CN100586273C - Method for inducing corn haploid and multi-embryo using high oil type inducing series - Google Patents
Method for inducing corn haploid and multi-embryo using high oil type inducing series Download PDFInfo
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- CN100586273C CN100586273C CN200710178370A CN200710178370A CN100586273C CN 100586273 C CN100586273 C CN 100586273C CN 200710178370 A CN200710178370 A CN 200710178370A CN 200710178370 A CN200710178370 A CN 200710178370A CN 100586273 C CN100586273 C CN 100586273C
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Abstract
The invention discloses a method using high-oil inducement series to induce the haploid and the polyembryony body of the corn. The method inducing the haploid and the polyembryony body of the corn provided by the invention is to use high-oil corn haploid inducement series as the father parent to pollinate the female parent, to obtain the first-generation grain and gain the haploid and the polyembryony body of the corn. The method not only has the advantages that the breeding process is simplified, the breeding course is accelerated, the purity of the breed is improved, etc. in the traditionalhaploid breeding; but also can use the genetic effect of the oil constituent to improve the identification efficiency of the haploid, and at the same can improve the inducement rate of the polyembryony body.
Description
Technical field
The present invention relates to that a kind of to utilize the parthenogenesis of high oil type corn to induce be the method for inducing maize monoploid and polyembryony body.
Background technology
Corn is the different flower of monoecism, carry out amphigenetic crop.Mainly be to utilize the amphigenetic mode selecting and breeding corn of corn inbred line, the combination of preparation corn, propagating corn crossbreed in the corn breeding work.But in the process of cultivating corn inbred line, must be through the selfing and the selection in too much generation.Usually kind of seed selection needs the time of last ten year, needs to consume great amount of manpower and material resources and financial resources.Therefore how shortening breeding process, improving breeding efficiency is the target that breeding men are pursued always.
Corn monoploid is the product of gametophytic apomixis, comprises etheogenesis and parthenogenesis two big classes.The monoploid that obtains is carried out artificial doubling, only need a generation just can obtain zygoid, shortening the breeding cycle greatly.Since (Guha S in 1964, Maheshwari SC.In vitro production of embryos fromantherof Dature[J] .Nature, 1964,204:497) disclosed after approach can obtain monoploid by experiment the method that the method by group training approach seed selection inbred line is considered to get a good chance of through anther culture monoploid.But because the anther culture difficulty is big, the technology complicated operation, and the genotype dependence is very strong, and the not necessarily good inbred line of the inbred line that selects, therefore be difficult to scale utilization in breeding.
Parthenogenesis monoploid can produce by biotic induce and abiotic inducing as approach such as chemical inductions, but abiotic problem of inducing and a lot of difficulties of the same existence of flower pesticide group training limit its application in breeding.Biotic induce utilizes the biotic induce material to make exactly and induces object generation monoploid.Coe (Coe E H.A line of maize withhigh haploid frequency[J] .Am Nat, 1959,93:381-382) found that in nineteen fifty-nine it is Stock6 that the corn parthenogenesis is induced.Constantly transformed on its basis afterwards and imported Navajo (ACR-nj) genetic marker gene, and can identify monoploid comparatively quickly and easily, became possibility thereby make biotic induce monoploid carry out haploid breeding by seed color and plant color.But in application in practice, also find, the inductivity of Stock6 still very low (being about 1%), and have a lot of serious defectives, to responsive to temperature, self-fruitfulness is poor as male flower, the fringe Maize Ear Rot is serious, the Navajo genetic marker a little less than.Therefore, novel the inducing of the higher inductivity of seed selection is to be the key that biology is led the monoploid technology.
Another restriction of biotic induce monoploid technology is haploid evaluation efficient.Present haploid evaluation mainly depends on A1A2C1C2BP1R-nj dominant marker system, promptly judges according to endosperm, embryo, plant color.It at first sorts out possible monoploid seed according to seed Navajo mark property (by the A1A2C1C2R-nj Gene Handling), passes through the true and false that plant color in seedling stage (by the A1A2BP1 Gene Handling) is determined haplobiont again.But find that in practice the genotype selectivity of this Mk system is more intense, it is very big that different material markings is expressed difference, and the material that has almost can not be identified with seed Navajo mark; And different environmental conditions is also very big to the expression influence of mark.Therefore it is also very important to haploid breeding with efficient evaluation monoploid to develop the mark that makes new advances.
Normal seed has only an embryo, polyembryonic seed then can be on a seed give birth to two and even more plumule, as twin embryo etc., the plantation back forms two embryo seedlings or three embryo seedlings etc.Polyembryonic seed is derived from the apomixis of corn, and this type of material can be used for genetic breeding research, as research corn reprogenetics and heterosis utilization thereof etc.But, the frequency of the spontaneous generation of polyembryony seedling is quite low, Yang Wenpeng etc. are once to the dissimilar corns statistics of germinateing, the frequency of the spontaneous generation polyembryony of corn seedling only is 0.058% (Yang Wenpeng, Bai Xiaoguang, Chen Zehui, the preliminary observation of the two embryo seedlings of corn, [J] Guizhou agricultural science, 1991 the 6th phases).Methods such as the thing of people's medication afterwards (as dimethyl sulfoxide (DMSO)), ion irradiation are carried out inducing of polyembryony seedling, but effect is all undesirable.Rarely has report by hybridized induction with the method that obtains a large amount of polyembryony seedlings.
Summary of the invention
The purpose of this invention is to provide that a kind of program is simple, speed is fast, and can induce the method for corn monoploid and polyembryony body in a large number.
The method of inducing maize monoploid provided by the present invention and polyembryony body is to make male parent with high oil type corn haploid inducing line to give maternal pollination, and results cross-pollinated seed obtains corn monoploid and polyembryony body.
Wherein, described corn monoploid can be identified as follows: to described cross-pollinated seed by means of the xenia effect of color mark and oil content and utilize the oil content of measuring single seed to combine and identify monoploid.Concrete grammar is as follows: in the described cross-pollinated seed, aleurone layer is that not have the seed that purple mark and oil content content is lower than the oil content content of a described maternal selfing generation be the monoploid seed for purple, plumule position.
Described polyembryony body can be identified as follows: described cross-pollinated seed is carried out the embryo examination and germinate identifying the polyembryony body.
Described female parent can be inbred line, basic population or the crossbreed that meets breeding objective.
Basic population described in the said method refers to the maize population that formed after by the certain way mixed pollination by a plurality of inbred lines or kind.
Crossbreed described in the said method refers to single cross hybrid, triple hybrid or double cross hybrid.
Described polyembryony body is meant that seed has polyembryony thereby the back seed of appearance that germinates grows two strains or the above seedling of two strains.
Described high oil type haploid inducing line male parent refers to that oil content is higher than high oil type haploid inducing line more than 6% such as agricultural university's height and lures and be etc. can obtain from country of China Agricultural University corn improvement center.
In the described step (2), identify monoploid as follows: in the seed that described step (1) is gathered in the crops, aleurone layer is that not have the seed that purple mark and oil content content is lower than described maternal selfing seed oil content content be the monoploid seed for purple, plumule position.
The used high oil type of the present invention induces cording that the tangible more Navajo mark than Stock6 is arranged, and has kept ABP1 purple plant mark.In addition, its oil content higher (more than 6%) has significant xenia effect, and the male parent effect value can reach 0.38.Therefore can identify monoploid at an easy rate in conjunction with seed color, seed oil content, three marks of plant color.
Produce haploid procedure of breeding with the traditional biological induced parthenogenesis and compare, the present invention has (1) and utilizes the haploid induction rate of selected high oil type haploid inducing line higher (average 6%, part material can up to 11%); (2) outside the depigmentation, oil content can be used as new genetic marker, and general oil content is more stable and have tangible xenia effect, and cross-pollinated seed embryo face is bigger, and monoploid seed embryo face is less relatively, helps range estimation identification monoploid; (3) can utilize simple grain oil content determining instrument such as NMR (NMR), near infrared spectrometer (NIR) etc. to measure oil content, monoploid is lower than the hybridization seed owing to oil content, thereby can be differentiated.This method also differentiates that with three kinds of monoploid such as seed color, seed oil content, plant color mark combines when improving the haploid induction rate, improved the monoploid identification efficiency.Simultaneously, method of the present invention has also improved the induction frequency of polyembryony body, and the induction frequency that makes the polyembryony mutant is up to 0.1%-0.21%.
Description of drawings
Fig. 1 for the present invention used high oil type haploid inducing line seed selection process schematic diagram
Embodiment
Experimental technique among the following embodiment if no special instructions, is conventional method.
Embodiment 1, utilize high oil type haploid inducing line inducing maize monoploid
One, experiment material
High oil type haploid inducing line can obtain from country of China Agricultural University corn improvement center.
Luring No. 2 with the high oil of high oil type haploid inducing line agricultural university is example, its selection as shown in Figure 1:
Stock6 draws from american corn heredity cooperative society.The north high oil of agricultural university (BHO) is that a high oil is improved colony, and the embryo face is very big, once finds the monoploid of spontaneous generation in the field.Dozens of individual plant and Stock6 are hybridized in the nineteen ninety-five selection BHO colony, select a wherein the darkest original combined that the hybridization fruit ear as choosing is of seed Navajo mark.Utilize this combination to backcross 1-2 time with Stock6 again, form F
1Selfing, (BC once backcrosses
1), (BC backcrosses for twice
2) 3 choosings are basic population.The offspring handles and is undertaken by pedigree method, carries out selfing, the test cross seed selection of 6 generations continuously.Each emphasis of selecting from generation to generation is: haploid induction rate, seed Navajo mark, plant ABP1 mark, the big embryo proterties of seed, pollen amount, fecundity and disease resistance etc., syncaryon magnetic resonance oil content is measured later on, further oil content and inductivity is selected.Final seed selection becomes that inductivity height, mark property are good, good the inducing of economical character is that the high oil of agricultural university lures No. 2, and oil content reaches more than 7%.This cording has the tangible more Navajo mark than Stock6, and has kept ABP1 purple plant mark, and pollen amount is big, and disease resistance is good.The xenia effect of oil content is obvious, and effect value can reach 0.38.
Lure No. 2 and Stock6 to carry out the comparison of inductivity to high oil, select for use and combine 695, lure No. 2 and Stock6 does male parent respectively and induces, hybridize 10 fringes and add up inductivity with high oil as maternal, the result shows that it is 8% that high oil lures No. 2 inductivity, and the inductivity of Stock6 has only 0.9%.High in addition oil lures No. 2 mark also obviously to be better than Stock6.
Corn inbred line 1145 is high resistance to bacterial wilt inbred line, can obtain from country of China Agricultural University corn improvement center;
Corn inbred line Y331 is high sense bacterial wilt inbred line, can obtain from country of China Agricultural University corn improvement center;
Corn inbred line 178 can obtain from country of China Agricultural University corn improvement center;
Corn inbred line 8701 can obtain from Agricultural University Of He'nan;
Two, inducing maize monoploid
Randomised block design is adopted in experiment, and this experiment comprises 12 processing.
Experiment is established five haploid inductions and is handled, the male parent that each haploid induction is handled is high oil and lures No. 2, the female parent of haploid induction processing 1 is corn hybrid seed 1145 * Y331, the female parent of haploid induction processing 2 is a corn inbred line 178, the female parent of haploid induction processing 3 is a corn inbred line 8701, the female parent of haploid induction processing 4 is corn inbred line Y331, and the female parent of haploid induction processing 5 is a corn inbred line 1145.Lure the maternal material pollination of 10 strains of giving above-mentioned each processing for No. 2 respectively, results seed with high oil.
Five control treatment are established in experiment, control treatment 1 is carried out selfing for corn hybrid seed 1145 * Y331, control treatment 2 is carried out selfing for corn inbred line 178, control treatment 3 is carried out selfing for corn inbred line 8701, control treatment 4 is carried out selfing for corn inbred line Y331, and control treatment 5 is carried out selfing for corn inbred line 1145.The results seed.Each control treatment 5 strain.
In the hybridization fruit ear that haploid induction is handled, comprise heterozygosis seed and monoploid.Two kinds of methods are adopted in haploid evaluation respectively, i.e. tracer method and oil content determination method.Tracer method is exactly to differentiate according to the Navajo mark of cross-pollinated seed plumule, and aleurone layer is that the cross-pollinated seed of purple, purple plumule is the heterozygosis seed, and aleurone layer is that the cross-pollinated seed of purple, no purple plumule is a monoploid.The oil content determination method is exactly to judge monoploid in conjunction with the oil content of seed, and aleurone layer is that purple, plumule position do not have purple, and grain oil content is lower than the monoploid that is of maternal selfing seed.Because it is the oil content height that test lures with height, has stronger xenia effect, therefore the oil content with the selfing seed is a standard, is higher than the selfing seed and is the double fertilization seed, is lower than the selfing seed and is monoploid.
A kind of seed type only appears in the selfing fruit ear in control treatment, and promptly aleurone layer is yellow.
In control treatment 1 to 5 these 5 processing, 50 self progeny's seeds are got in each processing.Selected seed is carried out carrying out oil content mensuration according to the method that Song Tongming describes with NMR, and (Song Tongming, pulse Magnetic Resonance Imaging instrument is to the quick mensuration of crop seed oil content, Acta Agronomica Sinica, 1989,15 (2): 160-166).
In order to verify the reliability of above-mentioned evaluation monoploid method, monoploid seed in each haploid induction processing is planted in nutritive cube, and the inductivity statistics is carried out according to plant color and form in the back of emerging, and wherein monoploid is green, plant is short and small, and blade is than upper punch; Non-monoploid is purple, and plant is tall and big.Haploid induction rate=monoploid kernal number/(monoploid kernal number+heterozygosis kernal number) * 100%.For further evaluation, therefrom spot-check 30 samples and carried out tip of a root compressing tablet, examine under a microscope the number of cell chromosome.Checking is the result show, in 7695 hybridization seeds that all combinations are induced, obtains 545 monoploid altogether, and average inductivity is 7.08%.
The checking result shows that haploid induction processing 1 obtains 10 hybridization fruit ears altogether, amounts to 3000 purple aleurone layer seeds, and wherein monoploid is 180.Use tracer method and identify that 212 monoploid (aleurone layer is the cross-pollinated seed of purple, no purple plumule) are arranged, identify efficient 85%.These 212 monoploid of identifying with tracer method are carried out oil content with NMR to be measured; Control treatment 1 is got 50 self progeny's seeds and is carried out oil content mensuration with NMR.The result shows the oil content content of the control treatment 1 selfing seed (mean value ± SD) that is 3.85% ± 0.15%, in 212 monoploid with this tracer method evaluation, the seed oil content has 194 to be lower than 4%, the oil content of these 194 seeds is 3.36% ± 0.27% (mean value ± SD).The efficient of identifying that the oil content tracer method is described is 93%.
The checking result shows that haploid induction processing 2 obtains 10 hybridization fruit ears altogether, amounts to 700 purple aleurone layer seeds, wherein 35 monoploid.Use tracer method and identify that 50 monoploid are arranged, identify efficient 70%.These 50 monoploid of identifying with tracer method are carried out oil content with NMR to be measured; Control treatment 2 is got 50 self progeny's seeds and is carried out oil content mensuration with NMR.The result shows the oil content content of the control treatment 2 selfing seeds (mean value ± SD) that is 4.13% ± 0.13%, in 50 monoploid with this tracer method evaluation, the seed oil content has 39 to be lower than 4.26%, the oil content of these 39 seeds is 3.63% ± 0.32% (mean value ± SD).The efficient of identifying that the oil content tracer method is described is 90%.
The checking result shows that haploid induction processing 3 obtains 10 hybridization fruit ears altogether, amounts to 928 purple aleurone layer seeds, wherein 80 monoploid.Use tracer method and identify that 100 monoploid are arranged, identify efficient 80%.These 100 monoploid of identifying with tracer method are carried out oil content with NMR to be measured; Control treatment 3 is got 50 self progeny's seeds and is carried out oil content mensuration with NMR.The result shows the oil content content of the control treatment 3 selfing seeds (mean value ± SD) that is 3.75% ± 0.18%, in 100 monoploid with this tracer method evaluation, the seed oil content has 91 to be lower than 3.93%, the oil content of these 91 seeds is 3.29% ± 0.37% (mean value ± SD).The efficient of identifying that the oil content tracer method is described is 88%.
The checking result shows that haploid induction processing 4 obtains 10 hybridization fruit ears altogether, amounts to 994 purple aleurone layer seeds, wherein 50 monoploid.Use tracer method and identify that 79 monoploid are arranged, identify efficient 63%.These 79 monoploid of identifying with tracer method are carried out oil content with NMR to be measured; Control treatment 4 is got 50 self progeny's seeds and is carried out oil content mensuration with NMR.The result shows the oil content content of the control treatment 4 selfing seeds (mean value ± SD) that is 3.28% ± 0.16%, in 79 monoploid with this tracer method evaluation, the seed oil content has 53 to be lower than 3.44%, the oil content of these 53 seeds is 2.98% ± 0.21% (mean value ± SD).The efficient of identifying that the oil content tracer method is described is 94%.
The checking result shows that haploid induction processing 5 obtains 10 hybridization fruit ears altogether, amounts to 2073 purple aleurone layer seeds, wherein 200 monoploid.Use tracer method and identify that 250 monoploid are arranged, identify efficient 80%.These 250 monoploid of identifying with tracer method are carried out oil content with NMR to be measured; Control treatment 6 is got 50 self progeny's seeds and is carried out oil content mensuration with NMR.The result shows the oil content content of the control treatment 6 selfing seeds (mean value ± SD) that is 4.01% ± 0.21%, in 250 monoploid with this tracer method evaluation, the seed oil content has 222 to be lower than 4.22%, the oil content of these 222 seeds is 3.53% ± 0.32% (mean value ± SD).The efficient of identifying that the oil content tracer method is described is 90%.
Oil content result shows at cross-pollinated Navajo mark and shows tangible material, utilizes mark to select more than haploid rate of accuracy reached to 85%, but it is higher than tracer method to survey oil process, and up to 94%, the two differs 9%.The Navajo marker color performance of Y331 etc. is more shallow, therefore selecting monoploid by the plumule color just is subjected to certain limitation, accuracy rate has only 63.23%, and the oil content determination method is because target is the oil content of embryo, be not subjected to the influence of shade, so approaching with the accuracy rate on the strong mark background, surpass tracer method nearly 30%.As seen, survey oil process and can overcome the shortcoming that the mark instability is brought, higher accuracy rate is all arranged under various conditions.This shows that the oil content determination method is to differentiate the effective ways of weak marker material.
Embodiment 2, to utilize high oil type to induce be inducing maize polyembryony body
One, test material
High oil lures and can obtain from country of China Agricultural University corn improvement center for No. 2;
High-oil hybrid 5598 can obtain from country of China Agricultural University corn improvement center;
Crossbreed agricultural university 108 can obtain from China Agricultural University;
Zheng Dan 958 can obtain from Henan Academy of Agricultural Sciences.
Two, test is handled
6 processing, completely random block design are established in test.
The test processing is established three polyembryony seedlings and is induced processing, it is that high oil lures No. 2 that each polyembryony seedling is induced the male parent of processing, it is crossbreed 5598 that the polyembryony seedling is induced the female parent of processing 1, and it is crossbreed agricultural university 108 that the polyembryony seedling is induced the female parent of processing 2, and it is Zheng Dan 958 that the polyembryony seedling is induced the female parent of processing 3.Lure the maternal pollination of 10 strains of giving above-mentioned each processing for No. 2 respectively, results seed with high oil.
Test is handled and is established 3 control treatment, and control treatment 1 is 5598 selfings, and control treatment 2 is agricultural university's 108 selfings, and control treatment 3 is Zheng Dan 958 selfings.Each handles selfing 10 strains, the results seed.
Seed after the results is germinateed.
The polyembryony seedling induces processing 1 to obtain 10 fruit ears altogether, amounts to 5157, and the back statistics polyembryony seedling that germinates has 11 strains, and inducing polyembryony seedling frequency is 0.21%, and wherein 10 strains are pair embryo seedlings, and 1 strain is three embryo seedlings; The polyembryony seedling induces processing 2 to obtain 10 fruit ears altogether, amounts to 4000, and the back statistics polyembryony seedling that germinates has 4 strains, and complete is two embryo seedlings, and the frequency of inducing the polyembryony seedling is 0.1%; The polyembryony seedling is induced and is handled 3194 of 3 coinductions, 10 fringes, and the back statistics polyembryony seedling that germinates is 4 strains, is two embryo seedlings entirely, and the frequency of inducing the polyembryony seedling is 0.15%.
Control treatment 1 obtains 10 fringes altogether, amounts to 5000, does not find the polyembryony seedling after the germination; Control treatment 2 obtains 10 fringes altogether, amounts to 3900, does not find the polyembryony seedling after the germination; Control treatment 3 obtains 10 fringes altogether, amounts to 3500, does not find the polyembryony seedling after the germination.
Handle and control treatment from inducing, it is the induction frequency that can improve the polyembryony seedling widely that high oil type is induced.
Claims (5)
1, a kind of method of inducing maize polyembryony body is to make male parent with high oil type corn haploid inducing line to give maternal pollination, and results cross-pollinated seed obtains corn polyembryony body.
2, method according to claim 1 is characterized in that: described polyembryony body is identified as follows: described cross-pollinated seed is carried out the embryo examination and germinate identifying the polyembryony body.
3, method according to claim 2 is characterized in that: described female parent is inbred line, basic population or the crossbreed that meets breeding objective.
4, method according to claim 3 is characterized in that: described basic population is the maize population that forms by behind a plurality of inbred lines or the kind mixed pollination.
5, method according to claim 4 is characterized in that: described crossbreed is single cross hybrid, triple hybrid or double cross hybrid.
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| CN101377481B (en) * | 2008-10-06 | 2012-04-18 | 中国农业大学 | Method for identifying corn parthenogenesis haploidy |
| CN102334447A (en) * | 2010-07-23 | 2012-02-01 | 沈天民 | Cultivation method of maize inbred line |
| CN103081797B (en) * | 2011-10-27 | 2014-11-05 | 中国农业大学 | Method for inducing corn haploid |
| CN103081802B (en) * | 2011-10-27 | 2014-05-14 | 中国农业大学 | Method for auxiliary identification of corn haploid induction line |
| CN102972285B (en) * | 2012-12-05 | 2014-03-26 | 湖南杂交水稻研究中心 | Breeding method of hybrid crops |
| CN103999769B (en) * | 2014-06-06 | 2016-06-01 | 山东省农业科学院玉米研究所 | A kind of corn haploid induction method |
| AU2016331021B2 (en) * | 2015-10-02 | 2022-10-27 | Keygene N.V. | Method for the production of haploid and subsequent doubled haploid plants |
| 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 |
| CN108668890B (en) * | 2018-04-08 | 2021-06-08 | 河南农业大学 | Method for improving correct recognition rate of corn haploid |
| EP3784031A4 (en) * | 2018-04-27 | 2022-01-26 | Monsanto Technology LLC | Methods for genotyping haploid embryos |
| CN109122292A (en) * | 2018-08-25 | 2019-01-04 | 吉林农业科技学院 | A kind of breeding method of waxy corn DH system |
| CN112005878B (en) * | 2020-08-24 | 2022-06-21 | 中国农业大学 | Method for rapidly breeding corn haploid induction line and application thereof |
| 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 |
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