CN116472954A - Method for creating clubroot-resistant cabbage pure line - Google Patents
Method for creating clubroot-resistant cabbage pure line Download PDFInfo
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- CN116472954A CN116472954A CN202310516699.6A CN202310516699A CN116472954A CN 116472954 A CN116472954 A CN 116472954A CN 202310516699 A CN202310516699 A CN 202310516699A CN 116472954 A CN116472954 A CN 116472954A
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- 240000007124 Brassica oleracea Species 0.000 title claims abstract description 71
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 title claims abstract description 64
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- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 title claims abstract description 64
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- 230000001404 mediated effect Effects 0.000 claims abstract description 7
- 230000001086 cytosolic effect Effects 0.000 claims abstract description 5
- 238000012216 screening Methods 0.000 claims description 11
- 241000196324 Embryophyta Species 0.000 claims description 8
- 101000708283 Oryza sativa subsp. indica Protein Rf1, mitochondrial Proteins 0.000 claims description 8
- 235000011302 Brassica oleracea Nutrition 0.000 claims description 7
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 5
- 241000220259 Raphanus Species 0.000 claims description 5
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- LKCWBDHBTVXHDL-RMDFUYIESA-N amikacin Chemical compound O([C@@H]1[C@@H](N)C[C@H]([C@@H]([C@H]1O)O[C@@H]1[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O1)O)NC(=O)[C@@H](O)CCN)[C@H]1O[C@H](CN)[C@@H](O)[C@H](O)[C@H]1O LKCWBDHBTVXHDL-RMDFUYIESA-N 0.000 description 3
- 229960004821 amikacin Drugs 0.000 description 3
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Classifications
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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
- A01C—PLANTING; SOWING; FERTILISING
- A01C21/00—Methods of fertilising, sowing or planting
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- 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
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- 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
- A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers
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- 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
- A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
- A01H1/122—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- A01H1/1245—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance
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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
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
A method for creating a clubroot-resistant cabbage pure line relates to the technical field of plant breeding, and comprises the following steps: hybridizing with the selfing line material with excellent characters as female parent and the fertility restoring line of cabbage with clubroot resistance and Ogura CMS variety as male parent; selecting a strain which does not contain a restoring gene Rfo, contains an clubroot resistance gene CRb and has excellent comprehensive properties from the hybrid seeds for free microspore culture; the method for creating the clubroot resistant cabbage pure line has the advantages that aiming at the current situation that the clubroot resistant cabbage commercial varieties are almost of Ogura type cytoplasmic male sterile line in current production, the fertility restoring gene Rfo is quickly transferred into the clubroot resistant cabbage commercial varieties through an agrobacterium-mediated method, excellent gene polymerization is carried out by using the obtained restoring line and excellent cabbage inbred line materials, molecular marker assisted selection, free microspore culture and other biological breeding technical means are combined for quickly creating the clubroot resistant cabbage pure line materials, and a breeding material foundation is provided for cultivating the clubroot resistant cabbage new varieties with independent intellectual property rights.
Description
Technical Field
The invention relates to the technical field of plant breeding, in particular to a method for creating a cabbage pure line resistant to clubroot.
Background
Common head cabbage (Brassica oleracea L.var.capitata) is simply called cabbage, is a worldwide important vegetable, and is also one of the main leaf vegetable types in China. The cabbage has rich nutrition, strong adaptability and stress resistance, high yield, storage and transportation resistance, and the like, is widely planted in various places in China, and basically stabilizes the sowing area to 1350 ten thousand mu (90 ten thousand hm) by 2021 2 )。
At present, clubroot is a main stream disease in cabbage production. Clubroot is a very infectious soil disease that is caused by the infestation of the genus clubroot (Plasmodiophora brassicae woron.) by brassicaceae plants. Once the disease occurs, cabbage starts to die from the seedling stage, and commercial leaf heads are difficult to form, thus causing serious economic loss to manufacturers. The disease is difficult to overcome by methods such as rotation cultivation, chemical control and the like, and the cultivation of the clubroot-resistant variety is the most effective method. In domestic production and scientific research breeding, clubroot is a novel disease, the storage of anti-clubroot resources is insufficient, the research basis is weaker, and the variety of the anti-clubroot cabbage with independent intellectual property rights is not yet proposed in China at present.
The Brussels of the first-sweet 336 cabbage variety introduced by the first-up (China) investment Limited company has high clubroot resistance, and CRb genes contained in the Brussels show high resistance to physiological races 2, 4 and 8 of the clubroot, so that the Brussels are excellent resistant resources. However, first glycine 336 is an Ogura type cytoplasmic male sterile (cytoplasmic male sterility, CMS) hybrid, whose strain is not pollen, resulting in the inability of the resistance gene in sterile material to be isolated for use. Moreover, more than 80% of the current cabbage commercial varieties are Ogura CMS type hybrid seeds, and innovation and utilization of germplasm resources are greatly limited. Thus, the creation of the cabbage Ogura CMS restorer is significant for sustainable use of germplasm resources.
Since the Ogura CMS type restorer line is only present in radish, it can only be introduced into cabbage by distant hybridization or transgenic means. However, the creation of restorer lines by distant hybridization requires multiple generations of backcrossing and continuous molecular marker assisted selection to obtain fertility restoration materials with genetic background of cabbage, which has large workload and long period and greatly limits the breeding process. The fertility restoring gene Rfo is quickly transferred into commercial varieties with excellent characters by an agrobacterium-mediated method, the obtained restoring line is utilized to realize barrier-free utilization of excellent commercial variety materials, excellent pure lines are quickly created by combining biological breeding means such as excellent gene polymerization hybridization, molecular marker assisted selection, free microspore culture technology and the like, and the process of cabbage germplasm resource innovation and clubroot-resistant cabbage germplasm source cultivation with independent intellectual property can be greatly accelerated.
Novel matters of the invention
The invention aims to provide a method for creating a clubroot resistant cabbage pure line, which aims at the current situation that the common clubroot resistant cabbage commercial varieties are of the Ogura type in current production, and the fertility restoration gene Rfo is quickly transferred into the common clubroot resistant cabbage commercial varieties by an agrobacterium-mediated method, and the obtained restoration line and an excellent cabbage inbred line material are utilized to perform excellent gene polymerization, so that molecular marker assisted selection, free microspore culture and other biological breeding technical means are combined to quickly create the clubroot resistant cabbage pure line material, thereby providing a breeding material foundation for cultivating the new cabbages with independent intellectual property rights.
In order to achieve the above purpose, the present invention provides the following technical solutions: the method is characterized in that: comprising the following steps: hybridizing with the selfing line material with excellent characters as female parent and the fertility restoring line of cabbage with clubroot resistance and Ogura CMS variety as male parent; and selecting a strain which does not contain a restoring gene Rfo, contains an clubroot resistance gene CRb and has excellent comprehensive properties from the hybrid seeds, and culturing the isolated microspores to obtain the cabbage double haploid pure line material with clubroot resistance and excellent properties.
Preferably, obtaining the cabbage inbred line material with excellent S1 property: is obtained by continuous multi-generation selfing separation and screening of cabbage materials with excellent characters.
Preferably, the S2 cabbage is resistant to clubroot and is obtained from the fertility restorer line of the Ogura CMS variety: the fertility restorer gene Rfo of the radish Ogura CMS is integrated into the cabbage variety of the anti-clubroot Ogura CMS by using an agrobacterium-mediated method to obtain the Ogura CMS restorer line.
Preferably, S3 contains no restorer gene Rfo, screening of excellent strains against clubroot: in the plant balling period, examining commodity characters such as spherical, color, center column length and the like, simultaneously identifying the existence of clubroot resistance by amplifying a clubroot resistance gene CRb fragment, and screening strains with excellent characters and the clubroot resistance; in the flowering period of plants, whether a restorer gene Rfo exists or not is identified by amplifying a herbicide resistance marker gene Bar and an orgura cytoplasmic sterility gene orf138 fragment; and selecting an excellent strain without the restorer gene Rfo and resistant to clubroot by combining screening and identification of the plant in the balling period and the flowering period.
Preferably, the obtaining of S4 clubroot-resistant and excellent-trait cabbage doubled haploid stock material is carried out by episomal cultivation of a strain selected from the hybrid containing the clubroot-resistant gene CRb, excellent-trait and no restorer gene Rfo.
Preferably, 400-fold liquid of a Mi Ka calcium were sprayed onto pre-sampled flower buds 2 days prior to sampling by free microspore culture.
Preferably, cabbage doubled haploid pure line materials with excellent properties and clubroot resistance are screened from DH lines obtained by culture of free microspores.
Compared with the prior art, the novel beneficial effects of the invention are as follows:
1. the method for creating the clubroot-resistant cabbage pure line contains the clubroot-resistant CRb gene, has good comprehensive commodity, and provides a material basis for cultivating new clubroot-resistant cabbage varieties with independent intellectual property rights.
2. Compared with a conventional cabbage vegetable free microspore culture system, the free microspore culture method for creating the clubroot-resistant cabbage pure line increases the treatment of spraying 400 times of liquid amikacin to the flower buds 2 days before the free microspore culture sampling, and the treatment improves the plant pollen quantity and pollen activity and obviously improves the embryogenic rate of the cabbage free microspore culture.
Amikacin is a novel quick-acting sugar alcohol calcium fertilizer product with high quality for Korean modern specialty companies. The amikacin contains conchiolin active calcium, can effectively stabilize the structure of cell walls and cell membranes, and can strengthen the cell elasticity; and proper amount of deep sea fish protein and H.T active enzyme factor are added, so that the plant can absorb more rapidly and the utilization rate is high.
3. The method for creating the clubroot-resistant cabbage pure line realizes comprehensive application of efficient biological breeding technologies such as fertility restoration, excellent gene polymerization hybridization, molecular marker assisted selection, free microspore culture and the like, remarkably improves cabbage breeding efficiency and also improves cabbage breeding level in China.
Detailed Description
The technical solutions of the novel embodiments of the present invention will be clearly and completely described, and it is apparent that the described embodiments are only some embodiments, but not all embodiments, of the novel embodiments of the present invention, and all other embodiments obtained by those skilled in the art without making creative efforts are within the scope of novel protection of the present invention.
Example 1:
the embodiment particularly provides a method for creating a clubroot-resistant cabbage pure line DHC05011, which comprises the following steps:
1. obtaining the cabbage inbred line material with excellent properties: is excellent backbone inbred line C09429 obtained by continuous 6-generation inbred separation screening.
2. Obtaining the fertility restorer line of the excellent variety Ogura CMS for resisting clubroot of the cabbage: integrating a fertility restorer gene Rfo of the radish Ogura CMS into a cabbage variety XG336 containing an clubroot resisting CRb gene and the Ogura CMS by using an agrobacterium-mediated method to obtain Ogura CMS restorer lines C-1 to C-15;
3. the self-bred line material 'C09429' of excellent character cabbage is used as female parent, ogura CMS restorer line 'C-6' is used as male parent to make bud-stage pollination hybridization so as to obtain the seed CC6F of C09429×C-6 hybrid 1 ；
4. At the junctionStage of globus, from CC6F 1 Of the 90 strains grown, 20 strains containing the clubroot-resistant CRb gene and having excellent properties are selected; during anthesis, CC6F is selected from 1 Of the 20 strains of the hybrid, 11 strains CC6F without the restorer gene Rfo were selected and identified 1 -01～CC6F 1 -11；
6. For CC6F 1 -05, culturing free microspores with an embryo yield of 7.5 microspores per bud, obtaining 60 DH lines;
7. DH lines containing clubroot-resistant CRb gene and flowers with excellent properties were selected from 60 DH lines and named 'DHC05011'.
Example 2:
the embodiment particularly provides a method for creating a clubroot-resistant cabbage pure line DHCT05037, which comprises the following steps:
1. obtaining the cabbage inbred line material with excellent properties: is excellent backbone inbred line C09429 obtained by continuous 6-generation inbred separation screening.
2. Obtaining the fertility restorer line of the excellent variety Ogura CMS for resisting clubroot of the cabbage: integrating a fertility restorer gene Rfo of the radish Ogura CMS into a cabbage variety XG336 containing an clubroot resisting CRb gene and the Ogura CMS by using an agrobacterium-mediated method to obtain Ogura CMS restorer lines C-1 to C-15;
3. the self-bred line material 'C09429' of excellent character cabbage is used as female parent, ogura CMS restorer line 'C-6' is used as male parent to make bud-stage pollination hybridization so as to obtain the seed CC6F of C09429×C-6 hybrid 1 ；
4. During the balling period, from CC6F 1 Of the 90 strains grown, 20 strains containing the clubroot-resistant CRb gene and having excellent properties are selected; during anthesis, CC6F is selected from 1 Of the 20 strains of the hybrid, 11 strains CC6F without the restorer gene Rfo were selected and identified 1 -01～CC6F 1 -11；
6. For CC6F 1 -05 free microspore culture, spraying a 400-fold solution of a Mi Ka calcium on pre-sampled flower buds 2 days before sampling the free microspore culture; the microspore embryo emergence rate is 13.4 per bud, and 70 DH lines are obtained;
7. DH lines containing clubroot-resistant CRb gene and flowers with good properties were selected from 70 DH lines and named 'DHCT05037'.
While there has been shown and described what are at present considered to be novel principles, main features and advantages of the present invention, it will be apparent to those skilled in the art that the present invention is not limited to the details of the foregoing exemplary embodiments, but may be embodied in other specific forms without departing from the spirit or essential characteristics thereof; the present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.
Claims (7)
1. A method for creating a clubroot-resistant cabbage clone, which is characterized by comprising the following steps: comprising the following steps: hybridizing with the selfing line material with excellent characters as female parent and the fertility restoring line of cabbage with clubroot resistance and Ogura CMS variety as male parent; and selecting a strain which does not contain a restoring gene Rfo, contains an clubroot resistance gene CRb and has excellent comprehensive properties from the hybrid seeds, and culturing the isolated microspores to obtain the cabbage double haploid pure line material with clubroot resistance and excellent properties.
2. The method for creating a clubroot-resistant brassica oleracea clone according to claim 1, wherein: obtaining the cabbage inbred line material with excellent S1 property: is obtained by continuous multi-generation selfing separation and screening of the fertile cabbage materials with excellent characters.
3. The method for creating a clubroot-resistant brassica oleracea clone according to claim 1, wherein: s2, obtaining a fertility restorer line of the cabbage anti-clubroot Ogura CMS variety: the fertility restorer gene Rfo of the radish Ogura CMS is integrated into the cabbage variety of the anti-clubroot Ogura CMS by using an agrobacterium-mediated method to obtain the Ogura CMS restorer line.
4. The method for creating a clubroot-resistant brassica oleracea clone according to claim 1, wherein: s3, screening of excellent strains without restoring gene Rfo and clubroot resisting gene CRb: in the plant balling period, examining commodity characters such as spherical, color, center column length and the like, simultaneously identifying the existence of clubroot resistance by amplifying a clubroot resistance gene CRb fragment, and screening strains with excellent characters and the clubroot resistance; in the flowering period of plants, whether a restorer gene Rfo exists or not is identified by amplifying a herbicide resistance marker gene Bar and an orgura cytoplasmic sterility gene orf138 fragment; and selecting an excellent strain without the restoring gene Rfo and with the clubroot resistance gene CRb by combining screening and identification of the plant in the balling period and the flowering period.
5. The method for creating a clubroot-resistant brassica oleracea clone according to claim 1, wherein: s4, obtaining cabbage double haploid pure line material with clubroot resistance and excellent characters, and carrying out free microspore culture on a strain which is selected from hybrid seeds, has excellent characters and does not contain a restoring gene Rfo.
6. The method for creating a clubroot-resistant brassica oleracea clone according to claim 1, wherein: 400-fold solution of a Mi Ka calcium were sprayed onto pre-sampled flower buds 2 days prior to sampling by free microspore culture.
7. The method for creating a clubroot-resistant brassica oleracea clone according to claim 1, wherein: screening out the cabbage dihaploid pure line material with excellent quality and resisting clubroot from DH strain obtained by culturing free microspores.
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| CN202310516699.6A CN116472954A (en) | 2023-05-09 | 2023-05-09 | Method for creating clubroot-resistant cabbage pure line |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116724878A (en) * | 2023-05-09 | 2023-09-12 | 江苏省农业科学院 | Method for creating high-glucoraphanin broccoli pure line |
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| US20050262583A1 (en) * | 2002-07-26 | 2005-11-24 | Syngenta Participations Ag | Clubroot resistant brassica oleracea plants |
| CN102283102A (en) * | 2011-07-11 | 2011-12-21 | 镇江瑞繁农艺有限公司 | Method for breeding disease resistant cabbage and producing seeds |
| CN114568294A (en) * | 2022-01-25 | 2022-06-03 | 华中农业大学 | Method for breeding clubroot-resistant variety based on brassica napus Ogu-CMS restorer line |
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| US20050262583A1 (en) * | 2002-07-26 | 2005-11-24 | Syngenta Participations Ag | Clubroot resistant brassica oleracea plants |
| CN102283102A (en) * | 2011-07-11 | 2011-12-21 | 镇江瑞繁农艺有限公司 | Method for breeding disease resistant cabbage and producing seeds |
| CN114568294A (en) * | 2022-01-25 | 2022-06-03 | 华中农业大学 | Method for breeding clubroot-resistant variety based on brassica napus Ogu-CMS restorer line |
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| CN116724878A (en) * | 2023-05-09 | 2023-09-12 | 江苏省农业科学院 | Method for creating high-glucoraphanin broccoli pure line |
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