Method for evaluating and screening salt tolerance of triticale germplasm resources in germination period

Abstract

The invention discloses a method for evaluating and screening salt tolerance of triticale germplasm resources in a germination stage, and belongs to the technical field of evaluation and screening of plant salt tolerance, salt stress concentration screening: triticale seeds are treated by NaCl solutions with different concentrations, indexes such as survival rate, plant height and overground part fresh weight are measured, the NaCl solution with the concentration of 200 mmol. L <-1 > is determined as the salt tolerance evaluation appropriate concentration, and the salt stress concentration is determined as the salt tolerance evaluation appropriate concentration. Salt tolerance evaluation test: carrying out salt stress treatment by adopting a 200mmol. L <-1 > NaCl solution, determining phenotypic indexes such as survival rate, plant height, fresh weight of overground part, root length and chlorophyll content, and physiological and biochemical indexes such as catalase, peroxidase and superoxide dismutase activity and malondialdehyde content, and carrying out principal component analysis, membership function analysis and clustering analysis to obtain a salt tolerance evaluation result. Calculating a salt tolerance comprehensive evaluation value, and screening salt tolerance triticale germplasm resources.

Description

Technical Field

[0001] The present invention relates to a method for evaluating and screening plant salt tolerance, in particular to a method for evaluating and screening salt tolerance of triticale germplasm resources during the germination period, and belongs to the technical field of plant salt tolerance evaluation and screening. Background Art

[0002] The salt stress concentration settings in existing studies vary greatly, with some studies using 100-150 mmol·L -1 NaCl solution is used as a stress concentration, but the phenotypic differences of different varieties of triticale at this concentration are not significant, making it difficult to accurately distinguish the salt tolerance. For example, studies have shown that when the NaCl concentration is lower than 200 mmol·L -1 When the concentration exceeds 250mmol·L -1 When the concentration is set at 0, most varieties will have a survival rate of 0, which cannot meet the needs of large-scale germplasm screening. This arbitrary concentration setting leads to a lack of comparability between different research results, which restricts the standardization of salt tolerance evaluation technology.

[0003] Traditional salt tolerance evaluation mostly relies on a single phenotypic indicator, such as survival rate or plant height, but such indicators are easily interfered with by environmental factors and cannot fully reflect the salt tolerance mechanism of plants. For example, when only survival rate is used as an indicator, important traits such as root growth and chlorophyll content may be ignored; and changes in a single physiological indicator are also difficult to accurately characterize the overall salt tolerance of the plant. In addition, existing studies have insufficiently integrated phenotypic indicators with physiological and biochemical indicators, and have not formed a multi-dimensional evaluation system. For example, the change pattern of malondialdehyde content as an indicator of membrane lipid peroxidation under extreme salt stress has not been fully applied to salt tolerance grading, resulting in inaccurate judgment of the salt tolerance limit of germplasm. Summary of the Invention

[0004] The main purpose of the invention is to provide a method for evaluating and screening the salt tolerance of triticale germplasm resources during the germination period.

[0005] The purpose of the present invention can be achieved by adopting the following technical solutions:

[0006] The method for evaluating and screening salt tolerance of triticale germplasm resources during the germination period comprises the following steps:

[0007] Salt stress concentration screening: Triticale seeds were treated with different concentrations of NaCl solution, and the survival rate, plant height and aboveground fresh weight of the seeds were measured. The concentration of 200mmol·L -1 NaCl solution is the appropriate concentration for salt tolerance evaluation;

[0008] Salt tolerance evaluation test: using 200mmol·L -1The plants were treated with NaCl solution for salt stress, and phenotypic indices such as survival rate, plant height, fresh weight of aboveground parts, root length, and chlorophyll content, as well as physiological and biochemical indices such as catalase, peroxidase, superoxide dismutase activities, and malondialdehyde content were measured.

[0009] Multivariate data analysis: Through principal component analysis, membership function analysis and cluster analysis, the comprehensive evaluation value of salt tolerance was calculated and salt-tolerant triticale germplasm resources were screened.

[0010] Preferably, the salt stress concentration screening is specifically as follows: 8 triticale germplasm materials are selected and 0, 50, 100, 150, 200, 250, 300 mmol·L -1 The survival rate, plant height and aboveground fresh weight of the plant were measured 14 days after sowing. The 200mmol·L -1 NaCl was the optimal concentration for evaluation.

[0011] Preferably, in the salt tolerance evaluation test, a matrix of vermiculite and nutrient soil mixed in a volume ratio of 1:1 is used, a control group and a salt treatment group are set, 10 seeds are sown in each pot, 3 times are repeated, and the seeds are germinated in an artificial culture room. The soil is watered every other day to keep it moist, and various indicators are measured after 14 days.

[0012] Preferably, the phenotypic indicator determination includes:

[0013] Calculation of survival rate: number of germinated seeds on the 14th day / number of test seeds × 100%;

[0014] Relative plant height was calculated as: treated plant height / control plant height × 100%;

[0015] Relative aboveground fresh weight was calculated as: aboveground fresh weight of treatment / aboveground fresh weight of control × 100%;

[0016] Root length measurement: Use a ruler to measure the length of the seedling roots.

[0017] Preferably, the physiological and biochemical index determination includes:

[0018] Relative chlorophyll content: measured using a chlorophyll meter;

[0019] Catalase activity: measured by UV absorption method;

[0020] Peroxidase activity: measured using a kit;

[0021] Superoxide dismutase activity: measured by microassay;

[0022] Malondialdehyde content: determined by thiobarbituric acid colorimetry.

[0023] Preferably, the principal component analysis is specifically as follows: performing principal component analysis on the salt tolerance coefficient, extracting comprehensive indicators with characteristic values ​​greater than 1 and cumulative contribution rates greater than 80%, and calculating the weights of each principal component. The principal component weight calculation formula is:

[0024]

[0025] Where: j = 1, 2, ..., n;

[0026] Weight W j Indicates the importance of the jth principal component;

[0027] p j represents the contribution rate of the jth principal component.

[0028] Preferably, the membership function analysis is specifically: using the membership function formula:

[0029] U(X ij )=(X ij -X jmin ) / (X jmax -X jmin );

[0030] Where: U represents the membership function value;

[0031] X ij represents the jth principal component value of the i-th accession;

[0032] X jmax represents the maximum value of the jth principal component;

[0033] X jmin represents the minimum value of the j-th principal component.

[0034] Preferably, triticale germplasm materials are clustered based on relative aboveground fresh weight, relative survival rate and relative plant height indicators to screen salt-tolerant core germplasm.

[0035] Preferably, the salt tolerance grade classification standard is: 0.80≤D value≤1.00 is high salt tolerance, 0.60≤D value<0.80 is salt tolerance, 0.40≤D value<0.60 is intermediate, 0.20≤D value<0.40 is salt intolerant, and 0.00≤D value<0.20 is salt sensitive.

[0036] Preferably, salt tolerance is evaluated by CAT activity in roots, POD and SOD activities in leaves, and extreme salt tolerance is evaluated by MDA content.

[0037] Beneficial technical effects of the present invention:

[0038] The present invention provides a method for evaluating and screening salt tolerance of triticale germplasm resources during the germination period, explores the appropriate salt concentration for evaluating salt tolerance of triticale, and determines that 200mmol·L -1 The NaCl solution was used as the treatment condition for this screening test, which provided a certain theoretical basis for the subsequent salt tolerance screening experiment of triticale. -1 Under NaCl stress, 103 triticale germplasm accessions were evaluated and screened for salt tolerance. Principal component analysis, membership function analysis, and cluster analysis ultimately identified two salt-tolerant accessions, QT-5 and LJ-65, and two salt-sensitive accessions, M90 and M36, providing germplasm resources for triticale salt tolerance breeding. The experiment also provided preliminary insights into triticale's salt tolerance mechanisms in terms of reactive oxygen species scavenging, enriching the indicators for evaluating triticale salt tolerance and expanding research on the physiological and biochemical mechanisms of triticale salt tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a cluster analysis diagram of triticale germplasm salt tolerance screening according to a preferred embodiment of the method for evaluating and screening triticale germplasm salt tolerance during the germination period of the present invention;

[0040] Figure 2 A preferred embodiment of the method for evaluating and screening salt tolerance of triticale germplasm resources during germination period according to the present invention Figure 2 a represents the comparative analysis of germination potential of triticale with different salt tolerance, Figure 2 b shows a comparative analysis of the survival rates of triticale with different salt tolerances. ST stands for salt tolerance, SM for salt tolerance intermediate, and SS for salt sensitivity.

[0041] Figure 3 A preferred embodiment of the method for evaluating and screening salt tolerance of triticale germplasm resources during germination period according to the present invention Figure 3 a represents the phenotypic comparison of triticale with different salt tolerance, Figure 3 b represents the comparative analysis of plant height of triticale with different salt tolerance, Figure 3 c represents the comparative analysis of fresh weight of aboveground parts of triticale with different salt tolerance. Figure 3 d shows the comparative analysis of root length of triticale with different salt tolerance;

[0042] Figure 4 A preferred embodiment of the method for evaluating and screening salt tolerance of triticale germplasm resources during germination period according to the present invention Figure 4 a represents the comparative analysis of CAT activity in leaves of triticale with different salt tolerance, Figure 4 b shows the comparative analysis of CAT activity in the roots of triticale with different salt tolerance. Figure 4c represents the comparative analysis of POD activity in leaves of triticale with different salt tolerance, Figure 4 d shows the comparative analysis of POD activity in the roots of triticale with different salt tolerance;

[0043] Figure 5 A preferred embodiment of the method for evaluating and screening salt tolerance of triticale germplasm resources during germination period according to the present invention Figure 5 a represents the comparative analysis of SOD activity in leaves of triticale with different salt tolerance, Figure 5 b represents the comparative analysis of SOD activity in the roots of triticale with different salt tolerance. Figure 5 c represents the comparative analysis of MDA content in leaves of triticale with different salt tolerance, Figure 5 d shows the comparative analysis of MDA content in the roots of triticale with different salt tolerance. DETAILED DESCRIPTION

[0044] In order to make the technical solution of the present invention more clear and specific to those skilled in the art, the present invention is further described in detail below with reference to embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0045] Test materials;

[0046] The 103 triticale germplasm resources used in this experiment are detailed in Appendix 1.

[0047] Test methods;

[0048] The experiment was carried out in the Grassland Laboratory of the College of Forestry and Grassland of Ningxia University, using a mixture of vermiculite and nutrient soil (v:v = 1:1) for cultivation.

[0049] Screening of suitable concentration for salt tolerance evaluation of triticale;

[0050] Eight triticale germplasm materials harvested that year were randomly selected, and seeds with full grains and uniform size were selected. The seeds were washed with distilled water to remove dust. On the day of sowing, the triticale was watered with different concentrations of 0, 50, 100, 150, 200, 250, and 300 mmol·L -1 The seeds were treated with salt stress using NaCl solution. Ten seeds were sown in each pot, with three replicates for each treatment, and then placed in an artificial culture room for germination.

[0051] Thereafter, the triticale was watered every other day to keep the soil moist. 14 days after sowing, the survival rate, plant height, and aboveground fresh weight of the plant were measured. The differences in these indicators among the varieties under different concentrations of NaCl stress were compared and analyzed to screen the appropriate NaCl solution concentration for evaluating triticale salt tolerance.

[0052] Evaluation and screening of salt-tolerant germplasm resources of triticale;

[0053] Vermiculite and nutrient soil were mixed evenly in a 1:1 ratio and placed in a small pot. A control group and a salt treatment group were set up. The same volume of distilled water and 200 mmol·L -1 NaCl solution infiltration.

[0054] Seeds with plump, uniform grains were selected for sowing. Dust was removed by washing with distilled water. Ten seeds were sown per pot, with three replicates per treatment. The cultivation conditions were the same as those used for screening for salt tolerance. Every other day, the triticale plants were watered to maintain soil moisture. Fourteen days after sowing, indicators such as survival rate, plant height, aboveground fresh weight, and chlorophyll content were measured.

[0055] Analysis of differences between salt-tolerant and salt-sensitive germplasm materials of triticale;

[0056] The selected salt-tolerant and salt-sensitive triticale germplasm materials were cultivated in a mixture of nutrient soil and vermiculite (v:v = 1:1) in the artificial climate chamber of the Science and Technology Building. A control group and a salt treatment group were set up. Three small pots were planted in each treatment for each variety. Seeds with full grains and uniform size were selected for planting in each pot. The control group was irrigated with distilled water, and the salt treatment group was irrigated with the same volume of 200mmol·L -1 NaCl solution, and then watered every other day to keep the soil moist. Phenotypic indicators such as survival rate, plant height, fresh weight of aboveground parts, and root length, as well as physiological and biochemical indicators such as chlorophyll content, peroxidase activity, and malondialdehyde content were measured 14 days after sowing.

[0057] Methods for determining morphological indicators;

[0058] The plant height at the seedling stage was measured with a ruler, and the average value of the plant height of 6 plants was taken for each germplasm material; the fresh weight of the aboveground part was weighed, and the average value of the aboveground fresh weight of 9 plants was taken for each germplasm material.

[0059] Survival rate (SR) = number of germinated seeds on the 14th day / number of test seeds × 100%;

[0060] Relative plant height (%) = treated plant height / control plant height × 100%;

[0061] Relative aboveground fresh weight (%) = treatment aboveground fresh weight / control aboveground fresh weight × 100%;

[0062] Methods for measuring physiological indicators;

[0063] relative chlorophyll content (SPAD);

[0064] The chlorophyll content of the top fully expanded seedling leaves was measured using a chlorophyll meter (TYS-B, Top Cloud Zhejiang Agricultural Science and Technology Co., Ltd.), with 6 replicates for each germplasm material.

[0065] Catalase (CAT);

[0066] Catalase activity is determined using the UV absorption method. Based on the strong absorption of H₂O₂ at 240nm, catalase can decompose hydrogen peroxide, causing the absorbance of the reaction solution to decrease over time. Catalase activity can be determined by measuring the rate of change in absorbance.

[0067] Malondialdehyde content (MDA);

[0068] The thiobarbituric acid (TBA) colorimetric method uses the absorbance of the colored compound formed by the reaction of malondialdehyde and thiobarbituric acid at 530 nm to assess the malondialdehyde content.

[0069] peroxidase (POD) activity;

[0070] The POD activity was determined using a kit from Solebaugh Beijing Technology Co., Ltd. POD catalyzes H2O2 to oxidize a specific substrate and has characteristic light absorption at 470 nm. The absorbance was measured using a spectrophotometer and the POD activity was calculated using a formula.

[0071] Superoxide dismutase (SOD) activity;

[0072] The absorbance was measured by micro-method using a superoxide dismutase activity detection kit from Solebau Technology Co., Ltd., and the SOD activity was calculated using the formula.

[0073] 2.4 Data Analysis

[0074] Excel 2019 was used for data statistics and organization, and the mean, standard deviation, etc. were calculated. SPSS 26 and Origin 2024 were used for multivariate analysis such as variance analysis, correlation analysis, membership function analysis, and principal component analysis. Origin 2024 was used for drawing.

[0075] Membership function calculation formula:

[0076] U(X ij )=(X ij -X jmin ) / (X jmax -X jmin )(1)

[0077] Where: U represents the membership function value; Xij represents the jth principal component value of the i-th accession; X jmax Xjmin represents the maximum value of the j-th principal component; Xjmin represents the minimum value of the j-th principal component.

[0078] The formula for calculating the principal component weight is:

[0079]

[0080] Where: j = 1, 2, ..., n; weight W j Indicates the importance of the jth principal component;

[0081] p j represents the contribution rate of the jth principal component.

[0082] Calculation formula for comprehensive evaluation of salt tolerance D value:

[0083]

[0084] Where: D value represents the comprehensive evaluation value of salt tolerance of the i-th triticale germplasm material under salt stress conditions.

[0085] Screening of the optimal concentration for salt tolerance evaluation of triticale;

[0086] As shown in Table 1, under different concentrations of salt stress treatment, the plant height, aboveground fresh weight, survival rate and other traits of the eight triticale varieties decreased to varying degrees. The various trait indicators showed a trend of first decreasing, then increasing, and then decreasing again with the change of salt stress concentration, and the amplitude of change also increased with the increase of salt stress concentration.

[0087] Comparison of the coefficients of variation of physiological indicators under different concentrations of salt stress revealed that at 300mmol·L -1 The coefficient of variation of plant height, fresh weight and survival rate reached the highest under NaCl solution stress, and the total coefficient of variation of the three indicators was 174.9%. The larger the coefficient of variation, the more obvious the phenotypic differences between different triticale varieties under the salt concentration stress.

[0088] But at 250 and 300 mmol·L -1 Under NaCl stress, the survival rate of some varieties was 0, which affected the subsequent large-scale screening of salt-tolerant triticale germplasm materials. The plant height, survival rate and aboveground fresh weight of different varieties of triticale under different salt stress were comprehensively analyzed, and the average value and coefficient of variation were finally determined. -1 NaCl solution is the optimal solution concentration for evaluating triticale salt tolerance. Under this concentration of NaCl stress, triticale not only has certain growth characteristics, but also has obvious phenotypic differences between different triticale varieties.

[0089] Table 1 Phenotypic traits of triticale under different concentrations of NaCl stress;

[0090]

[0091]

[0092] Principal component analysis of salt tolerance evaluation of 103 triticale germplasms

[0093] Use 200mmol·L -1 The salt tolerance of 103 triticale germplasm resources was evaluated using NaCl solution, and the seed survival rate, plant height, aboveground fresh weight, root length and relative chlorophyll content under control and salt stress conditions were determined.

[0094] In order to more accurately evaluate the salt tolerance of triticale germplasm materials, principal component analysis (PCA) was used to analyze the salt tolerance coefficients of five indicators and reconstruct them into a new set of independent comprehensive indicators. PCA analysis can also determine the weight of each comprehensive indicator, which can fully reflect the comprehensive indicator that plays a dominant role among the various indicators of triticale.

[0095] The comprehensive evaluation value of salt tolerance of each triticale variety was obtained by calculating the membership function, thereby comprehensively evaluating the salt tolerance of triticale varieties.

[0096] As shown in Table 2, based on the extraction principle of comprehensive index characteristic value > 1 or comprehensive index cumulative contribution rate > 80%, principal component analysis (PCA) extracted a total of three comprehensive indicators, and the weights of the three comprehensive indicators were calculated using formula (2). These three comprehensive indicators were used to conduct membership function analysis on 103 triticale germplasm materials to comprehensively evaluate the salt tolerance of triticale.

[0097] Table 2 Principal component analysis of evaluation indices for salt tolerance of triticale;

[0098]

[0099] Membership function analysis of salt tolerance evaluation of 103 triticale germplasm materials;

[0100] The membership function analysis was performed on the relative survival rate, relative plant height and relative aboveground fresh weight of 103 triticale germplasm materials. The membership function values ​​and D values ​​were calculated using formulas (1) (3). The germplasm materials were divided into five salt tolerance grades according to the D values.

[0101] As shown in Table 3, the D values ​​of the 103 triticale germplasm materials ranged from 0.93 to 0.18. The germplasm materials were divided into five salt tolerance levels according to the D values: 0.80≤D value≤1.00, level 1 high salt tolerance; 0.60≤D value<0.80, level 2 salt tolerance; 0.40≤D value<0.60, level 3 intermediate;

[0102] 0.20≤D value<0.40, level 4 salt-intolerant; 0.00≤D value<0.20, level 5 salt-sensitive. One highly salt-tolerant material was identified as LJ-26;

[0103] Four level 2 salt-tolerant materials were identified, namely LJ-8, QT-37, LJ-42, and LJ-27; 75 level 3 intermediate materials, 22 level 4 salt-intolerant materials, and one level 5 salt-sensitive material, namely QT-22.

[0104] Table 3 Membership function analysis of salt tolerance evaluation of triticale germplasm materials;

[0105]

[0106]

[0107] Table 3

[0108]

[0109] Table 3

[0110]

[0111] Cluster analysis of 103 triticale germplasm materials;

[0112] In order to more accurately screen out core germplasms related to salt tolerance of triticale, cluster analysis was performed on relative aboveground fresh weight, relative survival rate and relative plant height of 103 triticale germplasm materials.

[0113] like Figure 1 As shown in the figure, the red and blue areas indicate higher and lower index values, respectively. Varieties that are close to each other show similar performance in multiple measured indicators (survival rate, plant height, and aboveground fresh weight). Among them, LJ-65, QT-41, LJ-79, LJ-9, QT-44, QT-5, LJ-25, LJ-63, M47, QT-42, M38, and M14 are located in the white to red area, indicating that under salt stress, the germplasm materials can still perform well in indicators such as survival rate, plant height, and aboveground fresh weight, indicating that they have a certain degree of salt tolerance. However, the five triticale germplasm materials, LJ-77, LJ-11, M36, QT-4, and M90, are mainly located in the blue area, indicating that their various indicators perform poorly under salt stress treatment and show a certain degree of salt sensitivity. Based on the results of membership function and cluster analysis, the salt-tolerant triticale germplasm materials QT-5 and LJ-65 were finally screened out; the salt-sensitive germplasm materials M90 and M36; and the intermediate materials QT-41 and LJ-25 that showed relatively stable performance in both screenings.

[0114] Comparative analysis of germination indexes of triticale with different salt tolerance;

[0115] The above six varieties of triticale germplasm materials were selected to analyze the differences in phenotypic indicators and physiological and biochemical indicators of triticale germplasms with different salt tolerance in response to salt stress. -1After 4 days of NaCl solution stress treatment, the number of germinations of triticale germplasm materials was counted and the germination potential was calculated.

[0116] like Figure 2 As shown in a, the germination potential of triticale with different salt tolerances decreased significantly (P<0.05), and salt stress significantly inhibited the germination of triticale.

[0117] After salt stress treatment, the germination potential of the salt-tolerant germplasm QT-5 decreased by 18.5% compared with the control, and the germination potential of LJ-65 decreased by 26.2% compared with the control; the germination potential of the other triticale germplasms QT-41, LJ-25, M90 and M36 decreased by 30%, 49%, 53% and 79% respectively after salt stress treatment compared with the control.

[0118] After 14 days of salt stress treatment, the survival rate of salt-tolerant germplasm QT-5 was higher than that of the control ( Figure 2 b) LJ-65's survival rate decreased by 17% compared to the control; the survival rates of the intermediate accessions QT-41 and LJ-25 were slightly lower than the control. The survival rate of the salt-sensitive accession M90 was significantly reduced (P < 0.05), decreasing by 43% compared to the control, and the survival rate of M36 was reduced by 23% compared to the control. The results indicate that salt stress affects triticale growth and development during the early germination phase primarily by affecting germination potential and survival rate. Salt-tolerant accessions exhibited some resistance to salt stress during germination. Compared to germination potential, triticale survival rate increased significantly after 14 days of salt stress treatment. In particular, salt-tolerant and intermediate accessions, despite delayed germination due to salt stress, maintained a high survival rate.

[0119] Comparative analysis of phenotypic indicators of triticale germplasm materials with different salt tolerance;

[0120] Salt stress treatment has different effects on the phenotypes of triticale germplasms with different salt tolerance ( Figure 3 a) The effect on salt-tolerant triticale was not obvious from the phenotypic point of view. The plant height of intermediate triticale germplasm after salt treatment was lower than that of the control group. The plant height and growth vigor of salt-sensitive triticale after salt stress were significantly inferior to those of the control group.

[0121] Comparison of plant height among triticale germplasms with different salt tolerance ( Figure 3 b) showed that the salt-tolerant germplasms QT-5 and LJ-65 decreased by 15% and 12% respectively compared with the control group, the intermediate germplasms QT-41 and LJ-25 decreased by 17% and 25% respectively compared with the control group, and the salt-sensitive germplasms M90 and M36 decreased by 31% and 30% respectively compared with the control group.

[0122] Comparison of fresh weight of different varieties ( Figure 3c) It was found that the aboveground fresh weight of salt-tolerant germplasms QT-5 and LJ-65 decreased by 40% and 26% respectively compared with the control group, the intermediate germplasms QT-41 and LJ-25 decreased by 33% and 24% respectively compared with the control group, and the salt-sensitive germplasms M90 and M36 decreased by 48% and 44% respectively compared with the control group. Among them, the varieties showing resistance to salt stress also had a relatively large decrease in aboveground fresh weight, indicating that salt stress significantly affected the aboveground fresh weight of triticale.

[0123] Comparison of root length of different salt-tolerant germplasms ( Figure 3 d) showed that the salt-tolerant germplasms QT-5 and LJ-65 decreased by 21% and 15% respectively compared with the control group, the intermediate germplasms QT-41 and LJ-25 decreased by 36% and 26% respectively compared with the control group, and the salt-sensitive germplasms M90 and M36 decreased by 37% and 42% respectively compared with the control group. Figure 3 As shown in Figures 3b, 3c, and 3d, salt stress treatment had a significant effect on growth indicators of triticale, including plant height, fresh weight, and root length (P < 0.05). The reduction in plant height and root length of salt-tolerant germplasm was significantly smaller than that of salt-sensitive germplasm, while the reduction in aboveground fresh weight of the three salt-tolerant germplasms was relatively obvious, indicating that plant height is a better indicator for evaluating salt tolerance of triticale than root length.

[0124] Comparative analysis of physiological parameters of triticale germplasms with different salt tolerance;

[0125] Catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) are important enzymes that reduce the damage of reactive oxygen species (ROS) to plants. They form an enzyme system that protects plant cells from adverse environmental damage, working synergistically to combat these effects. However, their activity varies. Malondialdehyde (MDA) levels are a key indicator of the extent of plasma membrane damage and the severity of membrane peroxidation.

[0126] like Figure 4As shown in (a) and (b), under salt stress, catalase (CAT) activities in leaves of triticale germplasms with different salt tolerance increased compared with the control, except for the salt-tolerant germplasm QT-5, which decreased compared with the control. LJ-65 increased by 82.7%, QT-41 increased by 120%, LJ-25 increased by 164%, M90 increased by 22%, and M36 increased by 51%. In the roots, catalase activities in the roots of salt-tolerant germplasms QT-5, LJ-65, and intermediate germplasm LJ-25 increased by 15%, 13%, and 37%, respectively, compared with the control. However, catalase activities in salt-sensitive germplasms M90, M36, and intermediate germplasm QT-41 decreased by 45%, 44%, and 28%, respectively, compared with the control. The experimental results show that the activity of CAT in triticale leaves cannot well indicate the salt tolerance of the germplasm, while the higher the CAT activity in the root system, the stronger the salt tolerance of the germplasm.

[0127] like Figure 4 As shown in Figures c and 4d, under salt stress, peroxidase (POD) activity in leaves of triticale germplasms QT-5, LJ-65, QT-41, and LJ-25 increased by 127%, 193%, 20%, and 50%, respectively, compared with the control. POD activity in M90 and M36 decreased by 8% and 3%, respectively. After salt stress, POD activity in roots of QT-5, LJ-65, QT-41, LJ-25, and M36 increased by 36%, 93%, 13%, 57%, and 54%, respectively, compared with the control. POD activity in the roots of M90 decreased by 2%, respectively. POD activity indicates a plant's ability to withstand adverse environmental stress. Salt-tolerant and intermediate germplasms showed increased POD activity in both leaves and roots, while salt-sensitive germplasms showed decreased POD activity in leaves and both increased and decreased POD activity in roots. These results suggest that POD activity in leaves can be used as an evaluation indicator.

[0128] like Figure 5As shown in Figures 5a and 5b, under salt stress, superoxide dismutase (SOD) activity in the leaves of QT-5, LJ-65, and LJ-25 increased by 120%, 39%, and 5%, respectively, compared with the control, while that in QT-41, M90, and M36 decreased by 39%, 74%, and 56%, respectively. In the roots, salt-tolerant accessions QT-5 and LJ-65 increased by 10% and 18%, respectively, compared with the control. However, the SOD activity in the roots of the remaining accessions QT-41, LJ-25, M90, and M36 decreased by 3%, 1%, 37%, and 29%, respectively, compared with the control. These results indicate that the magnitude of change in SOD activity in the leaves of the three triticale varieties with varying salt tolerance was greater than that in the roots, but the accuracy of the evaluation and identification of intermediate triticale varieties decreased.

[0129] like Figure 5 As shown in Figures c and 5d, after salt stress treatment, malondialdehyde (MDA) content in the leaves of triticale germplasm materials QT-5, LJ-65, and QT-41 decreased by 9.5%, 12%, and 14%, respectively, compared with the control, while MDA content in the leaves of LJ-25, M90, and M36 increased by 21%, 60%, and 74%, respectively. MDA content in the roots of QT-5, LJ-65, and LJ-25 decreased by 18%, 14%, and 7.6%, respectively, compared with the control, while MDA content in the roots of QT-41, M90, and M36 increased by 11.5%, 110%, and 73%, respectively, compared with the control. The level of MDA content indicates the degree of damage to the plant's plasma membrane. The experimental results showed that the MDA content in the leaves and roots of the two salt-tolerant germplasms decreased, the MDA content in the leaves and roots of the two salt-sensitive germplasms increased, but the changes in the intermediate germplasms were different. The MDA content in the leaves of QT-41 decreased, and the MDA content in the roots increased, while the MDA content in the leaves of LJ-25 increased, and the MDA content in the roots decreased. This shows that MDA content can be used as an effective indicator to evaluate the extreme salt tolerance phenotype of triticale.

[0130] The budding stage of a plant, as the starting point of its growth and development, is the most vulnerable and sensitive period of life. Therefore, in experiments exploring plant salt tolerance, budding traits such as survival rate, root length, and fresh weight are often used as indicators for screening salt-tolerant plants. This experiment screened a large number of triticale germplasm materials. Using salt solutions of varying concentrations for screening was time-consuming and labor-intensive. Therefore, eight randomly selected samples from a total of 103 triticale germplasm materials were used for salt tolerance assessment and concentration-adapted screening.

[0131] Set 0-200mmol·L -1The salt tolerance of 7 triticale germplasm materials was screened by using NaCl solution as salt stress. The test showed that with the increase of NaCl concentration, the relative bud length, relative bud dry weight, relative root length, relative root dry weight, relative survival rate and other indicators of seeds all decreased significantly. The test also found that the 7 triticale materials had a significant effect on the salt tolerance of the seeds under the NaCl stress concentration of 160-200mmol·L -1 The difference in relative survival rate was not significant when the concentration of salt stress was higher than 200mmol·L -1 It is recommended to set higher salt stress concentrations in the experiment to identify the salt tolerance of triticale germplasm materials.

[0132] Use 200mmol·L -1 and 300mmol·L -1 NaCl solution was used to treat 29 triticale germplasm materials at the seedling stage with high concentration salt stress, and one triticale germplasm material with salt tolerance and four salt-sensitive germplasm materials were selected. -1 The appropriate concentration of NaCl solution was screened for salt tolerance evaluation of 8 randomly selected triticale germplasm materials harvested that year. The survival rate, plant height, and aboveground fresh weight were measured and analyzed for significant differences and coefficient of variation. Finally, 200mmol·L -1 NaCl solution was used as the stress concentration for the subsequent evaluation of triticale salt tolerance.

[0133] The salt tolerance of plants cannot be evaluated by a single indicator, because salt tolerance is a very complex comprehensive trait. Different indicators reflect different information. Using multiple indicators to comprehensively evaluate the strength of plant salt tolerance is more comprehensive and more convincing.

[0134] The study also believes that a single indicator is insufficient for evaluating plant salt tolerance, and conducted principal component analysis on multiple indicators such as survival rate, fresh weight, root length, etc., and screened out three main components as comprehensive indicators for salt tolerance evaluation. Membership function analysis and cluster analysis were performed on 55 Hengmai varieties (lines) of triticale, and 14 extremely salt-tolerant Hengmai varieties were screened out.

[0135] Studies have shown that using multiple indicators to jointly evaluate the salt tolerance of plants can reasonably and effectively screen out plants with high salt tolerance. This study converted 8 indicators in the bud stage and 6 indicators in the seedling stage into independent comprehensive indicators through principal component analysis, avoiding information overlap between indicators, and used membership function analysis and cluster analysis to grade the salt tolerance of the tested wheat materials. Finally, the materials Zhongzuo 60115 and Jimai No. 1 that showed high salt tolerance in both periods were screened out.

[0136] When studying rice salt tolerance, it was found that salt tolerance is a complex trait determined by multiple trait indicators. Using a single indicator of a certain period to evaluate and identify rice salt tolerance has limitations. Therefore, the experiment used principal component analysis to integrate multiple single indicators into a small number of comprehensive indicators, and calculated the corresponding principal component values ​​and membership function values. This not only took into account the correlation between indicators but also solved the problem of indicator information overlap, making the obtained rice salt tolerance comprehensive evaluation D value more scientific. Subsequently, cluster analysis was used to classify rice germplasm resources with different salt tolerance, and 51 rice germplasm resources were divided into 4 categories. Finally, the rice variety with the best salt tolerance, Haiyou No. 5, was selected. This experiment used 200mmol·L -1 Salt stress treatment with NaCl solution on 103 triticale accessions revealed varying degrees of suppression in indicators such as survival rate, plant height, aboveground fresh weight, and chlorophyll content, indicating that different parts of triticale respond differently to salt stress during the germination period. Therefore, this study evaluated the salt tolerance of 103 triticale accessions twice using comprehensive indicators, including principal component analysis, membership function analysis, and cluster analysis. Ultimately, two salt-tolerant accessions, QT-5 and LJ-65, two intermediate accessions, QT-41 and LJ-25, and two salt-sensitive accessions, M90 and M36, were selected. However, this study used NaCl solution only in the laboratory to screen for salt-tolerant triticale accessions. Whether these accessions can maintain salt tolerance in the field under mixed saline-alkali stress and complex soil composition requires further field testing.

[0137] Salt stress can cause problems such as ion imbalance, metabolic disorders, structural damage, and water shortage in plants. Plants have evolved corresponding adaptation methods in the long-term changing natural environment. Although salt stress can seriously affect the survival of plants, some plants have adapted to the saline-alkali environment. Therefore, screening salt-alkali tolerant plants, exploring the physiological and biochemical mechanisms of salt-alkali tolerance in plants, digging deep into the key genes for salt-alkali tolerance, and cultivating new salt-alkali tolerant plant varieties are effective ways to improve saline-alkali land by "changing crops to suitable places". This study used 200mmol·L -1Triticale was subjected to salt stress treatment with NaCl solution to screen for salt-tolerant, intermediate, and salt-sensitive triticale germplasms and further explore the salt tolerance mechanisms of triticale. Studies have found that salt stress significantly inhibits plant germination potential, survival rate, vigor index, and germination index, and also prolongs seed germination time. The main phenotypic effects on plants are slower growth, fewer new branches, and reduced plant height. This experiment found that under salt stress, the survival rate of salt-tolerant triticale QT-5 increased compared to the control, indicating that certain salt concentrations can promote germination in certain varieties. The aboveground fresh weight of salt-tolerant triticale QT-5 decreased more significantly than that of intermediate triticale varieties, while the root length of salt-tolerant triticale decreased less significantly than that of intermediate triticale and salt-sensitive triticale. The results showed that under salt stress, the aboveground parts of triticale are more sensitive to salt stress than the roots. The study suggests that the leaves of triticale resist salt stress before the roots, and this resistance mechanism protects the root structure and function of triticale, giving it a stronger ability to withstand salt stress. Therefore, this study suggests that root length is a more suitable indicator for evaluating triticale salt tolerance than aboveground fresh weight. Better root growth indicates a stronger ability of the aboveground parts to resist salt stress, indicating a higher salt tolerance of the germplasm.

[0138] ROS exists in the form of signaling molecules in plant physiological mechanisms, regulating plant growth and development, hormone signals, programmed cell death, and the plant's response to abiotic and biotic stresses. When it accumulates too much in the plant body, it will cause oxidative stress to the plant, destroy the plant cell structure, damage the plant's DNA, proteins and other biological macromolecules, and lead to plant cell death. Therefore, eliminating excess ROS in the body is an important physiological mechanism for plants to cope with stress.

[0139] This study found that, under salt stress, CAT activity in leaves significantly increased regardless of triticale's resistance, while CAT activity in roots varied depending on the triticale's salt tolerance. Conversely, when triticale varieties with varying salt tolerance were subjected to salt stress, the changes in POD and SOD activity in leaves were more pronounced than those in roots. Changes in MDA content were more pronounced in both leaves and roots of both highly salt-tolerant and salt-sensitive triticale accessions, but less pronounced in leaves and roots of intermediate accessions. In summary, this study suggests that root CAT activity and leaf POD and SOD activity can be used as screening indicators for evaluating triticale salt tolerance, while changes in MDA content can be an effective indicator for evaluating extreme salt tolerance.

[0140] The above is only a further 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 can make equivalent replacements or changes based on the technical solutions and concepts of the present invention within the scope disclosed by the present invention, which fall within the scope of protection of the present invention.

Claims

1. A method for evaluating and screening salt tolerance of triticale germplasm resources during the germination period, characterized by: The following steps are involved: Salt stress concentration screening: Triticale seeds were treated with different concentrations of NaCl solution, and the survival rate, plant height and aboveground fresh weight of the seeds were measured. The concentration of 200mmol·L -1 NaCl solution is the appropriate concentration for salt tolerance evaluation; Salt tolerance evaluation test: using 200mmol·L -1 The plants were treated with NaCl solution for salt stress, and phenotypic indices such as survival rate, plant height, fresh weight of aboveground parts, root length, and chlorophyll content, as well as physiological and biochemical indices such as catalase, peroxidase, superoxide dismutase activities, and malondialdehyde content were measured. Multivariate data analysis: Through principal component analysis, membership function analysis and cluster analysis, the comprehensive evaluation value of salt tolerance was calculated and salt-tolerant triticale germplasm resources were screened.

2. The method for evaluating and screening salt tolerance of triticale germplasm resources during the germination period according to claim 1, characterized in that: The salt stress concentration screening was specifically as follows: 8 triticale germplasm materials were selected and the concentrations of 0, 50, 100, 150, 200, 250 and 300 mmol·L -1 The survival rate, plant height and aboveground fresh weight of the plant were measured 14 days after sowing. The 200mmol·L -1 NaCl was the optimal concentration for evaluation.

3. The method for evaluating and screening salt tolerance of triticale germplasm resources during the germination period according to claim 1, characterized in that: In the salt tolerance evaluation test, a matrix of vermiculite and nutrient soil mixed in a volume ratio of 1:1 was used, a control group and a salt treatment group were set up, 10 seeds were sown in each pot, and 3 replicates were performed. The seeds were germinated in an artificial culture room, and the soil was watered every other day to keep it moist. Various indicators were measured after 14 days.

4. The method for evaluating and screening salt tolerance of triticale germplasm resources during the germination period according to claim 1, characterized in that: The phenotypic indicator determination includes: Calculation of survival rate: number of germinated seeds on the 14th day / number of test seeds × 100%; Relative plant height was calculated as: treated plant height / control plant height × 100%; Relative aboveground fresh weight was calculated as: aboveground fresh weight of treatment / aboveground fresh weight of control × 100%; Root length measurement: Use a ruler to measure the length of the seedling roots.

5. The method for evaluating and screening salt tolerance of triticale germplasm resources during germination period according to claim 1, characterized in that: The physiological and biochemical index determination includes: Relative chlorophyll content: measured using a chlorophyll meter; Catalase activity: measured by UV absorption method; Peroxidase activity: measured using a kit; Superoxide dismutase activity: measured by microassay; Malondialdehyde content: determined by thiobarbituric acid colorimetry.

6. The method for evaluating and screening salt tolerance of triticale germplasm resources during germination period according to claim 1, characterized in that: The principal component analysis is specifically as follows: performing principal component analysis on the salt tolerance coefficient, extracting comprehensive indicators with characteristic values ​​greater than 1 and cumulative contribution rates greater than 80%, and calculating the weights of each principal component. The principal component weight calculation formula is: Where: j = 1, 2, ..., n; Weight W j Indicates the importance of the jth principal component; p j represents the contribution rate of the jth principal component.

7. The method for evaluating and screening salt tolerance of triticale germplasm resources during the germination period according to claim 1, characterized in that: The membership function analysis is specifically as follows: through the membership function formula: U(X ij )=(X ij -X jmin ) / (X jmax -X jmin ); Where: U represents the membership function value; X ij represents the jth principal component value of the i-th accession; X jmax represents the maximum value of the jth principal component; X jmin represents the minimum value of the j-th principal component.

8. The method for evaluating and screening salt tolerance of triticale germplasm resources during the germination period according to claim 1, characterized in that: Based on relative aboveground fresh weight, relative survival rate and relative plant height, triticale germplasm materials were clustered and salt-tolerant core germplasm was screened.

9. The method for evaluating and screening salt tolerance of triticale germplasm resources during germination period according to claim 1, characterized in that: The salt tolerance grade classification standard is: 0.80≤D value≤1.00 is high salt tolerance, 0.60≤D value<0.80 is salt tolerance, 0.40≤D value<0.60 is intermediate, 0.20≤D value<0.40 is salt intolerant, and 0.00≤D value<0.20 is salt sensitive.

10. The method for evaluating and screening salt tolerance of triticale germplasm resources during the germination period according to claim 1, characterized in that: Salt tolerance was evaluated by CAT activity in roots and POD and SOD activities in leaves, and extreme salt tolerance was evaluated by MDA content.

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