A tissue culture and detoxification method of amomum tsao-ko

Abstract

This invention discloses a tissue culture detoxification method for Amomum villosum, comprising the following steps: S1, selecting robust Amomum villosum plants, using young stem segments as explants, and inoculating them into callus induction medium after disinfection to induce callus formation; wherein the callus induction medium comprises: MS + 2,4-D 0.8-1.2 mg / L, 6-BA 0.4-0.6 mg / L and chicoric acid 3-5 μmol / L; S2, heat treatment: cutting the callus obtained in step S1 into small pieces and placing them in a sterile water bath at 40±2℃ for 15-20 min; S3, differentiation culture: inoculating the heat-treated callus into a differentiation medium to induce tissue differentiation; wherein the differentiation medium comprises: 1 / 2 MS + 6-BA 1.2-1.8 mg / L + TDZ 0.04–0.06 mg / L; S4, Rooting Induction: The clustered shoots obtained after differentiation culture are cut into single shoots without callus tissue and inoculated into rooting medium; wherein the rooting medium comprises: 1 / 2 MS + IBA 1.0–1.4 mg / L + NAA 0.2–0.4 mg / L + brassinolide 0.04–0.07 mg / L. The method described in this invention can effectively improve the success rate of tomato spotted wilt virus elimination during grass fruit tissue culture.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically, it relates to a tissue culture detoxification method for cardamom. Background Technology

[0002] Amomum tsao-ko Crevostet Lemaire is a perennial herb belonging to the genus Amomum in the family Zingiberaceae. The essential oil extracted from the fruit of Amomum tsao-ko is used in the pharmaceutical and fragrance industries. Amomum tsao-ko is a popular seasoning and a traditional Chinese medicine. Wild Amomum tsao-ko grows in subtropical rainforests and is mainly distributed in parts of Yunnan, Guangxi and Guizhou in my country, as well as parts of northern Vietnam and Laos.

[0003] Currently, in order to enable cardamom plants to bear fruit as soon as possible, the method of propagation by division is often used for transplanting. However, this transplanting method can easily lead to the accumulation of tomato wilt virus in the plant, resulting in cardamom chlorosis and affecting the yield of cardamom. Summary of the Invention

[0004] In order to overcome the problems existing in the prior art, the present invention proposes a tissue culture detoxification method for cardamom.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution: A tissue culture detoxification method for cardamom includes the following steps: S1. Select robust cardamom plants and use young stem segments as explants. After disinfection, inoculate them into callus induction medium to induce callus formation. The callus induction medium includes: MS + 2,4-D 0.8-1.2 mg / L, 6-BA 0.4-0.6 mg / L and chicoric acid 3-5 μmol / L. S2, heat treatment: After cutting the callus tissue obtained in step S1 into small pieces, place it in a sterile water bath at 40±2℃ for 15-20 minutes. S3, Differentiation Culture: Callus tissue treated with a heat bath was inoculated into differentiation medium to induce tissue differentiation; the differentiation medium consisted of: 1 / 2 MS + 6-BA 1.2-1.8 mg / L + TDZ 0.04-0.06 mg / L; S4, Rooting Induction: The clustered shoots obtained after differentiation culture were cut into single shoots without callus tissue and inoculated into rooting medium; wherein the rooting medium consisted of: 1 / 2MS + IBA 1.0-1.4 mg / L + NAA 0.2-0.4 mg / L + brassinolide 0.04-0.07 mg / L.

[0006] Furthermore, in step S1, the explant is disinfected by soaking it in 70% ethanol for 30 seconds, then soaking it in 0.2% mercuric chloride solution for 10-12 minutes, and finally rinsing it with sterile water 3-5 times.

[0007] Furthermore, the culture conditions for inducing callus formation in step S1 are: dark culture at 25±2℃.

[0008] Furthermore, in the three steps, the conditions for differentiation culture are: temperature 25±2℃, 12h of light per day, and alternating light and dark culture with a light intensity of 2200~3200lx.

[0009] Furthermore, in step S4, the conditions for rooting culture are: temperature 25±2℃, 12h of light per day, and alternating light and dark conditions with a light intensity of 2200~3200lx.

[0010] Through the above technical solution, the present invention can achieve at least the following beneficial effects: The method described in this invention can effectively improve the success rate of virus elimination of tomato spotted wilt virus during grass fruit tissue culture. Detailed Implementation

[0011] Unless otherwise stated, all materials and reagents used in this invention are commercially available. Example 1

[0012] A tissue culture detoxification method for cardamom includes the following steps: S1. Select robust cardamom plants and use young stem segments of cardamom plants as explants. After disinfection, inoculate them into callus induction medium to induce callus formation. The callus induction medium includes: MS + 2,4-D 1 mg / L, 6-BA 0.5 mg / L and chicoric acid 4 μmol / L. S2, heat treatment: After cutting the callus tissue obtained in step S1 into small pieces, place it in a sterile water bath at 40±2℃ for 17 minutes. S3, Differentiation Culture: Callus tissue treated with heat bath was inoculated into differentiation medium to induce tissue differentiation; the differentiation medium consisted of: 1 / 2 MS + 6-BA 1.5 mg / L + TDZ 0.05 mg / L; S4, Rooting Induction: The clustered shoots obtained after differentiation culture were cut into single shoots without callus tissue and inoculated into rooting medium; wherein the rooting medium consisted of: 1 / 2MS + IBA 1.2mg / L + NAA 0.3mg / L + brassinolide 0.05mg / L.

[0013] In step S1, the explants are disinfected by soaking them in 70% ethanol for 30 seconds, then in 0.2% mercuric chloride solution for 11 minutes, and finally rinsing them four times with sterile water.

[0014] The culture conditions for inducing callus formation in step S1 are: dark culture at 25±2℃.

[0015] Among them, the differentiation culture conditions in the three steps are: temperature 25±2℃, 12h of light per day, and alternating light and dark culture with a light intensity of 2700lx.

[0016] In step S4, the conditions for rooting culture are: temperature 25±2℃, 12 hours of light per day, and alternating light and dark conditions with a light intensity of 2700lx. Example 2

[0017] A tissue culture detoxification method for cardamom includes the following steps: S1. Select robust cardamom plants and use young stem segments of cardamom plants as explants. After disinfection, inoculate them into callus induction medium to induce callus formation. The callus induction medium includes: MS + 2,4-D 0.8 mg / L, 6-BA 0.4 mg / L and chicoric acid 3 μmol / L. S2, heat treatment: After cutting the callus tissue obtained in step S1 into small pieces, place it in a sterile water bath at 40±2℃ for 15 minutes. S3, Differentiation Culture: Callus tissue treated with heat bath was inoculated into differentiation medium to induce tissue differentiation; the differentiation medium consisted of: 1 / 2 MS + 6-BA 1.2 mg / L + TDZ 0.046 mg / L; S4, Rooting Induction: The clustered shoots obtained after differentiation culture were cut into single shoots without callus tissue and inoculated into rooting medium; wherein the rooting medium consisted of: 1 / 2MS + IBA 1.0mg / L + NAA 0.2mg / L + brassinolide 0.04mg / L.

[0018] In step S1, the explant is disinfected by soaking it in 70% ethanol for 30 seconds, then soaking it in 0.2% mercuric chloride solution for 10 minutes, and finally rinsing it three times with sterile water.

[0019] The culture conditions for inducing callus formation in step S1 are: dark culture at 25±2℃.

[0020] Among them, the differentiation culture conditions in the three steps are: temperature 25±2℃, 12h of light per day, and alternating light and dark culture with a light intensity of 2200lx.

[0021] In step S4, the conditions for rooting culture are: temperature 25±2℃, 12 hours of light per day, and alternating light and dark conditions with a light intensity of 2200lx. Example 3

[0022] A tissue culture detoxification method for cardamom includes the following steps: S1. Select robust cardamom plants and use young stem segments of cardamom plants as explants. After disinfection, inoculate them into callus induction medium to induce callus formation. The callus induction medium includes: MS + 2,4-D 1.2 mg / L, 6-BA 0.6 mg / L and chicoric acid 5 μmol / L. S2, heat treatment: After cutting the callus tissue obtained in step S1 into small pieces, place it in a sterile water bath at 40±2℃ for 20 minutes. S3, Differentiation Culture: Callus tissue treated with heat bath was inoculated into differentiation medium to induce tissue differentiation; the differentiation medium consisted of: 1 / 2MS + 6-BA 1.8 mg / L + TDZ 0.06 mg / L; S4, Rooting Induction: The clustered shoots obtained after differentiation culture were cut into single shoots without callus tissue and inoculated into rooting medium; the rooting medium consisted of: 1 / 2MS + IBA 1.4mg / L + NAA 0.4mg / L + brassinolide 0.07mg / L.

[0023] In step S1, the explant is disinfected by soaking it in 70% ethanol for 30 seconds, then soaking it in 0.2% mercuric chloride solution for 12 minutes, and finally rinsing it 5 times with sterile water.

[0024] The culture conditions for inducing callus formation in step S1 are: dark culture at 25±2℃.

[0025] Among them, the differentiation culture conditions in the three steps are: temperature 25±2℃, 12h of light per day, and alternating light and dark culture with a light intensity of 3200lx.

[0026] In step S4, the conditions for rooting culture are: temperature 25±2℃, 12 hours of light per day, and alternating light and dark conditions with a light intensity of 3200lx. Comparative Example 1

[0027] The difference between Comparative Example 1 and Example 1 is that the callus induction culture medium does not contain chicoric acid. Comparative Example 2

[0028] The difference between Comparative Example 2 and Example 1 is that no heat treatment is performed. Comparative Example 3

[0029] The callus tissues obtained after heat treatment in Examples 1 to 3 and Comparative Example 1, as well as the callus tissue obtained in Comparative Example 2, were tested using a tomato spotted wilt virus kit (purchased from Beijing Zhongjian Baotai Biotechnology Co., Ltd.), and the detoxification success rate was calculated. The results are shown in Table 1 below: Example 1 86.32 Example 2 84.77 Example 3 88.51 Comparative Example 1 65.21 Comparative Example 2 71.34 As can be seen from Table 1 above, the detoxification method of the present invention can effectively improve the success rate of detoxification of tomato spotted wilt virus during the tissue culture process of cardamom.

[0030] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A method for detoxifying cardamom through tissue culture, characterized in that: It includes the following steps: S1. Select robust cardamom plants and use young stem segments as explants. After disinfection, inoculate them into callus induction medium to induce callus formation. The callus induction medium includes: MS + 2,4-D 0.8-1.2 mg / L, 6-BA 0.4-0.6 mg / L and chicoric acid 3-5 μmol / L. S2, heat treatment: After cutting the callus tissue obtained in step S1 into small pieces, place it in a sterile water bath at 40±2℃ for 15-20 minutes. S3, Differentiation Culture: Callus tissue treated with a heat bath was inoculated into differentiation medium to induce tissue differentiation; the differentiation medium consisted of: 1 / 2 MS + 6-BA 1.2-1.8 mg / L + TDZ 0.04-0.06 mg / L; S4, Rooting Induction: The clustered shoots obtained after differentiation culture were cut into single shoots without callus tissue and inoculated into rooting medium; wherein the rooting medium consisted of: 1 / 2MS + IBA 1.0-1.4 mg / L + NAA 0.2-0.4 mg / L + brassinolide 0.04-0.07 mg / L.

2. The method for tissue culture detoxification of cardamom according to claim 1, characterized in that: In step S1, the explant is disinfected by soaking it in 70% ethanol for 30 seconds, then soaking it in 0.2% mercuric chloride solution for 10-12 minutes, and finally rinsing it with sterile water 3-5 times.

3. The method for tissue culture detoxification of cardamom according to claim 1, characterized in that: The culture conditions for inducing callus formation in step S1 are: dark culture at 25±2℃.

4. The method for tissue culture detoxification of cardamom according to claim 1, characterized in that: In step S3, the differentiation culture conditions are: temperature 25±2℃, 12h of light per day, and alternating light and dark culture with a light intensity of 2200~3200lx.

5. The method for tissue culture detoxification of cardamom according to claim 1, characterized in that: In step S4, the conditions for rooting culture are: temperature 25±2℃, 12h of light per day, and alternating light and dark conditions with a light intensity of 2200~3200lx.