Centella Asiatica in vitro preservation method

Abstract

This invention discloses a method for in vitro preservation of Centella asiatica, comprising the following steps: obtaining Centella asiatica seedlings and inoculating them into a third culture medium for in vitro preservation culture. The third culture medium comprises 1 / 2 MS, 0.01-0.03 mg / L 6-benzylaminopurine, 0.6-1.4 mg / L quinacrine, 0.2-0.8 mg / L paclobutrazol, and 0.2-0.8 g / L activated carbon. The method for obtaining Centella asiatica seedlings is as follows: obtaining sterile explants of Centella asiatica and inoculating them into a first culture medium for induction culture to obtain induced shoots. These induced shoots are then inoculated into a second culture medium for seedling strengthening culture to obtain Centella asiatica seedlings. This invention, by changing the culture conditions and culture medium composition, can effectively extend the in vitro preservation time of Centella asiatica.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology. More specifically, this invention relates to a method for in vitro preservation of Centella asiatica. Background Technology

[0002] Centella asiatica (L.) Urban. is a herbaceous plant belonging to the genus Centella in the family Apiaceae. The dried whole plant is used medicinally. According to records, Centella asiatica is bitter and pungent in taste, and cold in nature. It has the effects of clearing heat and dampness, detoxifying and reducing swelling, and is used for damp-heat jaundice, carbuncles, and sores. It is a commonly used traditional Chinese medicine. Centella asiatica has been widely used in medicine, health food, and cosmetics, attracting numerous companies to produce it. Its market prospects are broad, and it has become a hot topic for researchers. Introduction, cultivation, and artificial propagation have become new ways to effectively utilize Centella asiatica resources. However, existing technologies have short in vitro preservation times for tissue culture seedlings, necessitating a tissue culture method that can extend the in vitro preservation time of Centella asiatica. Summary of the Invention

[0003] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0004] Another objective of this invention is to provide a method for in vitro preservation of Centella asiatica, which can effectively extend the in vitro preservation time of Centella asiatica by changing the culture conditions and culture medium composition.

[0005] To achieve these objectives and other advantages of the present invention, a method for in vitro preservation of Centella asiatica is provided, comprising the following steps:

[0006] Centella asiatica seedlings were obtained and inoculated into a third culture medium for in vitro preservation culture. The third culture medium consisted of 1 / 2 MS, 0.03 mg / L 6-benzylaminopurine, 0.6-1.4 mg / L quinacrine, 0.2-0.8 mg / L paclobutrazol, and 0.2-0.8 g / L activated carbon.

[0007] Preferably, the method for obtaining Centella asiatica bottle seedlings is as follows: obtain sterile explants of Centella asiatica, inoculate the sterile explants of Centella asiatica into a first culture medium for induction culture to obtain induced shoots, and then inoculate the induced shoots into a second culture medium for seedling strengthening culture to obtain Centella asiatica bottle seedlings.

[0008] Preferably, the first culture medium comprises MS, 0.2-0.8 mg / L 6-benzylaminopurine, and 0.1-0.3 mg / L naphthaleneacetic acid, wherein the culture temperature is 22-26℃, the light intensity is 1200-1800 LX, the daily light duration is 10-14 h, and the culture time is 21 days.

[0009] Preferably, the second culture medium comprises MS, 0.1-0.5 mg / L 6-benzylaminopurine, 0.02-0.08 mg / L naphthaleneacetic acid, and 0.5-1.5 g / L activated carbon, wherein the culture temperature is 22-26℃, the light intensity is 1200-1800 LX, and the daily light duration is 10-14 h.

[0010] Preferably, before transplanting the Centella asiatica seedlings to the third culture medium, the roots of the seedlings are soaked in a sterile soaking solution for 8 hours. The sterile soaking solution is prepared as follows: Take the petals of Rehmannia glutinosa and crush them to obtain a powder. Add 20 times the amount of water to the powder and reflux for 6-7 hours. Filter to obtain a filtrate. Add 0.2 times the total mass of aloe vera, 0.2 times the total mass of Ficus pumila flowers, and 0.7 times the total mass of olive leaves to the filtrate. Place it in a non-closed fermentation environment at 22-23℃ for 48 hours. Then, add 0.1 times the total mass of Bacillus subtilis solution and 0.2 times the total mass of kombucha solution to the system for closed fermentation for 5 days. Finally, ferment in a non-closed environment for 2 days to obtain the soaking solution. Filter and sterilize the soaking solution using a 0.22μm microporous membrane to obtain a sterile soaking solution.

[0011] Preferably, before soaking the seedlings in the sterile soaking solution, a portion of the sterile soaking solution is concentrated to obtain a concentrated solution, which is then filtered and sterilized using a 0.22μm microporous membrane. The concentrated solution is then added to the third culture medium, where the concentration of the concentrated solution in the third culture medium is 1mg / L.

[0012] Preferably, the culture conditions in the in vitro culture medium include: for the first month of culture in the in vitro culture medium, the daytime culture temperature is 20-22℃, the light intensity is 1000 LX, and the nighttime culture temperature is 10-11℃; for the second month onwards, the culture temperature is 18-20℃, the light intensity is 1200 LX, and the nighttime culture temperature is 12-14℃; for the fourth month onwards, the culture temperature is 18-20℃, the light intensity is 1800 LX, and the nighttime culture temperature is 15-17℃; and for the seventh month onwards, the daytime culture temperature is 22-23℃, the light intensity is 1500 LX, and the nighttime culture temperature is 17-19℃.

[0013] The present invention has at least the following beneficial effects:

[0014] This invention extends the in vitro preservation time of Centella asiatica seedlings to 281 days by adding quinquefoliol and paclobutrazol to the third culture medium. It also effectively prolongs the in vitro preservation time of Centella asiatica by soaking the seedlings in an infusion containing aloe vera, olive leaves, and sycamore flowers, and by adding the concentrated solution to the third culture medium. The synergistic effect of aloe vera, olive leaves, and sycamore flowers is necessary to significantly alter the in vitro preservation time. Furthermore, this invention effectively prolongs the in vitro preservation time of Centella asiatica by altering the temperature difference between day and night and correspondingly changing the light intensity. Finally, this application effectively improves the transplant survival rate by soaking the seedlings in an infusion containing aloe vera, olive leaves, and sycamore flowers, and by adding the concentrated solution to the third culture medium.

[0015] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Detailed Implementation

[0016] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0017] <Example 1>

[0018] The method for in vitro preservation of Centella asiatica includes the following steps:

[0019] Centella asiatica seedlings were obtained and inoculated into a third culture medium for in vitro preservation culture. The third culture medium consisted of 1 / 2 MS, 0.01 mg / L 6-benzylaminopurine, 0.6 mg / L quinacrine, 0.2 mg / L paclobutrazol, and 0.2 g / L activated carbon.

[0020] The method for obtaining Centella asiatica bottle seedlings is as follows: obtain sterile explants of Centella asiatica, inoculate the sterile explants of Centella asiatica into the first culture medium for induction culture to obtain induced shoots, and then inoculate the induced shoots into the second culture medium for seedling strengthening culture to obtain Centella asiatica bottle seedlings.

[0021] The method for obtaining sterile explants of Centella asiatica is as follows: Take tender branches of Centella asiatica with axillary buds, remove the leaves and cut them into 1cm long stem segments with axillary buds. Soak the stem segments in a 1% detergent solution for 10 minutes, rinse for 20 minutes, then rinse three times with sterile water, sterilize in a 75% ethanol solution for 30 seconds, rinse three times with sterile water, soak in a 0.1% HgCl2 solution for 7-8 minutes, and rinse three times with sterile water, each time for 3-5 minutes. Finally, use sterile paper to absorb the moisture from the surface of the stem segments to obtain sterile explants of Centella asiatica.

[0022] One to two drops of Tween-20 were added to every 500 mL of HgCl2 solution.

[0023] The first culture medium consisted of MS, 0.2 mg / L 6-benzylaminopurine, and 0.1 mg / L naphthaleneacetic acid. The culture temperature was 22℃, the light intensity was 1200 LX, the daily light duration was 10-14 h, and the culture time was 21 days.

[0024] The second culture medium consisted of MS, 0.1 mg / L 6-benzylaminopurine, 0.02 mg / L naphthaleneacetic acid, and 0.5 g / L activated carbon. The culture temperature was 22℃, the light intensity was 1200 LX, and the daily light duration was 10 h.

[0025] <Example 2>

[0026] The method for in vitro preservation of Centella asiatica includes the following steps:

[0027] Centella asiatica seedlings were obtained and inoculated into a third culture medium for in vitro preservation culture. The third culture medium consisted of 1 / 2 MS, 0.03 mg / L 6-benzylaminopurine, 1.4 mg / L quinacrine, 0.8 mg / L paclobutrazol, and 0.8 g / L activated carbon.

[0028] The method for obtaining Centella asiatica bottle seedlings is as follows: obtain sterile explants of Centella asiatica, inoculate the sterile explants of Centella asiatica into the first culture medium for induction culture to obtain induced shoots, and then inoculate the induced shoots into the second culture medium for seedling strengthening culture to obtain Centella asiatica bottle seedlings.

[0029] The method for obtaining sterile explants of Centella asiatica is as follows: Take tender branches of Centella asiatica with axillary buds, remove the leaves and cut them into 1cm long stem segments with axillary buds. Soak the stem segments in a 1% detergent solution for 10 minutes, rinse for 20 minutes, then rinse three times with sterile water, sterilize in a 75% ethanol solution for 30 seconds, rinse three times with sterile water, soak in a 0.1% HgCl2 solution for 7-8 minutes, and rinse three times with sterile water, each time for 3-5 minutes. Finally, use sterile paper to absorb the moisture from the surface of the stem segments to obtain sterile explants of Centella asiatica.

[0030] One to two drops of Tween-20 were added to every 500 mL of HgCl2 solution.

[0031] The first culture medium consisted of MS, 0.8 mg / L 6-benzylaminopurine, and 0.3 mg / L naphthaleneacetic acid. The culture temperature was 26 °C, the light intensity was 1800 LX, the daily light duration was 14 h, and the culture time was 21 days.

[0032] The second culture medium consisted of MS, 0.5 mg / L 6-benzylaminopurine, 0.08 mg / L naphthaleneacetic acid, and 1.5 g / L activated carbon. The culture temperature was 26 °C, the light intensity was 1800 LX, and the daily light duration was 14 h.

[0033] <Example 3>

[0034] The method for in vitro preservation of Centella asiatica includes the following steps:

[0035] Centella asiatica seedlings were obtained and inoculated into a third culture medium for in vitro preservation culture. The third culture medium consisted of 1 / 2 MS, 0.02 mg / L 6-benzylaminopurine, 1 mg / L quinacrine, 0.5 mg / L paclobutrazol, and 0.5 g / L activated carbon.

[0036] The method for obtaining Centella asiatica bottle seedlings is as follows: obtain sterile explants of Centella asiatica, inoculate the sterile explants of Centella asiatica into the first culture medium for induction culture to obtain induced shoots, and then inoculate the induced shoots into the second culture medium for seedling strengthening culture to obtain Centella asiatica bottle seedlings.

[0037] The method for obtaining sterile explants of Centella asiatica is as follows: Take tender branches of Centella asiatica with axillary buds, remove the leaves and cut them into 1cm long stem segments with axillary buds. Soak the stem segments in a 1% detergent solution for 10 minutes, rinse for 20 minutes, then rinse three times with sterile water, sterilize in a 75% ethanol solution for 30 seconds, rinse three times with sterile water, soak in a 0.1% HgCl2 solution for 7-8 minutes, and rinse three times with sterile water, each time for 3-5 minutes. Finally, use sterile paper to absorb the moisture from the surface of the stem segments to obtain sterile explants of Centella asiatica.

[0038] One to two drops of Tween-20 were added to every 500 mL of HgCl2 solution.

[0039] The first culture medium consisted of MS, 0.05 mg / L 6-benzylaminopurine, and 0.2 mg / L naphthaleneacetic acid. The culture temperature was 24 °C, the light intensity was 1500 LX, the daily light duration was 12 h, and the culture time was 21 days.

[0040] The second culture medium consisted of MS, 0.3 mg / L 6-benzylaminopurine, 0.05 mg / L naphthaleneacetic acid, and 1 g / L activated carbon. The culture temperature was 24℃, the light intensity was 1500 LX, and the daily light duration was 12 h.

[0041] <Example 4>

[0042] The method for in vitro preservation of Centella asiatica includes the following steps:

[0043] The method for obtaining Centella asiatica bottle seedlings is as follows: obtain sterile explants of Centella asiatica, inoculate the sterile explants of Centella asiatica into the first culture medium for induction culture to obtain induced shoots, and then inoculate the induced shoots into the second culture medium for seedling strengthening culture to obtain Centella asiatica bottle seedlings.

[0044] Centella asiatica seedlings were obtained and inoculated into a third culture medium for in vitro preservation culture. The third culture medium consisted of 1 / 2 MS, 0.012 mg / L 6-benzylaminopurine, 1 mg / L quinacrine, 0.5 mg / L paclobutrazol, and 0.5 g / L activated carbon.

[0045] The method for obtaining sterile explants of Centella asiatica is as follows: Take tender branches of Centella asiatica with axillary buds, remove the leaves and cut them into 1cm long stem segments with axillary buds. Soak the stem segments in a 1% detergent solution for 10 minutes, rinse for 20 minutes, then rinse three times with sterile water, sterilize in a 75% ethanol solution for 30 seconds, rinse three times with sterile water, soak in a 0.1% HgCl2 solution for 7-8 minutes, and rinse three times with sterile water, each time for 3-5 minutes. Finally, use sterile paper to absorb the moisture from the surface of the stem segments to obtain sterile explants of Centella asiatica.

[0046] One to two drops of Tween-20 were added to every 500 mL of HgCl2 solution.

[0047] The first culture medium consisted of MS, 0.05 mg / L 6-benzylaminopurine, and 0.2 mg / L naphthaleneacetic acid. The culture temperature was 24 °C, the light intensity was 1500 LX, the daily light duration was 12 h, and the culture time was 21 days.

[0048] The second culture medium consisted of MS, 0.3 mg / L 6-benzylaminopurine, 0.05 mg / L naphthaleneacetic acid, and 1 g / L activated carbon. The culture temperature was 24℃, the light intensity was 1500 LX, and the daily light duration was 12 h.

[0049] Before transplanting Centella asiatica seedlings to the third culture medium, soak the roots of the seedlings in a sterile soaking solution for 8 hours, then rinse three times with clean water. Repeat the soaking-rinsing operation three times. The sterile soaking solution is prepared as follows: crush Rehmannia glutinosa petals to obtain a powder, add 20 times the amount of water to the powder and reflux for 6-7 hours, filter, and obtain the filtrate; add 0.2 times the total mass of the filtrate of aloe vera, 0.2 times the total mass of the filtrate of Ficus microcarpa flowers, and 0.7 times the total mass of the filtrate of aloe vera. Double the amount of olive leaves were placed in a non-closed fermentation at 22-23℃ for 48 hours. Then, 0.1 times the total mass of the filtrate of Bacillus subtilis liquid and 0.2 times the total mass of the filtrate of kombucha liquid were added to the system for closed fermentation for 5 days. Finally, non-closed fermentation was carried out for 2 days. After the non-closed fermentation was completed, the system was sterilized at 120℃ for 15 minutes. After cooling and centrifugation, the supernatant was collected to obtain the soaking liquid. The soaking liquid was filtered and sterilized using a 0.22μm microporous membrane to obtain a sterile soaking liquid.

[0050] Before soaking Centella asiatica seedlings in sterile soaking solution, a portion of the sterile soaking solution was concentrated to obtain a concentrate. The concentrate was then filtered and sterilized using a 0.22μm microporous membrane. The concentrate was then added to the third culture medium, where the concentration of the concentrate was 1mg / L.

[0051] The conditions for culture in in vitro culture medium are as follows: During the first month of culture in in vitro culture medium, the daytime culture temperature is 20-22℃, the light intensity is 1000 LX, and the nighttime culture temperature is 10-11℃; from the second month onwards, the culture temperature is 18-20℃, the light intensity is 1200 LX, and the nighttime culture temperature is 12-14℃; from the fourth month onwards, the culture temperature is 18-20℃, the light intensity is 1800 LX, and the nighttime culture temperature is 15-17℃; from the seventh month onwards, the daytime culture temperature is 22-23℃, the light intensity is 1500 LX, and the nighttime culture temperature is 17-19℃.

[0052] <Comparative Example 1>

[0053] In vitro culture was performed using the method described in Example 3, except that the third culture medium in step three included: 1 / 2 MS, 0.012 mg / L 6-benzylaminopurine, and 0.5 g / L activated carbon.

[0054] <Comparative Example 2>

[0055] In vitro culture was carried out using the method described in Example 4, except that the Centella asiatica seedlings were directly transplanted into the third culture medium (i.e., the third culture medium contained a concentrated solution).

[0056] <Comparative Example 3>

[0057] In vitro culture was performed using the method described in Example 4, except that the sterile soaking solution was prepared as follows: Prepared Rehmannia glutinosa petals were crushed to obtain a powder. 20 times the volume of water was added to the powder, and the mixture was refluxed for 6-7 hours. The mixture was then filtered to obtain a filtrate. 0.05 times the total mass of Bacillus subtilis solution and 0.2 times the total mass of kombucha solution were added to the filtrate for sealed fermentation for 5 days, followed by 2 days of unsealed fermentation to obtain the soaking solution. The soaking solution was then filtered and sterilized using a 0.22 μm microporous membrane to obtain a sterile soaking solution.

[0058] <Comparative Example 4>

[0059] In vitro culture was performed using the method described in Example 4, except that the preparation method of the soaking solution was as follows: Prepared Rehmannia glutinosa petals were crushed to obtain a powder. 20 times the amount of water was added to the powder, and the mixture was refluxed for 6-7 hours. The mixture was then filtered to obtain a filtrate. Aloe vera (0.2 times the total mass of the filtrate) and olive leaves (0.7 times the total mass of the filtrate) were added to the filtrate, and the mixture was placed in a non-closed fermentation environment at 20-23°C for 48 hours. Then, Bacillus subtilis solution (0.05 times the total mass of the filtrate) and kombucha solution (0.2 times the total mass of the filtrate) were added to the system for closed fermentation for 5 days. Finally, non-closed fermentation was performed for another 2 days (after which the mixture was sterilized at 120°C for 15 minutes) to obtain the soaking solution. The soaking solution was then filtered and sterilized using a 0.22 μm microporous membrane to obtain a sterile soaking solution.

[0060] <Comparative Example 5>

[0061] In vitro culture was performed using the method described in Example 4, except that the preparation method of the soaking solution was as follows: The petals of Rehmannia glutinosa were crushed to obtain a powder. 20 times the amount of water was added to the powder, and the mixture was refluxed for 6-7 hours. The mixture was then filtered to obtain a filtrate. 0.2 times the total mass of the filtrate of Pauillac flowers and 0.7 times the total mass of the filtrate of olive leaves were added to the filtrate. The mixture was then placed in a non-closed fermentation environment at 20-23°C for 48 hours. Then, 0.05 times the total mass of the filtrate of Bacillus subtilis solution and 0.2 times the total mass of the filtrate of kombucha solution were added to the system for closed fermentation for 5 days. Finally, non-closed fermentation was performed for another 2 days to obtain the soaking solution. The soaking solution was then filtered and sterilized using a 0.22 μm microporous membrane to obtain a sterile soaking solution.

[0062] <Comparative Example 6>

[0063] In vitro culture was performed using the method described in Example 4, except that the preparation method of the soaking solution was as follows: Prepared Rehmannia glutinosa petals were crushed to obtain a powder. 20 times the amount of water was added to the powder, and the mixture was refluxed for 6-7 hours. The mixture was then filtered to obtain a filtrate. Aloe vera and Ficus pumila flowers (0.2 times the total mass of the filtrate) were added to the filtrate, and the mixture was placed in a non-closed fermentation environment at 20-23°C for 48 hours. Then, Bacillus subtilis solution (0.05 times the total mass of the filtrate) and kombucha solution (0.2 times the total mass of the filtrate) were added to the system for closed fermentation for 5 days. Finally, non-closed fermentation was performed for another 2 days to obtain the soaking solution. The soaking solution was then filtered and sterilized using a 0.22 μm microporous membrane to obtain a sterile soaking solution.

[0064] <Comparative Example 7>

[0065] In vitro culture was performed using the method described in Example 4, except that no concentrate was added to the third culture medium.

[0066] <Comparative Example 8>

[0067] The in vitro culture was carried out using the method described in Example 4, except that the culture conditions in the third culture medium were as follows: the daytime culture temperature was 23-25℃, the light intensity was 1500LX, and the nighttime culture temperature was 17-19℃.

[0068] In the above embodiments and comparative examples, aloe vera, paulownia flowers, and olive leaves were all pulverized and added to the filtrate.

[0069] <Experimental Results>

[0070] In vitro culture was carried out using the experimental methods of Examples 3, 4, and Comparative Examples 1-8.

[0071] 1. Ex vivo preservation time

[0072] The in vitro preservation time was defined as the time when more than half of the seedlings in the third culture medium died, as shown in Table 1.

[0073] Table 1. Ex vivo preservation time

[0074]

[0075] Comparative analysis of the data from Example 3 and Comparative Example 1 shows that the present invention can prolong the in vitro preservation time of Centella asiatica seedlings by adding quinacrine and paclobutrazol to the third culture medium.

[0076] Comparative analysis of the data from Example 4 and Comparative Example 2 shows that the present invention can effectively prolong the in vitro preservation time of Centella asiatica seedlings by soaking the roots of the seedlings in a sterile soaking solution.

[0077] Comparative analysis of the data from Example 4 and Comparative Example 7 shows that the present invention can effectively prolong the in vitro preservation time of Centella asiatica seedlings by adding a concentrated solution to the third culture medium.

[0078] Comparative analysis of the data from Example 4 and Comparative Examples 3-6 shows that the present invention uses an infusion containing aloe vera, olive leaves, and paulownia flowers for soaking and adds the concentrated solution to the third culture medium to effectively prolong the in vitro preservation time of Centella asiatica. Moreover, the synergistic effect of aloe vera, olive leaves, and paulownia flowers can significantly change the in vitro preservation time.

[0079] Comparative analysis of the data from Example 4 and Comparative Example 8 shows that the present invention can effectively extend the in vitro preservation time of Centella asiatica by changing the temperature difference between day and night and the corresponding change in light intensity.

[0080] 2. Survival rate of Centella asiatica after transplanting

[0081] The Centella asiatica seedlings preserved in Examples 3, 4, and Comparative Examples 2-6 were transplanted. The survival rate was calculated on the 30th day after transplanting. The results are shown in Table 2. The survival rate is: (surviving plants one month after transplanting / total number of transplanted plants) × 100%.

[0082] Table 2 shows the transplant survival rate.

[0083] Group Survival rate Example 3 80％ Example 4 98％ Comparative Example 2 85％ Comparative Example 3 83％ Comparative Example 4 87％ Comparative Example 5 85％ Comparative Example 6 90％

[0084] By comparing the survival rates of tissue culture seedlings preserved in Examples 3, 4, and Comparative Examples 2-6 in Table 2 after transplanting, this application can effectively improve the transplant survival rate by using an infusion containing aloe vera, olive leaves, and paulownia flowers, and by adding the concentrated solution to the third culture medium.

[0085] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.

Claims

1. A method for in vitro preservation of Centella asiatica, characterized in that, Includes the following steps: Centella asiatica seedlings were obtained and inoculated into a third culture medium for in vitro preservation culture. The third culture medium consisted of the following components: 1 / 2 MS, 0.01-0.03 mg / L 6-benzylaminopurine, 0.6-1.4 mg / L quinacrine, 0.2-0.8 mg / L paclobutrazol, and 0.2-0.8 g / L activated carbon. Before transplanting Centella asiatica seedlings to the third culture medium, the roots of the seedlings were soaked in a sterile soaking solution for 8 hours. The sterile soaking solution was prepared as follows: Crushed petals of Rehmannia glutinosa were added to the crushed material, and water was added to the crushed material and refluxed for 6-7 hours. The mixture was then filtered to obtain a filtrate. Aloe vera, Ficus pumila flowers, and olive leaves (0.2 times the total mass of the filtrate) were added to the filtrate, and the mixture was placed in a non-closed fermentation environment at 22-23℃ for 48 hours. Then, Bacillus subtilis solution (0.1 times the total mass of the filtrate) and kombucha solution (0.2 times the total mass of the filtrate) were added to the system for closed fermentation for 5 days. Finally, the mixture was fermented in a non-closed environment for 2 days to obtain the soaking solution. The soaking solution was then filtered and sterilized using a 0.22 μm microporous membrane to obtain a sterile soaking solution. Before soaking Centella asiatica seedlings in sterile soaking solution, a portion of the sterile soaking solution was concentrated to obtain a concentrate. The concentrate was then filtered and sterilized using a 0.22 μm microporous membrane. The concentrate was then added to the third culture medium, where the concentration of the concentrate was 1 mg / L. The culture conditions in the third medium included: for the first month, the daytime culture temperature was 20-22℃, the light intensity was 1000 LX, and the nighttime culture temperature was 10-11℃; for the second month onwards, the daytime culture temperature was 18-20℃, the light intensity was 1200 LX, and the nighttime culture temperature was 12-14℃; for the fourth month onwards, the daytime culture temperature was 18-20℃, the light intensity was 1800 LX, and the nighttime culture temperature was 15-17℃; and for the seventh month onwards, the daytime culture temperature was 22-23℃, the light intensity was 1500 LX, and the nighttime culture temperature was 17-19℃.

2. The method for in vitro preservation of Centella asiatica as described in claim 1, characterized in that, The method for obtaining Centella asiatica bottle seedlings is as follows: obtain sterile explants of Centella asiatica, inoculate the sterile explants of Centella asiatica into the first culture medium for induction culture to obtain induced shoots, and then inoculate the induced shoots into the second culture medium for seedling strengthening culture to obtain Centella asiatica bottle seedlings.

3. The method for in vitro preservation of Centella asiatica as described in claim 2, characterized in that, The first culture medium consisted of MS, 0.2-0.8 mg / L 6-benzylaminopurine, and 0.1-0.3 mg / L naphthaleneacetic acid. The culture temperature was 22-26℃, the light intensity was 1200-1800 LX, the daily light duration was 10-14 h, and the culture time was 21 days.

4. The method for in vitro preservation of Centella asiatica as described in claim 2, characterized in that, The second culture medium consists of MS, 0.1-0.5 mg / L 6-benzylaminopurine, 0.02-0.08 mg / L naphthaleneacetic acid, and 0.5-1.5 g / L activated carbon. The culture temperature is 22-26℃, the light intensity is 1200-1800 LX, and the daily light duration is 10-14 h.

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