A method for evaluating the salt tolerance of mulberry germplasm resources and screening salt-tolerant germplasm.

Abstract

This invention discloses a method for evaluating the salt tolerance of mulberry germplasm resources and screening salt-tolerant germplasm, belonging to the field of germplasm resource evaluation and screening technology. The method includes sampling and storage of experimental materials, explant preparation, disinfection, culture, salt tolerance identification, evaluation of salt tolerance using membership functions and cluster analysis, and comprehensive analysis of the salt tolerance of mulberry winter buds. This invention identifies the salt tolerance of mulberry planting resources collected from different regions, using the bud germination status index and the growth rate of relatively strong buds as evaluation indicators for screening a large number of mulberry materials for salt tolerance identification. Membership functions and cluster analysis are used for comprehensive evaluation of salt tolerance, screening out different salt-tolerant germplasm and providing excellent germplasm resources for subsequent salt tolerance gene discovery and salt-tolerant variety breeding.

Description

Technical Field

[0001] This invention belongs to the field of germplasm resource evaluation and screening technology, specifically relating to a method for evaluating the salt tolerance of mulberry germplasm resources and screening salt-tolerant germplasm. Background Technology

[0002] Mulberry (Morus L.) is a perennial deciduous tree or shrub belonging to the genus Morus in the family Moraceae. Widely distributed in my country, it possesses significant nutritional, medicinal, economic, and social value. Mulberry trees are cold-hardy, salt-tolerant, heat-tolerant, and drought-resistant, making them a valuable genetic resource for abiotic stress tolerance. Their role in ecological restoration, such as afforestation of barren mountains, desertification control, and soil erosion management, is increasingly prominent, making them a key pioneer species in vegetation restoration and reconstruction projects in fragile ecological zones. In recent years, soil salinization has become increasingly serious, posing a significant constraint on global environmental and agricultural development. Statistics show that irrigated and arable land worldwide suffers from varying degrees of salinization, with a trend of increasing annually, thus posing a serious threat to the sustainability of global crop production and food security. To mitigate the impact of soil salinization, various measures have been taken to improve and utilize saline-alkali land. Afforestation is one of the biological measures for improving saline-alkali land, not only improving the environment and inhibiting soil salinization but also directly utilizing saline-alkali land for forestry and fruit production, thereby increasing the productivity of saline-alkali land. Therefore, screening for salt-tolerant genetic resources is the foundation for breeding salt-tolerant varieties, and it is of great significance for the cultivation of salt-tolerant mulberry trees and the utilization of saline-alkali land.

[0003] Currently, research on salt tolerance in mulberry trees, both domestically and internationally, mainly focuses on field identification or laboratory soil cultivation methods. Chen et al., members of the research group, previously screened the salt tolerance of 14 mulberry F1 hybrids using different concentrations of NaCl, finding that 0.9% NaCl was the optimal concentration for evaluating salt tolerance, and selected the hybrid combination from the Anzhen × Xinghai Nei Shuangyou parent as the best in terms of salt tolerance. Liu Xueqin et al. used 12g / L NaCl and 400g / L PEG6000 to simulate saline and arid environments, screening the salt tolerance of 13 mulberry hybrid F1 generations. They selected Guisangyou 62 as suitable for coastal saline-alkali land, and Guisangyou 12 and Cesha-Za 6 as suitable for areas with perennial drought and severe salinization. Zhang Heyu et al., through the evaluation of salt tolerance of 12 mulberry species, found that Huasang and Baisang were highly salt-tolerant. Currently, research indicates that tissue culture is one of the most effective methods for identifying plant salt tolerance. However, there are relatively few reports on the use of mulberry winter bud tissue culture for salt tolerance identification. Foreign scholars have used tissue culture to screen salt-tolerant mulberry germplasm materials with genotypes such as C776, S1, SV1, S1635, C763, G2, G3, G4, S34, and S13 from isolated axillary buds. Zhang Heyu et al. found through the salt tolerance culture of winter buds from different mulberry varieties that Ruisang, 9001, and Husang 197 exhibited strong salt tolerance, and this result was largely consistent with the results of field salt tolerance identification. Therefore, the isolated winter bud tissue culture method can be applied to the identification and evaluation of salt tolerance in mulberry varieties or lines. Studies have also found that an appropriate NaCl concentration can effectively reflect the response of mulberry winter buds to salt stress, ensuring the reliability and validity of the experimental results. Currently, the comprehensive evaluation of salt tolerance of germplasm resources mainly adopts methods such as correlation analysis, principal component analysis, membership function analysis, and cluster analysis. These methods can effectively analyze the differences in salt tolerance among different germplasm resources. Summary of the Invention

[0004] The purpose of this invention is to provide a method for evaluating the salt tolerance of mulberry germplasm resources and screening salt-tolerant germplasm.

[0005] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:

[0006] A method for evaluating the salt tolerance of mulberry germplasm resources and screening salt-tolerant germplasm includes the following steps:

[0007] (1) Sampling and storage of experimental materials: The optimal sampling period for mulberry trees is from the time they have passed through deep dormancy until before the sap begins to flow. To ensure the consistency of winter bud development among different germplasm materials, sampling should not be performed after the winter buds have sprouted. For the sampling trees in Chengde, Hebei Province, no summer pruning was performed in the previous year to allow the mulberry buds to complete the flower bud differentiation process. One-year-old healthy branches with plump wood, full winter buds, no dead shoots, and no pests or diseases were cut from each region. The lower 1 / 3 of the branches were cut off and discarded. The remaining parts were cut into small sections of 25-30 cm, placed in self-sealing bags, and stored at 0-2℃ for later use.

[0008] (2) Explant preparation: Hold the branch segment upside down and use a scalpel to cut off the leaf scar along the upper edge of the leaf scar, down to the xylem; after cutting off all the leaf scars on the branch segment, hold the branch segment upright and use a scalpel to remove the secondary buds and the scales on the lower part of the bud, then use tweezers to gently peel off the outer scales to expose the first layer of young leaves, cut off the bud completely, put it in a clean petri dish, cover the dish, and sterilize it within 2 hours;

[0009] (3) Disinfection: Place different winter bud materials into small baskets made of copper wire mesh, soak them in sterile water for 10 minutes, remove the baskets, drain the water, and disinfect them in 70% ethanol for 20 seconds. Rinse them 2-3 times with sterile water, then disinfect them in 0.1% mercuric chloride solution for 6 minutes. Rinse them 4 times with sterile water. Then place the baskets on sterile gauze. Once there are no water droplets left in the baskets and the winter bud materials in the baskets, they can be inoculated.

[0010] (4) Start culture: Inoculate different winter bud materials into the pre-culture medium, inoculate 45-50 explants into each material, and inoculate no more than 6 explants into each culture bottle, and culture for 7 days;

[0011] (5) Salt tolerance assessment: Winter shoots of the test materials were placed on 1 / 2 MS solid medium containing 150 mmol / L NaCl or distilled water (control), and placed in an incubator at a temperature of (25±2)℃, a light intensity of 2000-3000 lx, and a day / night photoperiod of 14h / 10h. Five winter shoots were placed in each bottle, and three bottles were used as one treatment, with three replicates. After 14 days of culture, the number of dead, withered, maintained, budded, and leaf-opening explants in the winter shoots was counted to calculate the germination status index. Strong shoots were selected and placed in a new control and sterile medium containing 1 / 2 MS containing NaCl. After 14 days of culture, the weight of the winter shoot explants was measured, and the growth of the relatively strong shoots was calculated. The germination status index of the six germplasm materials on the 14th day after the first subculture and the growth of the relatively strong shoots on the 14th day after the second subculture were measured under different salt stress concentrations.

[0012] (6) Salt tolerance evaluation using membership functions and cluster analysis: The membership function values ​​of the two indicators, germination state index and relative strong bud growth, in step (5) are calculated to obtain the membership function values ​​of the two indicators. Then, the membership function values ​​of the two indicators are averaged to obtain the average membership function value. Salt tolerance is graded according to the size of the membership function value. At the same time, cluster analysis is performed based on the size of the average membership function value.

[0013] (7) Comprehensive analysis of salt tolerance of winter buds of mulberry trees.

[0014] Furthermore, the experimental materials in step (1) were taken from salt-tolerant materials from the south, ancient mulberry trees from the coastal areas of Hebei and Tianjin, wild mulberry trees from the coastal areas of Hebei and Tianjin, and materials preserved in the planting resource nursery of the Sericulture Research Institute.

[0015] Furthermore, the culture medium for pre-culturing in step (4) is: 1 / 2MS + 6-BA (2mg / L) + sucrose (15g / L) + agar (6g / L).

[0016] Furthermore, the germination status index formula in step (5) is: germination status index (%) = (number of buds that have fallen off * 1 + number of leaves that have opened * 2 - number of dead buds * 2 - number of withered buds * 1) / (total number of buds) * 100%; the formula for the growth of the relatively strong bud is: the growth of the relatively strong bud (%) = (fresh weight after 14 days of second transfer culture in salt-tolerant treatment / fresh weight after 14 days of second transfer culture in control) * 100%.

[0017] Furthermore, in step (5), the original experimental data were analyzed using Microsoft Excel 2013 software to calculate the mean of each treatment.

[0018] Furthermore, the membership formulas for the germination state index and the relative strong bud growth in step (6) are as follows:

[0019] Salt tolerance coefficient = Measured value under treatment conditions / Measured value under control conditions (1)

[0020] Membership function

[0021] anti-membership function

[0022] In the formula, U ij The membership function value of the j-th index (j=1,2,…,) of the i-th germplasm material. ij Let X be the salt tolerance coefficient of the j-th index of the i-th germplasm material. min X maxThese are the minimum and maximum values ​​of the salt tolerance coefficient, the j-th index, among all tested germplasm materials. Calculate the membership values ​​of the germination state index and the relative strong shoot growth. If the measured index is positively correlated with salt tolerance, use the membership function formula (2) to calculate; if it is negatively correlated, use the inverse membership function formula (3) to calculate.

[0023] Furthermore, the salt tolerance grading criteria in step (6) are as follows: when the average membership function value is 0.9651 to 0.9844, it is a highly salt-tolerant planting material; when the average membership function value is 0.7619 to 0.8274, it is a strongly salt-tolerant germplasm material; when the average membership function value is 0.488 to 0.7508, it is a moderately salt-tolerant germplasm material; when the average membership function value is 0.1037 to 0.446, it is a salt-sensitive germplasm material; and when the average membership function value is 0.0000 to 0.217, it is a highly salt-sensitive germplasm material.

[0024] Furthermore, in step (6), the cluster analysis is performed using SPSS 23.0 software. The cluster analysis utilizes the method of linking between groups, and the measurement interval is clustered using squared Euclidean distance, and the distance is recalibrated.

[0025] Beneficial effects

[0026] This invention provides a method for evaluating the salt tolerance of mulberry germplasm resources and screening salt-tolerant germplasm. This invention identifies the salt tolerance of mulberry planting resources collected from different regions, using the winter bud germination status index and the growth of relatively strong buds as evaluation indicators for screening a large number of mulberry materials for salt tolerance identification. Membership function and cluster analysis methods are used for comprehensive evaluation of salt tolerance, and different salt-tolerant germplasm are screened out, providing excellent germplasm resources for subsequent salt tolerance gene discovery and salt-tolerant variety breeding. Attached Figure Description

[0027] Figure 1 Cluster diagram of salt tolerance of in vitro winter bud germination for 63 mulberry germplasm materials; Note: The range of numbers in the figure represents the salt tolerance ranking (average membership function value) range of various germplasm materials. Detailed Implementation

[0028] The technical solution of the present invention will be further described below with reference to specific embodiments, but it is not limited thereto.

[0029] This invention discloses a method for evaluating the salt tolerance of mulberry germplasm resources and screening salt-tolerant germplasm, comprising the following steps:

[0030] (1) Collection and storage of experimental materials: The optimal time for sampling is from the time the mulberry tree has passed through its deep dormancy period until the sap begins to flow. To ensure the consistency of the winter bud development of different germplasm materials, sampling should not be carried out after the winter buds have sprouted. The sampling trees in Chengde, Hebei Province, were not subjected to summer pruning in the previous year so that the mulberry buds could complete the flower bud differentiation process. One-year-old healthy branches with full wood, plump winter buds, no dead branches, and no pests or diseases were cut off. About 1 / 3 of the lower part of the branch was cut off and discarded. The remaining part was cut into small sections of 25-30cm, put into self-sealing bags, and stored at 0-2℃ for later use. The 63 test materials included 6 salt-tolerant materials from the south, 7 ancient mulberry single trees from the Hebei-Tianjin coastal area, 10 wild mulberry single trees from the Hebei-Tianjin coastal area, and 40 materials preserved in the planting resource nursery of the Sericulture Research Institute. The source of the materials is detailed in Table 1.

[0031] Table 1. Sources of winter buds from mulberry trees used in the tests.

[0032]

[0033]

[0034]

[0035] (2) Explant preparation: Holding the branch segment upside down, use a scalpel to remove the leaf scars along the upper edge of the leaf scars, down to the xylem; after removing all the leaf scars on the branch segment, hold the branch segment upright and use a scalpel to remove the secondary buds and the lower scales of the bud, then gently peel off the outer scales with tweezers to expose the first layer of young leaves, cut off the bud intact, place it in a clean petri dish, cover the dish, 2

[0036] Disinfection must be carried out within 24 hours;

[0037] (3) Disinfection: Place different winter bud materials into small copper wire mesh baskets, immerse them in sterile water for 10 minutes, remove the baskets, drain the water, transfer them to 70% ethanol for 20 seconds for disinfection, rinse 2-3 times with sterile water, and then transfer them to 0.1% ethanol for disinfection.

[0038] Disinfect in mercuric chloride solution for 6 minutes, rinse 4 times with sterile water, then place the basket on sterile gauze. Once there are no water droplets remaining in the basket and the winter bud material in the basket, it can be inoculated.

[0039] (4) Culture: Different winter bud materials were inoculated into pre-culture medium, with 45-50 explants per material and no more than 6 explants per culture flask. Cultured for 7 days. The pre-culture medium was: 1 / 2 MS + 6-

[0040] BA (2 mg / L) + sucrose (15 g / L) + agar (6 g / L);

[0041] (5) Salt tolerance assessment: Winter shoots of the test material were placed on 1 / 2 MS solid medium containing 150 mmol / L NaCl or distilled water (control). They were placed in an incubator at a temperature of (25±2)℃, a light intensity of 2000-3000 lx, and a day / night photoperiod of 14h / 10h. Five winter shoots were placed in each bottle, and three bottles were used as one treatment, with three replicates. After 14 days of culture, the number of dead, withered, maintained, budded, and leaf-opened winter shoot explants was counted to calculate the germination status index (Formula 1). Strong shoots were selected and placed in fresh control and sterile 1 / 2 MS medium containing NaCl. After 14 days of culture, the weight of the winter shoot explants was measured, and the growth of the relatively strong shoots was calculated.

[0042] Germination status index of 6 germplasm materials on day 14 after the first transfer under different salt stress concentrations, and relative strong shoot growth on day 14 after the second transfer (Formula 2);

[0043] Germination Status Index (%) = (Number of buds that have fallen off * 1 + Number of leaves that have opened * 2 - Number of dead buds * 2 - Number of withered buds * 1) / (Total number of buds)

[0044] *2)*100%(One)

[0045] Relative strong shoot growth rate (%) = (fresh weight of salt-tolerant treatment after 14 days of second transfer culture / fresh weight of control after 14 days of second transfer culture) * 100% (II)

[0046] (6) Salt tolerance evaluation using membership functions and cluster analysis: The membership function values ​​of the germination state index and the relative strong bud growth in step (5) are calculated to obtain the membership function values ​​of the two indicators. The membership function values ​​of the two indicators are then averaged to obtain the average membership function value. Salt tolerance is graded according to the size of the membership function value. At the same time, cluster analysis is performed based on the size of the average membership function value.

[0047] The membership function formulas for the germination state index and the relative strong bud growth are as follows:

[0048] Salt tolerance coefficient = Measured value under treatment conditions / Measured value under control conditions (1)

[0049] Membership function

[0050] anti-membership function

[0051] In the formula, U ij The membership function value of the j-th index (j=1,2,…,) of the i-th germplasm material. ij Let X be the salt tolerance coefficient of the j-th index of the i-th germplasm material. min X maxThese are the minimum and maximum values ​​of the salt tolerance coefficient of the j-th index among all tested germplasm materials. Calculate the membership values ​​of the two indices, germination state index and relative strong shoot growth. If the measured index is positively correlated with salt tolerance, use the membership function formula (2) to calculate; if it is negatively correlated, use the inverse membership function formula (3) to calculate.

[0052] (7) Comprehensive analysis of salt tolerance of winter buds of mulberry trees.

[0053] Results and Analysis

[0054] Response of winter buds of different mulberry planting materials to salt stress

[0055] Under 150 mmol / L NaCl stress, the germination status index and relative strong bud growth of winter buds of 63 mulberry germplasm materials were statistically analyzed (see Table 2). The results showed that, comparing the relative strong bud growth of each sample, Yingjiaoting No. 1 (0012) from Qinhuangdao, Hebei Province, had the highest at 91.57%, followed by Taiwan Qingpi (0006) and J3-3 (00062), at 86.27% and 81.39%, respectively. Tengsang (0034) from Chengde, Hebei Province, had the lowest relative strong bud growth, at only 15.58%. The germination status index results showed that Taiwan Qingpi (0006) and Yingjiaoting No. 1 (0012) had the highest germination status indices under salt stress, at 91.11% and 88.89%, respectively, while Tengsang (0034) had the lowest, at only 19.79%. Based on the two indicators, it was found that Taiwan Green Peel (0006) and Yingjiaoting No. 1 (0012) had higher germination status index and relatively strong bud growth, indicating that their winter buds showed a high level of germination ability and vitality under salt stress, thus having good salt tolerance during the bud stage. In contrast, the winter buds of Tengsang (0034) had the weakest salt tolerance.

[0056] Table 2 Germination traits of detached winter buds of mulberry under salt stress

[0057]

[0058]

[0059]

[0060] Statistical analysis of the coefficients of variation of various indicators of the tested materials

[0061] Statistical analysis of the coefficient of variation of two indicators of winter buds from 63 mulberry germplasm materials (Table 3) revealed that, after salt stress, the average values ​​of the germination status index and the relative strong bud growth of winter buds from different mulberry germplasm resources were 65.07% and 51.07%, respectively. The germination status index ranged from 9.38% to 100.00%, and the relative strong bud growth ranged from 12.62% to 96.22%. The coefficients of variation for these two indicators were relatively large, at 25.51 and 35.37, respectively. This indicates that there are significant differences in the germination status index and the relative strong bud growth of winter buds from different mulberry germplasm resources under salt stress. Both indicators can well reflect the germination status of winter buds under salt stress and can serve as indicators for salt tolerance identification.

[0062] Table 3. Descriptive statistics and analysis of variance of salt tolerance indices of 63 mulberry germplasm materials under salt stress.

[0063]

[0064] Membership function and cluster analysis were used to evaluate the salt tolerance of mulberry germplasm materials during the bud stage.

[0065] To comprehensively evaluate the salt tolerance of mulberry germplasm materials during the bud stage, the membership function values ​​of two indicators—germination state index and relative strong bud growth—were averaged to obtain 63 membership values ​​(Tables 4 and 5). Simultaneously, based on the magnitude of the average membership function value, cluster analysis was performed using the between-group linkage method based on Euclidean squared distance. Figure 1 When the Euclidean distance was between 3 and 4, the tested mulberry germplasm materials were divided into 5 groups. The first group had a membership function mean ranging from 0.9651 to 0.9844, consisting of 2 germplasm materials, accounting for 3.2% of the tested materials. These materials had the highest relative values ​​of each trait under salt stress and were considered highly salt-tolerant germplasm materials. The second group had a membership function mean ranging from 0.7619 to 0.8274, consisting of 10 germplasm materials, accounting for 15.9%. These materials had relatively high relative values ​​of each trait and were considered highly salt-tolerant germplasm materials. The third group had a membership function mean of... The first group, with a membership function mean ranging from 0.488 to 0.7508, comprises 31 germplasm materials, accounting for 49.2% of the total. These materials exhibit moderate relative values ​​for each trait, classifying them as moderately salt-tolerant. The second group, with a membership function mean ranging from 0.1037 to 0.446, comprises 15 germplasm materials, accounting for 23.8% of the total. These materials exhibit low relative values ​​for each trait, classifying them as salt-sensitive. The third group, with a membership function mean ranging from 0.0000 to 0.217, comprises 5 germplasm materials, accounting for 7.9% of the total. These materials exhibit very low relative values ​​for each trait, classifying them as highly salt-sensitive. Among these, the top five mulberry germplasm resources in terms of salt tolerance are 0012, 0006, 0007, 0062, and 0033.

[0066] Table 4. Membership values ​​of salt tolerance for winter bud germination in mulberry germplasm materials

[0067]

[0068]

[0069] Table 5. Salt tolerance classification of 63 mulberry germplasm resources

[0070] Classification Salt-tolerant types Membership value range quantity Percentage / % I Salt tolerance 0.9651～0.9844 2 3.2 II Strong salt tolerance 0.7619～0.8274 10 15.9 III Moderate salt tolerance 0.488～0.7508 31 49.2 IV Salt sensitivity 0.1037～0.446 15 23.8 V Salt sensitivity 0.0000～0.217 5 7.9

[0071] Comprehensive analysis of salt tolerance of mulberry winter buds

[0072] Cluster analysis based on salt tolerance during bud stage ( Figure 1 The list of 63 mulberry salt tolerance classifications (Table 6) shows that salt-tolerant (medium-tolerant) and above salt-tolerant resources account for 68.3% of the tested resources. Among them, the two germplasm resources, Taiwan Qingpi (0006) from Hangzhou, Zhejiang and Yingjiaoting No. 1 (0012) from Qinhuangdao, Hebei, have high values ​​of all traits under salt stress and the best overall salt tolerance, making them extremely salt-tolerant materials. Yongqing No. 6 (0058), Huanglu self-pollinated-B1 (0039), Huanglu (0060), Shengli B1 (0054), and Tengsang (0034) are also included. The salt tolerance of winter buds from all germplasm resources was poor, indicating that they were highly salt-sensitive germplasm materials. Among them, Tengsang (0034) had a germination state index and a membership value of 0 for both the germination state index and the relative strong bud growth, and its winter bud salt tolerance ranked 63rd, showing the worst salt tolerance. Meanwhile, seven ancient mulberry single-tree materials and ten wild mulberry single-tree materials from the Hebei-Tianjin coastal area all showed above the moderately salt-tolerant germplasm materials, indicating that the materials from the saline-alkali land surrounding the Hebei-Tianjin coastal area have good salt tolerance and can provide excellent parental sources for mulberry salt tolerance breeding.

[0073] Table 6. Classification of salt tolerance during the seedling stage of 63 mulberry germplasm materials

[0074]

[0075] It should be noted that the above embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

Claims

1. A method for evaluating the salt tolerance of mulberry germplasm resources, characterized in that, Includes the following steps: (1) Sampling and storage of experimental materials: The optimal time for sampling is from the time the mulberry tree passes through the deep dormancy period until the sap begins to flow. In order to ensure the consistency of the winter bud development of different germplasm materials, it is important to avoid sampling after the winter buds sprout. Select healthy one-year-old branches from each region that are woody, have plump winter buds, are free of dead shoots and pests. Cut off about 1 / 3 of the lower part of the branch and discard it. Cut the remaining part into 25-30cm sections, put them into self-sealing bags, and store them at 0-2℃ for later use. (2) Explant preparation: Hold the branch segment upside down and use a scalpel to cut off the leaf scar along the upper edge of the leaf scar, down to the xylem; after cutting off all the leaf scars on the branch segment, hold the branch segment upright and use a scalpel to remove the secondary buds and the scales on the lower part of the bud, then use tweezers to gently peel off the outer scales to expose the first layer of young leaves, cut off the bud completely, put it in a clean petri dish, cover the petri dish, and sterilize it within 2 hours; (3) Disinfection: Place different winter bud materials into small baskets made of copper wire mesh, soak them in sterile water for 10 minutes, remove the baskets, drain the water, and disinfect them in 70% ethanol for 20 seconds. Rinse them 2-3 times with sterile water, then disinfect them in 0.1% mercuric chloride solution for 6 minutes. Rinse them 4 times with sterile water. Then place the baskets on sterile gauze. Once there are no water droplets left in the baskets and the winter bud materials in the baskets, they can be inoculated. (4) Start culture: Inoculate different winter bud materials into the pre-culture medium, inoculate 45-50 explants into each material, and inoculate no more than 6 explants into each culture bottle, and culture for 7 days; (5) Salt tolerance identification: The winter shoots of the test materials were placed on 1 / 2 MS solid medium containing 150 mmol / L NaCl and distilled water, respectively, and placed in an incubator at a temperature of 25±2℃, a light intensity of 2000~3000lx, and a day / night photoperiod of 14h / 10h. Five winter shoots were placed in each bottle, and three bottles were used as one treatment, with three replicates. After 14 days of culture, the number of dead, withered, maintained, budded, and leaf-opened winter shoot explants were counted to calculate the germination status index. Strong shoots were selected and placed in a new sterile 1 / 2 MS solid medium containing 150 mmol / L NaCl and distilled water. After 14 days of culture, the fresh weight of the winter shoot explants was measured, and the growth of the relatively strong shoots was calculated. The germination status index of the six germplasm materials on the 14th day after the first transfer and the growth of the relatively strong shoots on the 14th day after the second transfer were measured under different salt stress concentrations. (6) Salt tolerance is evaluated using membership functions and cluster analysis: The membership function values ​​of the germination state index and the relative strong bud growth in step (5) are calculated to obtain the membership function values ​​of the two indicators. The membership function values ​​of the two indicators are then averaged to obtain the average membership function value. Salt tolerance is graded according to the size of the membership function value. At the same time, cluster analysis is performed based on the size of the average membership function value. (7) Comprehensive analysis of salt tolerance of winter buds of mulberry trees; The germination status index formula in step (5) is: Germination status index (%) = (number of buds that have fallen off * 1 + number of leaves that have opened * 2 - number of dead buds * 2 - number of withered buds * 1) / (total number of buds) * 100%; The formula for the growth of relatively strong buds is: Relative strong bud growth (%) = (fresh weight after 14 days of second transfer culture in salt-tolerant treatment / fresh weight after 14 days of second transfer culture in control) * 100%; The membership formulas for the germination state index and the relative strong bud growth in step (6) are as follows: Salt tolerance coefficient = Measured value under treatment conditions / Measured value under control conditions (1) Membership function anti-membership function In the formula, U ij The membership function value of the j-th index of the i-th germplasm material, X ij Let X be the salt tolerance coefficient of the j-th index of the i-th germplasm material. min X max These are the minimum and maximum values ​​of the salt tolerance coefficient of the j-th index among all tested germplasm materials; calculate the membership values ​​of the two indices, germination state index and relative strong bud growth. If the measured index is positively correlated with salt tolerance, use the membership function formula (2) to calculate; if it is negatively correlated, use the inverse membership function formula (3) to calculate. The salt tolerance grading criteria in step (6) are as follows: when the average membership function value is 0.9651 to 0.9844, it is a very salt-tolerant planting material; when the average membership function value is 0.7619 to 0.8274, it is a strongly salt-tolerant germplasm material; when the average membership function value is 0.488 to 0.7508, it is a moderately salt-tolerant germplasm material; when the average membership function value is 0.1037 to 0.446, it is a salt-sensitive germplasm material; when the average membership function value is 0.0000 to 0.217, it is a highly salt-sensitive germplasm material.

2. The method for evaluating the salt tolerance of mulberry germplasm resources according to claim 1, characterized in that, The experimental materials in step (1) were taken from salt-tolerant materials from the south, ancient mulberry trees from the Hebei-Tianjin coastal area, wild mulberry trees from the Hebei-Tianjin coastal area, and materials preserved in the planting resource nursery of the Sericulture Research Institute.

3. The method for evaluating the salt tolerance of mulberry germplasm resources according to claim 1, characterized in that, The culture medium for pre-culturing in step (4) is: 1 / 2 MS + 6-BA + sucrose + agar.

4. The method for evaluating the salt tolerance of mulberry germplasm resources according to claim 3, characterized in that, The concentration of 6-BA was 2 mg / L, the concentration of sucrose was 15 g / L, and the amount of agar used was 6 g / L.

5. The method for evaluating the salt tolerance of mulberry germplasm resources according to claim 1, characterized in that, In step (5), the original experimental data were analyzed using Microsoft Excel 2013 software, and the mean of each treatment was calculated.

6. The method for evaluating the salt tolerance of mulberry germplasm resources according to claim 1, characterized in that, In step (6), cluster analysis is performed using SPSS 23.0 software. The cluster analysis utilizes the method of linking between groups, and the measurement interval is clustered using squared Euclidean distance, and the distance is recalibrated.

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