Culture method of rice callus
By designing a special culture medium and culture method, using N6 culture medium supplemented with JA, GA3 and KT, and combining the culture steps of darkness and red and blue light irradiation, the problem of low rice callus induction rate was solved, and efficient and stable callus culture was achieved.
Patent Information
- Application Number
- CN202511017100.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-23
- Publication Date
- 2025-09-23
AI Technical Summary
The induction rate of rice callus in existing technologies is low, resulting in poor efficiency and quality of the propagation process.
A specific composition of N6 culture medium was used to add different concentrations of JA, GA3 and KT, combined with dark culture, shaking culture and red and blue light irradiation culture methods, and rice callus tissue was gradually induced through three stages of induction culture medium.
It significantly improved the induction success rate and proliferation multiples of callus tissue, shortened the culture cycle, and improved the quality of callus tissue.
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Figure BDA0005513528680000081
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of plant tissue culture, and more particularly to a method for culturing rice callus. Background Art
[0002] Rice is a plant of the genus Oryza in the grass family, native to China and India. Its grains are known as paddy rice. Hulled rice is called brown rice, and the bran layer removed from brown rice is milled and milled. Rice is the staple food for half the world's population. As the world's population continues to grow and arable land continues to decrease, improving rice yield and quality is a key approach to addressing the current food crisis.
[0003] Rice callus culture offers a novel approach to rice propagation, bypassing the limitations of traditional propagation methods and significantly shortening the reproductive cycle. Furthermore, this technique provides important research materials for fields such as rice genetic engineering and secondary metabolite production. However, the callus induction rate in rice callus culture remains very low. Summary of the Invention
[0004] The present invention aims to overcome the defects in the above-mentioned background technology and provides a method for culturing rice callus.
[0005] In order to achieve the above object, the present invention is implemented through the following technical solutions:
[0006] A method for culturing rice callus comprises the following steps:
[0007] Inoculating sterile rice seeds into a 1# induction medium for a first-stage culture, transferring the first-stage culture product into a 2# induction medium for a second-stage culture, transferring the second-stage culture product into a 3# induction medium for a third-stage culture, and obtaining callus tissue;
[0008] Among them, 1# induction medium is N6 medium supplemented with 0.1-0.4 mg / L JA, 0.8-1.2 mg / L KT, 10-30 g / L sucrose, and 5-10 g / L agar; 2# induction medium is N6 medium supplemented with 0.5-1 mg / L JA, 1.5-2 mg / L GA3, 2-3.5 mg / L KT, and 10-30 g / L sucrose; 3# induction medium is N6 medium supplemented with 0.5-1 mg / L JA, 1.5-2 mg / L GA3, 2-3.5 mg / L KT, 10-30 g / L sucrose, and 5-10 g / L agar.
[0009] In the preferred embodiment, the first stage of culture is carried out under dark conditions at a temperature of 22 to 25° C. for 1 to 3 days.
[0010] In the preferred embodiment, the conditions for the second stage of culture are: culture on a shaking platform at a rotation speed of 80-150 rpm for 8-12 days, a temperature of 24-28°C, a light duration of 8-12 h / d, and a light intensity of 1800-2500 lx.
[0011] Under the preferred scheme, the conditions for the third stage of culture are: temperature of 26-30°C, red and blue light irradiation, illumination time of 12-16h / d, light intensity of 1500-3000lx, and culture for 15-20d.
[0012] In the preferred embodiment, the wavelength ratio of red light to blue light is (4-8):1.
[0013] In a preferred embodiment, the pH value of the three culture stages is 5.8 to 6.5.
[0014] 7. The method for culturing rice callus according to claim 1, characterized in that the rice seeds are pretreated before induction culture, comprising soaking and disinfecting the rice seeds in 75% ethanol solution and 2% sodium hypochlorite solution in sequence, and then rinsing the rice seeds with sterile water 2 to 4 times to obtain sterile rice seeds.
[0015] The beneficial effects of the present invention are:
[0016] The present invention establishes a more efficient, simple, safe and stable rice callus culture system by designing a special culture medium and culture method, shortens the culture period of callus tissue, significantly improves the induction success rate and proliferation multiples of callus tissue, and improves the quality of callus tissue. DETAILED DESCRIPTION
[0017] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.
[0018] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. The experimental methods in the examples are conventional methods unless otherwise specified. Where specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, for which the manufacturer is not specified, are all commercially available conventional products.
[0019] Example 1
[0020] A method for culturing rice callus comprises the following steps:
[0021] (1) Select mature rice seeds with full grains and no mold. Under sterile conditions, remove the rice husks, taking care to keep the embryo intact. Place the hulled seeds in a triangular flask, first add a 75% ethanol solution to cover the seeds, seal, and soak for 15 minutes. Discard the ethanol solution, then add a 2% sodium hypochlorite solution to cover the seeds, seal, and soak for 2 hours to sterilize the seeds. During this period, shake the triangular flask appropriately to sterilize the rice seeds. After 2 hours of disinfection, rinse with sterile water three times, place the sterile rice seeds in a new triangular flask, add sterile water to cover the seeds, and store in a sealed container at 4°C for later use.
[0022] (2) Under sterile conditions, sterile rice seeds were inoculated into induction medium #1 at 22°C. After culturing in the dark for 3 days, the culture product was transferred to induction medium #2 at 25°C, with 10 hours of light per day at a light intensity of 1800 lx, and cultured on a shaking table at a speed of 150 rpm for 8 days. After culture on a shaking table, the culture product was transferred to induction medium #3 at 28°C, with red and blue light irradiation for 16 hours per day at a light intensity of 3000 lx, and a red to blue light wavelength ratio of 6:1. The culture was cultured for 20 days to induce callus tissue.
[0023] Among them, the pH of the above culture process is 5.8; 1# induction medium is N6 medium supplemented with 0.2 mg / L JA, 0.8 mg / L KT, 10 g / L sucrose, and 5 g / L agar; 2# induction medium is N6 medium supplemented with 0.5 mg / L JA, 1.5 mg / L GA3, 2 mg / L KT, and 30 g / L sucrose; 3# induction medium is N6 medium supplemented with 0.5 mg / L JA, 1.5 mg / L GA3, 2 mg / L KT, 30 g / L sucrose, and 5 g / L agar.
[0024] Example 2
[0025] A method for culturing rice callus comprises the following steps:
[0026] (1) Select mature rice seeds with full grains and no mold. Under sterile conditions, remove the rice husks, taking care to keep the embryo intact. Place the hulled seeds in a triangular flask, first add a 75% ethanol solution to cover the seeds, seal, and soak for 15 minutes. Discard the ethanol solution, then add a 2% sodium hypochlorite solution to cover the seeds, seal, and soak for 2 hours to sterilize the seeds. During this period, shake the triangular flask appropriately to sterilize the rice seeds. After 2 hours of disinfection, rinse with sterile water three times, place the sterile rice seeds in a new triangular flask, add sterile water to cover the seeds, and store in a sealed container at 4°C for later use.
[0027] (2) Under sterile conditions, sterile rice seeds were inoculated into induction medium #1 at 22°C. After culturing in the dark for 3 days, the culture product was transferred to induction medium #2 at 25°C, with 10 hours of light per day at a light intensity of 1800 lx, and cultured on a shaking table at a speed of 150 rpm for 8 days. After culture on a shaking table, the culture product was transferred to induction medium #3 at 28°C, with red and blue light irradiation for 16 hours per day at a light intensity of 3000 lx, and a red to blue light wavelength ratio of 6:1. The culture was cultured for 20 days to induce callus tissue.
[0028] Among them, the pH of the above culture process is 5.8; 1# induction medium is N6 medium supplemented with 0.2 mg / L JA, 0.8 mg / L KT, 10 g / L sucrose, and 5 g / L agar; 2# induction medium is N6 medium supplemented with 0.5 mg / L JA, 1.5 mg / L GA3, 3.5 mg / L KT, and 30 g / L sucrose; 3# induction medium is N6 medium supplemented with 0.5 mg / L JA, 1.5 mg / L GA3, 3.5 mg / L KT, 30 g / L sucrose, and 5 g / L agar.
[0029] Example 3
[0030] A method for culturing rice callus comprises the following steps:
[0031] (1) Select mature rice seeds with full grains and no mold. Under sterile conditions, remove the rice husks, taking care to keep the embryo intact. Place the hulled seeds in a triangular flask, first add a 75% ethanol solution to cover the seeds, seal, and soak for 15 minutes. Discard the ethanol solution, then add a 2% sodium hypochlorite solution to cover the seeds, seal, and soak for 2 hours to sterilize the seeds. During this period, shake the triangular flask appropriately to sterilize the rice seeds. After 2 hours of disinfection, rinse with sterile water three times, place the sterile rice seeds in a new triangular flask, add sterile water to cover the seeds, and store in a sealed container at 4°C for later use.
[0032] (2) Under sterile conditions, sterile rice seeds were inoculated into induction medium #1 at 22°C. After culturing in the dark for 3 days, the culture product was transferred to induction medium #2 at 25°C, with 10 hours of light per day at a light intensity of 1800 lx, and cultured on a shaking table at a speed of 150 rpm for 8 days. After culture on a shaking table, the culture product was transferred to induction medium #3 at 28°C, with red and blue light irradiation for 16 hours per day at a light intensity of 3000 lx, and a red to blue light wavelength ratio of 6:1. The culture was cultured for 20 days to induce callus tissue.
[0033] The pH of the above culture process is 5.8; the 1# induction medium is an N6 medium supplemented with 0.2 mg / L JA, 0.8 mg / L KT, 10 g / L sucrose, and 5 g / L agar; the 2# induction medium is an N6 medium supplemented with 0.5 mg / L JA, 2 mg / L GA3, 2 mg / L KT, and 30 g / L sucrose; and the 3# induction medium is an N6 medium supplemented with 0.5 mg / L JA, 2 mg / L GA3, 2 mg / L KT, 30 g / L sucrose, and 5 g / L agar.
[0034] Example 4
[0035] A method for culturing rice callus comprises the following steps:
[0036] (1) Select mature rice seeds with full grains and no mold. Under sterile conditions, remove the rice husks, taking care to keep the embryo intact. Place the hulled seeds in a triangular flask, first add a 75% ethanol solution to cover the seeds, seal, and soak for 15 minutes. Discard the ethanol solution, then add a 2% sodium hypochlorite solution to cover the seeds, seal, and soak for 2 hours to sterilize the seeds. During this period, shake the triangular flask appropriately to sterilize the rice seeds. After 2 hours of disinfection, rinse with sterile water three times, place the sterile rice seeds in a new triangular flask, add sterile water to cover the seeds, and store in a sealed container at 4°C for later use.
[0037] (2) Under sterile conditions, sterile rice seeds were inoculated into induction medium #1 at 22°C. After culturing in the dark for 3 days, the culture product was transferred to induction medium #2 at 25°C, with 10 hours of light per day at a light intensity of 1800 lx, and cultured on a shaking table at a speed of 150 rpm for 8 days. After culture on a shaking table, the culture product was transferred to induction medium #3 at 28°C, with red and blue light irradiation for 16 hours per day at a light intensity of 3000 lx, and a red to blue light wavelength ratio of 6:1. The culture was cultured for 20 days to induce callus tissue.
[0038] Among them, the pH of the above culture process is 5.8; 1# induction medium is N6 medium supplemented with 0.2 mg / L JA, 0.8 mg / L KT, 10 g / L sucrose, and 5 g / L agar; 2# induction medium is N6 medium supplemented with 0.5 mg / L JA, 2 mg / L GA3, 3.5 mg / L KT, and 30 g / L sucrose; 3# induction medium is N6 medium supplemented with 0.5 mg / L JA, 2 mg / L GA3, 3.5 mg / L KT, 30 g / L sucrose, and 5 g / L agar.
[0039] Example 5
[0040] A method for culturing rice callus comprises the following steps:
[0041] (1) Select mature rice seeds with full grains and no mold. Under sterile conditions, remove the rice husks, taking care to keep the embryo intact. Place the hulled seeds in a triangular flask, first add a 75% ethanol solution to cover the seeds, seal, and soak for 15 minutes. Discard the ethanol solution, then add a 2% sodium hypochlorite solution to cover the seeds, seal, and soak for 2 hours to sterilize the seeds. During this period, shake the triangular flask appropriately to sterilize the rice seeds. After 2 hours of disinfection, rinse with sterile water three times, place the sterile rice seeds in a new triangular flask, add sterile water to cover the seeds, and store in a sealed container at 4°C for later use.
[0042] (2) Under sterile conditions, sterile rice seeds were inoculated into induction medium #1 at 22°C. After culturing in the dark for 3 days, the culture product was transferred to induction medium #2 at 25°C, with 10 hours of light per day at a light intensity of 1800 lx, and cultured on a shaking table at a speed of 150 rpm for 8 days. After culture on a shaking table, the culture product was transferred to induction medium #3 at 28°C, with red and blue light irradiation for 16 hours per day at a light intensity of 3000 lx, and a red to blue light wavelength ratio of 6:1. The culture was cultured for 20 days to induce callus tissue.
[0043] Among them, the pH of the above culture process is 5.8; 1# induction medium is N6 medium supplemented with 0.2 mg / L JA, 0.8 mg / L KT, 10 g / L sucrose, and 5 g / L agar; 2# induction medium is N6 medium supplemented with 1 mg / L JA, 1.5 mg / L GA3, 2 mg / L KT, and 30 g / L sucrose; 3# induction medium is N6 medium supplemented with 1 mg / L JA, 1.5 mg / L GA3, 2 mg / L KT, 30 g / L sucrose, and 5 g / L agar.
[0044] Example 6
[0045] A method for culturing rice callus comprises the following steps:
[0046] (1) Select mature rice seeds with full grains and no mold. Under sterile conditions, remove the rice husks, taking care to keep the embryo intact. Place the hulled seeds in a triangular flask, first add a 75% ethanol solution to cover the seeds, seal, and soak for 15 minutes. Discard the ethanol solution, then add a 2% sodium hypochlorite solution to cover the seeds, seal, and soak for 2 hours to sterilize the seeds. During this period, shake the triangular flask appropriately to sterilize the rice seeds. After 2 hours of disinfection, rinse with sterile water three times, place the sterile rice seeds in a new triangular flask, add sterile water to cover the seeds, and store in a sealed container at 4°C for later use.
[0047] (2) Under sterile conditions, sterile rice seeds were inoculated into induction medium #1 at 22°C. After culturing in the dark for 3 days, the culture product was transferred to induction medium #2 at 25°C, with 10 hours of light per day at a light intensity of 1800 lx, and cultured on a shaking table at a speed of 150 rpm for 8 days. After culture on a shaking table, the culture product was transferred to induction medium #3 at 28°C, with red and blue light irradiation for 16 hours per day at a light intensity of 3000 lx, and a red to blue light wavelength ratio of 6:1. The culture was cultured for 20 days to induce callus tissue.
[0048] Among them, the pH of the above culture process is 5.8; 1# induction medium is N6 medium supplemented with 0.2 mg / L JA, 0.8 mg / L KT, 10 g / L sucrose, and 5 g / L agar; 2# induction medium is N6 medium supplemented with 1 mg / L JA, 1.5 mg / L GA3, 3.5 mg / L KT, and 30 g / L sucrose; 3# induction medium is N6 medium supplemented with 1 mg / L JA, 1.5 mg / L GA3, 3.5 mg / L KT, 30 g / L sucrose, and 5 g / L agar.
[0049] Example 7
[0050] A method for culturing rice callus comprises the following steps:
[0051] (1) Select mature rice seeds with full grains and no mold. Under sterile conditions, remove the rice husks, taking care to keep the embryo intact. Place the hulled seeds in a triangular flask, first add a 75% ethanol solution to cover the seeds, seal, and soak for 15 minutes. Discard the ethanol solution, then add a 2% sodium hypochlorite solution to cover the seeds, seal, and soak for 2 hours to sterilize the seeds. During this period, shake the triangular flask appropriately to sterilize the rice seeds. After 2 hours of disinfection, rinse with sterile water three times, place the sterile rice seeds in a new triangular flask, add sterile water to cover the seeds, and store in a sealed container at 4°C for later use.
[0052] (2) Under sterile conditions, sterile rice seeds were inoculated into induction medium #1 at 22°C. After culturing in the dark for 3 days, the culture product was transferred to induction medium #2 at 25°C, with 10 hours of light per day at a light intensity of 1800 lx, and cultured on a shaking table at a speed of 150 rpm for 8 days. After culture on a shaking table, the culture product was transferred to induction medium #3 at 28°C, with red and blue light irradiation for 16 hours per day at a light intensity of 3000 lx, and a red to blue light wavelength ratio of 6:1. The culture was cultured for 20 days to induce callus tissue.
[0053] Among them, the pH of the above culture process is 5.8; 1# induction medium is N6 medium supplemented with 0.2 mg / L JA, 0.8 mg / L KT, 10 g / L sucrose, and 5 g / L agar; 2# induction medium is N6 medium supplemented with 1 mg / L JA, 2 mg / L GA3, 2 mg / L KT, and 30 g / L sucrose; 3# induction medium is N6 medium supplemented with 1 mg / L JA, 2 mg / L GA3, 2 mg / L KT, 30 g / L sucrose, and 5 g / L agar.
[0054] Example 8
[0055] A method for culturing rice callus comprises the following steps:
[0056] (1) Select mature rice seeds with full grains and no mold. Under sterile conditions, remove the rice husks, taking care to keep the embryo intact. Place the hulled seeds in a triangular flask, first add a 75% ethanol solution to cover the seeds, seal, and soak for 15 minutes. Discard the ethanol solution, then add a 2% sodium hypochlorite solution to cover the seeds, seal, and soak for 2 hours to sterilize the seeds. During this period, shake the triangular flask appropriately to sterilize the rice seeds. After 2 hours of disinfection, rinse with sterile water three times, place the sterile rice seeds in a new triangular flask, add sterile water to cover the seeds, and store in a sealed container at 4°C for later use.
[0057] (2) Under sterile conditions, sterile rice seeds were inoculated into induction medium #1 at 22°C. After culturing in the dark for 3 days, the culture product was transferred to induction medium #2 at 25°C, with 10 hours of light per day at a light intensity of 1800 lx, and cultured on a shaking table at a speed of 150 rpm for 8 days. After culture on a shaking table, the culture product was transferred to induction medium #3 at 28°C, with red and blue light irradiation for 16 hours per day at a light intensity of 3000 lx, and a red to blue light wavelength ratio of 6:1. The culture was cultured for 20 days to induce callus tissue.
[0058] Among them, the pH of the above culture process is 5.8; 1# induction medium is N6 medium supplemented with 0.2 mg / L JA, 0.8 mg / L KT, 10 g / L sucrose, and 5 g / L agar; 2# induction medium is N6 medium supplemented with 1 mg / L JA, 2 mg / L GA3, 3.5 mg / L KT, and 30 g / L sucrose; 3# induction medium is N6 medium supplemented with 1 mg / L JA, 2 mg / L GA3, 3.5 mg / L KT, 30 g / L sucrose, and 5 g / L agar.
[0059] Example 9
[0060] A method for culturing rice callus comprises the following steps:
[0061] (1) Select mature rice seeds with full grains and no mold. Under sterile conditions, remove the rice husks, taking care to keep the embryo intact. Place the hulled seeds in a triangular flask, first add a 75% ethanol solution to cover the seeds, seal, and soak for 15 minutes. Discard the ethanol solution, then add a 2% sodium hypochlorite solution to cover the seeds, seal, and soak for 2 hours to sterilize the seeds. During this period, shake the triangular flask appropriately to sterilize the rice seeds. After 2 hours of disinfection, rinse with sterile water three times, place the sterile rice seeds in a new triangular flask, add sterile water to cover the seeds, and store in a sealed container at 4°C for later use.
[0062] (2) Under sterile conditions, sterile rice seeds were inoculated into induction medium #1 at 22°C. After culturing in the dark for 3 days, the culture product was transferred to induction medium #2 at 25°C, with 10 hours of light per day at a light intensity of 1800 lx, and cultured on a shaking table at a speed of 150 rpm for 8 days. After culture on a shaking table, the culture product was transferred to induction medium #3 at 28°C, with red and blue light irradiation for 16 hours per day at a light intensity of 3000 lx, and a red to blue light wavelength ratio of 4:1. The culture was cultured for 20 days to induce callus tissue.
[0063] Among them, the pH of the above culture process is 5.8; 1# induction medium is N6 medium supplemented with 0.2 mg / L JA, 0.8 mg / L KT, 10 g / L sucrose, and 5 g / L agar; 2# induction medium is N6 medium supplemented with 1 mg / L JA, 1.5 mg / L GA3, 2 mg / L KT, and 30 g / L sucrose; 3# induction medium is N6 medium supplemented with 1 mg / L JA, 1.5 mg / L GA3, 2 mg / L KT, 30 g / L sucrose, and 5 g / L agar.
[0064] Example 10
[0065] A method for culturing rice callus comprises the following steps:
[0066] (1) Select mature rice seeds with full grains and no mold. Under sterile conditions, remove the rice husks, taking care to keep the embryo intact. Place the hulled seeds in a triangular flask, first add a 75% ethanol solution to cover the seeds, seal, and soak for 15 minutes. Discard the ethanol solution, then add a 2% sodium hypochlorite solution to cover the seeds, seal, and soak for 2 hours to sterilize the seeds. During this period, shake the triangular flask appropriately to sterilize the rice seeds. After 2 hours of disinfection, rinse with sterile water three times, place the sterile rice seeds in a new triangular flask, add sterile water to cover the seeds, and store in a sealed container at 4°C for later use.
[0067] (2) Under sterile conditions, sterile rice seeds were inoculated into induction medium #1 at 22°C. After culturing in the dark for 3 days, the culture product was transferred to induction medium #2 at 25°C, with 10 hours of light per day at a light intensity of 1800 lx, and cultured on a shaking table at a speed of 150 rpm for 8 days. After culture on a shaking table, the culture product was transferred to induction medium #3 at 28°C, with red and blue light irradiation for 16 hours per day at a light intensity of 3000 lx, and a red to blue light wavelength ratio of 8:1. The culture was cultured for 20 days to induce callus tissue.
[0068] Among them, the pH of the above culture process is 5.8; 1# induction medium is N6 medium supplemented with 0.2 mg / L JA, 0.8 mg / L KT, 10 g / L sucrose, and 5 g / L agar; 2# induction medium is N6 medium supplemented with 1 mg / L JA, 1.5 mg / L GA3, 2 mg / L KT, and 30 g / L sucrose; 3# induction medium is N6 medium supplemented with 1 mg / L JA, 1.5 mg / L GA3, 2 mg / L KT, 30 g / L sucrose, and 5 g / L agar.
[0069] Comparative Example 1
[0070] This comparative example is basically the same as Example 5, except that JA is not added to the 2# induction medium and the 3# induction medium.
[0071] Comparative Example 2
[0072] This comparative example is basically the same as Example 5, except that GA3 is not added to the 2# induction medium and the 3# induction medium.
[0073] Comparative Example 3
[0074] This comparative example is basically the same as Example 5, except that KT is not added to the 2# induction medium and the 3# induction medium.
[0075] Comparative Example 4
[0076] A method for culturing rice callus comprises the following steps:
[0077] (1) Select mature rice seeds with full grains and no mold. Under sterile conditions, remove the rice husks, taking care to keep the embryo intact. Place the hulled seeds in a triangular flask, first add a 75% ethanol solution to cover the seeds, seal, and soak for 15 minutes. Discard the ethanol solution, then add a 2% sodium hypochlorite solution to cover the seeds, seal, and soak for 2 hours to sterilize the seeds. During this period, shake the triangular flask appropriately to sterilize the rice seeds. After 2 hours of disinfection, rinse with sterile water three times, place the sterile rice seeds in a new triangular flask, add sterile water to cover the seeds, and store in a sealed container at 4°C for later use.
[0078] (2) Under sterile conditions, sterile rice seeds were inoculated into 3# induction medium at 25°C, with 16 hours of light per day and a light intensity of 2000 lx for 35 days.
[0079] The pH of the above culture process was 5.8; the 3# induction medium was N6 medium supplemented with 1 mg / L JA, 1.5 mg / L GA3, 2 mg / L KT, 30 g / L sucrose, and 5 g / L agar.
[0080] Table 1 Results of callus induction and proliferation from rice seeds
[0081]
[0082] As shown in Table 1, Examples 1 to 8 differ in the mass concentrations of JA, GA3, and KT added to the 2# induction medium and the 3# induction medium. Comparing the results of Examples 1 to 8, it can be seen that the callus induction success rate and callus proliferation multiples were the best when 1 mg / L JA, 1.5 mg / L GA3, and 2 mg / L KT were added to the N6 medium (Example 5). The difference between Examples 5 and 9 to 10 is that the wavelength ratio of red and blue light was different. Comparing the culture results of Examples 5 and 9 to 10, it can be seen that the callus induction success rate and callus proliferation multiples were still the best when the culture process of Example 5 was used. The culture conditions of Comparative Examples 1 to 3 are substantially the same as those of Example 5, except that only two components of JA, GA3, and KT are added to the 2# induction medium and the 3# induction medium in Comparative Examples 1 to 3. Comparison of the culture results of Examples 1 to 10 and Comparative Examples 1 to 3 shows that after culture in the 2# induction medium and the 3# induction medium provided by the present invention, the callus induction success rate and the callus proliferation multiple are significantly better than those of Comparative Examples 1 to 3. The difference between Example 5 and Comparative Example 4 is that Comparative Example 4 does not undergo dark culture and suspension culture. Comparison of the culture results of Examples 1 to 10 and Comparative Example 4 shows that the callus induction success rate and proliferation multiple obtained by the culture method of the present invention are significantly better than those of Comparative Example 4.
[0083] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.
Claims
1. A method for culturing rice callus, characterized in that: The steps include: Inoculating sterile rice seeds into a 1# induction medium for a first-stage culture, transferring the first-stage culture product into a 2# induction medium for a second-stage culture, transferring the second-stage culture product into a 3# induction medium for a third-stage culture, and obtaining callus tissue; Among them, 1# induction medium is N6 medium supplemented with 0.1-0.4 mg / L JA, 0.8-1.2 mg / L KT, 10-30 g / L sucrose, and 5-10 g / L agar; 2# induction medium is N6 medium supplemented with 0.5-1 mg / L JA, 1.5-2 mg / L GA3, 2-3.5 mg / L KT, and 10-30 g / L sucrose; 3# induction medium is N6 medium supplemented with 0.5-1 mg / L JA, 1.5-2 mg / L GA3, 2-3.5 mg / L KT, 10-30 g / L sucrose, and 5-10 g / L agar.
2. The method for culturing rice callus according to claim 1, wherein: The first stage of culture conditions are: in the dark, at a temperature of 22-25°C, for 1-3 days.
3. The method for culturing rice callus according to claim 1, wherein: The conditions for the second stage of culture are: shaking culture at a speed of 80-150 rpm for 8-12 days, a temperature of 24-28°C, a light duration of 8-12 h / d, and a light intensity of 1800-2500 lx.
4. The method for culturing rice callus according to claim 1, wherein: The conditions for the third stage of culture are: temperature of 26-30°C, red and blue light irradiation, illumination time of 12-16h / d, light intensity of 1500-3000lx, and culture for 15-20d.
5. The method for culturing rice callus according to claim 4, characterized in that: The wavelength ratio of red and blue light is (4~8):
1.
6. The method for culturing rice callus according to any one of claims 1 to 5, characterized in that: The pH value of the three culture stages was 5.8 to 6.
5.
7. The method for culturing rice callus according to claim 1, wherein: Before induction culture, the rice seeds are pretreated, including soaking and disinfecting the rice seeds in 75% ethanol solution and 2% sodium hypochlorite solution in sequence, and then washing the rice seeds with sterile water for 2 to 4 times to obtain sterile rice seeds.