CN111183900A - Method for efficiently obtaining aseptic safflower seedlings - Google Patents
Method for efficiently obtaining aseptic safflower seedlings Download PDFInfo
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- CN111183900A CN111183900A CN202010059960.0A CN202010059960A CN111183900A CN 111183900 A CN111183900 A CN 111183900A CN 202010059960 A CN202010059960 A CN 202010059960A CN 111183900 A CN111183900 A CN 111183900A
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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
- A01H4/001—Culture apparatus for tissue culture
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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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Abstract
The invention discloses a method for efficiently obtaining a safflower aseptic seedling. The method comprises the steps of material selection, fruit treatment, seed disinfection, inoculation and culture: firstly, husking seeds, then utilizing sodium hypochlorite solution and alcohol to carry out disinfection and sterilization, cleaning, then inoculating the seeds on MS conventional culture medium, and culturing to obtain the safflower aseptic seedlings. The invention can obtain a large amount of safflower aseptic seedlings, can overcome the problems of large contamination rate and the like of explants in the process of obtaining the aseptic seedlings, can greatly shorten the time of emergence, improve the rate of emergence, save the cost and time, improve the economic benefit, and has the advantages of high efficiency, low cost, no pollution, safety to human bodies, environment and the like.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a method for obtaining a safflower aseptic seedling.
Background
Carthami flos is dry flower of Carthamus tinctorius L of Compositae, and is called grass Carthamus tinctorius L, Woodward Variegatus (L.) Druce, and radix seu folium Linderae Strychnifoliae. It is pungent and warm in property, enters heart and liver meridians, and is a good herb for activating blood and dredging meridians, removing blood stasis and relieving pain. The chemical components of safflower mainly comprise flavonoids, alkaloids, polyacetylenes, spermidine, lignans, sesquiterpenes, organic acids, sterols, alkyl glycols, polysaccharides and other components. The compounds in safflower have wide pharmacological activity, not only have certain effect on cardiovascular and cerebrovascular diseases, nervous system, immune system and the like, but also have various physiological activities of anti-inflammation, analgesia, anti-tumor, antioxidation and the like. The safflower oil is edible oil which is recognized in the world and has the functions of eating, health care and beauty treatment.
Just as safflower has so many chemical components and functions of treatment and health care, it has been more and more widely used. Studies show that traditional mercuric chloride (HgCl) is mostly adopted in aseptic seedling culture of safflower2) The disinfection method can only disinfect the surface of the plant generally, has stronger disinfection capability on bacteria than fungi and is ineffective on spores, so the disinfection range is smaller; moreover, the paint is easy to corrode metal instruments, has the defects of heavy metal residue and the like, and is easy to cause harm and pollution to human bodies and the environment. Moreover, the safflower seeds are not only hard in shell but also weak in gaps between the pericarp and the seed coat; the outer layer contains a layer of thick-walled stable wax, which is very easy to grow bacteria. The main reason that the safflower has low germination rate and is difficult to obtain stable aseptic seedlings at present is that the safflower has high germination rate.
Can be used as main raw material of various medicines such as safflower oil, safflower injection, etc. Most of safflower in the current market is wild resource, so that the artificial planting is less, and the market demand is difficult to meet. The application and development of the biotechnology of safflower in China are slow, so that the method is very necessary for efficiently inducing the low-toxicity and safe aseptic safflower seedlings. In addition, the acquisition of sterile explants is an important link of plant tissue culture, and can be used for protoplast fusion, cell culture, in-vitro rapid propagation, plant transgenosis and other aspects. Particularly, successful induction and related application of good sterile seedlings obtained by shelling treatment of safflower seeds and disinfection by using a sodium hypochlorite solution are not reported at home and abroad at present.
Disclosure of Invention
The invention aims to provide a method for efficiently obtaining aseptic safflower seedlings. The invention can obtain a large amount of safflower aseptic seedlings, can overcome the problems of large contamination rate of explants in the process of obtaining the aseptic seedlings and the like, can greatly shorten the time of emergence, improve the rate of emergence, save the cost and time, improve the economic benefit, and has the characteristics of high efficiency, low cost, no pollution, safety to human bodies, environment and the like.
The technical scheme of the invention is as follows: a method for efficiently obtaining aseptic seedlings of safflower comprises the steps of material selection, fruit treatment, seed disinfection, inoculation and culture: firstly, husking seeds, then utilizing sodium hypochlorite solution and alcohol to carry out disinfection and sterilization, cleaning, then inoculating the seeds on MS conventional culture medium, and culturing to obtain the safflower aseptic seedlings.
In the method for efficiently obtaining the aseptic seedlings of the safflower, the seeds are hard, full, shrinkage-free and mature safflower seeds.
In the method for efficiently obtaining the sterile safflower seedlings, the seeds are seeds produced by plants with the age of 3-5 months.
The method for efficiently obtaining the sterile safflower seedlings comprises the following steps:
(1) selecting materials: picking up the newly harvested mature safflower seeds;
(2) seed and fruit treatment: peeling off the shell of the picked seeds by using tweezers;
(3) seed disinfection and sterilization: disinfecting the seeds without shells by using a sodium hypochlorite solution, and cleaning by using sterile water; disinfecting the seeds with alcohol, cleaning with sterile water, and blotting water stain on sterilized filter paper;
(4) inoculation and culture: inoculating the sterilized seeds into an MS culture medium, and then culturing in a tissue culture room to obtain the safflower aseptic seedlings.
In the method for efficiently obtaining the sterile seedlings of safflower, in the step (2), when the shells of the seeds are peeled off, the embryos are not damaged, and the complete seed coats are kept.
In the method for efficiently obtaining the aseptic seedlings of safflower, in the step (3), the seeds without shells are sterilized by 3% sodium hypochlorite solution for 5-10min and washed by sterile water for 2-4 times; sterilizing the seeds with 75% alcohol for 1-2min, and washing with sterile water for 2-4 times.
In the method for efficiently obtaining the aseptic seedlings of safflower, in the step (3), the seeds without shells are soaked in a 3% sodium hypochlorite solution and slightly shaken for disinfection for 5-10 min; soaking the seeds with 75% alcohol, and slightly shaking for sterilization for 1-2 min;
the 3% sodium hypochlorite solution is obtained by diluting 30% sodium hypochlorite reagent commonly used in the market by 10 times; the 75% alcohol is obtained by mixing a common absolute ethyl alcohol reagent on the market with sterile water in a ratio of 3: 1;
in the method for efficiently obtaining the aseptic seedlings of safflower, in the step (4), the culture temperature of the tissue culture room is 25 +/-2 ℃, the illumination time is 14 +/-2 hours under the long-day condition, and the illumination intensity is 1500 lx.
In the method for efficiently obtaining the aseptic seedlings of safflower, in the step (4), the culture time of the tissue culture room is 7-10 days.
Compared with the prior art, the invention has the following beneficial effects:
1. in the prior art, traditional mercuric chloride (HgCl) is mostly adopted for aseptic seedling culture of safflower2) The disinfection method can only disinfect the surface of the plant generally, has stronger disinfection capability on bacteria than fungi and is ineffective on spores, so the disinfection range is smaller; moreover, the paint is easy to corrode metal instruments, has the defects of heavy metal residue and the like, and is easy to cause harm and pollution to human bodies and the environment. In the invention, seeds are peeled and then disinfected by sodium hypochlorite solution, wherein the sodium hypochlorite is a strong oxidant and can kill a plurality of microorganisms including bacterial propagules, cell spores, fungi, mycobacteria, hepatitis viruses, various infectious disease virus bacteria and the like. The sterilization mechanism of the bactericide on microorganisms is as follows: sodium hypochlorite is hydrolyzed to form hypochlorous acid which acts on mycoprotein, and the hypochlorous acid not only can act with cell walls, but also invades cells to generate oxidation with protein or destroy phosphate dehydrogenase of the protein due to small molecules and no electric charge, so that the cell death is caused by sugar metabolism disorder; the strong oxidizability denatures proteins of bacteria and viruses, thereby causing death of pathogenic microorganisms. Has the advantages of high efficiency, low cost and no toxicityPollution, safety to human body and environment, etc. Moreover, the safflower seed obtained by shelling treatment not only can reduce the risk of high mixed bacteria pollution rate caused by seed hulls in the past, but also shortens the disinfection time, and the obtained safflower aseptic seedling has the advantages of tidy seedling emergence, convenient culture, high plant vitality, fast growth and the like, and particularly, successful induction and related application of shelling treatment of the safflower seed and disinfection by utilizing a sodium hypochlorite solution to obtain a good aseptic seedling are not reported at home and abroad at present.
2. The invention utilizes ripe seeds of safflower, after shelling treatment, and then utilizes sodium hypochlorite solution and alcohol to sterilize the ripe seeds, and after inoculating the ripe seeds to a culture medium, the material is placed in a tissue culture room for culture, and seedlings can emerge in about one week, and the beneficial effects are as follows: according to the method, an operation of removing the shell of the seeds is added before the seeds are sterilized, so that the contamination rate can be reduced, and the risk that the contamination rate of mixed bacteria is high and constant due to the seed shells is reduced; meanwhile, the germination speed of the emergence of the safflower aseptic seedlings is increased; moreover, the disinfection time is shortened by the safflower seeds obtained by shelling treatment, and the obtained aseptic safflower seedlings have the advantages of regular emergence of seedlings, convenient culture, high plant vitality, quick growth and the like. Finally, the invention solves the problems of heavy metal residue, high mixed bacteria rate and low germination rate caused by the traditional mercury raising reagent disinfection, and can sterilize by one-time operation. After the seeds are sterilized, the seeds can quickly grow on a conventional MS culture medium. Finally, the method of the invention has simple operation, low price of the used reagent and short time.
Experiments prove that:
in the embodiments 1-3 of the present invention, 60 sterilized seeds are obtained and inoculated in the MS culture medium, and then cultured in the tissue culture room, and the culture results are shown in the following table 1:
TABLE 1 culture results of examples 1 to 3
In conclusion, the invention can obtain a large amount of safflower aseptic seedlings, can overcome the problems of large contamination rate of explants in the process of obtaining the aseptic seedlings and the like, can greatly shorten the time of emergence, improve the rate of emergence, save the cost and time, improve the economic benefit, and has the advantages of high efficiency, low cost, no pollution, safety to human bodies, environment and the like.
Detailed Description
The present invention is further illustrated by the following examples, which are not to be construed as limiting the invention.
Example 1: a method for efficiently obtaining aseptic seedlings of safflower comprises the following steps:
(1) selecting materials: picking up the newly harvested mature safflower seeds; the seeds are hard, full and non-shrinkage safflower seeds in a mature period, and the seeds are seeds produced by plants of 3 months old;
(2) seed and fruit treatment: peeling the picked seeds by using tweezers, wherein when the shells of the seeds are peeled, embryos are not damaged, and complete seed coats are reserved;
(3) seed disinfection and sterilization: sterilizing the seeds without the shells with 3% sodium hypochlorite solution for 5min, and washing with sterile water for 2 times; sterilizing the seeds with 75% alcohol for 1min, washing with sterile water for 2 times, and blotting water stain on sterilized filter paper;
(4) inoculation and culture: inoculating the sterilized seeds into an MS culture medium, and then culturing for 7 days in a tissue culture room to obtain the safflower aseptic seedlings; the culture temperature of the tissue culture room is 23 ℃, the illumination time is set to be 12h under the long-day condition, and the illumination intensity is 1500 lx.
Example 2: a method for efficiently obtaining aseptic seedlings of safflower comprises the following steps:
(1) selecting materials: picking up the newly harvested mature safflower seeds; the seeds are hard, full and non-shrinkage safflower seeds in a mature period, and the seeds are seeds produced by plants with the age of 4 months;
(2) seed and fruit treatment: peeling the picked seeds by using tweezers, wherein when the shells of the seeds are peeled, embryos are not damaged, and complete seed coats are reserved;
(3) seed disinfection and sterilization: sterilizing the seeds without the shells with 3% sodium hypochlorite solution for 8min, and washing with sterile water for 3 times; sterilizing the seeds with 75% alcohol for 1.5min, washing with sterile water for 3 times, and blotting the seeds with sterile filter paper;
(4) inoculation and culture: inoculating the sterilized seeds into an MS culture medium, and then culturing for 8 days in a tissue culture room to obtain the safflower aseptic seedlings; the culture temperature of the tissue culture room is 25 ℃, the illumination time is 14h under the long-day condition, and the illumination intensity is 1500 lx.
Example 3: a method for efficiently obtaining aseptic seedlings of safflower comprises the following steps:
(1) selecting materials: picking up the newly harvested mature safflower seeds; the seeds are hard, full and non-shrinkage safflower seeds in a mature period, and the seeds are seeds produced by plants of 5 months old;
(2) seed and fruit treatment: peeling the picked seeds by using tweezers, wherein when the shells of the seeds are peeled, embryos are not damaged, and complete seed coats are reserved;
(3) seed disinfection and sterilization: sterilizing the seeds without the shells with 3% sodium hypochlorite solution for 10min, and washing with sterile water for 4 times; sterilizing the seeds with 75% alcohol for 2min, washing with sterile water for 4 times, and blotting the seeds with sterile filter paper;
(4) inoculation and culture: inoculating the sterilized seeds into an MS culture medium, and then culturing for 10 days in a tissue culture room to obtain the safflower aseptic seedlings; the culture temperature of the tissue culture room is 27 ℃, the illumination time is set to be 16h under the long-day condition, and the illumination intensity is 1500 lx.
Claims (8)
1. A method for efficiently obtaining aseptic seedlings of safflower comprises the steps of material selection, fruit treatment, seed disinfection, inoculation and culture, and is characterized in that: firstly, husking seeds, then utilizing sodium hypochlorite solution and alcohol to carry out disinfection and sterilization, cleaning, then inoculating the seeds on MS conventional culture medium, and culturing to obtain the safflower aseptic seedlings.
2. The method for efficiently obtaining the sterile safflower seedlings according to claim 1, which is characterized in that: the seeds are hard, full, shrinkage-free and mature safflower seeds.
3. The method for efficiently obtaining the sterile safflower seedlings according to claim 1, which is characterized in that: the seeds are seeds produced by plants with the age of 3-5 months.
4. The method for efficiently obtaining sterile seedlings of safflower as claimed in any one of claims 1 to 3, wherein: the method comprises the following steps:
(1) selecting materials: picking up the newly harvested mature safflower seeds;
(2) seed and fruit treatment: peeling off the shell of the picked seeds by using tweezers;
(3) seed disinfection and sterilization: disinfecting the seeds without shells by using a sodium hypochlorite solution, and cleaning by using sterile water; disinfecting the seeds with alcohol, cleaning with sterile water, and blotting water stain on sterilized filter paper;
(4) inoculation and culture: inoculating the sterilized seeds into an MS culture medium, and then culturing in a tissue culture room to obtain the safflower aseptic seedlings.
5. The method for efficiently obtaining the sterile safflower seedlings according to claim 4, wherein the method comprises the following steps: in the step (2), when the seed shells are peeled off, the embryos are not damaged, and the complete seed coats are reserved.
6. The method for efficiently obtaining the sterile safflower seedlings according to claim 4, wherein the method comprises the following steps: in the step (3), the seeds without shells are disinfected for 5-10min by 3% sodium hypochlorite solution and washed for 2-4 times by sterile water; sterilizing the seeds with 75% alcohol for 1-2min, and washing with sterile water for 2-4 times.
7. The method for efficiently obtaining the sterile safflower seedlings according to claim 4, wherein the method comprises the following steps: in the step (4), the culture temperature of the tissue culture room is 25 +/-2 ℃, the illumination time is 14 +/-2 h under the long-day condition, and the illumination intensity is 1500 lx.
8. The method for efficiently obtaining the sterile safflower seedlings according to claim 4, wherein the method comprises the following steps: in the step (4), the culture time of the tissue culture room is 7-10 days.
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Application publication date: 20200522 |