Method for efficiently inducing genomic variation in rice microspore sexual cells

Abstract

A method for efficiently inducing genomic variation in rice microspore sexual cells, relating to the technical field of plant cell engineering. In the method, a rice male gamete (early uninucleate microspore) is used as a mutagenic material, it is attempted, for the first time, to irradiate rice microspore sexual cells with heavy ions (4 Gy) and γ rays (10 Gy), experimental technologies such as fluorescence microscope observation, FDA-PI dyeing, and immunofluorescence detection are taken into account to determine three indicators comprising a target period for irradiation sample sampling, an appropriate heavy ion / γ ray radiation dose, and an appropriate sampling time after the end of radiation, and a relatively complete rice microspore sexual cell mutagenesis system is established. The method involves clear indicators, strong operability, can support batch mutagenesis operations, and can provide a technical system support for the efficient batch production of rice sexual cell mutants.

Description

Method for efficiently inducing genome mutation of rice microspore cells TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant cell engineering, and particularly relates to a method for efficiently inducing genome mutation of rice microspore cells. BACKGROUND

[0002] Mutation breeding is an effective breeding method for creating high-quality genes that do not exist in nature and safely using them in breeding by irradiating plant materials with rays to induce mutations in plants and then expanding the range of breeding materials that can be screened. In mutation breeding, gamma rays and heavy ions are the two most commonly used irradiation mutation methods.

[0003] Gamma rays are a kind of ionizing radiation with high penetration ability and strong killing ability to cells, and are the most commonly used in various plant mutation technologies. The range of materials that can be selected for irradiation is relatively the most complete, from seeds, bulbs (corms, bulbs and tubers, etc.), scions to whole vegetative bodies (whole plants), buds (flower buds, branch buds and axillary buds, etc.), pollen and callus, etc. Unlike chemical mutagens, gamma rays mainly induce SNPs, InDels and SVs. Gamma ray irradiation has the advantages of simple operation, convenience, fastness and low cost.

[0004] Heavy ions refer to positively charged atomic nuclei after nitrogen, carbon and other atoms are stripped or partially stripped of their outer electrons. Heavy ion mutagenesis has the biological advantages of small damage repair effect, high relative biological effect, large transmission energy density and good energy deposition spatial resolution, and can achieve good mutation effect under the condition of high survival rate of mutation materials. It has been reported that the total mutation efficiency induced by heavy ion beam radiation is about 10 times higher than that of common low-energy rays, and the efficiency of chromosomal mutation rearrangement and induced point mutation is 13-14 times higher than that of common low-energy rays.

[0005] Microspore culture is an effective method for obtaining haploid or double haploid plants. As haploids, microspores are easy to induce gene mutations and double haploids, and thus quickly obtain pure lines of excellent mutant genes and speed up the breeding process, which has good application prospects in plant breeding and basic research.

[0006] Therefore, creating a method for efficiently inducing genome mutation of rice microspore cells is beneficial to creating a large number of mutations in rice haploid cells, thereby enriching the source of mutation genes for mutation breeding and speeding up the process of mutation breeding.

[0007] SUMMARY

[0008] To solve the above technical problems, the application provides a method for efficiently inducing genome variation of rice microspore cells.

[0009] To achieve the above purpose, the application adopts the following technical scheme:

[0010] The application provides a method for efficiently inducing genome variation of rice microspore cells, comprising the following steps:

[0011] S1, culturing anthers using a modified N6 medium, and then separating microspores therefrom as mutagenesis objects;

[0012] S2, selecting microspores at a tetrad stage or a mononuclear early stage, and irradiating and mutagenizing the microspores using heavy ions and / or gamma rays;

[0013] S3, sampling and detecting genome variation 12 hours after the irradiation ends.

[0014] Preferably, the anthers in the step S1 are taken from rice ears at a differentiation stage of 6-8.

[0015] Preferably, the modified N6 medium in the step S1 has the following formula:

[0016] Further preferably, the modified N6 medium in the step S1 has the following formula:

[0017] Preferably, the microspores at the mononuclear early stage are selected in the step S2 for irradiation mutagenesis.

[0018] Preferably, when heavy ions are used for irradiation mutagenesis in the step S2, the irradiation energy of the heavy ions is 75-85 MeV / u, the dose rate is 0.8-1.2 Gy / min, and the dose gradient is 3-5 Gy.

[0019] Further preferably, the irradiation energy of the heavy ions is 80.55 MeV / u, the dose rate is 1 Gy / min, and the dose gradient is 4 Gy.

[0020] Preferably, when using γ-rays for irradiation mutagenesis in step S2, the irradiation source is... 60 Co irradiation source, dose rate 0.50–0.70 Gy / min, dose gradient 8–12 Gy.

[0021] More preferably, the dose rate of the irradiation source is 0.66 Gy / min, and the dose gradient is 10 Gy.

[0022] The beneficial effects of this invention are:

[0023] The most significant innovation of this invention lies in:

[0024] This invention uses rice male gametes (early mononuclear microspores) as mutagenic material and, for the first time, attempts to irradiate rice microspore sex cells with heavy ions (4 Gy) and gamma rays (10 Gy). Combining fluorescence microscopy, FDA-PI staining, and immunofluorescence detection, three key indicators were determined: the target sampling period, the appropriate heavy ion radiation dose, and the appropriate sampling time after irradiation. A relatively complete rice microspore sex cell mutagenesis system was established. This invention has clear technical indicators, strong operability, and allows for batch mutagenesis operations, providing technical support for the efficient and large-scale creation of rice sex cell mutants.

[0025] II. Other innovative aspects of this invention are reflected in:

[0026] (1) In previous studies, the rice materials used for radiation-induced mutation breeding were mostly diploid materials such as seeds. Since diploid materials are prone to heterozygous mutations, it is still necessary to obtain homozygous lines with mutant genes through methods such as line separation and homozygous aggregation, which is time-consuming and laborious.

[0027] This invention uses rice microspore cells for mutagenesis. Since microspores are haploid materials, there is no problem of heterozygous mutation in the mutagenesis results, and there is no need to carry out the breeding step of gene homozygosity. By means of chromosome doubling or hybridization with "haploid inducible lines", homozygous lines of mutant genes can be obtained quickly.

[0028] (2) Previous studies on the selection of radiation mutagenesis materials for rice sex cells have rarely gone into the determination of the specific microspore development stage, especially considering the influence of the cell wall on the mutagenesis effect. As a result, the particle radiation is blocked by the cell wall, which has an adverse effect on the mutagenesis effect on the genome.

[0029] This invention provides a more detailed supplement to the technical system of heavy ion mutagenesis of rice microspores, explores the mutagenic effects of different types of radiation (heavy ion radiation and gamma rays), clarifies the morphological characteristics of microspores at different developmental stages, determines that heavy ion radiation has a better mutagenic effect on early mononuclear microspores, and establishes an experimental system for inducing genomic variations in rice microspore sex cells.

[0030] (3) Previous studies have mostly disinfected the young rice panicles and directly extracted the anthers and isolated the microspores, with little anther culture. In this culture process, it is considered to improve the culture medium formula to increase the anther activity, so as to facilitate the extraction of microspores with higher growth viability for mutagenesis.

[0031] This invention discloses an improved N6 liquid culture medium for culturing retrieved anthers, enhancing the viability of microspores. The medium's formulation fully considers the anthers' requirements for macro- and micro-elements, iron salts, and organic matter, while additionally adding nutrients such as serine, glutamine, and inositol to promote rapid anther recovery and growth, thereby facilitating the extraction of highly viable microspores and ensuring a certain survival rate of the material after radiation treatment.

[0032] (4) Previous studies have rarely studied the sampling time after radiation mutagenesis treatment. Many studies take samples immediately after radiation for subsequent experiments. However, there are few studies on whether DNA double-strand breaks (DSBs) have occurred and been repaired in the genome after the organism is mutated. This makes it impossible to obtain suitable samples for subsequent studies when the mutagenesis recovery effect is optimal.

[0033] This invention uses phosphorylated γH2AX protein to label DSB sites, and combines immunofluorescence and FDA-PI staining techniques to track the repair of DSBs in microspores 1-12 hours after mutagenesis. Considering that a higher γH2AX expression level (reflected in the number of red fluorescent dots in the image) indicates more DSBs to be repaired, and vice versa, indicating fewer DSBs to be repaired and that DSB repair is essentially complete, the sampling time was chosen 12 hours after mutagenesis, when the red fluorescent dots had largely disappeared. This ensures that the site variations in the samples are truly stable.

[0034] (5) Previous studies have directly extracted and sequenced nucleic acids after inducing mutations in rice materials, with little histological analysis of the mutagenic effects of the materials before sequence detection, resulting in insufficient clarity of the overall mutation status of the materials in subsequent experiments.

[0035] This invention combines immunofluorescence and FDA-PI staining techniques to identify the occurrence and repair of DSBs 1-12 hours after mutagenesis using phosphorylated γH2AX protein labeling. This clarifies the real and stable site variations occurring inside microspores after mutagenesis, enabling more accurate subsequent sequencing analysis of the mutated materials. Attached Figure Description

[0036] Figure 1 shows the staining results of microspore cell wall tissue at different stages in Example 1 of the present invention;

[0037] Figure 2 shows the cell survival under heavy ion and γ-ray irradiation in Example 1 of the present invention, wherein: a. activity identification of early mononuclear microspores; b. activity identification of late mononuclear microspores;

[0038] Figure 3 shows the repair status of DSBs at different time points in Embodiment 1 of the present invention. Detailed Implementation

[0039] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The products, reagents, instruments, and equipment used in the following examples are all commercially available, and the methods used, unless otherwise specified, are consistent with conventional methods.

[0040] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0041] Example 1

[0042] I. Sampling of Irradiated Materials

[0043] Methods: Rice panicles at the 7th-8th stage of panicle differentiation were collected, surface-sterilized with 70% alcohol, wrapped in clean, damp gauze, and sealed at 4℃ for 7 days. After 7 days, the panicles were removed, sterilized with 3% sodium hypochlorite for 20 minutes, rinsed with sterile water, and the anthers were collected. The anthers were pre-cultured in modified N6 liquid medium for 3 days under 25℃ dark conditions at a density of 20 anthers / mL. After 3 days, the anthers were magnetically stirred at 1000 rpm until they became transparent, releasing uninucleate microspores at the periphery stage. The anther residue was filtered through a 200-mesh sieve, and the microspores were collected by centrifugation at 1000 rpm for 10 minutes. The microspores were then resuspended in fresh modified N6 liquid medium and dispensed into 3.5 cm diameter petri dishes at a density of 1×10⁻⁶. 4 The culture medium was prepared at 25°C and stored under sealed, sterile conditions. The modified N6 culture medium formulation is shown in Table 1.

[0044] Table 1. Details of the modified N6 culture medium formulation

[0045] Meanwhile, a method used in previous studies, where only disinfection was performed after harvesting rice panicles before directly collecting anthers and isolating microspores, was used as a control. Specifically, rice panicles at the 6th-7th stage of panicle differentiation were collected. Six anthers were isolated from the appropriate panicles, and the anthers were gently squeezed in 1×PBS (Phosphate-buffered saline) to release the microspores. The developmental stage of the microspores was observed under a microscope. Microspores in the early uninucleate stage were collected in 3.5 cm diameter petri dishes, and an appropriate amount of 1×PBS buffer was added to adjust the density to 1×10⁻⁶. 4 Quantity / mL, sealed and sterile at 4℃.

[0046] The viability of microspore cells obtained by the two methods was then identified using the FDA-PI fluorescence staining method. Under a fluorescence microscope, live cells appeared green, and dead cells appeared red. The FDA-PI staining method involved diluting the FDA (Fluorescein diacetate) stock solution 1000-fold with 1×PBS and the P (Ipropidium iodide) stock solution 2000-fold. The diluted FDA and PI were then mixed at a 1:1 ratio to prepare the FDA-PI working solution. 1 mL of cell suspension from each culture dish was transferred to a 1.5 mL centrifuge tube, centrifuged at 1000 rpm for 10 minutes, and the supernatant was discarded. 200 μL of the FDA-PI working solution was added, and the cells were stained at room temperature for 30 minutes. After washing three times with 1×PBS buffer, the cells were resuspended. 50 μL of cell suspension was aspirated from each tube and observed under a fluorescence microscope. Each tube was repeated three times, with 10 fields of view observed per replicate. Each field of view was photographed and recorded.

[0047] The improved N6 liquid culture medium of this invention enhances the viability of microspores when used to culture the retrieved anthers. The medium formulation fully considers the anthers' requirements for macro- and micro-elements, iron salts, and organic matter, while additionally adding nutrients such as serine, glutamine, and inositol to promote rapid anther recovery and growth, thereby facilitating the extraction of highly viable microspores and ensuring a certain survival rate of the material after radiation treatment.

[0048] II. Determine the target period for irradiation sample collection.

[0049] To determine the microspore stage without cell wall tissue suitable for single-cell genome sequencing, the cell wall tissue development of microspores at different developmental stages was observed using the japonica rice variety 02428. Microspores at different stages were stained with the fluorescent whitening agent VBL, resulting in a blue cell wall, which was observed under a fluorescence microscope. The specific method was as follows: To observe the cell wall tissue of microspores at different developmental stages, a 0.1% low-concentration solution of VBL was prepared using sterile 0.5M mannitol (pH=7.0). After complete dissolution, the solution was centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected. Microspore cells at different developmental stages were collected by centrifugation, and the sample was submerged in the fluorescent whitening agent. Staining was performed at room temperature for 5 minutes, followed by washing with 0.5M mannitol 3-4 times. The samples were then observed and photographed under a fluorescence microscope. The results are shown in Figure 1.

[0050] Staining results showed that microspores had obvious cell wall tissue during the pollen mother cell stage. No cell wall tissue was observed in the tetrad and early uninucleate stages. Cell wall tissue re-formed in the mid-uninucleate stage and remained present in microspores throughout subsequent stages. Therefore, the tetrad stage and early uninucleate stage can be used as sampling periods for single-cell sequencing. Considering the difficulty of isolating individual cells from the tetrad, this invention selected the early uninucleate stage as the target sampling period for single-cell sequencing (Figure 1).

[0051] III. Determining the appropriate heavy ion radiation dose

[0052] Microspore samples in the early mononuclear stage were irradiated with heavy ions using a high-energy ion beam provided by the Lanzhou Heavy Ion Research Facility at the Institute of Modern Physics, Chinese Academy of Sciences. 12 C 6+ Irradiation was performed at an energy of 80.55 MeV / u and a dose rate of 1 Gy / min, with four dose gradients of 2, 4, 8, and 16 Gy. The gamma-ray radiation was administered by the Guangzhou BGI Irradiation Center. 60 Co irradiation source, dose rate 0.66 Gy / min, five dose gradients of 5, 10, 20, 40, and 80 Gy were set, with each dose repeated in triplicate. Unirradiated samples were also prepared as controls.

[0053] The study aimed to explore the median lethal radiation dose for early mononuclear microspores as the optimal dose. Cell viability was assessed after irradiation with different doses, and cell survival rates under different irradiation methods and doses were statistically analyzed. The constructed dose-survival regression equation is as follows: For early mononuclear microspores irradiated with heavy ions, y = 100.82e -0.222x R 2 =0.9858, median lethal dose 3.16 Gy; early mononuclear microspores under gamma ray irradiation y = 92.5239e -0.059x R2 =0.9967, median lethal dose 10.43 Gy (y is survival rate, in %; x is heavy ion radiation dose, in Gy).

[0054] In actual operation, the dose closest to the theoretical value, as shown in Figure 2, was selected as the actual sampling dose: under heavy ion irradiation, 4 Gy irradiation of early mononuclear microspores resulted in a survival rate of 44.77%; under gamma ray irradiation, 10 Gy irradiation of early mononuclear microspores resulted in a survival rate of 52.17% (Figure 2). The actual survival rate was close to 50%, and the regression equation showed a high degree of agreement with the actual results.

[0055] IV. Determining the appropriate sampling time after radiation exposure.

[0056] The detection of plant genome variations needs to be performed after damage repair is completed. After DNA double-strand breaks (DSBs) occur, histone H2AX is phosphorylated at the Ser139 site to become γH2AX. γH2AX marks the DSB site and recruits cell cycle check and DNA repair factors to the damage site. Its protein level increases within minutes after the double-strand break and can be used as a biomarker for DSBs.

[0057] Immunofluorescence was used to observe the repair of DSBs at three time points: 1, 6, and 12 hours after irradiation. Red fluorescence indicates γH2AX staining results; more red fluorescent spots indicate more DSBs to be repaired. The specific procedure for immunofluorescence was as follows: early mononuclear microspores were flash-frozen in liquid nitrogen according to the treatment time, thawed, centrifuged, and fixed with 4% para-21 formaldehyde fixative for 30 minutes at room temperature. The cells were then washed three times with 1×PBS buffer for 5 minutes each time. 10 μL of 0.05% Triton-X-100 was added to the cell sample, and after 30 minutes, the cells were washed three times with 1×PBS. Cells were collected by centrifugation at 500 rpm for 10 minutes. An appropriate amount of cells was placed on a glass slide, covered with a coverslip, and flash-frozen in liquid nitrogen for 30 seconds. The coverslip was then quickly removed, and the cells were air-dried. The primary antibody was diluted 500-fold with antibody dilution buffer. 50 μL of the diluted primary antibody was added to the dried cell sample, and the slide was placed in a humidified chamber for hybridization at 37°C for 2 hours. Two hours later, the slide was washed three times with 1×PBS, 5 minutes each time. The secondary antibody was diluted 1000-fold with antibody dilution buffer, and 50 μL of the secondary antibody was added to the sample portion of the slide. The slide was placed in a humidified chamber and hybridized at 37°C for 1 hour. One hour later, the slide was washed three times with 1×PBS, 5 minutes each time, and then air-dried. 10 μL of DAP (I4',6-diamidino-2-phenylindole) staining solution was added to the dried slide, a coverslip was placed on top, and the slide was observed and photographed under a fluorescence microscope. The results are shown in Figure 3.

[0058] The results showed that all DSBs had been repaired 12 hours after the irradiation ended. Therefore, in order to detect all genomic variations, sampling needs to be performed 12 hours after the irradiation ended (Figure 3).

[0059] Based on the target sampling period, appropriate heavy ion / γ-ray radiation dose, and suitable sampling time after irradiation determined in this embodiment, the final mutagenesis results obtained by using heavy ion mutagenesis alone were as follows: the number of mutation sites in a single cell was between 36,041 and 41,152, and the mutagenesis rate was 0.0097-0.0110% (reference genome is Nipponbare genome MSU7.0, total base count 373,245,519 bp); the final mutagenesis results obtained by using γ-ray mutagenesis alone were as follows: the average number of mutation sites in a single cell was 11,336-12,272, and the mutagenesis rate reached 0.0030-0.0033%.

[0060] Comparative Example 1

[0061] This comparative example is a case study from the 2021 issue of the Journal of Plant Genetic Resources, titled "Phenological and genomic variation analysis of ion beam induced variant rice JD-1".

[0062] The specific process involves using a carbon ion beam provided by the Heavy Ion Research Facility in Lanzhou (HIRFL), Institute of Modern Physics, Chinese Academy of Sciences. 12 C 6+ Radiation-induced mutagenesis was conducted on the erect panicle variety Jindao 565 rice at a beam energy of 80 MeV / u, an irradiation dose rate of 60 Gy / min, and a cumulative irradiation dose of 80 Gy. M0 plants were planted in the field in Chengdu in April of that year and harvested together. In 2016, 10,000 M1 plants were planted, and one mutant with drooping panicles and other panicle traits was found, named JD-1. After multiple generations of self-pollination, JD-1 showed stable traits. During the peak tillering stage, leaves of wild-type Jindao 565 and the mutant JD-1 were collected, and DNA was extracted using the CTAB method and resequencing was performed.

[0063] The mutagenesis results showed that the mutant JD-1, compared with the wild-type Jindao 565, had a total of 22,067 mutation sites, including 18,639 single nucleotide polymorphisms (SNPs) and 3,428 small insertion-deletion sites (InDels). The mutagenesis rate was 0.0059% (reference genome: Nipponbare genome MSU7.0, total base count 373,245,519 bp).

[0064] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for efficiently inducing genome mutation of rice microspore cells, characterized in that, The method comprises the following steps: S1, culturing anthers using a modified N6 medium, and then separating microspores in the anthers as mutagenesis objects; S2, selecting microspores at a tetrad stage or a single nucleus early stage, and irradiating and mutagenizing the microspores using heavy ions and / or γ rays; S3, sampling and detecting variations of genomes 12 hours after the irradiation ends.

2. The method of claim 1, wherein, The anthers in the step S1 are taken from rice ears at a differentiation stage of 6-8.

3. The method of claim 2, wherein, The improved N6 medium formula in the step S1 is as follows:

4. The method of claim 3, wherein, The microspores at a single nucleus early stage are selected in the step S2 for irradiation mutagenesis.

5. The method of claim 4, wherein, When the heavy ions are used for irradiation mutagenesis in the step S2, the irradiation energy of the heavy ions is 75-85 MeV / u, the dose rate is 0.8-1.2 Gy / min, and the dose gradient is 3-5 Gy.

6. The method of claim 5, wherein, The irradiation energy of the heavy ions is 80.55 MeV / u, the dose rate is 1 Gy / min, and the dose gradient is 4 Gy.

7. The method of claim 4, wherein, The irradiation mutagenesis in the step S2 uses a gamma ray as the irradiation source 60 Co irradiation source, dose rate is 0.50-0.70 Gy / min, dose gradient is 8-12 Gy.

8. The method of claim 7, wherein, The dose rate of the irradiation source is 0.66 Gy / min, and the dose gradient is 10 Gy.

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