CN106234209A - A kind of new corn haploid induction line and application thereof - Google Patents
A kind of new corn haploid induction line and application thereof Download PDFInfo
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Abstract
The invention belongs to gene engineering technology field, it is specifically related to a kind of new corn haploid induction line and application thereof, the present invention includes: with transgenic corns (♂) and haploid inducing line maternal (♀) hybridization as male parent of express fluorescent protein gene, anti insect gene and anti-herbicide gene, produce first-filial generation, through repeatedly backcrossing, screen and selfing, it is thus achieved that new corn haploid induction line.The present invention solves monoploid difficulty in former system and identifies, accuracy is the highest, and speed is slow, depends on the problems such as experienced research worker.Can carry out Rapid identification by simple device, it is not necessary to experienced research worker, accuracy is high, time saving and energy saving, can carry out large-scale monoploid production, be applied to corn breeding.Haploid inducing line in the present invention has quantity and the quality of pest-resistant, the feature of antiweed, beneficially field management and raising haploid induction simultaneously.
Description
Technical field
The invention belongs to gene engineering technology field, be specifically related to a kind of new corn haploid induction line generation and
Application.
Background technology
It is known that haplophyte genome comprises only a group chromosome, the most sterile.But pass through natural dyeing
Body doubles or after artificial doubling, can form dihaploid.Owing to chromosome doubling is by the genome of haplobiont times
Increase, two chromosome sets identical (pure and mild) of the liploid plant therefore formed.This is had any different in being formed by hybridization
Heterozygous diploid (, from female parent, one from male parent for a chromosome set).Dihaploid is due to the most pure and mild, therefore plant
It is good selfing line material in breeding, is used directly for preparing hybrid combination.
In the past, plant breeding scholar mainly carries out breeding by selection and hybridization.In plant breeding, the acquisition of selfing line
It it is the key of preparing hybrid combination.And generally this process will backcrossing or selfing through 6-8 generation, and the selfing line obtained only have
The pure and mild degree of 99%.Semen Maydis general one is only kind of a generation in the north, and 6-8 generation needs the time of 6-8, even if adding generation in south,
The most secondary, it is still desirable to the time of 3-4.During screening selfing line, generally require the substantial amounts of individuality of plantation, take big
The farmland of amount, expends substantial amounts of manpower and materials.The traditional method cycle is long, and workload is big, inefficiency.The application of monoploid technology
Can obvious shortening the breeding cycle, improve efficiency of selection, it is possible to reach 100% pure and mild degree, therefore general in corn breeding
All over using (Chang and Coe, 2009, Geiger and Gordillo, 2009, Zhang etc. 2008, Prasanna etc.
2012, Robert etc. 2005).
The method of haploid induction has genetic induction at present, manually modified, artificial induction, and pollen (medicine) is cultivated, and remote source is miscellaneous
Hand over and parthenogenesis etc..Semen Maydis monoploid is found in natural population the earliest, and Chase (1949) finds have in Semen Maydis natural population
Monoploid occurrence frequency less than 0.1%.Semen Maydis monoploid can be obtained in a large number, such as Chinese Academy of Sciences's heredity by Anther Culture
Institute 401 groups (1975) and Institute of Botany, Chinese Academy of Sciences (1978) report that Anther Culture obtains list that can be hereditary respectively
Times body.But external evoked method has significant limitation, as haploid induction is affected by plant genotype, in group training
It is easily generated somatic mutation, needs to expend substantial amounts of manpower and materials and (Barret etc. 2004, the Beckert etc. such as efficiency is low
1994).These limitations have had a strong impact on the application in Semen Maydis conventional breeding of the Semen Maydis monoploid technology.
The Haploid Breeding of Maize that is found to be of corn haploid induction line provides possibility.Initial Semen Maydis haploid induction
System derives from the Stock6 (Coe, 1959) that nineteen fifty-nine American scientist Ed Coe finds.Stock6 is as male parent and other Semen Maydiss
Material hybridizes, and has about 2% for monoploid in offspring.The whole chromosome of haploid genome both is from female parent material, because
Male parent chromosome is eliminated (Zhang etc. 2008) during forming monoploid.Haploid induced efficiency was once much to grind
The emphasis studied carefully, and obtained the biggest breakthrough.The higher monoploid of a series of induction frequency is defined through repeatedly transformation
Induction system, such as KMS and ZMS (Tyrnov and Zavalishina, 1984), WS14 (Lashermes and Beckert,
1988), KEMS (Sarkar etc. 1994), MHI and M751H (Eder and Chalyk, 2002), RWS (Rober etc. 2005),
UH400 (Chang and Coe, 2009), PK6 (Barret etc. 2008), HZI 1 (Zhang etc. 2008), CAUHOI (Chen
And Song, 2003), PHI (Rotarenco etc. 2010).The monoploid technology that is produced as that high efficiency induction is is at corn breeding
In large-scale application provide guarantee.
The another one research emphasis of corn haploid induction line is haploid screening system, the most how substantial amounts of two
Haploid individuality is picked out in times body filial generation.The monoploid screening system commonly used at present is to utilize R1-nj labelling.R1-
Nj is an allele of Semen Maydis anthocyanidin synthesis regulation gene R1, and the expression of R1-nj shows seed on corn kernel
Top endosperm be purple, and purple embryo.R1-nj can be as the screening haploid labelling of Semen Maydis.As haploid inducing line contains
Pure and mild R1-nj gene, and when derivative female parent is the seed without color, due to haploid embryo not through being fertilized or being subject to
After essence, male parent chromosome is excluded, and embryo does not has R1-nj gene, and for colourless, but endosperm is through fertilization, thus has R1-nj base
Because expressing, for purple.On the contrary, the embryo of normal diploid seed and endosperm all through fertilization, have R1-nj to express, thus
The most purpuric labelling.But this systematic difference is affected by following factor: the most derivative female parent material can not have pigment
Express, because this can cover R1-nj labelling；2.R1-nj gene express by environmental factors affected (Kebede et al.,
2011；Prigge et al.,2011；Prigge et al.,2012；2005)；3.R1-nj expression
By other anthocyanidin synthesis and controlling gene affected, as c1-I, c2-Idf and in-1D (Burr et al., 1996；Della
Vedova et al.,2005；Paz-Ares et al.,1990)；The most haploid qualification generally requires experienced person and enters
Row is identified.For the shortcoming overcoming R1-nj system, it is thus proposed that come by seed oil content or by Seedling Stage B1 and PL1 labelling
Selection markers as an alternative, but these systems also and unstable and accurately (Rotarenco et al., 2010；Rotarenco
et al.,2007)。
It addition, current haploid inducing line is much the most pest-resistant and herbicide, therefore in growth promoter and the hybridization of induction system
During, weeds management difficulty, it is susceptible to insect pest, the breeding of system and haploid induction are induced in impact.
Summary of the invention
It is contemplated that overcome the defect that said system induction and screening exist, it is provided that a kind of efficiently induction and screening Semen Maydis
Haploid method, is more accurately produced faster and identifies monoploid, and doubled by colchicine-induced haploid chromosomes
Form heritable dihaploid (DH), be applied to maize genetic breeding.
Technical solution of the present invention is as follows:
The generation of a kind of new corn haploid induction line and screening technique, principle is as follows:
Express fluorescent protein (EGFP) gene, anti insect gene (BT) and anti-herbicide gene simultaneously is built by genetic engineering
(Bar) carrier, and by agriculture bacillus mediated gene transformation by these 3 gene transformation to Semen Maydis, with this transgenic plant
For male parent, hybridize with corn haploid induction line RWS, and by repeatedly backcrossing in RWS female parent, screening expresses these 3 simultaneously
Gene and R-nj gene also have the individuality of haploid induction ability, and by 2 selfings, screening obtains pure and mild containing fluorescence egg
White gene, anti insect gene and the new haploid inducing line of anti-herbicide gene, the new Semen Maydis monoploid being the present invention lures
Lead and be.
With this induction system as male parent (♂), produce F1 generation seed with for maternal Semen Maydis (♀) hybridization that is induced, sprout F1 generation
Seed, observes EGFP gene expression in seed young root；The seed wherein expressing EGFP is normal diploid, does not express EGFP
Seed be monoploid, haploid chromosomes is identified, determine containing 10 chromosomes for monoploid, containing 20 chromosomes
For diploid, bypass diploid, by haplobiont through Colchicine process carry out chromosome doubling after transplant in greenhouse or
Field carries out selfing, the dihaploid after doubling can normal knot, i.e. obtain dihaploid pure lines.
Wherein haploid inducing line and filial generation all have pest-resistant characteristic, are effectively protected haploid inducing line
Growth, breed and for the quantity of haploid induction pollen, be also effectively protected filial generation from pest damage, add
Haploid quality and quantity.The new anti-herbicide gene in induction system can provide this induction to be the ability of antiweed,
Be conducive to removing weeds and other milpas in planting process, effectively carry out field management.
Further, the generation of a kind of new corn haploid induction line and screening technique, specifically comprise the following steps that
Step one, maternal with haploid inducing line RWS with the transgenic corns male parent (♂) of above-mentioned expression EGFP/Bt/Bar
(♀) hybridization produces first-filial generation F1；
Step 2, F1 backcross haploid inducing line RWS (♀) as male parent (♂), produce first backcross generation BC1；
The BC1 that R1-nj and EGFP/Bt/Bar is expressed in step 3, screening simultaneously is individual,
Step 4, with the BC1 of step 3 screening, individual as male parent, (♂) backcrosses haploid inducing line RWS (♀) again, produces
Raw second backcross generation BC2；
The BC2 that R1-nj and EGFP/Bt/Bar is expressed in step 5, screening simultaneously is individual,
Step 6, with the BC2 of step 5 screening, individual as male parent, (♂) RWS (♀) that again backcrosses produces third backcross generation
BC3；
Step 7, screening are expressed the BC3 individuality of R1-nj and EGFP/Bt/Bar simultaneously and are carried out selfing, produce BC3F1；
Step 8, screening are expressed the BC3F1 individuality of R1-nj and EGFP/Bt/Bar simultaneously and are carried out selfing, produce BC3F2,；
Step 9, the pure and mild individuality screened in BC3F2 are also bred, and are new pure and mild haploid inducing line；
Specifically it is described below:
1. by design expression vector clone's EGFP and Bt gene to (figure on the bacillus Expression carrier containing Bar gene
2), described expression vector contains three gene expression frames: the expression of EGFP gene, by 2x35S promoters driven, utilizes AMV to increase
The expression (Datla etc. 1993) of hadron sequence enchancer, utilizes NosT terminator terminator to transcribe；The expression of Bt gene is led to
Cross 2x35S promoters driven, utilize NosT terminator terminator to transcribe；The expression of herbicide-resistant gene Bar is opened by 2x35S
Mover drives, and utilizes NosT terminator terminator to transcribe, the labelling that Bar gene screens as corn transformation.
Wherein, LB and RB is respectively the left and right border sequence of Agrobacterium binary vector Bin19, and 2x35S is dual Brassica oleracea L. var. botrytis L.
Mosaic virus 35 S promoter, AMV is the expression for enhancing gene of the alfalfa mosaic virus sequence, and EGFP is that enhanced green is glimmering
Aequorin, NosT is gene transcriptional terminator, and Bt is Cry1Ab toxin gene, and Bar is anti-herbicide gene
(bialaphos resistance)。
2., by agriculture bacillus mediated gene transformation, obtain the stable transgenic corns expressing EGFP, Bt and Bar gene
(Fig. 3)
3., by repeatedly backcrossing and selfing is transferred in corn haploid induction line EGFP, Bt and Bar gene, formed new
Corn haploid induction line (Fig. 4)
4. with new induction system for male parent with other corn hybridizations, it is thus achieved that filial generation
5. by the monoploid in EGFP label screening filial generation
The LED flashlight producing uv excitation light can be utilized to be irradiated, the material expressing EGFP then in darkroom
In with the naked eye by the glasses of anti-ultraviolet carry out observe screening (Fig. 5)
6. double the haplobiont screened to obtain dihaploid (DH) (accompanying drawing by chromosome doubling technology
6)
7. dihaploid can be applied to corn breeding (accompanying drawing 7) as pure lines
Wherein the haploid screening of Semen Maydis is the expression of the EGFP gene in being by induction.Due to the institute in filial generation
Having normal diploid is all through double fertilization, and the most diplontic embryo and endosperm have the expression of EGFP gene.On the contrary, at single times
In body, owing to endosperm is the triploid of normal fertilization, therefore contain EGFP, Bar and Bt gene of paternal origin, therefore can examine
Measure the expression of green fluorescent protein and there is pest-resistant feature, and haploid embryo contains only the list group dyeing in maternal source
Body, therefore not from the transgene component of male parent, so not expressing EGFP.But the triploid embryo of all filial generation seeds
There is the expression of Bt gene in Ruzhong, and therefore filial generation all has pest-resistant feature, thus decreases the list caused because of insect pest
The loss of times body offspring, can increase the quality and quantity of monoploid offspring compared with former system.Isogeneous induction system is hybridized
The seed of offspring is sprouted, and in the radicle in detection embryo source, the expression of EGFP, can quickly determine whether for monoploid.EGFP
Detection can use fluorescence microscope, fluorescence anatomical lens is, or utilize the pocket lamp launching blue excitation light to irradiate in darkroom
Germination seed radicle fluorescence is excited to determine.The observation of fluorescence can use UV resistance glasses to observe by the naked eye.
Doubling monoploids in described step 6 is to process, by Colchicine, the seedling sprouted 4-5 days to carry out；To sprout also
Have the seedling of about 2 centimeter length plumules to excise in plumule 1 centimeters to expose growing point, simultaneously from seed about 1 centimeters by children
Root excises, and is immersed in the aqueous solution containing 0.04% Colchicine and 0.5%DMSO, the seedling of excision plumule and radicle in room
Temperature dark is soaked 12 hours, then rinse 1 hour with in tap water flowing water, plant in the hole tray containing peat soil, cultivating
Cultivating in case to 3 leaf phases, condition of culture is 16 hours every days of illumination, and 8 hours dark, illumination temperature 28 DEG C, dark temperature 25 DEG C.
Incubation time is 1--2 week.
The key point of the present invention
1. the corn haploid induction line of express fluorescent protein gene, anti insect gene and anti-herbicide gene
2. utilize new induction system to produce the haploid method of Semen Maydis
3. for detecting the haploid device of Semen Maydis by EGFP
Advantages of the present invention and good effect
Having efficiency by corn haploid induction line induction monoploid high, easily operate, do not limited by genotype, save time province
Power, it is easy to the advantages such as large-scale production.But the monoploid screening system in existing system can not identify list fast and accurately
Times body, and affected by E&H background, limit its application in corn breeding.The present invention is by the most single
Implanting EGFP labelling in times body induction system, before solving, in system, monoploid difficulty is identified, accuracy is the highest, and speed is slow, relies on
In problems such as experienced research worker.Rapid identification can be carried out, it is not necessary to experienced research worker by simple device,
Accuracy is high, time saving and energy saving, can carry out large-scale monoploid production, be applied to corn breeding.Lure for current monoploid
Leading is the easy problem by insect pest, and the present invention is by expressing anti insect gene so that induction system itself and the offspring hybridized with induction system
Seed all has pest-resistant advantage, can improve the growth promoter quality of induction system, and the seed after hybridizing has pest-resistant spy
Property, increase seed amount and quality；The management of weeds in growth course is tied up to, due to the induction system that this is new for haploid induction
There is the feature of antiweed, effectively can go to cut weeds and other milpas by spraying herbicide, be conducive to inducing system
Field management；Monoploid forms dihaploid (DH) after Colchicine process doubles, and these dihaploids are without turning base
Because of gene element, the breeding in Semen Maydis will can be directly utilized as pure lines.
The present invention utilizes transgenic technology, successfully by enhanced green fluorescent protein gene (EGFP) anti-herbicide gene
(Bar) express in Semen Maydis with Bt toxin gene, and by repeatedly backcrossing, EGFP, Bar and Bt gene is transferred to single times
Body is induced in system, and don't affects the efficiency of haploid induction, forms new corn haploid induction line.Both former induction system had been retained
High efficiency induces haploid advantage, overcomes again original system and utilizes the limitation of R1-nj marker gene, for quickly and accurately
Screening Semen Maydis monoploid provides a very effective method.Use the inducible system that this is new, the life of induction system can be improved
Long development quality, and the seed after hybridizing has pest-resistant characteristic, increases seed amount and quality；By filial generation is carried out
The expression analysis of EGFP, thus quickly and accurately filter out monoploid；Monoploid is formed after Colchicine process doubles
Dihaploid (DH), these dihaploids do not contain transgene component, will can directly utilize the breeding in Semen Maydis as pure lines.
Accompanying drawing explanation
Fig. 1 is the present invention Semen Maydis haploid induction with EGFP as labelling and the system diagram of monoploid qualification.
Wherein, induction system produces F1 generation seed (B) for the same hybridization of female parent of male parent (A), detects EGFP children after seed germination
Expression in root, the individuality having EGFP to express is normal diploid (C), and the individuality not expressing EGFP is monoploid (D), monoploid
Can pass through Chromosome Identification, containing 10 chromosomes, and normal diploid is containing 20 chromosomes.
Fig. 2 is EGFP/Bt/Bar expression vector schematic diagram of the present invention.
Wherein, LB and RB is respectively the left and right border sequence of Agrobacterium binary vector Bin19, and 2x35S is dual Brassica oleracea L. var. botrytis L.
Mosaic virus 35 S promoter, AMV is the expression for enhancing gene of the alfalfa mosaic virus sequence, and EGFP is that enhanced green is glimmering
Aequorin, NosT is gene transcriptional terminator, and Bt is Cry1Ab toxin gene, and Bar is anti-herbicide gene
(bialaphos resistance)。
Fig. 3 is the expression picture of corn gene callus EGFP of the present invention.
Wherein, the transgenic calli of EGFP is expressed in arrow instruction, and triangle is designated as non-transgenic callus.
Fig. 4 is that the present invention passes through to backcross and selfing is transferred to EGFP/Bt/Bar gene in corn haploid induction line RWS
Schematic diagram
Fig. 5 is the ultraviolet LED pocket lamp that detects for Semen Maydis monoploid of the present invention and anti-ultraviolet glasses.
Wherein, LED flashlight produces the uv excitation light of 395 nanometers and EGFP can be excited to send green fluorescence, and naked eyes lead to
Cross anti-ultraviolet glasses to observe in darkroom.
Fig. 6 is that haploid induction of the present invention doubles middle seedling radicle and the partial cutaway schematic view of plumule
Fig. 7 is that the heterozygote (left) in dihaploid pure lines (right) after chromosome doubling of the present invention and non-monoploid source is beautiful
Rice schematic diagram.
Detailed description of the invention
Further illustrating the present invention below in conjunction with drawings and Examples, following example will assist in those skilled in the art
Member is further appreciated by the present invention, but limits the present invention the most in any form.It should be pointed out that, other enforcement can also be used
Example, or the embodiment enumerated herein is carried out amendment structurally and functionally, without departing from the scope of the present invention and essence.
The structure of example 1 transgene carrier and the gene transformation of Semen Maydis
Express vector construction such as Fig. 2 of EGFP/Bt/Bar.Carrier contains three gene expression frames: the expression of EGFP gene
By 2x35S promoters driven, utilize the expression (Datla etc. 1993) of AMV enhancer sequence enchancer, utilize NosT to terminate
Sub-terminator is transcribed；The expression of Bt gene, by 2x35S promoters driven, utilizes NosT terminator terminator to transcribe；Anti-
The expression of herbicide resistance gene Bar, by 2x35S promoters driven, utilizes NosT terminator terminator to transcribe.Bar gene conduct
The labelling of corn transformation screening.
Carrier containing EGFP/Bt/Bar expression cassette is converted by freeze-thaw method (Weigel and Glazebrook, 2006)
Agrobacterium tumefaciems EHA101 bacterial strain (Hood et al., 1986).Agrobacterium tumefaciens mediated Semen Maydis HiII kind (Amstrong
Deng 1991) rataria converts the method with reference to (2002) such as Frame.The maize immature embryos of 1.5-2 mm in size after agroinfection,
Put co-culture culture medium (N6 a great number of elements and vitamin, 1.5mg/L 2,4-D, 0.7g/L L-proline, 30g/L sucrose,
0.5g/L 2-(4-morpholino)-ethane sulfonic acid (MES), 100 μ g/ml acetosyringones, 3g/L
Gelrite, pH 5.8) upper 23 DEG C of dark co-culture 3 days, proceed to maize calli screening culture medium (N6 a great number of elements and dimension
Raw element, 1.5mg/L 2,4-D, 0.7g/L L-proline, 30g/L sucrose, 0.5g/L 2-(4-morpholino)-ethane
Sulfonic acid (MES), 0.85mg/L silver nitrate, 100mg/L cefotaxime, 100mg/L vancomycin, 3g/L
Gelrite, Bialaphos, pH 5.8) formation of upper 28 DEG C of light culture callus induction, and proceed to fresh Semen Maydis in every 3 weeks
Successive transfer culture in callus screening culture medium.
In resistant calli, the expression of EGFP can screen (Fig. 3) by fluorescence anatomical lens inspection.
The transgenic calli expressing EGFP forwards division culture medium (MS culture medium a great number of elements and vitamin, 60g/L to
Sucrose, 100mg/L myo-inositol, and 3g/L gelrite, pH 5.8) on differentiate seedling, and be transplanted in greenhouse
Cultivating, transfer-gen plant is collected seed after selfing, with the hybridization of haploid inducing line and is backcrossed for next step.
Example 2 is by backcrossing and selfing is transferred to EGFP/Bt/Bar gene in corn haploid induction line RWS
The transgenic corns (♂) expressing EGFP/Bt/Bar produces first-filial generation with haploid inducing line RWS (♀) hybridization
F1；F1 as male parent (♂) backcross RWS (♀) produce first backcross generation BC1；Screening expresses R1-nj's and EGFP/Bt/Bar simultaneously
BC1 is individual, and (♂) RWS (♀) that backcrosses produces second backcross generation BC2 with this individuality as male parent；Screening simultaneously express R1-nj and
The BC2 of EGFP/Bt/Bar is individual, and (♂) RWS (♀) that backcrosses produces third backcross generation BC3 with this individuality as male parent；Screening is simultaneously
The BC3 individuality expressing R1-nj and EGFP/Bt/Bar carries out selfing, produces BC3F1；Screen 10 strains express simultaneously R1-nj and
The BC3F1 individuality of EGFP/Bt/Bar carries out selfing (wherein part is pure and mild body), produces BC3F2 (Fig. 4).If BC3F1 is individual
For pure and mild, BC3F2 offspring produced by its selfing should all express R1-nj and EGFP/Bt/Bar.10 strain BC3F1 individualities have
The self progeny of three strains expresses R1-nj and EGFP/Bt/Bar the most simultaneously, and for pure and mild body, its self progeny BC3F2 is as new
Pure and mild haploid inducing line.This offspring individual contains the genome of the former RWS haploid inducing line of 87% and pure and mild EGFP/
Bt/Bar gene, will carry out following haploid induction test and Insect resistance assay further.
Example 3 detects the device (Fig. 5) that Semen Maydis EGFP expresses
The detection of EGFP typically can be carried out under fluorescence microscope or fluorescence anatomical lens, but fluorescence microscope and anatomical lens
Expensive, the most portable, operation complexity, it is unfavorable for detecting Semen Maydis monoploid in a large number.For in detection corn kernel and seedling
EGFP express can utilize produce uv excitation light LED flashlight to express EGFP material be irradiated, then secretly
Room with the naked eye carries out observing screening by the glasses of anti-ultraviolet.
Example 4 utilizes the haploid test of haploid inducing line inducing maize containing EGFP/Bt/Bar labelling
As it is shown in figure 1, new haploid inducing line is hybridized with G1-1xB73 female parent material, gather in the crops F1 generation hybrid, pass through
EGFP labelling utilizes fluorescence microscope, anatomical lens or fluorescence excitation device (Fig. 5) screening monoploid, and true by chromosome observation
Determine the accuracy rate that monoploid is identified.In 153 seeds of F1 generation, 137 are had to have EGFP to express in radicle, other 16 nothings
EGFP expresses.36 individualities expressing EGFP and 12 individualities not expressing EGFP are carried out Chromosome Identification and find 36 expression
The individuality of EGFP is entirely diploid, and having 11 in 12 individualities not expressing EGFP is monoploid, and another one is non-whole
Times body, carries out the rate of accuracy reached of monoploid discriminating to 91.7% with EGFP labelling.
Example 5 induces sweet corn monoploid by the new haploid inducing line containing EGFP/Bt/Bar labelling
With new induction system as male parent, hybridize with the sweet corn hybrid on 5 markets, and filial generation is passed through
EGFP labelling carries out haploid screening, and data list table 1 in.183 individualities not expressing EGFP are screened out as possible
Monoploid individual, carried out Chromosome Identification to wherein 18, be found to have 13 for real monoploid, other for non-whole
Times body.Average haploid-induction 7.9%.
Table 1, super-sweet corn haploid induction
The new haploid inducing line pest resistance analysis of example 6
New haploid inducing line and former induction system compare and have pest-resistant advantage, new haploid inducing line after taking seed germination
Indoor Insect resistance assay is carried out with the seedling leaves of former induction system.Each culture dish puts 4 blades, with water-wet, is then placed in 30
Being less than the Ostrinia furnacalis larvae of 12 hours after head hatching, be positioned over temperature 26-28 degree, relative humidity is in the environment of 70%
Cultivate, within every 2 days, change a fresh corn blade, and observe larvae alive situation, statistics mortality rate (table 2).
In the case of tying up to not execute insecticide to the new haploid inducing line of field growing and former induction, female, tassel insect pest occurs
Situation carry out investigation find, the haploid inducing line containing Bt gene is female, tassel insect pest incidence rate substantially reduces, and draws because of insect pest
The rate that fractures the tassel risen substantially reduces.It is yield and the haploid induction of pollen that appreciable impact is induced in haploid induction by this
It it is the breeding of oneself.
The new corn haploid induction line Insect resistance assay of table 2.
Table 3. contrasts with without Bt gene haploid inducing line field insect pest incidence rate (meansigma methods) containing Bt gene
It is the pest-resistant performance of filial generation that example 7 is newly induced
Respectively with induction system new, old as male parent, hybridize with sweet corn Hybrid, in the case of not executing insecticide
To the pollination young ear worm of latter 20 days evil, a situation arises carries out field investigation, finds with the young fringe dye of the induction system hybridization containing Bt gene
Worm rate substantially reduces.Ripe filial generation kernal number component analysis is found, with average every fringe of the induction system hybridization containing Bt gene
Kernal number substantially increases.
Table 4. is (flat with sweet corn hybridization children's fringe field insect pest incidence rate with without Bt gene haploid inducing line containing Bt gene
Average) and filial generation kernal number
Example 8 is by chromosome doubling haplobiont output dihaploid
Doubling monoploids processes, by Colchicine, the seedling sprouted 4-5 days to be carried out.By sprouting and there are about 2 centimeter length plumules
Seedling excise in plumule 1 centimeters to expose growing point, simultaneously from seed about 1 centimeters, young root is being excised (Fig. 6).To cut
Except the seedling of plumule and radicle is immersed in the aqueous solution containing 0.04% Colchicine and 0.5%DMSO, soak in room temperature dark
Steep 12 hours, then rinse 1 hour with in tap water flowing water, plant in the hole tray containing peat soil, cultivate in incubator to 3
Ye Qi, condition of culture is 16 hours every days of illumination, and 8 hours dark, illumination temperature 28 DEG C, dark temperature 25 DEG C.Incubation time 1-2
Week.The seedling survival that a total of 55 strains processed is got off, and is transplanted to land for growing field crops, and it is (same that each plant educated carries out selfing
Time produce pollen and female fringe).There are 14 strain success selfings solid, gather in the crops after seed maturity.
Super-sweet corn major part contains pure and mild Sh2 gene (Tracy, 1997), seed gauffer depression (Fig. 7 is right).From
The monoploid of super-sweet corn female parent should contain Sh2 gene, and the dihaploid after chromosome doubling should contain pure and mild Sh2 gene, therefore
Retain the feature of seed gauffer depression.Owing to part seedling is aneuploid, therefore containing the Sh2 gene of heterozygosis, these are individual
Self progeny in can show the separation of Sh2 gene, self-bred progeny individuality shows as part seed gauffer depression (Sh2
Pure and mild) and the normal seed (Sh2 heterozygosis) of part (as Fig. 7 is left), these individualities are not dihaploid, the most i.e. lose
Fall.And real dihaploid will be retained and be used as pure lines and be used for breeding.In this is tested, produce altogether 7 double lists
Times body pure lines.
The foregoing is only presently preferred embodiments of the present invention, those skilled in the art know, in the essence without departing from the present invention
In the case of god and scope, these features and embodiment can be carried out various change or equivalent.It addition, the present invention's
Under teaching, can modify these features and embodiment to adapt to particular situation and material without departing from the present invention's
Spirit and scope.Therefore, the present invention is not limited to the particular embodiment disclosed, and the right of fallen with the application is wanted
Embodiment in the range of asking broadly falls into protection scope of the present invention.
Claims (8)
1. a new corn haploid induction line, it is characterised in that:
1) this new induction system express fluorescent protein gene, anti insect gene and anti-herbicide gene simultaneously；
2) with this induction system for male parent and any Semen Maydis hybridization of female parent, the offspring individuals endosperm of gained is the most all expressed above-mentioned 3
Gene；
3) with this induction system for male parent and any Semen Maydis hybridization of female parent, the part offspring individuals embryo of gained and plant are monoploid,
These individualities do not contain above-mentioned 3 genes；
4) without fluorescin in the seedling root formed after individual seeds is sprouted in above-mentioned 3) and bud, can be by exciting and observing
The device of fluorescin and other individualities distinguish；
5) above-mentioned 3) and 4) in monoploid individuality can pass through induced chromosome doubling techniques, form dihaploid (DH)；
6) dihaploid described in above-mentioned 5) is the pure and mild son that can educate, and can be applied to the hybridization of Semen Maydis as selfing line and educate
Kind.
A kind of new corn haploid induction line the most according to claim 1, it is characterised in that: it produces and screening technique
Operating procedure is as follows:
Step one, the transgenic corns male parent (♂) expressing EGFP/Bt/Bar and haploid inducing line RWS maternal (♀) hybridization product
Raw first-filial generation F1；
Step 2, F1 backcross haploid inducing line RWS (♀) as male parent (♂), produce first backcross generation BC1；
The BC1 that R1-nj and EGFP/Bt/Bar is expressed in step 3, screening simultaneously is individual,
Step 4, with the BC1 of step 3 screening, individual as male parent, (♂) backcrosses haploid inducing line RWS (♀) again, produces back
Hand over secondary BC2；
The BC2 that R1-nj and EGFP/Bt/Bar is expressed in step 5, screening simultaneously is individual,
Step 6, with the BC2 of step 5 screening, individual as male parent, (♂) RWS (♀) that again backcrosses produces third backcross generation BC3；
Step 7, screening are expressed the BC3 individuality of R1-nj and EGFP/Bt/Bar simultaneously and are carried out selfing, produce BC3F1；
Step 8, screening are expressed the BC3F1 individuality of R1-nj and EGFP/Bt/Bar simultaneously and are carried out selfing, produce BC3F2,；
Step 9, the pure and mild individuality screened in BC3F2 are also bred, and are new pure and mild haploid inducing line.
A kind of new corn haploid induction line the most according to claim 1, it is characterised in that: this induction cording has anti-
Worm, the feature of antiweed.
A kind of new corn haploid induction line the most according to claim 1, it is characterised in that: inducing with this is as male parent
Express anti insect gene with being induced the seed that Semen Maydis hybridization of female parent formed, and there is pest-resistant characteristic.
A kind of new corn haploid induction line the most according to claim 1, it is characterised in that: lure with the monoploid that this is new
Leading is can be hybridization of female parent with any corn material (selfing line and cenospecies) for male parent, induces Haploid production, and passes through
The selection markers of fluorescence protein gene carries out haploid screening.
A kind of new corn haploid induction line the most according to claim 1, it is characterised in that: described step is screened glimmering
Aequorin expression in seed young root, its screening implement can use fluorescence microscope, fluorescence anatomical lens, or in darkroom
The middle LED flashlight utilizing transmitting blue excitation light is irradiated and is excited germination seed radicle fluorescence to determine, the observation of fluorescence is permissible
UV resistance glasses are used to observe by the naked eye screening in darkroom.
A kind of new corn haploid induction line the most according to claim 1, it is characterised in that: above-mentioned induction also filters out
Monoploid can form dihaploid by chromosome doubling.
8. the application of a new corn haploid induction line, it is characterised in that: use the new jade described in claim 1--7
The Semen Maydis dihaploid that rice haploid inducing line is obtained is applied in maize genetic breeding.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106701803A (en) * | 2017-01-13 | 2017-05-24 | 中国农业大学 | Maize female haploid major inducible gene and application thereof |
| CN108220333A (en) * | 2018-03-28 | 2018-06-29 | 中国农业科学院作物科学研究所 | A kind of high-efficiency plant receptor Parthenogenesis haploid screening technique |
| CN109182371A (en) * | 2018-08-03 | 2019-01-11 | 吉林省农业科学院 | A kind of plant gene edit methods independent of genotype |
| CN112005878A (en) * | 2020-08-24 | 2020-12-01 | 中国农业大学 | Method for rapidly breeding corn haploid induction line and application thereof |
| KR20210072854A (en) * | 2019-12-09 | 2021-06-18 | 대한민국(농촌진흥청장) | System and Method for Detecting polyploid and haploid |
| CN113317197A (en) * | 2021-08-03 | 2021-08-31 | 中国农业科学院生物技术研究所 | Rapid chromogenic parthenogenesis induction line and application thereof in identification of corn haploid |
| CN116267583A (en) * | 2023-03-15 | 2023-06-23 | 北京市农林科学院 | Precise introduction and identification method for excellent characters of corn |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106701803A (en) * | 2017-01-13 | 2017-05-24 | 中国农业大学 | Maize female haploid major inducible gene and application thereof |
| CN106701803B (en) * | 2017-01-13 | 2019-03-08 | 中国农业大学 | Corn female parent monoploid main effect induced gene and application |
| CN108220333A (en) * | 2018-03-28 | 2018-06-29 | 中国农业科学院作物科学研究所 | A kind of high-efficiency plant receptor Parthenogenesis haploid screening technique |
| CN109182371A (en) * | 2018-08-03 | 2019-01-11 | 吉林省农业科学院 | A kind of plant gene edit methods independent of genotype |
| KR20210072854A (en) * | 2019-12-09 | 2021-06-18 | 대한민국(농촌진흥청장) | System and Method for Detecting polyploid and haploid |
| KR102274798B1 (en) | 2019-12-09 | 2021-07-09 | 대한민국(농촌진흥청장) | System and Method for Detecting polyploid and haploid |
| CN112005878A (en) * | 2020-08-24 | 2020-12-01 | 中国农业大学 | Method for rapidly breeding corn haploid induction line and application thereof |
| CN113317197A (en) * | 2021-08-03 | 2021-08-31 | 中国农业科学院生物技术研究所 | Rapid chromogenic parthenogenesis induction line and application thereof in identification of corn haploid |
| CN116267583A (en) * | 2023-03-15 | 2023-06-23 | 北京市农林科学院 | Precise introduction and identification method for excellent characters of corn |
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