Introduction

Low temperature stress is one of the most important factors that harm plant growth (Li et al. 2015). Tibet is located on the Qinghai-Tibet Plateau with an average altitude of more than 4000 m. It is known as the "roof of the world" and the "third pole" of the earth. Low temperature is one of the most important factors restricting plant growth (Miao et al., 2008). Under such climatic environment, currently the artificial grasses planted in Tibet are mainly gramineous species with strong stress resistance. Leguminous grasses have only a few varieties such as alfalfa and arrowhead pea, and the area is small (Cao et al., 2007). In addition, due to climate change and overloaded grazing, Tibet's natural grassland has been degraded, desertified, and soil erosion (Cao et al., 2007). Leguminous forages not only have higher nutritional quality, but also can improve soil fertility (Ma et al., 2019). Therefore, the selection of leguminous forages with strong cold resistance is essential to solve the above problems.

Melilotus Mill plants are annual or perennial legumes (Leguminosae) herbaceous plants. In my country, the sweet clover plants are mainly biannual. There are 3 species distributed in Tibet, namely Melilotus indicus and Melilotus indicus (M. officinalis) and white-flowered clover (M. albus), both of which are wild species, are mainly born in water ditches, grasslands or farmlands at an altitude of 2650～3700m. They are distributed in Baxiu, Linzhi, Milin, Central Tibet and Eastern Tibet. Zedang and other counties have almost no distribution in Nagqu and Ali (Ma et al., 2019). Melilotus not only contains high crude fat and crude protein, it is a good feed for feeding livestock, but also can inhibit the growth of weeds, increase soil fertility, adjust physical and chemical properties and improve soil structure. It is used to transform low-yield fields, control saline-alkali soils, and protect against wind. Ideal and fine grass for sand fixation and soil and water conservation (Ma et al., 2019; Tian et al., 2019).

A large number of research results indicate that changes in physiological indicators such as SS content, Pro content, MDA content, POD activity and SOD activity in plants are related to the cold resistance of plants, such as Fu Juanjuan (Fu, 2017), Zhang et al. (2016) and other studies found that the accumulation of higher soluble sugar and proline under low temperature stress can improve the low temperature tolerance of wild Elymus nutans in Tibet; Wen Riyu (Wen et al., 2019) and others believe that POD, SOD, etc. Enzymes participate in the process of removing reactive oxygen species and weaken their own damage to enhance the plant's resistance to low temperature stress. At present, there are few reports on the study of wild sweet clover in Tibet, most of which are about agronomic performance and cultivation management, and there is no research on its cold tolerance. Therefore, this experiment used Tibet's wild sweet clover and white clover as materials, studied the effects of low temperature stress on the seed germination and seedling physiological characteristics of the two types of sweet clover, and conducted a comprehensive evaluation of cold resistance, hoping to be the local wild clover The development and utilization of germplasm resources provide a scientific basis.

1 Results and Analysis

1.1 The effect of low temperature stress on seed germination characteristics

As the initial stage of plant life history, seed germination is of great significance to plant survival. Under low temperature stress, the quality of plant seed germination is an important index to measure the adaptability of the plant to low temperature environment. It can be seen from Table 1 that under 4℃, the germination rate, germination vigor, germination index and vigor index of the tested two materials, as well as the growth of radicle and embryo, all had a significant inhibitory effect (p<0.05). At 25℃ and 4℃, the germination indexes of Melilotus officinalis are higher than that of Melilotus officinalis, and the radicle and embryo grow faster than that of Melilotus officinalis, indicating that the seed quality of Melilotus officinalis is better under natural conditions. Good, and from the point of view of seed germination characteristics, Melilotus officinalis has stronger low temperature resistance.

Table 1 Effect of low temperature stress on seed germination characteristics Note: The difference between different lowercase letters of the same test material at different temperature treatments is significant (p <0.05), the same below

1.2 The influence of low temperature stress on the content of malondialdehyde

Under low temperature stress conditions, the change of MDA content can reflect the degree of peroxidation of plant membrane lipids. The higher the MDA content, the more severe the damage to the cell membrane. The results in Figure 1 show that with the intensification of low temperature stress, the MDA content of the two tested materials showed an increasing trend, but the magnitude of the change was different. Under 5℃ stress, the MDA content of the two materials increased, but both were not significantly different from the control (p>0.05); under 0℃ stress, the MDA content of M. officinalis was significantly higher than the control (p<0.05). It shows that the cell membrane of its leaves has begun to be damaged, and the difference of M.albus is not significant (p>0.05); under -5℃ stress, the MDA content of the two materials are significantly higher than the control (p<0.05); under -10℃ stress Below, the MDA content of the two materials reached the maximum treatment level. It can also be seen from the figure that under each temperature treatment, the MDA content of M. officinalis is higher than that of M. albus, and it is already significantly higher than that of the control under 0℃ stress (p<0.05), so from the indicator of MDA Look, M. albus have stronger cold resistance.

Figure 1 The effect of low temperature stress on the content of malondialdehyde in leaves

1.3 The effect of low temperature stress on soluble sugar content

It can be seen from Figure 2 that as the temperature decreases, the SS content of the two materials slowly increases and then decreases. Under the stress of 5℃, the SS content of M. albus was significantly higher than that of the control (p<0.05), and M. officinalis was also higher than that of the control, but the difference was not significant (p>0.05), and the content of M. albus increased significantly, which was 5.3. %; Under 0℃ stress, the SS content of the two materials was significantly higher than that of the control (p<0.05); under -5℃ stress, the SS content of the two materials continued to increase and reached the maximum of each treatment level , M. officinalis is 0.136μg·g-1, and M. albus is 0.145μg·g-1; under the stress of -10℃, the SS content of the two materials decreased, and there was no significant difference from the control (p> 0.05). In general, under the conditions of low temperature stress, the soluble sugar content in the leaves of M. albus increased faster, so on the index of SS, M. albus also showed stronger cold tolerance.

Figure 2 The effect of low temperature stress on the soluble sugar content in leaves

1.4 The influence of low temperature stress on proline content

It can be seen from Figure 3 that as the temperature decreases, the Pro content in the blades of the two materials increases first and then decreases. Under the stress of 5℃, the Pro content of M. officinalis was significantly higher than that of the control (p<0.05), and the content of M. albus was also higher than that of the control, but the difference was not significant (p>0.05); The content of Pro was significantly higher than that of the control (p<0.05); under the stress of -5℃, the content of Pro of the two materials were significantly higher than that of the control (p<0.05), and reached the maximum value of each treatment level. M. officinalis is 152.30 μg·g-1, and M. albus is 123.26μg·g-1; under -10℃ stress, the SS content of both materials decreased. Compared with -5℃, the decrease range was larger, and compared with The control difference was not significant (p>0.05). It can also be seen from the figure that during the entire low temperature stress process, the Pro content of the two materials increased significantly, and the Pro content of M. officinalis was higher than that of M. albus at all treatment levels.

Figure 3 The effect of low temperature stress on the content of proline in leaves

1.5 The influence of low temperature stress on peroxidase activity

It can be seen from Figure 4 that with the intensification of low temperature stress, the POD activity of the two test materials showed a trend of first increasing and then decreasing. Under 5℃ stress, the POD activities of the two materials were higher than the control, but the difference was not significant (p>0.05); under 0℃ stress, the POD activities of the two materials were significantly higher than the control (p<0.05), yellow The increase of M. officinalis was 22.36%, and that of M. albus was 31.57%; under -5℃stress, the Pro content of the two materials was significantly higher than that of the control (p<0.05), and reached the maximum of each treatment level. M. officinalis was 161.1 U·g-1·min-1, an increase of 32.73%, and M. albus was 216.1 U·g-1·min-1, an increase of 24.4%; under -10℃ stress, the SS content of the two materials Both of them dropped significantly, M. officinalis was 69.77 U·g-1·min-1, and M. albus was 73.83 U·g-1·min-1, and both were lower than the control.

Figure 4 Effect of low temperature stress on peroxidase activity

1.6 The effect of low temperature stress on the activity of superoxide dismutase

It can be seen from Figure 5 that with the intensification of low temperature stress, the SOD activity of the two test materials also increased first and then decreased. Under 5℃ stress, the POD activities of the two materials were higher than those of the control, but the difference was not significant (p>0.05); under 0℃ stress, the POD activity of M. albus was significantly higher than that of the control (p<0.05). At 16.87%, the POD activity of M. officinalis was also higher than that of the control, but the difference was not significant (p>0.05); under -5℃ stress, the Pro content of the two materials was significantly higher than that of the control, and reached the level of each treatment. The maximum value is 118.12 U·g-1 of M. officinalis, an increase of 19.78%, and M. albus is 134.46 U·g-1, an increase of 20.09%. Under -10℃ stress, the SS content of the two materials decreased, But all higher than the control.

Figure 5 Effect of low temperature stress on superoxide dismutase activity

1.7 Comprehensive evaluation of cold tolerance

Plant cold tolerance is a comprehensive trait controlled by multiple indicators. Evaluation using a single indicator has a certain degree of one-sidedness. Therefore, the comprehensive evaluation of multiple indicators can reflect the scientificity and accuracy of the results. In order to more accurately evaluate the cold resistance of the four forage grasses and reduce the judgment error of the experiment, this experiment calculated the membership function values of the five physiological indexes and four seed germination indexes of the two materials to obtain the average membership function value (Table 2), and then intuitively evaluate the cold resistance of the two test materials. Because the indexes of the two materials are quite different under the condition of 0℃ in this study, the indexes measured at 0℃ are used as the actual measured values for comprehensive evaluation of their cold resistance in seedling stage. The results show that the white flowers and clover has stronger cold resistance.

Table 2 Subordinate degree analysis of different measurement indexes of Melilotus

2 Discussion

2.1 The relationship between temperature and plant seed germination

Seed germination is the beginning of plant life history, and suitable temperature is one of the most important environmental factors it needs. Under low temperature conditions, whether seeds can germinate normally is the basis for studying the adaptability of plants to low temperature environments, and also for evaluating their cold resistance One of the important indicators of sex. The results of this experiment showed that the seed germination rate, germination vigor, germination index, vigor index, as well as the radicle and embryo length of the two tested materials were significantly lower than ck under the low temperature condition of 4℃, indicating that low temperature stress has an effect on the test. The material's seed germination and seedling growth have a certain inhibitory effect. This is basically consistent with the research reports of He Jiayuan (He Jiayuan et al., 2013), Zhao Yangjia (Zhao Yangjia et al., 2019) and others. Seed germination refers to the process in which mature seeds gradually form seedlings through a series of physiological and biochemical reactions under appropriate conditions (Han Jianguo, 2011). From this perspective, one of the reasons why low temperature stress inhibits seed germination may be that it hinders the activation of enzymes in plant seed cells, preventing a series of physiological reactions inside the seed, thereby reducing the germination rate.

2.2 The relationship between physiological characteristics and plant cold resistance

Under low temperature stress conditions, plants will accumulate a large number of active oxygen free radicals in their bodies, which will cause membrane lipid peroxidation and produce a large amount of membrane lipid peroxidation product MDA. The changes in MDA content can reflect the degree of peroxidation of plant membrane lipids. The higher the MDA content, the more severe the damage to the cell membrane, which can lead to cell death (Han Jianguo, 2011). Therefore, the change of MDA content is one of the important indicators for judging the degree of damage to plants by low temperature stress. The results of this experimental study showed that with the decrease of temperature, the MDA content of the two test materials showed an increasing trend, indicating that as the degree of low temperature stress intensified, the damage to their cell membranes continued to deepen. This is consistent with the findings of Zhang (2019), Chi et al. (2019) and others. Under low temperature stress conditions, a series of physiological and biochemical changes occur in plants to adapt to the low temperature environment, among which osmotic adjustment is one of the most important physiological mechanisms of cold resistance in plants (Han, 2011). Soluble sugar and proline are the main osmotic regulators in cells. Both can alleviate or resist the occurrence of low temperature damage under low temperature stress, and their content can reflect the ability of plants to adapt to low temperature environments. In this experiment, with the decrease of temperature, the contents of SS and Pro in the leaves of the two materials increased first and then decreased, indicating that under a certain range of low temperature treatment, the contents of soluble sugar and proline can be increased by the two materials. Respond to low temperature stress. However, when the temperature drops to -10℃, its content is greatly reduced, indicating that the cell structure of its leaves has been destroyed under the condition of -10℃, and normal physiological and biochemical reactions cannot be carried out to deal with the stress.

Low temperature stress disrupts the balance of active oxygen metabolism in plant leaf cells, resulting in excess active oxygen, attacking the membrane system, and harming cells (Han, 2011). Therefore, enhancing the ability of scavenging active oxygen is also one of the important physiological mechanisms for plants to adapt to low temperature environments. A large number of research results show that SOD and POD play an important role in the process of plant protection against low temperature stress. The higher the activity, the stronger the cold resistance of plants (Zhang et al., 2017). The results of this experiment showed that with the decrease of temperature, the leaves of the two materials increased first and then decreased, but the degree of change of different materials was different. Under different treatments, the activities of the two enzymes of Melilotus officinalis were higher than Melilotus officinalis. It shows that under low temperature stress conditions, Melilotus officinalis has a stronger ability to scavenge active oxygen and has stronger cold resistance, which is consistent with the results of the comprehensive evaluation of cold resistance.

2.3 Comprehensive evaluation of cold tolerance

The cold resistance of plants is a complex trait that is affected by many factors, and the identification of a single index has a certain degree of one-sidedness. Therefore, this study adopts the membership function method to analyze the membership function of the two test materials' SS content, Pro content, MDA content, POD activity, SOD activity and other physiological indicators and seed germination indicators to evaluate the cold resistance of the two materials. Based on a comprehensive evaluation, the results show that the cold resistance of white flowers and trees is higher than that of Melilotus officinalis.

3 Materials and Methods

3.1 Test materials

The wild materials for testing were collected from Dazi County, Lhasa City in September 2011. The collected seeds were air-dried and stored in a refrigerator at 4℃.

3.2 Test method

3.2.1 Seed germination test

The experiment was carried out in the Plateau Crop Cultivation and Cultivation Laboratory of Tibet Agriculture and Animal Husbandry University in September 2019. The paper germination method was used to select sweet clover seeds with full particles and the same size. The surface of the seeds was disinfected with 1% NaClO for 10 minutes. Rinse with distilled water, and after natural drying, place the seeds in a germination box with two layers of filter paper. The seed germination test was carried out in an artificial climate box at 4℃. Set light for 12h, dark for 12h, humidity throughout the day at 75%, temperature at 4℃ (low temperature stress) and 25℃ (control), 3 replicates for each treatment, and 50 seeds for each replicate. Change the distilled water every day during the germination period to keep the filter paper moist. After the seeds were placed in the artificial climate box for 24 hours, the number of germination of the seeds was observed and recorded every day. The radicle length reached half of the seed length as a sign of seed germination, and moldy seeds were removed every day.

The calculation formula of each index is as follows (Wang et al., 2016; Jia et al., 2020):

Germination rate of seeds (GR)=(n/N)×100%, where n is the number of seeds that will germinate normally, and N is the number of trial seeds;

Germination potential (GF)=the number of germination of seeds during the peak period of germination/total number of tested seeds×100%;

Germination index (GI)=∑(Gt/Dt) where: Gt is the number of seedlings on the t day; Dt is the corresponding number of seedlings;

Vigor index (VI)=GI×S; where S is the average shoot length.

3.2.2 Physiological test

Set 5℃, 0℃, -5℃, and -10℃ 4 treatment levels, with 25℃ as the control. The test materials used potting method to raise seedlings, choose 42×42×6 cm seedling trays, the cultivation substrate was mixed according to the field soil: dry sheep dung=9:1 ratio, each treatment was 3 repetitions, each repetition planted 1 plate, each plate was even Sow 200 seeds. The seedling raising stage is carried out in the glass greenhouse of the farm practice base of the Academy of Plant Sciences of Tibet Agriculture and Animal Husbandry College. When the seedlings grow to 5 leaf age, each treatment group is put into a high and low temperature alternating test box (KLT-4050) for low temperature stress treatment 12h, set the temperature of the high and low temperature alternating test box to 5℃, 0℃, -5℃ and -10℃, and put the control group in the 25℃ incubator for culture. After the treatment, an appropriate amount of leaves were taken to determine the physiological indicators.

The determination of plant leaf physiological indicators refers to the method in Zhang Zhiliang's "Plant Physiology Test Guide" (Zhang Zhiliang et al., 2009): The determination of malondialdehyde (MDA) uses thiobarbituric acid (TBA) color method, soluble sugar (SS) ) Using anthrone method, proline (Pro) by ninhydrin method, superoxide dismutase (SOD) activity by nitroblue tetrazolium (NBT) photochemical reduction method, peroxidase (POD) activity by guaiac Wood phenol method.

3.3 Comprehensive evaluation of cold resistance

The membership function method of fuzzy function is used to comprehensively evaluate the test materials (Zhao et al., 2020).

The calculation formula of membership function value: X(μ)=(X-Xmin)/(Xmax-Xmin);

Inverse membership function value calculation formula: X(v)=1-X(μ)

In the formula, X represents the measured value of the index, and Xmax and Xmin respectively represent the maximum and minimum values of a certain index of all test materials. If one of the indexes is negatively correlated with the cold resistance of plants, the conversion of the anti-membership function is used to calculate it. Resistance membership function value.

3.4 Data processing

DPS6.0 statistical analysis software was used for analysis of variance, the significance of differences between samples was tested by Duncan's new multiple range method, and Excel2016 software was used for graphing.

Authors’ contributions

Jia Xiang is the executive of the experimental design and experimental research of this research; Jia Xiang completed the data analysis and the writing of the first draft of the paper. Wang Xiangtao, Bao Saiyana and Wang Mingtao participated in the experimental design and analysis of the experimental results; Miao Yanjun was the project creator and the person in charge, guide experimental design, data analysis, thesis writing and revision. All authors read and approved the final manuscript.

Acknowledgements

This research was jointly funded by the key project of the Tibet Autonomous Region Department of Science and Technology (No. XZ201901NA03) and the Tibet Alpine Fine Forage Germplasm Resource Selection Platform (No. 533320005).

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