Name: whole-mount clearing and staining of arabidopsis flower organs and siliques
Uploaded: 2018-04-12T13:00:00.000+00:00
Duration: 9 min 17 s
Description: Watch this Scientific Journal Video about Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques at JoVE.com

This work was supported by the University of Zurich, an IEF Marie Curie Grant (grant no. TransEpigen-254797 to A.H.), an Advanced Grant of the European Research Council (grant no. MEDEA-250358 to U.G.), and a Research and Technology Development project (grant MecanX to U.G.) from SystemsX.ch, the Swiss Initiative in Systems Biology.

1. Flower and Silique Fixation
<ol>
	<li>Harvest flowers and siliques from plants synchronized at the opening of the first flower. 
	NOTE: Under the experimental conditions used here, plants start flowering approximately 21 days after transplantation from Murashige and Skoog (MS) plates to soil. Seeds are stratified for 3&#8211;4 days at 4 &#176;C and germinated/grown on MS plates at 22 &#176;C/16 h light and 18 &#176;C/8 h dark for 8 to 10 days, before transplanting the seedlings on nutrient-rich soil in pots kept under the same conditions (Figure 1). The number of replicates depends on the specific research objective, but a minimum of 5 inflorescences (from 5 individual plants) for each treatment is recommended. If flowers are the experimental unit, a minimum of 10 flowers per replicate is recommended.</li>
	<li>Cut inflorescences or flowers (Figure 1E-H) using small scissors (e.g., nail scissors) and place them immediately in a microcentrifuge tube (for single inflorescence/flower fixation) or a conical tube (for collective fixations) containing freshly made Carnoy&#39;s, methanol/acetic acid, or FPA50 fixatives (depending on the application, see below) on ice. 
	NOTE: Samples should be completely submerged in the fixative.</li>
	<li>Leave the tissue within the fixative for 4 h to overnight at 4 &#176;C. 
	NOTE: Vacuum infiltration can be used for speeding up the penetration of the fixative into the tissue, but this may be detrimental to preserving the structure of the tissue. The authors do not implement vacuum infiltration.</li>
	<li>Remove the fixative, add enough 70% ethanol to cover the samples, and return them to 4 &#176;C for at least 24 h; samples can stay indefinitely in this solution. After removing ethanol, proceed immediately to the next step; either dissection, or SDS clearing.</li>
</ol>
2. Dissection under the Stereomicroscope
<ol>
	<li>Put freshly made 70% ethanol in a watch maker&#39;s glass placed on a small Petri dish for support.</li>
	<li>Place the inflorescence/flower/silique on this freshly made fixative and dissect under the stereomicroscope using forceps and a syringe with a needle.
	<ol>
		<li>Use a watch maker&#39;s glass or similar device that allows the samples to be totally covered with ethanol (recommended) for the dissection of inflorescences, and for tearing apart floral organs of mature flowers (sepals, petals, and stamen can be dissected at this stage) to avoid the risk of samples drying out.</li>
	</ol>
	</li>
	<li>Dissect siliques and small flower buds on a slide as described below.
	<ol>
		<li>Put the watch maker&#39;s glass with pre-processed samples aside, and use a normal slide for the final dissection.</li>
		<li>Move the sample of interest to the slide and add 10 &#181;L of fresh 70% ethanol. Work swiftly on the final dissection and add 10 &#181;L of ethanol, if necessary, to keep the sample moist without excessive liquid.</li>
		<li>For releasing pollen grains from stamens, see step 5.1. For dissecting carpels and ovules, follow the procedure described in Figure 2. This procedure applies to all stages of development of the carpel, including the stage of development of green siliques. 
		NOTE: Do not add ethanol if there is sufficient ethanol carry-over from moving the samples; dissecting very small samples is much easier in a minimal amount of liquid. Always keep only the organs of interest (sepals, petals, stamens, carpels, ovules, seeds, or pollen grains) on the slide. This procedure will ensure a uniform thickness between the slide and coverslip, corresponding to the thickness of the organ under examination, resulting in a higher homogeneity, and thus efficiency for microscopic examinations (higher number of target specimens in the same focal plane).</li>
	</ol>
	</li>
	<li>Use a small piece of blotting paper to absorb and remove as much ethanol as possible. Quickly proceed to the next step (section 3, 4, or 6) before the sample dries out.</li>
</ol>
3. Chloral Hydrate-Based Clearing and Combined Clearing-staining
NOTE: Best results for chloral hydrate-based clearing are obtained with FPA50 fixed material.
<ol>
	<li>Place 20 &#181;L of clearing solution (chloral hydrate/glycerol, modified Hoyer&#39;s medium, Herr&#39;s 4&#189;, or Herr&#39;s IKI-4&#189; solutions) on the specimen-bearing slide. Using a pair of syringes with hypodermal needles, position the specimens as needed by reducing the space between them, and flipping those where the organ of interest faces downward. Remove any remaining air bubble using the needle.</li>
	<li>Lower a coverslip sideways and gently place it, pressing very gently, and wait until the clearing solution fills the space in between. Add minimal clearing solution, if needed, to fill the entire space under the coverslip.</li>
	<li>Place the slide on a slide holder and leave it under the fume hood. Proceed to microscopic observations after at least 4 h and during the following 4&#8211;5 days.</li>
</ol>
4. Combined Alexander Staining and Herr&#39;s 4&#189; Clearing of Anthers
NOTE: The original Alexander protocol is based on releasing pollen grains on the slide before staining. An efficient and more informative, modified Alexander staining and clearing procedure is the staining of mature pollen grains within mature but non-dehiscent anthers.
<ol>
	<li>Choose freshly harvested flower buds that are about to open with non-dehiscent anthers. 
	NOTE: Do not take younger flower buds because Alexander staining is intended for mature pollen grains and does not efficiently stain immature pollen grains. A minimum of 5 flowers per treatment harvested from different plants and inflorescences is recommended.</li>
	<li>Prepare a 96-well plate with enough Alexander&#39;s solution to submerge a flower bud. Expose the anthers by removing the sepals and petals and immerse them in Alexander&#39;s solution. Keep the samples under the fume hood for 1&#8211;3 h.</li>
	<li>Replace Alexander&#39;s solution with Herr&#39;s 4&#189; solution and place the multi-well plate back under the fume hood for an overnight incubation.</li>
	<li>Using forceps, gently move the cleared flower buds onto a new slide. Since some carry-over of Alexander&#39;s solution may occur, use a blotting paper to remove as much clearing solution as possible.</li>
	<li>Dissect the stamens and remove all other tissues. Follow the steps in section 3 using Herr&#39;s 4&#189; solution.</li>
</ol>
5. Removing the Exine from Pollen Grains
NOTE: We recommend using Carnoy&#39;s fixative for DAPI staining.
<ol>
	<li>Follow the fixation and dissection procedures described in sections 1 and 2. By now, one should have one to many stamens on the slide within a minimum amount of 70% ethanol.</li>
	<li>Use a blotting paper to remove excess ethanol, and add 5&#8211;10 &#181;L of DAPI solution. Using a couple of syringes with needles, release the pollen grains within that small amount of solution (e.g., use one syringe to immobilize the stamen and the other to release the pollen grains). Add another 5 &#181;L of DAPI if the solution dries out.</li>
	<li>Removing all debris of the stamen is a critical step, such that only pollen grains are left between the slide and the coverslip.</li>
	<li>Place a coverslip on the specimen-bearing slide by lowering it sideways and add the minimal amount of DAPI solution required to fill the space. Place an index finger on the coverslip, and without squashing too much make a quick, firm, and short sliding movement. 
	NOTE: To gauge the necessary force and amplitude of the sliding movement, this step should be practiced several times, checking the results each time under the microscope.</li>
	<li>Add DAPI solution if needed and place the slide in a humid box in the dark at 4 &#176;C for 15 min to 1 h. Proceed to observe samples under wide-field fluorescence or confocal microscopy within 24 h. Try to combine this procedure with SDS clearing to check whether further improvements can be obtained.</li>
</ol>
6. Sodium Dodecyl Sulfate (SDS) Clearing
NOTE: Depending on the floral organ to be analyzed, and on the researcher&#39;s skills to dissect very soft and small specimens, the SDS treatment can be carried out either before (for experienced researchers) or after the dissection step (for less experienced researchers) on methanol-acetic acid fixed material.
<ol>
	<li>Use a watch maker&#39;s glass, a concave slide, or a microcentrifuge tube to incubate dissected or non-dissected samples in 1% SDS and 0.2 N NaOH solution overnight at room temperature.</li>
	<li>Handle with care because the sample is very soft and has a transparent appearance. Rinse several times with water. 
	NOTE: Remnants of the SDS solution might lead to precipitation when adding the staining solution, e.g., aniline blue.</li>
	<li>For non-dissected samples, follow the dissection procedure described in section 2, using water instead of 70% ethanol. After dissecting the desired tissue, remove any remnants and debris, and any excess water with a blotting paper, then add 20&#8211;40 &#181;L of staining solution, and proceed with step 6.5.</li>
	<li>For dissected samples, carefully move the sample from the washing solution and place it onto a clean slide, and add 20&#8211;40 &#181;L of staining solution.</li>
	<li>Gently place a coverslip on the slide by lowering it sideways. Take care not to squash the preparation. If the tissue is too soft, place any thin separator in between the slide and the coverslip at its corners (e.g., tape or a broken coverslip), leaving a chamber-like space between slide and coverslip, such that the coverslip doesn&#39;t crush the specimen.</li>
	<li>Place all the slides within a humid box, cover it with aluminum foil, and place it at 4 &#176;C for at least 1 h. Proceed to observe the specimen using widefield fluorescence or confocal microscopy within 24 h.</li>
</ol>

The authors have nothing to disclose.

The existence of many flower buds within a single inflorescence of Arabidopsis, spanning all flower developmental stages, offers a unique opportunity for studies aimed at characterizing an effect of a treatment or a developmental feature simultaneously across the different stages of flower development. A good reference point between different individual plants is the opening of the first flower of the main inflorescence. Plants are treated in such a way that flowering is synchronized as much as possible (e.g...

Flowers are among the most important defining organs of angiosperms. Flowering plants appeared some 90&#8211;130 million years ago1, and diversified so fast that their rapid appearance was described as an &#34;abominable mystery&#34; by Charles Darwin2. The interests of plant researchers in flower development are diverse. Some research has focused on understanding the evolutionary origin of the flower as a whole, or the evolution of specific anatomical, structural, and functional properties of flowers3,4,5,6. The high variation in floral form and structure, as well as the modes of sexual and asexual reproduction relying on them, make the flower a highly complex structure. This has led to diverse efforts to characterize the anatomy and structural features of floral organs, using light and electron microscopical techniques that could be combined with genetic and molecular investigations7. Furthermore, as the source of fruits and seeds, flowers are of paramount importance for human and animal nutrition. Therefore, the characterization of flower and fruit development has many implications for applied research, including food security for an ever-increasing human population and ecological conservation strategies under a changing environment8,9,10.
Flower development in Arabidopsis starts with flower induction and the transformation of the vegetative meristem to an inflorescence (group of flowers) meristem. Flower primordia are initiated laterally on the flank of the inflorescence meristem11. The floral organ primordia form progressively in concentric whorls from the outside to the center of the flower, and eventually develop into sepals, petals, stamens, and carpels7. These floral organs fulfill distinct nutritive, protective, and functional (e.g., pollinator attraction) roles in different plant species, with the sexual organs sustaining the development of male and female gametophytes, respectively12,13. The gametophytes, in turn, each differentiate a pair of male (sperm) and female gametes (egg and central cell), which unite upon double fertilization to form the next generation, the zygote, and the primary endosperm, a terminal tissue supporting the development of the embryo14,15. Fruit and seed development support the growth, maturation, and preservation of the embryo and, eventually, its dispersal. Extensive research has been performed to characterize flower and embryo development in diverse plant species, especially in the model species Arabidopsis7,16,17.
Early microscopic analyses of flower development were based on time-consuming sample processing and observation techniques, such as paraffin or resin embedding and sectioning, combined with light or electron microscopy. These traditional microscopic techniques were often used in combination with molecular genetic investigations, such as microscopical analyses of mutants, the localization of RNA by in situ hybridization, or the immuno-detection of proteins. Recent progress in wide-field and confocal fluorescence microscopy, in state-of-the-art 3-D computer-aided image analyses, and the continuous refinement of molecular methods that can be used on minimally processed whole-mount specimens, has led to a need for efficient and minimal sample processing techniques that are preferentially amenable to quantitative analyses. In recent years, significant progress has been made in developing clearing techniques on whole-mount animal specimen. They render the sample transparent either by using aqueous urea- or sugar-based reagents (e.g., SCALE, SeeDB, CUBIC)18,19,20, or by selectively removing lipids (using the detergent SDS) after embedding samples in stable hydrogels; the removal of lipids can be achieved either by passive diffusion (e.g., modified CLARITY protocol21, PACT-PARS-RIMS22) or actively by electrophoresis (original CLARITY protocol23 and ACT-PRESTO24). Encouraged by this fast progress, some derived techniques are also emerging for use in plants.
In this methods paper focused on the model Arabidopsis, we describe the procedure for the proper dissection of flower buds, flowers, and young siliques, and the clearing of whole-mount samples for diverse staining and observation procedures using classical or a recent SDS-based clearing method. Examples for starch, callose, and chromatin staining are given. Although these procedures may need further improvements and adaptations when used with other species, we hope they will set the stage for further research on these simple but critical methods that are the starting point of many research projects.

<table><tbody><tr><td>&lt;strong&gt;Reagents and Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ethanol</td><td>Scharlau</td><td>ET00102500</td><td></td></tr><tr><td>Acetic Acid</td><td>Applichem</td><td>A3686,2500</td><td>100% Molecular biology grade</td></tr><tr><td>Glacial Acetic Acid</td><td>Sigma-Aldrich</td><td>320099</td><td>Molecular Biology Grade</td></tr><tr><td>Methanol</td><td>Scharlau</td><td>ME03062500</td><td></td></tr><tr><td>Formaldehyde Solution</td><td>Sigma-Aldrich</td><td>F1635</td><td></td></tr><tr><td>Propionic acid</td><td>Sigma-Aldrich</td><td>81910-250 ml</td><td></td></tr><tr><td>Chloral hydrate</td><td>Sigma-Aldrich</td><td>15307</td><td></td></tr><tr><td>Glycerol</td><td>Roth</td><td>3783.1</td><td></td></tr><tr><td>Gum arabic</td><td>Fluka</td><td>51198</td><td></td></tr><tr><td>Lactic acid</td><td>Fluka</td><td>69773</td><td></td></tr><tr><td>Phenol</td><td>Sigma-Aldrich</td><td>77607-250ML</td><td>We used liquid phenol (use the density to find the required volume for your solution)</td></tr><tr><td>Clove oil</td><td>Sigma-Aldrich</td><td>C8392-100ML</td><td></td></tr><tr><td>Xylene</td><td>Roth</td><td>4436.1</td><td></td></tr><tr><td>Iodine</td><td>Fluka</td><td>57665</td><td></td></tr><tr><td>Potassium iodide</td><td>Merck</td><td>5043</td><td></td></tr><tr><td>Malachite Green</td><td>Fluka</td><td>63160</td><td></td></tr><tr><td>Fuchsin acid</td><td>Fluka</td><td>84600</td><td></td></tr><tr><td>Orange G</td><td>Sigma</td><td>7252</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate</td><td>Sigma-Aldrich</td><td>L3771</td><td>Molecular Biology Grade</td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>71690</td><td></td></tr><tr><td>Sodium di-Hydrogen Phosphate</td><td>Applichem</td><td>A1047,1000</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma-Aldrich</td><td>S9763-1KG</td><td></td></tr><tr><td>Potassium phosphate</td><td>Sigma-Aldrich</td><td>04347</td><td></td></tr><tr><td>EDTA</td><td>Applichem</td><td>A2937,1000</td><td></td></tr><tr><td>Calcofluor</td><td>Sigma</td><td>F6259</td><td>Fluorescent brightener 28</td></tr><tr><td>Auramine</td><td>Chroma</td><td>10120</td><td></td></tr><tr><td>DAPI</td><td>Sigma</td><td>D9542</td><td>toxic</td></tr><tr><td>Triton-X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Aniline blue</td><td>Merck</td><td>1275</td><td></td></tr><tr><td>MS medium</td><td>Carolina</td><td>19-57030</td><td></td></tr><tr><td>Nutrient-rich substrate</td><td>Einheitserde</td><td>ED73</td><td></td></tr><tr><td>Watch maker&#039;s glass</td><td></td><td></td><td>No specific brand</td></tr><tr><td>15 ml falcon centrifuge tubes</td><td>VWR</td><td>62406-200</td><td></td></tr><tr><td>Dumond Forceps</td><td>Actimed</td><td>0208-5SPSF-PS</td><td></td></tr><tr><td>Forceps</td><td>DUMONT BIOLOGY</td><td>0108-5</td><td></td></tr><tr><td>Syringe</td><td>BD</td><td>BD Plastipak 300013</td><td>1 ml</td></tr><tr><td>Preparation needle</td><td>BD</td><td>BD Microlance 304000</td><td></td></tr><tr><td>Microscope slides</td><td>Thermo Scientific</td><td>10143562CE</td><td>cut edges</td></tr><tr><td>Coverslips</td><td>Thermo Scientific</td><td>DV40008</td><td></td></tr><tr><td>Humid box</td><td></td><td></td><td>A plastic box with damp paper towel and slide supports inside</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Fixatives&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Carnoy&#039;s (Farmer&#039;s) fixative</td><td></td><td></td><td>Absolute ethanol : glacial acetic acid, 3:1 (ml:ml)</td></tr><tr><td>Methanol/acetic acid fixative</td><td></td><td></td><td>50 % (v/v) methanol, 10 % (v/v) glacial acetic acid in deionized water</td></tr><tr><td>FPA50 fixative</td><td></td><td></td><td>Formalin, propionic acid, 50% ethanol; 5:5:90 (ml:ml:ml)</td></tr><tr><td>&lt;strong&gt;Clearing solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Chloral hydrate/glycerol</td><td></td><td></td><td>Chloral hydrate : glycerol : water, 8:1:2 (g:ml:ml). Can be used for all flower developmental stages and for silique development with DIC microscopy. The best fixative is the formaline based FPA50</td></tr><tr><td>Modified Hoyer</td><td></td><td></td><td>Gum arabic 7.5 g, chloral hydrate 100 g, glycerol 5 ml , water 30 ml. Can be used for all flower developmental stages and for silique development with DIC microscopy. The best fixative is the formaline based FPA50</td></tr><tr><td>Herr&#039;s 4&amp;frac12; clearing fluid</td><td></td><td></td><td>Lactic acid, chloral hydrate, phenol crystals, clove oil, xylene; 2:2:2:2:1, by weight. Can be used for all flower developmental stages (especially for stamen development) and for silique development with DIC microscopy. The best fixative is the formaline based FPA50</td></tr><tr><td>SDS/NaOH solution</td><td></td><td></td><td>Mix-dilute the the SDS and the NaOH stock solution to 1% SDS / 0.2 N NaOH (10x dilution). For all stages of flower and silique developmental stages. The best fixative is the methano/acetic acid fixative; the other two fixatives can also be used. Can be combined with calcofluor, auramine, DAPI, and aniline blue staining solution.</td></tr><tr><td>SDS stock solution</td><td></td><td></td><td>10 % (w/v) sodium dodecyl sulphate. Dissolve 10 g sodium dodecyl sulphate in 80 ml deionized water and make up to 100 ml with deionized water.</td></tr><tr><td>NaOH stock solution</td><td></td><td></td><td>2 N NaOH solution: dissolve 4 g of NaOH in 40 ml of deionized water and make up to 100 ml with deionized water</td></tr><tr><td>&lt;strong&gt;Combined clearing and staining solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Herr&#039;s IKI-4&amp;frac12;</td><td></td><td></td><td>To a standard 4&amp;frac12; (9 g in total) add: 100 mg iodine, 500 mg potassium iodide. This clearing solution can be used for all flower developmental stages and for silique development, either for increasing contrast or for characterizing starch dynamics. Use FPA50 for structural analysis and Carnoy&#039;s fixative for quantitative starch analysis.</td></tr><tr><td>Alexander staining</td><td></td><td></td><td>Ethanol 95% 10ml, malachite green (1% in 95% EtOH) 1 ml, fuchsin acid (1% in ddH2O) 5ml, orange G (1% in ddH2O) 0.5ml, phenol 5g, chloral hydrate 5g, glacial acetic acid 2ml, glycerol 25ml . This clearing/staining alone or in combination with Herr&#039;s 4&amp;frac12; solution can be used to evaluate pollen abortion in flowers with mature and tricellular pollen grains. It&#039;s used on freshly harved non-fixed material.</td></tr><tr><td>&lt;strong&gt;Staining solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Calcofluor solution</td><td></td><td></td><td>Calcofluor 0.007% in water (g:ml). Originally used as an optical brightner. Can be used for staining cellulose, carboxylated polysaccharides and callose in cell walls. Frequently used to stain the intine of the pollen grain. All three fixatives can be used with this solution.</td></tr><tr><td>Auramine solution</td><td></td><td></td><td>Auramine 0.01% in water (g:ml). This lipophilic fluorscent dye can be used for staining cuticles, cutin, and exine among others. All three fixatives can be used with this solution</td></tr><tr><td>Calcofluor-Auramine mixture</td><td></td><td></td><td>Auramine solution : Calcofluor solution, 3:1. Can be used for a combined staining by both solutions. Other proportions can be assayed maintaining a smaller proportion of calcofluor with respect to auramine.</td></tr><tr><td>DAPI solution</td><td></td><td></td><td>DAPI 0.4 ug/ml, 0.1 M sodium phosphate buffer (pH 7), 0.1% Triton-X-100, 1 mM EDTA. This solution can be used for staining chromosome spreads during male and female meiosis, and cell nuclei of any tissue. Frequently used for studying pollen grain development. Carnoy&#039;s and methanol/ acetic acid are the best fixatives for this solution. Formaldehyde-based fixatives such as FPA50 may interfere with the staining. Excitation in the UV and maximum emission around 461 nm.</td></tr><tr><td>Sodium phosphate buffer (0.1 M)</td><td></td><td></td><td>Proton receptor: 0.2 M Na2HPO4, proton donor: 0.2 M NaH2PO4, ratio proton donor / proton receptor: 1.364 ( for a pH 7)</td></tr><tr><td>Aniline blue solution</td><td></td><td></td><td>0.1% (w/v) aniline blue, 108 mM K3PO4 (pH 11), 2% glycerol. This solution can be used for staining callose and cellulose of many stages of development (e.g callose deposition in male and female terads, callose plugs in pollen tubes). Excitation in the UV and maximum emission around 455. It can also be excited at 514 nm with emission in the red for cell content staining.</td></tr></tbody></table>

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.

Arabidopsis belongs to the Brassicacea family, bearing inflorescences with bisexual flowers arranged in a corymb (Figure 1). Each flower has four sepals, four petals, six stamens (four long and two short), and a syncarpous gynoecium consisting of two congenitally fused carpels (Figure 1F-H) arranged in four concentric whorls25,26. Arabidopsis...

Watch this Scientific Journal Video about Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques at JoVE.com

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.

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, ...

whole-mount-clearing-staining-arabidopsis-flower-organs

Research

JoVE Journal

Developmental Biology

16.7K Views.  University of Zurich. 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.

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

Live Confocal Imaging of Developing Arabidopsis Flowers

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope.

Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers.

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Here, we describe the generation and application of a set of transgenic Arabidopsis thaliana lines enabling inducible, tissue-specific expression in the three main meristems, the shoot apical meristem, the root apical meristem, and the cambium.

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the ...

Genetics

Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis

The symplastic tracer 5(6)-carboxyfluorescein diacetate (CFDA) has been widely applied in living plants to demonstrate the intercellular connection, phloem transport and vascular patterning. This protocol shows bottom-to-top carboxyfluorescein (CF) movement in the Arabidopsis by using the root-cutting and the hypocotyl-pinching procedure respectively. These two different procedures result in different efficiencies of CF movement: about 91% appearance of CF in the shoots with the hypocotyl-pinching procedure, whereas only about 70% appearance of CF with the root-cutting procedure. The simple change of loading sites, resulting in significant changes in the mobile efficiency of this symplastic dye, suggests CF movement might be subject to the symplastic regulation, most probably by the root-hypocotyl junction.

Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis

The goal of this protocol is to show how to load the CFDA into different sites of the bottom parts of Arabidopsis. We then present the resulting distribution pattern of CF in the shoots.

The symplastic tracer 5(6)-carboxyfluorescein diacetate (CFDA) has been widely applied in living plants to demonstrate the intercellular connection, ...

Environment

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Biology

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.

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.

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

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

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 ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

Optical Clearing of Plant Tissues for Fluorescence Imaging

It is challenging to directly observe the internal structure of multi-layered and opaque plant specimens, without dissection, under a microscope. In addition, autofluorescence attributed to chlorophyll hampers the observation of fluorescent proteins in plants. For a long time, various clearing reagents have been used to make plants transparent. However, conventional clearing reagents diminish fluorescent signals; therefore, it has not been possible to observe the cellular and intracellular structures with fluorescent proteins. Reagents were developed that can clear plant tissues by removing chlorophyll while maintaining fluorescent protein stability. A detailed protocol is provided here for the optical clearing of plant tissues using clearing reagents, ClearSee (CS) or ClearSeeAlpha (CSA). The preparation of cleared plant tissues involves three steps: fixation, washing, and clearing. Fixation is a crucial step in maintaining the cellular structures and intracellular stability of fluorescent proteins. The incubation time for clearing depends on the tissue type and species. In Arabidopsis thaliana, the time required for clearing with CS was 4 days for leaves and roots, 7 days for seedlings, and 1 month for pistils. CS also required a relatively short time of 4 days to make the gametophytic leaves of Physcomitrium patens transparent. In contrast, pistils in tobacco and torenia produced brown pigment due to oxidation during CS treatment. CSA reduced the brown pigment by preventing oxidation and could make tobacco and torenia pistils transparent, although it took a relatively long time (1 or 2 months). CS and CSA were also compatible with staining using chemical dyes, such as DAPI (4',6-diamidino-2-phenylindole) and Hoechst 33342 for DNA and Calcofluor White, SR2200, and Direct Red 23 for the cell wall. This method can be useful for whole-plant imaging to reveal intact morphology, developmental processes, plant-microbe interactions, and nematode infections.

Here, a method is described for making plant tissues transparent while maintaining the stability of fluorescent proteins. This technique facilitates deep imaging of cleared plant tissues without physical sectioning.

It is challenging to directly observe the internal structure of multi-layered and opaque plant specimens, without dissection, under a microscope. In ...

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls requires cellular reprogramming and changes in the metabolism of the infected plant. This is necessary to establish a pathogen-oriented physiological sink toward which the host nutrients are redirected. For a complete understanding of this particular plant-pathogen interaction and the mechanisms by which host growth and development are subverted and repatterned, it is essential to track and observe the internal changes accompanying gall formation with cellular resolution. Methods combining fluorescent stains and fluorescent proteins are often employed to study anatomical and physiological responses in plants. Unfortunately, the large size of galls and their low transparency act as major hurdles in performing whole-mount observations under the microscope. Moreover, low transparency limits the employment of fluorescence microscopy to study clubroot disease progression and gall formation. This article presents an optimized method for fixing and clearing galls to facilitate epifluorescence and confocal microscopy for inspecting P. brassicae-infected galls. A tissue-clearing protocol for rapid optical clearing was used followed by vibratome sectioning to detect anatomical changes and localize gene expression with promoter fusions and reporter lines tagged with fluorescent proteins. This method will prove useful for studying cellular and physiological responses in other pathogen-triggered structures in plants, such as nematode-induced syncytia and root knots, as well as leaf galls and deformations caused by insects.

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae

The present protocol describes an optimized method for the histological observation of galls induced by Plasmodiophora brassicae. Vibratome sections of hypocotyls are cleared before fluorescence imaging to study the involvement of transcription factors and phytohormones during disease progression. This protocol overcomes resin embedding limitations, enabling in planta visualization of fluorescent proteins.

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls ...

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

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Chapters in this video

Live Confocal Imaging of Developing Arabidopsis Flowers

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::β-glucuronidase Reporter Gene Expression

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

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

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

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Optical Clearing of Plant Tissues for Fluorescence Imaging

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae