Introduction

The genus Allium has been extensively investigated due to its species diversity, ecological adaptations, and beneficial applications. Research has primarily focused on underground organs that function as storage structures for nutrients and moisture, facilitating vegetative reproduction and contributing to genetic stability in natural populations (Kamenetsky 1993). Allium species also exhibit notable adaptation to arid conditions, indicating a transition from vegetative propagation to seminal reproduction. Furthermore, their well-documented antimicrobial properties, which are attributed to compounds such as allicin, inhibit the growth of various microorganisms (Arifa et al. 2020).

A. taquetii, indigenous to the high-altitude regions of Baek-Un, Jiri, and Jeju Island, is referred to as “Halla-buchu” in Korea. The plant attains a height of up to 30 cm, possesses narrow edible leaves, and produces small red-violet flowers that are suitable for ornamental cultivation in rock gardens (The Plant List 2017). A. senescens (“Dume-buchu”), distributed across the Northern Hemisphere, is utilized locally for its tonic properties and exhibits umbels of small violet flowers (The Plant List 2017). A. spirale Willd. (“ChamDume-buchu”) is characterized by its helically twisted leaves and dark red flowers, enhancing its ornamental value (Lee and Lee 1997).

Polyploidization, or genome doubling, results in plants with more than two sets of chromosomes, which play a significant role in speciation, adaptation, and crop improvement (Yali 2022). Polyploids exhibit increased biomass, resilience, and phytochemical production (Hieu 2022). Although this process confers agricultural benefits, it can lead to complications in mammals, including congenital diseases in humans. In plants, polyploidy can arise through meiotic nonreduction (2n-gametes production) and can be manipulated to transfer genes for crop breeding (Sattler et al. 2016). The complexity of polyploid genomes presents challenges, but offers opportunities for crop improvement through modern biotechnological tools (Bharadwaj 2015;Fitzgerald et al. 2012). Combining polyploidy with genomic techniques offers promising avenues for improving crop traits, including ornamental features, such as color, size, and flower longevity (De 2017;Manzoor et al. 2018;Mayfield et al. 2011;Niazian and Nalousi 2020;Vamosi et al. 2007;Zhang et al. 2022). Antimitotic agents, such as colchicine and oryzalin, induce polyploidization by disrupting cell division. While colchicine is commonly employed, its use is associated with considerable toxicity and phytotoxic risks (Chalak and Legave 1996;Trojak-Goluch et al. 2021).

Our study presents a novel method for inducing polyploidization in three Allium species (A. taquetii, A. senescens, and A. spirale Willd.) native to Korea. By adjusting the colchicine treatment parameters, we effectively induced polyploidy in a controlled manner, thereby expanding the genetic diversity of the target species. This method eliminates the need for tissue culture facilities, streamlines the process, and enhances the scalability. We employed a multidisciplinary approach combining flow cytometry, chromosome counting, FISH analysis, and stomatal comparison to understand the genetic mechanisms of polyploid induction and its implications for ornamental trait improvement in the Allium species endemic to Korea.

Materials and Methods

Seed germination and in vivo colchicine treatment

Seeds of three Allium species (A. senescens, A. spirale Willd., and A. taquetii) were provided by the Korea National Arboretum and germinated in a petri dish on wet filter paper for three days at room temperature. Subsequently, each germinated seedling was transferred to a 96-well plate containing 5 μL of colchicine solution (0.2, 0.4, 0.6, 0.8, and 1.0%) for 6, 12, 24, 36, and 48 hrs (Fig. 1 and Table 1). After treatment, the seedlings were transferred to 9 cm² square pots and grown in a temperature range of 18 - 20°C, with a light period of 16 hrs in the greenhouse of Kyungpook National University.

Flow cytometry analysis

Approximately 1 - 2 cm of fresh leaves were collected and finely chopped using a razor blade and 500 μL of nuclei extraction solution was added to elute the nuclei. The nuclei were subsequently stained by adding 2000 μL of staining solution, and the ploidy level was estimated by analyzing the nuclear size using a flow cytometer.

Stomata observations

The methods for studying Stomata have been studied using the tape-peel approach (Lawrence et al. 2018). Stomata of control plants and colchicine-treated A. senescens and A. spirale Willd., and A. taquetii were examined to assess morphological changes. The epidermis was carefully separated using forceps and was placed on a microscope slide. The slides were then observed under a microscope (Olympus, BX53, Japan) at a magnification of 200X. The stomatal length and guard cell width were measured using CytoVision v. 2.6 (Leica Microsystems, Germany).

Somatic chromosomes preparation

The somatic chromosome slides were prepared as described by Kirov et al. (2014). Young root tips were collected in a 1M α-Bromonaphthalene solution for 4 hrs at 14°C and transferred to Carnoy’s solution (absolute ethanol to acetic acid (3:1, v/v)) and incubated for 24 hrs at room temperature. Roots were thoroughly washed in distilled water and incubated with digestion enzyme for 1 hr at 37°C. After removing the enzyme supernatant, 50 μL of Carnoy's solution was vortexed thoroughly and the suspension was incubated on ice for 3 min. The cell suspensions were then centrifuged at 12,000 rpm at 4°C for 3 minutes. After removing the supernatant, 25 μL of the dispersion solution (absolute ethanol to acetic acid (1:9, v/v) was added to resuspend the pellet. The cell suspension was then mounted on an ethanol-cleaned slide, which was steamed for 15 - 30 seconds in a hot water bath and air-dried. The somatic chromosomes were observed under a microscope (Olympus BX53, Japan).

Statistical analysis

To assess the variations in stomatal length and guard cell width between the control and polyploid groups across the three species, an independent sample t-test was conducted. This study utilized measurements from 30 stomata per group for each species, with separate analyses performed for individual species. The t-test was implemented with the assumption of unequal variances between the groups. The null hypothesis postulated no significant differences in the mean values of stomatal length and guard cell width between control and polyploids. Statistical significance was set at a p-value of 0.05. Independent sample t-tests were executed using Python (Python Software Foundation, v. 3.12.5), specifically by employing the ttest_ind function from the scipy.stats module. This function compares the means between the control and treatment groups, assuming unequal variances. Analyses were conducted using custom-developed Python scripts designed for data processing and statistical evaluation.

Fluorescence in situ hybridization (FISH)

FISH was performed according to the method described by Lim et al. (2001), with minor modifications. The slides were washed in 2X SSC buffer for 5 min and incubated with 100 μg･mL^-1 RNase A (10 mg･mL^-1) in 2X SSC solution in a humid chamber for 1hr at 37°C. The slides were washed three times in 2X SSC buffer for 5 min each and fixed with 4% paraformaldehyde for 10 min. The slides were then washed again in 2X SSC buffer for 15 min and dehydrated with 70%, 90%, and 100% ethanol. The hybridization mixture (40 μL of hybridization mixture including 100% formamide, 50% dextran sulfate, 20X SSC buffer, 10% sodium dodecyl sulfate, and 45S rDNA probe labeled with digoxigenin and herring sperm (12.5 ng･μL^-1) was mounted on a pre-treated slide, incubated in a humid chamber at 80°C for 2 min, and incubated in a humid chamber at 37°C for 16 hrs. For stringency washing, slides were washed with 2X SSC buffer and 0.1X SSC buffer for 5 min each and incubated in anti-digoxigenin FITC and blocking buffer (1:100 ratio) for 1 hr at 37°C. The slides were then dehydrated with 70%, 90%, and 100% ethanol, stained with 6-diamidino-2-phenylindole (DAPI) in VECTASHIELD solution (1:100 ratio), and observed under a fluorescence microscope. Cytogenetic results (e.g., chromosome length and FISH results) were analyzed using Cytovision v. 2.6 (Leica Microsystems, Germany), and the idiograms were constructed accordingly.

Results and Discussion

Phenotypic variation and plant growth

The influence of ploidy on phenotypic variation was examined by transplanting control plants and polyploids into plastic pots and cultivating them in a greenhouse for six months. As polyploids exhibited weaker growth and a slower rate of development, the average plant height was found to be lower in the polyploid group than in the control group (Fig. 2). The polyploidization of Allium species has revealed complex effects on plant growth and development. The observed differences between control plants and polyploid species show that polyploid plants exhibit less vigorous growth and slower development, leading to shorter plants than their control counterparts (Fig. 2). This phenomenon might be due to the increased cellular complexity and metabolic demands associated with polyploidy, which could temporarily reduce growth vigor as plants adapt to their new genetic status. However, similar to other studies, it has been reported that polyploid plants initially exhibit slower growth rates (Wu et al. 2023). Despite this reduction in height, polyploid plants may possess other beneficial traits that are not immediately apparent during early growth stages. For instance, polyploidy is often associated with enhanced stress tolerance, a larger cell size, and increased secondary metabolite production (Bharadwaj 2015;Dermen 1940;Huy et al. 2019;Levin 1983;Miguel and Leonhardt 2011;Okazaki 2005;Ramanna and Jacobsen 2003;Ramsey and Schemske 1998). These attributes could be advantageous for ornamental applications once plants overcome the initial growth lag. Additionally, our study demonstrated that in vivo induction of polyploidy in Allium species provides significant practical advantages over traditional in vitro methods (Farhadi et al. 2023). Specifically, in vivo methods reduce both time and labor and substantially decrease the amount of chemical agents required compared to conventional in vitro protocols, which often involve extensive and resource-intensive procedures. Future research should concentrate on the long-term cultivation and monitoring of polyploid Allium species to evaluate their overall growth performance, flowering behavior, and ornamental qualities. Understanding the full range of polyploidy effects will provide valuable insights for optimizing breeding programs and enhancing the horticultural value of these plants.

Stomatal characteristics and implications of polyploidization

The stomatal phenotypes of control plants and polyploids of the three Allium species are shown in (Fig. 3). Our findings indicated that the stomata, as well as the size and width of the guard cells, were larger in polyploids than in the control plants (Table 2). Specifically, the length of the control plant guard cell was 33.5 μm for A. senescens, whereas the polyploid was 45.2 μm. Similarly, the width was 11.9 μm for the control plants, compared to 15.1 μm for the polyploid plants. For A. spirale Willd., the guard cell length was 43.42 μm, whereas that of the polyploid was 54.19 μm. The width was 12.99 μm for the control plants compared to 18.3 μm for the polyploid. Lastly, for A. taquetii, the guard cell length was 42.85 μm, whereas of that the polyploid was 52.30 μm. The width was 5.68 μm for the control plants, compared to 9.98 μm for the polyploids. The results of the independent sample t-tests revealed significant differences in both stomatal length and guard cell width between the control and polyploids for all species analyzed. The t-statistics and corresponding p-values for each species are summarized in (Table 3). The p values for all species and measurements were statistically significant (p < 0.05), indicating substantial evidence to reject the null hypothesis of no difference between the control and treatment groups. The exceptionally low p-values (e.g., 1.05 × 10^-17 for A. senescens stomatal length) suggest a pronounced effect of polyploidy on stomatal and guard cell dimensions. Furthermore, the t-statistics, which quantify the magnitude of the difference between the two groups, were high, further corroborating the biological significance of these differences.

In addition to stomatal dimensions, stomatal density was also significantly affected by polyploidy. For A. senescens, the stomatal density was lower in polyploids (mean density = 1.20 per 100 μm²) than in controls (mean density = 2.00 per 100 μm²), with a t-statistic of 7.09, and a p-value of 2.05 × 10^-9 (Table 3). Similarly, polyploid A. taquetii plants showed reduced stomatal density, and the differences were statistically significant. In contrast, no significant difference in stomatal density was observed for A. spirale Willd., where a p-value of 0.104 did not reach statistical significance. These findings demonstrate that polyploidy leads not only to larger stomata and guard cells, but also to lower stomatal density in certain species, highlighting the distinct morphological impact of genome duplication.

The observed increase in stomatal size and guard cell dimensions in polyploid Allium species when compared to their control plant counterparts is a noteworthy phenotypic consequence of polyploidization (Fig. 3). Our results corroborate those of previous studies that reported larger stomata as a common feature of polyploid plants (Niazian and Nalousi 2020). The enlarged stomata in polyploids can be attributed to increased cell size, typically associated with higher ploidy levels resulting from genomic duplication. The larger stomata and guard cells have various physiological implications. For example, they can influence the gas exchange capacity and water use efficiency of plants. Larger stomatal pores may facilitate more efficient CO₂ uptake for photosynthesis, potentially enhancing plant growth and productivity under optimal conditions (Warner and Edwards 1993). However, this could also lead to increased water loss through transpiration, particularly under water stress conditions (Kelliher et al. 1980), which may necessitate further adaptive mechanisms or breeding strategies to ensure plant resilience under varying environmental conditions. Future research should explore the broader physiological and ecological effects of enlarged stomata in polyploid Allium species. Investigating the balance between improved photosynthetic capacity and potential water loss will be crucial for understanding the viability and sustainability of these plants as ornamentals. Furthermore, long-term studies assessing the adaptive responses of these polyploids to different environmental stresses will provide valuable insights into breeding programs aimed at developing robust ornamental cultivars.

Cytogenetic confirmation of polyploidization

Flow cytometry involves comparing the channel positions of peaks to determine the level of ploidy. In this study, the controls were set to channel 50 (Fig. 4A, C, and E). For polyploid plants, peaks were observed at channel 100 (Fig. 4B, D, and F), indicating that the amount of DNA in the polyploid plants was twice that in the control plants. One advantage of flow cytometry over the other methods in this study was its ability to identify polyploids and screen for polyploidy levels at an early stage of the experiment. The application of flow cytometry and chromosome analysis provided robust evidence confirming the successful induction of polyploidy in the three Allium species studied (Fig. 4). Flow cytometry, with its capacity to precisely measure DNA content, revealed clear peaks corresponding to control and polyploid plants, effectively demonstrating the doubling of the DNA content in polyploids. The efficiency of the method, which allows early-stage identification of polyploid individuals, underscores its value in breeding programs aimed at developing polyploid cultivars (Galbraith et al. 1983). Following this confirmation, the success of colchicine treatment in polyploidy induction was evaluated. Among the different concentrations and durations tested, the 0.2% solution applied for 3 hours achieved the highest success rate (Table 1).

The results of the chromosome analysis by FISH in (Fig. 5) are depicted as idiograms in (Fig. 6). Detailed characteristics of the chromosomes are presented in (Table 4). The number of chromosomes in A. senescens was 2n = 4x = 32. The 45S rDNA signal appears as a green signal. The number of signals was set to two. Somatic chromosome counting showed that the chromosome number of A. senescens octoploids was 2n = 8x = 64. The number of chromosomes in A. spirale Willd. was 2n = 4x = 32. The 45S rDNA signal appears as a green signal. The number of signals was set to two. Somatic chromosome counting showed that the chromosome number of A. spirale Willd. octoploids, 2n = 8x = 64. The number of chromosomes in A. taquetii is 2n = 2x = 16. The 45S rDNA signal was determined to be two. Somatic chromosome counting showed that the chromosome number of A. taquetii tetraploids was 2n = 4x = 32. The 45s rDNA loci on the long arm of chromosome #8 showed the same pattern in A. senesces, A. spirale Willd. and A. taquetii. Polyploid variants of these species also exhibited 45s rDNA loci on the long arm of chromosome #8. In A. taquetii diploids, one B chromosome was observed, whereas in A. taquetii tetraploids, two B chromosomes were observed. The chromosomal length of A. senescens was 103.34 ± 0.54 μm, with the shortest chromosome (Chr. #6) measuring 4.36 ± 6.86 μm and the longest chromosome (Chr. #1) measuring 7.31 ± 8.37 μm. For A. spirale Willd. the chromosomal length was 153.35 ± 0.98 μm, with the shortest chromosome (Chr. #7) at 2.99 ± 12.67 μm and the longest chromosome (Chr. #5) at 5.45 ± 11.08 μm. A. taquetii had a chromosomal length of 135.81 ± 0.99 μm, with the shortest chromosome (Chr. #4) 3.95 ± 12.26 μm and longest chromosome (Chr. #5) at 3.56 ± 16.27 μm.

Chromosome counting and fluorescence in situ hybridization (FISH) analysis further corroborated these findings by directly visualizing chromosome number and structure (Fig. 5 and Fig. 6). The consistent presence of double chromosome numbers in polyploids, along with the 45S ribosomal DNA (rDNA) loci, indicates successful chromosomal duplication without significant structural aberrations. This stability is critical for maintaining the genetic integrity and ensuring the potential for subsequent breeding and propagation. Observation of the B chromosomes in A. taquetii. and their increased number in polyploids suggest intriguing genetic dynamics (Fig. 5E). B chromosomes, often considered supernumerary and dispensable, can influence genome stability and plant development (Jones and Houben 2003). Their presence and increase in number following polyploidization warrants further investigation to understand their role and impact on the phenotype and adaptability of these plants.

The results of our cytogenetic analysis lend credence to the notion that polyploidization can serve as a potent means of developing novel ornamental cultivars of Allium. The stable chromosome counts and consistent 45S rDNA loci observed in these polyploids indicate that they may exhibit desirable traits, such as increased robustness and visual appeal, despite the initial growth lag that was observed. Future research should investigate the long-term stability and fertility of these polyploid lines, as well as their performance under different environmental conditions, to fully harness their ornamental potential.

Conclusion

This study successfully induced polyploidy in three Allium species, A. taquetii, A. senescens, and A. spirale Willd. after colchicine treatment, as confirmed by flow cytometry, chromosome counting, and FISH analysis. The polyploid plants exhibited doubling of DNA content with stable chromosomal structures, indicating successful genome duplication without significant structural abnormalities. Phenotypic alterations resulting from polyploidy included enlarged stomata and guard cells across all three species, which could potentially enhance the photosynthetic efficiency. However, reductions in stomatal density in some species suggest the need to balance improved gas exchange with potential water-use efficiency challenges. Although polyploid plants demonstrate reduced growth rates and diminished heights, these traits may be counterbalanced by long-term benefits, such as improved stress tolerance and enhanced ornamental characteristics. In conclusion, polyploidy is a valuable approach for improving the genetic diversity and ornamental traits of the Allium species. Subsequent research should explore the long-term stability and adaptability of these polyploids to realize their full potential for developing novel, resilient ornamental cultivars.