We acknowledge the U.S. Department of Agriculture (Grant 58-6406-1-017) for supporting this research. We also acknowledge the WKU Biotechnology Centre, Western Kentucky University, Bowling Green, KY, USA, and the Director, CSIR Central Institute of Medicinal and Aromatic Plants, Lucknow, India, for providing the instrument facilities and support (CSIR CIMAP manuscript communication no. CIMAP/PUB/2022/103).&#160;SS acknowledges the financial support from Saint Joseph&#39;s University, Philadelphia, USA.

The whole protocol is summarized schematically in Figure 1, showing all the essential steps involved in revealing the root system architecture (RSA) of plantlets. Protocol steps are given in detail below:
1. Arabidopsis seed surface sterilization
<ol>
	<li>Transfer a tiny scoop (approximately 100 seeds = approximately 2.5 mg) of seeds to a microfuge tube, and soak for 30 min in distilled water at room temperature (RT). This entire procedure is carried out in the aseptic condition.</li>
	<li>Briefly centrifuge the microfuge tube containing seeds at 500 x g for 5 s, using any tabletop centrifuge at RT to let the seeds settle down.</li>
	<li>Decant the water, add 700 &#181;L of 70% (v/v) ethanol, vortex for a few seconds, and spin. Repeat vortexing and spinning if required, but ensure the treatment time of 70% ethanol remains at 3 min.</li>
	<li>After 3 min, immediately rinse once with sterile water. Keep the ethanol washing step as timely as possible, as prolonged ethanol exposure decreases germination.</li>
	<li>Treat the seeds with diluted commercial bleach (4% v/v) with a drop of Tween-20 for 7 min. Mix the seeds with bleach solution by inverting the tubes rapidly 8-12 times, followed by a brief centrifuge (500 x g for 5 s at RT). Froth is seen appearing in the tube.</li>
	<li>Decant the supernatant using a 1 mL pipette and rinse the seeds with at least five washes with sterile water, following the same vortexing procedure.</li>
	<li>Leave the surface sterilized seeds in water and incubate for 2-3 days at 4 &#176;C for stratification10.</li>
</ol>
2. Setting a hydroponic system for seed germination
<ol>
	<li>Half-fill a standard magenta box with distilled water and autoclave it. Autoclave the polycarbonate sheet (clear color and smooth texture) and cut 4 cm x 8 cm rectangles, with a midpoint notched more than halfway through the rectangle so that two rectangles may slot together to form an X shape10. Use this setup to hold the polypropylene mesh (6 cm x 6 cm squares of 250 &#181;m pore size, or depending upon the requirement) cut from 12x 24 inch sheets10. 
	NOTE: Polypropylene is highly resistant to acids, alkalis, and other chemicals; therefore, it has been opted. Autoclaving tends to distort polypropylene mesh; hence, it is recommended to be carried separately wrapped in aluminum foil. Typical autoclaving conditions of 16 min, 121 &#176;C, 15 psi, or 775 mm Hg are recommended.</li>
	<li>Add sterile half-MS basal media with vitamins + 1.5% (w/v) sucrose, as described by Shukla et al.1, to each box to reach the bottom edge of the polypropylene mesh in a laminar flow. All the procedures are carried out under aseptic conditions.</li>
	<li>Sow the surface-sterilized seeds on the mesh (250 &#181;m pore size) hydroponically and allow them to grow for 3 days.</li>
	<li>After 3 days, transfer the seedlings onto a mesh (500 &#181;m pore size) and allow them to grow for 2 days.</li>
	<li>After 2 days (total of 5 days), transfer the seedlings onto the control media (i.e., modified MS nutrient media1 containing 2.0 mM NH4NO3, 1.9 mM KNO3, 0.15 mM MgSO4&#183;7H2O, 0.1 mM MnSO4&#183;H2O, 3.0 &#181;M ZnSO4&#183;7H2O, 0.1 &#181;M CuSO4&#183;5H2O, 0.3 mM CaCl2&#183;2H2O, 5.0 &#181;M KI, 0.1 &#181;M CoCl2&#183;6H2O, 0.1 mM FeSO4&#183;7H2O, 0.1 mM Na2EDTA&#183;2H2O, 1.25 mM KH2PO4, 100 &#181;M H3BO3, 1 &#181;M Na2MoO4&#183;2H2O, 1.5% sucrose, 1.25 mM MES, pH 5.7 adjusted with 0.1 M MES [pH 6.1]) and to the experimental media (e.g., P- [0 mM] treatment; KH2PO4 is replaced with 0.62 mM K2SO4 from the control media composition as mentioned above1. For excess Pi treatments, the concentration of KH2PO4 is increased in modified MS medium [2.5, 5.0, 10.0, 20.0 mM]1) and let the seeds grow for 7 days. 
	&#8203;NOTE: A larger mesh pore size (500 &#181;m) facilitates the smooth picking of entire seedlings out without any damage or need of cutting at the hypocotyl. Plantlets grow under standard growth conditions (i.e., 16 h light/8 h dark photoperiod, 150 &#181;mol&#183;m-2&#183;s-1 light intensity, 60%-70% humidity) at 23 &#176;C.</li>
</ol>
3. Examination of RSA
<ol>
	<li>Prepare agar (1.1%) plates for root spreading (Petri plate size: 150 mm x 15 mm).</li>
	<li>Add 10-20 mL of autoclaved filtered tap water to the Petri plate, as mentioned above. Gently pull out the seedlings from the mesh (500 &#181;m) and submerge them in water on the plates.</li>
	<li>Gently spread each plantlets&#39; root in the water-filled plate with the help of a round watercolor art brush (sizes: no. 14, 16, 18, and 20). 
	NOTE: While carrying out the spreading of the root system, first get hold of the primary root and spread it into a straight line, as it serves as an axis. Then, spread the LRs symmetrically on each side of the primary root, wherever possible. After that, spread the second-order LR linked to the first-order LR. This spreading process is a kind of art; do it gently, slowly, like an artist drawing an image of the RSA.</li>
	<li>Tilt the plate slightly to remove the water. 
	&#8203;NOTE: At this point, the procedure can be paused by putting these spread plates at 4 &#176;C. Later, when image processing is required, take out the plates and place them at RT for a while. Wipe out the condensed water, and then the image can be processed conveniently.</li>
</ol>
4. Recording images for RSA
<ol>
	<li>Scan or photograph these Petri plates appropriately. 
	NOTE: For obtaining high-quality photographs, the 600 dpi resolution is recommended for scanning, and at least a 12 megapixel camera is recommended for photography.</li>
	<li>Measure the root system architecture traits using freely available ImageJ software (https://imagej.nih.gov/ij/index.html). To quickly follow the steps to measure the root length using ImageJ software, please refer to the example &#34;measuring DNA contour length&#34;&#8203;17. 
	NOTE: These steps are followed to measure the root lengths on pictures captured using a high-resolution scanner or camera.
	<ol>
		<li>Use a given distance of length to set the scale. The known distance of the scale bar in Figure 3 is 2 cm. Select the Straight Line tool from the ImageJ toolbar (fifth tool from the left). Use the Straight-Line tool to create a line selection that outlines the scale bar. Finish outlining by right-clicking, double-clicking, or clicking in the box at the beginning.</li>
		<li>Measure the length of the known scale bar in pixels using the Analyze &#62; Measure toolbar. Make a note of the pixel length.</li>
		<li>Open the Set Scale dialogue box by clicking the Set Scale tab in the Analyze tab. In the Distance in Pixels field, enter the pixel length (as noted above). Next, in the Known Distance field, enter the value, as shown by the scale bar (here, it is 20 mm). Set the Unit of Length as mm. The pixel-aspect ratio is 1.0. Now, the scale is specified by the x number of pixels per millimeter. To lock the scale for this particular image, click on OK.</li>
		<li>Create a line selection that outlines the root length using the Segmented Line tool. Finish outlining by right-clicking, double-clicking, or clicking in the box at the beginning. Click and drag the small black and white &#34;handles&#34; along the outline to adjust the line selection as needed.</li>
		<li>Use the Measure command under the Analyze tab of ImageJ to quantify the length of the root. To transfer the measured data to a spreadsheet, right-click on the Results window, select Copy All from the popup menu, switch to the spreadsheet, and then paste the data. 
		NOTE: As described above, set the scale using the known distance of the scale bar in the ImageJ set scale option. This gives the number of pixels per unit length. It is required to freshly set the scale every time, whenever a new image is being analyzed.</li>
	</ol>
	</li>
	<li>Measurement and calculation of RSA traits
	<ol>
		<li>Measure the primary root length between the hypocotyl junction to the root tip&#39;s end.</li>
		<li>Measure the first- and second-order LR length.</li>
		<li>Measure the branching zone (BZ) of the primary root. The branching zone of the primary root (BZPR) spans the first LR emerging point to the last LR emerging point.</li>
		<li>Record the number of LRs, which is the number of LRs originating within the boundary of the BZPR.</li>
		<li>Measure the average length of the first- and higher-order LRs. Derive the average length of the first-order LR (1&#176; LR) (centimeter per root) by dividing the total length of the 1&#176; LR by the total number of 1&#176; LRs.</li>
		<li>Measure the average length of the second-order LR. Calculate the average length of the second-order LR (2&#176; LR) by dividing the total length of the 2&#176; LR by the total number of 2&#176; LRs.</li>
		<li>Measure the 1&#176; LR density. Calculate the 1&#176; LR density (number of 1&#176; LRs per unit length of BZPR) by dividing the number of 1&#176; LRs by the length of the BZPR.</li>
		<li>Measure the 2&#176; LR density. Calculate the 2&#176; LR density by dividing the number of 2&#176; LRs by the length of the BZ of 1&#176; LRs (number of 2&#176; LRs per unit length of the BZ of 1&#176; lateral roots).</li>
		<li>Measure the total root length (TRL). This is the aggregate of the primary root, 1&#176; LR, and 2&#176; LR (and more if present) lengths.</li>
	</ol>
	</li>
</ol>
5. Root hair measurement
NOTE: Although the hydroponic system is not good at promoting root hair growth and development, despite being as robust as it is in solid growth media, it is still important to study it in the present context. Follow the steps below to analyze root hair development in a 5 mm section from the tip of the primary root of the seedlings.
<ol>
	<li>Chop off a 2 cm section of the primary root from the root tip.</li>
	<li>Mount the root section on a slide using 10% glycerol as a mounting medium.</li>
	<li>Place the slide under a stereo microscope.</li>
	<li>Use the axial carrier to visualize and capture images of the root hairs.</li>
	<li>Analyze the images to study the structure and characteristics of the root hairs using ImageJ software as earlier described.</li>
</ol>

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The authors declare no conflict of interest.

This work demonstrated mapping RSA utilizing simple laboratory equipment. Using this method, phenotypic alterations are recorded at the refined level. The benefit of this strategy is that the shoot portion never comes in contact with the media, so the phenotype of the plantlets is original. This method involves setting up a hydroponic system to grow plantlets as described in the protocol. Then, each plantlet is taken out intact and placed on an agar-filled Petri plate. The root system is then allowed to spread manually u...

The root system architecture (RSA), which is underground, is a vital organ for plant growth and productivity1,2,3. After the embryonic stage, plants undergo their most significant morphological changes. The way in which the roots grow in the soil greatly affects the growth of plant parts above ground. Root growth is the first step in germination. It is an informative trait as it uniquely responds to different available nutrients1,2,3,4. The RSA exhibits a high degree of developmental plasticity, which means that the environment is always used to make decisions about development2,5. Changes in the environment have made crop production more difficult in the present scenario. On a continuous basis, the RSA incorporates environmental signals into developmental choices5. As a result, a thorough understanding of the principles behind root development is essential for learning how plants respond to changing environments2,5.
The RSA senses varying nutrient concentrations and renders phenotypic alterations4,6,7,8,9,10,11,12. Studies suggest that root morphology/RSA is highly plastic compared to shoot morphology1,3. RSA trait mapping is highly effective in recording the effect of changing the surrounding soil environment1,11,12.
In general, discrepancies in the effect of various nutrient deficiencies on the root phenotype have been reported in many earlier studies3,11,13,14,15. For example, there are several contrasting reports on phosphate (Pi) starvation-induced changes in the number, length, and density of lateral roots (LRs). An increase in LR density has been reported under the Pi deficient condition6,8. In contrast, a decrease in LR density under Pi deficient conditions has also been reported by other authors3,13,16. One of the prominent causes of these inconsistencies is the use of the elemental contamination-prone gelling medium, which agar often contains10. Researchers typically grow their experimental plants on an agar-based plate system and record the root traits. Numerous RSA traits are frequently concealed or entrenched within the agar material and cannot be documented. Experiments linked to inducing nutrient deficiency, in which users often exclude one component totally from the medium, cannot be performed in elemental contamination-prone gelling medium11,14,15. Numerous nutrients are frequently present in significant amounts in the agar media, including P, Zn, Fe, and many more11,14,15. Furthermore, RSA growth is slower in agar-based media than in non-agar-based liquid medium. As a result, there is a need to establish an alternate non-agar-based approach for quantifying and qualitatively recording the phenotype of RSA. Consequently, the current method has been developed, in which plantlets are raised in a magenta box-based hydroponic system atop a polypropylene mesh supported by polycarbonate wedges1,10,11.
This study presents a detailed improvised version of the earlier method described by Jain et al.10. This strategy has been tuned for current demands in plant root biology and can also be used for plants like Alfalfa, other than model plants. The protocol is the primary way to measure the changes in RSA, and it only requires simple equipment. The present protocol illustrates how to phenotype several root features, such as primary and lateral roots in normal and modified medium (Pi deficient). Step-by-step directions and other helpful hints gleaned from the author&#39;s experiences are provided to help the researchers follow along with the methodologies offered in this method. The present study aims to provide a simple and effective method for revealing the entire root system of plants, including higher-order LRs. This method involves manually spreading the root system with a round watercolor art brush, allowing for precise control over the exposure of the roots1,10,11,12. It does not require expensive equipment or complicated software. This method has improved nutrient uptake and growth rate; plants have a nutrient-rich solution easily absorbed by their roots. The present method is suitable for researchers who wish to map the traits of a plant&#39;s root system in detail, particularly during early development (10-15 days after germination). It is suitable for small root systems, model plants like Arabidopsis and tobacco, and non-conventional plants like Alfalfa until their root system fits in the magenta boxes.
The steps for phenotypic analysis of RSA development in Arabidopsis are outlined in this protocol as follows: (1) the method of seed surface sterilization for plants (Arabidopsis), (2) the steps to set up the hydroponic system, followed by seed sowing on a medium, (3) procedure for taking out the complete seedings and spreading on the Petri plate for RSA analysis, (4) how to record the images for RSA, and (5) calculate important RSA parameters using ImageJ software.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Arabidospsis thaliana (Col 0)</td>
			<td>Lehle Seeds</td>
			<td>WT-02</td>
			<td>Columbia (Col-0**, no markers)*</td>
		</tr>
		<tr>
			<td>Art brushes</td>
			<td>Amazon or any other vendor</td>
			<td></td>
			<td>Water color round brush size no. 14 (8 mm), 16 (9.5 mm), 18 (12 mm), and 20 (14.2 mm)</td>
		</tr>
		<tr>
			<td>Automated Microscope with digital camera</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>LAS version 4.12.0, Leica Microsystems</td>
		</tr>
		<tr>
			<td>Imaging Software</td>
			<td>ImageJ</td>
			<td></td>
			<td>ImageJ V 
			&#160;1.8.0</td>
		</tr>
		<tr>
			<td>Magenta box GA-7</td>
			<td>Fisher Scientific&#160;</td>
			<td>50-255-176</td>
			<td></td>
		</tr>
		<tr>
			<td>Medicago sativa</td>
			<td>Johnny&#39;s Seeds</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri-plate (150 mm x 15 mm)</td>
			<td>USA Scientific</td>
			<td>8609-0215</td>
			<td>150 mm x 15 mm PS Petri Dish (https://www.usascientific.com)</td>
		</tr>
		<tr>
			<td>Photo camera</td>
			<td>Cannon or Nikon</td>
			<td></td>
			<td>Any high mega pixel (atleast 12 mega pixel per inch) camera on macro mode</td>
		</tr>
		<tr>
			<td>Plant-Agar</td>
			<td>Sigma-Aldrich</td>
			<td>A3301</td>
			<td>Agargel&#160; Suitable for plant tissue culture</td>
		</tr>
		<tr>
			<td>Polycarbonate Sheets</td>
			<td>Amazon</td>
			<td></td>
			<td>1 mm&#160; thick</td>
		</tr>
		<tr>
			<td>Polypropylene Mesh</td>
			<td>Amazon</td>
			<td></td>
			<td>Pore size 250 &#181;m, 500 &#181;m and 1000 &#181;m</td>
		</tr>
		<tr>
			<td>Scanner</td>
			<td>Epson</td>
			<td></td>
			<td>Epson Perfection V700 Photo (Scan at 600 dpi)</td>
		</tr>
	</tbody>
</table>

a simple protocol for mapping the plant root system architecture traits

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar tolerance to environmental challenges. An experimental protocol is presented for setting up the hydroponic system, plantlet growth, RSA spreading, and imaging. The approach used a magenta box-based hydroponic system containing polypropylene mesh supported by polycarbonate wedges. Experimental settings are exemplified by assessing the RSA of the plantlets under varying nutrient (phosphate [Pi]) supply. The system was established to examine the RSA of Arabidopsis, but it is readily adaptable to study other plants like Medicago sativa (Alfalfa). Arabidopsis thaliana (Col-0) plantlets are used in this investigation as an example to understand the plant RSA. Seeds are surface sterilized by treating ethanol and diluted commercial bleach, and kept at 4 &#176;C for stratification. The seeds are germinated and grown on a liquid half-MS medium on a polypropylene mesh supported by polycarbonate wedges. The plantlets are grown under standard growth conditions for the desired number days, gently picked out from the mesh, and submersed in water-containing agar plates. Each root system of the plantlets is spread gently on the water-filled plate with the help of a round art brush. These Petri plates are photographed or scanned at high resolution to document the RSA traits. The root traits, such as primary root, lateral roots, and branching zone, are measured using the freely available ImageJ software. This study provides techniques for measuring plant root characteristics in controlled environmental settings. We discuss how to (1) grow the plantlets, and collect and spread root samples, (2) obtain pictures of spread RSA samples, (3) capture the images, and (4) use image analysis software to quantify root attributes. The advantage of the present method is the versatile, easy, and efficient measurement of the RSA traits.

The different morphometric traits of root system architecture (RSA) are measured using simple laboratory tools, and the steps are depicted schematically in Figure 1. The details of the hydroponic setup demonstrate the protocol&#39;s potential in measuring the RSA (Figure 1 and Figure 2).
Given the observed differences in gelling agents, we used a hydroponic growing system to conduct all the studies<sup cl...

Watch this Scientific Journal Video about A Simple Protocol for Mapping the Plant Root System Architecture Traits at JoVE.com

A Simple Protocol for Mapping the Plant Root System Architecture Traits

We use simple laboratory tools to examine the root system architecture (RSA) of Arabidopsis and Medicago. The plantlets are grown hydroponically over mesh and spread using an art brush to reveal the RSA. Images are taken using scanning or a high-resolution camera, then analyzed with ImageJ to map traits.

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar ...

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Biology

2.4K Views.  Western Kentucky University. We use simple laboratory tools to examine the root system architecture (RSA) of Arabidopsis and Medicago. The plantlets are grown hydroponically over mesh and spread using an art brush to reveal the RSA. Images are taken using scanning or a high-resolution camera, then analyzed with ImageJ to map traits.

Video: A Simple Protocol for Mapping the Plant Root System Architecture Traits

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Lateral Root Inducible System in Arabidopsis and Maize

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is however tedious, because it occurs only in a few cells inside the root and in an unpredictable manner. To circumvent this problem, a Lateral Root Inducible System (LRIS) has been developed. By treating seedlings consecutively with an auxin transport inhibitor and a synthetic auxin, highly controlled lateral root initiation occurs synchronously in the primary root, allowing abundant sampling of a desired developmental stage. The LRIS has first been developed for Arabidopsis thaliana, but can be applied to other plants as well. Accordingly, it has been adapted for use in maize (Zea mays). A detailed overview of the different steps of the LRIS in both plants is given. The combination of this system with comparative transcriptomics made it possible to identify functional homologs of Arabidopsis lateral root initiation genes in other species as illustrated here for the CYCLIN B1;1 (CYCB1;1) cell cycle gene in maize. Finally, the principles that need to be taken into account when an LRIS is developed for other plant species are discussed.

Lateral Root Inducible System in Arabidopsis and Maize

The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize.

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

Plants responding to both intracellular and extracellular mechanical stimulations (or force signals) and develop special morphological changes, a called thigmomorphogenesis. In past decades, several signaling components have been identified and reported for being involved in the mechanotransduction (e.g., calcium ion binding proteins and jasmonic acid biosynthesis enzymes). However, the relatively slow pace of research in the study of force signaling or thigmomorphogenesis is largely attributed to two reasons: the requirement for laborious human hand-manipulated touch induction of thigmomorphogenesis and the force strength errors associated with people&#8217;s hand-touch. To enhance the efficiency of external force loading on a plant organism, an automatic touch-force loading machine was built. This robotic arm-driven hair brush touches provide a labor-saving and easily repeatable touch-force simulation, unlimited rounds of touch repetition and adjustable touch strength. This hair touch-force loading machine can be used for both large scale screening of touch-force signaling mutants and the phenomics study of plant thigmomorphogenesis. In addition, touch materials such as human hair, can be replaced with other natural materials like animal hair, silk threads and cotton fibers. The automated moving arms on the machine may be equipped with water sprinkling nozzles and air blowers to mimic the natural forces of rain drops and wind, respectively. By using this automatic hair touch-force loading machine in combination with the hand-performed cotton swab touching, we have investigated the touch response of two force signaling mutants, MAP KINASE KINASE 1 (MKK1) and MKK2 plants. The phenomes of the touch-force loaded wild type plants and two mutants were evaluated statistically. They have exhibited significant differences in touch response.

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

A gentle touch-force loading machine is built from human hair brushes, robotic arms and a controller. The hair brushes are driven by robotic arms installed on the machine and move periodically to apply touch-force on plants. The strength of machine-driven hair touches is comparable to that of manually applied touches.

Plants responding to both intracellular and extracellular mechanical stimulations (or force signals) and develop special morphological changes, a called ...

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, ...

Semihydroponic Glass Jar System for Exudate Analysis in Multiple Plant Species

A Versatile Glass Jar System for Semihydroponic Root Exudate Profiling

Root exudates shape the plant-soil interface, are involved in nutrient cycling&#160;and modulate interactions with soil organisms. Root exudates are dynamic and shaped by biological, environmental, and experimental conditions. Due to their wide diversity and low concentrations, accurate exudate profiles are challenging to determine, even more so in natural environments where other organisms are present, turning over plant-derived compounds and producing additional compounds themselves. The semihydroponic glass jar experimental system introduced here allows control over biological, environmental, and experimental factors. It allows the growth of various phylogenetically distinct plant species for up to several months with or without microbes, in a variety of different growth media. The glass-based design offers a low metabolite background for high sensitivity and low environmental impact as it can be reused. Exudates can be sampled nondestructively, and conditions can be altered over the course of an experiment if desired. The setup is compatible with mass spectrometry analytics and other downstream analytical procedures. In summary, we present a versatile growth system suited for sensitive root exudate analysis in a variety of conditions.

Author Spotlight: Exploring Plant-Microbe Interactions Through Root Exudates in a Novel Growth System 

We present a protocol for a glass-based, semihydroponic experimental system supporting the growth of a variety of phylogenetically distinct plants with or without microbes. The system is compatible with different growth media and permits nondestructive root exudate sampling for downstream analysis.

Root exudates shape the plant-soil interface, are involved in nutrient cycling&#160;and modulate interactions with soil organisms. Root exudates are ...

Agrobacterium-Mediated Hairy Root Induction and Regeneration in Plants

Hairy Root Transformation and Regeneration in Arabidopsis thaliana and Brassica napus

Hairy root transformation represents a versatile tool for plant biotechnology in various species. Infection by an Agrobacterium strain carrying a Root-inducing (Ri) plasmid induces the formation of hairy roots at the wounding site after the transfer of T-DNA from the Ri plasmid into the plant genome. The protocol describes in detail the procedure of the injection-based hairy root induction in Brassica napus DH12075 and Arabidopsis thaliana Col-0. The hairy roots may be used to analyze a transgene of interest or processed for the generation of transgenic plants. Regeneration medium containing cytokinin 6-benzylaminopurine (5 mg/L) and auxin 1-naphthaleneacetic acid (8 mg/L) successfully elicits shoot formation in both species. The protocol covers the genotyping and selection of regenerants and T1 plants to obtain plants carrying a transgene of interest and free of T-DNA from the Ri plasmid. An alternative process leading to the formation of a composite plant is also depicted. In this case, hairy roots are kept on the shoot (instead of the natural roots), which enables the study of a transgene in hairy root cultures in the context of the whole plant.

Author Spotlight: Optimizing Hairy Root-Based Transformation Protocols for Enhanced Efficiency in Brassicaceae

The protocol describes hairy root induction using Arabidopsis primary inflorescence stems and Brassica napus hypocotyls. The hairy roots can be cultured and used as explants to regenerate transgenic plants.