CN115176702B - Method for doubling chromosome of wheat haploid plant without medicament treatment and application - Google Patents
Method for doubling chromosome of wheat haploid plant without medicament treatment and application Download PDFInfo
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- CN115176702B CN115176702B CN202211033715.8A CN202211033715A CN115176702B CN 115176702 B CN115176702 B CN 115176702B CN 202211033715 A CN202211033715 A CN 202211033715A CN 115176702 B CN115176702 B CN 115176702B
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- 241000209140 Triticum Species 0.000 title claims abstract description 62
- 235000021307 Triticum Nutrition 0.000 title claims abstract description 57
- 241000196324 Embryophyta Species 0.000 title claims abstract description 55
- 238000000034 method Methods 0.000 title claims abstract description 29
- 239000003814 drug Substances 0.000 title claims abstract description 24
- 210000000349 chromosome Anatomy 0.000 title claims abstract description 21
- 238000005286 illumination Methods 0.000 claims abstract description 15
- 238000012258 culturing Methods 0.000 claims abstract description 11
- 238000009395 breeding Methods 0.000 claims abstract description 7
- 230000001488 breeding effect Effects 0.000 claims abstract description 7
- 239000003795 chemical substances by application Substances 0.000 claims 2
- IAKHMKGGTNLKSZ-INIZCTEOSA-N (S)-colchicine Chemical compound C1([C@@H](NC(C)=O)CC2)=CC(=O)C(OC)=CC=C1C1=C2C=C(OC)C(OC)=C1OC IAKHMKGGTNLKSZ-INIZCTEOSA-N 0.000 abstract description 30
- 229960001338 colchicine Drugs 0.000 abstract description 15
- 230000000694 effects Effects 0.000 abstract description 5
- 210000000745 plant chromosome Anatomy 0.000 abstract description 5
- 231100000331 toxic Toxicity 0.000 abstract description 4
- 230000002588 toxic effect Effects 0.000 abstract description 4
- 230000000052 comparative effect Effects 0.000 description 5
- 230000004069 differentiation Effects 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 230000006872 improvement Effects 0.000 description 4
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- 239000002609 medium Substances 0.000 description 3
- 229940079593 drug Drugs 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 230000003203 everyday effect Effects 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
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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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Abstract
The invention relates to a wheat haploid plant chromosome doubling method without medicament treatment, which comprises the steps of putting a wheat haploid plant into an artificial climate incubator, respectively carrying out light and dark alternate circulation culture under different temperature conditions, transplanting the wheat haploid plant into a greenhouse or a field after vernalization is completed until fructification is achieved, wherein the light and dark alternate circulation is two stages, the first culture stage is dark culture at 5 ℃ for 10 hours, illumination culture at 7 ℃ for 14 hours, and culture for 3 days; and in the second stage, culturing for 14 hours under illumination at 9.5-10.5 ℃, and culturing for 10 hours under dark at 3.5-4.0 ℃ until vernalization is completed. The invention controls the environment condition of wheat haploid plant culture to make the wheat haploid plant complete chromosome doubling, the doubling effect is better than the effect of using colchicine and other medicaments to treat, simultaneously, the toxic action of colchicine to operators, the pollution to environment and the dead seedling phenomenon caused by medicament treatment are avoided, the difficulty and the cost of wheat flower culture haploid breeding are reduced, and the popularization and the application are easy.
Description
Technical Field
The invention belongs to the technical field of culturing and cultivating new varieties by using wheat haploids, and particularly relates to a method for doubling chromosomes of a wheat haploid plant without medicament treatment and application.
Background
The haploid refers to an individual with gamete chromosome number, the haploid is generated by utilizing a wheat anther culture technology, and a homozygous diploid pure line with all genetic homogeneity is obtained by doubling, so that the method is an important way for quickly cultivating a new wheat variety and constructing a special genetic population. Obtaining haploid plants and carrying out chromosome doubling on the haploid plants are two key steps of success or failure of wheat haploid breeding, under natural conditions, the rate of forming doubled haploid plants by the haploid plants is very low, most of the doubled haploid plants cannot bear fruit and are difficult to apply to breeding and genetic research, and therefore, the chromosomes of the haploid plants are doubled into doubled haploids by means of artificial measures.
At present, the most effective doubling method is to treat tillering nodes in seedling stage by using colchicine, and a method of directly adding colchicine in the process of tissue culture is also available, and Cai Hua uses colchicine to carry out root soaking doubling treatment on haploid plants. The doubling efficiency of the methods is not too high and is not stable, and the death rate of the treated plants is higher due to the larger toxic effect of colchicine on the plants. 3238 Zxft 3238 et al, CN 3262 Zxft 3262A, "a method for doubling wheat haploid test-tube plant chromosomes", improves the method for doubling by colchicine and obtains better doubling effect. In summary, although the haploid doubling technology has been greatly improved, the basic means of doubling using drugs such as colchicine is still adopted, and the research content mainly focuses on the concentration of colchicine, the treatment site, the treatment time and the treatment temperature, the application of the doubling method in combination with dimethyl sulfoxide, and the treatment modes of root soaking, tillering treatment, in-flask treatment, field seedling taking treatment and the like, all of which can not release the use of drugs such as colchicine.
Considering the toxic action of colchicine and other medicaments on operators, the pollution to the environment and the dead seedling phenomenon caused by medicament treatment, the invention of the wheat haploid plant chromosome doubling method without medicament treatment has important significance.
Disclosure of Invention
The invention aims to solve the defect that the chromosome doubling of the existing wheat haploid plant needs medicament treatment, and provides a method for doubling the chromosome of the wheat haploid plant without medicament treatment.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention provides a method for doubling chromosomes of wheat haploid plants without medicament treatment, which comprises the following steps:
s1, artificial climate culture: putting a culture container with a wheat haploid plant into an artificial climate incubator for light-dark alternate cycle culture until vernalization is completed, wherein the light-dark alternate cycle is two stages, and the specific culture conditions are as follows: in the first stage, dark culture is carried out for 10 hours under the condition of 5 ℃, and illumination culture is carried out for 14 hours under the condition of 7 ℃ for 3 days; in the second stage, the culture is carried out for 14 hours under the temperature condition of 9.5 to 10.5 ℃ and in the dark for 10 hours under the temperature condition of 3.5 to 4.0 ℃ until the vernalization is finished;
s2, transplanting and culturing plants: and (4) transplanting the wheat plant which is subjected to vernalization in the S1 step into a greenhouse or a field, and normally growing until fructification.
As a further improvement of the invention, the light-dark alternate cycle culture conditions in S1 are as follows: culturing at 10 deg.C for 14 hr; the culture was carried out at 4.0 ℃ for 10 hours in the dark.
As a further improvement of the invention, the S1 midlet wheat haploid plant refers to a wheat complete haploid plant which is obtained by wheat anther culture or other ways and is not vernalized.
As a further improvement of the invention, said completion of vernalization in S2 means that the growth of stem elongation begins to occur in the wheat plant.
As a further improvement of the invention, the growth temperature of the greenhouse or the field in S2 is 10 to 27 ℃, and the illumination is 14 to 16 hours per day.
The invention also provides application of the wheat haploid plant chromosome doubling method without medicament treatment in wheat breeding, wheat variety resource creation and wheat transgenosis.
Adopt the produced beneficial effect of above-mentioned technical scheme to lie in:
1. the invention establishes a technology for doubling the chromosome of the wheat haploid plant by controlling the environmental condition of the culture of the wheat haploid plant without using colchicine and other medicaments for treatment, the doubling effect of the invention is not only better than the effect of using colchicine and other medicaments for treatment, but also can save a large amount of medicament purchasing cost, avoid the toxic action of colchicine on operators and the pollution to the environment, and also avoid the seedling death phenomenon caused by medicament treatment.
2. The method provided by the invention can be combined and applied when the chromosome doubling and vernalization treatment of the wheat haploid plant are improved until the vernalization action is completed, so that the treatment difficulty is reduced, the treatment link is reduced, and the time is shortened.
3. The method provided by the invention is simple and easy to learn, reduces the difficulty of wheat anther culture haploid breeding in this link, is easy to popularize and apply, and has great practical significance for accelerating wheat breeding speed, and increasing varieties.
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In order to more clearly illustrate the detailed description of the invention or the technical solutions in the prior art, the drawings that are needed in the detailed description of the invention or the prior art will be briefly described below.
FIG. 1 is a diagram illustrating the method of example 1 of the present invention for handling a fruit setting;
FIG. 2 shows the method of comparative example 1 according to the present invention for treating a setting condition;
FIG. 3 shows the condition of plants treated in example 1 and comparative example 1 of the present invention, wherein A is example 1,B is comparative example 1;
FIG. 4 shows the plant seed set for large scale application in example 2 of the present invention.
Detailed Description
The invention relates to a method for doubling chromosomes of wheat haploid plants without medicament treatment.
In some embodiments, a wheat haploid plant refers to a wheat haploid plant that has not been vernalized obtained by wheat anther culture or other means.
In some embodiments, the artificial climate culture is to put a culture container with a wheat haploid plant into an artificial climate incubator for light and dark alternate cycle culture until vernalization is completed, wherein the light and dark alternate cycle is two stages, and the specific culture conditions are as follows: in the first stage, dark culture is carried out for 10 hours at the temperature of 5 ℃, and illumination culture is carried out for 14 hours at the temperature of 7 ℃ for 3 days; in the second stage, the culture is carried out for 14 hours under the temperature condition of 9.5 to 10.5 ℃ and for 10 hours under the temperature condition of 3.5 to 4.0 ℃ in the dark until the vernalization is completed.
In some embodiments, the wheat plants after the vernalization are transplanted to a greenhouse or a field, the growth temperature is 10 to 27 ℃, and the illumination is 14 to 16 hours per day.
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in detail and fully with reference to the following embodiments.
The differentiation medium used in this example is a commonly used differentiation medium, for example, MS is used as a minimal medium to which KT and NAA are added at appropriate concentrations, and other media that can achieve differentiation of wheat callus may also be suitable for this application.
Example 1
Taking a wheat filial generation D72 as a test material, inoculating wheat anther on 6 days 5 and 6 months in 2020, starting to transfer callus to a differentiation culture medium on 16 days 11 and 16 months in 2020, differentiating plants under the conditions of 25 ℃ and 14 hours of illumination each day, and transferring the differentiated wheat haploid plants to the following culture medium: 1/2MS mass factors, MS trace elements, MS organic components, MS iron salts, 0.5mg/L NAA, 10g/L sucrose and 2.5 g/L plant gel, and the pH value is 5.8.
S1, artificial climate culture: starting at 2021 year, 2 month and 1 day, placing the plant in an artificial climate box for light-dark alternating circulation culture treatment, wherein the culture treatment condition is dark culture at 5 ℃ for 10 hours, and illumination culture at 7 ℃ for 14 hours; the treatment conditions were set to dark culture at 4 ℃ for 10 hours and light culture at 10 ℃ for 14 hours, starting at 2, 4 and 2021.
S2, transplanting and culturing plants: transplanting the seedlings into a greenhouse at 23 months and 2 years in 2021, carrying out cultivation management under the conditions of 10 to 27 ℃ (high day and low night) and 14 to 16 hours of illumination per day, and investigating fructification conditions after harvesting, wherein the fructification strain rate is 83%, and the plants are shown in fig. 1 and 3.
Example 2 chromosome doubling assay with multiple hybridization combinations
In 2021, haploid plants differentiated by flower cultivation are used for carrying out plant chromosome doubling in batches, and doubling materials are F1 generation haploid plants of a plurality of wheat hybridization combinations, and the method comprises the following steps: jinhe 9123X 9306, kenong 199X Jinhe 9123, jinhe 9123X Kenong 199, jinhe 9123X Jimai 22, jinhe 9123X Ji Mai, jimai 38X Jimai 22, kenong 199X 9306, kenong 199X Ji Mai, kenong 199X 1800-06, 9306X Kenong 199, etc.; and Jinhe 9123, and alternate 987 wheat varieties. Carrying out 7 batches of chromosome doubling on the obtained wheat haploid plants by using a light-dark alternating temperature changing method, wherein the processing conditions are light-dark alternating circulation culture in two stages, and the specific culture conditions are as follows: in the first stage, dark culture is carried out for 10 hours at the temperature of 5 ℃, and illumination culture is carried out for 14 hours at the temperature of 7 ℃ for 3 days; in the second stage, the culture is carried out for 14 hours under the condition of 10 ℃ in light and 10 hours under the condition of 4.0 ℃ in dark until the vernalization is completed.
And after the vernalization is finished, planting the seeds in a flowerpot in a greenhouse at the temperature of 10 to 27 ℃ for 14 to 16 hours per day, carrying out normal cultivation management, and harvesting after ripening. The results show that: the setting rate of 1 batch is 100%, the setting rate of 1 batch is 85%, the setting rate of 1 batch is 73%, the setting rate of 2 batches is 67%, the setting rate of 1 batch is 62%, the setting rate of 1 batch is 57%, and typical setting conditions are shown in FIG. 4.
Comparative example 1
Taking wheat filial generation D72 as a test material, inoculating wheat anther on 6 days 5 and 6 months in 2020, transferring callus onto a differentiation culture medium on 16 days 11 and 16 months in 2020, and differentiating plants under the conditions of temperature of 25 ℃ and illumination for 14 hours every day. Culturing differentiated wheat haploid plants in the dark at the temperature of 5 ℃ for 10 hours, culturing in the light at the temperature of 7 ℃ for 14 hours until vernalization, respectively adding 0.5ml of 2% dimethyl sulfoxide and 0.5% colchicine solution to the root of each seedling, treating at the temperature of 25 ℃ for 15 hours, cleaning, transplanting into a flowerpot of a greenhouse, culturing and managing at the temperature of 10 to 27 ℃ (high day and low night) and in the light of 14 to 16 hours per day, and investigating the fructification condition after harvesting, wherein the fructification rate is 32%, and the plants are shown in figures 2 and 3.
Comparative example 2
The material is as follows: jinhe 9123 and a anther culture haploid plant of Kenong 199 wheat variety, chromosome doubling is not carried out by medicament treatment and variable temperature treatment, vernalization treatment is carried out only, the vernalization treatment conditions comprise dark culture for 10 hours at the temperature of 8 ℃ and illumination culture for 14 hours, the vernalization is finished, the plant is planted in a flowerpot and grows in a greenhouse at the temperature of 10 to 27 ℃ (day high and night low) and illumination for 14 to 16 hours every day, no seed can be obtained, and the maturing rate is 0.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (6)
1. A method for doubling chromosomes of a wheat haploid plant without medicament treatment is characterized by comprising the following steps:
s1, artificial climate culture: putting a culture container with a wheat haploid plant into an artificial climate incubator for light-dark alternate cycle culture until vernalization is completed, wherein the light-dark alternate cycle is two stages, and the specific culture conditions are as follows: in the first stage, dark culture is carried out for 10 hours at the temperature of 5 ℃, and illumination culture is carried out for 14 hours at the temperature of 7 ℃ for 3 days; in the second stage, the culture is carried out for 14 hours under the condition of 9.5 to 10.5 ℃ and carried out for 10 hours in the dark under the condition of 3.5 to 4.0 ℃ until the vernalization is finished;
s2, transplanting and culturing plants: and (4) transplanting the wheat plant which is subjected to vernalization in the S1 step into a greenhouse or a field, and normally growing until fructification.
2. The method for doubling chromosomes of a wheat haploid plant not treated by a medicament, according to claim 1, wherein the culture conditions of the second stage of the alternating cycle of light and dark in S1 are as follows: culturing at 10 deg.C for 14 hr; the culture was carried out at 4.0 ℃ for 10 hours in the dark.
3. The method of claim 1, wherein the midlet haploid plant in S1 is a wheat haploid plant obtained by anther culture or other means without vernalization.
4. The method for doubling chromosomes of a wheat haploid plant not treated with an agent as claimed in claim 1, wherein after vernalization in S2 is completed, the wheat plant begins to grow with stem elongation.
5. The method for doubling chromosomes of wheat haploid plants not treated by a medicament as claimed in claim 1, wherein the growing temperature in a greenhouse or a field in S2 is 10 to 27 ℃, and the illumination time is 14 to 16 hours per day.
6. Use of the method of doubling a chromosome of a haploid plant of wheat without treatment with an agent of any one of claims 1~5 in wheat breeding, resource creation of a wheat variety, and wheat transgenesis.
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| CN102106255A (en) * | 2010-12-27 | 2011-06-29 | 河北科技大学 | Method for creating novel germ plasm of translocation line through anther culture of intergeneric hybrids between wheat and Leymus multicaulis |
| CN105340732A (en) * | 2015-12-03 | 2016-02-24 | 中国农业科学院作物科学研究所 | Chromosome doubling method for wheat haploid test-tube plants |
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Effective date of registration: 20231207 Address after: 050051 No. 598 Heping West Road, Hebei, Shijiazhuang Patentee after: Institute of biotechnology and food science, Hebei Academy of agricultural and Forestry Sciences Patentee after: Hebei Yuansheng Agricultural Development Co.,Ltd. Address before: 050051 No. 598 Heping West Road, Hebei, Shijiazhuang Patentee before: Institute of biotechnology and food science, Hebei Academy of agricultural and Forestry Sciences |