An inducer for improving female flower differentiation of xanthoceras sorbifolia bunge and its preparation method and application

Abstract

This invention belongs to the field of plant growth regulator technology, and relates to an inducer for improving female flower differentiation in *Xanthoceras sorbifolium*, its preparation method, and its application. The inducer, calculated per liter of aqueous solution, comprises 150-1200 mg of 6-benzyladenine, 10-50 mg of salicylic acid, 100-300 mg of choline chloride, 1.0-5.0 g of urea, 2.0-3.0 g of potassium dihydrogen phosphate, 0.5-2.0 g of boric acid, and 0.5-1.0 mL of emulsifier. This invention constructs a multi-component synergistic system for the directional regulation of flower bud sex differentiation, significantly increasing the number and proportion of female flowers in *Xanthoceras sorbifolium*, and reducing the flower bud abortion rate. Simultaneously, it improves the receptivity of the stigma of female flowers and the subsequent fruit setting rate, increasing yield per unit area, solving the industrial problem of "one fruit per thousand flowers" in *Xanthoceras sorbifolium*, and meeting the core needs of high-yield and high-efficiency cultivation and large-scale industrial development of *Xanthoceras sorbifolium*.

Description

Technical Field

[0001] This invention belongs to the field of plant growth regulator technology, and relates to an inducer for improving female flower differentiation in *Xanthoceras sorbifolium*, its preparation method, and its application. Background Technology

[0002] Sapindus mukorossi ( Xanthoceras sorbifolium Bunge *Xanthoceras sorbifolium*, belonging to the Sapindaceae family, is a rare and native deciduous oilseed tree species endemic to my country. Naturally distributed in Northeast, Northwest, and North my country, it possesses outstanding ecological and economic value: strong resilience, wide adaptability, and tolerance to drought and poor soil conditions make it a preferred species for windbreak, sand fixation, soil and water conservation, and ecological restoration in arid and semi-arid regions of northern my country. The kernels of *Xanthoceras sorbifolium* contain 50%–67% oil, rich in unsaturated fatty acids and functional components such as nervonic acid, making it a high-quality raw material for high-end edible vegetable oils, biodiesel, and biopharmaceutical products. It is a key characteristic woody oilseed tree species for national development, with broad prospects for industrialization. However, in actual production, due to its monoecious biological characteristics, the natural ratio of male to female flowers in *Xanthoceras sorbifolium* is severely imbalanced, with female flowers generally accounting for less than 5% of fruit-bearing plants. Coupled with high flower and fruit drop rates and low fruit set rates, this has created the industry-recognized problem of "one fruit for every thousand flowers," directly resulting in low yield per unit area and severely restricting the standardized, large-scale, and high-quality development of the industry. Therefore, the core technological requirement for breaking through the bottleneck of the development of the *Vernicia fordii* industry is to use safe and efficient technical means to directionally regulate the sex differentiation of flower buds and significantly increase the number and proportion of female flowers.

[0003] The application of plant growth regulators and nutrient preparations plays a crucial role in regulating the process of flower bud differentiation, adjusting the ratio of male to female flowers, and improving fruit set rate and fruit quality. These are commonly used techniques in the high-yield cultivation of economic forest crops. Existing research shows that spraying different plant growth regulators during the flowering period of Korla fragrant pear can effectively improve its fruit set rate and fruit quality. Studies on camellia oleifera have also found that spraying plant growth regulators in combination with nutrients during the flowering period can significantly improve the fruit set rate and oil content of camellia seeds, confirming the application value of the combination of growth regulators and nutrients in regulating the flowering and fruiting of woody oilseed species. In the cultivation research of *Xanthoceras sorbifolium*, existing techniques often use single plant growth regulators such as gibberellin and 6-benzylaminopurine (6-BA) for spraying to regulate its flower bud differentiation, confirming that exogenous growth regulators have a certain regulatory effect on the sex differentiation of *Xanthoceras sorbifolium* flower buds.

[0004] However, current technologies and products for regulating female flower differentiation in *Xanthoceras sorbifolium* still have significant shortcomings and industry gaps. Firstly, existing technologies mostly employ single plant growth regulators, targeting only a single point and increasing the number of female flowers by only 10%–30%, which is insufficient for high-yield cultivation. Furthermore, single regulators have a narrow effective concentration window; improper concentrations can easily lead to excessive vegetative growth, flower bud abortion, flower and fruit drop, and even weakened tree vigor, resulting in significant side effects. Secondly, existing compound formulations focus only on the combination of growth regulators, neglecting the rigid requirements of female flower differentiation and development for nutrients such as nitrogen, phosphorus, potassium, and boron. This fails to achieve synergistic effects of hormone regulation, nutrient supply, and physiological function enhancement, resulting in unstable regulatory effects, short-lasting effects, and easy female flower abortion, making stable application in actual production difficult. Thirdly, existing research defines the timing of regulator application in a general way, failing to achieve precise regulation during the critical period of *Xanthoceras sorbifolium* flower sex differentiation, and exhibiting poor adaptability to *Xanthoceras sorbifolium* under different site conditions. Therefore, the industry has not yet developed a scientifically formulated, synergistic, stable, side-effect-free, easy-to-operate, and scalable female flower differentiation inducer for *Xanthoceras sorbifolium*, which cannot meet the core industry needs of high-yield and high-efficiency cultivation of *Xanthoceras sorbifolium*. Summary of the Invention

[0005] The main objective of this invention is to overcome the deficiencies in the prior art and provide an inducer for improving female flower differentiation in *Xanthoceras sorbifolium*, its preparation method, and its application.

[0006] To achieve the above objectives, the specific technical solution is as follows: This invention provides an inducer to enhance female flower differentiation in *Xanthoceras sorbifolium*, comprising, per liter of aqueous solution, 150-1200 mg of 6-benzyladenine (6-BA), 10-50 mg of salicylic acid, 100-300 mg of choline chloride, 1.0-5.0 g of urea, 2.0-3.0 g of potassium dihydrogen phosphate, 0.5-2.0 g of boric acid, and 0.5-1.0 mL of emulsifier.

[0007] The main hormone used in this invention, 6-BA, is compatible with the hormone requirements for female flower differentiation in Sapindaceae plants such as Xanthoceras sorbifolium. Combined with salicylic acid, choline chloride, and nutrient components, and emulsified with an emulsifier for enhanced emulsification, a synergistic system is formed. Compared with existing technologies, the female flower induction effect is more stable. At the same time, it can also improve the receptivity of the female flower stigma and the subsequent fruit setting rate, effectively alleviating the problem of "abundant flowers but few fruits" in plants such as Xanthoceras sorbifolium, and achieving simultaneous increase in flowering, quality, and yield.

[0008] Furthermore, the inducer for promoting female flower differentiation in *Xanthoceras sorbifolium* comprises, per liter of aqueous solution, 400-800 mg of 6-benzyladenine, 20-40 mg of salicylic acid, 150-250 mg of choline chloride, 2-4 g of urea, 2.0-3.0 g of potassium dihydrogen phosphate, 0.5-2.0 g of boric acid, and 0.5-2.0 mL of emulsifier.

[0009] This invention, employing the aforementioned formula, further enhances the synergistic effect of its components, enabling precise regulation of hormone balance and nutrient supply during the differentiation of female flowers in *Xanthoceras sorbifolium*. Through scientific formulation, it achieves multi-dimensional synergy of "hormone regulation - nutrient supplementation - enhanced absorption," significantly increasing the proportion of female flower differentiation and improving female flower quality, laying a solid foundation for subsequent fruit setting. This effectively addresses the core issues of low female flower quantity and low fruit set rate in *Xanthoceras sorbifolium* production.

[0010] Furthermore, the inducer for promoting female flower differentiation in *Xanthoceras sorbifolium* comprises, per liter of aqueous solution, 600 mg of 6-BA, 30 mg of salicylic acid, 200 mg of choline chloride, and 3 g of urea, combined with fixed components of 2.5 g of potassium dihydrogen phosphate, 1.5 g of boric acid, and 1.0 mL of emulsifier.

[0011] The above-mentioned formulation is theoretically expected to increase the total number of female flowers per plant of *Xanthoceras sorbifolium* by 42.1% and the proportion of female flowers by 38.6% compared to the blank control group, while reducing the flower bud abortion rate by 62.0%. In the orthogonal experiment, treatment No. 13, which is closest to the optimal combination effect, showed an actual increase of 34.1% in the total number of female flowers per plant, a 30.2% increase in the proportion of female flowers, and a 53.1% reduction in the flower bud abortion rate compared to the blank control group. This verifies that the formulation system has a significant directional induction effect on the differentiation of female flowers in *Xanthoceras sorbifolium* and can effectively improve the problem of the imbalance between male and female flowers in *Xanthoceras sorbifolium*.

[0012] Furthermore, the emulsifier is selected from one or more ester-based nonionic surfactants.

[0013] Furthermore, the emulsifier is selected from one or more of Tween 80, Tween 20, Tween 60, and Tween 40, with Tween 20 being preferred.

[0014] Furthermore, the inducer for enhancing female flower differentiation in *Xanthoceras sorbifolium* also includes one or more of indolepyruvic acid (IPYA), tryptophan (TAM), and 2-O-β-D-glucosylsalicylic acid (SAG).

[0015] The addition of the above-mentioned substances to the inducer of this invention can further optimize the regulatory network of the inducer on the differentiation of female flowers in *Xanthoceras sorbifolium*. Specifically, indolepyruvate, as a precursor to auxin synthesis, can more effectively promote the initiation and development of female flower primordia by enhancing the biosynthesis and transport of auxin in the plant, and synergistically promoting cytokinin-like substances such as 6-BA. Tryptophan, as an important precursor to indole alkaloids, not only participates in regulating plant growth and development signaling pathways, but may also further increase the proportion of female flower differentiation by affecting the expression of genes related to floral organ development. Meanwhile, 2-O-β-D-glucosylsalicylic acid, as a glycosylated derivative of salicylic acid, has more stable chemical properties and a longer duration of action, which can continuously enhance the plant's resistance to stress, reduce the inhibitory effect of adverse environments on female flower differentiation, and its slowly released salicylic acid component can maintain a long-term promoting effect on female flower differentiation.

[0016] Further, per liter of aqueous solution, the indolepyruvic acid is 5-20 mg, tryptophan is 10-30 mg, and 2-O-β-D-glucosylsalicylic acid is 5-15 mg.

[0017] This invention constructs an efficient, stable and safe female flower differentiation induction system by precisely controlling the concentration range of each component. This allows the inducer to fully exert the synergistic effect of each component in practical applications, while also adapting to the growth needs of *Sapindus mukorossi* under different environmental conditions.

[0018] Furthermore, the inducer for promoting female flower differentiation in *Xanthoceras sorbifolium* is applied by spraying onto the male and female flower reproductive organs; preferably, the spraying time is 7 days before the development of the male and female flower reproductive organs.

[0019] The field appearance criteria for judging the sex organs of *Xanthoceras sorbifolium* 7 days before their development: when the spring temperature rises steadily to above 5℃, the flower buds of *Xanthoceras sorbifolium* break dormancy, the buds swell and show white, the scales loosen and crack, and the inflorescence rudimentary shape inside is clearly visible.

[0020] Furthermore, the spraying is to be carried out in the early morning (6:00-8:00) or late afternoon (18:00-20:00) when there is no wind; the air temperature during the spraying is 15-25℃, and the operation should be avoided in high temperature, strong sunlight, rainy days or windy weather.

[0021] Furthermore, the target of the spraying is a Sapindaceae tree (such as Xanthoceras sorbifolium) that is 3 years or older, grows vigorously, is free from pests and diseases, and has full flower buds. Priority is given to plants with a medium and short fruit branch ratio of ≥60%. For plants that are too weak or have severe excessive growth, the tree vigor should be adjusted through conventional pruning and water and fertilizer regulation before spraying.

[0022] Furthermore, the spraying method is as follows: spray the fruiting mother branches and plump flower buds in the upper and middle part of the tree canopy in a directional and uniform manner, focusing on spraying the axillary flower buds and terminal flower buds on the fruiting branches, spraying until the buds and branches are completely wet and the liquid is about to drip, avoiding missed spraying, double spraying or large amounts of liquid dripping.

[0023] Furthermore, regarding the re-spraying and additional spraying: if it rains within 4 hours after spraying, re-spray once within 1-2 hours after the rain stops, with the re-spray dosage being 1 / 2 of the original dosage; for plants with vigorous growth, re-spray once after the first spraying at an interval of 5 days, with the spraying dosage and operation specifications being the same as the first spraying.

[0024] Furthermore, the following post-spraying management measures are as follows: within 15 days before and after spraying, the spraying of gibberellin-type plant growth regulators, alkaline pesticides, and alkaline foliar fertilizers is prohibited to avoid antagonizing the induction effect or damaging the stability of the inducing agent components; during the spraying period, regular tree management should be carried out, such as pruning and thinning out overly dense branches, weak branches, and diseased and insect-infested branches in winter, and watering thoroughly before bud break in spring to ensure the supply of water and basic nutrients to the tree.

[0025] This invention also provides a method for preparing the above-mentioned inducer for enhancing female flower differentiation in *Xanthoceras sorbifolium*, comprising the following steps: (1) Add 6-BA and salicylic acid to 10-20 mL of anhydrous ethanol, stir to dissolve, and obtain the regulator stock solution; (2) Add urea, potassium dihydrogen phosphate, boric acid and choline chloride to 600-800 ml of deionized water, stir to dissolve, and obtain water-soluble nutrient mother liquor; (3) Add the regulator stock solution dropwise to the water-soluble nutrient stock solution while stirring; after the addition is complete, add Tween 20 and stir to mix. (4) Add deionized water to a final volume of 1L and stir for 3-5 minutes.

[0026] The inducing agent of this invention does not require complicated equipment, can be prepared and used immediately, and must be sprayed within 4 hours after preparation to avoid degradation of the active ingredients. After preparation, it can be stored at 4℃ for 2-3 months.

[0027] Further, indolepyruvic acid (IPYA), tryptophan (TAM), and 2-O-β-D-glucosylsalicylic acid (SAG) are added after step (3) is completed.

[0028] Compared with the prior art, the present invention has the following significant advantages: This invention provides a scientifically formulated, synergistic, and stable inducer for improving female flower differentiation in *Xanthoceras sorbifolium*. By constructing a multi-component synergistic system for the directional regulation of flower bud sex differentiation, it significantly increases the number and proportion of female flowers, reduces the flower bud abortion rate, solves the industrial problem of "one fruit per thousand flowers" in *Xanthoceras sorbifolium*, and meets the needs of large-scale, high-yield cultivation of *Xanthoceras sorbifolium*.

[0029] The formulation components of this invention are all agriculturally safe substances, with no high toxicity or residue, and no adverse side effects. They can enhance the tree's resistance to stress and are suitable for large-scale long-term application. At the same time, the formulation concentration can be flexibly adjusted according to the tree vigor and regional phenological stage. Optional components can be selected to further optimize the induction effect. No special supporting equipment is required, making it suitable for cultivation scenarios of different regions and tree vigor. It is easy to promote on a large scale and provides technical support for the high-quality and high-yield cultivation of *Sapindus mukorossi*.

[0030] This invention requires no complex equipment for preparation, has a simple field spraying process, is ready to use immediately after preparation, requires fewer spraying times, and has low labor and material costs. By applying it directionally during the critical window period of flower sex organ development in *Xanthoceras sorbifolium*, it achieves precise control over flower bud sex differentiation, significantly increases the number of female flowers and the proportion of female flowers on lateral branches, reduces flower bud abortion rate, and increases the yield per unit area of ​​*Xanthoceras sorbifolium*, thus meeting the core needs of high-yield and high-efficiency cultivation and large-scale industrial development of *Xanthoceras sorbifolium*. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0032] Figure 1 This is a morphological diagram of the flower bud development process corresponding to the key window period of female flower differentiation in *Sapindus mukorossi* according to the present invention. The left image shows the morphology of flower buds during the flowering induction stage; the middle image shows the morphology of flower buds during the flowering initiation stage; and the right image shows the anatomical structure of flower organs during the flower organ formation stage. Figure 2 This is the flowering state of *Xanthoceras sorbifolium* during its peak flowering period after the application of the inducing agent according to this invention. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0034] Unless otherwise specified in the embodiments of the present invention, the techniques or conditions described in the literature in this field or the product instructions shall be followed; if the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased through legitimate channels.

[0035] Description of reagents and materials used in this invention: 6-BA (6-benzyladenine), salicylic acid, choline chloride, urea, potassium dihydrogen phosphate, boric acid, Tween 20, and anhydrous ethanol were all analytical grade or agricultural grade reagents, purchased from Sinopharm Chemical Reagent Co., Ltd. The *Xanthoceras sorbifolium* plants used in the experiment were selected from 5-year-old, robust *Xanthoceras sorbifolium* trees planted in a 500-mu (approximately 33 hectares) *Xanthoceras sorbifolium* forest germplasm collection nursery in Qiaozicun Village, Xidi Town, Qitai County, Changji Prefecture, Xinjiang. This nursery collected *Xanthoceras sorbifolium* seedling germplasm from various regions of northern and southern Xinjiang. The nursery's coordinates are 89°40'20", 44°6'45", 89°40'22", 44°6'45", 89°40'20", 44°6'46", 89°40'22", 44°6'46". The nursery had consistent site conditions, uniform soil fertility, and convenient irrigation and drainage, and adopted conventional cultivation management methods.

[0036] The basic formula for all fixed components in this invention is: 15 mL anhydrous ethanol, 2.5 g potassium dihydrogen phosphate, 1.5 g boric acid, 1.0 mL Tween 20, and deionized water as solvent, to a final volume of 1 L.

[0037] Example 1 This embodiment provides a female flower differentiation inducer for *Xanthoceras sorbifolium*, its preparation method, and an orthogonal screening experiment of the optimal formulation. (1) Experimental design and material preparation To screen the optimal core formulation of inducing agents for regulating female flower differentiation in *Xanthoceras sorbifolium*, four experimental factors were set up: 6-BA, salicylic acid, choline chloride, and urea. Each factor was set with five concentration levels, as shown in Table 1. A standard orthogonal array was used to conduct the experiment, with an additional water-based blank control group (CK), for a total of 26 treatments.

[0038] Table 1 Experimental Factors and Levels Design Table

[0039] The experiment employed a randomized block design, with three biological replicates for each treatment. Each replicate included three fruiting trees of uniform growth, with selected trees meeting the following criteria: diameter at breast height (DBH), crown width, and branch quantity differing by ≤10%; full flower buds; and a proportion of medium to short fruiting branches ≥60%. Trees that were too weak or excessively vigorous were excluded. Protective rows were established between blocks to avoid marginal effects and cross-contamination of pesticide solutions. The control group (CK) was sprayed with 1L of deionized water containing 15mL anhydrous ethanol and 1.0mL Tween 20; all other procedures were identical to the treatment groups.

[0040] (2) Preparation of inducing agent solution All treatment solutions were prepared and used immediately on the day of application, with the ambient temperature controlled at 20-25℃, avoiding direct sunlight, and field spraying completed within 4 hours. The standardized preparation steps for 1L of solution are as follows: 1. Preparation of the conditioning stock solution: Accurately weigh the corresponding mass of 6-BA and salicylic acid according to the orthogonal array, add 15mL of anhydrous ethanol, and stir magnetically until completely dissolved to obtain a clear conditioning stock solution; 2. Preparation of nutrient stock solution: Accurately weigh the corresponding mass of choline chloride and urea according to the orthogonal array, as well as the fixed amount of potassium dihydrogen phosphate and boric acid, add 800 mL of room temperature deionized water, stir until completely dissolved, and obtain the nutrient stock solution. 3. Mixing and adjusting volume: Slowly add the conditioner stock solution to the nutrient stock solution dropwise while stirring at a constant speed; after the addition is complete, add 1.0 mL of Tween 20 and continue stirring for 8 min until the system is homogeneous; adjust the volume to 1 L with deionized water and stir again for 5 min to obtain the inducing agent for the corresponding treatment.

[0041] (3) Field spraying operation 1. Determination of the spraying window: When the spring temperature steadily rises above 5℃, the flower buds of *Xanthoceras sorbifolium* break dormancy, the buds swell and show white tips, and the scales loosen and crack. Randomly collect 10-15 plump flower buds from the upper and middle parts of the canopy daily. Observe under a stereomicroscope to confirm that the floret primordia differentiation is complete, the outline is clear, and there are no protruding morphological differentiations of the stamen and pistil primordia. This is the optimal spraying time. All treatments should be sprayed within this window period. The morphological diagram of the flower bud development process corresponding to the key window period of female flower differentiation in *Xanthoceras sorbifolium* is shown below. Figure 1 As shown; 2. Spraying Operation: Choose a windless early morning between 6:00 and 8:00 AM, with an air temperature of 15-25℃. Use the same backpack electric sprayer to spray the upper and middle fruiting branches and plump flower buds of the test trees in a directional and even manner, focusing on the axillary and terminal flower buds on the fruiting branches. Spray until the buds and branches are completely wetted and the solution is just about to drip. The amount of sprayed per tree should be consistent. When changing the treatment solution, thoroughly rinse the sprayer with clean water at least 3 times to avoid cross-contamination. 3. Supporting Management: Within 15 days before and after spraying, all test trees are prohibited from being sprayed with gibberellin-based plant growth regulators, alkaline pesticides, or alkaline foliar fertilizers. All management measures, including water and fertilizer application, pruning, and pest and disease control, must remain consistent throughout the entire treatment cycle. If rainfall occurs within 4 hours after spraying, all treatments should be re-sprayed once within 1-2 hours after the rain stops, with the re-spray dosage being half the original dosage.

[0042] (4) Indicator measurement and result analysis When the *Xanthoceras sorbifolium* enters its peak flowering period (when more than 50% of the flowers on the tree are open), all treatments were tested on the same day. The total number of female flowers differentiated per tree, the proportion of female flowers, and the flower bud abortion rate were counted. The average value of three replicates for each treatment was taken. The experimental results and range analysis are shown in Tables 2 and 3.

[0043] Table 2. Orthogonal Experimental Design and Results

[0044] Table 3. Range analysis results of the female flower percentage index

[0045] As shown in Table 2, a 1L system containing 400-800mg of 6-BA, 20-40mg of salicylic acid, 150-250mg of choline chloride, and 2-4g of urea, combined with fixed components of 2.5g of potassium dihydrogen phosphate, 1.5g of boric acid, and 1.0mL of Tween 20, theoretically, could increase the total number of female flowers per plant by more than 20% and the proportion of female flowers by more than 20% compared to the blank control group, while reducing the flower bud abortion rate by 30%. Actual measurements showed that the total number of female flowers per plant increased by 21.3-24.4% compared to the blank control group, the proportion of female flowers increased by 23.4-26.8%, and the flower bud abortion rate decreased by 33.4-40.5%, indicating a good directional induction effect on female flower differentiation in *Xanthoceras sorbifolium*, effectively improving the imbalance in the ratio of male to female flowers.

[0046] The range analysis results in Table 3 show that the order of influence of the four factors on the proportion of female flowers in *Xanthoceras sorbifolium* is: 6-BA > urea > choline chloride > salicylic acid. Among them, 6-BA is the core dominant factor regulating the differentiation of female flowers in *Xanthoceras sorbifolium*, urea is an important nutritional support and synergistic component, and choline chloride and salicylic acid are auxiliary regulatory components. Based on the effect values ​​of each factor level, the optimal core formula combination obtained by screening is A3-B3-C3-D3, that is, 1L of system contains 600mg of 6-BA, 30mg of salicylic acid, 200mg of choline chloride, and 3g of urea, combined with fixed components 2.5g of potassium dihydrogen phosphate, 1.5g of boric acid, and 1.0mL of Tween 20. The optimal combination theory is expected to increase the total number of female flowers per plant by 42.1% and the proportion of female flowers by 38.6% compared with the blank control group, while reducing the flower bud abortion rate by 62.0%. In the orthogonal experiment, treatment No. 13, which is closest to the effect of the optimal combination, showed that the total number of female flowers per plant increased by 34.1% and the proportion of female flowers by 30.2% compared with the blank control group, while reducing the flower bud abortion rate by 53.1%. This verifies that the formulation system has a significant directional induction effect on the differentiation of female flowers in *Xanthoceras sorbifolium*, and can effectively improve the problem of the imbalance between male and female flowers in *Xanthoceras sorbifolium*.

[0047] Application Examples Field application validation of the optimal inducer formulation on *Sapindus mukorossi* trees of different vigors (1) Preparation of experimental materials: Two treatment groups were set up for the experiment: the optimal formula treatment group (the optimal core formula combination A3-B3-C3-D3 obtained by range analysis in Example 1) and the blank control group (CK). The experiment was carried out on three types of Xanthoceras sorbifolium plants with weak, moderate, and vigorous tree vigor. Three biological replicates were set up for each treatment for each type of plant, and each replicate contained three trees. All selected plants were Xanthoceras sorbifolium fruiting trees that were more than 3 years old, and the tree age, site conditions, and basic cultivation management measures were completely consistent to ensure that the only variable in the experiment was the inducing agent treatment.

[0048] (2) Preparation and field application of the inducer: The optimal formulation of the inducer was prepared according to the unified preparation method in Example 1. The 1L system contained 600mg of 6-BA, 30mg of salicylic acid, 200mg of choline chloride, and 3g of urea, combined with fixed components of 2.5g of potassium dihydrogen phosphate, 1.5g of boric acid, and 1.0mL of Tween 20. The blank control group was sprayed with 1L of deionized water containing 15mL of anhydrous ethanol and 1.0mL of Tween 20 to eliminate the interference of solvents and emulsifiers on the experimental results. The determination of the spraying window period, the spraying operation specifications, and the supporting management measures throughout the cycle were completely consistent with those in Example 1. Among them, plants with excessive vigor were sprayed again after the first spraying, 5 days later, with the spraying dosage and operation specifications being exactly the same as the first spraying.

[0049] (3) Indicator Measurement and Result Analysis: After *Xanthoceras sorbifolium* entered its peak flowering period, all treatments were uniformly measured on the same day for three core indicators: total number of female flowers per plant, percentage of female flowers, and flower bud abortion rate. The average value of three biological replicates for each treatment was taken, and the results are shown in Table 4. The flowering status of *Xanthoceras sorbifolium* during its peak flowering period after spraying the inducing agent according to this invention is as follows: Figure 2 As shown.

[0050] Table 4. Induction effect of the optimal formula on *Sapindus mukorossi* fruit of different tree vigors.

[0051] The results showed that the optimal inducing agent formulation screened in this invention had a stable and significant directional induction effect on female flower differentiation in *Xanthoceras sorbifolium* trees of different vigor. Compared with the blank control group of the same vigor, the optimal formulation treatment significantly increased the number and proportion of female flowers, while greatly reducing the flower bud abortion rate. It showed good adaptability to trees with weak, medium, and vigorous vigor, and can meet the core requirements for high-yield cultivation of *Xanthoceras sorbifolium* under different site conditions and cultivation management modes.

[0052] Comparative Example 1 To verify the contribution of key hormones and related substances in the optimal inducing agent formulation to the induction of female flowers in *Xanthoceras sorbifolium*, this comparative experiment was designed. The *Xanthoceras sorbifolium* trees used in the experiment were all of medium vigor, consistent with the examples, totaling 15 trees, divided into 5 groups (each group replicated 3 times). Treatment methods for each group: Based on the optimal formulation, four key components—6-BA, salicylic acid, choline chloride, and Tween 20—were removed, with the optimal formulation treatment group serving as the control. The field spraying procedures, methods for measuring relevant indicators of female flowers, and result analysis methods for each group were consistent with the examples. The growth effect of female flowers in *Xanthoceras sorbifolium* without the corresponding hormones or substances is shown in Table 5.

[0053] Table 5. Growth effect of female flowers of *Sapindus mukorossi* when lacking corresponding hormones or substances.

[0054] Table 5 shows that in the absence of 6-BA, the total number of female flowers per plant and the proportion of female flowers were only 53.3% and 13.1%, respectively, and the flower bud abortion rate reached 15.7%. This may be because the absence of 6-BA cannot significantly suppress the expression of abortion genes, causing the ovary to still undergo programmed cell death. Related studies have shown that, in the case of the oilseed tree *Sapium sebiferum* (also known as Chinese tallow tree),... Sapium sebiferum Exogenous application of 6-BA significantly promoted female flower development and increased fruit quantity, and genes related to female flower development were also identified. SPATULA (SPT) , KANADI 2 (KAN2) , JAGGED (JAG) and cytochrome P450 78A9 (CYP79A9) The expression of was significantly upregulated.

[0055] In the absence of salicylic acid, the total number of female flowers per plant decreased by 13.7% (59.7 flowers) compared to the optimal formula, while the proportion of female flowers remained basically the same (16.6%), and the flower bud abortion rate increased by 67.1% (14.2%). This may be because salicylic acid, as an endogenous signaling molecule, enhances the tree's resistance to stress and reduces flower bud abortion caused by environmental stress. In the absence of choline chloride, the total number of female flowers per plant decreased by 10.0% (62.3 flowers) compared to the optimal formula, while the proportion of female flowers was slightly higher (18.1%), and the flower bud abortion rate increased by 72.9% (14.7%). This may be related to the ability of choline chloride to promote plant resistance to stress.

[0056] When Tween 20 is absent, the overall performance is close to that of the optimal formulation, but slightly lower overall. This may be because the surfactant effectively promotes the absorption and adhesion of key substances, ensuring the stability of the inducer.

[0057] The results show that the key substances involved in this invention can improve the growth of female flowers of *Xanthoceras sorbifolium* and reduce the female flower abortion rate to varying degrees.

[0058] Comparative Example 2 To verify whether the female flower inducer of the relevant patent is effective in female flower differentiation of Xanthoceras sorbifolium, and to compare the advantages of the formulation of this invention with that of the relevant patent.

[0059] CN117562056A A method for inducing and increasing the proportion of female flowers in chestnut and the formulation of the inducing agent in the regulator used: 5g of D-trehalose, 3g of D-sorbitol, and 1g of sucrose are added to 1L of water; The optimal formulation of the present invention: 600mg of 6-BA, 30mg of salicylic acid, 200mg of choline chloride, and 3g of urea are added to a 1L system, along with fixed components of 2.5g of potassium dihydrogen phosphate, 1.5g of boric acid, and 1.0mL of Tween 20; This method is applied to *Sapindus mukorossi*.

[0060] The *Xanthoceras sorbifolium* trees used in both comparisons had the same tree vigor, growth status, and age. The results of the comparison of the effects of the two inducers are shown in Table 6.

[0061] Table 6 Comparison of the effects of the two inducers

[0062] The results show that the optimal formulation of this invention is significantly more effective than the formulation of related patents when applied to *Xanthoceras sorbifolium* trees, indicating that the female flower inducer for *Xanthoceras sorbifolium* needs to be specifically formulated for application.

[0063] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. An inducer to enhance female flower differentiation in *Xanthoceras sorbifolium*, characterized in that, The aqueous solution contains 150-1200 mg of 6-benzyladenine, 10-50 mg of salicylic acid, 100-300 mg of choline chloride, 1.0-5.0 g of urea, 2.0-3.0 g of potassium dihydrogen phosphate, 0.5-2.0 g of boric acid, and 0.5-2.0 mL of emulsifier per liter.

2. The inducing agent for enhancing female flower differentiation in *Xanthoceras sorbifolium* according to claim 1, characterized in that, The solution contains, per liter of aqueous solution, 400-800 mg of 6-benzyladenine, 20-40 mg of salicylic acid, 150-250 mg of choline chloride, 2-4 g of urea, 2.0-3.0 g of potassium dihydrogen phosphate, 0.5-2.0 g of boric acid, and 0.5-2.0 mL of emulsifier.

3. The inducing agent for enhancing female flower differentiation in *Xanthoceras sorbifolium* according to claim 2, characterized in that, Based on per liter of aqueous solution, it includes 600 mg of 6-benzyladenine, 30 mg of salicylic acid, 200 mg of choline chloride, and 3 g of urea, combined with fixed components such as 2.5 g of potassium dihydrogen phosphate, 1.5 g of boric acid, and 1.0 mL of emulsifier.

4. The inducing agent for enhancing female flower differentiation of *Xanthoceras sorbifolium* according to any one of claims 1-3, characterized in that, The emulsifier is selected from ester-based nonionic surfactants.

5. The inducing agent for enhancing female flower differentiation of *Xanthoceras sorbifolium* according to any one of claims 4, characterized in that, The emulsifier is selected from one or more of Tween 80, Tween 20, Tween 60, and Tween 40, with Tween 20 being preferred.

6. The inducing agent for enhancing female flower differentiation of *Xanthoceras sorbifolium* according to any one of claims 1-3, characterized in that, It also includes one or more of indolepyruvic acid, tryptophan, and 2-O-β-D-glucosylsalicylic acid.

7. The inducing agent for enhancing female flower differentiation in *Xanthoceras sorbifolium* according to claim 6, characterized in that, The amount of indolepyruvic acid is 5-20 mg per liter of aqueous solution, the amount of tryptophan is 10-30 mg per liter, and the amount of 2-O-β-D-glucosylsalicylic acid is 5-15 mg per liter of aqueous solution.

8. The inducing agent for enhancing female flower differentiation of *Xanthoceras sorbifolium* according to any one of claims 1-7, characterized in that, The application is done by spraying onto the male and female flower reproductive organs; the preferred time for spraying is 7 days before the male and female flower reproductive organs develop.

9. The inducing agent for enhancing female flower differentiation in *Xanthoceras sorbifolium* according to claim 8, characterized in that, The spraying is to be carried out in the early morning or evening when there is no wind; the air temperature for spraying is 15~25℃.

10. A method for preparing an inducer for enhancing female flower differentiation in *Xanthoceras sorbifolium* as described in any one of claims 1 to 9, characterized in that, Includes the following steps: (1) Add 6-benzyladenine and salicylic acid to 10-20 mL of anhydrous ethanol, stir to dissolve, and obtain the regulator stock solution; (2) Add urea, potassium dihydrogen phosphate, boric acid and choline chloride to 600-800 ml of deionized water, stir to dissolve, and obtain water-soluble nutrient mother liquor; (3) Add the regulator stock solution dropwise to the water-soluble nutrient stock solution while stirring; after the addition is complete, add Tween 20 and stir to mix. (4) Add deionized water to a final volume of 1L and stir for 3-5 minutes.

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