CN105494082B - A kind of form based on maize immature embryos differentiates haploid method - Google Patents
A kind of form based on maize immature embryos differentiates haploid method Download PDFInfo
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- CN105494082B CN105494082B CN201510917519.0A CN201510917519A CN105494082B CN 105494082 B CN105494082 B CN 105494082B CN 201510917519 A CN201510917519 A CN 201510917519A CN 105494082 B CN105494082 B CN 105494082B
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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
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
Abstract
Haploid method is differentiated based on maize immature embryos form the invention discloses one kind.This method comprises the following steps：It pollinates by the use of corn haploid induction line as male parent to maternal, young fringe is taken to carry out stripping embryo after pollination, obtains maize immature embryos；The maize immature embryos are ranked up according to the ascending sequence of length or width or area, selected and sorted is preceding 20 30% maize immature embryos, obtains corn monoploid rataria.It is experimentally confirmed, the method of the present invention can be realized in maize immature embryos period to be selected haploid, the diploid rataria of a big chunk heterozygosis is eliminated before the expression of rataria color, it is advanced by the time that monoploid selects, improve the efficiency selected, breeding time is further shortened, subsequent cost is also saved, improves economic benefit.And there is method provided by the invention automation to select haploid prospect, accelerate the process of corn monoploid engineering breeding.
Description
Technical field
The invention belongs to biotechnologies, and in particular to one kind differentiates haploid method based on maize immature embryos form.
Background technology
Corn is the first big crop of China's cultivated area, and 2012, corn seeding area was more than 34,000,000 hm2.China
More than 97% corn seeding area uses hybrid maize (Li J, 2009).The selection and breeding of good inbred lines are that corn utilizes
Hybrid vigour, the basis of selection and breeding elite hybrid and key.But traditional breeding method needs 7~8 could obtain more from generation to generation
Stable self-mating system, and haploid breeding technology only needs 2 generations (Weber D F, 2014).Therefore, haploid breeding technology
As a kind of method for being quickly obtained pure lines by domestic and international many Seed company's scale applications, becoming can be with transgenosis skill
One of the three big core technology of modernization corn breeding that art, molecular mark technology compare favourably (Chen Shaojiang etc., 2009).
With the large-scale application of haploid breeding technology, further improving its efficiency and engineering seems particularly important.
Monoploid techniqueflow can be divided into following 4 links：1. haploid generation；2. haploid discriminating；It is 3. haploid
It doubles；4. the utilization of DH systems.Wherein, haploid induction is most important to the generation of DH systems (doubled haploid, DH),
Decide the quantity that monoploid obtains.Haploid discriminating is the important link of haploid breeding engineering, is differentiated
Accuracy rate, rate directly affect the scale application of haploid breeding technology.
The factorial praluction that DH systems are carried out by tissue culture approach is to realize that monoploid is engineered the another important way of breeding.
Existing haploid discrimination method is as included tip of a root tabletting chromosome counting, Flow cytometry analysis nucleic acid content, SSR and SNP
(the Antoine-Michard S and such as Markers for Detection, R1-nj color marks, seed oil label, kernel weight
Beckert M,1997；Chen Shaojiang etc., 2003；Barret P et al,2008；Melchinger A E et al,2013；Xu
X et al,2013；Barton J E et al,2014；Smelser A et al,2015).These methods are mainly to seed
It is preferable with plant effect, but under condition of tissue culture, how to realize that haploid quick and precisely selection there is no high efficiency method.
Invention content
It is an object of the present invention to provide a kind of methods for identifying or assisting in discriminating corn monoploid rataria.
It is provided by the invention identify or assisting in differentiate the method for corn monoploid rataria for it is following 1) or 2) or 3)：
1) it pollinates by the use of corn haploid induction line as male parent to maternal, young fringe is taken to carry out stripping embryo after pollination, obtains corn
Rataria；The maize immature embryos are ranked up according to the ascending sequence of width, the corn children that selected and sorted is preceding 20-30%
Embryo, obtains or candidate obtains corn monoploid rataria；
2) it pollinates by the use of corn haploid induction line as male parent to maternal, young fringe is taken to carry out stripping embryo after pollination, obtains corn
Rataria；The maize immature embryos are ranked up according to the ascending sequence of area, the corn children that selected and sorted is preceding 20-30%
Embryo, obtains or candidate obtains corn monoploid rataria；
3) it pollinates by the use of corn haploid induction line as male parent to maternal, young fringe is taken to carry out stripping embryo after pollination, obtains corn
Rataria；The maize immature embryos are ranked up according to the ascending sequence of length, the corn children that selected and sorted is preceding 20-30%
Embryo, obtains or candidate obtains corn monoploid rataria.
In the above method, the selected and sorted is preceding 30% maize immature embryos.
In the above method, the maize immature embryos length is 2.0~7.0mm.
In the above method, the corn haploid induction line lures the high oily height of No. 5 or agricultural university to lure No. 3 for agricultural university's height；The mother
This is corn inbred line or corn hybrid seed.
In the above method, the corn inbred line is B73 or Mo17；The corn hybrid seed is Zheng Dan 958 or B73/
Mo17；
The B73/Mo17 is using B73 as female parent, and Mo17 is the hybrid generation of male parent.
In the above method, the quantity of the maize immature embryos is 86-1734；The quantity of the maize immature embryos is specially 86,
189th, 428,335,411,474,1642 or 1734.
It is a further object to provide the new applications of the above method.
The present invention provides application of the above method in corn haplobiont is cultivated.
Final object of the present invention is to provide a kind of method for cultivating corn haplobiont.
It is provided by the invention cultivate corn haplobiont method include the following steps,
1) it pollinates by the use of corn haploid induction line as male parent to maternal, young fringe is taken to carry out stripping embryo after pollination, obtains corn
Rataria；The maize immature embryos are ranked up according to the ascending sequence of length or width or area, selected and sorted is preceding 20-
30% maize immature embryos, obtain or candidate obtains corn monoploid rataria；
2) the corn monoploid rataria on culture medium is doubled is doubled, cultivated, according to plant type and/or stalk face
Color sorting takes corn haplobiont.
It is experimentally confirmed：The method of the present invention can be realized in maize immature embryos period to be selected haploid, in rataria
The diploid rataria of a big chunk heterozygosis is eliminated before color expression, is advanced by the time that monoploid selects, raising is chosen
The efficiency of choosing, further shortens breeding time, also saves subsequent cost, improves economic benefit.And the present invention provides
Method have automation select haploid prospect, accelerate corn monoploid engineering breeding process.
Description of the drawings
Fig. 1 is the photo of monoploid rataria.Wherein, red arrow meaning is monoploid rataria.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Corn haploid induction line agricultural university height lure No. 5 (CAU5) be following embodiments in male parent, document " Dong, X.,
et al.(2014)."Marker-assisted selection and evaluation of high oil in vivo
haploid inducers in maize."Molecular Breeding 34(3):1147-1158. " disclosed in mistake, the public
It can be obtained from China Agricultural University.
Corn (Zea mays L.) cenospecies Zheng Dan 958 is the product of Beijing Si Nongzhong industry Co., Ltd, performs standard：
GB4404.1-2008。
It is the male parent in following embodiments that the high oily height of corn haploid induction line agricultural university, which lures No. 3 (CHOI3), in document " Dong
Sunrise, 2014, corn haploid induction gene qhir1 finely positionings and novel induction are election effects, doctoral thesis " disclosed in
It crosses, the public can obtain from China Agricultural University.
Corn inbred line B73 and Mo17 be following embodiments in female parent, document " Lee M, Sharopova N,
Beavis W D,et al.Expanding the genetic map of maize with the intermated B73×
Mo17(IBM)population[J].Plant molecular biology,2002,48(5-6):Disclosed in 453-461. "
It crosses, the public can obtain from China Agricultural University.
MS salt is the product of Shanghai Yu Han bio tech ltd, catalog number 140225.
B73/Mo17 be using B73 as female parent, Mo17 be paternal hybrid obtain F1 generation individual, document " Lee M,
Sharopova N,Beavis W D,et al.Expanding the genetic map of maize with the
intermated B73×Mo17(IBM)population[J].Plant molecular biology,2002,48(5-6):
Mistake disclosed in 453-461. ", the public can obtain from China Agricultural University.
Embodiment 1, a kind of maize immature embryos form that is based on differentiate haploid method
First, the discrimination method of monoploid rataria
1st, hybridize
(1) hybrid strain
Using B73 as female parent, using induction be CAU5 as male parent, hybridization, obtain hybrid generation A；
Using Mo17 as female parent, using induction be CAU5 as male parent, hybridization, obtain hybrid generation B；
Using B73 as female parent, using induction be CHOI3 as male parent, hybridization, obtain hybrid generation C；
Using Mo17 as female parent, using induction be CHOI3 as male parent, hybridization, obtain hybrid generation D.
(2) specific method of hybridization
Spring in 2015, the loose powder phase carried out field pollination work by the plantation of maternal and male parent in Bei Jingshang village experiment centre：Treat mother
After this material B73 and Mo17 major part filigree all sprouts, in it is excessive on the same day be respectively CAU5 and CHOI3 to female parent material by induction
B73 and Mo17 pollinate；It takes young fringe within 12-23 days after pollination, obtains the hybrid generation that immature embryo length is 2~7mm.
2nd, pseudohaploid rataria is selected
(1) stripping embryo is taken pictures
The young fringe of the hybrid generation A of a length of 2.0-7.0mm embryos of rataria, hybrid generation B, hybrid generation C, hybrid generation D are taken,
Second day stripping embryo is simultaneously taken pictures (young fringe can temporarily be put in 4 DEG C after fetching, but should not too long).It is as follows：Young fringe is shelled
Bract is gone, plucks net filigree, is inserted into tweezers from top, is entered super-clean bench after the disinfection of 75% alcohol mist, filled with clean large beaker
75% alcohol of 1500ml, corn is put into, is stirred evenly, and in disinfection a moment, during which corn agitation is several times.It takes out, treats after the completion of disinfection
Start to shell embryo after vaporized alcohol, obtain maize immature embryos, and place it in and double on culture medium, while with Stereo microscope to each
Culture dish is taken pictures.
(2) pseudohaploid is selected
According to the situation of taking pictures, the hybrid generation E that above-mentioned steps 1 obtain, hybrid generation F, hybrid generation G, miscellaneous is calculated respectively
Maize immature embryos and according to length are ranked up for the length of the maize immature embryos of H, ranking (are chosen for preceding 20% by jiao zi from small to large
Select 20% rataria group) and the rataria of preceding 30% (selecting 30% rataria group) do mark, as pseudohaploid rataria.
According to the situation of taking pictures, the hybrid generation E that above-mentioned steps 1 obtain, hybrid generation F, hybrid generation G, miscellaneous is calculated respectively
Maize immature embryos and according to width are ranked up for the width of the maize immature embryos of H, ranking (are chosen for preceding 20% by jiao zi from small to large
Select 20% rataria group) and the rataria of preceding 30% (selecting 30% rataria group) do mark, as pseudohaploid rataria.
According to the situation of taking pictures, the hybrid generation E that above-mentioned steps 1 obtain, hybrid generation F, hybrid generation G, miscellaneous is calculated respectively
Maize immature embryos and according to area are ranked up for the area of the maize immature embryos of H, are preceding 20% by ranking by jiao zi from small to large
The rataria of (selecting 20% rataria group) and preceding 30% (selecting 30% rataria group) does mark, as pseudohaploid rataria.
2nd, the identification of haplobiont
1st, monoploid rataria doubles：The pseudohaploid rataria (Fig. 1) that above-mentioned steps one are selected and remaining corn children
Doubling culture medium, (MS salt is 3.0g/L to embryo, sucrose 40g/L, agar 7.5g/L, Tween 80 are 1 ‰, dimethyl sulfoxide (DMSO) is
2%) it is doubled in, illumination cultivation (incubation time is 1-4 days, and temperature is 26-30 DEG C), the rataria after being doubled.Because of flower
Powder directly-sensing effect since heterozygous diploid contains the A1A2C1C2R1-nj genes from male parent, therefore can clearly be seen in embryo portion scultellum
It is expressed to purple pigment, and monoploid rataria is free of the dominant gene of male parent, therefore embryo portion scultellum is colourless, is being transferred to regeneration culture medium
Before regeneration, having by embryo portion scultellum colourless can eliminate most heterozygous diploids, remaining only a few heterozygote.
2nd, raw seedling：Rataria after doubling is transferred to regeneration culture medium (salt containing MS 3.0g/L, sucrose 30g/L, agar
Raw seedling in 7.5g/L).Notice that embryo is face-up at this time.28 DEG C of light cultures 3-5 days, subsequent normal illumination culture obtains hybrid generation
Seedling；After about 10-14 days, two leaf of seedling wholeheartedly when, move in culture bottle in time, in culture bottle be MS culture mediums (salt containing MS
3.0g/L, sucrose 30g/L, agar 7.5g/L).Seedlings root is flourishing after 7-10 days, four leaves wholeheartedly when, need to transplant seedlings in time to temperature
The suitable environment such as room carries out hardening.
3rd, hardening：The seedling of above-mentioned hybrid generation is moved in nutritive cube, hardening is carried out in suitable place such as greenhouse.It moves
Pay attention to rinsing well root culture medium during seedling, being put into Nutrition Soil, (Nutrition Soil is configured：Nutrition Soil：Vermiculite=3：1) in.Refining
Initial stage shade, liquid manure supply are paid attention between seedling stage, controlling disease can pour a water a little with carbendazim.
4th, it transplants：Hardening about treats that seedling starts restoration ecosystem after a week, and the 5-6 leaf phases select rainy weather or dusk, move
It plants into crop field or greenhouse flowerpot.Pay attention to field management, ensure the timely ample supply of liquid manure, when loose powder, carry out bagging, selfing
Pollination, obtains plant (dihaploid) after doubling monoploids.
5th, the identification of haplobiont
It is colourless since CAU5, CHOI3 induction are plant color itself, so haplobiont can not be distinguished with color
With heterozygous diploid plant, can corn haplobiont be chosen according to plant plant type at this time：Plant is tall and big or blade to hang down loosely be miscellaneous
Close liploid plant, and plant is short and small or blade on punching be haplobiont.
3rd, haploid efficiency is differentiated according to maize immature embryos form
According to the identification result of haplobiont, represented to select 20% rataria group with A；It is represented to select 30% rataria group with B；
Mistake selects heterozygous diploid rataria number/pseudohaploid rataria total number in rate=pseudohaploid rataria；Rate=leakage menu is selected in leakage
Times body rataria number/embryo sum.The monoploid based on 3 morphological indexs is counted respectively selecting leakage, rate and mistake is selected to select rate.
1st, carry out differentiating monoploid rataria that the results are shown in Table 1 based on area：As can be seen from the table, on the whole, it chooses
30% rataria group is selected to differentiate that the mistake of monoploid rataria selects a little higher than 20% rataria group of selecting of rate to be based on area discriminating list based on area
The mistake of times body rataria selects rate, but selects 30% rataria group and differentiate that the leakage of monoploid rataria is selected rate to be substantially less than and selected based on area
20% rataria group differentiates that rate is selected in the leakage of monoploid rataria based on area.
Table 1, the mistake based on area select rate and leakage to select rate statistical result
2nd, carry out differentiating monoploid rataria that the results are shown in Table 2 based on length：As can be seen from the table, on the whole, it chooses
30% rataria group is selected to differentiate that the mistake of monoploid rataria selects a little higher than 20% rataria group of selecting of rate to be based on length discriminating list based on length
The mistake of times body rataria selects rate, but selects 30% rataria group and differentiate that the leakage of monoploid rataria is selected rate to be substantially less than and selected based on length
20% rataria group differentiates that rate is selected in the leakage of monoploid rataria based on length.
Table 2, the mistake based on length select rate and leakage to select rate statistical result
3rd, carry out differentiating monoploid rataria that the results are shown in Table 3 based on width：As can be seen from the table, on the whole, it chooses
30% rataria group is selected to differentiate that the mistake of monoploid rataria selects a little higher than 20% rataria group of selecting of rate to be based on width discriminating list based on width
The mistake of times body rataria selects rate, but selects 30% rataria group and differentiate that the leakage of monoploid rataria is selected rate to be substantially less than and selected based on width
20% rataria group differentiates that rate is selected in the leakage of monoploid rataria based on width.
Table 3, the mistake based on width select rate and leakage to select rate statistical result
In summary：The present embodiment is hybridized using self-mating system B73 and Mo17 as female parent, obtains the rataria of hybrid generation,
It is proved by testing：The discrimination method of the monoploid rataria of the present invention can be used for the hybrid generation obtained using self-mating system as female parent
Monoploid rataria screening or assisting sifting.
Embodiment 2, the application for differentiating haploid method based on maize immature embryos form
First, the discrimination method of monoploid rataria
1st, hybridize
(1) hybrid strain
With Zheng Dan 958 for female parent, using induction be CAU5 as male parent, hybridization, obtain hybrid generation E；
With Zheng Dan 958 for female parent, using induction be CHOI3 as male parent, hybridization, obtain hybrid generation F；
Using B73/Mo17 as female parent, using induction be CAU5 as male parent, hybridization, obtain hybrid generation G；
Using B73/Mo17 as female parent, using induction be CHOI3 as male parent, hybridization, obtain hybrid generation H.
(2) specific method of hybridization
Spring in 2015, the loose powder phase carried out field pollination work by the plantation of maternal and male parent in Bei Jingshang village experiment centre：Treat mother
After this material Zheng Dan 958, B73/Mo17 major part filigrees all sprout, in it is excessive on the same day be respectively CAU5 and CHOI3 couples by induction
Female parent material Zheng Dan 958, B73/Mo17 pollinate；Take young fringe within 12-23 days after pollination, it is the miscellaneous of 2~7mm to obtain immature embryo length
Jiao zi generation.
2nd, pseudohaploid rataria is selected
(1) stripping embryo is taken pictures
The young fringe of the hybrid generation E of a length of 2.0-7.0mm embryos of rataria, hybrid generation F, hybrid generation G, hybrid generation H are taken,
Second day stripping embryo is simultaneously taken pictures (young fringe can temporarily be put in 4 DEG C after fetching, but should not too long).It is as follows：Young fringe is shelled
Bract is gone, plucks net filigree, is inserted into tweezers from top, is entered super-clean bench after the disinfection of 75% alcohol mist, filled with clean large beaker
75% alcohol of 1500ml, corn is put into, is stirred evenly, and in disinfection a moment, during which corn agitation is several times.It takes out, treats after the completion of disinfection
Start to shell embryo after vaporized alcohol, obtain maize immature embryos, and place it in and double on culture medium, while with Stereo microscope to each
Culture dish is taken pictures.
(2) pseudohaploid is selected
According to the situation of taking pictures, the hybrid generation E that above-mentioned steps 1 obtain, hybrid generation F, hybrid generation G, miscellaneous is calculated respectively
Maize immature embryos and according to length are ranked up for the length of the maize immature embryos of H, ranking (are chosen for preceding 20% by jiao zi from small to large
Select 20% rataria group) and the rataria of preceding 30% (selecting 30% rataria group) do mark, as pseudohaploid rataria.
According to the situation of taking pictures, the hybrid generation E that above-mentioned steps 1 obtain, hybrid generation F, hybrid generation G, miscellaneous is calculated respectively
Maize immature embryos and according to width are ranked up for the width of the maize immature embryos of H, ranking (are chosen for preceding 20% by jiao zi from small to large
Select 20% rataria group) and the rataria of preceding 30% (selecting 30% rataria group) do mark, as pseudohaploid rataria.
According to the situation of taking pictures, the hybrid generation E that above-mentioned steps 1 obtain, hybrid generation F, hybrid generation G, miscellaneous is calculated respectively
Maize immature embryos and according to area are ranked up for the area of the maize immature embryos of H, are preceding 20% by ranking by jiao zi from small to large
The rataria of (selecting 20% rataria group) and preceding 30% (selecting 30% rataria group) does mark, as pseudohaploid rataria.
2nd, the identification of haplobiont
1st, monoploid rataria doubles：Pseudohaploid rataria that above-mentioned steps one are selected and remaining maize immature embryos are being added
In times culture medium (MS salt is 3.0g/L, sucrose 40g/L, agar 7.5g/L, Tween 80 are 1 ‰, dimethyl sulfoxide (DMSO) 2%)
It is doubled, illumination cultivation (incubation time is 1-4 days, and temperature is 26-30 DEG C), the rataria after being doubled.Because of xenia
Effect since heterozygous diploid contains the A1A2C1C2R1-nj genes from male parent, therefore can be clearly apparent purple in embryo portion scultellum
Pigment is expressed, and monoploid rataria is free of the dominant gene of male parent, therefore embryo portion scultellum is colourless, is being transferred to regeneration culture medium regeneration
Before, having by embryo portion scultellum colourless can eliminate most heterozygous diploids, remaining only a few heterozygote.
2nd, raw seedling：Rataria after doubling is transferred to regeneration culture medium (salt containing MS 3.0g/L, sucrose 30g/L, agar
Raw seedling in 7.5g/L).Notice that embryo is face-up at this time.28 DEG C of light cultures 3-5 days, subsequent normal illumination culture obtains hybrid generation
Seedling；After about 10-14 days, two leaf of seedling wholeheartedly when, move in culture bottle in time, in culture bottle be MS culture mediums (salt containing MS
3.0g/L, sucrose 30g/L, agar 7.5g/L).Seedlings root is flourishing after 7-10 days, four leaves wholeheartedly when, need to transplant seedlings in time to temperature
The suitable environment such as room carries out hardening.
3rd, hardening：The seedling of above-mentioned hybrid generation is moved in nutritive cube, hardening is carried out in suitable place such as greenhouse.It moves
Pay attention to rinsing well root culture medium during seedling, being put into Nutrition Soil, (Nutrition Soil is configured：Nutrition Soil：Vermiculite=3：1) in.Refining
Initial stage shade, liquid manure supply are paid attention between seedling stage, controlling disease can pour a water a little with carbendazim.
4th, it transplants：Hardening about treats that seedling starts restoration ecosystem after a week, and the 5-6 leaf phases select rainy weather or dusk, move
It plants into crop field or greenhouse flowerpot.Pay attention to field management, ensure the timely ample supply of liquid manure, when loose powder, carry out bagging, selfing
Pollination, obtains plant (dihaploid) after doubling monoploids.
5th, the identification of haplobiont
It is colourless since CAU5, CHOI3 induction are plant color itself, so haplobiont can not be distinguished with color
With heterozygous diploid plant, can corn haplobiont be chosen according to plant plant type at this time：Plant is tall and big or blade to hang down loosely be miscellaneous
Close liploid plant, and plant is short and small or blade on punching be haplobiont.
3rd, haploid efficiency is differentiated according to maize immature embryos form
According to the identification result of haplobiont, represented to select 20% rataria group with A；It is represented to select 30% rataria group with B；
Mistake selects the number of heterozygous diploid in rate=pseudohaploid)/pseudohaploid rataria total number；Leakage selects rate=leakage to select monoploid rataria
Number/embryo sum.The monoploid based on 3 morphological indexs is counted respectively selecting leakage, rate and mistake is selected to select rate.
1st, carry out differentiating monoploid rataria that the results are shown in Table 4 based on area：As can be seen from the table, on the whole, it chooses
30% rataria group is selected to differentiate that the mistake of monoploid rataria selects a little higher than 20% rataria group of selecting of rate to be based on area discriminating list based on area
The mistake of times body rataria selects rate, but selects 30% rataria group and differentiate that the leakage of monoploid rataria is selected rate to be substantially less than and selected based on area
20% rataria group differentiates that rate is selected in the leakage of monoploid rataria based on area.
Table 4, the mistake based on area select rate and leakage to select rate statistical result
2nd, carry out differentiating monoploid rataria that the results are shown in Table 5 based on length：As can be seen from the table, on the whole, it chooses
30% rataria group is selected to differentiate that the mistake of monoploid rataria selects a little higher than 20% rataria group of selecting of rate to be based on length discriminating list based on length
The mistake of times body rataria selects rate, but selects 30% rataria group and differentiate that the leakage of monoploid rataria is selected rate to be substantially less than and selected based on length
20% rataria group differentiates that rate is selected in the leakage of monoploid rataria based on length.
Table 5, the mistake based on length select rate and leakage to select rate statistical result
3rd, carry out differentiating monoploid rataria that the results are shown in Table 6 based on width：As can be seen from the table, on the whole, it chooses
30% rataria group is selected to differentiate that the mistake of monoploid rataria selects a little higher than 20% rataria group of selecting of rate to be based on width discriminating list based on width
The mistake of times body rataria selects rate, but selects 30% rataria group and differentiate that the leakage of monoploid rataria is selected rate to be substantially less than and selected based on width
20% rataria group differentiates that rate is selected in the leakage of monoploid rataria based on area.
Table 6, the mistake based on width select rate and leakage to select rate statistical result
In summary：The present embodiment is hybridized using cenospecies Zheng Dan 958 and B73/Mo17 as female parent, obtains hybrid generation
Rataria, pass through test prove：The method of the present invention can be used for the monoploid of hybrid generation obtained using cenospecies as female parent
The screening of rataria.
Claims (7)
1. a kind of identify or assisting in the method for differentiating corn monoploid rataria, for it is following 1) or 2) or 3)：
1) it pollinates by the use of corn haploid induction line as male parent to maternal, young fringe is taken to carry out stripping embryo after pollination, obtains maize immature embryos；
The maize immature embryos are ranked up according to the ascending sequence of width, selected and sorted is the maize immature embryos of preceding 20-30%, is obtained
To or candidate obtain corn monoploid rataria；
2) it pollinates by the use of corn haploid induction line as male parent to maternal, young fringe is taken to carry out stripping embryo after pollination, obtains maize immature embryos；
The maize immature embryos are ranked up according to the ascending sequence of area, selected and sorted is the maize immature embryos of preceding 20-30%, is obtained
To or candidate obtain corn monoploid rataria；
3) it pollinates by the use of corn haploid induction line as male parent to maternal, young fringe is taken to carry out stripping embryo after pollination, obtains maize immature embryos；
The maize immature embryos are ranked up according to the ascending sequence of length, selected and sorted is the maize immature embryos of preceding 20-30%, is obtained
To or candidate obtain corn monoploid rataria；
Young fringe is taken after the pollination to take within 12-23 days young fringe after pollination；
The corn haploid induction line lures No. 5 for agricultural university's height；It is described maternal for corn inbred line or corn hybrid seed.
2. according to the method described in claim 1, it is characterized in that：The selected and sorted is preceding 30% maize immature embryos.
3. method according to claim 1 or 2, it is characterised in that：The maize immature embryos length is 2.0~7.0mm.
4. method according to claim 1 or 2, it is characterised in that：The corn inbred line is B73 or Mo17；The jade
Rice cenospecies is Zheng Dan 958 or B73/Mo17；
The B73/Mo17 is using B73 as female parent, and Mo17 is the hybrid generation of male parent.
5. method according to claim 1 or 2, it is characterised in that：The quantity of the maize immature embryos is 86-1734.
6. application of any method in corn haplobiont is cultivated in claim 1-5.
7. a kind of method for cultivating corn haplobiont, includes the following steps：
1) it pollinates by the use of corn haploid induction line as male parent to maternal, young fringe is taken to carry out stripping embryo after pollination, obtains maize immature embryos；
The maize immature embryos are ranked up according to the ascending sequence of length or width or area, selected and sorted is preceding 20-30%
Maize immature embryos, obtain or candidate obtain corn monoploid rataria；
2) the corn monoploid rataria on culture medium is doubled is doubled, cultivated, selected according to plant type and/or stalk color
Take corn haplobiont；
Young fringe is taken after the pollination to take within 12-23 days young fringe after pollination；
The corn haploid induction line lures No. 5 for agricultural university's height；It is described maternal for corn inbred line or corn hybrid seed.
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