Induction method of illicium verum callus

Abstract

According to the method for inducing the illicium verum callus, the optimal induction method for rapidly growing and loose callus is screened out by exploring the optimal hormone factors and conditions for inducing the illicium verum callus, and key technical support is provided for research of an illicium verum secondary metabolite accumulation mechanism and establishment of a genetic transformation system. The invention relates to mercury bichloride, alcohol, an MS basic culture medium, 6-benzyladenine 6-BA, naphthylacetic acid NAA and zeatin ZT. ZT is one of common cytokinin for tissue culture, has strong bud induction capability, can improve the proliferation efficiency of aseptic seedlings and buds, and provides a large amount of excellent materials for callus induction.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for inducing star anise callus. Background Art

[0002] Star anise (Illicium verumHook.f.) belongs to the genus Illicium of the Magnoliaceae family, also known as star anise. It is an important economic tree species with both medicinal and edible properties in southern my country. It is mainly distributed in western Guangxi, southern Fujian, Yunnan, and Guangdong. The main parts of star anise are fruits and branches and leaves. According to the pharmacopoeia, its fruit is spicy and warm in nature. It enters the liver, kidney, spleen, and stomach meridians, and has the effects of warming yang and dispersing cold, regulating qi and relieving pain. Star anise was first recorded in Wang Yan's "Boji Fang" in the Song Dynasty, calling it "shipped fennel" and used in Jinhua Pills, Muxiang Tongzhen Powder, Xiaoding Chen Pills and other prescriptions. Modern pharmacological studies have shown that star anise mainly contains phenylpropanoids, flavonoids, phenolic acids and other ingredients, and has anti-inflammatory, analgesic, antiviral, and anti-tumor effects.

[0003] Star anise is usually propagated by seeds under natural conditions. Because the seeds contain oil and the seed coat is very thin, it is easy to lose germination ability. At the same time, the offspring of seedlings are very easy to mutate, and it is difficult to ensure the quality of the offspring. Plant tissue culture is carried out under artificial conditions. It uses plant organs, tissues, and cells to induce and cultivate complete plants under suitable conditions. Compared with traditional breeding and propagation methods, it has many incomparable advantages. In addition, the growth conditions of star anise are relatively harsh, and there are certain requirements for air humidity, ambient temperature, sunshine time, soil topography, etc. Due to the harsh growth environment, the output of star anise has not been high worldwide. The influence of factors such as climate, pests and diseases will also cause changes in the annual output of star anise. Specifically, because star anise likes warm and humid climates, if it encounters individual wind and frost years, the actual output and product quality of star anise will be greatly reduced. In recent years, the market demand for star anise has continued to increase, and the price has gradually increased. In addition, wild resources are scarce and artificial management is extensive. Therefore, tissue culture regeneration is an effective means to alleviate the current contradiction between supply and demand. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for inducing star anise callus, by exploring the best hormone factors and conditions for inducing star anise callus, screening out the best induction method for fast-growing, loose callus, and providing key technical support for the study of the accumulation mechanism of star anise secondary metabolites and the establishment of a genetic transformation system. To solve the above technical problems, the present invention provides a method for inducing star anise callus, comprising the following steps:

[0005] (1) Disinfection of explants: First, clean the dirt on the surface of the explants with a detergent solution for 10 minutes, place them in a beaker, cover them with gauze and rinse them with running water for 30 minutes, then move them to an ultra-clean workbench, soak the seeds in 75% v / v alcohol for 30 seconds, rinse them with sterile water for 3-5 times, and then soak them in 0.1% v / v HgCl2 for 8, 10, and 15 minutes respectively, soak them in sterile water for 3-5 times, dry the surface moisture of the seeds with sterile filter paper and then inoculate them, wherein the sterile water is distilled water sterilized by high temperature and high pressure; the explants are stem segments or terminal buds of star anise.

[0006] (2) Obtaining sterile seedlings: The explants obtained in step (1) were inoculated into MS induction medium and cultured at a temperature of 25±2°C and a light intensity of 20 to 30 μmol / (m 2 ·s), and the culture began to germinate for 8 to 15 days under the condition of a light intensity of 12h / d, and then sterile seedlings grew out, wherein the MS induction medium was supplemented with 0.5mg / L 6-benzyladenine 6-BA, 0.5mg / L naphthyl acetate NAA, 0.2mg / L zeatin ZT, 30g / L sucrose, and 5g / L agar, and the pH value of the medium was 5.8;

[0007] (3) Effect of culture medium on callus induction: The sterile buds obtained in step (2) were inoculated into MS basic culture medium and cultured at a temperature of 25±2°C and a light intensity of 20 to 30 μmol / (m 2 ·s), and cultured for 25 to 30 days under the condition of a light intensity of 12 h / d, gradually producing loose callus tissue, wherein 0.5 mg / L 6-benzyladenine 6-BA, 1 mg / L naphthyl acetate NAA, 30 g / L sucrose, and 5 g / L agar are added to the MS basic medium, and the pH value of the medium is 5.8.

[0008] The advantages and beneficial effects of the present invention are:

[0009] 1. By taking star anise seeds, stem segments or terminal buds for callus induction, a large number of star anise callus with good growth can be cultivated in a short time, providing key technical support for the study of the accumulation mechanism of star anise secondary metabolites and the establishment of a genetic transformation system.

[0010] 2. The present invention relates to mercuric chloride, alcohol, MS basic culture medium, 6-benzyladenine 6-BA, naphthyl acetic acid NAA, and zeatin ZT. ZT is one of the commonly used cytokinins in tissue culture, has a strong bud induction ability, can improve the proliferation efficiency of sterile seedlings, and provide a large amount of high-quality materials for callus induction.

[0011] 3. The induction rate of the star anise callus obtained by the culture method of the present invention reaches more than 80% after 40 days of culture. The generated callus grows fast and most of them are light green granular loose callus.

[0012] 4. At present, the research on the tissue culture system of star anise remains at the stage of cluster bud induction, and the problem of star anise callus induction is still unresolved, which seriously limits the efficiency of star anise callus culture and is difficult to meet the needs of large-scale production. It has not been applied to factory production practice so far. The present invention explores the best hormone factors and conditions for star anise callus induction, screens out the best induction method for fast-growing, loose callus, and provides key technical support for the research on the accumulation mechanism of star anise secondary metabolites and the establishment of a genetic transformation system. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a morphological photograph of the callus induced by the present invention at 8 minutes;

[0014] Figure 2 This is a morphological photograph of the callus induced by the present invention at 10 minutes;

[0015] Figure 3 This is a morphological photograph of the callus induced by the present invention at 15 minutes; DETAILED DESCRIPTION

[0016] The present invention is further described in detail below by way of examples. These examples are only used to illustrate the present invention and do not limit the protection scope of the present invention.

[0017] A method for inducing star anise callus tissue comprises the following steps:

[0018] (1) Disinfection of explants: First, use a detergent solution to clean the dirt on the surface of the explant for 10 minutes, place it in a beaker, cover it with gauze and rinse it with running water for 30 minutes, then move it to an ultra-clean workbench, soak the seeds in 75% v / v alcohol for 30 seconds, rinse it with sterile water 3-5 times, and then soak it in 0.1% v / v HgCl2 for 8, 10, and 15 minutes respectively, soak it in sterile water 3-5 times, use sterile filter paper to absorb the moisture on the surface of the seeds and then inoculate them, wherein the sterile water is distilled water sterilized by high temperature and high pressure; the explant is a bud stem segment or terminal bud of star anise.

[0019] (2) Obtaining sterile seedlings: The explants obtained in step (1) were inoculated into MS induction medium and cultured at a temperature of 25±2°C and a light intensity of 20 to 30 μmol / (m 2·s), and the culture began to germinate for 8 to 15 days under the condition of a light intensity of 12h / d, and then sterile seedlings grew out, wherein the MS induction medium was supplemented with 0.5mg / L 6-benzyladenine 6-BA, 0.5mg / L naphthyl acetate NAA, 0.2mg / L zeatin ZT, 30g / L sucrose, and 5g / L agar, and the pH value of the medium was 5.8;

[0020] (3) Effect of culture medium on callus induction: The sterile buds obtained in step (2) were inoculated into MS basic culture medium and cultured at a temperature of 25±2°C and a light intensity of 20 to 30 μmol / (m 2 ·s), and cultured for 25 to 30 days under the condition of a light intensity of 12 h / d, gradually producing loose callus tissue, wherein 0.5 mg / L 6-benzyladenine 6-BA, 1 mg / L naphthyl acetate NAA, 30 g / L sucrose, and 5 g / L agar are added to the MS basic medium, and the pH value of the medium is 5.8.

[0021] The applicant compared the above method with the existing star anise callus induction method, as shown in the following table:

[0022]

[0023] It can be seen from the above table that the callus rate, contamination rate and sterile explant acquisition rate of the existing induction method 1 and the existing induction method 2 at several similar time points are not as good as the induction method described in the present invention, and from the callus morphology, it can be seen that the callus induced by the induction method of the present invention presents the texture of embryonic callus after 10 minutes, grows rapidly and has strong division.

Claims

1. A method for inducing callus of star anise, characterized in that: The following steps are involved: (1) Disinfection of explants: First, use a detergent solution to clean the dirt on the surface of the explant for 10 minutes, place it in a beaker, cover it with gauze and rinse it with running water for 30 minutes, then move it to an ultra-clean workbench, soak the seeds in 75% v / v alcohol for 30 seconds, rinse it with sterile water 3-5 times, and then soak it in 0.1% v / v HgCl2 for 8, 10, and 15 minutes respectively, soak it in sterile water 3-5 times, use sterile filter paper to absorb the moisture on the surface of the seeds and then inoculate them, wherein the sterile water is distilled water sterilized by high temperature and high pressure; the explant is a bud stem segment or terminal bud of star anise. (2) Obtaining sterile seedlings: The explants obtained in step (1) were inoculated into MS induction medium and cultured at a temperature of 25±2°C and a light intensity of 20 to 30 μmol / (m 2 ·s), and the culture began to germinate for 8 to 15 days under the condition of a light intensity of 12h / d, and then sterile seedlings grew out, wherein the MS induction medium was supplemented with 0.5mg / L 6-benzyladenine 6-BA, 0.5mg / L naphthyl acetate NAA, 0.2mg / L zeatin ZT, 30g / L sucrose, and 5g / L agar, and the pH value of the medium was 5.8; (3) Effect of culture medium on callus induction: The sterile buds obtained in step (2) were inoculated into MS basic culture medium and cultured at a temperature of 25±2°C and a light intensity of 20 to 30 μmol / (m 2 ·s), and cultured for 25 to 30 days under the condition of a light intensity of 12 h / d, gradually producing loose callus tissue, wherein 0.5 mg / L 6-benzyladenine 6-BA, 1 mg / L naphthyl acetate NAA, 30 g / L sucrose, and 5 g / L agar are added to the MS basic medium, and the pH value of the medium is 5.8.

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