Millet mutation breeding method and application thereof

Abstract

The invention relates to the technical field of mutation breeding, and particularly discloses a millet mutation breeding method and application thereof. The mutation breeding method for the millet comprises the following steps: (1) irradiating dry seeds of the millet by using a heavy ion beam to obtain M1-generation seeds; the energy is 78-82 MeV / u, the energy transfer linear density LET is 29-31 KeV / mu m, the irradiation dose is 150-200 Gy, the dose rate is 38-42 Gy / min, and the irradiation time is 42-47 min; (2) planting M1-generation seeds, and carrying out single-plant seed harvesting and threshing on all survival plants to obtain M2-generation seeds; (3) planting M2-generation seeds, and performing single-plant seed harvesting and threshing on all variant plants to obtain M3-generation seeds; and (4) planting M3-generation seeds of each variant plant, performing single-plant seed harvesting on plants with stable characters, and performing threshing to obtain M4-generation seeds. The method provided by the invention can improve the mutation rate of foxtail millet, is rich in offspring variation variety, shortens the breeding period, and improves the breeding efficiency.

Description

Technical Field

[0001] The invention relates to the technical field of mutation breeding, in particular to a millet mutation breeding method and application thereof. Background Art

[0002] Millet Setaria italica ) is a crop with a long history of cultivation. It occupies an important position in dryland agricultural production and has important significance in the history of crop cultivation. As a dominant crop in dryland agricultural areas, millet is rich in balanced nutrition and has strong health functions. It is suitable for the consumption needs of consumers, especially urban consumers, for "high quality, nutrition, health care and convenience". It is a nutritionally superior crop. With the gradual recognition of the nutritional value of grains, high-yield and high-quality millet varieties have become the favorite of farmers and consumers. However, the high-quality millet varieties currently planted in production are relatively single, and diseases and insect pests are serious, which greatly restricts the development of the millet industry. Therefore, with the development of millet production and the expansion of market demand, new requirements have been put forward for the selection and breeding of new millet varieties.

[0003] Millet breeding is mainly based on traditional line selection and hybrid breeding, which has a long cycle and low efficiency. In addition, hybrid breeding is greatly affected by parental materials. The selection of excellent parental materials is extremely important, but the selection range of parental materials is narrow and there is a lack of variant materials, which makes the genetic basis of millet narrow. Heavy ion radiation mutagenesis technology is a powerful tool for plant germplasm innovation. Compared with irradiation technologies such as X-rays, 60Co-γ rays, and electron beams, heavy ion radiation mutagenesis technology has biological advantages such as high energy transfer line density, high relative biological effect, small post-damage repair effect, and good spatial resolution of energy deposition. It can induce genetic variation in seeds and obtain a higher mutation rate under a higher survival rate. Most variant traits are stable quickly, which can accelerate the breeding process and help increase the diversity of genetic resources. However, the research and application of heavy ion radiation mutagenesis in millet is still blank. Summary of the invention

[0004] One of the purposes of the present invention is to provide a millet mutation breeding method with high mutation rate under the condition of high survival rate.

[0005] The present invention provides a millet mutation breeding method, which comprises: (1) Use heavy ion beam to irradiate millet dry seeds to obtain M1 generation seeds; the energy is 78-82MeV / u, the linear energy transfer density LET=29-31KeV / μm, the irradiation dose is 150-200Gy, the dose rate is 38-42Gy / min, and the irradiation time is 42-47min; (2) planting the M1 generation seeds, harvesting all surviving plants individually at the harvest time, and threshing to obtain M2 generation seeds; (3) planting the M2 generation seeds, harvesting all the mutant plants individually at the harvest time, and threshing to obtain M3 generation seeds; (4) Plant the M3 generation seeds of each variant plant, harvest the plants with stable traits individually at the harvest time, and thresh them to obtain the M4 generation seeds.

[0006] The present invention has found a millet mutation breeding method that can produce a high mutation rate under the condition of a high survival rate by exploring the parameters of millet heavy ion beam irradiation. The offspring has abundant mutant materials and many beneficial types, and excellent mutant materials with increased yield, lodging resistance, staying green, early maturity, good rice quality, etc. can be screened out. At the same time, extreme mutants with research value such as leaf color, awn color, and ear type variation can be obtained, which is beneficial to accelerate the millet breeding process.

[0007] In the present invention, millet varieties with wide adaptability and excellent comprehensive characteristics are preferably used as irradiation materials. The time for loading and changing samples during heavy ion beam irradiation is 10 minutes.

[0008] Preferably, in step (1) of the present invention, two irradiation doses (such as 150 Gy and 200 Gy) are used to irradiate millet dry seeds respectively to obtain two M1 generation seeds prepared by different irradiation doses. In this case, the parents of the M2 generation seeds in step (2) include two M1 generations prepared by different irradiation doses.

[0009] In step (1) of the method of the present invention, dry millet seeds are first spread in a single layer in a sulfuric acid paper bag, which is then sealed and irradiated with a heavy ion beam.

[0010] In the specific operation, you can first spread the dry millet seeds in a single layer on a 20-25cm 2 The cells were placed in a square sulfuric acid paper bag, sealed with invisible tape on all sides, and then placed in a cell culture dish with a diameter of 60 mm and irradiated with a heavy ion beam.

[0011] The present invention has found that due to the small size and weight of millet seeds, static electricity is easily generated when they are placed in irradiated culture dishes during irradiation sample preparation, causing the seeds to adhere to the dish wall and dish cover, making it impossible to achieve single-layer flat irradiation, thereby affecting the mutagenesis effect. Through experimental exploration, the present invention has found that placing seeds in sulfuric acid paper bags can effectively improve the mutagenesis effect.

[0012] In step (1) of the method of the present invention, the number of seeds irradiated by heavy ion beam is 600-750.

[0013] In the method of the present invention, the type of heavy ion beam is 20 Ne 10+ , 12 C 6+ , 16 O 8+ One of the ions, preferably a carbon ion.

[0014] In steps (2) to (4) of the method of the present invention, the plant spacing when planting seeds is 10-20 cm, and the row spacing is 30-40 cm.

[0015] In steps (2) to (4) of the method of the present invention, wild millet is planted as a control.

[0016] In step (3) of the method of the present invention, the M3 generation seeds are marked according to the variant traits of the variant plants.

[0017] In step (4) of the method of the present invention, 280-350 plants of each variant plant are planted.

[0018] Step (4) of the method of the present invention also includes investigating the uniformity and yield of the plants with stable traits at the seedling stage, heading stage and maturity stage.

[0019] After step (4), technicians in this field may also conduct yield, quality, and resistance identification tests on the obtained excellent stable variant strains, further detect genetic stability using ISSR molecular markers, and further conduct tasting and variety comparison tests and multi-point regional production tests on the stable strains.

[0020] The present invention also provides application of the method in improving the mutation rate of millet induced breeding.

[0021] The beneficial effects of the present invention are at least: The present invention utilizes specific heavy ion beam conditions to induce millet mutation, which can increase the mutation rate while maintaining a high survival rate, and produce abundant mutant materials in offspring, thus providing a new technical approach for breeding high-quality, high-yield, multi-resistant and widely adaptable millet new varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the breeding process of new millet varieties induced by heavy ion radiation.

[0023] Figure 2 These are some mutant phenotypes of millet induced by heavy ion irradiation. DETAILED DESCRIPTION

[0024] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0025] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available or prepared according to conventional methods in the art unless otherwise specified.

[0026] Example 1 This embodiment provides a method for heavy ion beam irradiation millet mutation breeding (see the breeding process diagram Figure 1 ), as follows: (1) In 2018, the millet variety "Yugu 24" with wide adaptability was selected as the material for heavy ion irradiation; (2) Using the Heavy Ion Research Facility (HIRFL) of the Institute of Modern Physics, Chinese Academy of Sciences, several dried seeds of Yugu 24 were carefully selected and spread in a single layer on a 25 cm 2 The seeds were placed in a square sulfuric acid paper bag, sealed with invisible tape on all sides, and then placed in a cell culture dish with a diameter of 60 mm. Two irradiation dose groups were set up, with three repetitions in each group, and a total of 650 seeds in each dose. Carbon ion beam was used for irradiation, with an energy of 80 MeV / u, a linear energy transfer density of LET=30 KeV / μm, irradiation doses of 150 Gy and 200 Gy, respectively, a dose rate of 40 Gy / min, an irradiation time of 45 min, and a loading and changing time of 10 min; (3) The M1 generation seeds irradiated in step (2) were used as controls, and the "Yugu 24" seeds in step (1) were sown normally with a plant spacing of 20 cm and a row spacing of 40 cm. Field investigation showed that the physiological damage was serious, and the survival rate was about 68% for the irradiation dose of 150 Gy, and about 53% for the irradiation dose of 200 Gy. Maturity variation occurred and was marked with a label number. All surviving plants were harvested, threshed and preserved to obtain the M2 generation; (4) In mid-April 2019, all M2 generation plants were planted in rows (plant spacing 20 cm, row spacing 40 cm), totaling 3,100 plants. At the same time, the control "Yugu 24" seeds were planted. The M2 generation showed obvious trait separation, with a total mutation rate of 10.23%. During the entire growth period, the plants with mutant phenotypes were numbered and labeled compared with the control. At the harvest time, 317 mutant plants were obtained according to the variable traits such as leaf color, awn color, panicle type, plant height, growth period, etc. and with reference to the control. All mutant plants were harvested, threshed, labeled and stored, and indoor seed testing was carried out to obtain the M3 generation; (5) In mid-April 2020, the M3 generation (317 mutant plants harvested) continued to be planted in rows (plant spacing 20 cm, row spacing 40 cm), with about 300 plants of each mutant plant planted, and the control "Yugu 24" was planted at the same time. The field survey of the variant traits of the M3 generation was carried out throughout the growth period, and the phenotypes at the seedling stage, heading stage and maturity stage were investigated. For the M3 generation materials with stable traits (i.e., the uniformity of each phenotype at each stage was >90%), field records were made and marked. In this example, 15 strains with stable traits were finally harvested (representative photos are shown in Figure 2 ), including 2 yellow leaf color variants (M1), 2 striped leaf color variants (M2), 3 awn color variants, 2 early maturity variants (M4-M5), 1 dwarfing variant, 2 lemma color variants (M6-M7) and 3 panicle variant materials (M8-M9). The stable strains at each maturity stage were sampled and measured for yield according to the plot, and harvested according to the strains; the M3 generation variant plants with separated traits were numbered and listed, and the individual plants were harvested and threshed at maturity, and tested indoors to obtain the M4 generation.

[0027] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A millet mutation breeding method, characterized in that: include: (1) Use heavy ion beam to irradiate millet dry seeds to obtain M1 generation seeds; the energy is 78-82MeV / u, the linear energy transfer density LET=29-31KeV / μm, the irradiation dose is 150-200Gy, the dose rate is 38-42Gy / min, and the irradiation time is 42-47min; (2) planting the M1 generation seeds, harvesting all surviving plants individually at the harvest time, and threshing to obtain M2 generation seeds; (3) planting the M2 generation seeds, harvesting all the mutant plants individually at the harvest time, and threshing to obtain M3 generation seeds; (4) Plant the M3 generation seeds of each variant plant, harvest the plants with stable traits individually at the harvest time, and thresh them to obtain the M4 generation seeds.

2. The method according to claim 1, characterized in that In step (1), dry millet seeds are first spread in a single layer in a sulfuric acid paper bag, which is then sealed and irradiated with a heavy ion beam.

3. The method according to claim 1 or 2, characterized in that: In step (1), the number of seeds irradiated by the heavy ion beam is 600-750.

4. The method according to any one of claims 1 to 3, characterized in that: The heavy ion beam type is 20 Ne 10+ , 12 C 6+ , 16 O 8+ One of them.

5. The method according to any one of claims 1 to 4, characterized in that: In steps (2) to (4), the spacing between plants when planting seeds is 10-20 cm, and the spacing between rows is 30-40 cm.

6. The method according to any one of claims 1 to 5, characterized in that: In steps (2) to (4), wild-type millet was planted as a control.

7. The method according to any one of claims 1 to 6, characterized in that: In step (3), the M3 generation seeds are labeled according to the variant traits of the variant plants.

8. The method according to any one of claims 1 to 7, characterized in that: In step (4), 280-350 plants of each variant are planted.

9. The method according to any one of claims 1 to 8, characterized in that: Step (4) also includes investigating the uniformity and yield of plants with stable traits at the seedling stage, heading stage and maturity stage.

10. Use of the method according to any one of claims 1 to 9 in improving the mutation rate of millet induced breeding.

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