CN115176703A - Bacteriostatic agent, culture medium sterilization method using same and application - Google Patents
Bacteriostatic agent, culture medium sterilization method using same and application Download PDFInfo
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- CN115176703A CN115176703A CN202210829497.2A CN202210829497A CN115176703A CN 115176703 A CN115176703 A CN 115176703A CN 202210829497 A CN202210829497 A CN 202210829497A CN 115176703 A CN115176703 A CN 115176703A
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- 230000001954 sterilising effect Effects 0.000 title claims abstract description 91
- 239000001963 growth medium Substances 0.000 title claims abstract description 72
- 239000000022 bacteriostatic agent Substances 0.000 title claims abstract description 68
- 238000004659 sterilization and disinfection Methods 0.000 title claims abstract description 67
- 238000000034 method Methods 0.000 title claims abstract description 35
- OPQARKPSCNTWTJ-UHFFFAOYSA-L copper(ii) acetate Chemical compound [Cu+2].CC([O-])=O.CC([O-])=O OPQARKPSCNTWTJ-UHFFFAOYSA-L 0.000 claims abstract description 25
- TWFZGCMQGLPBSX-UHFFFAOYSA-N Carbendazim Natural products C1=CC=C2NC(NC(=O)OC)=NC2=C1 TWFZGCMQGLPBSX-UHFFFAOYSA-N 0.000 claims abstract description 17
- JNPZQRQPIHJYNM-UHFFFAOYSA-N carbendazim Chemical compound C1=C[CH]C2=NC(NC(=O)OC)=NC2=C1 JNPZQRQPIHJYNM-UHFFFAOYSA-N 0.000 claims abstract description 17
- 239000006013 carbendazim Substances 0.000 claims abstract description 17
- YBHILYKTIRIUTE-UHFFFAOYSA-N berberine Chemical compound C1=C2CC[N+]3=CC4=C(OC)C(OC)=CC=C4C=C3C2=CC2=C1OCO2 YBHILYKTIRIUTE-UHFFFAOYSA-N 0.000 claims abstract description 16
- 229940093265 berberine Drugs 0.000 claims abstract description 16
- QISXPYZVZJBNDM-UHFFFAOYSA-N berberine Natural products COc1ccc2C=C3N(Cc2c1OC)C=Cc4cc5OCOc5cc34 QISXPYZVZJBNDM-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000005708 Sodium hypochlorite Substances 0.000 claims abstract description 13
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims abstract description 13
- -1 oxypyrron Chemical compound 0.000 claims abstract description 5
- 238000011109 contamination Methods 0.000 claims description 28
- 238000001816 cooling Methods 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- 239000002609 medium Substances 0.000 claims description 7
- 239000008367 deionised water Substances 0.000 claims description 6
- 229910021641 deionized water Inorganic materials 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 239000006870 ms-medium Substances 0.000 claims description 5
- 230000000249 desinfective effect Effects 0.000 claims description 3
- 230000000694 effects Effects 0.000 abstract description 14
- 230000035784 germination Effects 0.000 abstract description 11
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 7
- 238000002474 experimental method Methods 0.000 abstract description 7
- 230000012010 growth Effects 0.000 abstract description 7
- 241000894006 Bacteria Species 0.000 abstract description 6
- 235000002566 Capsicum Nutrition 0.000 description 10
- 241000196324 Embryophyta Species 0.000 description 10
- 239000006002 Pepper Substances 0.000 description 10
- 241000722363 Piper Species 0.000 description 10
- 235000016761 Piper aduncum Nutrition 0.000 description 10
- 235000017804 Piper guineense Nutrition 0.000 description 10
- 235000008184 Piper nigrum Nutrition 0.000 description 10
- 238000011081 inoculation Methods 0.000 description 6
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical group [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 4
- 229910052802 copper Inorganic materials 0.000 description 4
- 239000010949 copper Substances 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 3
- 238000002791 soaking Methods 0.000 description 3
- 230000001629 suppression Effects 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- 206010020649 Hyperkeratosis Diseases 0.000 description 2
- 244000061456 Solanum tuberosum Species 0.000 description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
- 239000003899 bactericide agent Substances 0.000 description 2
- 230000003385 bacteriostatic effect Effects 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 230000017074 necrotic cell death Effects 0.000 description 2
- 230000008635 plant growth Effects 0.000 description 2
- 238000004161 plant tissue culture Methods 0.000 description 2
- 230000008467 tissue growth Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 239000003905 agrochemical Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- GTCAXTIRRLKXRU-UHFFFAOYSA-N carbamic acid methyl ester Natural products COC(N)=O GTCAXTIRRLKXRU-UHFFFAOYSA-N 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 229910001410 inorganic ion Inorganic materials 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 239000002917 insecticide Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Developmental Biology & Embryology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a bacteriostatic agent, a culture medium sterilization method for avoiding tissue culture seedling pollution by utilizing the broad spectrum of the bacteriostatic agent and application thereof, wherein the bacteriostatic agent comprises the following components: carbendazim, oxypyrron, copper acetate, sodium hypochlorite and berberine. According to the invention, a large number of experiments are carried out to obtain the bacteriostatic agent, the antibacterial effect of the bacteriostatic agent is doubled, the bacteriostatic agent can ensure no bacteria growth for 15 days even if a culture medium is exposed in the air, and the bacteriostatic agent has a certain promotion effect on plant germination and seedling growth.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a bacteriostatic agent, a culture medium sterilization method for preventing tissue culture seedling from being polluted by utilizing the broad spectrum of the bacteriostatic agent and application.
Background
The MS culture medium is designed by Murashige and Skoog for tobacco cell culture in 1962, and is currently the most widely used and common culture medium in plant tissue culture. The MS culture medium has higher inorganic salt and ion concentration, and can provide mineral nutrition required by plant tissue growth while promoting the plant callus growth.
The MS culture medium is prepared, sterilized and inoculated to plants under the aseptic condition according to strict operation specifications, the sterilization requirement is high, the sterilization time and pressure are strictly controlled during autoclaving, and the culture medium is not completely sterilized, so that the culture medium is easily infected by different bacteria, and the aim of culturing the callus of the plant explant is not fulfilled.
The existing MS culture medium sterilization method is difficult to determine the optimal sterilization time, and the following problems can occur in the practical application process:
1) The short sterilization time causes incomplete sterilization, thereby causing the pollution of the tissue culture seedlings;
2) The pH of the medium is lowered due to the excessively long sterilization time, so that the medium cannot be solidified.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a culture medium sterilization method which can prevent tissue culture seedlings from being polluted in a broad spectrum manner.
In a first aspect, the present invention provides a bacteriostatic agent comprising the following components: carbendazim, oxypyrron, copper acetate, sodium hypochlorite and berberine.
Preferably, the bacteriostatic agent comprises the following components in parts by weight:
10-15 parts of carbendazim, 200-250 parts of carbendazim, 250-350 parts of oxypyrron, 15-20 parts of copper acetate, 80-120 parts of sodium hypochlorite and 80-120 parts of berberine.
Preferably, the bacteriostatic agent comprises the following components in parts by weight:
12 parts of carbendazim, 250 parts of carbendazim, 300 parts of Yipeilong, 20 parts of copper acetate, 100 parts of sodium hypochlorite and 100 parts of berberine.
In a second aspect, the present invention provides a method for sterilizing a culture medium to avoid contamination of a tissue culture seedling by using a broad spectrum of the above bacteriostatic agent, the method comprising the steps of:
s1, preparation: dissolving an MS culture medium in deionized water, and adding a bacteriostatic agent, wherein the weight ratio of the bacteriostatic agent to the MS culture medium is 1:1000, parts by weight;
s2, high-pressure steam sterilization: sterilizing the MS culture medium added with the bacteriostatic agent under high pressure by an autoclave;
s3, cooling: closing the sterilization pot after the sterilization time is over, exhausting and cooling, taking out after cooling to a proper temperature, wherein the cooling time is not suitable to be overlong;
s4, ultraviolet sterilization: and (3) disinfecting the sterile operating platform by using alcohol, starting an ultraviolet lamp to sterilize the sterile operating platform, taking the MS culture medium subjected to high-pressure sterilization out of the sterilizing pot, and immediately transferring the MS culture medium to the sterile operating platform for ultraviolet sterilization.
Preferably, the MS medium in S1 is prepared by using Coolaber modified medium at the temperature of 60-80 ℃.
Preferably, the sterilization temperature in the S2 is 121 ℃, the sterilization time is 15min, and the air pressure is more than 100kPa.
Preferably, the S3 is taken out after being cooled to 45-65 ℃, and the cooling time is not more than 10 minutes.
Preferably, in the step S4, 75% alcohol is used for sterilization, an ultraviolet lamp is turned on to sterilize the sterile operating table for 10-15min, and then the MS culture medium after autoclaving is taken out of the sterilization pot and immediately transferred to the sterile operating table for ultraviolet sterilization, wherein the sterilization time is 10-15min.
In a third aspect, the invention also provides application of the bacteriostatic agent, and the bacteriostatic agent is applied to a culture medium sterilization method for widely avoiding tissue culture seedling pollution.
The difference between the invention and the prior art is that the invention achieves the following technical effects:
according to the invention, a large number of experiments are carried out to obtain the bacteriostatic agent, the antibacterial effect of the bacteriostatic agent is doubled, no bacteria can grow for 15 days even if a culture medium is exposed in the air, and the bacteriostatic agent has a certain promotion effect on plant germination and seedling growth; the optimal sterilization method of the culture medium is obtained by selecting the preparation temperature of the culture medium, adding a specially-prepared bacteriostatic agent after preparation, and controlling the high-pressure sterilization time, the cooling time and the ultraviolet sterilization time, so that the optimal sterilization method avoids the tissue culture seedling pollution caused by the culture medium colony residue; the method has the advantages of good sterilization effect, thorough sterilization, low pollution rate of less than 5 percent, and capability of effectively avoiding the pollution of tissue culture seedlings without influencing the quality of a culture medium.
Drawings
FIG. 1 shows the effect of the method for sterilizing a medium of Experimental example 1 of the present invention on the suppression of contamination of a cultured seedling. In fig. 1, A1 is the sterilization method used in example 1 of the present invention, and B1 is the conventional sterilization method used for the same tissue culture seedlings.
FIG. 2 shows the effect of the method for sterilizing a culture medium on the suppression of the contamination of a cultured seedling according to Experimental example 2 of the present invention. In fig. 2, A2 is the sterilization method used in experimental example 2, and B2 is the conventional sterilization method used for the same tissue culture seedlings.
FIG. 3 shows the effect of the method for sterilizing a culture medium on the suppression of the contamination of a cultured seedling according to Experimental example 3 of the present invention. In fig. 3, A3 is the sterilization method used in experimental example 3, and B3 is the conventional sterilization method used for the same tissue culture seedlings.
FIG. 4 shows the effect of bacteriostatic on the inhibition of tissue culture seedling contamination in Experimental example 4. In fig. 4, D1 is the case of control D1 contamination, D2 is the case of control D2 contamination, and D3 is the case of control D3 contamination.
FIG. 5 is a graph showing the inhibitory effect of copper acetate on the contamination of tissue culture seedlings in Experimental example 5. In fig. 5, E1 is the case of control E1 contamination, E2 is the case of control E2 contamination, and E3 is the case of control E3 contamination.
FIG. 6 shows the inhibitory effect of bacteriostatic agent of Experimental example 6 on MS medium contamination. In fig. 6, F1 is the case of contamination of control F1, F2 is the case of contamination of control F2, and F3 is the case of contamination of control F3.
FIG. 7 shows the effect of the bacteriostatic agent of Experimental example 7 on the inhibition of tissue culture seedling contamination. In FIG. 7, G1 is the case of control G1 contamination, G2 is the case of control G2 contamination, and G3 is the case of control G3 contamination.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
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.
The terminology used in the embodiments of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the examples of the present invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
It should be understood that the term "and/or" as used herein is merely a relationship that describes an associated object, meaning that three relationships may exist, e.g., a and/or B, may represent: a exists alone, A and B exist simultaneously, and B exists alone. In addition, the character "/" herein generally indicates that the former and latter related objects are in an "or" relationship.
Wherein carbendazim is benzimidazole-2-yl methyl carbamate. The plant-cultivating agent is produced by Shanghai Yuguan Biotechnology limited company, and the batch number of the product is 141218,100ml. Yipelong: shanghai C is manufactured by Chemicals, inc., product number EY0013, 435 mg/bottle.
Example 1
The embodiment of the invention provides a bacteriostatic agent. The bacteriostatic agent comprises the following components in parts by weight: 12 parts, 250 parts of carbendazim, 300 parts of Yipelong, 20 parts of copper acetate, 100 parts of sodium hypochlorite and 100 parts of berberine.
The components are mixed and then added with deionized water to be constant volume of 100ml for use.
Example 2
The embodiment of the invention provides a bacteriostatic agent. The bacteriostatic agent comprises the following components in parts by weight:
10 parts of carbendazim, 200 parts of carbendazim, 250 parts of Yipeilong, 15 parts of copper acetate, 80 parts of sodium hypochlorite and 80 parts of berberine.
Example 3
The embodiment of the invention provides a bacteriostatic agent. The bacteriostatic agent comprises the following components in parts by weight:
15 parts of carbendazim, 250 parts of carbendazim, 350 parts of Yipelong, 20 parts of copper acetate, 120 parts of sodium hypochlorite and 120 parts of berberine.
Experimental example 1
The embodiment of the invention provides a culture medium sterilization method capable of avoiding tissue culture seedling pollution in a broad spectrum manner. The method comprises the following steps:
(1) Soaking pepper seeds in 2% sodium hypochlorite for 20min, and washing with distilled water;
(2) Weighing the MS culture medium according to a corresponding proportion, dissolving the MS culture medium in deionized water at 80 ℃, and adding a bacteriostatic agent, wherein the weight ratio of the bacteriostatic agent to the MS culture medium is 1:1000, parts by weight;
(3) Sterilizing the MS culture medium added with the bacteriostatic agent for 15min at 121 ℃ in an autoclave under 100kPa;
(4) Closing the sterilizing pot after the high-pressure sterilization is finished, exhausting and cooling, taking out after the temperature is reduced to 65 ℃, wherein the cooling time is not more than 10min;
(5) Sterilizing the sterile operating table with 75% alcohol, starting an ultraviolet lamp to sterilize the sterile operating table for 15min, taking out the autoclaved MS culture medium from the sterilizing pot, and immediately transferring the MS culture medium to the sterile operating table for ultraviolet sterilization for 15min;
(6) The culture medium was placed in a sealed room for thirty days after sterilization to observe contamination, and the results are shown in FIG. 1.
The components and the proportion of the bacteriostatic agent in the embodiment of the invention are the same as those in the embodiment 1.
Experimental example 2
The embodiment of the invention provides a culture medium sterilization method capable of avoiding tissue culture seedling pollution in a broad spectrum manner. The method comprises the following steps:
(1) Soaking pepper seeds in 2% sodium hypochlorite for 20min, and washing with distilled water;
(2) Weighing according to the corresponding proportion of the MS culture medium, dissolving in deionized water at 80 ℃, and adding a bacteriostatic agent, wherein the weight ratio of the bacteriostatic agent to the MS culture medium is 1:1000;
(3) Sterilizing the MS culture medium added with the bacteriostatic agent for 15min at 121 ℃ in an autoclave under 100kPa;
(4) Closing the sterilization pot after the high-pressure sterilization is finished, exhausting and cooling, taking out after the temperature is reduced to 65 ℃, wherein the cooling time is not more than 10min;
(5) Sterilizing the sterile operating table with 75% alcohol, starting an ultraviolet lamp to sterilize the sterile operating table for 15min, taking out the autoclaved MS culture medium from the sterilizing pot, and immediately transferring the MS culture medium to the sterile operating table for ultraviolet sterilization for 15min;
(6) After sterilization, the sterilized pepper seeds were transferred to a clean bench for culture medium inoculation, and the contamination was observed ten days after inoculation, and the results are shown in fig. 2.
The components and the proportion of the bacteriostatic agent provided by the embodiment of the invention are the same as those of the bacteriostatic agent provided by the embodiment 1.
Experimental example 3
The embodiment of the invention provides a culture medium sterilization method capable of avoiding tissue culture seedling pollution in a broad spectrum manner. The method comprises the following steps:
(1) Soaking and cleaning potato explants for three times by using sterile water after disinfecting the potato explants by using sodium hypochlorite;
(2) Weighing according to the corresponding proportion of the MS culture medium, dissolving in deionized water at 80 ℃, and adding a bacteriostatic agent, wherein the weight ratio of the bacteriostatic agent to the MS culture medium is 1:1000, parts by weight;
(3) Sterilizing the MS culture medium added with the bacteriostatic agent for 15min at 121 ℃ in an autoclave under 100kPa;
(4) Closing the sterilizing pot after the high-pressure sterilization is finished, exhausting and cooling, taking out after the temperature is reduced to 65 ℃, wherein the cooling time is not more than 10min;
(5) Sterilizing the sterile operation table by using 75% alcohol, starting an ultraviolet lamp to sterilize the sterile operation table for 15min, taking out the MS culture medium subjected to high-pressure sterilization from a sterilization pot, and immediately transferring the MS culture medium to the sterile operation table for ultraviolet sterilization for 15min;
(6) After sterilization, the soaked explants were transferred to a clean bench, after drying, medium inoculation was performed, and the contamination was observed ten days after inoculation, and the results are shown in fig. 3.
The components and the proportion of the bacteriostatic agent in the embodiment of the invention are the same as those in the embodiment 1.
Experimental example 4
In this example, the germination of pepper seeds was observed experimentally, and the pepper seeds were cultured at 20 ℃ for 12 hours in light and 12 hours in darkness for 10 days, and 3 control groups D1 (without the bacteriostatic agent of example 1), D2 (with the bacteriostatic agent of example 1), and D3 (with the bacteriostatic agent of example 1, but with copper acetate and berberine deleted) were provided. The contamination was observed ten days after inoculation and the results are shown in FIG. 4.
Experimental example 5
In this example, the germination conditions of pepper seeds were experimentally observed, and when the pepper seeds were cultured for 10 days under the conditions of 20 ℃ and 12 hours of light and 12 hours of dark, 3 control groups, E1 (no bacteriostatic agent, no copper sulfate added), E2 (no bacteriostatic agent, 2% by weight of culture medium added copper acetate), and E3 (no bacteriostatic agent, 5% by weight of culture medium added copper acetate), were set. The contamination was observed ten days after inoculation, and the results are shown in FIG. 5.
The germination experiment is carried out on seeds by adding different copper acetate, and experimental data show that trace addition of copper acetate is harmless to plants, excessive addition of copper acetate can cause damage to tissue culture plants, and the germination rate is reduced. Meanwhile, the copper acetate is unexpectedly found to have a better bacteriostatic action.
Experimental example 6
In this example, experimental observation was performed on the MS medium contamination inhibition effect by using the bacteriostatic agent, and 3 control groups F1 (without adding the bacteriostatic agent of example 1), F2 (with the bacteriostatic agent of example 1), and F3 (with the bacteriostatic agent of example 1, but with copper acetate and berberine deleted) were set in the MS medium sterilization treatment. The contamination was observed for ten days, and the results are shown in FIG. 6. The no copper acetate sample added produced a mildew spot.
Experimental example 7
In this example, the germination conditions of pepper seeds were experimentally observed, and 3 control groups G1 (only copper acetate was added), G2 (no bacteriostatic agent was added), and G3 (bacteriostatic agent of example 1 was added) were set, and cultured for 10 days at 20 ℃ under 12h light and 12h dark, and the results are shown in fig. 7.
Wherein, the culture medium without the added bacteriostatic agent has slight growth of bacteria, and the germination of pepper seeds is inhibited and partially necrotized in the culture medium with only the added copper acetate (after the growth of bacteria, the germination is dug out for better observation). The pepper seeds in the fully-added culture medium grow strongly and are infected by sterile colonies.
It should be noted that copper acetate can be used as a bactericide and an insecticide in agricultural chemicals and the prior art, but those skilled in the art also know that copper acetate is a heavy metal which has a plant growth inhibitory effect and a negative effect while acting as a bactericide. While the MS culture medium is copper-containing element, the copper-containing inhibiting bacteria cannot be added additionally, otherwise, excessive copper causes plant necrosis or influences tissue growth. In addition, the inventor conducts a copper stress germination experiment to record convenient photographing, the culture medium is not sealed, a certain antibacterial effect on the culture medium is discovered unexpectedly after ten days, plant necrosis is caused by excessive addition, and the test proves that the culture medium has a good antibacterial effect and a certain promotion effect on plant growth at a concentration of 5 ml/L. The invention overcomes the technical prejudice.
The inventor of the invention proves that the antibacterial effect of the berberine is doubled by adding two components of copper acetate and berberine at the same time through a large number of experiments, and particularly the whole proportion of the antibacterial agent can ensure that the berberine is not opened for 15 days even if the berberine is exposed in the air. Moreover, the bacteriostatic agent has certain promotion effect on plant germination and seedling growth.
According to the invention, through a large number of experiments, the optimal sterilization method of the culture medium is obtained by selecting the preparation temperature of the culture medium, adding a specially-prepared bacteriostatic agent after preparation, and controlling the high-pressure sterilization time, the cooling time and the ultraviolet sterilization time, so that the tissue culture seedling pollution caused by the culture medium colony residue is avoided; the method has the advantages of good sterilization effect, thorough sterilization and less than 5% of pollution, and can effectively avoid the pollution of tissue culture seedlings without influencing the quality of the culture medium.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.
Claims (9)
1. A bacteriostatic agent is characterized by comprising the following components: carbendazim, yipeuron, copper acetate, sodium hypochlorite and berberine.
2. The bacteriostatic agent according to claim 1, which is characterized by comprising the following components in parts by weight:
10-15 parts of carbendazim, 200-250 parts of carbendazim, 250-350 parts of yipelong, 15-20 parts of copper acetate, 80-120 parts of sodium hypochlorite and 80-120 parts of berberine.
3. The bacteriostatic agent according to claim 2, which comprises the following components in parts by weight:
12 parts of carbendazim, 250 parts of carbendazim, 300 parts of Yipelong, 20 parts of copper acetate, 100 parts of sodium hypochlorite and 100 parts of berberine.
4. A method of sterilizing a culture medium using a bacteriostatic agent according to any one of claims 1 to 3, comprising the steps of:
s1, preparation: dissolving an MS culture medium in deionized water, and adding a bacteriostatic agent, wherein the weight ratio of the bacteriostatic agent to the MS culture medium is 1:1000;
s2, high-pressure steam sterilization: sterilizing the MS culture medium added with the bacteriostatic agent under high pressure by an autoclave;
s3, cooling: closing the sterilizing pot after the sterilizing time is over, exhausting and cooling, and taking out after cooling to a proper temperature;
s4, ultraviolet sterilization: and (3) disinfecting the sterile operating platform by using alcohol, starting an ultraviolet lamp to sterilize the sterile operating platform, taking the MS culture medium subjected to high-pressure sterilization out of the sterilizing pot, and immediately transferring the MS culture medium to the sterile operating platform for ultraviolet sterilization.
5. The method as claimed in claim 4, wherein the MS medium in S1 is prepared by using Coolaber modified medium at a temperature of 60-80 ℃.
6. The method for sterilizing a medium according to claim 4, wherein the sterilization temperature in S2 is 121 ℃, the sterilization time is 15min, and the air pressure is more than 100kPa.
7. The method for sterilizing the culture medium according to claim 4, wherein the S3 is taken out after being cooled to 45-65 ℃, and the cooling time is not more than 10 minutes.
8. The method for sterilizing a culture medium according to claim 4, wherein the sterilization in S4 is performed by using 75% alcohol, the ultraviolet lamp is turned on to sterilize the sterile operating table for 10-15min, and the autoclaved MS culture medium is taken out of the autoclave and then transferred to the sterile operating table for ultraviolet sterilization for 10-15min.
9. Use of a bacteriostatic agent according to any one of claims 1 to 3 in the sterilization of a culture medium for broad spectrum avoidance of contamination by tissue culture plantlets.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111657143A (en) * | 2020-06-24 | 2020-09-15 | 广西壮族自治区中国科学院广西植物研究所 | Passion fruit detoxification and rapid propagation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111657143A (en) * | 2020-06-24 | 2020-09-15 | 广西壮族自治区中国科学院广西植物研究所 | Passion fruit detoxification and rapid propagation method |
Non-Patent Citations (4)
| Title |
|---|
| 扁红英等: "次氯酸钠对白刺开放式组培苗生理特性的影响", 草业科学, vol. 39, no. 2 * |
| 薛寒青等: "不同抗生素对生姜组织培养中姜芽脱菌效果的研究", 北方园艺, no. 6 * |
| 颜彩燕等: "3 种抑菌剂对马铃薯试管苗生长的影响及抑菌效果", 乡村科技, no. 2, pages 1 - 1 * |
| 魏少征: "农药安全卫生技术", 化学工业出版社, pages: 119 - 120 * |
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