CN117158321A - Tissue culture propagation-expanding explant treatment and disinfection method for fritillaria thunbergii - Google Patents
Tissue culture propagation-expanding explant treatment and disinfection method for fritillaria thunbergii Download PDFInfo
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- CN117158321A CN117158321A CN202311361922.0A CN202311361922A CN117158321A CN 117158321 A CN117158321 A CN 117158321A CN 202311361922 A CN202311361922 A CN 202311361922A CN 117158321 A CN117158321 A CN 117158321A
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- soaking
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- sterilizing
- tissue culture
- seed balls
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- 238000004659 sterilization and disinfection Methods 0.000 title claims abstract description 24
- 238000000034 method Methods 0.000 title claims abstract description 19
- 241001547125 Fritillaria thunbergii Species 0.000 title claims description 6
- 241000935235 Fritillaria meleagris Species 0.000 claims abstract description 19
- 230000006698 induction Effects 0.000 claims abstract description 5
- 238000002791 soaking Methods 0.000 claims description 37
- 230000001954 sterilising effect Effects 0.000 claims description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 14
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 14
- 230000008859 change Effects 0.000 claims description 12
- 229960002523 mercuric chloride Drugs 0.000 claims description 12
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 12
- 238000011010 flushing procedure Methods 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000008223 sterile water Substances 0.000 claims description 6
- 239000008399 tap water Substances 0.000 claims description 6
- 235000020679 tap water Nutrition 0.000 claims description 6
- 238000009423 ventilation Methods 0.000 claims description 6
- 210000003097 mucus Anatomy 0.000 claims description 4
- 229920000742 Cotton Polymers 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 238000004506 ultrasonic cleaning Methods 0.000 claims description 3
- 238000005520 cutting process Methods 0.000 claims description 2
- 239000001963 growth medium Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 7
- 239000000645 desinfectant Substances 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 3
- 238000004134 energy conservation Methods 0.000 abstract description 2
- 239000007788 liquid Substances 0.000 abstract description 2
- 230000009467 reduction Effects 0.000 abstract description 2
- 230000004083 survival effect Effects 0.000 abstract description 2
- 239000002699 waste material Substances 0.000 abstract description 2
- 230000035784 germination Effects 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 description 2
- 230000000249 desinfective effect Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 206010011224 Cough Diseases 0.000 description 1
- 241000234280 Liliaceae Species 0.000 description 1
- 206010036790 Productive cough Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000002845 discoloration Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 210000003802 sputum Anatomy 0.000 description 1
- 208000024794 sputum Diseases 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of disinfection of fritillary tissue culture explants, and particularly discloses a fritillary tissue culture propagation-enlarging explant treatment disinfection method. The method can effectively improve the survival rate of the tissue culture explant and the success rate of the induction culture, the activity and the quality of the seed balls can also be improved, the higher success rate of the induction culture can effectively improve the production efficiency, the disinfection method can greatly reduce the usage amount of disinfectant, reduce the production amount of disinfection waste liquid, and play a role in energy conservation and emission reduction.
Description
Technical Field
The invention relates to the technical field of disinfection of fritillary tissue culture explants, in particular to a method for treating and disinfecting a fritillary tissue culture propagation-expanding explant.
Background
The fritillary bulb is a plant for a plurality of years of crude drugs in the lily family, is used as a medicament by bulbs, has the effects of relieving cough, reducing sputum, clearing heat and moistening lung, is a famous "Zheeight Chinese medicinal material in Zhejiang province, and has three hundred years of artificial cultivation history so far. However, in artificial cultivation, the bulb is adopted for the production of the fritillary bulb, the propagation coefficient is low and is about 1.6-1.8 times, the seed consumption is extremely large, the seed consumption is 500 kg/mu, the low propagation coefficient and the high seed consumption are caused, the production land and the seed reserving area are increased, the production cost is increased, meanwhile, the fritillary bulb is seriously degraded in quality due to long-term nutrition propagation, the bulb yield is reduced, and the infection of plant viruses is also the main cause of the degradation of the fritillary bulb.
Tissue culture can obviously improve the reproduction rate of the fritillary bulb, and the growth cycle is obviously shortened. However, considering the problem of virus infection, and the performance of the traditional explant disinfection means is unsatisfactory, the success rate of the traditional disinfection method is low, and the tissue culture of the explant is greatly influenced, so that the development of a method for treating and disinfecting the explant by tissue culture propagation of fritillaria thunbergii is necessary.
Disclosure of Invention
The invention aims at solving the problems and provides a method for treating and sterilizing a tissue culture propagation-expanding explant of fritillaria thunbergii.
In order to achieve the above purpose, the specific method steps of the scheme are as follows:
s1, healthy seed balls of fritillary bulb in the current year are selected, the seed balls are primarily cleaned by using a hand sanitizer or a washing powder solution, and then the surfaces are cleaned by tap water;
s2, peeling the surfaces of the seed balls, and flushing with flowing tap water;
s3, soaking the seed balls with the surfaces removed and cleaned in warm water until no obvious mucus exists on the surfaces of the seed balls, and then fishing out;
s4, after the seed balls are fished out, sucking the surface moisture of the seed balls in an ultra-clean workbench by using a sterile cotton towel, and placing the seed balls in the ventilation state of the ultra-clean workbench for 4-6 hours;
s5, soaking the seed balls which are placed through ventilation in alcohol for disinfection, taking out, and flushing with sterile water;
s6, soaking and sterilizing the cleaned seed balls in mercuric chloride or sodium hypochlorite solution for the first time; after the seed ball is taken out, observing the external color change condition of the seed ball, discarding the seed ball with the external color change area exceeding 20 percent and with the external color change area smaller than 20 percent, cutting off the external color change part of the seed ball by a sterilized scalpel, and placing the seed ball in mercuric chloride or sodium hypochlorite solution again for secondary soaking sterilization;
s7, washing the seed balls sterilized by the mercuric chloride and the sodium hypochlorite with sterile water for 3-4 times, and inoculating the seed balls to an induction culture medium for culture.
Further, preferably, the temperature of the warm water is 45 ℃, the soaking time of the warm water is 30 minutes, the soaking time can be properly increased, and the soaking time is not more than 50 minutes, wherein the soaking time is based on that no obvious mucus exists on the surfaces of fritillary bulbs.
Further, preferably, the alcohol concentration is 75%, and the alcohol soaking time is 30 to 50 seconds.
Further, preferably, the concentration of the mercuric chloride solution is 0.1%, the concentration of the sodium hypochlorite solution is 0.3-0.5%, and the first time of soaking and sterilizing the mercuric chloride and the sodium hypochlorite solution is 15-20 minutes, and the second time is 8-15 minutes.
Further, during the second soaking and disinfection, ultrasonic cleaning is started, the temperature in the soaking container is controlled to be 0-5 ℃ and the holding time is controlled to be 6-10 minutes, and then the temperature in the soaking container is controlled to be 60-70 ℃ and the holding time is controlled to be 2-3 minutes.
The invention has the beneficial effects that: the invention relates to a method for treating and sterilizing a tissue culture propagation explant of fritillary bulb, which adopts the conventional cleaning and sterilizing measures, and adopts the steps of washing with flowing water, soaking treatment with warm water, standing and blowing in an ultra-clean bench, soaking and sterilizing with mercuric chloride and sodium hypochlorite, peeling off the outer disinfection and color-changing cortex, soaking and sterilizing twice again and the like to comprehensively treat the tissue culture explant, so that the survival rate of the tissue culture explant can be effectively improved, the success rate of the induced culture of the explant after the sterilization treatment is greatly improved, and the activity and quality of a seed ball can be improved. The invention can greatly reduce the usage amount of disinfectant, reduce the production amount of disinfectant waste liquid, play a role in energy conservation and emission reduction, and effectively improve the production efficiency with higher success rate of induced culture.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to fall within the scope of the invention.
Example 1
In one embodiment of the invention, a method for treating and sterilizing a tissue-cultured and propagated explant of fritillaria thunbergii comprises the following steps:
s1, healthy seed balls of fritillary bulb in the current year are selected, the seed balls are primarily cleaned by using a hand sanitizer or a washing powder solution, and then the surfaces are cleaned by tap water;
s2, peeling the surfaces of the seed balls, and flushing with flowing tap water;
s3, soaking the seed balls with the surfaces removed and cleaned in warm water at 45 ℃ until no obvious mucus is on the surfaces of the seed balls, and then fishing out the seed balls, wherein the soaking time of the seed balls is 30 minutes;
s4, after the seed balls are fished out, sucking water on the surface of the seed balls in an ultra-clean workbench by using a sterile cotton towel, and placing the seed balls in a ventilation state of the ultra-clean workbench for 5 hours;
s5, soaking the seed balls subjected to ventilation placement in 75% alcohol for sterilization for 40 seconds, taking out, and flushing with sterile water;
s6, soaking and sterilizing the cleaned seed balls in 0.1% mercuric chloride or 0.4% sodium hypochlorite solution for the first time, wherein the time for the first soaking and sterilizing is 15 minutes; after the seed ball is taken out, observing the external color change condition of the seed ball, discarding the seed ball with the external color change area exceeding 20% and the external color change area smaller than 20%, removing the external color change part of the seed ball by using a sterilized scalpel, soaking and sterilizing the seed ball in 0.1% mercuric chloride or 0.4% sodium hypochlorite solution for the second time, starting ultrasonic cleaning when the seed ball is soaked and sterilized for the second time, controlling the temperature in a soaking container to be 5 ℃ for 8 minutes, and then controlling the temperature in the soaking container to be 65 ℃ for 2 minutes;
s7, washing the seed balls sterilized by the mercury chloride and the sodium hypochlorite for 3 times by using sterile water, and inoculating the seed balls to an induction medium for culture.
Example two
In this example, disinfection and tissue culture experiments were performed using the conventional method and the method of the present invention, respectively, and the specific results are as follows:
| project | Traditional disinfection treatment mode A | Disinfection treatment mode B of this embodiment |
| Number of explants | 20 seed balls | 20 seed balls |
| Number of successful disinfection | 3 pieces of | 12 |
| Success rate of disinfection | 15％ | 60％ |
| Number of normal germination | 2 | 10 |
| Germination rate | 10％ | 50％ |
| Culturing for 50 days | 4-5 bud balls | 30-35 bud balls |
| Germination rate | 20-25％ | 150-175％ |
| Subculture 45 days of germination | 8-10 | 60-100 |
To sum up: the invention provides a method for treating and sterilizing a tissue culture propagation explant of fritillary bulb, which utilizes comprehensive treatment of steps of running water flushing, warm water soaking treatment, standing and blowing in an ultra-clean bench, mercury chloride and sodium hypochlorite soaking sterilization, and peeling off the outer disinfection and discoloration cortex, and the like.
Finally, it should be noted that: the foregoing description is only illustrative of the preferred embodiments of the present invention, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described, or equivalents may be substituted for elements thereof, and any modifications, equivalents, improvements or changes may be made without departing from the spirit and principles of the present invention.
Claims (5)
1. The method for treating and sterilizing the fritillary bulb tissue culture propagation explant is characterized by comprising the following steps of:
s1, healthy seed balls of fritillary bulb in the current year are selected, the seed balls are primarily cleaned by using a hand sanitizer or a washing powder solution, and then the surfaces are cleaned by tap water;
s2, peeling the surfaces of the seed balls, and flushing with flowing tap water;
s3, soaking the seed balls with the surfaces removed and cleaned in warm water until no obvious mucus exists on the surfaces of the seed balls, and then fishing out;
s4, after the seed balls are fished out, sucking the surface moisture of the seed balls in an ultra-clean workbench by using a sterile cotton towel, and placing the seed balls in the ventilation state of the ultra-clean workbench for 4-6 hours;
s5, soaking the seed balls which are placed through ventilation in alcohol for disinfection, taking out, and flushing with sterile water;
s6, soaking and sterilizing the cleaned seed balls in mercuric chloride or sodium hypochlorite solution for the first time; after the seed ball is taken out, observing the external color change condition of the seed ball, discarding the seed ball with the external color change area exceeding 20 percent and with the external color change area smaller than 20 percent, cutting off the external color change part of the seed ball by a sterilized scalpel, and placing the seed ball in mercuric chloride or sodium hypochlorite solution again for secondary soaking sterilization;
s7, washing the seed balls sterilized by the mercuric chloride and the sodium hypochlorite with sterile water for 3-4 times, and inoculating the seed balls to an induction culture medium for culture.
2. The method for treating and sterilizing a tissue culture propagation-expanding explant of fritillary bulb according to claim 1, wherein the temperature of the warm water in the step S3 is 45 ℃, and the soaking time is 30-50 minutes.
3. The method for treating and sterilizing a tissue culture propagation-expanding explant of fritillaria thunbergii according to claim 1, wherein the alcohol concentration in the step S5 is 75%, and the soaking and sterilizing time is 30-50 seconds.
4. The method for treating and sterilizing the tissue culture propagation explant of fritillary bulb according to claim 1, wherein in the step S6, the concentration of the mercuric chloride solution is 0.1%, the concentration of the sodium hypochlorite solution is 0.3-0.5%, the first soaking and sterilizing time is 15-20 minutes, and the second soaking and sterilizing time is 8-15 minutes.
5. The method for treating and sterilizing the tissue culture propagation explant of fritillary bulb according to claim 4, wherein the ultrasonic cleaning is started in the second soaking and sterilizing step, the temperature in the soaking container is controlled to be 0-5 ℃ for 6-10 minutes, and then the temperature in the soaking container is controlled to be 60-70 ℃ for 2-3 minutes.
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| CN202311361922.0A CN117158321A (en) | 2023-10-20 | 2023-10-20 | Tissue culture propagation-expanding explant treatment and disinfection method for fritillaria thunbergii |
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| CN202311361922.0A CN117158321A (en) | 2023-10-20 | 2023-10-20 | Tissue culture propagation-expanding explant treatment and disinfection method for fritillaria thunbergii |
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|---|---|---|---|---|
| CN103283601A (en) * | 2013-05-31 | 2013-09-11 | 常熟市佳盛农业科技发展有限公司 | Method for rapidly propagating Hupeh fritillary by utilizing tissue culture technology |
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2023
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Patent Citations (6)
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