CN114375832A - Method for quickly obtaining aseptic eichhornia crassipes seedlings - Google Patents
Method for quickly obtaining aseptic eichhornia crassipes seedlings Download PDFInfo
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- CN114375832A CN114375832A CN202111499985.3A CN202111499985A CN114375832A CN 114375832 A CN114375832 A CN 114375832A CN 202111499985 A CN202111499985 A CN 202111499985A CN 114375832 A CN114375832 A CN 114375832A
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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
- A01H4/008—Methods for regeneration to complete plants
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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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Abstract
The invention discloses a method for quickly obtaining an eichhornia crassipes aseptic seedling, which comprises the steps of pretreating an eichhornia crassipes plant to obtain a degerminated body; inserting the bud body into a solid culture medium, culturing at 28 ℃ in a dark mode, transferring the bud body into the solid culture medium to culture until a new bud grows out, cutting off a blackening and ageing part, transferring the bud body into the solid culture medium, culturing at 28 ℃ in the dark mode, culturing in the solid culture medium until the new bud grows out on a stolon, cutting off the stolon at the base part of the new bud by using a dissecting knife, removing the stolon at the base part of the new bud, transferring the bud body into the solid culture medium to culture until the new bud grows out on the stolon; repeating the previous step; removing the mother plant after the stolons, cutting off old leaves and black rot tissues, transferring the mother plant into a liquid culture medium 1 for culture, removing the black rot tissues and redundant stolons if the stolons grow out, transferring the mother plant into the liquid culture medium 1, and transferring the mother plant into a liquid culture medium 2; circularly culturing to obtain sterile filial generation eichhornia crassipes. The method effectively removes the bacterial pollution of the eichhornia crassipes, obtains aseptic seedlings with larger population quantity in a shorter time, and has strong replication performance and easy operation.
Description
Technical Field
The invention relates to the technical field of water hyacinth tissue culture, in particular to a method for quickly obtaining water hyacinth aseptic seedlings.
Background
Eichhornia crassipes (Eichhornia crassipes) is also called water hyacinth, is a floating aquatic plant, can be used as feed and fertilizer, has extremely strong pollution resistance, can effectively remove N, P, K, phenol, cyanogen, organic matters, heavy metals and other pollutants in water, can inhibit algae from breeding, and has very important functions in sewage purification and eutrophic water body treatment. However, abundant and various microorganisms are grown on the surface of the eichhornia crassipes plant, and when the eichhornia crassipes plant is used as a test material to research a purification mechanism, the purification effect of the eichhornia crassipes plant cannot be proved to be generated by the action of the microorganisms on the surface of the eichhornia crassipes. Therefore, the aseptic seedlings obtained by the tissue culture technology are used as scientific research materials and are an important link in the research of the purification mechanism of the aseptic seedlings.
However, the water hyacinth can not leave the water surface to survive, the bud points of the plants are wrapped layer by layer, and the tender parts are often mucus which is difficult to remove, so that in the process of culturing the aseptic seedlings, microorganisms, especially bacteria, on the surfaces of the plants are difficult to completely remove, and the real aseptic seedlings are difficult to obtain.
Therefore, how to provide a method for rapidly obtaining the aseptic eichhornia crassipes seedlings is a problem to be solved urgently by the technical personnel in the field.
Disclosure of Invention
In view of the above, the invention provides a rapid propagation method for rapidly obtaining aseptic seedlings of eichhornia crassipes.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for quickly obtaining an eichhornia crassipes aseptic seedling comprises the following steps:
1) pretreatment: pretreating the eichhornia crassipes plant to obtain a disinfected and air-dried bud body;
2) tissue culture: inserting the bud body obtained in the step 1) into a solid culture medium, culturing for 12h in the dark at 28 ℃, transferring into the solid culture medium, and culturing until a new bud grows out, wherein the culture time L: d is 12: 12;
3) and (3) tissue culture again: taking out the eichhornia crassipes explants from a super clean bench, cutting off blackened and aged parts by using a scalpel, transferring the cut parts into a solid culture medium, culturing the cut parts in the dark at the temperature of 28 ℃ for 12 hours, and then, carrying out the culture in the dark for 12 hours: d is 12: 12 in a solid medium;
4) transferring: culturing until the stolons grow up to new buds, cutting off the new buds with a dissecting knife, removing the stolons at the base parts of the new buds, transferring the new buds into a solid culture medium at the temperature of 28 ℃, and culturing the new buds in a culture medium at the temperature of L: d is 12: culturing in an environment of 12 till new buds grow on the stolons; repeating the step 3);
5) and (3) proliferation: removing the mother plant after stolons, cutting off old leaves and black rot tissues, transferring the cut-off mother plant into a liquid culture medium 1, and culturing at the temperature of 28 ℃, L: d is 12: culturing for 30 days in an environment of 12 days, then transferring, if stolons grow out, cutting by using a scalpel, removing black rot tissues and redundant stolons, transferring into a liquid culture medium 1, and transferring the stock plant into a liquid culture medium 2;
6) and (3) circulating culture: repeatedly and circularly culturing to obtain sterile filial generation Eichhornia crassipes.
As a preferable embodiment of the above-mentioned technical means, in the step 1), the pretreatment specifically includes the steps of:
11) taking back the eichhornia crassipes with the stolons, culturing in clear water for 1 week, removing the stolons, cutting off leaves at a position 2-3cm away from the stem base, removing the roots, cleaning with clear water, and completely drying in a ventilated place;
12) removing the redundant petioles and stems, only leaving the bud with the largest middle part, not damaging the bud in the middle part when cutting the root, cleaning for 1-2 times by using a detergent, and air-drying;
13) after the buds are air-dried, the buds are surface-sterilized for 30s by using alcohol with the concentration of 75%, the buds are transferred to a superclean bench for operation, the buds are taken out and washed by sterile water, and the sterilized absorbent paper is dried;
14) adding 0.1% mercuric chloride into Tween-20, sterilizing for 5min, sealing in a super clean bench, cleaning with ultrasonic vibration for 2.5min, washing with sterile water for 3 times, drying with sterilized absorbent paper, and air drying on a sterile tray;
15) the excess petiole and stem tissues were excised, leaving the largest bud in the middle.
As a preferable technical scheme of the technical scheme, in the step 14), the amount of mercuric chloride is 4 drops, and the amount of tween is 100 ml.
As a preferable technical solution of the above technical solution, the formula of the solid medium includes: NH (NH)4NO3 825mg/L、KNO3 950mg/L、MgSO4.7H2O 185mg/L、KH2PO4 85mg/L、CaCl2.2H2O 220mg/L、KI 0.83mg/L、H3BO3 6.2mg/L、MnSO4.4H2O 22.3mg/L、ZnSO4.7H2O 8.6mg/L、Na2MoO4.2H2O 0.25mg/L、CuSO4.5H2O 0.025mg/L、CoCl2.6H2O 0.025mg/L、FeSO4.7H2O 13.9mg/L、Na2-EDTA.2H2O, inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, nicotinic acid 0.5mg/L, hydrolyzed milk protein 250mg/L, 6-benzylaminopurine 3mg/L, 1.5% sucrose, 0.65% agar, and pH 5.8.
As a preferable technical scheme of the technical scheme, the formula of the liquid culture medium 1 comprises: NH (NH)4NO3 825mg/L、KNO3950mg/L、MgSO4.7H2O 185mg/L、KH2PO4 85mg/L、CaCl2.2H2O 220mg/L、KI 0.83mg/L、H3BO36.2mg/L、MnSO4.4H2O 22.3mg/L、ZnSO4.7H2O 8.6mg/L、Na2MoO4.2H2O 0.25mg/L、CuSO4.5H2O 0.025mg/L、CoCl2.6H2O 0.025mg/L、FeSO4.7H2O 13.9mg/L、Na2-EDTA.2H2O, inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, nicotinic acid 0.5mg/L, 6-benzylaminopurine 1mg/L, 1% sucrose, and pH 6.5.
As a preferable technical scheme of the technical scheme, the formula of the liquid culture medium 2 comprises: NH (NH)4NO3 825mg/L、KNO3950mg/L、MgSO4.7H2O 185mg/L、KH2PO4 85mg/L、CaCl2.2H2O 220mg/L、KI 0.83mg/L、H3BO36.2mg/L、MnSO4.4H2O 22.3mg/L、ZnSO4.7H2O 8.6mg/L、Na2MoO4.2H2O 0.25mg/L、CuSO4.5H2O 0.025mg/L、CoCl2.6H2O 0.025mg/L、FeSO4.7H2O 13.9mg/L、Na2-EDTA.2H2O, inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, nicotinic acid 0.5mg/L, 1% sucrose, and pH 6.5.
As a preferable embodiment of the above-mentioned means, in the steps 2) to 4), the light intensity at the time of culturing in the solid medium is 5000 lux.
As a preferable embodiment of the above-mentioned means, the light intensity at the time of culturing in the liquid medium 1 and the liquid medium 2 is 6000 lux.
The method has the advantages that the bacterial pollution of the eichhornia crassipes can be effectively cleared, and because the bacteria are difficult to remove in the tissue culture process of the eichhornia crassipes, the bacterial pollution is gradually shown and accumulated along with the increase of the subculture quantity, the probability of the bacterial pollution of the eichhornia crassipes is reduced in the previous treatment process, and the survival rate and the quality are improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a diagram of a mature plant of Eichhornia crassipes taken in step 2) of example 1;
FIG. 2 is a diagram showing the explant obtained in step 3) of example 1;
FIG. 3 is a drawing showing a tissue culture obtained in step 5) of example 1;
FIG. 4 is a diagram of a sterile progeny seedling obtained in step 9) of the example.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. 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 embodiment of the invention provides a method for rapidly propagating an eichhornia crassipes aseptic seedling.
Example 1:
the first step is as follows: collecting mature plants of the Eichhornia crassipes with good growth vigor;
secondly, putting the mixture into clear water for culturing for 5 days; see FIG. 1;
the third step: taking back the eichhornia crassipes with stolons, culturing in clear water for 1 week, and treating about 12 hours before the experiment: removing stolons, cutting off leaves at a position 2-3cm away from the base of the stem, removing roots, cleaning with clear water, and completely drying in a ventilated place; see FIG. 2;
the fourth step: after being taken back to the laboratory, the redundant petioles and stems are removed, only the bud with the largest middle part is left, the bud is washed for 1 to 2 times by using a detergent, and the bud is placed in a fume hood for air drying.
The third step: after the buds are air-dried, the buds are surface-sterilized for 30s by using alcohol with the concentration of 75%, the buds are transferred to a super clean bench for operation, the buds are taken out and washed once by using sterile water, and the sterilized absorbent paper is sucked to be dry.
The fourth step: 100ml of 0.1% mercuric chloride is added with Tween-204 drops before use, the mixture is sealed in a super clean bench, continuously oscillated, surface sterilized for 5min, ultrasonically vibrated and cleaned for 2.5min, washed with sterile water for 3 times after treatment, blotted by sterile absorbent paper and dried on a sterile tray.
The fifth step: cutting off redundant petioles and stem tissues of the air-dried buds, only keeping the buds with the largest middle length of about 1cm, and inserting the buds into a sterilized solid culture medium. Controlling the temperature at 28 ℃, culturing in the dark for 12 hours, and transferring to the environment with the illumination intensity of 5000lux and the illumination duration of 12 hours for culturing; see FIG. 3;
and a sixth step: after the buds grow out, taking out the eichhornia crassipes explants from a super clean bench, cutting off blackened and aged parts by using a scalpel, and transferring the blackened and aged parts into a sterilized solid culture medium. The temperature is controlled at 28 ℃, the culture is carried out in the dark for 12 hours, and the culture is carried out in the environment with the illumination intensity of 5000lux and the illumination duration of 12 hours.
The seventh step: the transferred seedlings are cultured for 20d to grow into complete plants. Continuing culturing for 10 days until the stolons grow up to new buds, cutting off with a dissecting knife, removing the stolons at the base of the new buds, and culturing in a sterilized solid culture medium. And (3) cutting off the mother plant after the stolons are cut off, cutting off old leaves and black rot tissues, and transferring the cut-off mother plant into a liquid culture medium 1 for culture.
Eighth step: transferring the bud body to a solid culture medium, culturing in an environment with the temperature of 28 ℃, the illumination intensity of 5000lux and the illumination duration of 12h, growing stolons for 29d, and repeating the seventh step. Culturing the plant transferred into liquid at 28 deg.C under the condition of illumination intensity of 6000lux and illumination duration of 12h, transferring after 30d, if any stolons grow out, cutting off with scalpel, removing black rot tissue and redundant stolons, transferring into liquid culture medium 1, and transferring the stock plant into liquid culture medium 2.
The ninth step: through the steps, sterile filial generation eichhornia crassipes can be obtained through repeated circulating culture, and the plant can be used as an experimental material after stably growing on a hormone-free sterilization culture medium, as shown in figure 4;
the solid culture medium formula comprises: NH (NH)4NO3 825mg/L、KNO3 950mg/L、MgSO4.7H2O 185mg/L、KH2PO4 85mg/L、CaCl2.2H2O 220mg/L、KI 0.83mg/L、H3BO3 6.2mg/L、MnSO4.4H2O 22.3mg/L、ZnSO4.7H2O 8.6mg/L、Na2MoO4.2H2O 0.25mg/L、CuSO4.5H2O 0.025mg/L、CoCl2.6H2O 0.025mg/L、FeSO4.7H2O 13.9mg/L、Na2-EDTA.2H2O, inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, nicotinic acid 0.5mg/L, hydrolyzed milk protein 250mg/L, 6-benzylaminopurine 3mg/L, 1.5% sucrose, 0.65% agar, and pH 5.8.
The liquid culture medium 1 comprises the following components: NH (NH)4NO3 825mg/L、KNO3 950mg/L、MgSO4.7H2O 185mg/L、KH2PO4 85mg/L、CaCl2.2H2O 220mg/L、KI 0.83mg/L、H3BO3 6.2mg/L、MnSO4.4H2O 22.3mg/L、ZnSO4.7H2O 8.6mg/L、Na2MoO4.2H2O 0.25mg/L、CuSO4.5H2O 0.025mg/L、CoCl2.6H2O 0.025mg/L、FeSO4.7H2O 13.9mg/L、Na2-EDTA.2H2O, inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, nicotinic acid 0.5mg/L, 6-benzylaminopurine 1mg/L, 1% sucrose, and pH 6.5.
The liquid culture medium 2 comprises the following formula: NH (NH)4NO3 825mg/L、KNO3 950mg/L、MgSO4.7H2O 185mg/L、KH2PO4 85mg/L、CaCl2.2H2O 220mg/L、KI 0.83mg/L、H3BO3 6.2mg/L、MnSO4.4H2O 22.3mg/L、ZnSO4.7H2O 8.6mg/L、Na2MoO4.2H2O 0.25mg/L、CuSO4.5H2O 0.025mg/L、CoCl2.6H2O 0.025mg/L、FeSO4.7H2O 13.9mg/L、Na2-EDTA.2H2O, inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, nicotinic acid 0.5mg/L, 1% sucrose, and pH 6.5.
Example 2:
the first step is as follows: collecting mature plants of the Eichhornia crassipes with good growth vigor;
secondly, putting the mixture into clear water for culturing for 5 days;
the third step: selecting a stout plant of Eichhornia crassipes, removing redundant petioles and stems, only leaving the bud with the largest middle, cleaning for 1-2 times by using a detergent, and air-drying in a fume hood.
The fourth step: after the buds are air-dried, the buds are surface-sterilized for 30s by using alcohol with the concentration of 75%, the buds are transferred to a super clean bench for operation, the buds are taken out and washed once by using sterile water, and the sterilized absorbent paper is sucked to be dry.
The fifth step: 100ml of 0.1% mercuric chloride is added with Tween-204 drops before use, the mixture is sealed in a super clean bench, continuously oscillated, surface sterilized for 8min, washed with sterile water for 3 times after treatment, sterilized absorbent paper is blotted dry, and the mixture is put on a sterile tray for air drying.
And a sixth step: cutting off redundant petioles and stem tissues of the air-dried buds, only keeping the buds with the largest middle length of about 1cm, and inserting the buds into a sterilized solid culture medium. The temperature is controlled at 28 ℃, the culture is carried out in the dark for 12 hours, and the culture is carried out in the environment with the illumination intensity of 5000lux and the illumination duration of 12 hours.
The seventh step: after the buds grow out, taking out the eichhornia crassipes explants from a super clean bench, cutting off the blackened and aged parts by using a scalpel, cutting off the parts as far away from the culture medium as possible if bacterial pollution exists, and transferring the parts into a sterilized solid culture medium. The temperature is controlled at 28 ℃, the culture is carried out in the dark for 12 hours, and the culture is carried out in the environment with the illumination intensity of 5000lux and the illumination duration of 12 hours.
Eighth step: continuously generating bacterial contamination in the culture process, and adopting the method of the seventh step for subculturing.
The ninth step: the transferred seedlings are cultured for 20d to grow into complete plants. Continuing culturing for 10 days until the stolons grow up to new buds, cutting off with a dissecting knife, removing the stolons at the base of the new buds, and culturing in a sterilized solid culture medium. And (3) cutting off the mother plant after the stolons are cut off, cutting off old leaves and black rot tissues, and transferring the cut-off mother plant into a liquid culture medium 1 for culture.
The tenth step: transferring the bud body to a solid culture medium, culturing in an environment with the temperature of 28 ℃, the illumination intensity of 5000lux and the illumination duration of 12h, growing stolons for 29d, and repeating the seventh step. Culturing the plant transferred into liquid at 28 deg.C under the condition of illumination intensity of 6000lux and illumination duration of 12h, transferring after 30d, if any stolons grow out, cutting off with scalpel, removing black rot tissue and redundant stolons, transferring into liquid culture medium 1, and transferring the stock plant into liquid culture medium 2.
The eleventh step: through the steps, sterile filial generation eichhornia crassipes can be obtained through repeated circulating culture, and the plant can be used as an experimental material after stably growing on a hormone-free sterilization culture medium.
The solid culture medium formula comprises: NH (NH)4NO3 825mg/L、KNO3 950mg/L、MgSO4.7H2O 185mg/L、KH2PO4 85mg/L、CaCl2.2H2O 220mg/L、KI 0.83mg/L、H3BO3 6.2mg/L、MnSO4.4H2O 22.3mg/L、ZnSO4.7H2O 8.6mg/L、Na2MoO4.2H2O 0.25mg/L、CuSO4.5H2O 0.025mg/L、CoCl2.6H2O 0.025mg/L、FeSO4.7H2O 13.9mg/L、Na2-EDTA.2H2O, inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, nicotinic acid 0.5mg/L, hydrolyzed milk protein 250mg/L, 6-benzylaminopurine 3mg/L, 1.5% sucrose, 0.65% agar, and pH 5.8.
The liquid culture medium 1 comprises the following components: NH (NH)4NO3 825mg/L、KNO3 950mg/L、MgSO4.7H2O 185mg/L、KH2PO4 85mg/L、CaCl2.2H2O 220mg/L、KI 0.83mg/L、H3BO3 6.2mg/L、MnSO4.4H2O 22.3mg/L、ZnSO4.7H2O 8.6mg/L、Na2MoO4.2H2O 0.25mg/L、CuSO4.5H2O 0.025mg/L、CoCl2.6H2O 0.025mg/L、FeSO4.7H2O 13.9mg/L、Na2-EDTA.2H2O, inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, nicotinic acid 0.5mg/L, 6-benzylaminopurine 1mg/L, 1% sucrose, and pH 6.5.
The liquid culture medium 2 comprises the following formula: NH (NH)4NO3 825mg/L、KNO3 950mg/L、MgSO4.7H2O 185mg/L、KH2PO4 85mg/L、CaCl2.2H2O 220mg/L、KI 0.83mg/L、H3BO3 6.2mg/L、MnSO4.4H2O 22.3mg/L、ZnSO4.7H2O 8.6mg/L、Na2MoO4.2H2O 0.25mg/L、CuSO4.5H2O 0.025mg/L、CoCl2.6H2O 0.025mg/L、FeSO4.7H2O 13.9mg/L、Na2-EDTA.2H2O, inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, nicotinic acid 0.5mg/L, 1% sucrose, and pH 6.5.
In example 2, the disinfection of eichhornia crassipes explants was not ideal, and only 43% of the explants were free of fungal and bacterial contamination in the first culture, 21% of the number of non-contaminated tissue culture seedlings in the second culture, and only 8% of the number of non-contaminated tissue culture seedlings in the third culture. Example 1 is guided by the patent technology, and the number of explants which are completely polluted by bacteria and fungi can reach more than 60% through multi-generation culture.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (8)
1. A method for quickly obtaining an eichhornia crassipes aseptic seedling is characterized by comprising the following steps:
1) pretreatment: pretreating the eichhornia crassipes plant to obtain a disinfected and air-dried bud body;
2) tissue culture: inserting the bud body obtained in the step 1) into a solid culture medium, culturing for 12h in the dark at 28 ℃, transferring into the solid culture medium, and culturing until a new bud grows out, wherein the culture time L: d is 12: 12;
3) and (3) tissue culture again: taking out the eichhornia crassipes explants from a super clean bench, cutting off blackened and aged parts by using a scalpel, transferring the cut parts into a solid culture medium, culturing the cut parts in the dark at the temperature of 28 ℃ for 12 hours, and then, carrying out the culture in the dark for 12 hours: d is 12: 12 in a solid medium;
4) transferring: culturing until the stolons grow up to new buds, cutting off the new buds with a dissecting knife, removing the stolons at the base parts of the new buds, transferring the new buds into a solid culture medium at the temperature of 28 ℃, and culturing the new buds in a culture medium at the temperature of L: d is 12: culturing in an environment of 12 till new buds grow on the stolons; repeating the step 3);
5) and (3) proliferation: removing the mother plant after stolons, cutting off old leaves and black rot tissues, transferring the cut-off mother plant into a liquid culture medium 1, and culturing at the temperature of 28 ℃, L: d is 12: 12, culturing in an environment of 12, if stolons grow out, cutting off by using a scalpel, removing black rot tissues and redundant stolons, transferring into a liquid culture medium 1, and transferring the stock plant into a liquid culture medium 2;
6) and (3) circulating culture: repeatedly and circularly culturing to obtain sterile filial generation Eichhornia crassipes.
2. The method for rapidly obtaining the eichhornia crassipes sterile seedlings according to claim 1, wherein in the step 1), the pretreatment specifically comprises the following steps:
11) taking back the eichhornia crassipes with the stolons, culturing in clear water for 1 week, removing the stolons, cutting off leaves at a position 2-3cm away from the stem base, removing the roots, cleaning with clear water, and completely drying in a ventilated place;
12) removing the redundant petioles and stems, only leaving the bud with the largest middle part, not damaging the bud in the middle part when cutting the root, cleaning for 1-2 times by using a detergent, and air-drying;
13) after the buds are air-dried, the buds are surface-sterilized for 30s by using alcohol with the concentration of 75%, the buds are transferred to a superclean bench for operation, the buds are taken out and washed by sterile water, and the sterilized absorbent paper is dried;
14) adding 0.1% mercuric chloride into Tween-20, sterilizing for 5min, sealing in a super clean bench, cleaning with ultrasonic vibration for 2.5min, washing with sterile water for 3 times, drying with sterilized absorbent paper, and air drying on a sterile tray;
15) the excess petiole and stem tissues were excised, leaving the largest bud in the middle.
3. The method for rapidly obtaining the eichhornia crassipes sterile seedlings according to claim 2, wherein in the step 14), the amount of mercuric chloride is 4 drops, and the amount of tween is 100 ml.
4. The method for rapidly obtaining the eichhornia crassipes sterile seedlings according to claim 1, wherein the solid medium formula comprises: NH (NH)4NO3825mg/L、KNO3950mg/L、MgSO4.7H2O185mg/L、KH2PO485 mg/L、CaCl2.2H2O 220mg/L、KI 0.83mg/L、H3BO36.2mg/L、MnSO4.4H2O 22.3mg/L、ZnSO4.7H2O 8.6mg/L、Na2MoO4.2H2O 0.25mg/L、CuSO4.5H2O 0.025mg/L、CoCl2.6H2O 0.025mg/L、FeSO4.7H2O 13.9mg/L、Na2-EDTA.2H2O, inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, nicotinic acid 0.5mg/L, hydrolyzed milk protein 250mg/L, 6-benzylaminopurine 3mg/L, 1.5% sucrose, 0.65% agar, and pH 5.8.
5. The method for rapidly obtaining the eichhornia crassipes sterile seedlings according to claim 1, wherein the formula of the liquid culture medium 1 comprises: NH (NH)4NO3825mg/L、KNO3950mg/L、MgSO4.7H2O185mg/L、KH2PO485 mg/L、CaCl2.2H2O 220mg/L、KI 0.83mg/L、H3BO36.2mg/L、MnSO4.4H2O 22.3mg/L、ZnSO4.7H2O 8.6mg/L、Na2MoO4.2H2O 0.25mg/L、CuSO4.5H2O 0.025mg/L、CoCl2.6H2O 0.025mg/L、FeSO4.7H2O 13.9mg/L、Na2-EDTA.2H2O, inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, nicotinic acid 0.5mg/L, 6-benzylaminopurine 1mg/L, 1% sucrose, and pH 6.5.
6. The method for rapidly obtaining the eichhornia crassipes sterile seedlings according to claim 1, wherein the liquid culture medium 2 comprises the following formula: NH (NH)4NO3825mg/L、KNO3950mg/L、MgSO4.7H2O185mg/L、KH2PO485 mg/L、CaCl2.2H2O 220mg/L、KI 0.83mg/L、H3BO36.2mg/L、MnSO4.4H2O 22.3mg/L、ZnSO4.7H2O 8.6mg/L、Na2MoO4.2H2O 0.25mg/L、CuSO4.5H2O 0.025mg/L、CoCl2.6H2O 0.025mg/L、FeSO4.7H2O 13.9mg/L、Na2-EDTA.2H2O, inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, nicotinic acid 0.5mg/L, 1% sucrose, and pH 6.5.
7. The method for rapidly obtaining the eichhornia crassipes sterile seedlings according to claim 1, wherein the illumination intensity in the solid culture medium in the steps 2) -4) is 5000 lux.
8. The method for rapidly obtaining the eichhornia crassipes sterile seedlings according to claim 1, wherein the illumination intensity during the culture in the liquid culture medium 1 and the liquid culture medium 2 is 6000 lux.
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