CN104488694A - Method for rapidly cultivating transgene maize inbred line - Google Patents
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Abstract
The invention belongs to the crop breeding field, and concreatly relates to a method for rapidly cultivating transgene maize inbred line. The method comprises the following steps: 1)operating transgene base material: performing hybridization of a transgene positive material and an excellent maize inbred line; 2)performing hybrid induction: performing hybridization of an induction line and the transgene base material; 3)performing haploid selection: selecting a haploid plant through grain and field plant; 4) doubling haploid: using a natural or an artificial chemical doubling method for doubling the haploid plant and inbreeding; and 5)transgene DH line identification: performing a PCR level detection on the obtained DH line, and confirming the transgene maize inbred line containing the target gene. The method greatly shortens a cultivation process of the transgene maize inbred line.
Description
Technical field
The invention belongs to crop breeding field, be specially a kind of method of quickly breeding transgenic corn self-bred line.
Background technology
Gene engineering and transgenosis, as a kind of new method of crops genetic improvement, greatly provide breeding work quality and efficiency.Since the insect-resistant transgenic corn BT176 developed by Syngenta Co., Ltd plants from 1996 as the first transgenic corns commercialization in the world, genetically modified crops cultivated area continues to increase, 1.752 hundred million hectares of 2013 are increased to from 1996 1,700,000 hectares, more than 100 times are added in 18 years, 2013 world markets of being only transgenic seed are worth and just reach 15,600,000,000 dollars, account for 35% of the 45000000000 dollars of business Seed Markets in the whole world.This growth makes transgenic technology become in modern agriculture history with fair margin of profit and applies crop technology the most rapidly.At present, genetically modified crops mainly concentrate on the seven large class crops such as soybean, corn, cotton.Corn is one of world's Three major grain crops, important grain, warp, raises crop, 2013, and global transgenic corns cultivated area is 5,740 ten thousand hectares, accounts for 32% of the total cultivated area of global corn.But transgenic corns is not also planted by China, based on cotton, pawpaw.
For the strategic demand ensureing food security and development Biology Breeding industry, within 2008, State Council approved implements genetically modified organism rearing new variety key special subjects, final goal to cultivate a collection of Resistant, degeneration-resistant, high-quality, highly efficient and productive great genetically modified organism new varieties, improve the research of agriculture genetically modified organism and industrialization integral level, for China's agricultural sustainable development provides strong science and technology support.This, for the enhancing agricultural science and technology capability of independent innovation, promotes China's Biology Breeding level, promotes growth of agricultural efficiency and increasing peasant income, improve China's Affecting Agricultural International Competitiveness, have great strategic significance.
The cultivation of transgenic corns new varieties is comprehensive system engineerings, setting up good receptor system is the most important thing that maize genetic transforms, by the restriction of Genetic Transformation in Higher Plants technology, the transformation efficiency of existing genetic transforming method is not high, as the agriculture bacillus mediated rataria genetic transforming method be most widely used in corn, due to genotype restriction, most Inbred Lines promoted mainly of producing cannot induce embryo callus, and the susceptibility infected Agrobacterium is extremely low, therefore cannot be directly used in maize genetic and transforms.Current domestic and international application widely corn gene recipient genotypes mainly concentrates on a few material such as Hi-II, A188, H99, these materials contain the recessive gene controlling embryo callus height inductivity, susceptible Agrobacterium, have higher genetic transformation efficiency as acceptor material.But these economical characters being easy to the acceptor material transformed are poor.Therefore, these minority transgenic corns acceptor materials that conversion need be utilized to obtain and Key Inbred Lines in Maize carry out backcross transformation, general need backcross 6-7 generation, again more than selfing 3 generation, final could formulate out there is excellent economical character and import the new inbred line of transgenic corns of foreign gene, this very long breeding process has greatly delayed the research and development speed of transgenic corns breeding.Therefore, a kind of method setting up quickly breeding transgenic corn self-bred line becomes the combustion adjuvant accelerating transgenic corns rearing new variety.
In recent years, haploid breeding technology has progressively become the guardian technique of China's corn conventional breeding.Double haploid (doubled haploid, DH) technology be the one of selecting and breeding corn inbred line the soonest, the easiest, economic, practical and direct method.Its basic procedure is the haplobiont with biotic induce, obtains through artificial or Natural double the liploid plant isozygotied, more therefrom screening is newly.Its main feature is choosing is that speed is fast, only needs 2 generations can obtain stable pure lines (traditional inbred line breeding need more than 7 generations).At present, progressively large-scale application in ordinary maize inbred line breeding.And also do not attempt in transgenic corn self-bred line seed selection.Based on the needs of transgenic corns breeding and the advantage of haploid breeding technology, monoploid technology is applied to transgenic corn self-bred line and cultivates, significant for the cultivation accelerating transgenic corn self-bred line, possess good application prospect.
Summary of the invention
The object of this invention is to provide a kind of method of quickly breeding transgenic corn self-bred line.
The method of a kind of quickly breeding transgenic corn self-bred line provided by the present invention, comprises the steps:
(1) transgenosis basic material assembly
Utilize the hereditary basis material of hybridized induction unisexual reproduction method therefrom seed selection transgenic corn self-bred line can be containing the hybrid combination proceeding to the objective trait genes such as pest-resistant, antiweed, crossbreed (comprising single cross hybrid, triple hybrid, double cross hybrid) or each types of populations (comprising synthetic cross variety etc.) or by its derivative F2, F3 for colony etc., obtain by the kind matter containing intended transgenic proterties and the material such as Elite inbred, each types of populations are carried out hybridization under isolation condition.Containing the kind matter of intended transgenic proterties by the acquisition such as agrobacterium-mediated transformation, particle bombardment.Basic material will have high yield, high-quality, disease-resistant degeneration-resistant, the good characteristic such as comprehensive agronomy proterties is good.
(2) hybridized induction
Under isolation condition, by the transgenosis basic material of assembly and the plantation of induction system, transgenosis basic material is 100 strains, join with 4: 1 and broadcast induction system, usually select Stock6, lucky high lure No. 3, agricultural university is high lures the induction systems such as series, the induction system as far as possible selecting the florescence close with basic material, or the measure such as take that a point 2-3 phase is sowed, guarantee the two flower synchronization, complete hybridization smoothly.
Inducement crossbreeding Pollination Modes is pollinated identical with conventional hybridization.To be induced transgenosis basic material for female parent, after transgenosis basic material has just exposed female fringe, sulfuric acid paper bag is used to isolate female fringe artificial bagging immediately, and after its extraction filigree grows to 5cm (or weaving silk 3 days) left and right, do male parent with haploid inducing line, get its fresh pollen (bagging the previous day) and pollinate.During thermal extremes weather pollination time before morning 11:00 as well.Gather in the crops after hybridization fruit ear maturation and keep properly.
(3) monoploid mirror choosing
Monoploid seed is identified: select autumn seed aleurone layer to be that black or mauve corn ear are gathered in the crops, carry out threshing after natural drying 20-30 days.According to seed phenotypic character (seed top and plumule color), monoploid is tentatively reflected choosing, endosperm aleurone layer painted (black or aubergine), embryo clypeole colourless (clypeole is inverted-triangular, cave inward obviously) be pseudohaploid seed.
As adopted artificial chemistry method for doubling to carry out secondary qualification, when pseudohaploid young shoot growing to 1-2cm, eliminating radicle length and sturdy, that growing way is strong young shoot, selecting the young shoot that radicle is very thin, growing way is small and weak.
Monoploid field confirms: in field, pseudohaploid plant grows to before and after the 8-10 leaf phase, differentiates monoploid according to plant ABP1 purple mark and morphological feature.All seedling leaf sheaths and blade are purple, the tall and big sturdy plant of plant is all eliminated, and seedling growth is slow, plant height is low, blade is less, narrow, short and compared with upper punch, leaf look shallow, can be confirmed to be haplobiont, carry out doubling process.
(4) doubling monoploids
Doubling monoploids has nature and artificial doubling two kinds of methods.
Natural doubling rate is lower, and generally between 1%-5%, different basic materials, ecotope, implantation time all can have impact to clear-cutting forestland rate.The clear-cutting forestland of fertility can be relied on just can to realize self-fertility for the monoploid genotype that loose powder rate is higher.Selection isolation condition is good, physical features is smooth, and fertility better plot plantation monoploid, waters in time and carry out field precision management.When haplobiont tassel has flower pesticide to expose and has obvious pollen to produce, get its flower pesticide and carry out meticulous pollination self, strictly close the bag overlapping female fringe after pollination, avoid foreign pollen to pollute.
The monoploid genotype very low to Natural doubling rate must carry out artificial doubling process.Main method for doubling has method for soaking seed, leaching root method, leaching bud method, injection, field spray herbicide etc.Here leaching (cutting) bud method and leaching root method is introduced.
Namely leaching (cutting) bud method soaks the method for the young shoot cut through with colchicine solution.When the young shoot that monoploid seed is sprouted grows to 2 ~ 3cm, manually cut most advanced and sophisticated about the 1-2mm of coleoptile, bud is soaked 9 hours with the mixed solution of colchicine 0.06% and dimethyl sulfoxide (DMSO) 5%, use clean water again 1.5 hours, put into germination dish and carry out slow seedling process, slow seedling is after 2 days, selection isolation condition is good, physical features is smooth, transplanting seedlings in the better plot of fertility, transplants according to spacing in the rows 20cm, waters in time and carry out field precision management.
Namely leaching root method soaks haploid plantlets root with colchicine solution.Can carry out in young shoot phase or Seedling Stage, the haploid plantlets root system of 3 leaf phases is soaked 24 hours in the colchicine solution of 0.05%, after rinsing process with clear water, seedling replanting is good in isolation condition, physical features is smooth, the better plot of fertility, transplant according to spacing in the rows 20cm, water in time and carry out field precision management.
When pollinating, putting pouch in time female fringe, carefully also repeatedly observing the tassel of each individual plant, get pollen in time, find tassel loose powder, pollinate (being advisable with at 9 .-12 in the morning) at once.General each individual plant is pollinated and is advisable for 2 times, 1 time that pollen amount is large.
(5) transgenosis DH system qualification
After selfing fruit ear maturation or pollination after about 35d, results seed.The coming season or the double haploid seed precise sowing will obtained in green house, if plant growing way neat and consistent, seed, without separation, meets inbred line properties and characteristics, can confirm as the DH inbred line of isozygotying.
Growing period, gets every part of fresh blade 100mg of DH system, shreds the EP pipe putting into 2ml, high-flux tissue grinder is utilized to grind, then use TIANGEN plant genome DNA to extract the DNA that kit extracts sample, dissolve with the ddH2O of 100ul ,-20 DEG C save backup.Utilize Nanodrop2000 ultraviolet specrophotometer to measure the DNA concentration of model, DNA solution is diluted to 25ng/ μ L-50ng/ μ L ,-20 DEG C of preservations according to the concentration recorded.
Sample DNA after dilution, positive sample, negative control, blank are carried out pcr amplification.PCR reaction system 25 μ L, comprise 10 × PCR buffer solution (containing Mg2+) 2.5 μ L, 2.5mmolL-1 dNTPs2 μ L (often kind of dNTP final concentration is 200 μ LmolL-1), 10 μm of each 0.5 μ L of olL-1 primer (final concentration is 200nmolL-1), 5U μ L-1rTaq archaeal dna polymerase 0.125 μ L, template DNA 2 μ L (100ng), adding ultra-pure water to final volume is 25 μ L.
PCR response procedures: 94 DEG C of sex change 5min; (94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 30s to carry out 35 cyclic amplification reactions.According to the Tm value of the primer sequence of different target character gene, the annealing temperature that PCR reacts suitably can be changed); 72 DEG C extend 10min.The pcr amplification product agarose gel electrophoresis of 2% detects and gel imaging photograph observation, and as detected, object pcr amplification product band can be defined as positive transgenic inbred line.
Current, genetically modified organism rearing new variety key special subjects is just in implementation process, and target is that characterization and evaluation goes out the outstanding transgenosis new germ plasm of a collection of objective trait.Adopt traditional backcross transformation, two rings choosings are that method obtains homozygous transgenic inbred line and generally needs 7 generations more than, and per in generation, need carry out a large amount of Molecular Detection, therefore how fast backcross transformation thus to select homozygous transgenic inbred line extremely urgent.The present invention can be applied to and obtain transgenosis inbred line fast, only needs 2 generations.
Accompanying drawing explanation
Fig. 1 reflect choosing the pseudohaploid seed turning MON89034 gene
The PCR of Fig. 2 MON89034 gene detects
The protein level of Fig. 3 MON89034 gene detects
1：DH7；2：DH9；3：DH15；4：DH17
The inbred line fruit ear turning MON89034 gene that Fig. 4 obtains
A left side is DH7; The right side is DH9
Below in conjunction with accompanying drawing, preferred embodiment of the present invention is described in detail, can be easier to make advantages and features of the invention be readily appreciated by one skilled in the art, thus more explicit defining is made to protection scope of the present invention.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
Embodiment 1:
1, for examination material
Carry out hybridization with good inbred lines lucky 201000 with the material containing MON89034 gene and obtain F1.With parthenogenesis induction, to be that agricultural university is high lure No. 3 (being provided by China Agricultural University) for male parent to second Growing season, with transgenic hybrid combination F1 plant for female parent is hybridized, autumn, the F1 fruit ear seed mirror to results selected monoploid, identified pseudohaploid seed 226 (Fig. 1).
2, doubling monoploids
Adopt the leaching bud method that chemistry doubles, medicament uses colchicine (Colchicine, COL) and dimethyl sulfoxide (DMSO) (Dimethyl sulfoxide, DMSO), method is the same, obtain the strain of pseudohaploid seedling 205, and be transplanted to the field of isolation, determine the strain of accurate haplobiont 190 by field test, carry out pollination self 40 fringe summer, gather in the crops selfing fruit ear 30 fringe autumn, kernal number is between 3-50 grain, and threshing is preserved.
3, PCR detects
Choose the wherein 4 parts of DH systems (DH7, DH9, DH15, DH17) obtaining seed more to plant in greenhouse, when five leaf phases, get blade and carry out genes of interest detection.Detection method performs by the regulation of National Standard of the People's Republic of China (Ministry of Agriculture No. 1861 bulletin-4-2012) [the pest-resistant corn 89034 of genetically modified plants and products thereof composition detection and derived varieties qualitative method thereof].
Result shows: DH7 and DH9 detects target gene amplified production band, and DH15 and DH17 does not detect target gene amplified production band.
1:Marker DL2000; 2: blank; 3: positive control; 4: negative control; 5:DH7; 6:DH9; 7:DH15; 8:DH17
4, protein expression detects
Get while blade carries out PCR detection, separately get a part of blade and carry out protein expression detection.Protein expression detects and adopts detection GMOs test strips, selects AS 003 BG of Envirologix company of the U.S., LS, test item Cry1Ab.
(1) leaf tissue method is extracted
A) 2 layers of blade are cut with off-line pipe lid, bottom cone disposed tubes; B) 20-30 second is ground with cone; C) draw 0.5ml extract and pour centrifuge tube into; D) grinding is repeated.
(2) ELISA test strip method
A) test strips is placed in grinding liquid; B) within 5 minutes; occur that control line shows that this test paper has function; if sample contains Cry1Ab gene; article 2, line (detection line) will appear between control line and boundary belt within 5 minutes, and any clear discernible pink colour detection line all will be considered to positive.
Result shows (Fig. 3): testing result is consistent with Molecular Detection result, and detection line appears in DH7 and DH9, and detection line does not appear in DH15 and DH17.
Thus, confirm that DH7 and DH9 is transgenosis selfing (DH) and is, its fruit ear of results (Fig. 4) after ripe.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (8)
1. a method for quickly breeding transgenic corn self-bred line, comprises the steps:
(1) transgenosis basic material assembly
(2) hybridized induction
(3) monoploid mirror choosing
(4) doubling monoploids
(5) transgenosis DH system qualification.
2. the method for claim 1, it is characterized in that: described material assembly process is, the hereditary basis material utilizing hybridized induction unisexual reproduction method therefrom seed selection transgenic corn self-bred line can be pest-resistant containing proceeding to, the hybrid combination of the objective trait genes such as antiweed, crossbreed (comprises single cross hybrid, triple hybrid, double cross hybrid) or each types of populations (comprising synthetic cross variety etc.) or by its derivative F2, F3 is for colony etc., by by the kind matter containing intended transgenic proterties and Elite inbred, the materials such as each types of populations carry out hybridization and obtain under isolation condition.Containing the kind matter of intended transgenic proterties by the acquisition such as agrobacterium-mediated transformation, particle bombardment.Basic material will have high yield, high-quality, disease-resistant degeneration-resistant, the good characteristic such as comprehensive agronomy proterties is good.
3. the method for claim 1, it is characterized in that: described hybridized induction process is: under isolation condition, by the transgenosis basic material of assembly and the plantation of induction system, transgenosis basic material 100 strain, joins with 4: 1 and broadcasts induction system, select Stock6, lucky high lure No. 3, agricultural university is high lures the induction systems such as series, the induction system as far as possible selecting the florescence close with basic material, or take a point 2-3 phase to sow measure, guarantee the two flower synchronization, complete hybridization smoothly;
Inducement crossbreeding Pollination Modes is pollinated identical with conventional hybridization.To be induced transgenosis basic material for female parent, after transgenosis basic material has just exposed female fringe, sulfuric acid paper bag is used to isolate female fringe artificial bagging immediately, and after its extraction filigree grows to 5cm (or weaving silk 3 days) left and right, do male parent with haploid inducing line, get its fresh pollen (bagging the previous day) and pollinate.During thermal extremes weather pollination time before morning 11:00 as well.Gather in the crops after hybridization fruit ear maturation and keep properly.
4. the method for claim 1, is characterized in that: monoploid seed is identified: select autumn seed aleurone layer to be that black or mauve corn ear are gathered in the crops, carry out threshing after natural drying 20-30 days.According to seed phenotypic character (seed top and plumule color), monoploid is tentatively reflected choosing, endosperm aleurone layer painted (black or aubergine), embryo clypeole colourless (clypeole is inverted-triangular, cave inward obviously) be pseudohaploid seed;
As adopted artificial chemistry method for doubling to carry out secondary qualification, when pseudohaploid young shoot growing to 1-2cm, eliminating radicle length and sturdy, that growing way is strong young shoot, selecting the young shoot that radicle is very thin, growing way is small and weak;
Monoploid field confirms: in field, pseudohaploid plant grows to before and after the 8-10 leaf phase, differentiates monoploid according to plant ABP1 purple mark and morphological feature.All seedling leaf sheaths and blade are purple, the tall and big sturdy plant of plant is all eliminated, and seedling growth is slow, plant height is low, blade is less, narrow, short and compared with upper punch, leaf look shallow, can be confirmed to be haplobiont, carry out pollination self process.
5. the method for claim 1, is characterized in that: adopt Natural double or double based on the artificial chemistry soaking (cutting) bud method.
Different basic materials, ecotope, implantation time all can have impact to clear-cutting forestland rate.The clear-cutting forestland of fertility can be relied on just can to realize self-fertility for the monoploid genotype that loose powder rate is higher.Selection isolation condition is good, physical features is smooth, and fertility better plot plantation monoploid, waters in time and carry out field precision management.When haplobiont tassel has flower pesticide to expose and has obvious pollen to produce, get its flower pesticide and carry out meticulous pollination self, strictly close the bag overlapping female fringe after pollination, avoid foreign pollen to pollute.
The monoploid genotype very low to Natural doubling rate must carry out artificial doubling process.Main method for doubling has method for soaking seed, leaching root method, leaching bud method, injection, field spray herbicide etc.Here leaching (cutting) bud method and leaching root method is introduced.
Namely leaching (cutting) bud method soaks the method for the young shoot cut through with colchicine solution.When the young shoot that monoploid seed is sprouted grows to 2 ~ 3cm, manually cut most advanced and sophisticated about the 1-2mm of coleoptile, bud is soaked 9 hours with the mixed solution of colchicine 0.06% and dimethyl sulfoxide (DMSO) 5%, use clean water again 1.5 hours, put into germination dish and carry out slow seedling process, slow seedling is after 2 days, selection isolation condition is good, physical features is smooth, transplanting seedlings in the better plot of fertility, transplants according to spacing in the rows 20cm, waters in time and carry out field precision management.
Namely leaching root method soaks haploid plantlets root with colchicine solution.Can carry out in young shoot phase or Seedling Stage, the haploid plantlets root system of 3 leaf phases is soaked 24 hours in the colchicine solution of 0.05%, after rinsing process with clear water, seedling replanting is good in isolation condition, physical features is smooth, the better plot of fertility, transplant according to spacing in the rows 20cm, water in time and carry out field precision management.
When pollinating, putting pouch in time the female fringe of monoploid, carefully also repeatedly observing the tassel of each individual plant, get pollen in time, find tassel loose powder, pollinate (being advisable with at 9 .-12 in the morning) at once.General each individual plant is pollinated and is advisable for 2 times, 1 time that pollen amount is large.
6. the method for claim 1, is characterized in that: after selfing fruit ear maturation or pollination after about 35d, results seed.The double haploid seed precise sowing that the coming season will obtain in green house, if plant growing way neat and consistent, seed, without separation, meets inbred line properties and characteristics, can confirm as the DH inbred line of isozygotying;
Growing period, gets every part of fresh blade 100mg of DH system, shreds the EP pipe putting into 2ml, utilize high-flux tissue grinder to grind, and then uses TIANGEN plant genome DNA to extract the DNA of kit extraction sample, with the ddH of 100ul
2o dissolves, and-20 DEG C save backup.Utilize Nanodrop2000 ultraviolet specrophotometer to measure the DNA concentration of model, DNA solution is diluted to 25ng/ μ L-50ng/ μ L ,-20 DEG C of preservations according to the concentration recorded;
Sample DNA after dilution, positive sample, negative control, blank are carried out pcr amplification.
7. the method for claim 1, it is characterized in that: PCR reaction system 25 μ L, comprise 10 × PCR buffer solution (containing Mg2+) 2.5 μ L, 2.5mmolL-1dNTPs 2 μ L (often kind of dNTP final concentration is 200 μ LmolL-1), 10 μm of each 0.5 μ L of olL-1 primer (final concentration is 200nmolL-1), 5U μ L-1rTaq archaeal dna polymerase 0.125 μ L, template DNA 2 μ L (100ng), adding ultra-pure water to final volume is 25 μ L.
8. the method for claim 1, is characterized in that: PCR response procedures: 94 DEG C of sex change 5min; (94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 30s to carry out 35 cyclic amplification reactions.According to the Tm value of the primer sequence of different target character gene, the annealing temperature that PCR reacts suitably can be changed); 72 DEG C extend 10min.The pcr amplification product agarose gel electrophoresis of 2% detects and gel imaging photograph observation, and as detected, object pcr amplification product band can be defined as positive transgenic inbred line.
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| CN104798681A (en) * | 2015-05-14 | 2015-07-29 | 阜阳金种子玉米研究所 | Novel summer corn SRP class group selfing line breeding method |
| CN105046108A (en) * | 2015-07-07 | 2015-11-11 | 中国农业大学 | Selfing line SSR (Simple Sequence Repeat) and phenotype information based corn hybrid combination method and system |
| CN105660381A (en) * | 2016-02-25 | 2016-06-15 | 安徽省农业科学院烟草研究所 | Method for producing large-scale induction pollen sources in maize haploid breeding |
| CN108377899A (en) * | 2018-02-13 | 2018-08-10 | 吉林省农业科学院 | Haploid breeding method is induced using yellowish green seedling marker assisted selection corn hybridization |
| CN112616649A (en) * | 2020-12-14 | 2021-04-09 | 四川省农业科学院作物研究所 | Rapid cultivation method of transgenic maize inbred line by efficient nitrogen utilization |
| CN114342800A (en) * | 2022-01-05 | 2022-04-15 | 吉林省农业科学院 | Double induction method for rapidly breeding waxy haploid induction line |
| CN115633635A (en) * | 2022-10-09 | 2023-01-24 | 武汉市农业科学院 | Method for creating sweet corn DH line based on haploid breeding technology |
| CN117016372A (en) * | 2023-03-15 | 2023-11-10 | 北京市农林科学院 | Method for creating excellent corn germplasm and application thereof |
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| CN104798681A (en) * | 2015-05-14 | 2015-07-29 | 阜阳金种子玉米研究所 | Novel summer corn SRP class group selfing line breeding method |
| CN105046108A (en) * | 2015-07-07 | 2015-11-11 | 中国农业大学 | Selfing line SSR (Simple Sequence Repeat) and phenotype information based corn hybrid combination method and system |
| CN105046108B (en) * | 2015-07-07 | 2017-12-05 | 中国农业大学 | Corn hybridization compound formulation and system based on self-mating system SSR and phenotypic information |
| CN105660381A (en) * | 2016-02-25 | 2016-06-15 | 安徽省农业科学院烟草研究所 | Method for producing large-scale induction pollen sources in maize haploid breeding |
| CN108377899A (en) * | 2018-02-13 | 2018-08-10 | 吉林省农业科学院 | Haploid breeding method is induced using yellowish green seedling marker assisted selection corn hybridization |
| CN108377899B (en) * | 2018-02-13 | 2021-11-05 | 吉林省农业科学院 | Breeding method for auxiliary selection of corn hybridization induced haploid by yellow-green seedling marker |
| CN112616649A (en) * | 2020-12-14 | 2021-04-09 | 四川省农业科学院作物研究所 | Rapid cultivation method of transgenic maize inbred line by efficient nitrogen utilization |
| CN112616649B (en) * | 2020-12-14 | 2022-03-01 | 四川省农业科学院作物研究所 | Rapid cultivation method of transgenic maize inbred line by efficient nitrogen utilization |
| CN114342800A (en) * | 2022-01-05 | 2022-04-15 | 吉林省农业科学院 | Double induction method for rapidly breeding waxy haploid induction line |
| CN115633635A (en) * | 2022-10-09 | 2023-01-24 | 武汉市农业科学院 | Method for creating sweet corn DH line based on haploid breeding technology |
| CN117016372A (en) * | 2023-03-15 | 2023-11-10 | 北京市农林科学院 | Method for creating excellent corn germplasm and application thereof |
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| CN104488694B (en) | 2020-04-21 |
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