CN116195513A - Method for sterilizing orchid explant - Google Patents
Method for sterilizing orchid explant Download PDFInfo
- Publication number
- CN116195513A CN116195513A CN202211647583.8A CN202211647583A CN116195513A CN 116195513 A CN116195513 A CN 116195513A CN 202211647583 A CN202211647583 A CN 202211647583A CN 116195513 A CN116195513 A CN 116195513A
- Authority
- CN
- China
- Prior art keywords
- orchid
- explant
- seconds
- explants
- nano silver
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Links
- 241000233855 Orchidaceae Species 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 25
- 230000001954 sterilising effect Effects 0.000 title claims abstract description 21
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 29
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims abstract description 23
- 241000894006 Bacteria Species 0.000 claims abstract description 17
- 239000010453 quartz Substances 0.000 claims description 27
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 27
- 230000005855 radiation Effects 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 3
- 229910052709 silver Inorganic materials 0.000 claims description 2
- 239000004332 silver Substances 0.000 claims description 2
- 238000002791 soaking Methods 0.000 abstract 1
- 239000008223 sterile water Substances 0.000 description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 15
- 241000196324 Embryophyta Species 0.000 description 10
- 230000001678 irradiating effect Effects 0.000 description 9
- 238000005406 washing Methods 0.000 description 8
- 239000000645 desinfectant Substances 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 5
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 210000001161 mammalian embryo Anatomy 0.000 description 3
- 239000005708 Sodium hypochlorite Substances 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000357 carcinogen Toxicity 0.000 description 1
- 239000003183 carcinogenic agent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 229960002523 mercuric chloride Drugs 0.000 description 1
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 230000001699 photocatalysis Effects 0.000 description 1
- 238000007146 photocatalysis Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
Classifications
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Apparatus For Disinfection Or Sterilisation (AREA)
Abstract
The invention discloses a method for sterilizing orchid explants, which comprises the steps of rapidly and completely soaking fresh cut orchid explants in a transparent vessel containing nano silver sol under a normal bacteria environment, and then taking out the explants after the transparent vessel is irradiated for 10-3000 seconds under ultraviolet rays or visible light, thus completing sterilization. The invention can carry out sterilization operation in a bacteria environment, greatly improves the convenience and efficiency of operation, and overcomes the complicated limitation that the explant is cut off and sterilized in a bacteria-free environment in the prior art.
Description
Technical Field
The invention belongs to the field of nano materials, and particularly relates to application of nano silver sol in orchid explant disinfection.
Background
The tissue culture propagation of orchid by adopting the explant has become the most important propagation mode in orchid industry. However, in the propagation process, the problems of explant pollution caused by incomplete disinfection, explant damage caused by excessive use of disinfectants, and the like are easy to occur. Therefore, how to select a proper disinfectant and a using method thereof becomes a key technical bottleneck for restricting the tissue culture of orchid.
The related art adopts disinfectants such as ethanol, sodium hypochlorite, hydrogen peroxide and the like, the antibacterial spectrum of the disinfectants is relatively narrow, and incomplete disinfection is easy to occur; meanwhile, the disinfectant needs longer time to disinfect, which is easy to damage the explant and affects the later-period reproduction. In order to overcome the defects of the disinfectant, the related technology adopts mercury chloride to disinfect the explant. However, mercury chloride is a highly toxic and strong carcinogen, is easy to pollute the environment and poses serious threat to the health of operators. In addition, the above related art requires excision, sterilization and subsequent propagation of the explant in a sterile clean box, and the operation is inconvenient and cumbersome.
In summary, a disinfectant with high efficiency, safety and broad antibacterial spectrum is selected, and a simple and easy disinfection method is adopted, so that the method becomes a key technology which is urgently needed to be solved in the field of orchid explant disinfection.
Disclosure of Invention
The main object of the present invention is to provide a flow regulator.
In order to solve the problems, the invention provides a orchid explant disinfection method, which is realized by the following technical scheme;
(1) The concentration of the nano silver sol was adjusted to 1mg/L to 1000mg/L (calculated as silver content, hereinafter calculated in the same manner) with sterile water or ultrapure water, and placed in a transparent vessel; the transparent vessel can pass ultraviolet rays;
(2) Under the normal bacteria environment, the fresh cut orchid explant is quickly and completely soaked in the transparent vessel containing the nano silver sol;
(3) And (3) irradiating the quartz vessel for 10-3000 seconds under ultraviolet or visible light, and taking out the explant to finish disinfection. For example, 20 seconds, 40 seconds, 60 seconds, 120 seconds, 180 seconds, 240 seconds, 300 seconds, 360 seconds, etc.
The normal bacteria environment is that the air is in the indoor or outdoor environment with normal circulation.
Preferably, the nano silver sol refers to nano silver particles with the particle size of 1nm-100nm and a compound thereof;
preferably, the ultraviolet rays have a radiation wavelength of 10-400nm;
preferably, the visible light has a radiation wavelength of 400-800nm;
preferably, the transparent vessel comprises a quartz vessel.
Preferably, the intensity of the ultraviolet light and the visible light is 1.65X10 13 ～1.98×10 18 photons/sec/cm 3 。
Preferably, the irradiation is performed under ultraviolet or visible light for 60 seconds to 600 seconds.
The orchid explant disinfection method can kill all pathogenic microorganisms which are endogenous or exogenous in orchid explants. When the invention is adopted for sterilizing the orchid explant, the rate of bacterial contamination of the explant in the later tissue culture is not more than 0.15%, thus laying a foundation for large-scale orchid tissue culture.
The invention has the following technical effects and advantages:
(1) The high-efficiency and short-time disinfection effect is realized, and the damage to the explant is avoided while the disinfection effect is ensured;
(2) The environmental pollution and health threat problems of the traditional mercury chloride are overcome;
(3) The sterilization operation can be performed in a bacteria environment, so that the convenience and efficiency of the operation are greatly improved, and the complicated limitation that the removal and sterilization of the explant are performed in a sterile environment in the prior art is overcome.
Detailed Description
Hereinafter, the present invention will be described in detail with reference to examples. It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be combined with each other.
Example 1
The embodiment provides a method for sterilizing orchid explants, in the embodiment, nano silver sol with the concentration of 50mg/L is placed in a quartz vessel; cutting orchid bud from plant base under normal bacteria environment, washing with sterile water, immediately placing into the quartz vessel, and irradiating the quartz vessel under ultraviolet light for 300 seconds (irradiation intensity is 1.65X10) 16 photons/sec/cm 3 ) Taking out the new buds to finish disinfection.
Example 2
The embodiment provides a method for sterilizing orchid explants, in this embodimentIn the example, nano silver sol with the concentration of 100mg/L is placed in a quartz vessel; cutting stem tip of orchid from plant under normal bacteria environment, washing with sterile water, immediately placing in the quartz vessel, and irradiating the quartz vessel under ultraviolet light for 100 seconds (irradiation intensity is 3.3X10) 16 photons/sec/cm 3 ) And taking out the stem tip to finish disinfection.
Example 3
The embodiment provides a method for sterilizing orchid explants, in the embodiment, nano silver sol with the concentration of 200mg/L is placed in a quartz vessel; cutting roots of orchid from plants under normal bacteria environment, washing with sterile water, immediately placing in the quartz vessel, and irradiating the quartz vessel with ultraviolet light for 50 seconds (irradiation intensity of 1.65X10) 17 photons/sec/cm 3 ) And taking out the root to finish disinfection.
Example 4
The embodiment provides a method for sterilizing orchid explants, in the embodiment, nano silver sol with the concentration of 400mg/L is placed in a quartz vessel; cutting leaves of orchid under normal bacteria environment, washing with sterile water, immediately placing in the quartz vessel, and irradiating the quartz vessel under visible light for 50 seconds (irradiation intensity of 2.0X10) 18 photons/sec/cm 3 ) And taking out the blade, thus finishing disinfection.
Example 5
The embodiment provides a method for sterilizing orchid explants, in the embodiment, nano silver sol with the concentration of 500mg/L is placed in a quartz vessel; cutting stem of orchid from plant under normal bacteria environment, washing with sterile water, immediately placing in the quartz vessel, and irradiating the quartz vessel under visible light for 200 seconds (irradiation intensity of 2.0X10) 17 photons/sec/cm 3 ) And taking out the pedicel to finish disinfection.
Example 6
In this example, 500mg/L nano silver sol was placed in a quartz vesselThe method comprises the steps of carrying out a first treatment on the surface of the Cutting stem of orchid from plant under normal bacteria environment, washing with sterile water, immediately placing in the quartz vessel, and irradiating the quartz vessel under ultraviolet light for 100 seconds (irradiation intensity of 2.0X10) 17 photons/sec/cm 3 ) And taking out the pedicel to finish disinfection.
Example 7
The embodiment provides a method for sterilizing orchid explants, in the embodiment, nano silver sol with the concentration of 600mg/L is placed in a quartz vessel; cutting young orchid embryo from plant under normal bacteria environment, washing with sterile water, immediately placing into the quartz vessel, and irradiating the quartz vessel under ultraviolet light for 150 seconds (irradiation intensity is 1.0X10) 18 photons/sec/cm 3 ) And taking out the pedicel to finish disinfection.
Example 8
The embodiment provides a method for sterilizing orchid explants, in the embodiment, nano silver sol with the concentration of 900mg/L is placed in a quartz vessel; cutting young orchid embryo from plant under normal bacteria environment, washing with sterile water, immediately placing into the quartz vessel, and irradiating the quartz vessel under ultraviolet light for 20 seconds (irradiation intensity is 3.0X10) 17 photons/sec/cm 3 ) And taking out the pedicel to finish disinfection.
Using the above examples 1850 explants (250 per example 1-7, 100 per example 8) were sterilized and then inoculated in normal medium, and after 7 days only 2 contaminated explants were observed with a contamination rate of 0.108%.
In the following comparative examples, 100 explants were taken for each comparative example.
Comparative example 1:
under normal bacteria environment, the young orchid embryo is cut off from the plant, washed by sterile water, soaked by 75% alcohol for 30s, disinfected by 10% sodium hypochlorite, washed by sterile water for 3-5 times, inoculated in culture medium, and observed after 7 days, the pollution rate is 57%.
Comparative example 2:
under normal bacteria environment, the orchid peduncles are cut off from plants, washed by sterile water, soaked by 75% alcohol for 30s, sterilized by 0.5% mercuric chloride, washed by sterile water for 3-5 times, inoculated in a culture medium, and observed after 7 days, the pollution rate is 43%.
Comparative example 2:
under normal bacteria environment, the orchid peduncles are cut off from plants, washed by sterile water, soaked by 75% alcohol for 30s, sterilized by 10% hydrogen peroxide, washed by sterile water for 3-5 times, inoculated in a culture medium, and observed after 7 days, the pollution rate is 60%.
It should be noted that the parameters used in the preparation in the above examples are only for illustration and are not limiting to the application, and those skilled in the art can flexibly select the preparation parameters according to actual needs. The nano silver sol can adopt one of all nano silver sols in the related technology and a mixture thereof, and the core of the invention is to fully integrate the photocatalysis characteristic and the disinfection performance of the nano silver sol.
The foregoing description is only illustrative of the preferred embodiments of the present invention, and therefore should not be taken as limiting the scope of the invention, for all changes and modifications that come within the meaning and range of equivalency of the claims and specification are therefore intended to be embraced therein.
Claims (8)
1. The orchid explant disinfection method is characterized in that under the normal bacteria environment, the freshly cut orchid explant is quickly and completely soaked in a transparent vessel containing nano silver sol, the transparent vessel can pass through ultraviolet rays, and then the transparent vessel is irradiated for 10 seconds to 3000 seconds under the ultraviolet rays or visible light, and the explant is taken out, so that disinfection is completed.
2. The method of sterilizing orchid explants according to claim 1, wherein the concentration of the nanosilver sol is 1mg/L to 1000mg/L calculated as silver content.
3. The method of sterilizing orchid explants according to claim 1, wherein the nano silver in the nano silver sol has a particle size of 1-100nm.
4. The method of claim 1, wherein the transparent vessel is a quartz vessel.
5. The method of sterilization of orchid explants according to claim 1, wherein the ultraviolet radiation has a wavelength of 10-400nm.
6. The method of sterilization of orchid explants according to claim 1, wherein the visible light radiation has a wavelength of 400-800nm.
7. The method of claim 1, wherein the ultraviolet or visible light has a light intensity of 1.65x10 13 ～1.98×10 18 photons/sec/cm 3 。
8. The method of sterilization of orchid explants according to claim 1, wherein the irradiation is performed under ultraviolet or visible light for 60 seconds to 600 seconds.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211647583.8A CN116195513B (en) | 2022-12-21 | 2022-12-21 | Method for sterilizing orchid explant |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211647583.8A CN116195513B (en) | 2022-12-21 | 2022-12-21 | Method for sterilizing orchid explant |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116195513A true CN116195513A (en) | 2023-06-02 |
| CN116195513B CN116195513B (en) | 2024-07-19 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211647583.8A Active CN116195513B (en) | 2022-12-21 | 2022-12-21 | Method for sterilizing orchid explant |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116195513B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106973796A (en) * | 2017-06-01 | 2017-07-25 | 广东鼎峰生态农业开发有限公司 | A kind of tissue cultivating and seedling method of Idesia polycarpa |
| CN109601386A (en) * | 2019-01-24 | 2019-04-12 | 北京农业职业学院 | The sterilization method of bud explant is cut in a kind of state orchid stem tip tissue culture |
| WO2021258738A1 (en) * | 2021-01-14 | 2021-12-30 | 中国科学院华南植物园 | Tissue culture and rapid propagation method for high-quality hippeastrum vittatum seedlings |
| CN114532227A (en) * | 2022-03-07 | 2022-05-27 | 上海交通大学 | Method for inducing and proliferating calluses of agapanthus radicis roots and tips |
-
2022
- 2022-12-21 CN CN202211647583.8A patent/CN116195513B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106973796A (en) * | 2017-06-01 | 2017-07-25 | 广东鼎峰生态农业开发有限公司 | A kind of tissue cultivating and seedling method of Idesia polycarpa |
| CN109601386A (en) * | 2019-01-24 | 2019-04-12 | 北京农业职业学院 | The sterilization method of bud explant is cut in a kind of state orchid stem tip tissue culture |
| WO2021258738A1 (en) * | 2021-01-14 | 2021-12-30 | 中国科学院华南植物园 | Tissue culture and rapid propagation method for high-quality hippeastrum vittatum seedlings |
| CN114532227A (en) * | 2022-03-07 | 2022-05-27 | 上海交通大学 | Method for inducing and proliferating calluses of agapanthus radicis roots and tips |
Non-Patent Citations (3)
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|---|
| 何国庆 等: "《环境工程微生物实验教程》", 中国农业大学出版社, pages: 139 * |
| 徐正伟 等: "《纳米科技探索：科普与实验.上册》", 30 June 2022, 苏州大学出版社, pages: 48 - 49 * |
| 黄崇杏 等: "《可持续包装=Sustainable Packaging》", 31 March 2022, 中国农业大学出版社, pages: 184 * |
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| Publication number | Publication date |
|---|---|
| CN116195513B (en) | 2024-07-19 |
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