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. 2026 Jul 8;26:1308. doi: 10.1186/s12870-026-09456-z

In vitro induction of tetraploids and their morphological and phenolic acid content in Cnidium officinale

Hui Yeong Jeong 1,2, Ji Ah Kim 1, Ho Jun Son 1,✉, Jun Won Kang 2,✉
PMCID: PMC13445674  PMID: 42420843

Abstract

Background

Cnidium officinale (Makino) Kitag (C. officinale; 2n = 2x = 22) is a medicinal plant that exhibits species-wide infertility due to meiotic instability and abnormal embryo sac development, resulting in exclusive vegetative propagation. This reproductive limitation restricts genetic diversity and breeding opportunities. Polyploidization is an effective breeding strategy for generating novel germplasm with altered morphology and enhanced metabolite production. This study aimed to induce tetraploids in C. officinale and evaluate their morphological and phytochemical characteristics.

Methods

In vitro shoots were treated with colchicine or oryzalin at various concentrations using liquid and solid culture systems. Ploidy levels were determined by flow cytometry and confirmed through somatic chromosome counting. Morphological traits, stomatal characteristics, and phenolic acid contents were compared between diploids and tetraploids.

Results

Colchicine successfully induced tetraploids, whereas oryzalin was ineffective under the tested conditions. The highest induction rate (40%) was achieved by treating shoots with 20 mg/L colchicine for 48 h in liquid Murashige and Skoog medium. A total of 28 tetraploid plants were identified from 300 colchicine treated explants. Subsequent ploidy monitoring during repeated subcultures confirmed 11 plants as stable tetraploids. Chromosome counting of a representative stable tetraploid verified chromosome doubling from 2n = 22 to 2n = 44. Tetraploid exhibited typical polyploid characteristics, including significantly increased stomatal length and reduced stomatal density. Under in vitro conditions, tetraploids developed significantly thicker petioles than diploids. Following acclimatization, tetraploid plants showed significantly greater leaf length and area than diploids. Quantitative analysis of phenolic acid content revealed a significantly higher ferulic acid content in tetraploid fibrous roots (3.49 ± 0.14 µg/mL) compared with diploids.

Conclusions

This study provides the first report of colchicine-mediated tetraploid induction in C. officinale. Future research should focus on evaluating the agronomic performance of tetraploid plants under greenhouse and field conditions, examining the long-term stability of metabolite production, and clarifying the molecular basis of metabolite biosynthesis associated with polyploidization.

Keywords: Cnidium officinale, Tetraploid, Colchicine, In vitro induction, Phenolic acid, Morphology

Background

C. officinale is propagated asexually, limiting trait improvement through traditional breeding. C. officinale exhibits severely reduced sexual fertility, including low seed set associated with meiotic irregularities and abnormal embryo sac development. These reproductive limitations hinder conventional breeding and necessitate clonal propagation through vegetative organs [1]. Elevated temperature and water deficit conditions negatively affect plant establishment, rhizome biomass accumulation, and survival in C. officinale. Environmental stress may also alter the accumulation of bioactive secondary metabolites, potentially reducing medicinal quality and commercial value [1–2]. The pharmacological properties of C. officinale are largely attributed to diverse secondary metabolites, including phenolic acids such as ferulic acid and chlorogenic acid, as well as phthalide compounds such as ligustilide [3–4].

Polyploid induction has frequently been associated with enhanced biomass production, stress tolerance, and altered secondary metabolite accumulation in medicinal plants, although these responses are species- and genotype-dependent [5]. Polyploidization can influence secondary metabolism through multiple mechanisms, including increased gene dosage, altered transcriptional regulation, and changes in metabolic flux across biosynthetic pathways. These genomic and regulatory modifications may lead to quantitative or qualitative changes in metabolite accumulation [6, 7]. For instance, polyploid induction enhances secondary metabolite accumulation, including tropane alkaloids in Hyoscyamus muticus, podophyllotoxin in Linum album, artemisinin in Artemisia annua, wedelolactone in Eclipta alba, and trans-teupolioside in Ajuga reptans [6, 7–10]. Recently, several studies have used autotetraploids to increase essential oil content in medicinal plants such as Thymus vulgaris [11], Melissa officinalis [12], and cumin [13]. These findings highlight the effectiveness of polyploid induction in enhancing the medicinal value of plants.

Artificial polyploidization, which duplicates entire chromosome sets, often enhances morphological, physiological, and biochemical traits, improving crop productivity, quality, and resistance to biotic and abiotic stresses [14]. Polyploidization can be induced using various antimitotic agents, most commonly colchicine and oryzalin. Colchicine is widely used to induce polyploidy in plants such as Gerbera [15], Lippia alba [16], Polemonium caeruleum [17], and Cymbidium aloifolium [18] because of its effectiveness in chromosome doubling. In contrast, oryzalin is preferred for its lower toxicity and higher affinity for plant tubulins, making it a safer alternative. Studies show its effectiveness in inducing polyploidy in plants such as Escallonia [19], banana [20], Zea mays [21], and Limonium sinuatum [22]. Both agents are effective for inducing autotetraploids with desirable traits. In vitro polyploidization involves applying antimitotic agents to plant tissues under sterile, controlled conditions. This method enables faster polyploid development with lower mortality and fewer chimeras than in vivo methods [23, 24]. Various explants, including shoot apical meristems, seeds, nodal segments, and leaf segments, can be used [25, 26]. In vitro systems also provide precise control of culture conditions, enabling efficient and reproducible polyploidization. Flow cytometry is widely used to confirm polyploid induction and determine ploidy levels because of its accuracy and efficiency. This technique enables rapid quantification of nuclear DNA content in plant cells, facilitating identification of polyploid individuals [27, 28]. It is also used to determine ploidy levels in plants treated with colchicine and oryzalin, ensuring accurate detection of genome duplication [29, 30].

Autotetraploids (2n = 4x) enhance crucial plant traits and can serve as parents in triploid breeding systems, facilitating crop improvement [31–32]. For instance, autotetraploidy induction increases morphological traits such as leaf size [33, 34], stem thickness [35], stomatal and guard cell size [36, 37], flower size [38, 39], seed size [40], and bark and wood layer thickness [41]. It also improves physiological traits, including chlorophyll content [42], and tolerance to stresses such as drought [43, 44] and pathogens [45].

However, autotetraploids often exhibit dwarfism after transfer ex vitro, hindering plant growth and development [46]. This dwarfism may result from altered expression of genes regulating hormones such as gibberellin, auxin, brassinolide, and jasmonic acid, and can be partially mitigated by supplemental hormone treatments [47, 48]. For example, colchicine-induced polyploids in Populus and Tobacco show reduced growth, which was partially alleviated by hormone treatments [49, 50].

In C. officinale, autotetraploids have previously been induced using oryzalin, and their morphological and phytochemical characteristics have been partially described [51]. However, colchicine-mediated tetraploid induction has not yet been reported in this species, and direct comparisons between colchicine and oryzalin as chromosome-doubling agents remain limited.

Therefore, this study aimed to establish an efficient colchicine-based protocol for in vitro autotetraploid induction in C. officinale and to evaluate the resulting morphological traits and phenolic acid contents. In addition, the effectiveness of oryzalin treatment was assessed under the experimental conditions used in this study.

This study aims to develop an optimized protocol for in vitro autotetraploidy induction in C. officinale to facilitate the breeding of new cultivars with improved morphological traits and enhanced phenolic acid production, ultimately increasing pharmaceutical value and economic benefits for growers.

Results

Tetraploid induction and verification

Colchicine-mediated tetraploid induction

Among 300 colchicine-treated explants, 28 putative tetraploids and 7 mixoploids were initially identified by flow cytometry, while the remaining regenerants were diploid (Table 1). Subsequent ploidy assessment during repeated subcultures revealed that only 11 tetraploids remained cytogenetically stable, whereas some putative tetraploids reverted to diploid or mixoploid states. Plant survival decreased markedly with increasing colchicine concentration, particularly above 100 mg/L, and no plants survived at concentrations ≥ 200 mg/L. The highest tetraploid induction efficiencies were observed at 20 mg/L (40%) and 50 mg/L (30%), indicating these concentrations as optimal for colchicine-mediated tetraploid induction in C. officinale.

Table 1.

Effects of colchicine concentration on polyploid induction in C. officinale after the second subculture

Treatment Colchicine Additional
treatments		 No. of
plants		 Survival rate
(%)		 TT
(%)		 No. of
TTs		 MX
(%)		 No. of
MXs
Conc.
(mg/L)		 duration
(h)
Cont. - - - 20 100 - - - -
Co1 1 48 - 20 80 - - - -
Co5 5 48 - 20 75 5 1 - -
Co10 10 48 - 20 95 10 2 10 2
Co20 20 48 - 20 95 40 8 20 4
Co30 30 48 - 20 100 10 2 5 1
Co50 50 48 - 20 75 30 6 - -
Co80 80 48 - 20 85 10 2 - -
Co100 100 48 - 20 20 10 2 - -
Co100.S10 100 48 10% sucrose 20 20 5 1 - -
Co100.D2 100 48 2% DMSO 20 25 15 3 - -
Co100.20 m 100 48 Dried for 20 min 20 10 5 1 - -
Co200 200 48 - 20 - - - - -
Co500 500 48 - 20 - - - - -
Co1000 1000 48 - 20 - - - - -
Total - - - 300 - - 28 - 7
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Cont. Control, Co Colchicine, S10 10% sucrose, D2 2% DMSO, DMSO dimethyl sulfoxide, 20m Dried for 20 min, No. Number, Conc. Concentration, TT tetraploid, MX mixoploid

Oryzalin-mediated tetraploid induction

No tetraploids or mixoploids were obtained from 480 explants treated with oryzalin in liquid medium for 48 h (Table 2). All plants died at concentrations ≥ 200 mg/L. Although oryzalin is generally considered more plant-specific and less toxic than colchicine, it did not induce tetraploids in C. officinale under liquid-medium conditions. In contrast, five mixoploids were obtained from 180 explants after oryzalin treatment on solid medium (Table 3).

Table 2.

Effects of oryzalin concentration on polyploid induction in C. officinale after the second subculture

Treatment Oryzalin Additional
treatments		 No. of plants Survival rate
(%)		 TT
(%)		 No. of
TTs		 MX
(%)		 No. of
MXs
Conc.
(mg/L)		 Duration
(h)
Cont. - -  - 20 - - - - -
Oy1 1 48  - 20 - - - - -
Oy5a 5 48  - 20 90 - - - -
Oy10a 10 48  - 20 90 - - - -
Oy20 20 48  - 20 90 - - - -
Oy30 30 48  - 20 90 - - - -
Oy50 50 48  - 20 100 - - - -
Oy80 80 48  - 20 100 - - - -
Oy100 100 48  - 20 100 - - - -
Oy100.S10 100 48 10% sucrose 20 90 - - - -
Oy100.D2 100 48 2% DMSO 20 95 - - - -
Oy100.20 m 100 48 Dried for 20 min 20 95 - - - -
Oy200 200 48  - 20 100 - - - -
Oy500 500 48  - 20 100 - - - -
Oy1000 1000 48  - 20 90 - - - -
Total - - - 300 - - - - -
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Cont. Control, Oy Oryzalin, S10 10% sucrose, D2 2% DMSO, DMSO dimethyl sulfoxide, 20m Dried for 20 min, No. Number, Conc. Concentration, TT tetraploid, MX mixoploid

Table 3.

Effects of oryzalin concentration (based on treatment duration) on polyploid induction in C. officinale after the second subculture

Treatment Oryzalin No. of plants Survival rate
(%)		 TT
(%)		 No. of
TTs		 MX
(%)		 No. of
MXs
Conc. (mg/L) Time
(Days)
Cont. - - 20 85 - - -
Oy1.5 1.5 7 20 85 - - -
Oy3.5 3.5 7 20 75 - - -
Oy5b 5 7 20 80 - - 10 2
Oy7 7 7 20 85 - - 5 1
Oy10b 10 7 20 80 - - 10 2
Cont. - - 15 100 - - - -
Oy6 6 14 15 13.3 - - - -
Oy8 8 14 15 0 - - - -
Oy10c 10 14 15 0 - - - -
Total - - 180 - - - - 5
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Cont. Control, Oy Oryzalin, No. Number, Conc. Concentration, TT tetraploid, MX mixoploid

Ploidy identification

Representative histograms of diploid, mixoploid, and tetraploid samples are presented in Fig. 1. The flow cytometric analysis of 300 colchicine-treated and 480 oryzalin-treated plantlets revealed a peak at PE-A 20 for diploids and at PE-A 40 for tetraploids (Fig. 1A). The tetraploid peak, approximately twice that of diploids, confirmed chromosome doubling. Mixoploids exhibited peaks at both PE-A 20 and 40 (Fig. 1B), indicating the coexistence of diploid and tetraploid cells. The G0/G1 peak CVs were below 5% in all representative samples (diploid: 4.82% and 4.75%; mixoploid: 4.84% and 4.58%; tetraploid: 4.73%).

Fig. 1.

Fig. 1

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Flow cytometric analysis of ploidy levels in C. officinale. Histograms show tetraploid (A) and mixoploid (B) profiles, with the upper and lower boxes representing the gated region and number of analyzed cells over time

Flow cytometry was performed at each subculture to monitor ploidy stability, yielding 11 tetraploids and 10 mixoploids. Some tetraploids reverted to diploid or mixoploid, highlighting the need for periodic screening. Mixoploids were separated into diploids or tetraploids through repeated subculture at 4-week intervals, with resolution into diploids or tetraploids typically occurring by the third subculture, indicating that shoot proliferation effectively resolves mixoploidy.

Chromosome analysis confirmed successful doubling in tetraploids (2n = 44) compared to diploids (2n = 22) (Fig. 2).

Fig. 2.

Fig. 2

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Chromosome numbers in diploid (A: C-8) and tetraploid (B: Co30_1–1) of C. officinale after the sixth subculture (scale bar = 50 μm, 1000×)

Comparison of diploid and tetraploid characteristics

Morphology

During in vitro growth, tetraploids had significantly thicker petioles (Tables 4 and 5; Fig. 3), but root number, root length, and leaf traits did not differ. The survival rate of each set of five diploids and tetraploids was 100%. Two months after acclimatization, tetraploids exhibited significantly greater leaf length and area than diploids (Tables 6 and 7; Fig. 4), although they produced significantly fewer leaves overall.

Table 4.

Comparison of in vitro growth traits between diploid and tetraploid C. officinale

Ploidy Petiole length
(cm)		 Petiole diameter
(mm)		 Root number Root length
(cm)
Diploid 5.11 ± 1.25 1.27 ± 0.12 13.67 ± 1.53 12.37 ± 3.02
Tetraploid 5.37 ± 1.16 1.83 ± 0.13* 11.33 ± 0.58 8.56 ± 0.43
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* t-test, p < 0.05 (n = 3)

Table 5.

Comparison of in vitro leaf traits between diploid and tetraploid C. officinale

Ploidy Leaf number Leaf length
(cm)		 Leaf width
(cm)		 Leaf area
(cm2)
Diploid 5.58 ± 2.10 2.82 ± 0.05 3.48 ± 0.33 0.57 ± 0.16
Tetraploid 4.08 ± 0.88 3.12 ± 0.45 3.53 ± 0.61 0.83 ± 0.24
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Fig. 3.

Fig. 3

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Comparison of growth (A, C) and leaf characteristics (B, D: scale bar = 1 cm) in diploid (A, B: C-12) and tetraploid (C, D: Co100.S10_6) C. officinale after the fourth subculture

Table 6.

Comparison of leaf growth traits in diploid and tetraploid C. officinale after acclimatization

Ploidy Petiole length
(cm)		 Petiole diameter
(mm)		 Root number Root length
(cm)
Diploid 7.42 ± 0.69 1.29 ± 0.23 30.00 ± 2.00 5.70 ± 4.82
Tetraploid 7.57 ± 0.78 1.81 ± 0.25 23.33 ± 2.89 7.91 ± 0.69
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Table 7.

Comparison of leaf growth characteristics in diploid and tetraploid C. officinale after acclimatization

Ploidy Leaf number Leaf length
(cm)		 Leaf width
(mm)		 Leaf area
(cm2)
Diploid 22.33 ± 1.15* 2.80 ± 0.08 3.49 ± 0.65 4.04 ± 0.67
Tetraploid 9.67 ± 3.51 3.61 ± 0.23* 4.49 ± 0.43 5.66 ± 0.70*
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*t-test, p < 0.05 (=3)

Fig. 4.

Fig. 4

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Comparison of plant architecture and growth traits in diploid (A, C: C-8) and tetraploid (B, D: Co100_4 − 2) C. officinale after 2 months of acclimatization (bar = 1 cm)

Stomatal structure

Tetraploids had significantly longer stomata and significantly lower stomatal density than diploids (Table 8; Fig. 5).

Table 8.

Comparison of stomatal traits in diploid and tetraploid C. officinale in vitro

Ploidy Average number of stomata
per magnification field
(x400)		 Stomata length
(µm)		 Stomata width
(µm)
Diploid 17.47 ± 4.16* 29.31 ± 3.70 24.44 ± 6.24
Tetraploid 7.47 ± 3.35 46.66 ± 6.82* 38.10 ± 6.61
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* t-test, p < 0.05 (n = 3)

Fig. 5.

Fig. 5

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Comparison of stomatal traits between control (A: C-12) and tetraploid (B: Co100.S10_6) C. officinale after the fourth subculture (scale bar = 50 μm)

Compound analysis

Quantitative UPLC analysis showed excellent linearity (R² > 0.999) for chlorogenic and ferulic acids, with a limit of detection and limit of quantification of 0.01–0.02 µg/mL. Relative standard deviation values for ferulic and chlorogenic acids were 0.07–0.19% and 0.03–0.09%, respectively, confirming method reliability (Tables 9 and 10).

Table 9.

Calibration parameters (linear regression, LOD, LOQ) for ferulic and chlorogenic acids in diploid and tetraploid C. officinale

Compound Regression equation Correlation coefficient (r) Range
(µg/mL)		 LOD
(µg/mL)		 LOQ
(µg/mL)
Ferulic acid y = 3818.6x − 244.25 0.9999 1–100 0.01 0.02
Chlorogenic acid y = 2237.8x -247.86 1 1–500 0.01 0.02
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LOD limit of detection, LOQ limit of quantification

Table 10.

Intra- and inter-day precision (concentration and RSD) for ferulic and chlorogenic acids in diploid and tetraploid C. officinale

Compound Conc.
(µg/mL)		 Intra-day a (n = 3) Inter-day b (n = 3)
Conc.
found
(µg/mL)		 RSD
(%)		 Conc.
found
(µg/mL)		 RSD
(%)
Ferulic acid 5 4.96 0.11 4.96 0.13
25 25.67 0.07 25.67 0.07
50 52.27 0.16 52.30 0.19
Chlorogenic acid 5 4.84 0.09 4.84 0.09
25 25.90 0.04 25.88 0.03
50 52.43 0.06 52.50 0.06
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RSD relative standard deviation

aSample was analyzed three times daily

bSample was analyzed daily for 3 consecutive days

Ferulic acid in fibrous roots was significantly higher in tetraploids (3.49 ± 0.14 µg/mL) than in diploids (2.64 ± 0.27 µg/mL) (Table 11), whereas chlorogenic acid showed no significant difference.

Table 11.

Comparison of major compounds in diploid and tetraploid C. officinale

Compound Ferulic acid
(µg/mL)		 Chlorogenic acid
(µg/mL)
Diploid 2.64 ± 0.27 2.30 ± 1.10
Tetraploid 3.49 ± 0.14* 4.01 ± 2.67
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* t-test, p < 0.05 (n = 3)

Methods

Plant material

Plant materials of C. officinale were obtained from a cultivation field in Subi-myeon, Yeongyang-gun, Gyeongsangbuk-do, Republic of Korea. The plant materials were formally identified by Dr. Ho Jun Son, Gangwon national university in Korea. A voucher specimen (specimen numbers: FMRC-191008-07-02–4528 A) was deposited at the Herbarium of the Forest Medicinal Resources Research Center (FMRC), which is affiliated with the National Institute of Forest Science in Korea. The materials used in this study were obtained from cultivated stocks; therefore, no specific permission or license was required for plant collection.

Buds from selected elite rhizomes were utilized as explants for shoot induction. Explants were washed under running tap water to remove surface debris, surface-sterilized in 70% ethanol for 2 min, and rinsed three times with sterile distilled water. Subsequently, explants were treated with 4% sodium hypochlorite (NaOCl) for 30 min and washed at least five times with sterile distilled water. After bud excision, explants were cultured on Murashige and Skoog (MS) medium with 100 mg/L tetracycline to eliminate contamination. Cells were cultured in 90 × 10 mm Petri dishes (SPL Life Science Inc., Pocheon, Korea).

In vitro culture conditions

The basal medium comprised MS medium supplemented with 3% sucrose and 0.3% gelrite. The medium pH was adjusted to 5.7 before autoclaving at 121 °C for 15 min (HS-50200, Hanshin Medical Corporation, Incheon, Korea). Glass bottles (H: 13.0 cm × W: 7.7 cm) served as culture vessels. Cultures were subcultured every 4 weeks after multiplication. Culture conditions were maintained in a controlled environment (Korea Science and Technology Industry, Suwon, Korea) at 25 ± 1 °C with 45 ± 2% relative humidity. A 16-h photoperiod was provided using white fluorescent light (70 µmol m⁻² s⁻¹; FHF32W/865, OSRAM, Munich, Germany).

Tetraploid induction and verification

Colchicine and oryzalin mediated tetraploid induction

To induce autotetraploidy, in vitro cultured C. officinale plants were treated with various concentrations of colchicine and oryzalin (Duchefa, The Netherlands) under different methods and treatment durations. For explants, shoots from in vitro C. officinale were excised into approximately 1–2 cm segments, with roots entirely removed. Liquid MS medium supplemented with filter-sterilized antimitotic solutions (oryzalin, colchicine) at 0, 1, 5, 10, 20, 30, 50, 80, 100, 200, 500, and 1000 mg/L was used for tetraploid induction. Cell wall penetration was attempted using three methods under 100 mg/L concentration of oryzalin and colchicine: 10% sucrose application replacing 3% sucrose, pre-treatment drying for 20 min, and adding 2% dimethyl sulfoxide (DMSO, Sigma-Aldrich, USA). Each treatment was conducted for 48 h, using 20 explants, with a total of 300 explants used across all treatments. After shaking (180 rpm, 25 ± 1 ℃, dark), the explants were washed thrice with distilled water, blot-dried with sterilized filter paper, and transferred to solid MS medium. In a separate treatment, in vitro plantlets cultured on solid MS medium were exposed to oryzalin (0, 1.5, 3.5, 5, 7, 10 mg/L) for 7 days using 20 explants per treatment, with a total of 120 explants included in the experiment. Additionally, explants (n = 15 per concentration) were cultured on solid MS medium, with a total of 60 explants used across all treatments, containing 6, 8, and 10 mg/L oryzalin for 14 days to evaluate a broader concentration range. Post-treatment, all explants were subcultured on fresh solid MS medium at 4-week intervals as previously described above.

Liquid and solid media were adjusted to pH 5.7 before autoclaving, supplemented with 3% sucrose, autoclaved at 121 ℃ for 15 min, and filter-sterilized antimitotic solutions were added. The solid medium was also supplemented with 0.3% gelrite (Duchefa, The Netherlands). Stock solutions of antimitotic agents were prepared by dissolving colchicine in water and oryzalin in DMSO.

Ploidy identification

The polyploidy level of C. officinale was determined using a flow cytometer (CytoFLEX, Beckman Coulter Inc., Fullerton, CA, USA). Using the CyStain PI Absolute P Kit (Partec, Germany), nuclei were extracted from 1 cm² leaf discs of C. officinale and Nicotiana tabacum cv. Xanti (internal reference standard) in 200 µL of extraction buffer. The solution was filtered through a 30 μm filter (CellTrics™, Partec, Germany). Subsequently, 800 µL of staining solution was added to the filtered solution for nuclear DNA staining, determining relative genome size and ploidy level. The samples were incubated in darkness for 5 min at room temperature, then analyzed using CytoFLEX. At least 5000 nuclei were analyzed per sample. For accurate flow cytometric analysis, a gate was established on the SSC-A versus PE-A dot plot to exclude debris and background noise. Only events with PE-A values of ≥ 10 were selected for subsequent analyses.

Chromosome counts were determined using the direct method for diploids and tetraploids. To confirm chromosome numbers, root tip meristems (~ 7 mm) from diploids and tetraploids, previously identified using flow cytometry, were collected between 08:00 and 09:00, coinciding with peak mitotic activity. Root tips were pretreated in distilled water at 4 °C for 24 h, then fixed in Carnoy’s solution (99% EtOH: acetic acid = 3:1) for 6 h. After fixation, samples were washed thrice with distilled water, treated with 1 N HCl at 60 °C for 30 s to soften cell walls, and subsequently rinsed again thrice with distilled water. Root tips were stained with 1% aceto-carmine, gently squashed on microscope slides, and observed under a light microscope (BX53, Olympus, Tokyo, Japan).

Comparison of diploid and tetraploid characteristics

Morphology

Morphological characteristics of diploid and tetraploid plants were compared under controlled in vitro conditions (25 ± 1 ℃, 45 ± 2%). Parameters including plant height, number of leaves, leaf length and width, leaf area, petiole diameter, number of roots, and root length were measured using 4-week-old plants, with three replicates per parameter, using a digital vernier caliper (CD-20APX, Mitutoyo Corp., Kanagawa, Japan).

After ploidy verification, each set of five diploids and tetraploids was subcultured three times and acclimatized for further study. In vitro plantlets were thoroughly washed and transplanted into 10 cm plastic pots containing an artificial soil mixture (peat moss: perlite: vermiculite = 1:1:1) and irrigated weekly. Immediately after acclimatization, the temperature was maintained at 25 ± 1 °C with 90% relative humidity, which was gradually reduced to 60% after 2 weeks under a 16-h photoperiod at 70 µmol m⁻² s⁻¹ light intensity. After 4 weeks, plants were transferred to the greenhouse for 30 days, and growth parameters were analyzed after 2 months of ex vitro cultivation.

Under controlled greenhouse conditions (25 ± 1 ℃, 45 ± 2%), the growth characteristics of diploids and tetraploids were evaluated. Plant height, number of leaves, leaf length and width, leaf area, petiole diameter, number of roots, and root length of 8-week-old plants were measured (n = 3) using a digital vernier caliper (CD-20APX, Mitutoyo Corp., Kanagawa, Japan).

Stomatal structure

Stomatal traits such as length, width, and density were measured using the leaf epidermis. To isolate the epidermal layer, expanded leaves were coated with colorless varnish, and the layer was subsequently peeled off using tweezers. The separated epidermal layers were mounted on glass slides, and fixed sections were observed and documented using a light microscope with a camera (DMI8, Leica, Germany). Images were analyzed using ImageJ software.

Compound analysis

Aerial parts and fibrous roots of in vitro-grown plants were separated, freeze-dried, and stored at room temperature with silica gel. Dried samples (10 mg) were extracted using 1 mL of 100% methanol, sonicated for 1 h, and centrifuged at 8,000 rpm for 10 min (LABOGENE, 1248). Before analysis, the supernatant was filtered through a 0.2-µm membrane filter (Whatman). The samples were analyzed using a Shimadzu Nexera X2 UHPLC system (Shimadzu Co., Kyoto, Japan). The system comprised two LC-30AD pumps, a DGU-20A5R degasser, an SIL-30AC autosampler, a CTO-20 A column oven, a CBM-20 A system controller, and an SPD-M20A PDA detector. The analysis utilized an Acquity UPLC HSS T3 column Agriculture 2025, 15, 1950 4 of 15 (part number 186003539, C18, 2.1 mm × 100 mm, 1.8 μm, 100Å, Waters Co., Milford, MA, USA) within a column oven at 30 ℃. The mobile phase consisted of water with 0.1% formic acid (A) and ACN with 0.1% formic acid (B), with the following gradient conditions: initial to 2 min, 20% B; 4 min, 55% B; 10 to 14 min, 85% B. The flow rate was 1 mL/min, the injection volume was 2 µL, and detection was performed at 280 nm using a photodiode array detector. Peak areas were quantified using Empower Software 2.0 and expressed in µg/mL. The extract samples were analyzed at UV 280 nm. The data were processed using LabSolutions software (version 5.82, Shimadzu, Kyoto, Japan), and the samples were analyzed in triplicate and expressed as mean values. Precision was assessed using the relative standard deviation (RSD, %) for repeatability and reproducibility. The validation samples were tested three times at three different concentrations,

Statistical analysis

Morphological traits were evaluated using three independent biological replicates (three individual plants) for each ploidy level. Plant length was measured once for each plant. All other morphological traits, including root length, root diameter, petiole length, petiole diameter, leaf length, and leaf width, were measured three times on each individual plant, and the average value was used for statistical analysis. The polyploid induction experiment was conducted using a single batch of 20 explants for each treatment. Therefore, survival and tetraploid induction frequencies are presented as descriptive data without statistical comparison. Morphological and biochemical analyses were performed using three independent biological replicates. Mean values were analyzed using Student’s t-test, and differences were considered statistically significant at P < 0.05. All results are presented as means ± standard deviation (means ± SD). Statistical analyses were performed using SPSS 25.0 software (SPSS Inc, IBM, USA).

Discussion

In C. officinale, colchicine effectively induced tetraploid, whereas oryzalin showed limited effectiveness under similar conditions. Flow cytometric analysis and chromosome counting clearly distinguished diploid from tetraploid individuals, confirming chromosome doubling.

The differential effectiveness of colchicine and oryzalin observed in this study is likely attributable to their distinct modes of interaction with microtubules and their uptake efficiency in plant tissues. Colchicine binds to tubulin dimers and inhibits microtubule polymerization, thereby effectively disrupting spindle formation during mitosis [52] In contrast, oryzalin, a dinitroaniline herbicide, binds specifically to plant α-tubulin with high affinity but may exhibit limited cellular uptake depending on tissue type and exposure conditions [53]. Furthermore, differences in tissue permeability and explant sensitivity may have contributed to the observed outcomes. The explants used in this study may have allowed more efficient penetration or retention of colchicine compared to oryzalin, particularly under the given treatment duration. It is also possible that the exposure period or concentration of oryzalin was insufficient to induce stable chromosome doubling, as oryzalin often requires optimized conditions tailored to species and tissue type. Therefore, the superior performance of colchicine in this study is not solely attributable to experimental variability or genetic background but can be mechanistically explained by differences in microtubule-binding dynamics, intracellular accessibility, and explant-specific responsiveness.

The highest tetraploid induction efficiencies were observed at 20 mg/L (40%), suggesting that moderate colchicine concentrations are optimal for chromosome doubling in C. officinale. While survival rates decreased markedly with increasing colchicine concentration, concentrations > 100 mg/L caused severe toxicity, and no plants survived at ≥ 200 mg/L. Some plants died during subculture, likely owing to antimitotic-induced toxicity or physiological imbalance [54]. These findings are consistent with previous studies demonstrating that colchicine effectively induces polyploidy but can cause severe phytotoxic effects at high concentrations owing to its strong inhibition of spindle fiber formation during mitosis [15–17].

Oryzalin, generally considered more plant-specific and less toxic than colchicine [19–21], did not induce stable tetraploids under liquid-medium conditions in this study. While previous studies reported tetraploid induction at 4.0 mg/L oryzalin for 2 days [51], no tetraploids were obtained under identical conditions, likely owing to differences in laboratory conditions or genetic background. Only a few mixoploids were produced with oryzalin application on solid medium, suggesting that discrepancies may result from differences in treatment conditions, explant physiological status, or genetic background of the plant material.

Mixoploidy is a major challenge during polyploid induction, necessitating repeated ploidy screening [55]. Mixoploidy occurs because antimitotic agents often affect only a portion of the meristematic cells, producing chimeric tissues with cells having different chromosome numbers [56]. In this study, repeated subculture effectively reduced mixoploidy, as some mixoploid individuals gradually separated into either diploid or tetraploid plants over successive cycles. Similar findings were reported in other species, where repeated subculture or regeneration mitigated mixoploidy [57, 58].

Despite successful tetraploid induction, some plants died during subculture, likely owing to residual toxicity of antimitotic agents or physiological imbalances caused by altered chromosome numbers. In addition, several tetraploid plants reverted to diploid or mixoploid states, indicating that ploidy stability could not be assumed immediately after induction. These results highlight the importance of long-term ploidy monitoring using flow cytometry to select genetically stable polyploid lines suitable for further propagation and breeding.

Cytological analysis confirmed chromosome doubling in tetraploid plants, with chromosome numbers increasing from 2n = 22 in diploids to 2n = 44 in tetraploids, consistent with observations in Hibiscus syriacus polyploids [59].

During in vitro growth, tetraploids had significantly thicker petioles than diploids. Organ thickness is a typical morphological response to polyploidization and is generally associated with enlargement of individual cells [35]. The morphological changes observed in the present study were generally consistent with those reported by Kim et al. [51] for oryzalin-induced tetraploid C. officinale. Both studies reported increased petiole thickness, enlarged stomata, reduced plant height, and enhanced accumulation of root secondary metabolites. For example, Kim et al. [51] reported an approximately 39% increase in petiole diameter in tetraploids, whereas the colchicine-induced tetraploids in this study exhibited a 44% increase. Similar increases in stomatal size and dwarf growth characteristics were also observed. These similarities suggest that the observed phenotypic changes are primarily associated with genome doubling itself rather than the specific antimitotic agent used. Therefore, the principal contribution of the present study is the establishment of a colchicine-mediated tetraploid induction system for C. officinale and the characterization of ferulic acid accumulation in the resulting tetraploid plants.

After acclimatization, tetraploids also exhibited greater leaf length and leaf area than diploids, consistent with findings in several previous studies [33, 34]. However, tetraploids exhibited fewer leaves and reduced growth compared with diploids, indicating a dwarf phenotype. Dwarfism is frequently observed in tetraploids of Populus alba, Allium spp., and Escallonia spp [19, 46, 49]., with similar growth reduction observed in this study. Moreover, oryzalin-induced mixoploids exhibited narrower leaves than diploids, suggesting that partial polyploidization can also affect plant morphology. These phenotypic variations further highlight the complexity of cellular and developmental responses following chromosome doubling.

Stomatal characteristics further support these findings. Tetraploids exhibited increased stomatal and guard cell size, consistent with the established relationship between genome size and cell dimensions in polyploid plants [36, 37]. In addition, significantly longer stomata indicate that chromosome doubling led to increased cell size and enhanced organ thickness.

Polyploidization is often associated with higher secondary metabolite accumulation, likely owing to gene dosage effects and enhanced metabolic capacity [6, 8]. Similar increases in secondary metabolites have been observed in tetraploids of Linum album, Artemisia annua, and Ajuga reptans [7, 9, 10], suggesting that tetraploidization enhances metabolite accumulation in C. officinale. These findings indicate that tetraploidization may also enhance metabolite accumulation in C. officinale, an important medicinal plant valued for its bioactive compounds. Therefore, the tetraploid lines developed in this study may provide valuable genetic resources for improving the pharmacological quality of this species.

One limitation of this study is the relatively small number of biological replicates used for morphological and biochemical analyses. Although technical replication reduced measurement error, the limited number of independent biological replicates may have reduced the statistical power to detect differences in traits with relatively high biological variability, such as chlorogenic acid content. Therefore, the non-significant difference in chlorogenic acid content should be interpreted with caution.

Overall, colchicine treatment combined with repeated in vitro subculture and ploidy screening is effective for generating stable tetraploid plants of C. officinale. The study also highlights the importance of optimizing antimitotic treatments and monitoring long-term ploidy stability to ensure successful polyploid breeding. These findings provide a foundation for future research aimed at evaluating the agronomic traits and metabolite profiles of tetraploid C. officinale lines.

Conclusion

This study successfully established an effective in vitro system for polyploid induction in C. officinale and generated tetraploid plants through colchicine treatment. The induced tetraploids were confirmed by flow cytometric analysis and morphological characterization.

Colchicine was a highly effective antimitotic agent, achieving the highest induction rate of 40% at 20 mg/L in liquid medium, followed by 30% at 50 mg/L. A total of 28 tetraploid plants were identified from 300 colchicine treated explants (9.8%). In solid-medium oryzalin treatments, approximately 10% mixoploids were obtained at 5 and 10 mg/L, whereas no tetraploids were induced in liquid-medium oryzalin treatments. The overall rate of oryzalin treated mixoploid is 5 of 480 (1.0%).

Chemical analysis demonstrated that tetraploid plants showed increased accumulation of ferulic acid compared with diploid plants, suggesting that chromosome doubling may influence specific metabolite biosynthesis.

Tetraploid plants exhibited distinct morphological alterations, including enlarged leaves and thicker petioles, indicating that polyploidization induced clear phenotypic variation in C. officinale.

Although tetraploid plants showed improved ferulic acid accumulation and distinct leaf morphological characteristics, reduced vegetative growth was also observed. These results suggest that polyploid breeding in C. officinale may involve trade-offs between biomass productivity and phytochemical improvement.

This study is the first to report colchicine-mediated chromosome doubling in C. officinale and the resulting significant morphological and functional changes.

Further studies are needed to evaluate the agronomic performance of tetraploid lines under greenhouse and field conditions, assess the stability of metabolite production across generations, and investigate the molecular mechanisms underlying metabolite biosynthesis in polyploid plants.

Acknowledgements

Not applicable.

Authors’ contributions

Conceptualization, H.-Y.J. ,J.-A.K., H.-J.S. and J.-W.K.; methodology, H.-Y.J., J.-A.K. and J.-W.K.; software, H.-Y.J.; validation, H.-Y.J.; formal analysis, H.-Y.J.; investigation, H.-Y.J.; resources, H.-Y.J.; data curation, H.-Y.J.; writing—original draft preparation, H.-Y.J.; writing—review and editing, J.-A.K.; H.-J.S and J.-W.K.; visualization, H.-Y.J.; supervision, J.-A.K., H.-J.S. and J.-W.K.; project administration, H.-J.S. and J.-W.K. ; funding acquisition, H.-J.S. ; All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the National Institute of Forest Science (grant number FP0802-2023-01-2026).

Data availability

All data generated and/or analyzed during this study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

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Contributor Information

Ho Jun Son, Email: shj7400@korea.kr.

Jun Won Kang, Email: jwkang15@knu.ac.kr.

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Data Availability Statement

All data generated and/or analyzed during this study are available from the corresponding author on reasonable request.

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