Abstract
Background
Polyploidy is also important in evolution of plants, and improvement of crops through increase in morphological, physiological, and biochemical characteristics. The objective of the current investigation was to induce polyploidy in one of the varieties of Brassica rapa namely chinensis with colchicine and to determine its effect on germination, growth, cytological behavior, minerals composition and nuclear DNA content.
Results
In vitro results were performed on the mature seeds of five diverse genotypes to be treated with 0.05, 0.08, 0.1, and 0.2 percent colchicine after 0, 24 and 48 h treatment. Parameters of germination such as days to germination and germination percentage were recorded daily. The colchicine level and duration of the treatment had a significant effect on all parameters (p < 0.05). The best responses were realized with optimal treatment (0.08% 24 h) which included early germination (2.33 days), maximum seedling height (5.75 cm), increased root length (6.06 cm), improved biomass accumulation, and stomatal enlargement to a maximum of 31.76 µm. The maximum efficiency of polyploid induction was at 0.2% with a 24 h time yielding 58.67% with lower survival rates and greater cytological anomalies. Mitotic index decreased to 21.2 to 8.0 at 0.2% and 48 h and abnormal frequency of mitosis was raised to 75.0. The nutrient accumulation was better with the optimum treatment as mineral analysis indicated with potassium (38,000 mg/kg), calcium (22,000 mg/kg), and phosphorus (2,600 mg/kg). The successful polyploid induction was supported by the fact that flow cytometric analysis of 30,270 nuclei showed that two groups of cells were present, which are diploid (46.4%) and endoreduplicated (53.6%), with an endoreduplication index of 53.6%.
Conclusion
Colchicine (0.08% in 24 h) usage optimally produces polyploidy in Brassica rapa var. chinensis, which increases germination, growth, and mineral composition. Five different genotypes have been used, which makes sure that there will be no genotype-specific findings. Flow cytometry had verified a successful chromosome doubling with definite results of both diploid and endoreduced populations. The results give a basis through which better polyploid lines can be developed, and further field tests should be conducted to determine agronomic potential.
Graphical Abstract
Keywords: Polyploidy induction, Colchicine, Cytological analysis, Stomatal traits, Mineral composition, And vegetative growth parameter
Introduction
Agriculture is a key to food security and economic growth of the world, especially in developing countries, where approximately 40–45 percent of the population is directly or indirectly reliant on agricultural processes [7]. By 2050, the world will have 9.7 billion people, which will increase the demand of the nutritious and high-yielding crops as well as the crops that are stress-tolerant [11]. Traditional breeding methods do not always suffice to address them,hence, new genetic and cytogenetic methods like polyploidy induction have become rather significant in the crop improvement programs [6, 41]. Polyploidy refers to the situation whereby an organism has more than two sets of complete chromosomes in its nucleus. Whereas the diploid plants have a 2n = 2 × chromosome set, polyploids can be 3x, 4 × and beyond [15]. It has been estimated that about 47–70 per cent of all flowering plants have undergone one or more polyploidization episodes in their evolutionary history. Polyploidy has been a core part of evolution and speciation in plants, as well as genetic diversification [26]. Polyploid plants tend to have high morphological and physiological traits, such as larger cell size, bigger leaves, stems, more chlorophyll, and high biotic and abiotic stress tolerance [9]. Artificial polyploidy is usually induced by the use of antimitotic agents that disrupt the formation of spindle fibres in the course of mitosis. The most widely used of these agents is colchicine because of its effectiveness and reliability [34]. Colchicine is an alkaloid naturally occurring, and is mostly extracted out of Colchicum autumnale and Gloriosa superba. It works by binding to β-tubulin which suppresses the polymerization of microtubules and halts the process of cell division at the metaphase stage. Consequently, the segregation of chromosomes does not occur and the daughter cells double the chromosomes [28, 30]. Colchicine is commonly used in the range of 0.01–0.5 percent and ranges of 6–48 h in the field of plant polyploidization, depending on the type of plant and tissue, as well as method of application [33, 45]. Nonetheless, there is a need to optimize on concentration and exposure period because when increased doses are administered they can lead to decreased survival of the plants or abnormal growth. Brassica is a type of vegetable, oilseeds, and condiments genus in the family Brassicaceae that includes a number of economically significant crops that are grown all over the world [32]. The Brassica rapa var. chinensis, also known as pak choi or Chinese cabbage is a popular type of leafy vegetables, especially in East and Southeast Asia. It is a diploid with a chromosome number of 2n = 20 (AA genome) and a short growth cycle of about 50–60 days [42]. Pak choi thrives in temperatures of 13–21 C and is considered to be a fast-growing and skilled plant. Pak choi is high in nutritional vitamins and minerals. It is a source of about 20 kcal of energy, 45 mg of vitamin C, 4468 IU of vitamin A, 105 mg of calcium, and 1.2g of dietary fiber per 100 g of fresh leaves [24, 44]. However, it has potential to improve its yield, biomass and stress tolerance although it has a nutritional and economic significance. Polyploid induction in Brassica rapa var. chinensis is likely to enhance agronomic characteristics, including leaf area, fresh biomass, chlorophyll level, and the general plant vitality with the so-called gigas effect, traditionally used in polyploid plants [1, 18]. In addition, the induction of polyploidy in Brassica rapa var. chinensis with colchicine may be more tolerant to environmental stresses, and hence, suitable to sustainable vegetable production via colchicine induction can be examined in the present study [18, 25].Thus, the primary aim of the present study is to induce polyploidy in Brassica rapa var. chinensis by using colchicine and to determine how the various concentrations of colchicine The results of the study are useful in conducting future experiments to assess the productivity and quality of pak choi in the field.
Materials and methods
Plant material, seed selection and explant preparation
The seeds of five different genotypes of Brassica rapa var. chinensis were sourced in various locations within Peshawar, Pakistan, one of them being the Nuclear institute of food and Agriculture (NIFA), the University of Agriculture Peshawar (Department of Plant Breeding and Genetics), PCSIR Laboratories Complex, the Institute of Biotechnology and Genetic Engineering (IBGE), and the Agricultural Research Institute (ARI), Tarnab. Only healthy full grown seeds were chosen and further experimentation was done. Opening of pods was done with a sharp knife rinsed with sterility to prevent damage to seeds. The seeds were also divided into mature, immature and shriveled seeds and only the healthy and fully matured seeds were used in further experimentation and the rest of the seeds were discarded. Surface sterilization was done to remove microbial contamination that was usually experienced in in vitro conditions. Sodium hypochlorite (NaOCl) was used at concentration of 0, 5 and 10 percent and incubated with the seed in duration of 1, 5 and 10 min. The treated seeds were washed using sterile distilled water three times and then tested on viability and germination. Light green embryos were counted as viable seeds and the percent germination was determined using the normal formula. The experiment was designed in a randomized complete block version (RCBD) whereby there were three biological replicates per treatment. In every biological replicate, there were 10 seeds and this formed 30 seeds in total (3 replicates of 10 seeds), each. These 30 seeds per treatment were used to record all the parameters of germination, growth, biomass and all the survival parameters.
Colchicine treatment and polyploidy induction
Smooth seeds were then surface sterilized with 70 percent ethanol in 5 min and then 10 percent sodium hypochlorite in 10 min and then washed with a lot of autoclaved distilled water. Induction of polyploidy in seeds was then done by placing the seed in various concentrations of colchicine (0.05, 0.08, 0.1 and 0.2 percent) over a period of 24 and 48 h. Dimethyl sulfoxide (DMSO) was added to each solution in few drops in order to facilitate the penetration of colchicine. All the procedures were carried out in a biosafety cabinet so as to provide aseptic conditions. Seeds were washed and left to heal after treatment before being left to culture. The efficiency of polyploidy induction was determined by the colchicine concentration and exposure time, which was subsequently validated by morphological, cytological and molecular methodology.
Media preparation and in vitro culturing conditions
The preparation of the Murashige and Skoog (MS) medium was done according to the standard protocol with the agar (8 g/L) to cement the medium and the pH adjusted to 5.8. About 10 mL medium was inoculated into all test tubes and autoclaved at 121o C and 1520 min. The 0 h seeds were treated and controlled then cultured on MS medium in an aseptic manner. The cultures were kept in a growth room at 25 O C with a light intensity of about 2500 lx. The germination of seeds, growth of seedlings were also observed frequently to assess the effects of colchicine treatment on seedling survival, growth behavior, and polyploid induction.
Transplantation, hardening and survival assessment
Plantlets that germinated and developed were carefully clipped off test tubes and transplanted into pots with soil to acclimatize them. Polythene bags were used to cover the plants during a period of one week to ensure that the plants were kept moist and to avoid transplant shock. Plants were then gradually put under natural environmental conditions after hardening. Germination was observed and the survival rates were noted at the early stages and the first two weeks. The percentage of survival was obtained by dividing the 30 seeds initially cultured in each of the treatments (10 seeds per replicate × 3 replicates) with the following formula: Survival rate (percent) = (Surviving plants/30) × 100.
Morphological evaluation and structural parameters
Morphological evaluation was conducted to identify differences between control (0 h) and colchicine-treated plants. Parameters such as plant height, leaf length, leaf width, leaf thickness, and overall vigor were recorded at early seedling stages. Stomatal characteristics were examined using the nail varnish technique, and stomatal size and density were measured under a light microscope. Polyploid plants generally exhibited thicker leaves, larger stomata, reduced stomatal density, and increased leaf size compared to control (0 h). Floral traits such as flower size, petal size, and flower density were also observed, with polyploid plants showing enhanced floral characteristics compared to control (0 h).
Cytological and genotoxicity analysis
Cytological analysis was carried out to confirm polyploidy and assess genotoxic effects. For each treatment, 10 root tips (3–4 root tips per replicate) were collected and examined. A minimum of 1000 cells per treatment were scored to calculate mitotic index (MI%) and abnormal mitotic frequency (AMF%) using the following formulae:
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Chromosomal abnormalities such as lagging chromosomes, bridges, stickiness, micronuclei, and C-metaphase were recorded. Reduced MI and more AMF was a good sign of genotoxic stress.
Molecular confirmation and flow cytometry analysis
Polyploidy was confirmed by the molecular method of flow cytometry to determine the content of nuclear DNA. The nuclei isolation was done using Galbraith buffer on young leaf tissue (about 100 mg) and then filtered with a 30 µm nylon mesh. Propidium iodide (PI) and RNase were used to stain DNA and remove RNA interference. Analysis of samples was done by CytoFLEX flow cytometer. Diploid plants only had one G1 peak (2n DNA content), and polyploid plants had peaks with relatively twice the intensity of the fluorescence (4n). Regenerated plant DNA content was found to be consistent with repeated measurements.
Statistical analysis
A two-way analysis of variance (ANOVA) in SPSS (version 26.0) was used to assess the primary impact of colchicine concentration and treatment period as well as interaction between the two on each of the measured parameters. In cases where ANOVA showed significant interaction (p < 0.05), the means of all combinations of treatments (concentration × duration) were compared by the use of Fisher Least Significant Difference (LSD) post-hoc test. The various superscript letters a, b, c, d, e, f used in the tables reveal that there exists significant differences between these means of interaction at p < 0.05. This method will make sure that main effects along with interaction effects are well taken into consideration in comparisons of the means.
Results
Germination response and early establishment
The concentration and the treatment time of colchicine had statistically significant effect on the germination behavior of Brassica rapa var. chinensis (Table 1).
Table 1.
Effect of colchicine concentration (% w/v) and treatment duration (h) on survival percentage (%) of Brassica rapa var. chinensis
| Colchicine concentration (% w/v) | Survival at 0 h (%) | Survival at 24 h (%) | Survival at 48 h (%) | Mean survival (%) | Reduction vs control (%) |
|---|---|---|---|---|---|
| Control 0 h | 55.00 ± 1.73 a | 56.00 ± 2.00 a | 54.33 ± 1.53 a | 55.11 | 0.00 |
| 0.05 | 43.67 ± 1.53 c | 51.67 ± 1.53 b | 49.00 ± 1.00 b | 48.11 | 7.00 |
| 0.08 | 44.33 ± 1.15 c | 44.00 ± 1.73 c | 41.00 ± 1.00 c | 43.11 | 12.00 |
| 0.10 | 42.67 ± 1.53 c | 36.67 ± 1.53 d | 32.33 ± 1.53 d | 37.22 | 17.89 |
| 0.20 | 49.33 ± 1.53 b | 28.00 ± 1.00 e | 25.00 ± 1.00 f | 34.11 | 21.00 |
The values are mean SD ± 3 of three biological replicates (n = 3). The notations of the letters in the same column have shown a substantial difference in treatment combinations (concentration x duration) at p = 0.05 (LSD test following significance interaction of ANOVA)
The Germination time was also significantly different among treatments (p < 0.05), with the Germination time of 2.33 days at 0.08% × 24 h to 5.67 days in control. The germination of the seeds was faster, on the time of optimal treatment (0.08% × 24 h) with germination that was achieved in about 3 days compared with control, and with higher concentration of the treatment (0.2%) the germination was delayed indicating a response that is dose dependent (Fig. 1). Analysis of variance showed that concentration of colchicine, duration of exposure, and the interaction of the two had a significant effect on the days to germination (p < 0.05), indicating that moderate colchicine concentration and the duration of exposure to colchicine significantly contributed to the early seedling establishment, but high concentration reduced seed germination.
Fig. 1.
Germination time of Brassica rapa var. chinensis seeds treated with different colchicine concentrations and durations. Bars represent mean ± SD (n = 3). Different letters above bars indicate significant differences among minerals at each concentration (p < 0.05; two-way ANOVA followed by LSD test)
Seedling growth and root development
Seedling height and root length of Brassica rapa var. chinensis exhibited marked numerical variation in response to different colchicine concentrations and treatment durations (Fig. 2).
Fig. 2.
Growth parameters of Brassica rapa var. chinensis under different colchicine treatments. A Quantitative comparison of seedling height and root length at 24 h treatment. B Visual comparison of plant height between diploid and polyploid plants. C Leaf length comparison between one-week-old diploid and polyploid seedlings showing enhanced leaf growth in polyploid individuals (3.7–5.2 mm) compared to diploids (3.0–4.4 mm)
Mean seedling height varied from 2.84 cm at 0.2% to 4.70 cm at 0.08% colchicine. Compared to the control 0 h (3.02–3.09 cm), seedlings treated with 0.08% colchicine showed an increase of approximately 1.68 cm, highlighting enhanced vegetative growth. With respect to treatment duration, the highest mean seedling height was recorded at 24 h (4.35 cm), which was 1.26 cm taller than the control at 0 h (3.09 cm) and 0.56 cm taller than the 48 h treatment (3.79 cm). The interaction effect further amplified growth, where the maximum seedling height (5.75 cm) was observed at 0.08% × 24 h, exceeding the minimum height (2.87 cm) recorded at control 0 h by 2.88 cm, or nearly twofold. Root length increased from 2.47 cm at 0.2% to 4.86 cm at 0.08% colchicine. Across durations, the highest mean root length was observed at 24 h (4.05 cm), compared to 3.72 cm at 48 h and 2.66 cm at 0 h. The longest roots (6.06 cm at 0.08% × 24 h) were 4.14 cm longer than the shortest roots (1.92 cm at 0.2% × 24 h). Overall, seedling height and root length ranged from 2.84–5.75 cm and 1.92–6.06 cm, respectively, demonstrating a strong growth-promoting effect of moderate colchicine concentration (Fig. 3). The ANOVA test used indicated that the concentration of colchicine, duration, and the interaction of the two factors had a significant effect on seedling growth and roots development (p < 0.05).
Fig. 3.
Seedling height and root length of Brassica rapa var. chinensis under different colchicine concentrations at 24 h treatment. Bars represent mean ± SD (n = 3). Different letters above bars indicate significant differences among minerals at each concentration (p < 0.05; two-way ANOVA followed by LSD test)
Biomass accumulation and vegetative growth parameters (Lab)
Root and shoot biomass of Brassica rapa var. chinensis responded significantly to different colchicine concentrations and treatment durations (Tables 2 and 3). Mean root biomass ranged from 0.02 g in control to 0.05 g at 0.08% colchicine. Root biomass ranged from 0.01 g to 0.06 g across treatments highlighting the strong stimulatory effect of optimized colchicine exposure. Across treatment durations, root biomass increased from 0.02 g at control (0 h) to 0.04 g at 24 h, before slightly declining to 0.03 g at 48 h, indicating that 24 h exposure was the most effective. Shoot biomass increased from 0.10 g at 0.2% to 0.15 g at 0.08% colchicine. The 0.08% treatment also indicated better shoot vigor since it increased by 0.04 g compared to the control 0 h (0.11 g) (Fig. 3). In terms of duration, the mean biomass of the shoot was highest at 24 h (0.14 g), then at 48 h (0.13 g) and lastly at 0 h (0.11 g). The biomass of the shoot was 0.09 g to 0.18 g between treatments. In general, the biomass accumulation was 0.01–0.06g of root biomass and 0.09–0.18g of shoot biomass, which showed that the biomass had increased significantly in moderate levels of colchicine treatment. The statistical analysis has confirmed that the concentration of colchicine, the length of treatment, and the interaction between treatment length and colchicine concentration was significant in influencing the biomass accumulation (p < 0.05) (Fig. 4).
Table 2.
Types of chromosomal abnormalities observed in root tip cells of Brassica rapa var. chinensis under different colchicine concentrations
| Colchicine Concentration | Stickiness | Bridges | Lagging Chromosomes | Micronuclei | C-Metaphase | Total Abnormalities (%)* |
|---|---|---|---|---|---|---|
| Control (0%) | + | + | + | + | - | 2.44 |
| 0.05% | + | + | + | + | + | 10.78 |
| 0.08% | + | + | + | + | + | 20.33 |
| 0.10% | + | + | + | + | + | 36.78 |
| 0.20% | + | + | + | + | + | 50.11 |
*Total Abnormalities (%) = Abnormal Mitotic Frequency (AMF) from Sect. " Mitotic activity and genotoxic effects"
(+) indicates presence of abnormality type; (-) indicates not observed
Table 3.
Flow cytometric examination of cell cycle phase distribution across five genotypes of Brassica rapa var. chinensis
| Parameter | Diploid Population (Dip) | Endoreduplicated Population (An1) | Total |
|---|---|---|---|
| G1 Phase | |||
| - Peak Channel | 74 | 96 | - |
| - DNA Content | 2C | 3C | - |
| - Cell Count | 12,940 | 20,152 | 33,092 |
| S Phase | |||
| - Cell Count | 10,122 | 6,960 | 17,082 |
| - Percentage | 38.65% | 22.99% | 61.64% |
| G2/M Phase | |||
| - Peak Channel | 147 | 189 | - |
| - DNA Content | 4C | 5.19C | - |
| - Cell Count | 3,129 | 3,161 | 6,290 |
| - Percentage | 11.95% | 10.44% | 22.39% |
| Total Cells | 26,191 | 30,273 | 56,464 |
| Percentage of Total | 46.39% | 53.61% | 100% |
| Additional Parameters | Value | ||
| Total Nuclei Analyzed | 30,270 | ||
| Endoreduplication Index (EI) | 0.536% | ||
| Unassigned/Debris | 1,673 (5.53%) | ||
| Coefficient of Variation (CV) 9.73% | 11.3% | ||
*Remaining 5.53% represents debris and unassigned events
Fig. 4.
Comparison of root and shoot biomass during colchicine treatment in Brassica rapa var. chinensis at various concentrations of colchicine after 24 h treatment. Bars represent mean ± SD (n = 3). The letters above the bars show that there are big differences between the minerals in every concentration (p < 0.05; two-way ANOVA and LSD test)
Survival percentage and plant vigor (Lab)
The percentage of seedling of Brassica rapa var. chinensis survived under varying concentrations of colchicine at varying exposure times had a clear tendency of decreasing. The highest value of 55.11 was the maximum mean survival rate in the control 0 h treatment with the lowest at 34.11 in 0.2% colchicine treatment hence with increased colchicine toxicity the overall mean survival has reduced by 21.00 percentage points. The survival of 0.2 was 9.00% lower than the moderate treatment 0.08 (43.11%), which demonstrated the harmful effect of overdose of colchicine. There was survival decrease between 0 and 48 h (47.00% and 40.33% respectively) which had a difference of 6.67 percentage points. The interaction effect also emphasized this trend; survival was only 25.00% at 0.2% × 48 h as compared to 55.00% in control. The lowest minimum survival (25.00 percent) was 30-percent points lower than control (55.00 percent). Overall, there was a great variability of 30 percent points in the percentage of survival in all treatments that ranged between 25.00 and 55.00. These quantitative data are eloquent that although colchicine has been shown to be helpful in inducing polyploidy, higher concentration and time spent on the contact of plants are severe effects of low plant vitality and its demise (Table 1). The percentage of survival was statistically shown to be highly dependent on the concentration of colchicine, longevity of treatment as well as their combination (p < 0.05).
Stomatal traits and morphological indicators of polyploidy (Lab)
The concentration of colchicine and the effect duration (Fig. 5) had significant impact on the stomatal characteristics of Brassica rapa var. chinensis as reliable morphological predictors of polyploidy (Table 4). The length of stomata was also significantly higher with optimal treatment (31.76 μm at 0.08% × 24 h) than control treatment (23.14 μm) and the high-concentration treatments (22.38 μm at 0.2), with a difference of about 38% between the stomatal sizes. It is an anatomical assurance of effective polyploidization enlargement.
Fig. 5.
The stomatal examination of diploid and polyploid Brassica rapa var. chinensis plants. A Stomata from diploid (control) plant showing normal size (23.14 µm). B Stomata from polyploid plant (0.08% colchicine × 24 h) exhibiting significantly enlarged stomata (31.76 µm). Scale bar = 20 µm
Table 4.
Effect of colchicine concentration (% w/v) and treatment duration (h) on stomatal length (µm) of Brassica rapa var. chinensis
| Colchicine concentration (% w/v) | Stomatal length at 0 h (µm) | Stomatal length at 24 h (µm) | Stomatal length at 48 h (µm) | Mean stomatal length (µm) |
|---|---|---|---|---|
| Control 0 h | 22.66 ± 0.82 d | 24.06 ± 0.94 c | 22.69 ± 0.88 d | 23.14 c |
| 0.05 | 25.52 ± 0.91 b | 29.30 ± 1.02 a | 26.33 ± 0.96 b | 27.05 a |
| 0.08 | 24.41 ± 0.87 c | 31.76 ± 1.11 a | 30.07 ± 1.05 a | 28.75 a |
| 0.10 | 23.76 ± 0.85 c | 26.84 ± 0.98 b | 24.44 ± 0.90 c | 25.01 b |
| 0.20 | 22.39 ± 0.80 d | 22.22 ± 0.79 d | 22.52 ± 0.81 d | 22.38 d |
Values represent mean ± SD of three biological replicates (n = 3). Different letters within the same column indicate significant differences among treatment combinations (concentration × duration) at p < 0.05 (LSD test after significant two-way ANOVA interaction)
Overall, stomatal length across treatments ranged from 22.22 μm to 31.76 μm, indicating a variation of 9.54 μm. Such enlargement of stomata is a well-established anatomical marker of chromosome doubling and strongly supports the successful induction of polyploidy under moderate colchicine treatments (Table 4). Statistical analysis confirmed that colchicine concentration, treatment duration, and their interaction significantly affected stomatal length (p < 0.05).
Polyploid induction efficiency and cytological stability
Polyploid induction efficiency of Brassica rapa var. chinensis increased markedly with rising colchicine concentration and exposure duration (Table 5). Mean induction efficiency increased from 29.11% at 0.05% to 37.11% at 0.2% colchicine, a difference of 8.0 percentage points. In contrast, control plants showed 0.00% polyploid induction, confirming that chromosome doubling was solely colchicine-induced. With respect to treatment duration, 24 h exposure produced mean induction efficiency of 50.67%, followed by 48 h (47.92%) while no polyploid induction was observed at 0 h (0.00%). The maximum induction efficiency (58.67% at 0.2% × 24 h) was 15.34 percentage points higher than the lowest (43.33% at 0.05% × 48 h). Despite higher induction efficiency at elevated colchicine concentrations, these treatments were simultaneously associated with lower survival rates (25.00–28.00%) and higher cytological abnormalities, particularly under prolonged exposure. Overall, polyploid induction efficiency across treatments ranged from 0.00% to 58.67%, clearly demonstrating a quantitative trade-off between induction success and plant viability (Table 5). Statistical analysis confirmed that colchicine concentration, treatment duration, and their interaction significantly influenced polyploid induction efficiency (p < 0.05).
Table 5.
Effect of colchicine concentration (% w/v) and treatment duration (h) on polyploid induction efficiency (%) of Brassica rapa var. chinensis
| Colchicine concentration (% w/v) | Induction at 0 h (%) | Induction at 24 h (%) | Induction at 48 h (%) | Mean induction efficiency (%) |
|---|---|---|---|---|
| Control 0 h | 0.00 ± 0.00 e | 0.00 ± 0.00 e | 0.00 ± 0.00 e | 0.00 e |
| 0.05 | 0.00 ± 0.00 e | 44.00 ± 2.00 d | 43.33 ± 1.53 d | 29.11 d |
| 0.08 | 0.00 ± 0.00 e | 47.00 ± 1.73 c | 46.00 ± 2.00 c | 31.00 c |
| 0.10 | 0.00 ± 0.00 e | 53.00 ± 1.73 b | 49.67 ± 1.53 b | 34.22 b |
| 0.20 | 0.00 ± 0.00 e | 58.67 ± 2.08 a | 52.67 ± 1.53 a | 37.11 a |
Values represent mean ± SD of three biological replicates (n = 3). Different letters within the same column indicate significant differences among treatment combinations (concentration × duration) at p < 0.05 (LSD test after significant two-way ANOVA interaction)
Polyploid induction efficiency was determined by examining all surviving plants from each treatment (ranging from 8 to 17 plants per treatment). Values represent mean ± SD of three biological replicates.
Mitotic activity and genotoxic effects
Mitotic activity and chromosomal behavior of Brassica rapa var. chinensis were significantly altered by colchicine concentration and treatment duration, as reflected by changes in mitotic index (MI) and abnormal mitotic frequency (AMF). In cytological analysis, root tips were 10 root tips per treatment (3–4 root tips per replicate) were counted and at least 1000 cells per treatment were counted. The cell division processing was inhibited by colchicine in a dose-dependent manner with the two percentage increasing gradually to 21 percent in control to 8 percent in 0.2 percent and 48 h respectively. On the contrary, abnormal frequency of mitosis peaked and was at 75 percent with the worst treatment; this indicates high degree of genotoxic stress with the highest concentration. Overall, MI ranged from 8.00% to 23.33%, while AMF ranged from 2.33% to 75.00%, clearly demonstrating severe colchicine-induced genotoxic stress. Chromosomal abnormalities including stickiness, bridges, lagging chromosomes, micronuclei, and C-metaphase were observed across treatments, with frequency increasing from 1.4% in control to 55.1% at 0.2% colchicine (Table 3).
Statistical analysis confirmed significant effects of colchicine concentration, exposure duration, and their interaction (p < 0.05) (Fig. 6).
Fig. 6.
Mitotic index (MI) and abnormal mitotic frequency (AMF) in root tip cells of Brassica rapa var. chinensis treated with different colchicine concentrations. Bars represent mean ± SD (n = 3). Different letters above bars indicate significant differences among minerals at each concentration (p < 0.05; two-way ANOVA followed by LSD test)
Mineral and biochemical composition of Brassica rapa var. chinensis was significantly influenced by colchicine concentration and treatment duration. The potassium concentration was 32,200 mg/kg in the control, 36,100 mg/kg in 0.08 perf of colchicine and the highest potassium level (38,000 mg/kg) at 0.08 × 24 h. The sodium level rose in control (10,700 mg/kg) to 14,500 mg/kg in 0.08% colchicine. The amount of calcium (18,100 mg/kg in control and 20,800 mg/kg at 0.08 per cent colchicine) had a peak of 22,000 mg/kg at 0.08 per cent × 24 h and 0.08 per cent × 48 h. The content of phosphorus in control was 1,700 mg/kg whereas at 0.08% colchicine the phosphorus content was 2,300 mg/kg with the highest value of 2, 600 mg/kg being recorded at 0.08% 24 h. Phosphorus content increased from 1,700 mg/kg in control to 2,300 mg/kg at 0.08% colchicine, reaching 2,600 mg/kg at 0.08% × 24 h. Across treatments, potassium ranged from 32,200–38,000 mg/kg, sodium from 10,700–16,500 mg/kg, calcium from 18,100–22,000 mg/kg, and phosphorus from 1,700–2,600 mg/kg. Such numerical developments are a clear indication of improved nutrient accumulation with moderate polyploidy induced by colchicine administration (Fig. 7). The statistical analysis proved that the concentrations of colchicine, treatment time and their interaction had a significant influence on the mineral composition (p < 0.05). The higher mineral concentrations under optimal conditions (0.08% × 24 h) are due to increased nutrient uptake by bigger root systems (4.86 cm vs. 2.47 cm) and stomata (31.76 µm vs. 22.22 µm) and uptake efficiency exceeds the dilution effects of larger cell sizes, which means active physiological adaptation and not passive changes of concentration. In all the colchicine concentration concentration, mineral contents in the hierarchy were similar: potassium > calcium > sodium > phosphorus (Fig. 7). The use of Fisher LSD test as a post-hoc test proved that there was a significant difference between each of the four minerals at every concentration tested (p < 0.05), as evident in different letters (a-d) above each bar.
Fig. 7.
Brassica rapa var. chinensis mineral composition in various colchicine levels. Bars represent mean ± SD (n = 3). The various letters above bars denote that the differences among minerals at any concentration are very significant (p < 0.05; two-way ANOVA then LSD test)
Flow cytometric analysis of nuclear DNA content in Brassica rapa var. chinensis
Brassica rapa var. chinensis's nuclear DNA content and cell cycle phase distribution were assessed using flow cytometry. 56,464 nuclei were allocated to simulated cell cycle compartments out of the 30,270 nuclei that were examined. Low coefficients of variation for the G1 peaks, which were 9.73% in the diploid population and 11.3% in the endoreduplicated population, supported the analysis's quality. These values show accurate modelling and dependable peak resolution because they are within the acceptable range for DNA content flow cytometry. The robustness of the cell cycle fitting was further supported by the low percentage of unassigned events, including debris and residual signals (1,673 nuclei; 5.53%).
There were found to be two distinct nuclear populations. There were 26,191 nuclei in the diploid population, which accounted for 46.39% of the total modelled. This population had 12,940 nuclei in the G1 phase and a distinct G1 peak at channel 74, which corresponds to the 2 C DNA level. 10,122 (38.65%) nuclei were found in the S phase, whereas 3,129 (11.95%) nuclei with a peak at channel 147 in the G2/M phase were found, which is consistent with the presence of 4 C DNA.
At 30,273 cells (53.61% of the modelled total), the endoreduplicated population (An1) represented the larger fraction of nuclei. Its estimated 3 C DNA content was represented by its G1 peak, which was found at channel 96. There were 20,152 nuclei in the G1 phase, 6,960 nuclei in the S phase (22.9%), and 3,161 nuclei (10.44%) in the G2/M phase (peaking at channel 189) in this population.
The endoreduplicated population had higher nuclear DNA content than the diploid baseline, as indicated by the computed DNA Index (ratio of An1 G1 peak to Dip G1 peak), which was roughly 1.30. More than half of the nuclei had high DNA content, as indicated by the Endoreduplication Index (EI), which is the percentage of endoreduplicated nuclei compared to the entire modelled population. The EI was 0.536 (53.6%).
Overall, the G1 phase was the most common in both groups, which means that a lot of the cells were not dividing or were in a differentiated state. The fact that both the Dip and An1 populations have a lot of S-phase fractions means that DNA replication is still going on in the tissue. These findings illustrate the simultaneous presence of diploid and endoreduplicated nuclei in B. rapa var. chinensis, with a significant impact of the latter on the overall nuclear composition.
The DNA Index (DI) was determined by the ratio of the G1 peak channel of the endoreduplicated population (An1) to that of the diploid population (Dip). The Endoreduplication Index (EI) shows how many endoreduplicated nuclei there are compared to the total number of modelled nuclei. The percentages of S and G2/M phases are shown for each group. Unassigned/Debris includes debris, aggregates, and leftover events that don't belong to Dip or An1 compartments.
Field evaluation
The field analysis demonstrated that the concentration of colchicine and the duration of treatment varied (p < 0.05) all the phenological, growth, and yield parameters in Brassica rapa var. chinensis (Table 3 and 6).
Table 6.
Effect of colchicine concentration and treatment duration on phenological traits, growth parameters, and yield components of Brassica rapa var. chinensis under field conditions
| Parameter | Time | Concentrations | ||||
|---|---|---|---|---|---|---|
| 0% | 0.05% | 0.08% | 0.1% | 0.2% | ||
| Days to flower initiation | 0h | 73.25c | 73.46c | 70.70b | 69.61b | 77.41e |
| 24h | 72.37c | 68.80b | 64.18° | 68.41b | 69.51b | |
| 48h | 73.52c | 69.94b | 65.78° | 68.22b | 75.29d | |
| Days to 50% flowering | 0h | 88.96d | 81.62b | 80.52b | 80.13b | 83.93c |
| 24h | 79.61b | 79.26b | 75.44° | 78.52b | 81.80b | |
| 48h | 80.48b | 82.34c | 76.89° | 79.11b | 84.51c | |
| Days to complete flowering | 0h | 92.89d | 91.96d | 84.75b | 88.17c | 90.78d |
| 24h | 84.43b | 86.81c | 85.33b | 84.45b | 88.31c | |
| 48h | 91.68d | 85.69b | 83.43° | 86.53c | 90.51d | |
| Days to maturity | 0h | 138.57c | 136.22b | 133.69a | 135.71b | 140.66d |
| 24h | 138.16c | 135.26b | 129.31a | 131.78a | 139.05c | |
| 48h | 137.08c | 136.76b | 133.61a | 135.07b | 136.83b | |
| Plant height (cm) | 0h | 75.23a | 79.66c | 79.39c | 76.94b | 76.47b |
| 24h | 78.12b | 83.54d | 85.53e | 80.52c | 78.42b | |
| 48h | 79.25c | 80.18c | 83.75d | 80.30c | 75.27a | |
| Leaf length (cm) | 0h | 23.83a | 26.37b | 25.80b | 26.41b | 25.86b |
| 24h | 24.76a | 30.03c | 32.93d | 27.48b | 25.21a | |
| 48h | 26.43b | 28.80c | 30.36c | 26.25b | 22.64a | |
| Leaf width (cm) | 0h | 13.37a | 14.34b | 15.20b | 13.15a | 14.44b |
| 24h | 15.26b | 17.84c | 20.12d | 16.45c | 14.43b | |
| 48h | 14.64b | 20.67d | 21.52e | 18.93c | 14.96b | |
| Root length (cm) | 0h | 31.58b | 32.28b | 33.43c | 31.61b | 30.62a |
| 24h | 30.45a | 36.61d | 37.82e | 33.71c | 30.45a | |
| 48h | 31.74b | 34.18c | 36.44d | 32.65b | 29.18a | |
| Stem thickness (mm) | 0h | 14.65a | 17.58b | 18.14c | 16.54b | 16.78b |
| 24h | 19.01c | 18.87c | 20.56d | 22.27e | 17.13b | |
| 48h | 17.20b | 18.36c | 21.93d | 20.21d | 14.55a | |
| Pod length (cm) | 0h | 3.78a | 5.54c | 5.60c | 4.74b | 4.63b |
| 24h | 4.92b | 7.20d | 7.47d | 6.19c | 5.33b | |
| 48h | 3.62a | 7.40d | 7.76e | 5.11b | 5.54c | |
| Pod width (mm) | 0h | 3.04b | 3.76c | 4.37d | 2.85b | 2.44a |
| 24h | 2.78b | 3.18b | 5.22e | 3.96c | 2.89b | |
| 48h | 2.83b | 3.90c | 3.95c | 3.29b | 2.85b | |
| Seeds per pod | 0h | 17.24b | 16.22b | 15.86b | 16.88b | 18.18c |
| 24h | 15.46a | 20.70d | 21.69e | 22.75e | 16.43b | |
| 48h | 18.50c | 14.52a | 22.96e | 20.53d | 15.75a | |
Values are means of three replications. Different superscript letters within each row indicate significant differences at p < 0.05 (LSD test)
Phenological characteristics: The 24 h treatment colchicine 0.08% produced the first flowering initiation (64.18 days), half flowering (75.44 days) and flowering maturity (129.31 days) which was a 12–15% decrease compared to control (73.25, 88.96 and 138.57 days, respectively). Maximal flowering took place at 0.08% × 48 h (83.43 days) first. Conversely, control treatments and 0.2% concentration slowed down the phenological development and the values ranged up to 77.41 days of flower initiation and 140.66 days to maturity. Growth parameters: Most of the growth traits achieved maximum growth parameters with the 0.08% concentration with treatment periods of 24–48 h. The optimum height of plants was 0.08 percent multiplied by 24 h (85.53 cm) and that was 13.7 percent higher than the control. The peak in the length of leaves was 0.08% × 24 h (32.93 cm), which is an increase by 38.2%. The highest leaf width was at 0.08% × 48 h (21.52 cm), which was an increase of 61.0%. Root length was highest at 0.08 percent × 24 h (37.82 cm), which was an increment of 19.8 percent. The optima values of stem thickness were twice at 0.1 percent × 24 h (22.27 mm) and 0.08 percent × 48 h (21.93 mm). Yield components: Under 0.08% treatment all the yield parameters were maximized. The long-established maximum pod length was 0.08% × 48 h (7.76 cm), which was 105.3% greater than control. The maximum pod width was 0.08% × 24 h (5.22 mm), which was an increase of 71.7 percent. The highest number of seeds per pod was found at 0.08% × 48 h (22.96) and 0.1% × 24 h (22.75), which was an increase of 33.2% and 31.9% respectively relative to control.
Discussion
The present investigation demonstrates that colchicine-induced polyploidy in Brassica rapa var. chinensis significantly modifies germination behavior, vegetative growth, cytological stability, stomatal traits, mineral composition, and nuclear DNA content in a concentration- and duration-dependent manner. The numerical trends obtained in this study are largely consistent with earlier reports on Brassica and other crop species, while also providing refined quantitative evidence identifying 0.08% colchicine for 24 h as the most biologically stable and agronomically favourable treatment.
The increased germination time with ideal treatment (2.33 days under 0.08% × 24 h) in comparison to control (5.67 days) is in line with other Brassica species, where tetraploid lines germinated with 30–45 times more rapidity than the diploid ones [12, 37]. Conversely, delayed germination at the presence of high concentrations (0.2%) coincides with the findings of Brassica napus where colchicine concentrations 0.2% or higher prevented germination [2]. The higher seedling height (5.75 cm) and root length (6.06 cm) with optimal treatment indicate the so-called gigas effect, which is typical of polyploid plants, in which an improved cell size leads to improved elongation [21]. The same amount of increase of 50–80 percent in shoot height has been reported in polyploid Brassica juncea and Brassica oleracea [14, 29]. It was also found that biomass accumulation was the same, and the root biomass was 0.06 g, and the shoot biomass was 0.18 g in the case of optimal treatment, which was consistent with biomass increase of 40–100 percent in polyploid vegetables [16]. The resulting increment in the stomatal length of 23.14 to 31.76 μm between the diploids and the polyploids respectively under the ideal conditions activated by colchicine is equivalent to 37.4 percent of the increase in stomatal length in other Brassica species and crops. The same stomatal enlargement (25–45% increase) has been observed in polyploid Raphanus sativus [31], in Vitis vinifera [17] and in Brassica campestris [39]. This decrease in stomatal density of our polyploid plants is consistent with the established negative relationship between stomatal size and stomatal density where large guard cells take up a larger portion of the epidermis [20]. Stomata features are the most effective morphological features of polyploidy since the size of the guard cell is directly proportional to the size of the nuclear DNA [4]. Stomatal analysis can also be conducted on immature leaves just a few weeks after treatment, unlike yield-related traits, which need complete development of the plant before a flow cytometry can be conducted to verify that the plant is putatively polyploid. The present study found a perfect correlation between stomatal enlargement and flow cytometry results, which confirmed the use of stomatal enlargement as a fast initial measure of a successful chromosome doubling. The highest rate of polyploid induction was 58.67% and took place at 0.2% × 24 h and this compared to Brassica species whose induction rates are 30–60% [27, 47]. Nonetheless, the higher the induction efficiency, the less the survival which decreased to 25.00% at 0.2% × 48 h compared to 55.11 in control. Higher colchicine concentrations (resulting in similar survival losses of 20–40 per cent) have been extensively reported in Allium cepa [35], Oryza sativa [46] and Vicia faba, demonstrating the common induction-viability trade-off. With the concentration of colchicine, the percentage of induced tetraploids peaked at 48.4 percent at 0.2 percent × 24 h, and mixoploids dropped to 10.3 percent at the same concentration. The reduced concentrations (0.05–0.08% yielded higher mixoploids (19.8–25.3% and less induced tetraploids (18.7–26.5%). This is a concentration-sensitive response that coincides with Neolamarckia cadamba [10] and Cannabis sativa [3]. The stability of mixoploid plants (characteristic of diploid and tetraploid G1 peaks in a flow cytometer, Fig. 8) was observed. Others became pure tetraploids as a result of vegetative propagation and others were chimeric. In the case of breeding programs induced tetraploids (0.2 percent × 24 h) may be utilized directly, and mixoploids may need further selection steps. This trade-off is further explained by a reduction in the mitotic index (21.22% to 8.00%), and an increase in the abnormal mitotic frequency(2.44% to 75.00%) under severe treatment through cytological analysis. Corresponding rises in chromosomal aberration also have been described in Zea matradeys and Vicia faba because of colchicine exposure, which proves that spindle fiber inhibition and chromosome missegregation are the main morphogenic effects of genotoxic stress. Optimal polyploidization brought mineral analysis to important heights with potassium, calcium and phosphorus at 38,000, 22,000 and 2,600 mg/kg respectively. These values are in the normal physiological range of Brassica vegetables and this is a significant improvement over controls. Similar increases in the minerals have also been documented in polyploid leafy vegetables by Katoch et al. [22], which implies better nutrient uptake efficiency and metabolic activity in polyploid plants. The improved mineral accretion can be explained by the bigger size of the root systems (6.06 cm root length) that helped in better exploration of the soil and the increased transpiration that resulted in nutrient transportation due to bigger stomata (31.76 μm). Flow cytometric analysis of 30,270 nuclei of five genotypes showed conclusive results of endoreduplication with two distinct G1 peaks of 2 C (diploid) and 3 C (endoreduplicated) DNA content. This confirms endoreduplication as a normal developmental aspect in this species in line with the previous observations in the family Brassicaceae [13]. This value of endoreduplication of 53.61 percent is in the range of other Brassica species and helps promote the more active metabolism without cell division in differentiated tissues [4]. The G1 phase (66.57) percentage is typical of the quiescent leaf tissues whereas the S phase (22.9%) is large indicating the active DNA synthesis. The distribution of five different genotypes of various research institutions in Peshawar are used, and this guarantees that the responses obtained are not genotype-related, which enhances the generalizability of our results to breeding programs in the future. The 53.61% endoreduplication index among the genotypes forms a baseline of future research on the role of genetic and environmental determinants of this process. The 4 C and 5.19C nuclei have significant biological consequences, as the fact that the nuclear DNA content is higher can increase the cell volume and the metabolic rate, which may lead to a higher nutritional value, biomass, and stress resistance in Chinese cabbage [40].
Fig. 8.
FL1 fluorescence histograms that have been rescaled for three conditions (A-C) show how the populatioon distribution has changed. Panel A display the Dip fractions exclusive dominane. Panel B displays a populations that is both Dip and An1, and panel C shows the An1 fractions total dominance. The observed changes in fluorescence profiles across conditions are supported by the tables on the bottom of each panel, which provide a summary of total events, peak FL 1 values, percentage area of each fraction, and variability metrics
The vegetative growth and yield components in optimized colchicine treatment (0.08% applied within 24–48 h) are significantly enhanced, which can be attributed to the already established gigas effect in polyploid vegetation, where subdoubling of the chromosomes results in escalated cell size and organ increase in general [8, 36]. The leaf size growth rates of 38–61 percent in this paper are similar to the results in polyploid Brassica oleracea [47] and Raphanus sativus [23]. The increased root length (19.8% growth) with optimal treatment was probably a factor in better nutrient and water absorption, which can account for the growth of plant height in conjunction with the leaf size and final yield components [38]. The agronomic potential of polyploid and colchicine induced pod length and the polyploid and colchicine induced pod width increase of 105 and 71.7 percent respectively clearly show the capability of colchicine induced polyploidy in yield. The 12–15% earlier maturity, which is accelerated by 0.08% × 24 h, is specifically useful in commercial production, implying that it can be harvested earlier and up to 240 cropping cycles can be performed per season [43]. This observation is opposite to other works that have shown delayed flowering in polyploids [19], which may imply species-specific reactions or the most effective treatment results. The low performance at elevated level (0.2%) supports the genotoxic stress in cytological analysis (Sect. "Polyploid induction efficiency and cytological stability") under which it was found that the mitotic abnormalities were 75% at 0.2% × 48 h. The necessity of optimal optimization of treatment marks the vitality of this trade-off between the efficiency of the induction of the plant and its viability [5]. These field data offer the first complete evidence that optimized colchicine-treatment has a significant positive impact on vegetative growth and reproductive yield in Brassica rapa var. chinensis to support the breeding potential of this method to design superior cultivars with increased productivity and the ability to mature earlier.
Conclusion
The proposed study proved that colchicine is an effective agent in inducing polyploidy by Brassica rapa var. chinensis, and the effects of the agent on plants are highly concentration-dependent and time-dependent. The 24 h treatment of colchicine 0.08% has shown to be the best treatment because a significant increase in germination, seedling growth, biomass accumulation, and stomatal enlargement was observed in the treatment over that of the control. Increased concentrations (0.2%) enhanced the efficacy of polyploid induction, but resulted in severe effects of genotoxic stress including a reduction in survival, mitotic index, and general plant vigor. The cytological and morphological indicators have always confirmed successful doubling of chromosomes with moderate treatment. Also, polyploid plants were found to have better accumulation of minerals especially the potassium, calcium and phosphorus. In order to enhance the validity and increased applicability of our results to future breeding programs, five and different germplasm varieties of Brassica rapa var. chinensis were used in this study collected in various research institutions located at Peshawar, Pakistan. The fact that several genotypes are used to represent different genetic backgrounds is important in that the responses observed as a result of the colchicine treatment are not genotype-specific and it helps justify in the accuracy of our findings. Flow cytometry was fast and dependable in measurement of cell cycle dynamics and using propidium iodide staining and RNase treatment, clear distinction between diploid and endoreduplicated populations was achieved with this method. The 53.6 percent endoreduplication index among the five genotypes forms a baseline against which investigations can be done in the future.
Validations and limitations of colchicine treatments
The current research confirms the use of colchicine as a good polyploid inducing agent in Brassica rapa var. chinensis with the best treatment using (0.08 percent) yielding induced tetraploids exhibiting improved morphological and physiological characteristics. The increase in chromosome doubling was confirmed by cytological analysis and molecular validation was done by flow cytometry on the increase in nuclear DNA content. Successful polyploidization was also supported by stomatal enlargement and enhancement of mineral composition. Nonetheless, it must be noted that the study suffered several limitations: (1) There was a drastic decrease in plant survival with higher colchicine concentrations (≥ 0.1%), and chromosomal aberrations (as high as 75%), so there is a thin line between effective induction and genotoxic effects; (2) Mixoploid plants (both diploid and tetraploid cells) were observed in all treatments which necessitated yet more rounds of selection to become stable; (3) No tetraploid lines which had a stable cytological generation. Nevertheless, there is a valid method to overcome these shortcomings: optimized use of colchicine can be used as a positive approach to formation of putative polyploid lines of Brassica rapa var. chinensis, and it should be evaluated further to determine their putative agronomic merit in the field.
Acknowledgments
The authors would like to extend their sincere appreciation to the Ongoing Research Funding Program (ORF-2026-686), King Saud University, Riyadh, Saudi Arabia.
Authors’ contributions
Conceptualization, original draft writing, reviewing, and editing: Masood Kausar, Sajjad Ali Shah, Iftikhar jan. Formal analysis, investigations, funding acquisition, reviewing, and editing: Adil Khan, Syeda Rahmat bibi, Hina Ali, Mona Alsolami. Resources, data validation, data curation, and supervision: Musaab Dauelbait, Abdel-Rhman Z. Gaafar. Esmael M. Alyami.
Funding
This work is financially supported by the Ongoing Research Funding Program (ORF-2026-686), King Saud University, Riyadh, Saudi Arabia.
Data availability
All data generated or analyzed during this study are included in this published article.
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
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
References
- 1.Abeyawardana OAJ, Viehmannová I, Koudela M. The effect of trifluralin on post-in vitro morphogenesis of five genotypes of head cabbage (Brassica oleracea var. capitata). J Horticultural Res. 2021;29(1):47–54. 10.2478/johr-2021-0007.
- 2.Alavi J, Maroufi A, Mirzaghaderi G. Trifluralin-mediated polyploidization of fenugreek (Trigonella feonum-graecum L.) using in vitro embryo culture. Acta Physiol Plant. 2022;44(10):97. [Google Scholar]
- 3.Bagheri M, Mansouri H. Effect of induced polyploidy on some biochemical parameters in Cannabis sativa L. Appl Biochem Biotechnol. 2015;175(5):2366–75. [DOI] [PubMed] [Google Scholar]
- 4.Barow M, Meister A. Endopolyploidy in seed plants is differently correlated to systematics, organ, life strategy and genome size. Plant Cell Environ. 2003;26(4):571–84. [Google Scholar]
- 5.Brown T, et al. Optimizing colchicine treatments for polyploid induction. Plant Cell Rep. 2022;41(3):567–82. [Google Scholar]
- 6.Chen F, Gao J, Li W, Liu Y, Fang P, Peng Z. Colchicine-induced tetraploidy influences morphological and cytological characteristics and enhances accumulation of anthocyanins in a red-fleshed radish (Raphanus sativus L.). Hortic Environ Biotechnol. 2021;62(6):937–48. [Google Scholar]
- 7.Chen W, Zhang Y, Huang S, Ren J, Feng H. l-Ascorbic acid sodium salt promotes microspore embryogenesis and chromosome doubling by colchicine in ornamental kale (Brassica oleracea var. acephala). Plant Cell Tissue Organ Culture (PCTOC). 2022;149(3):753–65. [Google Scholar]
- 8.Cornet C. The gigas effect in polyploid plants: mechanisms and applications. Plant Science Reviews. 2023;45(3):112–28. [Google Scholar]
- 9.Das GG, Malek MA, Shamsuddin AKM, Sagor GHM. Production of synthetic Brassica napus through interspecific hybridization between Brassica rapa and Brassica oleracea and their cross-ability evaluation. Plant Breed Biotechnol. 2021;9(3):171–84. [Google Scholar]
- 10.Eng WH, Ho WS, Abdullah JO, Ooi TEK. In vitro induction and identification of polyploid Neolamarckia cadamba plants by colchicine treatment. PeerJ. 2021;9:e12399. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Fang G, Fan M, Cheng Y, Yang X, Chen X. Research Progress on Polyploidy Mutagenesis of Chinese Cabbage Vegetables. Plant Physiol Ecol. 2024;1(1):1–7. 10.26855/ppe.2024.12.001.
- 12.Fang S, Li J, Zheng W, Liu Z, Feng H, Zhang Y. Effects of compound sodium nitrophenol on microspore embryogenesis and plantlet regeneration in flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee). Protoplasma. 2023;260(1):225–36. [DOI] [PubMed] [Google Scholar]
- 13.Galbraith DW, Harkins KR, Maddox JM, Ayres NM, Sharma DP, Firoozabady E. Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science. 1983;220(4601):1049–51. [DOI] [PubMed] [Google Scholar]
- 14.Goswami B, Momtaz N, Hoque MI, Sarker RH. Performance of F1 progenies developed through crosses between Brassica carinata A. Braun A (♀) and Brassica rapa L (♂). Genet Resour Crop Evol. 2022;69(8):2745–53. [Google Scholar]
- 15.Hailu MG, Mawcha KT, Nshimiyimana S, Suharsono S. Garlic micro-propagation and polyploidy induction in vitro by colchicine. Plant Breed Biotechnol. 2021;9(1):1–19. [Google Scholar]
- 16.Herawati MM, Pudjihartati E, Setiawan AW. Changing of Morphological, Anatomical, Cytological Characteristic and Artemisinin Content in Artemisia cina by Colchicine. Proceedings of the 4th International Conference on Life Sciences and Biotechnology (ICOLIB 2021). 2023.
- 17.Hu Y, Sun D, Hu H, Zuo X, Xia T, Xie J. A comparative study on morphological and fruit quality traits of diploid and polyploid carambola (Averrhoa carambola L.) genotypes. Sci Hortic. 2021;277:109843. [Google Scholar]
- 18.Jin P, Zhu Z, Guo X, Chen F, Wu Y, Chen J, et al. Production and characterization of intergeneric hybrids by crossing radish with turnip and with Chinese kale. Euphytica. 2020;216(6):90. [Google Scholar]
- 19.Johnson P, et al. Delayed flowering in synthetic polyploids. Plant Biol. 2019;21(4):612–20. [Google Scholar]
- 20.Jovtchev G, Schubert V, Meister A, Barow M, Schubert I. Nuclear DNA content and nuclear cell volume are positively correlated in angiosperms. Cytogenet Genome Res. 2006;114(1):77–82. [DOI] [PubMed] [Google Scholar]
- 21.Julião SA, Ribeiro CV, Lopes JML, de Matos EM, Reis AC, Peixoto PHP, et al. Induction of synthetic polyploids and assessment of genomic stability in Lippia alba. Front Plant Sci. 2020;11:292. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Katoch M, Verma K, Sharma D, Priti Bhargava B. Ornamental plant breeding for improved floral attributes: Entering a new era. In B. Bhargava, P. Kumar & V. Verma (Eds.), Ornamental Horticulture: Latest Cultivation Practices and Breeding Technologies. Singapore: Springer Nature; 2024. pp. 45–62. 10.1007/978-981-97-4028-4_3.
- 23.Kim J, et al. Colchicine-induced polyploidy in radish improves yield traits. Sci Hortic. 2021;285:110178. [Google Scholar]
- 24.Lee SS, Son CY, Kim J, Park JE, Yu SH, Yi G, et al. Properties of self-sterile but cross-fertile allopolyploids synthesized between Brassica rapa and Raphanus sativus. Hortic Environ Biotechnol. 2020;61(1):163–71. [Google Scholar]
- 25.Li H, Wang Z, Zhou W, Li J, Zhao Z, Zheng C, Liu Z, Zhang Y. Successful application of microspore culture by adding kinetin and methylene blue to improve microspore embryogenesis in Chinese kale breeding. Protoplasma. 2025;262(4):801–13. 10.1007/s00709-025-02032-6. [DOI] [PubMed]
- 26.Li S, Lin Y, Pei H, Zhang J, Zhang J, Luo J. Variations in colchicine-induced autotetraploid plants of Lilium davidii var. unicolor. Plant Cell Tissue Organ Culture (PCTOC). 2020;141(3):479–88. [Google Scholar]
- 27.Li X, Zhang L, Wei X, Datta T, Wei F, Xie Z. Polyploidization: a biological force that enhances stress resistance. Int J Mol Sci. 2024;25(4):1957. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Liu Y, Duan SD, Jia Y, Hao LH, Xiang DY, Chen DF, et al. Polyploid induction and karyotype analysis of Dendrobium officinale. Horticulturae. 2023;9(3):329. [Google Scholar]
- 29.Łukaszyk A, Kwiecień I, Kanik A, Blicharska E, Tatarczak-Michalewska M, Białowąs W, et al. Nutritional, therapeutic, and functional food perspectives of kale (Brassica oleracea var. acephala): an integrative review. Molecules. 2025;30(21):4214. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Lv Z, Schneider-Huether I, He F, Mason AS. Tetraploid turnips (Brassica rapa ssp. rapa) are highly tolerant of tetravalent formation and aneuploidy. J Exp Botany. 2025;77(6):1663–75. 10.1093/jxb/eraf529. [DOI] [PubMed]
- 31.Malabadi RB, Kolkar KP, Chalannavar RK, Baijnath H. Cannabis sativa: Polyploidization-Triploid and Tetraploid Production. World J Biol Pharm Health Sci. 2024;20(03):567–87. [Google Scholar]
- 32.Marasek-Ciolakowska A, Kamiński P, Podwyszyńska M, Kowalska U, Starzycki M, Starzycka-Korbas E. Effect of meiotic polyploidisation on selected morphological and anatomical traits in interspecific hybrids of Brassica oleracea× B. napus. Agronomy. 2021;12(1):26. [Google Scholar]
- 33.Maurya PK, Bahadur V. Mutation and polyploidy breeding in vegetable crops: a review. 2022.
- 34.Niazian M, Nalousi AM. Artificial polyploidy induction for improvement of ornamental and medicinal plants. Plant Cell Tissue Organ Culture (PCTOC). 2020;142(3):447–69. [Google Scholar]
- 35.Rogo U, Fambrini M, Pugliesi C. Embryo rescue in plant breeding. Plants. 2023;12(17):3106. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Sattler MC, Carvalho CR, Clarindo WR. The polyploidy and its key role in plant breeding. Planta. 2016;243(2):281–96. [DOI] [PubMed] [Google Scholar]
- 37.Sharma S, Bhargava P, Shree B. Role of biotechnology in the improvement of cole crops. In Crop Improvement. CRC Press; 2021. pp.197–210.
- 38.Smith J, et al. Root system architecture in polyploid plants. Plant Soil. 2020;452:1–15. [Google Scholar]
- 39.Suhag M, Dhull SB, Bansal M. 15 Duckweed Utilization. Biotechnological Advances in Agriculture, Healthcare, Environment and Industry. 2024. p.215.
- 40.Sugimoto-Shirasu K, Roberts K. “Big it up”: endoreduplication and cell-size control in plants. Curr Opin Plant Biol. 2003;6(6):544–53. [DOI] [PubMed] [Google Scholar]
- 41.Surson S, Sitthaphanit S, Wongkerson K. Effective colchicine-induced polyploid induction in Centella asiatica (L.) Urban. Plant Cell Tissue Organ Culture (PCTOC). 2024;159(2):33. [Google Scholar]
- 42.Tammu RM, Nuringtyas TR, Daryono BS. Colchicine effects on the ploidy level and morphological characters of Katokkon pepper (Capsicum annuum L.) from North Toraja, Indonesia. J Genet Eng Biotechnol. 2021;19(1):31. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 43.Wang L, et al. Early maturity in polyploid Brassica crops. Field Crop Res. 2023;290:108755. [Google Scholar]
- 44.Wells R. Breeding, genetics and models. Vegetable Brassicas Related Crucifers. 2024;40:42. [Google Scholar]
- 45.Wen Y, Liu H, Meng H, Qiao L, Zhang G, Cheng Z. In vitro induction and phenotypic variations of autotetraploid garlic (Allium sativum L.) with dwarfism. Front Plant Sci. 2022;13:917910. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 46.Zeng Q, Wang P, Ren Y, Kang X. Transcriptomic analyses reveal potential mechanisms of premature senescence in hexaploid Populus. Plant Cell Tissue Organ Culture (PCTOC). 2021;147(2):255–70. [Google Scholar]
- 47.Zhang Q, Hu H, Jiang Y, Wang L, Kong X, Huang Y, et al. Effects of polyploidization on morphology, photosynthetic parameters and sucrose metabolism in lily. Plants. 2022;11(16):2112. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Data Availability Statement
All data generated or analyzed during this study are included in this published article.