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 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.</li>
	<li>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.</li>
	<li>Prepare Petri dishes (single use) and filter paper (medium speed qualitative filter paper), both with a diameter of 9 cm. 
	&#8203;NOTE: The material of the Petri dish can be changed; however, the diameter of the Petri dish and filter paper must be the same.</li>
</ol>
2. Seed soaking and preparation for germination
<ol>
	<li>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.</li>
	<li>For seed disinfection, soak selected pepper seeds in 0.2% KMnO4 solution for 15 min and then rinse five times with distilled water.</li>
	<li>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. 
	&#8203;NOTE: The soaking time of seeds for different crops may vary.</li>
</ol>
3. Seed germination and seedling growth
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#176;C and 80% air humidity for germination in the dark.</li>
	<li>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.</li>
	<li>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. 
	&#8203;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.</li>
</ol>
4. Measurement and calculation of indicators
<ol>
	<li>Determination of the seed germination indexes
	<ol>
		<li>Determine the germination rate daily after sowing, with the radicle breaking seed coat reaching half the seed diameter length as the germination marker.</li>
		<li>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) &#215; 100 
		Germination potential (%) = (number of normal germinated seeds on day 3 after sowing/number of tested seeds) &#215; 100 
		Relative salt rate (%) = (control germination rate - treatment germination rate)/control germination rate &#215; 100 
		calculated using the seed germination rate on day 7 after sowing 
		Germination index (GI) = &#8721; [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 
		&#8203;where S is the root length</li>
	</ol>
	</li>
	<li>Determination of the seedling growth index
	<ol>
		<li>On day 14 after sowing, randomly select 10 representative seedlings from each Petri dish and measure the root length and hypocotyl length.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Determine the antioxidant enzyme activity, malondialdehyde (MDA) level, and proline (Pro) content in pepper as follows.
	<ol>
		<li>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 &#176;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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>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) 
		&#8203;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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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&#39;s correlation analysis to investigate the correlation between seed germination and seedling physiological indexes of pepper under compound salt stress.</li>
</ol>

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The authors declare no conflicts of interest.

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><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&amp;amp;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&amp;nbsp; 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 (KMnO&lt;sub&gt;4&lt;/sub&gt;</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&amp;nbsp;</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>&amp;nbsp;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 ...

analysis-effect-compound-salt-stress-on-seed-germination-salt

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

4.3K Views.  Gannan Normal University. 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. 

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

Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester fabric and glue can be folded and glued to construct a strip of compartments that house seeds and identifying information, while allowing contact with soil leachate, water, microorganisms, and ambient temperature. Strips may be constructed with a wide range of compartment numbers and sizes and allow the researcher to house a variety of genotypes within a single species, different species, or seeds that have experienced different treatments. As opposed to individual seed packets, strips are more easily retrieved as a unit. While replicate packets can be included within a strip, different strips can act as blocks or can be retrieved at different times for observation of seed behavior over time. We used a high temperature glue gun to delineate compartments and sealed the strips once the seed and tags identifying block and removal times were inserted. The seed strips were then buried in the field at the desired depth, with the location marked for later removal. Burrowing animal predators were effectively excluded by use of a covering of metal mesh hardware cloth on the soil surface. After the selected time interval for burial, strips were dug up and seeds were assessed for germination, dormancy and mortality. While clearly dead seeds can often be distinguished from ungerminated living ones by eye, dormant seeds were conclusively identified using a standard Tetrazolium chloride colorimetric test for seed viability.

Here we present a protocol for assessing seed survivorship, germination and dormancy under field conditions using buried, labeled seed strips and tetrazolium chloride (TZ) viability testing.

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester ...

Environment

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a significant increase and decrease in guard cell volume, respectively. Because shuttle transport of ions and water occurs between guard cells and larger neighboring epidermal cells during stomatal movement, the spaced distribution of plant stomata is considered an optimal distribution for stomatal movement. Experimental systems for perturbing the spaced pattern of stomata are useful to examine the spacing pattern's significance. Several key genes associated with the spaced stomatal distribution have been identified, and clustered stomata can be experimentally induced by altering these genes. Alternatively, clustered stomata can be also induced by exogenous treatments without genetic modification. In this article, we describe a simple induction system for clustered stomata in Arabidopsis thaliana seedlings by immersion treatment with a sucrose-containing medium solution. Our method is easy and directly applicable to transgenic or mutant lines. Larger chloroplasts are presented as a cell biological hallmark of sucrose-induced clustered guard cells. In addition, a representative confocal microscopic image of cortical microtubules is shown as an example of intracellular observation of clustered guard cells. The radial orientation of cortical microtubules is maintained in clustered guard cells as in spaced guard cells in control conditions.

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

The goal of this protocol is to demonstrate how to induce clustered stomata in cotyledons of Arabidopsis thaliana seedlings by immersion treatment with a sugar-containing medium solution and how to observe intracellular structures such as chloroplasts and microtubules in the clustered guard cells using confocal laser microscopy.

Stomatal movement mediates plant gas exchange, which is essential for photosynthesis and transpiration. Stomatal opening and closing are accomplished by a ...

An Effective Inoculation Method for Phytophthora capsici on Black Pepper Plants

Piper nigrum L. (black pepper) is a typical woody vine that is an economically important spice crop across the world. Black pepper production is significantly impacted by root rot disease caused by Phytophthora capsici, which has seriously influenced the industry development as a "choke point" problem. However, the molecular genetic mechanism of resistance in black pepper is unclear, leading to slow progress in the development of new black pepper varieties. An effective inoculation and precise sampling system for Phytophthora capsici on black pepper plants is essential for studying this plant-pathogen interaction. The main aim of this study is to demonstrate a detailed methodology where the basal head of black pepper is inoculated with Phytophthora capsici, while also providing a reference for the inoculation of woody vine plants. The basal head of the black pepper plant was pinpricked to damage it, and mycelial pellets covered the three holes to retain the moisture so the pathogen could infect the plant well. This method provides a better way of solving the instability that is caused by traditional inoculation methods including soil drench or root dipping. It also provides a promising means for studying the mode of action between plants and other soil-borne plant pathogens in agricultural precision breeding.

An Effective Inoculation Method for Phytophthora capsici on Black Pepper Plants

Pinpricking the basal head of the black pepper plant is a brief and time-saving method to damage it. Here, we provided detailed steps with a video for infecting black pepper plants.

Piper nigrum L. (black pepper) is a typical woody vine that is an economically important spice crop across the world. Black pepper production is ...

Genetics

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of dormant seeds or that of nondormant seeds irradiated by a far red (FR) light pulse. In order to further gain insight into the molecular genetic mechanisms underlying the germination repressive activity exerted by the endosperm, a &#34;seed coat bedding&#34; assay (SCBA) was devised. The SCBA is a dissection procedure physically separating seed coats and embryos from seeds, which allows monitoring the growth of embryos on an underlying layer of seed coats. Remarkably, the SCBA reconstitutes the germination repressive activities of the seed coat in the context of seed dormancy and FR-dependent control of seed germination. Since the SCBA allows the combinatorial use of dormant, nondormant and genetically modified seed coat and embryonic materials, the genetic pathways controlling germination and specifically operating in the endosperm and embryo can be dissected. Here we detail the procedure to assemble a SCBA.

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

We present the procedure to assemble a seed coat bedding assay (SCBA) from Arabidopsis thaliana seeds. The SCBA was shown to be a powerful tool to explore genetically and in vitro how the endosperm controls seed germination in dormant seeds and in response to light cues. The SCBA is in principle applicable under any situation where the endosperm is suspected to influence embryonic growth.

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

Dry Root Rot Disease Assays in Chickpea: a Detailed Methodology

Dry root rot (DRR) disease is an emerging biotic stress threat to chickpea cultivation around the world. It is caused by a soil-borne fungal pathogen, Rhizoctonia bataticola. In the literature, comprehensive and detailed step-by-step protocols on disease assays are sparse. This article provides complete details on the steps involved in setting up a blotting paper technique for quickly screening genotypes for resistance to DRR. The blotting paper technique is easy and less expensive. Another method, based on the sick pot approach, is a mimic of natural infection and can be applied to study the interacting components&#8212;plant, pathogen, and environment&#8212;involved in the disease triangle.
Moreover, in nature, DRR occurs mostly in rainfed chickpea cultivation areas, where soil moisture recedes as crop growth advances. Drought stress is known to predispose chickpea plants to DRR disease. Pathomorphological and molecular understanding of plant-pathogen interaction under drought stress can pave the way for the identification of elite DRR-resistant varieties from the chickpea germplasm pool. This article provides a stepwise methodology for the preparation of a sick pot and subsequent disease assay. Overall, the information presented herein will help researchers prepare R. bataticola fungal inoculum, maintain this pathogen, set up the blotting paper technique, prepare sick culture and sick pot, and assess pathogen infection in chickpea plants.

This study presents methodologies to study the pathomorphological and molecular mechanisms underlying chickpea&#8211;Rhizoctonia bataticola interaction. The blotting paper method is useful to rapidly study chickpea genotype responses, while the sick pot-based method can be used to simultaneously impose drought and R. bataticola infection and screen for tolerant genotypes.

Dry root rot (DRR) disease is an emerging biotic stress threat to chickpea cultivation around the world. It is caused by a soil-borne fungal pathogen, ...

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant productivity and survival. As such, understanding the mechanisms by which stomata develop and pattern has tremendous agronomic value. This paper describes two phenotypic methods using Arabidopsis cotyledons that can be used to characterize the genes controlling stomatal development and patterning. Presented first are procedures for analyzing the stomatal phenotypes using toluidine blue O-stained cotyledons. This method is fast and reliable and does not require the use of epidermal peels, which are widely used for phenotypic analyses but require specialized training. Due to the presence of multiple cysteine residues, the identification and generation of bioactive EPF peptides that have a role in stomatal development have been challenging. Thus, presented second is a procedure used to identify stomatal ligands and monitor their biological activity by bioassays. The main advantage of this method is that it produces reproducible data relatively easily while reducing the amount of peptide solution and the time required to characterize the role of the peptides in controlling stomatal patterning and development. Overall, these well-designed protocols enhance the efficiency of studying the potential stomatal regulators, including cysteine-rich secretory peptides, which require highly complex structures for their activity.

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

This paper describes two phenotyping methods without the use of epidermal peels to characterize the genes controlling stomatal development. The first method demonstrates how to analyze a stomatal phenotype using a toluidine blue O-stained plant epidermis. The second method describes how to identify stomatal ligands and monitor their biological activities.

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant ...

Production of Arbuscular Mycorrhizal (AM) Fungal Inoculum and Phenotypic Evaluation of Rice and AM Symbiosis Under Saline Conditions

Rice (Oryza sativa L.) is a vital food crop for more than half of the global population. However, its growth is severely impacted by saline soils, which present a significant challenge to crop production worldwide. Arbuscular mycorrhizal (AM) fungi, which form mutualistic symbiotic relationships with over 90% of agricultural plants and 80% of terrestrial plant species, have been shown to enhance the salt tolerance of rice plants. AM fungi are obligate symbionts that cannot complete their life cycle without a host root. Therefore, effectively utilizing plants to produce AM fungal inoculum is crucial for advancing research in this field. In this study, we present a series of robust methods that begin with generating sand inoculum containing spores of Rhizophagus irregularis using Allium tuberosum L. These methods include inoculating rice seedlings with the sand inoculum, analyzing the growth phenotype of mycorrhizal rice, and quantifying fungal colonization levels using trypan blue staining under salt stress. These approaches can efficiently generate AM fungal inoculum for further investigation into how AM symbiosis enhances the salinity tolerance of rice.

This article describes a protocol for generating arbuscular mycorrhizal (AM) fungi inoculum to investigate AM-enhanced salt stress tolerance in rice.

Rice (Oryza sativa L.) is a vital food crop for more than half of the global population. However, its growth is severely impacted by saline soils, which ...

Preparation of Intact Tissue for Microscopic Analysis of the Endosperm Cell Layer in Developing and Mature Arabidopsis Seeds

In Arabidopsis seeds, the endosperm, a single layer of living cells located between the embryo and the testa, plays a critical role in regulating seed maturation, dormancy, and germination. Microscopic analysis of intact endosperm cells is essential for understanding the physiological functions of the endosperm at cellular and molecular levels. However, sample preparation has been challenging due to the small size of Arabidopsis seeds and the location of the endosperm cell layer beneath the testa. This article details the preparation of intact endosperm cell layer samples suitable for microscopic observation and analysis in both developing and mature seeds. This method enables the observation of large areas and numerous intact endosperm cells without requiring fixation or sectioning. Additionally, the protocol utilizes only standard laboratory equipment, such as injection needles, precision forceps, and stereo microscopes. This approach successfully enables high-resolution live-cell imaging of fluorescent signals, such as green fluorescent protein (GFP), in intact endosperm cells. This method allows for the observation of intracellular localization and movement of various proteins, as well as the morphology of organelles, in the endosperm cells of different Arabidopsis mutants. This protocol contributes to the elucidation of novel endosperm functions and expands the potential for cellular and molecular studies of this essential tissue.

This protocol describes the preparation of intact samples of the endosperm cell layer in Arabidopsis thaliana seeds. The method requires only common laboratory equipment, such as an injection needle and precision forceps, and enables high-resolution fluorescent live-cell imaging of endosperm cells in both developing and mature seeds.

In Arabidopsis seeds, the endosperm, a single layer of living cells located between the embryo and the testa, plays a critical role in regulating seed ...

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

Summary

Explore More Videos

Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions

An Induction System for Clustered Stomata by Sugar Solution Immersion Treatment in Arabidopsis thaliana Seedlings

An Effective Inoculation Method for Phytophthora capsici on Black Pepper Plants

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

Measuring Spatial and Temporal Ca²⁺ Signals in Arabidopsis Plants

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Dry Root Rot Disease Assays in Chickpea: a Detailed Methodology

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

Production of Arbuscular Mycorrhizal (AM) Fungal Inoculum and Phenotypic Evaluation of Rice and AM Symbiosis Under Saline Conditions

Preparation of Intact Tissue for Microscopic Analysis of the Endosperm Cell Layer in Developing and Mature Arabidopsis Seeds