Method for creating drought-resistant new blue fescue strain based on < 60 > Co-gamma radiation mutagenesis

Abstract

The invention provides a method for creating a novel drought-resistant blue fescue strain based on < 60 > Co-gamma radiation mutagenesis, and belongs to the technical field of mutation breeding. The method for directionally screening the drought-resistant mutagenic mutant of the festuca arundinacea comprises the following steps: taking embryonic callus of the festuca arundinacea as an irradiation material, and performing < 60 > Co-gamma radiation mutagenesis with the dose of a radiation mutagenic agent being 30-50Gy; after mutagenesis is completed, recovery culture, differentiation culture and rooting culture are carried out, 20%-30% of PEG6000 is added into a differentiation culture medium, and drought-resistant directional screening is carried out. The callus is subjected to radiation treatment, and in the differentiation culture stage, PEG6000 is added as the selection pressure of drought resistance screening, so that the culture process of the drought-resistant variety of the festuca arundinacea can be greatly accelerated.

Description

Technical Field

[0001] The present invention belongs to the technical field of mutation breeding and specifically relates to a method based on 60 A method for creating new drought-resistant strains of blue fescue by Co-γ radiation mutagenesis. Background Art

[0002] Drought stress is one of the abiotic stresses that restricts ecological benefits and affects the ornamental value of plants. At present, due to the impact of global warming, the degree of drought in various regions of China has worsened. Drought in various regions is not limited by region. Whether it is humid, semi-humid, arid, or semi-arid areas in the north or south, there are different degrees of drought. According to statistics, the frequency of drought disasters in my country accounts for about 1 / 3 of the total frequency of climate disasters, ranking first among all disasters.

[0003] Blue fescue (Festuca glauca), also known as silver fescue, is an evergreen herb belonging to the Poaceae family and the genus Fescue. It is a cool-season ornamental grass with blue leaves. It has a beautiful plant shape and is often used as the edging of flower beds and flower mirrors in garden landscapes. It can also be used as a potted ornamental plant. However, blue fescue has poor drought tolerance and usually requires a lot of irrigation during maintenance, which greatly increases its breeding costs. On the other hand, studies have also found that planting drought-resistant and low-water-demand ornamental grass varieties can greatly save water. In addition, blue fescue is native to Europe, and it is difficult to obtain germplasm resources in China, which is another obstacle to its promotion and application in China. Therefore, cultivating new blue fescue strains with strong drought resistance is of great significance in the development of the ornamental grass seed industry.

[0004] Radiation mutagenesis has the advantages of being simple, safe, having a high mutation rate, stable mutant traits, and a short breeding cycle. It has now become an effective means to improve germplasm traits and create new germplasm, and is widely used in various fields. 60 Co-γ rays are an important radiation source and have been used in the breeding of lawn grasses and crops, and many new varieties with excellent traits have been bred. There are no reports on the research of radiation-induced mutagenesis of blue fescue in China. 60 The Co-γ-ray radiation induced mutagenesis breeding technology has been used to cultivate drought-resistant mutants of blue fescue. On the one hand, it has greatly expanded the germplasm resources of blue fescue, and on the other hand, it has provided technical support for the cultivation of water-saving and drought-resistant blue fescue. Summary of the invention

[0005] In view of the above-mentioned problems existing in the current blue fescue, the present invention provides a 60 A method for creating a new drought-resistant strain of blue fescue by Co-γ radiation mutagenesis. 60 Co-γ radiation induced the embryonic callus of blue fescue to produce drought-resistant mutants. After being cultured in a special culture medium and cultivation matrix, the germination rate and survival rate of the mutants were guaranteed, and a blue fescue mutant with strong drought resistance was cultivated.

[0006] The present invention is based on 60 The method for creating a new drought-resistant strain of blue fescue by Co-γ radiation mutagenesis comprises the following steps:

[0007] S1: Obtaining callus tissue: After sterilizing blue fescue seeds, inoculate them in the induction medium to induce callus tissue; the specific treatment method of blue fescue seeds is as follows: wrap the mature blue fescue seeds to be sterilized with gauze, place them under running water for 3 hours, put the seeds in a 50mL sterilized beaker, add 50% sulfuric acid on the clean bench, stir for 10 minutes to remove the husk, sterilize with saturated sodium hypochlorite for 10 minutes, sterilize with 70% ethanol for 1 minute, and rinse with sterile water for 4 to 5 times. After the liquid on the surface of the seeds is dried with sterilized filter paper, cut them longitudinally along the groove (the cut surface contacts the culture medium), inoculate them in the induction culture medium, and culture them in the dark at 25°C for 30 days.

[0008] S2: Callus subculture: The callus cultured in S1 was transferred to the subculture medium to obtain granular embryonic callus; the callus grown for 30 days was transferred to the subculture medium, cultured in the dark at 25°C, the culture medium was replaced every 10 days, and the total culture was 30 days to obtain granular embryonic callus.

[0009] S3: Irradiation treatment: The granular embryonic callus obtained in S2 was subjected to 60 Co-γ ray irradiation, and then transferred to callus recovery medium; granular embryonic callus cut into 2-3 mm small pieces, transferred to sterile culture dishes to receive 60 Co-γ-ray irradiation. After irradiation, the cells were transferred to callus recovery medium and cultured in the dark at 25°C for 20 days.

[0010] S4: Polyethylene glycol stress: The callus tissue recovered from S3 was transferred to a differentiation medium containing polyethylene glycol for differentiation culture; the concentration of PEG6000 was 20-30%. The culture conditions were: temperature 25°C, photoperiod 16.0h, and culture for 20 days.

[0011] S5: Rooting and transplanting: The rootless seedlings differentiated and cultured in S4 were transferred to the rooting medium for 25-30 days, and the seedlings produced a large number of adventitious roots. The bottle cap was opened for hardening. After 3 days of hardening, the seedlings were transplanted into a 128-hole tray filled with a substrate (50% peat soil + 50% vermiculite) and placed in a greenhouse to continue to grow into seedlings.

[0012] The invention also comprises a drought resistance identification step: selecting physiological indicators such as relative water content (RWC), relative electrical conductivity (REC), malondialdehyde (MDA), chlorophyll and proline (Pro) content for drought resistance identification.

[0013] Furthermore, the induction medium in S1 is: MS medium supplemented with 2,4-D, NAA, sucrose, and agar, and the added amounts of 2,4-D, NAA, sucrose, and agar are 8 mg / L, 0.5 mg / L, 30.0 g / L, and 8.0 g / L, respectively.

[0014] Furthermore, the S2 subculture medium is: MS medium + 2,4-D (2.0 mg / L) + NAA (0.5 mg / L) + acid hydrolyzed casein (0.5 g / L) + agar (8.0 g / L).

[0015] Furthermore, in S3 60 The Co-γ ray radiation dose is 5-70Gy, and the radiation dose rate is 1Gy / min.

[0016] Furthermore, the recovery medium in S3 is: MS medium + 2,4-D (2.0 mg / L) + NAA (0.5 mg / L) + acid hydrolyzed casein (0.5 g / L) + agar (8.0 g / L).

[0017] Furthermore, the differentiation medium in S4 is: MS medium + 6-BA (2.0 mg / L) + KT (0.5 mg / L) + NAA (0.1 mg / L) + sucrose (30.0 g / L) + agar (8.0 g / L).

[0018] Furthermore, the differentiation culture conditions in S4 are: temperature 25°C, photoperiod 16.0h, culture 20 days. After 15-20 days of culture, rootless seedlings are gradually formed.

[0019] Furthermore, the rooting medium in S5 is: 1 / 2MS medium + NAA (0.5 mg / L) + sucrose (30.0 g / L) + agar (8.0 g / L).

[0020] Furthermore, the greenhouse environment culture conditions in S5 are: relative humidity 65%, PAR 1500 μmol / m 2 / s, photoperiod of 16.0h, and average temperature of 30℃ / 25℃ (day / night).

[0021] The present invention also provides a new drought-resistant strain of blue fescue, which is based on the 60 The method of creating a new drought-resistant strain of blue fescue by Co-γ radiation mutagenesis was used.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1) The present invention applies radiation mutagenesis technology to the cultivation of drought-resistant blue fescue. By determining the radiation dose, a drought-resistant blue fescue mutant is successfully cultivated, which has stronger drought resistance than the existing blue fescue and is more suitable for planting in a water-deficient environment. The drought-resistant blue fescue mutant has stronger drought resistance and survival ability in water-deficient land than the existing blue fescue.

[0024] 2) The step of screening target radiation mutants in the prior art is generally carried out after the cultivation is completed. Since radiation mutagenesis will produce a large number of mutant offspring, the screening work is cumbersome, the workload is large, and a large amount of manpower and material resources are consumed. The present invention introduces the screening step in the cultivation stage, and the drought resistance is screened after the radiation-induced callus mutation, and the most suitable PEG screening pressure is determined, which ensures that the blue fescue seedlings regenerated after screening have drought resistance and can root and grow normally, effectively reducing labor input and accelerating the seedling cultivation process, which is very suitable for industrial production.

[0025] 3) The present invention overcomes the existing plant radiation mutagenesis methods which mainly use the whole plant or tissues and organs of different parts of the plant as materials, and the radiation mutagenesis often results in recessive mutants and chimeras. Under in vitro culture conditions, granular embryonic callus with high frequency plant regeneration ability is selected as the material for 60 Co-γ-ray irradiation treatment, regenerating plants from irradiated cells, is beneficial for obtaining induced homozygotes, reducing induced chimeras, and improving the breeding efficiency of radiation-induced mutagenesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Correlation analysis between 60Co-γ irradiation dose and callus mortality;

[0027] Figure 2 Changes in leaf water content of blue fescue during PEG treatment;

[0028] Figure 3 Malondialdehyde content in blue fescue leaves during PEG treatment;

[0029] Figure 4 Relative conductivity of blue fescue leaves during PEG treatment;

[0030] Figure 5 Proline content in blue fescue leaves during PEG treatment;

[0031] Figure 6 Chlorophyll content of blue fescue leaves during PEG treatment;

[0032] Figure 7 Embryogenic callus of blue fescue before irradiation treatment;

[0033] Figure 8Blue fescue callus after radiation mutagenesis;

[0034] Fig. 9 Regenerated shoots formed after differentiation by PEG treatment;

[0035] Fig.10 Blue fescue mutants grown in a greenhouse. DETAILED DESCRIPTION

[0036] Combination Figure 1-10 The technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] Example 1

[0038] A kind of 60 The method for creating a new drought-resistant strain of blue fescue by Co-γ radiation mutagenesis includes the following experimental schemes and steps:

[0039] 1.1 Test materials

[0040] The experiment was conducted in April 2023. The experimental material was mature seeds of blue fescue "Festina", provided by Jiangsu Agricultural and Animal Husbandry Science and Technology Vocational College. The radiation source was 60 Co-γ-ray source, irradiation treatment was carried out in the Irradiation Center of Nanjing University of Aeronautics and Astronautics.

[0041] 1.2 Callus preparation

[0042] Wrap the mature seeds of blue fescue to be sterilized with gauze, soak them under running water for 3 hours, put the seeds in a 50mL sterilized beaker, add 50% sulfuric acid on a clean bench, stir for 10 minutes to remove the husks, sterilize with saturated sodium hypochlorite for 10 minutes, disinfect with 70% ethanol for 1 minute, and rinse with sterile water 4 to 5 times. After absorbing the liquid on the surface of the seeds with sterilized filter paper, cut them longitudinally along the groove (the cut surface contacts the culture medium), inoculate them in the induction medium, and culture them in the dark at 25°C for 30 days. The induction medium used is: MS medium + 2,4-D (8 mg / L) + NAA (0.5 mg / L) + sucrose (30.0 g / L) + agar (8.0 g / L). The callus tissue is then transferred to the subculture liquid culture medium and cultured in the dark at 25°C. The culture medium is replaced every 10 days, and the total culture is 30 days to obtain granular embryonic callus tissue, such as Figure 6 shown.

[0043] The subculture medium used was: MS medium + 2,4-D (2.0 mg / L) + NAA (0.5 mg / L) + acid hydrolyzed casein (0.5 g / L) + agar (8.0 g / L).

[0044] 1.3 Radiation treatment

[0045] Select a portion of the granular embryonic callus and cut it into 2-3 mm small pieces and transfer it to a sterile culture dish to receive 60 Co-γ-ray irradiation, 10Gy radiation dose, 1Gy / min radiation dose rate, 10 culture dishes per treatment group, 6 pieces of granular embryonic callus tissue of the same texture per culture dish, 4 replicates. After irradiation treatment, transfer to callus recovery medium and culture in the dark at 25℃ for 20 days. Figure 7 shown.

[0046] The recovery medium used was: MS medium + 2,4-D (2.0 mg / L) + NAA (0.5 mg / L) + acid hydrolyzed casein (0.5 g / L) + agar (8.0 g / L).

[0047] 1.4 PEG6000 screening of mutants

[0048] The callus tissue that recovered normal growth after radiation mutagenesis was transferred to a differentiation medium containing 20% ​​PEG6000 to simulate drought and perform differentiation culture. The culture conditions were: temperature 25°C, photoperiod 16.0h, and culture for 20 days. After 15-20 days of culture, rootless seedlings gradually formed. Figure 8 shown.

[0049] The differentiation medium used was: MS medium + 6-BA (2.0 mg / L) + KT (0.5 mg / L) + NAA (0.1 mg / L) + sucrose (30.0 g / L) + agar (8.0 g / L).

[0050] 1.5 Rooting and transplanting

[0051] After the rootless seedlings were transferred to the rooting medium and cultured for 25-30 days, a large number of adventitious roots were produced. The seedlings were hardened by opening the bottle cap. After 3 days of hardening, they were transplanted into a 128-hole tray filled with a substrate (50% peat soil + 50% vermiculite) and placed in a greenhouse to continue to cultivate into seedlings. Greenhouse environmental culture conditions: relative humidity 65%, PAR 1500μmol / m 2 / s, photoperiod 16.0h, average temperature 30℃ / 25℃ (day / night). Fig. 9 shown.

[0052] The rooting medium used was: 1 / 2MS medium + NAA (0.5 mg / L) + sucrose (30.0 g / L) + agar (8.0 g / L).

[0053] Example 2

[0054] A kind of 60 The method for creating a new drought-resistant strain of blue fescue by Co-γ radiation mutagenesis is different from that in Example 1 in that:

[0055] 1.3 Radiation treatment: Granular embryonic callus received 60 Co-γ-ray irradiation, radiation dose of 20 Gy;

[0056] 1.4 PEG6000 screening of mutants: The recovered callus tissue was transferred to the differentiation medium containing 20% ​​PEG6000.

[0057] Example 3

[0058] A kind of 60 The method for creating a new drought-resistant strain of blue fescue by Co-γ radiation mutagenesis is different from that in Example 1 in that:

[0059] 1.3 Radiation treatment: Granular embryonic callus received 60 Co-γ-ray irradiation, radiation dose of 30 Gy;

[0060] 1.4 PEG6000 screening of mutants: The recovered callus tissue was transferred to the differentiation medium containing 20% ​​PEG6000.

[0061] Example 4

[0062] A kind of 60 The method for creating a new drought-resistant strain of blue fescue by Co-γ radiation mutagenesis is different from that in Example 1 in that:

[0063] 1.3 Radiation treatment: Granular embryonic callus received 60 Co-γ-ray irradiation, radiation dose of 40 Gy;

[0064] 1.4 PEG6000 screening of mutants: The recovered callus tissue was transferred to the differentiation medium containing 20% ​​PEG6000.

[0065] Example 5

[0066] A kind of 60 The method for creating a new drought-resistant strain of blue fescue by Co-γ radiation mutagenesis is different from that in Example 1 in that:

[0067] 1.3 Radiation treatment: Granular embryonic callus received 60 Co-γ-ray irradiation, radiation dose of 50 Gy;

[0068] 1.4 PEG6000 screening of mutants: The recovered callus tissue was transferred to a differentiation medium containing 30% PEG6000.

[0069] Example 6

[0070] A kind of 60 The method for creating a new drought-resistant strain of blue fescue by Co-γ radiation mutagenesis is different from that in Example 1 in that:

[0071] 1.3 Radiation treatment: Granular embryonic callus received 60 Co-γ-ray irradiation, radiation dose of 60 Gy;

[0072] 1.4 PEG6000 screening of mutants: The recovered callus tissue was transferred to a differentiation medium containing 30% PEG6000.

[0073] Example 7

[0074] A kind of 60 The method for creating a new drought-resistant strain of blue fescue by Co-γ radiation mutagenesis is different from that in Example 1 in that:

[0075] 1.3 Radiation treatment: Granular embryonic callus received 60 Co-γ-ray irradiation, radiation dose of 70 Gy;

[0076] 1.4 PEG6000 screening of mutants: The recovered callus tissue was transferred to a differentiation medium containing 30% PEG6000.

[0077] Example 8

[0078] A kind of 60 The method for creating a new drought-resistant strain of blue fescue by Co-γ radiation mutagenesis is different from that in Example 1 in that:

[0079] 1.3 Radiation treatment: Granular embryonic callus received 60 Co-γ-ray irradiation, radiation dose of 40 Gy;

[0080] 1.4 PEG6000 screening of mutants: The recovered callus tissue was transferred to the differentiation medium containing 0% PEG6000.

[0081] Example 9

[0082] A kind of 60 The method for creating a new drought-resistant strain of blue fescue by Co-γ radiation mutagenesis is different from that in Example 1 in that:

[0083] 1.3 Radiation treatment: Granular embryonic callus received 60Co-γ-ray irradiation, radiation dose of 40 Gy;

[0084] 1.4 PEG6000 screening of mutants: The recovered callus tissue was transferred to the differentiation medium containing 10% PEG6000.

[0085] Example 10

[0086] A kind of 60 The method for creating a new drought-resistant strain of blue fescue by Co-γ radiation mutagenesis is different from that in Example 1 in that:

[0087] 1.3 Radiation treatment: Granular embryonic callus received 60 Co-γ-ray irradiation, radiation dose of 40 Gy;

[0088] 1.4 PEG6000 screening of mutants: The recovered callus tissue was transferred to a differentiation medium containing 30% PEG6000.

[0089] Embodiment 11

[0090] A kind of 60 The method for creating a new drought-resistant strain of blue fescue by Co-γ radiation mutagenesis is different from that in Example 1 in that:

[0091] 1.3 Radiation treatment: Granular embryonic callus received 60 Co-γ-ray irradiation, radiation dose of 40 Gy;

[0092] 1.4 PEG6000 screening of mutants: The recovered callus tissue was transferred to the differentiation medium containing 40% PEG6000.

[0093] Comparative Example 1

[0094] 1.1 Test materials

[0095] The experiment was conducted in April 2023. The experimental material was mature seeds of blue fescue "Festina", provided by Jiangsu Agricultural and Animal Husbandry Science and Technology Vocational College. The radiation source was 60 Co-γ-ray source, irradiation treatment was carried out in the Irradiation Center of Nanjing University of Aeronautics and Astronautics.

[0096] 1.2 Callus preparation

[0097] Wrap the mature seeds of blue fescue to be sterilized with gauze, place them under running water and brew for 3 hours, put the seeds in a 50mL sterilized beaker, add 50% sulfuric acid and stir for 10 minutes on the clean bench to remove the husk, sterilize with saturated sodium hypochlorite for 10 minutes, sterilize with 70% ethanol for 1 minute, and rinse with sterile water 4 to 5 times. After the liquid on the surface of the seeds is absorbed by sterilized filter paper, they are cut longitudinally along the groove (the cut surface contacts the culture medium), inoculated in the induction culture medium, and cultured in the dark at 25°C for 30 days. The induction culture medium used is: MS culture medium + 2,4-D (8mg / L) + NAA (0.5mg / L) + sucrose (30.0g / L) + agar (8.0g / L). Then the callus tissue is transferred to the subculture liquid culture medium, cultured in the dark at 25°C, and the culture medium is replaced every 10 days. The total culture is 30 days to obtain granular embryonic callus tissue. The subculture medium used was: MS medium + 2,4-D (2.0 mg / L) + NAA (0.5 mg / L) + acid hydrolyzed casein (0.5 g / L) + agar (8.0 g / L).

[0098] 1.3 Differentiation culture

[0099] The callus of blue fescue was used as the material, transferred to the differentiation medium, and continued to be cultured and observed. The culture conditions during the differentiation culture were: temperature 25℃, photoperiod 16.0h, and culture for 20 days. After 15-20 days of culture, rootless seedlings gradually formed. The differentiation medium used was: MS medium + 6-BA (2.0mg / L) + KT (0.5mg / L) + NAA (0.1mg / L) + sucrose (30.0g / L) + agar (8.0g / L).

[0100] After the rootless seedlings were transferred to the rooting medium and cultured for 25-30 days, a large number of adventitious roots were produced. The seedlings were hardened by opening the bottle cap. After 3 days of hardening, they were transplanted into a 128-hole tray filled with a substrate (50% peat soil + 50% vermiculite) and placed in a greenhouse to continue to cultivate into seedlings. Greenhouse environmental culture conditions: relative humidity 65%, PAR 1500μmol / m 2 / s, photoperiod 16.0h, average temperature 30℃ / 25℃ (day / night). The rooting medium used was: 1 / 2MS medium + NAA (0.5mg / L) + sucrose (30.0g / L) + agar (8.0g / L).

[0101] The following results and analysis were obtained through Examples 1-11 and Comparative Example 1:

[0102] 2.1 Effects of radiation dose on callus differentiation rate and seedling formation of blue fescue

[0103] Through the above examples and comparative examples, the callus differentiation rate, seedling rate and mortality rate of different radiation treatments were calculated, and the radiation half-lethal dose (LD50) was obtained according to the mortality rate, that is, the radiation dose that causes 50% callus death. Half-lethal dose (LD50): With different radiation doses x as independent variables and mortality rates y under different doses as dependent variables, the linear regression equation y=a+bx and the formula LD50=(50-a) / b were used for calculation.

[0104] Callus differentiation rate (%) = number of callus differentiation / number of inoculated callus blocks × 100%

[0105] Seedling rate (%) = number of regenerated plants / number of inoculated callus blocks × 100%

[0106] Mortality (%) = number of dead calli / number of inoculated callus blocks × 100%

[0107] Table 1 Callus differentiation rate, mortality rate and seedling rate of blue fescue treated with different radiation doses

[0108]

[0109]

[0110] It can be seen from Table 1 that the callus differentiation rate and seedling rate at low doses (0-10Gy) are higher than those at 0Gy (Table 1), indicating that low-dose irradiation has a stimulating effect on callus growth, but this stimulating effect gradually disappears with the extension of time. With the increase of radiation dose (20-70Gy), the callus differentiation rate and seedling rate decreased (Table 1). From the seedling rate, it can be seen that 30-50Gy is the dose-sensitive area for callus mutagenesis of blue fescue, among which 40Gy is the best radiation dose.

[0111] 2.2 60 Determination of the half-lethal dose of Co-γ irradiated blue fescue callus

[0112] With the increase of radiation dose, the mortality rate of callus tissue showed an upward trend. When the dose was greater than 30Gy, the mortality rate increased sharply. When the dose increased to 40Gy, the mortality rate of callus tissue was close to 50%. When the dose continued to increase to 50Gy, the mortality rate of callus tissue exceeded 2 / 3 (Table 1). With the increase of radiation dose, the color of some callus tissue gradually darkened, from green to brown, and the time for the callus tissue to turn brown was accelerated, and finally the callus tissue gradually died. Through the correlation and regression analysis of radiation dose and mortality rate, the linear equation was obtained as y=1.4968x-11.825, and the correlation coefficient R 2 =0.9644, indicating that there is a positive correlation between the mortality of blue fescue callus and the irradiation dose ( Figure 1). The median lethal dose (LD50) of Pinellia ternata callus was calculated based on the linear regression equation, which was 41.30 Gy.

[0113] 2.3 Effects of different PEG6000 screening pressures on callus differentiation of blue fescue

[0114] The higher the PEG concentration, the lower the water potential of the culture medium, and the greater the drought stress intensity on the blue fescue callus. With the increase of PEG concentration, the blue fescue callus showed a trend of becoming more seriously affected by drought stress. When the PEG concentration was 20-30%, the differentiation rate of blue fescue callus was between 38.4-24.3%, which is a suitable PEG concentration screening pressure. Compared with comparative example 1, the differentiation rate of blue fescue callus at 30% PEG was close to half of the differentiation rate of the control. It can be seen that the 30% PEG concentration is the semi-lethal concentration for the differentiation of blue fescue callus.

[0115] Table 2 Callus differentiation rate, mortality rate and seedling rate of blue fescue under different PEG screening pressure

[0116]

[0117] 2.4 Phenotypic identification of mutants

[0118] Relative water content is an indicator that directly measures the water content in plant leaves. On the one hand, it can partially reflect the drought resistance of plants, and on the other hand, it can also reflect the impact of drought stress on plants. Figure 2 As shown in the figure, there was no significant difference among the three groups of materials (mutants dt1, dt2 and WT) on the 0th day of drought. After 14 days of drought, the relative water content of mutants dt1, dt2 and WT was significantly reduced compared with the control (0th day of drought), but the RWC of mutants dt1 and dt2 was significantly higher than that of WT. This result shows that after experiencing drought stress, the water retention capacity of mutants dt1 and dt2 is stronger than that of WT.

[0119] Relative conductivity (EL) and malondialdehyde (MDA) content are important physiological indicators for measuring the degree of cell membrane damage. The higher the EL and MDA, the more serious the cell membrane damage. Figure 3 and 4As shown in the figure, under normal conditions, there was no significant difference in EL and MDA content among the three groups of materials (mutants dt1, dt2 and WT). After 15 days of PEG drought treatment, the EL content of the leaves of mutants dt1, dt2 and WT increased significantly, and the EL content of mutant dt1 and dt2 leaves was significantly lower than that of WT. Similarly, after 15 days of drought stress treatment, the MDA content of mutant dt1, dt2 and WT leaves increased significantly, and the MDA content of dt1 and dt2 leaves was significantly lower than that of WT. The above results show that after drought stress treatment, the degree of cell membrane lipid damage in mutants dt1 and dt2 is significantly lower than that of WT.

[0120] Free proline is one of the most important osmotic regulating substances in plants. It plays an important role in maintaining the water content of plant cells. The change of its content is closely related to the drought tolerance of plants. In drought-tolerant plants, the proline content is significantly higher than that of drought-intolerant varieties. Figure 5 It can be seen that after PEG drought stress treatment, the proline content in the leaves of the mutants dt1 and dt2 was significantly higher than that of WT. The results showed that the mutants dt1 and dt2 had a stronger ability to maintain cell water content and had stronger cold resistance.

[0121] Under drought stress conditions, leaves will lack water and cause chlorophyll decomposition and a decrease in content, thus hindering photosynthesis. Chlorophyll content is negatively correlated with drought stress, and the higher the degree of drought, the faster the chlorophyll content decreases. The longer the crop is exposed to drought, the greater the decrease in chlorophyll content. Figure 6 It can be seen that after PEG drought stress treatment, the chlorophyll content of the leaves of the mutants dt1 and dt2 was significantly higher than that of WT. The results showed that compared with WT, the mutants dt1 and dt2 had stronger photosynthesis ability and stronger drought resistance.

[0122] Comprehensive changes in relative water content, relative conductivity, malondialdehyde content, chlorophyll content and free proline content showed that the mutant had stronger drought resistance.

[0123] It should be noted that, in this article, positional terms such as top, bottom, left, right, etc. are used only for convenience of description. Moreover, the terms "include", "comprises", or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article, or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, or article.

[0124] Of course, the above description is only a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It should be noted that all equivalent substitutions and obvious deformation forms made by any technician familiar with the field under the guidance of this specification fall within the essential scope of this specification and should be protected by the present invention.

Claims

1. A method based on 60 The method for creating a new drought-resistant strain of blue fescue by Co-γ radiation mutagenesis is characterized in that: The steps include: S1: Obtaining callus tissue: After sterilizing blue fescue seeds, inoculate them into induction medium to induce callus tissue; S2: Callus subculture: The callus cultured in S1 is transferred to the subculture medium to obtain granular embryonic callus; S3: Irradiation treatment: The granular embryonic callus obtained in S2 was subjected to 60 Co-γ-ray irradiation followed by transfer to callus recovery medium; S4: polyethylene glycol stress: the callus tissue recovered from S3 was transferred to the differentiation medium containing polyethylene glycol for differentiation culture; S5: Rooting and transplanting: The rootless seedlings differentiated and cultured in S4 are transferred to rooting medium for culture. After a large number of adventitious roots are produced, they are cultured into seedlings in the greenhouse.

2. According to claim 1, based on 60 The method for creating a new drought-resistant strain of blue fescue by Co-γ radiation mutagenesis is characterized by: The induction medium in S1 is: MS medium supplemented with 2,4-D, NAA, sucrose, and agar, wherein the added amounts of 2,4-D, NAA, sucrose, and agar are 8 mg / L, 0.5 mg / L, 30.0 g / L, and 8.0 g / L, respectively.

3. According to claim 1 based on 60 The method for creating a new drought-resistant strain of blue fescue by Co-γ radiation mutagenesis is characterized by: The S2 subculture medium is: MS medium + 2,4-D (2.0 mg / L) + NAA (0.5 mg / L) + acid hydrolyzed casein (0.5 g / L) + agar (8.0 g / L).

4. The method according to claim 1 or 3 60 The method for creating a new drought-resistant strain of blue fescue by Co-γ radiation mutagenesis is characterized by: The S2 subculture method is: culturing in the dark at 25° C., replacing the culture medium every 10 days, and culturing for a total of 30 days.

5. The method according to claim 1 60 The method for creating a new drought-resistant strain of blue fescue by Co-γ radiation mutagenesis is characterized by: The S3 60 The Co-γ ray radiation dose is 30-50Gy.

6. The method according to claim 1 or 5 60 The method for creating a new drought-resistant strain of blue fescue by Co-γ radiation mutagenesis is characterized by: The recovery medium in the S3 is: MS medium + 2,4-D (2.0 mg / L) + NAA (0.5 mg / L) + acid hydrolyzed casein (0.5 g / L) + agar (8.0 g / L).

7. The method according to claim 1 60 The method for creating a new drought-resistant strain of blue fescue by Co-γ radiation mutagenesis is characterized by: The differentiation medium in the S4 is: MS medium + 6-BA (2.0 mg / L) + KT (0.5 mg / L) + NAA (0.1 mg / L) + sucrose (30.0 g / L) + agar (8.0 g / L).

8. The method according to claim 1 or 7. 60 The method for creating a new drought-resistant strain of blue fescue by Co-γ radiation mutagenesis is characterized by: The concentration of polyethylene glycol in the S4 is 20-30%.

9. The method according to claim 1 60 The method for creating a new drought-resistant strain of blue fescue by Co-γ radiation mutagenesis is characterized by: The S5 rooting medium is: 1 / 2MS medium + NAA (0.5 mg / L) + sucrose (30.0 g / L) + agar (8.0 g / L).

10. A new drought-resistant strain of blue fescue, characterized in that: Based on claims 1 to 9 60 The method of creating a new drought-resistant strain of blue fescue by Co-γ radiation mutagenesis was used.

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