Analysis of Effect of Compound Salt Stress on Seed Germination and Salt Tolerance Analysis of Pepper (Capsicum annuum L.)

Summary

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.

Abstract

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, MgSO_4, 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.

Introduction

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 salinization^2,3. 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 induction^10,11,12. However, soil salt damage is mainly due to Na⁺, Ca²⁺, Mg²⁺, Cl^-, CO₃^2-, and SO₄^2- 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.

Protocol

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

1. Prepare crop seeds for cultivars-Hongtianhu 101 with strong salt tolerance and Xinxiang 8 with low tolerance.
2. Prepare 0.2% KMnO₄ solution as a seed disinfection reagent. First, weigh 4.0 g of KMnO₄, and then add 2,000 mL of distilled water.
   NOTE: Potassium permanganate is usually unstable due to its strong oxidation; accordingly, it is prepared immediately before use.
3. Prepare the mixed salts using seven salts, including sodium carbonate, sodium bicarbonate, sodium chloride, calcium chloride, magnesium chloride, magnesium sulfate, and sodium sulfate¹³. Add the same molar amount of each, which successively account for 14.8%, 11.7%, 8.2%, 15.5%, 13.3%, 16.7%, and 19.8% of the total mass ratio of the mixed salts, respectively.
4. Prepare Petri dishes (single use) and filter paper (medium speed qualitative filter paper), both with a diameter of 9 cm.
   ​NOTE: The material of the Petri dish can be changed; however, the diameter of the Petri dish and filter paper must be the same.

2. Seed soaking and preparation for germination

1. For seed optimization, select pepper seeds with consistent size and full particles from each variety, with an average diameter of 4.2 mm and 3.7 mm for Hongtianhu 101 and Xinxiang 8 seeds, respectively. Calculate the total number of seeds selected according to the test workload.
2. For seed disinfection, soak selected pepper seeds in 0.2% KMnO₄ solution for 15 min and then rinse five times with distilled water.
3. For seed soaking, transfer the sterilized seeds to distilled water and allow them to soak for 24 h. Rinse the seeds several times with distilled water and dry for further use.
   ​NOTE: The soaking time of seeds for different crops may vary.

3. Seed germination and seedling growth

1. Prepare six concentrations of the mixed salts: 0 (control), 3, 5, 10, 15, and 20 g/L. Measure the conductivity of the salt solution using a conductivity meter; the solution conductivity EC values are 0.092, 3.05, 4.73, 8.33, 11.53, and 15.22 ms/cm, respectively.
2. For seed preparation, evenly place 40 pepper seeds in a Petri dish with two layers of filter paper. Prepare the seeds for six experimental treatments and repeat each treatment five times.
3. For seed germination, add a suitable amount of the six mixed salt concentrations to the Petri dish to ensure the filter paper is kept moist. Place the seeds in an air incubator at 28 °C and 80% air humidity for germination in the dark.
4. After seed germination, allow the seedlings to continue to grow in light (light intensity of approximately 450 Lux; light cycle of 12/12h) in the incubator for 14 days after sowing. The temperature and humidity at the seedling growth stage must be the same as those used at the germination stage.
5. Replenish the solution in the culture dish every 12 h to retain a moist filter paper, and completely wash the filter paper every 24 h with the corresponding concentration of the mixed salt solution to keep a constant mixed salt concentration in the Petri dish.
   ​NOTE: The amount of salt solution added to wet seeds can be adjusted according to the seed germination and growth stages. Many methods are available for maintaining a constant concentration of salt solutions in culture dishes. In addition to the methods described in this experiment, the strategy of adding distilled water by weight can be used.

4. Measurement and calculation of indicators

1. Determination of the seed germination indexes
   1. Determine the germination rate daily after sowing, with the radicle breaking seed coat reaching half the seed diameter length as the germination marker.
   2. Calculate the germination rate, germination potential, relative salt rate, germination index, and seed germination vigor index using the following formulas:
      Germination rate (%) = (number of normal germinated seeds on day 7 after sowing/number of tested seeds) × 100
      Germination potential (%) = (number of normal germinated seeds on day 3 after sowing/number of tested seeds) × 100
      Relative salt rate (%) = (control germination rate - treatment germination rate)/control germination rate × 100
      calculated using the seed germination rate on day 7 after sowing
      Germination index (GI) = ∑ [Gt/Dt]
      where Gt refers to the seed germination number at a period of time (t) after sowing and Dt refers to the corresponding germination days
      Seed germination vigor index (VI) = GI x S
      ​where S is the root length
2. Determination of the seedling growth index
   1. On day 14 after sowing, randomly select 10 representative seedlings from each Petri dish and measure the root length and hypocotyl length.
   2. Use a knife to divide the pepper seedlings into two parts: radicle and aboveground parts. Remove the water from the seedlings by wiping, and weigh the seedlings separately to determine the fresh weight.
3. Determine the antioxidant enzyme activity, malondialdehyde (MDA) level, and proline (Pro) content in pepper as follows.
   1. To preserve the pepper seedlings, select representative whole pepper seedlings (approximately 24.0 g) from each treatment on day 14 after sowing. After removing the surface water, immediately freeze the seedlings in liquid nitrogen for 1 min and store them in a refrigerator at an ultra-low temperature (-80 °C).
      NOTE: The sample number of pepper seedlings stored in the ultra-low temperature refrigerator should be sufficient, in case some indicators need to be retested.
   2. Retrieve approximately 1.0 g of seedling sample from each treatment collected in triplicate. Place the seedling sample in a centrifuge tube, add liquid nitrogen, and grind the sample using a grinding rod to determine the physiological indexes of the seedlings. The determined indexes and the measurement scheme are shown below.
   3. Determine the seedling protective enzyme activity (peroxidase [POD], catalase [CAT], superoxide dismutase [SOD]), malondialdehyde (MDA), and proline (Pro) contents using a commercially available kit (spectrophotometry-based) for each factor¹⁴.
      NOTE: Earlier observations revealed no difference in salt stress between the 15 and 20 g/L mixed salt concentrations. As a result, only five salt concentrations (0, 3, 5, 10, and 15 g/L) are measured.
4. Comprehensive evaluation of salt tolerance using the membership function method
   NOTE: The membership function uses a fuzzy mathematics method, which converts qualitative evaluation into quantitative evaluation¹⁵, to evaluate a variety of physiological indexes affected by salt damage.
   1. Calculate the value of the membership function using the following formula by Zhoubin Liu et al.¹⁵:
      Ri = (Xi - Xmin)/(Xmax - Xmin)
      If a trait is negatively correlated with salt tolerance, calculate the inverse membership function using:
      Ri = 1 - (Xi - Xmin)/(Xmax - Xmin)
      ​Accumulate the membership values of each physiological index, where Xi is the measured value of a certain trait, Xmax and Xmin are the maximum and minimum values for Xi, respectively, and Ri is the membership value of that trait.
   2. Include the following relevant indicators: seed germination characteristics (germination potential, germination rate, germination index, and seed germination vigor index), seedling growth characteristics at the germination stage (root length, hypocotyl length, root fresh weight, and fresh weight aboveground), MDA, Pro, and protective enzyme activity (CAT, POD, SOD) for membership function value calculation. The membership function values are obtained from each indicator.
5. Use spreadsheet and SPSS software (version 22.0) to analyze and process the test data and apply the least significant difference (LSD) method for multiple comparisons to identify significant differences. Use Pearson's correlation analysis to investigate the correlation between seed germination and seedling physiological indexes of pepper under compound salt stress.

Results

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 relat...

Discussion

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 s...

Materials

Name	Company	Catalog Number	Comments
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)