CN111642392A - Sterilization method for surface of dahlia explant - Google Patents
Sterilization method for surface of dahlia explant Download PDFInfo
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- CN111642392A CN111642392A CN202010355528.6A CN202010355528A CN111642392A CN 111642392 A CN111642392 A CN 111642392A CN 202010355528 A CN202010355528 A CN 202010355528A CN 111642392 A CN111642392 A CN 111642392A
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- explant
- dahlia
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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
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- Developmental Biology & Embryology (AREA)
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Abstract
The invention provides a dahlia explant surface sterilization method, which is characterized by comprising the following steps: collecting the current year healthy receptacle of the dahlia in the month of 4, washing the receptacle with washing powder, and rinsing in running water for 30 min. Transferring into sterile room, sterilizing receptacle with 70% ethanol solution for 15-30s, washing with sterile water for 2-3 times (1-2 min each time), sterilizing with 0.1% mercuric chloride solution for 8-10min, repeatedly washing with sterile water for 3-5 times (1-2 min each time), sucking water with absorbent paper, and inoculating into culture medium. The callus obtained by the sterilization method has high induction rate, low pollution rate, relatively simple operation and easy realization. Compared with the prior art, the method is improved, an ideal explant can be cultured, and the method has good adaptability and high survival rate in tissue culture.
Description
Technical Field
The invention relates to a method for sterilizing the surface of a plant explant, belonging to the technical field of plant tissue culture.
Background
Dahlia (Dahlia pinnata Cav.), namely margarita, peonies, aster, Dahlia, pachyrhizus, and the like, belong to the family Compositae and the genus Dahlia, are perennial herbs, and have huge rod-shaped root tubers. The stem is upright and has multiple branches, and the flower is a common herb flower in developed countries in Europe and America. Because the plant is beautiful, the flower shape of the flower is famous and various, and is rich and colorful, the adaptability to the environment is strong, and the plant is often applied to gardens.
Dahlia propagation traditionally employs cutting propagation and plant division propagation. The cutting propagation generation has large variation and is difficult to maintain the excellent characters of the original variety; the propagation coefficient of the plant is low, and the requirement of industrial production cannot be met.
On the basis of previous research, the invention takes the tender receptacle as an explant to carry out tissue culture so as to quickly realize the supply of a large number of seedlings.
Disclosure of Invention
The invention aims to provide a dahlia explant surface sterilization method, which has the advantages that the callus obtained by the sterilization method is high in induction rate, low in pollution rate, relatively simple to operate and easy to realize.
It comprises the following steps:
1) selection of explants: collecting the current-year healthy receptacle of the dahlia in 4 months;
2) pretreatment of explants: washing the surface dirt of the explant by using a washing powder solution;
3) and (3) disinfection of explants: transferring into sterile room, and sterilizing with 70% ethanol solution and 0.1% mercuric chloride solution.
The sterilization method comprises the step 1) of selecting explants, and preferably healthy plants with current-year healthy receptacle of dahlia as explants. The current-year healthy receptacle is selected as the explant, which is beneficial to quickly forming a large amount of callus, has low pollution rate, is easy to form cluster buds, has strong buds and high quality, and is an ideal explant material.
The sterilization method comprises the following steps of 2) pretreating explants, cleaning dirt on the surfaces of the explants by using a washing powder solution with the concentration of 10-20ppm, and then washing the surface of the explants clean by using tap water.
The sterilization method comprises the steps of 3) sterilizing explants, transferring the explants to a sterile room, sterilizing receptacle on an ultra-clean workbench for 15-30s by using 70% ethanol solution, washing the receptacle with sterile water for 2-3 times, sterilizing the receptacle with 0.1% mercuric chloride solution for 8-10min, repeatedly washing the receptacle with sterile water for 3-5 times, and inoculating the receptacle to a culture medium after absorbing water with absorbent paper.
After each disinfection, the bottles were rinsed with sterile water to remove the mercuric chloride completely. The materials are fully stirred during disinfection and rinsing, so that all parts of the materials are fully contacted with the disinfectant or sterile water. Preferably, the product is rinsed with sterile water for 3-5 times, each time for at least 2 min.
After the explants are washed by sterile water, the water is absorbed by absorbent paper and then the explants are inoculated in a culture medium.
The sterilization operation is carried out on an ultra-clean workbench in a sterile room.
Compared with the prior art, the explant sterilization method provided by the invention is improved, an ideal explant can be cultured, the explant has good adaptability in tissue culture, can quickly form a large amount of callus, is low in pollution rate, is easy to form cluster buds, is robust in buds and is high in quality.
Detailed Description
The following description will explain embodiments of the present invention by referring to application examples of the method for tissue culture sterilization of plants of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
1. Preparing a disinfection vessel:
selecting a 250ml plastic tissue culture bottle, cleaning, and sterilizing with high-pressure steam for later use.
2. Preparation of a disinfectant:
autoclaved sterile water, 70% ethanol solution, 75% ethanol solution, 0.1% mercuric chloride solution, 0.2% mercuric chloride solution, 0.3% mercuric chloride solution.
3. Preparation of the instrument: ,
sterilizing filter paper, scalpel, scissors, tweezers and absorbent paper.
Example (b):
collecting the current year healthy receptacle of the dahlia in the month of 4, washing the receptacle with washing powder, and rinsing in running water for 30 min. Transferring into sterile room, sterilizing receptacle with 70% ethanol solution for 15-30s, washing with sterile water for 2-3 times (1-2 min each time), sterilizing with 0.1% mercuric chloride solution for 8-10min, repeatedly washing with sterile water for 3-5 times (1-2 min each time), sucking water with absorbent paper, and inoculating into culture medium. Inoculating the seeds, and culturing at the temperature of 22 +/-2 ℃ under the illumination intensity of 1500-2000 lx for 14h every day. After the receptacle is inoculated for 18 days, a large amount of callus is formed, namely the callus is differentiated into new buds.
Experimental example 1
The embodiment provides a surface sterilization method of dahlia explants, which comprises the following steps:
1) selection of explants: selecting a current-year healthy receptacle of dahlia;
2) pretreatment of explants: soaking the clothes washing powder with the concentration of 20ppm in water for 15min, and washing the clothes washing powder for 30min in running water under tap water;
3) and (3) disinfection of explants: soaking and sterilizing with 70% ethanol solution for 30s, and soaking and sterilizing with 0.1% mercuric chloride solution for 10 min; rinsing with sterile water for 5 times, each for 2 min; and then averagely cutting the explant into 3-4 small blocks on a clean bench and inoculating the small blocks into a prepared culture medium to obtain the explant.
Experimental example 2
The embodiment provides a surface sterilization method of dahlia explants, which comprises the following steps:
1) selection of explants: selecting a current-year healthy receptacle of dahlia;
2) pretreatment of explants: soaking the clothes washing powder with the concentration of 20ppm in water for 15min, and washing the clothes washing powder for 30min in running water under tap water;
3) and (3) disinfection of explants: soaking and sterilizing with 70% ethanol solution for 30s, and soaking and sterilizing with 0.2% mercuric chloride solution for 10 min; rinsing with sterile water for 5 times, each for 2 min; and then averagely cutting the explant into 3-4 small blocks on a clean bench and inoculating the small blocks into a prepared culture medium to obtain the explant.
Experimental example 3
The embodiment provides a surface sterilization method of dahlia explants, which comprises the following steps:
1) selection of explants: selecting a current-year healthy receptacle of dahlia;
2) pretreatment of explants: soaking the clothes washing powder with the concentration of 20ppm in water for 15min, and washing the clothes washing powder for 30min in running water under tap water;
3) and (3) disinfection of explants: soaking and sterilizing with 75% ethanol solution for 30s, and soaking and sterilizing with 0.3% mercuric chloride solution for 10 min; rinsing with sterile water for 5 times, each for 2 min; and then averagely cutting the explant into 3-4 small blocks on a clean bench and inoculating the small blocks into a prepared culture medium to obtain the explant.
Test examples
The dahlia explants prepared in the above embodiments are inoculated into a culture medium, and the culture is carried out after inoculation at the temperature of 22 +/-2 ℃, the illumination intensity is 1500-2000 lx, and the dahlia explants are irradiated for 14h every day. After 18 days of culture, the culture was observed.
The culture medium comprises 6-BA 2.0 mg.L-1 + KT 4.0 mg.L-1 + NAA 0.5 mg.L-1 +3.0g/L sucrose +0.7g/L agar, and the pH value is 5.8.
The detection result is as follows:
according to the comprehensive experiment result, the dahlia selects receptacle as an explant, and is inoculated in a formula culture medium after disinfection treatment, wherein 6-BA 2.0 mg.L-1 + KT 4.0 mg.L-1 + NAA 0.5 mg.L-1 +3.0g/L sucrose +0.7g/L agar can quickly form a large amount of callus, and the pollution rate is low.
Claims (7)
1. A dahlia explant surface sterilization method is characterized in that: the method comprises the following steps:
1) selection of explants: collecting the current-year healthy receptacle of the dahlia in 4 months;
2) pretreatment of explants: washing the surface dirt of the explant by using a washing powder solution;
3) and (3) disinfection of explants: transferring into sterile room, and sterilizing with 70% ethanol solution and 0.1% mercuric chloride solution.
2. The method for sterilizing the surface of a dahlia explant according to claim 1, wherein: step 1) collecting the current-year healthy receptacle of the dahlia in 4 months.
3. The method for sterilizing the surface of a dahlia explant according to claim 2, wherein: and 3) sterilizing the ethanol solution for 15-30 s.
4. The method for sterilizing the surface of a dahlia explant according to claim 3, wherein: and 3) sterilizing the mercuric chloride solution for 8-10 min.
5. The method for sterilizing the surface of a dahlia explant according to any one of claims 1-4, wherein: step 3) sterilization of the explant: the explants were sterilized with 70% ethanol solution for 15-30s and then with 0.1% mercuric chloride solution for 8-10 min.
6. The method for sterilizing the surface of a dahlia explant according to any one of claims 1-5, wherein: step 2) pretreating the explant, washing the explant by using a washing powder solution with the concentration of 10-20ppm, and then washing the explant by using tap water.
7. The method for sterilizing the surface of a dahlia explant according to any one of claims 1-6, wherein: rinsing with sterile water for 3-5 times (not less than 2min each time).
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| Application Number | Priority Date | Filing Date | Title |
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| CN202010355528.6A CN111642392A (en) | 2020-04-29 | 2020-04-29 | Sterilization method for surface of dahlia explant |
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| CN202010355528.6A CN111642392A (en) | 2020-04-29 | 2020-04-29 | Sterilization method for surface of dahlia explant |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| USPP11526P (en) * | 1999-01-19 | 2000-09-26 | Ruigrok; Frank N. G. | Dahlia plant named `Ruivi` |
| CN105028214A (en) * | 2015-09-10 | 2015-11-11 | 宋立胜 | Efficient expanding propagation method for dahlia toxin-free seedlings |
| CN105123524A (en) * | 2015-09-10 | 2015-12-09 | 宋立胜 | Germplasm preservation method of dahlia tissue culture propagation |
| CN105210864A (en) * | 2015-09-10 | 2016-01-06 | 宋立胜 | A kind of acquisition methods of dahlia detoxic seedling |
| CN107094628A (en) * | 2017-06-03 | 2017-08-29 | 合肥雪立歆农业科技有限公司 | A kind of cultural method of dahlia |
-
2020
- 2020-04-29 CN CN202010355528.6A patent/CN111642392A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| USPP11526P (en) * | 1999-01-19 | 2000-09-26 | Ruigrok; Frank N. G. | Dahlia plant named `Ruivi` |
| CN105028214A (en) * | 2015-09-10 | 2015-11-11 | 宋立胜 | Efficient expanding propagation method for dahlia toxin-free seedlings |
| CN105123524A (en) * | 2015-09-10 | 2015-12-09 | 宋立胜 | Germplasm preservation method of dahlia tissue culture propagation |
| CN105210864A (en) * | 2015-09-10 | 2016-01-06 | 宋立胜 | A kind of acquisition methods of dahlia detoxic seedling |
| CN107094628A (en) * | 2017-06-03 | 2017-08-29 | 合肥雪立歆农业科技有限公司 | A kind of cultural method of dahlia |
Non-Patent Citations (2)
| Title |
|---|
| OTANI,YUKO等: "Highly efficient system for plant regeneration from leaf and stem explants in Dahlia", 《PLANT BIOTECHNOLOGY》 * |
| 韦三立: "大丽花茎段的离体快速繁殖", 《北京农业大学学报》 * |
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