Wheat EMS mutation breeding method

Abstract

The invention relates to the technical field of mutation breeding, and particularly discloses a wheat EMS mutation breeding method which comprises the following steps: S1, seed pretreatment: soaking wheat seeds in water to activate embryo tissues; s2, EMS mutagenesis treatment: performing EMS mutagenesis on the wheat seeds by regulating and controlling the concentration of a mutagenic agent, the treatment time and the operation environment; s3, seed washing and germination determination: washing with running water to remove residual EMS, and then determining germination potential and germination rate; s4, physiological property detection of the seedlings: measuring physiological and biochemical indexes of the wheat seedlings under different mutagenesis conditions, and optimizing mutagenesis parameters; by optimizing wheat EMS mutagenesis conditions, the mutation point rate is increased, more mutant single plants are obtained, excellent wheat germplasm is created, rich germplasm resources are provided for cultivating new wheat varieties, and the breeding period is shortened; abundant mutation materials are provided for mining of candidate genes and research of functional genes, and a foundation is laid for research of functional genomics.

Description

Technical Field

[0001] The invention belongs to the technical field of mutation breeding, and in particular relates to a wheat EMS mutation breeding method. Background Art

[0002] Ethyl methyl sulfone (EMS) is a common alkylating agent composed of four elements: C, H, O, and S, with a molecular formula of C3H8O3S. In the 1950s, scientist Cormack first discovered that EMS could induce plant mutations. Since then, EMS has been widely used in mutation breeding by plant researchers and has achieved a lot of research results.

[0003] EMS-induced mutations are mainly completed in two steps. First, the N-7 position of guanine (G) is alkylated to become a positively charged quaternary ammonium group, resulting in two genetic effects: one is a conversion mutation, in which the alkylated guanine (G) no longer pairs with cytosine (C), causing the G≡C base pair to become a T=A base pair; the other is a transversion mutation. After the N-7 position of guanine is alkylated, the glycosidic bond breaks, causing depurination, and the position is missing. In the subsequent DNA molecule replication process, the four bases may enter their complementary positions and cause substitution. If base substitution occurs in the polypeptide coding region, it may affect the codons and cause changes in transcription and translation of genetic information, replacing the original amino acid with one amino acid; a stop codon may also occur to interrupt the synthesis of the polypeptide chain, and the original protein cannot be formed and completely loses a certain biological activity. In addition, mutagens can also react with the phosphate of the nucleoside structure to form esters and cut the nucleotides from the phosphate and sugar molecules, resulting in chromosome deletions.

[0004] The wide application range, high probability of mutagenesis and high mutation efficiency, namely "one wide and two highs" can accurately summarize the mutagenesis advantages of EMS. Compared with other mutagens, EMS has a higher mutagenesis frequency, produces relatively more dominant mutants, and is easier to screen. EMS mutagenesis has little effect on chromosomal teratogenesis, mainly causing a higher frequency of point mutations, which can improve certain traits and quality of crops.

[0005] In recent years, EMS mutagenesis technology has been studied and applied on a large scale at home and abroad. The research on mutagenesis breeding using EMS lags far behind model plants such as Arabidopsis and rice. The most important prerequisite for successful mutagenesis is to determine the optimal mutagenesis dose. The mutagenesis conditions of different crops are not the same. The present invention determines that the mutagenesis population using the optimal treatment combination conditions can produce more excellent variations while maintaining the characteristics of the original variety. This method improves the application in wheat breeding.

[0006] In this regard, the inventors proposed a wheat EMS mutation breeding method to solve the above problems. Summary of the invention

[0007] The object of the present invention is to provide a wheat EMS mutation breeding method to solve the problems raised in the above background technology.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] A wheat EMS mutation breeding method comprising:

[0010] S1. Seed pretreatment: soak wheat seeds in water to activate embryo tissue;

[0011] S2. EMS mutagenesis: wheat seeds were subjected to EMS mutagenesis by regulating the mutagen concentration, treatment time and operating environment;

[0012] S3, seed washing and germination determination: after washing with running water to remove residual EMS, the germination potential and germination rate were determined;

[0013] S4. Detection of physiological characteristics of seedlings: Determine the physiological and biochemical indicators of wheat seedlings under different mutagenesis conditions, optimize mutagenesis parameters, and screen suitable mutagenesis treatment conditions.

[0014] Preferably, large, full and undamaged wheat seeds are soaked in distilled water at room temperature for 12 hours to allow the seeds to reach an optimal water absorption state.

[0015] Preferably, the EMS mutagenesis treatment comprises:

[0016] Prepare five concentrations of EMS solutions: 0.3%, 0.6%, 0.9%, 1.2%, and 1.5%;

[0017] Set five processing times: 6 hours, 8 hours, 10 hours, 12 hours, and 14 hours;

[0018] Operate on ice and place in an oscillator with a speed controlled at 60-115 r / min to avoid volatilization of the mutagen and improve processing accuracy.

[0019] Preferably, the germination assay measures the germination potential on the 4th day after sowing, records the germination rate on the 8th day, and uses a concentration and time combination with a lethality close to 50% as the appropriate mutagenesis condition.

[0020] Preferably, the concentration and time combination for 50% lethality is 1.2%, 12 hours or 1.5%, 10 hours.

[0021] Preferably, the detection of physiological characteristics of seedlings includes:

[0022] Determination of proline (Pro), malondialdehyde (MDA) content and superoxide dismutase (SOD), peroxidase (POD) activities;

[0023] It was found that when the EMS concentration was 1.2% and the treatment time was 12 hours, the Pro and MDA contents increased significantly, and the SOD and POD activities reached the highest, indicating that this was the optimal mutagenesis treatment condition.

[0024] Preferably, the optimization of mutagenesis parameters includes optimizing EMS concentration and treatment time to increase mutation rate, obtain more excellent germplasm resources, and provide material support for the breeding of new wheat varieties and functional gene research.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention optimizes wheat EMS mutagenesis conditions, improves the mutation point rate, obtains more mutant individual plants, creates excellent wheat germplasm, provides rich germplasm resources for breeding new wheat varieties, and shortens the breeding period; provides rich mutation materials for the mining of candidate genes and the research of functional genes, and lays a foundation for the research of functional genomics. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A flow chart of a wheat EMS mutation breeding method of the present invention;

[0028] Figure 2 This is an experimental diagram of seed germination under 1.2% EMS concentration for 12 hours of treatment according to the present invention;

[0029] Figure 3 This is an experimental diagram of malondialdehyde content under different treatments in Example 1 of the present invention;

[0030] Figure 4 This is a graph showing the proline content under different treatments in Example 1 of the present invention;

[0031] Figure 5 This is a graph of SOD activity under different treatments in Example 1 of the present invention;

[0032] Figure 6 This is a diagram of POD activity under different treatments in Example 1 of the present invention. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Embodiment 1:

[0035] See also Figures 1 to 6 As shown, a wheat EMS mutation breeding method comprises:

[0036] like Figure 1 As shown, five concentrations of EMS solution were prepared: 0.3%, 0.6%, 0.9%, 1.2%, and 1.5%, and distilled water was added as a control. Select large, plump, and undamaged wheat seeds of the season, and soak the seeds in a centrifuge tube filled with distilled water at room temperature for 12 hours. The pre-soaked seeds are sensitive to the absorption of mutagens, and the absorption rate of mutagens reaches the most suitable rate, so that more mutagens enter the embryonic tissue in the shortest time. Set up 5 concentration gradients and 5 time gradients. After the wheat seeds are drained, add five concentrations of EMS phosphate buffer solution, such as 0.3%, 0.6%, 0.9%, 1.2%, and 1.5%, and distilled water as a control in the centrifuge tubes. This operation is performed on ice to reduce the volatilization of EMS and ensure the accuracy of the experiment. After sealing, wrap it in a black plastic bag (dark treatment to prevent contamination and light decomposition of EMS), place it on a shaker for oscillation, and induce treatment for 6h, 8h, 10h, 12h, and 14h respectively. Set a control for each time treatment, and set the rotation speed to 60-115r / min. Pour the induced seeds of different concentration gradients into a gauze bag and rinse them under tap water for 3-12h to remove the residual EMS on the seed embryo and prevent the mutagen from having adverse effects on the seeds. After rinsing, dry the seeds for a short time and sow them in a culture dish. Measure the germination potential on the 4th day after sowing, and record the germination rate on the 8th day. Use a drug close to the semi-lethal dose as the appropriate dose for the formal experiment. Figure 2 As shown, the statistical germination rate determined that the treatments of 1.2%, 12 h and 1.5%, 10 h were closest to 50% lethality;

[0037] Table 1 below shows the seed germination rate and germination potential under different treatments.

[0038]

[0039]

[0040] Table 1

[0041] Seedling physiological characteristics detection:

[0042] The physiological characteristics of wheat seedlings under different concentrations and time treatments were determined, and the changes in the proline (Pro) and malondialdehyde (MDA) contents and superoxide dismutase (SOD) and peroxidase (POD) activities of wheat seedlings under different EMS concentrations and time induction were preliminarily verified. Compared with the control, the MDA and Pro contents in wheat leaves increased significantly under EMS treatment, and increased with the increase of EMS concentration and time, but when the concentration reached 1.5%, the MDA and Pro contents showed a downward trend with the increase of time ( Figure 3 , Figure 4 ). This indicates that too high a concentration and too long a treatment time will cause irreversible damage to wheat seedlings.

[0043] EMS is equivalent to an adverse environment that affects the germination and growth of wheat seeds. With the increase of EMS concentration and time, the increase of SOD and POD activities showed a trend of first increasing and then decreasing. When the EMS concentration was 1.2% and the treatment time was 12h, the SOD activity reached the highest level and the POD activity was also at a high level ( Figure 5 , Figure 6 ).

[0044] The determination of physiological indices of wheat seedlings further proved that the wheat seedlings could adapt to the damage of EMS when the concentration was 1.2% and the treatment time was 12h, which was the best treatment condition.

[0045] As can be seen from the above, through the present invention, the EMS mutagenesis conditions of wheat are optimized, the mutation point rate is increased, more mutant individual plants are obtained, and excellent wheat germplasm is created, which provides rich germplasm resources for cultivating new wheat varieties and shortens the breeding period; it provides rich mutant materials for the mining of candidate genes and the research of functional genes, and lays a foundation for the research of functional genomics.

[0046] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples, unless they are contradictory.

[0047] In the drawings of the embodiments disclosed in the present invention, only the structures related to the embodiments disclosed in the present invention are involved, and other structures can refer to the general design. In the absence of conflict, the same embodiment and different embodiments of the present invention can be combined with each other.

[0048] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A wheat EMS mutation breeding method, characterized in that: The following steps are involved: S1. Seed pretreatment: soak wheat seeds in water to activate embryo tissue; S2. EMS mutagenesis: wheat seeds were subjected to EMS mutagenesis by regulating the mutagen concentration, treatment time and operating environment; S3, seed washing and germination determination: after washing with running water to remove residual EMS, the germination potential and germination rate were determined; S4. Detection of physiological characteristics of seedlings: Determine the physiological and biochemical indicators of wheat seedlings under different mutagenesis conditions, optimize mutagenesis parameters, and screen suitable mutagenesis treatment conditions.

2. A wheat EMS mutation breeding method according to claim 1, characterized in that, The seed pretreatment comprises: soaking wheat seeds with large, full and undamaged grains in distilled water at room temperature for 12 hours to make the seeds reach an optimal water absorption state.

3. A wheat EMS mutation breeding method according to claim 1, characterized in that, The EMS mutagenesis treatment includes: Prepare five concentrations of EMS solutions: 0.3%, 0.6%, 0.9%, 1.2%, and 1.5%; Set five processing times: 6 hours, 8 hours, 10 hours, 12 hours, and 14 hours; Operate on ice and place in an oscillator with a speed controlled at 60-115 r / min to avoid volatilization of the mutagen and improve processing accuracy.

4. A wheat EMS mutation breeding method according to claim 1, characterized in that, The germination assay measures germination potential on the 4th day after sowing, records germination rate on the 8th day, and uses a concentration and time combination that is close to 50% lethal as the appropriate mutagenesis condition.

5. A wheat EMS mutation breeding method according to claim 4, characterized in that, The concentration and time combinations for 50% lethality were 1.2%, 12 hours or 1.5%, 10 hours.

6. A wheat EMS mutation breeding method according to claim 1, characterized in that, The seedling physiological characteristics detection comprises: Determination of proline (Pro), malondialdehyde (MDA) content and superoxide dismutase (SOD), peroxidase (POD) activities; It was found that when the EMS concentration was 1.2% and the treatment time was 12 hours, the Pro and MDA contents increased significantly, and the SOD and POD activities reached the highest, indicating that this was the optimal mutagenesis treatment condition.

7. A wheat EMS mutation breeding method according to claim 1, characterized in that: The optimization of mutagenesis parameters includes optimizing EMS concentration and treatment time to increase mutation rate and obtain more excellent germplasm resources.

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