Methods for preserving monk fruit in vitro

Abstract

This invention discloses a method for in vitro preservation of monk fruit, comprising the following steps: inoculating monk fruit seedlings into an in vitro culture medium for in vitro preservation culture, wherein the in vitro culture medium comprises 1 / 2 MS, 0.01-0.03 mg / L 6-benzylaminopurine, 0.6-1.4 mg / L quinacrine, 0.05-0.15 mg / L dextrin, 0.2-0.8 g / L activated carbon, 0.3-0.7 mg / L white tea extract, 0.01-0.03 mg / L amino acids, 0.01-0.03 mg / L brassinolide, and 0.5-1.5 mg / L eucalyptus leaf infusion. This invention, by adding white tea extract and eucalyptus leaf infusion to the in vitro culture medium, can effectively extend the in vitro preservation time of monk fruit.

Description

Technical Field

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

[0002] Monk fruit is a dioecious plant, with a male-to-female ratio of 3:7 inoculated seeds. It is necessary to observe flowering after sowing to distinguish the male and female offspring. Therefore, the preservation of monk fruit germplasm mainly relies on tissue culture. Tissue culture technology has become an important means for the industrialization of monk fruit. Although factory production has been achieved, in the conventional monk fruit tissue culture process, except for the spring planting season when seedlings need propagation, rooting, and transplanting, the tissue culture seedlings are in a seed preservation state at other times. When using conventional MS medium for in vitro preservation, the tissue culture seedlings grow too quickly or even age, requiring transfer after about one month. This leads to the problem of rapid growth and the need for repeated subculturing, which is not conducive to the long-term preservation of monk fruit germplasm. 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 monk fruit, which can effectively extend the in vitro preservation time of monk fruit by adding white tea liquid and eucalyptus leaf infusion to the in vitro culture medium.

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

[0006] Luo Han Guo (Siraitia grosvenorii) seedlings were inoculated into an in vitro culture medium for in vitro preservation and culture. The in vitro culture medium included 1 / 2 MS, 0.01-0.03 mg / L 6-benzylaminopurine, 0.6-1.4 mg / L quinacrine, 0.05-0.15 mg / L dextrin, 0.2-0.8 g / L activated carbon, 0.3-0.7 mg / L white tea extract, 0.01-0.03 mg / L amino acids, 0.01-0.03 mg / L brassinolide, and 0.5-1.5 mg / L eucalyptus leaf infusion. The white tea extract was prepared by soaking white tea in water at 30 times its total mass for 3 hours, filtering and concentrating to obtain a first concentrate, and then filtering and sterilizing the first concentrate using a 0.22 μm microporous membrane to obtain the white tea extract.

[0007] Preferably, the method for obtaining Luo Han Guo (monk fruit) bottle seedlings is as follows: obtain sterile explants of Luo Han Guo, inoculate the sterile explants of Luo Han Guo into an induction medium for induction culture to obtain induced buds, and inoculate the induced buds into a seedling strengthening medium for seedling strengthening culture to obtain Luo Han Guo bottle seedlings.

[0008] Preferably, the induction medium includes MS, 0.5 mg / L 6-benzylaminopurine, and 0.2 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 seedling culture medium includes 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, 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 23-25℃, the light intensity is 1500LX, and the nighttime culture temperature is 10-11℃; for the second month onwards, the culture temperature is 23-25℃, the light intensity is 1500LX, and the nighttime culture temperature is 12-14℃; for the fourth month onwards, the culture temperature is 20-22℃, the light intensity is 1500LX, 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 1500LX, and the nighttime culture temperature is 17-19℃.

[0011] The preparation method of eucalyptus leaf infusion is as follows: add 20 times the total mass of eucalyptus leaves to an ethanol solution with a mass fraction of 50% and heat at 70°C for 3 hours. Filter and concentrate to obtain a second concentrate. Then, filter and sterilize the second concentrate using a 0.22μm microporous membrane to obtain eucalyptus leaf infusion.

[0012] The present invention has at least the following beneficial effects:

[0013] This application demonstrates that adding white tea liquid and eucalyptus leaf infusion to the in vitro culture medium can effectively extend the in vitro preservation time of monk fruit, extending the in vitro preservation time of monk fruit seedlings to 503 days. Comparative analysis shows that only the simultaneous addition of both white tea liquid and eucalyptus leaf infusion to the in vitro culture medium can significantly extend the in vitro preservation time of monk fruit. This invention also extends the in vitro preservation time of monk fruit by gradually reducing the temperature difference between day and night. Furthermore, the simultaneous addition of white tea liquid and eucalyptus leaf infusion to the in vitro culture medium can effectively improve the survival rate of monk fruit seedlings after transplanting, and the effect of adding both white tea liquid and eucalyptus leaf infusion is significantly better than adding either white tea liquid or eucalyptus leaf infusion alone.

[0014] 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

[0015] 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.

[0016] <Example 1>

[0017] The method for preserving monk fruit in vitro includes the following steps:

[0018] Luo Han Guo (Siraitia grosvenorii) seedlings were inoculated into an in vitro culture medium for in vitro preservation and culture. The in vitro culture medium included 1 / 2 MS, 0.01 mg / L 6-benzylaminopurine, 0.6 mg / L quinacrine, 0.05 mg / L dextrin, 0.2 g / L activated carbon, 0.3 mg / L white tea extract, 0.01 mg / L amino acids, 0.01 mg / L brassinolide, and 0.5 mg / L eucalyptus leaf infusion. The white tea extract was prepared by soaking white tea in water at 30 times its total mass for 3 hours, filtering and concentrating to obtain a first concentrate, and then filtering and sterilizing the first concentrate using a 0.22 μm microporous membrane to obtain the white tea extract.

[0019] The method for obtaining Luo Han Guo (monk fruit) bottle seedlings is as follows: obtain sterile explants of Luo Han Guo, inoculate the sterile explants into an induction medium for induction culture to obtain induced buds, and then inoculate the induced buds into a seedling strengthening medium for seedling strengthening culture to obtain Luo Han Guo bottle seedlings.

[0020] The method for obtaining sterile explants of monk fruit is as follows: Take tender branches of monk fruit 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 surface moisture of the stem segments to obtain sterile explants of monk fruit.

[0021] One to two drops of Tween-20 were added to every 500 mL of HgCl2 solution.

[0022] The induction medium consisted of MS, 0.5 mg / L 6-benzylaminopurine, and 0.2 mg / L naphthaleneacetic acid. The culture temperature was 22℃, the light intensity was 1200 LX, the daily light duration was 10 h, and the culture time was 21 days.

[0023] The seedling 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.

[0024] The preparation method of eucalyptus leaf infusion is as follows: add 20 times the total mass of eucalyptus leaves to an ethanol solution with a mass fraction of 50% and heat at 70°C for 3 hours. Filter and concentrate to obtain a second concentrate. Then, filter and sterilize the second concentrate using a 0.22μm microporous membrane to obtain eucalyptus leaf infusion.

[0025] In this embodiment, the culture conditions for preservation in the in vitro culture medium adopt existing technology, namely, the culture temperature is 21-23℃, the light intensity is 1500LX, and the light duration is 9h per day.

[0026] <Example 2>

[0027] The method for preserving monk fruit in vitro includes the following steps:

[0028] Luo Han Guo (Siraitia grosvenorii) seedlings were inoculated into an in vitro culture medium for in vitro preservation and culture. The in vitro culture medium included 1 / 2 MS, 0.03 mg / L 6-benzylaminopurine, 1.4 mg / L quinacrine, 0.15 mg / L dextrin, 0.8 g / L activated carbon, 0.7 mg / L white tea extract, 0.03 mg / L amino acids, 0.03 mg / L brassinolide, and 1.5 mg / L eucalyptus leaf infusion. The white tea extract was prepared by soaking white tea in water at 30 times its total mass for 3 hours, filtering and concentrating to obtain a first concentrate, and then filtering and sterilizing the first concentrate using a 0.22 μm microporous membrane to obtain the white tea extract.

[0029] The method for obtaining Luo Han Guo (monk fruit) bottle seedlings is as follows: obtain sterile explants of Luo Han Guo, inoculate the sterile explants into an induction medium for induction culture to obtain induced buds, and then inoculate the induced buds into a seedling strengthening medium for seedling strengthening culture to obtain Luo Han Guo bottle seedlings.

[0030] The method for obtaining sterile explants of monk fruit is as follows: Take tender branches of monk fruit 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 surface moisture of the stem segments to obtain sterile explants of monk fruit.

[0031] One to two drops of Tween-20 were added to every 500 mL of HgCl2 solution.

[0032] The induction medium consisted of MS, 0.5 mg / L 6-benzylaminopurine, and 0.2 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.

[0033] The seedling 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℃, the light intensity was 1800 LX, and the daily light duration was 14 h.

[0034] The preparation method of eucalyptus leaf infusion is as follows: add 20 times the total mass of eucalyptus leaves to an ethanol solution with a mass fraction of 50% and heat at 70°C for 3 hours. Filter and concentrate to obtain a second concentrate. Then, filter and sterilize the second concentrate using a 0.22μm microporous membrane to obtain eucalyptus leaf infusion.

[0035] In this embodiment, the culture conditions for preservation in the in vitro culture medium adopt existing technology, namely, the culture temperature is 21-23℃, the light intensity is 1500LX, and the light duration is 9h per day.

[0036] <Example 3>

[0037] The method for preserving monk fruit in vitro includes the following steps:

[0038] Luo Han Guo (Siraitia grosvenorii) seedlings were inoculated into an in vitro culture medium for in vitro preservation and culture. The in vitro culture medium included 1 / 2 MS, 0.02 mg / L 6-benzylaminopurine, 1 mg / L quinacrine, 0.1 mg / L dextrin, 0.5 g / L activated carbon, 0.5 mg / L white tea extract, 0.02 mg / L amino acids, 0.02 mg / L brassinolide, and 1 mg / L eucalyptus leaf infusion. The white tea extract was prepared by soaking white tea in water at 30 times its total mass for 3 hours, filtering and concentrating to obtain a first concentrate, and then filtering and sterilizing the first concentrate using a 0.22 μm microporous membrane to obtain the white tea extract.

[0039] The method for obtaining Luo Han Guo (monk fruit) bottle seedlings is as follows: obtain sterile explants of Luo Han Guo, inoculate the sterile explants into an induction medium for induction culture to obtain induced buds, and then inoculate the induced buds into a seedling strengthening medium for seedling strengthening culture to obtain Luo Han Guo bottle seedlings.

[0040] The method for obtaining sterile explants of monk fruit is as follows: Take tender branches of monk fruit 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 surface moisture of the stem segments to obtain sterile explants of monk fruit.

[0041] One to two drops of Tween-20 were added to every 500 mL of HgCl2 solution.

[0042] The induction medium consisted of MS, 0.5 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.

[0043] The seedling 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.

[0044] The preparation method of eucalyptus leaf infusion is as follows: add 20 times the total mass of eucalyptus leaves to an ethanol solution with a mass fraction of 50% and heat at 70°C for 3 hours. Filter and concentrate to obtain a second concentrate. Then, filter and sterilize the second concentrate using a 0.22μm microporous membrane to obtain eucalyptus leaf infusion.

[0045] In this embodiment, the culture conditions for preservation in the in vitro culture medium adopt existing technology, namely, the culture temperature is 21-23℃, the light intensity is 1500LX, and the light duration is 9h per day.

[0046] <Example 4>

[0047] The method for preserving monk fruit in vitro includes the following steps:

[0048] Luo Han Guo (Siraitia grosvenorii) seedlings were inoculated into an in vitro culture medium for in vitro preservation and culture. The in vitro culture medium included 1 / 2 MS, 0.02 mg / L 6-benzylaminopurine, 1 mg / L quinacrine, 0.1 mg / L dextrin, 0.5 g / L activated carbon, 0.5 mg / L white tea extract, 0.02 mg / L amino acids, 0.02 mg / L brassinolide, and 1 mg / L eucalyptus leaf infusion. The white tea extract was prepared by soaking white tea in water at 30 times its total mass for 3 hours, filtering and concentrating to obtain a first concentrate, and then filtering and sterilizing the first concentrate using a 0.22 μm microporous membrane to obtain the white tea extract.

[0049] The method for obtaining Luo Han Guo (monk fruit) bottle seedlings is as follows: obtain sterile explants of Luo Han Guo, inoculate the sterile explants into an induction medium for induction culture to obtain induced buds, and then inoculate the induced buds into a seedling strengthening medium for seedling strengthening culture to obtain Luo Han Guo bottle seedlings.

[0050] The method for obtaining sterile explants of monk fruit is as follows: Take tender branches of monk fruit 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 surface moisture of the stem segments to obtain sterile explants of monk fruit.

[0051] One to two drops of Tween-20 were added to every 500 mL of HgCl2 solution.

[0052] The induction medium consisted of MS, 0.5 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.

[0053] The seedling 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.

[0054] The conditions for culture in in vitro culture medium are as follows: for the first month of culture, the daytime culture temperature is 23-25℃, the light intensity is 1500 LX, and the nighttime culture temperature is 10-11℃; for the second month onwards, the culture temperature is 23-25℃, the light intensity is 1500 LX, and the nighttime culture temperature is 12-14℃; for the third month onwards, the culture temperature is 20-22℃, the light intensity is 1500 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℃.

[0055] The preparation method of eucalyptus leaf infusion is as follows: add 20 times the total mass of eucalyptus leaves to an ethanol solution with a mass fraction of 50% and heat at 70°C for 3 hours. Filter and concentrate to obtain a second concentrate. Then, filter and sterilize the second concentrate using a 0.22μm microporous membrane to obtain eucalyptus leaf infusion.

[0056] <Comparative Example 1>

[0057] The in vitro preservation was performed using the method described in Example 4, except that the in vitro culture medium included: 1 / 2 MS, 0.02 mg / L 6-benzylaminopurine, 1 mg / L quinacrine, 0.1 mg / L pyridoxine, 0.5 g / L activated carbon, 0.02 mg / L amino acids, and 0.02 mg / L brassinolide.

[0058] <Comparative Example 2>

[0059] In vitro preservation was performed using the method described in Example 4, except that the in vitro culture medium included: 1 / 2 MS, 0.02 mg / L 6-benzylaminopurine, 1 mg / L quinacrine, 0.1 mg / L dextrin, 0.5 g / L activated charcoal, 0.5 mg / L white tea liquid, 0.02 mg / L amino acids, and 0.02 mg / L brassinolide. The white tea liquid was prepared by soaking white tea in water at 30 times its total mass for 3 hours, filtering and concentrating, and then sterilizing by filtration through a 0.22 μm microporous membrane to obtain the white tea liquid.

[0060] <Comparative Example 3>

[0061] In vitro preservation was performed using the method described in Example 4, except that the in vitro culture medium included 1 / 2 MS, 0.02 mg / L 6-benzylaminopurine, 1 mg / L quinacrine, 0.1 mg / L dextrin, 0.5 g / L activated carbon, 0.02 mg / L amino acids, 0.02 mg / L brassinolide, and 1 mg / L eucalyptus leaf infusion. The eucalyptus leaf infusion was prepared by adding 20 times the total mass of eucalyptus leaves to a 50% ethanol solution and placing it at 70°C for 3 hours. The solution was then filtered, concentrated, and sterilized by filtration through a 0.22 μm microporous membrane.

[0062] <Experimental Characterization>

[0063] 1. Ex vivo preservation time

[0064] The in vitro preservation time was the time when half of the seedlings in the in vitro culture medium died, as shown in Table 1.

[0065] Table 1 shows the in vitro preservation time.

[0066]

[0067]

[0068] Comparative analysis of the experimental data of Example 4 and Comparative Examples 1-3 in Table 1 shows that adding white tea liquid and eucalyptus leaf infusion to the in vitro culture medium can effectively prolong the in vitro preservation time of monk fruit. Furthermore, comparative analysis shows that only by adding both white tea liquid and eucalyptus leaf infusion to the in vitro culture medium can the in vitro preservation time of monk fruit be significantly extended. Comparative analysis of Examples 3 and 4 shows that the present invention can effectively prolong the in vitro preservation time of monk fruit by changing the temperature difference between day and night in stages.

[0069] 2. Survival rate of Luo Han Guo tissue culture seedlings after transplanting

[0070] The seedlings preserved in Examples 3, 4, and Comparative Examples 1-3 were cultured in in vitro culture medium for six months and then transplanted. The survival rate was counted 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%.

[0071] Table 2 Survival Rate

[0072] Group Survival rate Example 3 93％ Example 4 98.5％ Comparative Example 1 86％ Comparative Example 2 88％ Comparative Example 3 86.5％

[0073] Comparative analysis of the survival rate experiments in Example 4 and Comparative Examples 1-3 in Table 2 shows that the present application can effectively improve the survival rate of Luo Han Guo seedlings after transplanting by simultaneously adding white tea liquid and eucalyptus leaf infusion to the in vitro culture medium. Moreover, the effect of adding white tea liquid and eucalyptus leaf infusion at the same time is significantly better than adding white tea liquid or eucalyptus leaf infusion alone. Comparative analysis of Example 3 and Example 4 shows that the transplant survival rate can be improved by changing the temperature difference between day and night in stages.

[0074] 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 monk fruit, characterized in that, Includes the following steps: Luo Han Guo (Siraitia grosvenorii) seedlings were inoculated into an in vitro culture medium for in vitro preservation and culture. The in vitro culture medium consisted of 1 / 2 MS, 0.01-0.03 mg / L 6-benzylaminopurine, 0.6-1.4 mg / L quinacrine, 0.05-0.15 mg / L dextrin, 0.2-0.8 g / L activated carbon, 0.3-0.7 mg / L white tea extract, 0.01-0.03 mg / L amino acids, 0.01-0.03 mg / L brassinolide, and 0.5-1.5 mg / L eucalyptus leaf infusion. The white tea extract was prepared by soaking white tea in water at 30 times its total mass for 3 hours, filtering and concentrating to obtain a first concentrate, and then filtering and sterilizing the first concentrate using a 0.22 μm microporous membrane to obtain the white tea extract. The conditions for culture in in vitro culture medium are as follows: For the first month of culture, the daytime temperature is 23-25℃, the light intensity is 1500 LX, and the nighttime temperature is 10-11℃; for the second month onwards, the daytime temperature is 23-25℃, the light intensity is 1500 LX, and the nighttime temperature is 12-14℃; for the fourth month onwards, the daytime temperature is 20-22℃, the light intensity is 1500 LX, and the nighttime temperature is 15-17℃; and for the seventh month onwards, the daytime temperature is 22-23℃, the light intensity is 1500 LX, and the nighttime temperature is 17-19℃. The preparation method of eucalyptus leaf infusion is as follows: add 20 times the total mass of eucalyptus leaves to an ethanol solution with a mass fraction of 50% and heat at 70°C for 3 hours. Filter and concentrate to obtain a second concentrate. Then, filter and sterilize the second concentrate using a 0.22 μm microporous membrane to obtain eucalyptus leaf infusion.

2. The method for in vitro preservation of monk fruit as described in claim 1, characterized in that, The method for obtaining Luo Han Guo (monk fruit) bottle seedlings is as follows: obtain sterile explants of Luo Han Guo, inoculate the sterile explants into an induction medium for induction culture to obtain induced buds, and then inoculate the induced buds into a seedling strengthening medium for seedling strengthening culture to obtain Luo Han Guo bottle seedlings.

3. The method for in vitro preservation of monk fruit as described in claim 2, characterized in that, The induction medium consisted of MS, 0.5 mg / L 6-benzylaminopurine, and 0.2 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 monk fruit as described in claim 2, characterized in that, The seedling culture medium consisted 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 was 22-26℃, the light intensity was 1200-1800 LX, and the daily light duration was 10-14 h.

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