CN106818458A - The method that corn friction standing grain monosomic addition line is cultivated using corn allopolyploid - Google Patents
The method that corn friction standing grain monosomic addition line is cultivated using corn allopolyploid Download PDFInfo
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- CN106818458A CN106818458A CN201710053008.8A CN201710053008A CN106818458A CN 106818458 A CN106818458 A CN 106818458A CN 201710053008 A CN201710053008 A CN 201710053008A CN 106818458 A CN106818458 A CN 106818458A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
Abstract
The invention belongs to Wide_cross in Plant breeding method field, the method the invention discloses corn friction standing grain monosomic addition line is cultivated using corn allopolyploid.The method is with corn allohexaploid (Tripsazea creammaize T.；2n=76) it is nonrecurrent parent, with corn (Zea mays L.) for recurrent parent hybridizes, chromosome number and genome are carried out to solid offspring into identification and is selected, it is 21 finally to obtain total chromosome number, the corn friction standing grain monosomic addition line that wherein maize chromosome is 20, friction standing grain chromosome number is 1.The present invention is that the excellent genes of the standing grain that rubs are imported into corn to provide a kind of effective way；Secondly, it is of the invention for gene functional research provides good material；Monosomic addition line chromosome stability of the present invention is good, can stably entail offspring；The inventive method is simple, and the time is short, efficiency high.
Description
Technical field
The invention belongs to Wide_cross in Plant breeding method field, and in particular to cultivate beautiful using corn allopolyploid
The method of rice-friction standing grain monosomic addition line.
Background technology
Crop Wild related germplasm has many excellent genes that can be utilized for raise crop and characteristic, such as Resistant
Property, resistance, high-quality etc., but due to reproduction isolation, these excellent genes and characteristic are difficult to be utilized by raise crop.Being will
These foreign genes are imported into raise crop, are bred as a series of addition line, and substitution line or translocation line are abundant crop genetics
A kind of important means of resource.Alien addition line refers to the aneuploid that external source kind chromosome is addition of in species protogene group
Material.According to the number and species of additional chromosome, alien addition line can be divided into：Monomer alien addition line, disome are heterologous attached
Plus be, dual MCU system addition line, double disomic addition lines and multiple addition line etc..Wherein most research and use value are monomers
Alien addition line and disome alien addition line.Mark accurately can be assigned to single external source by Monosomic Alien Addition Line with high throughput
On chromosome, and each monomer alien addition line can be counted as a corresponding exogenous chromosome library, can be relatively outer
Mark position between source chromosome and respective homoreceptor chromosome, also allow for study homologue impregnation mechanism and
Pairing state (Tan Guang pavilions .HEREDITAS (Beijing), 2008,30 (1):35-45).Many plants are formulated at present
Alien addition line, such as Allium cepa imported into (Masayoshi Shigyo*YT .Genes Genet in shallot
Sys.1996,71:363–371)；Oryza officinalis chromosome imported into the (.Theoretical such as Tan G and in paddy rice
applied genetics.2005,111(8):1585-1595)；Ae.speltoides chromosome is imported into wheat
(.Theoretical and Applied Genetics.2000,101 such as Friebe B (1-2):51-58)；Maize chromosome
(.Plant such as Kynast RG physiology.2001,125 (3) in importeding into oat:1216-1227).Additionally, in rape
Substantial amounts of addition line (Struss D etc., PlantBreeding.1991,106 (3) are also obtain during species intermolecular hybrid:
209-214)。
Being isolated due to reproduction between common cultivation corn (Zea mays L.) and its nearly edge species causes distant hybridization to compare
Difficulty, thus be difficult to the desirable genes of nearly edge species and imported into common cultivation corn.Mangelsdof etc. by dliploid or
Tetraploid finger-like friction standing grain is awarded on maize ear after mixing with the pollen of corn by cutting short filigree, resulting hybrid seed
The culture of embryo is carried out, corn-friction standing grain hybrid F is successfully obtained for the first time in the world in nineteen twenty-ninel(Mangelsdorf PC
Deng .Journal of Heredity.1931,22 (11):329-343).Thereafter many scholars have carried out friction standing grain chromosome and have led
Enter the research in corn, its offspring is essentially all to be saved by embryo and inducing somatic formation acquisition (Molina MdC etc.
.Electronic Journal of Biotechnology.2006,9(3):276-280).Therefore, reproduction isolation is to cause jade
Rice is difficult by the principal element of its nearly edge species desirable genes.
MTF-1 (Tripsazea creammaize are T.2n=76.) is that Sichuan Agricultural University is bred as by distant hybridization
Comprising the dyeing of a full set of maize chromosome group (20), gama grass genome (36) and tetraperennial teosinte
The corn allohexaploid (the expensive Sichuan Agricultural Universities Master's thesis of Su Yue, 2009) of body group (20), is that a nature is not deposited
New species.Because MTF-1 have corn, tetraperennial teosinte and friction standing grain a full set of chromosome and its female can
The characteristic educated, enables MTF-1 and corn, tetraperennial teosinte or friction standing grain cross-fertile, after producing fertile hybridization
Generation.It is the hybridization such as bridge material and cultivated maize with MTF-1 using this characteristic of MTF-1, distant hybridization can be overcome to hinder
Hinder, so as to the excellent genes of corn Spherical scanning are transferred in cultivated maize.
Through retrieval, without the report found about cultivating corn-friction standing grain monosomic addition line using corn allopolyploid.
The content of the invention
Low with its Related species material distant hybridization success rate for current corn, the germplasm of its nearly edge species is difficult by
The problems such as, cultivating the purposes that corn-friction standing grain added monomer is fastened present invention aim at corn allopolyploid is provided.
Another object of the present invention is to provide a kind of using corn allopolyploid cultivation corn-friction standing grain monosomic addition line
Method.
Realize that technical scheme is as follows：
Corn allopolyploid is cultivating the application that corn-friction standing grain added monomer is fastened.
Described corn allopolyploid refers to corn allohexaploid MTF-1 (Tripsazea creammaize T.；
2n=76) or its Derivative line.
Described MTF-1 (Tripsazea creammaize T.；It is 2n=76) that Sichuan Agricultural University passes through distant hybridization
What is be bred as contains corn (Zea mays L.), tetraperennial teosinte (Zea perennis, 2n=40) and friction standing grain
Corn allohexaploid material (the expensive Sichuan of Su Yue of a full set of chromosome of (Tripsacum dactyloides L., 2n=72)
Agriculture university's Master's thesis, 2009).
Described Derivative line refer to be with MTF-1 it is maternal, it is perennial with corn (Zea mays are L.2n=20), tetraploid
Class corn (Zea perennis, 2n=40) or friction standing grain (Tripsacum dactyloides L., 2n=72) are that male parent is miscellaneous
Hand over, after selection is comprising corn (Zea mays L.), three species full set chromosomes of tetraperennial teosinte and friction standing grain
In generation (2n=76), the new allohexaploid of gained is the Derivative line of MTF-1.
The genome of described corn-friction standing grain monosomic addition line is into including 20 maize chromosomes and 1 friction standing grain
Chromosome.
The method that corn-friction standing grain monosomic addition line is cultivated using corn allopolyploid, including with heterologous six times of corn
Body (Tripsazea creammaize T.；It is 2n=76) female parent, with corn (Zea mays L.) as paternal hybrid, harvests knot
Real seed, obtains F1Generation；Then to F1Into being identified, selective staining body sum is 45~55 and friction standing grain to the genome in generation
Chromosome number is the F of 1~181For material；Again with selected F1It is female parent for material, is returned by male parent of corn, obtains BC1
Generation；To BC1In generation, carries out genome into being identified, then selective staining body sum is 21~30 and friction standing grain chromosome number
It is the BC of 1~51On behalf of female parent, continue to be returned as male parent with corn, obtain BC2Generation；Selection BC2Total chromosome number is 21 in generation,
The material that wherein maize chromosome is 20, friction standing grain chromosome number is 1, as corn-friction standing grain monosomic addition line.
Corn allohexaploid described in the above method refers to MTF-1 (Tripsazea creammaize T.；2n=
Or its Derivative line 76).
The method that above-mentioned utilization corn allopolyploid cultivates corn-friction standing grain monosomic addition line, it is comprised the following steps that：
(1), since late March, point 10 batches of maize plantings (Zea mays L., 2n=20), 20 plants every batch, between every batch
Every 3 days；The first tenday period of a month in April are by corn allohexaploid (Tripsazea creammaize T.；2n=76) plant in flowerpot；
(2), by corn allohexaploid female fringe bagging before spinning, and its filigree is cut short to 2~3cm before pollination,
Then pollinated to corn allohexaploid female fringe by male parent of corn, each female fringe at least repeats pollination 3 times, every time after pollination all
Marked on bagging, seed is harvested when ripe, obtain F1Generation；
(3), April next year, by the F obtained by step (2)1Be planted in the nutritive cube of growth cabinet for seed, 28 DEG C,
Humidity is vernalization under conditions of 70%, and plantation is transplanted into flowerpot during plant strain growth to 21 heart of leaf；During plant to be planted plant height >=30cm,
In the fine day morning 10：00~12:00 and temperature be higher than 25 DEG C when, take the tip of a root, using Chromosome Pressing Technology identification body cell contaminate
Colour solid number, identifies genome into each individual plant at least counts 50 cells using hybridization in situ technique；Selective staining body number
Mesh is 45~55 and friction standing grain chromosome number is that the plant of 1~18 is cooked female parent, the bagging before female fringe spinning, is cut after spinning
Short filigree is pollinated to 1~2cm by male parent of corn, and every repetition pollination in 2 days 1 time after being pollinated at the 1st time, each female fringe is extremely
It is few to repeat pollination 3 times, seed is harvested when ripe, obtain BC1Generation；Wherein described corn is equally planted by stages with step (1)；
(4), according to the BC obtained by the method plantation described in step (3)1Generation, and according to the method described in step (3)
Using chromosome compressing tablet and hybridization in situ technique to BC1Identified for root tip chromosomes number and composition；Selective staining body is total
Number is for 21~30 and friction standing grain chromosome number is the BC of 1~51For plant；With corn as male parent to selected BC1Dai Zhi
Strain pollination, harvests to obtain BC2Generation；
(5), BC is planted according to the method described in step (3)2Generation, using chromosome compressing tablet to BC2For root tip chromosomes number
Mesh identified, BC2There was only the two types of 20 and 21 for dyeing material body number；
(6) chromosome, is carried out to the plant residue tip of a root that chromosome number in step (5) is 21 using flame seasoning
Film-making, selective staining body is in mid-term and decentralization tip of a root combustion piece higher carries out double-colored genomic in situ hybridization, respectively land used
Gaoxin and biotin labeling corn and friction standing grain total genomic dna, identification genome into；Selection root tip cell chromosome number
It is 21, and the material wherein containing 20 maize chromosomes and 1 friction standing grain chromosome, as corn-friction standing grain monomer is attached
Plus be.
The place of the plantation described in the above method is Sichuan or the ecotope similar to Sichuan.
Corn allohexaploid described in above method step (1) or (2) refers to MTF-1 (Tripsazea
creammaize T.；2n=76) or its Derivative line.
Described MTF-1 (Tripsazea creammaize are T.2n=76.) is the bag that Sichuan Agricultural University is bred as
Containing a full set of maize chromosome group (20), gama grass genome (36) and tetraperennial teosinte chromosome
The corn allohexaploid of group (20), is the non-existent new species of nature.
MTF-1 is Sichuan Agricultural University with the tetraploid corn (Zea mays are L.4n=40) quoted from the U.S. and tetraploid
Intergeneric hybrid F between gama grass (or finger-like friction standing grain, Tripsacum dactyloides L., 2n=72)1
It is female parent, is the intergeneric hybrid (Su Yue that paternal hybrid is bred as with tetraperennial teosinte (Zea perennis, 2n=40)
Your Sichuan Agricultural Universities Master's thesis, 2009).
MTF-1 (Tripsazea creammaize T.) is perennial, and vegetative propagation, plant is healthy and strong, uprightly grows thickly, root
System is flourishing, and plant winter resistance is strong.
MTF-1 is mainly carried out numerous by asexual reproduction methods such as point pocket breeding, stalk cuttage or other vegetative manners
Grow.
Described Derivative line refers to MTF-1 as maternal, with corn (Zea mays L.), tetraperennial teosinte
Or friction standing grain is paternal hybrid, selection includes corn, three species full set chromosomes of tetraperennial teosinte and friction standing grain
Offspring (2n=76), the new allohexaploid of gained is its Derivative line.
Sichuan Agricultural University ensures from the present patent application in 20 years, MTF-1 (Tripsazea to be provided to the public
Creammaize is T.2n=76) propagating materials, the public can obtain the biomaterial from Sichuan Agricultural University.
Corn described in the above method refers to common cultivation corn (Zea mays L.), and described corn can be miscellaneous
Hand over kind, self-mating system, local varieties or collective species etc..
Total chromosome number described in above method step (3) is preferably 45~50；Described friction standing grain chromosome number is excellent
Elect 1~10 as.
The specification of the flowerpot described in above method step (1) or (3) is bore 40cm, 35cm high.
The method of the chromosome compressing tablet described in above method step (3), (4) or (5) is：In the fine day morning 10：00~
12:00 and temperature when being higher than 25 DEG C, take the tip of a root, and with α-bromonaphthalene saturated aqueous solution pretreatment 3h, the pretreated tip of a root is existed
The hypotonic 30min of distilled water is used at room temperature, then with Fresh fixative (methyl alcohol：Glacial acetic acid=3：1) fixed more than 12h.Again will be solid
The tip of a root after fixed is placed in 70% absolute ethyl alcohol, in being stored in 4 DEG C overnight：The fixer on the tip of a root is cleaned with distilled water, with etc.
6% cellulase (Yakult) and 1% pectase (Yakult) of mixing are measured, tip of a root 25h, the tip of a root after enzymolysis are digested at 37 DEG C
Being dyeed with the carbolfuchsin after improvement carries out compressing tablet, and microphotograph simultaneously counts chromosome number.
The methods such as chromosome compressing tablet, in situ hybridization described in above method step (3), (4) or (5) can be found in Su Yuegui
Master's thesis (Su Yuegui, Sichuan Agricultural University's Master's thesis, 2009).
Flame seasoning described in above method step (6) referring to Su Yuegui Master's thesis (Su Yuegui, sichuan agriculture
University's Master's thesis, 2009).
The present invention has the advantage that and good effect：(1), the present invention is the dysgenesia for overcoming corn distant hybridization, is
The excellent genes of the standing grain that rubs are imported into corn and provides a kind of effective way, enrich corn germ plasm resource, be conducive to choosing
Educate breakthrough corn variety.(2), corn of the present invention-friction standing grain monosomic addition line is good for gene functional research is provided
Material.(3) corn-friction standing grain monosomic addition line chromosome stability, using the inventive method incubation is good, can stably lose
Offspring is transmitted to, for permanently effective utilization provides guarantee.(4), the present invention is beautiful by bridge material selective breeding of corn allohexaploid
With methods such as traditional distant hybridization simply, the time is short, efficiency high, workload are small for rice-friction standing grain monosomic addition line Billy.
Brief description of the drawings
The plant photo of Fig. 1, MTF-1.
The F of Fig. 2, corn and MTF-11For somatic chromosome in situ hybridization collection of illustrative plates；Wherein blue is friction standing grain chromosome,
Light green color is maize chromosome, and baby pink is friction standing grain chromosome.
The BC of Fig. 3, corn and MTF-11For conventional corn chromosome (2n=20) microphoto
The BC of Fig. 4, corn and MTF-11Corn-friction standing grain monosomic addition line chromosome (2n=20+1) micro- photograph in generation
Piece.
The BC of Fig. 5, corn and MTF-11It is in situ for corn-friction standing grain monosomic addition line (2n=20+1) somatic chromosome
Hybridization collection of illustrative plates；Wherein arrow show friction standing grain chromosome.
The BC of Fig. 6, corn and MTF-12Conventional corn chromosome (2n=20) microphoto in generation.
The BC of Fig. 7, corn and MTF-12Middle corn-friction standing grain monosomic addition line chromosome (2n=20+1) microphoto.
The BC of Fig. 8, corn and MTF-12Corn-friction standing grain monosomic addition line (2n=20+1) body cell in situ hybridization in generation
Collection of illustrative plates；Wherein arrow show friction standing grain chromosome.
Specific embodiment
Embodiment 1：The initiative of corn-friction standing grain monosomic addition line
Carry out as follows：
(1), in early April, 2012 in Sichuan Agricultural University Chengdu school district by MTF-1 (Tripsazea creammaize
T., 2n=76) (see Fig. 1) is planted in flowerpot (flowerpot specification be bore 40cm, 35cm high)；Since late March, divide 10
Batch, the 3 days maize planting self-mating system Mo17 (Zea mays L., 2n=20) in every batch of interval, every batch is planted 20 plants.
(2), by MTF-1 female fringe baggings before spinning, and cut short to 2 MTF-1 filigrees after the spinning of MTF-1 female fringes~
3cm, then pollinates by male parent of Mo17 to MTF-1, and each female fringe at least repeats pollination 3 times, every time after pollination all on bagging
Mark, seed is harvested when ripe, obtain F1Generation.
(3), in April, 2013, the F that step (2) is obtained1It is planted in the nutritive cube of growth cabinet for seed, 28
DEG C, humidity is vernalization under conditions of 70%, transplants to flowerpot that (flowerpot specification is bore 40cm, high during plant strain growth to 21 heart of leaf
Plantation, finally gives 30 plants of F in 35cm)1For plant；During plant to be planted plant height >=30cm, in the fine day morning 10：00~12:00 and
When temperature is higher than 25 DEG C, the tip of a root is taken, (chromosome compressing tablet and hybridization in situ technique are joined using chromosome compressing tablet and hybridization in situ technique
See Su Yuegui, Sichuan Agricultural University's Master's thesis, 2009) identification of cell chromosome number and composition (described chromosome compressing tablet
Method be：Tip of a root α-bromonaphthalene saturated aqueous solution is pre-processed into 3h, the pretreated tip of a root is low with distilled water at room temperature
30min is oozed, then with Fresh fixative (methyl alcohol：Glacial acetic acid=3：1) fixed more than 12h.The tip of a root after fixation is placed in again
In 70% absolute ethyl alcohol, in being stored in 4 DEG C overnight：The fixer on the tip of a root is cleaned with distilled water, with 6% fiber of mixed in equal amounts
Plain enzyme (Yakult) and 1% pectase (Yakult), tip of a root 2.5h is digested at 37 DEG C, and the card after tip of a root improvement after enzymolysis is precious
Moral training carries out compressing tablet), microphotograph simultaneously counts chromosome number, and each individual plant at least counts 50 cells, selective staining
Body number is that (T, P, M represent friction standing grain to the plant of 45 (see Fig. 2), its genome as 16T+11P+18M (1MP) respectively
Chromosome, teosinte chromosome, maize chromosome, MP represent corn and teosinte translocation chromosome)；In selected F1For plant
Bagging before female fringe spinning, cuts short filigree to 1~2cm after spinning, (Mo17 is planted in batches as step (1)) with Mo17 as male parent
To selected F1For plant pollination, pollinated every repetition in 2 days after being pollinated at the 1st time, pollinated 3 times altogether, every time all in set after pollination
Marked on bag, seed is harvested when ripe, obtain BC1Generation.
(4), in April, 2014, BC is planted according to the method described in step (3)1Generation, and it is miscellaneous using chromosome compressing tablet and original position
Friendship technology (chromosome compressing tablet and hybridization in situ technique are referring to Su Yuegui, Sichuan Agricultural University's Master's thesis, 2009) identification of cell
Chromosome number and composition, according to BC1Chromosome distribution pattern from BC1Selective staining body bar number levels off to corn simultaneously in family
And (chromosome number is 21,20M+1T, and M represents maize chromosome, and T represents friction standing grain dyeing to comprise only friction standing grain chromosome
Body, is shown in Fig. 4, Fig. 5) individuality set a microcommunity, with Mo17 be male parent to selected plant pollination, seed is harvested when ripe, obtain
BC2Generation.
(5) in, April next year, BC is planted according to step (3) methods described2Generation, using Chromosome Pressing Technology to BC2The dye in generation
Colour solid number identified, as a result shows BC2There was only 20 (see Fig. 6) and the two types of 21 for dyeing material body type
(see Fig. 7).
(6) dyeing system, is carried out to the plant residue tip of a root that chromosome bar number in step (5) is 21 using flame seasoning
Piece, selective staining body be in mid-term and decentralization it is higher the tip of a root combustion piece carry out double-colored genomic in situ hybridization (referring to Su Yuegui,
Sichuan Agricultural University's Master's thesis, 2009), respectively with digoxin and biotin labeling corn and friction standing grain total genomic dna, mirror
Determine genome into；Selection plant root tip cell chromosome number is 21, and is wherein rubbed containing 20 maize chromosomes, 1
Wipe the material (see Fig. 8) of standing grain chromosome, as corn-friction standing grain monosomic addition line.
Claims (10)
1. corn allopolyploid is cultivating the application that corn-friction standing grain added monomer is fastened.
2. application according to claim 1, it is characterised in that described corn allopolyploid refers to heterologous six times of corn
Body MTF-1 (Tripsazea creammaize T.；2n=76) or its Derivative line.
3. application according to claim 2, it is characterised in that described Derivative line refers to MTF-1 as maternal, with corn
(Zea mays are L.2n=20), tetraperennial teosinte (Zea perennis, 2n=40) or friction standing grain (Tripsacum
Dactyloides L., 2n=72) it is paternal hybrid, selection includes corn (Zea mays L.), tetraperennial teosinte
With the offsprings (2n=76) of friction standing grain three species full set chromosome, the new allohexaploid of gained is the derivative of MTF-1
System.
4. the method that corn-friction standing grain monosomic addition line is cultivated using corn allopolyploid, it is characterised in that heterologous with corn
Hexaploid (Tripsazea creammaize T.；It is 2n=76) female parent, with corn (Zea mays L.) as paternal hybrid, receives
Solid seed is obtained, F is obtained1Generation；Then to F1, into being identified, selective staining body sum is for 45~55 and rubs for the genome in generation
Wipe the F that standing grain chromosome number is 1~181For material；Again with selected F1It is female parent for material, is returned by male parent of corn, obtains
BC1Generation；To BC1In generation, carries out genome into being identified, then selective staining body sum is 21~30 and friction standing grain chromosome
Number is the BC of 1~51On behalf of female parent, continue to be returned as male parent with corn, obtain BC2Generation；Selection BC2Total chromosome number is in generation
21, the material that wherein maize chromosome is 20, friction standing grain chromosome number is 1, as corn-friction standing grain monosomic addition line.
5. method according to claim 4, it is characterised in that described corn allohexaploid refers to MTF-1
(Tripsazea creammaize T.；2n=76) or its Derivative line.
6. the method that corn-friction standing grain monosomic addition line is cultivated using corn allopolyploid, it is characterised in that its specific steps
It is as follows：
(1), since late March, point 10 batches of maize plantings (Zea mays L., 2n=20), 20 plants every batch, every batch of interval 3
My god；The first tenday period of a month in April are by corn allohexaploid (Tripsazea creammaize T.；2n=76) plant in flowerpot；
(2), by corn allohexaploid female fringe bagging before spinning, and its filigree is cut short to 2~3cm before pollination, then
Pollinated to corn allohexaploid female fringe by male parent of corn, each female fringe at least repeats pollination 3 times, every time all in set after pollination
Marked on bag, seed is harvested when ripe, obtain F1Generation；
(3), April next year, by the F obtained by step (2)1It is planted in the nutritive cube of growth cabinet for seed, in 28 DEG C, humidity
It is vernalization under conditions of 70%, plantation is transplanted into flowerpot during plant strain growth to 21 heart of leaf；During plant to be planted plant height >=30cm, fine
Its morning 10：00~12:00 and temperature be higher than 25 DEG C when, take the tip of a root, using Chromosome Pressing Technology identify somatic chromosome
Number, identifies genome into each individual plant at least counts 50 cells using hybridization in situ technique；Selective staining body number is
45~55 and friction standing grain chromosome number is that the plant of 1~18 is cooked female parent, the bagging before female fringe spinning, flower is cut short after spinning
Silk is pollinated to 1~2cm by male parent of corn, and every repetition pollination in 2 days 1 time after being pollinated at the 1st time, each female fringe is at least weighed
Pollinate 3 times again, seed is harvested when ripe, obtain BC1Generation；Wherein described corn is equally planted by stages with step (1)；
(4), according to the BC obtained by the method plantation described in step (3)1Generation, and according to the method described in step (3) using dye
Colour solid compressing tablet and hybridization in situ technique are to BC1Identified for root tip chromosomes number and composition；Selective staining body sum is 21
~30 and the BC that standing grain chromosome number is 1~5 that rubs1For plant；With corn as male parent to selected BC1For plant pollination,
Harvest to obtain BC2Generation；
(5), BC is planted according to the method described in step (3)2Generation, using chromosome compressing tablet to BC2Enter for root tip chromosomes number
Row identification, BC2There was only the two types of 20 and 21 for dyeing material body number；
(6) dyeing system, is carried out to the plant residue tip of a root that chromosome number in step (5) is 21 using flame seasoning
Piece, selective staining body is in mid-term and decentralization tip of a root combustion piece higher carries out double-colored genomic in situ hybridization, and land used is high respectively
Pungent and biotin labeling corn and friction standing grain total genomic dna, identification genome into；Selection root tip cell chromosome number be
21, and the material wherein containing 20 maize chromosomes and 1 friction standing grain chromosome, as corn-friction standing grain added monomer
System.
7. method according to claim 6, it is characterised in that the corn allohexaploid described in its step (1) or (2)
Refer to MTF-1 (Tripsazea creammaize T.；2n=76) or its Derivative line.
8. method according to claim 6, it is characterised in that described corn refers to common cultivation corn (Zea mays
L.)；Described common cultivation corn refers to cenospecies, self-mating system, local varieties or collective species.
9. method according to claim 6, it is characterised in that the total chromosome number described in its step (3) is 45~50；
Described friction standing grain chromosome number is 1~10.
10. method according to claim 6, it is characterised in that the chromosome compressing tablet described in its step (3), (4) or (5)
Method be：In the fine day morning 10：00~12:00 and temperature when being higher than 25 DEG C, take the tip of a root, and with α-bromonaphthalene saturated aqueous solution
Pretreatment 3h, uses the hypotonic 30min of distilled water, then with Fresh fixative (methyl alcohol at room temperature by the pretreated tip of a root：Ice second
Acid=3：1) fixed more than 12h.The tip of a root after fixation is placed in 70% absolute ethyl alcohol again, in being stored in 4 DEG C overnight：Use double steamings
Water cleans the fixer on the tip of a root, with 6% cellulase (Yakult) and 1% pectase (Yakult) of mixed in equal amounts, 37
DEG C enzymolysis tip of a root 2.5h, the tip of a root after enzymolysis carries out compressing tablet with the carbolfuchsin dyeing after improvement, and microphotograph simultaneously counts dyeing
Body number.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710053008.8A CN106818458B (en) | 2017-01-24 | 2017-01-24 | Corn-friction standing grain monosomic addition line method is cultivated using corn allopolyploid |
Applications Claiming Priority (1)
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107568059A (en) * | 2017-10-30 | 2018-01-12 | 四川农业大学 | The method that low temperature resistant germination corn variety is cultivated using corn allopolyploid |
| CN110972930A (en) * | 2019-11-22 | 2020-04-10 | 四川农业大学 | Method for cultivating high-fertility heterotetraploid corn |
| CN116034866A (en) * | 2023-01-10 | 2023-05-02 | 四川农业大学 | Method for cultivating dwarf corn variety by utilizing corn heterohexaploid |
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| CN103609428A (en) * | 2013-11-14 | 2014-03-05 | 四川农业大学 | Method for cultivating corn allopolyploid by using unreduced gamete characteristic of tripsacum dactyloides |
| CN104946644A (en) * | 2015-07-17 | 2015-09-30 | 四川农业大学 | Molecular marker for corn tripsacum monosome addition line nucleic male sterility genes and application thereof |
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| CN103609428A (en) * | 2013-11-14 | 2014-03-05 | 四川农业大学 | Method for cultivating corn allopolyploid by using unreduced gamete characteristic of tripsacum dactyloides |
| CN104946644A (en) * | 2015-07-17 | 2015-09-30 | 四川农业大学 | Molecular marker for corn tripsacum monosome addition line nucleic male sterility genes and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107568059A (en) * | 2017-10-30 | 2018-01-12 | 四川农业大学 | The method that low temperature resistant germination corn variety is cultivated using corn allopolyploid |
| CN110972930A (en) * | 2019-11-22 | 2020-04-10 | 四川农业大学 | Method for cultivating high-fertility heterotetraploid corn |
| CN110972930B (en) * | 2019-11-22 | 2022-04-22 | 四川农业大学 | Method for cultivating high-fertility heterotetraploid corn |
| CN116034866A (en) * | 2023-01-10 | 2023-05-02 | 四川农业大学 | Method for cultivating dwarf corn variety by utilizing corn heterohexaploid |
| CN116034866B (en) * | 2023-01-10 | 2024-04-09 | 四川农业大学 | Method for cultivating dwarf corn variety by utilizing corn heterohexaploid |
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