NL2029158B1 - Method for evaluating salt tolerance of wolfberry - Google Patents

Abstract

The disclosure discloses a method for evaluating salt tolerance of wolfberry, which relates to the field of the adversity adaptability evaluation of wolfberry. In the method, the seedlings of wolfberry are treated with salt stress and without salt stress, the leaves of the seedlings are taken for lipid molecules detection and analysis. The type number and/or content of lipid molecules in the salt stress treatment group that have significantly changed compared to the no salt stress treatment group are used as the evaluation index of salt tolerance. The analysis of lipid molecule content can accurately reflect the salt tolerance level between different wolfberry varieties, which has important guiding significance for the breeding of stress tolerance of wolfberry and has wide application prospects in the promotion of vegetation recovery, desertification prevention and saline-alkali land utilization.

Description

METHOD FOR EVALUATING SALT TOLERANCE OF WOLFBERRRY Technical Field The present disclosure relates to the field of the adversity adaptability evaluation of wolfberry, especially relates to a method for evaluating salt tolerance of wolfberry. Background Wolfberry, a perennial deciduous shrub, belongs to the medicinal and food homologous species, distributes in Ningxia, Gansu, Qinghai, Xinjiang, Tibet and other regions of China. The fruits and leaves of wolfberry contain rich nutrients and effective substances, which are commonly used Chinese medicinal materials and also very important health-care products. The research on the breeding, planting and functional components of wolfberry has been widely concerned in recent years.

Saline-alkali soil refers to the soil with a large amount of soluble salt under the action of natural factors and human factors. At present, soil salinization is a key factor that restricts agricultural production and economic development. Wolfberry is a plant with strong saline-alkali resistance and drought tolerance, and is suitable for planting in saline-alkali, drought and deserts. Wolfberry is one of the pioneer tree species for vegetation recovery, desertification prevention and saline-alkali land utilization in northwest China. It is of great practical significance to improve the growth state of wolfberry under the extreme environment such as saline-alkali land, and to cultivate and select the varieties with high salt tolerance.

The adverse effects of excessive soluble salt in soil on plants are referred to as salt damage, and the tolerance capacity of plants to salt damage is called salt tolerance. Currently, it is commonly used in the prior art to evaluate the salt tolerance of different varieties of wolfberry by observing the growth states of seedlings and the seed germination rate under different salt stress conditions. Although the differences in salt tolerance of different varieties of wolfberry by the above methods can be visually displayed, the evaluation of salt tolerance of wolfberry is relatively rough and lacks accurate analysis.

Summary The present disclosure provides a new evaluation method of salt tolerance of wolfberry, so as to solve the defects that the method for evaluating the salt tolerance of wolfberry in the prior art is relatively rough and lacks accurate analysis.

The present disclosure provides a method for evaluating salt tolerance of wolfberry, the seedlings of wolfberry are respectively treated with salt stress (adding NaCl} and without salt stress (not adding NaCl), the leaves of the seedlings are taken for lipid molecules detection and analysis. The number and/or content of lipid molecules in the salt stress treatment group that have significantly changed compared to the no salt stress treatment group are used as the evaluation index of salt tolerance.

Specifically, the method includes: Cutting the seedlings of wolfberry varieties to be evaluated in the culture medium without NaCl and the culture medium with NaCl for cultivation, which are respectively the control group and the experimental group. The culture medium is a MS culture medium, containing 4.23¢g/L of macro elements, 85.29mg/L of trace elements and 103.1mg/L of vitamin; taking the leaves of cultured seedlings in the control group and the experimental group to detect the content of lipid molecules in the leaves, and obtaining the types of lipid molecules with significant content changes in the experimental group, compared with the control group. The content of lipid molecules in leaves was detected by the following steps: (1) taking the powder of the ground leaves and adding into the preheated isopropyl alcohol, heating, then adding the full-fat internal standard, chloroform and ultra-pure water, swirling, centrifuging, and taking the first supernatant; (2) adding the mixture of chloroform and methanol (containing BHT) into the remaining liquid after the first supernatant is taken in step (1), swirling, centrifuging, and taking the second supernatant; (3) mixing the first supernatant and the second supernatant, then adding KCI in it, swirling, centrifuging, and taking the subnatant, blow-drying with nitrogen, re-dissolving with isopropyl alcohol, and filtering to obtain a to-be-tested solution; evaluating the salt tolerance of wolfberry according to the number and/or content of lipid molecules with significant changes in content, wherein, the fewer the type number of lipid molecules with significant changes in content, the less the change in content, the higher the salt tolerance of wolfberry.

The method further comprises: taking the leaves of cultured seedlings of the control group and the experimental group, and detecting the content of abscisic acid and/or jasmonic acid in the leaves; evaluating the salt tolerance of wolfberry according to the significance of changes in the content of abscisic acid and/or jasmonic acid in the leaves of the experimental group compared with the control group, wherein, the more significant the content change of abscisic acid and/or jasmonic acid, the higher the salt tolerance of wolfberry.

The present disclosure has the following advantages:

1. In the evaluation method of salt tolerance of wolfberry provided by the present disclosure, the seedlings of wolfberry are respectively treated with salt stress (adding NaCl) and without salt stress (not adding NaCl), the leaves of the seedlings are taken for lipid molecules detection and analysis. The number and/or content of lipid molecules in the salt stress treatment group that have significantly changed compared to the no salt stress treatment group are used as the evaluation index of salt tolerance. The analysis of lipid molecule content based on salt stress treatment and no salt stress treatment can accurately reflect the salt tolerance level between different wolfberry varieties, which has important guiding significance for the breeding of stress tolerance of wolfberry and has wide application prospects in the promotion of vegetation recovery, desertification prevention and saline-alkali land utilization in northwest China.

2. This evaluation method further comprises the steps of evaluating the salt tolerance of wolfberry using abscisic acid and/or jasmonic acid. By using the significant changes in the content of abscisic acid and/or jasmonic acid in the leaves of the salt stress treatment group relative to the no salt stress treatment group as the evaluation index of salt tolerance, which can assist in the analysis of the salt tolerance between different wolfberry varieties. It is beneficial to provide further guidance for the adversity resistance breeding of wolfberry. Brief Description of Fig.s In order to more clearly explain the specific embodiments of the present invention or the technical solutions in the prior art, the following will make a brief introduction of the drawings needed in the specific embodiments or prior art. It is obviously that the attached drawings in the following description are part embodiments of the present invention. For those skilled in the art, other drawings based on these drawings can be obtained on the basis of the following drawings without creative work.

Fig. 1 is a comparison diagram of the number of lipid molecules types in the experimental group and the control group in Embodiment 1 of the present disclosure; Fig. 2 is a comparison diagram of the number of lipid molecules under different lipid subgroups in the experimental group and the control group in Embodiment 1 of the present disclosure; Fig. 3 is a graph showing changes in the content of different lipid molecules in experimental group 1 and control group 1 in Embodiment 1 of the present disclosure; Fig. 4 is a graph showing changes in the content of different lipid molecules in experimental group 2 and the control group 2 in Embodiment 1 of the present disclosure; Fig. 5 is a comparison diagram of ABA content in the experimental groups and the control groups in Embodiment 2 of the present disclosure; Fig. 6 is a comparison diagram of JA content in the experimental groups and the control groups in Embodiment 2 of the present disclosure; Fig. 7 is a comparison diagram of SA content in the experimental groups and the control groups in Embodiment 2 of the present disclosure; Fig. 8 is a comparison diagram of the growth states of seedlings under different salt stress conditions in Embodiment 3 of the present disclosure; Fig. 9 is a trend chart of the germination rate in the experimental groups and the control groups over time in Embodiment 4 of the present disclosure; Fig. 10 is a scale chart of the germination rate in the experimental groups and the control groups on the 24th day of culture in Embodiment 4 of the present disclosure. Detailed Description The following embodiments are provided for a better understanding of the present disclosure. They are not limited to the best embodiment, and do not limit the content and protection scope of the present disclosure. Any product identical or similar to the present disclosure obtained under the inspiration of the disclosure or by combining the features of present disclosure and other prior art, falls within the protection scope of the present disclosure.

The MS culture medium used in the embodiments was purchased from Rishui Biology Co., LTD. The formula was as follows: macro elements (constant elements) 4.23¢/L, trace elements

85.29mg/L, vitamin 103.1mg/L.

Abscisic acid internal standard used in the embodiments: [2H6]-abscisic acid; jasmonic acid internal standard: [2H5]-jasmonic acid; salicylic acid internal standard:[2H4]-salicylic acid; they were all purchased from Shanghai Zhenzhun Bio- technology Co., LTD.

The full-fat internal standard used in the embodiments was a mixture of thirteen internal standards of heavy-hydrogen lipids, with each concentration is 100 pg/mL, purchased from Avanti Corporation, and the Product Number is 330731-1EA.

It can be carried out according to the conventional experimental steps described in the literature in this art if the specific experimental steps or conditions are not indicated in the embodiments. The raw materials or instruments used are all commercially available conventional products, including but not limited to the raw materials or instruments used in the embodiments of the present disclosure.

Embodiment 1 Lipid analysis was used to evaluate the salt tolerance of Lycium Chinese and Lycium Ruthenicum, the method is as follows: (1) Fresh twigs {branches without lignification, cut from wolfberry trees in spring) were cut from Lycium Chinese trees and Lycium Ruthenicum trees. The length of the twigs was about 8 cm. The twigs were disinfected with mercury (0.01% mercury chloride) in an ultra-clean workbench, washed with sterile water and aired, and then cut in MS culture medium to culture for 3 weeks in the greenhouse (photoperiod was 10h light and 14h dark, temperature was 25°C, humidity was 50%). Then the twigs were cut again into about 5 cm in length for aseptic propagation in an ultra-clean workbench. The seedlings with the same stem diameter on the twigs about 5 cm in length were cut for subsequent operations.

(2) The seedlings of Lycium Chinese obtained in step (1) were respectively cut in MS culture medium without NaCl and MS culture medium with NaCl (NaCl concentration was 150mmol/L) to culture for 21 days in the greenhouse {photoperiod was 10h light and 14h dark, temperature was 25°C, humidity was 50%), as control group 1 (LC-mock) and experimental group 1 (LC-NaCl). The seedlings of Lycium Ruthenicum obtained in step (1) were respectively cut in MS culture medium without NaCl and MS culture medium with NaCl (NaCl concentration was 150mmol/L) to 5 cluture for 21 days in the greenhouse (photoperiod was 10h light and 14h dark, temperature was 25°C, humidity was 50%), as control group 2 (LR-mock) and experimental group 2 (LR-NaCl). The leaves of the seedlings of the above groups were cut from the petioles and were quickly frozen in liquid nitrogen, and then stored in a refrigerator at -80°C for later use. The content of lipid molecules in the leaves was detected: the leaves pre-frozen in the refrigerator at -80°C were added with liquid nitrogen, and then ground into uniform powder; 20 mg powder was weighed and added with 3mL of isopropyl alcohol preheated to 75°C, heated for 15 min, added with 100 LL of full-fat internal standard with a concentration of 10 ug/mL, 1.5mL chloroform and 0.6mL ultra-pure water, swirled for 1h, centrifuged, and the first supernatant was taken. After the first supernatant was taken, the remaining liquid was added with 4 mL of the mixture of chloroform and methanol (containing 0.01%BHT) with a volume ratio of 2:1, swirled for 30 min, centrifuged, and the second supernatant was taken, the above steps were repeated for three times, the extracting solution was obtained by mixing the first supernatant and the second supernatant, 1mL of KCI with a concentration of 1 mol/L was added into the extracting solution, swirled for 1h, centrifuged, then the subnatant was taken and blown dried with nitrogen, re- dissolved with 1 mL of isopropyl alcohol, and filtered with 0.22 um organic filter membrane to obtain a to-be-tested solution. (3) Instrument detection: an ultra-high performance liquid chromatography-mass spectrometry method was adopted, wherein, chromatography system: Shimadzu UPLC LC-30A; chromatographic column: Phenomenex Kinetics C18 column (100 x 2.1 mm, 2.6 ym); injection volume: 1 pL; flow rate: 0.4 mL/min; column temperature: 60°C; temperature of a sample chamber: 4°C; Mobile phase A: a mixture of water, methanol and acetonitrile with a volume ratio of 1: 1: 1, containing 5 mmol/L ammonium acetate; mobile phase B phase: a mixture of isopropyl alcohol and acetonitrile with a volume ratio of 5: 1, containing 5 mmol/L ammonium acetate; gradient elution conditions: Omin—0.5min, A phase: B phase = 80: 20, V/V; 0.5 min—1.5 min, A phase: B phase = 60: 40; 1.5 min—3.0 min, A phase: B phase = 40: 60; 3.0 min—13.0 min; A phase : B phase = 80: 20; 13.0 min—17.0 min; A phase: B phase = 80: 20; mass spectrometry system: AB Sciex TripleTOF 6600; ESI ion source: positive mode; the mass range of mass spectrum acquisition was m/z 100-1200; mass spectrometry conditions: curtain gas: 35.000 psi; ion source gas 1: 50.000; ion source gas 2: 50.00; ionSpray voltage:

5500.00V; ion source temperature: 600°C. (4) Statistical analysis of data: sigmaplot software was used for statistical analysis of data,

and t-test was used to analyze the significance of differences in the content of lipid molecules among the groups (*: p<0.05, significant difference; **: p<0.01, extremely significant difference). (5) Result analysis:

(DAs shown in Fig. 1, the effects of salt stress on the number of lipid molecules types in leaves of Lycium Chinese and Lycium Ruthenicum were statistically analyzed.

According to the comparison between the control group 1 and the experimental group 1 (LC-mock vs.

LC-NaCl), it can be seen that salt stress induced significant changes in the content of 36 kinds of lipid molecules in the leaves of Lycium Chinese, among which the content of 33 kinds of lipid molecules increased significantly and the content of 3 kinds of lipid molecules decreased significantly.

According to the comparison between the control group 2 and the experimental group 2 (LR-mock vs.

LR-NaCl), it can be seen that salt stress only caused significant changes in the content of 3 kinds of lipid molecules in the leaves of Lycium Ruthenicum, and all of them were up-regulated.

According to the comparison between the control group 1 and the control group 2 (LC-mock vs.

LR-mock) and the comparison between the experimental group 1 and the experimental group 2 (LC-NaCl vs.

LR-NaCl), compared with Lycium Chinese, most of the lipid molecules with significant changes in content in the leaves of Lycium Ruthenicum were up-regulated no matter under normal growth conditions or high salt stress conditions.

The salt tolerance evaluation of wolfberry by the number of lipid molecules types with significant changes in content need fewer number of the lipid molecules types for resisting salt stress, but with the stronger salt tolerance.

It was concluded that the salt tolerance of Lycium Ruthenicum was higher than that of Lycium Chinese. (2) Lipid molecules contain fatty acid (FA), lysophosphatidic acid (LPA), phosphatidylcholine (PC), lysophosphatidylcholine (LPC), phosphatidylethanolamine (PE), phosphatidyl glycerol (PG), diglyceride (DG), digalactosyl diglyceride (DGDG), monogalactosyl diglyceride (MGDG), sulphoquinovosyl diglyceride (SQDG), triglyceride (TG) and other subgroups.

As shown in Fig. 2, the effects of salt stress on the number of lipid molecules types in leaves of Lycium Chinese and Lycium Ruthenicum in different subgroups were statistically analyzed.

It can be seen that salt stress induced the most changes in the number molecules types under the triglyceride (TG) in the leaves of Lycium Chinese, followed by the diglyceride (DG), and only a few kinds of lipid molecules content in other subgroups changed.

For the leaves of Lycium Ruthenicum, salt stress only induced significant changes in the content of two phosphatidylethanolamine (PE) molecules and one igalactose diglyceride (DGDG) molecule.

It also was concluded that the salt tolerance of Lycium Ruthenicum was higher than that of Lycium Chinese. @As shown in Fig. 3 (FA: fatty acid; LPA: lysophosphatidic acid; LPC: lysophosphatidylcholine; LPE: lysophosphatidyl ethanolamine; PA: phosphatidic acid; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PG: phosphatidyl glycerol; PI:

phosphatidylinositol, PMeOH: phosphatidyl methanol; PS: phosphatidylserine; DG: diglyceride; DGDG: digalactosyl diglyceride; MGDG: monogalactosyl diglyceride; SQDG: sulphoquinovosyl diglyceride; TG: triglyceride), the effects of salt stress on the content of lipid molecules in the leaves of Lycium Chinese were statistically analyzed.

According to the comparison between the control group 1 and the experimental group 1 (LC-mock vs.

LC-NaCl), it can be seen that salt stress induced significant changes in the content of 36 kinds of lipid molecules in the leaves of Lycium Chinese, among which the content of 22 kinds of triglycerides (TG) was up-regulated, and the content of 7 kinds of lipid molecules changed more than twice, the content of a kind of fatty acid (FA) was up to the maximum, which was more than 3 times.

Only the content of 3 kinds of diglyceride (DG) was down-regulated, and the down-regulated range was about 1 time.

As shown in Fig. 4, the effects of salt stress on the content of lipid molecules in leaves of Lycium Ruthenicum were statistically analyzed.

According to the comparison between the control group 2 and the experimental group 2 (LR-mock vs.

LR-NaCl), it can be seen that salt stress only induced significant changes in the content of 3 kinds of lipid molecules in leaves of Lycium Ruthenicum, and all of them were up-regulated, including two phosphatidylethanolamine (PE) and one digalactosyl diglyceride (DGDG). The up-regulated range of these three kinds of lipid molecules was at a low level, only 1.0-1.4 times.

In summary, the content of lipid molecules with significantly changed content in leaves of Lycium Ruthenicum has a lower change multiple than that of Lycium Chinese.

Therefore, the salt tolerance of Lycium Ruthenicum was higher than that of Lycium Chinese from the analysis of content change.

Embodiment 2 Hormonal analysis was used to evaluate the salt tolerance of Lycium Chinese and Lycium Ruthenicum, the method is as follows: (1)The leaves pre-frozen in the refrigerator at -80°C in Embodiment 1 were taken; abscisic acid (ABA), jasmonic acid (JA) and salicylic acid (SA) in the leaves were extracted: liquid nitrogen was added to the leaves, and then the leaves were ground into uniform powder. 100 mg of powder was weighed and added with 100 pL each of the internal standard of ababolic acid, jasmonic acid and salicylic acid, 1 mL of extraction reagent (a mixture of methanol, water and formic acid with a volume ratio of 15:4:1), was added to perform extraction, impurities were removed by centrifugation, supernatant was then taken to obtain an extracting solution.

The extracting solution was centrifuged, removed supernatant to retain the precipitate, the precipitate was dissolved in methanol aqueous solution with the mass fraction of 80%, and then filtered with a 0.22 um polytetrafluoroethylene (PTFE) filter membrane after complete dissolution, the filtered solution (the to-be-tested solution) was placed in a sample bottle for next instrument analysis. (2)Instrument detection: the to-be-tested solution obtained in step (1) was detected by Ultra- high liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and the content of abscisic acid, jasmonic acid and salicylic acid were determined by internal standard method: chromatography system: Shim-pack UFLC SHIMADAZU CBM30A; chromatographic column: Waters ACQUITY UPLC HSS T3 C18 (1.8 um, 2.1mmx100mm); mobile phase:aqueous phase of ultra-pure water containing 0.05% formic acid; the organic phase is acetonitrile containing 0.05% formic acid; gradient elution conditions: O min, ultra-pure water: acetonitrile = 95:5, V/V; 0 min—1.0 min, ultra-pure water: acetonitrile = 95:5, V V; 1.0 min->8.0 min, ultra-pure water: acetonitrile = 5: 95, V/V; 8.0 min—9.0 min, ultra-pure water: acetonitrile = 5: 95, V/V; 9.0 min — 9.1 min, ultra-pure water: acetonitrile = 95:5, V/V; 9.1 min — 12.0 min, ultra-pure water: acetonitrile = 95:5, V/V, flow rate: 0.35 mL/min; column temperature:40°C; Injection volume: 2 HL; mass spectrometry system: Applied Biosystems 6500 Quadrupole Trap; mass spectrometry conditions: electrospray ion source temperature: 500°C; mass spectral voltage: 4500 V; curtain gas: 35 psi; collision induced ionization parameters: medium.

Finally, Analyst1.6.1 was used to process and analyze the mass spectrum data to obtain the qualitative and quantitative analysis data of hormones.

(3) Statistical analysis of data: sigmaplot software was used for statistical analysis of data, and t-test was used to analyze the significance of differences in hormones content among the groups (*: p<0.05, significant difference; **: p<0.01, extremely significant difference).

(4)Result analysis: As shown in Fig. 5, the content of abscisic acid (ABA) in leaves of Lycium Chinese (LC) in the experimental group (150 mM NaCl) increased compared with that of the control group (mock), and it can be seen that salt stress induced an increase in the content of abscisic acid (ABA) in the leaves of Lycium Chinese, but not a significant difference. The content of abscisic acid (ABA) in leaves of Lycium Ruthenicum in the experimental group (150 mM NaCl) also increased compared with that of the control group (mock), and it can be seen that salt stress induced an extremely significant increase in the content of abscisic acid (ABA) in the leaves of Lycium Ruthenicum (p<0.01). In summary, the content change of abscisic acid in leaves of Lycium Ruthenicum had a higher significance than that of Lycium Chinese. Therefore, the salt tolerance of Lycium Ruthenicum was higher than that of Lycium Chinese. This was the same as the evaluation result of Embodiment 1.

As shown in Fig. 8, the content of jasmonic acid (JA) in leaves of Lycium Chinese (LC) in the experimental group (150 mM NaCl) decreased compared with that of the control group (mock), and it can be seen that salt stress induced a significant decrease in the content of jasmonic acid (JA) in the leaves of Lycium Chinese(p<0.05). The content of jasmonic acid (JA) in leaves of Lycium Ruthenicum in the experimental group (150 mM NaCl) decreased compared with that of the control group (mock), and it can be seen that salt stress induced an extremely significant decrease in the content of jasmonic acid (JA) in the leaves of Lycium Ruthenicum (p<0.01). In summary, the content change of jasmonic acid in leaves of Lycium Ruthenicum had a higher significance than that of Lycium Chinese. Therefore, the salt tolerance of Lycium Ruthenicum was higher than that of Lycium Chinese. This was the same as the evaluation result of Embodiment

1. As shown in Fig. 7, the content of salicylic acid (SA) in leaves of Lycium Chinese (LC) in the experimental group (150 mM NaCl) increased compared with that of the control group (mock), and it can be seen that salt stress induced an increase in the content of salicylic acid (SA) in the leaves of Lycium Chinese, but not a significant difference. The content of salicylic acid (SA) in leaves of Lycium Ruthenicum in the experimental group (150 mM NaCl) decreased compared with that of the control group (mock), and it can be seen that salt stress induced a decrease in the content of salicylic acid (SA) in the leaves of Lycium Ruthenicum, but also not a significant difference. Therefore, salicylic acid was not selected as an index to evaluate the salt tolerance of wolfberry.

Embodiment 3 The salt tolerance of Lycium Chinese and Lycium Ruthenicum was evaluated by observing the growth states of seedlings under different salt stress conditions, so as to verify the reliability of the method of Embodiment 1 and Embodiment 2. The method was as follows: The seedlings cut after aseptic propagation in step (1) of Embodiment 1 were taken, the seedlings of Lycium Chinese and Lycium Ruthenicum were respectively cut in MS culture medium containing different concentrations of NaCl (0 mmol/L, 150 mmol/L, 200 mmol/L, 250 mmol/L, 300 mmol/L) to culture for 21 days in the greenhouse (photoperiod was 10h light and 14h dark, temperature was 25°C, humidity was 50%), the seedlings were separated and taken out from the MS culture medium together with their roots, the culture medium adhered to the roots of seedlings was washed, the water on the seedlings was dried with absorbent paper, and then the seedlings were photographed for observation.

As shown in Fig. 8, 150 mmol/L NaCl stress begun to inhibit the growth of seedlings, but it did not cause lethal effects on seedlings. The stress damage to seedlings became more serious with the increase of NaCl concentration. It can also be seen that the seedlings of Lycium Chinese were more seriously damaged than that of Lycium Ruthenicum under the same salt stress conditions, so it was concluded that the salt tolerance of Lycium Ruthenicum was higher than that of Lycium Chinese, which was the same as the evaluation result of Embodiments 1 and 2. Therefore, the evaluation method provided by the present disclosure was reliable and could be used to evaluate the salt tolerance of different wolfberry varieties.

Embodiment 4 The salt tolerance of Lycium Chinese and Lycium Ruthenicum was evaluated by calculating the seed germination rate under different salt stress conditions, so as to verify the reliability of the method of Embodiment 1 and Embodiment 2. The method was as follows: dried fruits of Lycium Chinese and Lycium Ruthenicum were artificially crushed, and seeds were separated from the pulp by flowing water, plump seeds with the same size were selected for subsequent experiments. Seeds were surface-sterilized with 0.1% sodium hypochlorite solution for 10 minutes, and then rinsed with sterile water 5 to 6 times, and blown dried in an ultra-clean workbench. Dried seeds of Lycium Chinese and Lycium Ruthenicum were planted in MS culture medium containing no NaCl and 150 mmol/L NaCl respectively, and cultured in a refrigerator with 4°C for 3 days, then cultured in the greenhouse (photoperiod was 10h light and 14h dark, temperature was 25°C, humidity was 50%). The statistics of seed germination rate of Lycium Chinese control group (LC-mock), experimental group of Lycium Chinese (LC-150mM NaCl), control group of Lycium Ruthenicum (LR-mock) and experimental group of Lycium Ruthenicum IO (LR-150mM NaCl) was performed every day from the third day to the 24th day of planting, and the ratio of seed germination rate of the 24th day in the experimental group/control group of Lycium Chinese and Lycium Ruthenicum was calculated respectivel. Sigmaplot software was used for statistical analysis of data, and t-test was used to analyze the significance of differences in the ratio of seed germination rate of Lycium Chinese and Lycium Ruthenicum (*: p<0.05, significant difference; **: p<0.01, extremely significant difference).

As shown in Fig. 9 and Fig. 10, the seed germination rate of Lycium Chinese and Lycium Ruthenicum was decreased in different degrees under salt stress conditions, and the inhibition degree of seed germination rate of Lycium Chinese was higher than that of Lycium Ruthenicum, so it was concluded that the salt tolerance of Lycium Ruthenicum was higher than that of Lycium Chinese, which was the same as the evaluation results of Embodiment 1 and Embodiment 2. Therefore, it was further proved that the evaluation method provided by this present disclosure was reliable and could be used to evaluate the salt tolerance of different wolfberry varieties.

Obviously, the above embodiments are only used to clearly describe the examples given, and are not intended to limit the mode of embodiments. Other changes or modifications in different forms can be made based on the above description for those skilled in the art. It is not necessary and possible to list all the embodiments here. The apparent change or variation derived from it is still within the protective scope of this present disclosure.

The following numbered clauses include examples that are contemplated and nonlimiting: Clause 1. A method for evaluating salt tolerance of wolfberry, comprising: cutting seedlings of wolfberry to be evaluated in a culture medium without NaCl and a culture medium with NaCl to culture, respectively as a control group and an experimental group; the culture medium is a MS culture medium, the MS culture medium comprises the following components: 4.23 g/L of macro elements, 85.29 mg/L of trace elements and 103.1 mg/L of vitamin; taking leaves of the seedlings after culturing in the control group and the experimental group, detecting a content of lipid molecules in the leaves, obtaining types of the lipid molecules with significant changes of content in the experimental group compared with the control group; the content of the lipid molecules in the leaves is detected by the following steps: (1) taking a power of ground leaves, and adding into a preheated isopropyl alcohol, heating, adding a full-fat internal standard, a chloroform and an ultra-pure water, swirling, centrifuging, and taking a first supernatant; (2) adding a mixture of chloroform and methanol containing BHT into a remaining liquid after the first supernatant in step (1) is taken, swirling, centrifuging, and taking a second supernatant; (3) mixing the first supernatant and the second supernatant, adding KCl in it, swirling, centrifuging, taking subnatant, blow-drying with nitrogen, re-dissolving with isopropyl alcohol, and filtering to obtain a to-be-tested solution; evaluating the salt tolerance of wolfberry according to a type number and/or a content of lipid molecules with significant changes in content, the fewer the type number of lipid molecules with significant changes in content, the less the change in content, the higher the salt tolerance of wolfberry.

Clause 2. The method for evaluating salt tolerance of wolfberry according to clause 1, wherein, in the culture medium with NaCl, a concentration of NaCl is 100~200 mmol/L.

Clause 3. The method for evaluating salt tolerance of wolfberry according to clause 1, wherein, in the culture medium with NaCl, a concentration of NaCl is 150 mmol/L.

Clause 4. The method for evaluating salt tolerance of wolfberry according to one of clauses 1-3, wherein, performing the culture in a greenhouse for 21 days, with a photoperiod is 10h light and 14h dark, a temperature is 25°C, a humidity is 50~70%.

Clause 5. The method for evaluating salt tolerance of wolfberry according to one of clauses 1-3, wherein, using a t-test to determine types of lipid molecules with significant changes in content in the experimental group compared with the control group.

Clause 6. The method for evaluating salt tolerance of wolfberry according to one of clauses 1-3, wherein, the method further comprises: taking leaves of the seedlings after culturing in the control group and the experimental group, detecting a content of abscisic acid and/or jasmonic acid in the leaves; evaluating the salt tolerance of wolfberry according to the significance of changes in the content of abscisic acid and/or jasmonic acid in the leaves of the experimental group compared with the control group, the higher the content of abscisic acid and/or jasmonic acid, the higher the salt tolerance of wolfberry.

Clause 7. The method for evaluating salt tolerance of wolfberry according to clause 6, wherein, a method for detecting the content of abscisic acid and/or jasmonic acid in the leaves, further comprises: (1) taking leaves and adding liquid nitrogen to grind into uniform powder; (2) adding an internal standard into the uniform powder obtained in step (1), adding an extraction reagent to obtain an extracting solution;

(3) concentrating, re-dissolving and filtering the extracting solution obtained in step (2) to obtain a to-be-tested solution; (4) detecting the to-be-tested solution obtained in step (3) by using an ultra-high performance liquid chromatography-mass spectrometry method, determining the content of abscisic acid or jasmonic acid by an internal standard method.

Claims (7)

Conclusions A method for evaluating the salt tolerance of wolfberry, comprising: cutting wolfberry seedlings for evaluation in a culture medium without NaCl and in a culture medium with NaCl for cultivation, respectively as a control group and an experimental group; the culture medium is an MS culture medium, the MS culture medium comprises the following components: 4.23 g/L macro elements, 85.29 mg/L trace elements and 103.1 mg/L vitamin; taking leaves from the seedlings after cultivation in the control group and the experimental group, detecting a content of lipid molecules in the leaves, obtaining types of lipid molecules with significant changes of content in the experimental group as compared with the control group; the content of the lipid molecules in the leaves is detected by the following steps: (1) taking a powder of crushed leaves and adding it to a pre-warmed isopropyl alcohol, heating, adding a full-fat internal standard, a chloroform and an ultrapure water, vortexing , centrifuging and taking a first supernatant; (2) adding a mixture of chloroform and methanol containing BHT to a residual liquid after taking the first supernatant in step (1), vortexing, centrifuging and taking a second supernatant; (3) mixing the first supernatant and the second supernatant, adding KCl thereto, vortexing, centrifuging, taking subnatant, blow drying with nitrogen, redissolving with isopropyl alcohol and filtering to obtain a solution to be tested; evaluating wolfberry salt tolerance based on a type number and/or content of lipid molecules with significant changes in content, the less the type number of lipid molecules with significant changes in content, the less the change in content, the higher the salt tolerance of wolfberry. The method for evaluating the salt tolerance of wolfberry according to claim 1, wherein, in the culture medium containing NaCl, a concentration of NaCl is 100-200 mmol/L. The method for evaluating the salt tolerance of wolfberry according to claim 1, wherein in the culture medium containing NaCl, a concentration of NaCl is 150 mmol/L. A method for evaluating the salt tolerance of wolfberry according to any one of claims 1 to 3, wherein, carrying out the cultivation in a greenhouse for 21 days, with a photoperiod of 10 hours of light and 14 hours of darkness, a temperature of 250C, and a humidity 50-70%. A method for evaluating wolfberry salt tolerance according to any one of claims 1 to 3, wherein, using a t-test to determine types of lipid molecules with significant changes in content in the experimental group as compared to the control group. The method for evaluating wolfberry salt tolerance according to any one of claims 1 to 3, the method further comprising: taking leaves from the seedlings after cultivation in the control group and the experimental group, detecting a content of abscisic acid and/or jasmonic acid in the leaves; assessing the salt tolerance of wolfberry according to the significance of changes in the content of abscisic acid and/or jasmonic acid in the leaves of the experimental group compared to the control group, the higher the content of abscisic acid and/or jasmonic acid, the higher the salt tolerance of wolfberry. The method for evaluating the salt tolerance of wolfberry according to claim 6, wherein a method for detecting the abscisic acid and/or jasmonic acid content in the leaves further comprises: (1) taking leaves and adding liquid nitrogen to form grind a uniform powder; (2) adding an internal standard to the uniform powder obtained in step (1), adding an extraction reagent to obtain an extraction solution; (3) concentrating, redissolving and filtering the extraction solution obtained in step (2) to obtain a solution to be tested; (4) detecting the solution to be tested obtained in step (3) by an ultra-high performance liquid chromatography-mass spectrometry method, wherein the content of abscisic acid or jasmonic acid is determined by an internal standard method.