A kind of 60 Method for breeding salt-tolerant Miscanthus sinensis mutants induced by Co-γ radiation

Abstract

The present invention belongs to the field of agricultural planting technology and specifically relates to a method based on 60 The method for cultivating salt-tolerant Miscanthus sinensis mutants induced by Co-γ radiation comprises the following steps: S1 seed material disinfection: drying Miscanthus sinensis seeds and then soaking them, washing them after soaking, adding a disinfectant to soak and disinfect them, washing and removing water after disinfection to obtain sterile seed materials; S2 radiation mutagenesis: inoculating the sterile seed materials onto a germination medium, selecting 60 Co-γ radiation induces mutation in seed material, and the mutated seed material is transplanted into a screening medium for screening and cultivation to germinate salt-tolerant Miscanthus sinensis seedlings; S3 Miscanthus sinensis seedling cultivation: the salt-tolerant Miscanthus sinensis seedlings are transplanted into a seedling-strengthening medium for cultivation until the seedling buds emerge and then transplanted; S4 Miscanthus sinensis seedling transplanting: the salt-tolerant Miscanthus sinensis seedlings in S3 are transplanted into a cultivation medium for cultivation to obtain salt-tolerant Miscanthus sinensis mutants. The Miscanthus sinensis cultivated by the present invention is cultured in a special culture medium and cultivation medium to ensure the germination rate and survival rate of the mutants, thereby cultivating a Miscanthus sinensis mutant with strong salt tolerance.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural planting technology and specifically relates to a method based on 60 A method for cultivating salt-tolerant Miscanthus sinensis mutants induced by Co-γ radiation. Background Art

[0002] Miscanthus sinensis is an excellent ornamental grass with advantages such as fast growth, high yield, long flower spikes, and beautiful leaves. As a lignocellulosic crop with the greatest development potential, Miscanthus sinensis has attracted considerable attention from scholars both domestically and internationally. Cultivating high-quality Miscanthus germplasm from existing germplasm is a prerequisite for efficient cultivation and industrialization.

[0003] Miscanthus has well-developed underground rhizomes, reaching depths exceeding 1 meter, and possesses a strong soil-holding capacity. Furthermore, its woody rhizomes make it more tolerant to salt stress than other herbs. Therefore, Miscanthus is also cultivated in saline-alkali lands, helping to control and improve their ecology.

[0004] In summary, the cultivation of a new salt-tolerant and high-yielding Miscanthus sinensis variety is of great significance in the comprehensive utilization of saline-alkali land. Summary of the Invention

[0005] In view of the above-mentioned problems existing in the current Miscanthus, the present invention provides a 60 A method for cultivating salt-tolerant Miscanthus sinensis mutants induced by Co-γ radiation. 60 Co-γ radiation induced salt-tolerant mutants were produced. Using a special culture medium and cultivation matrix, the germination rate and survival rate of the mutants were guaranteed, resulting in the development of a Miscanthus sinensis mutant with strong salt tolerance. The specific technical solution is as follows:

[0006] First, the present invention provides a 60 The method for cultivating salt-tolerant Miscanthus sinensis mutants induced by Co-γ radiation is characterized by comprising the following steps:

[0007] S1 Seed material disinfection: drying the Miscanthus seeds and then soaking them, washing them after soaking, adding a disinfectant to soak and disinfect them, and washing and removing water after disinfection to obtain sterile seed materials;

[0008] S2 radiation mutagenesis: Inoculate the sterile seed material onto the germination medium and select 60 Co-γ radiation is used to induce mutation in seed materials, and the mutated seed materials are transferred to a screening medium for screening and cultivation to germinate salt-tolerant Miscanthus seedlings.

[0009] S3 Miscanthus seedling cultivation: The salt-tolerant Miscanthus seedlings were transplanted into the seedling-strengthening culture medium and cultured until the buds of the seedlings grew out before transplanting;

[0010] S4 transplanting Miscanthus seedlings: transplanting the salt-tolerant Miscanthus seedlings from S3 into a cultivation medium to obtain salt-tolerant Miscanthus mutants. Furthermore, in step S1, the Miscanthus seeds are dried at a temperature of 60-65° C. for 8-10 hours.

[0011] The soaking solution is 75% ethanol solution, and the soaking time is 1 to 2 minutes;

[0012] The disinfectant is a sodium hypochlorite solution, the chloride ion concentration of the sodium hypochlorite solution is 0.057%, and the disinfection time is 30 to 45 minutes.

[0013] Furthermore, the germination medium in step S2 includes MS medium, 0.1-0.2 mg / L NAA, 0.5-2.0 mg / L 6-BA, 2.0-5.0 mg / L GA3, 30.0 g / L sucrose and 8.0 g / L agar, and the pH value of the germination medium is 5.8; the germination medium is sterilized.

[0014] Furthermore, in step S2, the radiation dose is 250 to 350 Gy, and the dose rate is 1 min / Gy.

[0015] Furthermore, the screening culture medium in step S2 includes MS culture medium, 0.1-0.2 mg / L NAA, 0.5-2.0 mg / L 6-BA, 2.0-5.0 mg / L GA3, 30.0 g / L sucrose, 8.0 g / L agar and 20-30 g / L NaCl, the pH is 5.8, and the germination time is 20-30 days.

[0016] Furthermore, the seedling-strengthening culture medium in step S3 includes MS culture medium, 0.5 mg / L 6-BA, 0.1 mg / L IBA, 30.0 g / L sucrose, and 8.0 g / agar, and the pH value of the seedling-strengthening culture medium is 5.8; the culture temperature is 25-30° C., and the photoperiod is 16 h.

[0017] Furthermore, in step S4, the cultivation medium includes 33% peat soil, 33% vermiculite, and 33% perlite.

[0018] Furthermore, in step S4, the planting temperature is 25-30°C, the relative humidity is 65%, and the PAR is 800 μmol / m 2 / s, and the photoperiod is 16h.

[0019] Furthermore, step S4 also includes a salt-tolerant Miscanthus mutant evaluation step: transplanting the salt-tolerant Miscanthus mutant into a hydroponic box, adding hydroponic nutrient solution to culture and observe the phenotype of the Miscanthus mutant, and selecting relative water content, relative conductivity and wilting rate as indicators to evaluate the salt tolerance of the Miscanthus mutant.

[0020] Furthermore, the hydroponic nutrient solution includes Hoagland culture solution and 20 g / L NaCl, and the culture time is 15 days.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1) The present invention applies radiation mutagenesis technology to cultivate salt-tolerant Miscanthus sinensis. By determining the radiation dose, the invention successfully cultivates a salt-tolerant Miscanthus sinensis mutant that exhibits stronger salt tolerance than existing Miscanthus sinensis and is more suitable for cultivation in saline-alkali soils. This salt-tolerant Miscanthus sinensis mutant exhibits stronger salt resistance and survival ability in saline-alkali soils than existing Miscanthus sinensis. Furthermore, the invention introduces new germination, seedling growth, and cultivation media at each stage of cultivation to ensure the survival rate of the Miscanthus sinensis mutant, making the invention highly practical.

[0023] 2) In the prior art, screening for suitable radiation mutants is generally performed after cultivation is completed. Since radiation mutagenesis produces a large number of mutant offspring, the screening process is very tedious and requires a lot of manpower and material resources. However, the present invention introduces a screening step during the cultivation stage, screening for salt tolerance immediately after radiation-induced seed mutations, and determines the optimal NaCl screening pressure. This ensures that the germinated Miscanthus seedlings after screening have salt tolerance and can root and grow normally, effectively reducing labor input and accelerating seedling cultivation, making it very suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a graph showing the relative water content test results in Experimental Example 4 of the present invention;

[0025] Figure 2 This is a graph showing the wilting rate test results in Experimental Example 4 of the present invention;

[0026] Figure 3 This is a graph showing the relative conductivity test results in Experimental Example 4 of the present invention. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with embodiments.

[0028] Example 1

[0029] This embodiment provides a 60 The method for cultivating salt-tolerant Miscanthus sinensis mutants induced by Co-γ radiation comprises the following steps:

[0030] S1 Seed material disinfection: drying the Miscanthus seeds and then soaking them, washing them after soaking, adding a disinfectant to soak and disinfect them, and washing and removing water after disinfection to obtain sterile seed materials;

[0031] Specifically, 5,000 'Early Hybrids' Miscanthus seeds were pre-dried at 60°C for 8 hours before use. The dried seeds were soaked in 75% ethanol for 1 minute, washed twice with distilled water, and then soaked in 50 mL of sodium hypochlorite for 30 minutes. The sodium hypochlorite solution was prepared by mixing a sodium hypochlorite aqueous solution with an available chlorine concentration of 5.6% with water in a volume ratio of 1:98. The seeds were shaken 2-3 times and then rinsed three times with sterile water. The sterile seed material was then dried with sterile filter paper.

[0032] S2 radiation mutagenesis: Inoculate the sterile seed material onto the germination medium and select 60 Co-γ radiation is used to induce mutation in seed materials, and the mutated seed materials are transferred to a screening medium for screening and cultivation to germinate Miscanthus seedlings;

[0033] Specifically, the sterile Miscanthus seeds obtained in step S1 were inoculated into a germination medium. The pH of the germination medium was 5.8, and the germination medium was prepared by adding 0.1 mg / L NAA, 0.5 mg / L 6-BA, 2.0 mg / L GA3, 30.0 g / L sucrose, and 8.0 g / L agar to the MS medium, stirring well, and sterilizing. 60 Miscanthus seeds were treated with Co-γ radiation at a dose rate of 1 min / Gy.

[0034] Miscanthus sinensis seeds that had undergone radiation-induced mutation were transplanted and germinated in a screening medium containing MS medium, 0.1 mg / L NAA, 0.5 mg / L 6-BA, 2.0 mg / L GA3, 30.0 g / L sucrose, 8.0 g / L agar, and 20 g / L NaCl at a pH of 5.8. Salt-tolerant Miscanthus sinensis seedlings germinated after 25 days of culture in the screening medium.

[0035] S3 Miscanthus seedling cultivation: Miscanthus seedlings were transplanted into seedling culture medium and cultured until the buds of Miscanthus seedlings grew to 5 cm in height before transplanting;

[0036] Specifically, the Miscanthus sinensis seedlings obtained in step S2 were transferred to a seedling growth medium with a pH of 5.8, specifically, 0.5 mg / L 6-BA, 0.1 mg / L IBA, 30.0 g / L sucrose, and 8.0 g / L agar were added to the MS medium. The culture environment was a 25°C tissue culture room with a photoperiod of 16.0 h. When the green buds grew to 5.0 cm in height, they were ready for transplanting.

[0037] S4 transplanting Miscanthus seedlings: The Miscanthus seedlings in S3 were transplanted into the cultivation medium to obtain salt-tolerant Miscanthus mutants.

[0038] Specifically, the Miscanthus sinensis seedlings obtained in step S3 were transferred to 128-hole trays in a greenhouse, and the greenhouse environment was set at a relative humidity of 65% and a PAR of 800 μmol / m 2 / s, a photoperiod of 16h, and an average temperature of 30℃ / 25℃ (day / night). The cultivation medium includes 33% peat soil + 33% vermiculite + 33% perlite, and finally a salt-tolerant Miscanthus sinensis mutant is obtained.

[0039] The obtained salt-tolerant Miscanthus sinensis mutants were transferred to a hydroponic culture box for hydroculture. The hydroponic nutrient solution was Hoagland's culture medium supplemented with 20 g / L NaCl. The phenotype of the Miscanthus sinensis mutants was observed after 15 days of culture. Relative water content, relative conductivity and wilting rate were selected as indicators to quickly evaluate the salt tolerance of the Miscanthus sinensis mutants.

[0040] Experimental Example 1: Effect of Radiation Dose on Miscanthus Seed Germination

[0041] Experimental purpose: To verify the effects of different radiation doses on the germination rate and germination status of Miscanthus seeds in step S2 radiation mutagenesis

[0042] Experimental Method: The sterile Miscanthus sinensis seeds obtained in step S1 were inoculated onto the germination medium described in step S2, with 100 seeds per dish. Five radiation doses of 100, 250, 300, 350, and 500 Gy (at a dose rate of 1 min / Gy) were selected for treatment. The germination rate of the seeds was tested after 15 days. The test results are shown in Table 1 below, where germination rate = number of germinated Miscanthus sinensis seeds / total number of inoculated seeds × 100%.

[0043] Table 1: Effects of different radiation doses on the germination of Miscanthus seeds

[0044]

[0045] Analysis of the experimental results: As shown in the table above, at a radiation dose of 300 Gy, the germination rate of Miscanthus seeds approached 50%, reaching the semi-lethal dose. After mutation, most seeds germinated normally, indicating the optimal radiation dose. At radiation doses of 250 Gy and 350 Gy, the germination rates were approximately 60% and 40%, respectively, and most seeds also germinated normally. At lower radiation doses, such as 100 Gy, the germination rate of Miscanthus seeds approached 100%, indicating that the vast majority of seeds did not mutate due to radiation. At even lower radiation doses, such as 500 Gy, the germination rate was only around 20%, and the majority of seedlings failed to root, indicating that most seeds could no longer be cultivated normally.

[0046] Experimental Example 2: Screening of salt-tolerant Miscanthus seedlings

[0047] Experimental purpose: To screen out salt-tolerant Miscanthus seedlings at the germination stage

[0048] Experimental method: The sterile Miscanthus seeds obtained in step S1 were used as materials and inoculated onto the germination medium described in step S2, with 100 seeds per dish. The irradiation treatment was performed at a radiation dose of 300 Gy and a dose rate of 1 min / Gy. After treatment, the obtained mutant strains were transferred to the screening medium for germination, and an orthogonal experiment was designed to explore the effects of different hormone combinations on Miscanthus seed germination. At the same time, the germination rate of the irradiated seeds was tested by designing different concentration gradients of NaCl in the screening medium. The test results are shown in Tables 2 and 3 below, where germination rate = number of Miscanthus seeds germinated / total number of seeds inoculated × 100%.

[0049] Table 2: Effects of different hormones on Miscanthus germination rate

[0050]

[0051] Table 3: Effects of different NaCl concentrations on the germination rate of Miscanthus mutant seedlings

[0052]

[0053] Analysis of experimental results: As can be seen from the above table, the most suitable culture medium is 0.1-0.2 mg / L NAA, 0.5-2.0 mg / L 6-BA, and 2.0-5.0 mg / L GA3, and adding 20-30 g / L NaCl to the screening culture medium is close to reaching the screening critical value. The Miscanthus seeds screened at this concentration already have excellent salt tolerance.

[0054] Experimental Example 3: Effect of cultivation medium on the survival rate of salt-tolerant Miscanthus sinensis mutants

[0055] Experimental purpose: To verify the effect of different cultivation media on the survival rate of salt-tolerant Miscanthus sinensis mutants in step S4

[0056] Experimental method: The Miscanthus sinensis seedlings obtained in step S3 were transferred to 128-hole trays in a greenhouse. The greenhouse environment was 65% relative humidity and 800 μmol / m 2 / s, a 16.0h photoperiod, and an average temperature of 30°C / 25°C (day / night). Different cultivation media were selected to observe their effects on the transplant survival rate of salt-tolerant Miscanthus mutant seedlings. The experimental results are summarized in Table 3 below, where transplant survival rate = number of surviving Miscanthus seeds / total number of transplanted seedlings × 100%.

[0057] Table 3: Effects of different cultivation media on the survival rate of transplanted Miscanthus mutant seedlings

[0058]

[0059] Analysis of the experimental results: As can be seen from the table above, the use of the cultivation medium provided by the present invention ensures a 100% survival rate for the Miscanthus sinensis mutant seedlings, enhancing the success rate and practical application value of the cultivation method of the present invention. However, when a single component, such as vermiculite, peat, fine sand, or perlite, is used as the cultivation medium, the survival rate of the Miscanthus sinensis mutant seedlings decreases significantly. Even when the cultivation medium is replaced with 33% peat, 33% vermiculite, and 33% fine sand, the survival rate of the Miscanthus sinensis mutant seedlings is still lower than that of the cultivation medium formula provided by the present invention.

[0060] Experimental Example 4: Evaluation of salt tolerance of Miscanthus sinensis mutants

[0061] Experimental purpose: Rapidly evaluate the endurance performance of the Miscanthus sinensis mutant cultivated by the present invention

[0062] Experimental method: The salt-tolerant Miscanthus sinensis mutants (sd1, sd2 and sd3) cultivated by the present invention and the normally cultivated Miscanthus sinensis (wt) were transferred to a hydroponic culture box for hydroculture. The hydroponic nutrient solution was MS culture medium and 20g / L NaCl. The Miscanthus sinensis phenotype was observed for 15 days, and its relative conductivity, relative water content and wilting rate were tested. The experimental results are shown in the attached figure. Figure 1 , Attachment Figure 2 and attached Figure 3 shown.

[0063] Analysis of experimental results: The salt-tolerant Miscanthus mutant cultivated using the present invention achieved a relative moisture content of over 70% after treatment with 20g / L NaCl; the wilting rate was below 55%, with a minimum of only around 35%; and the relative electrical conductivity was around 60%. Normal Miscanthus has a relative moisture content of approximately 60%, indicating that the salt-tolerant Miscanthus mutant cultivated using the present invention has a stronger ability to retain moisture than normal Miscanthus; the wilting rate of normal Miscanthus is over 70%, indicating that the salt-tolerant Miscanthus mutant cultivated using the present invention is more suitable for growing in high-salt environments than normal Miscanthus; and the electrical conductivity of normal Miscanthus is over 80%, indicating that the salt-tolerant Miscanthus mutant cultivated using the present invention absorbs less sodium chloride than normal Miscanthus. In summary, the salt-tolerant Miscanthus mutant cultivated using the present invention has stronger salt tolerance than normal Miscanthus and is more suitable for cultivation in harsh saline-alkali soils.

[0064] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be viewed as exemplary and non-restrictive in all respects. Furthermore, it should be understood that although this specification is described in terms of implementation methods, it does not encompass only one technical solution. This narrative is provided for clarity only, and those skilled in the art should consider the specification as a whole. The technical solutions in the embodiments may also be appropriately combined to form other implementation methods that are understandable to those skilled in the art.

Claims

1. A method based on 60 The method for cultivating salt-tolerant Miscanthus sinensis mutants induced by Co-γ radiation is characterized by: The following steps are involved: S1: Seed material disinfection: dry the Miscanthus seeds and soak them, wash them after soaking, add disinfectant to disinfect them, wash and remove water after disinfection to obtain sterile seed materials; S2: Radiation mutagenesis: Inoculate the sterile seed material onto the germination medium and select 60 Co-γ radiation is used to induce mutation in seed materials, and the mutated seed materials are transferred to a screening medium for screening and cultivation to germinate salt-tolerant Miscanthus seedlings, wherein the germination medium comprises MS medium, 0.1-0.2 mg / L NAA, 0.5-2.0 mg / L 6-BA, 2.0-5.0 mg / L GA3, 30.0 g / L sucrose, and 8.0 g / L agar, the germination medium has a pH of 5.8, and the germination medium is sterilized. The radiation dose is 250-350 Gy at a dose rate of 1 min / Gy; The screening medium consists of MS medium, 0.1-0.2 mg / L NAA, 0.5-2.0 mg / L 6-BA, 2.0-5.0 mg / L GA3, 30.0 g / L sucrose, 8.0 g / L agar and 20-30 g / L NaCl, the pH of the screening medium is 5.8, and the germination time is 20-30 days; S3: Miscanthus sinensis seedling cultivation: salt-tolerant Miscanthus sinensis seedlings were transplanted into a seedling cultivation medium and cultured until the seedling buds grew out, wherein the seedling cultivation medium consisted of MS medium, 0.5 mg / L 6-BA, 0.1 mg / L 1 BA, 30.0 g / L sucrose, and 8.0 g / L agar, and the pH value of the seedling cultivation medium was 5.8; the culture temperature was 25-30°C, and the photoperiod was 16 h; S4: Transplanting Miscanthus sinensis seedlings: Transplant the salt-tolerant Miscanthus sinensis seedlings in S3 into a cultivation medium to obtain salt-tolerant Miscanthus sinensis mutants.

2. A method according to claim 1 60 The method for cultivating salt-tolerant Miscanthus sinensis mutants induced by Co-γ radiation is characterized by: In step S1, the Miscanthus seeds are dried at a temperature of 60 to 65° C. and for a drying time of 8 to 10 hours; the soaking solution is a 75% ethanol solution, and the soaking time is 1 to 2 minutes; the disinfectant is a sodium hypochlorite solution, the chloride ion concentration of the sodium hypochlorite solution is 0.057%, and the disinfection time is 30 to 45 minutes.

3. A method based on claim 2 60 The method for cultivating salt-tolerant Miscanthus sinensis mutants induced by Co-γ radiation is characterized by: In step S4, the cultivation matrix includes 33% peat soil, 33% vermiculite, and 33% perlite.

4. A method according to claim 3 based on 60 The method for cultivating salt-tolerant Miscanthus sinensis mutants induced by Co-γ radiation is characterized by: In step S4, the planting temperature is 25-30°C, the relative humidity is 65%, and the PAR is 800 μmol / m 2 / s, and the photoperiod is 16h.

5. A method based on claim 4 60 The method for cultivating salt-tolerant Miscanthus sinensis mutants induced by Co-γ radiation is characterized by: The step S4 also includes a salt-tolerant Miscanthus mutant evaluation step: transplanting the salt-tolerant Miscanthus mutant into a hydroponic box, adding hydroponic nutrient solution to culture and observe the phenotype of the Miscanthus mutant, and selecting relative water content, relative conductivity and wilting rate as indicators to evaluate the salt tolerance of the Miscanthus mutant.

6. A method according to claim 5 60 The method for cultivating salt-tolerant Miscanthus sinensis mutants induced by Co-γ radiation is characterized by: The hydroponic nutrient solution includes Hoagland culture solution and 20g / L NaCl, and the culture time is 15 days.

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