Biological characteristics of flowers and examination of pollen viability at different developmental stages of Epimedium sagittatum( Sieb. et Zucc. )Maxim

Biological characteristics of flowers and examination of pollen viability at different developmental stages of Epimedium sagittatum( Sieb. et Zucc. )Maxim | 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 Biological characteristics of flowers and examination of pollen viability at different developmental stages of Epimedium sagittatum( Sieb. et Zucc. )Maxim Jianglong He, Lixin Pei, Baoyu Ji, Hai-bo Wang, Hua Zhong, Chengming Dong, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3599092/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract To gain a deeper understanding of the flowering pattern and reproductive characteristics of Epimedium sagittatum , to enrich the research on the flower development of Epimedium sagittatum and its reproductive regulation, and to screen the methods suitable for the rapid detection of pollen viability of Epimedium sagittatum and to promote its cross-breeding. The characteristics of its flower parts were observed, recorded and measured, and the pollen viability of Epimedium sagittatumwas determined by five methods, including TTC staining, I 2 -KI staining, red ink staining, peroxidase method and in vitro germination method. The flowering process of Epimedium sagittatum can be divided into five stages: calyx dehiscence, bract spathe, petal outgrowth, pollen dispersal, and pollination and withering. The results of I 2 -KI staining and peroxidase method were significantly higher than those of other methods; the in vitro germination method was intuitive and accurate, but the operation was complicated and time-consuming; the red ink staining method was easy to operate and had obvious staining effect, and the results were the closest to those of the in vitro germination method; and it was found that the pollen of Epimedium sagittatum was not as effective as the in vitro germination method at the bud stamen stage, the flower stigma and the flower bud. It was also found that the pollen viability and germination rate of Epimedium sagittatum pollen were higher in the three periods of bud spitting, petal adductor and pollen dispersal. Comparing the five methods, the red ink staining method was found to be a better method for the rapid detection of pollen viability; the best pollination periods of Epimedium sagittatum were the bud stamen stage, petal adductor stage, and pollen dispersal stage of flowers at the peak of bloom. This study on the flowering and fruiting pattern of Epimedium sagittatum , and the related mechanism of sexual reproduction, can be used as a reference for the next step of research on the breeding of Epimedium sagittatum . Epimedium sagittatum Different developmental stages Floral characteristics Pollen viability Determination method Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Epimedium sagittatum (Sieb. et Zucc.) Maxim. (also known as “three branches and nine leaves grass”) is one of the most important traditional medicinal plants in China. It belongs to Epimedium , a perennial herbaceous plant in the Berberidaceae family (Wen et al.,2022). First recorded in Shennong's Classic of Materia Medica, it was listed as a top grade herb (Xu et al.,2020) with the effects of reinforcing kidney yang, strengthening muscles and bones, dispelling rheumatism, etc. It is often used to treat kidney yang deficiency, osteoporosis and other diseases (Wei et al.,2023; Wang et al.,2023). With the continuous in-depth research on Epimedium in modern science, the latest research has found that it also has anti-tumor effects, especially on patients with advanced liver cancer (Li et al.,2021). This has led to an increasing demand for Epimedium , and wild resources cannot meet the demand, resulting in rising prices. In order to obtain a large amount of Epimedium , artificial cultivation has begun. Currently, Epimedium seeds are expensive. In the cultivation process, it was discovered that although Epimedium sagittatum has abundant flowering, its reproductive ability is low, the seed setting rate is low, and the germination rate is extremely low. Therefore, there are few seedlings in natural populations (Cui et al.,2022). It can be seen from the above that the weak reproductive ability and low seed germination rate of Epimedium sagittatum have always been a problem in Epimedium cultivation. Studies have found that the size of pollen viability directly affects the pollination and fertilization processes of plants, which is closely related to the yield and quality of plant seeds (Bai et al.,2023). Testing the viability of Epimedium sagittatum pollen at different flowering stages before pollination can understand the viability, development and physiological characteristics of Epimedium sagittatum pollen, and grasp the morphology of sterile pollen, providing a theoretical basis for artificial breeding (Li et al.,2023; Xu et al.,2012). Therefore, researching the viability and germination characteristics of Epimedium sagittatum pollen is very necessary to improve pollination breeding efficiency and reasonably carry out breeding to increase seed germination rate and obtain high-quality Epimedium sagittatum seeds. On this basis, it is of great value and significance to establish scientific and effective pollination methods for the future, accelerate the breeding process of excellent Epimedium sagittatum varieties, shorten the differentiation period between varieties, and thus enhance the competitiveness of Epimedium sagittatum germplasm. At the same time, it can provide a basis for the construction of the Epimedium sagittatum breeding system under artificial cultivation conditions and production application, thereby promoting the realization of maximized economic benefits in the large-scale production of Epimedium sagittatum. However, research and reports on pollen viability detection of this species have not been seen so far. This paper systematically studies the flowering dynamics and floral characteristics of Epimedium sagittatum . It also determines the pollen viability at different developmental stages and screens suitable methods for rapid detection of pollen viability in Epimedium sagittatum. The aim is to deeply understand its flowering habits and reproductive characteristics, enrich data on flower development and reproductive regulation of Epimedium sagittatum , explore the limiting factors affecting its breeding process, discuss its breeding habits and pollination methods, and explore ways to improve the pollen viability of Epimedium sagittatum . This study aims to reveal the flowering mechanism of Epimedium sagittatum , clearly understand the physiological and biochemical characteristics of its pollen at different developmental stages, grasp the changes in pollen viability, thereby determine the optimal pollination time and pollen testing method. The purpose is to provide preliminary research basis for exploring artificial assisted fertilization technology, improving seed viability, guiding artificial cultivation management for high yield and efficiency, simplifying production procedures, and selecting new varieties in the future. This will provide preliminary research basis for renewing and reproductive biological studies as well as selecting new varieties of this species. 2. Experimental Materials and Methods 2.1 Experimental Materials 2.1.1 Sample Collection The test site was located in the Epimedium plantation base in Pingyu County, Henan Province, at east longitude 114°61' and north latitude 32°96'. The test materials were Epimedium sagittatum seeded and nursed in October 2021 with good growth after routine field management. Pollen was collected at the peak flowering period on sunny mornings from 9:00–10:00. Flowers at different blooming stages were collected and brought back to the lab for pollen extraction and testing. 2.2 Experimental Methods 2.2.1 Observation of Floral Organ Characteristics, Flowering Habits and Dynamics From March to June 2023, 120 Epimedium sagittatum plants were randomly selected and tagged in the base. The date when the first flower bloomed was recorded as the initial flowering period, the period when over 50% of flowers bloomed was recorded as peak flowering period, and the date when the last flower bloomed was recorded as final flowering period. At peak flowering, 60 freshly bloomed single flowers were randomly selected every day and observed 6 times. Observations were made every 2 hours from initial to full bloom until withering. The collected fresh flowers were placed under a SZX2-ILLTQ stereo microscope to measure and record the dimensions of each floral part. 2.2.2 Pollen Viability Testing 2.2.2.1 Triphenyl Tetrazolium Chloride (TTC) Staining TTC is an oxidation-reduction dye with a standard oxidation potential of 80mV. It dissolves into a colorless solution in water. After reduction, it forms the red insoluble triphenyl formazan. Its reduction amount can indicate dehydrogenase activity. This substance is relatively stable and not easily oxidized, so TTC is widely used in enzyme experiments to determine pollen viability. Referring to Liang Lu (Ling et al.,2022) et al.'s method with improvements, 1–2 drops of 0.5% TTC solution were dropped onto a concave slide. Pollen was sprinkled into the solution, mixed well, covered with a coverslip, and placed in a 35°C incubator for 1h. After taking out and microscopic examination, viable pollen appeared red, weakly viable pollen appeared light red, non-viable or sterile pollen was colorless. 2.2.2.2 I 2 -KI Staining The starch content in pollen can be used as a criterion to judge the level of pollen development. Normal mature pollen is mostly spherical with high starch content and can be stained blue by I 2 -KI solution. Underdeveloped pollen is mostly deformed and generally contains no or little starch. It usually does not contain starch or contains relatively less starch and will not be stained or will be stained yellow or yellowish brown by I 2 -KI solution. According to pollen staining, pollen viability can be identified. Referring to Song Jing (Song et al.,2022) et al.’s method with improvements, 1–2 drops of I 2 -KI solution were dropped onto a concave slide. Pollen was sprinkled into the solution, mixed well, covered with a coverslip, and stained for 5 min before microscopic examination. 2.2.2.3 Red Ink Staining The protoplasm of viable pollen cells has selective permeability and selective absorption capacity for external substances, such as red ink dyes cannot enter the cells and the pollen cannot be stained. However, the protoplasmic membrane of lifeless pollen cells will lose this activity, dyes will enter the cells and stain them. Therefore, pollen viability can be judged by whether pollen grains are stained. Referring to Wang Jinhua (Wang et al.,2022) et al.’s method with improvements, 5% red ink solution was prepared, 1–2 drops were dropped onto a concave slide, pollen was sprinkled into the solution, mixed well, covered with a coverslip, and examined under the microscope immediately. Viable pollen was not stained while non-viable pollen was stained red. 2.2.2.4 Peroxidase Method Peroxidase is contained in pollen. Pollen with higher viability has stronger peroxidase activity. Peroxidase forms a complex with oxidants. The activated hydrogen peroxide in the complex can oxidize phenolic compounds. Pollen viability can be judged by the color change. Viable pollen is purple-red, while pollen with weaker viability or no viability is light red or colorless. Referring to Fu Qinchao (Fu et al.,2015) et al.’s method with improvements, 1 drop of aromatic amine coloring solution (0.5% aniline dye solution : 0.5% α-naphthol dye solution : aromatic buffer = 1:1:1) and 0.1% hydrogen peroxide each were dropped onto a concave slide. Pollen was sprinkled into the solution, mixed well, covered with a coverslip, and kept at 30°C for 15 min before microscopic examination. 2.2.2.5 In Vitro Germination Normal mature pollen grains have strong viability. They can germinate and grow under suitable culture conditions. Germination number can be directly observed and counted under the microscope to calculate germination rate and determine pollen viability. Referring to Liu Xinyu (Liu et al.,2022) et al.’s method with improvements, prepared pollen germination culture medium was dropped onto concave slides. Pollen was sprinkled into the medium, mixed well, covered with coverslips, and placed in a germination box lined with wet filter paper. It was cultured in a 25°C incubator and examined under the microscope every 1 hour to count germinated pollen. Pollen with pollen tube length exceeding pollen diameter was considered as germinated. Germination rate (%) = (number of germinated pollen grains / total number of pollen grains) × 100% 2.3 Data Statistics Three slides were prepared for each treatment and observed under a BX53F2 optical microscope. 5 visual fields were selected on each slide, the total number of pollen grains counted was no less than 500. Pollen viability was calculated: Pollen viability (%) = (number of viable pollen / total number of pollen) × 100% 2.4 Data Processing The experimental data obtained were processed using Excel 2020 and SPSS 26 software. 3. Results and Analysis 3.1 Comprehensive Characteristics of Epimedium sagittatum Flowers 3.1.1 Biological Characteristics Epimedium sagittatum is a long-day shade-loving plant(Figure 1 ). The flowering period is from March to June each year. The inflorescence is an indefinite panicle with numerous branches on the inflorescence axis. Each branch has three flowers and the terminal has one flower. The calyx has two whorls of four sepals each. The outer whorl is green with purple spots while the inner whorl is white. After blooming, the outer calyx falls off and the inner calyx opens up. The four petals are yellow in color. The four stamens grow out of the bud before the pistil. The style is green and the ovary is green with purple spots. After pollination, the ovary enlarges and the style gradually elongates, exceeding the stamens. 3.1.2 Floral Morphology Epimedium sagittatum has a terminal panicle inflorescence, Flower organ parameter display (Table 1 )10–35 cm long, with 20–50 flowers. The single flower is a complete flower without nectaries. The calyx has two whorls of four sepals each. The first whorl is green with purple spots, with 1 pair narrowly ovate and 1 pair oblong-ovate. The second whorl is white. After fully opening, the first whorl of sepals falls off. The lobes are triangular and reflexed. The inflorescence axis, pedicels and calyx surface all have purple spots. The petals are yellow or pale yellow, with 4 petals in a saccate, oblong or obovate shape. There are 4 stamens with yellow anthers in a cruciform position and purple-red filaments. There is 1 pistil with a purple-red cylindrical style and dark green stigma. When mature, the stigma surface secretes to adsorb pollen. The stigma position is about 8 mm higher than the anthers. The ovary is semi-inferior and spherical with 6–7 locules(Figure 2 ). Table 1 Organ parameters of Epimedium sagittatum flowers Floral Organ Parameters Size/mm Standard Deviation/mm Petal length 2.24 0.14 Petal width 1.52 0.18 Filament length 3.69 0.19 Style length 2.05 0.21 Flower diameter 5.80 1.10 Calyx diameter 5.17 0.86 Ovary length 1.55 0.26 3.1.3 Flowering Dynamics Observation The flowering period of Epimedium sagittatum is concentrated in March to June, with peak flowering in April. The overall flowering pattern shows concentrated mass flowering. It takes 2–4 hours from bud split to complete opening of the corolla. The average flowering period of the population is about 120 days, the flowering period of a single inflorescence is 10–20 days, and the duration of a single flower is 2–5 days. Through observation, it was found that the blooming process of a single Epimedium sagittatum flower can be divided into 5 stages (Fig. 3 ): ( 1 ) Sepal splitting stage - the bud swells and the sepals gradually split, exposing the internal tissues; ( 2 ) Stamen protrusion stage - the stamens break through the bud and are exposed outside; ( 3 ) Petal expansion stage - the petals push open the bud and unfold outwards, at this time the outer sepals fall off; ( 4 ) Peak pollen release stage - the flower is fully open, the pistil and stamens are completely exposed, and the anthers begin to release pollen; ( 5 ) Flower withering stage - the petal color gradually fades, the anthers begin to brown, and gradually wither and fall off, the ovary swells, and the style elongates. It takes about 1 hour for the Epimedium sagittatum calyx to fully split. Then the stamens protrude through the bud with the stamen cluster spreading out. After about 1 hour, the yellow anthers begin to release pollen. At this time, the flower starts to bloom. After 1–2 hours, it fully blooms and enters the peak flowering period. At this time, pollen release decreases and the anthers begin to fall off. After blooming for 1–2 days, there is no significant change in the style, and the edges of the petals begin to wither. On the 2nd day after blooming, the filaments become dry and withered, on the 3rd day after blooming, the petals begin to fall off, the filaments become withered and curved shortened, the anthers gradually turn brown and begin to shrivel and fall off, the ovary begins to swell, and the style begins to elongate and gradually turns brown. On the 5th day after blooming, all petals have fallen off, the ovary sits on the fruit and gradually swells. 3.2 Comparison of Different Pollen Viability Testing Methods 3.2.1 TTC Staining The TTC staining effect on Epimedium sagittatum pollen is shown in Fig. 4 B. After TTC staining, all pollen turned yellowish brown, and no red pollen was observed. The pollen viability was 0, indicating that TTC staining does not easily stain Epimedium sagittatum pollen. Therefore, it is not suitable for determining the viability of Epimedium sagittatum pollen. 3.2.2 I 2 -KI Staining The I 2 -KI staining effect on Epimedium sagittatum pollen viability is shown in Fig. 4 C. Viable pollen with high starch content is stained red-brown, while non-viable pollen with low starch content is not stained and appears yellow-brown. The pollen viability measured by I 2 -KI staining at 5 stages was 75.26%, 74.30%, 93.56%, 96.54% and 95.85%, respectively. The pollen viability was lowest at the stamen protrusion stage at 74.30%; and highest at the peak pollen release stage at 96.53% (Table 2 ). The I 2 -KI staining is fast, simple to operate, and the difference between viable and non-viable pollen is obvious. It is suitable for determining the viability of Epimedium sagittatum pollen. Table 2 Statistics of pollen viability at different developmental stages of Epimedium sagittatum were examined by I 2 -KI staining Parameters Sepal splitting stage Stamen protrusion stage Petal expansion stage Peak pollen release stage Flower withering stage Average value(%) 75.26 74.30 93.56 96.54 95.85 Maximum value(%) 90.52 94.44 100.00 100.00 100.00 Minimum value(%) 43.55 50.00 90.59 88.89 93.38 Standard deviation 17.85 14.89 3.192 3.485 2.084 3.2.3 Red Ink Staining The red ink staining effect on Epimedium sagittatum pollen viability is shown in Fig. 4 D. Viable pollen has selective permeability of the cell membrane and cannot be stained by red ink, while non-viable pollen has complete permeability and is stained red. The pollen viability measured by red ink staining at the 5 developmental stages of Epimedium sagittatum was 84.73%, 44.99%, 24.30%, 31.76%, and 10.26%, respectively. The pollen viability was lowest at the flower withering stage, with the minimum value of 10.26%; and highest at the sepal splitting stage, with the maximum value of 84.73% (Table 3 ). Red ink staining is fast, simple to operate, and the difference between viable and non-viable pollen is obvious. It is suitable for determining the viability of Epimedium sagittatum pollen. Table 3 Statistics of pollen viability at different developmental stages of Epimedium sagittatum were examined by red ink staining Parameters Sepal splitting stage Stamen protrusion stage Petal expansion stage Peak pollen release stage Flower withering stage Average value(%) 84.73 44.99 24.30 31.76 10.26 Maximum value(%) 96.43 61.11 48.48 50.00 20.83 Minimum value(%) 66.67 29.45 11.76 0.00 0.00 Standard deviation 10.01 8.33 10.83 25.22 7.79 3.2.4 Peroxidase Method The peroxidase staining effect on Epimedium sagittatum pollen viability is shown in Fig. 4 E. Viable pollen has strong peroxidase activity and reacts with oxidants to appear purple-red, while pollen with weak or no activity appears light red or colorless. The pollen viability measured by the peroxidase method at the 5 developmental stages of Epimedium sagittatum was 99.23%, 81.51%, 99.30%, 19.93%, and 13.17%, respectively. The pollen viability was lowest at the flower withering stage, with the minimum value of 13.17%; and highest at the petal expansion stage, with the maximum value of 99.30% (Table 4 ). This method is slower, and the difference between viable and non-viable pollen is obvious, but it is easily interfered by other parts of the flower and is not suitable for determining the viability of Epimedium sagittatum pollen. Table 4 Statistics of pollen viability of Epimedium sagittatum by peroxidase method Parameters Sepal splitting stage Stamen protrusion stage Petal expansion stage Peak pollen release stage Flower withering stage Average value(%) 99.23 81.51 99.30 19.93 13.17 Maximum value(%) 100.00 88.52 100.00 41.18 50.00 Minimum value(%) 96.67 68.24 96.23 5.71 0.00 Standard deviation 1.438 6.075 1.398 15.08 14.69 3.2.5 In Vitro Pollen Germination Test The germination of Epimedium sagittatum pollen on the germination medium is shown in Fig. 4 F. The 1–8 hour germination rates of pollen at 5 developmental stages of Epimedium sagittatum are shown in Table 5 . In vitro germination can directly observe pollen germination. When Epimedium sagittatum pollen at each stage was cultured by in vitro germination for 1–7 hours, due to the different pollen conditions, the time required for germination was also different and could not fully reflect the pollen germination. When the pollen was germinated for 7–8 hours, the basically fully germinated pollen with stronger activity, and the germination rate was relatively stable. The measured pollen viability at the 5 developmental stages of Epimedium sagittatum was 20.35%, 48.14%, 43.81%, 53.86% and 7.13%, respectively. The pollen viability was lowest at the flower withering stage, with the minimum value of 7.13%; and highest at the stamen protrusion stage, with the maximum value of 48.13% (Table 5 ). The in vitro pollen germination test results are relatively accurate, but this method requires higher cultivation temperature, nutrient composition, and pH value, and takes a longer time. Table 5 In vitro germination rate of Epimedium sagittatum pollen from 1 to 8 h at different stages Time Sepal splitting stage Stamen protrusion stage Petal expansion stage Peak pollen release stage Flower withering stage 1h 0.00% 3.26% 0.93% 0.00% 0.00% 2h 1.21% 10.31% 4.13% 1.30% 0.00% 3h 7.33% 20.51% 15.11% 5.61% 0.13% 4h 10.51% 35.70% 17.53% 31.67% 6.57% 5h 15.67% 46.74% 35.79% 36.21% 6.77% 6h 20.31% 48.12% 43.74% 53.76% 6.98% 7h 20.33% 48.13% 43.76% 53.78% 7.04% 8h 20.35% 48.14% 43.81% 53.86% 7.13% Standard deviation 9.15 4.73 6.83 6,75 3.15 3.2.6 Comparison of Different Pollen Viability Testing Methods Pollen viability directly affects the results of fertilization and fruit setting rate after hybridization pollination. Varieties with higher pollen viability are preferred as male parents, which is more conducive to the successful breeding of new varieties (Yin et al.,2021). Currently, there are many methods for determining pollen viability, but most of the pollen testing methods obtain higher pollen viability than the actual viability. Due to the simple operation, less test time consumption, and ability to determine pollen viability in a short time, pollen staining methods are widely used in pollen viability detection of various plants (Liang et al.,2022). Du Wenwen et al. concluded through the determination of pollen viability in Camellia that pollen staining was more suitable than I 2 -KI staining and TTC staining (Du et al.,2021). Ren Feiyan et al. used TTC staining, MTT staining, Alexander staining, I 2 -KI staining, red ink staining and in vitro germination to determine the pollen viability of Morus alba, and believed that Alexander staining and I 2 -KI staining were more suitable (Ren et al.,2021). Liu Chengqin et al. used TTC staining, acridine red staining, I 2 -KI staining, peroxidase staining and in vitro pollen germination to determine the pollen viability of Cuscuta chinensis, and believed that I 2 -KI staining was the most suitable method for determining the viability of Cuscuta chinensis pollen (Liu et al.,2021). In addition, in vitro culture is another method for determining pollen viability. This method can directly observe pollen germination and gives more reliable results than pollen staining methods. It can be seen that different detection methods are suitable for different plants. The results of this test found that in vitro germination can directly observe pollen germination. In this test, the pollen of Epimedium sagittatum at five developmental stages can germinate well after 8 hours of cultivation, and the germination rate is relatively stable. It is the preferred method for determining pollen viability, but this method has complex operation and long time consumption, and is not suitable for rapid detection of pollen viability. The staining method has the advantages of being fast and simple. It can reflect the viability of Epimedium sagittatum pollen to some extent, but is greatly affected by pollen characteristics. Different staining methods are suitable for different plants. TTC staining did not stain Epimedium pollen, and the measured pollen viability values were all 0, so pollen viability comparison analysis was not performed. As can be seen from Table 6 , the pollen viability values measured by different methods vary greatly, with the order of peroxidase method > I 2 -KI staining > pollen germination > red ink staining. Significance analysis showed extremely significant differences among the 5 methods. The results of I 2 -KI staining and peroxidase method were significantly higher than red ink staining and in vitro germination. The results of red ink staining were closer to in vitro germination, and the staining effect was good and the operation was simple. It is the most suitable method for rapid detection of Epimedium sagittatum pollen viability. Table 6 Comparison of results of different assays for pollen vitality in Epimedium sagittatum Determination method Sepal splitting stage Stamen protrusion stage Petal expansion stage Peak pollen release stage Flower withering stage Pollen germination method 20.35 48.14 43.81 53.86 7.13 Peroxidase method 99.23 81.51 99.30 19.93 13.17 Red ink staining method 84.73 44.99 24.30 31.76 10.26 I 2 -KI staining method 75.26 74.30 93.56 96.54 95.85 TTC staining method 0.00 0.00 0.00 0.00 0.00 3.3 Analysis of Pollen Viability at Different Developmental Stages The results of this test found that the pollen viability of Epimedium sagittatum at different stages has extremely significant differences. From the results of the pollen germination test, it can be seen that the pollen germination rates of Epimedium sagittatum were higher during the stamen protrusion stage, petal expansion stage and peak pollen release stage. At this time, the pollen of Epimedium sagittatum has better pollination, but from the results of the pollen staining method, it can be seen that the pollen viability was also very high during the sepal splitting stage. Combined with microscopic observation, it was found that although the pollen viability was high at this time, the deformity rate was higher, up to more than 70%. If pollination succeeded at this time, it may lead to low germination rate of pollinated seeds. Therefore, the optimal pollination period of Epimedium sagittatum should be the stamen protrusion stage, petal expansion stage and peak pollen release stage. Through the pollen germination test, it was found that the pollen germination rate of Epimedium sagittatum was low, and the highest germination rate was only 53.86%. From the flowering process of Epimedium sagittatum (Fig. 3 ), it can be seen that the flowering of Epimedium sagittatum is protandrous with stamens protruding first and then pistil exposed for pollination. The pollen viability was stronger when the stamens protruded through the buds. Due to the different protruding times of stamens and pistils, the optimal pollination time may be missed. Combined with the investigation of local climate and ecology, due to temperature reasons, wild pollinating insects such as bees and butterflies in Pingyu, Henan Province were not active from March to April, making cross-pollination relatively difficult, resulting in poor fruit setting rate and seed germination rate of Epimedium sagittatum . Therefore, means such as artificial beekeeping can be adopted to increase the success rate of cross-pollination of Epimedium sagittatum , so as not to miss the optimal pollination time, thus enabling Epimedium sagittatum to breed high-viability, high-quality seeds. 4. Conclusion In summary, the flowering period of Epimedium sagittatum is from March to June each year, with peak flowering in April. Its flowers are in panicles. Through observation, it was found that the blooming process of a single Epimedium sagittatum flower can be divided into 5 stages: sepal splitting stage, stamen protrusion stage, petal expansion stage, peak pollen release stage and flower withering stage. Experiments found that in vitro germination was the most accurate and effective method for detecting the pollen viability of Epimedium sagittatum. TTC staining, peroxidase method and I 2 -KI staining were not suitable for determining pollen viability. Red ink staining had simple operation and obvious staining effect and was a good method for rapid detection of pollen viability. Among them, the detection results of red ink staining were closest to those of in vitro germination, making it the most suitable method for rapid detection of Epimedium sagittatum pollen viability. Combined with the flowering process and pollen viability at different developmental stages of Epimedium sagittatum , it was found that the optimal pollination period of Epimedium sagittatum should be the stamen protrusion stage, petal expansion stage and peak pollen release stage. It was also found that the pollen viability of Epimedium sagittatum was low, and the different protruding times of stamens and pistils may lead to missing the optimal pollination time. In addition, due to local climatic reasons, wild pollinating insects such as bees and butterflies were not active, resulting in lower cross-pollination rates and poorer fruit setting rate and seed germination rate of Epimedium sagittatum. Therefore, it is proposed to use artificial beekeeping to achieve cross-pollination and avoid missing the optimal pollination time, thereby increasing the success rate of cross-pollination of Epimedium sagittatum . The flowering results and related sexual reproduction mechanisms of Epimedium sagittatum in this study can provide reference for further research on the breeding of Epimedium sagittatum , in order to lay the foundation for hybrid breeding and cultivation promotion of Epimedium sagittatum . Declarations Author Contributions: B.J. and J.H. conceived and designed the experiments, J.H. and X.L. participated in the execution of the experiments, J.H. analysed the experiments, L.P. and J.H. supervised and completed the writing, P.L. and H.W. assisted in the design of the figures and tables, H.Z. reviewed the references, C.D. and S.C. reviewed the manuscript and provided valuable comments. All authors read and approved the published version of the manuscript Funding: Supported by the State Drug Administration Key Laboratory Open Project (KF202105) Acknowledgments: The authors would like to sincerely thank the School of Pharmacy, Henan University of Traditional Chinese Medicine for their support. as well as the financial support and open projects from the State Drug Administration Key Laboratory. Conflicts of Interest: The authors declare no conflict of interest for the manuscript submission. All authors have seen and approved the final version of the manuscript being submitted. We warrant that the article is the authors’ original work, hasn’t received prior publication and isn’t under consideration for publication elsewhere. References Wen, C. M., Zhang, C. W., Xu, R.,Xu, C. Q.,Shen, G. R.,Suo, F. M., & Guo, B. L. (2022) Rapid prediction of flavonoid content in Epimedium sagittatum by infrared spectroscopy. Chinese Journal of Traditional Chinese Medicine , 47(22),6020-6026. DOI:10.19540/j.cnki.cjcmm.20220809.102 Xu, Y. Q., Li, R. Q., Zhang, H. Y.,Li, F. Q.,Jiang, Y., & Huang, X. F. (2020) Distribution of Epimedium sagittatum and its research progress on quality characteristics. Chinese Herbal Medicine , 51(23),6119-6132. DOI: https://kns.cnki.net/kcms/detail/detail.aspx?FileName=ZCYO202023027&DbName=DKFX2020 Wei, J. J., Zhang, J. K., Li, M.,Xie, S. S.,Tao, S. Q.,Yang, Y.,Yang, M.,Zhu, D. H.,Zheng, X. K., & Feng, W. S. (2023)A new flavonoid glycoside from Epimedium sagittatum. Acta Pharmaceutica Sinica , 58(01),180-185. DOI:10.16438/j.0513-4870.2022-1028 Wang, D., Pei, L. X., Ji, B. Y.,Wang, H. B.,Yang, L. L.,Zhong, H., & Dong, C. M. (2023)Optimum Induction Conditions of Epimedium Sagittatum Callus and its Browning Causes. Molecular Plant Breeding .DOI:1-10[2023-07-20].http ://kns.cnki.net/kcms/detail/46.1068.S.20230216.1055.008.html Li, H., Liu, Y. J., Jiang, W.,Xue, J. H., Cheng, Y. N.,Wang, J. Y.,Yang, R. X., & Zhang, X. W. (2021)Icaritin promotes apoptosis and inhibits proliferation by down-regulating AFP gene expression in hepatocellular carcinoma. BMC cancer , 21(1),318.DOI:10.1186/s12885-021-08043-9 Cui, X. P., Yan, B. B., Wan, X. F.,Sun, J. H.,Yang, J.,Sun, K., Wang, T. L., & Zhang, Y.(2022) Research Progress and Prospect of Cultivation of Medicinal Plants of Epimedii Folium. Chinese Modern Chinese Medicine, 24(04),705-714.DOI:10.13313/j.issn.1673-4890.20201028001. Bai, J. Y., Zhang, X. M., & Gao, Y. K. (2023)Studies on flowering characteristics and breeding system of Hemerocallis minor. Journal of China Agricultural University , 28(06),146-152. DOI: https://kns.cnki.net/kcms/detail/detail.aspx?FileName=NYDX202306013&DbName=CJFQ2023 Li, M. C., Jiang, F. F., Huang, L. B., Wang, H., Song, W. Q., Zhang, X. X., Zhang, Y. L.,& Niu, L. X. (2023). Optimization of In Vitro Germination, Viability Tests and Storage of Paeonia ostii Pollen. Plants , 12(13), 2460. DOI:10.3390/plants12132460 Xu, L., Liu, C. L., Wang, H. D., & Chen, K. L. (2012). Study on the pollen viability and stigma receptivity of Chrysanthemum morifolium'Fubaiju'. Zhong yao cai= Zhongyaocai= Journal of Chinese Medicinal Materials , 35(10), 1546-1550. Oct;35(10):1546-1550. PMID: 23627116. Liang, L.,Kong, D. Z., Liu, Z. X.,Li, H. C.,Liu, Q. Q.,Liu, F. L.,Zhang, D. S., & Tian, D. K. (2022) Comparative analysis of detection methods for lotus pollen viability. Jiangsu Agricultural Sciences ,50(02),131-136. DOI:10.15889/j.issn.1002-1302.2022.02.022. Song, J., Wang, J., Du, C.,Li, J.,Yu, X.,Luo, Z. H.,Pan, Y., & Zhang, P. Y.(2022)Analysis of Pollen Vitality Differences in Provenances of Peony Diannan at Different Altitudes. Journal of Plant Physiology , 58(09),1675-1684. DOI:10.13592/j.cnki.ppj.100069. Wang, J. H., Zhang, J. Q., Cheng, L. Q.,Lv, J. W., Hu, T. H., Rao, Q. L., Jiang, M., & Wang, J. (2022)Comparison of different methods for measuring peanut pollen viability. Seed ,41(08),126-130. DOI:10.16590/j.cnki.1001-4705.2022.08.126 Fu, Q. C., Wang, J., Zhang, X. Y., Liu, C., & Liu, F. (2015) Study on Detection Methods for Pollen Viability of Sinojackia sacocarpa L.Q.Luo. Molecular Plant Breeding , 13(05),1146-1150. DOI:10.13271/j.mpb.013.001146 Liu, X., Du, C. X., Zi, J., Xiao, Y. L., Yan, J.,Li, C. H.,Chen, D. D.,Wang, S. B., & Liang, Q. Z. (2022)Effects of temperature stress on pollen germination of mango "Renong 1". Fruit Trees in South China , 51(03),70-73+78. DOI:10.13938/j.issn.1007-1431.20210334 Yin, S. H., Wang, K., Huang, X. X.,Li, S. B., & Cheng, X. M.(2021) Evaluation of Cross Affinity Between Distant and Intervarietal Varieties of Old Chinese Rose. Northern Horticulture , (18),81-87.DOI:https://kns.cnki.net/kcms/detail/detail.aspx?FileName=BFYY202118013&DbName=CJFQ2021 Du, W. W., Duan, Q., Yang, N., Cai, J. J.,Lin, P. S., Ma, L. L., Wang, X. N., Jia, W. J., & Cui, G. F.(2021) Screening of Test Methods and Determination of Pollen Viability of 30 Varieties of Foliage Begonia. Southwest Agricultural Journal , 34(07),1521-1527. DOI:10.16213/j.cnki.scjas.2021.7.023 Ren, F. Y., Chen, X., & Wang, S. Q(2021)Comparative Study on the Determination Methods of Pollen Viability of Sonneratia ovata. Seed , 40(03),35-39. DOI:10.16590/j.cnki.1001-4705.2021.03.035 Liu, C. Q., Zhao, X. W., Kang, Z. Y., Ding, J. J.,Sun, Y. X., & Wang, J. J. (2021)Comparison of five methods for measuring pollen vigor of Tengguan vines. Journal of Yunnan Agricultural University (Natural Science), 36(01),179-182. https://kns.cnki.net/kcms2/article/abstract Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-3599092","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":249643745,"identity":"31a22898-a709-44d0-9cf5-43eed431b85a","order_by":0,"name":"Jianglong He","email":"","orcid":"","institution":"Henan University of Traditional Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jianglong","middleName":"","lastName":"He","suffix":""},{"id":249643746,"identity":"d7032006-bc8c-4805-be9f-bc9128856e9d","order_by":1,"name":"Lixin Pei","email":"","orcid":"","institution":"Henan University of Traditional Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lixin","middleName":"","lastName":"Pei","suffix":""},{"id":249643747,"identity":"bf91063f-4fd0-4f4a-94e8-e4d3ba02fb19","order_by":2,"name":"Baoyu Ji","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYBAC+/aGxAcJBjZybOwNRGox4Dnw2OBBRZoxP88BYrVIJD6TfHDmUOLMGQlEajHnOZxskNh2wNjg5uONNxhqbKIJarFsb0t8kNh2R87gdlqxBcOxtNwGgnrOnAHZ8szY4HaOmQRjw2EitNzI/yaR2HY4ccPNM0RqMbiRkCaRcOYw0Ps8RGqR7DmQbJAADmSgXxKI8Qs/e0Piwx/gqDy88caHGhsi/ILsSIkEUpRDtJCqYxSMglEwCkYGAABAhEkHdVwJjAAAAABJRU5ErkJggg==","orcid":"","institution":"Henan University of Traditional Chinese Medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Baoyu","middleName":"","lastName":"Ji","suffix":""},{"id":249643748,"identity":"76c48a41-ef0f-4a2f-81f0-968f64f1d515","order_by":3,"name":"Hai-bo Wang","email":"","orcid":"","institution":"Henan University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hai-bo","middleName":"","lastName":"Wang","suffix":""},{"id":249643749,"identity":"b50ab4bc-ec5e-44cf-aab7-217d4c2fc870","order_by":4,"name":"Hua Zhong","email":"","orcid":"","institution":"Rural Agriculture Bureau of Pingyu County","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hua","middleName":"","lastName":"Zhong","suffix":""},{"id":249643750,"identity":"5c0fa27f-25a7-4bd0-9eb4-fe114c53d0b4","order_by":5,"name":"Chengming Dong","email":"","orcid":"","institution":"Henan University of Traditional Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chengming","middleName":"","lastName":"Dong","suffix":""},{"id":249643751,"identity":"72a143b4-66a0-441c-88c3-d9026566ce4e","order_by":6,"name":"Suiqing Chen","email":"","orcid":"","institution":"Henan University of Traditional Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Suiqing","middleName":"","lastName":"Chen","suffix":""},{"id":249643752,"identity":"c71f72b2-44a4-4ab1-8e1f-9737cc2326e0","order_by":7,"name":"Xiuqing Li","email":"","orcid":"","institution":"Henan University of Traditional Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiuqing","middleName":"","lastName":"Li","suffix":""},{"id":249643753,"identity":"649eb4bc-3e44-409a-8dbe-30530c642907","order_by":8,"name":"Panpan Li","email":"","orcid":"","institution":"Henan University of Traditional Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Panpan","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2023-11-12 08:44:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3599092/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3599092/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":46674171,"identity":"c17f4d47-24bd-4c19-a22e-19481daf3203","added_by":"auto","created_at":"2023-11-17 19:01:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1018279,"visible":true,"origin":"","legend":"\u003cp\u003eFlowering pictures of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3599092/v1/7fcf39d3f3687d6f7db78e53.png"},{"id":46674172,"identity":"c7cb33c8-db02-430d-ad00-da666131c1a0","added_by":"auto","created_at":"2023-11-17 19:01:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":568743,"visible":true,"origin":"","legend":"\u003cp\u003eLocal plot of \u003cem\u003eEpimedium\u003c/em\u003e \u003cem\u003esagittatum \u003c/em\u003eflowers\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3599092/v1/27498d885891dd82df74d79c.png"},{"id":46674170,"identity":"b64de5ff-1063-4452-a6d0-950ada153ba2","added_by":"auto","created_at":"2023-11-17 19:01:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":663853,"visible":true,"origin":"","legend":"\u003cp\u003eBlossom process of \u003cem\u003eEpimedium\u003c/em\u003e \u003cem\u003esagittatum\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3599092/v1/9b5b5d8456f9072acfd5b00d.png"},{"id":46674173,"identity":"d10b603e-5085-4d5f-98e1-af0455533569","added_by":"auto","created_at":"2023-11-17 19:01:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2521394,"visible":true,"origin":"","legend":"\u003cp\u003ePollen grains under the different activity assays\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3599092/v1/eecca1153d94c587d347cb3a.png"},{"id":47069841,"identity":"bd50124f-ebea-4d2a-b978-333c9a2b141f","added_by":"auto","created_at":"2023-11-25 15:52:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4535607,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3599092/v1/9f2f5937-2063-4217-bc9b-6719f81eee40.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biological characteristics of flowers and examination of pollen viability at different developmental stages of Epimedium sagittatum( Sieb. et Zucc. )Maxim","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cem\u003eEpimedium sagittatum\u003c/em\u003e (Sieb. et Zucc.) Maxim. (also known as \u0026ldquo;three branches and nine leaves grass\u0026rdquo;) is one of the most important traditional medicinal plants in China. It belongs to \u003cem\u003eEpimedium\u003c/em\u003e, a perennial herbaceous plant in the Berberidaceae family (Wen et al.,2022). First recorded in Shennong's Classic of Materia Medica, it was listed as a top grade herb (Xu et al.,2020) with the effects of reinforcing kidney yang, strengthening muscles and bones, dispelling rheumatism, etc. It is often used to treat kidney yang deficiency, osteoporosis and other diseases (Wei et al.,2023; Wang et al.,2023). With the continuous in-depth research on \u003cem\u003eEpimedium\u003c/em\u003e in modern science, the latest research has found that it also has anti-tumor effects, especially on patients with advanced liver cancer (Li et al.,2021). This has led to an increasing demand for \u003cem\u003eEpimedium\u003c/em\u003e, and wild resources cannot meet the demand, resulting in rising prices. In order to obtain a large amount of \u003cem\u003eEpimedium\u003c/em\u003e, artificial cultivation has begun. Currently, \u003cem\u003eEpimedium\u003c/em\u003e seeds are expensive. In the cultivation process, it was discovered that although \u003cem\u003eEpimedium sagittatum\u003c/em\u003e has abundant flowering, its reproductive ability is low, the seed setting rate is low, and the germination rate is extremely low. Therefore, there are few seedlings in natural populations (Cui et al.,2022).\u003c/p\u003e \u003cp\u003eIt can be seen from the above that the weak reproductive ability and low seed germination rate of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e have always been a problem in \u003cem\u003eEpimedium\u003c/em\u003e cultivation. Studies have found that the size of pollen viability directly affects the pollination and fertilization processes of plants, which is closely related to the yield and quality of plant seeds (Bai et al.,2023). Testing the viability of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e pollen at different flowering stages before pollination can understand the viability, development and physiological characteristics of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e pollen, and grasp the morphology of sterile pollen, providing a theoretical basis for artificial breeding (Li et al.,2023; Xu et al.,2012). Therefore, researching the viability and germination characteristics of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e pollen is very necessary to improve pollination breeding efficiency and reasonably carry out breeding to increase seed germination rate and obtain high-quality \u003cem\u003eEpimedium sagittatum\u003c/em\u003e seeds. On this basis, it is of great value and significance to establish scientific and effective pollination methods for the future, accelerate the breeding process of excellent \u003cem\u003eEpimedium sagittatum\u003c/em\u003e varieties, shorten the differentiation period between varieties, and thus enhance the competitiveness of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e germplasm. At the same time, it can provide a basis for the construction of the \u003cem\u003eEpimedium sagittatum\u003c/em\u003e breeding system under artificial cultivation conditions and production application, thereby promoting the realization of maximized economic benefits in the large-scale production of \u003cem\u003eEpimedium sagittatum.\u003c/em\u003e However, research and reports on pollen viability detection of this species have not been seen so far.\u003c/p\u003e \u003cp\u003eThis paper systematically studies the flowering dynamics and floral characteristics of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e. It also determines the pollen viability at different developmental stages and screens suitable methods for rapid detection of pollen viability in Epimedium sagittatum. The aim is to deeply understand its flowering habits and reproductive characteristics, enrich data on flower development and reproductive regulation of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e, explore the limiting factors affecting its breeding process, discuss its breeding habits and pollination methods, and explore ways to improve the pollen viability of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e. This study aims to reveal the flowering mechanism of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e, clearly understand the physiological and biochemical characteristics of its pollen at different developmental stages, grasp the changes in pollen viability, thereby determine the optimal pollination time and pollen testing method. The purpose is to provide preliminary research basis for exploring artificial assisted fertilization technology, improving seed viability, guiding artificial cultivation management for high yield and efficiency, simplifying production procedures, and selecting new varieties in the future. This will provide preliminary research basis for renewing and reproductive biological studies as well as selecting new varieties of this species.\u003c/p\u003e"},{"header":"2. Experimental Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Experimental Materials\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.1 Sample Collection\u003c/h2\u003e \u003cp\u003eThe test site was located in the \u003cem\u003eEpimedium\u003c/em\u003e plantation base in Pingyu County, Henan Province, at east longitude 114\u0026deg;61' and north latitude 32\u0026deg;96'. The test materials were \u003cem\u003eEpimedium sagittatum\u003c/em\u003e seeded and nursed in October 2021 with good growth after routine field management. Pollen was collected at the peak flowering period on sunny mornings from 9:00\u0026ndash;10:00. Flowers at different blooming stages were collected and brought back to the lab for pollen extraction and testing.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Experimental Methods\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Observation of Floral Organ Characteristics, Flowering Habits and Dynamics\u003c/h2\u003e \u003cp\u003eFrom March to June 2023, 120 \u003cem\u003eEpimedium sagittatum\u003c/em\u003e plants were randomly selected and tagged in the base. The date when the first flower bloomed was recorded as the initial flowering period, the period when over 50% of flowers bloomed was recorded as peak flowering period, and the date when the last flower bloomed was recorded as final flowering period. At peak flowering, 60 freshly bloomed single flowers were randomly selected every day and observed 6 times. Observations were made every 2 hours from initial to full bloom until withering. The collected fresh flowers were placed under a SZX2-ILLTQ stereo microscope to measure and record the dimensions of each floral part.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Pollen Viability Testing\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section4\"\u003e \u003ch2\u003e2.2.2.1 Triphenyl Tetrazolium Chloride (TTC) Staining\u003c/h2\u003e \u003cp\u003eTTC is an oxidation-reduction dye with a standard oxidation potential of 80mV. It dissolves into a colorless solution in water. After reduction, it forms the red insoluble triphenyl formazan. Its reduction amount can indicate dehydrogenase activity. This substance is relatively stable and not easily oxidized, so TTC is widely used in enzyme experiments to determine pollen viability. Referring to Liang Lu (Ling et al.,2022) et al.'s method with improvements, 1\u0026ndash;2 drops of 0.5% TTC solution were dropped onto a concave slide. Pollen was sprinkled into the solution, mixed well, covered with a coverslip, and placed in a 35\u0026deg;C incubator for 1h. After taking out and microscopic examination, viable pollen appeared red, weakly viable pollen appeared light red, non-viable or sterile pollen was colorless.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section4\"\u003e \u003ch2\u003e2.2.2.2 I\u003csub\u003e2\u003c/sub\u003e-KI Staining\u003c/h2\u003e \u003cp\u003eThe starch content in pollen can be used as a criterion to judge the level of pollen development. Normal mature pollen is mostly spherical with high starch content and can be stained blue by I\u003csub\u003e2\u003c/sub\u003e-KI solution. Underdeveloped pollen is mostly deformed and generally contains no or little starch. It usually does not contain starch or contains relatively less starch and will not be stained or will be stained yellow or yellowish brown by I\u003csub\u003e2\u003c/sub\u003e-KI solution. According to pollen staining, pollen viability can be identified. Referring to Song Jing (Song et al.,2022) et al.\u0026rsquo;s method with improvements, 1\u0026ndash;2 drops of I\u003csub\u003e2\u003c/sub\u003e-KI solution were dropped onto a concave slide. Pollen was sprinkled into the solution, mixed well, covered with a coverslip, and stained for 5 min before microscopic examination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section4\"\u003e \u003ch2\u003e2.2.2.3 Red Ink Staining\u003c/h2\u003e \u003cp\u003eThe protoplasm of viable pollen cells has selective permeability and selective absorption capacity for external substances, such as red ink dyes cannot enter the cells and the pollen cannot be stained. However, the protoplasmic membrane of lifeless pollen cells will lose this activity, dyes will enter the cells and stain them. Therefore, pollen viability can be judged by whether pollen grains are stained. Referring to Wang Jinhua (Wang et al.,2022) et al.\u0026rsquo;s method with improvements, 5% red ink solution was prepared, 1\u0026ndash;2 drops were dropped onto a concave slide, pollen was sprinkled into the solution, mixed well, covered with a coverslip, and examined under the microscope immediately. Viable pollen was not stained while non-viable pollen was stained red.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section4\"\u003e \u003ch2\u003e2.2.2.4 Peroxidase Method\u003c/h2\u003e \u003cp\u003ePeroxidase is contained in pollen. Pollen with higher viability has stronger peroxidase activity. Peroxidase forms a complex with oxidants. The activated hydrogen peroxide in the complex can oxidize phenolic compounds. Pollen viability can be judged by the color change. Viable pollen is purple-red, while pollen with weaker viability or no viability is light red or colorless. Referring to Fu Qinchao (Fu et al.,2015) et al.\u0026rsquo;s method with improvements, 1 drop of aromatic amine coloring solution (0.5% aniline dye solution : 0.5% α-naphthol dye solution : aromatic buffer\u0026thinsp;=\u0026thinsp;1:1:1) and 0.1% hydrogen peroxide each were dropped onto a concave slide. Pollen was sprinkled into the solution, mixed well, covered with a coverslip, and kept at 30\u0026deg;C for 15 min before microscopic examination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section4\"\u003e \u003ch2\u003e2.2.2.5 In Vitro Germination\u003c/h2\u003e \u003cp\u003eNormal mature pollen grains have strong viability. They can germinate and grow under suitable culture conditions. Germination number can be directly observed and counted under the microscope to calculate germination rate and determine pollen viability. Referring to Liu Xinyu (Liu et al.,2022) et al.\u0026rsquo;s method with improvements, prepared pollen germination culture medium was dropped onto concave slides. Pollen was sprinkled into the medium, mixed well, covered with coverslips, and placed in a germination box lined with wet filter paper. It was cultured in a 25\u0026deg;C incubator and examined under the microscope every 1 hour to count germinated pollen. Pollen with pollen tube length exceeding pollen diameter was considered as germinated.\u003c/p\u003e \u003cp\u003eGermination rate (%) = (number of germinated pollen grains / total number of pollen grains) \u0026times; 100%\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Data Statistics\u003c/h2\u003e \u003cp\u003eThree slides were prepared for each treatment and observed under a BX53F2 optical microscope. 5 visual fields were selected on each slide, the total number of pollen grains counted was no less than 500. Pollen viability was calculated:\u003c/p\u003e \u003cp\u003ePollen viability (%) = (number of viable pollen / total number of pollen) \u0026times; 100%\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Data Processing\u003c/h2\u003e \u003cp\u003eThe experimental data obtained were processed using Excel 2020 and SPSS 26 software.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Analysis","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Comprehensive Characteristics of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e Flowers\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1 Biological Characteristics\u003c/h2\u003e \u003cp\u003e \u003cem\u003eEpimedium sagittatum\u003c/em\u003e is a long-day shade-loving plant(Figure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The flowering period is from March to June each year. The inflorescence is an indefinite panicle with numerous branches on the inflorescence axis. Each branch has three flowers and the terminal has one flower. The calyx has two whorls of four sepals each. The outer whorl is green with purple spots while the inner whorl is white. After blooming, the outer calyx falls off and the inner calyx opens up. The four petals are yellow in color. The four stamens grow out of the bud before the pistil. The style is green and the ovary is green with purple spots. After pollination, the ovary enlarges and the style gradually elongates, exceeding the stamens.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2 Floral Morphology\u003c/h2\u003e \u003cp\u003e \u003cem\u003eEpimedium sagittatum\u003c/em\u003e has a terminal panicle inflorescence, Flower organ parameter display (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)10\u0026ndash;35 cm long, with 20\u0026ndash;50 flowers. The single flower is a complete flower without nectaries. The calyx has two whorls of four sepals each. The first whorl is green with purple spots, with 1 pair narrowly ovate and 1 pair oblong-ovate. The second whorl is white. After fully opening, the first whorl of sepals falls off. The lobes are triangular and reflexed. The inflorescence axis, pedicels and calyx surface all have purple spots. The petals are yellow or pale yellow, with 4 petals in a saccate, oblong or obovate shape. There are 4 stamens with yellow anthers in a cruciform position and purple-red filaments. There is 1 pistil with a purple-red cylindrical style and dark green stigma. When mature, the stigma surface secretes to adsorb pollen. The stigma position is about 8 mm higher than the anthers. The ovary is semi-inferior and spherical with 6\u0026ndash;7 locules(Figure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\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\u003eOrgan parameters of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e flowers\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFloral Organ Parameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSize/mm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStandard Deviation/mm\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePetal length\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePetal width\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFilament length\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStyle length\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlower diameter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCalyx diameter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOvary length\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.26\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=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e3.1.3 Flowering Dynamics Observation\u003c/h2\u003e \u003cp\u003eThe flowering period of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e is concentrated in March to June, with peak flowering in April. The overall flowering pattern shows concentrated mass flowering. It takes 2\u0026ndash;4 hours from bud split to complete opening of the corolla. The average flowering period of the population is about 120 days, the flowering period of a single inflorescence is 10\u0026ndash;20 days, and the duration of a single flower is 2\u0026ndash;5 days. Through observation, it was found that the blooming process of a single \u003cem\u003eEpimedium sagittatum\u003c/em\u003e flower can be divided into 5 stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003e): (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Sepal splitting stage - the bud swells and the sepals gradually split, exposing the internal tissues; (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) Stamen protrusion stage - the stamens break through the bud and are exposed outside; (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) Petal expansion stage - the petals push open the bud and unfold outwards, at this time the outer sepals fall off; (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) Peak pollen release stage - the flower is fully open, the pistil and stamens are completely exposed, and the anthers begin to release pollen; (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) Flower withering stage - the petal color gradually fades, the anthers begin to brown, and gradually wither and fall off, the ovary swells, and the style elongates.\u003c/p\u003e \u003cp\u003eIt takes about 1 hour for the \u003cem\u003eEpimedium sagittatum\u003c/em\u003e calyx to fully split. Then the stamens protrude through the bud with the stamen cluster spreading out. After about 1 hour, the yellow anthers begin to release pollen. At this time, the flower starts to bloom. After 1\u0026ndash;2 hours, it fully blooms and enters the peak flowering period. At this time, pollen release decreases and the anthers begin to fall off. After blooming for 1\u0026ndash;2 days, there is no significant change in the style, and the edges of the petals begin to wither. On the 2nd day after blooming, the filaments become dry and withered, on the 3rd day after blooming, the petals begin to fall off, the filaments become withered and curved shortened, the anthers gradually turn brown and begin to shrivel and fall off, the ovary begins to swell, and the style begins to elongate and gradually turns brown. On the 5th day after blooming, all petals have fallen off, the ovary sits on the fruit and gradually swells.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Comparison of Different Pollen Viability Testing Methods\u003c/h2\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1 TTC Staining\u003c/h2\u003e \u003cp\u003eThe TTC staining effect on \u003cem\u003eEpimedium sagittatum\u003c/em\u003e pollen is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eB. After TTC staining, all pollen turned yellowish brown, and no red pollen was observed. The pollen viability was 0, indicating that TTC staining does not easily stain \u003cem\u003eEpimedium sagittatum\u003c/em\u003e pollen. Therefore, it is not suitable for determining the viability of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e pollen.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2 I\u003csub\u003e2\u003c/sub\u003e-KI Staining\u003c/h2\u003e \u003cp\u003eThe I\u003csub\u003e2\u003c/sub\u003e-KI staining effect on \u003cem\u003eEpimedium sagittatum\u003c/em\u003e pollen viability is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eC. Viable pollen with high starch content is stained red-brown, while non-viable pollen with low starch content is not stained and appears yellow-brown. The pollen viability measured by I\u003csub\u003e2\u003c/sub\u003e-KI staining at 5 stages was 75.26%, 74.30%, 93.56%, 96.54% and 95.85%, respectively. The pollen viability was lowest at the stamen protrusion stage at 74.30%; and highest at the peak pollen release stage at 96.53% (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The I\u003csub\u003e2\u003c/sub\u003e-KI staining is fast, simple to operate, and the difference between viable and non-viable pollen is obvious. It is suitable for determining the viability of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e pollen.\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\u003eStatistics of pollen viability at different developmental stages of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e were examined by I\u003csub\u003e2\u003c/sub\u003e-KI staining\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSepal splitting stage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eStamen protrusion stage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePetal expansion stage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePeak pollen release stage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eFlower withering stage\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAverage value(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e74.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e93.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e96.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e95.85\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaximum value(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e90.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e94.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMinimum value(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e43.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e50.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e90.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e88.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e93.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStandard deviation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e14.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.192\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.485\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.084\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=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e3.2.3 Red Ink Staining\u003c/h2\u003e \u003cp\u003eThe red ink staining effect on \u003cem\u003eEpimedium sagittatum\u003c/em\u003e pollen viability is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eD. Viable pollen has selective permeability of the cell membrane and cannot be stained by red ink, while non-viable pollen has complete permeability and is stained red. The pollen viability measured by red ink staining at the 5 developmental stages of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e was 84.73%, 44.99%, 24.30%, 31.76%, and 10.26%, respectively. The pollen viability was lowest at the flower withering stage, with the minimum value of 10.26%; and highest at the sepal splitting stage, with the maximum value of 84.73% (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Red ink staining is fast, simple to operate, and the difference between viable and non-viable pollen is obvious. It is suitable for determining the viability of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e pollen.\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\u003eStatistics of pollen viability at different developmental stages of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e were examined by red ink staining\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSepal splitting stage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStamen protrusion stage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePetal expansion stage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePeak pollen release stage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFlower withering stage\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAverage value(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e84.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e31.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaximum value(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e96.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e48.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e20.83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMinimum value(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStandard deviation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.79\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=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e3.2.4 Peroxidase Method\u003c/h2\u003e \u003cp\u003eThe peroxidase staining effect on \u003cem\u003eEpimedium sagittatum\u003c/em\u003e pollen viability is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eE. Viable pollen has strong peroxidase activity and reacts with oxidants to appear purple-red, while pollen with weak or no activity appears light red or colorless. The pollen viability measured by the peroxidase method at the 5 developmental stages of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e was 99.23%, 81.51%, 99.30%, 19.93%, and 13.17%, respectively. The pollen viability was lowest at the flower withering stage, with the minimum value of 13.17%; and highest at the petal expansion stage, with the maximum value of 99.30% (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This method is slower, and the difference between viable and non-viable pollen is obvious, but it is easily interfered by other parts of the flower and is not suitable for determining the viability of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e pollen.\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\u003eStatistics of pollen viability of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e by peroxidase method\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSepal splitting stage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStamen protrusion stage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePetal expansion stage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePeak pollen release stage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFlower withering stage\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAverage value(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e81.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaximum value(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e88.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e41.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e50.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMinimum value(%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e96.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e68.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStandard deviation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.438\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.075\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.398\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14.69\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=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e3.2.5 In Vitro Pollen Germination Test\u003c/h2\u003e \u003cp\u003eThe germination of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e pollen on the germination medium is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eF. The 1\u0026ndash;8 hour germination rates of pollen at 5 developmental stages of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e are shown in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. In vitro germination can directly observe pollen germination. When \u003cem\u003eEpimedium sagittatum\u003c/em\u003e pollen at each stage was cultured by in vitro germination for 1\u0026ndash;7 hours, due to the different pollen conditions, the time required for germination was also different and could not fully reflect the pollen germination. When the pollen was germinated for 7\u0026ndash;8 hours, the basically fully germinated pollen with stronger activity, and the germination rate was relatively stable. The measured pollen viability at the 5 developmental stages of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e was 20.35%, 48.14%, 43.81%, 53.86% and 7.13%, respectively. The pollen viability was lowest at the flower withering stage, with the minimum value of 7.13%; and highest at the stamen protrusion stage, with the maximum value of 48.13% (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The in vitro pollen germination test results are relatively accurate, but this method requires higher cultivation temperature, nutrient composition, and pH value, and takes a longer time.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIn vitro germination rate of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e pollen from 1 to 8 h at different stages\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSepal splitting stage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStamen protrusion stage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePetal expansion stage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePeak pollen release stage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFlower withering stage\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.00%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.26%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.93%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.00%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.21%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.31%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.13%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.30%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.33%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.51%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.11%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.61%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.13%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.51%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35.70%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.53%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e31.67%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.57%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15.67%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46.74%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35.79%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e36.21%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.77%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20.31%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48.12%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43.74%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e53.76%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.98%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20.33%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48.13%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43.76%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e53.78%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.04%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20.35%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48.14%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43.81%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e53.86%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.13%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStandard deviation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6,75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.15\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=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e3.2.6 Comparison of Different Pollen Viability Testing Methods\u003c/h2\u003e \u003cp\u003ePollen viability directly affects the results of fertilization and fruit setting rate after hybridization pollination. Varieties with higher pollen viability are preferred as male parents, which is more conducive to the successful breeding of new varieties (Yin et al.,2021). Currently, there are many methods for determining pollen viability, but most of the pollen testing methods obtain higher pollen viability than the actual viability. Due to the simple operation, less test time consumption, and ability to determine pollen viability in a short time, pollen staining methods are widely used in pollen viability detection of various plants (Liang et al.,2022). Du Wenwen et al. concluded through the determination of pollen viability in Camellia that pollen staining was more suitable than I\u003csub\u003e2\u003c/sub\u003e-KI staining and TTC staining (Du et al.,2021). Ren Feiyan et al. used TTC staining, MTT staining, Alexander staining, I\u003csub\u003e2\u003c/sub\u003e-KI staining, red ink staining and in vitro germination to determine the pollen viability of Morus alba, and believed that Alexander staining and I\u003csub\u003e2\u003c/sub\u003e-KI staining were more suitable (Ren et al.,2021). Liu Chengqin et al. used TTC staining, acridine red staining, I\u003csub\u003e2\u003c/sub\u003e-KI staining, peroxidase staining and in vitro pollen germination to determine the pollen viability of Cuscuta chinensis, and believed that I\u003csub\u003e2\u003c/sub\u003e-KI staining was the most suitable method for determining the viability of Cuscuta chinensis pollen (Liu et al.,2021). In addition, in vitro culture is another method for determining pollen viability. This method can directly observe pollen germination and gives more reliable results than pollen staining methods. It can be seen that different detection methods are suitable for different plants.\u003c/p\u003e \u003cp\u003eThe results of this test found that in vitro germination can directly observe pollen germination. In this test, the pollen of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e at five developmental stages can germinate well after 8 hours of cultivation, and the germination rate is relatively stable. It is the preferred method for determining pollen viability, but this method has complex operation and long time consumption, and is not suitable for rapid detection of pollen viability. The staining method has the advantages of being fast and simple. It can reflect the viability of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e pollen to some extent, but is greatly affected by pollen characteristics. Different staining methods are suitable for different plants. TTC staining did not stain \u003cem\u003eEpimedium\u003c/em\u003e pollen, and the measured pollen viability values were all 0, so pollen viability comparison analysis was not performed. As can be seen from Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, the pollen viability values measured by different methods vary greatly, with the order of peroxidase method\u0026thinsp;\u0026gt;\u0026thinsp;I\u003csub\u003e2\u003c/sub\u003e-KI staining\u0026thinsp;\u0026gt;\u0026thinsp;pollen germination\u0026thinsp;\u0026gt;\u0026thinsp;red ink staining. Significance analysis showed extremely significant differences among the 5 methods. The results of I\u003csub\u003e2\u003c/sub\u003e-KI staining and peroxidase method were significantly higher than red ink staining and in vitro germination. The results of red ink staining were closer to in vitro germination, and the staining effect was good and the operation was simple. It is the most suitable method for rapid detection of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e pollen viability.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of results of different assays for pollen vitality in \u003cem\u003eEpimedium sagittatum\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDetermination method\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSepal splitting stage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStamen protrusion stage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePetal expansion stage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePeak pollen release stage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFlower withering stage\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePollen germination method\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e53.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePeroxidase method\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e81.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRed ink staining method\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e84.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e31.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI\u003csub\u003e2\u003c/sub\u003e-KI staining method\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e74.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e93.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e96.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e95.85\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTTC staining method\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\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 \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Analysis of Pollen Viability at Different Developmental Stages\u003c/h2\u003e \u003cp\u003eThe results of this test found that the pollen viability of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e at different stages has extremely significant differences. From the results of the pollen germination test, it can be seen that the pollen germination rates of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e were higher during the stamen protrusion stage, petal expansion stage and peak pollen release stage. At this time, the pollen of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e has better pollination, but from the results of the pollen staining method, it can be seen that the pollen viability was also very high during the sepal splitting stage. Combined with microscopic observation, it was found that although the pollen viability was high at this time, the deformity rate was higher, up to more than 70%. If pollination succeeded at this time, it may lead to low germination rate of pollinated seeds. Therefore, the optimal pollination period of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e should be the stamen protrusion stage, petal expansion stage and peak pollen release stage. Through the pollen germination test, it was found that the pollen germination rate of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e was low, and the highest germination rate was only 53.86%. From the flowering process of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003e), it can be seen that the flowering of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e is protandrous with stamens protruding first and then pistil exposed for pollination. The pollen viability was stronger when the stamens protruded through the buds. Due to the different protruding times of stamens and pistils, the optimal pollination time may be missed. Combined with the investigation of local climate and ecology, due to temperature reasons, wild pollinating insects such as bees and butterflies in Pingyu, Henan Province were not active from March to April, making cross-pollination relatively difficult, resulting in poor fruit setting rate and seed germination rate of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e. Therefore, means such as artificial beekeeping can be adopted to increase the success rate of cross-pollination of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e, so as not to miss the optimal pollination time, thus enabling \u003cem\u003eEpimedium sagittatum\u003c/em\u003e to breed high-viability, high-quality seeds.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn summary, the flowering period of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e is from March to June each year, with peak flowering in April. Its flowers are in panicles. Through observation, it was found that the blooming process of a single \u003cem\u003eEpimedium sagittatum\u003c/em\u003e flower can be divided into 5 stages: sepal splitting stage, stamen protrusion stage, petal expansion stage, peak pollen release stage and flower withering stage. Experiments found that in vitro germination was the most accurate and effective method for detecting the pollen viability of \u003cem\u003eEpimedium sagittatum.\u003c/em\u003e TTC staining, peroxidase method and I\u003csub\u003e2\u003c/sub\u003e-KI staining were not suitable for determining pollen viability. Red ink staining had simple operation and obvious staining effect and was a good method for rapid detection of pollen viability. Among them, the detection results of red ink staining were closest to those of in vitro germination, making it the most suitable method for rapid detection of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e pollen viability. Combined with the flowering process and pollen viability at different developmental stages of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e, it was found that the optimal pollination period of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e should be the stamen protrusion stage, petal expansion stage and peak pollen release stage. It was also found that the pollen viability of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e was low, and the different protruding times of stamens and pistils may lead to missing the optimal pollination time. In addition, due to local climatic reasons, wild pollinating insects such as bees and butterflies were not active, resulting in lower cross-pollination rates and poorer fruit setting rate and seed germination rate of Epimedium sagittatum. Therefore, it is proposed to use artificial beekeeping to achieve cross-pollination and avoid missing the optimal pollination time, thereby increasing the success rate of cross-pollination of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e. The flowering results and related sexual reproduction mechanisms of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e in this study can provide reference for further research on the breeding of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e, in order to lay the foundation for hybrid breeding and cultivation promotion of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eB.J. and J.H. conceived and designed the experiments, J.H. and X.L. participated in the execution of the experiments, J.H. analysed the experiments, L.P. and J.H. supervised and completed the writing, P.L. and H.W. assisted in the design of the figures and tables, H.Z. reviewed the references, C.D. and S.C. reviewed the manuscript and provided valuable comments. All authors read and approved the published version of the manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eSupported by the State Drug Administration Key Laboratory Open Project (KF202105)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eThe authors would like to sincerely thank the School of Pharmacy, Henan University of Traditional Chinese Medicine for their support. as well as the financial support and open projects from the State Drug Administration Key Laboratory.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare no conflict of interest for the manuscript submission. All authors have seen and approved the final version of the manuscript being submitted. We warrant that the article is the authors\u0026rsquo; original work, hasn\u0026rsquo;t received prior publication and isn\u0026rsquo;t under consideration for publication elsewhere.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWen, C. M., Zhang, C. W., Xu, R.,Xu, C. Q.,Shen, G. R.,Suo, F. M., \u0026amp; Guo, B. L. (2022) Rapid prediction of flavonoid content in Epimedium sagittatum by infrared spectroscopy. \u003cem\u003eChinese Journal of Traditional Chinese Medicine\u003c/em\u003e, 47(22),6020-6026. DOI:10.19540/j.cnki.cjcmm.20220809.102\u003c/li\u003e\n\u003cli\u003eXu, Y. Q., Li, R. Q., Zhang, H. Y.,Li, F. Q.,Jiang, Y., \u0026amp; Huang, X. F. (2020) Distribution of Epimedium sagittatum and its research progress on quality characteristics.\u003cem\u003eChinese Herbal Medicine\u003c/em\u003e, 51(23),6119-6132. 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H.,Pan, Y., \u0026amp; Zhang, P. Y.(2022)Analysis of Pollen Vitality Differences in Provenances of Peony Diannan at Different Altitudes.\u003cem\u003eJournal of Plant Physiology\u003c/em\u003e, 58(09),1675-1684. DOI:10.13592/j.cnki.ppj.100069.\u003c/li\u003e\n\u003cli\u003eWang, J. H., Zhang, J. Q., Cheng, L. Q.,Lv, J. W., Hu, T. H., Rao, Q. L., Jiang, M., \u0026amp; Wang, J. (2022)Comparison of different methods for measuring peanut pollen viability.\u003cem\u003eSeed\u003c/em\u003e,41(08),126-130. DOI:10.16590/j.cnki.1001-4705.2022.08.126\u003c/li\u003e\n\u003cli\u003eFu, Q. C., Wang, J., Zhang, X. Y., Liu, C., \u0026amp; Liu, F. (2015) Study on Detection Methods for Pollen Viability of Sinojackia sacocarpa L.Q.Luo. \u003cem\u003eMolecular Plant Breeding\u003c/em\u003e, 13(05),1146-1150. DOI:10.13271/j.mpb.013.001146\u003c/li\u003e\n\u003cli\u003eLiu, X., Du, C. X., Zi, J., Xiao, Y. L., Yan, J.,Li, C. H.,Chen, D. D.,Wang, S. B., \u0026amp; Liang, Q. Z. (2022)Effects of temperature stress on pollen germination of mango \u0026quot;Renong 1\u0026quot;. \u003cem\u003eFruit Trees in South China\u003c/em\u003e, 51(03),70-73+78. DOI:10.13938/j.issn.1007-1431.20210334\u003c/li\u003e\n\u003cli\u003eYin, S. H., Wang, K., Huang, X. X.,Li, S. B., \u0026amp; Cheng, X. M.(2021) Evaluation of Cross Affinity Between Distant and Intervarietal Varieties of Old Chinese Rose.\u003cem\u003eNorthern Horticulture\u003c/em\u003e, (18),81-87.DOI:https://kns.cnki.net/kcms/detail/detail.aspx?FileName=BFYY202118013\u0026amp;DbName=CJFQ2021\u003c/li\u003e\n\u003cli\u003eDu, W. W., Duan, Q., Yang, N., Cai, J. J.,Lin, P. S., Ma, L. L., Wang, X. N., Jia, W. J., \u0026amp; Cui, G. F.(2021) Screening of Test Methods and Determination of Pollen Viability of 30 Varieties of Foliage Begonia. \u003cem\u003eSouthwest Agricultural Journal\u003c/em\u003e, 34(07),1521-1527. DOI:10.16213/j.cnki.scjas.2021.7.023\u003c/li\u003e\n\u003cli\u003eRen, F. Y., Chen, X., \u0026amp; Wang, S. Q(2021)Comparative Study on the Determination Methods of Pollen Viability of Sonneratia ovata.\u003cem\u003eSeed\u003c/em\u003e, 40(03),35-39. DOI:10.16590/j.cnki.1001-4705.2021.03.035\u003c/li\u003e\n\u003cli\u003eLiu, C. Q., Zhao, X. W., Kang, Z. Y., Ding, J. J.,Sun, Y. X., \u0026amp; Wang, J. J. (2021)Comparison of five methods for measuring pollen vigor of Tengguan vines.\u003cem\u003eJournal of Yunnan Agricultural University (Natural Science),\u003c/em\u003e36(01),179-182. https://kns.cnki.net/kcms2/article/abstract\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Epimedium sagittatum, Different developmental stages, Floral characteristics, Pollen viability, Determination method","lastPublishedDoi":"10.21203/rs.3.rs-3599092/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3599092/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo gain a deeper understanding of the flowering pattern and reproductive characteristics of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e, to enrich the research on the flower development of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e and its reproductive regulation, and to screen the methods suitable for the rapid detection of pollen viability of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e and to promote its cross-breeding. The characteristics of its flower parts were observed, recorded and measured, and the pollen viability of \u003cem\u003eEpimedium sagittatumwas\u003c/em\u003e determined by five methods, including TTC staining, I\u003csub\u003e2\u003c/sub\u003e-KI staining, red ink staining, peroxidase method and in vitro germination method. The flowering process of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e can be divided into five stages: calyx dehiscence, bract spathe, petal outgrowth, pollen dispersal, and pollination and withering. The results of I\u003csub\u003e2\u003c/sub\u003e-KI staining and peroxidase method were significantly higher than those of other methods; the in vitro germination method was intuitive and accurate, but the operation was complicated and time-consuming; the red ink staining method was easy to operate and had obvious staining effect, and the results were the closest to those of the in vitro germination method; and it was found that the pollen of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e was not as effective as the in vitro germination method at the bud stamen stage, the flower stigma and the flower bud. It was also found that the pollen viability and germination rate of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e pollen were higher in the three periods of bud spitting, petal adductor and pollen dispersal. Comparing the five methods, the red ink staining method was found to be a better method for the rapid detection of pollen viability; the best pollination periods of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e were the bud stamen stage, petal adductor stage, and pollen dispersal stage of flowers at the peak of bloom. This study on the flowering and fruiting pattern of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e, and the related mechanism of sexual reproduction, can be used as a reference for the next step of research on the breeding of \u003cem\u003eEpimedium sagittatum\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Biological characteristics of flowers and examination of pollen viability at different developmental stages of Epimedium sagittatum( Sieb. et Zucc. )Maxim","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-11-17 19:01:12","doi":"10.21203/rs.3.rs-3599092/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b095f37e-c1ff-4276-bed5-12ffdec5c7b0","owner":[],"postedDate":"November 17th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-12-21T03:59:15+00:00","versionOfRecord":[],"versionCreatedAt":"2023-11-17 19:01:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3599092","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3599092","identity":"rs-3599092","version":["v1"]},"buildId":"ApUGefWb6u5IBVtyqm6d5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}