The authors are grateful to Dr. Jie Le and Dr. Xiufen Song for their helpful suggestions on differential interference contrast microscopy and conventional clearing method, respectively. This research was financed by the National Natural Science Foundation of China (31870299) and the Youth Innovation Promotion Association of the Chinese Academy of Sciences. Figure&#8197;2 was created with BioRender.com.

1. Preparation of solutions
<ol>
	<li>Prepare FAA fixative by adding 2.5 mL of 37% formaldehyde, 2.5 mL of glacial acetic acid, and 45 mL of 70% ethanol in a 50 mL centrifuge tube. Vortex and store it at 4 &#176;C. Freshly prepare FAA fixative just before use. 
	CAUTION: 37% formaldehyde is corrosive and potentially carcinogenic if exposed or inhaled. The fixative must be performed in a fume hood while wearing appropriate personal protective equipment.</li>
	<li>Prepare clearing solution by adding 5 mL of 100% glycerol, 40 g chloral hydrate, and 10 mL of distilled water in a 100 mL glass bottle wrapped with tin foil. Dissolve using a magnetic stirrer overnight at room temperature. The prepared solution can be stored at 4 &#176;C for up to 6 months. 
	CAUTION: Chloral hydrate is carcinogenic and has a pungent smell. Perform the experiment in a fume hood and wear appropriate personal protective equipment. Chloral hydrate is easy to deliquesce in the air and should not be stored in large quantities. The clearing solution can decompose when exposed to light and should be kept away from light.</li>
	<li>Prepare the disinfectant solution by adding 10 mL of 6% sodium hypochlorite, 40 mL distilled water, and 50 &#181;L Tween-20 in a 50 mL centrifuge tube. Vortex and store it at room temperature. Freshly prepare the disinfectant solution just before use. 
	NOTE:&#160;The effective chlorine content of sodium hypochlorite that has been placed for a long time may decrease, and the content of 6% sodium hypochlorite can be increased according to the actual situation.&#160;</li>
</ol>
2. Seed collection
<ol>
	<li>Sow tomato seeds (S. lycopersicum L. cv. Micro-Tom, see Table of Materials) in 8 cm &#215; 8 cm &#215; 8 cm square basins full with a 1:1 mixture of flower nutrient soil and substrate (v/v) (see Table of Materials), and grow in a growth room with 16/8 h light/dark periods at 24 &#177; 2 &#176;C (day) and 18 &#177; 2 &#176;C (night), 60%-70% relative humidity, and a light intensity of 4,000 Lux. Approximately 6 weeks later, plants entered the flowering stage.</li>
	<li>Tag the flowering date of independent flowers at anthesis (opening angle of the petals is 90&#176;), and record the day after flowering (DAF).</li>
	<li>Harvest the fruits from 3 to 23 DAF tomato plants and immediately put them on ice. Divide fruits (seeds or embryos) from 3 to 23 DAF into early (3-10 DAF), middle (11-16 DAF), and late (17-23 DAF) fruits (seeds or embryos). 
	NOTE: Do not collect too many fruits at a time and ensure that the seeds in each fruit are stripped within 1 h for further treatment.</li>
	<li>For early fruits, break the fruit and put it on a slide, and collect fresh seeds carefully with precision forceps under the stereomicroscope (see Table of Materials) at 1x to 4x magnification. For middle and late fruits, break the fruit and directly collect fresh seeds using precision forceps.</li>
</ol>
3. Chloral hydrate-based clearing of seeds
NOTE: Conventional35 and optimized protocols were compared in this study for their seed clearing efficiency.
<ol>
	<li>Clearing using a conventional protocol
	<ol>
		<li>Place fresh seeds (obtained in step 2.4) immediately in a 2 mL centrifuge tube containing 1.5 mL FAA fixative and incubate on an orbital shaker (5 rpm, see Table of Materials) for 4 h at room temperature.</li>
		<li>Dehydrate these seeds in an ethanol series of 70%, 95%, and 100% ethanol (v/v) for 1 h each on an orbital shaker (5 rpm).</li>
		<li>Place the seeds in 3-5 drops of clearing solution (~100 &#181;L) on the slide and gently cover the sample with a coverslip. Replace the slide with a single concave slide (see Table of Materials) for middle and late seeds.</li>
		<li>Keep these slides or single concave slides at room temperature for 30 min (3 DAF), 2 h (6 DAF), 1 day (9 DAF), 3 days (12 DAF), or 7 days (14 to 22 DAF) depending on the developmental stages of the seed material (the younger the materials, the faster the clearing speed).</li>
		<li>Observe the samples with a differential interference contrast (DIC) microscope equipped with a digital camera (see Table of Materials) at 10x, 20x, and 40x magnification. Adjust and optimize the transmitted light brightness, DIC slider, and condenser aperture in real time for each sample and capture images.</li>
	</ol>
	</li>
	<li>Clearing using the optimized protocol
	<ol>
		<li>Place fresh seeds (obtained in step 2.4) directly into a 2 mL centrifuge tube containing 1.5 mL disinfectant solution (step 1.3). 
		NOTE: The number of seeds collected in the 2 mL centrifuge tube varies according to the developmental stage of the seeds. Details are listed in Table 1.</li>
		<li>Incubate the samples on an orbital shaker (30 rpm) at room temperature for 3 to 50 min until the innermost seed coat outline is clearly visible. Discard the disinfectant solution. 
		NOTE: The incubation time required depends on the seed developmental stage (the later the seeds, the longer will be the incubation time). Details are listed in Table 1.</li>
		<li>For middle and late seeds, transfer the seeds onto a slide and remove the seed mucilage with forceps and a dissecting needle under a stereomicroscope at 1x to 4x magnification. Transfer the seeds back into the original centrifuge tube using forceps. 
		NOTE: This step is not necessary for early seeds.</li>
		<li>Wash the seeds 5x with 1.5 mL deionized water, 10 s each time. Discard the deionized water.</li>
		<li>Add a clearing solution of 2 &#215; the volume of the seeds, followed by vacuum treatment for 0 to 50 min (Table 1) with a vacuum pump (see Table of Materials). Intermittent vacuum treatment is performed with each vacuum treatment for 10 min spaced at 10 min intervals.</li>
		<li>Replace with a fresh clearing solution of 2 &#215; the volume of the seeds. Keep the 2 mL centrifuge tube containing the seeds at room temperature and protected from light for 30 min to 7 days to facilitate clearing (Table 1). During the incubation, for late embryos, replace the clearing solution daily with fresh solution and subject the seeds to vacuum treatment for 10 min.</li>
		<li>Prepare the cleared seeds on slides or single-concave slides and observe with the DIC microscope equipped with a digital camera at 10x, 20x, and 40x magnification. Adjust and optimize the transmitted light brightness, DIC slider, and condenser aperture in real time for each sample and capture images.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest to disclose.

Compared to mechanical sectioning, the clearing technology is more advantageous for three-dimensional imaging as it retains the integrity of plant tissues or organs16. Conventional clearing protocols are often limited to small samples due to easier penetration of chemical solutions. Tomato seed is a problematic sample for tissue clearing because it is about 70 times larger than an Arabidopsis seed in size and has more permeability barriers. The Arabidopsis seed coat is composed o...

Tomato (S. lycopersicum L.) is one of the most important vegetable crops around the world, with an output of 186.8 million tons of fleshy fruits from 5.1 million hectares in 20201. It belongs to the large Solanaceae family with about 2,716 species2, including many commercially important crops such as eggplant, peppers, potato, and tobacco. The cultivated tomato is a diploid species (2n = 2x = 24) with a genome size of approximately 900 Mb3. For a long time, great effort has been made toward tomato domestication and breeding by selecting desirable traits from wild Solanum spp. There are over 5,000 tomato accessions listed in the Tomato Genetics Resource Center and more than 80,000 germplasm of tomatoes are stored worldwide4. The tomato plant is perennial in the greenhouse and propagates by seeds. A mature tomato seed consists of three major compartments: a full-grown embryo, residual cellular-type endosperm, and a hard seed coat5,6 (Figure 1A). After double fertilization, the development of cellular-type endosperm precedes the development of zygotes. At ~5-6 days after flowering (DAF), two-celled proembryo is first observed when the endosperm consists of six to eight nuclei7. In Solanum pimpinellifolium, the embryo approaches its final size after 20 DAF, and seeds are viable for germination after 32 DAF8. As the embryo develops, the endosperm is gradually absorbed and only a small amount of endosperm remains in the seed. The residual endosperm consists of micropylar endosperm surrounding the radicle tip, and lateral endosperm in the rest of the seed9,10. The outer seed coat is developed from thickened and lignified outer epidermis of the integument, and with the dead layers of integument remnants, they form a hard shell to protect the embryo and endosperm5.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64445/64445fig01.jpg" /> 
Figure 1: Schematic representation of a mature seed in Solanum lycopersicum and Arabidopsis thaliana. (A) Longitudinal anatomy of a mature tomato seed. (B) Longitudinal anatomy of a mature Arabidopsis seed. A mature tomato seed is approximately 70 times larger in size than an Arabidopsis seed. Scale bars = (A) 400 &#181;m, (B) 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64445/64445fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Production of high-quality tomato seeds depends on the coordination between the embryo, the endosperm, and the maternal seed components11. Dissecting key genes and networks in seed development requires a deep and full-track phenotypic recording of mutant seeds. Conventional embedding-sectioning techniques, such as the semi-thin section and paraffin section, are widely applied to tomato seeds to observe the local and finer structures of the embryo12,13,14,15. However, analyzing the seed development from thin sections is usually laborious and lacks z-axis spatial resolution. In comparison, tissue clearing is a fast and efficient method to pinpoint the developmental stage of embryo defects that are most likely to occur16. The clearing method reduces the opaqueness of internal tissue by homogenizing the refractive index with one or more biochemical agents16. Whole tissue clearing allows observation of a plant tissue structure without destroying its integrity, and the combination of clearing technology and three-dimensional imaging has become an ideal solution to obtain information on the morphology and developmental state of a plant organ17,18. Over the years, seed clearing techniques have been used in various plant species, including Arabidopsis thaliana, Hordeum vulgare, and Beta vulgaris19,20,21,22,23.&#160;Among these, the whole-mount ovule clearing technology has been an efficient approach to studying seed development of Arabidopsis, due to its small size, 4-5 layers of the seed coat cell, and the nuclear-type endosperm24,25. With the continuous updating of different clearing mixtures, such as the emergence of Hoyer&#39;s solution26, internal structures of the barley ovule were imaged with a high degree of clarity although its endosperm makes up the bulk of the seeds. Embryogenesis of sugar beet can be observed by clearing combined with vacuum treatment and softening with hydrochloric acid19. Nonetheless, unlike the species mentioned above, embryological observations by clearing protocols in tomato seeds have not been reported. This prevents detailed investigation into the embryonic and seed development of tomatoes.
Chloral hydrate is commonly used as a clearing solution that allows the immersed tissues and cells to be displayed on different optical planes, and substantially preserves the cells or tissue components27,28,29. Chloral hydrate-based clearing protocol has been successfully used for the whole-mount clearing of seeds to observe the embryo and endosperm of Arabidopsis21,28. However, this clearing solution is not efficient in clearing tomato seeds, which are more impermeable than Arabidopsis seeds. The physical barriers include: (1) the tomato integument has nearly 20 cell layers at 3 to 15 DAF30,31, (2) the tomato endosperm is cellular-type, not nuclear-type32, and (3) tomato seeds are about 70 times larger in size33,34 and (4) produce large amounts of seed coat mucilage, which blocks the penetration of clearing reagents and affects the visualization of embryo cells.
Therefore, this report presents an optimized chloral hydrate-based clearing method for whole-mount clearing of tomato seeds at different stages, which allows deep imaging of the embryo development process (Figure 2).

<table><tbody><tr><td>1,000 &amp;micro;L pipette</td><td>GILSON</td><td>FA10006M</td><td></td></tr><tr><td>1,000 &amp;micro;L pipette tips</td><td>Corning</td><td>T-1000-B</td><td></td></tr><tr><td>2 ml centrifuge tube</td><td>Axygen</td><td>MCT-200-C</td><td></td></tr><tr><td>37% formaldehyde</td><td>DAMAO</td><td>685-2013</td><td></td></tr><tr><td>5,000 &amp;micro;L pipette</td><td>Eppendorf</td><td>3120000275</td><td></td></tr><tr><td>5,000 &amp;micro;L pipette tips</td><td>biosharp</td><td>BS-5000-TL</td><td></td></tr><tr><td>50 ml centrifuge tube</td><td>Corning</td><td>430829</td><td></td></tr><tr><td>Absolute Ethanol</td><td>BOYUAN</td><td>678-2002</td><td></td></tr><tr><td>Bottle glass</td><td>Fisher</td><td>FB800-100</td><td></td></tr><tr><td>Chloral Hydrate</td><td>Meryer</td><td>M13315-100G</td><td></td></tr><tr><td>Coverslip</td><td>Leica</td><td>384200</td><td></td></tr><tr><td>DIC microscope</td><td>Zeiss</td><td>Axio Imager A1</td><td>10x, 20x and 40x magnification</td></tr><tr><td>Disinfectant</td><td>QIKELONGAN</td><td>17-9185</td><td></td></tr><tr><td>Dissecting needle</td><td>Bioroyee</td><td>17-9140</td><td></td></tr><tr><td>Flower nutrient soil</td><td>FANGJIE</td><td></td><td></td></tr><tr><td>Forceps</td><td>HAIOU</td><td>4-94</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>BOYUAN</td><td>676-2007</td><td></td></tr><tr><td>Glycerol</td><td>Solarbio</td><td>G8190</td><td></td></tr><tr><td>Magnetic stirrer</td><td>IKA</td><td>RET basic</td><td></td></tr><tr><td>Micro-Tom</td><td>Tomato Genetics Resource Center</td><td>LA3911</td><td></td></tr><tr><td>Orbital shaker</td><td>QILINBEIER</td><td>QB-206</td><td></td></tr><tr><td>Seeding substrate</td><td>PINDSTRUP</td><td>LV713/018-LV252</td><td>Screening:0-10 mm</td></tr><tr><td>Single concave slide</td><td>HUABODEYI</td><td>HBDY1895</td><td></td></tr><tr><td>Slide</td><td>Leica</td><td>3800381</td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>S8 APO</td><td>1x to 4x magnification</td></tr><tr><td>Tin foil</td><td>ZAOWUFANG</td><td>613</td><td></td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P1379</td><td></td></tr><tr><td>Vacuum pump</td><td>SHIDING</td><td>SHB-III</td><td></td></tr><tr><td>Vortex meter</td><td>Silogex</td><td>MX-S</td><td></td></tr></tbody></table>

an efficient clearing protocol for the study of seed development in tomato (solanum lycopersicum l.)

Tomato (Solanum lycopersicum L.) is one of the major cash crops worldwide. The tomato seed is an important model for studying genetics and developmental biology during plant reproduction. Visualization of finer embryonic structure within a tomato seed is often hampered by seed coat mucilage, multi-cell-layered integument, and a thick-walled endosperm, which needs to be resolved by laborious embedding-sectioning. A simpler alternative is to employ tissue clearing techniques that turn the seed almost transparent using chemical agents. Although conventional clearing procedures allow deep insight into smaller seeds with a thinner seed coat, clearing tomato seeds continues to be technically challenging, especially in the late developmental stages.
Presented here is a rapid and labor-saving clearing protocol to observe tomato seed development from 3 to 23 days after flowering when embryonic morphology is nearly complete. This method combines chloral hydrate-based clearing solution widely used in Arabidopsis with other modifications, including the omission of Formalin-Aceto-Alcohol (FAA) fixation, the addition of sodium hypochlorite treatment of seeds, removal of the softened seed coat mucilage, and washing and vacuum treatment. This method can be applied for efficient clearing of tomato seeds at different developmental stages and is useful in full monitoring of the developmental process of mutant seeds with good spatial resolution. This clearing protocol may also be applied to deep imaging of other commercially important species in the Solanaceae.

When tomato seeds were cleared using a conventional method as in Arabidopsis, dense endosperm cells blocked the visualization of early tomato embryos at 3 DAF and 6 DAF (Figure 3A,B). As the total volume of the embryo increased, a globular embryo was barely distinguishable at 9 DAF (Figure 3C). However, as the seed size continued to increase, its permeability decreased, resulting in a fuzzy heart embryo at 12 DAF (F...

Watch this Scientific Journal Video about An Efficient Clearing Protocol for the Study of Seed Development in Tomato Solanum lycopersicum L. at JoVE.com

An Efficient Clearing Protocol for the Study of Seed Development in Tomato Solanum lycopersicum L.

The tomato seed is an important model for studying genetics and developmental biology during plant reproduction. This protocol is useful for clearing tomato seeds at different developmental stages to observe the finer embryonic structure.

An Efficient Clearing Protocol for the Study of Seed Development in Tomato (Solanum lycopersicum L.)

Tomato (Solanum lycopersicum L.) is one of the major cash crops worldwide. The tomato seed is an important model for studying genetics and developmental ...

an-efficient-clearing-protocol-for-study-seed-development-tomato

Research

JoVE Journal

Developmental Biology

3.9K Views.  Chinese Academy of Sciences. Deze methode kan onderzoekers helpen om snel en moeiteloos abnormale perioden van tomatenembryo-ontwikkeling te vinden. Het belangrijkste voordeel van dit protocol is een behandeling van tomatenzaden met natriumhypochloriet die een basis biedt voor de observatie van tomatenembryo's. Begin met de chemische bereiding en oogst van de Solanum lycopersicum vruchten drie tot 23 dagen na de bloei of DAF.Zet de vruchten onmiddellijk op ijs en verdeel ze in vroege, middelste en late vruchten, g...

Video: An Efficient Clearing Protocol for the Study of Seed Development in Tomato Solanum lycopersicum L.

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important plant parasitic nematodes. The microscopic second-stage juvenile (J2), molted once in the egg, is the infective stage. The J2s hatch from the eggs, move freely in the soil within a film of water, and locate root tips of suitable plant species. After penetrating the plant root, they migrate towards the vascular cylinder where they establish a feeding site and initiate feeding using their stylets. The multicellular feeding site is comprised of several enlarged multinuclear cells called 'giant cells' which are formed from cells that underwent karyokinesis (repeated mitosis) without cytokinesis. Neighboring pericycle cells divide and enlarge in size giving rise to a typical gall or root knot, the characteristic symptom of root-knot nematode infection. Once feeding is initiated, J2s become sedentary and undergo three additional molts to become adults. The adult female lays 150-250 eggs in a gelatinous matrix on or below the surface of the root. From the eggs new infective J2s hatch and start a new cycle. The root-knot nematode life cycle is completed in 4-6 weeks at 26-28&deg;C.
Here we present the traditional protocol to infect plants, grown in pots, with root-knot nematodes and two methods for high-throughput assays. The first high-throughput method is used for plants with small seeds such as tomato while the second is for plants with large seeds such as cowpea and common bean. Large seeds support extended seedling growth with minimal nutrient supplement. The first high throughput assay utilizes seedlings grown in sand in trays while in the second assay plants are grown in pouches in the absence of soil. The seedling growth pouch is made of a 15.5 x 12.5cm paper wick, folded at the top to form a 2-cm-deep trough in which the seed or seedling is placed. The paper wick is contained inside a transparent plastic pouch. These growth pouches allow direct observation of nematode infection symptoms, galling of roots and egg mass production, under the surface of a transparent pouch. Both methods allow the use of the screened plants, after phenotyping, for crossing or seed production. An additional advantage of the use of growth pouches is the small space requirement because pouches are stored in plastic hanging folders arranged in racks.

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Two distinct methods to screen plants with root-knot nematodes are described. The described approaches include high-throughput screens with nematodes in a nondestructive manner facilitating the use of these plants in breeding programs.

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important ...

Immunology and Infection

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease

Ralstonia solanacearum is a devastating soil borne vascular pathogen that can infect a large range of plant species, causing an important threat to agriculture. However, the Ralstonia model is considerably underexplored in comparison to other models involving bacterial plant pathogens, such as Pseudomonas syringae in Arabidopsis. Research targeted to understanding the interaction between Ralstonia and crop plants is essential to develop sustainable solutions to fight against bacterial wilt disease but is currently hindered by the lack of straightforward experimental assays to characterize the different components of the interaction in native host plants. In this scenario, we have developed a method to perform genetic analysis of Ralstonia infection of tomato, a natural host of Ralstonia. This method is based on Agrobacterium rhizogenes-mediated transformation of tomato roots, followed by Ralstonia soil-drenching inoculation of the resulting plants, containing transformed roots expressing the construct of interest. The versatility of the root transformation assay allows performing either gene overexpression or gene silencing mediated by RNAi. As a proof of concept, we used this method to show that RNAi-mediated silencing of SlCESA6 in tomato roots conferred resistance to Ralstonia. Here, we describe this method in detail, enabling genetic approaches to understand bacterial wilt disease in a relatively short time and with small requirements of equipment and plant growth space.

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease

Here, we present a versatile method for tomato root transformation followed by inoculation with Ralstonia solanacearum to perform straightforward genetic analysis for the study of bacterial wilt disease.

Ralstonia solanacearum is a devastating soil borne vascular pathogen that can infect a large range of plant species, causing an important threat to ...

Genetics

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, especially, in plant reproductive biology. However, plant morphological, anatomical, and ultrastructural analyses traditionally involve time-consuming embedding and sectioning procedures for bright field, scanning, and electron microscopy. Recent progress in confocal fluorescence microscopy, state-of-the-art 3-D computer-aided microscopic analyses, and the continuous refinement of molecular techniques to be used on minimally processed whole-mount specimens, has led to an increased demand for developing efficient and minimal sample processing techniques. In this protocol, we describe techniques for properly dissecting Arabidopsis flowers and siliques, basic clearing techniques, and some staining procedures for whole-mount observations of reproductive structures.

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

In this protocol, we describe techniques for the proper dissection of Arabidopsis flowers and siliques, some basic clearing techniques, and selected staining procedures for whole-mount observations of reproductive structures.

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, ...

Inoculation Strategies to Infect Plant Roots with Soil-Borne Microorganisms

The rhizosphere harbors a highly complex microbial community in which plant roots are constantly challenged. Roots are in close contact with a wide variety of microorganisms, but studies on soil-borne interactions are still behind those performed on aboveground organs. Although some inoculation strategies for infecting model plants with model root pathogens are described in the literature, it remains difficult to get a comprehensive methodological overview. To address this problem, three different root inoculation systems are precisely described that can be applied to gain insights into the biology of root-microbe interactions. For illustration, Verticillium species (namely, V. longisporum and V. dahliae) were employed as root invading model pathogens. However, the methods can be easily adapted to other root colonizing microbes - both pathogenic and beneficial. By colonizing the plant xylem, vascular soil-borne fungi such as Verticillium spp. exhibit a unique lifestyle. After root invasion, they spread via the xylem vessels acropetally, reach the shoot, and elicit disease symptoms. Three representative plant species were chosen as model hosts: Arabidopsis thaliana, economically important oilseed rape (Brassica napus), and tomato (Solanum lycopersicum). Step-by-step protocols are given. Representative results of pathogenicity assays, transcriptional analyses of marker genes, and independent confirmations by reporter constructs are shown. Furthermore, the advantages and disadvantages of each inoculation system are thoroughly discussed. These proven protocols can assist in providing approaches for research questions on root-microbe interactions. Knowing how plants cope with microbes in the soil is crucial for developing new strategies to improve agriculture.

This protocol presents a detailed summary of strategies to inoculate plant roots with soil-borne microbes. Exemplified for the fungi Verticillium longisporum and Verticillium dahliae, three different root infection systems are described. Potential applications and possible downstream analyses are highlighted, and advantages or disadvantages are discussed for each system.

The rhizosphere harbors a highly complex microbial community in which plant roots are constantly challenged. Roots are in close contact with a wide ...

Environment

Tomato Analyzer: A Useful Software Application to Collect Accurate and Detailed Morphological and Colorimetric Data from Two-dimensional Objects

Measuring fruit morphology and color traits of vegetable and fruit crops in an objective and reproducible way is important for detailed phenotypic analyses of these traits. Tomato Analyzer (TA) is a software program that measures 37 attributes related to two-dimensional shape in a semi-automatic and reproducible manner1,2. Many of these attributes, such as angles at the distal and proximal ends of the fruit and areas of indentation, are difficult to quantify manually. The attributes are organized in ten categories within the software: Basic Measurement, Fruit Shape Index, Blockiness, Homogeneity, Proximal Fruit End Shape, Distal Fruit End Shape, Asymmetry, Internal Eccentricity, Latitudinal Section and Morphometrics. The last category requires neither prior knowledge nor predetermined notions of the shape attributes, so morphometric analysis offers an unbiased option that may be better adapted to high-throughput analyses than attribute analysis. TA also offers the Color Test application that was designed to collect color measurements from scanned images and allow scanning devices to be calibrated using color standards3. 
TA provides several options to export and analyze shape attribute, morphometric, and color data. The data may be exported to an excel file in batch mode (more than 100 images at one time) or exported as individual images. The user can choose between output that displays the average for each attribute for the objects in each image (including standard deviation), or an output that displays the attribute values for each object on the image. TA has been a valuable and effective tool for indentifying and confirming tomato fruit shape Quantitative Trait Loci (QTL), as well as performing in-depth analyses of the effect of key fruit shape genes on plant morphology. Also, TA can be used to objectively classify fruit into various shape categories. Lastly, fruit shape and color traits in other plant species as well as other plant organs such as leaves and seeds can be evaluated with TA.

Tomato Analyzer (TA) quantifies attributes of two dimensional shapes and color in a reproducible and accurate manner. A step-by-step procedure for obtaining high quality digitalized images of tomato fruit, morphological and color analyses of these images and several applications using the data generated through this software are described.

Measuring fruit morphology and color traits of vegetable and fruit crops in an objective and reproducible way is important for detailed phenotypic ...

Biology

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...

Bioengineering

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Automated Stomatal Response Measurement to Bacterial Invasion in Arabidopsis

Direct Observation and Automated Measurement of Stomatal Responses to Pseudomonas syringae pv. tomato DC3000 in Arabidopsis thaliana

Stomata are microscopic pores found in the plant leaf epidermis. Regulation of stomatal aperture is pivotal not only for balancing carbon dioxide uptake for photosynthesis and transpirational water loss but also for restricting bacterial invasion. While plants close stomata upon recognition of microbes, pathogenic bacteria, such as Pseudomonas syringae pv. tomato DC3000 (Pto), reopen the closed stomata to gain access into the leaf interior. In conventional assays for assessing stomatal responses to bacterial invasion, leaf epidermal peels, leaf discs, or detached leaves are floated on bacterial suspension, and then stomata are observed under a microscope followed by manual measurement of stomatal aperture. However, these assays are cumbersome and may not reflect stomatal responses to natural bacterial invasion in a leaf attached to the plant. Recently, a portable imaging device was developed that can observe stomata by pinching a leaf without detaching it from the plant, together with a deep learning-based image analysis pipeline designed to automatically measure stomatal aperture from leaf images captured by the device. Here, building on these technical advances, a new method to assess stomatal responses to bacterial invasion in Arabidopsis thaliana is introduced. This method consists of three simple steps: spray inoculation of Pto mimicking natural infection processes, direct observation of stomata on a leaf of the Pto-inoculated plant using the portable imaging device, and automated measurement of stomatal aperture by the image analysis pipeline. This method was successfully used to demonstrate stomatal closure and reopening during Pto invasion under conditions that closely mimic the natural plant-bacteria interaction.

Author Spotlight: Advancing Stomatal Research with Automated Aperture Measurement

Here, we present a simple method for direct observation and automated measurement of stomatal responses to bacterial invasion in Arabidopsis thaliana. This method leverages a portable stomatal imaging device, together with an image analysis pipeline designed for leaf images captured by the device.

Stomata are microscopic pores found in the plant leaf epidermis. Regulation of stomatal aperture is pivotal not only for balancing carbon dioxide uptake ...

A Strategy to Validate the Role of Callose-mediated Plasmodesmal Gating in the Tropic Response

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The establishment of an auxin gradient is a key event leading to phototropism and gravitropism. Previously, polar auxin transport (PAT) was shown to establish an auxin gradient in different cellular domains of plants. However, Han et al. recently demonstrated that for proper auxin gradient formation, plasmodesmal callose-mediated symplasmic connectivity between the adjacent cells is also a critical factor. In this manuscript, the strategy to elucidate the role of particular genes, which can affect phototropism and gravitropism by altering the symplasmic connectivity through modulating plasmodesmal callose synthesis, is discussed. The first step is to screen aberrant tropic responses from 3-day-old etiolated seedlings of mutants or over-expression lines of a gene along with the wild type. This preliminary screening can lead to the identification of a range of genes functioning in PAT or controlling symplasmic connectivity. The second screening involves the sorting of candidates that show altered tropic responses by affecting symplasmic connectivity. To address such candidates, the movement of a symplasmic tracer and the deposition of plasmodesmal callose were examined. This strategy would be useful to explore new candidate genes that can regulate symplasmic connectivity directly or indirectly during tropic responses and other developmental processes.

This article describes the methods for screening the genes controlling plasmodesmal permeability and hence auxin gradient during tropic response. This includes the measurement of the degree of tropic response in hypocotyl of Arabidopsis thaliana and checking plasmodesmal permeability by 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) loading and finally callose level assessment.

An Efficient Clearing Protocol for the Study of Seed Development in Tomato (Solanum lycopersicum L.)

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