Efficient tissue culture and rapid propagation seedling growing method for xanthoceras sorbifolia bunge

Abstract

The invention relates to the technical field of seedling raising, and discloses an efficient tissue culture and rapid propagation seedling raising method for xanthoceras sorbifolia bunge, which comprises the following steps: selecting and pretreating explants, selecting current-year robust branches of the xanthoceras sorbifolia bunge, cutting stem segments with axillary buds as the explants, cleaning, trimming and disinfecting the explants, performing surface disinfection on the pretreated explants, and performing tissue culture and rapid propagation seedling raising on the pretreated explants to obtain the xanthoceras sorbifolia bunge. When the Xanthoceras sorbifolia Bunge is subjected to efficient tissue culture and rapid propagation seedling culture, an operation standard considering thorough sterilization and tissue activity protection is established by standardizing an explant selection standard and executing a step-by-step surface disinfection procedure, so that the microbial contamination rate caused by incomplete disinfection of the explant can be reduced, meanwhile, tissue activity damage caused by excessive disinfection is reduced, and the survival rate of the Xanthoceras sorbifolia Bunge is improved. Therefore, sufficient and healthy sterile initial materials are guaranteed to be obtained, the problems of high primary culture pollution rate and induction starting failure are solved, and a reliable foundation is laid for establishment of a whole tissue culture and rapid propagation system.

Description

Technical Field

[0001] This invention relates to the field of seedling technology, specifically to a method for efficient tissue culture and rapid propagation of *Xanthoceras sorbifolium* seedlings. Background Technology

[0002] *Xanthoceras sorbifolium*, a deciduous shrub or small tree belonging to the genus *Xanthoceras sorbifolium* in the family Sapindaceae, is an excellent ornamental tree species. Its stems or branches and leaves can be used medicinally, possessing the effects of dispelling wind and dampness, reducing swelling and relieving pain. Mature *Xanthoceras sorbifolium* has a well-developed, deep, and widely distributed root system, making it a pioneer tree species for windbreak and sand fixation, small watershed management, and other purposes. The kernels have a high oil content and are an industrial raw material for high-grade lubricants and cosmetics. In addition, fresh leaves of *Xanthoceras sorbifolium* can be made into tea, while the seed coat and pericarp can be made into activated carbon.

[0003] Currently, in the practical operation of rapid propagation of Xanthoceras sorbifolium tissue culture, due to the endophytic bacteria carried by the explants and their complex surface structure, it is difficult to achieve a balance between thorough sterilization and protection of tissue activity when performing disinfection treatment using conventional disinfectant concentration and time combinations. When disinfection is incomplete or the explants are excessively damaged, the initial contamination rate will remain high or induction initiation will fail, resulting in insufficient sterile material. At the same time, during the proliferation culture stage, the hormone ratio and culture mode of conventional culture media are relatively fixed and cannot dynamically adapt to the growth differences that occur in different genotypes or clones during the culture process. This can lead to low proliferation coefficients in some superior lines, weak growth of clustered shoots, or physiological abnormalities such as vitrification. Furthermore, there is a lack of effective means to regulate growth abnormalities. During the rooting and transplanting stages, because tissue culture seedlings are in a high-humidity, constant-temperature, heterotrophic environment for a long time, their morphological structure and physiological functions differ from those of seedlings grown from seed. Conventional rooting induction and hardening methods often result in poor root development, sparse root hairs, or long recovery periods after transplanting and large fluctuations in survival rates, further affecting the overall efficiency and economic benefits of rapid propagation technology through tissue culture.

[0004] Therefore, a highly efficient tissue culture method for rapid propagation of *Xanthoceras sorbifolium* seedlings is proposed to address the aforementioned problems. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a highly efficient tissue culture method for rapid propagation of *Sapindus mukorossi* seedlings, solving the problem mentioned in the background art where conventional combinations of disinfectant concentration and time are difficult to balance between thorough sterilization and protection of tissue activity.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for efficient tissue culture and rapid propagation of *Xanthoceras sorbifolium* seedlings, comprising the following steps: Step 1: Explant selection and pretreatment. Select healthy current-year branches of *Xanthoceras sorbifolium*, cut stem segments with axillary buds as explants, and clean and prune them. Step 2: Disinfection treatment, the surface of the pretreated explants is disinfected to remove microbial contamination; Step 3: Preparation of primary culture medium; preparation of solid culture medium for primary induction. Step 4: Inoculation and primary culture. The sterilized explants are inoculated onto the primary culture medium and primary induction culture is carried out under controlled conditions. Step 5: Propagation culture. Transfer the clustered or adventitious shoots obtained from the initial culture into the propagation culture medium for further propagation. Step Six: Rooting Culture. Transfer the proliferated rootless seedlings into a rooting culture medium to induce root formation. Step 7: Hardening off and transplanting. After hardening off the tissue culture seedlings with complete roots, transplant them into the seedling substrate. In step three, the primary culture medium is prepared from the following components: MS basal medium, 6-benzyl adenine 0.5-2.0 mg / L, naphthaleneacetic acid 0.1-0.5 mg / L, sucrose 25-35 g / L, agar 5-7 g / L, and pH adjusted to 5.7-5.9; In step five, the proliferation medium is prepared from the following components: MS basal medium, 6-benzyl adenine 1.0-3.0 mg / L, naphthaleneacetic acid 0.1-0.5 mg / L, sucrose 25-35 g / L, agar 5-7 g / L, and pH adjusted to 5.7-5.9; In step six, the rooting medium is prepared from the following components: 1 / 2 MS basal medium, indolebutyric acid 0.5-1.5 mg / L, sucrose 15-25 g / L, agar 5-7 g / L, and pH adjusted to 5.7-5.9.

[0007] Preferably, in step one, the healthy current-year branches of *Sapindus mukorossi* are semi-lignified branches with a diameter of 0.3-0.6 cm. The length of the cut stem segments is 1.0-2.0 cm, and each stem segment retains 1-2 axillary buds. After pruning, the stem segments are rinsed with running water for 30-60 minutes to remove surface impurities.

[0008] Preferably, in step two, the disinfection process includes the following steps: placing the pretreated explant in a clean bench, first immersing it in a 70-75% ethanol solution for 30-60 seconds for preliminary disinfection, then immersing it in a 0.1-0.2% mercuric chloride solution or a 2-4% sodium hypochlorite solution for 8-15 minutes for disinfection, and finally rinsing it with sterile distilled water 3-5 times to remove residual disinfectant.

[0009] Preferably, in step three, the preparation method of the primary culture medium includes: dissolving MS basic culture medium powder in distilled water, adding the plant growth regulator solution of 6-benzyl adenine and naphthaleneacetic acid, then adding sucrose and agar, stirring and mixing evenly, adjusting the pH to 5.7-5.9, dispensing into culture containers, and autoclaving at 121°C for 15-20 minutes.

[0010] Preferably, in step four, the initial culture conditions include: a culture temperature of 24-26℃, a light intensity of 2000-3000 lux, a photoperiod of 14-16 hours of light / 8-10 hours of darkness, and a culture time of 20-35 days, until the explants induce clustered buds or adventitious buds.

[0011] Preferably, in step five, the conditions for proliferation culture include: a culture temperature of 24-26℃, a light intensity of 2000-3000 lux, a photoperiod of 14-16 hours of light / 8-10 hours of darkness, a culture cycle of 25-35 days, and a proliferation coefficient of 3-5 times per generation. During the proliferation process, the clustered buds are cut into small segments with buds and then transferred to fresh proliferation culture medium for subculture.

[0012] Preferably, in step six, the conditions for rooting culture include: a culture temperature of 24-26℃, a light intensity of 2000-3000 lux, a photoperiod of 14-16 hours of light / 8-10 hours of darkness, and a culture time of 15-25 days, until roots of 2-3 cm in length are induced at the base of the seedlings. The rooting culture medium also contains 0.5-1.0 g / L of activated carbon to promote robust root growth.

[0013] Preferably, in step seven, the hardening-off and transplanting of seedlings includes the following steps: when the rooted seedlings grow to 3-5cm in height, the culture container is moved to a natural light environment for hardening-off for 3-5 days. Then, the tissue culture seedlings are taken out, the root culture medium is washed off, and they are transplanted into a seedling substrate made of vermiculite, perlite and peat moss in a volume ratio of 1:1:1. Initially, the humidity is maintained at 80-90% and the temperature at 20-25℃. After 7-10 days of recovery, normal management is carried out.

[0014] Preferably, the primary culture medium, the proliferation culture medium, and the rooting culture medium all contain organic additives, which are 50-100 ml / L of coconut juice or 50-100 g / L of banana puree. The organic additives are added aseptically after the culture medium has been sterilized and cooled to 50-60°C.

[0015] Preferably, the relative humidity of the culture environment in steps four, five and six is ​​controlled at 60-70%, and a breathable sealing film is used to cover the culture container to promote gas exchange. The entire tissue culture process is carried out under aseptic conditions, and the explant inoculation density is 3-5 stem segments per bottle.

[0016] Compared with the prior art, the present invention provides a method for efficient tissue culture and rapid propagation of *Sapindus mukorossi* seedlings, which has the following beneficial effects: 1. In this invention, when conducting efficient tissue culture and rapid propagation of Xanthoceras sorbifolium seedlings, by standardizing the selection criteria for explants and implementing a step-by-step surface disinfection procedure, an operational standard that balances thorough sterilization with the protection of tissue viability is established. This reduces the microbial contamination rate of explants due to incomplete disinfection, while also minimizing tissue viability damage caused by excessive disinfection. This ensures the acquisition of sufficient, healthy, sterile initial material, solves the problems of high contamination rate in primary culture and induction initiation failure, and lays a reliable foundation for the establishment of the entire tissue culture rapid propagation system.

[0017] 2. In this invention, when conducting efficient tissue culture and rapid propagation of *Xanthoceras sorbifolium* seedlings, by adopting optimized culture medium formulas for each stage of primary induction, proliferation and rooting culture, and by accurately controlling light, temperature and photoperiod environmental parameters, suitable physicochemical conditions are provided for tissue growth and differentiation at different culture stages. This enables the proliferation culture to obtain higher proliferation coefficients and stronger clustered or adventitious buds, reduces the occurrence of vitrification physiological abnormalities, and solves the problems of poor adaptability, low proliferation efficiency and uneven seedling quality of conventional methods.

[0018] 3. In this invention, when conducting efficient tissue culture and rapid propagation of *Xanthoceras sorbifolium* seedlings, specific growth regulators and auxiliary components are introduced into the rooting medium, and a systematic hardening and transplanting management process is established. This promotes the induction and development of robust root systems in tissue culture seedlings, and alleviates the physiological transition stress from heterotrophic to autotrophic seedlings through a gradual acclimatization process. This improves the survival rate and early growth potential of transplanted seedlings, and solves the key technical bottlenecks of poor rooting quality, long transplanting adaptation period, and unstable survival rate of tissue culture seedlings in conventional methods. Attached Figure Description

[0019] Figure 1 This is a flowchart of a method for efficient tissue culture and rapid propagation of *Sapindus mukorossi* seedlings according to the present invention. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Example 1: A method for efficient tissue culture and rapid propagation of *Sapindus mukorossi* seedlings includes the following steps: Step 1: Explant selection and pretreatment. Select healthy current-year branches of *Xanthoceras sorbifolium*, cut stem segments with axillary buds as explants, and clean and prune them. Step 2: Disinfection treatment, the surface of the pretreated explants is disinfected to remove microbial contamination; Step 3: Preparation of primary culture medium; preparation of solid culture medium for primary induction. Step 4: Inoculation and primary culture. The sterilized explants are inoculated onto the primary culture medium and primary induction culture is carried out under controlled conditions. Step 5: Propagation culture. Transfer the clustered or adventitious shoots obtained from the initial culture into the propagation culture medium for further propagation. Step Six: Rooting Culture. Transfer the proliferated rootless seedlings into a rooting culture medium to induce root formation. Step 7: Hardening off and transplanting. After hardening off the tissue culture seedlings with complete roots, transplant them into the seedling substrate. In step three, the primary culture medium was prepared from the following components: MS basal medium, 6-benzyl adenine 0.5 mg / L, naphthaleneacetic acid 0.1 mg / L, sucrose 25 g / L, agar 5 g / L, and pH adjusted to 5.7; In step five, the proliferation medium is prepared from the following components: MS basal medium, 1.0 mg / L of 6-benzyl adenine, 0.1 mg / L of naphthaleneacetic acid, 25 g / L of sucrose, 5 g / L of agar, and pH adjusted to 5.7; In step six, the rooting medium is prepared from the following components: 1 / 2 MS basal medium, indolebutyric acid 0.5 mg / L, sucrose 15 g / L, agar 5 g / L, and pH adjusted to 5.7.

[0022] In step one, the healthy, current-year shoots of *Xanthoceras sorbifolium* are semi-lignified branches with a diameter of 0.3 cm. The cut stem segments are 1.0 cm long, with one axillary bud retained on each segment. After pruning, rinse with running water for 30 minutes to remove surface impurities.

[0023] In step two, the disinfection process includes the following steps: the pretreated explants are placed in a clean bench, first soaked in 70% ethanol solution for 30 seconds for preliminary disinfection, then soaked in 0.1% mercuric chloride solution or 2% sodium hypochlorite solution for 8 minutes for disinfection, and finally rinsed 3 times with sterile distilled water to remove residual disinfectant.

[0024] In step three, the preparation method of the primary culture medium includes: dissolving MS basic culture medium powder in distilled water, adding a plant growth regulator solution of 6-benzyl adenine and naphthaleneacetic acid, then adding sucrose and agar, stirring and mixing evenly, adjusting the pH to 5.7, dispensing into culture containers, and autoclaving at 121°C for 15 minutes.

[0025] In step four, the conditions for the initial culture include: a culture temperature of 24℃, a light intensity of 2000 lux, a photoperiod of 14 hours of light / 10 hours of darkness, and a culture time of 20 days, until the explants induce clustered shoots or adventitious shoots.

[0026] In step five, the conditions for proliferation culture include: the culture temperature is set at 24℃, the light intensity is 2000 lux, the photoperiod is 14 hours of light / 10 hours of darkness, the culture cycle is 25 days, and the proliferation coefficient of each generation reaches 3 times. During the proliferation process, the clustered buds are cut into small segments with buds and then transferred to fresh proliferation medium for subculture.

[0027] In step six, the conditions for rooting culture include: a culture temperature of 24℃, a light intensity of 2000 lux, a photoperiod of 14 hours of light / 10 hours of darkness, and a culture time of 15 days, until roots of 2 cm in length are induced at the base of the seedlings. Activated carbon of 0.5 g / L is also added to the rooting medium to promote robust root growth.

[0028] Step 7, hardening off and transplanting, includes the following steps: When the rooted seedlings grow to 3cm in height, move the culture container to a natural light environment for hardening off for 3 days. Then, take out the tissue culture seedlings, wash off the culture medium from the roots, and transplant them into a seedling substrate made of vermiculite, perlite and peat moss in a volume ratio of 1:1:1. Initially, maintain a humidity of 80% and a temperature of 20℃. After 7 days of recovery, normal management can begin.

[0029] Organic additives were added to the primary culture medium, proliferation culture medium and rooting culture medium. The organic additives were 50 ml / L of coconut juice or 50 g / L of banana puree. They were added aseptically after the culture medium was sterilized and cooled to 50°C.

[0030] In steps four, five, and six, the relative humidity of the culture environment was controlled at 60%, and the culture containers were covered with a breathable sealing film to promote gas exchange. The entire tissue culture process was carried out under aseptic conditions, and the explant inoculation density was 3 stem segments per bottle.

[0031] Example 2: A method for efficient tissue culture and rapid propagation of *Sapindus mukorossi* seedlings includes the following steps: Step 1: Explant selection and pretreatment. Select healthy current-year branches of *Xanthoceras sorbifolium*, cut stem segments with axillary buds as explants, and clean and prune them. Step 2: Disinfection treatment, the surface of the pretreated explants is disinfected to remove microbial contamination; Step 3: Preparation of primary culture medium; preparation of solid culture medium for primary induction. Step 4: Inoculation and primary culture. The sterilized explants are inoculated onto the primary culture medium and primary induction culture is carried out under controlled conditions. Step 5: Propagation culture. Transfer the clustered or adventitious shoots obtained from the initial culture into the propagation culture medium for further propagation. Step Six: Rooting Culture. Transfer the proliferated rootless seedlings into a rooting culture medium to induce root formation. Step 7: Hardening off and transplanting. After hardening off the tissue culture seedlings with complete roots, transplant them into the seedling substrate. In step three, the primary culture medium was prepared from the following components: MS basal medium, 1.5 mg / L of 6-benzyl adenine, 0.3 mg / L of naphthaleneacetic acid, 30 g / L of sucrose, 6 g / L of agar, and pH adjusted to 5.8; In step five, the proliferation medium is prepared from the following components: MS basal medium, 6-benzyl adenine 2.0 mg / L, naphthaleneacetic acid 0.3 mg / L, sucrose 30 g / L, agar 6 g / L, and pH adjusted to 5.8; In step six, the rooting medium is prepared from the following components: 1 / 2 MS basal medium, indolebutyric acid 1.0 mg / L, sucrose 20 g / L, agar 6 g / L, and pH adjusted to 5.8.

[0032] In step one, the healthy current-year branches of *Xanthoceras sorbifolium* are semi-lignified branches with a diameter of 0.45cm. The stem segments cut are 1.5cm long, and each stem segment retains 1.5 axillary buds. After pruning, rinse with running water for 45 minutes to remove surface impurities.

[0033] In step two, the disinfection process includes the following steps: the pretreated explants are placed in a clean bench and first soaked in 73% ethanol solution for 45 seconds for preliminary disinfection, then soaked in 0.15% mercuric chloride solution or 3% sodium hypochlorite solution for 11 minutes for disinfection, and finally rinsed 4 times with sterile distilled water to remove residual disinfectant.

[0034] In step three, the preparation method of the primary culture medium includes: dissolving MS basic culture medium powder in distilled water, adding a plant growth regulator solution of 6-benzyl adenine and naphthaleneacetic acid, then adding sucrose and agar, stirring and mixing evenly, adjusting the pH to 5.8, dispensing into culture containers, and autoclaving at 121°C for 17 minutes.

[0035] In step four, the conditions for the initial culture include: a culture temperature of 25°C, a light intensity of 2500 lux, a photoperiod of 15 hours of light / 9 hours of darkness, and a culture time of 27 days, until the explants induce clustered shoots or adventitious shoots.

[0036] In step five, the conditions for proliferation culture include: the culture temperature is set at 25℃, the light intensity is 2500 lux, the photoperiod is 15 hours of light / 9 hours of darkness, the culture cycle is 30 days, and the proliferation coefficient of each generation reaches 4 times. During the proliferation process, the clustered buds are cut into small segments with buds and then transferred to fresh proliferation medium for subculture.

[0037] In step six, the conditions for rooting culture include: a culture temperature of 25℃, a light intensity of 25 lux, a photoperiod of 15 hours of light / 9 hours of darkness, and a culture time of 20 days, until roots of 2.5 cm in length are induced at the base of the seedlings. Activated carbon of 0.7 g / L is also added to the rooting medium to promote robust root growth.

[0038] Step 7, hardening off and transplanting, includes the following steps: When the rooted seedlings grow to 4cm in height, move the culture container to a natural light environment for hardening off for 4 days. Then, take out the tissue culture seedlings, wash off the culture medium from the roots, and transplant them into a seedling substrate made of vermiculite, perlite and peat moss in a volume ratio of 1:1:1. Initially, maintain a humidity of 85% and a temperature of 23℃. After 8 days of recovery, normal management can begin.

[0039] Organic additives were added to the primary culture medium, proliferation culture medium and rooting culture medium. The organic additives were 70 ml / L of coconut juice or 70 g / L of banana puree. They were added aseptically after the culture medium was sterilized and cooled to 55°C.

[0040] In steps four, five, and six, the relative humidity of the culture environment was controlled at 65%, and the culture containers were covered with a breathable sealing film to promote gas exchange. The entire tissue culture process was carried out under aseptic conditions, and the explant inoculation density was 4 stem segments per bottle.

[0041] Example 3: A method for efficient tissue culture and rapid propagation of *Sapindus mukorossi* seedlings includes the following steps: Step 1: Explant selection and pretreatment. Select healthy current-year branches of *Xanthoceras sorbifolium*, cut stem segments with axillary buds as explants, and clean and prune them. Step 2: Disinfection treatment, the surface of the pretreated explants is disinfected to remove microbial contamination; Step 3: Preparation of primary culture medium; preparation of solid culture medium for primary induction. Step 4: Inoculation and primary culture. The sterilized explants are inoculated onto the primary culture medium and primary induction culture is carried out under controlled conditions. Step 5: Propagation culture. Transfer the clustered or adventitious shoots obtained from the initial culture into the propagation culture medium for further propagation. Step Six: Rooting Culture. Transfer the proliferated rootless seedlings into a rooting culture medium to induce root formation. Step 7: Hardening off and transplanting. After hardening off the tissue culture seedlings with complete roots, transplant them into the seedling substrate. In step three, the primary culture medium was prepared from the following components: MS basal medium, 6-benzyl adenine 2.0 mg / L, naphthaleneacetic acid 0.5 mg / L, sucrose 35 g / L, agar 7 g / L, and pH adjusted to 5.9; In step five, the proliferation medium is prepared from the following components: MS basal medium, 6-benzyl adenine 3.0 mg / L, naphthaleneacetic acid 0.5 mg / L, sucrose 35 g / L, agar 7 g / L, and pH adjusted to 5.9; In step six, the rooting medium is prepared from the following components: 1 / 2 MS basal medium, indolebutyric acid 1.5 mg / L, sucrose 25 g / L, agar 7 g / L, and pH adjusted to 5.9.

[0042] In step one, the healthy current-year branches of the Chinese tallow tree are semi-lignified branches with a diameter of 0.6cm. The stem segments cut are 2.0cm in length, and each stem segment retains 2 axillary buds. After pruning, rinse with running water for 60 minutes to remove surface impurities.

[0043] Step two includes the following disinfection process: the pretreated explants are placed in a clean bench and first soaked in 75% ethanol solution for 60 seconds for initial disinfection, then soaked in 0.2% mercuric chloride solution or 4% sodium hypochlorite solution for 15 minutes for disinfection, and finally rinsed 5 times with sterile distilled water to remove residual disinfectant.

[0044] In step three, the preparation method of the primary culture medium includes: dissolving MS basic culture medium powder in distilled water, adding a plant growth regulator solution of 6-benzyl adenine and naphthaleneacetic acid, then adding sucrose and agar, stirring and mixing evenly, adjusting the pH to 5.9, dispensing into culture containers, and autoclaving at 121°C for 20 minutes.

[0045] In step four, the conditions for the initial culture include: a culture temperature of 26°C, a light intensity of 3000 lux, a photoperiod of 16 hours of light / 8 hours of darkness, and a culture time of 35 days, until the explants induce clustered shoots or adventitious shoots.

[0046] In step five, the conditions for proliferation culture include: the culture temperature is set at 26℃, the light intensity is 3000 lux, the photoperiod is 16 hours of light / 8 hours of darkness, the culture cycle is 35 days, and the proliferation coefficient of each generation reaches 5 times. During the proliferation process, the clustered buds are cut into small segments with buds and then transferred to fresh proliferation medium for subculture.

[0047] In step six, the conditions for rooting culture include: a culture temperature of 26℃, a light intensity of 3000 lux, a photoperiod of 16 hours of light / 8 hours of darkness, and a culture time of 25 days, until roots of 3 cm in length are induced at the base of the seedlings. Activated carbon of 1.0 g / L is also added to the rooting medium to promote robust root growth.

[0048] Step 7, hardening off and transplanting, includes the following steps: When the rooted seedlings grow to 5cm in height, move the culture container to a natural light environment for hardening off for 5 days. Then, take out the tissue culture seedlings, wash the culture medium from the roots, and transplant them into a seedling substrate made of vermiculite, perlite and peat moss in a volume ratio of 1:1:1. Initially, maintain a humidity of 90% and a temperature of 25℃. After 10 days of recovery, normal management can begin.

[0049] Organic additives were added to the primary culture medium, proliferation culture medium and rooting culture medium. The organic additives were 100 ml / L of coconut juice or 100 g / L of banana puree. They were added aseptically after the culture medium was sterilized and cooled to 60°C.

[0050] In steps four, five, and six, the relative humidity of the culture environment was controlled at 70%, and the culture containers were covered with a breathable sealing film to promote gas exchange. The entire tissue culture process was carried out under aseptic conditions, and the explant inoculation density was 5 stem segments per bottle.

[0051] Comparative Example 1: The difference between this comparative example and Example 1 is that this comparative example did not use a step-by-step disinfection procedure when disinfecting the explants; it only used a 2% sodium hypochlorite solution for immersion disinfection for 15 minutes.

[0052] Comparative Example 2 differs from Example 2 in that 6-benzyl adenine and naphthaleneacetic acid plant growth regulators were not added when preparing the proliferation culture medium in this comparative example.

[0053] Comparative Example 3 differs from Example 3 in that: after the tissue culture seedlings rooted, no hardening-off treatment was performed in this comparative example, and the tissue culture seedlings were directly taken out of the culture container and transplanted into the seedling substrate.

[0054] Comparative Example 4 differs from Example 3 in that MS basal medium was used instead of 1 / 2 MS basal medium in the preparation of the rooting medium, and indolebutyric acid and activated carbon were not added.

[0055] Key performance indicators of the *Xanthoceras sorbifolium* tissue culture seedlings obtained in Examples 1-3 and Comparative Examples 1-4 were tested. The test items and methods are as follows: To calculate the contamination rate of the first-generation culture, the number of obvious bacterial spots or pus-like substances on the surface of the explants was observed and recorded daily within 20 days after inoculation, and the percentage of contaminated explants out of the total inoculated explants was calculated. The proliferation coefficient was determined by counting the number of adventitious buds or clustered buds produced in each inoculation unit at the end of the proliferation culture cycle and comparing it with the initial number of inoculated buds to calculate the average proliferation multiple. Observation of rooting rate and root morphology: At the end of the rooting culture cycle, count the number of rootless seedlings that have grown roots longer than two centimeters, calculate the rooting percentage, and observe whether the root system is robust and whether the root hairs are well-developed. To calculate the survival rate of transplanted seedlings, the number of seedlings that survived and showed new growth was counted 30 days after transplanting the tissue culture seedlings into the seedling substrate, and the survival percentage was calculated.

[0056] The key test data of Examples 1-3 and Comparative Examples 1-4 are recorded in the table below:

[0057] By comparing and analyzing the data in the table, it can be seen that the rapid propagation of *Xanthoceras sorbifolium* seedlings using the processes in Examples 1-3 had a significantly lower initial culture contamination rate than Comparative Example 1, while its proliferation coefficient, rooting rate, and transplant survival rate were significantly higher than Comparative Examples 2, 3, and 4. This indicates that by standardizing explant selection criteria and implementing a step-by-step surface disinfection procedure, an operational standard was established that balances thorough sterilization with the protection of tissue viability. This reduces the microbial contamination rate of explants due to incomplete disinfection, while also minimizing tissue viability damage caused by excessive disinfection. This ensures the acquisition of sufficient, healthy, sterile initial material, solving the problems of high initial culture contamination rate and induction initiation failure. This lays a reliable foundation for the establishment of the entire rapid tissue culture propagation system. Furthermore, by employing optimized culture medium formulations for each stage of initial induction, proliferation, and rooting culture, and... By precisely controlling the environmental parameters of light, temperature, and photoperiod, suitable physicochemical conditions are provided for tissue growth and differentiation at different culture stages. This enables proliferation culture to obtain higher proliferation coefficients and more robust clustered or adventitious buds, reducing the occurrence of vitrification physiological abnormalities. It solves the problems of poor adaptability, low proliferation efficiency, and uneven seedling quality of conventional methods. By introducing specific growth regulators and auxiliary components into the rooting medium and establishing a systematic seedling hardening and transplanting management process, the induction and development of robust root systems in tissue culture seedlings are promoted. The gradual acclimatization process alleviates the physiological transition stress of tissue culture seedlings from heterotrophic to autotrophic, thereby improving the survival rate and early growth potential of tissue culture seedlings after transplanting. This solves the key technical bottlenecks of poor rooting quality, long transplanting adaptation period, and unstable survival rate of tissue culture seedlings in conventional methods.

[0058] By comparing and analyzing the relevant data in the table, it can be seen that the efficient tissue culture rapid propagation method of *Xanthoceras sorbifolium* provided by this invention has significant advantages in controlling pollution, promoting proliferation, inducing rooting, and improving transplant survival rate. This indicates that the efficient tissue culture rapid propagation method of *Xanthoceras sorbifolium* provided by this invention has superior comprehensive performance.

[0059] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0060] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for efficient tissue culture and rapid propagation of *Sapindus mukorossi* seedlings, characterized in that, Includes the following steps: Step 1: Explant selection and pretreatment. Select healthy current-year branches of *Xanthoceras sorbifolium*, cut stem segments with axillary buds as explants, and clean and prune them. Step 2: Disinfection treatment, the surface of the pretreated explants is disinfected; Step 3: Preparation of primary culture medium; preparation of solid culture medium for primary induction. Step 4: Inoculation and primary culture. The sterilized explants are inoculated onto the primary culture medium and primary induction culture is carried out under controlled conditions. Step 5: Propagation culture. Transfer the clustered or adventitious shoots obtained from the initial culture into the propagation culture medium for further propagation. Step Six: Rooting Culture. Transfer the proliferated rootless seedlings into a rooting culture medium to induce root formation. Step 7: Hardening off and transplanting. After hardening off the tissue culture seedlings with complete roots, transplant them into the seedling substrate. In step three, the primary culture medium is prepared from the following components: MS basal medium, 6-benzyl adenine 0.5-2.0 mg / L, naphthaleneacetic acid 0.1-0.5 mg / L, sucrose 25-35 g / L, agar 5-7 g / L, and pH adjusted to 5.7-5.9; In step five, the proliferation medium is prepared from the following components: MS basal medium, 6-benzyl adenine 1.0-3.0 mg / L, naphthaleneacetic acid 0.1-0.5 mg / L, sucrose 25-35 g / L, agar 5-7 g / L, and pH adjusted to 5.7-5.9; In step six, the rooting medium is prepared from the following components: 1 / 2 MS basal medium, indolebutyric acid 0.5-1.5 mg / L, sucrose 15-25 g / L, agar 5-7 g / L, and pH adjusted to 5.7-5.

9.

2. The method for efficient tissue culture and rapid propagation of *Sapindus mukorossi* seedlings according to claim 1, characterized in that, In step one, the healthy current-year branches of *Xanthoceras sorbifolium* are semi-lignified branches with a diameter of 0.3-0.6 cm. The cut stem segments are 1.0-2.0 cm long, and each stem segment retains 1-2 axillary buds. After pruning, the stem segments are rinsed with running water for 30-60 minutes.

3. The method for efficient tissue culture and rapid propagation of *Sapindus mukorossi* seedlings according to claim 1, characterized in that, In step two, the disinfection process includes the following steps: the pretreated explants are placed in a clean bench and first soaked in 70-75% ethanol solution for 30-60 seconds for preliminary disinfection, then soaked in 0.1-0.2% mercuric chloride solution or 2-4% sodium hypochlorite solution for 8-15 minutes for disinfection, and finally rinsed with sterile distilled water 3-5 times.

4. The method for efficient tissue culture and rapid propagation of *Sapindus mukorossi* seedlings according to claim 1, characterized in that, In step three, the preparation method of the primary culture medium includes: dissolving MS basic culture medium powder in distilled water, adding the plant growth regulator solution of 6-benzyl adenine and naphthaleneacetic acid, then adding sucrose and agar, stirring and mixing evenly, adjusting the pH to 5.7-5.9, dispensing into culture containers, and autoclaving at 121°C for 15-20 minutes.

5. The method for efficient tissue culture and rapid propagation of *Sapindus mukorossi* seedlings according to claim 1, characterized in that, In step four, the initial culture conditions include: a culture temperature of 24-26℃, a light intensity of 2000-3000 lux, a photoperiod of 14-16 hours of light / 8-10 hours of darkness, and a culture time of 20-35 days, until the explants induce clustered buds or adventitious buds.

6. The method for efficient tissue culture and rapid propagation of *Sapindus mukorossi* seedlings according to claim 1, characterized in that, In step five, the conditions for proliferation culture include: a culture temperature of 24-26℃, a light intensity of 2000-3000 lux, a photoperiod of 14-16 hours of light / 8-10 hours of darkness, a culture cycle of 25-35 days, and a proliferation coefficient of 3-5 times per generation. During the proliferation process, the clustered buds are cut into small segments with buds and then transferred to fresh proliferation culture medium for subculture.

7. The method for efficient tissue culture and rapid propagation of *Sapindus mukorossi* seedlings according to claim 1, characterized in that, In step six, the conditions for rooting culture include: a culture temperature of 24-26℃, a light intensity of 2000-3000 lux, a photoperiod of 14-16 hours of light / 8-10 hours of darkness, and a culture time of 15-25 days, until roots of 2-3 cm in length are induced at the base of the seedlings. The rooting culture medium also contains 0.5-1.0 g / L of activated carbon.

8. The method for efficient tissue culture and rapid propagation of *Sapindus mukorossi* seedlings according to claim 1, characterized in that, In step seven, the hardening-off and transplanting of seedlings includes the following steps: when the rooted seedlings grow to 3-5cm in height, move the culture container to a natural light environment for hardening-off for 3-5 days. Then, take out the tissue culture seedlings, wash the culture medium from the roots, and transplant them into a seedling substrate made of vermiculite, perlite and peat moss in a volume ratio of 1:1:

1. Initially, maintain a humidity of 80-90% and a temperature of 20-25℃. After 7-10 days of recovery, normal management can begin.

9. The method for efficient tissue culture and rapid propagation of *Sapindus mukorossi* seedlings according to claim 1, characterized in that, Organic additives are added to the primary culture medium, proliferation culture medium and rooting culture medium. The organic additives are 50-100 ml / L of coconut juice or 50-100 g / L of banana puree. They are added aseptically after the culture medium has been sterilized and cooled to 50-60°C.

10. The method for efficient tissue culture and rapid propagation of *Xanthoceras sorbifolium* seedlings according to claim 1, characterized in that, In steps four, five, and six, the relative humidity of the culture environment is controlled at 60-70%, and the culture container is covered with a breathable sealing film. The entire tissue culture process is carried out under aseptic conditions, and the explant inoculation density is 3-5 stem segments per bottle.

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