CN112369431A

CN112369431A - Plant tissue culture explant disinfectant and preparation method and application method thereof

Info

Publication number: CN112369431A
Application number: CN202011236937.0A
Authority: CN
Inventors: 王桂兰; 王英; 陈娜; 张红心; 陈超
Original assignee: Tangshan Normal University
Current assignee: Tangshan Xinyanuo Biotechnology Co ltd
Priority date: 2020-11-09
Filing date: 2020-11-09
Publication date: 2021-02-19
Anticipated expiration: 2040-11-09
Also published as: CN112369431B

Abstract

The disinfectant for the explant for plant tissue culture is characterized by being a purified wood vinegar solution of casuarina equisetifolia. The disinfectant has good killing effect on bacteria and fungi on explants in plant tissue culture, has small damage and inhibition effect on the explants, has no pollution to the environment, is simple and quick in preparation method, convenient in use process and suitable for large-scale popularization.

Description

Plant tissue culture explant disinfectant and preparation method and application method thereof

Technical Field

The invention relates to the field of plant tissue culture explant disinfection, belongs to the technical field of application of purified wood vinegar, and particularly relates to a plant tissue culture explant disinfectant, and a preparation method and a use method of the disinfectant.

Background

In the plant tissue culture technology, whether the disinfection of the plant explant is successful or not is the technical key of the primary culture of tissue culture. The common explant disinfectant comprises mercuric chloride (mercuric chloride), sodium hypochlorite, calcium hypochlorite and other chemical disinfectants, wherein the mercuric chloride has good effect as the explant disinfectant, but has strong toxicity, is harmful to human and livestock and pollutes the environment. Sodium hypochlorite and calcium hypochlorite are chlorine-containing disinfectants with strong corrosivity and strong oxidizing property, have good disinfection effect, but have strong stimulation to respiratory tract of users and great pollution to environment. Therefore, how to provide an environmentally friendly and efficient agent for sterilizing plant tissue culture explants is a subject of constant research and search by those skilled in the art.

The pyroligneous is an organic liquid component obtained by condensing and separating gas generated by plant pyrolysis and carbonization, wherein the organic chemical component accounts for 10-20%, mainly acetic acid, the content accounts for about 50% of the total amount of the organic matters, and other organic chemical components are phenols, alcohols, aldehydes and the like. In the middle of the 17 th century, wood vinegar was produced by the wood dry distillation industry in europe, and by the 80 s of the 18 th century, wood vinegar was introduced in japan. In the 40 s of the 20 th century, wood vinegar was first valued in the countries of the United states, Japan, Korea, etc. The pyroligneous liquor is mainly used in the fields of forestry, fruit industry, garden gardening and the like in the United states; used mainly in agriculture, food, medicine, etc. in japan; the compound fertilizer is mainly used for promoting the growth of crops and preventing and treating diseases and insect pests in Taiwan; however, in the continental area of china, research and application of the method is relatively late, and the application of the method mainly focuses on component analysis, purification, agriculture and other aspects. The previous research shows that the wood vinegar has certain inhibiting effect on fungi and bacteria and broad-spectrum bacteriostasis effect, and has no toxic effect on animals and human bodies, so that the wood vinegar has wide application prospect in the aspects of microbial prevention and control and the like. Therefore, the application of wood vinegar in sterilization and disinfection is an important research topic for those skilled in the art.

Disclosure of Invention

Aiming at the technical defects mentioned in the background technology, the invention provides an environment-friendly and efficient disinfectant for plant tissue culture explants, a preparation method of the disinfectant and a use method of the disinfectant.

The technical scheme adopted by the invention is as follows: the disinfectant for the explant for plant tissue culture is characterized by being a purified wood vinegar solution of casuarina equisetifolia.

The purified wood vinegar solution of casuarina equisetifolia contains 1.4-2.4% of organic acid, 0.9-1.9% of phenols, 0.8-1.8% of ketones, 1.7-2.7% of esters and the balance of water.

The disinfectant is casuarina equisetifolia purified wood vinegar, and the preparation process of the casuarina equisetifolia purified wood vinegar is as follows:

s1: selecting branches of the casuarina equisetifolia trees, and carrying out anaerobic thermal cracking at the temperature of 300-600 ℃;

s2: cooling and standing the smoke generated in the step S1 to obtain wine red liquid wood vinegar stock solution;

s3: filtering and distilling the wood vinegar stock solution obtained in the step S2 to obtain purified wood vinegar solution, namely the purified wood vinegar solution of the casuarina equisetifolia.

In step S1, the branches of the casuarina equisetifolia tree are cut into about 5 cm, which are fresh or dry branches of the casuarina equisetifolia tree.

In step S2, the standing time is at least 6 months.

The use method of the disinfectant for the plant tissue culture explants is characterized in that the disinfectant is a purified wood vinegar solution of casuarina equisetifolia; the using steps are as follows:

s1, washing the plant tissue explant to be disinfected with clear water for 15-20 minutes, and placing the explant on a superclean workbench for later use;

s2, soaking the explants cleaned in the step S1 in 75% alcohol for 30 seconds;

s3, pouring off alcohol, and soaking the explant obtained in the step S2 for 6-10 minutes by using the diluted disinfectant;

s4, taking out the soaked explant, and washing with sterile water for 3-4 times.

In step S3, the dilution factor of the disinfectant is 4 times.

Compared with the prior art, the disinfectant disclosed by the invention has a good killing effect on bacteria and fungi on explants in plant tissue culture, has small damage and inhibition effect on the explants, has no pollution to the environment, is simple and rapid in preparation method, convenient in use process and suitable for large-scale popularization.

Detailed Description

The plant tissue culture explant disinfectant disclosed by the invention is a purified wood vinegar solution of casuarina equisetifolia. The disinfectant used in the explant disinfection method is the purified wood vinegar of the casuarina equisetifolia. Casuarina equisetifolia (academic name:Casuarina equisetifoliaforst.) is an evergreen tree of the genus phellodendron of the family phellodendron. Casuarina equisetifolia is originally produced in northeastern Australia, northern Australia and Pacific islands near beaches and sand dunes. Taiwan, Fujian, Guangdong, Guangxi provinces, coastal areas and south sea islands are all cultivated. The casuarina equisetifolia grows rapidly and germinates strongly, and has the characteristics of drought resistance, sand resistance and salt and alkali resistance due to the deep and wide root system, so that the casuarina equisetifolia becomes an excellent pioneer tree species for wind prevention and sand fixation of tropical seashore. Ma Huang can be used as good firewood, because the bark contains tannin 11% -18%, the purity can reach 80% -85%, also can be used as the industrial raw material of tannin extract. The branches and leaves of casuarina equisetifolia can be used as feed for livestock or medicine, and the seeds can be used as feed for chicken.

The organic components of the casuarina equisetifolia purified wood vinegar used as the disinfectant for the explant of plant tissue culture comprise 1.4-2.4% of organic acid, 0.9-1.9% of phenols, 0.8-1.8% of ketones, 1.7-2.7% of esters and the balance of water, and account for more than 90%.

The preparation process of the casuarina equisetifolia purified wood vinegar liquid is as follows:

s1: selecting branches of the casuarina equisetifolia trees, and carrying out anaerobic thermal cracking at the temperature of 300-600 ℃; the branches of the casuarina equisetifolia trees are fresh or dry branches of the casuarina equisetifolia trees which are cut into about 5 centimeters.

S2: cooling and standing the smoke generated in the step S1 to obtain wine red liquid wood vinegar stock solution; the standing time is more than 6 months.

S3: distilling and filtering the wood vinegar stock solution obtained in the step S2 to obtain purified wood vinegar solution, namely the purified wood vinegar solution of the casuarina equisetifolia.

The application method of the plant tissue culture explant disinfectant comprises the following steps:

s2, discharging the explants cleaned in the step S1 in 75% alcohol for 30-45 seconds;

s3, pouring off alcohol, and soaking the explant obtained in the step S2 for 6-10 minutes by using the diluted disinfectant; the dilution factor of the disinfectant is 1-4 times. Preferably, the disinfectant is diluted by a factor of 4, i.e. the concentration of disinfectant is 25% when used. The principle of the disinfection of the explant in the plant tissue culture is that all fungi on the explant are killed, plant tissue cells cannot be injured, the cell injury is reduced to the maximum extent, and the cost is reduced, so that the dilution multiple for the disinfection of the explant in the plant tissue culture is 4 times.

The present invention will be described in detail with reference to specific examples.

Example 1

The PDA culture medium containing 10%, 12.5%, 16.7%, 25% and 50% (volume ratio) of the purified wood vinegar is used to inoculate three kinds of fungi such as cladosporium sphaericum, aspergillus oryzae and aspergillus terreus, and four kinds of bacteria such as Pantoea agglomerans, Bacillus endophyticus, Bacillus dyscrasiae and Pseudomonas putida. The purified wood vinegar liquid with different concentrations has different inhibiting effects on seven kinds of bacteria, wherein the concentration of 25 percent or more has complete killing effect on three kinds of fungi and four kinds of bacteria (the specific disinfection condition is shown in table 1).

Example 2

The 25% purified wood vinegar (dilution multiple of 4 times is conventional usage) is used for treating different explants such as rhodiola sachalinensis leaf, ginkgo leaf, goose down stem and African violet leaf with epidermal hair, and can completely kill various fungi of the explants within 6-10min of disinfection time, and sterile explants can be obtained. The wood vinegar is treated for 10min under the same condition, the disinfection effect of the wood vinegar on explants is the same as that of sodium hypochlorite and mercury bichloride, and the wood vinegar is harmless to human bodies and free of pollution to the environment (the disinfection methods and the environmental protection conditions of different disinfectants are shown in table 2).

TABLE 2 explant Disinfection method and environmental conditions

Disinfectant species	Treatment time (min)	Degree of environmental pollution	Degree of injury to human body in tissue culture operation
				Casuarina equisetifolia wood vinegar	6-10	-	-
Sodium hypochlorite	6-10	+	++
				Calcium hypochlorite	6-10	++	+++
Mercuric chloride	6-10	+++	+++

Note: "-" indicates no contamination and no harm; "+" indicates light; "+ +" indicates medium; "+ + + +" indicates heavy.

Claims

1. The disinfectant for the explant for plant tissue culture is characterized by being a purified wood vinegar solution of casuarina equisetifolia.

2. The disinfectant for plant tissue culture explants according to claim 1, wherein the casuarina equisetifolia purified pyroligneous solution contains 1.4-2.4% of organic acids, 0.9-1.9% of phenols, 0.8-1.8% of ketones, 1.7-2.7% of esters, and the balance of water.

3. The method for preparing a disinfectant for explant of plant tissue culture according to claim 1, wherein said disinfectant is a purified pyroligneous solution of casuarina equisetifolia, which is prepared by the following steps:

4. The method for preparing a disinfectant for explant of plant tissue culture according to claim 3, wherein in step S1, the branch of Ephedra distachya tree is cut to about 5 cm.

5. The method for preparing a disinfectant for plant tissue culture explants of claim 3, wherein the standing time in step S2 is at least 6 months.

6. A method of using the disinfectant for plant tissue culture explants prepared by the method of any one of claims 3 to 5, wherein the disinfectant is a purified pyroligneous solution of Ephedra sinica Stapf; the using steps are as follows:

s2, soaking the explants cleaned in the step S1 in 75% alcohol for 30 seconds;

7. The method of using a disinfectant for plant tissue culture explants of claim 6, wherein the dilution factor of the disinfectant in step S3 is 4-fold.