CN110754299A

CN110754299A - Method for bottle cultivation of poria cocos sclerotia

Info

Publication number: CN110754299A
Application number: CN201911219996.4A
Authority: CN
Inventors: 邹娟; 顾磊; 朱娅展; 高佳乐; 彭海钰; 王婷; 胡兴
Original assignee: Huaihua University
Current assignee: Huaihua University
Priority date: 2019-12-03
Filing date: 2019-12-03
Publication date: 2020-02-07
Anticipated expiration: 2039-12-03
Also published as: CN110754299B

Abstract

The invention discloses a method for bottle cultivation of poria cocos sclerotia. The invention uses bottle cultivation tuckahoe sclerotium and uses sawdust (can be fir sawdust and miscellaneous sawdust) as the growing carrier of the tuckahoe sclerotium, realizes the indoor cultivation technology of the tuckahoe sclerotium without using polyethylene bags and pine, provides a method for the research of the formation of the tuckahoe sclerotium, provides a technology for the production of the sclerotium, is beneficial to fully utilizing the tailing of various timbers, is environment-friendly and pollution-free, and can bring good ecological effect.

Description

Method for bottle cultivation of poria cocos sclerotia

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for bottle cultivation of poria cocos sclerotia.

Background

At present, methods adopted for cultivating poria sclerotium include a bag cultivation method (a poria bag cultivation method disclosed by CN 101731100A), a segment cultivation method (patents CN110419385A, CN108934761A and the like), raw material cultivation depending on pine groves (a poria raw material cultivation method disclosed by patent CN 105309201A) and the like, the segment cultivation method needs pine wood, not only has high cost, but also can cause the damage of pine forest resources; the raw material cultivation needs the pine pocket remained after the felling. The bag cultivation method can adopt wood chips as a culture medium, so that the problems are solved, but the bag cultivation method has the problems that a fresh poria sclerotium block needs to be inoculated as the primordium of the new sclerotium besides the poria cocos strain, the raw material of the fresh poria sclerotium block is limited, the cultivation steps are complicated, and if the fresh poria sclerotium block is not inoculated as the primordium of the new sclerotium through the experiment of the inventor, the sclerotium formation rate is only about 60 percent, and white pollution exists. In addition, the planting methods all need to be completely buried in soil, and a three-dimensional modern cultivation system is difficult to form.

At present, the research on the forming mechanism of poria cocos sclerotia belongs to the problem of the current research, which is that the external factors inducing the formation of the poria cocos sclerotia are not reported for a while, and the modern three-dimensional bottle cultivation technology is not adapted to appear at present.

Disclosure of Invention

In order to solve the technical problem, the invention discloses a method for bottle cultivation of poria cocos sclerotia. The invention uses bottle cultivation tuckahoe sclerotium and uses wood chips (can be fir chips and miscellaneous wood chips) as the tuckahoe sclerotium growth carrier, realizes the cultivation technology of the tuckahoe sclerotium without using polyethylene bags, tuckahoe sclerotium and pine, is beneficial to fully using the tailing of various woods, reduces the cultivation cost, is environment-friendly and pollution-free, and can bring good ecological effect.

The purpose of the invention is realized by the following technical scheme:

a method for bottle cultivation of sclerotium of Poria cocos comprises the following steps:

step one, culturing tuckahoe strains in a solid culture medium to obtain tuckahoe mycelia;

inoculating the poria cocos mycelia in a poria cocos sclerotium culture medium until the poria cocos mycelia overgrow the poria cocos sclerotium culture medium, and placing the poria cocos sclerotium culture medium in a tissue culture bottle for later use;

sterilizing the sand at high temperature to obtain sterile sand, cooling the sterile sand, placing the cooled sterile sand into a tissue culture bottle, covering a poria cocos sclerotium culture medium with the covering height of 4-6cm, and then placing the poria cocos sclerotium culture medium at 28-30 ℃ for dark culture for 10-15 days to enable poria cocos hyphae in the poria cocos sclerotium culture medium to grow into fine sand by 1-3cm for later use;

and step four, adding soil into the tissue culture bottle, covering the tissue culture bottle on sterile sand, wherein the height of a covered soil layer is 3-5cm, then placing the tissue culture bottle in a condition of 25-35 ℃ for dark culture, supplementing water to the soil on the uppermost layer of the tissue culture bottle every other week, and culturing for more than 60 days until small sclerotia with the particle size of 3-8mm grows out in the poria cocos sclerotia culture medium in the bottle, the epidermis is brown, and the internal tissues are beige white, so that the poria cocos sclerotia is obtained.

In a further improvement, the solid culture medium is a potato dextrose agar culture medium.

In a further improvement, the poria cocos sclerotium culture medium comprises a substrate and water; the matrix comprises 78-80% of sawdust, 15-19% of bran, 1-3% of corn, 1-3% of white granulated sugar, 0.5-1.5% of magnesium sulfate and 0.2-0.5% of calcium sulfate by weight.

In a further improvement, the mass ratio of the matrix to the water is 1: 3; adding the matrix and water into 1/3 of the volume of the tissue culture bottle, sterilizing at 121 deg.C for 2h, and cooling to obtain sterile Poria sclerotium culture medium.

Further improvement, in the third step, the sand is subjected to dry heat sterilization at the temperature of 160-170 ℃ for 2 hours to obtain the sterile sand.

In a further improvement, in the first step, the tuckahoe strain is a tuckahoe strain which is separated from the tuckahoe sclerotium tissue; taking small pieces of tissue of 0.5cm x 0.5cm from sclerotium of Poria cocos by tissue separation method, placing in potato glucose agar culture medium, and culturing at 25-28 deg.C to obtain Poria cocos mycelium for use.

Further improvement, the soil is the soil in the depth of 5-10cm of the masson pine forest.

Drawings

FIG. 1 is a flow chart of the present invention;

FIG. 2 is a graph comparing example 1 with comparative example 4, wherein the left side of the graph is a culture graph of comparative example 4, and the right side of the graph is a culture graph of example 1.

FIGS. 3 and 4 are diagrams of cultured Poria sclerotium of the present invention, which is indicated by arrows.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.

Example 1

A method for culturing sclerotium of Poria cocos by bottle cultivation comprises inducing and culturing sclerotium of Poria cocos with sand and soil, wherein the raw material of the solid culture medium of sclerotium of Poria cocos comprises sawdust (such as pine sawdust, China fir sawdust and miscellaneous sawdust), bran, corn, white sugar, magnesium sulfate, calcium sulfate and water. The addition amount of each raw material is 78-80% of sawdust, 15-19% of bran, 1-3% of corn, 1-3% of white granulated sugar, 0.5-1.5% of magnesium sulfate and 0.2-0.5% of calcium sulfate; sand needs dry heat sterilization; the soil is 5-10cm of the masson pine forest soil layer; the Poria strain is prepared by tissue separating Poria sclerotium;

taking small pieces of tissues from poria cocos sclerotia by adopting a tissue separation method, placing the small pieces of tissues in a potato glucose agar culture medium, and culturing at 25-28 ℃ to obtain poria cocos mycelia for later use;

adding wood chips, bran, corn, white granulated sugar, magnesium sulfate, calcium sulfate and water into a tissue culture bottle, wherein the material-liquid ratio is 1:3, the loading amount is 1/3 with the volume, sterilizing at 121 ℃ for 2h, cooling to obtain a sterile poria cocos sclerotium culture medium, inoculating a cake containing poria cocos hyphae with the diameter of 5-8mm, carrying out dark culture at 25-28 ℃, and culturing for 15-20 days to ensure that the poria cocos hyphae grow over the solid culture medium for later use;

step three, sand, 160-sand 170 ℃ dry heat sterilization is carried out for 2h, the sand is cooled, the sand is added into the tissue culture bottle full of poria cocos hyphae, the tissue culture bottle is covered with a poria cocos sclerotium culture medium, the coverage height is 4-6cm, dark culture is carried out at the temperature of 28-30 ℃, and the tissue culture bottle is cultured for 10-15 days, so that the poria cocos hyphae in the tissue culture bottle grow into fine sand for 1-3cm for later use;

step four, taking soil at a position of 5-10cm of the pinus massoniana forest, adding the soil into the tissue culture bottle in the step three, covering the soil on sterile fine sand, wherein the height of a covered soil layer is 3-5cm, placing the soil at the temperature of 25-35 ℃ for dark culture, supplementing tap water to the soil on the uppermost layer of the tissue culture bottle every other week, culturing for more than 60 days, growing small sclerotium with the particle size of 3-8mm in the poria cocos sclerotium culture medium in the bottle, enabling the skin to be brown, and enabling internal tissues to be beige.

According to the method, 89 of 100 bottles are cultured, the poria cocos sclerotia is formed, and the success rate of culturing the poria cocos sclerotia reaches about 90%, so that guidance is provided for bottle-culture type three-dimensional culturing of the poria cocos sclerotia.

Comparative example 1: on the same basis as the rest of the procedure of example 1, the covered sand was changed to crude sand (not subjected to dry heat sterilization at 160-170 ℃), and formation of sterile nuclei was found.

Comparative example 2: on the same basis as the rest of the procedure of example 1, no soil was covered and formation of sterile nuclei was found.

Comparative example 3: on the same basis as the rest of the steps of example 1, sand was not covered and formation of sterile nuclei was found;

comparative example 4: on the same basis as the rest of the procedure of example 1, neither sand nor soil was covered, and formation of sterile nuclei was found.

Comparative example 5: on the basis of the same steps as the rest of the tuckahoe bag material cultivation method disclosed in CN101731100A, fresh tuckahoe sclerotium blocks are not inoculated to serve as 'primordium' of new sclerotium, eight cellars are planted, each cellars has five bags, only 25 bags form sclerotium, and the formation rate is only about 60%.

While embodiments of the invention have been described above, it is not limited to the applications set forth in the specification and the embodiments, which are fully applicable to various fields of endeavor for which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the specific details shown and described herein without departing from the general concept defined by the claims and their equivalents.

Claims

1. A method for bottle cultivation of sclerotium of Poria cocos is characterized by comprising the following steps:

2. The method for bottle cultivation of Poria sclerotium according to claim 1, wherein the solid medium is potato dextrose agar.

3. The method for bottle cultivation of Poria cocos sclerotia according to claim 1, wherein the Poria cocos sclerotia medium comprises a matrix and water; the matrix comprises 78-80% of sawdust, 15-19% of bran, 1-3% of corn, 1-3% of white granulated sugar, 0.5-1.5% of magnesium sulfate and 0.2-0.5% of calcium sulfate by weight.

4. The method for bottle cultivation of Poria cocos sclerotia according to claim 3, wherein the mass ratio of the matrix to water is 1: 3; adding the matrix and water into 1/3 of the volume of the tissue culture bottle, sterilizing at 121 deg.C for 2h, and cooling to obtain sterile Poria sclerotium culture medium.

5. The method for bottle cultivation of sclerotium of Poria cocos according to claim 1, wherein the sterile sand is obtained by dry heat sterilization of sand 160-.

6. The method for bottle cultivation of Poria cocos sclerotia according to claim 1, wherein the soil is a 5-10cm deep soil of Pinus massoniana forest.