CN116326480A - Method for wheat breeding by combining heavy ions with tissue culture - Google Patents
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- 241000209140 Triticum Species 0.000 title claims abstract description 41
- 235000021307 Triticum Nutrition 0.000 title claims abstract description 41
- 238000009395 breeding Methods 0.000 title claims abstract description 32
- 230000001488 breeding effect Effects 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 19
- 150000002500 ions Chemical class 0.000 title claims abstract description 16
- 241000196324 Embryophyta Species 0.000 claims abstract description 17
- 230000005855 radiation Effects 0.000 claims abstract description 11
- 238000010884 ion-beam technique Methods 0.000 claims abstract description 10
- 210000001161 mammalian embryo Anatomy 0.000 claims description 10
- 230000004069 differentiation Effects 0.000 claims description 9
- 238000003306 harvesting Methods 0.000 claims description 9
- 230000006698 induction Effects 0.000 claims description 9
- 230000009418 agronomic effect Effects 0.000 claims description 7
- 238000005286 illumination Methods 0.000 claims description 7
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- 238000012360 testing method Methods 0.000 abstract description 6
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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
-
- 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
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
-
- 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
- A01H4/002—Culture media for tissue culture
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/14—Measures for saving energy, e.g. in green houses
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- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
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Abstract
The invention belongs to the technical field of wheat mutation breeding, and particularly discloses a method for breeding wheat by combining heavy ions with tissue culture, which adopts a method combining C ion beam radiation mutation with tissue culture to obtain mutant plants with original excellent properties and quality and short plant height, M 2 S 1 The generation is basically stable, can enter a district-based test and enter a variety approval program, so that the breeding period is greatly shortened, and the wheat breeding efficiency is improved.
Description
Technical Field
The invention relates to the technical field of wheat mutation breeding, in particular to a method for breeding wheat by combining heavy ions with tissue culture.
Background
Wheat is one of the main food crops. The Heilongjiang province is an important spring wheat producing area in China, and the wheat production has important significance for meeting the dietary requirements of people, guaranteeing the national grain safety, optimizing the planting industry structure and establishing a scientific rotation system. Due to the lack of excellent and heterogeneous resources, conventional hybridization breeding is mostly adopted in breeding, the period is long, the method is relatively lagged, and breakthrough varieties with high yield, good quality and strong stress resistance are not yet bred. Heavy ion beams are a novel radiation source and have important roles in the field of life science research. Tissue culture can also produce a large number of somaclonal variants. The combination of heavy ion radiation technology and tissue culture can more effectively improve the variation frequency of the somaclonal, expand the variation spectrum, create more selection opportunities, shorten the breeding period and open a new way for creating excellent germplasm resources with rich types and breeding new varieties of wheat.
The method for breeding wheat seeds by site-directed mutagenesis by heavy ion beams disclosed in application number CN03134291.4 utilizes the unique advantages of small lateral divergence of heavy ions and high spatial resolution of energy loss, adopts ion beams with different energies and different injection amounts, and injects different depths into different parts of wheat embryo parts (radicle and embryo) and endosperm. The method can also carry out mutation breeding on wheat, but adopts ion beams with different energies and different injection amounts to inject different depths into different parts of wheat embryo parts (radicle and embryo) and endosperm, and has high operation precision and large workload.
15-676 strain wheat is an excellent strain bred by the unit, has high yield, good quality and good disease resistance and stress resistance, but has high plant height, and due to uncertainty of climate, lodging can occur in large-area planting, mechanical harvest is affected, and yield is reduced. Therefore, a breeding method for improving the breeding efficiency of the wheat of the 15-676 strain is needed to be provided.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for breeding wheat by combining heavy ions with tissue culture.
In order to achieve the above purpose, the invention is implemented according to the following technical scheme:
a method for wheat breeding by combining heavy ions with tissue culture, comprising the following steps:
s1, seed treatment: treating the wheat pure seed by using a C ion beam generated by a heavy ion accelerator, wherein the radiation dose is 80Gry;
s2, inoculating young embryo: freezing the seeds subjected to radiation treatment in a refrigerator at the temperature of-20 ℃ for 7d, and sowing the seeds in the field; after wheat flowers 14d, picking wheat ears, peeling young embryo, soaking in 70% alcohol under aseptic condition for 30s, washing with aseptic water for 2 times, and performing surface sterilization; then 0.1% HgCl 2 Sterilizing for 7-8min, rinsing with sterile water for 3 times, inoculating into induction culture medium,10 young embryos are placed in each triangular flask;
s3, inducing and differentiating culture of the callus: placing the inoculated triangular flask in a culture room at 23+/-1 ℃ for dark culture, and investigating the healing condition after dark culture for about 10 days; transferring embryogenic callus to a differentiation medium of MS+0.5mg/L KT for carrying out illumination differentiation culture in a culture chamber at 23+/-1 ℃ after primary subculture, and investigating differentiation conditions;
s4, culturing regenerated seedlings: transplanting the differentiated green seedlings into a pot, placing the pot in a greenhouse for continuous culture, and performing conventional management until the green seedlings are ripe; harvesting and threshing according to the strain after the mature to obtain M 1 S 0 A strain;
S5、M 2 S 1 group construction and agronomic trait investigation: m harvested in greenhouse 1 S 0 The seeds are sown on demand according to plant lines, and the seeds of the wheat pure line are sown on demand as a contrast; normal management and fertility investigation are carried out; selecting plants according to a set breeding target in a mature period; m to be selected after harvesting 2 S 1 And (5) checking the strain, and examining agronomic characters.
Further, the induction medium in the step S2 is MS medium +2mg/L2.4-D +30g/L sucrose +8g/L agar, and the pH of the induction medium is 5.8.
Further, the illumination intensity of the culture room in the step S3 is 2000lx, and the culture is carried out for 12 hours by daily illumination.
Further, in the step S5, the plant acts 5cm, the small row spacing is 15cm, and the large row spacing is 40cm.
Compared with the prior art, the invention adopts the method of combining C ion beam radiation mutagenesis and tissue culture to obtain the mutant plant which keeps the original excellent properties and quality and has short plant height, M 2 S 1 The generation is basically stable, can enter a district-based test and enter a variety approval program, so that the breeding period is greatly shortened, and the wheat breeding efficiency is improved.
Drawings
FIG. 1 is a diagram of 15-676M obtained according to the present invention 2 S 1 Field performance of the mutant lines with 15-676 controls.
FIG. 2 is a graph of 15-676M 2 S 1 OffspringAnd its control electropherograms.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific embodiments described herein are for purposes of illustration only and are not intended to limit the invention.
The test materials used in the examples below were wheat inbred seeds 15-676 and the corresponding controls. The breeding process of the wheat pure line seed 15-676 variety specifically comprises the following steps: parental combination, parental source, breeding method, generation, characteristic description, etc.) Long Fumai 5676 is the machine precision dibbling of the next year in 2013, the academy of agricultural sciences of the black dragon river, which uses the Dragon's foot 09-594 as the female parent and the Dragon's foot 10-891 as the male parent. And selecting the separated offspring according to a pedigree method, and carrying out tracking analysis on sedimentation values of the high generation and disease identification. In 2018, the high-yield disease-resistant variety with strong tendons is selected in F6 generation. The variety code Dragon root 18-676 is a new variety of strong gluten wheat. The test of the Heilongjiang province area is participated in 2022, and the yield is increased by 6.9 percent compared with 19 grams of drought. 2. Variety standard (characteristic) Long Fumai 5676, new wheat variety spring, emergence to mature growth days about 85 days. The seedlings are semi-upright, she Xichang, light green in color, strong in tillering force and good in luxuriance. The plant height is about 95cm, the stalk is strong, the lodging resistance is good, and the later yellowing is good. There are mango, huang Ke, red grain, cutin, oval grain, thousand grain weight about 37.8 g, volume weight 812 g/liter. Quality analysis results: the analysis by the quality supervision and inspection test center (Harbin) of grains and products of the agricultural department shows that the protein content is 16.4%, the wet gluten is 34.6%, the water absorption is 59.2%, the forming time is 12.5 minutes, the stabilizing time is 21.6 minutes, and the evaluation value is 85. Inoculation identification result: high stem rust and leaf rust resistance, and light scab root rot. The yield in 2019-2021 identifies that the average mu yield is 421.3 kg, and the yield is increased by 12.8% compared with the control g-upland 19. In the 7-point off-site identification test in 2020, the average yield per mu is 317.46 kg, and the average yield is 9.52% compared with the average yield of the control g-run 19. In a 7-point off-site identification test in 2021, the average yield per mu is 321.46 kg, and the average yield is 9.99% compared with that of a control g-run 19. 3. The long-noded wheat 5676 has the advantages of drought resistance in the early stage, moisture resistance in the later stage, more tillers, good flourishing property, lodging resistance, high yield, stalk resistance, leaf rust resistance and other excellent characteristics. The agronomic traits are shown in table 1.
TABLE 1
However, in actual planting, the wheat pure line seeds 15-676 are high, and due to uncertainty of climate, lodging can occur in large-area planting, mechanical harvest is affected, and yield is reduced.
Based on the above, the embodiment provides a method for breeding wheat by combining heavy ions with tissue culture, which comprises the following specific steps:
s1, seed treatment: treating wheat seed with C ion beam produced by modern physical heavy ion accelerator (HIRFL) with radiation dose of 80Gry;
s2, inoculating young embryo: freezing the seeds subjected to radiation treatment in a refrigerator at the temperature of-20 ℃ for 7d, and sowing the seeds in the field; after wheat flowers 14d, picking wheat ears, peeling young embryo, soaking in 70% alcohol under aseptic condition for 30s, washing with aseptic water for 2 times, and performing surface sterilization; then 0.1% HgCl 2 Sterilizing for 7-8min, rinsing with sterile water for 3 times, inoculating into an induction culture medium, inoculating 10 young embryos into each triangular flask, wherein the induction culture medium is MS culture medium +2mg/L2.4-D +30g/L sucrose +8g/L agar, and the pH of the induction culture medium is 5.8;
s3, inducing and differentiating culture of the callus: placing the inoculated triangular flask in a culture room at 23+/-1 ℃ for dark culture, and investigating the healing condition after dark culture for about 10 days; transferring embryogenic callus to a differentiation medium of MS+0.5mg/L KT for one time after secondary culture, and carrying out illumination differentiation culture in a culture chamber with illumination intensity of 2000lx at 23+/-1 ℃ for 12 hours per day; investigation of differentiation conditions;
s4, culturing regenerated seedlings: transplanting the differentiated green seedlings into a pot, placing the pot in a greenhouse for continuous culture, and performing conventional management until the green seedlings are ripe; harvesting and threshing according to the strain after the mature to obtain M 1 S 0 A strain;
S5、M 2 S 1 group construction and agronomic trait investigation: m harvested in greenhouse 1 S 0 The seeds are sowed according to plant rows, and the wheat pure line seeds are sowed as a contrast, the plant rows are 5cm, the small row spacing is 15cm, and the large row spacing is 40cm; normal management and fertility investigation are carried out; the mature period is selected according to the established breeding target. M to be selected after harvesting 2 S 1 And (3) strain test, and performing obvious analysis on the differences of the agronomic characters by using SPSS 26.0 software.
The actual analysis shows that: m is M 2 S 1 The strain height was significantly lower than that of the control group (p=0.000<0.01 An average difference of 11.74cm, the difference being statistically significant. The tillering number and the main grain number are 0.39 and 4.14 more than the control, the main grain weight and the single plant grain weight are 0.17g and 0.78g respectively, and the specific results are shown in Table 2 (the difference has no statistical significance).
TABLE 2
Note that: experimental results to
The expression P < 0.05 and P < 0.01.
15-676M 2 S 1 The field performance of the mutant strain compared with 15-676 is shown in FIG. 1, and the left side strain in FIG. 1 acts as 15-676M 2 S 1 Mutant line, right strain behavior 15-676 control. After harvesting, seeds were selected to determine HMW-GS subunit composition, see Table 3, and the electropherograms were shown in FIG. 2.
TABLE 3 Table 3
| Strain of strain | HMW-GS subunit composition |
| 15-676M 2 S 1 -1 | 2*,7+9,5+10 |
| 15-676M 2 S 1 -2 | 2*,7+9,5+10 |
| 15-676M 2 S 1 -3 | 2*,7+9,5+10 |
| 15- |
2*,7+9,5+10 |
| 15-676M 2 S 1 -4 | 2*,7+9,5+10 |
| 15-676M 2 S 1 -5 | 2*,7+9,5+10 |
| 15-676M 2 S 1 -6 | 2*,7+9,5+10 |
As can be seen from table 3 and fig. 2: mutant 15-676M 2 S 1 The offspring seeds all had the same subunits as the control and all contained 5+10 premium subunits.
For review, a method of combining C ion beam radiation mutagenesis and tissue culture is adopted to obtain mutant plants with original excellent properties and quality and reduced plant height, M 2 S 1 The generation is basically stable, can enter a district-based test and enter a variety approval program, so that the breeding period is greatly shortened, and the wheat breeding efficiency is improved.
The technical scheme of the invention is not limited to the specific embodiment, and all technical modifications made according to the technical scheme of the invention fall within the protection scope of the invention.
Claims (4)
1. A method for wheat breeding by combining heavy ions with tissue culture, which is characterized by comprising the following steps:
s1, seed treatment: treating the wheat pure seed by using a C ion beam generated by a heavy ion accelerator, wherein the radiation dose is 80Gry;
s2, inoculating young embryo: freezing the seeds subjected to radiation treatment in a refrigerator at the temperature of-20 ℃ for 7d, and sowing the seeds in the field; after wheat flowers 14d, picking wheat ears, peeling young embryo, soaking in 70% alcohol under aseptic condition for 30s, washing with aseptic water for 2 times, and performing surface sterilization; then 0.1% HgCl 2 Sterilizing for 7-8min, rinsing with sterile water for 3 times, inoculating into induction culture medium, and placing 10 young embryos in each triangular flask;
s3, inducing and differentiating culture of the callus: placing the inoculated triangular flask in a culture room at 23+/-1 ℃ for dark culture, and investigating the healing condition after dark culture for about 10 days; transferring embryogenic callus to a differentiation medium of MS+0.5mg/L KT for carrying out illumination differentiation culture in a culture chamber at 23+/-1 ℃ after primary subculture, and investigating differentiation conditions;
s4, culturing regenerated seedlings: transplanting the differentiated green seedlings into a pot, placing the pot in a greenhouse for continuous culture, and performing conventional management until the green seedlings are ripe; harvesting and threshing according to the strain after the mature to obtain M 1 S 0 A strain;
S5、M 2 S 1 group construction and agronomic trait investigation: m harvested in greenhouse 1 S 0 The seeds are sown on demand according to plant lines, and the seeds of the wheat pure line are sown on demand as a contrast; normal management and fertility investigation are carried out; selecting plants according to a set breeding target in a mature period; m to be selected after harvesting 2 S 1 And (5) checking the strain, and examining agronomic characters.
2. The method for breeding wheat by combining heavy ions with tissue culture according to claim 1, wherein the induction medium in the step S2 is MS medium +2mg/L2.4-D +30g/L sucrose +8g/L agar, and the pH of the induction medium is 5.8.
3. The method for breeding wheat by combining heavy ions with tissue culture according to claim 1, wherein the illumination intensity of the culture room in the step S3 is 2000lx, and the culture is performed for 12 hours per day.
4. The method for breeding wheat by combining heavy ions with tissue culture according to claim 1, wherein the plant acts by 5cm when the seeds are on demand in the step S5, the small row spacing is 15cm, and the large row spacing is 40cm.
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| WO2023005883A1 (en) * | 2021-07-28 | 2023-02-02 | 西南大学 | Breeding method of blue marked two-line hybrid wheat system and use |
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