CN106613985A - Method for rapidly creating double-haploid homozygous progeny of transgenic maize - Google Patents
Method for rapidly creating double-haploid homozygous progeny of transgenic maize Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention discloses a method for rapidly creating a double-haploid homozygous progeny of transgenic maize. Compared with the prior art, Dongyou No. 1 of a maize haploid inducing line is used as a male parent to pollinate a transgenic maize hybrid, a haploid young embryo is subjected to tissue culture, and is subjected to chromosome doubling by using colchicines, then a transgenic double-haploid maize plant is cultured, and transgenic double-haploid seeds are harvested. The method can be used for rapid homozygosis of backcross transformation progeny of the transgenic maize, and can also be used for quickly establishing a maize double-haploid group to carry out genetic analysis of important characters of the maize.
Description
Technical field
The present invention relates to plant biotechnology field, more particularly to a kind of quick initiative transgenic corns dihaploid homozygosis
The method of offspring.
Background technology
Corn (Zea mays) is important grain and forage crop, and in China's maize sown area most first has been risen
Position, per unit area yield and total producing keep second, be only second to paddy rice (Li Shaokun, 2010).Simultaneously because corn is also to carry out genetic research
Good material with the development of World Economics, the raising of living standard, people are to the demand trend of corn in rigidity increasing
It is long.Using hybrid vigour, the basic and crucial seed selection good inbred lines of seed selection superior corn cenospecies.Traditional breeding is excellent
Self-mating system is to assemble after basic material that continuous selfing 5～6 is more than generation from quality germplasm according to breeding objective, and proterties is stable
After carry out combining ability test, assemble cross combination further according to Heterosis.Corn monoploid is referred to gametophyte dyeing
The plant of body number, can to a certain extent overcome traditional breeding way to exist not using Haploid Breeding of Maize technology
Foot.Carrying out breeding using monoploid research can shorten the time of generation homozygous line.
Corn haploid induction line Stock6 for commonly using at present is that Ed Coe (1950) have found that this is self progeny
In can produce about 2.52% haplobiont.Control Kernel trait and plant are imported with selfing twice afterwards and through being returned twice
The Dominant selectable marker of strain proterties (purple blade, stalk, tassel and flower pesticide), the dominant pigment gene of 90% individuality is up to pure
Close, genotype is ABPICR-njy, it has two mark properties：Seed is dapple, strain blade and leaf sheath are in purple, is
By complementary genes control.Phenotype is that fruit ear seed is white Hard grain type, and there are purple patch and purple plumule, plant in seed top
Blade with darkviolet, stalk, tassel and flower pesticide, with triple proterties mark.Stock6 induces the principle of Haploid production
Clear, a sperm is combined with polar core in double Process of Insemination, and another sperm can not combine to form insemination ovum with ovum.
It is used as the derivative breeding examination material of male parent in breeding practice and makees maternal, about 3% haploid embryo.Stock6 is lured with hybridization
Lead maternal egagametophyte and form the haploid ability of high-frequency, meet the basic demand of haploid breeding, so that corn list times
Physical culture kind is changed into a kind of breeding method of reality.But find that Stock6 has many defects, such as pollen amount in actual applications
Seldom or not loose powder, self-fruitfulness are very poor, QTL mapping is serious, the expression of seed Navajo genetic marker is weaker.
Foreign countries pass through the method for crossbreeding and improvement from some new induction systems have been gone out derived from Stock6, such as France
ZMS of SW14, Muscovite Krasnodar Markers and Moldova etc..These new induction systems not only conform to locality
Ecological environment, and inductivity has further raising.Hence with value well beyond Stock6.
China is later using the research development of haploid inducing line Breeding of Inbred Lines, and haploid breeding system is all to improve
Based on Stock6, the haploid inducing line of seed selection is less.China Agricultural University Liu Zhi increase (1998) with High Oil Corn Populations BHO and
Stock6 hybridizes, and Jing inbreeding of more generation and test cross select agricultural university's height and lure No. 1 haploid inducing line, its inductivity to may be up to 5.8%.
The just tall and erect of Jilin Academy of Agricultural Science is waited from continuous 6 generation test cross and selection in the filial generation of Stodk6 and M278 within 2007, is educated
It is No. 3 to lure into high-frequency haploid inducing line Ji height, and the haploid induction result for there be not Genotype to 20 shows, its list
Times body inductivity between 5.50%～15.94%, average out to 10.40%.
In system/program is selected using haploid inducing line, monoploid individuality must double to become dliploid and could produce and can educate
Offspring.Haploid double frequency is directly connected to the efficiency of haploid breeding.People are ground in a large number to inducing monoploid
Study carefully, and achieve certain achievement.According to Chase, haploid Natural double frequency is 10% or so；Zabirova etc. and
The data of Shatskaya shows that the monoploid Natural doubling rate of many materials is less than 5%, and some materials do not occur Natural double.
The haplobiont Natural doubling rate that induction is produced is very low, just need to can obtain more DH systems through artificial doubling, is educated with meeting
Planting needs.Early in nineteen fifty-two Chase it is proposed that the scutellum node for injecting seedling with colchicine solution is doubled, Gayen exists
Use method for soaking seed within 1994, make plus multiplying power has reached 18%.However, up to the present the haploid chemistry of corn adds multiplying power
Still than relatively low.Need to explore more efficiently method for doubling for this, improve plus multiplying power, to carrying for corn monoploid artificial doubling
For a kind of effective ways.
The genetic transformation efficiency of corn is relatively low, generally below 5%, and with stronger genotype-independent, regeneration frequency compared with
It is high, that be suitable for induction II type embryo callus is corn hybrid seed HiII.Generally acknowledged corn genetic transformation method is Agrobacterium
The rataria of HiII is infected, through evoked callus, regeneration plant is differentiated.Afterwards by the HiII offsprings of transgenosis and common jade
6-8 generations are returned after rice hybridization between selfed lines, in selfing 2-3 generations, target gene are proceeded in conventional corn self-mating system, the test period is long,
Time and effort consuming.Using haploid induction technology, the HiII offsprings of transgenosis are carried out with the F1 generation of conventional corn hybridization between selfed lines
Haploid induction further doubles into zygoid, can greatly accelerate the speed of genotype homozygosis.
The content of the invention
The purpose of the present invention is that and provide that a kind of quick initiative transgenic corns are double single times in order to solve the above problems
The method of body homozygosis offspring.
The present invention is achieved through the following technical solutions above-mentioned purpose：
The present invention includes：
Test material：Corn haploid induction line east lures No. 1, L7 and JY3, is agricultural college of Northeast Agricultural University corn problem
Group seed selection, with R-nj marks；For creating haploid F1 cenospecies, its female parent is to turn corn arginase ZmARG genes
The T2 of corn HiII is conjunction 344 for strain, male parent；The above-mentioned ordinary maize material public can be from agricultural college of Northeast Agricultural University corn
Seminar obtains；
Test method：
The plantation of material：Using RANDOMIZED BLOCK DESIGN, 3 repetitions, duplicate rows area, the long 5m of row, spacing in the rows 30cm, line width 70cm,
Every 17 plants of row；Field management standard in two places is consistent, and with conventional corn field is produced；5 meters of row length, spacing in the rows 30cm, line-spacing 70cm；Hybridization
Plant and once sow, the time is April 29 days then, induction 3 phases of system point are sowed, and the time is respectively April 29 days then, May 7 and 5
The moon 11；
Pollination：The pollination of F1 cenospecies is given with induction system, be listed record pollination time, each induction system at least awards 10 plants；
The experiment process of tissue cultures：
Culture medium：
(1) screening and culturing medium：MS fluid nutrient medium+100uM ABA；
(2) culture medium is doubled：The colchicine of MS fluid nutrient medium+2mg/L asparagine+1mg/L BAP+0.05%；
(3) growth medium：MS solid mediums；
Method of operating：
(A) sterilization of young fringe
The 18d after pollination respectively, 20d take young fringe, peel off bract, and sterilize 8min, 0.1%HgCl in 75% ethanol2Disappear
Malicious 6min, aseptic water washing 3 times；
(B) rataria is stripped：
Rataria is taken, scultellum is placed in down in culture dish on the aseptic filter paper infiltrated with screening and culturing medium, 28 DEG C, 16h illumination/
8 hours dark, cultivate 24h；
(C) monoploid rataria is doubled：
Pick out the non-pigmented monoploid rataria of scultellum, scultellum is inoculated into upward in culture dish with doubling culture medium leaching
On the aseptic filter paper of profit, 26 DEG C of dark culturings 24h；
(D) IMMATURE EMBRYOS CULTURE：
Rataria is transferred in grown cultures bottle, scultellum downwards, 26 DEG C of light cultures 1 week, afterwards 26 DEG C of optical cultures 2-3 weeks are straight
It is complete to root development；Per bottle is inoculated with 15 or so；
(E) transplant：
By in little transplantation of seedlings to soil matrix, cultivate in greenhouse, haplobiont is differentiated according to leaf sheath color, until into
Ripe, strict selfing harvests seed；
(F) the PCR detections of dihaploid：
The STb gene of Leaves of Maize Seedlings is extracted using CTAB a small amount of method；Due to ZmARG genes be corn endogenous gene, root
According to promoter Ubi sequence and objective gene sequence design primer, primer ARG-F is located at promoter Ubi interior sequences, primer ARG-
R is located inside objective gene sequence, and primer size is 775bp；Riddled basins are antiweed glyphosate gene, and product is big
It is little for 522bp；Primer sequence is as follows：
ARG-F:5’-CGCCCTTCTTTGGTGAT-3’
ARG-R:5’-CCCTGCCTTCATACGCT-3’
PCR response procedures are, 94 DEG C of 5min, 94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min, 72 DEG C of 8min, 32 circulations；1%
Agarose gel electrophoresis detection pcr amplification product；
Data statistical approach:
Haploid inductivity=monoploid rataria number/inoculation rataria number * 100%；Plus multiplying power=solid strain number/regrowth
Number * 100%；Edit is carried out to test data using Excel 2010, using statistical analysis software SPSS Statistics
Independent sample T inspections and variance analysis program are further analyzed in 17.0.
Further, the leaf sheath color in the step (E) differentiates that the leaf sheath color of haplobiont is green leaf sheath.It is described
In step (E) during selfing, if loose powder is difficult, need to needle flower pesticide release pollen with pin.
The beneficial effects of the present invention is：
The present invention is a kind of method of quick initiative transgenic corns dihaploid homozygosis offspring, compared with prior art,
Present invention corn haploid induction line east lures No. 1 to pollinate to transgenic corns cenospecies for male parent, and picking monoploid rataria enters
Row tissue cultures, carry out genome and double using colchicine to monoploid rataria, and then it is beautiful to cultivate transgenosis dihaploid
Rice plant, and harvest transgenosis dihaploid seed.The present invention can be used for the quick homozygosis of transgenic corns backcross transformation offspring,
The quick foundation of corn Double-haploid population is can also be used for, the genetic analysis of corn important character is carried out.
Description of the drawings
The PCR amplification figures of target gene ZmARG in Fig. 1 corn regeneration plants.
In figure：M:Trans2K DNA Marker；P:Positive control；W:Blank；N:Negative control；1-8:Transgenosis
Plant.
Specific embodiment
Below in conjunction with the accompanying drawings the invention will be further described：
First, corn haploid induction line east lures the biological characteristics sex investigation of No. 1
Test material：
Corn haploid induction line east agriculture lures No. 1, and control material is L7 and JY3 for induction, is Northeast Agricultural University's agriculture
Corn seminar of institute seed selection, with R-nj marks.The above-mentioned corn material public can be from agricultural college of Northeast Agricultural University corn class
Topic group is obtained.
Test method：
The plantation of material：
Test material is planted in Northeast Agricultural University proving ground, using RANDOMIZED BLOCK DESIGN, 3 repetitions, and 3 row areas, OK
Long 5m, spacing in the rows 30cm, line width 70cm, often 17 plants of row.Field management produces field with conventional corn.
Field investigation project and investigation method：
Standard is reached as the date in each period using the whole district 80%, but for the abnormal plant of field emergence will reject not
Meter.Investigation index and investigation standard are as follows.
Tasseling stage：Expose 3～5cm of top in plant tassel tip；
Florescence：Plant tassel starts loose powder；
Reel off raw silk from cocoons the phase：The filigree of plant female fringe stretches out 2cm or so from bract；
It is seeded into the number of days of the phase of reeling off raw silk from cocoons：It is the number of days for being seeded into the phase of reeling off raw silk from cocoons；
It is seeded into the number of days of loose powder phase：It is the number of days for being seeded into florescence；
The loose powder duration：It is from there is the number of days that pollen terminates to loose powder；
Pollen amount：The number of plant loose powder；
Plant height：Milk stage, selects at random 5 plants to measure plant top respectively to ground level in every cell；
Ear height：Milk stage, selects at random 5 plants to measure respectively from corn base portion (part of basseting) to corn in every cell
The distance of topmost the first fringe fringe portion bottom (disregarding corn Miho handle length)；
Tassel branch number：Select the sum of 5 plants of all branches for measuring tassel male inflorescence respectively at random per cell；
Investigation result：
Field investigation the results are shown in Table 1.It can be seen that it is phase with other two inductions that corn haploid induction line Dong Nonggao lures No. 1
Than, advantage is very prominent, with it is precocious, pollen amount is big, plant height is suitable, the florescence is long the features such as.It is adapted for corn list times
The initiative of body colony and Haploid Breeding of Maize are used.
Corn haploid induction line Dong Nonggao of table 1 lures the biological character of No. 1 to investigate
2nd, corn haploid induction line east lures the inducing effect of No. 1 to analyze
Test material：
Corn haploid induction line east lures No. 1, and control material is L7 and JY3 for induction, is Northeast Agricultural University's agronomy
Corn seminar of institute seed selection, with R-nj marks.Female parent material is corn hybrid seed east agriculture 254.The above-mentioned corn material public can
Obtain from corn seminar of agricultural college of Northeast Agricultural University.
Test method：
The plantation of material：
Test material is planted in Northeast Agricultural University proving ground, using RANDOMIZED BLOCK DESIGN, 3 repetitions, and duplicate rows area, OK
Long 5m, spacing in the rows 30cm, line width 70cm, often 17 plants of row.Field management standard in two places is consistent, and with conventional corn field is produced.5 meters of rows
It is long, spacing in the rows 30cm, line-spacing 70cm.Cenospecies is once sowed, and the time is on April 29th, 2015, and induction 3 phases of system point are sowed, the time
On April 29th, 1, May 7 and May 11.
Pollination：
Cenospecies pollination is given with induction system, be listed record pollination time, each induction system at least awards 10 plants.
The acquisition of monoploid seed and the calculating of inductivity：
The phenotype according to of both hybridization grain endosperm color and embryo color is differentiated after corn ear fully maturation, is used
To distinguish dliploid and monoploid and hybridization dliploid seed.The purple embryo of silkworms grain in purple top, Bai Dingzi embryo of silkworms grains are the two of normal hybridisation
Times body；Bai Dingbai embryos, are by pollen contamination or defective seed；The white embryo grain in purple top, is pseudohaploid.
Result of the test：
Result of the test is shown in Table 2.In from table, 3 corn inductions are the 34 plants of milpas that pollinated altogether, the fruit that will be harvested
After fringe list fringe threshing, according to seed embryo and the color of endosperm, the seed for selecting the white endosperm in wherein purple top is monoploid seed.Wherein
East lures No. 1 haploid-induction to be up to 11.7%, is significantly higher than other two induction systems.
The comparison of the different haploid inducing line inducing effects of table 2
Note：* the significant difference in 0.05 level is represented
3rd, transgenic corns homozygosis offspring's strain is created using corn haploid induction line
1st, include
Test material：Corn haploid induction line east lures No. 1, L7 and JY3, is agricultural college of Northeast Agricultural University corn problem
Group seed selection, with R-nj marks；For creating haploid F1 cenospecies, its female parent is to turn corn arginase ZmARG genes
The T2 of corn HiII is conjunction 344 for strain, male parent；The above-mentioned ordinary maize material public can be from agricultural college of Northeast Agricultural University corn
Seminar obtains；
Test method：
The plantation of material：Using RANDOMIZED BLOCK DESIGN, 3 repetitions, duplicate rows area, the long 5m of row, spacing in the rows 30cm, line width 70cm,
Every 17 plants of row；Field management standard in two places is consistent, and with conventional corn field is produced；5 meters of row length, spacing in the rows 30cm, line-spacing 70cm；Hybridization
Plant and once sow, the time is April 29 days then, induction 3 phases of system point are sowed, and the time is respectively April 29 days then, May 7 and 5
The moon 11；
Pollination：The pollination of F1 cenospecies is given with induction system, be listed record pollination time, each induction system at least awards 10 plants；
The experiment process of tissue cultures：
Culture medium：
(1) screening and culturing medium：MS fluid nutrient medium+100uM ABA；
(2) culture medium is doubled：The colchicine of MS fluid nutrient medium+2mg/L asparagine+1mg/L BAP+0.05%；
(3) growth medium：MS solid mediums；
Method of operating：
(A) sterilization of young fringe
The 18d after pollination respectively, 20d take young fringe, peel off bract, and sterilize 8min, 0.1%HgCl in 75% ethanol2Disappear
Malicious 6min, aseptic water washing 3 times；
(B) rataria is stripped：
Rataria is taken, scultellum is placed in down in culture dish on the aseptic filter paper infiltrated with screening and culturing medium, 28 DEG C, 16h illumination/
8 hours dark, cultivate 24h；
(C) monoploid rataria is doubled：
Pick out the non-pigmented monoploid rataria of scultellum, scultellum is inoculated into upward in culture dish with doubling culture medium leaching
On the aseptic filter paper of profit, 26 DEG C of dark culturings 24h；
(D) IMMATURE EMBRYOS CULTURE：
Rataria is transferred in grown cultures bottle, scultellum downwards, 26 DEG C of light cultures 1 week, afterwards 26 DEG C of optical cultures 2-3 weeks are straight
It is complete to root development；Per bottle is inoculated with 15 or so；
(E) transplant：
By in little transplantation of seedlings to soil matrix, cultivate in greenhouse, haplobiont is differentiated according to leaf sheath color, until into
Ripe, strict selfing harvests seed；
(F) the PCR detections of dihaploid：
The STb gene of Leaves of Maize Seedlings is extracted using CTAB a small amount of method；Due to ZmARG genes be corn endogenous gene, root
According to promoter Ubi sequence and objective gene sequence design primer, primer ARG-F is located at promoter Ubi interior sequences, primer ARG-
R is located inside objective gene sequence, and primer size is 775bp；Riddled basins are antiweed glyphosate gene, and product is big
It is little for 522bp；Primer sequence is as follows：
ARG-F:5’-CGCCCTTCTTTGGTGAT-3’
ARG-R:5’-CCCTGCCTTCATACGCT-3’
PCR response procedures are, 94 DEG C of 5min, 94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min, 72 DEG C of 8min, 32 circulations；1%
Agarose gel electrophoresis detection pcr amplification product；
Data statistical approach:
Haploid inductivity=monoploid rataria number/inoculation rataria number * 100%；Plus multiplying power=solid strain number/regrowth
Number * 100%；Edit is carried out to test data using Excel 2010, using statistical analysis software SPSS Statistics
Independent sample T inspections and variance analysis program are further analyzed in 17.0.
Specifically, the leaf sheath color in the step (E) differentiates that the leaf sheath color of haplobiont is green leaf sheath.It is described
In step (E) during selfing, if loose powder is difficult, need to needle flower pesticide release pollen with pin.
Result of the test：
Dihaploid induction result：
The rataria of picking transgenosis F1 generation cenospecies, selects monoploid rataria therein and doubles to process according to color, passes through
Method for tissue culture obtains regeneration liploid plant.The results are shown in Table 3.
The result of the tissue culture method of table 3 difference haploid inducing line induction
Target gene PCR testing results：
ZmARG gene corn offspring's regrowths are turned as material with above-mentioned acquisition, blade is extracted with CTAB a small amount of method total
DNA.With DNA as positive control, with ddH2O is blank, right with non-transgenic corn seed DNA as negative control
Genes of interest ZmARG enters performing PCR detection.PCR results show there are 160 plants of ZmARG bases for amplifying 775bp in 191 plants of regrowths
Because of specific fragment, its amplified band position is consistent with DNA positive control, and negative control and blank do not expand shaping
Band, is shown in Fig. 1, is tentatively obtained using tissue cultures auxiliary haploid induction method and turns ZmARG gene backcross transformation offsprings.
The general principle and principal character and advantages of the present invention of the present invention has been shown and described above.The technology of the industry
Personnel it should be appreciated that the present invention is not restricted to the described embodiments, the simply explanation described in above-described embodiment and specification this
The principle of invention, without departing from the spirit and scope of the present invention, the present invention also has various changes and modifications, these changes
Change and improvement is both fallen within scope of the claimed invention.The claimed scope of the invention by appending claims and its
Equivalent thereof.
Claims (3)
1. a kind of method of quick initiative transgenic corns dihaploid homozygosis offspring, it is characterised in that：Including
Test material：Corn haploid induction line east lures No. 1, L7 and JY3, is the choosing of corn seminar of agricultural college of Northeast Agricultural University
Educate, with R-nj marks；For creating haploid F1 cenospecies, its female parent is to turn corn arginase ZmARG gene corns
The T2 of HiII is conjunction 344 for strain, male parent；The above-mentioned ordinary maize material public can be from agricultural college of Northeast Agricultural University corn problem
Group is obtained；
Test method：
The plantation of material：Using RANDOMIZED BLOCK DESIGN, 3 repetitions, duplicate rows area, the long 5m of row, spacing in the rows 30cm, line width 70cm is often gone
17 plants；Field management standard in two places is consistent, and with conventional corn field is produced；5 meters of row length, spacing in the rows 30cm, line-spacing 70cm；Cenospecies one
Secondary sowing, the time is April 29 days then, and induction system point 3 phases sowing, the time is respectively April 29 days then, May 7 and May 11
Day；
Pollination：The pollination of F1 cenospecies is given with induction system, be listed record pollination time, each induction system at least awards 10 plants；
The experiment process of tissue cultures：
Culture medium：
(1) screening and culturing medium：MS fluid nutrient medium+100uM ABA；
(2) culture medium is doubled：The colchicine of MS fluid nutrient medium+2mg/L asparagine+1mg/L BAP+0.05%；
(3) growth medium：MS solid mediums；
Method of operating：
(A) sterilization of young fringe
The 18d after pollination respectively, 20d take young fringe, peel off bract, and sterilize 8min, 0.1%HgCl in 75% ethanol2Sterilization
6min, aseptic water washing 3 times；
(B) rataria is stripped：
Rataria is taken, scultellum is placed in down in culture dish on the aseptic filter paper infiltrated with screening and culturing medium, and 28 DEG C, 16h illumination/8 are little
When it is dark, cultivate 24h；
(C) monoploid rataria is doubled：
The non-pigmented monoploid rataria of scultellum is picked out, scultellum is inoculated into upward in culture dish to use and doubles culture medium infiltration
On aseptic filter paper, 26 DEG C of dark culturings 24h；
(D) IMMATURE EMBRYOS CULTURE：
Rataria is transferred in grown cultures bottle, scultellum downwards, 26 DEG C of light cultures 1 week, afterwards 26 DEG C of optical cultures 2-3 weeks are until root
Development is complete；Per bottle is inoculated with 15 or so；
(E) transplant：
By in little transplantation of seedlings to soil matrix, cultivate in greenhouse, haplobiont is differentiated according to leaf sheath color, until it is ripe, sternly
Lattice selfing, harvests seed；
(F) the PCR detections of dihaploid：
The STb gene of Leaves of Maize Seedlings is extracted using CTAB a small amount of method；Because ZmARG genes are corn endogenous gene, according to opening
Mover Ubi sequences and objective gene sequence design primer, primer ARG-F is located at promoter Ubi interior sequences, primer ARG-R positions
Inside objective gene sequence, primer size is 775bp；Riddled basins be antiweed glyphosate gene, primer size
For 522bp；Primer sequence is as follows：
ARG-F:5’-CGCCCTTCTTTGGTGAT-3’
ARG-R:5’-CCCTGCCTTCATACGCT-3’
PCR response procedures are, 94 DEG C of 5min, 94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min, 72 DEG C of 8min, 32 circulations；1% fine jade
Sepharose electrophoresis detection pcr amplification product；
Data statistical approach:
Haploid inductivity=monoploid rataria number/inoculation rataria number * 100%；Plus multiplying power=solid strain number/regrowth number *
100%；Edit is carried out to test data using Excel 2010, using statistical analysis software SPSS Statistics
Independent sample T inspections and variance analysis program are further analyzed in 17.0.
2. the method for quick initiative transgenic corns dihaploid homozygosis offspring according to claim 1, it is characterised in that：
Leaf sheath color in the step (E) differentiates that the leaf sheath color of haplobiont is green leaf sheath.
3. the method for quick initiative transgenic corns dihaploid homozygosis offspring according to claim 1, it is characterised in that：
In the step (E) during selfing, if loose powder is difficult, need to needle flower pesticide release pollen with pin.
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| CN113455378A (en) * | 2021-08-04 | 2021-10-01 | 北大荒垦丰种业股份有限公司 | Breeding method of corn haploid induction line and application thereof |
| CN114946639A (en) * | 2022-06-08 | 2022-08-30 | 中国农业大学 | Application of two wheat induction systems in wheat haploid immature embryo and seed identification |
| CN115067209A (en) * | 2022-07-21 | 2022-09-20 | 北京市农林科学院 | Method for improving doubling efficiency of corn haploid by using zeatin |
| CN115104525A (en) * | 2022-06-28 | 2022-09-27 | 北京市农林科学院 | Breeding method combining haploid breeding with conventional breeding and induced selection of corn and application of breeding method |
| CN117461557A (en) * | 2022-07-21 | 2024-01-30 | 北京市农林科学院 | Efficient doubling method for corn haploid |
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2016
- 2016-12-29 CN CN201611244164.4A patent/CN106613985A/en active Pending
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| CN113455378A (en) * | 2021-08-04 | 2021-10-01 | 北大荒垦丰种业股份有限公司 | Breeding method of corn haploid induction line and application thereof |
| CN114946639A (en) * | 2022-06-08 | 2022-08-30 | 中国农业大学 | Application of two wheat induction systems in wheat haploid immature embryo and seed identification |
| CN115104525A (en) * | 2022-06-28 | 2022-09-27 | 北京市农林科学院 | Breeding method combining haploid breeding with conventional breeding and induced selection of corn and application of breeding method |
| CN115067209A (en) * | 2022-07-21 | 2022-09-20 | 北京市农林科学院 | Method for improving doubling efficiency of corn haploid by using zeatin |
| CN117461557A (en) * | 2022-07-21 | 2024-01-30 | 北京市农林科学院 | Efficient doubling method for corn haploid |
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