CN102763591B - Atrac tylodes macrocephala koidz conservation method and re-cultivating method - Google Patents

Abstract

The invention discloses a preservation method and a recultivation method of Atractylodes macrocephala. The preservation method provided by the present invention comprises the following steps: (1) taking the shoot tips of Atractylodes macrocephala tissue culture seedlings, and pre-cultivating them in a pre-culture solution; the pre-culture solution is MS liquid medium containing 0.3mol/L sucrose; ( 2) Take the shoot tips that have completed step (1), and perform pretreatment in the pretreatment solution; the pretreatment solution is MS liquid medium containing 2.0mol/L glycerol and 0.4mol/L sucrose; (3) Take the completed The stem tip of step (2) is treated in a vitrification protective agent; the preparation method of the vitrification protective agent is: 3.26mol glycerin, 2.42mol ethylene glycol, 1.9mol dimethyl sulfoxide and 0.4mol sucrose and Mix the MS liquid medium to 1L; (4) Put the shoot tips that have completed step (3) into liquid nitrogen for storage. The method provided by the invention has the advantages of low cost, simple operation and high plant regeneration rate, and is an effective way for in vitro preservation of Atractylodes macrocephala germplasm resources.

Description

A kind of preservation method and recultivation method of Atractylodes macrocephala

technical field

The invention relates to a preservation method and a recultivation method of Atractylodes macrocephala.

Background technique

Atractylodes macrocephala Koidz. is a plant of the genus Atractylodes in the family Asteraceae. Its rhizome is used as medicine and is an important medicinal plant. Due to the high market demand, long-term human excavation and environmental damage, the wild resources of Atractylodes macrocephala are scarce. At present, artificial cultivation is mainly used to ensure that the resources meet the market demand. Introduced and cultivated in most places in my country, Yuqian in Zhejiang and southern Anhui mountainous areas are used as authentic places.

At present, Atractylodes macrocephala seeds or tuberous rhizomes are often used for preservation and propagation in the preservation garden or seed propagation garden through field planting. The use of seeds for propagation is prone to variation, and the phenomenon of species degeneration and germplasm mixing may occur; it is difficult to obtain a large number of germplasm resources in a short period of time for root tuber propagation. Moreover, preservation by field planting not only occupies arable land, but also requires a lot of manpower and material resources for cultivation and management. Natural disasters such as drought, flood, low temperature, heat damage, pests and diseases may also lead to loss of germplasm resources. save. In order to preserve the germplasm resources, research on in vitro tissue culture and rapid propagation of Atractylodes macrocephala has been carried out in China, and tissue culture media and culture conditions suitable for germination, multiplication, rooting, etc. have been obtained, and a large number of tissue culture seedlings can be obtained in a short period of time. However, in tissue subculture, multiple subcultures may also lead to genetic variation, and at the same time, regular subculture requires manpower and material resources. Therefore, it is still necessary to find the most suitable long-term preservation method.

Contents of the invention

The object of the present invention is to provide a preservation method and a recultivation method of Atractylodes macrocephala.

The invention provides a method for preserving Atractylodes macrocephala, which comprises the following steps in turn:

(1) Take the shoot tips of Atractylodes macrocephala tissue culture seedlings, and pre-culture them in the pre-culture solution; the pre-culture solution is MS liquid medium containing 0.3mol/L sucrose;

(2) Take the shoot tips that have completed step (1), and perform pretreatment in the pretreatment solution; the pretreatment solution is MS liquid medium containing 2.0mol/L glycerol and 0.4mol/L sucrose;

(3) Take the stem tips that have completed step (2), and process them in a vitrification protective agent; the preparation method of the vitrification protective agent is: mix 3.26mol glycerin, 2.42mol ethylene glycol, 1.9mol dimethyl Mix sulfone and 0.4mol sucrose with MS liquid medium to 1L;

(4) Put the shoot tips that have completed step (3) into liquid nitrogen for storage.

In step (1), the stem tip may be a stem tip of 1.5-2mm.

The tissue-cultured seedlings of Atractylodes Rhizoma Atractylodes Rhizoma can be specifically 2-month-old tissue-cultured seedlings of Atractylodes Rhizoma.

In step (1), the pre-cultivation condition may be: dark culture at 25° C. for 1-4 days (specifically, 3 days).

In step (2), the pretreatment may be: immerse the shoot tip after step (1) in the pretreatment solution at room temperature for 30 minutes (shaking treatment is preferred). Specifically, the room temperature may be 20-30°C (such as 25°C).

In step (3), the treatment may be: immersing the shoot tip completed in step (2) in the vitrification protectant at 0° C. for 60 minutes.

The step (4) includes the following steps: after the step (3) is completed, the vitrification agent containing the shoot tip is added dropwise on the aluminum foil, and then the aluminum foil is transferred to a freezing tube filled with liquid nitrogen, and then the The cryopreservation tubes were put into liquid nitrogen for preservation. Each drop of the vitrification agent containing the shoot tip can be specifically 16 microliters. Each drop of the vitrification agent containing the shoot tips may specifically contain 5 of the shoot tips.

The present invention also protects a method for re-cultivating Atractylodes Rhizome preserved by any of the above methods, comprising the following steps: re-cultivating Atractylodes Rhizome preserved by any of the above methods to obtain Atractylodes Rhizome plants.

The re-cultivation includes the following steps: the thawed Atractylodes macrocephala shoot tip is first cultured in the MS semi-solid medium containing 0.3mol/L sucrose in dark for 1 day, and then cultured in the medium containing 0.3mg/L 6-BA, 0.02mg/L NAA and 0.1mg/L GA ₃ MS solid medium were cultured in dark for 5-7 days (shoot tips turned green and showed signs of sprouting), and then transferred to MS solid medium for normal light culture.

The re-culturing method also includes the step of washing the Atractylodes Rhizoma Rhizoma Rhizoma Rhizome preserved by any of the above-mentioned methods with MS liquid medium containing 1.2 mol/L sucrose before re-culturing.

The effect of the pre-cultivation: it can reduce the free water content of the shoot tip tissue cells, increase the content of protective substances such as soluble sugar, thereby improving the frost resistance of the shoot tip, reducing or avoiding freezing damage, and improving the plant survival rate.

The effect of the pretreatment: further reduce the water content of the shoot tip tissue cells, so as to avoid rapid dehydration in the higher concentration vitrification protective agent.

The effect of the vitrifying agent: add the vitrifying agent to make the stem tip tissue cells reach a vitrified state during the cooling process, so as to avoid the formation of ice crystals in the cells to damage the cell membrane.

The invention provides a cryopreservation method of Atractylodes Rhizoma Rhizome and a method for recultivating the genes stored in the cryopreservation to finally obtain Atractylodes Rhizome plants. By adopting the method provided by the invention for preservation and recultivation, the plant regeneration rate can reach more than 60%. The method provided by the invention is low in cost, easy to operate, and has high plant regeneration rate, avoiding the risk of germplasm variation or destruction caused by natural disasters in field preservation, and the risk of genetic trait variation or pollution caused by multiple subcultures in tissue culture preservation. , is an effective way to preserve Atractylodes macrocephala germplasm resources in vitro.

Description of drawings

Figure 1 is a photograph of the tissue cultured seedlings.

Figure 2 is a photo of the shoot tip.

Fig. 3 is the aluminum foil strip after dripping the PVS2 vitrification protective agent containing the stem tip.

Fig. 4 is a photo of 7 days after being transferred to MS solid medium and cultured under normal light.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent companies unless otherwise specified. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

6-BA is 6-(benzylamino)purine, and its structural formula is shown in formula (I).

NAA, that is, 1-naphthalene acetic acid, its structural formula is shown in formula (II).

GA ₃ , namely gibberellin A ₃ , the chemical name is 2,4a,7-trihydroxy-1-methyl-8-methylene gibberellin-3-ene-1,10-dicarboxylic acid-1,4a -Lactone, the structural formula is shown in formula (Ⅲ).

IBA, that is, 3-indole-butyric acid, its structural formula is shown in formula (IV).

MS liquid medium: sucrose 30g, 10×macroelement 100ml (10×macroelement formula: KNO ₃ 19.00g, NH ₄ NO ₃ 16.5g, MgSO ₄ 7H ₂ O 3.7g, KH ₂ PO ₄ 1.7g, CaCl ₂ ·2H ₂ O 4.4g, add water to make up to 1L), 100×trace element 10ml (100×trace element formula: MnSO ₄ ·H ₂ O 1.69g, ZnSO ₄ ·7H ₂ O 0.86g, H ₃ BO ₃ 0.62g , KI 0.085g, NaMoO ₄ 2H ₂ O 0.025g, CuSO ₄ 5H ₂ O 0.0025g, CoCl ₂ 6H ₂ O0.0025g, add distilled water to 1L), 100× iron salt 10ml (100× iron salt Recipe: FeSO ₄ 7H ₂ O 1.39g, Na ₂ -EDTA 1.86g, add distilled water to 500ml), 200×organic matter 5ml (200×organic matter formula: glycine 0.2g, B ₁ 0.04g, B ₆ 0.05g, Niacin 0.05g, add distilled water to 500ml), 100× inositol 10ml (100× inositol formula: inositol 5g, add distilled water to 500ml), pH 5.8, distilled water to 1L.

MS solid medium: the formula is the same as MS liquid medium, after adding 8.5g of agar, the volume is adjusted to 1L, pH5.8.

MS semi-solid medium: the formula is the same as MS liquid medium, after adding 5-7g of agar, the volume is adjusted to 1L, pH5.8.

Atractylodes macrophylla tissue culture seedlings: The public can obtain them from the Institute of Crop Science, Chinese Academy of Agricultural Sciences; references: Liang Xiaomin, Zhu Zhaohui, Hu Ximao, Research on in vitro rapid propagation of Atractylodes macrocephala shoot tips, Anhui Agricultural Sciences, 2009, 37 (35): 17356 -17357.

Embodiment 1, the cryopreservation of Atractylodes Rhizome

1. Reagent formulations

Pre-culture medium: MS liquid medium containing 0.3mol/L sucrose.

Pretreatment solution: MS liquid medium containing 2.0mol/L glycerol and 0.4mol/L sucrose.

Vitrification protectant: 3.26mol glycerol, 2.42mol ethylene glycol, 1.9mol dimethyl sulfoxide and 0.4mol sucrose were thoroughly mixed with MS liquid medium to 1L.

2. Ultra-low temperature preservation of Atractylodes macrocephala

Take 2-month-old Atractylodes macrocephala tissue-cultured seedlings, peel off the outer leaves layer by layer until the remaining 1-2 leaf primordium surrounds the shoot apex meristem, and cut out 1.5-2mm shoot tips (a total of 100 shoot tips were obtained), Soak in the pre-culture solution for 3 days in the dark at 25°C, then soak in the pretreatment solution for 30 minutes with shaking at 25°C (normal light), then soak in the vitrification protectant for 60 minutes at 0°C (normal light), and then in each Add 4 drops of PVS2 vitrification agent containing shoot tips (16 microliters per drop, which contains 5 shoot tips) on a sterile aluminum foil strip of 5 mm × 20 mm, and finally transfer the aluminum foil strip to a freezer filled with liquid nitrogen. into the tube and stored in liquid nitrogen.

See Figure 1 for photos of 2-month-old Atractylodes macrocephala tissue-cultured seedlings. A photo of the cut shoot tip is shown in Figure 2. The aluminum foil strip after dripping the PVS2 vitrification protective agent containing the shoot tip is shown in Figure 3.

3. Statistics on recultivation, survival rate and regeneration rate

1. Take out the cryopreservation tube in liquid nitrogen, then take out the aluminum foil strip in the cryopreservation tube, wash it twice with MS liquid medium containing 1.2mol/L sucrose at 25°C (10min each time), and take the shoot tip.

2. The shoot tip of step 1 was first cultured in dark for 1 day in MS semi _- solid medium containing 0.3mol/L sucrose; Culture in MS solid medium in dark for 5-7 days (the shoot tips can be observed to turn green and have signs of sprouting); then transfer to MS solid medium for normal light culture, and normal light growth can be observed after 15 days of normal light culture For surviving plants, the plants that grew leaves and roots when cultured under normal light for 30 days were regarded as regenerated plants.

See Figure 4 for photos after being transferred to MS solid medium and cultured under normal light for 7 days.

Plant survival rate = number of surviving plants ÷ number of shoot tips × 100%. Where the number of shoot tips = 100.

Plant regeneration rate = number of regenerated plants ÷ number of shoot tips × 100%. Where the number of shoot tips = 100.

Carry out three repeated tests, the plant survival rate of each test is respectively 66%, 93%, 67%, and the average value of the plant survival rate is 75.5%; The plant regeneration rate of each test is respectively 60%, 67% and 60%. %, that is, the average plant regeneration rate was 62.2%.

Embodiment 2, the cryopreservation of Atractylodes macrocephala

1. Reagent formulations

All the same as Step 1 of Example 1.

2. Ultra-low temperature preservation of Atractylodes macrocephala

The time of dark cultivation at 25° C. in the pre-culture solution is 1 day, 2 days, 3 days, 4 days or 5 days respectively, and the others are the same as step 2 of Example 1.

3. Statistics on recultivation, survival rate and regeneration rate

Same as Step 3 of Example 1.

The experiments were repeated three times, and the results were averaged.

When cultured in the dark at 25°C for 1 day in the pre-culture solution, the average plant survival rate of the three experiments was 69.8%. The average plant regeneration rate of the three experiments was 54.9%.

When cultured in the dark at 25°C for 2 days in the pre-culture solution, the average plant survival rate of the three experiments was 66.0%. The average plant regeneration rate of the three experiments was 52.6%.

When cultured in the dark at 25°C for 3 days in the pre-culture solution, the average plant survival rate of the three experiments was 75.6%. The average plant regeneration rate of the three experiments was 62.2%.

When cultured in the dark at 25°C for 4 days in the pre-culture solution, the average plant survival rate of the three experiments was 57.1%. The average plant regeneration rate of the three experiments was 46.5%.

When cultured in the dark at 25°C for 5 days in the pre-culture solution, the average plant survival rate of the three experiments was 34.4%. The average plant regeneration rate of the three experiments was 23.3%.

Claims (3)

1. A method for preserving Atractylodes macrocephala, comprising the steps successively: (1) Take the shoot tips of Atractylodes macrocephala tissue culture seedlings, and pre-culture them in the pre-culture solution; the pre-culture solution is MS liquid medium containing 0.3mol/L sucrose; the conditions of the pre-culture are: 25°C in the dark Cultivate for 1-4 days; the shoot tip is a shoot tip of 1.5-2mm; (2) Take the shoot tips that have completed step (1), and perform pretreatment in the pretreatment solution; the pretreatment solution is MS liquid medium containing 2.0mol/L glycerol and 0.4mol/L sucrose; the pretreatment The method is: soaking the stem tip after completing the step (1) in the pretreatment solution at room temperature for 30 minutes; (3) Take the stem tips that have completed step (2), and process them in a vitrification protective agent; the preparation method of the vitrification protective agent is: mix 3.26mol glycerin, 2.42mol ethylene glycol, 1.9mol dimethyl Mix sulfone and 0.4mol sucrose with MS liquid medium to 1 L; the treatment is: soak the shoot tip after completing step (2) in the vitrification agent at 0°C for 60 minutes; (4) Put the shoot tips that have completed step (3) into liquid nitrogen for storage. 2. The method according to claim 1, characterized in that: the step (4) comprises the following steps: after completing the step (3), dripping the vitrification agent containing the stem tip onto the aluminum foil, and then The aluminum foil was transferred to a cryovial filled with liquid nitrogen, and then the cryovial was put into liquid nitrogen for storage. 3. The method for re-cultivating the Atractylodes Rhizome preserved by the method according to claim 1 or 2, comprising the steps of: re-culturing the Atractylodes Rhizome preserved by the method according to claim 1 or 2, to obtain Atractylodes Rhizome plants; The re-cultivation comprises the following steps: the thawed Atractylodes macrocephala shoot tip is first cultured in the MS semi-solid medium containing 0.3mol/L sucrose in dark for 1 day, and then cultured in the medium containing 0.3mg/L6-BA, 0.02mg/L NAA and 0.1mg/L GA ₃ MS solid medium for dark culture for 5-7 days, and then transferred to MS solid medium for light culture; The method also includes the step of washing the preserved Atractylodes Rhizoma Atractylodes Rhizome with 1.2 mol/L sucrose liquid medium before recultivation.

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