The paper below presents a protocol for measuring seed germination, seedling growth, and physiological indexes of two pepper varieties with salinity tolerance differences in response to six mixed salt concentrations. This protocol can be used to evaluate the salt tolerance of pepper varieties.
To determine the salt tolerance and physiological mechanism of pepper (Capsicum annuum L.) at the germination stage, the Hongtianhu 101 and Xinxiang 8 varieties, which have large differences in salt tolerance, are employed as the study materials. Six mixed salt concentrations of 0, 3, 5, 10, 15, and 20 g/L derived using equal molar ratios of Na2CO3, NaHCO3, NaCl, CaCl2, MgCl2, MgSO4, and Na2SO4 are used. To determine their effects, the related indexes of seed germination, seedling growth, and physiology are measured, and salt tolerance is comprehensively evaluated using membership function analysis. The results show that as the mixed salt concentration increases, the germination potential, germination index, germination rate, seed germination vigor index, root length, and root fresh weight of the two cultivars significantly decrease, whereas the relative salt rate gradually increases. The hypocotyl length and fresh weight aboveground increase first and then decrease, while the malondialdehyde (MDA), proline (Pro) content, catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) activity decrease and then increase. The germination potential, germination index, germination rate, seed germination vigor index, root length, root fresh weight, MDA and Pro content, and CAT activity of the Hongtianhu 101 seeds are higher than those of Xinxiang 8 for all salt concentrations employed here. However, hypocotyl length, fresh weight aboveground, and relative salt rate are lower in Hongtianhu 101 than in Xinxiang 8. The comprehensive evaluation of salt tolerance reveals that the total weighted values of the two membership function indexes increase first and then decrease as the mixed salt concentration increases. Compared with 5 g/L, which has the highest membership function value, the index under salt concentrations of 3 g/L, 10 g/L, and 15 g/L decreases by 4.7%-11.1%, 25.3%-28.3%, and 41.4%-45.1%, respectively. This study provides theoretical guidance for the breeding of salt-tolerant varieties of pepper and an analysis of the physiological mechanisms involved in salt tolerance and salt-tolerant cultivation.
Salinity is a major limiting factor for crop productivity worldwide1. At present, nearly 19.5% of the world's irrigated land and 2.1% of dry land are affected by salinity, and approximately 1% of agricultural land degenerates into saline-alkali land every year. By 2050, 50% of arable land is expected to be affected by salinization2,3. In addition to natural factors, such as natural rock weathering and salty rainwater near or around the coast, rapid surface evaporation, low rainfall, and unreasonable agricultural management methods have exacerbated the process of soil salinization. Soil salinization inhibits the growth of plant roots and reduces the absorption and transportation of water and nutrients from the plant roots to the leaves. This inhibition results in physiological water shortages, nutritional imbalances, and ion toxicity, which lead to reduced crop productivity and a complete loss of crop yield. The salinization of cultivated land is gradually becoming one of the most critical abiotic stress factors affecting global agricultural food production4. Salt stress reduces the arable land available for agriculture, which may result in a significant imbalance between the supply and demand of future agricultural products. Therefore, exploring the effects of soil salinization on crop growth and physiological and biochemical mechanisms is conducive for breeding salt-tolerant varieties, the sustainable utilization of saline soil, and the safety of agricultural products.
Pepper (Capsicum annuum L.) is planted worldwide owing to its high nutritional and medicinal value. For example, capsaicin is an alkaloid responsible for the spicy flavor of pepper. Capsaicin can be used for pain relief, weight loss, improving cardiovascular, gastrointestinal tract, and respiratory systems, and in several other applications5. Pepper is also rich in bioactive substances, especially different antioxidant compounds (carotenoids, phenolics, and flavonoids) and vitamin C6. Currently, pepper is reported to be the vegetable crop with the largest cultivation area in China, with an annual planting area of more than 1.5 x 106 ha, thereby accounting for 8%-10% of the total vegetable planting area in China. The pepper industry has become one of the largest vegetable industries in China and has the highest output value7. However, pepper cultivation is often subjected to a variety of biological (pests and fungi) and abiotic stresses, especially salt stress, which has a direct negative impact on seed germination, growth, and development, resulting in the reduction of pepper fruit yield and quality8.
Seed germination is the first stage of interaction between plants and the environment. Seed germination is highly sensitive to fluctuations in the surrounding media, especially soil salt stress, which may exert reversed effects on physiology and metabolism, and eventually disorder the normal growth, development, and morphogenesis of crops9. In previous studies, pepper seed germination and seedling growth under salt stress were extensively investigated; however, most studies used NaCl as the only salt for stress induction10,11,12. However, soil salt damage is mainly due to Na+, Ca2+, Mg2+, Cl–, CO32-, and SO42- ion toxicity generated by the dissociation of sodium, calcium, and magnesium salts. Owing to the synergy and antagonism between ions, the effects of mixed salt and single salt on crop growth and development may be quite different. However, the corresponding characteristics of pepper seed germination and growth in mixed salt are still unclear. Therefore, two pepper varieties with remarkable differences in salt tolerance are used as materials in this study. Analyzing the effects of different salt concentrations on pepper seed germination, growth, and physiological and biochemical indexes after equimolar mixing of seven salts can reveal the response mechanism of pepper seed germination to salinity stress. It can also provide a theoretical basis for cultivating strong pepper seedlings, as well as high yield and high-quality cultivation in saline cultivated land.
NOTE: Here, we present a protocol for assessing the response characteristics and internal mechanisms of pepper seed germination and seedling growth under different mixed salt stresses, which can serve as a reference method for seed salt tolerance evaluation.
1. Experimental preparation
2. Seed soaking and preparation for germination
3. Seed germination and seedling growth
4. Measurement and calculation of indicators
Seed germination characteristics
As the mixed salt concentration increases, the germination potential and germination index of Hongtianhu 101 and Xinxiang 8 decreases significantly. Both cultivars have a sharp decline in salt concentrations from 0-3 g/L, and a slow and steady decline for salt concentrations from 3-20 g/L (Figure 1A,B). The germination rate of the two varieties gradually decreases as the mixed salt concentrations increase, and the relative salt rate for the varieties gradually increases. There is no significant difference in the germination rate and relative salt rate at salt concentrations of 3-15 g/L. However, the difference is significant at all other salt concentrations (Figure 1C,D). Regarding salt tolerance between the two varieties, the germination potential, germination index, and germination rate of Hongtianhu 101 seeds with increasing mixed salt concentrations are higher than those of Xinxiang 8, whereas the relative salt rate is lower than that of Xinxiang 8.
As the mixed salt concentration increases (0-15 g/L), the seed germination vigor index of the two varieties decreases significantly. When the mixed salt concentration is 15 g/L, the seed germination vigor index of Hongtianhu 101 and Xinxiang 8 decreases by 91.0% and 94.6%, respectively, compared with that of the control. Of note, the decrease is not significant when the mixed salt concentration further increases from 15 to 20 g/L. The seed germination vigor index of Hongtianhu 101 is higher than that of Xinxiang 8 at each mixed salt concentration level (Figure 1E).
Seedling growth characteristics
As the mixed salt concentration increases (0-15 g/L), the root length and root fresh weight of Hongtianhu 101 and Xinxiang 8 decrease significantly. When the mixed salt concentration is 15 g/L, the root length of Hongtianhu 101 and Xinxiang 8 decreases by 89.4% and 91.1%, respectively, and the root fresh weight decreases by 81.7% and 71.2%, respectively, compared to those of the control. However, when the salt concentration is 15-20 g/L, the root length and root fresh weight of the two varieties does not change significantly (Figure 2A,C). The root length and root fresh weight of Hongtianhu 101 are generally higher than those of Xinxiang 8 with increasing mixed salt levels, with obvious differences at concentrations ranging from 0 to 10 g/L.
The hypocotyl length and fresh weight aboveground of the two varieties increase and then decrease with increasing mixed salt concentration. Both indexes reach their highest values at a salt concentration of 5 g/L. Similarly, when the salt concentration is 15-20 g/L, the hypocotyl length and fresh weight aboveground of the two varieties decrease slightly. Based on the varietal differences, the hypocotyl length and fresh weight aboveground of Xinxiang 8 are higher than those of Hongtianhu 101 at each salt concentration (Figure 2B,D).
Membrane lipid peroxidation and osmotic adjustment substance content
As the mixed salt concentration increases, the MDA and Pro contents of the two varieties decrease and then increase. The MDA and Pro contents reach their lowest values at concentrations of 5 g/L and 3 g/L, respectively (Figure 3A,B). The MDA content decreases slightly at salt concentrations of 0-5 g/L and increases rapidly at 10 g/L. Compared with the 5 g/L treatment, the MDA content following the 10 g/L treatment increases by 59.9%-64.8%, and then remains unchanged. The MDA content of Hongtianhu 101 is higher than that of Xinxiang 8 at different salt concentrations (Figure 3A). The decrease in Pro content at 0-3 g/L is not significant and little difference is found between the two varieties. When the salt concentration is 3-15 g/L, the Pro content of Xinxiang 8 slowly increases and remains relatively stable, whereas the Pro content of Hongtianhu 101 increases rapidly. Compared to 3 g/L, the Pro content of Hongtianhu 101 significantly increases by 440.2% at 15 g/L (Figure 3B).
Protective enzyme activity
As the mixed salt concentration increases, the CAT, POD, and SOD activities of the seedlings of Hongtianhu 101 and Xinxiang 8 decrease and then increase, with the lowest values obtained at a concentration of 3 g/L (Figure 4A-C). The CAT and POD activities of the two varieties vary slightly at salt concentrations of 0-5 g/L, and the difference between them is small. Thereafter, the CAT and POD activities of the two varieties increase significantly with increasing salt concentration. Further, the CAT and POD activities of Hongtianhu 101 are higher than those of Xinxiang 8, and the difference between them increases gradually (Figure 4A,B). The SOD activity of the two varieties varies slightly at salt concentrations from 0-10 g/L, and then increases rapidly. The SOD activity of Xinxiang 8 is higher than that of Hongtianhu 101 at salt concentrations from 0-10 g/L; for the remaining concentrations, its activity is lower than that of Hongtianhu 101 (Figure 4C).
Correlation analysis of the seed germination indexes of pepper and comprehensive evaluation of salt stress
Correlation analysis (Table 1) reveals that the root length and root fresh weight for the seedling growth characteristic indexes are significantly positively correlated with the germination indexes (germination potential, germination rate, germination index, seed germination vigor index, etc.); however, no explicit relevance is found between the hypocotyl length, fresh weight aboveground, and germination indexes. A significant negative correlation is found between the indexes of seedling growth characteristics and the activity of protective enzymes (CAT, POD, and SOD). A significant negative correlation is also found between the hypocotyl length and the contents of MDA and Pro, and between the fresh weight of the shoot and the MDA content. With exception to the significant correlation between seed germination vigor index and protective enzyme activity, no significant correlation is found between the germination characteristic indexes and seedling physiological indexes (protective enzyme activity and MDA and Pro content).
The salt tolerance of the two pepper varieties under compound salt stress is evaluated using the membership function method for multiple traits. As the pepper seedlings are not subjected to salt stress when the mixed salt concentration is 0 g/L, their membership function value is not calculated. As a result, only the treatment with salt stress is assessed using membership function analysis. MDA is found to be negatively correlated with the salt tolerance of pepper seedlings and is calculated using the inverse membership function method; other indexes are calculated using the membership function method. Table 2 shows that as the mixed salt concentration increases, the total weighted values of each index function of the two varieties increase and then decrease, ultimately reaching a maximum at a salt concentration of 5 g/L. Compared with the 5 g/L treatment, the values obtained with the 3 g/L, 10 g/L, and 15 g/L salt concentration treatments decrease by 4.7%-11.1%, 25.3%-28.3%, and 41.4%-45.1%, respectively. Therefore, the salt tolerance of pepper subjected to 5 g/L, 3 g/L, 10 g/L, and 15 g/L salt concentration treatments can be categorized as best, second best, bad and worst, respectively.
Figure 1: Effects of increasing mixed salt concentrations on seed germination of pepper. (A), (B), (C), (D), and (E) represent the response characteristics of pepper seed germination potential, germination index, germination rate, relative salt rate, and seed germination vigor index to compound salt stress, respectively. Different lowercase letters in the figure indicate significant differences between treatments, which are analyzed by the Tukey's multiple range test (p < 0.05). Error bars indicate standard deviations (n = 5). Please click here to view a larger version of this figure.
Figure 2: Effects of increasing mixed salt concentrations on seed germination and morphological indexes of pepper. (A), (B), (C), and (D) represent the response characteristics of pepper seedling root length, hypocotyl length, root fresh weight, and fresh weight aboveground to compound salt stress, respectively. Different lowercase letters in the figure indicate significant differences between treatments, which are analyzed by the Tukey's multiple range test (p < 0.05). Error bars indicate standard deviation (n = 5). Please click here to view a larger version of this figure.
Figure 3: Effects of increasing mixed salt concentrations on MDA and Pro content of pepper seedlings. (A) and (B) represent the response characteristics of pepper seedling MDA and Pro content to compound salt stress, respectively. Different lowercase letters in the figure indicate significant differences between treatments, which are analyzed by the Tukey's multiple range test (p < 0.05). Error bars indicate standard deviation (n = 3). Please click here to view a larger version of this figure.
Figure 4: Effects of increasing mixed salt concentrations on CAT, POD, and SOD activities of pepper seedlings. (A), (B), and (C) represent the response characteristics of pepper seedling CAT, POD, and SOD activities to compound salt stress, respectively. Different lowercase letters in the figure indicate significant differences between treatments, which are analyzed by the Tukey's multiple range test (p < 0.05). Error bars indicate standard deviation (n = 3). Please click here to view a larger version of this figure.
Table 1: Correlation analysis between pepper germination and physiological indicators under compound salt stress (n = 30). Pearson's correlation analysis is used to investigate the correlation between seed germination and seedling physiological indexes of pepper under compound salt stress. * p < 0.05; ** p < 0.01. Please click here to download this Table.
Table 2: The weighted membership function value of pepper germination and seedlings' physiological indicators under mixed salt stress. Please click here to download this Table.
This research method comprises four key steps that affect the accuracy of the experimental results. First, owing to the poor dissolution of mixed salts caused by the increased solute content in high salt concentration solutions, and the low solubility of reagents such as calcium chloride, which are more difficult to solubilize in water, the weighed reagents must be fully ground in a mortar. Further, the reagents must be dissolved via ultrasonic waves before determining the capacity. Second, the configured salt solution must be completely shaken each time and added to the Petri dish for use. Third, the Petri dishes must retain a suitable water layer after addition of the salt solution, and the water status of each Petri dish must be relatively consistent. Finally, light conditions must be consistent after seed germination.
In this study, the number of test seeds in a single Petri dish can be adjusted by varying the diameter of the selected Petri dish. According to the specific situation of soil salt stress in different planting areas, the proportion of each single salt addition could be adjusted to enable consistency with the actual situation of salt stress in the local soil. While this method is practical, for smaller seeds (such as rape, ice plant, and amaranth) or larger seeds (such as sword beans and broad beans), problems such as operational difficulties in the determination of seedling length and fresh weight, or the availability of very few seeds in a single culture dish to repeat the data, lead to difficulties in studying salt tolerance using this method.
The method described here is used to determine the seed germination characteristics and seedling growth under different mixed salt concentrations and reveal the change mechanism through internal physiological enzyme activity, which is of great significance for objectively evaluating the salt tolerance characteristics of seeds. This technology can provide a technical reference for salt tolerance evaluation of other crops. Seed germination and seedling growth are the stages in which crops are most sensitive to salt stress. Thus, this method can effectively provide a reference for salt-tolerant cultivation and crop breeding.
In most crops, salt stress can inhibit seed germination and seedling growth under biotic stress. Such inhibition may be due to a reduction in seed water uptake by reducing the osmotic potential of the salt solution under saline conditions. Salt ion stress and toxicity may change the activity of protective enzymes (POD, CAT, SOD, etc.) and protein metabolism during seed germination, and destroy endogenous hormone balance11,16. Zhani et al. reported that the germination process was mainly changed by a reduced germination rate and prolonged germination under salt (NaCl) stress. Further, a significant difference was found in the germination rate among different varieties (10%-50%) at a salt concentration of 8 g/L17. The present study also reveals that as the mixed salt concentration increases, the germination potential, germination index, germination rate, and seed germination vigor index of Hongtianhu 101 and Xinxiang 8 decrease significantly, and the relative salt rate increases gradually. According to Patanè et al., although salt stress prolonged the germination time of sweet sorghum seeds, increasing salt stress had an adverse effect on the final germination of seeds18. However, related studies suggest that NaCl treatment can promote pepper seed germination and growth19, which may be related to differences in salt concentration levels.
Increasing the level of salt stress has a significant effect on the growth of pepper seedlings, and based on this study, the root length and root fresh weight of the two varieties decreased significantly with increasing mixed salt concentrations. This finding is similar to that of Mirosavljević et al., who suggested that root length and root weight decreased as salt stress increased, and the differences among the treatments were significant20. This result indicates that the root system was placed in the soil medium in direct contact with the soil solution, and the root length and root weight were more sensitive to NaCl osmotic stress. Root length and root weight are key indicators of plant response to salt stress. According to this study, as salt concentration increases, the hypocotyl length and fresh weight of the aboveground parts increase and then decrease, with maximum values achieved at 5 g/L. Khan et al. also suggested that as salinity (NaCl) increases (0-9 ms/cm), the shoot length of pepper increases first and then decreases, with the best performance obtained at 3 ms/cm12,21. The conductivity of salt concentration in this study was 4.73 ms/cm when the value of seedling hypocotyl length and fresh weight aboveground were the highest, which is higher than the value reported by Khan et al.21. This result might be due to the high tolerance of pepper aboveground to compound salt stress compared with single salt stress.
Salt stress not only hinders crop growth but also causes significant physiological changes in plants. Salt stress can increase the levels of reactive oxygen species (ROS). If ROS are not cleared in time, membrane lipid peroxidation and oxidative stress, which can cause serious damage to the plant cell membrane, may occur. MDA is the final metabolite of membrane lipid peroxidation, and intracellular MDA concentration is often used as an indicator to assess the degree of damage to plants under stress22. In the present study, as the mixed salt concentration increases, the MDA content of the seedlings of the two varieties decreases first and then increases. Of note, the decrease is not significant at salt concentrations ranging from 0-5 g/L. However, a rapid increase is observed from 5-10 g/L. Thereafter, the values remain unchanged, indicating that the degree of membrane lipid peroxide of pepper seeds changes from general, to rapidly increasing, to stable. Salt stress is speculated to have a serious impact on cell membrane permeability when the mixed salt concentration is greater than 10 g/L. Guzmán-Murillo et al. reported a similar conclusion of a decrease and then an increase in the membrane lipid peroxide level in sweet pepper seedlings as the NaCl concentration increased (0-50 nmol/L). Further, the level of lipid peroxidation was lowest at 25 nmol/L NaCl23.
Plants have evolved several strategies for coping with salt stress. On the one hand, crops enhance protein stability and membrane integrity by increasing osmotic adjustment substances, such as proline, and reduce the loss of intracellular water, thereby improving salt tolerance24. In the present study, as the mixed salt concentration increases, the Pro content of Xinxiang 8 and Hongtianhu 101 seedlings decreases first and then increases. Notably, the decrease is not significant at concentrations of 0-3 g/L and the difference between the two varieties is not significant, aligning with the experimental results of Muchate et al25. The Pro content of the Hongtianhu 101 pepper variety with good salt tolerance also increases rapidly at salt concentrations of 3-15 g/L. Compared with 3 g/L, the Pro content of 15 g/L significantly increases by 440.2%, whereas the Pro content of Xinxiang 8 with general salt tolerance increases slowly and maintains a relatively stable level at salt concentrations of 3-15 g/L. The scavenging effect of the antioxidant enzymes on ROS induced by salt stress has proven to be the main component of crop defense mechanisms. The activities of CAT, POD, and SOD have been reported to increase under different salt-stress environments, thereby improving its salt tolerance25,26. Chen et al. showed that as NaCl concentration increases, the activities of SOD, POD, and CAT in tomato seed germination increases gradually, with no significant difference in each index following treatment with 0-50 nmol/L NaCl27. This study also shows that as the mixed salt concentration increases, the activities of CAT, POD, and SOD in the seedlings of Hongtianhu 101 and Xinxiang 8 decrease and then increase. At low salt concentrations (0-3 g/L), the activity of antioxidant enzymes does not change significantly, and a further increase in salt concentration improves salt tolerance.
The adaptability and response characteristics of crops to salt stress are mainly reflected in morphology, structure, physiological ecology, etc. A comprehensive evaluation of compound salt stress is carried out using the membership function value method for multiple traits. This study shows that as the mixed salt concentration increases, the total weighted value of the function value increases first and then decreases. The two varieties reach a maximum at 5 g/L, leading to the best salt tolerance effect. Compared with the 5 g/L treatment, the weighted values following treatment with the salt concentrations of 3 g/L, 10 g/L, and 15 g/L decrease by 4.7%, 25.3%, and 41.4%, respectively, for Hongtianhu 101, and 11.1%, 28.3%, and 45.1%, respectively, for Xinxiang 8. Such findings indicate that the salt tolerance of Hongtianhu 101 is higher than that of Xinxiang 8.
This study provides a comprehensive description of the effects of different concentrations of compound salt stress on pepper seed germination and its physiological enzyme activities, and a technical reference for research on salt tolerance in other crops. Pepper seedlings are less affected by salt stress under simulated mixed salt concentrations (0-5 g/L). Seed germination, radicle elongation, and seedling morphogenesis of pepper are significantly inhibited under high salt stress (>5 g/L), causing serious membrane lipid peroxidation in pepper seedlings. Crops reduce the adverse effects of salt stress by increasing their Pro content and enhancing the activity of protective enzymes (CAT, POD, and SOD). Under salt stress, the proline content and protective enzyme activity of Hongtianhu 101 seedlings are higher, the degree of membrane lipid peroxidation is lower, and seed germination and seedling growth are more obvious than those of Xinxiang 8.
The authors have nothing to disclose.
This work was supported by the Science and Technology Department of Jiangxi Province (20203BBFL63065) and the General Project of Science and Technology Research Project of Jiangxi Education Department (GJJ211430). We would like to thank Editage (www.editage.cn) for English language editing.
Calcium chloride | Shanghai Experiment Reagent Co., Ltd.,China | Analytical reagent | |
Centrifugal machine | Shanghai Luxianyi Centrifuge Instrument Co., Ltd., China | TGL-16M | |
Centrifuge tube | None | None | |
Conductivity meter | Shanghai Instrument&Electronics Science Instrument Co., Ltd., China | DDSJ-308F | |
Constant temperature and humidity box | Ningbo Laifu Technology Co., Ltd.,China | PSX-280H | |
Digital display vernier caliper | Deli Group Co., Ltd.,China | DL90150 | |
Electronic balance | Mettler Toledo Instruments (Shanghai) Co., Ltd.,China | ME802E/02 | |
Filter paper | Hangzhou Fuyang North Wood Pulp and Paper Co., Ltd.,China | GB/T1914-2017 | |
Grinding rod | None | None | |
Hongtianhu 101 | Seminis Seed (Beijing) Co., Ltd.,China | 11933955/100147K1-137 | |
Ice machine | Shanghai Kehuai Instrument Co., Ltd., China | IM150G | |
Liquid nitrogen | None | None | |
Magnesium chloride | Tianjin Kermel Chemical Reagent Co., Ltd.,China | Analytical reagent | |
Magnesium sulfate | Tianjin Kermel Chemical Reagent Co., Ltd.,China | Analytical reagent | |
Petri dish | Jiangsu Yizhe Teaching Instrument Co., Ltd.,China | I-000163 | |
Pocket knife | None | None | |
Potassium permanganate (KMnO4 | Xilong Scientific Co.,Ltd.,China | Analytical reagent | |
Pure water equipment | Sichuan Youpu Ultrapure Technology Co., Ltd.,China | UPT-I-20T | |
Sodium bicarbonate | Xilong Scientific Co.,Ltd.,China | Analytical reagent | |
Sodium carbonate | Xilong Scientific Co.,Ltd.,China | Analytical reagent | |
Sodium chloride | Xilong Scientific Co.,Ltd.,China | Analytical reagent | |
Sodium sulfate | Xilong Scientific Co.,Ltd.,China | Analytical reagent | |
Test kit | Suzhou Keming, Biotechnology Co., Ltd, Suzhou.,China | Spectrophotometer method | |
Ultra-low temperature freezer | SANYO Techno Solution TottoriCo.,Ltd. | MDF-382 | |
Ultraviolet visible spectrophotometer | Shanghai Precision Scientific Instrument Co., Ltd., China | 760CRT | |
Xinxiang 8 | Jiangxi Nongwang High Tech Co., Ltd.,China | GPD Pepper 2017(360013) |