Tissue culture and rapid propagation method for leaves of xanthoceras sorbifolia bunge

Abstract

The invention provides a leaf tissue culture and rapid propagation method of xanthoceras sorbifolia bunge. The method comprises the steps of leaf explant preparation, induced differentiation culture, multiplication culture, rooting culture, seedling hardening and transplanting. According to the method, the shiny-leaved yellowhorn leaves are used as explants, adventitious buds are directly induced, a callus induction stage is not needed, the rapid propagation efficiency is improved, and the seedling formation speed is high. And on the basis of the invention, gene transformation can be carried out by utilizing agrobacterium tumefaciens mediation or other methods.

Description

Technical Field

[0001] This invention belongs to the field of tissue culture technology, specifically relating to a method for rapid propagation of Xanthoceras sorbifolium leaves through tissue culture. Background Technology

[0002] *Xanthoceras sorbifolium* Bunge is a deciduous shrub or small tree belonging to the genus *Xanthoceras* in the family Sapindaceae. Its wild distribution is mainly in the Loess Plateau of temperate and warm-temperate climate zones, where wild resources exhibit high diversity. To ensure varietal consistency, *Xanthoceras sorbifolium* is primarily propagated through grafting and cuttings. However, these techniques are limited by a shortage of parent material and are cumbersome, resulting in low propagation efficiency and failing to address the social needs for seedlings and rapid propagation of different varieties. Therefore, establishing an efficient propagation technology system for *Xanthoceras sorbifolium* is a crucial challenge that urgently needs to be addressed to rapidly increase seedling production. Summary of the Invention

[0003] The technical problem this invention aims to solve is to address the shortcomings of the prior art by providing a rapid propagation method for *Xanthoceras sorbifolium* leaves via tissue culture. This method allows *Xanthoceras sorbifolium* leaves to directly redifferentiate into adventitious buds, saving a step compared to leaf differentiation into callus tissue and callus tissue differentiation into adventitious buds, thus improving rapid propagation efficiency. Furthermore, based on this invention, gene transformation can be performed using Agrobacterium-mediated transformation or other methods.

[0004] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for rapid propagation of *Xanthoceras sorbifolium* by leaf tissue culture, the method being as follows: S1. Preparation of leaf explants: In April, young leaves (healthy leaves free from pests and diseases) sprouting from the field of *Sapindus mukorossi* were selected. The compound leaves were cut into single leaves, the surface was brushed with a brush, and then placed on a clean bench. They were treated with a 75% ethanol aqueous solution for 30 seconds, then rinsed twice with sterile water, and then treated with a 0.5% sodium dichloroisocyanurate aqueous solution for 5 minutes. Finally, they were rinsed five times with sterile water to obtain the sterilized explants. S2, Induced Differentiation Culture: The sterilized explants obtained in S1 were placed on an inoculation tray. Using a blade, wounds were made on the underside of the explant leaves perpendicular to the main vein, every 1.5 cm. The explants were then inoculated into the differentiation induction medium with the underside of the leaves facing down. 1 to 3 explants were inoculated per bottle. After 25 to 28 days of induction and differentiation culture, a large number of adventitious buds were produced on the petioles, veins, and wounds. Some of the adventitious buds could reach a length of more than 1.5 cm, thus obtaining the induced differentiated explants. The differentiation-inducing medium was prepared by adding the following raw materials to the modified DKW medium at the following final concentrations: 6-benzylaminopurine 0.5 mg / L, indolebutyric acid 1.0 mg / L and indoleacetic acid (IAA) 0.05 mg / L; The modified DKW medium is prepared by adding the following raw materials to distilled water at the following final concentrations and adjusting the pH to 5.8-6.0: 3.81 g / L DKW medium dry powder, 5 mL / L calcium salt solution, 1 g / L ammonium nitrate, 30 g / L sucrose, and 6.5 g / L agar; S3, Proliferation Culture: The explants obtained from S2 after induced differentiation were transferred to the proliferation medium with the leaf back facing up. Two explants were inoculated per bottle for proliferation culture to obtain clustered adventitious buds. Alternatively, the leaves of adventitious buds with a height greater than 1.5 cm from the adventitious buds obtained from S2 were removed along the stem, cut into stem segments with a length of 1.5 cm, transferred to the proliferation medium, and two explants were inoculated per bottle for proliferation culture to obtain clustered adventitious buds. The proliferation medium was prepared by adding the following raw materials to DKW medium at the following final concentrations: 0.75 mg / L of 6-benzylaminopurine and 0.75 mg / L of indolebutyric acid; The DKW culture medium is prepared by adding the following raw materials to distilled water at the final concentration and adjusting the pH to 5.8-6.0: 3.81 g / L DKW culture medium dry powder, 5 mL / L calcium salt solution, 30 g / L sucrose, and 6.5 g / L agar; S4. Rooting Culture: The adventitious buds with a height greater than 1.5 cm from the leaves that induced differentiation in S2 were cut off along the stem and cut into stem segments with a length of 1.5 cm. These segments were then transferred to the rooting medium, with 2 segments inoculated per bottle for rooting culture to obtain rooted seedlings of *Xanthoceras sorbifolium*. Alternatively, select adventitious buds with a height greater than 1.5cm from the clustered adventitious buds obtained in S3, remove the leaves along the stem, cut the stem segments with a length of 1.5cm, transfer them to the rooting medium, inoculate 2 buds per bottle, and carry out rooting culture to obtain rooted seedlings of Xanthoceras sorbifolium. The rooting medium is prepared by adding the following raw materials to the DKW basic medium at the following final concentrations: indolebutyric acid 0.5 mg / L, naphthaleneacetic acid 0.2 mg / L and sucrose 30 g / L; The DKW basic culture medium is prepared by adding the following raw materials to distilled water at the final concentration and adjusting the pH to 5.8-6.0: 3.81 g / L DKW culture medium dry powder, 5 mL / L calcium salt solution, and 6.5 g / L agar; S5. Hardening off and transplanting: The *Xanthoceras sorbifolium* seedlings obtained in S4 were transferred to a greenhouse for hardening off. They were first acclimatized for 3 days under natural conditions at a temperature of 25℃~28℃ and a humidity of 80%, and then allowed to acclimatize for another 3 days with the caps off. After cleaning the culture medium from the roots, they were transplanted into the substrate in seedling trays. After new leaves emerged, they were further cultivated in the greenhouse for 30 days under conditions of a temperature of 25℃~35℃ and a humidity of 35%~80%, and then transplanted to the field.

[0005] Preferably, the induction differentiation culture time in S2 is 25-28 days, and the culture conditions are as follows: first, dark culture for 7 days, with alternating dark culture conditions of 25℃ for 14 hours and 18℃ for 10 hours; then, light-dark alternating culture, with light conditions of 14 hours, light intensity of 3000 lx, and temperature of 25℃; and dark conditions of 10 hours and temperature of 18℃.

[0006] Preferably, the proliferation culture time in S3 is 21-25 days, and the culture is carried out by alternating light and dark. The light conditions are: light time of 14 hours, light intensity of 3000 lx, and temperature of 25°C; the dark conditions are: dark time of 10 hours and temperature of 18°C.

[0007] Preferably, the rooting culture time in S4 is 25-28 days, and the culture conditions are as follows: first, dark culture is carried out for 7 days, and the dark culture conditions are: alternating between 25℃, 14h and 18℃, 10h; then, light-dark alternating culture is carried out, and the light conditions are: light time of 14h, light intensity of 3000lx, and temperature of 25℃; the dark conditions are: dark time of 10h and temperature of 18℃.

[0008] Preferably, the matrix in S5 is a mixture of peat moss, vermiculite and perlite in a mass ratio of 1:1:1.

[0009] Compared with the prior art, the present invention has the following advantages: 1. This invention uses leaves of *Sapindus mukorossi* as explants, which requires fewer explant materials than using axillary buds or embryos as explants, and is more conducive to the rapid propagation of new varieties with limited parent material.

[0010] 2. This invention uses leaves to induce adventitious buds, which involves fewer steps and higher efficiency compared to the process of leaves differentiating into callus tissue and then into adventitious buds. A large number of transplantable tissue culture seedlings can be produced in as little as 3 months, which reduces the cultivation time by at least 1 month compared to the callus tissue approach.

[0011] The present invention will be further described in detail below with reference to the embodiments. Detailed Implementation

[0012] Example 1

[0013] This embodiment describes the optimization of the culture medium for the rapid propagation method of *Xanthoceras sorbifolium* via leaf tissue culture. The method is as follows: S1. Preparation of leaf explants: Select healthy leaves of *Xanthoceras sorbifolium* free from pests and diseases, cut the compound leaves into single leaves, gently brush the surface with a soft brush, place them on a clean bench, treat with a 75% ethanol aqueous solution for 30 seconds, then rinse twice with sterile water, then treat with a 0.5% sodium dichloroisocyanurate (NADCC) aqueous solution for 5 minutes, and then rinse five times with sterile water to obtain the disinfected explants; This embodiment also optimizes the disinfection methods and effects of leaf explants at different times during the preparation of explants, as shown in Table 1.

[0014] Table 1. Methods and effects of explant disinfection at different stages According to Table 1, the most suitable time for field explant disinfection is April, that is, from the early to the late leaf expansion stage of *Xanthoceras sorbifolium*. Select newly sprouted tender leaves, cut compound leaves into single leaves, and lightly brush the surface with a soft brush before use. On a clean bench, treat with 75% alcohol (75% ethanol aqueous solution) for 30 seconds, rinse twice with sterile water, then treat with 0.5% sodium dichloroisocyanurate (NADCC) aqueous solution for 5 minutes, and rinse five times with sterile water.

[0015] The hydroponic germination method involves cutting healthy branches after the leaves of the *Xanthoceras sorbifolium* have fallen. After brushing off surface dust, the branches are placed in purified water for germination treatment, with 1% GA3 added to the water. The suitable period for hydroponic germination is relatively long, from November to March, and sterilization is relatively easy, resulting in high rates of bacterial contamination and survival. This invention recommends using young leaves sprouting in the field. Young leaves for hydroponic germination are more readily available, and hydroponic germination can serve as a supplementary method to increase the availability of materials.

[0016] S2, Induced Differentiation Culture: The sterilized explants obtained in S1 were placed on an inoculation tray. Using a blade, wounds were made on the underside of the explant leaves perpendicular to the main vein, every 1.5 cm. The explants were then inoculated into the differentiation induction medium with the underside of the leaves facing down. 1 to 3 explants were inoculated per bottle and cultured for 25 to 28 days to induce differentiation. A large number of adventitious buds were produced in the petiole, veins and wounds. Some of the adventitious buds could reach a length of more than 1.5 cm, and the induced differentiated explants were obtained. The differentiation-inducing medium was prepared by adding the following raw materials to the modified DKW medium at the following final concentrations: 6-benzylaminopurine (6-BA) 0.5 mg / L, indolebutyric acid (IBA) 1.0 mg / L and indoleacetic acid (IAA) 0.05 mg / L; The modified DKW medium is prepared by adding ammonium nitrate to the DKW medium; The modified DKW medium is prepared by adding the following raw materials to distilled water at the following final concentrations and adjusting the pH to 5.8-6.0: 3.81 g / L DKW medium dry powder, 5 mL / L calcium salt solution, 1 g / L ammonium nitrate, 30 g / L sucrose, and 6.5 g / L agar; DKW culture medium powder and calcium salt solution were purchased commercially from Coollab.

[0017] The induced differentiation culture period was 25-28 days, and the culture conditions were as follows: first, a 7-day dark culture was performed, with alternating dark culture conditions of 25℃ for 14 hours and 18℃ for 10 hours; then, a light-dark alternating culture was performed, with light conditions of 14 hours at a light intensity of 3000 lx and a temperature of 25℃; and dark conditions of 10 hours at a temperature of 18℃. This embodiment also optimizes the induction differentiation effect of different induction differentiation media in the induction differentiation culture, as shown in Table 2.

[0018] Comprehensive index value = germination rate (%) × average sprout length (cm); Table 2 Differentiation induction effects of different culture media According to Table 2, WY6 is the most effective culture medium for inducing differentiation, exhibiting the highest comprehensive index value, highest differentiation rate, and best growth status of differentiated shoots. It is the optimal formulation among the induction differentiation media. Multiple verifications have confirmed this formulation as the optimal one.

[0019] S3, Proliferation Culture: The explants obtained from S2 after induced differentiation were transferred to the proliferation medium with the leaf abaxial surface facing up. Two explants were inoculated into each bottle for proliferation culture to obtain clusters of adventitious shoots. Alternatively, remove the leaves from the adventitious buds with a height greater than 1.5 cm obtained from S2 along the stem, cut them into stem segments with a length of 1.5 cm, transfer them to the proliferation medium, inoculate 2 per bottle for proliferation culture, and obtain clustered adventitious buds; The proliferation medium was prepared by adding the following raw materials to DKW medium at the following final concentrations: 6-benzylaminopurine (6-BA) 0.75 mg / L and indolebutyric acid (IBA) 0.75 mg / L; The DKW culture medium is prepared by adding the following raw materials to distilled water at the final concentration and adjusting the pH to 5.8-6.0: 3.81 g / L DKW culture medium dry powder, 5 mL / L calcium salt solution, 30 g / L sucrose, and 6.5 g / L agar; DKW culture medium powder and calcium salt solution were purchased commercially from Coollab. The proliferation culture time was 21-25 days, with alternating light and dark conditions. The light conditions were: 14 hours of light, 3000 lx light intensity, and 25°C; the dark conditions were: 10 hours of darkness and 18°C. This embodiment also optimizes the proliferation effect of different proliferation media in proliferation culture, as shown in Table 3.

[0020] Proliferation coefficient = number of effective adventitious buds ÷ number of inoculated explants. Effective adventitious buds are those with a length ≥ 0.5 cm.

[0021] Table 3. Proliferation effect of different culture media According to Table 3, WG10 was selected as the proliferation medium because it has the highest proliferation coefficient, the longest average bud length, and the highest germination rate, making it the optimal formulation among proliferation mediums.

[0022] S4. Rooting Culture: The adventitious buds with a height greater than 1.5 cm from the leaves that induced differentiation in S2 were cut off along the stem and cut into stem segments with a length of 1.5 cm. These segments were then transferred to the rooting medium, with 2 segments inoculated per bottle for rooting culture to obtain rooted seedlings of *Xanthoceras sorbifolium*. Alternatively, select adventitious buds with a height greater than 1.5cm from the clustered adventitious buds obtained in S3, remove the leaves along the stem, cut the stem segments with a length of 1.5cm, transfer them to the rooting medium, inoculate 2 buds per bottle, and carry out rooting culture to obtain rooted seedlings of Xanthoceras sorbifolium. The rooting medium is prepared by adding the following raw materials to DKW basic medium at the following final concentrations: indolebutyric acid (IBA) 0.5 mg / L, naphthaleneacetic acid (NAA) 0.2 mg / L, and sucrose 30 g / L; the DKW basic medium is prepared by adding the following raw materials to distilled water at the following final concentrations and adjusting the pH to 5.8-6.0: DKW medium dry powder 3.81 g / L, calcium salt solution 5 mL / L, and agar 6.5 g / L; The rooting culture period was 25-28 days, and the culture conditions were as follows: first, dark culture was carried out for 7 days, with alternating dark culture conditions of 25℃ for 14 hours and 18℃ for 10 hours; then, light-dark alternating culture was carried out, with light conditions of 14 hours, light intensity of 3000 lx, and temperature of 25℃; and dark conditions of 10 hours and temperature of 18℃. This embodiment also optimizes the rooting effect of different rooting media in rooting culture, as shown in Table 4.

[0023] Comprehensive index value = number of effective roots ÷ number of inoculated explants; Effective roots are those with a root length ≥ 0.5 cm. Table 4. Rooting effects of different rooting media Table 4 shows that WPM is made from the following raw materials: 19.91 g / L WPM medium powder + 1 ml / L calcium salt solution + 6.5 g / L agar. The WPM medium powder and calcium salt solution mentioned above are from the brand Coollabo. 1 / 2MS is prepared from the following raw materials: 2.289 g / L of 1 / 2MS medium powder + 6.5 g / L of agar. The 1 / 2MS medium powder is from the brand Cooler Master. DKW (pH 5.8-6.0) is made from the following ingredients: 3.81 g / L DKW culture medium powder, 5 mL / L calcium salt solution, and 6.5 g / L agar (the above DKW culture medium powder and calcium salt solution are from the brand Coollabo). According to Table 4, GW7 is the culture medium formulation with the highest comprehensive index value.

[0024] S5. Hardening off and transplanting: The *Xanthoceras sorbifolium* seedlings obtained in S4 were transferred to a greenhouse for hardening-off. They were first acclimatized for 3 days under natural conditions at a temperature of 25℃~28℃ and a humidity of 80%, and then allowed to acclimatize for another 3 days with the caps off. After cleaning the culture medium from the roots, they were transplanted into the substrate (a mixture of peat moss, vermiculite, and perlite in a mass ratio of 1:1:1) in seedling trays. After new leaves appeared, they were continued to be cultivated in the greenhouse for 30 days under conventional management (temperature of 25℃~35℃ and humidity of 35%~80%) before being transplanted to the field. The survival rate of transplanted seedlings can reach over 95%. Example 2

[0025] The rapid propagation method of *Xanthoceras sorbifolium* by leaf tissue culture in this embodiment is as follows: S1. Preparation of leaf explants: In April, healthy leaves of *Sapindus mukorossi* free from pests and diseases were selected. The compound leaves were cut into single leaves, the surface was lightly brushed with a soft brush, and then placed on a clean bench. The leaves were treated with a 75% ethanol aqueous solution for 30 seconds, then rinsed twice with sterile water, and then treated with a 0.5% sodium dichloroisocyanurate aqueous solution for 5 minutes. Finally, the leaves were rinsed five times with sterile water to obtain the sterilized explants. S2, Induced Differentiation Culture: Place the sterilized explants obtained in S1 on an inoculation tray. Make a cut every 1.5 cm on the underside of the explant leaf perpendicular to the main vein. Inoculate the explant leaf with the underside facing down in the induction differentiation medium. Inoculate 1 to 3 explants per bottle. After 25 days of induction differentiation culture, adventitious buds will be generated in the petiole, leaf vein and wound, and the inducing differentiated explants will be obtained. The differentiation-inducing medium was prepared by adding the following raw materials to the modified DKW medium at the following final concentrations: 6-benzylaminopurine 0.5 mg / L, indolebutyric acid 1.0 mg / L and indoleacetic acid 0.05 mg / L; The modified DKW medium is prepared by adding the following raw materials to distilled water at the final concentration and adjusting the pH to 5.8: 3.81 g / L DKW medium dry powder, 5 mL / L calcium salt solution, 1 g / L ammonium nitrate, 30 g / L sucrose, and 6.5 g / L agar; The induced differentiation culture time was 25 days, and the culture conditions were as follows: first, dark culture for 7 days, with alternating dark culture conditions of 25℃ for 14 hours and 18℃ for 10 hours; then, light-dark alternating culture was performed, with light conditions of 14 hours of light, light intensity of 3000 lx, and temperature of 25℃; and dark conditions of 10 hours of darkness and temperature of 18℃. S3, Proliferation Culture: The explants obtained from S2 after induced differentiation were transferred to the proliferation medium with the leaf abaxial surface facing up. Two explants were inoculated into each bottle for proliferation culture to obtain clusters of adventitious shoots. The proliferation medium was prepared by adding the following raw materials to DKW medium at the following final concentrations: 0.75 mg / L of 6-benzylaminopurine and 0.75 mg / L of indolebutyric acid; The DKW culture medium is prepared by adding the following raw materials to distilled water at the final concentration and adjusting the pH to 5.8: 3.81 g / L DKW culture medium dry powder, 5 mL / L calcium salt solution, 30 g / L sucrose, and 6.5 g / L agar; The proliferation culture lasted for 21 days, and was carried out under alternating light and dark conditions. The light conditions were: 14 hours of light, 3000 lx light intensity, and 25°C temperature; the dark conditions were: 10 hours of darkness and 18°C ​​temperature. S4. Rooting Culture: Select adventitious buds with a height greater than 1.5cm from the clustered adventitious buds obtained in S3, remove the leaves along the stem, cut the stem segments with a length of 1.5cm, transfer them to the rooting culture medium, inoculate 2 buds per bottle, and carry out rooting culture to obtain rooted seedlings of Xanthoceras sorbifolium. The rooting medium is prepared by adding the following raw materials to the DKW basic medium at the following final concentrations: indolebutyric acid 0.5 mg / L, naphthaleneacetic acid 0.2 mg / L and sucrose 30 g / L; The DKW basic culture medium is prepared by adding the following raw materials to distilled water at the final concentration and adjusting the pH to 5.8: 3.81 g / L DKW culture medium dry powder, 5 mL / L calcium salt solution, and 6.5 g / L agar; The rooting culture period was 25 days, and the culture conditions were as follows: first, a 7-day dark culture was conducted, with alternating dark conditions of 25℃ for 14 hours and 18℃ for 10 hours; then, a light-dark alternating culture was conducted, with light conditions of 14 hours at a light intensity of 3000 lx and a temperature of 25℃; and dark conditions of 10 hours at a temperature of 18℃. S5. Hardening off and transplanting: The *Xanthoceras sorbifolium* seedlings obtained in S4 were transferred to a greenhouse for hardening-off. They were first acclimatized for 3 days under natural conditions at 25°C and 80% humidity, and then allowed to acclimatize for another 3 days with the caps off. After the culture medium was washed off the roots, they were transplanted into the substrate in seedling trays. After new leaves appeared, they were further cultivated in the greenhouse for 30 days at 25°C and 35% humidity before being transplanted to the field. The substrate was a mixture of peat moss, vermiculite, and perlite in a mass ratio of 1:1:1.

[0026] The transplant survival rate in this embodiment reached 95%. The rapid propagation method in this embodiment requires 107 days. Example 3

[0027] The rapid propagation method of *Xanthoceras sorbifolium* by leaf tissue culture in this embodiment is as follows: S1. Preparation of leaf explants: In April, healthy leaves of *Sapindus mukorossi* free from pests and diseases were selected. The compound leaves were cut into single leaves, the surface was lightly brushed with a soft brush, and then placed on a clean bench. The leaves were treated with a 75% ethanol aqueous solution for 30 seconds, then rinsed twice with sterile water, and then treated with a 0.5% sodium dichloroisocyanurate aqueous solution for 5 minutes. Finally, the leaves were rinsed five times with sterile water to obtain the sterilized explants. S2, Induced Differentiation Culture: The sterilized explants obtained in S1 were placed on an inoculation tray. Using a blade, wounds were made on the underside of the explant leaves perpendicular to the main vein, every 1.5 cm. The explants were inoculated into the induction differentiation medium with the underside of the leaves facing down. 1 to 3 explants were inoculated per bottle. After 28 days of induction differentiation culture, adventitious buds were generated in the petiole, veins and wounds, and the inducing differentiated explants were obtained. The differentiation-inducing medium was prepared by adding the following raw materials to the modified DKW medium at the following final concentrations: 6-benzylaminopurine 0.5 mg / L, indolebutyric acid 1.0 mg / L and indoleacetic acid 0.05 mg / L; The modified DKW medium is prepared by adding the following raw materials to distilled water at the final concentration and adjusting the pH to 6.0: 3.81 g / L DKW medium dry powder, 5 mL / L calcium salt solution, 1 g / L ammonium nitrate, 30 g / L sucrose, and 6.5 g / L agar; The induced differentiation culture time was 28 days, and the culture conditions were as follows: first, dark culture was carried out for 7 days, with alternating dark culture conditions of 25℃ for 14 hours and 18℃ for 10 hours; then, light-dark alternating culture was carried out, with light conditions of 14 hours, light intensity of 3000 lx, and temperature of 25℃; and dark conditions of 10 hours and temperature of 18℃. S3, Proliferation Culture: Leaves were removed from adventitious buds with a height greater than 1.5 cm from those obtained in S2, and the buds were cut into stem segments with a length of 1.5 cm. The segments were then transferred to the proliferation medium, and two segments were inoculated into each bottle for proliferation culture to obtain clustered adventitious buds. The proliferation medium was prepared by adding the following raw materials to DKW medium at the following final concentrations: 0.75 mg / L of 6-benzylaminopurine and 0.75 mg / L of indolebutyric acid; The DKW medium is prepared by adding the following raw materials to distilled water at the final concentration and adjusting the pH to 6.0: 3.81 g / L DKW medium dry powder, 5 mL / L calcium salt solution, 30 g / L sucrose, and 6.5 g / L agar; The proliferation culture lasted for 25 days, and was carried out under alternating light and dark conditions. The light conditions were: 14 hours of light, 3000 lx light intensity, and 25°C temperature; the dark conditions were: 10 hours of darkness and 18°C ​​temperature. S4. Rooting Culture: Select adventitious buds with a height greater than 1.5cm from the clustered adventitious buds obtained in S3, remove the leaves along the stem, cut the stem segments with a length of 1.5cm, transfer them to the rooting culture medium, inoculate 2 buds per bottle, and carry out rooting culture to obtain rooted seedlings of Xanthoceras sorbifolium. The rooting medium is prepared by adding the following raw materials to the DKW basic medium at the following final concentrations: indolebutyric acid 0.5 mg / L, naphthaleneacetic acid 0.2 mg / L and sucrose 30 g / L; The DKW basic culture medium is prepared by adding the following raw materials to distilled water at the final concentration and adjusting the pH to 6.0: 3.81 g / L DKW culture medium dry powder, 5 mL / L calcium salt solution, and 6.5 g / L agar; The rooting culture period was 28 days, and the culture conditions were as follows: first, a 7-day dark culture was conducted, with alternating dark conditions of 25℃ for 14 hours and 18℃ for 10 hours; then, a light-dark alternating culture was conducted, with light conditions of 14 hours at a light intensity of 3000 lx and a temperature of 25℃; and dark conditions of 10 hours at a temperature of 18℃. S5. Hardening off and transplanting: The *Xanthoceras sorbifolium* seedlings obtained in S4 were transferred to a greenhouse for hardening-off. They were first acclimatized for 3 days under natural conditions at 28℃ and 80% humidity, and then allowed to acclimatize for another 3 days with the caps off. After the culture medium on the roots was cleaned, they were transplanted into the substrate in seedling trays. After new leaves appeared, they were further cultivated in the greenhouse for 30 days at 35℃ and 80% humidity before being transplanted to the field. The substrate was a mixture of peat moss, vermiculite, and perlite in a mass ratio of 1:1:1.

[0028] The transplant survival rate in this embodiment reached 95%. The cultivation period in this embodiment was 117 days. Example 4

[0029] The rapid propagation method of *Xanthoceras sorbifolium* by leaf tissue culture in this embodiment is as follows: S1. Preparation of leaf explants: In April, healthy leaves of *Sapindus mukorossi* free from pests and diseases were selected. The compound leaves were cut into single leaves, the surface was lightly brushed with a soft brush, and then placed on a clean bench. The leaves were treated with a 75% ethanol aqueous solution for 30 seconds, then rinsed twice with sterile water, and then treated with a 0.5% sodium dichloroisocyanurate aqueous solution for 5 minutes. Finally, the leaves were rinsed five times with sterile water to obtain the sterilized explants. S2, Induced Differentiation Culture: Place the sterilized explants obtained in S1 on an inoculation tray. Make a cut every 1.5 cm on the underside of the explant leaf perpendicular to the main vein. Inoculate the explant leaf with the underside facing down in the induction differentiation medium. Inoculate 1 to 3 explants per bottle. After 25 days of induction differentiation culture, adventitious buds will be generated in the petiole, leaf vein and wound, and the inducing differentiated explants will be obtained. The differentiation-inducing medium was prepared by adding the following raw materials to the modified DKW medium at the following final concentrations: 6-benzylaminopurine 0.5 mg / L, indolebutyric acid 1.0 mg / L and indoleacetic acid 0.05 mg / L; The modified DKW medium is prepared by adding the following raw materials to distilled water at the final concentration and adjusting the pH to 5.9: 3.81 g / L DKW medium dry powder, 5 mL / L calcium salt solution, 1 g / L ammonium nitrate, 30 g / L sucrose, and 6.5 g / L agar; The induced differentiation culture time was 25 days, and the culture conditions were as follows: first, dark culture for 7 days, with alternating dark culture conditions of 25℃ for 14 hours and 18℃ for 10 hours; then, light-dark alternating culture was performed, with light conditions of 14 hours of light, light intensity of 3000 lx, and temperature of 25℃; and dark conditions of 10 hours of darkness and temperature of 18℃. S3, Rooting Culture: The adventitious buds with a height greater than 1.5 cm from the leaves that induced differentiation in S2 were cut off along the stem and cut into stem segments with a length of 1.5 cm. These segments were then transferred to the rooting medium, with 2 segments inoculated per bottle for rooting culture to obtain rooted seedlings of *Xanthoceras sorbifolium*. The rooting medium is prepared by adding the following raw materials to the DKW basic medium at the following final concentrations: indolebutyric acid 0.5 mg / L, naphthaleneacetic acid 0.2 mg / L and sucrose 30 g / L; The DKW basic culture medium is prepared by adding the following raw materials to distilled water at the final concentration and adjusting the pH to 5.9: 3.81 g / L DKW culture medium dry powder, 5 mL / L calcium salt solution, and 6.5 g / L agar; The rooting culture period was 25 days, and the culture conditions were as follows: first, a 7-day dark culture was conducted, with alternating dark conditions of 25℃ for 14 hours and 18℃ for 10 hours; then, a light-dark alternating culture was conducted, with light conditions of 14 hours at a light intensity of 3000 lx and a temperature of 25℃; and dark conditions of 10 hours at a temperature of 18℃. S4. Hardening off and transplanting: The *Xanthoceras sorbifolium* seedlings obtained in S3 were transferred to a greenhouse for hardening-off. They were first acclimatized for 3 days under natural conditions at 26℃ and 80% humidity, and then allowed to acclimatize for another 3 days with the caps off. After the culture medium was washed off the roots, they were transplanted into the substrate in seedling trays. After new leaves appeared, they were further cultivated in the greenhouse for 30 days at 30℃ and 60% humidity before being transplanted to the field. The substrate was a mixture of peat moss, vermiculite, and perlite in a mass ratio of 1:1:1.

[0030] The transplant survival rate in this embodiment reached 95%, and the cultivation period in this embodiment was 86 days.

[0031] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method for rapid propagation of *Sapindus mukorossi* through leaf tissue culture, characterized in that, The method is as follows: S1. Preparation of leaf explants: Select healthy leaves of *Xanthoceras sorbifolium* free from pests and diseases, cut the compound leaves into single leaves, brush the surface with a brush, place them on a clean bench, treat with a 75% ethanol aqueous solution for 30 seconds, then rinse twice with sterile water, then treat with a 0.5% sodium dichloroisocyanurate aqueous solution for 5 minutes, and then rinse five times with sterile water to obtain the disinfected explants. S2, Induced Differentiation Culture: Place the sterilized explants obtained in S1 on an inoculation tray. Make a cut every 1.5 cm on the underside of the explant leaf perpendicular to the main vein. Inoculate the explant leaf with the underside facing down in the induction differentiation medium. Inoculate 1 to 3 explants per bottle. After 25 to 28 days of induction differentiation culture, adventitious buds will be generated in the petiole, leaf vein and wound, and the inducing differentiated explants will be obtained. The differentiation-inducing medium was prepared by adding the following raw materials to the modified DKW medium at the following final concentrations: 6-benzylaminopurine 0.5 mg / L, indolebutyric acid 1.0 mg / L and indoleacetic acid 0.05 mg / L; The modified DKW medium is prepared by adding the following raw materials to distilled water at the final concentration and adjusting the pH to 5.8-6.0: 3.81 g / L DKW medium dry powder, 5 mL / L calcium salt solution, 1 g / L ammonium nitrate, 30 g / L sucrose, and 6.5 g / L agar; S3, Proliferation Culture: The explants obtained from S2 after induced differentiation were transferred to the proliferation medium with the leaf back facing up. Two explants were inoculated per bottle for proliferation culture to obtain clustered adventitious buds. Alternatively, the leaves of adventitious buds with a height greater than 1.5 cm from the adventitious buds obtained from S2 were removed along the stem, cut into stem segments with a length of 1.5 cm, transferred to the proliferation medium, and two explants were inoculated per bottle for proliferation culture to obtain clustered adventitious buds. The proliferation medium was prepared by adding the following raw materials to DKW medium at the following final concentrations: 0.75 mg / L of 6-benzylaminopurine and 0.75 mg / L of indolebutyric acid; The DKW culture medium is prepared by adding the following raw materials to distilled water at the final concentration and adjusting the pH to 5.8-6.0: 3.81 g / L DKW culture medium dry powder, 5 mL / L calcium salt solution, 30 g / L sucrose, and 6.5 g / L agar; S4. Rooting Culture: The adventitious buds with a height greater than 1.5 cm from the leaves that induced differentiation in S2 were cut off along the stem and cut into stem segments with a length of 1.5 cm. These segments were then transferred to the rooting medium, with 2 segments inoculated per bottle for rooting culture to obtain rooted seedlings of *Xanthoceras sorbifolium*. Alternatively, select adventitious buds with a height greater than 1.5cm from the clustered adventitious buds obtained in S3, remove the leaves along the stem, cut the stem segments with a length of 1.5cm, transfer them to the rooting medium, inoculate 2 buds per bottle, and carry out rooting culture to obtain rooted seedlings of Xanthoceras sorbifolium. The rooting medium is prepared by adding the following raw materials to the DKW basic medium at the following final concentrations: indolebutyric acid 0.5 mg / L, naphthaleneacetic acid 0.2 mg / L and sucrose 30 g / L; The DKW basic culture medium is prepared by adding the following raw materials to distilled water at the final concentration and adjusting the pH to 5.8-6.0: 3.81 g / L DKW culture medium dry powder, 5 mL / L calcium salt solution, and 6.5 g / L agar; S5. Hardening off and transplanting: The *Xanthoceras sorbifolium* seedlings obtained in S4 were transferred to a greenhouse for hardening off. They were first acclimatized for 3 days under natural conditions at a temperature of 25℃~28℃ and a humidity of 80%, and then allowed to acclimatize for another 3 days with the caps off. After cleaning the culture medium from the roots, they were transplanted into the substrate in seedling trays. After new leaves appeared, they were further cultivated in the greenhouse for 30 days under conditions of a temperature of 25℃~35℃ and a humidity of 35%~80%, and then transplanted to the field.

2. The method for rapid propagation of *Xanthoceras sorbifolium* by leaf tissue culture according to claim 1, characterized in that, The induction differentiation culture time described in S2 is 25-28 days, and the culture conditions are as follows: first, dark culture for 7 days, with alternating dark culture conditions of 25℃ for 14 hours and 18℃ for 10 hours; then, light-dark alternating culture, with light conditions of 14 hours, light intensity of 3000 lx, and temperature of 25℃; and dark conditions of 10 hours and temperature of 18℃.

3. The method for rapid propagation of *Xanthoceras sorbifolium* by leaf tissue culture according to claim 1, characterized in that, The proliferation culture time described in S3 is 21-25 days, with alternating light and dark culture. The light conditions are: 14 hours of light, 3000 lx light intensity, and 25°C temperature; the dark conditions are: 10 hours of darkness and 18°C ​​temperature.

4. The method for rapid propagation of *Xanthoceras sorbifolium* by leaf tissue culture according to claim 1, characterized in that, The rooting culture time described in S4 is 25-28 days, and the culture conditions are as follows: first, dark culture for 7 days, with alternating dark culture conditions of 25℃ for 14 hours and 18℃ for 10 hours; then, light-dark alternating culture, with light conditions of 14 hours, light intensity of 3000 lx, and temperature of 25℃; and dark conditions of 10 hours and temperature of 18℃.

5. The method for rapid propagation of *Xanthoceras sorbifolium* by leaf tissue culture according to claim 1, characterized in that, The matrix described in S5 is a mixture of peat moss, vermiculite, and perlite in a mass ratio of 1:1:1.

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