CN108935082A - A kind of method for breeding haploidy of Ms2ms2 genotype wheat - Google Patents
A kind of method for breeding haploidy of Ms2ms2 genotype wheat Download PDFInfo
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- CN108935082A CN108935082A CN201811122946.XA CN201811122946A CN108935082A CN 108935082 A CN108935082 A CN 108935082A CN 201811122946 A CN201811122946 A CN 201811122946A CN 108935082 A CN108935082 A CN 108935082A
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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
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
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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
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention belongs to variety of crops breeding technique field, in particular to a kind of method for breeding haploidy of Ms2ms2 genotype wheat, including dwarf male sterile wheat plant is chosen as female parent, sweet-waxy maizes are as male parent, hybridization；It takes monoploid rataria to be inoculated in the MS culture medium that a great number of elements halves after the culture of wheat hybridizing plant and cultivates emergence；Haplobiont doubles to handle, and the Ms2Ms2 genotype group of acquisition obtains Ms2ms2 genotypic crossing F1 generation group with the fertile paternal hybrid of wheat as maternal.Ms2ms2 genotype F1 generation next year of the present invention can be hybridized with corn, and into the haploid breeding program of a new round, breeder can determine the number for recycling Ms2Ms2 genotype according to breeding objective.Dwarf male sterile wheat haploid breeding and compound breeding, the multi-parent strains crossbreeding technology such as pyramiding breeding can be combined, continuous output wheat homozygosis ms2ms2 genotype strain forms an efficient scale breeding platform.Breeding cost can be saved, breeding efficiency is improved, shortens the breeding time limit.
Description
Technical field
The invention belongs to variety of crops breeding technique field, in particular to a kind of monoploid of Ms2ms2 genotype wheat
Breeding method.
Background technique
In recent years, it is widely studied using chromosome null method initiative doubled haploid (Double Haploid, DH),
Wherein, corn is graduallyd mature with wheat hybridizing induction monoploid technology, it has also become the higher approach of doubled haploid generation efficiency
One of.Haploid breeding technology can obtain homozygous recombinant in a generation, shorten the breeding time limit, improve breeding efficiency, have
Huge Breeding Application and basic research value.However, wheat is manually gone in corn and wheat hybridizing induction monoploid program
Hero, it is time-consuming and laborious, it is at high cost, limit the large-scale application of the technology.
Tai-gu dominant male sterile wheat is the dominant genic male sterile mutant that China is found in wheat for the first time, is pole in wheat breeding
Its precious germ plasm resource.Tai-gu dominant male sterile wheat contains rare dominant male sterile gene Ms2, offspring according to 1:1 ratio
Separation, and sterile plant belongs to " non-pollen type ", sterility is stablized, and open pollination outcrossing seed-setting rate is high.By heredity exchange Ms2
It is combined together with the short dwarf gene Rht10 for becoming No.1, close linkage, exchange rate has been bred as dwarf male sterile wheat less than 0.18%.
Utilization in breeding has formed a set of based on recurrent selection, the breeding method of number of ways integrated use.
Extensive Hybrid Problems are solved using dwarf male sterile wheat, use the quick homozygous filial generation of doubled haploid technology, contracting
In short breeding cycle 3-4 generation, builds up dwarf male sterile wheat high-efficient breeding platform, is a big innovation of wheat breeding.Currently, by dwarf male sterile wheat
It is conjointly employed in breeding practice with doubled haploid technology to have not been reported, a few studies relate only to dwarf male sterile wheat and generate single times
The technological layers such as the frequency of body.
In addition, the short Ms2Ms2 genotype that loses of homozygosis of acquisition accounts for half in dwarf male sterile wheat and corn hybridization offspring, it can not
Self-fertility, if rejecting will be serious waste.Therefore, the pollination of other Wheat Pollens can be used directly, obtain dwarf male sterile wheat
The Combination nova F1 generation group of Ms2ms2 genotype can enter the haploid breeding program of a new round with corn pollination again after plantation,
It totally can shorten the first cross time, achieve the effect that make full use of and improve efficiency.
Summary of the invention
Ms2ms2 genotype F1 generation of the present invention, no anther, glume opening is big, is easily accepted by foreign pollen, and outcrossing seed-setting rate is high,
Hybridization or not whole fringe direct cross after heading 5 days can be done when applied to breeding after simply cutting grain husk, can be carried out large-scale direct
Outcrossing tissue culture crossbreeding is extraordinary wheat breeding material.
The present invention provides a kind of method for breeding haploidy of Ms2ms2 genotype wheat, comprising the following steps:
S1, First Year early October choose mature, full dwarf male sterile wheat seed sowing, obtain dwarf male sterile wheat plant；The
2 years, sweet-waxy maizes are sowed in greenhouse mid-January to late Febuary, obtain plant；
S2, using dwarf male sterile wheat plant in S1 as maternal, plant as male parent, female parent heading is awarded after 2-6 days by fringe
With paternal pollen, bagging isolation after pollination pollinates after 20-24h, sprays 2,4- dichlorphenoxyacetic acid, arabinogalactan-proteins
With the mixed solution of dimethyl sulfoxide, it is primary to spray the mixed solution after 20-24h again, obtains wheat hybridizing plant；
Wheat hybrid plant culture 16-18 days in S2 are taken out monoploid rataria from fringe and are inoculated in equipped with a large amount of members by S3
In the culture apparatus for the MS culture medium that element halves, sealing, dark culture 2-3 days at 4 DEG C, then relative humidity be 50%-70%,
Then dark culture 3-5 days at 22-25 DEG C is obtained with 14h illumination and 10h dark culture alternate cycles processing culture until to emerging
Wheat Haploid seedling；
S4 removes culture apparatus sealing when Wheat Haploid miaoye the piece number >=3 piece, plant height 4-7cm in S3, and addition is gone out
Bacterium water slow seedling 2-3 days, root culture medium is washed away, selects the Wheat Haploid transplantation of seedlings of robust growth to containing nutrient matrix and vermiculite
Pot for growing seedlings in, wherein in pot for growing seedlings the weight ratio of nutrient matrix and vermiculite be 2:1, be put into 3-6 DEG C of vernalization room vernalization 25-35d,
Obtain vernalization Wheat Haploid seedling；
S5 moves into 3-5d at 12-14 DEG C when vernalization Wheat Haploid seedling tiller number reaches 1-3 in S4, cleans vernalization
Wheat Haploid seedling root cuts root, so that root depth is retained 2-4cm, is dipped in the mixing of colchicin and dimethyl sulfoxide
In liquid, the tiller node not 1-2cm under liquid level, ventilation lucifugal handles 5h at 25 DEG C, and clear water is transplanted after rinsing overnight to pot for growing seedlings
In, cultivate 10-15d at 12-14 DEG C, then balled transplanting into flowerpot to solid maturation, obtain wheat doubled haploid plant；
S6, Meccah times small in S5 haplobiont is filtered out by Markers for Detection and phenotypic evaluation with short bar and
The Ms2Ms2 genotype of male-sterile character is as female parent, after heading 2-3 days, the whole fringe of grain husk is cut, using common wheat as father
This is hybridized, and Ms2ms2 genotypic crossing F1 generation is obtained.
Preferably, in S2 2,4- dichlorphenoxyacetic acid, arabinogalactan-proteins and dimethyl sulfoxide 1L mixed solution
Preparation method are as follows: 20ml dimethyl sulfoxide, 2, the 4- dichlorphenoxyacetic acid of 100mg and 2g arabinogalactan-proteins are mixed
It closes, adds distilled water to 1L, mix.
Preferably, in S5 colchicin and dimethyl sulfoxide mixed liquor preparation method are as follows: every mass per liter concentration be 0.1%
Colchicin in be added 20ml dimethyl sulfoxide, mix.
It preferably, will be through treated the Wheat Haploid transplantation of seedlings of colchicin and dimethyl sulfoxide mixed liquor to educating in S5
The mixture of the nutrient matrix and vermiculite that are 1:2 with weight ratio when in seedling alms bowl wraps up vernalization Wheat Haploid seedling root.
Compared with prior art, beneficial effects of the present invention:
Ms2ms2 genotype F1 generation glume opening of the present invention is big, is easily accepted by foreign pollen, and outcrossing rate is high, when being applied to breeding
The whole fringe of grain husk or not whole fringe direct cross after heading 5 days are simply cut, and common wheat needs to take considerable time that whole fringe (is cut grain husk and gone
Except anther), therefore large-scale directly outcrossing tissue culture crossbreeding can be carried out, it is extraordinary wheat breeding material；Monoploid
The ms2ms2 fertile plant that plant obtains after doubling can be directly entered primary strain qualification test；The short bar infertility of the Ms2Ms2 of acquisition
Strain can directly assemble Combination nova Ms2ms2 genotype F1 generation group, save the time for assembling Combination nova a season, recycle, save
Anther link is removed, quick hybridization is convenient for, is suitable for scale and operates, save breeding cost, improve breeding efficiency, shorten breeding year
Limit.
Specific embodiment
Several specific embodiments of the invention are described in detail below, it is to be understood that protection scope of the present invention
It is not limited by the specific implementation, experimental method used in embodiment is conventional method unless otherwise specified, real
Material used in example is applied, is commercially available.
Embodiment 1
A kind of method for breeding haploidy of Ms2ms2 genotype wheat, comprising the following steps:
S1, Henan Xinxiang, First Year October 7 choose mature, full dwarf male sterile wheat seed and are seeded in field, obtains
Dwarf male sterile wheat plant；Second year, January 15,5 days 2 months, 20 days 2 months, the interval sowing sweet-waxy maizes in greenhouse obtain corn
Plant enables the florescence of dwarf male sterile wheat and sweet-waxy maizes to meet.
S2, early April in the same year, using dwarf male sterile wheat plant in S1 as maternal, plant as male parent, after female parent heading
It simply cuts grain husk within 2 days, awards after 1 day by fringe with paternal pollen, bagging isolation after pollination, pollinate after 20h, spray 2,4- Dichlorophenoxy second
The mixed solutions of acid, arabinogalactan-proteins and dimethyl sulfoxide, it is primary to spray mixed solution after 20h again, and it is miscellaneous to obtain wheat
Hand over plant；
Wherein, the preparation of the 1L mixed solution of 2,4- dichlorphenoxyacetic acid, arabinogalactan-proteins and dimethyl sulfoxide
Method are as follows: 20ml dimethyl sulfoxide, 2, the 4- dichlorphenoxyacetic acid of 100mg and 2g arabinogalactan-proteins are mixed, steaming is added
Distilled water is mixed to 1L.
S3, by wheat hybrid plant culture 16 days in S2,2 leaves of band cut fringe, take out seed through 70% alcohol of volumetric concentration
30s is sterilized, then with mass concentration is 0.1%HgCl2Sterilize 10min, aseptic water washing 3 times, each 1min, takes out monoploid children
Embryo is inoculated in the culture apparatus of the MS culture medium halved equipped with a great number of elements, sealing, dark culture 2 days at 4 DEG C, then opposite
Humidity is then to be cultivated with 14h illumination and the processing of 10h dark culture alternate cycles up to emergence dark culture 3 days at 50%, 22 DEG C,
Obtain Wheat Haploid seedling.S4 removes culture apparatus sealing when Wheat Haploid miaoye the piece number >=3 piece, plant height 4cm in S3,
It is added 8ml aqua sterilisa slow seedling 2 days, washes away root culture medium, select the Wheat Haploid transplantation of seedlings of robust growth to containing Nutrient medium
In the pot for growing seedlings of matter and vermiculite, wherein the weight ratio of nutrient matrix and vermiculite is 2:1 in pot for growing seedlings, is put into 3 DEG C of vernalization room vernalization
25d obtains vernalization Wheat Haploid seedling.
S5 moves into 3d at 12 DEG C when vernalization Wheat Haploid seedling tiller number reaches 1 in S4, cleans vernalization wheat list
Times body seedling root cuts root, so that root depth is retained 2cm, is dipped in the mixed liquor of colchicin and dimethyl sulfoxide, tiller
The node not 1cm under liquid level, ventilation lucifugal handles 5h at 25 DEG C, and clear water is transplanted into pot for growing seedlings after rinsing overnight, trained at 12 DEG C
Support 10d, then balled transplanting into flowerpot to solid maturation, obtain wheat doubled haploid plant；
Wherein, the preparation method of colchicin and dimethyl sulfoxide mixed liquor are as follows: the autumn waters -- limid eyes that every mass per liter concentration is 0.1%
The dimethyl sulfoxide of 20ml is added in celestial alkali, mixes；
By through treated the Wheat Haploid transplantation of seedlings of colchicin and dimethyl sulfoxide mixed liquor to being equipped with educating for soil
The mixture of the nutrient matrix and vermiculite that are 1:2 with weight ratio when in seedling alms bowl wraps up vernalization Wheat Haploid seedling root, Nutrient medium
Matter selects conventional wheat seedling medium on the market, such as abundant sub- seedling medium of scape agricultural dark fund.
S6, Meccah times small in S5 haplobiont is filtered out by Markers for Detection and phenotypic evaluation with short bar and
The Ms2Ms2 genotype of male-sterile character after heading 2 days, simply cuts the whole fringe of grain husk as maternal, using common wheat as
Male parent is hybridized, and Ms2ms2 genotypic crossing F1 generation is obtained.
Embodiment 1 hybridizes through dwarf male sterile wheat and sweet-waxy maizes, and it is pure to obtain 21 Doubled haploid lines (ms2ms2 genotype)
Close fertile seed, 12 direct cross assemble it is short lose combination (Ms2ms2 genotype) seed, and demonstrate,proved by Markers for Detection
Real cross combination contains Taigu Genic Male-Sterile Gene.Moreover, the Wheat Haploid plant of this test double rate reach 90% with
On, single plant doubles solid and the solid acquisition of pollinating seed and differs from 3 to 155, and it is miscellaneous to provide enough Ms2ms2 genotype
Hand over progeny population.
Embodiment 2
A kind of method for breeding haploidy of Ms2ms2 genotype wheat, comprising the following steps:
S1, Henan Xinxiang, First Year October 7 choose mature, full dwarf male sterile wheat seed sowing, obtain it is short lose it is small
Wheat plant；Second year 5 days 2 months, sows sweet-waxy maizes in greenhouse, obtains plant, makes dwarf male sterile wheat and sweet-waxy maizes
Florescence can meet.
S2, mid-April in the same year, using dwarf male sterile wheat plant in S1 as maternal, plant as male parent, after female parent heading
The whole fringe of grain husk is simply cut within 3 days, is awarded after 1 day by fringe with paternal pollen, bagging isolation after pollination, after pollination for 24 hours, sprays 2,4- dichloro-benzenes
The mixed solution of fluoroacetic acid, arabinogalactan-proteins and dimethyl sulfoxide, for 24 hours after to spray mixed solution again primary, obtain small
Wheat hybrid plant；
Wherein, the preparation of the 1L mixed solution of 2,4- dichlorphenoxyacetic acid, arabinogalactan-proteins and dimethyl sulfoxide
Method are as follows: 20ml dimethyl sulfoxide, 2, the 4- dichlorphenoxyacetic acid of 100mg and 2g arabinogalactan-proteins are mixed, steaming is added
Distilled water is mixed to 1L.
S3, by wheat hybrid plant culture 18 days in S2,2 leaves of band cut fringe, take out seed through 70% alcohol of volumetric concentration
30s is sterilized, then with mass concentration is 0.1%HgCl2Sterilize 10min, aseptic water washing 5 times, each 1min, takes out monoploid children
Embryo is inoculated in the culture apparatus of the MS culture medium halved equipped with a great number of elements, sealing, dark culture 3 days at 4 DEG C, then opposite
Humidity is then to be cultivated with 14h illumination and the processing of 10h dark culture alternate cycles up to emergence dark culture 5 days at 70%, 25 DEG C,
Obtain Wheat Haploid seedling.
S4 removes culture apparatus sealing, 10ml is added when Wheat Haploid miaoye the piece number >=3 piece, plant height 7cm in S3
Aqua sterilisa slow seedling 3 days, root culture medium is washed away, selects the Wheat Haploid transplantation of seedlings of robust growth to containing nutrient matrix and vermiculite
Pot for growing seedlings in, wherein in pot for growing seedlings the weight ratio of nutrient matrix and vermiculite be 2:1, be put into 4 DEG C of vernalization room vernalization 35d, obtain
Vernalization Wheat Haploid seedling.
S5 moves into 5d at 14 DEG C when vernalization Wheat Haploid seedling tiller number 3 in S4, cleans vernalization Wheat Haploid
Seedling root cuts root, so that root depth is retained 4cm, is dipped in the mixed liquor of colchicin and dimethyl sulfoxide, tiller node
The not 2cm under liquid level, ventilation lucifugal handles 5h at 25 DEG C, and clear water is transplanted into pot for growing seedlings after rinsing overnight, cultivated at 14 DEG C
15d, then balled transplanting into flowerpot to solid maturation, obtain wheat doubled haploid plant；
Wherein, the preparation method of colchicin and dimethyl sulfoxide mixed liquor are as follows: the autumn waters -- limid eyes that every mass per liter concentration is 0.1%
The dimethyl sulfoxide of 20ml is added in celestial alkali, mixes；
By through treated the Wheat Haploid transplantation of seedlings of colchicin and dimethyl sulfoxide mixed liquor to being equipped with educating for soil
The mixture of the nutrient matrix and vermiculite that are 1:2 with weight ratio when in seedling alms bowl wraps up vernalization Wheat Haploid seedling root.
S6, Meccah times small in S5 haplobiont is filtered out by Markers for Detection and phenotypic evaluation with short bar and
The Ms2Ms2 genotype of male-sterile character after heading 3 days, simply cuts the whole fringe of grain husk as maternal, using common wheat as
Male parent is hybridized, and Ms2ms2 genotypic crossing F1 generation is obtained.
Embodiment 2 hybridizes through dwarf male sterile wheat and sweet-waxy maizes, and it is pure to obtain 45 Doubled haploid lines (ms2ms2 genotype)
Close fertile seed, 38 direct cross assemble it is short lose combination (Ms2ms2 genotype) seed, and demonstrate,proved by Markers for Detection
Real cross combination contains Taigu Genic Male-Sterile Gene.
Embodiment 3
A kind of method for breeding haploidy of Ms2ms2 genotype wheat, comprising the following steps:
S1, First Year October 15 choose mature, full dwarf male sterile wheat seed sowing, obtain dwarf male sterile wheat plant；The
2 years, 20 days 2 months, sweet-waxy maizes were sowed in greenhouse, plant is obtained, enables the florescence of dwarf male sterile wheat and sweet-waxy maizes
It meets.
S2, April in the same year, early and middle ten days, maternal to ear using dwarf male sterile wheat plant in S1 as maternal, plant as male parent
It afterwards after 5 days, awards by fringe with paternal pollen, bagging isolation after pollination, pollinates after 22h, spray 2,4- dichlorphenoxyacetic acid, Arab
The mixed solution of gala glycoprotein and dimethyl sulfoxide, it is primary to spray mixed solution after 23h again, obtains wheat hybridizing plant；
Wherein, the preparation of the 1L mixed solution of 2,4- dichlorphenoxyacetic acid, arabinogalactan-proteins and dimethyl sulfoxide
Method are as follows: 20ml dimethyl sulfoxide, 2, the 4- dichlorphenoxyacetic acid of 100mg and 2g arabinogalactan-proteins are mixed, steaming is added
Distilled water is mixed to 1L.
S3, by wheat hybrid plant culture 17 days in S2,2 leaves of band cut fringe, take out seed through 70% alcohol of volumetric concentration
30s is sterilized, then with mass concentration is 0.1%HgCl2Sterilize 10min, aseptic water washing 4 times, each 1min, takes out monoploid children
Embryo is inoculated in the culture apparatus of the MS culture medium halved equipped with a great number of elements, sealing, dark culture 3 days at 4 DEG C, then opposite
Humidity is then to be cultivated with 14h illumination and the processing of 10h dark culture alternate cycles up to emergence dark culture 4 days at 60%, 23 DEG C,
Obtain Wheat Haploid seedling.
S4 removes culture apparatus sealing when Wheat Haploid miaoye the piece number >=3 piece, plant height 5cm in S3, and 9ml is added and goes out
Bacterium water slow seedling 2 days, washes away root culture medium, selects the Wheat Haploid transplantation of seedlings of robust growth to containing nutrient matrix and vermiculite
In pot for growing seedlings, wherein the weight ratio of nutrient matrix and vermiculite is 2:1 in pot for growing seedlings, is put into 4 DEG C of vernalization room vernalization 28d, obtains the spring
Change Wheat Haploid seedling.
S5 moves into 4d at 13 DEG C when vernalization Wheat Haploid seedling tiller number 2 in S4, cleans vernalization Wheat Haploid
Seedling root cuts root, so that root depth is retained 3cm, is dipped in the mixed liquor of colchicin and dimethyl sulfoxide, tiller node
The not 1cm under liquid level, ventilation lucifugal handles 5h at 25 DEG C, and clear water is transplanted into pot for growing seedlings after rinsing overnight, cultivated at 13 DEG C
13d, then balled transplanting into flowerpot to solid maturation, obtain wheat doubled haploid plant；
Wherein, the preparation method of colchicin and dimethyl sulfoxide mixed liquor are as follows: the autumn waters -- limid eyes that every mass per liter concentration is 0.1%
The dimethyl sulfoxide of 20ml is added in celestial alkali, mixes；
By through treated the Wheat Haploid transplantation of seedlings of colchicin and dimethyl sulfoxide mixed liquor to being equipped with educating for soil
The mixture of the nutrient matrix and vermiculite that are 1:2 with weight ratio when in seedling alms bowl wraps up vernalization Wheat Haploid seedling root.
S6, Meccah times small in S5 haplobiont is filtered out by Markers for Detection and phenotypic evaluation with short bar and
The Ms2Ms2 genotype of male-sterile character after heading 3 days, simply cuts the whole fringe of grain husk as maternal, using common wheat as
Male parent is hybridized, and Ms2ms2 genotypic crossing F1 generation is obtained.
Embodiment 3 hybridizes through dwarf male sterile wheat and sweet-waxy maizes, and it is pure to obtain 37 Doubled haploid lines (ms2ms2 genotype)
Close fertile seed, 32 direct cross assemble it is short lose combination (Ms2ms2 genotype) seed, and demonstrate,proved by Markers for Detection
Real cross combination contains Taigu Genic Male-Sterile Gene.
It should be noted that the step method used in claims of the present invention is same as the previously described embodiments, in order to anti-
It only repeats, description of the invention preferred embodiment, once a person skilled in the art knows basic creative general
It reads, then additional changes and modifications can be made to these embodiments.So it includes preferred real that the following claims are intended to be interpreted as
It applies example and falls into all change and modification of the scope of the invention.
Obviously, various changes and modifications can be made to the invention without departing from essence of the invention by those skilled in the art
Mind and range.In this way, if these modifications and changes of the present invention belongs to the range of the claims in the present invention and its equivalent technologies
Within, then the present invention is also intended to include these modifications and variations.
Claims (4)
1. a kind of method for breeding haploidy of Ms2ms2 genotype wheat, which comprises the following steps:
S1, First Year early October choose mature, full dwarf male sterile wheat seed sowing, obtain dwarf male sterile wheat plant；Second
Year, sweet-waxy maizes are sowed in greenhouse mid-January to late Febuary, obtain plant；
S2, using dwarf male sterile wheat plant in S1 as maternal, plant as male parent, female parent heading is awarded after 2-6 days by fringe with father
This pollen, bagging isolation after pollination pollinate after 20-24h, spray 2,4- dichlorphenoxyacetic acid, arabinogalactan-proteins and two
The mixed solution of methyl sulfoxide, it is primary to spray the mixed solution after 20-24h again, obtains wheat hybridizing plant；
Wheat hybrid plant culture 16-18 days in S2 are taken out monoploid rataria and are inoculated in the MS halved equipped with a great number of elements by S3
It in the culture apparatus of culture medium, seals, dark culture 2-3 days at 4 DEG C, then in the case where relative humidity is 50%-70%, 22-25 DEG C
Dark culture 3-5 days, then with 14h illumination and 10h dark culture alternate cycles processing culture until emergence, obtains Wheat Haploid
Seedling；
S4 removes culture apparatus sealing, aqua sterilisa is added when Wheat Haploid miaoye the piece number >=3 piece, plant height 4-7cm in S3
Slow seedling 2-3 days, root culture medium is washed away, selects the Wheat Haploid transplantation of seedlings of robust growth to educating containing nutrient matrix and vermiculite
In seedling alms bowl, wherein the weight ratio of nutrient matrix and vermiculite is 2:1 in pot for growing seedlings, is put into 3-6 DEG C of vernalization room vernalization 25-35d, is obtained
Vernalization Wheat Haploid seedling；
S5 moves into 3-5d at 12-14 DEG C when vernalization Wheat Haploid seedling tiller number reaches 1-3 in S4, cleans vernalization wheat
Monoploid seedling root cuts root, so that root depth is retained 2-4cm, is dipped in the mixed liquor of colchicin and dimethyl sulfoxide,
The tiller node not 1-2cm under liquid level, ventilation lucifugal handles 5h at 25 DEG C, and clear water transplants the 12- into pot for growing seedlings after rinsing overnight
Cultivate 10-15d at 14 DEG C, then balled transplanting into flowerpot to solid maturation, obtain wheat doubled haploid plant；
S6 filters out with short bar and male Meccah times small in S5 haplobiont by Markers for Detection and phenotypic evaluation
The Ms2Ms2 genotype of sterile character after heading 2-3 days, cuts clever whole fringe as maternal, using common wheat as male parent into
Row hybridization, obtains Ms2ms2 genotypic crossing F1 generation.
2. the method for breeding haploidy of Ms2ms2 genotype wheat as described in claim 1, which is characterized in that 2,4- bis- in S2
The preparation method of the 1L mixed solution of chlorophenoxyacetic acid, arabinogalactan-proteins and dimethyl sulfoxide are as follows: by 20ml dimethyl
Sulfoxide, 2, the 4- dichlorphenoxyacetic acid of 100mg and the mixing of 2g arabinogalactan-proteins, add distilled water to 1L, mix.
3. the method for breeding haploidy of Ms2ms2 genotype wheat as described in claim 1, which is characterized in that colchicum in S5
The preparation method of alkali and dimethyl sulfoxide mixed liquor are as follows: the two of 20ml is added in the colchicin that every mass per liter concentration is 0.1%
Methyl sulfoxide mixes.
4. the method for breeding haploidy of Ms2ms2 genotype wheat as described in claim 1, which is characterized in that will be through the autumn in S5
The battalion for being 1:2 with weight ratio when treated the Wheat Haploid transplantation of seedlings of tazettine and dimethyl sulfoxide mixed liquor is into pot for growing seedlings
The mixture for supporting matrix and vermiculite wraps up vernalization Wheat Haploid seedling root.
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Cited By (1)
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| CN111670803A (en) * | 2020-07-03 | 2020-09-18 | 襄阳市农业科学院 | Method for breeding improved descendant DH new strain of dwarf-male-sterile wheat by chromosome disappearance method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102487816A (en) * | 2011-11-18 | 2012-06-13 | 河北省农林科学院旱作农业研究所 | Wheat double haploid production method by wheat-and-maize distant hybridization |
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