Xanthoceras sorbifolia bunge pollen and preparation method and application thereof

Abstract

The application provides a Xing-guan fruit pollen and a preparation method and application thereof, and belongs to the technical field of commercial pollen preparation.The preparation method of the Xing-guan fruit pollen provided by the application comprises the following steps: drying male inflorescences in the middle flowering stage at 35 DEG C for 36 hours, crushing and sieving, and collecting the sieved substance as the pollen.The preparation method of the Xing-guan fruit pollen provided by the application can obtain a large amount of pollen with high activity, can meet the pollen amount required by artificial assisted pollination or mechanical pollination of Xing-guan fruit, and is helpful to realize large-area high-yield and stable yield of Xing-guan fruit.The preparation method provided by the application can not only obtain a large amount of Xing-guan fruit pollen with high activity in a short time, but also can maintain high activity of the pollen in a long-term storage process, and promotes the development of Xing-guan fruit commercial pollen.

Description

Technical Field

[0001] This invention belongs to the field of commercial pollen preparation technology, and particularly relates to a type of *Sapindus mukorossi* pollen, its preparation method, and its application. Background Technology

[0002] Sapindus mukorossi ( Xanthoceras sorbifolium *Bunge* is a species of *Sapindaceae* family, genus *Sapindus*. Xanthoceras *Xanthoceras sorbifolium* is a deciduous shrub or small tree, also known as Chinese privet, papaya, *Xanthoceras sorbifolium*, cliff papaya, and *Xanthoceras sorbifolium*. In production, *Xanthoceras sorbifolium* suffers from low fruit set and unstable yield. This is mainly due to its reproductive biology: under natural conditions, it relies primarily on insect and wind pollination, and the pollination process is easily affected by the environment, resulting in low natural pollination efficiency, typically less than 30%. Furthermore, *Xanthoceras sorbifolium* is a monoecious plant with separate male and female flowers, exhibiting self-incompatibility, further limiting the fruit set rate. In addition, its stigma is of the dry stigma type, meaning the pollination period is mainly concentrated on the day of flowering and one day after flowering, resulting in a short effective pollination window. Artificial pollination is an effective way to improve the pollination rate, fruit set rate, and yield of *Xanthoceras sorbifolium*. Obtaining sufficient, highly viable pollen is the core prerequisite for ensuring the standardized implementation of artificial pollination techniques. Simultaneously, by collecting pollen artificially for targeted pollination, the cross-pollination rate can be precisely controlled, further enhancing the yield and quality improvement effects. However, there are some technical challenges in collecting pollen from *Xanthoceras sorbifolium*: the inflorescence of *Xanthoceras sorbifolium* is a raceme, which develops apically. The flowers open sequentially from the base to the top along the inflorescence axis, resulting in significant heterogeneity in the development process and pollen shedding time of different male flowers within the same inflorescence. This makes it difficult to achieve centralized and batch collection of pollen, resulting in low pollen collection efficiency and seriously restricting the large-scale promotion and widespread application of artificial pollination technology in the cultivation and production of *Xanthoceras sorbifolium*.

[0003] Therefore, solving the technical challenges of collecting pollen from *Xanthoceras sorbifolium* and developing efficient pollen collection technology can provide sufficient high-vitality pollen for artificial pollination. Artificial pollination can significantly improve the pollination rate of *Xanthoceras sorbifolium*, thereby increasing the fruit set rate and yield, which is an important technical foundation for achieving high and stable yields.

[0004] Pollen viability, defined as "the ability of pollen grains to transport sperm cells to the embryo sac after compatibility pollination" and "the ability of pollen grains to persist in the environment while maintaining their germination ability and producing pollen tubes with pointed growth on an acceptable stigma," is a core indicator for measuring the developmental quality of male gametophytes and pollination and fertilization potential, and is crucial for breeding and seed production. In hybridization breeding, pollen viability detection is an indispensable part, and rapid determination of pollen viability before artificial pollination has significant guiding significance for breeding work. Currently, many methods exist for determining pollen viability, but these methods lack universality for different species. Therefore, studying the pollen viability of a specific species requires first screening for a suitable method for measuring that species' pollen viability. Different staining methods target different points, resulting in significant differences in the accuracy and inter-species applicability of their results. Some methods suffer from blurred staining boundaries, susceptibility to environmental interference, and the likelihood of false positives or false negatives. Therefore, screening for suitable and precise staining methods for specific species is a prerequisite for pollen viability research. Currently, in studies on pollen viability of Xanthoceras sorbifolium, different scholars use inconsistent staining methods, and there is a lack of systematic screening of optimal viability detection methods for Xanthoceras sorbifolium pollen characteristics, which to some extent affects the reliability and comparability of pollen viability-related research results. Summary of the Invention

[0005] In view of this, one of the objectives of this invention is to provide a suitable method for determining the pollen viability of *Xanthoceras sorbifolium*, with accurate results and no false positives or false negatives, thus providing precise and reliable technical support for subsequent pollen viability testing.

[0006] The second objective of this invention is to provide a method for preparing *Xanthoceras sorbifolium* pollen, which can obtain a large quantity of highly viable pollen, meeting the pollen requirements for artificial or mechanical pollination of *Xanthoceras sorbifolium*, and helping to achieve large-scale, high-yield, and stable production of *Xanthoceras sorbifolium*.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for determining the pollen viability of *Xanthoceras sorbifolium*, including the in vitro germination liquid culture medium method and / or the TTC staining method; The in vitro germination liquid culture medium method includes the following steps: placing *Xanthoceras sorbifolium* pollen in a liquid culture medium and culturing it at 24℃~26℃ for 3~4 hours, and calculating the pollen germination rate; the liquid culture medium includes 150 g / L sucrose, 0.15 g / L boric acid and 0.2 g / L calcium nitrate, and the pH value of the liquid culture medium is 7.0; The TTC staining method includes the following steps: staining the pollen of *Xanthoceras sorbifolium* with 0.5% TTC staining solution; viable pollen is stained red, while inactive pollen is not stained and appears yellow; and the percentage of viable pollen is counted.

[0008] Preferably, when performing TTC staining, the staining temperature is 35℃~40℃ and the staining time is 15 min~30 min.

[0009] This invention also provides the application of the above-mentioned determination method in the preparation of *Xanthoceras sorbifolium* pollen.

[0010] The present invention also provides a method for preparing pollen from *Xanthoceras sorbifolium*, comprising the following steps: drying male inflorescences at mid-flowering stage at 35°C for 36 hours, pulverizing and sieving, and collecting the sieved material as pollen.

[0011] Preferably, the mid-flowering period is when 45% to 55% of the flower buds of the male inflorescence have opened; the male inflorescence is a lateral male inflorescence.

[0012] Preferably, the method further includes a step of measuring the viability of the prepared Xanthoceras sorbifolium pollen using the above-described method.

[0013] Preferably, the sieving is performed by sequentially passing through 80-mesh, 150-mesh, and 300-mesh sieves.

[0014] The present invention also provides *Xanthoceras sorbifolium* pollen prepared by the above preparation method.

[0015] The present invention also provides the application of the above-mentioned Xanthoceras sorbifolium pollen in any of the following: (1) artificial pollination of Xanthoceras sorbifolium; (2) hybridization breeding of Xanthoceras sorbifolium; (3) preparation of Xanthoceras sorbifolium pollination aid.

[0016] Preferably, the varieties of *Sapindus mukorossi* include Zhongshi No. 9, Zhongshi No. 4 and / or Zhongshi No. 1.

[0017] The beneficial effects of this invention are: The method for preparing *Xanthoceras sorbifolium* pollen provided by this invention can obtain large quantities of highly viable commercial pollen, meeting the pollen requirements for artificial or mechanical pollination of *Xanthoceras sorbifolium*, and contributing to large-scale, high-yield, and stable production of *Xanthoceras sorbifolium*. The preparation method provided by this invention not only allows for the rapid and large-scale production of highly viable *Xanthoceras sorbifolium* pollen but also maintains high viability during long-term storage, promoting the development of commercial *Xanthoceras sorbifolium* pollen. Artificial pollination using pollen prepared by this invention can significantly improve the fruit set rate of *Xanthoceras sorbifolium*. The efficient pollen collection technology system for *Xanthoceras sorbifolium* established by this invention has good feasibility for field application and yield-increasing effects.

[0018] The method for determining the viability of *Xanthoceras sorbifolium* pollen provided by this invention provides a standard method for the quantitative determination of *Xanthoceras sorbifolium* pollen viability, which can accurately reflect the true viability of *Xanthoceras sorbifolium* pollen. It not only provides a reliable technical means for pollen quality evaluation in the subsequent collection and screening and drying process optimization of *Xanthoceras sorbifolium* pollen, but also provides a methodological reference for the determination of pollen viability of other plants.

[0019] This invention focuses on the entire process of collecting *Xanthoceras sorbifolium* pollen, from selecting viability testing methods, analyzing developmental patterns, determining the collection period, optimizing drying processes, to pulverizing and sieving to obtain pollen. Furthermore, verification through artificial pollination shows that it can significantly improve the fruit set rate of *Xanthoceras sorbifolium*. This invention constructs a highly efficient pollen collection technology system for *Xanthoceras sorbifolium*, providing a feasible technical solution to the industry problem of "one fruit per thousand flowers," and is of great significance for promoting the high-quality development of the *Xanthoceras sorbifolium* industry. Attached Figure Description

[0020] Figure 1 Range analysis of orthogonal experiments for germination rate.

[0021] Figure 2 The image shows the TTC staining effect of Xanthoceras sorbifolium pollen at 30 ℃. Each number represents the temperature of the colorimetric reaction. A, B, C, D, E, and F indicate the staining time of Xanthoceras sorbifolium pollen as 5 min, 10 min, 15 min, 30 min, 1 h, and 10 h, respectively.

[0022] Figure 3 The image shows the TTC staining effect of *Xanthoceras sorbifolium* pollen at 35 ℃. Each number represents the temperature of the colorimetric reaction. A, B, C, D, E, and F indicate the staining time of *Xanthoceras sorbifolium* pollen as 5 min, 10 min, 15 min, 30 min, 1 h, and 10 h, respectively.

[0023] Figure 4 The image shows the TTC staining effect of *Xanthoceras sorbifolium* pollen at 40 ℃. Each number represents the temperature of the colorimetric reaction. A, B, C, D, E, and F indicate the staining time of *Xanthoceras sorbifolium* pollen as 5 min, 10 min, 15 min, 30 min, 1 h, and 10 h, respectively.

[0024] Figure 5 Microscopic images of fresh pollen viability of *Xanthoceras sorbifolium* determined by different methods are shown. A represents the TTC staining method, B the Alexandrite staining method, C the acetic acid-carmine staining method, and D the liquid culture medium method.

[0025] Figure 6 Microscopic images of pollen viability of *Sapindus mukorossi* stored at room temperature for one year, determined by different methods. A represents the TTC staining method, B the Alexandrite staining method, C the acetic acid-carmine staining method, and D the liquid culture medium method.

[0026] Figure 7 The images depict the external morphology of the lateral male inflorescences of *Xanthoceras sorbifolium* during their development. S1-S8 were collected on April 29, April 30, May 1, May 3, May 4, May 5, May 6, and May 8, 2025, respectively; the scale bar is 2 cm.

[0027] Figure 8 The image shows the external morphology of the male flower of *Xanthoceras sorbifolium* during its development, with a scale bar of 1 cm.

[0028] Figure 9 The images depict the external morphology of the anthers during the development of male flowers of *Xanthoceras sorbifolium*. A represents the bud stage, where the filaments have not yet elongated and the anthers have not yet dehisced. B shows the florets opening, with elongated filaments and dehisced anthers. C shows the anthers dehiscing in sequence (3, 2, 3). D shows all anthers dehiscing and releasing pollen. E shows pollen decreasing and the filaments beginning to turn red. F shows the anthers having almost no pollen left and the filaments turning purplish-red. The scale bar is 1 mm.

[0029] Figure 10 The images show the internal pollen development state of the lateral male inflorescences during the S1 stage. S1 indicates that the material is a lateral male inflorescence during the S1 stage. A, C, and E represent the internal morphology of the anthers and pollen grains inside the top, middle, and basal buds of the material, respectively, magnified 328 times. B, D, and F represent the internal morphology of the anthers and pollen grains inside the top, middle, and basal buds of the material, respectively, magnified 820 times.

[0030] Figure 11 The images show the internal pollen development of lateral male inflorescences during the S2 stage. S2 indicates that the material is a lateral male inflorescence during the S2 stage. A, C, and E represent the internal morphology of anthers and pollen grains magnified 328 times by electron magnification in the top, middle, and basal buds of the material, respectively. B, D, and F represent the internal morphology of anthers and pollen grains magnified 820 times by electron magnification in the top, middle, and basal buds of the material, respectively.

[0031] Figure 12 The images show the internal pollen development of lateral male inflorescences during the S3 stage. S3 indicates that the material is a lateral male inflorescence during the S3 stage. A, C, and E represent the internal morphology of anthers and pollen grains inside the top, middle, and basal buds of the material, respectively, magnified 328 times. B, D, and F represent the internal morphology of anthers and pollen grains inside the top, middle, and basal buds of the material, respectively, magnified 820 times.

[0032] Figure 13 The images show the internal pollen development state of the lateral male inflorescence at stage S4. S4 indicates that the material is a lateral male inflorescence at stage S4. A, C, and E represent the internal morphology of the anthers and pollen grains inside the top, middle, and basal buds of the material at 328x electron magnification, respectively. B, D, and F represent the internal morphology of the anthers and pollen grains inside the top, middle, and basal buds of the material at 820x electron magnification, respectively.

[0033] Figure 14The images show the internal pollen development of lateral male inflorescences at stage S5. S5 indicates that the material is a lateral male inflorescence at stage S5. A, C, and E represent the internal morphology of anthers and pollen grains inside the top, middle, and basal buds of the material at 328x electron magnification, respectively. B, D, and F represent the internal morphology of anthers and pollen grains inside the top, middle, and basal buds of the material at 820x electron magnification, respectively.

[0034] Figure 15 The images show the internal pollen development of lateral male inflorescences at stage S6. S6 indicates that the material is a lateral male inflorescence at stage S6. A, C, and E represent the internal morphology of anthers and pollen grains inside the top, middle, and basal buds of the material at 328x electron magnification, respectively. B, D, and F represent the internal morphology of anthers and pollen grains inside the top, middle, and basal buds of the material at 820x electron magnification, respectively.

[0035] Figure 16 The images show the internal pollen development state of the lateral male inflorescence at stage S7, where S7 indicates that the material is a lateral male inflorescence at stage S7, A represents the internal morphology of the anthers and pollen grains inside the apical bud of the material at 328x electron magnification, and B represents the internal morphology of the anthers and pollen grains inside the apical bud of the material at 820x electron magnification.

[0036] Figure 17 The variation in pollen viability of lateral male inflorescences at different stages is shown, where different lowercase letters indicate significant differences in pollen viability of lateral male inflorescences at different stages (P<0.05).

[0037] Figure 18 The figure represents the variation in pollen count of lateral male inflorescences at different time periods, where different capital letters indicate extremely significant differences in pollen count of lateral male inflorescences at different time periods (P<0.01).

[0038] Figure 19 The figure shows the changes in soluble protein content of anthers in lateral male inflorescences at different stages. Different lowercase letters indicate significant differences in soluble protein content of anthers in lateral male inflorescences at different stages (P<0.05).

[0039] Figure 20 The changes in SOD activity of anthers in lateral male inflorescences at different stages are shown. Different lowercase letters indicate significant differences in SOD activity of anthers in lateral male inflorescences at different stages (P<0.05).

[0040] Figure 21 The changes in POD activity of anthers in lateral male inflorescences at different stages are shown. Different lowercase letters indicate significant differences in POD activity of anthers in lateral male inflorescences at different stages (P<0.05).

[0041] Figure 22The changes in CAT activity of anthers in lateral male inflorescences at different stages are shown. Different lowercase letters indicate significant differences in CAT activity of anthers in lateral male inflorescences at different stages (P<0.05).

[0042] Figure 23 The variation of MDA content in the anthers of lateral male inflorescences at different stages is shown. Different lowercase letters indicate significant differences in MDA content in the anthers of lateral male inflorescences at different stages (P<0.05).

[0043] Figure 24 The images show microscopic views of pollen viability determined by sieving and TTC staining. A and B are microscopic views of pollen obtained by passing it through 80, 150, and 300 mesh sieves respectively, after adding water. C and D are microscopic views of pollen viability determined by TTC staining.

[0044] Figure 25 The data were categorized into natural pollination and natural pollination combined with artificial pollination. Groups A and B were both natural pollination (CK): the blue arrows indicate that the stigmas of the female flowers only accept natural pollination; Groups C and D were both natural pollination combined with artificial pollination (T): the red arrows indicate that the female flowers accept pollen sprayed by artificial means in addition to natural pollination. The scale bar is 3 cm.

[0045] Figure 26 The fruit clusters are 15 days after pollination of the lateral male inflorescences. The fruits indicated by the red arrows are enlarged fruits, and the fruits indicated by the blue arrows are unenlarged fruits. The scale bar is 3 cm.

[0046] Figure 27 The fruit set of Zhongshi No. 4 under different treatments is shown. A and B represent the fruit set of Zhongshi No. 4 15 days after natural pollination, while C and D represent the fruit set of Zhongshi No. 4 under natural pollination combined with artificial pollination. The scale bar is 3 cm. Detailed Implementation

[0047] This invention provides a method for determining the viability of *Xanthoceras sorbifolium* pollen, including an in vitro germination liquid culture medium method and / or a TTC staining method. The in vitro germination liquid culture medium method includes the following steps: placing *Xanthoceras sorbifolium* pollen in a liquid culture medium and culturing it at 24℃~26℃ for 3~4 hours, and counting the pollen germination rate. The liquid culture medium includes 150 g / L sucrose, 0.15 g / L boric acid, and 0.2 g / L calcium nitrate, and the pH value of the liquid culture medium is 7.0. The TTC staining method includes the following steps: staining *Xanthoceras sorbifolium* pollen with 0.5% TTC staining solution; viable pollen is stained red, while inactive pollen is not stained and appears yellow; counting the percentage of viable pollen.

[0048] Screening and optimizing methods for measuring pollen viability in *Xanthoceras sorbifolium* is a prerequisite for studying changes in pollen viability at different stages of *Xanthoceras sorbifolium* inflorescences, and is of great significance for obtaining effective pollen viability data. In vitro pollen culture is a relatively accurate method for measuring pollen viability and is also the most widely used method in laboratories. However, this method is only useful after optimizing the pollen germination medium for each species being evaluated; different staining methods also have different principles, and no single method is universally applicable to all species. Therefore, this chapter first optimizes the liquid culture medium for *Xanthoceras sorbifolium* pollen, then uses different staining methods to measure the viability of two batches of *Xanthoceras sorbifolium* pollen with different viability, compares the differences in results from different staining methods, and then screens a viability measurement method suitable for the characteristics of *Xanthoceras sorbifolium* pollen, which can provide accurate and reliable technical support for subsequent pollen viability detection work.

[0049] This invention does not specifically limit the source of each raw material in the liquid culture medium. In this invention, when performing TTC staining, the preferred staining temperature is 35℃~40℃, more preferably 36℃~38℃, and the preferred staining time is 15 min~30 min, more preferably 20 min~25 min. In this invention, when performing TTC staining, preferably 1~2 drops of 0.5% TTC staining solution are added to a glass slide. A single flower of *Xanthoceras sorbifolium* is taken, the petals are removed, and the anthers are immersed in the staining solution. The mixture is gently stirred to ensure the pollen is fully and evenly released into the staining solution. A coverslip is then placed in an oven, and the mixture is observed and counted under an optical microscope at regular intervals. Viable pollen is stained red, while inactive pollen remains unstained and appears yellow.

[0050] This invention also provides the application of the above-mentioned determination method in the preparation of *Xanthoceras sorbifolium* pollen.

[0051] The present invention also provides a method for preparing pollen from *Xanthoceras sorbifolium*, comprising the following steps: drying male inflorescences at mid-flowering stage at 35°C for 36 h, pulverizing and sieving, and collecting the sieved material as pollen.

[0052] In this invention, the mid-flowering period refers to the time when 45% to 55% of the buds of the male inflorescence have opened, more preferably when 50% of the buds of the male inflorescence have opened; the male inflorescence is preferably a lateral male inflorescence. The drying temperature and drying time specified in this invention can effectively remove moisture from the male inflorescence, thus facilitating subsequent pulverization and sieving, and can also effectively ensure that the pollen maintains a high viability. This invention does not have a specific limitation on the pulverization method; conventional pulverization methods in the art can be used. In this invention, the sieving is preferably performed sequentially through 80-mesh, 150-mesh, and 300-mesh sieves. The entire preparation process of this invention does not require additional removal of the inflorescence axis, greatly simplifying the preparation process. In the preparation method of this invention, a preferred step is to measure the viability of the prepared Xanthoceras sorbifolium pollen using the above-mentioned measurement method. Measuring the viability of the collected pollen can accurately determine the quality of the collected pollen; the pollen viability of the sieved pollen should be >60%. Using pollen of this standard can achieve better fruit setting results, laying the foundation for subsequent artificial pollination.

[0053] This invention also provides *Xanthoceras sorbifolium* pollen prepared by the above-described method. The optimal storage temperature for the *Xanthoceras sorbifolium* pollen prepared using the method of this invention is preferably -80℃ or -20℃. The *Xanthoceras sorbifolium* pollen prepared using the method of this invention can be used as commercial *Xanthoceras sorbifolium* pollen.

[0054] This invention uses *Xanthoceras sorbifolium* as material and focuses on the core objective of "efficient pollen collection technology." First, it screens pollen viability measurement methods. Then, it observes the developmental status of anthers and pollen within the lateral male inflorescences at different stages before and after flowering. Combining changes in pollen viability and quantity, it selects the optimal collection period. After optimizing inflorescence drying conditions, it verifies the technology through artificial pollination. This establishes a complete efficient pollen collection technology system for *Xanthoceras sorbifolium* using lateral male inflorescences as material, providing a scientific basis and technical support for artificial pollination and hybridization breeding.

[0055] The present invention also provides the application of the above-mentioned Xanthoceras sorbifolium pollen in any of the following: (1) artificial pollination of Xanthoceras sorbifolium; (2) hybridization breeding of Xanthoceras sorbifolium; (3) preparation of pollination adjuvants for Xanthoceras sorbifolium. In the present invention, the preferred varieties of Xanthoceras sorbifolium include Zhongshi No. 9, Zhongshi No. 4 and / or Zhongshi No. 1.

[0056] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0057] Unless otherwise specified, the following embodiments are all conventional methods.

[0058] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0059] Example 1 Screening of suitable methods for determining pollen viability of *Xanthoceras sorbifolium* 1. Materials and Methods 1.1 Overview of the Experimental Area The experimental site is located at the Hongshawo Desertification Comprehensive Control Experimental Station in Ganzhou District, Zhangye City, Gansu Province. Situated in the central part of the Hexi Corridor, on the southern edge of the Badain Jaran Desert, and on the northern foothills of the Qilian Mountains (100°06′-100°52′ E, 38°32′-39°24′ N), it lies in the transitional zone between the Qinghai-Tibet Plateau and the Mongolian Plateau. The climate is temperate continental, characterized by persistent drought and low rainfall, with an annual precipitation of 93.9 mm.

[0060] 1.2 Test Materials The selected sample trees were all 8-year-old wild-type seedling *Xanthoceras sorbifolium* trees from the Hongshawo Desertification Integrated Prevention and Control Experimental Station. The selected sample trees had uniform growth, were vigorous, and free from pests and diseases. Suitable single flowers or inflorescences were collected from each tree as experimental materials. Fresh pollen was the pollen from the newly dehisced anthers of male flowers, and pollen stored at room temperature (25℃) for one year was used as a completely inactivated pollen control.

[0061] 1.3 Test Methods 1.3.1 Optimization of the optimal liquid culture medium for pollen germination of Xanthoceras sorbifolium Using fresh pollen from *Xanthoceras sorbifolium* as experimental material, the culture medium was optimized using L9(3) 4 Orthogonal design to study sucrose, boric acid, and Ca 2+ Four factors, including concentration and pH, may affect the germination of *Xanthoceras sorbifolium* pollen. The effects of different levels of liquid culture medium on the in vitro germination of *Xanthoceras sorbifolium* pollen were investigated. No other substances were present. Specific formulations are shown in Table 1. Different treatment media were applied to grooved glass slides. The petals of freshly harvested individual flowers were removed, and the anthers were immersed in the media with gentle stirring. A moist filter paper was placed in the petri dish to maintain humidity, and the slide was then placed on the filter paper. After incubation at 25°C for 4 hours, pollen germination was observed under an optical microscope. Five fields of view were taken from each slide to calculate the germination rate. The germination criterion was that the pollen tube length exceeded twice the pollen grain diameter. The experiment was repeated three times. Analysis of variance and range were used to screen suitable media for *Xanthoceras sorbifolium* pollen germination. Germination rate = (number of germinated pollen grains in the field of view / total number of pollen grains in the field of view) × 100%.

[0062] Table 1. Optimization orthogonal design table for optimal liquid culture medium for pollen germination.

[0063] 1.3.2 Comparison of different methods for determining pollen viability In vitro culture method: The germination rate of fresh pollen and completely inactivated pollen control was determined using optimized liquid culture medium.

[0064] Staining methods: The TTC staining method is used to detect pollen viability in the same *Xanthoceras sorbifolium* pollen. Add 1-2 drops of 0.5% TTC staining solution to a glass slide. Remove the petals from a single flower, immerse the anthers in the staining solution, and gently stir to ensure the pollen is fully and evenly released. Cover with a coverslip and place in ovens at different temperatures. Observe and count the results under an optical microscope at regular intervals. Viable pollen is stained red, while inactive pollen remains unstained. Select the optimal temperature and time for subsequent measurements. The acetic acid-carmine staining method is similar to the TTC staining method, except that staining at 25°C for 30 minutes is followed by microscopic observation. Viable pollen is stained red, while inactive or aborted pollen is stained lightly or colorless. The Alexander staining method involves staining at 25°C for 5-10 hours, which will stain viable pollen purplish-red and inactive pollen green.

[0065] 1.4 Data Processing Data were processed using IBM SPSS Statistics 22.0 software, and ANOVA and Duncan's multiple comparison test were performed to examine the significance of differences between different levels of each factor; Excel 2021 software was used for graphing.

[0066] 2 Results and Analysis 2.1 Optimization of the optimal liquid culture medium for pollen germination of Xanthoceras sorbifolium The results are shown in Table 2 and Figure 1 As shown, the effects of sucrose, boric acid, calcium nitrate, and pH on the pollen germination rate of *Xanthoceras sorbifolium* at all levels were extremely significant. Multiple comparisons showed that sucrose at a concentration of 150 g / L was most effective for the germination of *Xanthoceras sorbifolium* pollen in liquid culture medium, followed by 200 g / L, with 100 g / L being the least effective. All concentrations showed highly significant effects on pollen germination. Boric acid at a concentration of 0.15 g / L showed the best pollen germination effect, significantly better than concentrations of 0.05 g / L and 0.1 g / L. Calcium nitrate at a concentration of 0.2 g / L was the most effective for pollen germination among the three concentrations, significantly better than the 0.1 g / L concentration. The pollen germination rate without calcium nitrate was significantly lower than at the 0.1 g / L concentration. A pH of 7.0 was optimal, significantly better than pH 6.5. At pH 6.0, the pollen germination rate was significantly lower than at pH 6.5.

[0067] Table 2. Multiple comparisons and range analysis for screening liquid culture media for *Xanthoceras sorbifolium* pollen germination.

[0068] Note: K1 represents the sum of observations at different levels of the same factor, k1 represents the average of observations at different levels of the same factor, and R represents the range; different capital letters indicate the significance of the difference at the 0.01 level, the same applies below.

[0069] From Table 2 and Figure 1 It was found that, under the four-factor, three-level conditions of sucrose (100 g / L, 150 g / L, and 200 g / L), boric acid (0.05 g / L, 0.1 g / L, and 0.15 g / L), calcium nitrate (0 g / L, 0.1 g / L, and 0.2 g / L), and pH values ​​of 6.0, 6.5, and 7.0, calcium nitrate was the main factor affecting the germination of *Xanthoceras sorbifolium* pollen in the liquid medium. The other factors, in descending order of influence, were sucrose, pH, and boric acid. The optimal combination of the four factors was 150 g / L sucrose, 0.15 g / L boric acid, 0.2 g / L calcium nitrate, and pH 7.0. This group of media was not included in the designed nine experimental groups. Using the above optimal liquid medium, the germination rate of *Xanthoceras sorbifolium* pollen was 85.25%.

[0070] 2.2 Comparison of different methods for determining pollen viability of *Xanthoceras sorbifolium* 2.2.1 Screening for the optimal temperature and time in the TTC staining method The results are as follows Figures 2-4 As shown, *Xanthoceras sorbifolium* pollen can be stained with 0.5% TTC staining solution at temperatures of 30℃, 35℃, and 40℃. Viable pollen is stained red, while inactive pollen is stained yellow. At 30℃, noticeable changes only appear after 1 hour of staining, and the color is relatively light. After 10 hours of staining, the pollen surroundings turn black, making it difficult to distinguish between viable and inactive pollen. This method is time-consuming and difficult to observe, therefore it is not suitable for determining *Xanthoceras sorbifolium* pollen viability using the TTC staining method at 30℃. At 35℃ and 40℃, *Xanthoceras sorbifolium* pollen can be stained rapidly. Pollen stained for 15 minutes can be distinguished by whether it is stained red, indicating viability. After 10 hours, the pollen surroundings also turn black. The staining effect is more obvious at 35℃. Therefore, it is recommended to use the TTC staining method at 35℃ for 15-30 minutes, as this method is fast and easily distinguishes between viable and inactive pollen.

[0071] 2.2.2 Comparison of different methods for determining pollen viability of *Xanthoceras sorbifolium* The results of pollen viability determination of fresh *Sapindus mukorossi* pollen using several staining methods are shown in Table 3. Figure 5As shown, TTC staining at 35℃ for 15-30 min stains viable pollen red, while inactive pollen remains unstained. Using this method, the viability of fresh *Xanthoceras sorbifolium* pollen was determined to be 89.73%. Alexandrine staining renders fresh pollen with intact cell membranes purplish-red, while pollen with ruptured membranes appears green. This method yielded a viability of 96.52% for fresh pollen. Acetaminophen binds to the chromatin-rich nuclei of viable pollen, resulting in a red color, while inactive pollen is stained lightly or colorless. Under a microscope, all fresh pollen from *Xanthoceras sorbifolium* was stained red, and this method yielded a viability of 100%. Compared to the culture method, the TTC staining results were slightly higher, but not significantly different. The Alexandrine staining and acetic acid-carmine staining results were significantly higher than the culture method.

[0072] Table 3. Determination of pollen viability of *Xanthoceras sorbifolium* using different methods

[0073] The viability of completely inactivated pollen controls stored at room temperature (25℃) for one year was then determined using liquid culture and different staining methods. The results are as follows: Figure 6 As shown, pollen cultured in liquid medium showed no pollen germination under a microscope; non-viable *Xanthoceras sorbifolium* pollen stained with 0.5% TTC appeared yellow under a microscope, indicating 0% pollen viability, consistent with the results obtained using the culture method; Alexandrine staining solution could still stain *Xanthoceras sorbifolium* pollen stored at room temperature for one year, showing a pollen viability of 95.72%, with a difference of less than 1% from the viability test results of fresh pollen; the staining results of acetocarmine were consistent with those of fresh pollen, with all pollen being stained red, indicating 100% pollen viability.

[0074] The above results indicate that sucrose concentration, boric acid concentration, calcium ion concentration, and pH all have a highly significant impact on the germination rate of *Xanthoceras sorbifolium* in liquid culture medium. The optimal liquid culture medium formula for *Xanthoceras sorbifolium* pollen was optimized to be 150 g / L sucrose, 0.15 g / L boric acid, 0.2 g / L calcium nitrate, and pH 7.0. Using this optimal liquid culture medium, the germination rate of fresh pollen was measured to be 85.26%. The viability of fresh pollen and pollen stored at room temperature for one year was determined using different staining methods. The TTC staining method showed no significant difference from the liquid culture method, making it suitable for rapid determination of *Xanthoceras sorbifolium* pollen viability. However, the Alexandrine staining method showed significantly higher viability of fresh pollen than the liquid culture method, and even for *Xanthoceras sorbifolium* pollen stored at room temperature for one year, it still showed 95.72% viability. This method cannot determine the true viability of *Xanthoceras sorbifolium* pollen and is unsuitable for its viability determination. The acetic acid-carmine staining method showed 100% staining for all types of *Xanthoceras sorbifolium* pollen, indicating that all pollen types could be stained, making it unsuitable for determining *Xanthoceras sorbifolium* pollen viability. Therefore, staining with 0.5% TTC solution at 35 ℃ for 15-30 min can stain viable pollen red, providing a rapid and relatively accurate method for determining *Xanthoceras sorbifolium* pollen viability.

[0075] Example 2 Morphological changes in the development of lateral male inflorescences and the development patterns of anthers and pollen in *Xanthoceras sorbifolium* Understanding the changes in the external morphology of the lateral male inflorescences of *Xanthoceras sorbifolium* and the developmental patterns of pollen at different stages helps to identify the period of pollen morphological maturity, which is a prerequisite for subsequent analysis of pollen quality changes.

[0076] 1. Materials and Methods 1.1 Test Materials The selected sample trees were all 8-year-old wild-type seedling *Sapindus mukorossi* trees from the Hongshawo Desertification Integrated Prevention and Control Experimental Station. The selected sample trees had uniform growth, were vigorous, and free from pests and diseases. Suitable single flowers or inflorescences from the middle of the crown of each tree were collected as experimental materials.

[0077] 1.2 Test Methods 1.2.1 Sampling Period Based on the local phenological period of *Xanthoceras sorbifolium*, lateral male inflorescences were collected every one to two days from 5 days before flowering at the base of the inflorescence (April 29, 2025, when the lateral male inflorescences were approximately 7 cm long) until 5 days after flowering (when more than 95% of the flowers were open). A total of eight periods were collected: April 29, April 30, May 1, May 3, May 4, May 5, May 6, and May 8, designated as periods S1-S8. During the experiment, the weather pattern exhibited typical characteristics of spring in arid regions: the early period was mainly sunny with a rapid rise in temperature, reaching a maximum of 30℃ on May 1. Subsequently, affected by a strong cold air mass, temperatures dropped and light rain occurred. The average high temperature was 23.4℃, and the average low temperature was 9.5℃, with significant overall temperature fluctuations.

[0078] 1.2.2 Sampling Method Thirty healthy, disease-free *Xanthoceras sorbifolium* trees with uniform vigor were selected, with six trees per biological replicate, for a total of five biological replicates. Three lateral male inflorescences from the middle of each tree were collected at eight different time points. Some were directly brought back for external morphological and stereomicroscopic observation, while others were fixed with FAA fixative (70% ethanol: glacial acetic acid: formaldehyde = 90:5:5, v / v / v) and brought back to the laboratory for routine paraffin section preparation.

[0079] 1.2.3 External morphology observation The appearance of each lateral male inflorescence was recorded with a camera, and its growth and development dynamics were observed in eight stages. The morphological changes of the anthers were then observed with a stereomicroscope.

[0080] 1.2.4 Observation of internal anatomical structure For lateral male inflorescences collected at different stages, buds or male flowers from the top, middle and base of the inflorescence that have not yet opened were prepared using conventional paraffin sections.

[0081] Paraffin section preparation: (1) Dehydration: Take the test material in FAA fixative and immerse it in 70% ethanol (2h), 85% ethanol (1.5h), 95% ethanol (1h), anhydrous ethanol (1h), and anhydrous ethanol (1h) in sequence to complete the dehydration treatment. (2) Transparency: Immerse the dehydrated material in an equal volume mixture of anhydrous ethanol and pure xylene (1h), pure xylene (1h), and pure xylene (1h) in sequence to complete the transparency treatment. (3) Wax impregnation and embedding: Place the material in pure xylene saturated with paraffin fragments and dry it overnight in an oven at 36.5℃. On the second day, the oven temperature is increased stepwise at 36.5℃, 40℃, 44℃, 48℃, 52℃, 56℃, 60℃, and 64℃, with an interval of 30 min between each level. Then, the first wax replacement is performed. After that, the pure paraffin is replaced every 4 hours. After a total of 3 replacements, the sample is embedded. (4) Trimming and sectioning: After the embedded wax block cools, trim it and section it using a Leica microtome. The section thickness is controlled to be 8-10 μm. (5) Mounting: Place the wax slide on a glass slide with adhesive, dry it, and then stain it. (6) Staining: Dewax the sections in pure xylene for 1 h, and then rehydrate them by mixing equal volumes of anhydrous ethanol and xylene (5 min), anhydrous ethanol (5 min), 95% ethanol (5 min), 85% ethanol (5 min), 70% ethanol (5 min), 50% ethanol (5 min), and distilled water (2 min). After staining with 1% safranin solution prepared with 85% ethanol for 2 hours, the slides were dehydrated in a gradient of distilled water, 50% ethanol, 70% ethanol, 85% ethanol, and 95% ethanol, with each gradient treatment lasting 20 seconds. Next, the slides were stained with 0.1% Fast Green solution prepared with 95% ethanol for 90 seconds, followed by dehydration with 95% ethanol and anhydrous ethanol for 20 seconds each. Clearing was then performed with an equal volume mixture of anhydrous ethanol and pure xylene, pure xylene, and pure xylene for 5 minutes each. Finally, the slides were mounted with neutral resin and dried. The slides can then be observed and photographed under an optical microscope.

[0082] 2 Results and Analysis 2.1 External morphological observation during the development of lateral male inflorescences of *Xanthoceras sorbifolium* The lateral male inflorescences of wild-type *Xanthoceras sorbifolium* exhibit significant morphological changes during their development from 5 days before to 5 days after flowering, such as... Figure 7As shown. In S1, the lower buds of the lateral male inflorescences begin to separate from the outer green scales. The main change in the inflorescence in S2 compared to S1 is the elongation of the inflorescence axis. In S3, the pedicels of the lower florets in the inflorescence elongate significantly. In S4, the inflorescence axis of the lateral male inflorescences elongates further, as do the pedicels of the florets. The buds at the base of the inflorescence show white tips, revealing petals about to open. The florets at the base of the lateral male inflorescence open in S5, with about 20% of the flowers already open. The petals of the opened florets are white with a yellowish-green base, and the anthers of 0-2 florets begin to dehisce and release pollen. In S6, approximately 50% of the florets... The small flowers open, with most of the petals still yellow at the base, and the base of the petals of 0-2 small flowers turning pink. The anthers of the small flowers at the base and lower middle part of the inflorescence have dehisced and released pollen. When the lateral male inflorescence develops to the S7 stage, about 75% of the small flowers on the inflorescence have opened, with most of the petals having yellow and pink bases. The petals of the small flowers at the base of the inflorescence axis have turned red at the base, and the anthers of more small flowers have dehisced and released pollen. By the S8 stage, more than 95% of the small flowers on the lateral male inflorescence have opened. The petals of the small flowers at the lower part of the inflorescence axis have red bases, and the petals have curled downwards and show a tendency to wilt. The petals of the small flowers in the middle part have pink or red bases and yellow upper parts.

[0083] Based on changes in external morphology, the development process of lateral male inflorescences can be divided into the following stages: Inflorescence elongation stage (S1-S2): The inflorescence axis of the lateral male inflorescence of *Xanthoceras sorbifolium* elongates, and the pedicels of the florets begin to elongate; Flower bud separation stage (S3-S4): The pedicels of the florets elongate significantly, and the flower buds separate from the green scales, while the inflorescence axis continues to elongate; Initial flowering stage (S5): About 20% of the florets of *Xanthoceras sorbifolium* bloom from the base or lower middle part of the inflorescence, and a small number of anthers dehisce and release pollen; Mid-flowering stage (S6): About 50% of the flowers bloom, and most of the anthers of the open flowers have released pollen; Full bloom stage (S7): About 75% of the flowers open, and the number of anthers releasing pollen further increases; Late flowering stage (S8): More than 95% of the flowers have opened, and only a few florets at the top of the inflorescence axis have anthers that have not dehisced and released pollen. The petals of the basal florets gradually wither, and some florets fall off.

[0084] The developmental dynamics of the florets in the lateral male inflorescences of *Xanthoceras sorbifolium* are as follows: Figure 8 and Figure 9As shown, the flower bud is nearly spherical to oval, with five fused sepals enclosing the inner petals and stamens. The petals are wrinkled, the stamen filaments have not yet elongated, and all eight anthers have not yet dehisced to release pollen. The bud stage lasts for a relatively long time. As development progresses, the bud gradually enlarges, reaching approximately 4.5 mm in transverse diameter and 5.5 mm in longitudinal diameter before flowering. The pistil is located at the base inside the stamens and gradually degenerates. When the bud first opens, the entire flower is semi-cup-shaped, with five separate petals, white at the top and yellowish-green at the base, extending to 1 / 2-2 / 3 of their total length. The stamen filaments elongate, and the anthers dehisce to release pollen, but not simultaneously. Three, two, and three of the eight anthers in each floret open sequentially. This stage lasts for a short time, about 12-24 hours. The degenerated pistil dries up at the base inside the stamens and is completely invisible. Subsequently, all anthers have released pollen, the petals are fully flattened, and the diameter of the single flower reaches its maximum, approximately 2-2.5 mm. At this stage, the base of the petals is yellowish-green or yellow, and the pollen content is relatively high. Under the influence of external factors such as wind, the pollen at the anthers gradually decreases. As the male flower continues to develop, the color of the petal base changes significantly, from yellow to pink to red, and finally to purplish-red. The petals curl back, the filaments also turn purplish-red, and the anthers become hollow and brownish-yellow and gradually droop. Eventually, the petals lose their luster, wither, wrinkle, and brown at the edges, and gradually fall off. The calyx and pedicel also turn yellow and soft, and are easily detached by touch.

[0085] 2.2 Observation on the development status of anthers and pollen during the development of lateral male inflorescences of *Xanthoceras sorbifolium* The anthers of *Xanthoceras sorbifolium* consist of four pollen sacs (two pairs), symmetrically separated and connected by a connective, resembling a butterfly shape. Pollen grains are produced within the pollen sacs. On the cross-section of the anther, from the outside in, the layers are: epidermis, anther endothelial wall (which later develops into a fibrous layer), middle layer, tapetum, and pollen grains. Using conventional paraffin sectioning, the structure and developmental characteristics of the anthers and pollen grains at the top, middle, and base of the lateral male inflorescences at different stages can be clearly observed under an optical microscope (see [link to article]). Figures 10-16 Significant differences exist in the developmental status of anthers and pollen in flower buds at different times and locations.

[0086] like Figure 10As shown in the paraffin sections of lateral male inflorescences at stage S1, the anthers at this stage have differentiated into a typical four-locular structure. The locules are nearly circular with clear and regular outlines. The four-layer structure of the locule wall—epidermis, inner wall, middle layer, and tapetum—is intact, with clear boundaries between the layers. In the anthers of the top buds, the thick tapetum cells are tightly packed, and the locules are filled with a large number of dispersed pollen grains without tetrad adhesion. The pollen grains are round and lack ornamentation. In the middle buds, the tapetum begins to degenerate, and some cells become vacuolated, providing nutrition for pollen development. The pollen grains are smaller and not significantly different from those in the top buds. The anther and pollen development of the basal buds represents the most advanced stage of development in the inflorescence at this time. Programmed cell death in the tapetum continues, but the cell structure and function of providing nutrition remain relatively intact. The pollen grains are larger than those in the upper part of the inflorescence.

[0087] like Figure 11 As shown, during the S2 stage, the developmental characteristics of the anthers and pollen of the flower buds in different parts of the inflorescence do not change much, and the tapetum gradually degenerates, continuously providing nutrition for the development of pollen grains.

[0088] When the lateral male inflorescences develop to the S3 stage ( Figure 12 The anthers of the top buds still have a relatively thick tapetum, but the degree of degeneration is higher than that of S1 and S2, and the pollen grains show no obvious morphological changes; the tapetum of the middle buds has degenerated significantly, the cell wall of the anther chamber has thickened fibrous layer, some pollen grains have increased in size, and the cytoplasm is thicker; the anthers of the basal buds show more significant changes, the tapetum has almost completely degenerated, the cell wall of the anther chamber has thickened fibrous layer, and large lip-shaped cells can be seen at the junction of the upper and lower pollen sacs, and there are also some pollen grains that have increased in size and have thick cytoplasm.

[0089] like Figure 13 In the S4 stage, the lateral male inflorescences already have mature pollen grains. The tapetum of the anthers in the top buds has further degenerated, but is still clearly visible. This continued degeneration provides nutrients for the maturation of pollen grains and the synthesis of pollen exine proteins, but the pollen grains are still immature. In the anthers of the middle buds, a small number of residual tapetum cells are still visible, and lip cells have formed. The junction of the upper and lower pollen sacs is thinned, and some pollen grains are mature. The immature pollen grains are small in size and their cell walls are not fully formed. The anther morphology of the basal buds has undergone more obvious changes. The tapetum has completely degenerated. The radial and tangential walls of the anther chamber cells have undergone band-like secondary thickening, and the specialized fiber layer contracts, generating mechanical force. During the longitudinal splitting at the junction of the two anther chambers, the upper and lower pollen sacs connect, forming two large pollen sacs on the left and right sides. Most of the pollen grains are already mature.

[0090] The lateral male inflorescences continue to develop until stage S5 (see...) Figure 14As the tapetum of the anthers in the top buds gradually degenerates, some pollen grains become significantly larger, but the tapetum has not yet fully performed its function. At this time, the pollen grains are close to maturity. The anthers and pollen grains of the middle and basal buds have similar morphological characteristics to those of the buds at the base of the lateral male inflorescences in the S4 stage. The tapetum of the anthers has completely degenerated, and the fibrous layer has shrunk, causing longitudinal splitting at the junction of the two anther chambers. The upper and lower pollen sacs have just connected, forming two large pollen sacs. The labiate cells are waiting to split open and release pollen. The pollen grains are full, and most of the pollen grains are mature.

[0091] like Figure 15 In the S6 stage, the anthers of the apical buds of the lateral male inflorescences still have some remnants of the tapetum layer, and most pollen grains have matured; the anthers of the middle male flowers have lost the septa of the adjacent pollen sacs, showing the morphology and structure of two large pollen sacs, the fiber layer has thickened and contracted, the pollen wall is about to split open to release pollen, and the internal pollen grains have also fully differentiated; the anthers of the basal male flowers have completely dehisced longitudinally, splitting open to release pollen, the anther wall only has the epidermis and fiber layer remaining, the pollen grains are plump, nearly spherical, with a three-pore structure, deeply stained, with thick cytoplasm, the pollen grains are mature and have the ability to pollinate.

[0092] During periods S7 and S8, the flowers are in full bloom and nearing the end of their blooming phases. Most of the flowers have already opened, except for a few buds at the top of the inflorescence, while the male flowers below have mostly split open and released pollen. Figure 16 Observing the unopened S7 flower buds, the tapetum of the anthers has degenerated, having fulfilled its functions of providing nutrients to the pollen grains, synthesizing pollen exine proteins, and synthesizing sporophytin. The fibrous layer has contracted, and the anthers are in the process of longitudinally dehiscing at the junction of the two chambers, with the two pollen sacs about to connect, preparing for subsequent dehiscence and pollen release. The pollen sacs are filled with mature pollen grains. The male flowers in the remaining parts have all dehisced and released pollen grains that are fully mature, dispersed by insects or wind.

[0093] As can be seen from the above, during pollen development, the external morphology of pollen grains is not significantly different in stages S1 to S4, and the development is mainly judged by the gradual degeneration of the tapetum and the continuous expansion of the anther chamber. In stages S5 to S8, the flower buds open, and the pollen grains gradually mature and dehisce to release pollen. During the same period, the development of flower buds in different parts of the lateral male inflorescence is asynchronous, with basal buds developing earlier than those in the middle, and middle buds developing earlier than those in the top, exhibiting a clear basal-to-apical development sequence.

[0094] The results above show that this invention, by observing the external morphology of the lateral male inflorescences of *Xanthoceras sorbifolium* before and after flowering, divides them into eight stages. S1-S2 is the inflorescence elongation stage, where the main morphological change is the elongation of the inflorescence axis. The bud separation stage (S3-S4) is characterized by the elongation of the inflorescence axis and the significant elongation of the pedicels of the florets, with the buds separating from the green scales. In the initial flowering stage (S5), about 20% of the florets of *Xanthoceras sorbifolium* bloom from the base or lower middle part of the inflorescence. In the mid-flowering stage (S6), about 50% of the flowers bloom, and the anthers at the base dehisce and release pollen. In the full bloom stage (S7), about 75% of the florets of the lateral male inflorescences have opened, and the amount of pollen-releasing anthers further increases. In the final flowering stage (S8), more than 95% of the flowers of *Xanthoceras sorbifolium* have opened, with only a few florets at the top of the inflorescence axis remaining without dehiscing and releasing pollen. The petals of the florets at the base gradually wither, and some male flowers fall off. When the flower buds first open, the base of the petals is yellowish-green and the anthers have not yet split open. As they develop, the filaments elongate, and the eight anthers split open in sequence into three, two, and three parts. After the pollen is released, the base of the petals gradually changes from yellowish-green to pink and then to red, and finally to purplish-red. The petals curl back, and the filaments also turn purplish-red.

[0095] By observing paraffin sections of flower buds from different parts of the lateral male inflorescence of *Xanthoceras sorbifolium* at eight developmental stages, the morphological characteristics and spatiotemporal patterns of anthers and pollen were clarified. The results showed significant spatiotemporal heterogeneity in the development of the lateral male inflorescence: temporally, from S1 to S8, there was a continuous process of gradual degeneration of the tapetum, continuous swelling of the anther chambers, and eventual dehiscence; spatially, within the same period, the development of basal buds consistently preceded that of the middle and upper buds. Based on the morphological characteristics of anthers and pollen at each stage, S6 was considered the optimal period for pollen collection, as inflorescences at this stage provide materials with high pollen maturity, stable viability, and high pollen retention.

[0096] Example 3 Analysis of the differences in pollen quality and quantity of lateral male inflorescences at different stages of *Xanthoceras sorbifolium* To prepare a large quantity of pollen suitable for artificial pollination, it is crucial to select a developmental stage of lateral male inflorescences with both high pollen quality and quantity. Through screening methods for measuring pollen viability and observing the developmental process of inflorescence pollen in Examples 1 and 2, the TTC staining method was selected as a rapid and accurate method for measuring pollen viability, and the developmental stages of lateral male inflorescences were defined. Currently, the variation patterns of the quality and quantity of lateral male inflorescences at different stages are still unclear. When preparing pollen using lateral male inflorescences at different stages, a period with both high pollen quantity and quality should be selected whenever possible.

[0097] This embodiment collects anthers from lateral male inflorescences at different stages and measures the pollen quantity, pollen viability, and pollen physiological indicators of the inflorescences at each stage. It clarifies the changing patterns of pollen viability, pollen quantity, and various physiological indicators, and selects inflorescences from the optimal collection period with good "quality" and "quantity" for pollen preparation. The measurement of various physiological indicators can reflect the metabolic activity and developmental status of pollen, and can explain the reasons for changes in pollen viability to a certain extent.

[0098] 1. Materials and Methods 1.1 Test Materials Thirty healthy, disease-free *Xanthoceras sorbifolium* trees with uniform vigor were selected. Six trees were grouped together as one biological replicate, for a total of five biological replicates. Lateral male inflorescences (i.e., lateral male inflorescences) of each tree were collected at various stages according to the sampling dates specified in 1.2.1 of Example 2 and immediately brought back to the laboratory for testing.

[0099] 1.2 Test Methods 1.2.1 Determination of pollen viability of lateral male inflorescences at different stages On each sampling day, one lateral male inflorescence was collected from each of 30 trees, with 6 lateral male inflorescences per biological replicate. All anthers from the upper, middle, and lower three buds of each inflorescence were placed in centrifuge tubes, and 0.5% TTC staining solution was added. The anthers in the centrifuge tubes were crushed with tweezers to release the pollen. The centrifuge tubes were then placed in a 35°C incubator for 15 min. After staining, the centrifuge tubes were removed, and the TTC staining solution mixed with the pollen was gently aspirated with a pipette and dropped onto a glass slide. A coverslip was then placed on the slide, and the slides were observed under an optical microscope. Five fields of view were observed on each slide, and the average value was taken to represent the pollen viability of that biological replicate. A total of 5 biological replicates were performed.

[0100] 1.2.2 Determination of pollen quantity in lateral male inflorescences at different stages Ten lateral male inflorescences were collected from each tree on each sampling date, with 60 lateral male inflorescences collected per group (i.e., per biological replicate), for a total of 5 biological replicates. The lateral male inflorescences were dried in an oven at 40°C for 24 h until constant weight. The flower buds were then ground and sieved (through 80, 150, and 300 mesh sieves, respectively), and the sieved material was collected as pollen. Finally, the pollen from each group (60 male inflorescences) was weighed.

[0101] 1.2.3 Differences in physiological indicators of pollen from lateral male inflorescences at different stages Three lateral male inflorescences were collected from each tree on each sampling day, with 18 lateral male inflorescences per biological replicate, for a total of 5 biological replicates. All anthers from the lateral male inflorescences were rapidly excised into cryovials, placed in liquid nitrogen, and brought back to the laboratory for analysis. Using a kit from Beijing Box Biotechnology Co., Ltd., tissue mass (g): extraction liquid volume (mL) was homogenized in an ice bath at a ratio of 1:5-10 (0.1 g of tissue was weighed and 1 mL of extraction liquid was added). The mixture was centrifuged at 8000 g, 4℃ for 10 min, and the supernatant was collected and placed on ice for analysis using a microplate reader to determine the soluble protein content, SOD, POD, CAT activities, and MDA content.

[0102] 1.3 Data Processing Data were processed using IBM SPSS Statistics 22.0 software. One-way ANOVA and Duncan's multiple comparison method were used to test the significance of differences in indicators at different time periods. Pearson correlation coefficient method was used to analyze the correlation between anther physiological indicators and pollen viability. Excel 2021 software was used for graphing.

[0103] 2 Results and Analysis 2.1 Changes in pollen viability of lateral male inflorescences at different stages Pollen viability is an important indicator of whether pollen can effectively participate in the pollination process, and it is crucial for breeding and seed production. The results of measuring the pollen viability of lateral male inflorescences at different stages are as follows: Figure 17 As shown, pollen viability exhibits a trend of first increasing and then decreasing during the development of lateral male inflorescences. During the inflorescence elongation stage (S1-S2), pollen viability is relatively low, only around 50%. By the bud separation stage (S3-S4), pollen viability significantly increases to approximately 70%. By the initial flowering stage (S5), pollen viability significantly increases to 83%, and pollen viability continues to gradually increase during the initial flowering stage (S5), mid-flowering stage (S6), and full bloom stage (S7), with no significant differences between these three stages. However, by the final flowering stage (S8), pollen viability of the lateral male inflorescences significantly decreases, showing no significant difference from stages S4 and S5. Among these, the pollen from lateral male inflorescences at stages S5, S6, and S7 exhibits higher pollen viability.

[0104] 2.2 Changes in pollen quantity of lateral male inflorescences at different stages 2.2.1 Changes in pollen count of lateral male inflorescences at different stages Pollen counts were measured in lateral male inflorescences at different stages, including both viable and non-viable pollen. Figure 18As shown, from S1 to S8, the pollen count of lateral male inflorescences showed a trend of first increasing and then decreasing. Specifically, the amount of pollen in the lateral male inflorescences during the inflorescence elongation period (S1-S2) and the bud separation period (S3-S4) is less than that in other periods. The amount of pollen in the lateral male inflorescences is the least in the S1 period. As development progresses, the amount of pollen shows a stable and slow upward trend, but the difference in pollen amount between the S1-S4 periods is still extremely significant. After the inflorescence blooms, from the bud separation period to the initial flowering period, the amount of pollen in the lateral male inflorescences increases sharply. The amount of pollen in the lateral male inflorescences in the S5 period is extremely significantly higher than that in the S4 period. The amount of pollen in the lateral male inflorescences in the mid-flowering period (S6) increases even more significantly, reaching the peak of the entire development process. Subsequently, the amount of pollen in the lateral male inflorescences in the full bloom period (S7) is extremely significantly less than that in the mid-flowering period. In the late flowering period (S8), the amount of pollen in the lateral male inflorescences is again extremely significantly less than that in the S7 period, decreasing to the level of the initial flowering period (S5), with no significant difference from the amount of pollen in the initial flowering period. Among them, the lateral male inflorescences in the S6 stage can produce more pollen.

[0105] 2.2.2 Changes in the number of effectively fertile pollen flowers in lateral male inflorescences at different stages Pollen viability and pollen quantity can respectively reflect the "quality" and "quantity" levels of pollen in lateral male inflorescences at different stages. Determining the pollen collection period using only a single indicator is often inaccurate. Multiplying the values ​​of pollen viability and pollen quantity allows us to calculate the quality of viable pollen in lateral male inflorescences at each stage. This indicator can then be used to comprehensively evaluate the effective fertile pollen quantity of lateral male inflorescences at different stages, i.e., effective fertile pollen quantity = pollen viability × pollen quantity. The results are shown in Table 4. The effective fertile pollen quantity of lateral male inflorescences at stage S6 was the highest and significantly higher than all other stages (P<0.01). The effective fertile pollen quantity of the remaining stages, from highest to lowest, was S7, S5, S8, S4, S3, S2, and S1. There were also highly significant differences between different stages. Therefore, the lateral male inflorescences in the S6 stage, which is the mid-flowering stage, can produce more effective and fertile pollen. In practical production, using the lateral male inflorescences in the S6 stage as material can produce more pollen materials that meet the requirements of artificial assisted pollination.

[0106] Table 4. Changes in the amount of effective fertile pollen from lateral male inflorescences at different time periods.

[0107] Note: Different capital letters indicate that the effective fertile pollen count of lateral male inflorescences differs significantly at different times (P<0.01).

[0108] 2.3 Changes in physiological parameters of anthers of lateral male inflorescences at different stages 2.3.1 Changes in the content of soluble protein in the anthers of lateral male inflorescences at different stages The change in soluble protein content in the anthers of the lateral male inflorescences of *Xanthoceras sorbifolium* as the inflorescence develops is as follows: Figure 19 As shown, the soluble protein content of anthers in lateral male inflorescences varied significantly at different developmental stages. From S1 to S8, the soluble protein content first increased, then decreased, and subsequently increased significantly again (P<0.05). During the inflorescence elongation stage (S1-S2), the soluble protein content significantly increased from 12.69 mg / g to 14.56 mg / g, then began to decrease during the bud separation stage (S3-S4), but remained at a relatively high level until the initial flowering stage (S5), when the soluble protein content plummeted to the lowest value of 10.18 mg / g throughout the entire cycle, significantly lower than at all other stages. Subsequently, it significantly increased again during the mid-flowering, full-blooming, and late-flowering stages (S6-S8), finally reaching a peak of 18.33 mg / g at the late-flowering stage, significantly higher than the soluble protein content at all other stages.

[0109] 2.3.2 Changes in SOD activity of anthers in lateral male inflorescences at different stages Changes in SOD activity of anthers in lateral male inflorescences at different stages, such as Figure 20 As shown, as the inflorescence develops, the SOD activity of the anthers of the lateral male inflorescences of *Xanthoceras sorbifolium* gradually increases. Its activity gradually increases during the S1-S7 period, and reaches a significant maximum at the end of the flowering period (S8), which is positively correlated with pollen maturity.

[0110] 2.3.3 Changes in POD activity of anthers in lateral male inflorescences at different stages Changes in POD activity, such as Figure 21 As shown, during the development of lateral male inflorescences, the POD activity of the anthers of lateral male inflorescences generally showed an increasing trend at different stages, with little change in value between adjacent stages, reaching the peak activity at the end of flowering (S8).

[0111] 2.3.4 Changes in CAT activity of anthers in lateral male inflorescences at different stages like Figure 22 As shown, the CAT activity of lateral male inflorescence anthers at different stages exhibited a trend of low activity in the early stage and a significant and continuous increase in the middle and late stages of inflorescence development. During the inflorescence elongation stage (S1-S2), CAT activity was at a low level, with no significant difference between the two stages. It increased significantly in stage S3, and then remained relatively stable during the bud separation stage and the initial flowering stage (S3-S5). After entering the full bloom stage (S6), CAT activity increased significantly again and continued to increase significantly with the development process, reaching a peak at the end of flowering (S8), which was significantly higher than other stages.

[0112] 2.3.5 Changes in MDA content of anthers in lateral male inflorescences at different stages Changes in MDA content of anthers in lateral male inflorescences at different stages, as shown in the figure Figure 23 As shown, the MDA content first increased and then decreased with the development of the inflorescence. The MDA content in stage S1 was 29.35 nmol / g. As the lateral male inflorescences developed, the MDA content of the anthers continued to increase slowly, reaching a peak of 34.85 nmol / g in stage S3, which was significantly higher than all other developmental stages. Then, the MDA content decreased rapidly in stages S4 and S5, reaching the lowest value of 22.21 nmol / g in stage S5. Subsequently, the MDA content increased and then tended to stabilize with no significant difference in the mid-flowering, full-blooming, and late-flowering stages (S6-S8).

[0113] 2.4 Correlation analysis between anther physiological indicators and pollen viability The results of the PART correlation analysis are shown in Table 5. The SOD, POD, and CAT activities of anthers were significantly positively correlated with pollen viability, with correlation coefficients of 0.864, 0.786, and 0.945, respectively (P<0.001). Among them, CAT activity had the strongest correlation with pollen viability. The soluble protein content and MDA content of anthers were weakly negatively correlated with pollen viability, but neither reached a statistically significant level. Therefore, there was no significant linear correlation between these two indicators and pollen viability.

[0114] Table 5. Correlation analysis between anther physiological indicators and pollen viability

[0115] The results show that there are significant or highly significant differences in pollen viability and pollen quantity in lateral male inflorescences at different stages, with an overall trend of first increasing and then decreasing as the lateral male inflorescences develop. Pollen viability in lateral male inflorescences is at a higher level during the initial flowering stage (S5), mid-flowering stage (S6), and full bloom stage (S7), significantly higher than other stages, reaching 83.04%, 84.45%, and 86.22%, respectively. Pollen quantity peaks at mid-flowering stage (S6), highly significantly higher than other stages. The effective fertile pollen quantity, obtained by multiplying the above two indicators, more directly reflects the pollen quality of lateral male inflorescences at different stages. The results show that lateral male inflorescences at mid-flowering stage (S6) can produce more effective fertile pollen, making this stage more suitable for pollen production compared to other stages. Soluble protein content initially fluctuated and decreased before steadily increasing during pollen development, reaching its lowest point at the initial flowering stage and peaking at the end of flowering. The activities of three antioxidant enzymes—SOD, POD, and CAT—all showed a continuous upward trend throughout inflorescence development, reaching their highest values ​​at the end of flowering. MDA content initially increased and then decreased, reaching its lowest point at the initial flowering stage (S5) before stabilizing. The changes in these physiological indicators clearly correlated with the inflorescence development process. This precise temporal pattern reveals the "golden period" for pollen collection from a physiological perspective, providing a crucial theoretical basis for efficient pollen production.

[0116] Example 4 Optimization of drying process for lateral male inflorescences of Xanthoceras sorbifolium 1. Materials and Methods 1.1 Test Materials Same as 1.1 in Example 3.

[0117] 1.2 Test Methods Nine lateral male inflorescences were collected from each of 30 trees on each sampling day, with 54 lateral male inflorescences per biological replicate, for a total of 5 biological replicates. The lateral male inflorescences were dried in ovens at 30, 35, and 40 °C, and the drying time was recorded. Pollen viability was measured every 12 hours until the inflorescences reached constant weight. Lateral male inflorescences in the mid-flowering stage were collected, dried completely at a suitable temperature, pulverized, and sieved to obtain pollen. The viability of the final pollen was determined using the TTC staining method, following the same procedure as in Example 3, 1.2.1.

[0118] 2 Results and Analysis 2.1 Effects of different drying temperatures on pollen viability of lateral male inflorescences at different stages Lateral male inflorescences of *Xanthoceras sorbifolium* from the elongation stage to the end of the flowering stage (S1-S8) were dried in ovens at 30 ℃, 35 ℃, and 40 ℃, respectively. Pollen viability was measured every 12 hours until the inflorescence reached constant weight. Before complete drying (e.g., 12 h), pollen clumps adhered to the sieve and could not be effectively sieved. The results are shown in Table 6. Drying to constant weight at different temperatures significantly affected the pollen viability of inflorescences at each stage, and this effect varied with different developmental stages.

[0119] After drying to constant weight at different temperatures, the pollen viability of inflorescences in the first three stages (S1-S3) was completely lost. It began to recover after stage S4. The changes in pollen viability of lateral male inflorescences after drying at different stages were basically consistent with the changes in pollen viability of fresh pollen at each stage, reaching a peak in stages S6-S7. Under drying conditions at 30 ℃, the time required for inflorescences to dry to constant weight was relatively long at all stages (approximately 72 h at stage S6), and pollen viability was generally lower than that of the 35 ℃ treatment group. Under drying conditions at 35 ℃, the time required for inflorescences to dry to constant weight was moderate at all stages (approximately 36 h at stage S6), and pollen viability remained at a high level. Under drying conditions at 40 ℃, the drying time was the shortest (approximately 24 h at stage S6), but pollen viability decreased significantly at all stages. During the S6 period (optimal collection period), the pollen viability of lateral male inflorescences remained at its highest level of 79.78% after complete drying at 35 ℃, which was significantly higher than that of pollen viability under the same drying condition of 40 ℃ (74.65%). There was no significant difference between the 30 ℃ treatment and the 30 ℃ treatment. At the same time, there was no significant difference between drying at 30 ℃ and 40 ℃.

[0120] Table 6. Pollen viability of fully dried lateral male inflorescences of *Sapindus mukorossi* at different drying temperatures at various stages.

[0121] 2.2 Comparison of the viability of dried and undried pollen Comparing the pollen viability at different stages after drying with the viability of fresh, undried pollen measured in Example 3, it was found that drying at 30-40 °C generally led to a decrease in pollen viability in the inflorescence, but the degree of decrease varied depending on the stage. Under drying conditions of 35 °C, the decrease in viability was greatest in stages S1-S3, with no viability after complete drying. The decrease was approximately 17% in stage S4, approximately 5%-7% in stages S5-S7, and approximately 2% in stage S8. Compared with fresh pollen, the rate of decrease in pollen viability gradually decreased as the inflorescence developed.

[0122] The viability of dried inflorescence pollen at different stages is basically consistent with that of fresh pollen. The pollen viability of inflorescences in the mid-flowering and full-blooming stages (S6-S7) is still the highest. The pollen viability does not increase during the drying process and there is no after-ripening phenomenon. The fresh pollen and safely dried pollen from the mid-flowering stage (S6) at the optimal collection period have high viability, which ensures the effectiveness of pollination materials.

[0123] 2.3 Effect of grinding and sieving on pollen viability To obtain *Xanthoceras sorbifolium* pollen, the completely dried inflorescences need to be pulverized and sieved. To clarify the effect of pulverization and sieving on pollen, lateral male inflorescences from stage S6 were collected and dried at 35℃ for 36 h. First, the inflorescences were pulverized, then passed through 80, 150, and 300 mesh sieves sequentially. The pollen that passed through all three meshes was then placed on a glass slide, water was added, and the results were observed under a microscope. Figure 24 As shown in A and B, different mesh sizes of sieves affect pollen purity. Using 80, 150, and 300 mesh sieves yields relatively pure pollen containing small amounts of other tissues. The viability of the sieved pollen was then determined using the TTC staining method, and the microscopic images are shown below. Figure 24 As shown in C and D, the pollen viability is 72.5%. The crushing process damages some pollen grains, reducing pollen viability by nearly 10% compared to the pollen before crushing and sieving. However, more than 70% of the pollen still maintains a high level of viability, which is sufficient to meet the needs of artificial pollination.

[0124] The above results indicate that complete drying at different temperatures significantly affects pollen viability. Pollen viability is highest after complete drying at 35℃, and is significantly higher than that after drying at 30℃ and 40℃ for most periods. The sensitivity of pollen from lateral male inflorescences at different developmental stages to temperature varies. By comparing the viability of fresh and completely dried pollen, it is generally found that as the lateral male inflorescence develops, the proportion of mature pollen increases, and the pollen's tolerance to high temperatures increases. Drying inflorescences to constant weight at 35℃ requires 36 or 48 hours, at 30℃ it requires 72 hours, and at 40℃ it requires 24 hours. The optimal collection period, i.e., mid-flowering stage (S6 stage), shows that pollen from lateral male inflorescences dried at 35℃ for 36 hours exhibits peak viability (79.78%), and this condition is considered the optimal drying method for inflorescences. Pollen from mid-flowering stage inflorescences completely dried at 35℃, after crushing and sieving, still retains a viability of approximately 70%, making it suitable for artificial pollination.

[0125] Example 5 Preparation and foliar spraying effect verification of Xanthoceras sorbifolium pollen 1. Materials and Methods 1.1 Test Materials The pollination experiment was conducted at the Hongshawo Desertification Integrated Control Experimental Station. Pollen used for pollination was obtained from *Xanthoceras sorbifolium* trees grown at the station. The maternal parents were two-year-old grafted trees ('Zhongshi No. 4', 'Zhongshi No. 9', and 'Zhongshi No. 1') grafted in 2023 at the station. Natural pollination (CK) was used as a control for all three varieties. Each maternal parent was a single-plant replicate, with three biological replicates for each treatment.

[0126] 1.2 Test Methods 1.2.1 Pollen Collection and Preparation Harvest the mid-flowering inflorescences of the *Xanthoceras sorbifolium* tree that are in good condition, dry them in an oven at 35 ℃ for 36 h, then crush the dried inflorescences in a pulverizer, and then pass them through 80 mesh, 150 mesh and 300 mesh sieves in sequence. Collect the sieve-passing material to obtain pollen for artificial pollination.

[0127] The sieved pollen and the dyeing auxiliary material, lycopodium powder, were mixed in a 1:15 ratio (adjust the ratio according to pollen viability) in a dry, clean container. The mixture was gently stirred with a clean, dry spoon, avoiding vigorous stirring or grinding to prevent damage to the pollen. After thorough mixing, the mixture was left at room temperature (in this experiment, it was sprayed immediately after mixing; otherwise, the pollen should be stored at a low temperature for later use).

[0128] 1.2.2 Pollination Test In a sunny, windless or lightly windy experimental environment with a temperature of around 25°C, from 9:00 AM to 11:00 AM, healthy, vigorous, and disease-free 'Zhongshi No. 4', 'Zhongshi No. 9', and 'Zhongshi No. 1' *Xanthoceras sorbifolium* trees were selected. Ten trees were selected as the parent plants for each pollination combination. Five trees were set up as the natural pollination group (CK) and the other five trees were set up as the experimental group (T). Natural pollination was combined with artificial pollination.

[0129] Pollen was placed in a pollination machine to artificially pollinate the three varieties of mother trees. At this time, the mother trees were approximately in their mid-to-peak flowering period. Figure 25 As shown, the stigmas of the female flowers in the natural pollination (CK) group were yellow and only accepted natural pollination; the natural pollination combined with artificial pollination (T) group showed the red color of the dye due to the artificial spraying of a mixture of pollen and dyeing agent. The two different treatments can be clearly distinguished macroscopically.

[0130] Fifteen days after pollination, the fruit clusters will appear as follows: Figure 26 As shown, after the first physiological fruit drop, the following statistics were collected 15 days after pollination: total number of fruits with enlarged ovaries, number of fruiting branches, fruit set rate (total number of fruits / number of female flowers × 100%), and ovary enlargement rate (number of fruits with significantly enlarged ovaries / number of female flowers × 100%).

[0131] 1.3 Statistical Analysis Data analysis was performed using IBM SPSS Statistics 22.0 software. When the data conformed to a normal distribution and had homogeneous variance, an independent samples t-test was conducted between the two groups, with results expressed as mean ± standard deviation. For data that did not conform to a normal distribution or had unequal variances, the Mann-Whitney U test was used to compare the significance level of differences between the two groups. Due to sample size limitations, only effect analysis was performed for statistically relevant indicators; no significance analysis was conducted. Lowercase letters after data related to fruit set rate indicate significance between treatments at the 0.05 level, while uppercase letters indicate significance between treatments at the 0.01 level.

[0132] 2 Results and Analysis 2.1 Effect of artificial pollination on the number of seeds produced Statistical analysis was performed on the data of three indicators—the number of fruits, the number of enlarged fruits, and the number of fruiting branches—15 days after pollination for 10 selected 'Zhongshi No. 4' trees (see Table 7). Natural pollination combined with artificial pollination resulted in varying degrees of increase in these three indicators for 'Zhongshi No. 4' trees 15 days after pollination. The number of fruits and enlarged fruits 15 days after pollination was more than double that of the naturally pollinated group. The number of fruits increased from 76.4 to 203, an increase of 165.71%; the number of enlarged fruits increased from 30.4 to 82.2, an increase of 170.39%; and the number of fruiting branches also increased significantly, from 19.2 to 34, an increase of 77.08%.

[0133] Table 7. Effects of artificial pollination on the number of seeds in 'Zhongshi No. 4' cultivar.

[0134] Note: CK refers to natural pollination, and T refers to natural pollination combined with artificial pollination.

[0135] The number of fruits, enlarged fruits, and fruiting branches were statistically analyzed 15 days after pollination for the selected 'Zhongshi No. 9' and 'Zhongshi No. 1' varieties, respectively. The results are shown in Tables 8 and 9. For 'Zhongshi No. 9', the results of all three indicators after natural pollination combined with artificial pollination were higher than those after natural pollination. The total number of fruits increased from 64.6 to 80.2, an increase of 24.22%; the number of enlarged fruits increased even more significantly from 43.8 to 63.6, an increase of 45.21%. For 'Zhongshi No. 1', the results of natural pollination combined with artificial pollination showed significant improvements in all indicators compared to natural pollination. The treatment resulted in a 3-5 fold increase in the total number of fruits, enlarged fruits, and fruiting branches 15 days after pollination, with improvements of 350.32%, 402.78%, and 243.75%, respectively.

[0136] Table 8. Effects of artificial pollination on the number of seeds in 'Zhongshi 9' cultivar.

[0137] Note: CK refers to natural pollination, and T refers to natural pollination combined with artificial pollination.

[0138] Table 9. Effects of artificial pollination on the number of seeds in 'Zhongshi No. 1' cultivar.

[0139] Note: CK refers to natural pollination, and T refers to natural pollination combined with artificial pollination.

[0140] 2.2 Effect of Artificial Pollination on Seed Setting Rate Fruit set rate is also an indicator that reflects fruit yield. Compared with the number of fruits, the fruit set rate has a smaller dispersion among different individual trees and is less affected by different individual trees. It can more accurately reflect the difference in fruit set under different treatments.

[0141] The initial fruit set rate of 'Zhongshi No. 4' under the two treatments (see Table 10) was tested for normality. The experimental group data did not meet the normal distribution, so the Mann-Whitney U test was performed as a nonparametric test. The p-value was 0.028, which is less than 0.05. The fruit set rate of 'Zhongshi No. 4' after 15 days of pollination with natural pollination combined with artificial pollination was significantly higher than that of natural pollination, increasing by 6.45 percentage points. The fruit enlargement rate of 'Zhongshi No. 4' after 15 days of pollination under the two treatments (see Table 10) conformed to a normal distribution and had homogeneity of variance. The independent samples t-test results showed that the p-value was less than 0.01. The fruit enlargement rate of 'Zhongshi No. 4' after 15 days of pollination with natural pollination combined with artificial pollination increased by 3.02 percentage points, which was significantly higher than that of natural pollination.

[0142] Table 10. Effects of artificial pollination on the seed setting rate of 'Zhongshi No. 4' cultivar.

[0143] Note: CK refers to natural pollination, and T refers to natural pollination combined with artificial pollination; lowercase letters after the data indicate the significance of the difference between treatments at the 0.05 level, and uppercase letters indicate the significance of the difference between treatments at the 0.01 level.

[0144] Similarly, the fruit set rate and fruit enlargement rate of 'Zhongshi No. 9' and 'Zhongshi No. 1' 15 days after pollination were statistically analyzed, and the results are shown in Tables 11 and 12. The fruit set rate and fruit enlargement rate of 'Zhongshi No. 9', with additional artificial pollination, were 0.15 and 1.32 percentage points higher than those of the naturally pollinated group, respectively, but the improvement was not statistically significant. 'Zhongshi No. 1', with artificial pollination, showed a 2.38 and 2.40 percentage point increase in fruit set rate and fruit enlargement rate compared to the naturally pollinated group, respectively. Compared to the naturally pollinated group without artificial pollination, both the fruit set rate and fruit enlargement rate were significantly improved 15 days after pollination.

[0145] Table 11 Effects of artificial pollination on fruit set rate of 'Zhongshi 9' cultivar

[0146] Note: CK refers to natural pollination, and T refers to natural pollination combined with artificial pollination; lowercase letters after the data indicate the significance of the difference between treatments at the 0.05 level, and uppercase letters indicate the significance of the difference between treatments at the 0.01 level.

[0147] Table 12 Effects of artificial pollination on fruit set rate of 'Zhongshi No. 1'

[0148] Note: CK refers to natural pollination, and T refers to natural pollination combined with artificial pollination; lowercase letters after the data indicate the significance of the difference between treatments at the 0.05 level, and uppercase letters indicate the significance of the difference between treatments at the 0.01 level.

[0149] In terms of inflorescence appearance, there are significant differences in fruit set between natural pollination and natural pollination combined with artificial pollination, such as... Figure 27 Taking 'Zhongshi No. 4' as an example, the number of fruits on the inflorescences that are naturally pollinated is small. However, artificial pollination can increase the number of fruits and thus increase the yield. Therefore, collecting inflorescences at the appropriate time, drying, crushing, and sieving them before artificial pollination has a direct and significant effect on increasing the yield of *Sapindus mukorossi*.

[0150] The above results indicate that pollen obtained by drying, crushing, and sieving collected mid-flowering inflorescences can increase the number and rate of fruits in 'Zhongshi No. 4', 'Zhongshi No. 9', and 'Zhongshi No. 1' through artificial pollination. The improvement effect varied slightly among different *Xanthoceras sorbifolium* varieties. For 'Zhongshi No. 9', the number of fruits enlarged 15 days after artificial pollination did not significantly increase compared to natural pollination, increasing from 43.8 to 63.6, a 45.21% increase. For 'Zhongshi No. 4', the number of fruits enlarged 15 days after artificial pollination increased from 30.4 to 82.2, more than double that under natural pollination. For 'Zhongshi No. 1', the number of fruits enlarged 15 days after artificial pollination increased from 14.4 to 70.4, more than four times that under natural pollination. The fruit enlargement rate 15 days after pollination is a better indicator of pollination effectiveness than the number of enlarged fruits. For 'Zhongshi No. 9', the rate increased from 6.66% to 7.98% after artificial pollination, an improvement of nearly 20%, though not significant. 'Zhongshi No. 4', however, saw a highly significant increase after artificial pollination, rising from 6.18% to 9.2%, an improvement of nearly 50%. 'Zhongshi No. 1' saw an increase from 2.58% to 4.98%, almost doubling its fruit enlargement rate 15 days after pollination, demonstrating a remarkable improvement.

[0151] Example 6 A method for determining the pollen viability of *Xanthoceras sorbifolium* using an in vitro germination liquid culture medium method, comprising the following steps: Liquid culture medium was dropped onto a grooved glass slide. The petals of freshly harvested *Xanthoceras sorbifolium* flowers were removed, and the anthers were immersed in the culture medium and gently stirred. A sheet of moist filter paper was placed in a petri dish to maintain humidity, and then the glass slide was placed on the filter paper. After incubation at 25°C for 4 hours, the pollen germination was observed under an optical microscope. Five fields of view were taken from each slide to calculate the germination rate. The germination standard was that the length of the pollen tube was more than twice the diameter of the pollen grain. This was repeated 3 times, and the germination rate was calculated as follows: germination rate = (number of germinated pollen grains in the field of view / total number of pollen grains in the field of view) × 100%. The liquid culture medium consisted of 150 g / L sucrose, 0.15 g / L boric acid, and 0.2 g / L calcium nitrate, and the pH of the liquid culture medium was 7.0.

[0152] Example 7 A method for determining the pollen viability of *Xanthoceras sorbifolium* using TTC staining, the steps of which are as follows: Add 2 drops of 0.5% TTC staining solution to a glass slide. Take a single flower of *Xanthoceras sorbifolium*, remove the petals, immerse the anthers in the staining solution, and gently stir to ensure that the pollen is fully and evenly released into the staining solution. Cover with a coverslip and place in a 35°C oven for staining for 15 minutes. Observe under an optical microscope at regular intervals and count the percentage of viable pollen. Viable pollen is stained red, while inactive pollen is not stained and appears yellow.

[0153] Example 8 A method for preparing pollen from *Xanthoceras sorbifolium*, comprising the following steps: The male inflorescences of *Xanthoceras sorbifolium* during the mid-flowering period were dried at 35℃ for 36 hours, pulverized, and then passed through 80-mesh, 150-mesh, and 300-mesh sieves. The sieved material was collected as pollen. The mid-flowering period refers to when 50% of the flower buds of the male inflorescence have opened.

[0154] Example 9 A method for preparing pollen from *Xanthoceras sorbifolium*, comprising the following steps: The male inflorescences of *Xanthoceras sorbifolium* during the mid-flowering period were dried at 35°C for 36 hours, pulverized, and then passed through 80-mesh, 150-mesh, and 300-mesh sieves. The sieved material was collected as pollen. The mid-flowering period refers to when 55% of the flower buds of the male inflorescence have opened. After obtaining the pollen, the pollen viability of *Xanthoceras sorbifolium* was determined using the method described in Example 7. If the pollen viability is >60%, it indicates that the collected pollen is qualified and can be used for subsequent artificial pollination.

[0155] Example 10 A method for preparing pollen from *Xanthoceras sorbifolium*, comprising the following steps: The male inflorescences of *Xanthoceras sorbifolium* during the mid-flowering period were dried at 35°C for 36 hours, pulverized, and then passed through 80-mesh, 150-mesh, and 300-mesh sieves. The sieved material was collected as pollen. The mid-flowering period refers to when 45% of the flower buds of the male inflorescences have opened. After obtaining the pollen, the pollen viability of *Xanthoceras sorbifolium* was determined using the method described in Example 6. If the pollen viability was >60%, it indicated that the collected pollen was qualified. The obtained *Xanthoceras sorbifolium* pollen was sealed in double-layered plastic bags and stored at -80°C.

[0156] Example 11 A method for preparing pollen from *Xanthoceras sorbifolium*, comprising the following steps: (1) Collection of male pollen: Collect pollen from the lateral male inflorescences of *Xanthoceras sorbifolium* during the flowering period; (2) The collected male inflorescences were dried in an oven at 35°C for 36 hours. This not only removed moisture to facilitate subsequent grinding, but also kept the pollen highly viable. (3) Grinding: Grind the food in a pulverizer; (4) Sieving: First, sieve the pollen once with an 80-mesh sieve, then sieve it once with a 150-mesh sieve, and finally sieve it once with a 300-mesh sieve to sieve out the pollen. Collect the sieved material, which is the pollen of *Sapindus mukorossi*.

[0157] (5) Pollen viability detection: The percentage of pollen germination was counted using the method in Example 6. Five fields of view were selected under a microscope to count the percentage of germinating pollen (percentage of germinating pollen = (number of germinating pollen grains in the field of view / total number of pollen grains in the field of view) × 100%) as the pollen viability identification result. The result was 71.93%, indicating that the pollen viability of *Xanthoceras sorbifolium* obtained by the above preparation method is high.

[0158] (6) Low temperature preservation: The obtained Sapindus mukorossi pollen is sealed in a double-layer plastic bag and stored at -20℃.

[0159] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing pollen from *Xanthoceras sorbifolium*, characterized in that, The process includes the following steps: drying the male inflorescences during the mid-flowering period at 35℃ for 36 hours, crushing and sieving them, and collecting the sieved material as pollen.

2. The preparation method according to claim 1, characterized in that, The mid-flowering period refers to the time when 45% to 55% of the flower buds in the male inflorescence have opened; the male inflorescence is a lateral male inflorescence.

3. The preparation method according to claim 1, characterized in that, The sieving process involves sequentially passing the material through 80-mesh, 150-mesh, and 300-mesh sieves.

4. The preparation method according to claim 1, characterized in that, It also includes a step of measuring the viability of the prepared Xanthoceras sorbifolium pollen.

5. The preparation method according to claim 4, characterized in that, Methods for determining the viability of prepared Xanthoceras sorbifolium pollen include in vitro germination liquid culture medium method and / or TTC staining method; The in vitro germination liquid culture medium method includes the following steps: placing *Xanthoceras sorbifolium* pollen in a liquid culture medium and culturing it at 24℃~26℃ for 3~4 hours, and calculating the pollen germination rate; the liquid culture medium includes 150 g / L sucrose, 0.15 g / L boric acid and 0.2 g / L calcium nitrate, and the pH value of the liquid culture medium is 7.

0.

6. The preparation method according to claim 5, characterized in that, The TTC staining method includes the following steps: staining the pollen of *Xanthoceras sorbifolium* with 0.5% TTC staining solution; viable pollen is stained red, while inactive pollen is not stained and appears yellow; and the percentage of viable pollen is counted.

7. The preparation method according to claim 5, characterized in that, When performing TTC staining, the staining temperature is 35℃~40℃ and the staining time is 15 min~30 min.

8. The *Xanthoceras sorbifolium* pollen prepared by the preparation method according to any one of claims 1 to 7.

9. The use of the *Xanthoceras sorbifolium* pollen according to claim 8 in any of the following, characterized in that: (1) Artificial pollination of Xanthoceras sorbifolium; (2) Hybrid breeding of Xanthoceras sorbifolium; (3) Preparation of pollination aids for Xanthoceras sorbifolium.

10. The application according to claim 9, characterized in that, The varieties of *Sapindus mukorossi* mentioned include Zhongshi No. 9, Zhongshi No. 4 and / or Zhongshi No. 1.

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