Method for detoxifying turmeric by combining indole-3-methanol with stem tip culture

Abstract

The invention discloses a rhizoma curcumae longae detoxification method by combining indole-3-methanol with stem tip culture, and belongs to the technical field of plant tissue culture detoxification seedling culture. The turmeric detoxification method comprises the following steps: (1) taking turmeric rhizomes as explants, and carrying out subculture to obtain turmeric tissue culture seedlings; (2) culturing the turmeric tissue culture seedling on an indole-3-methanol exogenous treatment culture medium to obtain a mature plant; and (3) cutting a stem tip from the mature plant, and culturing on a stem tip induced differentiation culture medium to obtain the turmeric virus-free tissue culture seedling. The turmeric detoxification tissue culture seedlings are cultured by applying the turmeric detoxification method combining indole-3-methanol exogenous treatment with one-time stem tip culture, the technical difficulty is low, the cost is low, the culture period is short, the regenerated plants are high in detoxification rate and strong in growth vigor, and a technical support is provided for large-scale production of the turmeric detoxification tissue culture seedlings.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture detoxification and seedling raising, in particular to a turmeric detoxification method combining indole-3-carbinol with stem tip culture. Background Art

[0002] Turmeric (Curcuma longa L.) is a plant of the genus Curcuma (Curcuma L.) of the Zingiberaceae family. Turmeric is mainly propagated by rhizomes. Turmeric is an important vegetable, spice and medicinal plant. The Compendium of Materia Medica states that turmeric can resist liver damage, lower blood pressure and blood lipids, and relieve pain, and has great medicinal value. Curcumin, a natural dual-purpose drug and food substance extracted from turmeric, can regulate human physiological functions and has a significant inhibitory effect on tumor cells. Turmeric and curcumin are listed as food additives by the World Health Organization and the Food and Agriculture Organization of the United Nations.

[0003] The main cultivation method of turmeric plants is rhizome propagation. Rhizome propagation belongs to the type of asexual reproduction, which has the advantages of maintaining the excellent genetic characteristics of the parents, rapid proliferation of plant numbers, and high stability in yield and quality. However, during long-term asexual reproduction, viruses can be transmitted through the vascular bundles and accumulated in the plant from generation to generation, leading to serious degeneration of the species. This degradation manifests itself in problems such as suppressed crop growth, morphological distortion, reduced yield and deterioration of quality. Turmeric is susceptible to cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV), which can cause mosaic, yellowing, reduced yield and other symptoms, significantly affecting the yield and quality of turmeric. It is of great significance to adopt effective measures to prevent and control turmeric virus diseases.

[0004] In order to reduce the huge harm caused by viruses to crop production, cutting off the spread of viruses from the source of seedlings and realizing the cultivation of turmeric virus-free seedlings is the only way for the rapid development of the turmeric industry. At present, virus-free seedlings are mainly obtained through biotechnology methods such as stem tip culture, heat treatment detoxification, anther culture, micrografting, callus culture and ultra-low temperature therapy. Among them, the stem 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. Therefore, improving the turmeric stem tip detoxification method and improving the detoxification efficiency can provide new directions and technical support for the large-scale production of turmeric detoxified tissue culture seedlings. Summary of the invention

[0005] The purpose of the present invention is to provide a turmeric detoxification method combining indole-3-methanol with shoot tip culture to solve the problems existing in the above-mentioned prior art. The present invention uses an indole-3-methanol exogenous treatment combined with a shoot tip culture turmeric detoxification method to culture turmeric detoxification tissue culture seedlings, which has low technical difficulty, low cost, short culture cycle, high detoxification rate of regenerated plants, strong growth, and provides technical support for the large-scale production of turmeric detoxification tissue culture seedlings.

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

[0007] The present invention provides a turmeric detoxification method combining indole-3-carbinol with shoot tip culture, comprising the following steps:

[0008] (1) taking turmeric rhizomes as explants and subculturing them to obtain turmeric tissue culture seedlings;

[0009] (2) culturing the turmeric tissue culture seedlings on an indole-3-methanol exogenously treated culture medium to obtain mature plants;

[0010] (3) cutting stem tips from the mature plants and culturing them on a stem tip induction differentiation medium to obtain turmeric virus-free tissue culture seedlings;

[0011] The indole-3-carbinol exogenous treatment medium is: MS+0.3-0.5 mg / L NAA+1.5-2.5 mg / L 6-BA+25-35 g / L sucrose+5-15 g / L agar+0.4-0.5 mg / L indole-3-carbinol, pH=5.5-6.0;

[0012] In step (3), the size of the stem tip is 0.15-0.3 mm.

[0013] Optionally, in step (1), the explant is disinfected with 2% chloramine T solution, alcohol, and 10% NaClO solution in sequence before being subcultured.

[0014] Furthermore, in step (1), the culture medium for subculture is: MS+0.3-0.5 mg / L NAA+1.5-2.5 mg / L 6-BA+25-35 g / L sucrose+5-15 g / L agar, pH=5.5-6.0.

[0015] Optionally, in step (1), the conditions for subculture are: at a temperature of 25°C-28°C, a light intensity of 2200-2500Lx, 12 hours of illumination per day, and subculture every 30 days.

[0016] Optionally, in step (2), the culture conditions on the indole-3-carbinol exogenous treatment medium are: at a temperature of 25° C.-28° C. and a light intensity of 2200-2500 Lx for 12 hours per day.

[0017] Optionally, in step (2), the culturing time is 40-50 days.

[0018] Furthermore, in step (3), the shoot tip induction differentiation medium is: MS+0.05-0.15 mg / L NAA+1.0-2.0 mg / L 6-BA+25-35 g / L sucrose+5-15 g / L agar, pH=5.5-6.0.

[0019] Optionally, in step (3), the culture conditions are: at a temperature of 25°C-28°C and a light intensity of 2200-2500Lx for 12 hours per day.

[0020] Optionally, in step (3), the culturing time is 35-45 days.

[0021] Optionally, the viruses removed in the turmeric detoxification method include cucumber mosaic virus and tobacco mosaic virus.

[0022] The present invention discloses the following technical effects:

[0023] The invention uses a turmeric detoxification method combining indole-3-methanol exogenous treatment with stem tip culture to culture turmeric detoxification tissue culture seedlings, has low technical difficulty, does not require expensive and precise instruments and equipment, has low cost, requires only one stem tip culture to obtain tissue culture seedlings with a high detoxification rate, and has a short culture cycle. The regenerated plants can obtain a high CMV detoxification rate and a TMV removal rate, can better maintain the excellent properties of the mother plant, and have strong growth potential, thereby providing technical support for the large-scale production of turmeric detoxification tissue culture seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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.

[0025] Figure 1 The electrophoresis diagram of the CMV detoxification effect of turmeric tissue culture seedlings; wherein, M is a marker; lane 1 is the mother plant of turmeric that has not been detoxified; lane 2 is a negative control (water); lanes 3-8 are detoxified turmeric tissue culture seedlings; the band at 100 bp is a primer dimer;

[0026] Figure 2The electrophoresis diagram of TMV detoxification effect of turmeric tissue culture seedlings; wherein, M is a marker; lane 1 is the mother plant of turmeric that has not been detoxified; lane 2 is a negative control (water); lanes 3-8 are detoxified turmeric tissue culture seedlings; the band at 100bp is a primer dimer. DETAILED DESCRIPTION

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] 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.

[0033] The Cas number of indole-3-carbinol used in the following examples of the present invention is 700-06-1.

[0034] The technical solution of the present invention is as follows:

[0035] Turmeric rhizomes without obvious diseases were selected as explants, which were rinsed under running water for 1 hour and then placed on cotton soaked with water to germinate in a light-shading environment; 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 then 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, surface disinfected with 10% NaClO for 15 minutes, rinsed with sterile water for 3-4 times, and the explants were inoculated on a subculture medium of MS+0.5 mg / L NAA+3.0 mg / L 6-BA+30 g / L sucrose+8 g / L agar (pH=5.8) to culture into tissue culture seedlings; the tissue culture seedlings were cultured at a temperature of 25°C-28°C and a light intensity of 2200-2500 Lx for 12 hours per day, and subcultured every 30 days.

[0036] After subculturing, the tissue culture seedlings were cultured on a medium of MS + 0.4 mg / L NAA + 2.0 mg / L 6-BA + 30 g / L sucrose + 8 g / L agar + 0.4 mg / L indole-3-methanol (pH = 5.8) for 40-50 days, at a temperature of 25°C-28°C and a light intensity of 2200-2500 Lx with 12 hours of illumination per day. Take mature plants, cut the stem tips of about 0.15-0.3 mm using a body dissecting microscope in an ultra-clean workbench, and culture them on MS+0.10 mg / L NAA+1.5 mg / L 6-BA+30 g / L sucrose+8 g / L agar (pH=5.8), and culture them at a temperature of 25°C-28°C and a light intensity of 2200-2500 Lx for 12 hours per day; the mature turmeric tissue culture seedlings formed after 35-45 days of stem tip culture are qualified after RT-PCR virus detection, and the virus removal is completed.

[0037] Example 1

[0038] The culture medium formula involved in this embodiment is as follows:

[0039] Turmeric subculture medium: MS + 0.4 mg / L NAA + 2.0 mg / L 6-BA + 30 g / L sucrose + 8 g / L agar (pH = 5.8).

[0040] Curcuma indole-3-carbinol exogenous treatment culture medium: MS + 0.4 mg / L NAA + 2.0 mg / L 6-BA + 30 g / L sucrose + 8 g / L agar + indole-3-carbinol (concentration to be determined) (pH = 5.8).

[0041] Curcuma longa shoot tip induction differentiation medium: MS+0.10 mg / L NAA+1.5 mg / L 6-BA+30 g / L sucrose+8 g / L agar (pH=5.8).

[0042] The turmeric detoxification method of the present embodiment, the specific operation process is as follows:

[0043] (1) Explant selection: Select turmeric rhizomes without obvious diseases as explants, rinse them under running water for 1 hour, place them on cotton soaked with water, and germinate them in a light-shaded environment;

[0044] (2) Explant treatment: Cut the explants with buds to a size of 0.3-0.5 cm, treat them in 2% chloramine T solution for 40 min, soak them in clean water and rinse them clean;

[0045] (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, surface disinfected with 10% NaClO for 15 minutes, and rinsed three times with sterile water. The explants were inoculated on a subculture medium of MS + 0.5 mg / L NAA + 3.0 mg / L 6-BA + 30 g / L sucrose + 8 g / L agar (pH = 5.8) to obtain tissue culture seedlings;

[0046] (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;

[0047] (5) Curcuma longa indole-3-methanol exogenous treatment: After subculture, the tissue culture seedlings were cultured on turmeric indole-3-methanol exogenous treatment medium containing 0, 0.2, 0.4, and 0.6 mg / L indole-3-methanol, respectively, for 45 days. The culture conditions were the same as those in step (4);

[0048] (6) Shoot tip culture: Take the mature plants obtained in step (5) and cut 0.2 mm, 0.5 mm and 1.0 mm stem tips using a dissecting microscope in a clean bench and culture them on a turmeric stem tip differentiation induction medium for 40 days. The culture conditions are the same as those in step (4);

[0049] (7) Regeneration efficiency and virus-free rate detection: The virus-free effect of the turmeric tissue culture seedlings obtained in step (6) was verified, and the effect indicators included the survival rate, regeneration rate and virus-free rate of the shoot tip. The virus-free rate was calculated as follows: (number of virus-free regenerated plants / total number of regenerated plants).

[0050] The viruses to be detected are CMV and TMV. The RT-PCR virus detection method is as follows: RNA is extracted from turmeric tissue culture seedlings. The extraction steps are carried out according to the instructions of the RNA extraction kit. RNA is detected using a micro-ultraviolet spectrophotometer. The RNA concentration is appropriate and the OD 260 / OD 280RNA with a value between 1.8 and 2.2 can be used for cDNA first-strand synthesis, and the specific operation is performed according to the instructions. The detection primers for CMV and TMV were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd., and the specific primer names and sequence information are shown in Table 1. The PCR reaction system is shown in Table 2. The PCR reaction program is: pre-denaturation at 94°C (3min); denaturation at 94°C (50s), annealing at 56°C (50s), and extension at 72°C (1min) for a total of 34 repetitions;

[0051] Extension at 72℃ (10min); storage at 4℃. PCR product detection method: 0.2g agarose, 20.0mL TAE buffer solution mixed, heated to boiling; drop 2.0μL nucleic acid dye. Pour into fixed plate well plate, solidify at room temperature. Spot 6μL per well on sugar gel; 180V constant voltage electrophoresis for 20min; chemical gel imager imaging observation.

[0052] Table 1 Detection primer information of CMV and TMV

[0053]

[0054] Table 2 PCR reaction system

[0055]

[0056]

[0057] Note: The reaction system was prepared at room temperature and the Blend Taq enzyme was placed on ice.

[0058] The results of the detoxification effect verification are shown in Table 3 and Figure 1-Figure 2 .

[0059] Table 3 Survival rate, regeneration rate and virus-free rate of shoot tips

[0060]

[0061] As can be seen from Table 3, compared with the conventional shoot tip culture detoxification method (0 mg / L indole-3-methanol), the use of indole-3-methanol for pretreatment can effectively improve the detoxification rate of CMV and TMV, indicating that the exogenous application of indole-3-methanol combined with shoot tip culture can effectively remove viruses from turmeric tissue culture seedlings, and the smaller the test shoot tip, the higher the detoxification rate. When the indole-3-methanol addition amount is 0.4 mg / L and 0.6 mg / L, when the shoot tip size is 0.2 mm, the detoxification rates of CMV and TMV are high, which are 28% to 30% and 100%, respectively, but under the treatment of 0.4 mg / L indole-3-methanol, the survival rate and regeneration rate of the shoot tip are high. Therefore, the detoxification efficiency of 0.4 mg / L indole-3-methanol treatment combined with 0.2 mm shoot tip culture is the best.

[0062] 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 turmeric by combining indole-3-carbinol with shoot tip culture, characterized in that: The steps include: (1) taking turmeric rhizomes as explants and subculturing them to obtain turmeric tissue culture seedlings; (2) culturing the turmeric tissue culture seedlings on an indole-3-methanol exogenously treated culture medium to obtain mature plants; (3) cutting stem tips from the mature plants and culturing them on a stem tip induction differentiation medium to obtain turmeric virus-free tissue culture seedlings; The indole-3-carbinol exogenous treatment medium is: MS+0.3-0.5 mg / L NAA+1.5-2.5 mg / L 6-BA+25-35 g / L sucrose+5-15 g / L agar+0.4-0.5 mg / L indole-3-carbinol, pH=5.5-6.0; In step (3), the size of the stem tip is 0.15-0.3 mm.

2. The turmeric detoxification method according to claim 1, characterized in that: In step (1), the explants are disinfected with 2% chloramine T solution, alcohol, and 10% NaClO solution in sequence, and then subcultured.

3. The turmeric detoxification method according to claim 1, characterized in that: In step (1), the culture medium for subculture is: MS+0.3-0.5 mg / L NAA+1.5-2.5 mg / L 6-BA+25-35 g / L sucrose+5-15 g / L agar, pH=5.5-6.

0.

4. The turmeric detoxification method according to claim 1, characterized in that: In step (1), the conditions for subculture are: at a temperature of 25°C-28°C, a light intensity of 2200-2500Lx, 12 hours of illumination per day, and subculture every 30 days.

5. The turmeric detoxification method according to claim 1, characterized in that: In step (2), the culture conditions on the indole-3-carbinol exogenous treatment medium are: at a temperature of 25° C.-28° C. and a light intensity of 2200-2500 Lx for 12 hours per day.

6. The turmeric detoxification method according to claim 1, characterized in that: In step (2), the culturing time is 40-50 days.

7. The turmeric detoxification method according to claim 1, characterized in that: In step (3), the shoot tip induction differentiation medium is: MS+0.05-0.15 mg / L NAA+1.0-2.0 mg / L 6-BA+25-35 g / L sucrose+5-15 g / L agar, pH=5.5-6.

0.

8. The turmeric detoxification method according to claim 1, characterized in that: In step (3), the culture conditions are: at a temperature of 25°C-28°C and a light intensity of 2200-2500Lx for 12 hours per day.

9. The turmeric detoxification method according to claim 1, characterized in that: In step (3), the culture time is 35-45 days.

10. The turmeric detoxification method according to claim 1, characterized in that: The viruses removed in the turmeric detoxification method include cucumber mosaic virus and tobacco mosaic virus.

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