Name: whole-mount clearing and staining of arabidopsis flower organs and siliques
Uploaded: 2018-04-12T13:00:00
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 kep...

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>Reagents and Materials</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&#39;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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fixatives</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carnoy&#39;s (Farmer&#39;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>Clearing solutions</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&#39;s 4&#189; 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>Combined clearing and staining solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Herr&#39;s IKI-4&#189;</td>
			<td></td>
			<td></td>
			<td>To a standard 4&#189; (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&#39;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&#39;s 4&#189; solution can be used to evaluate pollen abortion in flowers with mature and tricellular pollen grains. It&#39;s used on freshly harved non-fixed material.</td>
		</tr>
		<tr>
			<td>Staining solutions</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&#39;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, ...

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