Name: analysis of effect of compound salt stress on seed germination and salt tolerance analysis of pepper (capsicum annuum l.)
Uploaded: 2022-11-30T15:00:00
Description: Watch this Scientific Journal Video about Analysis of Effect of Compound Salt Stress on Seed Germination and Salt Tolerance Analysis of Pepper Capsicum annuum L. at JoVE.com

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.

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
<ol>
	<li>Prepare crop seeds for cultivars-Hongtianhu 101 with strong salt tolerance and Xinxiang 8 with low tolerance.</li>
	<li>Prepare 0.2% KMnO4 solution as a seed disinfection ...

<ol>
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X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exogenous+zeaxanthin+alleviates+low+temperature+combined+with+low+light+induced+photosynthesis+inhibition+and+oxidative+stress+in+pepper+(Capsicum+annuum+L.)+plants.">Exogenous zeaxanthin alleviates low temperature combined with low light induced photosynthesis inhibition and oxidative stress in pepper (Capsicum annuum L.) plants.</a> Current Issues in Molecular Biology. 44 (6), 2453-2471 (2022).</li><li>Liu, Z. B., Yang, B. Z., Ou, L. J., Zou, X. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+different+Ca2++spraying+period+on+alleviating+pepper+injury+under+the+waterlogging+stress.">The impact of different Ca2+ spraying period on alleviating pepper injury under the waterlogging stress.</a> Acta Horticulturae Sinica. 42 (8), 1487-1494 (2015).</li><li>Aloui, H., Souguir, M., Latique, S., Hannachi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germination+and+growth+in+control+and+primed+seeds+of+pepper+as+affected+by+salt+stress.">Germination and growth in control and primed seeds of pepper as affected by salt stress.</a> Cercet&#259;ri agronomice &#238;n Moldova. 47 (3), 83-95 (2014).</li><li>Zhani, K., Elouer, M. A., Aloui, H., Hannachi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+a+salt+tolerant+Tunisian+cultivar+of+chili+pepper+(Capsicum+frutescens).">Selection of a salt tolerant Tunisian cultivar of chili pepper (Capsicum frutescens).</a> EurAsian Journal of Biosciences. 6, 47-59 (2012).</li><li>Patan&#232;, C., Saita, A., Sortino, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+effects+of+salt+and+water+stress+on+seed+germination+and+early+embryo+growth+in+two+cultivars+of+sweet+sorghum.">Comparative effects of salt and water stress on seed germination and early embryo growth in two cultivars of sweet sorghum.</a> Journal of Agronomy and Crop Science. 199 (1), 30-37 (2013).</li><li>Smith, P. T., Cobb, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+germination+of+pepper+seed+by+priming+with+salt+solutions+and+water.">Accelerated germination of pepper seed by priming with salt solutions and water.</a> Hortscience. 26 (4), 417-419 (2019).</li><li>Mirosavljevi&#263;, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maize+germination+parameters+and+early+seedlings+growth+under+different+levels+of+salt+stress.">Maize germination parameters and early seedlings growth under different levels of salt stress.</a> Ratarstvo i Povrtarstvo. 50 (1), 49-53 (2013).</li><li>Khan, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormonal+priming+alleviates+salt+stress+in+hot+pepper+(Capsicum+annuum+L.).">Hormonal priming alleviates salt stress in hot pepper (Capsicum annuum L.).</a> Soil and Environment. 28 (2), 130-135 (2009).</li><li>Zhang, B. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+simulated+salinization+on+seed+germination+and+physiological+characteristics+of+muskmelon+seedlings.">Effects of simulated salinization on seed germination and physiological characteristics of muskmelon seedlings.</a> Chinese Journal of Tropical Crops. 41 (5), 912-920 (2020).</li><li>Guzm&#225;n-Murillo, M. A., Ascencio, F., Larrinaga-Mayoral, J. 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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...

Salinity is a major limiting factor&#160;for crop productivity worldwide1. At present, nearly 19.5% of the world&#39;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Calcium chloride</td>
			<td>Shanghai Experiment Reagent Co., Ltd.,China</td>
			<td>Analytical reagent</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugal machine</td>
			<td>Shanghai Luxianyi Centrifuge Instrument Co., Ltd., China</td>
			<td>TGL-16M</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tube</td>
			<td>None</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Conductivity meter</td>
			<td>Shanghai Instrument&#38;Electronics Science Instrument Co., Ltd., China</td>
			<td>DDSJ-308F</td>
			<td></td>
		</tr>
		<tr>
			<td>Constant temperature and humidity box</td>
			<td>Ningbo Laifu Technology Co., Ltd.,China</td>
			<td>PSX-280H</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital display vernier caliper</td>
			<td>Deli Group Co., Ltd.,China</td>
			<td>DL90150</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic balance</td>
			<td>Mettler Toledo Instruments (Shanghai) Co., Ltd.,China</td>
			<td>ME802E/02</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Hangzhou Fuyang North Wood Pulp and Paper Co., Ltd.,China</td>
			<td>GB/T1914-2017</td>
			<td></td>
		</tr>
		<tr>
			<td>Grinding rod</td>
			<td>None</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Hongtianhu&#160; 101</td>
			<td>Seminis Seed (Beijing) Co., Ltd.,China</td>
			<td>11933955/100147K1-137</td>
			<td></td>
		</tr>
		<tr>
			<td>Ice machine</td>
			<td>Shanghai Kehuai Instrument Co., Ltd., China</td>
			<td>IM150G</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td>None</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Tianjin Kermel Chemical Reagent Co., Ltd.,China</td>
			<td>Analytical reagent</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Tianjin Kermel Chemical Reagent Co., Ltd.,China</td>
			<td>Analytical reagent</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Jiangsu Yizhe Teaching Instrument Co., Ltd.,China</td>
			<td>I-000163</td>
			<td></td>
		</tr>
		<tr>
			<td>Pocket knife</td>
			<td>None</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium permanganate (KMnO4</td>
			<td>Xilong Scientific Co.,Ltd.,China</td>
			<td>Analytical reagent</td>
			<td></td>
		</tr>
		<tr>
			<td>Pure water equipment</td>
			<td>Sichuan Youpu Ultrapure Technology Co., Ltd.,China</td>
			<td>UPT-I-20T</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Xilong Scientific Co.,Ltd.,China</td>
			<td>Analytical reagent</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium carbonate</td>
			<td>Xilong Scientific Co.,Ltd.,China</td>
			<td>Analytical reagent</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Xilong Scientific Co.,Ltd.,China</td>
			<td>Analytical reagent</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium sulfate&#160;</td>
			<td>Xilong Scientific Co.,Ltd.,China</td>
			<td>Analytical reagent</td>
			<td></td>
		</tr>
		<tr>
			<td>Test kit</td>
			<td>Suzhou Keming, Biotechnology Co., Ltd, Suzhou.,China</td>
			<td>Spectrophotometer method</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-low temperature freezer</td>
			<td>SANYO Techno Solution TottoriCo.,Ltd.</td>
			<td>MDF-382</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultraviolet visible spectrophotometer</td>
			<td>Shanghai Precision Scientific Instrument Co., Ltd., China</td>
			<td>&#160;760CRT</td>
			<td></td>
		</tr>
		<tr>
			<td>Xinxiang 8</td>
			<td>Jiangxi Nongwang High Tech Co., Ltd.,China</td>
			<td>GPD Pepper 2017(360013)</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of effect of compound salt stress on seed germination and salt tolerance analysis of pepper (capsicum annuum l.)

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.

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...

Watch this Scientific Journal Video about Analysis of Effect of Compound Salt Stress on Seed Germination and Salt Tolerance Analysis of Pepper Capsicum annuum L. at JoVE.com

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

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.

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

To determine the salt tolerance and physiological mechanism of pepper (Capsicum annuum L.) at the germination stage, the Hongtianhu 101 and Xinxiang 8 ...

This study provides theoretical guidance for breeding salt tolerance pepper varieties and calibrating high yield and quality in saline-alkali soil. The experimentation period is 15 days with high efficiency, faster speed, good repeatability, and accurate results. This technology can provide technical references and theoretical guidance for the evaluation of salt tolerance of other crops.Desire preliminary expert to rather evaluate the salt tolerance of seeds. Begin by preparing the crop seeds for Hongtianhu 101 cultivar with strong salt tolerance, and Xinxiang 8 cultivar with low tolerance. Prepare 2000 milliliters of 0.2%potassium permanganate solution as a seed disinfectant reagent just before the use.Prepare the mixed salt solution using seven salts, including sodium carbonate, sodium bicarbonate, sodium chloride, calcium chloride, magnesium chloride, magnesium sulfate, and sodium sulfate. Then prepare single use, nine centimeter Petri dishes, and nine centimeter medium speed qualitative filter paper. Select pepper seeds with consistent size and full particles from each variety, with an average diameter of 4.2 millimeters for Hongtianhu 101 and 3.7 millimeters for Xinxiang 8 seeds.Calculate the total number of seeds selected according to the test workload. For seed disinfection, soak selected pepper seeds in 0.2%potassium permanganate solution. After 15 minutes, rinse the seeds five times with distilled water.Transfer the sterilized seeds to distilled water for 24 hours for soaking. Once done, rinse the seeds several times with distilled water before drying them for further use. Prepare six concentrations of the mixed salts and measure the conductivity of the salt solution, using a conductivity meter.In a Petri dish with two layers of filter paper, evenly place 40 pepper seeds. Prepare the seeds for six experimental treatments and five replicates. Add a suitable amount of the six mixed salt concentrations to the Petri dish for germination, to ensure the filter paper is moist.Place the seeds in an air incubator at 28 degrees Celsius and 80%air humidity for germination in the dark. After seed germination, allow the seedlings to grow for 14 days in approximately 450 lux light intensity. With the light cycle of 12 hour dark, and 12 hour light period in the incubator.Replenish the solution in the culture dish every 12 hours to retain a moist filter paper, and completely wash the filter paper every 24 hours with the corresponding concentration of the mixed salt solution, to keep a constant mixed salt concentration in the Petri dish. For the seed germination indexes, determine the germination rate, daily, after sowing with the radical breaking the seed coat, reaching half the seed diameter length as the germination marker. To determine the seedling growth index, randomly select 10 representative seedlings from each Petri dish on day 14 after sowing, and measure the root length and hypocotyl length.Use a knife to divide the pepper seedlings into radical and above ground parts. Remove the water from the seedlings by wiping, and weigh the seedlings separately to determine the fresh weight. To preserve the pepper seedlings, select approximately 24 grams of representative whole pepper seedlings from each treatment on day 14 after sowing.After removing the surface water, immediately freeze the seedlings in liquid nitrogen for one minute. And store them in a refrigerator at minus 80 degrees Celsius. Retrieve approximately one gram seedling sample from each treatment collected in triplicate.Immediately, 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, including the seedling protective enzyme activity, malondialdehyde, and proline contents. As the mixed salt concentration increased, the germination potential and germination index of Hongtianhu 101 and Xinxiang 8 decreased significantly.As the mixed salt concentrations increased, the germination rate of the two varieties gradually decreased, and the relative salt damage rate for the varieties increased. The seed germination vigor index of Hongtianhu 101 was higher than that of Xinxiang 8 at each mixed salt concentration level. At 15 grams per liter mixed concentration, the root length of Hongtianhu 101 and Xinxiang 8 decreased by 89.4%and 91.1%and the root fresh weight decreased by 81.7%and 71.2%respectively, compared to the control.Based on the varietal differences, the hypocotyl length, and fresh weight above ground of Xinxiang 8, were higher than Hongtianhu 101 at each salt concentration. At five and three grams per liter concentrations of malondialdehyde and proline contents reached their lowest values respectively. At the salt concentration of three to 15 grams per liter, the proline content of Xinxiang 8 slowly increased and remained relatively stable, whereas the proline content of Hongtianhu 101 increased rapidly.As the mixed salt concentration increased, the catalase, peroxidase, and superoxide dismutase activities of Hongtianhu 101 and Xinxiang 8 seedlings decreased, and then increased, with the lowest values obtained at three grams per liter. The catalase and peroxidase activities of Hongtianhu 101 were higher than Xinxiang 8, and the difference between them increased gradually. Keep a constant of mixed salt concentration in the Petri dish by retaining a moist filter paper and completely washing the filter paper.Researchers can innovate on the basis of this experiment, such as changing the ratio of different mixed salts to obtain more extensive and in-depth results.

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