Villous amomum fruit detoxification method through combination of protoporphyrin IX and secondary stem tip culture

Abstract

The invention discloses a villous amomum fruit detoxification method through combination of protoporphyrin IX and secondary stem tip culture, and belongs to the technical field of plant tissue culture detoxification seedling culture. The method comprises the following steps: cutting roots of fructus amomi as explants, and carrying out subculture to obtain tissue culture seedlings; after the tissue culture seedlings are treated by protoporphyrin IX, mature plants are obtained; cutting stem tips from the mature plants, inoculating the stem tips to a stem tip induced differentiation culture medium, and carrying out primary stem tip detoxification culture to obtain tissue culture plantlets; and repeating the steps (2)-(3) to finish secondary stem tip detoxification culture so as to obtain the detoxified tissue culture seedlings of the fructus amomi. The protoporphyrin IX is used for performing secondary stem tip culture detoxification on the fructus amomi tissue culture seedlings, the method is low in technical difficulty, does not need expensive and precise instruments and equipment, is low in cost and high in detoxification rate, regenerated plants can well maintain excellent characters of female parents and grow vigorously, high CMV detoxification rate and TMV removal rate can be obtained, and a new method is provided for cultivating detoxified fructus amomi.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture detoxification and seedling raising, in particular to a method for detoxification of Amomum villosum by combining protoporphyrin IX with secondary stem tip culture. Background Art

[0002] Amomum villosum Lour. is a perennial herbaceous plant of the genus Amomum in the family Zingiberaceae. Amomum villosum is an important crop for both medicinal and edible purposes, and can also be used as an important ornamental plant. In terms of edibility, Amomum villosum can be used as a seasoning vegetable, and the main edible part is the dried fruit. In terms of medicinal properties, "Yao Xing Lun" and "Kai Bao Ben Cao" record that Amomum villosum has the effects of removing dampness and promoting qi, warming the middle and stopping diarrhea, and calming the fetus. In terms of ornamental properties, the flowers can be appreciated in early summer and the fruits can be appreciated in midsummer.

[0003] The cultivation methods of Amomum villosum plants are mainly divided into seed propagation and division propagation. Amomum villosum has a strong ability to divide, can quickly form a group, develops quickly, and can bear fruit in two years with good management. Therefore, in actual production, division propagation is often used for production. Division propagation belongs to the type of asexual reproduction. This method has the advantages of maintaining the excellent genetic characteristics of the parents, rapid proliferation of plant numbers, and high stability of yield and quality. However, during long-term asexual reproduction, viruses can be transmitted through the vascular bundle and accumulate in the plant from generation to generation, resulting in serious degradation of the species. This degradation manifests itself in problems such as crop growth inhibition, morphological distortion, yield reduction, and quality deterioration. Amomum villosum is susceptible to cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV), which can cause mosaic, yellowing, and yield reduction, which significantly affects the yield and quality of Amomum villosum. It is of great significance to adopt effective measures to prevent and control virus diseases of Amomum villosum.

[0004] In order to reduce the great harm caused by viruses to crop production, the spread of viruses should be cut off from the source of seedlings, and the cultivation of virus-free seedlings of Amomum villosum is the only way for the rapid development of the Amomum villosum industry. At present, virus-free seedlings are mainly obtained through biotechnology methods such as shoot tip culture, heat treatment detoxification, anther culture, micrografting, callus culture, antiviral chemotherapy and ultra-low temperature therapy. Among them, the shoot tip detoxification method is the most commonly used and simplest method, but the detoxification efficiency is low; other methods are difficult to operate, time-consuming, and require high equipment and technology. The virus is unevenly distributed in the plant. There is usually no virus distribution in tissues with faster differentiation speed and tenderer cells such as the apical meristem of the shoot tip. Therefore, a certain detoxification rate can be obtained through shoot tip culture. The secondary shoot tip culture method is to peel off and culture the shoot tip again from the young regenerated seedlings obtained from the first shoot tip culture. The regenerated plants in the first shoot tip culture do not carry or carry low titer viruses. Therefore, a higher detoxification rate can be obtained through secondary shoot tip culture. The entire process is carried out in a sterile environment of tissue culture. Therefore, the virus-free varieties do not carry pathogens such as fungi and bacteria on the body surface. Summary of the invention

[0005] The purpose of the present invention is to provide a method for detoxifying Amomum villosum by combining protoporphyrin IX with secondary stem tip culture to solve the problems existing in the above-mentioned prior art. The present invention uses protoporphyrin IX to perform secondary stem tip culture detoxification on Amomum villosum tissue culture seedlings. The method has low technical difficulty, does not require expensive and precise instruments and equipment, has low cost, and has a high detoxification rate. The regenerated plants can better maintain the excellent traits of the mother plant, have strong growth potential, and can obtain a higher CMV detoxification rate and TMV detoxification rate, thereby providing a new method for cultivating detoxified Amomum villosum.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for detoxifying Amomum villosum by combining protoporphyrin IX with secondary stem tip culture, comprising the following steps:

[0008] (1) cutting the rhizomes of Amomum villosum as explants, and obtaining tissue culture seedlings through subculture;

[0009] (2) treating the tissue culture seedlings with protoporphyrin IX to obtain mature plants;

[0010] (3) cutting a 0.15-0.25 mm-sized stem tip from the mature plant under a dissecting microscope, inoculating it into a stem tip induction differentiation medium, performing a first stem tip virus-free culture, and obtaining a tissue culture plantlet;

[0011] (4) Repeat steps (2)-(3) to complete the second shoot tip virus-free culture to obtain Amomum villosum virus-free tissue culture seedlings.

[0012] Furthermore, the culture medium for the subculture is: MS+0.4-0.6 mg / L NAA+2.5-3.5 mg / L 6-BA+25-35 g / L sucrose+5-15 g / L agar, pH=5.5-6.0.

[0013] Furthermore, the conditions for the subculture are: temperature of 25°C to 28°C, light intensity of 2200Lx to 2500Lx, and 12 hours of light per day.

[0014] Furthermore, the culture medium for the protoporphyrin IX treatment is: MS+0.4-0.6 mg / L NAA+2.5-3.5 mg / L 6-BA+25-35 g / L sucrose+5-15 g / L agar+1.5-2.5 mg / L protoporphyrin IX, pH=5.5-6.0.

[0015] Furthermore, the conditions for the protoporphyrin IX treatment are: temperature of 25°C to 28°C, light intensity of 2200Lx to 2500Lx, and 12 hours of light per day.

[0016] Furthermore, the treatment time of protoporphyrin IX is 35 days to 45 days; furthermore, the treatment time of protoporphyrin IX is 40 days.

[0017] Furthermore, the shoot tip induction differentiation medium is: MS+0.08-0.12 mg / L NAA+0.8-1.5 mg / L 6-BA+25-35 g / L sucrose+5-15 g / L agar, pH=5.5-6.0.

[0018] Furthermore, the conditions for the first shoot tip virus-free culture are: temperature of 25°C to 28°C, light intensity of 2200Lx to 2500Lx, and 12 hours of light per day.

[0019] Optionally, the first time for the virus-free culture of the shoot tip is 35 days to 45 days; further optionally, the first time for the virus-free culture of the shoot tip is 40 days.

[0020] Optionally, the second time of the virus-free culture of the shoot tip is 35 days to 45 days; further optionally, the second time of the virus-free culture of the shoot tip is 40 days.

[0021] The present invention discloses the following technical effects:

[0022] The present invention uses protoporphyrin IX to perform secondary stem tip culture and virus removal on Amomum villosum tissue culture seedlings. After the Amomum villosum tissue culture seedlings are treated with protoporphyrin IX, a stem tip of about 0.2 mm is cut for first virus removal culture, and after some viruses are removed, the tissue culture plantlets are treated with protoporphyrin IX again, and the stem tip is cut for second virus removal culture, and most of the viruses can be removed. The method has low technical difficulty, does not require expensive and precise instruments and equipment, has low cost, high virus removal rate, and the regenerated plants can better maintain the excellent traits of the mother plant, have strong growth potential, and can obtain a higher CMV virus removal rate and TMV virus removal rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 is a schematic diagram of the structure of protoporphyrin IX;

[0025] Figure 2 RT-PCR gel electrophoresis of Amomum villosum tissue culture seedlings without CMV removal (obtained from the culture with a shoot tip size of 1.0 mm and a protoporphyrin IX treatment concentration of 0); M represents DNA Ladder 1500 Marker; 1 represents negative control water; 2-12 represent samples without virus removal;

[0026] Figure 3 The RT-PCR gel electrophoresis diagram of CMV virus in virus-free Amomum villosum tissue culture seedlings (cultivated with a stem tip size of 0.2 mm and treated with protoporphyrin IX at a concentration of 2 mg / L); M represents DNA Ladder 1500 Marker; 1 represents the positive control mother plant; 2-12 are virus-free samples;

[0027] Figure 4 The RT-PCR gel electrophoresis diagram of TMV virus in virus-free Amomum villosum tissue culture seedlings (cultivated with a stem tip size of 0.2 mm and treated with protoporphyrin IX at a concentration of 2 mg / L); M represents DNA Ladder 1500 Marker; 1 represents the positive control mother plant; 2-12 are virus-free samples;

[0028] Figure 5 The figure is the PCR gel electrophoresis of ISSR universal primer 811; M represents DNA Ladder 1500 Marker; 1 represents the positive control mother plant; 2-7 are virus-free regenerated plants;

[0029] Figure 6The figure is the PCR gel electrophoresis of ISSR universal primer 836; M represents DNA Ladder 1500 Marker; 1 represents the positive control mother plant; 2-7 are virus-free regenerated plants. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] Unless otherwise specified, other reagents or materials involved in the embodiments of the present invention can be purchased through conventional channels and used according to the product instructions, and are well known to the industry; the operating methods or detection methods involved, unless otherwise specified, are carried out according to conventional methods in the art.

[0036] The Cas number of protoporphyrin IX used in the following examples of the present invention is 553-12-8, and the structural formula is as follows: Figure 1 shown.

[0037] The technical solution of the present invention is as follows:

[0038] The rhizomes of Amomum villosum with vigorous growth and no obvious diseases were selected as explants, rinsed under running water for 1 hour, and then placed on cotton soaked with water to germinate in a light-shading environment; the explants with buds were cut to a size of 0.3-0.5 cm, and treated in a 2% chloramine T solution for 40 minutes; they were soaked in clean water and rinsed clean; the explants were transferred to a clean bench, rinsed with sterile water for 1-2 times, rinsed with alcohol for 30 seconds, rinsed with sterile water once, disinfected on the surface with 10% NaClO for 15 minutes, and rinsed with sterile water for 3-4 times, and the explants were inoculated on a subculture medium (MS+NAA 0.5 mg / L+6-BA 3.0 mg / L+sucrose 30 g / L+agar 8 g / L, pH=5.8) to make tissue culture seedlings; the tissue culture seedlings were cultured at a temperature of 25°C-28°C and a light intensity of 2200Lx-2500Lx with light exposure for 12 hours per day, and subcultured every 30 days.

[0039] The subcultured tissue culture seedlings were treated for 40 days at a temperature of 25°C-28°C and a light intensity of 2200Lx-2500Lx on Amomum villosum protoporphyrin IX treatment medium (MS+0.5mg / LNAA+3.0mg / L 6-BA+30g / L sucrose+8g / L agar+protoporphyrin IX2.0mg / L, pH=5.8), with 12 hours of light per day to obtain mature plants.

[0040] Take mature plants, cut 0.2mm stem tips with a body dissecting microscope in a clean bench, place in differentiation medium (MS+0.10mg / LNAA+1.0mg / L 6-BA+30g / L sucrose+8g / L agar, pH=5.8), and culture under the same culture conditions; just after the stem tips differentiated to form Amomum villosum tissue culture plantlets, they were treated with protoporphyrin IX, stem tip peeling and stem tip induction culture; after the second stem tip culture for 40 days, the mature Amomum villosum tissue culture seedlings were qualified by RT-PCR virus detection, and the virus was removed. The DNA polymorphism of the test tube mother plant and the virus-free regenerated plant of Amomum villosum was detected by ISSR. This method was obtained by a comparative test with 30 explants in each group and each culture medium. The stem tips detoxified by the secondary culture method had high survival rate and virus-free rate and genetic stability.

[0041] Example 1

[0042] The culture medium used in this example is as follows:

[0043] (1) Subculture medium: MS + 0.5 mg / L NAA + 3.0 mg / L 6-BA + 30 g / L sucrose + 8 g / L agar, pH = 5.8.

[0044] (2) Protoporphyrin IX treatment culture medium: MS + 0.5 mg / L NAA + 3.0 mg / L 6-BA + 30 g / L sucrose + 8 g / L agar + protoporphyrin IX 2.0 mg / L, pH = 5.8.

[0045] (3) Shoot tip induction differentiation medium: MS + 0.10 mg / L NAA + 1.0 mg / L 6-BA + 30 g / L sucrose + 8 g / L agar, pH = 5.8.

[0046] The specific detoxification culture process is as follows:

[0047] (1) Explant selection: Select rhizomes of Amomum villosum without obvious diseases as explants, rinse them under running water for 1 h, place the washed explants on cotton soaked with water, and germinate them in a light-shaded environment;

[0048] (2) Explant treatment: Cut the explants with buds to a size of 0.3-0.5 cm, soak them in 2% chloramine T solution, shake them on a shaker at 200 rpm for 40 min, soak them in clean water for 10 min, and then rinse them;

[0049] (3) Establishment of tissue culture seedlings: The explants were transferred to a clean bench, rinsed twice with sterile water, rinsed with alcohol for 30 seconds, rinsed once with sterile water, and transferred to a 10% NaClO solution for surface disinfection for 15 minutes, with continuous shaking. After surface disinfection, they were rinsed four times with sterile water. The explants were dried on sterile filter paper and inoculated on subculture medium to culture tissue culture seedlings.

[0050] (4) Propagation of tissue culture seedlings: The tissue culture seedlings were cultured at 25°C and 2500 Lx of light intensity for 12 h per day, and subcultured every 30 days;

[0051] (5) Protoporphyrin IX treatment: Select tissue culture seedlings with the same growth conditions after subculture and randomly divide them into two groups. One group is the control group, which is inoculated in a medium without protoporphyrin IX treatment, and the other group is the experimental group, which is inoculated on a medium treated with protoporphyrin IX. Both groups are placed at a temperature of 25°C and a light intensity of 2500Lx for 40 days, with light exposure for 12 hours per day, to obtain mature tissue culture plants;

[0052] (6) First shoot tip culture: In a clean bench, use a body dissecting microscope to cut 0.2 mm, 0.5 mm and 1.00 mm shoot tips from two groups of mature tissue culture plants, respectively. The cut shoot tips are placed in a shoot tip induction differentiation medium, and are illuminated for 12 hours per day at a temperature of 25°C and a light intensity of 2500 Lx for 40 days. The resulting Amomum villosum tissue culture seedlings are tested for viruses by RT-PCR, and the viruses to be tested are CMV and TMV. The survival rate, regeneration rate and virus-free rate of the first shoot tip culture are calculated. The virus-free rate is calculated as follows: (number of virus-free regenerated plants / total number of regenerated plants).

[0053] The RT-PCR virus detection process is as follows:

[0054] a. Extract RNA according to the instructions of the RNA extraction kit.

[0055] b. Use a micro-volume UV spectrophotometer to detect RNA. The RNA concentration is appropriate and the OD 260 / OD 280 RNA with a value between 1.8 and 2.2 can be used for cDNA first-strand synthesis, and the specific operation is carried out according to the instructions. The detection primers were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd., and the specific primer information is shown in Table 1. The PCR system is shown in Table 2. The PCR program is pre-denaturation at 94°C (3min); denaturation at 94°C (50s), annealing at 56°C (50s), extension at 72°C (1min) for a total of 34 times; extension at 72°C (10min); storage at 4°C. The steps for PCR product detection are: 0.2g agarose and 20.0mL TAE buffer are mixed and heated to boiling; 2.0μL nucleic acid dye is added. Pour into the fixed plate well plate and solidify at room temperature. 6μL is spotted in each well of the sugar gel; 180V constant voltage electrophoresis for 20min; chemical gel imager imaging observation.

[0056] Table 1 Primer sequences

[0057]

[0058] Table 2 PCR reaction system

[0059] Reagents Volume (μL) BlendTaq 0.1 2mM dNTPs 1.0 10×Blendtaqbuffer 1.0 20 μM upstream primer 0.1 20 μM downstream primer 0.1 cDNA template 1.0 Double distilled water 6.7 Total volume 10.0

[0060] Note: The reaction system was prepared at room temperature and the Blend Taq enzyme was placed on ice.

[0061] The statistical results are shown in Table 3.

[0062] Table 3 Survival rate, regeneration rate and virus-free rate of the first shoot tip culture

[0063]

[0064]

[0065] As can be seen from Table 3, protoporphyrin IX treatment can effectively promote the survival and regeneration of shoot tips, while enhancing the inhibition of viruses and increasing the virus-free rate. When protoporphyrin IX treatment is omitted, the size of the peeled shoot tip is proportional to the survival rate and virus-free rate of the shoot tip. The larger the shoot tip, the higher the survival rate and regeneration rate, and inversely proportional to the virus-free rate. The larger the shoot tip, the lower the virus-free rate of CMV and TMV. Compared with TMV, the virus-free rate of CMV is lower, and when the shoot tip size is 1.0 mm, CMV cannot be removed. When the shoot tip size is 0.5 mm, CMV cannot be removed without adding protoporphyrin IX.

[0066] (7) Second stem tip culture: After the stem tip differentiates into Amomum villosum tissue culture plantlets, select the tissue culture plantlets with a stem tip size of 0.2 mm after protoporphyrin IX treatment, and repeat steps (5) to (6); after the second stem tip culture for 40 days, the Amomum villosum tissue culture seedlings formed are tested for viruses by RT-PCR. The statistical results of the survival rate, regeneration rate and virus-free rate of the stem tip are shown in Table 4. Figure 2 , Figure 3 and Figure 4 . Figure 2 The results of CMV virus detection in Amomum villosum tissue culture seedlings obtained from 1.0 mm stem tips without protoporphyrin IX treatment; Figure 3 The results of CMV virus detection in Amomum villosum tissue culture seedlings obtained from 0.2 mm stem tips treated with 2 mg / L protoporphyrin IX; Figure 4 The results of TMV virus detection in Amomum villosum tissue culture seedlings obtained from 0.2 mm stem tips treated with 2 mg / L protoporphyrin IX.

[0067] Table 4 Survival rate, regeneration rate and virus-free rate of the second shoot tip culture

[0068]

[0069] From Table 4, Figure 2 , Figure 3 and Figure 4 It can be seen that, like the first shoot tip culture, protoporphyrin IX treatment can effectively promote the survival and regeneration of the shoot tip, while improving the inhibition of the virus and increasing the detoxification rate. Compared with the first shoot tip culture, the detoxification rate of CMV and TMV in the second shoot tip culture was significantly improved, and the bands could no longer be clearly observed in the CMV virus RT-PCR test results of the detoxified samples, indicating that the second shoot tip culture method of the present invention is a technology that can efficiently remove viruses, and the smaller the participating shoot tip, the higher the detoxification rate.

[0070] (8) Genetic stability detection: DNA polymorphism detection was performed on the test tube mother plants and virus-free regenerated plants of Amomum villosum using ISSR to determine the genetic stability of Amomum villosum detected by this method. DNA extraction was performed according to the instructions of the kit. ISSR primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the 811 and 836 ISSR universal primers designed for Columbia University are shown in Table 5. The PCR reaction system is shown in Table 6, and the PCR reaction procedure and PCR product detection are the same as step (6).

[0071] Table 5 Primer sequences

[0072] Primers Primer sequence (5'-3') 811 GAGAGAGAGAGAGAGAC(SEQ ID NO.5) 836 AGAGAGAGAGAGAGAGYA(SEQ ID NO.6)

[0073] Table 6 ISSR PCR reaction system

[0074] Components Concentration (μL) DNA template 1.0 Forward Primer 0.5 Reverse primer 0.5 T2×TaqPCRStarMix 10.0 <![CDATA[DEPC-ddH2O]]> 7.0

[0075] Test results are shown in Figure 5 and Figure 6 , neither primer detected polymorphic bands, indicating that the secondary shoot tip culture method treated with protoporphyrin IX would not cause genetic changes in Amomum villosum.

[0076] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for detoxifying Amomum villosum by combining protoporphyrin IX with secondary stem tip culture, characterized in that: The steps include: (1) cutting the rhizomes of Amomum villosum as explants, and obtaining tissue culture seedlings through subculturing; (2) treating the tissue culture seedlings with protoporphyrin IX to obtain mature plants; (3) cutting a 0.15-0.25 mm-sized stem tip from the mature plant, inoculating it into a stem tip induction differentiation medium, performing a first stem tip virus-free culture, and obtaining a tissue culture plantlet; (4) Repeat steps (2)-(3) to complete the second shoot tip virus-free culture to obtain Amomum villosum virus-free tissue culture seedlings.

2. The method for detoxifying Amomum villosum according to claim 1, characterized in that: The culture medium for the subculture is: MS+0.4-0.6 mg / L NAA+2.5-3.5 mg / L 6-BA+25-35 g / L sucrose+5-15 g / L agar, pH=5.5-6.

0.

3. The method for detoxifying Amomum villosum according to claim 1, characterized in that: The conditions for the subculture are: a temperature of 25°C to 28°C, a light intensity of 2200Lx to 2500Lx, and 12 hours of light per day.

4. The method for detoxifying Amomum villosum according to claim 1, characterized in that: The culture medium for protoporphyrin IX treatment is: MS+0.4-0.6 mg / L NAA+2.5-3.5 mg / L 6-BA+25-35 g / L sucrose+5-15 g / L agar+1.5-2.5 mg / L protoporphyrin IX, pH=5.5-6.

0.

5. The method for detoxifying Amomum villosum according to claim 1, characterized in that: The conditions for the protoporphyrin IX treatment are: temperature of 25°C to 28°C, light intensity of 2200Lx to 2500Lx, and 12 hours of light per day.

6. The method for detoxifying Amomum villosum according to claim 1, characterized in that: The protoporphyrin IX treatment time is 40 days.

7. The method for detoxifying Amomum villosum according to claim 1, characterized in that: The shoot tip induction differentiation medium is: MS+0.08-0.12 mg / L NAA+0.8-1.5 mg / L 6-BA+25-35 g / L sucrose+5-15 g / L agar, pH=5.5-6.

0.

8. The method for detoxifying Amomum villosum according to claim 1, characterized in that: The conditions for the first shoot tip virus-free culture are: temperature of 25°C to 28°C, light intensity of 2200Lx to 2500Lx, and 12 hours of light per day.

9. The method for detoxifying Amomum villosum according to claim 1, characterized in that: The time of the first shoot tip virus-free culture is 35d-45d.

10. The method for detoxifying Amomum villosum according to claim 1, characterized in that: The time of the second shoot tip virus-free culture is 35d-45d.

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