LU501051B1 - Method for Screening Saline-alkali Tolerant Wheat Germplasm at Seedling Stage Based on Aseptic Hydroponics Condition - Google Patents
Method for Screening Saline-alkali Tolerant Wheat Germplasm at Seedling Stage Based on Aseptic Hydroponics Condition Download PDFInfo
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Abstract
The invention discloses a method for screening saline-alkali tolerant wheat germplasm at seedling stage based on an aseptic hydroponic condition, which belongs to the technical field of agricultural science and technology. The method comprises the following steps: (1) preparing for sterilization; (2) seed planting and cultivation; (3) saline-alkali stress tolerance; (4) saline-alkali tolerance evaluation. The wheat saline-alkali resistant germplasm materials screened by this method can well meet the actual production demand, and solve the technical problem that the saline-alkali resistant germplasm screened by saline-alkali stress treatment with single salt or double salt method in the prior art cannot meet the actual production demand.
Description
DESCRIPTION HUS0T051 Method for Screening Saline-alkali Tolerant Wheat Germplasm at Seedling Stage Based on Aseptic Hydroponics Condition
TECHNICAL FIELD The invention relates to the technical field of agricultural science and technology, in particular to a method for screening saline-alkali resistant wheat germplasm at seedling stage based on an aseptic hydroponic condition.
BACKGROUND Wheat is an important food crop for the world population, and wheat yield is an important factor affecting world food security. However, soil salinization leads to the destruction of cultivated land resources and huge losses of agricultural production, which has become a worldwide ecological problem.
It is found that wheat has the characteristics of salt-alkali tolerance. Therefore, in order to make full use of saline-alkali land, increase wheat yield in saline-alkali land and alleviate food security problems, it is necessary to identify the salt-alkali tolerance of wheat germplasm, and select the salt-alkali tolerant wheat germplasm for wheat production in saline-alkali land, so as to increase wheat yield in saline-alkali land. Moreover, in order to understand the damage mechanism of saline-alkali stress on wheat and the response mechanism of wheat to saline-alkali stress, it is necessary to use saline-alkali resistant germplasm materials for follow-up mechanism research.
At present, according to the screening period, the methods of screening indoor saline-alkali tolerant germplasm mainly include identification of saline-alkali tolerance in bud stage and identification of saline-alkali tolerance in seedling stage. The main process of germination saline-alkali tolerance method is as follows: selecting undamaged seeds, disinfecting seeds, cleaning with sterilized distilled water for 3-5 times, spreading seeds in a petri dish with two layers of filter paper, adding saline-alkali treatment solution for stress treatment, and culturing in a light incubator at 25°C/20°C (daytime/night), 14 hours/10 hours (daytime/night), and 4000 LUX in daytime. The germination potential is investigated on the 3rd day of culture, and the indexes such as germination rate, root length, bud length, fresh root weight and fresh bud weight were investigated on the 7th day, and the salt and alkali tolerance are HUS01051 evaluated according to the indexes. This method mainly has the following problems:
1. After 7 days of cultivation, the germination rate of saline-alkali tolerant seeds is low or even some seeds do not germinate at all, and more tissues cannot be obtained for further research, such as physiological indexes.
2. There is not enough downward depth in the culture dish, which leads to the failure of normal physiological downward growth of the root system and the failure of normal vertical growth of the upper part of the root system, which affects the growth form of the plant.
3. Due to incomplete disinfection, bacterial contamination is easy to occur in the cultivation process, which leads to large errors and inaccurate test results.
Aiming at the above problems of identification of saline-alkali tolerance in bud stage, the common identification method of saline-alkali tolerance in seedling stage can solve the above problems 1 and 2, but it is also difficult to avoid the problem of bacterial pollution, which leads to abnormal plant growth and affects the subsequent research on physiological indexes and molecular mechanisms related to saline-alkali tolerance. In addition, the traditional identification of saline-alkali tolerance often uses the method of single salt or double salt to deal with saline-alkali stress. Research shows that saline-alkali soil contains not only single salt or double salt, but also other saline-alkali components. Therefore, the salt-tolerant germplasm screened by traditional technology cannot meet the actual production needs.
SUMMARY The purpose of the present invention is to provide a method for screening saline-alkali resistant wheat germplasm at seedling stage based on a sterile hydroponic condition, so as to solve the problems existing in the prior art. Through the screening method of the present invention, the saline-alkali resistant wheat germplasm materials can be accurately identified, which can better meet the actual production requirements.
In order to achieve the above objective, the present invention provides the following scheme:
The invention provides a method for screening saline-alkali tolerant wheat 750705) germplasm at seedling stage based on a sterile hydroponic condition, which comprises the following steps: (1) sterilization preparation: wheat seeds, quartz sand and culture boxes are selected and sterilized and dried respectively; (2) seed planting and cultivation: the treated seeds are planted in a cultivation box where quartz sand is placed, and nutrient solution containing disinfectant is poured in until the liquid level exceeds 0.5 cm of quartz sand for cultivation; (3) saline-alkali stress tolerance treatment: culturing till wheat seedling stage, setting treatment group and control group, wherein treatment group replaces the nutrient solution containing disinfectant with artificial seawater treatment solution containing disinfectant with salinity of 0.6 times of standard salinity for 10 days; (4) saline-alkali tolerance evaluation: through comparing the morphological indexes of wheat seedlings in the treatment group and the control group, the salt-alkali tolerance germplasm of wheat can be screened.
Further, the drying method of wheat seeds in step (1) is natural drying or drying in a 40°C oven until the water content reaches 13%; the wheat seeds are sterilized by soaking in 10% sodium hypochlorite for 5 min, and then cleaning with sterilized distilled water for 3-5 times.
Further, in step (1), the quartz sand or the culture box is sterilized and dried at 121°C for 15 min, and then put into an oven at 80°C for full drying.
Further, the planting density of the seeds in step (2) is 200-250 seeds per box; the ratio of disinfectant to nutrient solution is 2g/L.
Further, the culture conditions in step (2) are 25°C in the daytime, the illumination intensity is 4000 LUX for 14 h; hours; 20°C at night, for 10 h; the humidity is 30-50%, and the nutrient solution containing disinfectant is changed every 3 days.
Further, the artificial seawater with standard salinity in step (3) comprises 24 parts of sodium chloride, 4 parts of sodium sulfate, 0.7 part of potassium chloride, 0.2 part of sodium bicarbonate, 0.1 part of potassium bromide, 0.02-0.03 part of boric acid, 0.003 part of sodium fluoride, 0.05-0.06 part of magnesium chloride 1501081 hexahydrate, 0.01 part of calcium chloride dihydrate and 0.001 part of strontium chloride hexahydrate.
Further, in step (4), the morphological indexes are analyzed by data to obtain the comprehensive score value D, and the salt-alkali tolerant wheat germplasm in seedling stage is screened out according to the D value.
Further, the D value greater than or equal to 0.668 is a saline-alkali tolerant wheat germplasm.
The invention also provides the application of any of the above methods in the identification of saline-alkali tolerant wheat germplasm at seedling stage.
The invention discloses the following technical effects: The invention discloses a method of saline-alkali stress treatment of wheat by using saline-alkali formula of simulated artificial seawater and sterile hydroponics, and selects wheat saline-alkali resistant germplasm suitable for actual production by using membership function comprehensive evaluation method, which solves the technical problem that the selected saline-alkali resistant germplasm can't meet the actual production demand by using single-salt or double-salt method for saline-alkali stress treatment in the identification of saline-alkali resistance in the prior art.
BRIEF DESCRIPTION OF THE FIGURES In order to more clearly explain the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention, and for ordinary technicians in the field, other drawings can be obtained according to these drawings without paying creative efforts.
Fig. 1 1s an intergroup pedigree diagram of 15 wheat germplasm resources in Example 2; Fig. 2 is the overall growth performance of sensitive wheat and saline-alkali resistant wheat seedlings screened in Example 2;
Fig. 3 is a comparison of seedling growth performance between control group HUS01051 and treatment group of sensitive wheat and saline-alkali resistant wheat screened in Example 2; Fig. 4 is a comparison of seedling root growth between control group and treatment group of sensitive wheat and saline-alkali resistant wheat screened in Example 2.
DESCRIPTION OF THE INVENTION Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of some aspects, characteristics and embodiments of the present invention.
It should be understood that the terms used in this invention are only for describing specific embodiments, and are not used to limit the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by the ordinary technicians in the field of this invention.
Materials, instruments and reagents used in this invention can be obtained from commercial channels unless otherwise specified. Unless otherwise specified, the experimental methods used are the conventional experimental methods in this field.
Example 1 A method for screening saline-alkali tolerant wheat germplasm at seedling stage based on aseptic hydroponics conditions specifically comprises the following steps:
1. Seed selection: select undamaged wheat seeds to fully dry or dry in a 40°C oven until the water content of wheat seeds reaches 13%.
2. Preparation of quartz sand: the grain size of quartz sand used for hydroponics is 1-2 mm, rinse with clear water for 3-5 times to remove the floating and sinking of the surface layer, then put it into a sterilization bag and sterilize at 121°C for 15 min, then put it into an oven at 80°C for full drying.
. . . . LU501051
3. Preparation of culture box: choose a square high-temperature resistant plastic box with a diameter of 20 cm and a depth of 15 cm, put it into a sterilization bag and sterilize it at 121°C for 15 min, and then put it into an oven at 80°C for full drying.
4. Prepare nutrient solution mother liquor: Table 1 Hoagland nutrient solution formula Hoagland nutritive medium A macroelement (1 L, 100 x) Ingredient Weigh (g) KNO3 50.500 MgSO:.7H:0 49.300 KH:PO4 13.600 B calcium salt (1 L, 100 x) Ingredient Weigh (g) Ca(NO3)2.4H20 118 C microelement (1 L, 1000 x) Ingredient Weigh (g) H:BO:4 2.860 MnClz.4H:0 1.810 or MnSO4.H:0 3.090 ZnSO4.7H20 0.220 CuSO04.5H:0 0.125 H:MoO04 0.018 or H:MoO4.H20 0.020 or MoO; 0.160 Molysite (1 L, 100 x) Ingredient Weigh (g) FeSO4.7H:0 2.78 NaEDTA.2H,0 3.73 or NaFDTA 3.73
. . . . LU501051 Note: according to the weight of the concentrated solution, dilute to a constant volume of 1 L to obtain 100 times solution of A, 100 times solution of B, 1000 times solution of C and 100 times solution of iron salt.
5. Configure artificial seawater with standard salinity: Table 2 Formula of artificial seawater Molecul Name © eel a Mass (g) weight Sodium chloride (NaCl) 58.44 23.926 Sodium sulfate (NazSO4) 142.04 4.008 Potassium chloride (KCI) 74.56 0.677 Sodium bicarbonate (NaHCO3) 84 0.196 Potassium bromide (KBr) 119.01 0.098 Boric acid (H:BO3) 61.83 0.026 Karidium (NaF) 41.99 0.003 Magnesium chloride hexahydrate
203.33 0.053 (MgCla.6H:0) Calcium chloride dihydrate (CaCl,.2H,0) 147.03 0.01 Strontium hexachloride hydrate
266.64 0.001 (SrCl,.6H,0) Distilled water (H,O) Constant volume at room temperature to 1 L
6. Environmental disinfection: prepare 75% alcohol and put it in a watering can. On the day of planting, the room where the incubator is placed and the incubator will be disinfected by alcohol spray.
7. Seed disinfection: take a mask and sterile gloves when planting. First, soak and disinfect with 10% sodium hypochlorite for 5 minutes, and then clean with enough sterilized distilled water for 3-5 times.
8. Seed planting and cultivation: when planting, take a mask and a sterile glove, and put the sterilized and dried quartz sand in a sterilized and dried cultivation box with a depth of 10-12 cm, spread the sterilized seeds on the surface of quartz sand, and the planting density is 200-250 seeds per box, prepare nutrient solution containing 2 g/L carbendazim in proportion, and pour it into quartz sand, so that the liquid level exceeds the quartz sand by 0.5 cm and placing in an incubator for seedling culture;
culture conditions is as follows: the temperature was set at 25°C in the daytime, and HUS01051 the light intensity was 4000 LUX for 14 hours; 20°C at night, for 10 hours; humidity is about 40%. During the period, the nutrient solution containing carbendazim is changed every 3 days.
9. Saline-alkali stress tolerance: when the seedlings are cultivated to the stage of two leaves and one heart, saline-alkali stress is performed, and sterilized distilled water is used to prepare a treatment solution containing 2 g/L carbendazim with a saline-alkali concentration of 0.6 times the standard salinity (this treatment concentration is the appropriate concentration determined according to the previous preliminary test) (treatment group), and nutrient solution is used as control (control group). During the period, the treatment solution and nutrient solution containing 2 g/L. carbendazim are changed every 3 days.
10. Investigation of related characteristics: after 10 days of artificial seawater treatment with 0.6 times standard salinity, the indexes of treatment group and control group were measured, including seedling height, fresh root weight, fresh aboveground weight, yellowing rate of the first and second leaves, total root length, total root surface area and total root volume.
11. Data statistics: EXCEL is used to calculate the relative values of each variety under artificial seawater treatment with 0.6 times standard salinity, including: relative seedling height, relative root fresh weight, relative aboveground fresh weight, relative yellowing rate of the first and second leaves, relative total root length, relative total root surface area and relative total root volume, relative value (%)=treated value/reference valuex100%.
SPSS 22.0 statistical software is used to standardize the data and make principal component analysis, through principal component analysis, several principal components with larger characteristic value and contribution rate are selected.
Based on the scores of several principal components and their weights, a comprehensive score (D value) is obtained by membership function, which is calculated according to the following formula: u(Xj) = (Xj-Xmin)/(Xmax—Xmin) J = 1, 2, 3, ..., n (1)
Where p(X) represents the membership function value of the j-th comprehensive index, Xj represents the j-th comprehensive index value, Xmax represents the maximum value of the j-th comprehensive index and Xmin represents the minimum value of the j-th comprehensive index.
Wi=P/>"-1Pij=1,2,3,...,n(2) Where W; represent that weight of the J comprehensive index in all comprehensive indexes, and Pj is the contribution rate of the j comprehensive index of the variety.
D = 2-1 [nX)*Wilj=1,2,3, … n G) Type, D represents the comprehensive evaluation value of salt-alkali tolerance of each variety.
Finally, according to the comprehensive score (D value), cluster analysis is carried out to classify wheat germplasm into different saline-alkali tolerant types.
Example 2 Using the method of Example 1, 15 wheat germplasm resources are identified for salt and alkali tolerance at seedling stage, and the relative values of 7 indexes were calculated. The results are shown in Table 3: Table 3 Trait indexes of 15 germplasms under saline-alkali stress (%) Relative Total Relative abovegr Relativ Total relative Total Relative . e root relative yellowing rate of Na seedling ound root root fresh root Ist and 2nd me length fresh length surface weight volume leaves weight area R1 55.94 27.89 52.95 76.61 67.26 56.36 87.34 R2 64.96 43.06 64.46 76.59 72.03 63.36 75.64 R3 69.97 54.41 73.46 78.46 76.95 75.27 60.29 R4 53.09 25.72 51.96 72.66 64.21 54.05 90.34 RS 73.97 65.14 86.14 79.27 83.22 84.51 47.58 R6 148.80 24 87 50.81 73.88 62.88 52.22 91.06 R7 58.62 31.74 55.40 74.27 68.37 58.20 85.09 R8 72.15 65.06 85.35 79.45 82.50 84.27 48.00 R9 61.68 37.01 59.54 77.08 70.49 61.23 79.32 N 71.07 63.02 77.50 83.98 79.61 80.10 52.57 R1 1 70.10 56.78 73.98 82.27 78.45 75.14 55.36
R1 LU501051 2 71.85 63.19 80.00 79.13 80.58 81.43 52.49 R1 3 66.67 44.48 68.62 75.55 74.21 68.12 71.28 R1 4 48.52 23.47 50.64 75.28 60.96 50.41 92.22 R1 , 6930 50.70 70.32 70.73 75.45 70.61 61.55 Principal component analysis was conducted on 7 indexes by using relative values, and one principal component was obtained, with a contribution rate of 90.88%, which is related to 7 indexes (see Table 4). Furthermore, comprehensive scores (D values) of salt-alkali tolerance of 15 germplasm resources were obtained by membership function formula (see Table 5). Table 4 Characteristic root value, contribution rate and load matrix of principal components Principal constituent The first principal component Eigenvalue 6.361 Contribution rate 90.876 Cumulative contribution rate 90.876 Load factor Relative aboveground fresh 0.996 weight Relative root fresh weight 0.988 Total root length 0.709 Total heel surface area 0.992 Total relative root volume 0.995 Relative height of seedling 0.966 Relative yellowing rate of 1st and 0.902 2nd leaves Table 5 Comprehensive character index, u(X) and comprehensive evaluation value Name F1 u(X1) Comprehensive score value (D) R14 0.366 0 0 R6 0.36 0.019 0.017 R4 -0.256 0.062 0.056 R1 -2.813 0.182 0.166 R7 0.724 0.222 0.202 RO 1.138 0.361 0.328 R2 -0.29 0.454 0.413
R13 -0.908 0.531 0.483 LUS01051 R15 0.223 0.577 0.524 R3 -0.546 0.735 0.668 R11 1.087 0.818 0.743 R12 0.233 0.899 0.817 R10 0.66 0.923 0.839 R8 1.068 0.979 0.889 RS -0.598 1 0.909 Cluster analysis based on D value (Figure 1) showed that 15 wheat germplasms could be divided into 5 groups at Euclidean distance D’=6.5. The first group consists of 4 materials, accounting for 26.67% of the total, which are germplasm with strong salt-alkali tolerance, with D values ranging from 0.817 to 0.909, followed by RS, RS, R10 and R12. The second group consists of 2 materials, accounting for 13.33% of the total, which are saline-alkali tolerant germplasm, with D value of 0.668-0.743, followed by R11 and R3. The third group consists of 4 materials, accounting for 26.67% of the total, which is an intermediate germplasm with a D value of 0.328-0.524, followed by R15, R13, R2 and R9. The fourth group contains 2 materials, accounting for 13.33% of the total, which are saline-alkali sensitive germplasm, with D value of
0.166-0.202, followed by R7 and R1. The fifth group contains 3 materials, accounting for 20.00% of the total, which are strongly saline-alkali sensitive germplasm, with D value of 0.00-0.056, followed by R4, R6 and R14. Therefore, when the D value of salt-alkali related traits of wheat germplasm is greater than or equal to 0.668, it is selected as salt-alkali tolerant wheat germplasm.
Comparing the seedling growth performance of saline-alkali sensitive wheat germplasm with saline-alkali tolerant wheat germplasm (see Figure 2), the results showed that saline-alkali tolerant wheat could grow normally under high saline-alkali stress, while saline-alkali sensitive wheat showed yellow wilt. Comparing the control group and treatment group of the same germplasm (see Figure 3), the results showed that although the seedlings of saline-alkali resistant wheat germplasm were slightly wilted compared with the control group after being treated with saline-alkali stress, the wilting situation was significantly alleviated compared with saline-alkali sensitive wheat germplasm. At the same time, comparing the root conditions of the control 750705) group and the treatment group of sensitive germplasm, the results show that the root growth of sensitive wheat in the treatment group is slow, while the root growth of saline-alkali resistant wheat treatment group is not significantly different from that of the control group (as shown in Figure 4), which shows that the technical scheme of the invention can accurately identify the saline-alkali resistant wheat germplasm materials.
The above-mentioned embodiments only describe the preferred mode of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, all kinds of modifications and improvements made by ordinary technicians in the field to the technical scheme of the present invention should fall within the protection scope determined by the claims of the present invention.
Claims (9)
1. A method for screening saline-alkali tolerant wheat germplasm at seedling stage based on an aseptic hydroponic condition, characterized by comprising the following steps: (1) sterilization preparation: wheat seeds, quartz sand and culture boxes are selected and sterilized and dried respectively; (2) seed planting and cultivation: the treated seeds are planted in a cultivation box where quartz sand is placed, and nutrient solution containing disinfectant is poured in until the liquid level exceeds 0.5 cm of quartz sand for cultivation; (3) saline-alkali stress tolerance treatment: culturing till wheat seedling stage, setting treatment group and control group, wherein treatment group replaces the nutrient solution containing disinfectant with artificial seawater treatment solution containing disinfectant with salinity of 0.6 times of standard salinity for 10 days; (4) saline-alkali tolerance evaluation: through comparing the morphological indexes of wheat seedlings in the treatment group and the control group, the salt-alkali tolerance germplasm of wheat can be screened.
2. The method according to claim 1, characterized in that the drying method of wheat seeds in step (1) is natural drying or drying in a 40°C oven until the water content reaches 13%; the wheat seeds are sterilized by soaking in 10% sodium hypochlorite for 5 min, and then cleaning with sterilized distilled water for 3-5 times.
3. The method according to claim 1, characterized in that in step (1), the quartz sand or the culture box is sterilized and dried at 121°C for 15 min, and then put into an oven at 80°C for full drying.
4. The method according to claim 1, characterized in that the planting density of the seeds in step (2) is 200-250 seeds per box; the ratio of disinfectant to nutrient solution is 2 g/L.
5. The method according to claim 1, characterized in that the culture conditions in step (2) are 25°C in the daytime, the illumination intensity is 4000 LUX for 14 h; 20°C at night, for 10 h; the humidity is 30-50%, and the nutrient solution containing disinfectant is changed every 3 days.
6. The method according to claim 1, characterized in that the artificial seawater HUS01051 with standard salinity in step (3) comprises 24 parts of sodium chloride, 4 parts of sodium sulfate, 0.7 part of potassium chloride, 0.2 part of sodium bicarbonate, 0.1 part of potassium bromide, 0.02-0.03 part of boric acid, 0.003 part of sodium fluoride,
0.05-0.06 part of magnesium chloride hexahydrate, 0.01 part of calcium chloride dihydrate and 0.001 part of strontium chloride hexahydrate.
7. The method according to claim 1, characterized in that in step (4), the morphological indexes are analyzed by data to obtain the comprehensive score value D, and the salt-alkali tolerant wheat germplasm in seedling stage is screened out according to the D value.
8. The method according to claim 7, characterized in that the D value greater than or equal to 0.668 is a saline-alkali tolerant wheat germplasm.
9. The application of the method according to any one of claims 1-8 in the identification of saline-alkali tolerant wheat germplasm at seedling stage.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115672740A (en) * | 2022-09-21 | 2023-02-03 | 广东省农业科学院水稻研究所 | Experiment table for screening saline-alkali resistant germplasm of rice |
| CN117770138A (en) * | 2024-02-23 | 2024-03-29 | 潍坊市农业科学院(山东省农业科学院潍坊市分院) | Tissue culture method for creating moderate-severe saline-alkali resistant asparagus germplasm |
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2021
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115672740A (en) * | 2022-09-21 | 2023-02-03 | 广东省农业科学院水稻研究所 | Experiment table for screening saline-alkali resistant germplasm of rice |
| CN117770138A (en) * | 2024-02-23 | 2024-03-29 | 潍坊市农业科学院(山东省农业科学院潍坊市分院) | Tissue culture method for creating moderate-severe saline-alkali resistant asparagus germplasm |
| CN117770138B (en) * | 2024-02-23 | 2024-06-04 | 潍坊市农业科学院(山东省农业科学院潍坊市分院) | Tissue culture method for creating moderate-severe saline-alkali resistant asparagus germplasm |
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