CN104839015B - Breeding method of transgenic receptor of nucleoplasmic-interactive male-sterile line of corns and application of receptor in genetic transformation and descendant propagation - Google Patents
Breeding method of transgenic receptor of nucleoplasmic-interactive male-sterile line of corns and application of receptor in genetic transformation and descendant propagation Download PDFInfo
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Abstract
The invention relates to the technical fields of breeding of crops and particularly relates to a breeding method of a transgenic receptor of a nucleoplasmic-interactive male-sterile line of corns and application of the receptor in genetic transformation and descendant propagation. According to the breeding method, a transgenic receptor material with the male-sterile line is obtained, the labor cost in a propagation process of a transformation event is saved, and the pollution caused by different transformation events and the pollution of transgenic corns to a non-transgenic corn cultivated variety are avoided. The transgenic receptor material (being tested as a maintenance line of the corresponding male-sterile line) with high transformation efficiency is taken as a transformation object based on an existing nucleoplasmic-interactive male-sterile line of the corns and is transformed into the male-sterile line by virtue of multi-generation backcross and marker-assisted selection. The transgenic receptor material with the high transformation efficiency can be applied to genetic transformation, is pollinated and propagated by virtue of pollen of the maintenance line under natural conditions after the transformation events are obtained and then is subjected to character screening and genetic stability analysis.
Description
Technical field
The present invention relates to crop breeding technical field, more particularly to Semen Maydiss Cytoplasm male sterility transgene receptor
The application in genetic transformation and offspring's expanding propagation of breeding method and this receptor.
Background technology
Transgenic corns are exogenous gene to be imported in Maize genome by transgenic technology so as to possess mankind institute
Desired corn strain.Transformation event refer to exogenous gene import Maize genome during, specific exogenous gene
The event combined in specific chromosome, specific site with maize dna, the difference of the gene difference or binding site of importing are represented
Different transformation events, even identical gene, imports to the different loci of different chromosomes or same chromosome, its knot
Fruit also differs widely.In order to obtain, exogenous gene expression amount is high, purpose character is projected, the transformation event of inheritance stability, it is necessary to
Screen substantial amounts of transformation event.
At present, corn gene receptor is several normal selfing linies with high conversion efficiency or cenospecies, such as comprehensive 3 He
Comprehensive 31 (poplar meeting, kingdom's English, Dai Jingrui. maize elite inbred line is comprehensive 3, comprehensive 31 Study on Transformation. Journal of Agricultural Biotechnology,
2001,9(4):334～337), HiIIAB (Songstad D D, Armstrong C L, Peterson W L, et
al.Production of transgenic maize plant s and progeny by bombardment of HiⅡ
immature embryos.In Vitro Cellular&Developmental Biology,1996,32:179～183),
A188 and B73 (Zhang Rong, kingdom's English, Zhang Xiaohong, Zhao Huji. the foundation of Agrobacterium tumefaciens mediated maize genetic transformation system. agriculture
Industry biotechnology journal, 2001,99 (1):45～48), it is neat 319 (horse is beautiful. High Efficiency Regeneration System of Maize Inbred Immature Embryo is built
It is vertical. northwest agricultural journal, 2007,16 (6):85～89), P9-10 (Zhou Fengyong, Dai Jingrui, kingdom's English, Xie Youju, Cui Hongzhi,
Three heap of Guo. the foundation of corn inbred line P9-10 genetic conversion systems. Science Bulletin, 1998,43 (23):2517～2520) etc.,
The expanding propagation of the transformation event of these receptors take selfing or with normal selfing line be returned method, female tassel be required to bagging every
From, i.e., female fringe spinning before entangled with paper bag, prevent the pollen of other plant to be scattering in female fringe, before tassel loose powder, also use paper bag
Entangle, pollen dispersal is prevented to other plant.Treat that female fringe is spun, after tassel loose powder, take tassel pollen or normal selfing line flower again
Powder is pollinated to female fringe, completes pollinating process, and operating procedure is more and complicated.Due to the transformation event being related to it is more, one side work
Work amount is big, needs to expend substantial amounts of labour cost, and difficult quality guarantee；On the other hand due to the pollen containing transgene component
Inevitably be scattering in environment, the pollen dispersal of a transformation event is there is in the female fringe of other transformation events, made
Into mixing between different transformation events, huge trouble is brought to subsequent evaluation analysis.Meanwhile, in order to prevent transgenic corns
Pollen dispersal causes the pollution to non-transgenic corn cultivar in non-transgenic corn female fringe, brings huge in production
Big to lose, country is required extremely strictly, except requiring apart from upper isolation to the quarantine measures of transgenic corns growing area, also
There is isolation physically, this not only adds the construction cost of transgenic planting base, increased company's burden, simultaneously because
The base for meeting isolation condition is less, significantly limit the research and development process of China's transgenic corns.
Therefore, a kind of new transgenic acceptor is cultivated, had both can guarantee that transformation efficiency, and heavy pollination can be exempted and appointed
Business, saves labour cost, economizes on resources, moreover it is possible to prevent pollution between different transformation events and transgenic corns to non-transgenic
The pollution of maize culture kind, by with important economic implications and practical value.
The content of the invention
In view of this, the present invention provides a kind of breeding method of Semen Maydiss Cytoplasm male sterility transgene receptor and is somebody's turn to do
Application of the receptor in genetic transformation and offspring's expanding propagation.The method based on existing Semen Maydiss Cytoplasm male sterility system,
It is transformation object, Jing Guoduo with the transgenic acceptor (maintainer of the Jing tests for corresponding male sterility line) of high transformation efficiency
Generation backcrossing binding molecule marker assisted selection, quickly the transgene receptor material of high transformation efficiency within the relatively short time
Material transformation is into male sterile transgenic acceptor.
In order to realize foregoing invention purpose, the present invention provides technical scheme below：
The invention provides a kind of breeding method of Semen Maydiss Cytoplasm male sterility transgene receptor, including following step
Suddenly：
1) with Cytoplasm male sterility system as female parent, to be adapted to genetic transformation and the material as male sterility line maintainer
Expect for male parent, hybridization acquisition F1 generation fruit ear；
2) by step 1) the seed monoseeding of the F1 generation fruit ear that obtains, sterile plant is selected for female parent, with step 1 institute
Male parent backcrossing is stated, and BC1F1 is obtained for fruit ear；
3) from the beginning of BC1F1 generations, by molecular marker assisted selection, select acquisition background and be close to male parent described in step 1
Individual plant is returned, and obtains fruit ear of future generation, and the backcrossing through 4～5 generations is bred as male sterile transgenic acceptor.
Male sterility line, in referring to monoecious plant, stamen development is abnormal, it is impossible to produce functional pollen, but it
Stamen development it is normal, normal pollen can be received and fertilization, and male sterility can be entailed the plant lines of offspring,
It can be avoided well due to the pollution problem caused by pollen dispersal between different materials.Cytoplasm male sterility is by thin
Cytogene and nucleus gene interactive effect and the male sterility line that produces, Chinese patent CN201410190251,
Respectively describe in CN200510086942, CN200510011406, CN201210468807, CN201310751112 Oryza sativa L.,
The selection of Brassica campestris L, Caulis et Folium Brassicae capitatae, Cotton Gossypii and Semen Maydiss Cytoplasm male sterility system, is that the utilization of male sterility line has been established very
Good basis.The Cytoplasm male sterility kytoplasm of Semen Maydiss can be divided into S, T and C three types, T-shaped cytoplasmic male sterility category
In sporinite male sterility, can be infected by corn southern leaf blight T microspecies specialization, at present using fewer and feweri.S types cytoplasmatic male is not
Educate and belong to gametocyte male sterility, Sterile stability is poor.C-type cytoplasmic male sterility falls within sporinite male sterility, can
It is divided into tri- subgroups of CI, CII, CIII, this cytoplasmic sterility type pollen abortion morning in period, abortion are thorough, and sterility is steady
It is fixed, application at present more and more extensively, the C836G that such as has been reported that (Luo Hongbing, Huang Huang, Long Muhua, Liu Tangxing, Yang Youcai. inhomogeneity
Type mitochondrial DNA system Breeding for restoration lines. Agricultural University Of Hunan's journal, 2002,28 (3):202-205)、C48-2
(Xu Ke, Cao Moju, Zhu Yingguo, Pan Guangtang, Rong Tingzhao. corn C type cytoplasmic male sterile line C48-2 and its holding anchor line (string) grain
Body differential protein analysis. Acta Agronomica Sinica, 2008,34 (2):232-237), Cb37 (Shao Siquan, Li Yancong. corn C type male not
Educate the applied research for being. agricultural science and technology communicate .2014,74-75) etc. had more research and application.
The present invention is by the plasmagene of Cytoplasm male sterility system is by backcross transformation and binding molecule labelling is aided in
Selection is quickly introduced in the selfing line with high conversion efficiency, obtains the male sterility line with high conversion efficiency, should
Male sterility line can be used for genetic transformation, and the transgenic progeny for obtaining can be in isolation area with after maintainer spontaneous pollination breeding
In generation, reduce the labour cost of hard work task and great number during transgenic event expanding propagation, it is to avoid transgenic event
Between the pollution problem mixed with transgenic corns to non-transgenic corn cultivar.
It is an object of the invention to provide the method for cultivating Semen Maydiss Cytoplasm male sterility transgene receptor, using the party
The transgenic acceptor that method is cultivated is male sterile, save the work of artificial pollination during transgenic progeny expanding propagation into
This, it is often more important that pollution between different transgenic events and transgenic corns are avoided to non-transgenic corn cultivar
Pollution problem.
It is yet another object of the invention to provide Cytoplasm male sterility transgene receptor propagation method, with a small amount of labor
Power and cost provide sufficient transgenic acceptor.
It is yet another object of the invention to provide the expanding propagation method of transformation event, turns base with a small amount of labour force and cost expanding propagation
Because offspring is used for character analysis and safety evaluation test.
It is in some specific embodiments of the present invention, maternal described in breeding method to be selected from Maize Genetics
In Cooperation Stock Center preserving number be C437B, C437E, C437H, C537B, C537E, C537H, C657A,
The male sterility line of C657D, C657F, C836G or the Cytoplasm male sterility obtained through artificially breeding or natural mutation
System.
In some specific embodiments of the present invention, male parent described in breeding method is the selfing line of high transformation efficiency,
The male parent is selected from preserving number in National Plant Germplasm System is for the B73 of PI550473 or is adapted to conversion
Material.
In some specific embodiments of the present invention, the material for being adapted to conversion described in breeding method is comprehensive 31.
Present invention also offers the Semen Maydiss Cytoplasm male sterility transgene receptor that described breeding method is obtained.
Present invention also offers the Semen Maydiss Cytoplasm male sterility transgene receptor is converted and offspring in maize genetic
Application in expanding propagation.
In some specific embodiments of the present invention, the Semen Maydiss Cytoplasm male sterility transgene receptor is in jade
Maize genetic described in application in rice genetic transformation and offspring's expanding propagation is converted into and turns the Semen Maydiss Cytoplasm male sterility
Exogenous gene is imported the rataria and obtains male sterile transgenic progeny as acceptor material by the rataria of genetic recipient.
Present invention also offers the propagation method of described Semen Maydiss Cytoplasm male sterility transgene receptor, will be described
The same period sowing in the isolation area of Semen Maydiss Cytoplasm male sterility transgene receptor and maintainer, allows the pollen of the maintainer
Pollinate to raise up seed to the Semen Maydiss Cytoplasm male sterility transgene receptor under field conditions (factors).
In some specific embodiments of the present invention, the propagation method of transgenic progeny is by the transgenic progeny and guarantor
Hold and tie up to same period sowing in isolation area, allow the pollen of the maintainer to pollinate to the transgenic progeny under field conditions (factors).
The present invention is with 836 (Maize Genetics Cooperation Stock of c-type Cytoplasm male sterility system
Center preserving number C836G) and transgenic acceptor B73 (National Plant GermplasmSystem preserving numbers
PI550473 as a example by), male sterility line is not limited only to 836, other Cytoplasm male sterility systems, such as Maize in Semen Maydiss
In Genetics Cooperation Stock Center preserving number be C437B, C437E, C437H, C537B, C537E,
The male sterility line of C537H, C657A, C657D, C657F or other are mutual through matter-core that artificially breeding or natural mutation are obtained
Make male sterility line and can also reach the purpose of the present invention, thus it is also within the scope of the present invention.Transgenic acceptor
B73 is also not limited to, other transgenic acceptors for example comprehensive 31 etc. can also reach the purpose of the present invention in Semen Maydiss, therefore also exist
In protection scope of the present invention.
With c-type Cytoplasm male sterility system 836 as maternal, genotype is S (rfrf) to the present invention, with higher turn
The selfing line B73 for changing efficiency is male parent, and genotype is N (rfrf), is first hybridized, and is then returned as recurrent parent with B73
And binding molecule marker assisted selection, quickly by 836 transformation of c-type Cytoplasm male sterility system into high conversion efficiency
B73 backgrounds male sterility line and be used for the expanding propagation of genetic transformation and transformation generation, save labour cost, solve and turn base
Because of the pollution problem of mixing between event and transgenic corns to non-transgenic corn cultivar.
Description of the drawings
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
Accompanying drawing to be used needed for having technology description is briefly described.
Fig. 1 shows the tassel photo of normal Semen Maydiss and c-type Cytoplasm male sterility system 836；
Fig. 2 shows backcross progeny part seed Markers for Detection electrophoretogram；
Fig. 3 shows that cultivating corn C type Cytoplasm male sterility using backcross transformation and Molecular Marker Assisted Selection Technology turns
The Technology Roadmap of genetic recipient；
Fig. 4 shows partial transgenic offspring's Molecular Detection electrophoretogram.
Specific embodiment
The invention discloses the breeding method and this receptor of a kind of Semen Maydiss Cytoplasm male sterility transgene receptor are being lost
Conversion and the application in offspring's expanding propagation are passed, those skilled in the art can use for reference present disclosure, be suitably modified technological parameter realization.
Specifically, all similar replacements and change are apparent to those skilled in the art, and they are all
It is deemed to be included in the present invention.The method of the present invention and application are described by preferred embodiment, and related personnel is bright
Show off one's talent or competence in without departing from present invention, spirit and scope to method described herein and application be modified or suitably change with
Combination, realizes and applies the technology of the present invention.
Cytoplasm male sterility, not only needs Cytoplasm to have sterile gene S, and needs have homozygosis in nucleus
Sterile gene (rfrf), the two is present simultaneously, and plant can be made to show as male sterility.If cytogene is to educate N, no matter then
Karyogene is to educate (RfRf) or sterile (rfrf), all shows as male-fertile.Equally, gene can be educated as having in core
(RfRf) or (Rfrf), no matter then cytogene is to educate N or infertility S, also all show as male-fertile.It is this by core-matter
The male sterility line that interaction is formed, its genetic constitution are S (rfrf), it is impossible to produce normal pollen, but can be maternal as hybridization.
Maintainer N (rfrf) is hybridized with sterile line, and produced F1 remains to keep male sterility.
Corn C type Cytoplasm male sterility system 836 [genotype is S (rfrf)] comes from U.S. Maize
Genetics Cooperation Stock Center, preserving number is C836G, and corn inbred line B73 comes from the U.S.
National Plant Germplasm System, preserving number is PI550473, Jing tests, and B73 is 836 maintainer.
1 labelling title of table, position and sequence
The breeding method and this receptor of the Semen Maydiss Cytoplasm male sterility transgene receptor that the present invention is provided turns in heredity
Change and can be buied by market with raw materials used and reagent in the application in offspring's expanding propagation.
With reference to embodiment, the present invention is expanded on further：
The cultivation of 1 corn C type Cytoplasm male sterility transgene receptor of embodiment
The present invention is specifically described embodiment by taking the cultivation of B73S as an example.836 [S (rfrf)] belong to c-type matter-core interaction
Male sterility, is sporinite male sterility, and abortion morning in period, abortion is completely (Fig. 1).Intermission seeding sterile line 836 [S (rfrf)]
With selfing line B73 [N (rfrf)], selfing line B73 [N (rfrf)] is later than sterile line 836 [S (rfrf)] 3 days and sows, various 2 rows,
Row is long 4 meters, 20 centimetres of spacing in the rows, 60 centimetres of line-spacing.Normal field management is carried out after emerging, including watering, apply fertilizer, weeding, anti-
Disease and insect etc. is controlled, is treated that sterile line 836 [S (rfrf)] spins, during selfing line B73 [N (rfrf)] loose powder, with 836 [S (rfrf)] is
Female parent, with B73 [N (rfrf)] as paternal hybrid and harvests 10 F1 generation fruit ears, fruit ear numbering with harvest kernal number such as 1 institute of table
Show.Sow 10 F1 generation fruit ears lower season, and selfing line B73 [N (rfrf)] is sowed after 3 days, each 1 row of F1 generation fruit ear kind, from
Friendship is 2 row of B73 kinds, and row is long 4 meters, 20 centimetres of spacing in the rows, 60 centimetres of line-spacing.Treat that F1 generation is spun, selfing line B73 [N (rfrf)] loose powder
When, with F1 generation as female parent, be returned by recurrent parent of selfing line B73 [N (rfrf)], BC1F1 obtained for fruit ear, each head progeny row is received
5 BC1F1 are obtained for fruit ear, each fruit ear takes 50 seeds, be uniformly distributed on maize chromosome in 836 [S (rfrf)]
The SSR marker that there is polymorphism and B73 [N (rfrf)] between is detected (Fig. 2) that labelling title, position and sequence are shown in Table 2,
PCR reaction systems are (20 μ l)：Template DNA (836 or B73 genomic DNA) 1 μ l, 0.5 μ l of positive labelling, 0.5 μ of reverse labelling
2 μ l of 0.5 μ l of l, dNTP, 10 × PCR Buffer, 0.2 μ l of Taq enzyme, 15.3 μ l of ddH2O.PCR reaction conditions：94 DEG C of 5min,
94 DEG C of 30S, 56 DEG C of 30S, 72 DEG C of 30S, 35 circulations, 72 DEG C of 7min.Each BC1F1 picks out background closest to B73 for head progeny row
1 fringe (detecting marker bands most single fringe identical with B73 banding patterns of seed) continue sowing, single fringe that each head progeny row is selected in
Tokens statisticses result as shown in table 3, is returned with B73 [N (rfrf)] after spinning.It is later each to take 5 single fringes for each head progeny row, often
Individual single fringe takes 50 seeds, is further detected with the remaining labelling that there are polymorphic differences, and BC2F1, BC3F1, BC4F1 are substituted into
Menu fringe tokens statisticses result was returned for 4 generations as shown in table 4, table 5, table 6, filtered out background and the identical male sterilitys of B73
Individual plant 5-1-2-4,8-3-3-5,1-2-1-3-5,2-1-1-3-4,6-2-5-1-1,10-4-1-2-5 (Fig. 3).
2 sterile line 836 of table is counted with seed is harvested with the F1 generation fruit ear numbering of selfing line B73
| Fruit ear is numbered | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Harvest seed | 144 | 152 | 149 | 163 | 155 | 168 | 171 | 166 | 160 | 157 |
Table 3 is returned BC1F1 and counts for labelling similarity
| Fruit ear is numbered | 1-2 | 2-1 | 3-1 | 4-3 | 5-1 | 6-2 | 7-1 | 8-3 | 9-4 | 10-4 |
| BC1F1 | 79.1% | 81.3% | 80.7% | 78.0% | 82.6% | 77.9% | 81.3% | 82.6% | 78.8% | 80.7% |
Table 4 is returned BC2F1 and counts for labelling similarity
| Fruit ear is numbered | 1-2-1 | 2-1-1 | 3-1-4 | 4-3-2 | 5-1-2 | 6-2-5 | 7-1-3 | 8-3-3 | 9-4-3 | 10-4-1 |
| BC2F1 | 90.4% | 91.8% | 90.0% | 88.8% | 92.6% | 89.0% | 91.2% | 92.8% | 89.0% | 90.2% |
Table 5 is returned BC3F1 and counts for labelling similarity
Table 6 is returned BC4F1 and counts for labelling similarity
| Fruit ear is numbered | 1-2-1-3-5 | 2-1-1-3-4 | 3-1-4-2-2 | 4-3-2-1-5 | 6-2-5-1-1 | 7-1-3-5-3 | 9-4-3-4-3 | 10-4-1-2-5 |
| BC3F1 | 100.0% | 100.0% | 96.2% | 99.4% | 100.0% | 99.8% | 98.0% | 100.0% |
The male sterility line that 2 case study on implementation 1 of embodiment is obtained is used for genetic transformation
The method that the present invention infects maize immature embryos by Agrobacterium carries out genetic transformation.The male that embodiment 1 is obtained is not
Educate is that 5-1-2-4,8-3-3-5,1-2-1-3-5,2-1-1-3-4,6-2-5-1-1,10-4-1-2-5 are being isolated with maintainer B73
Same period interval sowing in area, per 3 row sterile line kind, 1 row maintainer, row is long 4 meters, 20 centimetres of spacing in the rows, 60 centimetres of line-spacing.After emerging
Normal field management is carried out, treats that sterile line spins, during maintainer B73 loose powder with the pollen of maintainer B73 under field conditions (factors)
Pollinate to sterile line, 9-11 days after pollination, take the Embryonic Ovule on pollination fruit ear seed, carry out Agrobacterium indoors and infect,
The rataria infected by Agrobacterium is placed on Selective agar medium carries out multi-turns screen, obtains kanamycin-resistant callus tissue, kanamycin-resistant callus tissue is regenerated
Seedling, obtains transgenic T0 for plant.
The method for infecting maize immature embryos by Agrobacterium carries out the detailed process of genetic transformation：
First, peel off rataria
1st, material prepares.The fruit ear of 9-11 days after pollinating is taken, is removed front end and is developed bad part, inserted from top with tweezers
Enter fruit ear, then fruit ear is put in beaker.
2nd, 70% ethanol disinfection 20 minutes is added in beaker, amount of alcohol there preferably was not fruit ear, in disinfecting process otherwise
When Mobile fruit cluster, allow fruit ear and ethanol to completely attach to, reach optimal Disinfection Effect.After sterilization terminates, take out fruit ear and erect
It is straight to place, allow the ethanol on fruit ear surface to flow out, prepare stripping embryo.
3rd, the fruit ear one end sterilized is placed on culture dish, the tweezers of other end insertion are fixed, and are reamed with scalpel
The top of seed, is careful not to cut rataria.
4th, be inserted between embryo and endosperm with the point of a knife of scalpel, then gently prize rataria upwards, and children is held up with point of a knife
Embryo, it is ensured that rataria be not subject to it is any damage, rataria is dipped in containing the hypertonic 2ml sterile centrifugation tubes for infecting liquid of 100 μM of AS
In, often pipe puts rataria 100 or so.
2nd, Agrobacterium is infected
1st, picking Agrobacterium positive colony is put into containing 5ml YEP (50mg/L Kan and 50mg/L Rif) culture medium
In 50ml centrifuge tubes, at a temperature of 28 DEG C, 75rpm shakes bacterium 2-4 hours.
2nd, the rataria in centrifuge tube is washed 2 times with the hypertonic liquid that infects of 100 μM of AS, is subsequently adding 1ml OD=0.3-
0.4 Agrobacterium bacterium solution, gently overturns and allows rataria and bacterium solution uniform contact, dark culturing 10 minutes, and guarantees that rataria all soaks
Bubble is in bacterium solution.
3rd, co-culture
1st, after infecting end, rataria is put on sterilized filter paper, the bacterium solution of embryo surface is blotted, then rataria is transferred to
Co-culture on base, make the plane contact media surface of rataria.
2nd, ParafilmTM culture dish is used, light culture 3 days under the conditions of 22 DEG C.
4th, postpone screening
1st, after co-culturing 3 days, rataria is transferred in delay screening culture medium, light culture 5 days.
5th, resistance screening
1st, after postponing screening 5 days, all of rataria is transferred to light culture two weeks on the Selective agar medium of 1mM glyphosates,
First round screening is carried out, light culture two weeks in the culture medium of 2mM glyphosates are proceeded to afterwards again, the second wheel screening is carried out.
6th, the regeneration of transfer-gen plant
1st, the maize calli through resistance screening is proceeded to into recovery media, renewal cultivation 20 days under dark condition.
2nd, the resistant calli after renewal cultivation is proceeded to into division culture medium, is first cultivated 3 days under dark condition,
Cultivated under illumination condition again.
3rd, the resistant plant for differentiating is proceeded to into root media, individual plant culture grows to two grades of root systems, is then transplanted to
Greenhouse, carries out normal management until pollination is solid.
Carry out the detection of genes of interest to transgenic regenerated plant, and count the transformation efficiency of each male sterility line, system
Meter result is as shown in table 7, it is seen that the transformation efficiency of sterile line 2-1-1-3-4 is higher, reaches 5.8%, and this is offspring neat
Cause, in addition to male sterility, plant height, Ear height, Tassel-Branch number, leaf color, leaf angle and B73 phenotypes are closest, and can be stable
Heredity, therefore be to be named as B73S by this, be the stability of selection-breeding preferably and transformation efficiency highest male sterility transgene receptor
Material.
Table 7 each male sterility line rataria transformation efficiency is counted
| Male sterility line | 5-1-2-4 | 8-3-3-5 | 1-2-1-3-5 | 2-1-1-3-4 | 6-2-5-1-1 | 10-4-1-2-5 |
| Transformation efficiency | 3.1% | 3.6% | 4.0% | 5.8% | 4.4% | 4.8% |
In 3 embodiment 2 of embodiment, the pollination of regenerated transgenic seedling is solid
Regenerated transgenic seedling in case study on implementation 2 is first carried out into the seedling exercising of 3-5 days in greenhouse, seedling exercising is transplanted to thing after terminating
First interval plantation has the isolation area of maintainer B73, is spaced kind generally by 3 row regenerated transgenic seedling, 1 row maintainer B73
Plant, row are long 4 meters, 20 centimetres of spacing in the rows, 60 centimetres of line-spacing, and maintainer was sowed once for generally 15 days in advance per 5 days, sowed three altogether
Batch, normal field management is carried out, treats that regenerated transgenic seedling spins, during maintainer B73 loose powder, with the pollen pair of maintainer B73
Transfer-gen plant completes spontaneous pollination solid.
Seen by the solid situation to 6 male sterility line transgenic progeny, setting percentage preferably, is to take 10 fringes to turn to each
Progeny carries out kernal number statistics, average as shown in table 8 per tassel seed number.
The average kernal number statistics of 8 male sterility line transgenic progeny of table, 10 fringe
The expanding propagation of transgenic progeny in 4 case study on implementation 3 of embodiment
By the transgenic progeny obtained in case study on implementation 3 by transformation event classification with maintainer B73 the same period in the isolation area
Interval sowing, by the sowing that is spaced of 3 row transgenic progeny, 1 row maintainer, row is long 4 meters, 20 centimetres of spacing in the rows, 60 lis of line-spacing
Rice.Normal field management is carried out, when growing into 5-6 leaf phases, Molecular Detection is carried out to transgenic progeny, partial detection is such as
Shown in Fig. 4, the transgenic positive plant for detecting retains, and non-positive plant is extracted, and treats that transgenic progeny is spun, maintainer B73
Spontaneous pollination solid is completed during loose powder, substantial amounts of seed is obtained and is analyzed for character screening and hereditary stability.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (5)
1. a kind of breeding method of Semen Maydiss Cytoplasm male sterility transgene receptor, it is characterised in that comprise the steps：
1) with Cytoplasm male sterility system as female parent, to be adapted to genetic transformation and material as male sterility line maintainer it is
Male parent, hybridization obtain F1 generation fruit ear；
2) by step 1) the seed monoseeding of the F1 generation fruit ear that obtains, sterile plant is selected for female parent, with father described in step 1
This backcrossing, obtains BC1F1 for fruit ear；
3) from the beginning of BC1F1 generations, by molecular marker assisted selection, select the individual plant for obtaining that background is close to male parent described in step 1
Backcrossing, obtains fruit ear of future generation, and the backcrossing through 4～5 generations is bred as male sterile transgenic acceptor；
It is described it is maternal selected from preserving number in Maize Genetics Cooperation Stock Center be C437B, C437E,
The male sterility line of C437H, C537B, C537E, C537H, C657A, C657D, C657F, C836G；
The male parent is the selfing line of high transformation efficiency, and the male parent is selected from National Plant Germplasm System
B73 or comprehensive 31 of the middle preserving number for PI550473.
2. the Semen Maydiss Cytoplasm male sterility transgene receptor that breeding method according to claim 1 is obtained is in Semen Maydiss
Application in genetic transformation and offspring's expanding propagation.
3. application according to claim 2, it is characterised in that the maize genetic is converted into will be the Semen Maydiss matter-core mutual
Make the rataria of male sterility transgene receptor as acceptor material, exogenous gene is imported into the rataria and obtains male sterile turn
Progeny.
4. the breeding of the Semen Maydiss Cytoplasm male sterility transgene receptor that breeding method according to claim 1 is obtained
Method, it is characterised in that the same period is broadcast in the isolation area by the Semen Maydiss Cytoplasm male sterility transgene receptor and maintainer
Kind, allow the pollen of the maintainer to come to Semen Maydiss Cytoplasm male sterility transgene receptor pollination under field conditions (factors)
Raise up seed.
5. the propagation method of the transgenic progeny for obtaining in application according to claim 3, it is characterised in that by described turn
The same period sowing in the isolation area of progeny and maintainer, allow the maintainer pollen under field conditions (factors) to the transgenic
Offspring pollinates.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101096676A (en) * | 2007-06-08 | 2008-01-02 | 南京农业大学 | Rice chlorophyll synthase mutant gene and its application in gene engineering |
| US7432427B1 (en) * | 2006-04-25 | 2008-10-07 | Monsanto Technology Llc | Plants and seeds of corn variety CV109921 |
| CN102443601A (en) * | 2010-10-12 | 2012-05-09 | 中国农业科学院生物技术研究所 | Corn genetic transformation method |
| CN103060369A (en) * | 2012-09-17 | 2013-04-24 | 西南大学 | Hybrid crop transgenic safety control method and gene deletion system for implementing same |
| CN104285776A (en) * | 2013-12-31 | 2015-01-21 | 北京市农林科学院 | Breeding method for corn male sterile line |
-
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7432427B1 (en) * | 2006-04-25 | 2008-10-07 | Monsanto Technology Llc | Plants and seeds of corn variety CV109921 |
| CN101096676A (en) * | 2007-06-08 | 2008-01-02 | 南京农业大学 | Rice chlorophyll synthase mutant gene and its application in gene engineering |
| CN102443601A (en) * | 2010-10-12 | 2012-05-09 | 中国农业科学院生物技术研究所 | Corn genetic transformation method |
| CN103060369A (en) * | 2012-09-17 | 2013-04-24 | 西南大学 | Hybrid crop transgenic safety control method and gene deletion system for implementing same |
| CN104285776A (en) * | 2013-12-31 | 2015-01-21 | 北京市农林科学院 | Breeding method for corn male sterile line |
Non-Patent Citations (1)
| Title |
|---|
| 玉米转基因育种的研究与进展;贾晓军等;《种子科技》;20091231(第8期);第20-23页 * |
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