CN104871958A - Method for breeding cabbage type yellow-seeded rape recessive genic male sterility temporary maintainer line - Google Patents
Method for breeding cabbage type yellow-seeded rape recessive genic male sterility temporary maintainer line Download PDFInfo
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Abstract
The invention provides a method for breeding a cabbage type yellow-seeded rape recessive genic male sterility temporary maintainer line. The method comprises the steps of (a) hybridizing a black-seeded recessive complete sterility strain as a female parent and a cabbage type yellow-seeded strain as a male parent, so as to obtain an F1 generation; (b) performing induced culture on isolated microspores of F1 plants by adopting a cabbage type rape microspore culture technique, so as to obtain a DH strain; (c) test-crossing the DH strain and sterile plants in a recessive homozygous two-type line, and identifying and selecting later generations, so as to obtain the cabbage type yellow-seeded rape genic male sterility temporary maintainer line. By adopting the method provided by the invention, the yellow-seeded recessive genic male sterility temporary maintainer line with homozygous traits and stable heredity can be quickly obtained, the method is simple, and raw materials are easy to obtain.
Description
Technical field
The present invention relates to crop breeding technical field, particularly relate to a kind of method that seed selection yellow-seeded brassica napus recessive nuclear sterile temporary protects system.
Background technology
Yellow-seeded brassica napus seed coat is thinner, seed oil content is high, oil is as clear as crystal, cake protein matter content is high, cellulose and polyphenol content is low, food utilization be worth high, significantly improve the commodity value of rapeseed oil and grouts, cultivate yellow-seeded brassica napus one of important goal becoming rapeseed breeding.Sweden professor Olsson (1960) have found yellow seed resource first from the cabbage type rape of Prof. Du Yucang.After this, West Germany Robbelen (1981), Jonsson (1983), Canadian Stefansson (1986) have also discovered yellow-seeded brassica napus resource.1975, Central China agricultural university Liu Houli professor found yellow-seeded brassica napus in China first from Wild cabbage type Interspecific Hybrid.After this, other unit domestic center (1980) as land-reclaimable in Shaanxi Province, Guizhou Agriculture College (1982), academy of agricultural sciences of Jiangsu Province (1984), oil plant institute of the Chinese Academy of Agricultural Sciences (1985) etc. have all carried out a large amount of application and basic research work to yellow-seeded brassica napus.
Compare with traditional breeding method, microspore culture can shortening the breeding cycle, improves breeding efficiency.Past adopts breeding by crossing to be bred as the kind of a genetic stability, needs very years.Breeding expert by hybridizing widely, backcrossing, then must carry out selfing, to ensure the homogeneity of new varieties.Now use microspores culture method, can F be utilized
1the microspore that generation produces is cultivated, and obtains haplobiont, then makes chromosome doubling obtain DH through colchicin, and breeding time can shorten several years, is specially adapted to be difficult to isozygotying of stable character, as isozygotying of cabbage type rape yellow seed proterties.Simultaneously breeding man utilizes DH to tie up in a less colony to select, greatly can improve the efficiency of conventional breeding.Such as in the breeding of rape low erucic acid, because canola is containing double recessive gene, the frequency that it occurs in double haploid is 1/4, and at usual F
2the frequency occurred in generation is 1/16.
The recessive complete sterile line WSL of black seed
1a is the male sterile line of Southwestern University's seed selection in 2013 sizing, and the qualification in April, 2013 by the variety certification committee of Chongqing City.The Breeding Process of this strain is from introduction Anhui oil 23 hybrids in 2006 and German NPZ company MSL sterile line material, the paired brother and sister of the isolated Sterile plants and fertile plants of Anhui oil 23 offspring were handed in 2007, according to Fertility segregation and Rescued virus, Anhui oil 23 offsprings and NPZ company MSL male sterile line phase mutual cross, differentiate that recessiveness is isozygotied dual purpose lines SL in 2008
1a × SL
1b, exchange fertile plant test cross determination temporary maintainer line WSL in 2009
1b.The continuous temporary maintainer line WSL of 2010-2012
1b and recessiveness are isozygotied the sterile strain SL of dual purpose lines
1a is hybridized, and check cross offspring is complete sterile line, and then is fixed to WSL
1a.This male sterile line flower is foresythia, and petal is little, gynoecium is normal, staminody is the withered shape of brown.Brassica type restorer SWU-05R derives from the two low strain that cabbage type rape many strains multiple cross offspring obtains through test cross and directive breeding, the qualification of SWU-05R in April, 2013 by the variety certification committee of Chongqing City.Utilize recessive complete sterility and restorer to produce crossbreed, the crossbreed " Chongqing oil 27 " of this sterile systematic breeding was authorized in 2012 by Chongqing City, spread in production.
The recessive complete sterile line of seed selection yellow-seeded brassica napus is an important channel of yellow seed rape hybridization pollination system, and obtaining yellow seed temporary maintainer line is prerequisite.But due to current temporary maintainer line WSL
1b is black seed, and temporary maintainer line controls above recessive gene by 2, needs extensively a large amount of test crosses.The yellow-seeded brassica napus that conventional breeding methods selects is all WSL
1the restorer of A, by the yellow seed temporary maintainer line of conventional breeding methods transformation, segregation population is large, and greatly, it is large that difficulty is implemented in field for test cross workload and test cross kind offspring Fertility identification workload.
Summary of the invention
In view of this, a kind of seed selection yellow-seeded brassica napus recessive nuclear sterile temporary is the object of the present invention is to provide to protect the method for system, method provided by the invention can acquired character be isozygotied fast, the yellow seed recessive nuclear sterile temporary of inheritance stability protects system, and method is simple, and starting material easily obtains.
The invention provides a kind of method that seed selection yellow-seeded brassica napus recessive nuclear sterile temporary protects system, comprising:
A) be maternal with the recessive complete sterility product of black seed, Brassica type product are that male parent is hybridized, and obtain F
1generation;
B) adopt Microspore of Brassica napus culture technique to the Isolated microspore Fiber differentiation of F1 generation plant, obtain DH strain;
C) by the sterile strain test cross in DH strain and recessive homozygous two strain, yellow-seeded brassica napus Genetic Sterility temporary maintainer line is obtained.
The present invention first with the recessive complete sterility of black seed for female parent, be paternal hybrid with Brassica type product, obtain F
1generation; Then adopt Microspore of Brassica napus culture technique to F
1for the Isolated microspore Fiber differentiation of plant, colchicin doubles to obtain DH strain; Select yellow seed DH strain and strain test cross sterile with recessive homozygous two-type line, test cross kind is 100% sterile, and corresponding yellow seed DH individual plant is yellow seed recessive nuclear sterile temporary of the present invention and protects system.Inheritance stability can be obtained fast by the present invention, yellow seed temporary maintainer line that proterties is isozygotied, for yellow-seeded brassica napus crossbreeding parent initiative provides new way.
In the present invention, the recessive complete sterility product of described black seed are WSL
1a is that Southwestern University's Wang Rui assistant researcher's seed selection forms, and the qualification in April, 2013 by the kind committee of Chongqing City.This male sterile line oil content 40.09%, erucic acid are 0.02%, sulphur glycosides 29.8umol/g, and lodging resistance is strong, and economical character is comparatively excellent.
Described Brassica type product are Brassica type restorer SWU-05R, are that Southwestern University Li Jiana professor seed selection forms, the qualification in April, 2013 by the variety certification committee of Chongqing City.This yellow seed strain oil content 41.44%, erucic acid 0%, sulphur resources 26.3umol/g, economical character is excellent.
According to well known to a person skilled in the art method, at the rape florescence with WSL
1a is maternal, take SWU-05R as paternal hybrid, obtains F
1for seed.
Then, adopt Microspore of Brassica napus culture technique to the Isolated microspore Fiber differentiation of F1 generation plant, obtain DH strain; Concrete grammar is as follows:
B1) with F
1be donor for plant, the florescence gets bud from brassica napus inflorescence, grinds, filters, centrifugal rear acquisition microspore sediment in microspore isolation medium;
B2) by step b1) the microspore sediment that obtains cultivates in containing the embryoid induction culture fluid of colchicin; Then by centrifugal for the culture obtained, the centrifugal microspore sediment obtained is cultured to visible embryoid in not containing the embryoid culture fluid of colchicin, forwards on shaking table and continues to be cultured to cotyledon period;
B3) by step b2) the cotyledon period embryoid that obtains proceeds in embryoid seedling medium and continues to be cultured to be a seedling;
B4) step b3) the seedling continued growth that obtains to the florescence, obtain DH strain.
By F
1f is obtained for seed plantation
1after plant, from the plant F of robust growth during bud stage
1on, win the bud of main inflorescence or primary branch inflorescence tip portion, its length is preferably 3.5 ~ 4.0mm.
In microspore isolation medium before grinding, first carry out disinfection and sterile water wash to bud, concrete operations are as follows:
By bud with 75% alcohol-pickled 0.5-lmin, aseptic water washing 3 times; Use the HgCl of 0.1% again
2soak 12min, aseptic water washing 3 times, each approximately 5min.
Ground in microspore isolation medium by bud, concrete operations are:
Bud after sterilization is proceeded in aseptic small beaker, adds little full sub-isolation medium, with glass syringe inner core extruding bud.
By the bud after grinding through filtering, filtrate being carried out centrifugal, obtaining microspore sediment.In the present invention, preferably through 300 object strainer filterings.Describedly centrifugally to be specially: filtrate being proceeded to volume with a scale is in the centrifuge tube of l0ml, and the centrifugal 3min of 1000rpm, outwells supernatant after centrifugal, rejoins microspore isolation medium, centrifugal 3 times, obtains microspore sediment.
In the present invention, described microspore isolation medium comprises: B5 macroelement, B5 trace element, B5 organic additive, FeNaEDTA 13.2mg.L
-1with sucrose 130g.L
-1(pH=6.0).Specifically, it comprises:
The KNO of 2500mg/L
3, 250mg/L MgSO
47H
2the CaCl of O, 150mg/L
22H
2(the NH of O, 134mg/L
4)
2sO
4, 150mg/L NaH
2pO
4h
2the H of KI, 3.0mg/L of O, 0.83mg/L
3bO
3, 10mg/L MnSO
44H
2the ZnSO of O, 2.0mg/L
47H
2the Na of O, 0.25mg/L
2moO
42H
2the CoCl of O, 0.025mg/L
26H
2the CuSO of O, 0.025mg/L
45H
2the sucrose of the puridoxine hydrochloride of the inositol of O, 100mg/L, the nicotinic acid of 1.0mg/L, 1.0mg/L, the Tyiamine Hd of 10mg/L, FeNaEDTA and 130g/L of 13.2mg/L; The pH value of described microspore isolation medium is 6.0.
After obtaining microspore sediment, added in the embryoid induction culture fluid containing colchicin and cultivate, the described embryoid induction liquid containing colchicin comprises: the KNO of 125mg/L
3, 125mg/L MgSO
4.7H
2the Ca of O, 500mg/L
4nO
3.4H
2the KH of O, 125mg/L
2pO
4, 26.4mg/L the MnSO of FeNaEDTA, 22.3mg/L
4.4H
2the H of O, 6.2mg/L
3bO
3, 8.6mg/L ZnSO
4.7H
2the CuSO of O, 0.025mg/L
4.5H
2the Na of O, 0.25mg/L
2moO
4.2H
2the CoCI of O, 0.025mg/L
2.6H
2the colchicin of the glutathione of the glycine of the puridoxine hydrochloride of the inositol of O, 100mg/L, the nicotinic acid of 5mg/L, 0.5mg/L, the thiamine hydrochloride of 0.5mg/L, 2mg/L, the folic acid of 0.5mg/L, the vitamin h 30mg/L of 0.05mg/L, the L-glutamine of 800mg/L, the Serine of 100mg/L, the sucrose of 130000mg/L and 10 ~ 50mg/L.In the present invention, microspore is being about 50,000/mL containing the density in the embryoid induction culture fluid of colchicin.
The present invention preferably under air-proof condition, in the insulating box of 32 DEG C light culture 1 ~ 2d; Then by centrifugal for the culture obtained, joined by the microspore sediment obtained and do not cultivate containing continuing in the embryoid culture fluid of colchicin, the described embryoid induction liquid not containing colchicin comprises: the KNO of 125mg/L
3, 125mg/L MgSO
4.7H
2the Ca of O, 500mg/L
4nO
3.4H
2the KH of O, 125mg/L
2pO
4, 26.4mg/L the MnSO of FeNaEDTA, 22.3mg/L
4.4H
2the H of O, 6.2mg/L
3bO
3, 8.6mg/L ZnSO
4.7H
2the CuSO of O, 0.025mg/L
4.5H
2the Na of O, 0.25mg/L
2moO
4.2H
2the CoCI of O, 0.025mg/L
2.6H
2the glutathione of the glycine of the puridoxine hydrochloride of the inositol of O, 100mg/L, the nicotinic acid of 5mg/L, 0.5mg/L, the thiamine hydrochloride of 0.5mg/L, 2mg/L, the folic acid of 0.5mg/L, the vitamin h 30mg/L of 0.05mg/L, the L-glutamine of 800mg/L, the Serine of 100mg/L and the sucrose of 130000mg/L.
The present invention preferably continues to cultivate under air-proof condition, under 25 DEG C of constant temperature in not containing the embryoid induction medium of colchicin, after being cultured to visible embryoid, forwarding on shaking table and continues to be cultured to cotyledon period.In the present invention, the temperature that shaking table is cultivated is 25 DEG C, and rotating speed is 60rpm.
After being cultured to cotyledon period, being proceeded in embryoid seedling medium and continue to be cultured to be a seedling.Described embryoid seedling medium comprises: B5, GA
30.1mg.L
-1, 6-BA 0.1mg.L
-1, sucrose 2% and agar 8g.L
-1(pH=5.8).Specifically, it comprises: the KNO of 2500mg/L
3, 250mg/L MgSO
47H
2the CaCl of O, 150mg/L
22H
2(the NH of O, 134mg/L
4)
2sO
4, 150mg/L NaH
2pO
4h
2the H of KI, 3.0mg/L of O, 0.75mg/L
3bO
3, 10mg/L MnSO
44H
2the ZnSO of O, 2.0mg/L
47H
2the Na of O, 0.25mg/L
2moO
42H
2the CoCl of O, 0.025mg/L
26H
2the CuSO of O, 0.025mg/L
45H
2the FeSO of Na2-EDTA, 27.8mg/L of O, 37.3mg/
47H
2the gibberellin of the puridoxine hydrochloride of the inositol of O, 100mg/L, the nicotinic acid of 1.0mg/L, 1.0mg/L, the Tyiamine Hd of 10mg/L, 0.1mg/L, the 6-benzyl aminoadenine of 0.1mg/L, the sucrose of 2% and the agar of 8g/L; The pH value of described embryoid seedling medium is 5.8.
The present invention is preferably cultured to be a seedling at 25 DEG C in embryoid seedling medium, and the plantlet of formation can be transplanted in soil, also can forward squamous subculture in fresh same medium to.The embryoid of seedling is not had to forward B to
5medium continues to cultivate, until develop into complete plantlet.After the Beginning of Winter, seedling is transplanted to field.
Be cultured to be a seedling in process, can according to circumstances selecting to cultivate in fast breeding culture medium and/or root media, to obtain seedling.In the present invention, described fast breeding culture medium comprises: MS, 6-BA 1.5mg.L
-1, NAA 0.1mg.L
-1, sucrose 30g.L
-1with agar 7g.L
-1(pH=5.8).Described root media comprises: 1/2MS, IBA0.5mg.L
-1, sucrose 30g.L
-1with agar 7g.L
-1(pH=5.8).
When seedling grows to the florescence, can obtain and double successfully to educate dliploid DH plant.This can be educated the sterile strain test cross in dliploid and recessive homozygous two strain, the test cross kind of acquisition is carried out Fertility identification when the florescence, the 100% sterile yellow seed recessive nuclear sterile temporary that is protects system.
When considering fertility, preferably consider seed color, the yellow seed recessive nuclear sterile temporary of seed selection protects system, and concrete grammar is as follows: this can be educated the selfing of dliploid strain bagging simultaneously, and with the sterile strain test cross in recessive homozygous two strain.The test cross kind of acquisition is carried out Fertility identification when the florescence, the fertility performance of record test cross kind.During seed maturity, investigate the seed color and luster of dliploid DH strain.If test cross kind is 100% sterile, corresponding male parent DH strain is yellow seed, then this yellow seed DH strain is yellow seed recessive nuclear sterile temporary of the present invention and protects system, and it is YSLB-1 that the yellow seed recessive nuclear sterile temporary of called after is protected.
In the present invention, described recessive homozygous two product are SL
1aB was Southwestern University Wang Rui assistant researcher from Anhui oil 23 hybrid generations and the mutual selection cross of German NPZ company MSL sterile material, and continuous multi-generation brother and sister friendship, was fixed to the recessive homozygous two-type line of genetic stability in 2009.SL
1aB AB line content of erucic acid 0.1%, sulphur resources 30.21umol/g.The present invention utilizes above-mentioned recessive homozygous two-type line SL
1sterile strain in AB carries out genotype identification to DH strain.
See Fig. 1, the schematic flow sheet of the selection that Fig. 1 provides for the embodiment of the present invention, with black seed recessiveness sterile (genotype is for ms3ms3Rfrf) for maternal, yellow seed strain (genotype is Ms3Ms3rfrf) is hybridized for male parent, and the F1 generation genotype of acquisition is Ms3ms3Rfrf and Ms3ms3rfrf.Get the bud of F1 generation plant, it doubles to obtain embryoid to cultivate induction in containing the medium of colchicin after separated free microspore, then cultivates in not containing the medium of colchicin and expands its quantity, finally obtain the dliploid doubled and can educate DH plant.Last with the sterile strain in recessive homozygous two strain and DH strain test cross, according to its fertility in conjunction with seed color and luster, obtain yellow-seeded brassica napus Genetic Sterility temporary maintainer line, its genotype is ms3ms3rfrf.
Tool of the present invention has the following advantages:
(1) because the recessive complete sterility of black seed is easy to obtain and collect with the crossbreed of yellow seed strain, not maintain secrecy restriction by the business of recessive homozygous two-type line, temporary maintainer line and yellow seed pure lines material, only need to the hybridization F of black seed recessiveness complete sterility and yellow seed strain
1the DH strain of cultivating for little full son carries out selecting and genotype identification, can the isozygoty yellow seed recessive nuclear sterile temporary guarantor of inheritance stability of acquired character be fast just.
(2) gene pool that Brassica type recessive nuclear sterile temporary protects system has been widened, for yellow-seeded brassica napus crossbreeding parent initiative provides a kind of new way.In addition, this method is applicable equally to the temporary maintainer line obtaining other objective trait fast, the temporary maintainer line of the proterties such as such as floorboard with high oil content, siliqua, applicable machine receipts.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below, apparently, accompanying drawing in the following describes is only embodiments of the invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to the accompanying drawing provided.
The schematic flow sheet of the selection that Fig. 1 provides for the embodiment of the present invention.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment 1
WAL
1a is the recessive complete sterility of the black seed of Wild cabbage type, and its genotype is ms3ms3Rfrf, is that Southwestern University's Wang Rui assistant researcher's seed selection forms, and the qualification in April, 2013 by the kind committee of Chongqing City.SWU-05R is Brassica type pure lines, and its genotype is Ms
3ms
3rfrf is that Southwestern University Li Jiana professor seed selection forms, the qualification in April, 2013 by the variety certification committee of Chongqing City.
The rape florescence in March, 2012 is with WAL
1a is maternal and male parent SWU-05R is hybridized, and obtains F
1for seed.
At the rape florescence in March, 2013, to F
1carry out microspores culture, obtain 150 embryoids altogether, obtain 125 individual plants through Fiber differentiation, in October, 2013 plants into land for growing field crops, identifies that doubling plant is 68 in March, 2014.Detailed process is as follows:
1, from the plant F of robust growth
1on, win the bud of main inflorescence or primary branch inflorescence tip portion 3.5 ~ 4mm, with the alcohol-pickled 0.5-lmin of 75%, aseptic water washing 3 times, then use the HgCl of 0.1%
2soak 12min, aseptic water washing 3 times, each approximately 5min;
2, the bud after sterilization is proceeded in aseptic small beaker, add microspore isolation medium, with glass syringe inner core extruding bud, again through 300 object strainer filterings, filtrate being proceeded to volume with a scale is in the centrifuge tube of l0ml, the centrifugal 3min of 1000rpm, supernatant is outwelled after centrifugal, rejoin microspore isolation medium, centrifugal 3 times, finally obtain microspore sediment.Embryoid induction liquid containing colchicin is added in microspore sediment, makes microspore density about 50,000/ml.Then the medium containing microspore is dispensed in culture dish and seals, and to be placed in the incubator of 32 DEG C after light culture 1-2d, carry out the centrifugal embryoid induction liquid culture fluid removed containing colchicin, and in culture dish, add the embryoid induction liquid not having colchicin, subsequently culture dish sealing is put into the incubator light culture of 25 DEG C.When there is macroscopic globular embryo in culture dish, then culture dish is put into shaking table concussion cultivation (60rpm, 25 DEG C).
3, when shaking the embryoid cultivated and growing cotyledon period, proceeded in embryoid seedling medium, and be placed in 25 DEG C and cultivate, the plantlet of formation can be transplanted in soil, also can forward squamous subculture in fresh same medium to.Do not have the embryoid of seedling can forward B5 medium to continue to cultivate, until develop into complete plantlet.After the Beginning of Winter, seedling is transplanted to field.After seedling, subculture, preserve with 1/2MS medium (i.e. fast breeding culture medium), root media when taking root, taking root is beginning in 20 ~ 30 days before transplanting, and therefore seedling before this needs succeeding preservation.
The medium that said process is used is as follows:
(1) microspore isolation medium (B5-13)
B5 macroelement+B5 trace element+B5 organic additive+FeNaEDTA 13.2mg.L
-1+ sucrose 130g.L
-1(pH=6.0)
(2) embryoid induction medium
NLN-13
Wherein, the composition of NLN-13 medium is as follows:
Macroelement: KNO
3125mg.L
-1, MgSO
4.7H
2o 125mg.L
-1, Ca
4nO
3.4H
2o500mg.L
-1, KH
2pO
4125mg.L
-1
Molysite: FeNaEDTA 26.4mg.L
-1
Trace element: MnSO
4.4H
2o 22.3mg.L
-1, H
3bO
36.2mg.L
-1, ZnSO
4.7H
2o8.6mg.L
-1, CuSO
4.5H
2o 0.025mg.L
-1, Na
2moO
4.2H
2o 0.25mg.L
-1, CoCI
2.6H
2o0.025mg.L
-1
Organic additive: inositol 100mg.L
-1, nicotinic acid 5mg.L
-1, puridoxine hydrochloride 0.5mg.L
-1, thiamine hydrochloride 0.5mg.L
-1, glycine 2mg.L
-1, folic acid 0.5mg.L
-1, vitamin h 0.05mg.L
-1, glutathione 30mg.L
-1, L-glutamine 800mg.L
-1, Serine 100mg.L
-1, sucrose 130000mg.L
-1
(3) embryoid seedling medium
B5+GA
30.1mg.L
-1+ 6-BA 0.1mg.L
-1+ sucrose 2%+ agar 8g.L
-1(pH=5.8)
(4) fast breeding culture medium
MS+6-BA 1.5mg.L
-1+ NAA 0.1mg.L
-1+ sucrose 30g.L
-1+ agar 7g.L
-1(pH=5.8)
(5) root media
1/2MS+IBA0.5mg.L
-1+ sucrose 30g.L
-1+ agar 7g.L
-1(pH=5.8)
In March, 2014 doubles the selfing of plant bagging simultaneously and homozygous two-type line SL to these 68
1sterile strain test cross in AB.Investigate the seed color and luster of 68 DH strain selfed seeds in May, 2014.In March, 2015 investigates the fertility performance of test cross kind, in conjunction with DH strain selfed seed color and luster, finds that there is two yellow seed DH strains and SL
1sterile strain test cross kind in AB shows as recessive complete sterility, and its genotype is ms
3ms
3rfrf.One of them yellow seed DH strain seed color and luster true yellow, growing way is normal, called after YSLB-1, is yellow-seeded brassica napus recessive nuclear sterile temporary described in the invention and protects system.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. seed selection yellow-seeded brassica napus recessive nuclear sterile temporary protects a method for system, it is characterized in that, comprising:
A) be maternal with the recessive complete sterility product of black seed, Brassica type product are that male parent is hybridized, and obtain F
1generation;
B) adopt Microspore of Brassica napus culture technique to the Isolated microspore Fiber differentiation of F1 generation plant, obtain DH strain;
C) by the sterile strain test cross in DH strain and recessive homozygous two strain, yellow-seeded brassica napus Genetic Sterility temporary maintainer line is obtained.
2. method according to claim 1, is characterized in that, described step a) in, the recessive complete sterility product of black seed are WSL
1a.
3. method according to claim 1, is characterized in that, after described step a) in, Brassica type product are SWU-05R.
4. method according to claim 1, is characterized in that, described step b) be specially:
B1) with F
1be donor for plant, the florescence gets bud from brassica napus inflorescence, grinds, filters, centrifugal rear acquisition microspore sediment in microspore isolation medium;
B2) by step b1) the microspore sediment that obtains cultivates in containing the embryoid induction culture fluid of colchicin; Then by centrifugal for the culture obtained, the centrifugal microspore sediment obtained is cultured to visible embryoid in not containing the embryoid culture fluid of colchicin, forwards on shaking table and continues to be cultured to cotyledon period;
B3) by step b2) the cotyledon period embryoid that obtains proceeds in embryoid seedling medium and continues to be cultured to be a seedling;
B4) step b3) the seedling continued growth that obtains to the florescence, obtain DH plant.
5. method according to claim 4, is characterized in that, described step b1) in, the length of described bud is 3.5 ~ 4.0mm;
Described filtration employing 300 mesh filter screen;
Described centrifugal rotating speed is 1000rpm, and the time is 3min;
Described microspore isolation medium comprises: the KNO of 2500mg/L
3, 250mg/L MgSO
47H
2the CaCl of O, 150mg/L
22H
2(the NH of O, 134mg/L
4)
2sO
4, 150mg/L NaH
2pO
4h
2the H of KI, 3.0mg/L of O, 0.83mg/L
3bO
3, 10mg/L MnSO
44H
2the ZnSO of O, 2.0mg/L
47H
2the Na of O, 0.25mg/L
2moO
42H
2the CoCl of O, 0.025mg/L
26H
2the CuSO of O, 0.025mg/L
45H
2the sucrose of the puridoxine hydrochloride of the inositol of O, 100mg/L, the nicotinic acid of 1.0mg/L, 1.0mg/L, the Tyiamine Hd of 10mg/L, FeNaEDTA and 130g/L of 13.2mg/L; The pH value of described microspore isolation medium is 6.0.
6. method according to claim 4, is characterized in that, described step b2) in, embryoid induction liquid comprises: the KNO of 125mg/L
3, 125mg/L MgSO
4.7H
2the Ca of O, 500mg/L
4nO
3.4H
2the KH of O, 125mg/L
2pO
4, 26.4mg/L the MnSO of FeNaEDTA, 22.3mg/L
4.4H
2the H of O, 6.2mg/L
3bO
3, 8.6mg/L ZnSO
4.7H
2the CuSO of O, 0.025mg/L
4.5H
2the Na of O, 0.25mg/L
2moO
4.2H
2the CoCI of O, 0.025mg/L
2.6H
2the sucrose of the vitamin h of the glycine of the puridoxine hydrochloride of the inositol of O, 100mg/L, the nicotinic acid of 5mg/L, 0.5mg/L, the thiamine hydrochloride of 0.5mg/L, 2mg/L, the folic acid of 0.5mg/L, 0.05mg/L, the glutathione of 30mg/L, the Glu of 800mg/L, the Serine of 100mg/L and 130000mg/L;
Described is 10 ~ 50mg/L containing the concentration of colchicin in the embryoid induction liquid of colchicin.
7. method according to claim 6, is characterized in that, by step b1) the microspore sediment that obtains containing in the embryoid induction culture fluid of colchicin, sealing, 32 DEG C of light culture 1 to 2 day, microspore density is 50,000/mL;
The centrifugal microspore sediment obtained not containing in the embryoid culture fluid of colchicin, sealing, 25 DEG C of light culture are to visible embryoid;
The temperature that described shaking table is cultivated is 25 DEG C, and rotating speed is 60rpm.
8. method according to claim 4, is characterized in that, described step b3) in, described embryoid seedling medium comprises: the KNO of 2500mg/L
3, 250mg/L MgSO
47H
2the CaCl of O, 150mg/L
22H
2(the NH of O, 134mg/L
4)
2sO
4, 150mg/L NaH
2pO
4h
2the H of KI, 3.0mg/L of O, 0.75mg/L
3bO
3, 10mg/L MnSO
44H
2the ZnSO of O, 2.0mg/L
47H
2the Na of O, 0.25mg/L
2moO
42H
2the CoCl of O, 0.025mg/L
26H
2the CuSO of O, 0.025mg/L
45H
2the Na of O, 37.3mg/
2the FeSO of-EDTA, 27.8mg/L
47H
2the gibberellin of the puridoxine hydrochloride of the inositol of O, 100mg/L, the nicotinic acid of 1.0mg/L, 1.0mg/L, the Tyiamine Hd of 10mg/L, 0.1mg/L, the 6-benzyl aminoadenine of 0.1mg/L, the sucrose of 2% and the agar of 8g/L; The pH value of described embryoid seedling medium is 5.8;
The temperature of described cultivation is 25 DEG C.
9. method according to claim 1, is characterized in that, described recessive homozygous two product are SL
1aB.
10. method according to claim 1, is characterized in that, described step c) be specially:
By the selfing of DH plant, and with the sterile strain test cross in recessive homozygous two strain, obtain yellow-seeded brassica napus Genetic Sterility temporary maintainer line.
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