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CN-121991879-A - Mint stem cell, isolated culture method and identification method

CN121991879ACN 121991879 ACN121991879 ACN 121991879ACN-121991879-A

Abstract

The invention provides a mint stem cell, a separation culture method and an identification method, wherein the separation culture method of the mint stem cell comprises the following steps: taking stem tip top meristem of peppermint as an explant, performing induction culture on the stem tip top meristem to generate embryogenic callus, and performing suspension culture on the embryogenic callus to obtain a mint stem cell suspension system. Solves the technical problems of unstable yield and quality of active ingredients of mint caused by the dependence on traditional agricultural planting in the prior art and lack of a specific and high-activity stem cell culture system aiming at mint. The method achieves the goal of obtaining high quality stem cells from a source through a series of carefully designed steps.

Inventors

  • LIU YUANZHU
  • XIAO LEYI
  • ZHAO TIANTIAN
  • RONG YUPING
  • SUN CHENG
  • WU XIAOYA
  • WANG CHAO
  • LI RUI

Assignees

  • 青岛言鼎生物医疗科技有限公司

Dates

Publication Date
20260508
Application Date
20260326

Claims (10)

  1. 1. A method for separating and culturing mint stem cells is characterized by comprising the following steps of taking stem tip top meristem of mint as an explant, carrying out induction culture on the stem tip top meristem to generate embryogenic callus, and carrying out suspension culture on the embryogenic callus to obtain a mint stem cell suspension system.
  2. 2. The isolated culture method of mint stem cells according to claim 1, wherein the diameter of the shoot apical meristem is 0.1-0.3 mm.
  3. 3. The isolated culture method of mint stem cells according to claim 1, wherein the stem tip apical meristem is subjected to surface sterilization treatment before induction culture.
  4. 4. The method according to claim 3, wherein the surface sterilization treatment comprises immersing the mint stem cells in 75% ethanol for 5-10 minutes and immersing the mint stem cells in 2-5% sodium hypochlorite solution for 8-15 minutes.
  5. 5. The isolated culture method of mint stem cells according to claim 1, wherein the stem tip apical meristem is inoculated on a solid induction culture medium for dark culture, wherein the solid induction culture medium is based on B5 or MS culture medium, the concentration of auxin 2, 4-dichlorophenoxyacetic acid is added, the concentration of cytokinin 6-benzylaminopurine is 0.1-0.5 mg/L, sucrose is also added, the concentration of sucrose is 20-30 g/L, the concentration of agar powder is 6-8 g/L, and the pH of the culture medium used for induction culture is 5.6-5.8.
  6. 6. The isolated culture method of mint stem cells according to claim 5, wherein the dark culture condition is that the temperature is 23+/-2 ℃ and the culture time is 20-30 days.
  7. 7. The isolated culture method of mint stem cells according to claim 1, wherein the culture medium for suspension culture is a liquid secondary culture medium, the liquid secondary culture medium is based on a B5 culture medium, and 2, 4-dichlorophenoxyacetic acid 0.5-2.0 mg/L, 6-benzylaminopurine 0.05-0.2 mg/L and sucrose 30 g/L are added, and the pH is adjusted to 5.6-5.8.
  8. 8. The method for isolated culture of mint stem cells according to claim 1, wherein the suspension culture is carried out at a temperature of 23+/-2 ℃ at a rotation speed of 100-120 rpm and continuously carried out for 3-5 times every 10-14 days under dark or dim light conditions.
  9. 9. A mint stem cell obtained by the isolated culture method of mint stem cells according to any one of claims 1-8.
  10. 10. A method of identifying mint stem cells according to claim 9, wherein the expression levels of stem cell marker genes WUS and CLV3 in mint stem cell suspension lines are detected and compared with the expression levels of WUS and CLV3 in mint mature leaf cells, and the mint stem cell suspension lines are such that the expression levels of WUS and CLV3 in the suspension cells are higher than in mature leaf tissue.

Description

Mint stem cell, isolated culture method and identification method Technical Field The invention relates to the technical field of plant tissue culture, in particular to mint stem cells, a separation culture method and an identification method. Background Peppermint is a perennial herb of the genus Mentha of the family Labiatae, which contains a variety of active ingredients, wherein menthol, menthone, flavonoids are widely used in the food additive, pharmaceutical, oral care, cosmetic and fragrance industries. At present, the production and extraction of the mint active ingredient mainly depend on traditional agricultural planting and chemical extraction, and the problems of strong dependence, long production period, easiness in environmental and seasonal influence, large fluctuation of the content of the active ingredient and the like exist, and the mint active ingredient has obvious blank in the cell level, especially in the stem cell research field. The plant stem cells are cells with undifferentiated characteristics and unlimited division capacity, can stably retain plant genetic information, can efficiently extract contained active ingredients through in vitro culture, and are important ways for solving the problems of plant resource shortage and unstable raw material quality. The existing plant stem cell culture technology usually takes differentiated root, stem and leaf tissues as explants, and stem cells are obtained through dedifferentiation of callus, so that the problems of weak cell division capability, easy degeneration variation, long culture period and the like exist. At present, related researches on mint mainly concentrate on cultivation, breeding, chemical component analysis, essential oil extraction process and the like, and researches on the field of mint stem cell culture are freshly reported. The existing plant stem cell culture technology is developed in medicinal plants such as ginseng, yew, saussurea involucrata and the like, and the culture system has species specificity and cannot be directly applied to mint. Further improvements and developments are therefore needed. Disclosure of Invention Aiming at overcoming various defects in the prior art, in order to solve the problems, a mint stem cell, a separation culture method and an identification method are provided, and the following technical scheme is provided: A method for separating and culturing mint stem cells comprises the following steps of taking stem tip top meristem of mint as an explant, carrying out induction culture on the stem tip top meristem to generate embryogenic callus, and carrying out suspension culture on the embryogenic callus to obtain a mint stem cell suspension system. Further, the diameter of the shoot tip apical meristem is 0.1-0.3 mm. Furthermore, the surface sterilization treatment is carried out before the induction culture of the stem tip apical meristem. Further, the surface sterilization treatment comprises the steps of firstly soaking the surface of the substrate in ethanol with the volume concentration of 75% for 5-10 minutes, and then soaking the surface of the substrate in sodium hypochlorite solution with the effective chlorine content of 2-5% for 8-15 minutes. Further, the stem tip top meristem is inoculated on a solid induction culture medium for dark culture, wherein the solid induction culture medium is based on B5 or MS culture medium, the concentration of auxin 2, 4-dichlorophenoxyacetic acid is 1.0-3.0 mg/L, cytokinin 6-benzylaminopurine is 0.1-0.5 mg/L, sucrose is also added 20-30 g/L, agar powder is 6-8 g/L, and the pH of the culture medium adopted in induction culture is 5.6-5.8. Further, the condition of the dark culture is that the temperature is 23+/-2 ℃ and the culture time is 20-30 days. Further, the culture medium for suspension culture is a liquid secondary culture medium, the liquid secondary culture medium is based on a B5 culture medium, and 0.5-2.0 mg/L of 2, 4-dichlorophenoxyacetic acid, 0.05-0.2 mg/L of 6-benzylaminopurine and 30 g/L of sucrose are added, and the pH is adjusted to 5.6-5.8. Further, the suspension culture condition is that the temperature is 23+/-2 ℃ and the rotating speed is 100-120 rpm, and the suspension culture is continuously carried out for 3-5 times every 10-14 days under dark or dim light conditions. In addition, the application also provides a mint stem cell suspension system obtained by the method. The application also provides an identification method of the mint stem cells, which is used for detecting and comparing the expression levels of stem cell marker genes WUS and CLV3 in mint stem cell suspension lines and mint mature leaf cells, wherein the expression levels of the WUS and CLV3 genes in the mint stem cell suspension lines are higher than those of mature leaf tissues, and then the mint stem cell suspension lines are stem cells. The identification method of the mint stem cells is characterized by detecting and comparing the expression le