns: linear for 'Biquinho', quadratic for others (Figure 3). Mean emergence time also increased with concentration and exposure duration, highlighting genotypic contrasts (Figure 4).

Trifluralin effects

Analysis of variance for the trifluralin treatments (Table 3) revealed a significant three-way interaction (G × C × T) only for mean emergence time (MET). Significant two-way interactions were observed between concentration and exposure time (C × T) for seed emergence (SE) and MET; between genotype and exposure time (G × T) for SE; and between genotype and concentration (G × C) for SE, MET, and the emergence speed index (ESI). Analysing factors independently, no significant differences were detected among genotypes. In contrast, concentration and exposure time significantly affected SE and MET, whereas only concentration had a significant effect on ESI.

Figure 3
Dose-response relationships between oryzalin concentrations and the seedlings emergence speed index (ESI) across four Capsicum genotypes. Areia, UFPB, 2023.

Figure 4
Effects of isolated factors on mean emergence time: (A) Capsicum genotype variation, (B) oryzalin concentration (%), and (C) exposure time (h). Areia, UFPB, 2023.

Table 3
Analysis of variance for seedling emergence (SE), emergence speed index (ESI), and mean emergence time (MET) of four Capsicum genotypes exposed to trifluralin. Areia, UFPB, 2023.

Source of variation	DF	SE	ESI	MET
Genotype (G)	3	0,01ns	0,01ns	0,09ns
Concentration (C)	4	2,19**	0,26**	34,05**
Exposure time (T)	3	0,53**	0,01ns	14,66**
G x C	12	0,05**	0,02**	1,37*
G x T	9	0,05*	0,01ns	0,97ns
C x T	12	0,21**	0,01ns	6,57**
G x C x T	36	0,03ns	0,01ns	1,07*
Residue	320	0,02	0,01	0,66
Total	399	-	-	-
CV (%)		16,91	9,76	59,2

• Significance levels: ^* p<0.05, ^** p<0.01, ^ns not significant (p>0.05).

Trifluralin caused more severe inhibition than oryzalin, with emergence nearly eliminated at ≥0.1% across genotypes (Table 3). Visual assessment revealed more severe morphological disturbances in response to trifluralin than oryzalin (Figure 5). The cultivar Floribela was more sensitive to trifluralin, producing fewer seedlings. Given the low mutation induction rate, no phenotypic alterations were observed in this genotype following trifluralin treatment. In contrast, the remaining genotypes produced seedlings exhibiting marked morphological modifications after trifluralin exposure, including UFPB-001 (one seedling), UFPB-004 (three seedlings), and cv. Biquinho (one seedling).

Figure 5
Dose and time-dependent morphological responses to trifluralin in Capsicum spp. A: UFPB-001 at 0.4% (48 h) showing complete root inhibition. B: cv. Biquinho at 0.05% (72 h) exhibiting leaf epinasty. C: UFPB-004 at 0.05% (24 h) with minor hypocotyl thickening. D: UFPB-004 at 0.05% (48 h) demonstrating progressive cotyledon deformation. Scale bar = 1 cm. Areia-PB, UFPB, 2023.

Abnormalities included intense leaf chlorosis, pronounced epinasty, and leaf vein deformations (Figure 5A). At higher concentrations (0.10%-0.20%), seedlings showed complete apical meristem collapse, absence of leaf development, and cotyledonary structure necrosis (Figure 5B). Morphological evaluation confirmed trifluralin induced more severe aberrations than oryzalin, suggesting stronger microtubule polymerization inhibition and consequently more intense antimitotic effects. Because trifluralin is the active ingredient of herbicides, its exposure induces severe phytotoxic stress in plants, particularly characterized by leaf chlorosis, pronounced epinasty, and growth stagnation in cereals and legumes (Sheval et al., 2008; Chowdhury et al., 2020).

The extreme toxicity of trifluralin (≥90% lethality at 0.1% for ‘Floribela’; Figure 5C) underscores its broad physiological impacts, including irreversible root meristem damage (Figure 5A) and loss of apical dominance evidenced by early seedling bifurcation (Figure 5D) (Alavi et al., 2022). Although some seedlings initially established, development ceased due to premature mortality (Figure 5A). This results from trifluralin-induced formation of root cells with abnormal chromosome sets and defective cell walls, producing short, thickened roots incapable of water and nutrient uptake (Guo et al., 2023).

Sheval et al. (2008) demonstrated that trifluralin-induced disruption of the microtubule cytoskeleton in Hordeum vulgare L. roots resulted in growth inhibition and abnormal root formation, characterized by defective cells and altered cell wall organization. These structural abnormalities triggered programmed cell death in root tissues, ultimately leading to seedling inviability. These findings are consistent with the severe phytotoxic effects observed in the present study, in which trifluralin exposure caused irreversible damage to the root meristem and arrested seedling development.

Karami et al. (2019) documented significant interactions (p<0.01) between trifluralin concentration and exposure time leading to morphological, anatomical, and physiological alterations in Matricaria chamomilla, corroborating this study's findings. Therefore, optimizing concentration and exposure time for each species and genotype is critical for developing efficient protocols to generate mutant plants with desirable traits or polyploid breeding parents in Capsicum improvement programs.

Emergence speed index dropped sharply from 0.05% dose, and mean emergence time increased proportionally with concentration. Some seedlings survived high doses, possibly reflecting transient polyploidization (Mortier et al., 2024) or tolerant subpopulations (Lovell et al., 2021). Prolonged MET in survivors suggests compensatory endoreduplication (Paige, 2018), resembling patterns in Populus spp. (Zeng, 2019) where lethal doses occasionally yielded regenerants.

General discussion

Oryzalin induced gradual, concentration-dependent inhibition, while trifluralin produced acute toxicity, particularly at meristems. Both agents significantly reduced seedling emergence (15.68% and 36.56%, respectively), slowed emergence speed, and caused characteristic morphological aberrations (Figsures 2, 5). These effects reflect β-tubulin polymerization inhibition and align with polyploidy-associated traits (Soltis & Soltis, 2016).

Although trifluralin was consistently more lethal, residual survival at high doses suggests opportunities for generating mutant or polyploid plants, supporting their dual role as phytotoxic stressors and potential breeding tools.

Conclusions

Although high concentrations of polyploidy-inducing agents (PIAs) universally compromised seedling establishment, the genotype-specific tolerance observed in commercial cultivars (cv. Floribela with oryzalin) highlights the interplay between genomic background and antimitotic resilience. This dichotomy positions these agents as dual-purpose tools: both for investigating stress adaptation mechanisms and for inducing polyploidy in breeding programs. However, the trade-offs between phytotoxicity and polyploidy induction necessitate optimized protocols, particularly for sensitive genotypes such as UFPB-004.

Future perspectives

Cytogenetic validation of polyploidy in survivors using flow cytometry or chromosome counting.

Molecular characterization of β-tubulin isoforms and identification of PIA-tolerance-associated proteins.

Dose-time optimization to balance polyploidy induction with seedling viability, based on nonlinear responses. These findings advance our understanding of chemical polyploidy induction during early plant development while emphasizing the need for genotype-tailored approaches in applied botany.

Acknowledgements

I would like to thank the Graduate Program in Agronomy at the Federal University of Paraíba. This work was carried out with the support of the Coordination for the Improvement of Higher Education Personnel, Brazil (CAPES), Funding Code 001.

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• Consent for publication
  All authors allow Horticultura Brasileira to publish the manuscript.
• Data availability
  Data will be made available upon request to the corresponding author.

Edited by

• Responsible editor
  Ana Cristina Portugal Pinto de Carvalho

Data availability

Data will be made available upon request to the corresponding author.

Publication Dates

• Publication in this collection
  25 May 2026
• Date of issue
  2026

History

• Received
  21 Oct 2025
• Accepted
  27 Feb 2026