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Effects of ploidy level on leaf morphology, stomata, and anatomical structure of Hibiscus syriacus L.

BMC Plant Biology volume 24, Article number: 1133 (2024) Cite this article

Abstract

Background

Hibiscus syriacus L. is a deciduous shrub with a strong environmental resistance and wide application prospects. The genetic background and ploidy levels of Hibiscus cultivars are complex, and polyploid breeding has long been an important method for developing new Hibiscus cultivars. However, the relationship of ploidy levels with leaf morphology, stomatal characteristics, and leaf anatomy remains unclear.

Results

This study analyzed three ploidy levels (triploid, tetraploid, and hexaploid) of Hibiscus syriacus. Flow cytometry confirmed the ploidy levels, and morphological traits were evaluated. Leaf length, leaf width, and petiole length decreased with increasing ploidy. Stomatal length, stomatal width, guard cell length, and guard cell width increased and stomatal number and density decreased with increasing ploidy. The hexaploids exhibited the highest midrib diameter and palisade tissue thickness values. Correlation analyses revealed that stomatal morphology served as a reliable marker for determining ploidy levels.

Conclusion

This study highlights the impact of varying ploidy levels on the leaf and stomatal morphologies and leaf anatomy of Hibiscus syriacus. These findings can provide theoretical guidance for improving Hibiscus cultivars in terms of stress resistance, adaptability, and ornamental traits, and for developing new cultivars with enhanced characteristics. Future research should focus on utilizing these morphological markers to optimize breeding strategies for Hibiscus cultivars.

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Introduction

Hibiscus syriacus L., commonly known as the Rose of Sharon, is a deciduous shrub that belongs to the mallow family, Malvaceae. Native to East Asia, it holds significant cultural importance in various countries, especially in Korea, where it is considered the national flower, symbolizing resilience and beauty. This plant has a rich cultural history and has been cultivated for thousands of years. Due to its wide range of uses [1], diverse flower colors, a continuous blooming period of up to three months from summer to fall, and strong resistance to environmental stresses such as drought, Hibiscus syriacus has become a popular choice for gardening and landscaping applications [2].

Despite its widespread use, the genetic background and ploidy levels of various Hibiscus syriacus cultivars remain complex and debated. There is ongoing discussion regarding the basic chromosomal count and ploidy variations within the species. Van Huylenbroeck et al. [3] detailed the ploidy of several hibiscus cultivars. They reported that ‘Ardens’, ‘Hamabo’, and ‘Red Heart’ are diploid variants, whereas ‘Diana’, ‘Helene’, and ‘Melrose’ are triploid types. Later, Lattier [4] proposed that hibiscus is mostly tetraploid (2n = 4x = 80), with a fundamental chromosomal number of x = 20. The enigma surrounding the number of chromosomes in hibiscus has also been resolved. Some earlier reports stated that hibiscus is diploid. However, in 2007 [4], it was reported that “diploid” is often used incorrectly instead of the accurate “tetraploid,” resulting in a confusion between the hexaploid (referred to as triploids) and octoploid cultivars (referred to as tetraploids).

Polyploid breeding has been widely used in Hibiscus cultivation to enhance the desirable traits, such as enhanced flower production, prolonged blooming, and sterility in hybrids [5]. A notable example is the hexaploid cultivar ‘Aphrodite’, developed by the US National Arboretum through colchicine-induced hybridization to reduce sterility and improve flower yield [6].

Polyploidy refers to the presence of two or more sets of chromosomes in a cell nucleus. Apart from being an important mechanism for species formation and adaptation to environmental changes [7], polyploidy is an important factor in plant breeding and improvement. Doubling or multiplication of the chromosome set in a plant cell nucleus often results in several significant changes, including alterations in morphology, physiology, nutritional ingredient, and ecological adaptations, potentially leading to the development of new beneficial traits [8,9,10,11]. Research on the relationship between polyploidy and morphology has gained traction with the progression of polyploid breeding methods. Several morphological studies have reported the “gigas” effect of polyploidy, including increased flower diameter, increased leaf area, decreased leaf aspect ratio, and expanded stomata [12,13,14]. Polyploidy has another significant implication: It allows the production of fertile interspecific hybrids and tetraploid hybrids from diploid × tetraploid crosses [13]. These hybrids increase genetic diversity, allowing plant breeders to combine the desirable traits from different species or ploidy levels, thereby enhancing their yield and adaptability. Polyploidy, therefore, has enormous promise in the field of plant breeding.

While polyploidy can enhance stress tolerance and adaptability in plants, the relationship between polyploidy and leaf anatomy remains a subject of interest. Research on the anatomical variations among polyploids has demonstrated that polyploidy improves resistance to both biotic and abiotic stresses [15,16,17]. When Li et al. [18] compared the fenestrated tissues and thicker upper and lower epidermis of tetraploid and diploid Lonicera japonica, they found that the tetraploid Lonicera japonica withstood drought more efficiently. After inducing Hibiscus syriacus with colchicine, Lee et al. [19] observed several morphological changes, such as alterations in leaf thickness, guard cell length, pollen grain diameter, etc.

The impact of ploidy on leaf and stomatal morphology has been confirmed in many plant species, typically related to changes in cell size, which, in turn, affects leaf thickness and stomatal size and density [20, 21]. Stomatal characteristics are crucial to plant physiology, regulating gas exchange and CO2 absorption [22], which, in turn, affects plant productivity and water use efficiency. In addition, stomatal structure is influenced by genetic factors linked to chromosomal composition, offering greater reliability in evaluating the effects of polyploidy and in the rapid determination of ploidy levels in plants [20, 23]. Ansari et al. [24] also reported that the counting stomatal guard cells and chloroplasts is an indirect and accurate method to evaluate the ploidy levels of alfalfa. Stomata and leaf anatomy of Hibiscus syriacus have been studied and documented in some studies [25, 26]. However, it is still unclear whether and to what degree the polyploidy of Hibiscus syriacus affects its leaf and stomatal morphology. This uncertainty underscores the need for further research to explore the relationship between ploidy levels and traits such as stomatal size and leaf morphology in Hibiscus.

The current study utilized three Hibiscus syriacus cultivars with distinct ploidy levels to investigate the impact of ploidy levels on leaf morphology, stomata, and anatomical structure of the cultivars. Our results provided breeders with certain morphological markers and offered scientific guidance for Hibiscus breeding. These findings could help the breeders select parents with desirable traits to cultivate new cultivars with improved stress resistance, adaptability, and ornamental characteristics, thereby promoting sustainable agricultural development.

Materials and methods

Plant materials

The cultivar information for Hibiscus syriacus was sourced from various references, including the Flora of China and other published literature. Field surveys and records show that, after undergoing environmental adaptation, the leaf traits of the experimental Hibiscus syriacus cultivars have stabilized and become relatively uniform. We selected 38 Hibiscus syriacus cultivars from Hunan Botanical Garden for ploidy identification. The selected cultivars could be categorized into four groups based on ploidy level. One of these groups comprised a low number of cultivars, and the morphological diversity was not representative, so it was excluded from this study.

Flow cytometry

Flow cytometry was employed to determine the ploidy level of Hibiscus syriacus cultivars, with the experimental procedures referenced from Sliwinsk et al. [27] Samples of approximately 0.5 cm2 area were cut from young leaves using a new double-sided blade and placed in a disposable Petri dish. Then, 300 µL of cell lysate (Precise-P, Sysmex) was added to the dish, and the sample was quickly chopped. Finally, 10 µL of aqueous polyvinyl pyrrolidone (PVP, Solarbio) solution with a mass fraction of 28.57% was added to the dish. The mixture was filtered into a sample tube using a 50-µm nylon filter, and 1200 µL of the DAPI staining solution (Precise-P, Sysmex) was added to the tube. The sample tube was then analyzed using a flow cytometer (Sysmex, Germany). Ploidy analysis images for each sample were generated using Flomax software (Sysmex, Germany).

Observation of leaf morphology

Four representative leaf traits, leaf length (LL), leaf width (LW), petiole length (LOP), and leaf aspect ratio (AL), were assessed to evaluate the leaf morphology of the selected cultivars. The leaf traits were observed between June 2023 and October 2023 from well-grown, healthy, and pest- and disease-free plants. Sampling was done at consistent time points and locations. As samples, ten mature leaves from the middle of the flowering branches were selected from each healthy plant and evaluated using vernier calipers with an accuracy of 0.001 cm. The average values were recorded. The leaf length and width ratio was calculated using the following formula:

$${\text{AL}} = {\text{LL}} /{\text{LW}}$$
(1)

Observation of stomatal morphology

The nail polish blotting method was used to observe the stomatal structure in the leaves as previously described by Pathoumthong et al. [28]. Briefly, three healthy mature leaves with similar growth sites were selected from each representative variety. Transparent nail polish was evenly applied to the abaxial surface of the leaves. The leaves were then left to stand for 5–10 min. Then, transparent adhesive tape was stuck to the back of the nail-painted leaves. Next, the tape was removed from the leaves and pasted on slides. The slides were observed using an optical microscope (OLYMPUS DP-71, Japan). A total of six indicators were selected to evaluate stomatal morphology: Stomatal number (SN), stomatal density (SD), stomatal length (SL), stomatal width (SW), guard cell length (GCL), and guard cell width (GCW). SN and SD were observed at 20x magnification. SL, SW, GCL, and GCW were observed at 100x magnification. For each variety, three fields of vision were randomly selected, and the number of stomata in SM (a microscope field of view area (0.24 mm2)) was counted. The data of SL, SW, GCL, and GCW were measured using the ruler tool in Photoshop 2021. The stomatal density was calculated using the following formula:

$${\text{SD}} = {\text{SN}} /{\text{SM}}$$
(2)

Observation of leaf anatomy

Leaf anatomy was assessed using the paraffin sectioning method as previously described by Chen et al. [29]. Briefly, the entire mature leaf sample was fixed with the FAA solution (70% alcohol: formaldehyde: acetic acid = 18:1:1). Next, 1 cm long and 0.5 cm wide rectangular piece was cut along both sides of the main vein. Each sample was subjected to an alcohol gradient dehydration of 30% (30 min), 50% (30 min), 70% (1 h), 85% (1 h), 95% (2 h), and 100% (5 h). Next, the sample was treated with xylene for 2 h, transferred into molten paraffin, and placed in a 60 °C oven overnight to facilitate paraffin infiltration. Then, the samples were embedded in paraffin wax and sectioned into 8–12 μm thick slices using a microtome (LEICA RM2235, Germany). The sections are pasted onto slides with distilled water and dewaxed in xylene for 20 min. The sections were then rehydrated through a graded alcohol series of 100% (5 min), 95% (5 min), 85% (5 min), 70% (5 min), 50% (5 min), and 30% (5 min). Next, the sections were rinsed with distilled water for 2 min, incubated in 1% safranine-O solution for 2 h, dyed with 0.5% fast green solution for 1 min, and sealed with a neutral tree resin. The slices were then observed under optical microscope (OLYMPUS DP-71, Japan). A total of 12 variables were evaluated, which are listed in Table 1. All observations were repeated thrice. The indicators in Table 1 were measured using the ruler tool in Photoshop 2021.

Table 1 Anatomical traits of Hibiscus syriacus leaf cross-section
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Statistical analysis

Excel 2021 was used to process the collected data, and the average values and standard error were obtained. SPSS 25.0 was used for the statistical analysis of the collected data. One-way analysis of variance (ANOVA) was used to analyze the significance of the difference at the level of p < 0.05. Spearman test was used for correlation analysis. Illustrations were generated using Origin 2021.

Results

Ploidy identification

The tetraploid hibiscus cultivars served as internal standards for analyzing the ploidy of the other cultivars to be examined. Flow cytometry was utilized in the first stage to determine the ploidy of the selected hibiscus cultivars. The results showed that the Hibiscus syriacus cultivars had four ploidy levels, namely diploid, triploid, tetraploid, and hexaploid. The diploid group contained only a few cultivars with no representative morphological diversity. Therefore, the remaining three ploidy levels were used for further experiments. Based on the peak value of the relative DNA content of the tetraploid control (which was 100), it can be concluded that the peaks of the triploid, tetraploid, and hexaploid were 75, 100, and 150, respectively. Consequently, the three ploidy levels of the test material were triploids (Fig. 1A), tetraploids (Fig. 1B), and hexaploids (Fig. 1C).

Fig. 1
figure 1

Flow cytometry of the samples with different ploidy levels

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Differences in leaf blade morphology

The leaf blades of the selected cultivars exhibited rhombic to triangular-ovate shapes, either three lobes or no lobe, varying depths, three main veins, an obtuse or pointed apex, a cuneate base, and irregularly serrated margins (Fig. 2A). Hibiscus syriacus leaves displayed varying morphologies, showing decreasing LL, LW, and LOP with increasing ploidy level. Triploids exhibited the highest values of LL, LW, and LOP (9.387, 6.276, and 2.598 cm, respectively). Furthermore, triploids and hexaploids exhibited significantly different LL, LW, and LOP (p < 0.05). However, with the exception of LOP, the differences between the LL and LW values for triploids and tetraploids were not significant (Fig. 2). This finding could be attributed to the fact that plant morphology can be influenced by ecological adaptive pressures. Triploids and tetraploids may have adapted to specific environmental conditions and reached a certain equilibrium. In contrast, hexaploids, with a greater genetic diversity, might have an enhanced ability to adapt to a wider range of environments, leading to more pronounced morphological differences.

We observed a substantial correlation between the ploidy levels of hibiscus plants and their LL, LW, and LOP. Further analyses showed that the mean AL values for triploids and tetraploids were comparable (1.502 and 1.507, respectively), while hexaploids exhibited a smaller mean AL value of 1.486. These ratios did not significantly differ across the various ploidy levels. Overall, the ploidy levels of the hibiscus cultivars impacted their leaf morphologies. These effects became more pronounced with rising ploidy levels.

Fig. 2
figure 2

Differences in the leaf morphologies of Hibiscus syriacus cultivars with different ploidy levels. A: Differences across different ploidy levels; B: Analysis of significant differences. LL: leaf length; LW: leaf width; LOP: petiole length; AL: LL/LW. (a) Triploid; (b) Tetraploid; (c) Hexaploid. The left Y-axis represents the trends in LL, LW, and LOP, while the right Y-axis represents the trend in AL. In bar charts with different lowercase letters “a” and “b,” if the letters differ between bars, it indicates a significant difference based on the analysis done using SPSS 25.0 followed by Duncan’s test (p < 0.05)

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Differences in stomatal structure

Stomata serve as a reliable indicator for determining the ploidy levels of different hibiscus cultivars. Our analyses revealed an irregular stomata distribution across different hibiscus cultivars. Stomata are characterized by two kidney-shaped guard cells surrounding them, creating a fusiform shape with respect to each other. The hexaploid cultivars exhibited significantly higher SL and SW values than tetraploids and triploids (Fig. 3).

Different hibiscus cultivars exhibited different stomatal shapes. Triploids exhibited the highest SN and SD. The triploids exhibited 7% and 28.11% higher SN than tetraploids and hexaploids, respectively. This result indicated a significant difference (p < 0.05) in the number of stomata between triploids and hexaploids (Fig. 4A). Furthermore, GCL, GCW, SL, and SW values increased with increasing ploidy levels. Thus, the hexaploids exhibited higher GCL, GCW, SL, and SW values than the tetraploids and triploids (Fig. 4B). Specifically, hexaploids had significantly higher SL and SW values (14.45% and 33.67%, respectively) and mildly higher GCL and GCW values than triploids (11.32% and 3.50%, respectively). Overall, our findings demonstrate that stomatal morphology varies markedly with ploidy. Higher ploidy levels were associated with lower SD and SN values but higher SL, SW, GCL, and GCW values.

Fig. 3
figure 3

Stomatal structure under 40x magnification. A: Triploid; B: Tetraploid; C: Hexaploid. SL: stomatal length; SW: stomatal width; GCL: guard cell length; GCW: guard cell width. GCW refers to the width of a pair of guard cells

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Fig. 4
figure 4

Differences in the stomatal morphologies of Hibiscus syriacus cultivars with different ploidy levels. SN: number of stomata; SD: stomatal density; SL: stomatal length; SW: stomatal width; GCL: guard cell length; GCW: guard cell width. The measurement unit for SN is “counts,” while the measurement unit for SD is “counts/mm2.” GCW refers to the width of a pair of guard cells. In bar charts with different lowercase letters “a” and “b,” if the letters differ between bars, it indicates a significant difference based on the analysis conducted using SPSS 25.0 followed by Duncan’s test (p < 0.05)

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Differences in leaf anatomy

The vascular bundle in the middle vein of Hibiscus syriacus is typically heart-shaped or nearly so, with cells containing transparent lenses (mainly cluster crystals) distributed around the vascular bundle. The xylem stained a distinct red color, distinguishing it from the phloem.

Our findings demonstrated that hexaploid leaves exhibited a substantially greater midrib diameter (DM) than triploid and tetraploid leaves (Fig. 5A, B, C). Upper epidermal cells, fenestrated tissue, spongy tissue, and lower epidermal cells make up the top-to-bottom structure of Hibiscus syriacus leaf tissues. Tufted crystal cells were also present in the leaf tissue. Notably, the blade thickness of triploid leaves was significantly greater than that of tetraploid and hexaploid leaves (Fig. 5D, E, F). In the selected plants, the fenestrated tissue was linked to the inner side of the upper epidermis and consists of cleanly organized long columnar cells. The spongy tissue was attached to the inner side of the lower epidermis and comprised irregularly shaped, loosely arranged, thin-walled cells with distinct gaps (Fig. 5).

Fig. 5
figure 5

Leaf anatomical structure. A–C show a magnification of 10x, while D–F show a magnification of 40x. A: Triploid midrib tissue structure; B: Tetraploid midrib tissue structure; C: Hexaploid midrib tissue structure; D: Triploid mesophyll tissue structure; E: Tetraploid mesophyll tissue structure; F: Hexaploid mesophyll tissue structure. DM: midrib diameter; XT: xylem thickness; PT: phloem thickness; LT: leaf thickness; UET: upper epidermis thickness; LET: lower epidermis thickness; PPT: palisade tissue thickness; SPT: sponge tissue thickness; NCC: number of cluster crystal cells

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Hibiscus cultivars with different ploidy levels exhibited varying leaf anatomies. However, none of the assessed variables showed significant differences across the ploidy levels. This finding showed that these traits are not effective indicators of the ploidy of hibiscus cultivars (Table 2). Cultivars with higher leaf thicknesses (LT) also exhibited comparatively higher fenestrated and spongy tissue thicknesses (SPT), indicating synergistic variation among them. The maximum values of upper epidermis thickness (UET), lower epidermal thickness (LET), and palisade tissue thickness (PPT) were non-triploid. Moreover, the mean LT values decreased with increasing ploidy (Table 2; Fig. 5). Furthermore, P/S and CTR values increased while SR values decreased with increasing ploidy levels. This finding suggested a correlation between the fenestrated tissue thickness and SPT, providing insights into the utility of these variables in identifying Hibiscus syriacus ploidy. Additionally, hexaploids exhibited the highest DM, UET, LET, PPT, P/S, and CTR values, which might be attributed to their ability to adapt to the environment.

Table 2 Differences in the leaf anatomies of Hibiscus syriacus cultivars at different ploidy levels
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Correlation of ploidy level with leaf and stomatal morphologies and leaf anatomy

In addition to the correlations among the indicators, we observed that the ploidy level exhibited varying degrees of correlations with leaf and stomatal morphologies and leaf anatomy (Fig. 6). Ploidy level negatively correlated with LL, LW, LOP, SN, SD, and SR, while positively correlating with SL, SW, GCL, UET, LET, and P/S (Fig. 6). Moreover, stomatal morphology was identified as a reliable marker for determining the ploidy level of hibiscus cultivars. Notably, the correlation between P/S and ploidy was as high as 0.82, suggesting that this variable could also serve as an indicator of the ploidy level in hibiscus cultivars. However, more studies are needed to validate these findings.

Fig. 6
figure 6

Analysis of correlation between each index and ploidy. * represents p ≤ 0.05, ** represents p ≤ 0.01. LL: leaf length; LW: leaf width; LOP: petiole length; AL: LL/LW; SN: number of stomata; SD: stomatal density; SL: stomatal length; SW: stomatal width; GCL: guard cell length; GCW: guard cell width; DM: midrib diameter; XT: xylem thickness; PT: phloem thickness; UET: upper epidermis thickness; LET: lower epidermis thickness; LT: leaf thickness; PPT: palisade tissue thickness; SPT: sponge tissue thickness; NCC: number of cluster crystal cells; P/S: ryoshika hoiby (PPT/SPT); CTR: leaf tissue structural tightness (PPT/LT) × 100; SR: leaf tissue structure laxity (SPT/LT) × 100; PY: ploidy. GCW refers to the width of a pair of guard cells

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Discussion

Ploidy level and leaf morphology

Alterations in plant ploidy usually cause corresponding changes in plant morphology, physiology, nutrient composition, and ecological adaptations and might lead to the emergence of new superior traits [30,31,32]. Polyploids often exhibit some distinctive characteristics. These changes include thicker leaves, larger flowers, thicker petals, different leaf and flower colors, longer flowering periods, and compact, dwarf plants [33]. For instance, homozygous tetraploids exhibit larger flowers and thicker leaves than the corresponding diploids [34]. Compared to diploids, the induced tetraploid Sorbus pohuashanensis exhibited significant morphological changes, such as thicker and larger leaves, as well as reduced leaf margin serration [35]. In contrast to these findings, our study observed that triploids exhibited larger LL and LW than tetraploids and hexaploids. Interestingly, Mo et al. [36] found that colchicine-induced tetraploid and octoploid Rhododendron exhibited smaller leaves compared to diploids, which is consistent with our findings. We propose three possible explanations for this result. Firstly, most hibiscus cultivars are tetraploid [4], and diploid Hibiscus cultivars are difficult to survive in the wild [37]. Therefore, different cultivars may have distinct genetic backgrounds, which can influence their growth performance in a polyploid state. This may result in some cultivars exhibiting different growth patterns as ploidy levels increase. Secondly, different cultivars exhibit varying rates of individual development [38]. These differences might affect the experimental outcomes due to the different developmental stages of different cultivars during sample collection. Finally, environmental conditions, such as light, humidity, and temperature, variably affect the development of leaf morphologies in different cultivars [39]. If polyploid plants encounter adverse environmental conditions such as water or light deficiency during growth, they may exhibit smaller leaves as an adaptive response to reduce transpiration or enhance their adaptability.

Ploidy level and stomatal morphology

Several studies have demonstrated that employing plant morphological traits to distinguish the ploidy levels is an easy and natural approach [40,41,42], significantly lessening the efforts of breeding personnel in determining plant ploidy on a broader scale. However, since not all identification indices are reliable, the accuracy of this approach is low. This approach is only ideal to make an educated guess about plant ploidy. Studies have shown that stomata and guard cells in leaves are regulated by chromosomes, and can preliminarily reflect the ploidy levels [4, 43]. With increasing ploidy, the defense cell length generally increases and SD generally decreases [44]. Several studies have reported the utility of stomatal morphology as a reliable indicator of plant ploidy [45,46,47]. Our findings reveal that hexaploids exhibited significantly higher SL, SW, GCL, and GCW compared to both triploids and tetraploids. In contrast, triploids had a higher SN and SD than tetraploids and hexaploids. These results align with previous studies that demonstrated polyploidy often leads to increased cell size [48], including stomata and guard cells [49], as a result of the additional chromosome sets. It is also indicated that stomatal morphology serves as a reliable marker for the ploidy level in Hibiscus cultivars. For instance, Auliya et al. [50] demonstrated that the stomata were oval in triploid banana cultivars but round in diploid banana cultivars (Musa acuminata ‘AAA’). The stomata in diploid banana cultivars were smaller than those in triploid banana cultivars. The polyploids induced in Glehnia littoralis through colchicine treatment also exhibited larger stomata and significantly reduced stomatal density [51].

The larger stomatal and guard cell sizes in hexaploids may be due to the increased genomic content and subsequent enlargement of individual cells [48], a common consequence of polyploidy. Moderate increases in stomatal size and aperture can lead to enhanced stomatal conductance [52], which in turn can improve photosynthetic efficiency [53]. Although larger stomata may increase the risk of water loss, polyploid plants can exhibit higher photosynthetic efficiency under conditions of adequate water supply and favorable temperatures. Plants can enhance resistance to abiotic stressors by regulating their stomata [4, 54], which might underlie the better environmental adaptability of polyploids [55].

Ploidy level and leaf anatomy

In addition to changing their morphology and appearance, polyploid plants also adapt better to their surroundings and are more resilient to abiotic stresses [56, 57]. Polyploids can modify their leaf anatomy to modify their own light and environmental tolerance. Previously, Jin et al. [58] reported that Malus spectabilis polyploid plants exhibited thicker leaf epidermis and fenestrated tissues, higher photosynthetic efficiency, and greater salinity tolerance. Polyploids were also shown to be more resilient to drought stress by Rao et al. [59] The research findings by Lin et al. [60] indicate that the palisade tissue of tetraploid Broussonetia papyrifera (L.) L’Hér.ex Vent is thicker. Our research results indicate that the palisade tissue thickness is greatest in hexaploids, which is consistent with findings from previous studies. However, the results of Yao et al. [61] showed that the leaf morphology and structure of polyploids Camellia sinensis exhibited significant differences, with more developed and larger xylem. In contrast to their findings, our results indicate that hexaploids exhibit greater DM compared to triploids and tetraploids, but XT and PT were not affected by ploidy levels. We hypothesize that this may be related to the variation in the expression of ploidy effects in different parts of the leaf [62]. The influence of ploidy on DM might be due to the fact that the midvein region is more involved in growth and expansion, while the XT and PT may relatively stably maintain their transport functions. This could result in certain parts, such as DM, being significantly affected by ploidy, whereas other parts, such as XT and PT, show less noticeable changes.

Based on the anatomical structure of Hibiscus leaves, the veins play a mechanical support role, while the palisade tissue is the main site of photosynthesis. The spongy tissue is closely arranged, with distinct intercellular spaces, which facilitates enhanced gas exchange and improves the efficiency of light energy utilization [63]. Cluster crystal cells are widely distributed in the mesophyll cells and leaf veins, serving various functions such as plant protection, heavy metal detoxification, and alleviation of stress from adverse environmental conditions [64].These characteristics are consistent with Hibiscus’ traits of being sun-loving, heat-tolerant, and highly resistant to environmental stress. Our results show that the vascular bundles of hexaploid Hibiscus syriacus are tightly arranged and the diameter of the conduit is large.These vascular bundles are responsible for transporting nutrients and water [65], further suggesting that polyploids are more adaptable to their surroundings. LT is closely related to SPT and PPT. Fenestrated tissues are the main site of photosynthesis in leaves, as their cells contain more chloroplasts. The elevated levels of photosynthetic products enhance water regulation in leaves. Previous studies have also shown that plants with thicker fenestrated tissues and higher P/S levels are more resistant to drought stress [66]. The current study showed that the hexaploids exhibited the thickest fenestrated tissue and the highest P/S value. These findings indicated that hexaploids are more adaptable to environmental changes and drought stress.

Breeding selection strategy

From a breeding program perspective, early identification of polyploidy or confirmation of ploidy levels in new breeding materials can significantly reduce the time required for selection and improve breeding efficiency. By investigating the leaf and stomatal morphologies, as well as leaf anatomy, of Hibiscus syriacus at different ploidy levels, we identified stomatal morphology as a reliable indicator that can assist in the preliminary identification of polyploidy in Hibiscus syriacus. Early-stage identification of polyploids through simple observation of stomata and guard cell structures can save considerable time in breeding selection. This can be followed by more precise identification of ploidy levels using flow cytometry and chromosome counting, which not only reduces the costs of identification but also enhances the overall breeding process. Our research results indicate that hexaploids showed excellent traits such as DM and CTR. These advantageous traits suggest that hexaploids could serve as excellent candidates for further breeding programs, particularly in enhancing stress tolerance. Of course, the importance of tetraploids should not be overlooked. The origin of the hexaploid variety ' Aphrodite ' is based on the tetraploid [6]. This underscores the importance of maintaining diversity in ploidy levels.

Conclusions

In conclusion, we investigated the variations among three ploidy levels of Hibiscus syriacus in terms of leaf and stomatal morphologies and leaf anatomy. LL, LW, and LOP gradually decreased with increasing ploidy. Stomatal morphology was identified to be a reliable marker for determining ploidy levels in hibiscus cultivars. SL, SW, GCL, and GCW increased and SN and SD decreased with increasing ploidy. The values of DM, UET, LET, PPT, P/S, and CTR in hexaploids are significantly higher across the three ploidy levels. These results are significant for optimizing Hibiscus syriacus breeding strategies. By understanding the relationship between ploidy and morphological traits, breeders can select cultivars with improved environmental adaptability and growth characteristics. In particular, hexaploids, which showed superior physiological traits, could be prioritized for cultivation in regions with variable climates. These findings will contribute to future breeding programs by guiding the selection of resilient, high-performance cultivars, ultimately enhancing agricultural practices and promoting sustainable production of Hibiscus syriacus.

This study has several limitations. First, it analyzed only three ploidy levels, which may not encompass all genetic variability within the cultivars. Second, while stomatal morphology is considered a reliable marker for ploidy determination, its applicability in different environments and across other hibiscus cultivars requires further validation. Looking forward, there are several areas for future research that remain unexplored. For instance, the relationship between polyploidy in Hibiscus syriacus and environmental factors warrants further investigation. Additionally, it would be valuable to explore how other physiological traits, such as flowering time and reproductive success, vary with ploidy levels.

Data availability

The data in this study can be provided to the corresponding authors according to reasonable requirements.

References

  1. Kang HC, Kim DY, Wang YG, et al. Expanded uses and trend of domestic and international research of Rose of Sharon (Hibiscus syriacus L.) as Korean national flower since the protection of new plant variety. J Korean Inst Landsc Archit. 2019;47(5):49–65.

    Article  Google Scholar 

  2. Areas G. Review on ornamental Rose of Sharon (Hibiscus syriacus L.): Assessment of decorativeness, invasiveness and ecosystem services in public. Sustainable Practices Hortic Landsc Archit, 2022;71.

  3. Van Huylenbroeck JM, De Riek J, De Loose M. Genetic relationships among Hibiscus syriacus, Hibiscus sinosyriacus and Hibiscus paramutabilis revealed by AFLP, morphology and ploidy analysis. Genet Resour Crop Evol. 2000;47(3):335–43.

    Article  Google Scholar 

  4. Lattier JD, Chen H, Contreras RN. Variation in genome size, ploidy, stomata, and rDNA signals in Althea. J Am Soc Hortic Sci. 2019;144(2):130–40.

    Article  CAS  Google Scholar 

  5. Van Laere K, Dewitte A, Van Huylenbroeck J, et al. Evidence for the occurrence of unreduced gametes in interspecific hybrids of Hibiscus. J Hortic Sci Biotechnol. 2009;84(2):240–7.

    Article  Google Scholar 

  6. Egolf DR. Aphrodite Rose of Sharon (Althea). HortScience. 1988;23(1):223–4.

    Article  Google Scholar 

  7. Huang G, Zhu YX. Plant polyploidy and evolution. J Integr Plant Biol. 2019;61(1):4–6.

    Article  PubMed  Google Scholar 

  8. Segraves KA, Anneberg TJ. Species interactions and plant polyploidy. Am J Bot. 2016;103(7):1326–35.

    Article  PubMed  Google Scholar 

  9. Haist G, Sidjimova B, Vladimirov V, et al. Morphological, cariological, and phytochemical studies of diploid and autotetraploid Hippeastrum Papilio plants. Planta. 2023;257(3):51.

    Article  CAS  PubMed  Google Scholar 

  10. Feng Z, Bi Z, Fu D, et al. A comparative study of morphology, photosynthetic physiology, and proteome between diploid and tetraploid watermelon (Citrullus lanatus L). Bioengineering. 2022;9(12):746.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Samatadze TE, Yurkevich OY, Khazieva FM, et al. Agro-morphological and cytogenetic characterization of colchicine-induced tetraploid plants of Polemonium caeruleum L. (Polemoniaceae). Plants. 2022;11(19):2585.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Sattler MC, Carvalho CR, Clarindo WR. The polyploidy and its key role in plant breeding. Planta. 2016;243(2):281–96.

    Article  CAS  PubMed  Google Scholar 

  13. Yan Z, Beibei W, Shuaizheng Q, et al. Ploidy and hybridity effects on leaf size, cell size and related genes expression in triploids, diploids and their parents in Populus. Planta. 2018;249(3):635–46.

    Google Scholar 

  14. Sabzehzari M, Hoveidamanesh S, Modarresi M, et al. Morphological, anatomical, physiological, and cytological studies in diploid and tetraploid plants of ispaghul (Plantago ovata Forsk). Genet Resour Crop Evol. 2020;67(1):129–37.

    Article  CAS  Google Scholar 

  15. Trojak-Goluch A, Kawka-Lipińska M, Wielgusz K, et al. Polyploidy in industrial crops: applications and perspectives in plant breeding. Agronomy. 2021;11(12):2574.

    Article  CAS  Google Scholar 

  16. Wójcik D, Marat M, Marasek-Ciołakowska A, et al. Apple autotetraploids—phenotypic characterisation and response to Drought stress. Agronomy. 2022;12(1):161.

    Article  Google Scholar 

  17. Tossi VE, Martínez Tosar LJ, Laino LE, et al. Impact of polyploidy on plant tolerance to abiotic and biotic stresses. Front Plant Sci. 2022;13:869423.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Li WD, Biswas DK, Xu H, et al. Photosynthetic responses to chromosome doubling in relation to leaf anatomy in Lonicera japonica subjected to water stress. Funct Plant Biol. 2009;36(9):783–92.

    Article  CAS  PubMed  Google Scholar 

  19. Lee SK, Kim CS. Studies on artificial polyploid forest trees XIII-some morphological and physiological characteristics of Colchitetraploid Hibiscus syriacus L. J Korean Soc for Sci. 1976;32(1):73–86.

    Article  Google Scholar 

  20. He Y, Sun Y, Zheng R, et al. Induction of tetraploid male sterile Tagetes erecta by colchicine treatment and its application for interspecific hybridization. Hortic Plant J. 2016;2(5):284–92.

    Article  Google Scholar 

  21. Qiao G, Liu M, Song K, et al. Phenotypic and comparative transcriptome analysis of different ploidy plants in Dendrocalamus latiflorus Munro. Front Plant Sci. 2017;8:1371.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Lawson T, Blatt MR. Stomatal size, speed, and responsiveness impact on photosynthesis and water use efficiency. Plant Physiol. 2014;164(4):1556–70.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Catalano C, Abbate L, Motisi A, et al. Autotetraploid emergence via somatic embryogenesis in Vitis vinifera induces marked morphological changes in shoots, mature leaves, and stomata. Cells. 2021;10(6):1336.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Ansari E, Khosrowshahli M, Etminan A, et al. Polyploidy induction and ploidy level determination in annual and perennial diploid Medicago species using the enumeration of chloroplasts of stomata guard cells. Cytol Genet. 2022;56(2):164–71.

    Article  Google Scholar 

  25. Rakhimov A, Yoziev L, Duschanova G. Structural features of the vegetative bodies of Hibiscus syriacus L. (Sev. Malvaceae Juss.) Growing under the conditions in Uzbekistan. BIO Web Conferences. 2021;30:04006.

    Article  Google Scholar 

  26. Jailan M, Sabah H, Zeinab A, et al. Botanical and genetic characterization of Hibiscus syriacus L. cultivated in Egypt. J Appl Pharm Sci. 2018;8(12):92–103.

    Article  Google Scholar 

  27. Sliwinska E, Loureiro J, Leitch IJ, et al. Application-based guidelines for best practices in plant flow cytometry. Cytometry A. 2021;101(9):749–81.

    Article  PubMed  Google Scholar 

  28. Pathoumthong P, Zhang Z, Roy SJ, et al. Rapid non-destructive method to phenotype stomatal traits. Plant Methods. 2023;19(1):36.

    Article  PubMed  PubMed Central  Google Scholar 

  29. Chen B, Wang C, Tian Y, et al. Anatomical characteristics of young stems and mature leaves of dwarf pear. Sci Hort. 2015;186:172–9.

    Article  Google Scholar 

  30. Baker RL, Yarkhunova Y, Vidal K, et al. Polyploidy and the relationship between leaf structure and function: implications for correlated evolution of anatomy, morphology, and physiology in Brassica. BMC Plant Biol. 2017;17(1):3.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Jiang Y, Liu S, Hu J, et al. Polyploidization of Plumbago auriculata Lam. In vitro and its characterization including cold tolerance. Plant Cell Tissue Organ Cult (PCTOC). 2020;140(2):315–25.

    Article  CAS  Google Scholar 

  32. Park CH, Park YE, Yeo HJ, et al. Comparative analysis of secondary metabolites and metabolic profiling between diploid and tetraploid Morus alba L. J Agric Food Chem. 2021;69(4):1300–7.

    Article  CAS  PubMed  Google Scholar 

  33. Manzoor A, Ahmad T, Bashir MA, et al. Silvestri C. Studies on colchicine induced chromosome doubling for enhancement of quality traits in ornamental plants. Plants. 2019;8(7):194.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Du J, Wang J, Wang T, et al. Identification and chromosome doubling of Fragaria Mandshurica and F. Nilgerrensis. Sci Hort. 2021;289:110507.

    Article  Google Scholar 

  35. Zhang Z, Zhang Y, Di Z, et al. Tetraploid induction with leaf morphology and sunburn variation in Sorbus pohuashanensis(hance) hedl. Forests. 2023;14(8):1589.

    Article  Google Scholar 

  36. Mo L, Chen J, Lou X, et al. Colchicine-induced polyploidy in Rhododendron Fortunei Lindl. Plants. 2020;9(4):424.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Zhitong L. Development and evaluation of diploid and polyploid Hibiscus moscheutos. HortScience. 2017;52(5):676–81.

    Article  Google Scholar 

  38. Qi M, Tingting G, Xianran L, et al. Phenotypic plasticity in Plant Height shaped by Interaction between Genetic Loci and diurnal temperature range. New Phytol. 2021;233(4):1768–79.

    Google Scholar 

  39. Garcia LM, Smith HJ, Lopez TA. Humidity and temperature effects on leaf morphology in varieties of tomato (Solanum lycopersicum). Environ Exp Bot. 2015;113(1):1–10.

    Google Scholar 

  40. Liu M, Liu P. Artificial autopolyploidization in jujube. Sci Hort. 2023;314:111916.

    Article  CAS  Google Scholar 

  41. Hartati S, Samanhudi OC, Cahyono O et al. Morphology of hybrid orchid induced by polyploidy using colchicine. IOP Conference Series: Earth and Environmental Science, 2023;1133(1): 012065.

  42. Tamayo-Ordóñez MC, Rodriguez-Zapata LC, Narváez-Zapata JA, et al. Morphological features of different polyploids for adaptation and molecular characterization of CC-NBS-LRR and LEA gene families in Agave L. J Plant Physiol. 2016;195:80–94.

    Article  PubMed  Google Scholar 

  43. Chepkoech E, Kinyua MG, Kiplagat O, et al. Assessment of the ploidy level diversity by chloroplast counts in stomatal guard cells of potato (Solanum tuberosum L.) mutants. Asian J Res Crop Sci. 2019;4(3):1–7.

    Article  Google Scholar 

  44. Padoan D, Mossad A, Chiancone B, et al. Ploidy levels in Citrus clementine affects leaf morphology, stomatal density and water content. Theoretical Experimental Plant Physiol. 2013;25(4):283–90.

    Article  Google Scholar 

  45. Kruthika HS, Rukmangada MS, Naik VG. Genome size, chromosome number variation and its correlation with stomatal characters for assessment of ploidy levels in a core subset of mulberry (Morus spp.) germplasm. Gene. 2023;881:147637.

    Article  CAS  PubMed  Google Scholar 

  46. Beck SL, Dunlop RW, Fossey A. Stomatal length and frequency as a measure of ploidy level in black wattle, Acacia mearnsii (De Wild). Bot J Linn Soc. 2003;141(2):177–81.

    Article  Google Scholar 

  47. John RA, Auxcilia J, Devarajan R, et al. Ploidy determination in banana hybrids through stomatal studies and chloroplast count. Int J Environ Clim Change. 2023;13(10):4210–9.

    Article  Google Scholar 

  48. Doyle JJ, Coate JE. Autopolyploidy: an epigenetic macromutation. Am J Bot. 2020;107(8):1097–100.

    Article  PubMed  Google Scholar 

  49. Catalano C, Abbate L, Motisi A, et al. Autotetraploid emergence via somatic embryogenesis in Vitis vinifera induces marked morphological changes in shoots, mature leaves, and stomata. Cells. 2021;10(6):1336.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Auliya I, Hapsari L, Azrianingsih R. Comparative study of leaf stomata profiles among different ploidy levels and genomic groups of bananas (Musa L.). IOP Conference Series: Earth and Environmental Science, 2019;391(1): 012037.

  51. Zhang X, Zheng Z, Wang J, et al. In vitro induction of tetraploids and their phenotypic and transcriptome analysis in Glehnia littoralis. BMC Plant Biol. 2024;24(1):439.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Monda K, Araki H, Kuhara S, et al. Enhanced stomatal conductance by a spontaneous Arabidopsis tetraploid, Me-0, results from increased stomatal size and greater stomatal aperture. Plant Physiol. 2016;170(3):1435–44.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Yin W, Yizhou W, Yanhong T, et al. Stomata conductance as a goalkeeper for increased photosynthetic efficiency. Curr Opin Plant Biol. 2022;70:102310.

    Article  Google Scholar 

  54. Park SI, Kim JJ, Shin SY, et al. ASR enhances environmental stress tolerance and improves grain yield by modulating stomatal closure in rice. Front Plant Sci. 2019;10:1752.

    Article  PubMed  Google Scholar 

  55. Deepo DM, Mazharul IM, Hwang YJ, et al. Chromosome and ploidy analysis of winter hardy Hibiscus species by FISH and flow cytometry. Euphytica. 2022;218(6):81.

    Article  CAS  Google Scholar 

  56. Hui T, Bao L, Shi X, et al. Grafting seedling rootstock strengthens tolerance to drought stress in polyploid mulberry (Morus alba L). Plant Physiol Biochem. 2024;208:108441.

    Article  CAS  PubMed  Google Scholar 

  57. Mangena P. Cell mutagenic autopolyploidy enhances salinity stress tolerance in leguminous crops. Cells, 2023;12(16): 2082.

  58. Jin Y, Zhao Y, Ai S, et al. Induction of polyploid Malus prunifolia and analysis of its salt tolerance. Tree Physiol. 2022;42(10):2100–15.

    CAS  PubMed  Google Scholar 

  59. Rao S, Tian Y, Xia X, et al. Chromosome doubling mediates superior drought tolerance in Lycium Ruthenicum via abscisic acid signaling. Hortic Res. 2020;7(1):40.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Lin J, Zhang B, Zou J, et al. Induction of tetraploids in Paper Mulberry (Broussonetia papyrifera (L.) L’Hér. Ex vent.) By colchicine. BMC Plant Biol. 2023;23(1):574.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Yao X, Qi Y, Chen H, et al. Study of Camellia sinensis diploid and triploid leaf development mechanism based on transcriptome and leaf characteristics. PLoS ONE. 2023;18(2):e0275652.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Tanaka Y, Matsuoka M, Shinozaki K. Effects of Polyploidy on Leaf vascular tissue development in Arabidopsis. Plant Physiol. 2017;173(3):1598–610.

    Google Scholar 

  63. Oguchi R, Onoda Y, Terashima I et al. Leaf anatomy and function. The leaf: a platform for performing photosynthesis, 2018;97–139.

  64. Khan MI, Pandith SA, Shah MA, et al. Calcium oxalate crystals, the plant ‘Gemstones’: insights into their synthesis and physiological implications in plants. Plant Cell Physiol. 2023;64(10):1124–38.

    Article  CAS  PubMed  Google Scholar 

  65. Lucas WJ, Groover A, Lichtenberger R, et al. The plant vascular system: evolution, development and functions. J Integr Plant Biol. 2013;55(4):294–388.

    Article  CAS  PubMed  Google Scholar 

  66. Zhu J, Cai D, Wang J, et al. Physiological and anatomical changes in two rapeseed (Brassica napus L.) genotypes under drought stress conditions. Oil Crop Sci. 2021;6(2):97–104.

    Article  Google Scholar 

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Acknowledgements

The authors extend their appreciation to College of Landscape Architecture, Central South University of Forestry and Technology.

Funding

This research was funded by the Key Discipline of the State Forestry Administration (LinRenFa [2016] No. 21), “Double First-Class” Cultivation Discipline of Hunan Province (XiangJiaoTong (2018) No. 469).

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Authors and Affiliations

  1. College of Landscape Architecture, Central South University of Forestry & Technology, Changsha, 410004, China

    Jingwen Zhang, Chuyu Cheng, Xinxin Zhang, Chen Zhang, Yazhi Zhao, Jing Xu & Xiaohong Wang

  2. Institute of Biodiversity, Hunan Academy of Forestry, Changsha, 410004, China

    Fen Xiao

  3. YeZi Biotechnology Company, Yiyang, 413000, China

    Shengqian Zhang

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  1. Jingwen Zhang
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  3. Fen Xiao
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  8. Shengqian Zhang
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  9. Xiaohong Wang
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Contributions

JZ: Conceptualization, investigation, writing first draft preparation, analysis, writing review and editing; CC: Investigation; software; formal analysis; writing review XW: Conceived and designed this research, writing review and editing; JX: Investigation; XZ & ZC: Software; FX & YZ: Writing review and editing. SZ: Funding acquisition. All authors have read and agreed to the published version of the manuscript.

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Correspondence to Xiaohong Wang.

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Zhang, J., Cheng, C., Xiao, F. et al. Effects of ploidy level on leaf morphology, stomata, and anatomical structure of Hibiscus syriacus L.. BMC Plant Biol 24, 1133 (2024). https://doi.org/10.1186/s12870-024-05778-y

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BMC Plant Biology

ISSN: 1471-2229

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