CN112753584A - Method for storing, backing up and doubling haploid plants cultured by anthers - Google Patents
Method for storing, backing up and doubling haploid plants cultured by anthers Download PDFInfo
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- CN112753584A CN112753584A CN202110225823.4A CN202110225823A CN112753584A CN 112753584 A CN112753584 A CN 112753584A CN 202110225823 A CN202110225823 A CN 202110225823A CN 112753584 A CN112753584 A CN 112753584A
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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
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
- A01H1/08—Methods for producing changes in chromosome number
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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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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention provides a haploid plant preservation backup and doubling method for anther culture, and belongs to the technical field of haploid plant culture. The preservation backup method of the anther culture haploid plant provided by the invention comprises the following steps: and after the haploid plant obtained by anther culture grows to 5-6 leaves, taking the top part of the haploid plant and placing the top part of the haploid plant in a culture medium for rooting to obtain a new haploid plant. The method for doubling the haploid plant cultured by the anther comprises the following steps: completely soaking the water absorbing medium in the doubling reagent, covering the growing point or axillary bud of the haploid plant, culturing in sealed dark for 12-24h, taking off the water absorbing medium, and recovering normal illumination period. The plant rapid propagation method is applied to backup preservation of the pepper anther culture haploid plant, the risk of plant loss caused by transplantation death of the anther culture plant or other burst factors is reduced, and the doubling efficiency is improved by doubling the haploid plant in a culture bottle.
Description
Technical Field
The invention belongs to the technical field of haploid plant culture, and particularly relates to a haploid plant preservation backup and doubling method for anther culture.
Background
Pure line materials can be obtained in a short time by utilizing an anther culture technology, the method is an important means for innovating germplasm resources, the years required by conventional cross breeding can be shortened, the seed selection efficiency is improved, the genotype types of various gamete combinations can be fully expressed, and the method has great potential in breeding. The research on pepper anther culture is earlier carried out and is greatly improved, and the research becomes one of the most important means for creating a new pepper material at present. However, the application of this technology to pepper still has some problems, such as complicated operation of the culture process, restriction of plant regeneration by genotype, etc. Therefore, each regenerated plant obtained by the anther culture technology is very precious, and if death occurs in the transplanting process or death occurs due to some biological or non-biological factors after transplantation survival, the early stage work can be short of one step. If the plants can be perfectly reserved and preserved in the tissue culture seedling stage, the risks can be avoided.
The haploid plant obtained by the anther culture technology only has a set of chromosomes, is highly sterile and can become available germplasm resources only by doubling. The natural multiplying power of the flower cultivated plant is not high, chemical mutagenesis is mostly carried out by adopting an artificial method, commonly used chemical mutagens comprise colchicine, p-dichlorobenzene, 8-hydroxyquinoline and the like, and the colchicine is most widely applied to the hot pepper. Common methods for colchicine plant materials include root soaking, seed soaking, injection, capillary methods, etc. The root soaking method needs to clean the plant root system and soak the plant root system in colchicine, so the required medicament amount is large, the cost is high, and the survival rate of the transplanted seedlings is influenced; the seed soaking is to directly soak seeds with colchicine solution, which is not suitable for doubling haploid plants; the injection method is to directly inject colchicine to the growth point of the stem tip of the plant by using an injector, which has requirements on the injection technology of operators and can damage the growth point of the plant due to improper operation. In addition, various treatments such as a coating method and a dropping method are available. The common point of the methods is that the treatment materials are plants which grow in an open environment after hardening or transplanting survival, the doubling effect is not ideal, and the high-efficiency application of the pepper anther culture technology is influenced.
Disclosure of Invention
In view of the above, the present invention aims to provide a convenient and fast method for storing, backing up and doubling haploid plants cultured with anthers, so as to reduce the risk of plant loss in anther culture and improve doubling efficiency.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a preservation and backup method of a haploid plant cultured by anther, which comprises the following steps: and after the haploid plant obtained by anther culture grows to 5-6 leaves, cutting off the top end part, placing the cut-off top end part in a culture medium for rooting to obtain a new haploid plant, and continuously culturing the haploid plant with the cut-off top end.
Preferably, the tip has 2 to 3 blades.
Preferably, the culture medium is MS + sucrose 30g/L + agar 7g/L, and the pH value is 5.8.
The invention also provides a method for doubling the haploid plant cultured by the anther, which comprises the following steps: after the water absorbing medium is completely soaked in the doubling reagent, the water absorbing medium is covered on the new haploid plant and/or the growing point or the axillary bud of the haploid plant after the top end is cut off in the method, the water absorbing medium is taken down after the cultivation for a certain time in a sealed dark place, and the normal illumination period is recovered.
Preferably, the doubling agents are 0.1% -0.2% colchicine solution and saturated 1, 4-dichlorobenzene solution.
Preferably, the volume ratio of the 0.1-0.2% colchicine solution to the saturated 1, 4-dichlorobenzene solution is 1:1-3: 2.
Preferably, the dark culture time is 12-24 h.
Preferably, the water absorbing medium comprises absorbent cotton and gauze.
Preferably, the gauze has 2-3 layers.
Preferably, the haploid plant comprises capsicum.
The invention has the beneficial effects that:
the invention applies the plant rapid propagation method to the backup preservation of the anther culture haploid plant, and reduces the risk of plant loss caused by transplantation death or other burst factors of the anther culture plant. The haploid plant doubling process is carried out in the culture bottle, the doubling is carried out in the tissue culture seedling stage, the relative humidity of air in the culture bottle is high, the air holes of the tissue culture seedling are completely opened, the absorption and utilization of a doubling reagent are facilitated, and the doubling efficiency can be improved.
Detailed Description
The invention provides a preservation and backup method of a haploid plant cultured by anther, which comprises the following steps: and after the haploid plant obtained by anther culture grows to 5-6 leaves, cutting off the top end part, placing the cut-off top end part in a culture medium for rooting to obtain a new haploid plant, and continuously culturing the haploid plant with the cut-off top end.
The preservation and backup method is not limited to haploid plants, and is also applicable to other plants cultured aseptically. The preservation and backup method of the present invention is preferably performed under aseptic conditions. The invention is not particularly limited to the type of haploid plant, and in the specific embodiment of the invention, hot pepper is taken as an example. The specific cutting mode of the invention is not particularly limited, and any cutting mode in the prior art can be adopted, such as cutting with a sterilized scalpel. In the present invention, the tip portion is preferably a tip portion with 2-3 blades. The specific type of the culture medium is not particularly limited in the invention, and any rooting culture medium conventional in the field can be adopted, and in the invention, the culture medium is preferably MS + sucrose 30g/L + agar 7g/L, and the pH value is 5.8.
The invention also provides a method for doubling the haploid plant cultured by the anther, which comprises the following steps: after the water absorbing medium is completely soaked in the doubling reagent, the water absorbing medium is covered on the new haploid plant and/or the growing point or the axillary bud of the haploid plant after the top end is cut off in the method, the water absorbing medium is taken down after the cultivation for a certain time in a sealed dark place, and the normal illumination period is recovered.
The doubling method for culturing the haploid plant by the anther preferably needs to be carried out under an aseptic condition, appliances, doubling reagents and the like required in the doubling process preferably need to be sterilized, the specific sterilization mode is not particularly limited, and the conventional sterilization mode in the field can be adopted. In addition, the whole process of the doubling method for culturing the haploid plant by the anther is carried out in a culture bottle, the doubling is carried out in a tissue culture seedling stage, the relative humidity of air in the tissue culture bottle is high, and the air holes of the tissue culture seedling are completely opened, so that the absorption and utilization of a doubling reagent are facilitated, and the doubling efficiency can be improved.
The invention has no special limitation on the type of the water absorbing medium, and can adopt the conventional water absorbing medium in the field. In a preferred embodiment of the present invention, the water absorbing medium is absorbent cotton or gauze. When the absorbent medium is gauze, the number of layers of gauze is preferably 2-3 layers, which helps to absorb the doubling agent. In the present invention, the doubling agent is preferably a 0.1% to 0.2% colchicine solution and a saturated 1, 4-dichlorobenzene solution, more preferably a 0.13% to 0.17% colchicine solution and a saturated 1, 4-dichlorobenzene solution; the volume ratio of the 0.1-0.2% colchicine solution to the saturated 1, 4-dichlorobenzene solution is preferably 1:1-3:2, more preferably 5:4-4: 3.
In the invention, the water absorbing medium can be taken by tweezers to cover the growing point or the axillary bud of the haploid plant, and then the water absorbing medium is taken and dripped on the absorbent cotton, so that the absorbent cotton is completely soaked and is tightly attached to the growing point and the axillary bud.
The sealing method of the present invention is not particularly limited, and the sealing of the culture flask with a sealing film is preferred. In the present invention, the dark culture time is preferably 12 to 24 hours, more preferably 15 to 21 hours, and still more preferably 17 to 19 hours. The illumination period is not particularly limited, and the conventional illumination period of the corresponding haploid plants in the field can be adopted.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Selecting pepper anther which grows to 6 leaves in a culture bottle to culture a haploid plant, uncovering the culture bottle on a clean bench, cutting off the top of 3 leaves by using sterilized forceps and a scalpel, inserting the cut top into a sterilized solid culture medium of MS, sucrose, 30g/L, agar, 7g/L and pH of 5.8 to culture until new roots grow, obtaining a new complete haploid plant, and continuously culturing the rest part of haploid plant.
Example 2
Two layers of gauze of 0.5cm × 2cm were soaked completely in doubling agent of 0.1% colchicine solution and saturated 1, 4-dichlorobenzene solution at volume ratio of 1:1, covered on the growing point of the new haploid plant in example 1, sealed and dark cultured for 24h, the gauze was removed, and the normal illumination period was recovered.
Example 3
After a proper amount of absorbent cotton is completely soaked in a doubling reagent of 0.2 percent colchicine solution and saturated 1, 4-dichlorobenzene solution in a volume ratio of 5:4, the mixture is covered on axillary buds of the rest haploid plants in the embodiment 1, sealed and dark cultivation is carried out for 15h, gauze is taken down, and the normal illumination period is recovered.
Example 4
After a proper amount of absorbent cotton is completely soaked in a doubling reagent of 0.13 percent colchicine solution and saturated 1, 4-dichlorobenzene solution in a volume ratio of 4:3, the mixture is covered on the growing points of the rest haploid plants in the example 1, sealed and dark culture is carried out for 17 hours, gauze is taken down, and the normal illumination period is recovered.
Example 5
(1) Plant preservation and backup: using 8 parts of anther culture plant as a material, selecting pepper anther which grows to 5 leaves in a culture bottle to culture a haploid plant, uncovering the culture bottle on a clean bench, cutting off the top of 2 leaves by using sterilized tweezers and a scalpel, inserting the cut leaves into a sterilized solid culture medium of MS + sucrose 30g/L + agar 7g/L and pH5.8 to culture until new roots grow out, and obtaining a new complete plant.
(2) Doubling of haploid plants: after the truncated plants in (1) had grown to 7d, the experiments were performed in two groups:
one group was doubled in flasks at a volume ratio of 3:2, mixing a 0.2 percent colchicine solution and a saturated 1, 4-dichlorobenzene solution (the solvent is alcohol) to prepare a doubling reagent, completely soaking two layers of 0.5cm multiplied by 2cm gauze in the doubling reagent, covering the two layers of gauze at the plant growing point with the top end cut off in the step (1), sealing a culture bottle by using a sealing film, taking down the gauze after culturing in the dark for 12 hours, recovering the normal illumination period, and continuously culturing in the culture bottle until hardening seedlings are transplanted.
And the other group is subjected to doubling treatment after the survival of the transplanted seedling, and the volume ratio is 3:2, mixing a 0.2 percent colchicine solution and a saturated 1, 4-dichlorobenzene solution to prepare a doubling reagent, completely soaking two layers of 0.5cm multiplied by 2cm gauzes in the doubling reagent, covering the positions of growing points of transplanted living plants, covering a layer of plastic film on the outside of the plants, culturing in the dark for 12 hours, taking down the gauzes, and recovering the normal illumination period.
After 1 month, the doubling efficiency is counted according to the seed setting condition, and the doubling rate (%) is equal to the number of the seed setting plants/the number of the doubling reagent treated plants multiplied by 100. As can be seen from Table 1, the doubling of the reagents during the tissue culture period is significantly higher than the doubling after transplantation.
TABLE 1 Single-fold doubling after different time-course doubling agent treatment
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A preservation and backup method for a haploid plant cultured by anther is characterized by comprising the following steps: and after the haploid plant obtained by anther culture grows to 5-6 leaves, cutting off the top end part, placing the cut-off top end part in a culture medium for rooting to obtain a new haploid plant, and continuously culturing the haploid plant with the cut-off top end.
2. The save backup method according to claim 1, wherein the tip has 2-3 blades.
3. The method for preserving backup according to claim 1, wherein the culture medium is MS + sucrose 30g/L + agar 7g/L, pH 5.8.
4. A method for doubling a haploid plant cultured by anthers is characterized by comprising the following steps: covering the growing point or axillary bud of the monoploid plant in the method of claim 1 after the water-absorbing medium is completely soaked in the doubling reagent, sealing and culturing in dark for a certain time, removing the water-absorbing medium, and recovering normal illumination period; wherein the haploid plant in the method of claim 1 is a new haploid plant and/or a post-apical excised haploid plant.
5. The doubling method of claim 4, wherein the doubling reagents are 0.1% -0.2% colchicine solution and saturated 1, 4-dichlorobenzene solution.
6. The doubling method according to claim 5, wherein the volume ratio of the 0.1% to 0.2% colchicine solution to the saturated 1, 4-dichlorobenzene solution is 1:1 to 3: 2.
7. The doubling method according to claim 4, wherein the dark cultivation is carried out for a period of 12-24 h.
8. The doubling method of claim 4, wherein the water absorbing medium comprises cotton wool and gauze.
9. The doubling method of claim 8, wherein the scrim is 2-3 layers.
10. Method for the conservation and backup of haploid plants according to any of claims 1-3 and for the doubling of haploid plants according to any of claims 4-9, characterized in that the haploid plants comprise hot pepper.
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| CN202110225823.4A CN112753584A (en) | 2021-03-01 | 2021-03-01 | Method for storing, backing up and doubling haploid plants cultured by anthers |
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| CN202110225823.4A CN112753584A (en) | 2021-03-01 | 2021-03-01 | Method for storing, backing up and doubling haploid plants cultured by anthers |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101161056A (en) * | 2007-09-28 | 2008-04-16 | 中国科学院新疆理化技术研究所 | A method for evoking yili caladium polyploid under culture in vitro |
| CN105638455A (en) * | 2014-11-14 | 2016-06-08 | 石河子大学 | Method for obtaining dry chilli haploid plant by anther culture and culture medium |
| CN111374048A (en) * | 2020-04-28 | 2020-07-07 | 北京市海淀区植物组织培养技术实验室 | Chromosome doubling method of pepper or eggplant haploid plant |
-
2021
- 2021-03-01 CN CN202110225823.4A patent/CN112753584A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101161056A (en) * | 2007-09-28 | 2008-04-16 | 中国科学院新疆理化技术研究所 | A method for evoking yili caladium polyploid under culture in vitro |
| CN105638455A (en) * | 2014-11-14 | 2016-06-08 | 石河子大学 | Method for obtaining dry chilli haploid plant by anther culture and culture medium |
| CN111374048A (en) * | 2020-04-28 | 2020-07-07 | 北京市海淀区植物组织培养技术实验室 | Chromosome doubling method of pepper or eggplant haploid plant |
Non-Patent Citations (1)
| Title |
|---|
| 静一等: ""加工型辣椒花药培养技术研究"", 《中国农学通报》 * |
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