阅读说明：本技术 一种胚芽细胞脱分化培养基质及其制备方法 (Embryonic germ cell dedifferentiation culture medium and preparation method thereof ) 是由 顾霆 于 2019-11-22 设计创作，主要内容包括：本发明公开了一种胚芽细胞脱分化培养基质,所述胚芽细胞脱分化培养基质的配方为：MS、改性琼脂6-8g/L、葡萄糖20-26g/L、2,4-二氯苯氧乙酸0.5-1.1mg/L、果糖0.5-1.1mg/L、营养液0.2-0.7mg/L、改性蒙脱土5-15g/L,pH值为5.6-5.8。本发明胚芽细胞脱分化培养基质在常规的基础上通过对琼脂进行改性,使琼脂被钼酸钠溶液改性后能够增强胞脱分化培养基质的营养基能,同时可提高基质的抗菌环境,营养液的添加使培养基质能够提供多种微量元素,从而提高胚芽细胞分化的程度,中草药发酵剂通过营养剂的形式添加,目的使植物在吸收营养时能够持续的处于灭菌的环境。(The invention discloses a germ cell dedifferentiation culture medium, which comprises the following components in percentage by weight: MS, 6-8g/L of modified agar, 20-26g/L of glucose, 0.5-1.1mg/L of 2, 4-dichlorophenoxyacetic acid, 0.5-1.1mg/L of fructose, 0.2-0.7mg/L of nutrient solution, 5-15g/L of modified montmorillonite, and pH value of 5.6-5.8. According to the germ cell dedifferentiation culture medium, agar is modified by a sodium molybdate solution on a conventional basis, so that the nutrient medium energy of the germ cell dedifferentiation culture medium can be enhanced, the antibacterial environment of the medium can be improved, the culture medium can provide various trace elements by adding a nutrient solution, the differentiation degree of the germ cells is improved, and a Chinese herbal medicine leavening agent is added in the form of a nutrient, so that plants can be continuously in a sterilized environment when absorbing nutrition.)

1. The culture medium for the dedifferentiation of the germ cells is characterized by comprising the following components in percentage by weight: MS, 6-8g/L of modified agar, 20-26g/L of glucose, 0.5-1.1mg/L of 2, 4-dichlorophenoxyacetic acid, 0.5-1.1mg/L of fructose, 0.2-0.7mg/L of nutrient solution, 5-15g/L of modified montmorillonite, and pH value of 5.6-5.8.

2. The embryonic bud cell dedifferentiation culture medium according to claim 1, wherein the modified agar is prepared by the method comprising: irradiating the agar by ultraviolet for 10-20min, then blending the agar and a sodium molybdate solution for 10-20min at the blending rotation speed of 200-280r/min, filtering after blending, and then drying.

3. The culture medium for dedifferentiating embryonic cells according to claim 2, characterized in that the power of the UV radiation is 20-100W.

4. The culture medium for dedifferentiation of germ cells according to claim 1, wherein the nutrient solution comprises the following raw materials in parts by weight: 1.2 to 1.3 portions of magnesium sulfate, 1.1 to 1.2 portions of sodium sulfate, 0.8 to 1.0 portion of zinc sulfate, 0.8 to 0.9 portion of copper sulfate and 0.2 to 0.5 portion of Chinese herbal medicine leaven.

5. The culture medium for dedifferentiation of germ cells according to claim 4, wherein the nutrient solution comprises the following raw materials in parts by weight: 1.25 parts of magnesium sulfate, 1.15 parts of sodium sulfate, 0.9 part of zinc sulfate, 0.85 part of copper sulfate and 0.35 part of Chinese herbal medicine leaven.

6. The culture medium for the dedifferentiation of germ cells according to claim 5, wherein the Chinese herbal medicine starter culture is prepared by adding honeysuckle, wormwood and dandelion in a boiling pot according to a weight ratio of 3:2:1 to boil, feeding the obtained juice and yeast into a fermentation tank together to perform fermentation treatment, wherein the fermentation temperature is 28-32 ℃, the fermentation time is 1-2 days, and the Chinese herbal medicine starter culture is obtained after the fermentation is finished.

7. The embryonic germ dedifferentiation culture medium according to claim 1, wherein the modified montmorillonite is prepared by adding montmorillonite into deionized water, performing ultrasonic dispersion on the mixture, drying the mixture, grinding the mixture for 1 to 3 times, and performing thermal activation treatment on the ground mixture to obtain the modified montmorillonite.

8. The embryonic cell dedifferentiation culture medium according to claim 7, wherein the heat activation treatment comprises the following specific steps: raising the temperature of montmorillonite to 150 deg.C at a speed of 2-5 deg.C/min, maintaining for 10min, then continuing to raise the temperature to 210 deg.C at a speed of 1 deg.C/min, continuing to maintain for 20-30min, and naturally returning to room temperature after the heat preservation is finished.

9. A method for preparing a culture medium for the dedifferentiation of germ cells according to any one of claims 1 to 8, comprising the following steps:

step one, weighing the following raw materials in parts by weight:

step two, sequentially adding MS, modified agar, glucose, 2, 4-dichlorophenoxyacetic acid, fructose and nutrient solution into a stirrer, starting the stirrer, stirring for 10-20min at the stirring speed of 700-;

and step three, adding the nutrient solution and the modified montmorillonite into the substrate A, continuing stirring at the low rotation speed of 100-200r/min for 2-5h, wherein the stirring temperature is 35-45 ℃, and after the stirring is finished, obtaining the germ cell dedifferentiation culture substrate.

Technical Field

The invention relates to the technical field of germ cells, in particular to a germ cell dedifferentiation culture medium and a preparation method thereof.

Background

Soybean is one of the important grain crops in China, has been cultivated for five thousand years, is called Shushu in ancient times, and is a crop with seeds containing rich plant protein, wherein northeast China is the main production area. Soybeans are most commonly used for making various bean products, extracting soybean oil, brewing soy sauce, and extracting proteins. Okara or soybeans ground into meal are also commonly used in livestock feed. The beans are commonly called soybeans. Glycine of Leguminosae family belongs to annual herb, and is 30-90 cm high. Soybean pods are hypertrophic, slightly curved, drooping, yellowish green, and densely covered with brownish yellow and hairy hair; 2-5 seeds, oval, nearly spherical, smooth seed coat, light green, yellow, brown and black, etc. The flowering period is 6-7 months, and the fruit period is 7-9 months.

The soybean germ cells need to use a culture medium in the dedifferentiation process, and the existing culture medium can provide nutrient substances in the dedifferentiation process of the germ cells, but has no antibacterial and sterilizing capabilities and easily influences the dedifferentiation effect of the germ cells.

Disclosure of Invention

The present invention aims to provide a culture medium for dedifferentiation of germ cells and a preparation method thereof, so as to solve the problems in the background art.

In order to achieve the purpose, the invention provides the following technical scheme:

the embryonic cell dedifferentiation culture medium comprises the following components in percentage by weight: MS, 6-8g/L of modified agar, 20-26g/L of glucose, 0.5-1.1mg/L of 2, 4-dichlorophenoxyacetic acid, 0.5-1.1mg/L of fructose, 0.2-0.7mg/L of nutrient solution, 5-15g/L of modified montmorillonite, and pH value of 5.6-5.8.

Preferably, the preparation method of the modified agar comprises the following steps: irradiating the agar by ultraviolet for 10-20min, then blending the agar and a sodium molybdate solution for 10-20min at the blending rotation speed of 200-280r/min, filtering after blending, and then drying.

Preferably, the power of the ultraviolet irradiation is 20-100W.

Preferably, the nutrient solution comprises the following raw materials in parts by weight: 1.2 to 1.3 portions of magnesium sulfate, 1.1 to 1.2 portions of sodium sulfate, 0.8 to 1.0 portion of zinc sulfate, 0.8 to 0.9 portion of copper sulfate and 0.2 to 0.5 portion of Chinese herbal medicine leaven.

Preferably, the nutrient solution comprises the following raw materials in parts by weight: 1.25 parts of magnesium sulfate, 1.15 parts of sodium sulfate, 0.9 part of zinc sulfate, 0.85 part of copper sulfate and 0.35 part of Chinese herbal medicine leaven.

Preferably, the preparation method of the Chinese herbal medicine starter comprises the steps of adding honeysuckle, wormwood and dandelion into a boiling pot according to the weight ratio of 3:2:1, boiling to obtain juice, feeding the juice and yeast into a fermentation tank together for fermentation treatment, wherein the fermentation temperature is 28-32 ℃, the fermentation time is 1-2 days, and after the fermentation is finished, obtaining the Chinese herbal medicine starter.

Preferably, the preparation method of the modified montmorillonite comprises the steps of adding montmorillonite into deionized water, carrying out dust feeding and ultrasonic dispersion, then drying, grinding for 1-3 times, and then carrying out thermal activation treatment to obtain the modified montmorillonite.

Preferably, the heat activation treatment comprises the following specific steps: raising the temperature of montmorillonite to 150 deg.C at a speed of 2-5 deg.C/min, maintaining for 10min, then continuing to raise the temperature to 210 deg.C at a speed of 1 deg.C/min, continuing to maintain for 20-30min, and naturally returning to room temperature after the heat preservation is finished.

The invention also provides a method for preparing the culture medium for the germ cell dedifferentiation, which comprises the following steps:

step one, weighing the following raw materials in parts by weight:

step two, sequentially adding MS, modified agar, glucose, 2, 4-dichlorophenoxyacetic acid, fructose and nutrient solution into a stirrer, starting the stirrer, stirring for 10-20min at the stirring speed of 700-;

and step three, adding the nutrient solution and the modified montmorillonite into the substrate A, continuing stirring at the low rotation speed of 100-200r/min for 2-5h, wherein the stirring temperature is 35-45 ℃, and after the stirring is finished, obtaining the germ cell dedifferentiation culture substrate.

Compared with the prior art, the invention has the following beneficial effects:

(1) according to the germ cell dedifferentiation culture medium, agar is modified by a sodium molybdate solution on a conventional basis, so that the nutrient medium energy of the germ cell dedifferentiation culture medium can be enhanced, the antibacterial environment of the medium can be improved, the culture medium can provide various trace elements by adding a nutrient solution, the differentiation degree of the germ cells is improved, and a Chinese herbal medicine leavening agent is added in the form of a nutrient agent, so that plants can be continuously in the sterile environment when absorbing nutrition, and the antibacterial ability is enhanced.

(2) The montmorillonite dispersion liquid can play an antibacterial function, has strong activity after modification treatment, and can improve the dispersion capacity of montmorillonite in matrix, thereby improving the antibacterial capacity.

(3) The culture medium of the embodiment 3 of the invention has strong antibacterial capacity to germ cells, the infection rate of the germ cells of the embodiment 3 is 0.11 percent, while the infection rate of the germ cells of the comparison example 2 is 1.23 percent, and the embodiment 3 has 1.22 percent of reduction compared with the comparison example 2, thereby having obvious reduction effect.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below with reference to specific embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.