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Article

Tetraploid Induction with Leaf Morphology and Sunburn Variation in Sorbus pohuashanensis (Hance) Hedl

by
Zeren Zhang
1,†,
Yan Zhang
1,†,
Zexin Di
1,
Ruili Zhang
1,
Yanjuan Mu
2,
Tao Sun
2,
Zhihui Tian
1,
Yizeng Lu
2,* and
Jian Zheng
1,*
1
School of Architecture, Beijing University of Agriculture, Beijing 102206, China
2
Shandong Provincial Center of Forest and Grass Germplasm Resources, Jinan 250102, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Forests 2023, 14(8), 1589; https://doi.org/10.3390/f14081589
Submission received: 28 June 2023 / Revised: 26 July 2023 / Accepted: 28 July 2023 / Published: 4 August 2023
(This article belongs to the Section Genetics and Molecular Biology)
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Abstract

:
Sorbus pohuashanensis (Hance) Hedl. is an important forestry species valued for its ornamental, medicinal, and ecological properties. Polyploidy breeding is an important method of germplasm innovation; however, polyploidy induction and phenotypic variation caused by chromosome doubling in S. pohuashanensis are poorly understood. In this study, S. pohuashanensis seeds were used to explore the effects of different colchicine concentrations, cold stratification times, and seeds from different sources on polyploidy induction. Ploidy levels of the regenerated plants were determined by flow cytometry. The results showed that the tetraploid induction effect of S. pohuashanensis seeds was significantly affected by colchicine concentration, and the highest tetraploid induction rate of 24.75% was achieved by immersion in 0.2% (w/v) colchicine for 48 h. After 2 years of induction, 77 tetraploid plants were obtained. Compared to diploids, tetraploid plants showed significant variations in plant height, leaf morphology (apical leaflet width, middle leaflet width), and diameter of the middle petiole. The stomatal size and chloroplast number increased with chromosome doubling whereas the stomatal number and density decreased. In addition, significant differences in the percentage of sunburn associated with ploidy changes were observed. This study provides a technique for tetraploid induction of S. pohuashanensis seeds, showing the variation in traits caused by polyploidization and the effect of chromosome doubling on sunburn resistance. Tetraploidy induction provides a new direction for S. pohuashanensis germplasm innovation.

1. Introduction

Sorbus L. belongs to the Maloideae subfamily of Rosaceae, and most species in the genus are deciduous trees or shrubs. There are approximately 258 species worldwide, naturally distributed across Asia, Europe, and North America [1]. Sorbus pohuashanensis is a known diploid species of Sorbus [2], common in North, Northwest, and Northeast China. The natural range of S. pohuashanensis is at an altitude of 1200–2000 m [3]. Moreover, because of its colorful autumn leaves and fruits, S. pohuashanensis has a high ornamental value and has attracted the attention of landscaping departments; in addition, the high contents of flavonoids, triterpenoids, anthocyanins, and cyanogenic glycosides in the fruits have potential health protection and medicinal value [4,5,6]. The introduction and cultivation of S. pohuashanensis at low altitudes is limited by abiotic stresses, particularly high temperature and sunlight [7,8]. Sunburn the plants’ leaves or fruits reduces their yield and affects their ornamental quality [7,9]. Furthermore, the intraspecific variation range of S. pohuashanensis is limited, and breeding improvement work has been insufficient; only one breeding method is currently available [10]. Therefore, increasing intraspecific variation through other breeding methods is necessary to enhance the abiotic stress resistance and ornamental value of S. pohuashanensis.
Polyploids have more than two complete sets of chromosomes, usually resulting from mitotic or meiotic errors, which occur widely in angiosperms [11,12]. Polyploidy, or whole-genome duplication (WGD), is an important driving force of plant evolution [13]. After chromosome doubling, these species often exhibit a faster growth rate, larger organs, increased yield and seed-setting rate, and stronger plant resistance [12,14,15]. Some studies have shown that triploid plants have far more growth advantages than diploids and display superior traits [16,17]. Therefore, polyploid breeding is commonly used as a method of germplasm innovation. For example, among the timber tree species, allotriploid Populus have a faster growth rate and higher volume growth than diploids [18]. Polyploidy in economically important tree species results in higher fruit yield and increased content of total sugars, soluble solids, and other substances in fruits [19,20]. However, not all changes due to polyploidization are beneficial. For example, some tetraploid species exhibit obvious dwarfing characteristics [21,22]. In addition, compared with diploids, some tetraploid species have lower photosynthetic rates and decreased carbohydrate synthesis and decomposition ability [23,24].
In 1937, Blakeslee successfully induced tetraploid Datura using colchicine [25], and scholars began to explore the artificial induction of polyploids. Based on treatment methods, artificial polyploid induction can be divided into physical induction (involving mechanical damage, extreme temperatures, radiation, etc.) and chemical induction (the use of colchicine, oryzalin, trifluralin, acenaphthene, etc.) [26]. Presently, polyploid induction by chemical reagents is the most common method, among which colchicine is the most widely used chemical reagent [27,28]. The same reagent with different induction materials involves different treatment methods. Plant seeds are the most easily obtained material, and the induction method is relatively simple; seeds are simply soaked in the reagent [29,30]. Notably, when using the same plant material and induction reagent to induce polyploidy, the induction effect may be affected by treatment time and concentration [29,30,31]. At present, studies on polyploidy within the Sorbus species have mainly focused on natural variations [2,32,33], and polyploid breeding has not been reported. Therefore, it is necessary to induce and explore the methods of tetraploid induction of S. pohuashanensis.
The morphological variation of vegetative organs caused by polyploidization is often a concern. The tetraploid Thymus persicus exhibited shorter roots and thicker stems than the diploids [34]. In addition, in Averrhoa carambola, triploid and tetraploid plants produced stronger branches and petioles, more leaflets, and thicker and larger leaves than diploids [35]. Many reports of polyploid morphological variation are related to the leaf traits. For example, leaf length and width are significantly greater in Populus polyploids than in diploids, and variations of leaf margins were also observed in tetraploid plants [36,37]. Leaf morphological and anatomical variations after polyploidization in Eucalyptus urophylla have also been observed [38]. Some species have been further explored for the possible causes of polyploid leaf changes. Cytology and gene expression analyses revealed that the leaf magnification of the Betula pendula tetraploid was related to cell expansion rather than cell number [39]. Thus, investigating the phenotypic variation of S. pohuashanensis tetraploids, especially leaf morphology, can help us better understand the variation in plant characteristics caused by polyploidization. However, polyploid breeding in S. pohuashanensis has not yet been reported.
In this study, we used S. pohuashanensis seeds to explore amethod of tetraploid induction. Polyploidy was induced by colchicine treatment of S. pohuashanensis seeds, and the ploidy level was determined by flow cytometry. Morphological and sunburn variations of S. pohuashanensis tetraploids were revealed. By artificially inducing tetraploidy in S. pohuashanensis, we expect to lay the foundation for artificial autopolyploid research on Sorbus and provide new germplasm resources for genetic improvement.

2. Materials and Methods

2.1. Plant Material

The seed induction tests of S. pohuashanensis were performed in 2020 and 2021. Natural provenances collected in Mount Tai, Tai’an City, Shandong Province, in 2018 (TS2018) and cultivated provenances collected in Shandong Provincial Center of Forest and Grass Germplasm Resources in 2021 (ZY2021) were selected as plant materials. All seeds were stored at 4 °C. S. pohuashanensis seeds have dormancy characteristics; thus, before the test, they were subjected to 1 g/L gibberellic acid processing for 24 h at room temperature and stored at 4 °C for cold stratification. All experiments in this study used randomized and complete block design.

2.2. Colchicine Treatment

In 2020, TS2018 seeds treated with cold stratification for 28 days were used as test materials. Colchicine solutions of 0.01%, 0.05%, 0.1%, and 0.2% were soaked for 12, 24, and 48 h, respectively, and a water-soaking treatment was used as the control (tap water was used). Each treatment was tested in triplicate, with each test containing 150 seeds.
In January 2020, the same seed materials were selected and treated at cold stratification times of 14 and 21 days and then treated for 48 h with various colchicine concentrations (0.1% and 0.2% w/v). After the treatment, the residual solution was cleaned with water and stored at 4 °C. All seeds were stored at room temperature after 28 days of stratification at low temperatures. Each treatment was repeated three times, with each test containing 100 seeds, and tap water was used as the control. After cold stratification, seeds were transferred into plastic trays (height: 7.5 cm; length: 37 cm; width: 30 cm) containing a 3:1 mixture of peat and vermiculite. The germination rate was calculated on the 20th day after sowing, and the seeds were transferred to a flowerpot (height: 29 cm; top width: 25.5 cm; bottom width: 22 cm) in April, with standard irrigation and fertilizer management (irrigation was conducted every three days on average, and compound fertilizer was applied regularly and uniformly).
In November 2021, based on the experimental results from 2020, to verify the feasibility of the tetraploid induction method of S. pohuashanensis, TS2018 and ZY2021 were selected as materials, and the experimental treatment process was repeated.

2.3. Ploidy Identification

Leaves of the plants (about 1 cm × 1 cm in size) were selected as samples. The samples were placed in a 90 mm plastic petri dish, and 1.5 mL of lysis buffer (kiwifruit buffer [40]) was added. Then the samples were chopped using a razor blade. Crude nuclei solution was filtered through CellTrics 30 μm disposable filters into a glass sample tube, and 100 μL DAPI (50 μg/mL) solution was added to the tube for staining. The sample tubes were loaded into the Cyflow® Ploidy analyzer (Sysmex Partec, Goerlitz, Germany) for chromosome ploidy analysis. Each sample contained at least 2000 nuclei. The induction rate was calculated based on the ploidy analysis. The ploidy levels of the plants induced in 2020 were determined twice; in 2021 and 2022, respectively, and those induced in 2021 were determined in 2022.

2.4. Plant Morphological Characteristics

The phenotypic traits of tetraploid S. pohuashanensis induced in 2020 were measured in 2021 and 2022, respectively. Three mature leaves were selected from the annual branches of each seedling, and sunburn of leaves was observed in August 2022. The leaf phenotypic traits are presented in Table 1 and Figure 1. The calculation formulas are as follows:
ALSI = ALL ÷ ALW
MLSI = MLL ÷ MLW
PS = number of sunburn leaves ÷ total number of leaves × 100%

2.5. Stomata Observation

Five tetraploids and five diploids were randomly selected, and five healthy and flat leaves were selected from each plant. The epidermal layer was peeled from the abaxial surface of the leaves along the vein. Then it was placed on a slide with water drops and covered with a coverslip. The samples were placed under a microscope (Zeiss Axio Scope.A1, Carl Zeiss AG, Oberkochen, Germany) with a 40× magnification objective for stomatal observation. Two microscope fields were randomly observed for each leaf, and five stomata were randomly selected in each field. ZEN blue edition software (ZEN 2.0; Carl Zeiss AG, Oberkochen, Germany) was used to measure stomatal length, width, density, and chloroplast number in the guard cells.

2.6. Statistical Analysis

Statistical analysis software (SPSS 21.0; SPSS Inc., Chicago, IL, USA) was used for significance and correlation analyses. Before analyses, normality and homogeneity tests of variance were conducted on the data, and arcsine transformation was performed on the percentage data. T-test, Kruskal-Wallis test, analysis of variance, and Duncan’s multiple test were used to analyze significance between the different treatment conditions. Pearson’s correlation coefficient analysis was used to determine the correlations between the phenotypic traits. Microsoft Excel 2019 (Microsoft Corp., Washington, DC, USA) was used for arcsine conversion and table construction. Principal component analysis (PCA) was performed using Origin 2022 software (Origin Lab, Northampton, MA, USA).

3. Results

3.1. Effect of Colchicine on Seed Germination

To investigate the optimal concentration and treatment time for colchicine, the effects of colchicine on seed germination of S. pohuashanensis were tested. The results showed that at low colchicine concentrations (w/v) (0.01% and 0.05%), the seed germination rate was not significantly different from that of the control. However, at 0.1% and 0.2% (w/v) colchicine treatments, the seed germination rates (33.51% and 28.98%, respectively) decreased significantly (Table 2). With the extension of colchicine treatment time, the germination rate decreased gradually, and significant differences in seed germination rate under the 12 h, 24 h, and 48 h treatment times were observed (Table 2). However, no significant difference was detected in the interaction between colchicine concentration and treatment time (Table A1). The results demonstrated that colchicine concentration and treatment time significantly affected seed germination. Under conditions of high concentration and long treatment time, the germination rate of S. pohuashanensis seeds remained considerable. To ensure that more tetraploid seedlings could be obtained, seedlings soaked in 0.1% and 0.2% (w/v) colchicine solution for 48 h were selected for subsequent experiments.

3.2. Tetraploid Induction and Determination

The results of ploidy analysis by flow cytometry are shown in Figure 2. No significant difference in survival rate, induction rate, or induction efficiency was observed between 14 days and 21 days of cold stratification (Table 3). In addition, there was no significant difference in induction rate at different cold stratification times, indicating that the change in stratification time did not significantly affect the induction effect (Table 3 and Table A2). However, significant differences in tetraploid induction were observed between the two colchicine treatments; the indices obtained with the 0.2% (w/v) treatment were higher than those obtained with the 0.1% (w/v) treatment (Table 3). Based on the variance analysis results, we observed that colchicine concentration significantly affected the tetraploid-induction rate of S. pohuashanensis (Table 3). Treatment with 0.2% (w/v) colchicine for 48 h yielded the best induction effect, with a tetraploid induction rate of 24.75% and an induction efficiency of 12.09%. Finally, we obtained 77 tetraploids of S. pohuashanensis seedlings (25 induced in 2020 and 52 induced in 2021).
Subsequently, we analyzed the differences in the tetraploid induction effect between natural provenances (TS2018) and artificially domesticated provenances (ZY2021) (Table 4). After colchicine treatment, we observed that colchicine concentration at 0.1% (w/v) and 0.2% (w/v) did not significantly affect the germination rate, consistent with previous experimental results on seed germination (Table 2). Natural provenances had significantly higher germination rates than cultivated provenances; however, the tetraploid induction rate did not increase (Table 4). No significant difference was observed between the two provenances, and no significant interaction was identified between colchicine concentration and provenance (Table A3). Interestingly, the seeds from both natural and cultivated provenances had higher tetraploid induction rates after the 0.2% (w/v) colchicine treatment (Table 4), indicating a more suitable condition for tetraploid induction.

3.3. Stomata Analysis

To further explore the relationship between tetraploid and diploid S. pohuashanensis, we observed and analyzed the stomatal characteristics of tetraploid and diploid leaves (Figure 3 and Table 5). The average length and width (37.10 and 24.44 μm) of stomata in tetraploid leaves were significantly longer than those of diploid leaves (28.53 and 19.56 μm), respectively, and more chloroplasts were discovered in tetraploid guard cells. However, the stomatal density per unit area of tetraploid leaves was significantly lower than that of diploid leaves. These results indicated that the abaxial epidermis of tetraploid leaves had larger stomata, lower stomatal density, and more chloroplasts.

3.4. Plant Morphological Characteristics

The morphological characteristics of 25 tetraploids and 20 diploid plants were observed for 2 consecutive years. We observed that the heights of the tetraploid plants were significantly lower than that of the diploid plants (Figure 4), and their leaves were morphologically different (Figure A1). We also observed that the ABL (annual branch length) of tetraploids was 9.91 cm shorter than that of diploids, indicating that the slow growth of new shoots was one of the reasons for the differences in the height of the tetraploid plants. In addition, the BD (basal diameter) of the tetraploid plants (12.28 mm) was thicker than that of the diploid plants (11.57 mm) in the first year; however, the difference was not significant. The observation results in the second year were similar; the BD of tetraploid plants was 13.08% thicker, and variance analysis revealed extremely significant differences between the diploid and tetraploid plants (Table 6). We also observed that ALW (apical leaflet width), MLW (middle leaflet width), ALSI (apical leaflet shape index), and MLSI (middle leaflet shape index) were significantly different between diploid and tetraploid plants. Tetraploid ALW and MLW were wider than that of diploid plants, whereas tetraploid ALSI and MLSI were smaller than that of diploid plants.
In addition, we included the DMP (diameter of middle petiole) and the PS (percentage of sunburn) in the 2022 observations (Figure 5 and Table 6) and discovered that tetraploid leaves showed higher PS and thicker DMP, which were significantly different from those of diploid leaves. The results indicated that polyploidization changed the size of plant organs and affected plant resistance.
Based on the correlation analysis, plant ploidy was negatively correlated with PH, ABL, ALSI, and MLSI and positively correlated with BD, ALW, MLW, DMP, and PS (Table 7). The phenotypes with correlation coefficients greater than 0.6 were MLSI, DMP, ALSI, ALW, and MLW (0.865, 0.777, 0.729, 0.643, and 0.605, respectively) (Table 7).
Subsequently, PCA was performed on the observations from 2022. As shown in Figure 6, scattered points representing individual plants were aggregated under the same ploidy, and significant separations between different ploidies revealed that plant ploidy differences could be well reflected in the morphological traits. Taking a score of greater than 0.6 as an indicator of the contribution strength of traits to the principal component, in the first principal component the traits with a score of more than 0.6 were DBL (0.882), CLL (0.869), CLW (0.791), MLL (0.784), MLW (0.741), ALL (0.728), CPL (0.664), and ALL (0.651). The traits that scored more than 0.6 in the second principal component were MLSI (0.915), ALSI (0.891), DMP (0.759), ALW (0.675), and MLW (0.613) (Table A4). Based on the correlation analysis, the characteristics with high ploidy correlation (ALW, MLW, ALSI, MLSI, and DMP) were mainly concentrated in the second principal component. Moreover, we noted that the scores of middle leaflet traits in the first and second principal components were higher than those of the apical leaflets (except for leaflet width in the second principal component). The distribution of each trait variable is shown by the green arrow in Figure 6, where the arrow directions of ALW, MLW, and DMP are highly consistent with the distribution directions of the tetraploid scatter points, and the arrows of ALSI and MLSI are in the same direction as the diploid scatter. In conclusion, ALW and MLW, leaf shape index, and DMP can be used as morphological characteristics to distinguish tetraploids from diploids, and changes in middle leaflet characteristics are more closely related to ploidy.
Figure 7 shows a boxplot created after the numerical standardization of each morphological character, representing the distribution of each character’s data. We observed that the data distribution of ALSI, MLSI, and DMP was extremely concentrated, and there was almost no data intersection between tetraploid and diploid plants within the data distribution range. WAL and WML followed, with relatively centralized data distribution and lesser data intersection between different ploidy plants within the data range. These results could explain the strong correlation between the three characteristics, ALSI, MLSI, and DMP, and plant ploidy in the correlation analysis. They are also consistent with the PCA results, which showed that the diploid scatter distribution direction was remarkably similar to the arrow direction of ALSI and MLSI. In contrast, the data for PH, BD, and ABL overlapped more frequently, as reflected in the correlation coefficients, showing a small correlation with ploidy (Table 7).
In summary, the results have demonstrated a close relationship between leaflet variation and ploidy. The degree of sunburn was also related to ploidy changes, suggesting that polyploidization reduces the ability of leaves to resist high temperature and sunlight.

4. Discussion

4.1. Colchicine Induced Tetraploids

In seed-induced tetraploids, characteristic differences between different plant seeds affect the plants’ tolerance to colchicine, and the concentration and time of colchicine treatment among different species differs; however, treatment concentrations of colchicine are mostly above 0.1% (w/v), and treatment time varies according to different seed tolerances, with the majority being 1–3 days [27]. Lycium barbarum seeds reached the maximum induction rate after treatment with 0.1% (w/v) colchicine for 48 h [41]. Taraxacum kok-saghyz showed similar results, and the best seed induction condition was achieved with 0.1% (w/v) colchicine for 48 h [30]. Catharanthus roseus seeds were soaked in 0.2% (w/v) colchicine for 24 h to obtain the highest induction rate [29]. In the present study, at 0.2% (w/v) colchicine treatment for 48 h, the germination rate of S. pohuashanensis seeds was 20.91%, and the tetraploid induction rate reached 24.75%. When the seeds of Platycladus orientalis [42], Betula platyphylla [31], and Catharanthus roseus [29] were treated with colchicine, they showed decreased germination rate (or increased mortality) with increasing treatment concentration or longer treatment time. Similar results were observed in this study, where excessive induction concentration and treatment time negatively affected seed growth and germination. The treatment of 0.2% (w/v) colchicine could increase the possibility of tetraploid induction; however, increased colchicine concentration causes cell toxicity, which leads to abnormal function of the cell, disrupts microtubule polymerization, and also affects cell division and growth [43,44]. Therefore, colchicine must be applied for a precise time and at a precise concentration.

4.2. Relationship between Phenotype and Polyploidy

The relationship between stomatal size and ploidy was reported as early as 1937 [45]. In 1990, Ho reported that the ploidy level of Zea mays L. could be determined by the number of chloroplasts in the guard cells [46]. Subsequently, Ewald also proved that chloroplast number could be used as an effective method to judge the ploidy level in Populus and Robinia pseudoacacia [47]. In addition, Rosa chinensis var. minima [48], Eriobotrya japonica [49], and Robinia pseudoacacia [50] all demonstrated the phenomenon of enlarged guard cells and increased chloroplast number in tetraploid plants. During cytological observation of S. pohuashanensis, we observed that tetraploid plants had larger guard cells, more chloroplasts, and fewer stomata per unit area than diploid plants. Chromosome doubling can change chloroplast number and stomatal size. Although the mechanism has not been elucidated, this study and many other studies support this hypothesis. Thus, the stomatal size and chloroplast number can serve as indices for distinguishing S. pohuashanensis tetraploids from diploids.
A common effect of polyploidy on the morphology of woody plants is observed with respect to height [22,51]. In this study, 25 autotetraploid plants were observed for 2 consecutive years. We observed that the height of tetraploid plants was significantly different from that of diploid plants; tetraploids exhibited obvious dwarfing characteristics (Figure 4). Previous studies have revealed that plants often exhibit retarded growth after polyploidization, accompanied by plant-type dwarfing [51]. This is also consistent with our observations of significant differences in annual branch length among the different ploidies in the second year, shorter annual branches in tetraploids. This finding demonstrates that tetraploid plants grow significantly slower than diploid plants, which ultimately leads to differences in plant height. The dwarf-type mechanism has been studied in several species. A decrease in growth- and elongation-related hormone levels has been associated with the dwarfing of plants after polyploidization, especially a decrease in indoleacetic acid (IAA), gibberellin (GA), and brassinosteroid (BR) levels [21,22]. The slow growth rate of the tetraploid plants may be associated with miRNA accumulation. The dwarf phenotype of tetraploid apples may be related to miRNA390 accumulation [22]. Similar results were obtained in the tetraploid Populus, where the expression of auxin- and GA-related miRNAs was significantly upregulated, negatively regulating hormone recognition and biosynthesis-related genes, resulting in significantly lower GA and IAA levels in tetraploids than in diploids [24]. In addition, the hybrid Liquidambar also showed a slow division rate and dormancy in the apical meristem of the tetraploid, which may be the direct cause of the shorter height of the tetraploid plants [21]. Based on these studies, the causes of dwarfing in tetraploid S. pohuashanensis may be related to the accumulation of genetic material and secretion level of endogenous hormones, which affect plant stem growth through direct or indirect (apical dormancy) pathways. In the case of economically important tree species, using polyploidy to breed dwarf stock can reduce plant height and improve plant resistance to maximize benefits. The cultivation of dwarf stocks enhances economic benefits [52,53].
In addition to plant height, we observed changes in leaf morphology. Correlation and principal component analyses revealed that the leaf width and leaf shape index were the most important different traits between tetraploid and diploid plants. A review of studies on leaf traits in polyploid plants showed interesting results: Zhang [37] compared the leaf morphology of diploid parents and offspring with that of triploid hybrid Populus offspring and observed that the leaf length-to-width ratio of triploids was significantly smaller than that of diploid plants. Similarly, Salix viminalis var. Energo leaves of different genotypes were significantly wider than those of diploid controls after polyploidization [54]. Liu [38] also noted that tetraploid leaf width increased more than the leaf length after polyploidization.
The effect of polyploidy on leaf width has also been reported in polyploid fruit trees [55]. The effect of increased ploidy on leaf width is not confined to woody plants. Taraxacum kok-saghyz [30], Cucumis sativus L. [56], Gerbera hybrida [57], and other horticultural plants have also demonstrated the phenomenon of increased leaf width and decreased leaf shape index caused by increased ploidy. Combined with previous studies and our results, we believe that the relationship between leaflet width, leaflet shape index, and polyploidy may be much closer than expected and can be used as an intuitive method to determine natural S. pohuashanensis ploidy levels in the future. However, further understanding of the molecular mechanisms and hormonal signals responsible for leaf shape changes as well as the possible causes of phenotypic changes is required.
The WUSCHEL-related homeobox (WOX) gene family is associated with the development of lateral leaf domains in several species. WOX homologous gene has been confirmed to alter leaf width in Oryza sativa [58], Hordeum vulgare L. [59], and Solanum lycopersicum [60]. In addition, Auxin response factors (ARFs) are involved in leaf expansion regulation [61]. Besides the gene family, miRNAs influence leaf width by participating in post-transcriptional gene regulation [62]. Several studies have shown that hormones are involved in cell expansion regulation in leaves [63]. Taking BR as an example, tobacco leaves showed obvious enlargement and expansion after the external application of 2,4-epibrassinolide, accompanied by increased secretion of endogenous hormones [64]. In conclusion, the phenotypic variation in S. pohuashanensis tetraploids may be influenced by development-related genes and hormone biosynthesis. However, the specific formation mechanism remains to be clarified.

4.3. Polyploidy and Plant Resistance

Polyploidization, or WGD, is a plant evolutionary strategy to avoid extinction in harsh environments [65]. The adaptation of polyploids to extreme temperatures has been explored in some species [66], and reports have been almost uniformly positive. Chen reported that the tetraploid Asparagus officinalis L. had better heat resistance than the diploid through long-term heat stress combined with physiological indices [67]. Similar results were obtained when heat stress resistance was compared between the tetraploid and diploid Dioscorea zingiberensis [68]. However, opposing results have also been reported. Under heat stress, the tetraploid Dendranthema nankingense showed lower semi-lethal temperatures than diploids, indicating that tetraploids have lower heat stress tolerance [69]. In the present study, we observed that the leaf sunburn resistance of the autotetraploid S. pohuashanensis was not better than that of diploid plants, and the percentage of sunburnt tetraploids was significantly higher than that of diploid plants. Studies have shown that for autopolyploids, the first generation after genome doubling does not experience genome restructuring or wide reorganization of gene expression; thus, the characters show little to no heterobeltiosis [70]. Changes in morphology, cytology, physiology, and molecular and biochemical pathways may eventually lead to an increased degree of sunburn in tetraploid leaves. In case of leaf trichomes, it is well know that leaf trichomes are a protective barrier against natural hazards including UV-B radiation [71], and it has the report that trichome density in tetraploid leaves was reduced to 70% of that in diploid leaves of Tanacetum parthenim [72]. We hypothesized that the change in leaf morphology might also increase the degree of tetraploid sunburn. Changes in leaf morphology have an effect on leaf surface temperature, and increasing leaf width raises leaf surface temperature [73]. The growth retardation and faster leaf senescence displayed by tetraploids may also be the reason for the aggravated sunburn phenomenon [21,24]. Based on our results, we believe that polyploid breeding cannot effectively solve the problems of low-altitude acclimation of S. pohuashanensis, and the relationship between polyploidy and the resistance of plants to heat stress requires further investigation.

5. Conclusions

We successfully induced autotetraploid in S. pohuashanensis by treating the seeds with colchicine. We observed that colchicine concentration plays a major role in the successful induction of tetraploid plants. Provenance and cold stratification time did not affect polyploidy induction in S. pohuashanensis. Through 2 years of morphological observation and correlation and principal component analyses, we identified significant changes in the leaf morphology of S. pohuashanensis. We concluded that the leaf width and DMP can be used to identify and distinguish the ploidy of S. pohuashanensis in the seedling stage. However, we observed that polyploidy does not reduce the damage caused by high temperatures and sunlight on the leaves of S. pohuashanensis in summer. This technique provides a baseline for breeding and germplasm innovation of S. pohuashanensis and the possibility of creating triploids of S. pohuashanensis.

Author Contributions

Z.Z. and Y.Z. conducted the experiments and wrote the manuscript. Z.D. participated in the experiments. J.Z. and Y.L. designed the experiments and revised the manuscript. R.Z., Y.M. and T.S. collected the materials. Z.T. participated in project administration. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by “R&D Program of Beijing Municipal Education Commission (KM202110020003)”, The Subject of Key R&D Plan of Shandong Province (Major Scientific and Technological Innovation Project) “Mining and Accurate Identification of Forest Tree Germplasm Resources” (2021LZGC023), Program of Beijing Agricultural University Young Teachers’ Research and Innovation Ability Enhancement Program (QJKC2022008) and National Key Research and Development Program (2017YFD0300401).

Data Availability Statement

Data is contained within the article.

Conflicts of Interest

The authors declare no conflict of interest.

Appendix A

Forests 14 01589 g0a1
Figure A1. Leaves of tetraploid and diploid. Pictures (a,b) show the leaves of tetraploid plants; pictures (c,d) show the leaves of diploid plants. Bar = 2 cm.
Figure A1. Leaves of tetraploid and diploid. Pictures (a,b) show the leaves of tetraploid plants; pictures (c,d) show the leaves of diploid plants. Bar = 2 cm.
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Table A1. Analysis of variance of seed germination rate of Sorbus pohuashanensis under different colchicine.
Table A1. Analysis of variance of seed germination rate of Sorbus pohuashanensis under different colchicine.
SOVdfMSFp-Value
Treatment time (h)21167.48580.5280.000 *
Colchicine concentration (%)4336.60123.2170.000 *
Colchicine Conc. × Treatment time810.1110.6970.691
Error3014.498
Note: df: degrees of freedom, MS: mean square, * Represents a significant difference at p < 0.05.
Table A2. Variance analysis of the effects of cold stratification days and colchicine concentration on seed induction of Sorbus pohuashanensis TS2018 in different years.
Table A2. Variance analysis of the effects of cold stratification days and colchicine concentration on seed induction of Sorbus pohuashanensis TS2018 in different years.
YearSOVPercentagedfMSFp-Value
2020Stratification time (d)Tetraploid (%)2266.8195.4490.100
Mixoploid (%)243.1260.8330.515
Induction percentage (%)2168.2411.8780.296
Concentration (%)Tetraploid (%)1115.1200.8140.418
Mixoploid (%)1112.8273.5070.134
Induction percentage (%)1216.8422.2330.209
2021Stratification time (d)Tetraploid (%)144.3890.2610.625
Mixoploid (%)1139.0451.3620.270
Induction percentage (%)1254.7891.0380.332
Concentration (%) (w/v)Tetraploid (%)11106.14917.3100.002 *
Mixoploid (%)1596.34910.5880.009 *
Induction percentage (%)11604.58414.5270.010 *
Note: df: degrees of freedom, MS: mean square, * Represents a significant difference at p < 0.05.
Table A3. Analysis of variance of seed induction of Sorbus pohuashanensis with different provenances and treatments.
Table A3. Analysis of variance of seed induction of Sorbus pohuashanensis with different provenances and treatments.
SOVPercentagedfMSFp-Value
ProvenancesTetraploid (%)150.4390.6280.446
germination rate (%)1637.49132.4040.000 *
Concentration (%) (w/v)Tetraploid (%)1491.88113.6200.004 *
germination rate (%)141.3550.5220.487
Provenances × ConcentrationTetraploid (%)116.2970.4430.524
germination rate (%)124.6381.5080.254
Note: df: degrees of freedom, MS: mean square, * Represents a significant difference at p < 0.05.
Table A4. Principal component analysis of morphological characters of tetraploid and diploid plants.
Table A4. Principal component analysis of morphological characters of tetraploid and diploid plants.
Principal Component
123
BDL0.8820.0950.108
CLL0.8690.2520.164
CLW0.7910.245−0.400
MLL0.7840.250−0.448
MLW0.741−0.613−0.125
ALL0.7280.152−0.127
CPL0.6640.4400.391
ALW0.651−0.6750.050
BD0.576−0.335−0.013
PH0.5560.5650.169
ABL0.4350.5540.551
DMP0.433−0.759−0.063
PS−0.043−0.3760.516
ALSI−0.1300.891−0.122
MLSI−0.1490.915−0.204
Eigenvalue5.7764.3321.246
Contribution rate (%)38.50528.8798.304
Cumulative contribution rate (%)38.50567.38475.688

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Figure 1. Schematic diagram of measuring indexes for leaf traits of Sorbus pohuashanensis.
Figure 1. Schematic diagram of measuring indexes for leaf traits of Sorbus pohuashanensis.
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Figure 2. Histograms of flow cytometric analysis of Sorbus pohuashanensis. (a) Diploid plant; (b) Tetraploid plant; (c) Mixoploid; (d) Diploid and tetraploid mixed samples.
Figure 2. Histograms of flow cytometric analysis of Sorbus pohuashanensis. (a) Diploid plant; (b) Tetraploid plant; (c) Mixoploid; (d) Diploid and tetraploid mixed samples.
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Figure 3. Stomata morphology of plants in Sorbus pohuashanensis. (a) Stomata morphology of diploid, bar = 50 μm; (b) stomata morphology of tetraploid, bar = 50 μm.
Figure 3. Stomata morphology of plants in Sorbus pohuashanensis. (a) Stomata morphology of diploid, bar = 50 μm; (b) stomata morphology of tetraploid, bar = 50 μm.
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Figure 4. Comparison of diploid and tetraploid plants Sorbus pohuashanensis. (a) Tetraploid and diploid plants were compared, tetraploid on the left and diploid on the right, bar = 15 cm; (b) Comparison of diploid and tetraploid plant populations, tetraploid on the left and diploid on the right, bar = 25 cm.
Figure 4. Comparison of diploid and tetraploid plants Sorbus pohuashanensis. (a) Tetraploid and diploid plants were compared, tetraploid on the left and diploid on the right, bar = 15 cm; (b) Comparison of diploid and tetraploid plant populations, tetraploid on the left and diploid on the right, bar = 25 cm.
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Figure 5. Sunburn phenomenon of diploid and tetraploid leaves of Sorbus pohuashanensis. Note: (a) Diploid sunburn condition; (b) Tetraploid sunburn condition; (c) Diploid leaves, bar = 3 cm; (d) Tetraploid leaves, bar = 3 cm.
Figure 5. Sunburn phenomenon of diploid and tetraploid leaves of Sorbus pohuashanensis. Note: (a) Diploid sunburn condition; (b) Tetraploid sunburn condition; (c) Diploid leaves, bar = 3 cm; (d) Tetraploid leaves, bar = 3 cm.
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Figure 6. Principal component analysis of morphological characters of tetraploid and diploid plants of Sorbus pohuashanensis. Values in parentheses indicate the percentage of total variance explained by each principal component. Red lines and red dots represent diploids; blue lines and blue dots represent tetraploids; green arrows represent different trait variables.
Figure 6. Principal component analysis of morphological characters of tetraploid and diploid plants of Sorbus pohuashanensis. Values in parentheses indicate the percentage of total variance explained by each principal component. Red lines and red dots represent diploids; blue lines and blue dots represent tetraploids; green arrows represent different trait variables.
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Figure 7. Box plot of tetraploid and diploid plant traits. Different colors represent different morphological traits; the square in the middle of the box represents the mean of the data.
Figure 7. Box plot of tetraploid and diploid plant traits. Different colors represent different morphological traits; the square in the middle of the box represents the mean of the data.
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Table 1. Plant morphological characteristics and abbreviations.
Table 1. Plant morphological characteristics and abbreviations.
CharacteristicsAbbreviation
Compound leaf lengthCLL
Compound leaf widthCLW
Common petiole lengthCPL
Diameter of middle petioleDMP
Distance between leafletsDBL
Apical leaflet lengthALL
Apical leaflet widthALW
Apical leaflet shape indexALSI
Middle leaflet lengthMLL
Middle leaflet widthMLW
Middle leaflet shape indexMLSI
Plant heightPH
Basal diameterBD
Annual branch lengthABL
Percentage of sunburnPS
Table 2. Seed germination rate of Sorbus pohuashanensis under different colchicine concentration and treatment time.
Table 2. Seed germination rate of Sorbus pohuashanensis under different colchicine concentration and treatment time.
Colchicine Concentration
(%) (w/v)		Treatment Time (h)
12 h24 h48 hMean
049.10 ± 1.41 abA41.92 ± 1.92 aB30.47 ± 1.22 abC40.50 ± 2.87 ab
0.0152.79 ± 1.86 aA40.65 ± 0.28 aB37.40 ± 0.47 aB43.61 ± 2.44 a
0.0549.48 ± 0.69 abA42.07 ± 0.42 aB32.50 ± 0.61 abC41.35 ± 2.48 ab
0.143.85 ± 2.19 bcA31.29 ± 1.76 bB25.38 ± 3.50 bcB33.51 ± 3.15 bc
0.239.21 ± 1.57 cA26.83 ± 1.21 bB20.91 ± 3.50 cB28.98 ± 3.05 c
Mean46.89 ± 1.48 A36.55 ± 1.79 B29.33 ± 1.86 C
Note: Data presented mean ± standard error (SE). Lowercase letters indicate significance between treatments within a column, and uppercase letters indicate significance between treatments within a row. Duncan’s multiple tests was used to test the significance of difference (p ≤ 0.05), and different letters represent significant differences between means.
Table 3. Effects of cold stratification time and colchicine concentration on polyploid induction of Sorbus pohuashanensis TS2018 in different years.
Table 3. Effects of cold stratification time and colchicine concentration on polyploid induction of Sorbus pohuashanensis TS2018 in different years.
TreatmentSurviving Rate (%)Tetraploid (%)Mixoploid (%)Induction Rate (%)Tetraploid Induction Efficiency (%)
Stratification time (d)1438.92 ± 2.15 a8.82 ± 3.00 a9.65 ± 3.41 a14.18 ± 4.30 a5.04 ± 1.68 a
2138.15 ± 2.82 a11.38 ± 5.25 a14.19 ± 4.58 a20.32 ± 7.34 a5.40 ± 2.39 a
Concentration (%) (w/v)043.82 ± 2.91 a0 ± 0 b0 ± 0 c0 ± 0 c0 ± 0 b
0.140.48 ± 0.83 a5.55 ± 2.67 b10.83 ± 2.39 b14.31 ± 1.33 b3.58 ± 1.72 b
0.231.32 ± 2.07 b24.75 ± 3.76 a24.93 ± 3.61 a37.44 ± 5.92 a12.09 ± 1.43 a
Note: Data presented mean ± standard error (SE). Lowercase letters represent the significance of Duncan’s multiple tests between each group of data. Surviving rate (%) = surviving seedlings/seeding no. Induction rate (%) = (tetraploids no. + mixoploids no.)/germinated seedlings. Tetraploid induction efficiency (%) = surviving rate (%) × tetraploid (%).
Table 4. Effects of different colchicine concentrations and provenances on polyploid induction and germination rate of Sorbus pohuashanensis.
Table 4. Effects of different colchicine concentrations and provenances on polyploid induction and germination rate of Sorbus pohuashanensis.
ProvenancesConcentration (%) (w/v)Tetraploid (%)Mixoploid (%)Induction Rate (%)Germination Rate (%)
TS20180.15.55 ± 2.67 b10.83 ± 2.39 c14.31 ± 1.33 c40.87 ± 0.78 a
0.224.75 ± 3.76 a24.93 ± 3.61 b37.44 ± 5.92 ab33.06 ± 1.04 b
ZY20210.19.76 ± 4.88 b22.91 ± 4.05 b26.41 ± 3.18 bc22.95 ± 3.38 c
0.224.9 ± 1.65 a40.08 ± 1.69 a50.42 ± 2.53 a22.10 ± 2.80 c
Note: Data presented mean ± standard error (SE). Lowercase letters represent the significance of Duncan’s multiple tests between columns (p < 0.05), and different letters represent significant differences between each other.
Table 5. Analysis of variance of stomatal length, width, density and chloroplast number in leaf epidermis of tetraploid and diploid plants of Sorbus pohuashanensis.
Table 5. Analysis of variance of stomatal length, width, density and chloroplast number in leaf epidermis of tetraploid and diploid plants of Sorbus pohuashanensis.
CharacteristicDiploidTetraploid
Stomatal length (μm)28.53 ± 0.4137.10 ± 0.46 **
Stomatal width (μm)19.56 ± 0.2724.44 ± 0.31 **
Stomatal density (number per mm2)156.37 ± 4.22 **105.27 ± 2.38
Chloroplast number in stomatal guard cells18.44 ± 0.2529.73 ± 0.45 **
Note: Data presented mean ± standard error (SE), ** represents significance reaching the level of p < 0.01.
Table 6. Analysis of variance of morphological characters of tetraploid and diploid plants of Sorbus pohuashanensis.
Table 6. Analysis of variance of morphological characters of tetraploid and diploid plants of Sorbus pohuashanensis.
CharacteristicFirst Year (2021)Second Year (2022)
DiploidTetraploidDiploidTetraploid
Plant height (cm)44.76 ± 2.18 **38.02 ± 3.3571.96 ± 3.82 **56.83 ± 3.39
Basal diameter (mm)11.57 ± 0.3412.28 ± 0.3913.53 ± 0.3615.3 ± 0.45 **
Compound leaf length (cm)19.82 ± 0.5218.43 ± 0.4116.29 ± 0.5116.47 ± 0.65
Compound leaf width (cm)10.63 ± 0.3710.08 ± 0.39.16 ± 0.328.72 ± 0.29
Common petiole length (mm)40.31 ± 1.3541.6 ± 4.0836.85 ± 1.7134.37 ± 1.93
Distance between leaflets (mm)23.86 ± 0.7724.33 ± 0.4921.93 ± 0.6622.74 ± 0.89
Aapical leaflet length (mm)52.65 ± 1.7147.59 ± 1.9441.48 ± 1.4241.33 ± 1.12
Aapical leaflet width (mm)20.23 ± 0.7124.18 ± 0.87 **20.08 ± 0.0625.93 ± 0.08 **
Apical leaflet shape index2.66 ± 0.07 **1.99 ± 0.062.08 ± 0.05 **1.62 ± 0.05
Middle leaflet length (mm)61.33 ± 3.2253.02 ± 1.4349.63 ± 1.5446.55 ± 1.5
Middle leaflet width (mm)20.8 ± 0.5125.07 ± 0.63 **21.93 ± 0.8127.63 ± 0.8 **
Middle leaflet shape index2.94 ± 0.14 **2.14 ± 0.062.28 ± 0.04 **1.7 ± 0.04
Annual branch length (cm)————32.93 ± 3.56 **23.02 ± 2.64
Diameter of middle petiole (mm)————1.52 ± 0.032.03 ± 0.05 **
Percentage of sunburn————44.08 ± 3.2553.24 ± 2.57 **
Note: Data presented mean ± standard error (SE); ** represents significance reaching the level of p < 0.01.
Table 7. Correlation analysis of plant polyploidy and morphological characters of Sorbus pohuashanensis.
Table 7. Correlation analysis of plant polyploidy and morphological characters of Sorbus pohuashanensis.
PloidyPHBDABLCLLCPLCLWDBLALLALWALSIMLLMLWMLSIDMPPS
Ploidy
PH−0.412 **
BD0.413 **0.178
ABL−0.329 *0.709 **0.083
CLL0.0320.549 **0.337 *0.489 **
CPL−0.1410.549 **0.1730.639 **0.843 **
CLW−0.1530.593 **0.301 *0.2860.665 **0.428 **
DBL0.1060.416 **0.449 **0.456 **0.870 **0.708 **0.601 **
ALL−0.0130.339 *0.373 *0.303 *0.614 **0.500 **0.545 **0.601 **
ALW0.643 **−0.0040.587 **−0.0180.349 *0.1390.2700.487 **0.496 **
ALSI−0.729 **0.303 *−0.321 *0.312 *0.1150.2540.141−0.0390.241−0.709 **
MLL−0.2110.574 **0.295 *0.2550.643 **0.392 **0.944 **0.643 **0.555 **0.2800.118
MLW0.605 **0.0960.560 **−0.0830.455 **0.1660.525 **0.553 **0.417 **0.869 **−0.654 **0.527 **
MLSI−0.865 **0.404 **−0.351 *0.358 *0.0560.1540.209−0.0500.015−0.728 **0.849 **0.251−0.679 **
DMP0.777 **−0.2310.420 **−0.2260.210−0.0950.2300.311 *0.1630.735 **−0.677 **0.1810.757 **−0.687 **
PS0.323 *−0.0790.011−0.015−0.059−0.155−0.144−0.103−0.1460.162−0.284−0.1830.176−0.337 *0.292
Note: * and ** indicate significant difference at p < 0.05 and p < 0.01 levels, respectively.
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Zhang, Z.; Zhang, Y.; Di, Z.; Zhang, R.; Mu, Y.; Sun, T.; Tian, Z.; Lu, Y.; Zheng, J. Tetraploid Induction with Leaf Morphology and Sunburn Variation in Sorbus pohuashanensis (Hance) Hedl. Forests 2023, 14, 1589. https://doi.org/10.3390/f14081589

AMA Style

Zhang Z, Zhang Y, Di Z, Zhang R, Mu Y, Sun T, Tian Z, Lu Y, Zheng J. Tetraploid Induction with Leaf Morphology and Sunburn Variation in Sorbus pohuashanensis (Hance) Hedl. Forests. 2023; 14(8):1589. https://doi.org/10.3390/f14081589

Chicago/Turabian Style

Zhang, Zeren, Yan Zhang, Zexin Di, Ruili Zhang, Yanjuan Mu, Tao Sun, Zhihui Tian, Yizeng Lu, and Jian Zheng. 2023. "Tetraploid Induction with Leaf Morphology and Sunburn Variation in Sorbus pohuashanensis (Hance) Hedl" Forests 14, no. 8: 1589. https://doi.org/10.3390/f14081589

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