Breeding method for improving stress tolerance of nostoc sphaeroids kutz

Abstract

The invention discloses a breeding method for improving stress tolerance of nostoc sphaeroids kutz. The breeding method comprises the steps of mutagenesis treatment of nostoc sphaeroids kutz, long-term laboratory adaptive evolution treatment by utilizing multiple adversity stress and the like. After the stress-tolerant common nostoc germplasm prepared by the method is subjected to standing culture for 50 days under the condition of 25 DEG C + 25 mol m <-2 > s <-1 > visible light, the dry matter cumulant is obviously increased by about 2 times; after being treated in a BG110 solution containing PEG6000 with the mass percent of 30% for 14 days, the survival rate is obviously increased by about 50%; after being treated in a BG110 solution containing 0.4 mol L <-1 > NaCl for 14 days, the survival rate is obviously increased by about 50%; the survival rate is obviously improved by about 50% after the culture medium is treated for 1 day under the condition of 1000 mol m <-2 > s <-1 >. The method is simple, good in effect and low in cost; the common nostoc germplasm is fast in growth, high in yield, strong in stress tolerance, good in stability, non-transgenic and easy to popularize.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and more particularly relates to a breeding method for improving the stress tolerance of kudzu vine. Background Art

[0002] Microalgae breeding techniques include mutation breeding, molecular breeding, and laboratory adaptive evolution breeding. (1) Mutation breeding is the use of physical or chemical factors to induce genetic variation in microalgae, and then targeted screening to cultivate target algae strains with excellent traits. Physical mutagenesis methods include ultraviolet rays, atmospheric pressure room temperature plasma, etc.; commonly used mutagens in chemical mutagenesis methods include alkylating agents and nucleic acid base analogs. Mutagenesis breeding has the advantages of high mutagenesis efficiency, wide mutation range, simple operation, low cost, and non-transgenic and easy to promote, but it also has problems such as randomness and poor targeting. (2) Molecular breeding is the application of molecular biology technology to breeding, directed control of gene expression, and the realization of site-directed mutation in microalgae breeding, which compensates for the non-directionality of mutation breeding, but is still affected by the lack of genetic background and the interaction between phenotypes. (3) Adaptive laboratory evolution technology (ALE) is to promote the adaptive natural evolution of algae strains by imposing artificial interference and controlling the growth environment in the laboratory. Short-term ALE, which typically requires only a few passages or tens of hours, is characterized by rapid results, a single evolutionary path, and a short timeframe. Long-term ALE, on the other hand, requires dozens or even hundreds of passages, using specific or incremental selection pressures to select strains with strong tolerance, rapid growth, and high yields of the target product. However, the application of these breeding techniques in kudzu vine has rarely been reported.

[0003] Chinese patent document CN201410269492.4 discloses a fast and efficient method for breeding kudzu vine, which obtains algae segments by inducing the rupture of kudzu vine spheres, obtains microspheres by culturing the algae segments, and finally uses the obtained microspheres for budding and reproduction; Chinese patent document CN201610946086.6 discloses a method for breeding kudzu vine heteromorphic cells by inducing multiple heteromorphic cells in series to obtain kudzu vine algae; Chinese patent document CN202310874907.X discloses a method for breeding kudzu vine heteromorphic cells by inducing multiple heteromorphic cells in series to obtain kudzu vine algae; Cultivation method: This invention uses selenium-rich hot spring water to prepare a complete nutrient culture medium, and at the same time adds disodium glycerophosphate to the culture medium to promote the growth and metabolic activity of kudzu rice to quickly produce seeds, and through sulfur-free induction and low-sulfur culture, increases the kudzu rice's ability to absorb selenium; Chinese patent document CN201711113508.2 discloses a production method for golden kudzu rice. This invention increases the proportion of carotenoids in kudzu rice cells by regulating nutrients, light intensity and light quality, making it appear golden yellow, enriching the variety of kudzu rice products and meeting market needs. The above breeding method involves the rapid propagation of kudzu rice and the cultivation of special kudzu rice rich in selenium or carotenoids. It is suitable for indoor factory-based kudzu rice cultivation, but does not involve kudzu rice stress tolerance breeding. Summary of the Invention

[0004] The purpose of the present invention is to overcome the above-mentioned defects of the prior art, give full play to the genetic potential of the kudzu vine, adopt long-term adaptive laboratory evolution technology combining multiple adverse stresses with mutagenesis, and provide a method for creating and breeding a non-transgenic, stress-tolerant kudzu vine germplasm that is suitable for indoor or outdoor rice field cultivation, has good effects, and can be promoted.

[0005] To achieve the above technical objectives, the present invention provides a breeding method for improving the stress tolerance of Miscanthus sibiricum, comprising the following steps: 1) Mutation treatment of kudzu vine: Homogenize kudzu vine, centrifuge, wash with BG110, add BG110 to make algae liquid, centrifuge and discard the supernatant, add pH=7 phosphate buffer and 5-6% ethyl methanesulfonate solution by weight, treat in the dark, add 5% sodium thiosulfate solution by weight to terminate the reaction, wash with BG110, suspend the algae liquid in BG110, protect from light, and store at 25℃+ 20µmol m -2 s -1 Under visible light conditions, the algae solution was spread on a BG110 plate. After the strain on the plate grew into a spherical explant, the kudzu seedlings were transferred to a BG110 liquid medium for further cultivation. 2) Long-term laboratory adaptive evolution using multiple stresses: The kudzu vine germplasm was transferred to BG110 medium and then placed in 100-500 μmol photons m-2 s -1 Visible light +1~ 2Wm -2 The culture was expanded under UV-B conditions until it dried naturally, and then the drying process was continued. Then BG110 medium was added and the above dry-wet cycle procedure was repeated for at least 15 times to select a stable genetic stress-tolerant Kudzu chinensis germplasm.

[0006] Preferably, the present invention provides a breeding method for improving the stress tolerance of Miscanthus truncatus, comprising the following steps: 1) Mutation treatment of kudzu vine: After homogenizing kudzu vine, centrifuge and wash three times with BG110. Add BG110 to adjust the algae solution density to OD 680 =0.3~0.4, take V1 volume of algae liquid, centrifuge and discard the supernatant, add pH=7 phosphate buffer and 5-6% mass percentage concentration of ethyl methanesulfonate solution, dark-treat for 6~12 hours, add equal volume mass percentage concentration of 5% sodium thiosulfate solution to terminate the reaction, wash with BG110 3 times, suspend the algae liquid in the V1 volume of BG110, protect from light for 12 hours, and store at 25℃+ 20µmol m -2 s -1 Cultivate under visible light for 6 to 14 days, dilute the algae solution, and apply it to BG110 plates. After the strain grows into a spherical protophyte with a diameter of 1 to 3 mm on the plate, transfer each Kudzu chinensis germplasm to 2 mL to 5 mL of BG110 liquid medium and continue culturing for 2 to 3 months. 2) Long-term laboratory adaptive evolution using multiple stresses: Transfer the germplasm of Miscanthus sibiricum to 50-200 mL BG110 medium and place it in 100-500 μmol photons m -2 s -1 Visible light +1~ 2 Wm -2 The culture was expanded statically under UV-B conditions until it dried naturally, and the drying treatment was continued for 0.5 to 3 months. Then 50 to 200 mL of BG110 medium was added and the above dry-wet cycle procedure was repeated 15 to 60 times to select a stable genetic stress-tolerant Kudzu chinensis germplasm.

[0007] The method of the invention is simple, effective and low in cost; the kudzu vine germplasm grows fast, has high yield, strong stress resistance and good stability, is non-transgenic and easy to promote. DETAILED DESCRIPTION

[0008] The technical scheme of the present invention is now described in detail with reference to the embodiments. It should be understood that the following embodiments are only intended to illustrate the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit and substance of the present invention, modifications or replacements made to the steps or conditions of the present invention are within the scope of the present invention.

[0009] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0010] In the following examples, the BG110 medium is a conventional nitrogen-free BG11 medium for cyanobacteria, which is composed of sodium carbonate, magnesium sulfate, calcium chloride, citric acid, ammonium ferric citrate and A5 trace elements.

[0011] Example 1 1) Mutation treatment of kudzu vine: After homogenization of vigorously growing kudzu vine, centrifuge and wash three times with fresh BG110. Adjust the algae density to OD680 = 0.3 with BG110. Take 5 mL of algae solution, centrifuge and discard the supernatant. Collect the algae pellet and add phosphate buffer (pH = 7) and 5-6% ethyl methanesulfonate solution by weight. Incubate in the dark for 6 hours. Terminate the reaction with an equal volume of 5% sodium thiosulfate solution by weight. Wash three times with fresh BG110. Resuspend the algae in 5 mL of fresh BG110 and incubate in the dark for 12 hours at 25°C + 20µmol m -2 s -1 After 14 days of static culture under visible light, the algae solution was diluted and plated on BG110 plates. Once the strains on the plates had grown to spherical explants with a diameter of 1 mm, each seed of the kudzu seedling was transferred to 3 mL of BG110 liquid medium and cultured for another 3 months.

[0012] 2) Long-term laboratory adaptive evolution using multiple stresses: The germplasm of Puerariae was transferred into 200 mL BG110 medium and the test tube was placed in 500 μmol photons m -2 s -1 Visible light + 2 Wm -2 Under UV-B conditions, the culture was expanded and placed statically for 1 month until it dried naturally, and then dried for 1 month. Then 200 mL of BG110 medium was added, and the above dry-wet cycle procedure was repeated 30 times. In about 3 years, a stable genetic stress-tolerant Pueraria lobata germplasm was bred.

[0013] 3) Germplasm stress tolerance evaluation: ① Compared with common kudzu vine germplasm, the stress-tolerant kudzu vine germplasm obtained in this example was heated to 25°C + 25 μmol m -2 s -1 After 50 days of static culture under visible light conditions, the cumulative amount of dry matter increased significantly by about 2 times; ② Compared with the common kudzu vine germplasm, the stress-tolerant kudzu vine germplasm obtained in this example was placed in a BG110 solution containing 30% by mass PEG6000 for 14 days, and the survival rate was significantly increased by about 50%; ③ Compared with the common kudzu vine germplasm, the stress-tolerant kudzu vine germplasm obtained in this example was placed in a 0.4 mol L -1The survival rate of the cultivars of Puerariae lobata was significantly increased by about 50% after being treated with BG110 solution of NaCl for 14 days. ④ Compared with the common cultivars of Puerariae lobata germplasm, the stress-tolerant cultivars of Puerariae lobata germplasm obtained in this example were placed in 1000 μmol m -2 s -1 The above test results confirm the stress tolerance of the new germplasm of Miscanthus sibiricum obtained in this example.

[0014] Example 2 1) Mutation treatment of kudzu vine: After homogenization of vigorously growing kudzu vine, centrifuge and wash three times with fresh BG110. Adjust the algae density to OD680 = 0.35 with BG110. Take 5 mL of algae solution, centrifuge and discard the supernatant. Collect the algae pellet and add phosphate buffer (pH = 7) and 5-6% ethyl methanesulfonate solution by weight. Incubate in the dark for 12 hours. Terminate the reaction with an equal volume of 5% sodium thiosulfate solution by weight. Wash three times with fresh BG110. Resuspend the algae in 5 mL of fresh BG110 and incubate in the dark for 12 hours at 25°C with 20 µmol m -2 s -1 After 6 days of static culture under visible light, the algae solution was diluted and plated on BG110 plates. Once the strains on the plates had grown to spherical explants with a diameter of 1 mm, each seed of the kudzu seedling was transferred to 2 mL of BG110 liquid medium and cultured for another 3 months.

[0015] 2) Long-term laboratory adaptive evolution using multiple stresses: Transfer the kudzu vine germplasm into 100 mL BG110 medium and place the test tube in 100 μmol photons m -2 s -1 Visible light + 1 Wm -2 Under UV-B conditions, the culture was expanded and placed statically for 1 month until it dried naturally, and then dried for 3 months. Then 100 mL of BG110 medium was added, and the above dry-wet cycle procedure was repeated 15 times. In about 5 years, a stable genetic stress-tolerant Pueraria lobata germplasm was bred.

[0016] 3) Germplasm stress tolerance evaluation: ① Compared with common kudzu vine germplasm, the stress-tolerant kudzu vine germplasm obtained in this example was heated to 25°C + 25 μmol m -2 s -1After 30 days of static culture under visible light conditions, the cumulative amount of dry matter increased significantly by about 0.5 times; ② Compared with the common kudzu germplasm, the stress-tolerant kudzu germplasm obtained in this example was placed in a BG110 solution containing 25% by mass PEG6000 for 10 days, and the survival rate was significantly increased by about 30%; ③ Compared with the common kudzu germplasm, the stress-tolerant kudzu germplasm obtained in this example was placed in a 0.2 mol L -1 The survival rate of the cultivar of Puerariae was significantly increased by about 30% after being treated with BG110 solution of NaCl for 10 days; ④ Compared with the common cultivar of Puerariae germplasm, the stress-tolerant cultivar of Puerariae germplasm obtained in this example was placed in 500 μmol m -2 s -1 The survival rate was significantly increased by about 30% under the above test results after treatment for 1 day under the above conditions. The stress tolerance characteristics of the new germplasm of Miscanthus sibiricum obtained in this example were confirmed.

[0017] Example 3 1) Mutation treatment of kudzu vine: After homogenization of vigorously growing kudzu vine, centrifuge and wash three times with fresh BG110. Adjust the algae density to OD680 = 0.4 with BG110. Take 5 mL of algae solution, centrifuge and discard the supernatant. Collect the algae pellet and add phosphate buffer (pH = 7) and 5-6% ethyl methanesulfonate solution by weight. Incubate in the dark for 8 hours. Terminate the reaction with an equal volume of 5% sodium thiosulfate solution by weight. Wash three times with fresh BG110. Resuspend the algae in 5 mL of fresh BG110 and incubate in the dark for 12 hours at 25°C + 20µmol m -2 s -1 After 10 days of static culture under visible light, the algae solution was diluted and plated on BG110 plates. Once the strains on the plates had grown to spherical protophytes with a diameter of 3 mm, each seed of the kudzu seedling was transferred to 5 mL of BG110 liquid medium and cultured for another 2 months.

[0018] 2) Long-term laboratory adaptive evolution using multiple stresses: Transfer the germplasm of Miscanthus sibiricum to 150 mL of BG110 medium and place the tube in a 200 μmol photons m -2 s -1 Visible light + 2 Wm -2 Under UV-B conditions, the culture was expanded and placed statically for 2 months until it dried naturally, and then dried for 2 months. Then 150 mL of BG110 medium was added, and the above dry-wet cycle procedure was repeated 30 times. In about 4 years, a stable genetic stress-tolerant Pueraria lobata germplasm was bred.

[0019] 3) Germplasm stress tolerance evaluation: ① Compared with common kudzu vine germplasm, the stress-tolerant kudzu vine germplasm obtained in this example was heated to 25°C + 25 μmol m -2s -1 After 40 days of static culture under visible light conditions, the cumulative amount of dry matter increased significantly by about 1 times; ② Compared with the common kudzu vine germplasm, the stress-tolerant kudzu vine germplasm obtained in this example was placed in a BG110 solution containing 28% by mass PEG6000 for 12 days, and the survival rate was significantly increased by about 40%; ③ Compared with the common kudzu vine germplasm, the stress-tolerant kudzu vine germplasm obtained in this example was placed in a 0.3 mol L -1 The survival rate of the cultivars was significantly increased by about 40% after being treated with BG110 solution containing 5% NaCl for 12 days. ④ Compared with the common cultivars of Puerariae, the stress-tolerant cultivars of Puerariae obtained in this example were placed in 700 μmol m -2 s -1 The survival rate was significantly increased by about 40% under the above test results after treatment for 1 day under the above conditions. The stress tolerance characteristics of the new germplasm of Miscanthus sibiricum obtained in this example were confirmed.

[0020] Example 4 1) Mutation treatment of kudzu vine: After the vigorously growing kudzu vine was homogenized, centrifuged, and washed three times with fresh BG110. BG110 was added to adjust the algae solution density to OD 680 =0.3. Take 5 mL of algae solution, centrifuge and discard the supernatant. Collect the algae pellet, add phosphate buffer (pH = 7) and a 5-6% by weight concentration of ethyl methanesulfonate solution, and keep in the dark for 6 hours. Add an equal volume of 5% by weight concentration of sodium thiosulfate solution to terminate the reaction. Wash three times with fresh BG110, resuspend the algae in 5 mL of fresh BG110, keep in the dark for 12 hours, and incubate at 25°C + 20µmol m -2 s -1 After 6-14 days of static culture under visible light, the algae solution was diluted and plated on BG110 plates. Once the strains on the plates had grown to spherical explants 3 mm in diameter, each seed of the kudzu seedling was transferred to 4 mL of BG110 liquid medium and cultured for another 2 months.

[0021] 2) Long-term laboratory adaptive evolution using multiple stresses: Transfer the kudzu vine germplasm into 50 mL BG110 medium and place the tube in a 300 μmol photons m -2 s -1 Visible light + 1.5 Wm -2 Under UV-B conditions, the culture was expanded and placed statically for half a month until it dried naturally, and then dried for half a month, and then 50 mL of BG110 culture medium was added. The above dry-wet cycle procedure was repeated 60 times. In about 5 years, a stable genetic stress-tolerant Pueraria lobata germplasm was bred.

[0022] 3) Germplasm stress tolerance evaluation: ① Compared with common kudzu vine germplasm, the stress-tolerant kudzu vine germplasm obtained in this example was heated to 25°C + 25 μmol m -2 s -1 After 45 days of static culture under visible light conditions, the cumulative amount of dry matter increased significantly by about 1.5 times; ② Compared with the common kudzu vine germplasm, the stress-tolerant kudzu vine germplasm obtained in this example was placed in a BG110 solution containing 25% by mass PEG6000 for 12 days, and the survival rate was significantly increased by about 45%; ③ Compared with the common kudzu vine germplasm, the stress-tolerant kudzu vine germplasm obtained in this example was placed in a 0.3 mol L -1 The survival rate of the cultivars was significantly increased by about 45% after being treated with BG110 solution of NaCl for 12 days. ④ Compared with the common cultivars of Puerariae, the stress-tolerant cultivars of Puerariae obtained in this example were placed in 700 μmol m -2 s -1 The survival rate was significantly increased by about 45% under the above test results after treatment for 1 day under the above conditions.

Claims

1. A breeding method for improving the stress tolerance of kudzu vine, characterized in that The steps include: 1) Mutation treatment of kudzu vine: Homogenize kudzu vine, centrifuge, wash with BG110, add BG110 to make algae liquid, centrifuge and discard the supernatant, add pH=7 phosphate buffer and 5-6% ethyl methanesulfonate solution by weight, treat in the dark, add 5% sodium thiosulfate solution by weight to terminate the reaction, wash with BG110, suspend the algae liquid in BG110, protect from light, and store at 25℃+ 20µmol m -2 s -1 Under visible light conditions, the algae solution was spread on a BG110 plate. After the strain on the plate grew into a spherical explant, the kudzu seedlings were transferred to a BG110 liquid medium for further cultivation. 2) Long-term laboratory adaptive evolution using multiple stresses: The kudzu vine germplasm was transferred to BG110 medium and then placed in 100-500 μmol photons m -2 s -1 Visible light +1~ 2Wm -2 The culture was expanded under UV-B conditions until it dried naturally, and then the drying process was continued. Then BG110 medium was added and the above dry-wet cycle procedure was repeated for at least 15 times to select a stable genetic stress-tolerant Kudzu chinensis germplasm.

2. A breeding method for improving the stress tolerance of Miscanthus truncatus as claimed in claim 1, characterized in that The steps include: 1) Mutation treatment of kudzu vine: After homogenizing kudzu vine, centrifuge and wash three times with BG110. Add BG110 to adjust the algae solution density to OD 680 =0.3~0.4, take V1 volume of algae liquid, centrifuge and discard the supernatant, add pH=7 phosphate buffer and 5-6% mass percentage concentration of ethyl methanesulfonate solution, dark-treat for 6~12 hours, add equal volume mass percentage concentration of 5% sodium thiosulfate solution to terminate the reaction, wash with BG110 3 times, suspend the algae liquid in the V1 volume of BG110, protect from light for 12 hours, and store at 25℃+ 20µmol m -2 s -1 Cultivate under visible light for 6 to 14 days, dilute the algae solution, and apply it to BG110 plates. After the strain grows into a spherical protophyte with a diameter of 1 to 3 mm on the plate, transfer each Kudzu seedling to 2 mL to 5 mL of BG110 liquid medium and continue culturing for 2 to 3 months. 2) Long-term laboratory adaptive evolution using multiple stresses: Transfer the germplasm of Miscanthus sibiricum to 50-200 mL BG110 medium and place it in 100-500 μmol photons m -2 s -1 Visible light +1~ 2 Wm -2 The culture was expanded statically under UV-B conditions until it dried naturally, and the drying treatment was continued for 0.5 to 3 months. Then 50 to 200 mL of BG110 medium was added and the above dry-wet cycle procedure was repeated 15 to 60 times to select a stable genetic stress-tolerant Kudzu chinensis germplasm.