Analysis of changes in morphological characters and drought resistance of tetraploid P. alba

Analysis of changes in morphological characters and drought resistance of tetraploid P. alba | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Analysis of changes in morphological characters and drought resistance of tetraploid P. alba liu yuanfu, wang xinyu, li siyuan, zhou yan, he ruihan, su chen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5751567/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Jul, 2025 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted 4 You are reading this latest preprint version Abstract Artificial induction of polyploids is an effective technique for plant breeding and genetic improvement. Understanding the changes in plant morphology after polyploidization is the key to studying the underlying physiological mechanisms of polyploid plant development. We obtained a tetraploid P. alba using colchicine induction and performed a characterization analysis on it. The results showed that the height and leaf area of the tetraploid plant were smaller than those of the diploid plant. The tetraploid plant have thicker leaves, higher chlorophyll contents, and larger but less dense stomata. Tetraploidization also resulted in significant changes in stem anatomy, including smaller xylem width and larger phloem width. In addition, we found that the tetraploid plants exhibited enhanced drought tolerance compared with the diploid parent. The results of our study not only revealed the structural and physiological changes in the tetraploid plants, but also provided valuable insights into the breeding of polyploid P. alba . P. alba tetraploid morphometric analysis drought tolerance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Key message In this study, the tetraploid P. alba was artificially induced, and its growth and physiological characteristics, as well as its drought resistance were analyzed. 1. Introduction Polyploidy denotes organisms possessing three or more complete sets of chromosomes within their cells (Van De Peer et al. 2021 ). It is a consequence of whole genome duplication (WGD). Polyploidization is one of the important ways of plant evolution. The induction of artificial polyploids is a common means of plant species formation and helps plants adapt to environmental cues (Domínguez-Delgado et al., 2021 ; Van De Peer et al., 2017 ). A common effect of WGD observed in polyploids is the alteration of morphological characteristics (Shin et al. 2017 ). For instance, polyploid Cypress Spurge (tetraploids and hexaploids) have significantly thicker stems compared to diploids. The tetraploids have more axillary vegetative shoots, and the hexaploids have wider and longer axillary vegetative shoots (Pungaršek and Frajman 2024 ). In rice, an increase in stomata size and a decrease in stomatal density are observed following polyploidization, affecting how they respond to light (Xiong 2022 ). Tetraploid muskmelon have significantly larger leaves and flowers, increased fruit weight, and increased soluble solids, soluble sugars, and vitamin C content in the fruit, and show better agronomic characteristics compared to diploid melons (Zhang et al. 2010 ). Drought is one of the most important abiotic stresses on plants induced by climate change, which greatly affects the morphological, biochemical and physiological levels of plants and reduces their yield and quality (Cohen et al. 2021 ). Numerous studies have shown that physiological characteristics are altered after WGD, such as growth rate, secondary metabolite production, and photosynthetic pigment content, resulting in polyploids having a greater tolerance to stress than diploids (Münzbergová and Haisel 2019 ; Wang et al. 2021 ; Castro et al. 2023 ). The leaves of tetraploid sour jujubes exhibit a denser cuticle layer and increased accumulation of leaf wax compared to their diploid counterparts. This enhanced cuticle and wax deposition can effectively decrease the permeability of the cuticle, thereby strengthening the leaves' resistance to drought conditions. (Li et al. 2024a ). Facing drought conditions, the triploid P. edulis demonstrated enhanced photosynthetic activity and increased chlorophyll fluorescence. Additionally, these plants maintained higher levels of soluble sugars, proteins, and proline, which are crucial for modulating osmotic balance within plant cells. (Su et al. 2024b ). Under drought conditions, a multitude of genes associated with photosynthetic processes exhibited altered expression patterns in tetraploid Citrus wilsonii. This genetic response aligns with the observed variations in photosynthetic parameters such as P n , gs, T r , C i and chlorophyll content in diploids and tetraploids, which could reflect that tetraploid Citrus wilsonii sustains a more robust photosynthetic capacity compared to diploids, potentially contributing to their improved drought tolerance. (Jiang et al. 2022 ). P. alba is one of the foundation species that make up the forest communities of the northern hemisphere and is widely distributed in Central Asia and Europe. It is characterized by rapid growth and can tolerate a variety of environmental stresses, such as drought, wind, salt and low temperatures(Hou et al. 2020 ). Polyploid breeding is now recognized as a significant approach for the generation of new poplar cultivars. (Kang and Wei 2022 ). In this study, we used colchicine to induce tetraploid P. alba artificially. Ploidy was detected by flow cytometry and fluorescence in situ hybridization (2n = 4x = 76). Diploid and tetraploid plants showed significant differences in growth characteristics, microstructure and photosynthetic properties. Further analysis showed that the tetraploid P. alba had stronger tolerance to drought stress. The results of this study can enrich the germplasm resources of poplar, and also provide a theoretical basis for poplar polyploid breeding and artificial polyploidization of forest trees. 2. Materials and methods 2.1 Plant material An elite diploid P. alba individual was collected from Xinjiang province. Leaves from the diploid poplar were sterilized and transferred to Murashige–Skoog (MS) medium (containing 0.5 mg/L 6-BA, 0.1 mg/L NAA, 25 g/L sucrose, and 6.5 g/L agar) for dedifferentiation. The vegetatively propagated plants were used for the subsequent tetraploid poplar induction. 2.2 Induction of tetraploid P. alba Young leaves of 1-month-old diploid plants were used as explants. The leaves were precultured on differentiation medium for 7 days, then transferred to medium containing 50 mg/L-100 mg/L colchicine for 2–4 days, and finally transferred to medium without colchicine. The induced 1-month-old plants were screened for morphology, and those that showed differences in morphology compared with diploid plants were screened as candidate tetraploids. 2.3 Plants growth conditions The plants were planted in a soil mixture (peat: vermiculite = 4:1), placed in a greenhouse and watered normally. Growing conditions in the greenhouse were 22–25°C, 1000–1500 lux, and 16/8 hours of light/dark. 2.4 Flow CytoMetry (FCM) Candidate polyploid plants with thick and short stems and dark leaf color were selected for the ploidy test and untreated diploid plants were used as control. The tips of young leaves were taken, and then 0.4 mL of Sysmex CyStain UV Precise P Nuclei Extraction Buffer was added and cut with a knife for about 1 min until chopped, and 1.6 mL of Sysmex CyStain UV Precise P DAPI staining solution was added to the chopped samples. Filter through a 30 µm Cell-Trics membrane and let stand for 1 min. Detection of samples using flow cytometry (CyFlow Cube, Germany), measuring at least 10,000 nuclei per sample. Samples were analyzed for peak values, coefficient of variation (CV) and relative ploidy index using Novo Express software. 2.5 Fluorescence in situ Hybridization (FISH) The FISH procedures were followed as described previously by Liu et al. ( 2021 ). The P. alba telomere repeat probe, (TTTAGGG)10, was used as FISH probes, root tip samples of 1 cm from both diploid and potential tetraploid plants were collected for chromosome analysis and slide preparation. The chromosome preparation involved denaturing in 0.2 M NaOH and 70% ethanol for 10 minutes, followed by a rinse in cold 70% ethanol at -20°C for an hour, and then air-drying. For each slide, the hybridization mixture (10 µL total volume) comprised 200 ng of labeled probe DNA, a 50% v/v formamide solution, 2 × SSC, 10% w/v dextran sulfate, and 0.2 µg of salmon sperm DNA. This mixture, along with the probes, was denatured in boiling water for 5 minutes and then plunged into ice water for 5 minutes. The hybridization process was carried out at 37°C overnight, with the experiment being independently repeated three times. 2.6 Morphological observation The diploid and tetraploid plants growing under the same conditions were selected, and the plant height and ground diameter of diploid and tetraploid plants were measured every 15 days for three consecutive months. Plant height and ground diameter were measured using a tape measure and an electronic vernier caliper, respectively. Leaf length, width, and area were measured for two-month-old leaves using Image J 1.46r. Root length and root thickness of three-month-old plants were measured using tape measure and vernier caliper. For the above growth parameters, 20 plants of each ploidy were measured as one biological replicate. 2.7 Histological and microscopic examination The diploid and tetraploid leaf longitudinal cross sections and stem transverse cross sections were prepared as described in Zhu et al. ( 2017 ). The samples were fixed in formaldehyde-acetic acid-ethanol solution for 24 h, then dehydrated in a gradient ethanol series and embedded in paraffin. The samples were sliced to a thickness of 10 µm using a rotary slicer (Leica RM 2245), and the slices were stained with senna and solid green. Micrographs were processed using scanner M8 (Precipoint) and ViewPoint (v.1.0.0.0, Precipoint, Freising, Germany) setup software. Thicknesses of the upper epidermis, lower epidermis, palisade tissue, spongy tissue, and leaf blades, as well as widths of xylem and phloem were calculated using Image J software. Stomatal density was determined by counting the number of stomata on the leaf subepidermis by examining a total of 20 fields of view using a scanning electron microscope (SEM). Stomatal length, stomatal width, guard cell length and width were calculated using Image J software. 2.8 Measurement of photosynthetic pigment contents and fluorescence kinetic parameters Leaf chlorophyll content was determined with reference to Li and Yi ( 2020 ). 0.1 g of plant leaves were ground with anhydrous acetone and then centrifuged to collect the supernatant and the absorbance was measured at 663 and 645 nm. Fluorescence kinetic parameters were measured with a portable fluorometer (PAM-2500, Walz), and plants were acclimatized in the dark for 30 min prior to the assay and then measured according to the manufacturer's instructions. 2.9 Determination of physiological indicators Proline (Pro), malondialdehyde (MDA), and soluble sugars (SS) content, peroxidase (POD) activity, superoxide dismutase (SOD) activity, and catalase (CAT) activity were determined using kits (Micro method; Grace Biotechnology, Su zhou, China). 3. Results 3.1 Generation of a tetraploid P. alba line Colchicine is a widely used mutagen that prevents microtubule formation in cells and doubles the number of chromosomes (Wu et al. 2023). After 2 days of colchicine treatment, the leaves of diploid poplar were cultured for 3 weeks on differentiation medium. Potential tetraploid shoots of P. alba were screened based on the morphological characteristics including thicker and greener leaves (Fig. 1 A). These candidate adventitious shoots were then transferred to 1/2 MS medium applied by 0.5 mg/L IBA 25 g/L sucrose and 6.5 g/L agar for rooting (Fig. 1 B-C). The ploidy levels of the selected plants were investigated by flow cytometry (FCM). The results showed that the diploid plant showed a peak at a relative fluorescence intensity value of 49. In contrast, the candidate tetraploid plants showed a peak at 93, which was nearly twice as high as the diploid plants (Fig. 1 D-F). To further confirm the ploidy level of the tetraploid plants, fluorescence in situ hybridization (FISH) was performed using a chromosome telomere probe. The results showed that the diploid and tetraploid plants contained 38 and 76 chromosomes, respectively (Fig. 1 G-H). Both the FCM and the FISH results confirmed that the tetraploid poplar was successfully induced by colchicine. 3.2 Growth differences between the diploid and tetraploid P. alba plants It is reported that polyploidization always alters the morphological characteristics of plants (Otto and Whitton 2000 ). We compared the growth of the diploid and tetraploid plants grown in greenhouse for 90 days to investigate their phenotypic characteristics. We found that the heights of the tetraploid plants were significantly lower than the diploid plants (Fig. 2 A). The height of the diploid and tetraploid plants at the 30th day was 18.30 and 10.88 cm, respectively. At the 60th day, the height of the diploid plants was 39.45 cm and that of the tetraploid plants was 24.43 cm. While at the 90th day, the height of the diploid and tetraploid plants reached 60.57 and 37.9 cm, respectively (Fig. 2 C). Overall, the height of the tetraploid plants was 37.43% lower than the diploid. However, during the 90 days of growth, the ground diameters of the tetraploid plants were similar to those of the diploid plants. At the 30th day, the ground diameter of the diploid and tetraploid plants was 2.21 and 2.39 mm, respectively. At the 60th day, the ground diameter of the diploid and tetraploid plants was 3.99 and 3.89 mm, respectively. At the 90th day, the ground diameter of the diploid and tetraploid plants reached 5.01 and 5.43 mm, respectively (Fig. 2 D). The above results indicated that polyploidization inhibits the growth of the P. alba plants, but may slightly promote the growth of ground diameter. We measured leaf characteristics of the diploid and tetraploid plants grown for 60 days (Fig. 2 B). The results showed that leaf length, leaf width and leaf area of the tetraploid plants were significantly smaller than those of the diploid plants ( P < 0.01). The leaf length and width of the tetraploid plants was 8.05 cm and 9.09 cm, while that of the diploid was 9.75 cm and 10.99 cm. The leaf area of the tetraploid plants was 47.16 cm 2 , which was 64.54 cm 2 in the diploid plants (Table 1 ).. Leaf index is the ratio of leaf length to width, which can reflect the growth condition and growth pattern of plant leaves (Wang et al. 2017), and there was no difference in leaf shape index between the two ploidy plants ( P > 0.05). The diploid and tetraploid P. alba also showed differences in root morphology. The roots of the diploid plants were significantly longer than the tetraploid plants ( P < 0.05, Fig. 2 E). The root length of the diploid plants was 28.02 cm, while that of the tetraploid plants was 19.61 cm (Fig. 2 F). While the root thickness in the two types of the plants were similar ( P > 0.05, Fig. 2 G). The root thickness of the diploid plants was 2.12 mm, and that of the tetraploid plants was 2.21 mm. Taken together, the results showed that the morphology of tetraploid P. alba was significant changed after chromosome doubling. Table 1 Comparison of morphological characteristics of diploid and tetraploid leaves Characteristics Diploid Tetraploid P value Leaf length (cm) 9.75 ± 0.66 8.0491 ± 0.60 0.000 Leaf width (cm) 10.99 ± 1.44 9.09 ± 1.10 0.006 Leaf area (cm 2 ) 64.54 ± 9.22 47.16 ± 4.01 0.000 Leaf shape index 0.90 ± 0.12 0.89 ± 0.08 0.867 3.3 Microscopic comparisons of the diploid and tetraploid P. alba plants We observed the leaf microstructures of the diploid and tetraploid plants (Fig. 3 A-B). We found that the leaves of the tetraploid plants were thicker than the diploids. The epidermal cells of the tetraploid plants were more neatly and closely arranged relative to the diploids. The leaf thickness of the tetraploid plants was 1.17 times of the diploid plants. But there was no significant difference in the thickness of upper and lower epidermal cells between the two ploidies of P. alba ( P > 0.05). The spongy tissue was 1.62 times thicker in the tetraploids than in the diploids. On the contrary, the palisade tissue was 1.17 times thicker in the diploids than in the tetraploids (Table 2 ). The results indicated that the thicken leaves of the tetraploid plants may be the consequence of enhanced spongy tissue. We found that the stoma size of the tetraploids was larger the diploids (Fig. 3 C-F). The stomata of the tetraploids were 1.53 times longer and 1.07 times wider than those of the diploids. In contrast, the stomatal density of the diploids was 1.72 times higher than that of the tetraploids. Moreover, the guard cells of the tetraploids were 1.35 times longer and 1.02 times wider than those of the diploids (Table 2 ). The results indicated that polyploidization has an effect on the stomatal characteristics of P. alba , with the tetraploid plants having larger stomata and guard cells and reduced stomatal density compared to the diploid plants, and this alteration may improve the plant's water utilization efficiency (Yao et al. 2023 ). By observing the anatomy of the stems of the two ploidies plants, we found that polyploidization also causes structural changes in the stems of P. alba . Compared with the stems of the diploid plants, the stems of tetraploid plants became thicker, in which the width of the xylem was reduced and the width of the phloem was increased (Fig. 3 G-H). The phloem width of the tetraploid plants was 0.30 mm, which was 1.76 times of the diploid plants, whose phloem was 0.17 mm in width. The ratio of phloem width to stem radius was 0.17 in the tetraploid plants and 0.08 in the diploid plants. In contrast, the xylem width of the diploid plants was 0.91 mm, which was 2.12 times of the tetraploid plants, whose xylem width was 0.43 mm. The ratio of xylem width to stem radius was 0.46 in the diploid plants and 0.24 in the tetraploid plants (Table 3 ). Table 2 Comparison of microstructural characteristics of diploid and tetraploid leaves Characteristics Diploid Tetraploid P value Thickness of upper epidermis (µm) 13.50 ± 2.01 12.39 ± 1.47 0.199 Thickness of lower epidermis (µm) 4.84 ± 0.74 5.09 ± 0.61 0.448 Thickness of palisade tissue (µm) 27.12 ± 2.29 23.20 ± 1.39 0.000 Thickness of spongy tissue (µm) 29.00 ± 1.80 46.99 ± 2.41 0.000 The ratio of palisade tissue to spongy tissue 0.94 ± 0.065 0.50 ± 0.04 0.000 Thickness of leaf (µm) 78.49 ± 2.05 92.16 ± 3.74 0.000 Stomatal length(µm) 9.91 ± 1.3 15.13 ± 0.82 0.000 Stomatal width (µm) 3.27 ± 0.23 3.50 ± 0.50 0.234 Stomatal density (Number per mm 2 ) 268.17 ± 12.92 156.01 ± 12.41 0.000 Guard cell length (µm) 16.04 ± 0.65 21.63 ± 2.96 0.000 Guard cell width(µm) 13.56 ± 0.54 13.95 ± 1.74 0.533 Table 3 Comparison of anatomical and structural characteristics of diploid and tetraploid stems Characteristics Diploid Tetraploid P value Xylem width(mm) 0.91 ± 0.05 0.43 ± 0.05 0.000 Phloem width(mm) 0.17 ± 0.03 0.30 ± 0.04 0.000 Xylem width/Stem radius 0.46 ± 0.03 0.24 ± 0.03 0.000 Phloem width/Stem radius 0.08 ± 0.01 0.17 ± 0.02 0.000 3.4 Photosynthetic comparisons of the diploid and tetraploid P. alba plants Chlorophyll helps plants to absorb light energy, thus directly affecting the strength of photosynthetic capacity (Wang and Grimm 2021 ). We found that the chlorophyll a , b and total chlorophyll contents in the tetraploid poplar were all higher than in the diploid poplar. The diploid poplar contained 1.05 mg/g chlorophyll a and 0.33 mg/g chlorophyll b . While the tetraploid poplar contained 1.24 mg/g chlorophyll a and 0.39 mg/g chlorophyll b . The total chlorophyll contents were 1.38 mg/g in the diploid plants and 1.64 mg/g in the tetraploid plants (Table 4 ). Enhanced photosynthesis promotes plant growth, thereby augmenting plant resistance (Shen et al. 2024 ). By comparing the fluorescence kinetic parameters of the two ploidies of P. alba , we found that except for the maximal efficiency of PSII photochemistry (Fv/Fm) which was no significant difference between the two ploidies plants ( P > 0.01 ), the photochemical quenching coefficient (qP), apparent electron transfer rate (ETR) and the actual photosynthetic efficiency of PSII (Y(II)), were greater in the tetraploid plants than in diploid plants ( P < 0.01, Table 4 ). These results suggest that the tetraploid plant possesses a superior photosynthetic capacity than the diploid plant. Table 4 Comparison of photosynthetic parameters in diploid and tetraploid plants Photosynthetic parameters Diploid Tetraploid P value Chlorophyll a (mg/g) 1.05 ± 0.07 1.24 ± 0.13 0.001 Chlorophyll b (mg/g) 0.33 ± 0.03 0.39 ± 0.05 0.009 Total chlorophyll(mg/g) 1.38 ± 0.10 1.64 ± 0.18 0.002 Fv/Fm 0.72 ± 0.003 0.73 ± 0.008 0.012 ETR 28.06 ± 0.19 28.32 ± 0.14 0.004 qP 0.72 ± 0.005 0.73 ± 0.004 0.005 Y(II) 0.87 ± 0.003 0.88 ± 0.004 0.000 3.5 The tetraploid plants exhibited enhanced drought tolerance Polyploidization can increase plant tolerance to abiotic stresses. Especially under water deficit conditions, drought tolerance of polyploid plants can be improved by altering the transpiration, photosynthetic rate, water utilization and morphology of plants (Maherali et al. 2009 ; Deng et al. 2012 ; Yang et al. 2014 ). Simulated drought stress was applied to the 60th days plants of the two ploidies by stopping watering for 9 days. We found that under drought treatment, the leaves of diploid plants showed more wilting and the degree of drought damage to diploid plants were more severe compared to tetraploid plants. (Fig. 4 A). With increasing drought duration, leaf water content decreased from 80.66–68.13% in the diploid plants and from 82.32–74.48% in the tetraploid plants (Fig. 4 B). Stem water content decreased from 78.97–64.06% in the diploid plants and from 80.80–74.93% in the tetraploid plants (Fig. 4 C). Leaves and stems of the tetraploid lost less water under drought conditions compared to the diploid. Malondialdehyde (MDA), a marker for membrane lipid peroxidation, indicates the extent of oxidative stress inflicted on cellular membranes. The level of MDA is often employed to measure how plants cope with various stressors. (Luo et al. 2023 ). After drought stress, the MDA content in the leaves of both plants showed an increasing trend with the drought duration. At the 9th day of drought stress, the MDA content in the leaves of diploid and tetraploid plants was 40.48 nmoL/g and 29.81 nmoL/g, respectively (Fig. 5 A). Compared with the diploid, the tetraploid suffered less damage to leaf membranes under drought stress and had stronger antioxidant capacity. Osmoregulatory substances such as proline (Pro) and soluble sugars (SS) can reduce the water potential of plant cells under drought conditions, thereby preventing cell dehydration to improve plant drought resistance (Ozturk et al. 2021 ; Huang et al. 2022b ). During drought stress, the Pro content and SS content of the tetraploid and diploid leaves gradually increased. At the 9th day of drought stress, the Pro content of diploid and tetraploid leaves was 62.87 µg/g and 122.03 µg/g, and the soluble sugar content was 36.46 mg/g and 46.67 mg/g (Fig. 5 B-C). The Pro content and SS content in the tetraploid leaves was 1.94 and 1.28 times higher than that of the diploid leaves, respectively. These results suggest that the tetraploid plants exhibit stronger osmotic adjustment and are more resistant to drought conditions than the diploid when subjected to drought stress. Peroxidase (POD), catalase (CAT) and superoxide dismutase (SOD) are important protective enzymes in plants and also reflect the ability of plants to scavenge reactive oxygen species (Li et al. 2024b ; Jin et al.). After 9 days of drought stress, all the enzyme activities in the tetraploid plant were higher than the diploid plant. The POD activity of the tetraploid leaves was essentially unchanged from 0–6 days, and increased significantly after the 6th day. The POD activity of the diploid leaves showed an increasing trend during drought. At the 9th day of drought, the POD activity in the diploid was 45.58 U/g and the POD activity in the tetraploid was 56.25 U/g, which was 1.37 times higher than that of the diploid (Fig. 5 D). The CAT activity in the leaves of different ploidies showed an increasing trend. At the 9th days of drought, the CAT activity in the diploid was 49.70 U/g and in the tetraploid was 72.46 U/g, which was 1.46 times higher than that of the diploid (Fig. 5 E). The activity of SOD showed a trend of increasing and then decreasing with the increase of drought stress time, and at the 9th day of drought stress, the SOD activity in the diploid leaves was 1113.32 U/g and in the tetraploid leaves was 2072.50 U/g, which was 1.86 times higher than that of the diploid (Fig. 5 F). Taken together, the results indicate that the tetraploid P. alba is more tolerant to drought than the diploid. 4. Discussion Compared to diploids, polyploids usually change the morphological characteristics of plants(Haist et al. 2023 ; Pungaršek and Frajman 2024 ; Su et al. 2024a ). In this study, we observed the phenotypic characteristics of the diploid and tetraploid P. alba. Plant height was significantly reduced in the tetraploid as compared to the diploid, which was similar to tetraploids of birch, apple, willow and Sorbus pohuashanensis (Mu et al. 2012 ; Dudits et al. 2016 ; Xue 2017 ; Zhang et al. 2023 ). Previous studies had found that among the differentially expressed genes in diploids and tetraploids, most of them are genes related to phytohormone signaling (Ren et al. 2022 ; Wu et al. 2024 ). Plant growth and development are regulated by endogenous hormones, and dwarfing of plants after polyploidization is associated with reduced levels of growth-related hormones. In poplar tetraploids, the expression of genes related to the regulation of indoleacetic acid (IAA), gibberellin (GA), and brassinosteroid (BR) synthesis was down-regulated with increasing leaf age (Xu et al. 2020 ). In addition to height, the morphology of the leaf also changed. We observed a smaller leaf area and deeper leaf color in the tetraploid leaves compared to the diploid leaves. Previous studies have suggested that leaf size increases with increasing ploidy, but this relationship is not linear (Kondorosi et al. 2000 ). For example, the triploid leaf size of atemoyas is larger than the diploid, but the leaf size of the tetraploid is smaller than the diploid (Losada et al. 2023 ). Regardless of leaf size, the color of leaves deepens after polyploidization, such as Rosa roxburghii (Wu et al. 2023), Populus (Zhang et al. 2020 ), Ziziphus jujuba Mill. (Cui et al. 2017 ) and Passiflora edulis Sims. Our observations indicated that the tetraploid plants had shorter root lengths compared to their diploid counterparts, a finding that aligns with earlier researc (Shao et al. 2003 ; Tavan et al. 2015 ). It may be the result of transcription factors and phytohormone regulation (Ren et al. 2021 ). Further research is needed to elucidate the mechanisms underlying the phenotypic differences in tetraploid P. alba . By observing the anatomy of the leaves of diploid and tetraploid P. alba , we found that the tetraploid leaves were thickened, palisade tissues were thinner and spongy tissues were thicker compared to the diploids. The tetraploid Lilium leaves exhibited a marked increase in the thickness of both the upper and lower epidermis and spongy tissues compared to diploid leaves. However, the thickness of the palisade tissues remained unchanged between the two ploidy levels, aligning with the outcomes of this investigation (Cao et al. 2018 ). In contrast, the palisade and spongy tissues of the leaves of tetraploid B. papyrifera (Lin et al. 2023 ) and Dendrobium cariniferum (Zhang and Gao 2021 ) were significantly thicker than those of the diploid leaves, suggesting that the increased thickness of palisade and spongy tissues in polyploidized plants is not absolutely. It may be influenced by individual genomic differences across species (Zhang et al. 2022 ). Stomatal length and width are commonly used as morphological markers for identifying potentially polyploid plants (Huang et al. 2022a ; Khan et al. 2023 ; Maisha et al. 2023 ). The increase in ploidy level is positively correlated with the size of plant stomata and negatively correlated with stomatal density (Widoretno 2016 ). This is consistent with our findings in tetraploid P. alba plants where we observed significantly longer and wider stomata and reduced stomatal density compared to diploids. Greener leaves and a greater number of stomata enhance photosynthesis, thus indirectly increasing plant tolerance to abiotic stresses. The anatomical structures of the two ploidy P. alba stems were also significantly different, with the tetraploid having a narrower width of xylem and a wider width of phloem, which differed from the results of previous studies. The number of secondary xylem cells is the same in diploid and tetraploid poplars , due to the fact that the size of xylem fiber cells in tetraploids is significantly larger than in diploids, resulting in wider xylem (Iwakawa et al. 2022 ). In tetraploid Citrus limonia, cortical cell area, xylem cell area, epidermal cell area, and cuticle thickness were significantly larger than in diploids (Allario et al. 2011 ). The reasons for the structural changes in the stem of tetraploid silver poplar need to be thoroughly investigated by subsequent experiments. Increased genetic diversity makes polyploids better adapted to extreme environments (Arrigo and Barker 2012 ). In our study, tetraploid P. alba had lower MDA content, higher water content, SS and Pro content, and higher SOD, POD, and CAT enzyme activities than diploids after drought stress.These results suggest that tetraploid P. alba possess enhanced tolerance to drought. Many studies in recent years have found tetraploid plants to be more resistant to unfavorable external environments, although many studies have been initiated to examine and characterize polyploid stress tolerance (Li et al. 2021 ; Jiang et al. 2022 ; Fakhrzad and Jowkar 2024 ). Nevertheless, a comprehensive understanding of the specific molecular pathways that confer their improved stress resistance remains scarce. This study's findings enhance our comprehension of the physiological and molecular factors that contribute to the improved stress resilience in polyploid P. alba . 5. Conclusion Through the induction of colchicine, tetraploid P. alba were obtained. Compared to diploid plants, significant changes were observed in the growth parameters, physiological structures, and photosynthetic parameters of tetraploid plants. Specifically, the tetraploid plants exhibited decreased height, reduced leaf area, darkened leaf color, increased leaf thickness, enlarged stomatal length and width and decreased stomatal density. The xylem of the stems narrowed and the phloem of the stems widened. Furthermore, we found that the tetraploid plants may exhibit stronger resistance to adverse external environments. Under drought stress, the tetraploid plants had increased contents of Pro and SS, higher activities of SOD, POD, and CAT, and lower MDA content compared to the diploid plants. The findings of this study can enrich the germplasm resources of poplar and provide a theoretical basis for polyploid breeding in tree species. Declarations Credit authorship contribution statement Yuanfu Liu: Writing original draft, Visualization, Validation, and Formal analysis. Xinyu Wang: Data curation, Experimental material arrangement. Siyuan Li: Identification and propagation of the plant materials. Yan Zhou and Ruihan He: Paraffin sections, physiological indices and photosynthetic parameters were determined. Su Chen: Conceptualization, Supervision, Writing – review & editing, Resources, Funding acquisition. Acknowledgments This work was supported by the National Key R&D Program of China (2021YFD2200800) and the Fundamental Research Funds for the Central Universities (2572022CG06). Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data Availability Data will be made available on request. References Allario T, Brumos J, Colmenero-Flores JM, et al (2011) Large changes in anatomy and physiology between diploid Rangpur lime (Citrus limonia) and its autotetraploid are not associated with large changes in leaf gene expression. 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Forests 14:1589. https://doi.org/10.3390/f14081589 Zhu Y, Song D, Xu P, et al (2017) A HD‐ZIP III gene, PtrHB4, is required for interfascicular cambium development in Populus Cite Share Download PDF Status: Published Journal Publication published 22 Jul, 2025 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted Reviewers agreed at journal 07 Jan, 2025 Reviewers invited by journal 07 Jan, 2025 Editor assigned by journal 02 Jan, 2025 First submitted to journal 02 Jan, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5751567","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":398785172,"identity":"8009bf49-eed9-4edc-8880-53210184bf4d","order_by":0,"name":"liu yuanfu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtElEQVRIiWNgGAWjYBACPgbGBx8qbOT4+dkbGx9+IEYLGwOz4YwzacaSM3sONxtLkKRlw4z0NgEeorRIJDM2HEgwkDCQfNjGIMFgJ6fbQKwWc+nEtgcFDMnGZgcIask//vjjjz91lrMT2w0kGA4kbiOsBeawmwfbJHhI03KDkVgtPI8hWiR7EoGBbECEX/jZobbwsx9/+PBDhZ0cQS0MAgnIPANCysHWEDR0FIyCUTAKRjwAADpfQwEhROUWAAAAAElFTkSuQmCC","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":true,"prefix":"","firstName":"liu","middleName":"","lastName":"yuanfu","suffix":""},{"id":398785173,"identity":"1dfd61c1-1876-480c-bb83-ac7a3a2dc5c8","order_by":1,"name":"wang xinyu","email":"","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"wang","middleName":"","lastName":"xinyu","suffix":""},{"id":398785174,"identity":"c58d27f0-e426-4df7-8b39-adf8ba398545","order_by":2,"name":"li siyuan","email":"","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"li","middleName":"","lastName":"siyuan","suffix":""},{"id":398785175,"identity":"b5ff551c-b6fe-4660-ac91-59443c9b89ff","order_by":3,"name":"zhou yan","email":"","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"zhou","middleName":"","lastName":"yan","suffix":""},{"id":398785176,"identity":"6ac5f326-3de9-4b3d-8068-d718cd28cc0d","order_by":4,"name":"he ruihan","email":"","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"he","middleName":"","lastName":"ruihan","suffix":""},{"id":398785177,"identity":"ad094b70-a124-4523-a3a4-c17c65717f8d","order_by":5,"name":"su chen","email":"","orcid":"https://orcid.org/0000-0002-8814-5444","institution":"Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"su","middleName":"","lastName":"chen","suffix":""}],"badges":[],"createdAt":"2025-01-02 11:55:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5751567/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5751567/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11240-025-03133-z","type":"published","date":"2025-07-22T15:57:12+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":73357091,"identity":"ef0b905a-d8ef-4248-9837-1a3d0a0ae802","added_by":"auto","created_at":"2025-01-09 08:10:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":13701833,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlow cytometry (FCM) and fluorescence in situ hybridization (FISH) of diploid and tetraploid\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e P. alba\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e (A-C) Comparison of diploid and suspected tetraploid poplar morphology, with the diploid on the left and the suspected tetraploid on the right.(A)\u003c/strong\u003e \u003cstrong\u003eCluster of buds.(B)\u003c/strong\u003e \u003cstrong\u003e7 d rooted seedlings(C)\u003c/strong\u003e \u003cstrong\u003e30 d rooted seedlings. FCM of the nuclear DNA content in (D) diploid and (E) tetraploid (F) Diploid and tetraploid leaves samples of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePopulus alba\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e L. Chromosomes prepared from (G) diploid (2n = 2× = 38) and (H) tetraploid (2n = 4× = 76) samples of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. alba\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e FISH using the telomeric repeat probe (green). Bar = 5 μm.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5751567/v1/b0b6cea8e42592acc5b62c20.png"},{"id":73357056,"identity":"3a674286-1283-4b96-9585-96af1f223f37","added_by":"auto","created_at":"2025-01-09 08:10:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":11864082,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological comparison between tetraploid and diploid \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. alba\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. (A) Plants at 90 days old. (B) Leaves of 60 days old plants. (C) Plant height. (D) Ground diameter. (E) Roots of the 90th day old plants. (F) Root length (G) Root thickness. Error bars indicate SE.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5751567/v1/32d34c3fa99d50f5a6218beb.png"},{"id":73357059,"identity":"c16e9a01-905e-4e39-8108-d2e4a8568777","added_by":"auto","created_at":"2025-01-09 08:10:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":42312392,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicroscopic comparison between tetraploid and diploid \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. alba\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. Microscopic observation using cross sections of diploid (A) and tetraploid (B) leaves. Scanning electron microscopic observations of stomata in diploid (C) and tetraploid (D) leaves at 500x and in diploid (E) and tetraploid (F) leaves at 1000x. Microscopic observation of cross sections of diploid (G) and tetraploid (H) stems. Three independent experiments were performed, and each showed similar results with a representative picture shown.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5751567/v1/9cb6d0eec92f3ebcb5891813.png"},{"id":73357067,"identity":"380d8f2c-17b4-48d9-8c29-b1ef0e50a278","added_by":"auto","created_at":"2025-01-09 08:10:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":9003072,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological changes in diploid and tetraploid \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. alba\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e under drought stress. (A) Morphological comparison of diploid and tetraploid \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. alba\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e under normal growth conditions and after 9 days of drought treatment. Comparison of (B) stem water content and (C) leaf water content of diploid and tetraploid \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. alba\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e under drought stress. Error bars indicate SE.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5751567/v1/d002212290769d5cbc58b881.png"},{"id":73357058,"identity":"921ef763-86a9-4768-b20e-499c8a7f579f","added_by":"auto","created_at":"2025-01-09 08:10:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1469070,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of physiological indices of diploid and tetraploid \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. alba\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e under drought stress. (A) MDA content (B) Proline content (C) Soluble sugars (D) POD activity (E) CAT activity. (F) SOD activity. Error bars indicate SE.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5751567/v1/c803cc584d5556e130e7a4ec.png"},{"id":87756682,"identity":"fdc26bf5-e9a6-48ec-b63a-648189b07e15","added_by":"auto","created_at":"2025-07-28 16:07:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":107180069,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5751567/v1/9403b3f0-e04e-4108-ae3b-801bc79cb88e.pdf"}],"financialInterests":"","formattedTitle":"Analysis of changes in morphological characters and drought resistance of tetraploid P. alba","fulltext":[{"header":"Key message","content":"\u003cp\u003eIn this study, the tetraploid\u0026nbsp;\u003cem\u003eP. alba\u003c/em\u003e was artificially induced, and its growth and physiological characteristics, as well as its drought resistance were analyzed.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003ePolyploidy denotes organisms possessing three or more complete sets of chromosomes within their cells (Van De Peer et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). It is a consequence of whole genome duplication (WGD). Polyploidization is one of the important ways of plant evolution. The induction of artificial polyploids is a common means of plant species formation and helps plants adapt to environmental cues (Dom\u0026iacute;nguez-Delgado et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Van De Peer et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). A common effect of WGD observed in polyploids is the alteration of morphological characteristics (Shin et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). For instance, polyploid Cypress Spurge (tetraploids and hexaploids) have significantly thicker stems compared to diploids. The tetraploids have more axillary vegetative shoots, and the hexaploids have wider and longer axillary vegetative shoots (Pungaršek and Frajman \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In rice, an increase in stomata size and a decrease in stomatal density are observed following polyploidization, affecting how they respond to light (Xiong \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Tetraploid muskmelon have significantly larger leaves and flowers, increased fruit weight, and increased soluble solids, soluble sugars, and vitamin C content in the fruit, and show better agronomic characteristics compared to diploid melons (Zhang et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDrought is one of the most important abiotic stresses on plants induced by climate change, which greatly affects the morphological, biochemical and physiological levels of plants and reduces their yield and quality (Cohen et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Numerous studies have shown that physiological characteristics are altered after WGD, such as growth rate, secondary metabolite production, and photosynthetic pigment content, resulting in polyploids having a greater tolerance to stress than diploids (M\u0026uuml;nzbergov\u0026aacute; and Haisel \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Castro et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The leaves of tetraploid sour jujubes exhibit a denser cuticle layer and increased accumulation of leaf wax compared to their diploid counterparts. This enhanced cuticle and wax deposition can effectively decrease the permeability of the cuticle, thereby strengthening the leaves' resistance to drought conditions. (Li et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e). Facing drought conditions, the triploid \u003cem\u003eP. edulis\u003c/em\u003e demonstrated enhanced photosynthetic activity and increased chlorophyll fluorescence. Additionally, these plants maintained higher levels of soluble sugars, proteins, and proline, which are crucial for modulating osmotic balance within plant cells. (Su et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e). Under drought conditions, a multitude of genes associated with photosynthetic processes exhibited altered expression patterns in tetraploid Citrus wilsonii. This genetic response aligns with the observed variations in photosynthetic parameters such as P\u003csub\u003en\u003c/sub\u003e, gs, T\u003csub\u003er\u003c/sub\u003e, C\u003csub\u003ei\u003c/sub\u003e and chlorophyll content in diploids and tetraploids, which could reflect that tetraploid Citrus wilsonii sustains a more robust photosynthetic capacity compared to diploids, potentially contributing to their improved drought tolerance. (Jiang et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eP. alba\u003c/em\u003e is one of the foundation species that make up the forest communities of the northern hemisphere and is widely distributed in Central Asia and Europe. It is characterized by rapid growth and can tolerate a variety of environmental stresses, such as drought, wind, salt and low temperatures(Hou et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Polyploid breeding is now recognized as a significant approach for the generation of new poplar cultivars. (Kang and Wei \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In this study, we used colchicine to induce tetraploid \u003cem\u003eP. alba\u003c/em\u003e artificially. Ploidy was detected by flow cytometry and fluorescence \u003cem\u003ein situ\u003c/em\u003e hybridization (2n\u0026thinsp;=\u0026thinsp;4x\u0026thinsp;=\u0026thinsp;76). Diploid and tetraploid plants showed significant differences in growth characteristics, microstructure and photosynthetic properties. Further analysis showed that the tetraploid \u003cem\u003eP. alba\u003c/em\u003e had stronger tolerance to drought stress. The results of this study can enrich the germplasm resources of poplar, and also provide a theoretical basis for poplar polyploid breeding and artificial polyploidization of forest trees.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Plant material\u003c/h2\u003e \u003cp\u003eAn elite diploid \u003cem\u003eP. alba\u003c/em\u003e individual was collected from Xinjiang province. Leaves from the diploid poplar were sterilized and transferred to Murashige\u0026ndash;Skoog (MS) medium (containing 0.5 mg/L 6-BA, 0.1 mg/L NAA, 25 g/L sucrose, and 6.5 g/L agar) for dedifferentiation. The vegetatively propagated plants were used for the subsequent tetraploid poplar induction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Induction of tetraploid \u003cem\u003eP. alba\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eYoung leaves of 1-month-old diploid plants were used as explants. The leaves were precultured on differentiation medium for 7 days, then transferred to medium containing 50 mg/L-100 mg/L colchicine for 2\u0026ndash;4 days, and finally transferred to medium without colchicine. The induced 1-month-old plants were screened for morphology, and those that showed differences in morphology compared with diploid plants were screened as candidate tetraploids.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Plants growth conditions\u003c/h2\u003e \u003cp\u003eThe plants were planted in a soil mixture (peat: vermiculite\u0026thinsp;=\u0026thinsp;4:1), placed in a greenhouse and watered normally. Growing conditions in the greenhouse were 22\u0026ndash;25\u0026deg;C, 1000\u0026ndash;1500 lux, and 16/8 hours of light/dark.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Flow CytoMetry (FCM)\u003c/h2\u003e \u003cp\u003eCandidate polyploid plants with thick and short stems and dark leaf color were selected for the ploidy test and untreated diploid plants were used as control. The tips of young leaves were taken, and then 0.4 mL of Sysmex CyStain UV Precise P Nuclei Extraction Buffer was added and cut with a knife for about 1 min until chopped, and 1.6 mL of Sysmex CyStain UV Precise P DAPI staining solution was added to the chopped samples. Filter through a 30 \u0026micro;m Cell-Trics membrane and let stand for 1 min. Detection of samples using flow cytometry (CyFlow Cube, Germany), measuring at least 10,000 nuclei per sample. Samples were analyzed for peak values, coefficient of variation (CV) and relative ploidy index using Novo Express software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Fluorescence \u003cem\u003ein situ\u003c/em\u003e Hybridization (FISH)\u003c/h2\u003e \u003cp\u003eThe FISH procedures were followed as described previously by Liu et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The \u003cem\u003eP. alba\u003c/em\u003e telomere repeat probe, (TTTAGGG)10, was used as FISH probes, root tip samples of 1 cm from both diploid and potential tetraploid plants were collected for chromosome analysis and slide preparation. The chromosome preparation involved denaturing in 0.2 M NaOH and 70% ethanol for 10 minutes, followed by a rinse in cold 70% ethanol at -20\u0026deg;C for an hour, and then air-drying. For each slide, the hybridization mixture (10 \u0026micro;L total volume) comprised 200 ng of labeled probe DNA, a 50% v/v formamide solution, 2 \u0026times; SSC, 10% w/v dextran sulfate, and 0.2 \u0026micro;g of salmon sperm DNA. This mixture, along with the probes, was denatured in boiling water for 5 minutes and then plunged into ice water for 5 minutes. The hybridization process was carried out at 37\u0026deg;C overnight, with the experiment being independently repeated three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Morphological observation\u003c/h2\u003e \u003cp\u003eThe diploid and tetraploid plants growing under the same conditions were selected, and the plant height and ground diameter of diploid and tetraploid plants were measured every 15 days for three consecutive months. Plant height and ground diameter were measured using a tape measure and an electronic vernier caliper, respectively. Leaf length, width, and area were measured for two-month-old leaves using Image J 1.46r. Root length and root thickness of three-month-old plants were measured using tape measure and vernier caliper. For the above growth parameters, 20 plants of each ploidy were measured as one biological replicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Histological and microscopic examination\u003c/h2\u003e \u003cp\u003eThe diploid and tetraploid leaf longitudinal cross sections and stem transverse cross sections were prepared as described in Zhu et al. (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The samples were fixed in formaldehyde-acetic acid-ethanol solution for 24 h, then dehydrated in a gradient ethanol series and embedded in paraffin. The samples were sliced to a thickness of 10 \u0026micro;m using a rotary slicer (Leica RM 2245), and the slices were stained with senna and solid green. Micrographs were processed using scanner M8 (Precipoint) and ViewPoint (v.1.0.0.0, Precipoint, Freising, Germany) setup software. Thicknesses of the upper epidermis, lower epidermis, palisade tissue, spongy tissue, and leaf blades, as well as widths of xylem and phloem were calculated using Image J software. Stomatal density was determined by counting the number of stomata on the leaf subepidermis by examining a total of 20 fields of view using a scanning electron microscope (SEM). Stomatal length, stomatal width, guard cell length and width were calculated using Image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Measurement of photosynthetic pigment contents and fluorescence kinetic parameters\u003c/h2\u003e \u003cp\u003eLeaf chlorophyll content was determined with reference to Li and Yi (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). 0.1 g of plant leaves were ground with anhydrous acetone and then centrifuged to collect the supernatant and the absorbance was measured at 663 and 645 nm. Fluorescence kinetic parameters were measured with a portable fluorometer (PAM-2500, Walz), and plants were acclimatized in the dark for 30 min prior to the assay and then measured according to the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Determination of physiological indicators\u003c/h2\u003e \u003cp\u003eProline (Pro), malondialdehyde (MDA), and soluble sugars (SS) content, peroxidase (POD) activity, superoxide dismutase (SOD) activity, and catalase (CAT) activity were determined using kits (Micro method; Grace Biotechnology, Su zhou, China).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Generation of a tetraploid \u003cem\u003eP. alba\u003c/em\u003e line\u003c/h2\u003e \u003cp\u003eColchicine is a widely used mutagen that prevents microtubule formation in cells and doubles the number of chromosomes (Wu et al. 2023). After 2 days of colchicine treatment, the leaves of diploid poplar were cultured for 3 weeks on differentiation medium. Potential tetraploid shoots of \u003cem\u003eP. alba\u003c/em\u003e were screened based on the morphological characteristics including thicker and greener leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). These candidate adventitious shoots were then transferred to 1/2 MS medium applied by 0.5 mg/L IBA 25 g/L sucrose and 6.5 g/L agar for rooting (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-C).\u003c/p\u003e \u003cp\u003eThe ploidy levels of the selected plants were investigated by flow cytometry (FCM). The results showed that the diploid plant showed a peak at a relative fluorescence intensity value of 49. In contrast, the candidate tetraploid plants showed a peak at 93, which was nearly twice as high as the diploid plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-F). To further confirm the ploidy level of the tetraploid plants, fluorescence \u003cem\u003ein situ\u003c/em\u003e hybridization (FISH) was performed using a chromosome telomere probe. The results showed that the diploid and tetraploid plants contained 38 and 76 chromosomes, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-H). Both the FCM and the FISH results confirmed that the tetraploid poplar was successfully induced by colchicine.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Growth differences between the diploid and tetraploid \u003cem\u003eP. alba\u003c/em\u003e plants\u003c/h2\u003e \u003cp\u003eIt is reported that polyploidization always alters the morphological characteristics of plants (Otto and Whitton \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). We compared the growth of the diploid and tetraploid plants grown in greenhouse for 90 days to investigate their phenotypic characteristics. We found that the heights of the tetraploid plants were significantly lower than the diploid plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The height of the diploid and tetraploid plants at the 30th day was 18.30 and 10.88 cm, respectively. At the 60th day, the height of the diploid plants was 39.45 cm and that of the tetraploid plants was 24.43 cm. While at the 90th day, the height of the diploid and tetraploid plants reached 60.57 and 37.9 cm, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Overall, the height of the tetraploid plants was 37.43% lower than the diploid. However, during the 90 days of growth, the ground diameters of the tetraploid plants were similar to those of the diploid plants. At the 30th day, the ground diameter of the diploid and tetraploid plants was 2.21 and 2.39 mm, respectively. At the 60th day, the ground diameter of the diploid and tetraploid plants was 3.99 and 3.89 mm, respectively. At the 90th day, the ground diameter of the diploid and tetraploid plants reached 5.01 and 5.43 mm, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The above results indicated that polyploidization inhibits the growth of the \u003cem\u003eP. alba\u003c/em\u003e plants, but may slightly promote the growth of ground diameter.\u003c/p\u003e \u003cp\u003eWe measured leaf characteristics of the diploid and tetraploid plants grown for 60 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The results showed that leaf length, leaf width and leaf area of the tetraploid plants were significantly smaller than those of the diploid plants (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The leaf length and width of the tetraploid plants was 8.05 cm and 9.09 cm, while that of the diploid was 9.75 cm and 10.99 cm. The leaf area of the tetraploid plants was 47.16 cm\u003csup\u003e2\u003c/sup\u003e, which was 64.54 cm\u003csup\u003e2\u003c/sup\u003e in the diploid plants (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).. Leaf index is the ratio of leaf length to width, which can reflect the growth condition and growth pattern of plant leaves (Wang et al. 2017), and there was no difference in leaf shape index between the two ploidy plants (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eThe diploid and tetraploid \u003cem\u003eP. alba\u003c/em\u003e also showed differences in root morphology. The roots of the diploid plants were significantly longer than the tetraploid plants (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). The root length of the diploid plants was 28.02 cm, while that of the tetraploid plants was 19.61 cm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). While the root thickness in the two types of the plants were similar (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). The root thickness of the diploid plants was 2.12 mm, and that of the tetraploid plants was 2.21 mm. Taken together, the results showed that the morphology of tetraploid \u003cem\u003eP. alba\u003c/em\u003e was significant changed after chromosome doubling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of morphological characteristics of diploid and tetraploid leaves\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDiploid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTetraploid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeaf length (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e9.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e8.0491\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeaf width (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e10.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e9.09\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeaf area (cm\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e64.54\u0026thinsp;\u0026plusmn;\u0026thinsp;9.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e47.16\u0026thinsp;\u0026plusmn;\u0026thinsp;4.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeaf shape index\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.867\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Microscopic comparisons of the diploid and tetraploid \u003cem\u003eP. alba\u003c/em\u003e plants\u003c/h2\u003e \u003cp\u003eWe observed the leaf microstructures of the diploid and tetraploid plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). We found that the leaves of the tetraploid plants were thicker than the diploids. The epidermal cells of the tetraploid plants were more neatly and closely arranged relative to the diploids. The leaf thickness of the tetraploid plants was 1.17 times of the diploid plants. But there was no significant difference in the thickness of upper and lower epidermal cells between the two ploidies of \u003cem\u003eP. alba\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The spongy tissue was 1.62 times thicker in the tetraploids than in the diploids. On the contrary, the palisade tissue was 1.17 times thicker in the diploids than in the tetraploids (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The results indicated that the thicken leaves of the tetraploid plants may be the consequence of enhanced spongy tissue.\u003c/p\u003e \u003cp\u003eWe found that the stoma size of the tetraploids was larger the diploids (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-F). The stomata of the tetraploids were 1.53 times longer and 1.07 times wider than those of the diploids. In contrast, the stomatal density of the diploids was 1.72 times higher than that of the tetraploids. Moreover, the guard cells of the tetraploids were 1.35 times longer and 1.02 times wider than those of the diploids (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The results indicated that polyploidization has an effect on the stomatal characteristics of \u003cem\u003eP. alba\u003c/em\u003e, with the tetraploid plants having larger stomata and guard cells and reduced stomatal density compared to the diploid plants, and this alteration may improve the plant's water utilization efficiency (Yao et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBy observing the anatomy of the stems of the two ploidies plants, we found that polyploidization also causes structural changes in the stems of \u003cem\u003eP. alba\u003c/em\u003e. Compared with the stems of the diploid plants, the stems of tetraploid plants became thicker, in which the width of the xylem was reduced and the width of the phloem was increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-H). The phloem width of the tetraploid plants was 0.30 mm, which was 1.76 times of the diploid plants, whose phloem was 0.17 mm in width. The ratio of phloem width to stem radius was 0.17 in the tetraploid plants and 0.08 in the diploid plants. In contrast, the xylem width of the diploid plants was 0.91 mm, which was 2.12 times of the tetraploid plants, whose xylem width was 0.43 mm. The ratio of xylem width to stem radius was 0.46 in the diploid plants and 0.24 in the tetraploid plants (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of microstructural characteristics of diploid and tetraploid leaves\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDiploid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTetraploid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThickness of upper epidermis (\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e13.50\u0026thinsp;\u0026plusmn;\u0026thinsp;2.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e12.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.199\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThickness of lower epidermis (\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.448\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThickness of palisade tissue (\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e27.12\u0026thinsp;\u0026plusmn;\u0026thinsp;2.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e23.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThickness of spongy tissue (\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e29.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e46.99\u0026thinsp;\u0026plusmn;\u0026thinsp;2.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThe ratio of palisade tissue to spongy tissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.065\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThickness of leaf (\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e78.49\u0026thinsp;\u0026plusmn;\u0026thinsp;2.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e92.16\u0026thinsp;\u0026plusmn;\u0026thinsp;3.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStomatal length(\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e9.91\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e15.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStomatal width (\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.234\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStomatal density (Number per mm\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e268.17\u0026thinsp;\u0026plusmn;\u0026thinsp;12.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e156.01\u0026thinsp;\u0026plusmn;\u0026thinsp;12.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGuard cell length (\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e16.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e21.63\u0026thinsp;\u0026plusmn;\u0026thinsp;2.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGuard cell width(\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e13.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e13.95\u0026thinsp;\u0026plusmn;\u0026thinsp;1.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.533\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of anatomical and structural characteristics of diploid and tetraploid stems\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDiploid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTetraploid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eXylem width(mm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhloem width(mm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eXylem width/Stem radius\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhloem width/Stem radius\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Photosynthetic comparisons of the diploid and tetraploid \u003cem\u003eP. alba\u003c/em\u003e plants\u003c/h2\u003e \u003cp\u003eChlorophyll helps plants to absorb light energy, thus directly affecting the strength of photosynthetic capacity (Wang and Grimm \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We found that the chlorophyll \u003cem\u003ea\u003c/em\u003e, \u003cem\u003eb\u003c/em\u003e and total chlorophyll contents in the tetraploid poplar were all higher than in the diploid poplar. The diploid poplar contained 1.05 mg/g chlorophyll \u003cem\u003ea\u003c/em\u003e and 0.33 mg/g chlorophyll \u003cem\u003eb\u003c/em\u003e. While the tetraploid poplar contained 1.24 mg/g chlorophyll \u003cem\u003ea\u003c/em\u003e and 0.39 mg/g chlorophyll \u003cem\u003eb\u003c/em\u003e. The total chlorophyll contents were 1.38 mg/g in the diploid plants and 1.64 mg/g in the tetraploid plants (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEnhanced photosynthesis promotes plant growth, thereby augmenting plant resistance (Shen et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). By comparing the fluorescence kinetic parameters of the two ploidies of \u003cem\u003eP. alba\u003c/em\u003e, we found that except for the maximal efficiency of PSII photochemistry (Fv/Fm) which was no significant difference between the two ploidies plants ( \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.01 ), the photochemical quenching coefficient (qP), apparent electron transfer rate (ETR) and the actual photosynthetic efficiency of PSII (Y(II)), were greater in the tetraploid plants than in diploid plants (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These results suggest that the tetraploid plant possesses a superior photosynthetic capacity than the diploid plant.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of photosynthetic parameters in diploid and tetraploid plants\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhotosynthetic parameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDiploid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTetraploid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChlorophyll \u003cem\u003ea\u003c/em\u003e(mg/g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChlorophyll \u003cem\u003eb\u003c/em\u003e(mg/g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.009\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal chlorophyll(mg/g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFv/Fm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.012\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eETR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e28.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e28.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.004\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eqP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eY(II)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5 The tetraploid plants exhibited enhanced drought tolerance\u003c/h2\u003e \u003cp\u003ePolyploidization can increase plant tolerance to abiotic stresses. Especially under water deficit conditions, drought tolerance of polyploid plants can be improved by altering the transpiration, photosynthetic rate, water utilization and morphology of plants (Maherali et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Deng et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Simulated drought stress was applied to the 60th days plants of the two ploidies by stopping watering for 9 days. We found that under drought treatment, the leaves of diploid plants showed more wilting and the degree of drought damage to diploid plants were more severe compared to tetraploid plants. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). With increasing drought duration, leaf water content decreased from 80.66\u0026ndash;68.13% in the diploid plants and from 82.32\u0026ndash;74.48% in the tetraploid plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Stem water content decreased from 78.97\u0026ndash;64.06% in the diploid plants and from 80.80\u0026ndash;74.93% in the tetraploid plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Leaves and stems of the tetraploid lost less water under drought conditions compared to the diploid.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMalondialdehyde (MDA), a marker for membrane lipid peroxidation, indicates the extent of oxidative stress inflicted on cellular membranes. The level of MDA is often employed to measure how plants cope with various stressors. (Luo et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). After drought stress, the MDA content in the leaves of both plants showed an increasing trend with the drought duration. At the 9th day of drought stress, the MDA content in the leaves of diploid and tetraploid plants was 40.48 nmoL/g and 29.81 nmoL/g, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Compared with the diploid, the tetraploid suffered less damage to leaf membranes under drought stress and had stronger antioxidant capacity.\u003c/p\u003e \u003cp\u003eOsmoregulatory substances such as proline (Pro) and soluble sugars (SS) can reduce the water potential of plant cells under drought conditions, thereby preventing cell dehydration to improve plant drought resistance (Ozturk et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e). During drought stress, the Pro content and SS content of the tetraploid and diploid leaves gradually increased. At the 9th day of drought stress, the Pro content of diploid and tetraploid leaves was 62.87 \u0026micro;g/g and 122.03 \u0026micro;g/g, and the soluble sugar content was 36.46 mg/g and 46.67 mg/g (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-C). The Pro content and SS content in the tetraploid leaves was 1.94 and 1.28 times higher than that of the diploid leaves, respectively. These results suggest that the tetraploid plants exhibit stronger osmotic adjustment and are more resistant to drought conditions than the diploid when subjected to drought stress.\u003c/p\u003e \u003cp\u003ePeroxidase (POD), catalase (CAT) and superoxide dismutase (SOD) are important protective enzymes in plants and also reflect the ability of plants to scavenge reactive oxygen species (Li et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e; Jin et al.). After 9 days of drought stress, all the enzyme activities in the tetraploid plant were higher than the diploid plant. The POD activity of the tetraploid leaves was essentially unchanged from 0\u0026ndash;6 days, and increased significantly after the 6th day. The POD activity of the diploid leaves showed an increasing trend during drought. At the 9th day of drought, the POD activity in the diploid was 45.58 U/g and the POD activity in the tetraploid was 56.25 U/g, which was 1.37 times higher than that of the diploid (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). The CAT activity in the leaves of different ploidies showed an increasing trend. At the 9th days of drought, the CAT activity in the diploid was 49.70 U/g and in the tetraploid was 72.46 U/g, which was 1.46 times higher than that of the diploid (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). The activity of SOD showed a trend of increasing and then decreasing with the increase of drought stress time, and at the 9th day of drought stress, the SOD activity in the diploid leaves was 1113.32 U/g and in the tetraploid leaves was 2072.50 U/g, which was 1.86 times higher than that of the diploid (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Taken together, the results indicate that the tetraploid \u003cem\u003eP. alba\u003c/em\u003e is more tolerant to drought than the diploid.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eCompared to diploids, polyploids usually change the morphological characteristics of plants(Haist et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Pungaršek and Frajman \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Su et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e). In this study, we observed the phenotypic characteristics of the diploid and tetraploid \u003cem\u003eP. alba.\u003c/em\u003e Plant height was significantly reduced in the tetraploid as compared to the diploid, which was similar to tetraploids of birch, apple, willow and Sorbus pohuashanensis (Mu et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Dudits et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Xue \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Previous studies had found that among the differentially expressed genes in diploids and tetraploids, most of them are genes related to phytohormone signaling (Ren et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Plant growth and development are regulated by endogenous hormones, and dwarfing of plants after polyploidization is associated with reduced levels of growth-related hormones. In poplar tetraploids, the expression of genes related to the regulation of indoleacetic acid (IAA), gibberellin (GA), and brassinosteroid (BR) synthesis was down-regulated with increasing leaf age (Xu et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In addition to height, the morphology of the leaf also changed. We observed a smaller leaf area and deeper leaf color in the tetraploid leaves compared to the diploid leaves. Previous studies have suggested that leaf size increases with increasing ploidy, but this relationship is not linear (Kondorosi et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). For example, the triploid leaf size of atemoyas is larger than the diploid, but the leaf size of the tetraploid is smaller than the diploid (Losada et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Regardless of leaf size, the color of leaves deepens after polyploidization, such as \u003cem\u003eRosa roxburghii\u003c/em\u003e (Wu et al. 2023), \u003cem\u003ePopulus\u003c/em\u003e (Zhang et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), \u003cem\u003eZiziphus jujuba\u003c/em\u003e Mill. (Cui et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and \u003cem\u003ePassiflora edulis\u003c/em\u003e Sims. Our observations indicated that the tetraploid plants had shorter root lengths compared to their diploid counterparts, a finding that aligns with earlier researc (Shao et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Tavan et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). It may be the result of transcription factors and phytohormone regulation (Ren et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Further research is needed to elucidate the mechanisms underlying the phenotypic differences in tetraploid \u003cem\u003eP. alba\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eBy observing the anatomy of the leaves of diploid and tetraploid \u003cem\u003eP. alba\u003c/em\u003e, we found that the tetraploid leaves were thickened, palisade tissues were thinner and spongy tissues were thicker compared to the diploids. The tetraploid \u003cem\u003eLilium\u003c/em\u003e leaves exhibited a marked increase in the thickness of both the upper and lower epidermis and spongy tissues compared to diploid leaves. However, the thickness of the palisade tissues remained unchanged between the two ploidy levels, aligning with the outcomes of this investigation (Cao et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In contrast, the palisade and spongy tissues of the leaves of tetraploid \u003cem\u003eB. papyrifera\u003c/em\u003e (Lin et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and \u003cem\u003eDendrobium cariniferum\u003c/em\u003e (Zhang and Gao \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) were significantly thicker than those of the diploid leaves, suggesting that the increased thickness of palisade and spongy tissues in polyploidized plants is not absolutely. It may be influenced by individual genomic differences across species (Zhang et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Stomatal length and width are commonly used as morphological markers for identifying potentially polyploid plants (Huang et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e; Khan et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Maisha et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The increase in ploidy level is positively correlated with the size of plant stomata and negatively correlated with stomatal density (Widoretno \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This is consistent with our findings in tetraploid \u003cem\u003eP. alba\u003c/em\u003e plants where we observed significantly longer and wider stomata and reduced stomatal density compared to diploids. Greener leaves and a greater number of stomata enhance photosynthesis, thus indirectly increasing plant tolerance to abiotic stresses. The anatomical structures of the two ploidy \u003cem\u003eP. alba\u003c/em\u003e stems were also significantly different, with the tetraploid having a narrower width of xylem and a wider width of phloem, which differed from the results of previous studies. The number of secondary xylem cells is the same in diploid and tetraploid \u003cem\u003epoplars\u003c/em\u003e, due to the fact that the size of xylem fiber cells in tetraploids is significantly larger than in diploids, resulting in wider xylem (Iwakawa et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In tetraploid Citrus limonia, cortical cell area, xylem cell area, epidermal cell area, and cuticle thickness were significantly larger than in diploids (Allario et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The reasons for the structural changes in the stem of tetraploid silver poplar need to be thoroughly investigated by subsequent experiments.\u003c/p\u003e \u003cp\u003eIncreased genetic diversity makes polyploids better adapted to extreme environments (Arrigo and Barker \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In our study, tetraploid \u003cem\u003eP. alba\u003c/em\u003e had lower MDA content, higher water content, SS and Pro content, and higher SOD, POD, and CAT enzyme activities than diploids after drought stress.These results suggest that tetraploid \u003cem\u003eP. alba\u003c/em\u003e possess enhanced tolerance to drought. Many studies in recent years have found tetraploid plants to be more resistant to unfavorable external environments, although many studies have been initiated to examine and characterize polyploid stress tolerance (Li et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Jiang et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Fakhrzad and Jowkar \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Nevertheless, a comprehensive understanding of the specific molecular pathways that confer their improved stress resistance remains scarce. This study's findings enhance our comprehension of the physiological and molecular factors that contribute to the improved stress resilience in polyploid \u003cem\u003eP. alba\u003c/em\u003e.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThrough the induction of colchicine, tetraploid \u003cem\u003eP. alba\u003c/em\u003e were obtained. Compared to diploid plants, significant changes were observed in the growth parameters, physiological structures, and photosynthetic parameters of tetraploid plants. Specifically, the tetraploid plants exhibited decreased height, reduced leaf area, darkened leaf color, increased leaf thickness, enlarged stomatal length and width and decreased stomatal density. The xylem of the stems narrowed and the phloem of the stems widened. Furthermore, we found that the tetraploid plants may exhibit stronger resistance to adverse external environments. Under drought stress, the tetraploid plants had increased contents of Pro and SS, higher activities of SOD, POD, and CAT, and lower MDA content compared to the diploid plants. The findings of this study can enrich the germplasm resources of poplar and provide a theoretical basis for polyploid breeding in tree species.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCredit authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYuanfu Liu:\u003c/strong\u003e Writing original draft, Visualization, Validation, and Formal analysis. \u003cstrong\u003eXinyu Wang:\u0026nbsp;\u003c/strong\u003eData curation, Experimental material arrangement. \u003cstrong\u003eSiyuan Li:\u003c/strong\u003e Identification and propagation of the plant materials. \u003cstrong\u003eYan Zhou and Ruihan He:\u0026nbsp;\u003c/strong\u003eParaffin sections, physiological indices and photosynthetic parameters were determined. \u003cstrong\u003eSu Chen:\u003c/strong\u003e Conceptualization, Supervision, Writing \u0026ndash; review \u0026amp; editing, Resources, Funding acquisition.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key R\u0026amp;D Program of China (2021YFD2200800) and the Fundamental Research Funds for the Central Universities (2572022CG06).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAllario T, Brumos J, Colmenero-Flores JM, et al (2011) Large changes in anatomy and physiology between diploid Rangpur lime (Citrus limonia) and its autotetraploid are not associated with large changes in leaf gene expression. 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Forests 14:1589. https://doi.org/10.3390/f14081589\u003c/li\u003e\n \u003cli\u003eZhu Y, Song D, Xu P, et al (2017) A HD‐ZIP III gene, PtrHB4, is required for interfascicular cambium development in Populus\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"P. alba, tetraploid, morphometric analysis, drought tolerance","lastPublishedDoi":"10.21203/rs.3.rs-5751567/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5751567/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eArtificial induction of polyploids is an effective technique for plant breeding and genetic improvement. Understanding the changes in plant morphology after polyploidization is the key to studying the underlying physiological mechanisms of polyploid plant development. We obtained a tetraploid \u003cem\u003eP. alba\u003c/em\u003e using colchicine induction and performed a characterization analysis on it. The results showed that the height and leaf area of the tetraploid plant were smaller than those of the diploid plant. The tetraploid plant have thicker leaves, higher chlorophyll contents, and larger but less dense stomata. Tetraploidization also resulted in significant changes in stem anatomy, including smaller xylem width and larger phloem width. In addition, we found that the tetraploid plants exhibited enhanced drought tolerance compared with the diploid parent. The results of our study not only revealed the structural and physiological changes in the tetraploid plants, but also provided valuable insights into the breeding of polyploid \u003cem\u003eP. alba\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Analysis of changes in morphological characters and drought resistance of tetraploid P. alba","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-09 08:10:38","doi":"10.21203/rs.3.rs-5751567/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-01-07T10:12:54+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-01-07T09:52:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-02T14:13:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2025-01-02T06:54:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b3ea9abd-8e99-47e1-a005-72f6f4f88111","owner":[],"postedDate":"January 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-28T15:59:50+00:00","versionOfRecord":{"articleIdentity":"rs-5751567","link":"https://doi.org/10.1007/s11240-025-03133-z","journal":{"identity":"plant-cell-tissue-and-organ-culture-pctoc","isVorOnly":false,"title":"Plant Cell, Tissue and Organ Culture (PCTOC)"},"publishedOn":"2025-07-22 15:57:12","publishedOnDateReadable":"July 22nd, 2025"},"versionCreatedAt":"2025-01-09 08:10:38","video":"","vorDoi":"10.1007/s11240-025-03133-z","vorDoiUrl":"https://doi.org/10.1007/s11240-025-03133-z","workflowStages":[]},"version":"v1","identity":"rs-5751567","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5751567","identity":"rs-5751567","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}