Name: an efficient clearing protocol for the study of seed development in tomato (solanum lycopersicum l.)
Uploaded: 2022-09-07T14:15:00
Description: Watch this Scientific Journal Video about An Efficient Clearing Protocol for the Study of Seed Development in Tomato Solanum lycopersicum L. at JoVE.com

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...

<ol>
	<li>. FAOSTAT Available from: <a target="_blank" href="https://www.fao.org/faostat/en/#data/QCL">https://www.fao.org/faostat/en/#data/QCL</a> (2022)</li><li>Olmstead, R. G., Bohs, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+summary+of+molecular+systematic+research+in+Solanaceae:+1982-2006.">A summary of molecular systematic research in Solanaceae: 1982-2006.</a> Acta Horticulturae. 745, 255-268 (2007).</li><li>Consortium, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tomato+genome+sequence+provides+insights+into+fleshy+fruit+evolution.">The tomato genome sequence provides insights into fleshy fruit evolution.</a> Nature. 485 (7400), 635-641 (2012).</li><li>Ebert, A. W., Chou, Y. 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E., Jurgens, G., Hulskamp, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryo+and+endosperm+development+is+disrupted+in+the+female+gametophytic+capulet+mutants+of+Arabidopsis.">Embryo and endosperm development is disrupted in the female gametophytic capulet mutants of Arabidopsis.</a> Genetics. 162 (4), 1911-1925 (2002).</li><li>Kataoka, K., Uemachi, A., Yazawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fruit+growth+and+pseudoembryo+development+affected+by+uniconazole,+an+inhibitor+of+gibberellin+biosynthesis,+in+pat-2+and+auxin-Induced+parthenocarpic+tomato+fruits.">Fruit growth and pseudoembryo development affected by uniconazole, an inhibitor of gibberellin biosynthesis, in pat-2 and auxin-Induced parthenocarpic tomato fruits.</a> Scientia Horticulturae. 98 (1), 9-16 (2003).</li><li>de Jong, M., Wolters-Arts, M., Feron, R., Mariani, C., Vriezen, W. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,000 &#181;L pipette</td>
			<td>GILSON</td>
			<td>FA10006M</td>
			<td></td>
		</tr>
		<tr>
			<td>1,000 &#181;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 &#181;L pipette</td>
			<td>Eppendorf</td>
			<td>3120000275</td>
			<td></td>
		</tr>
		<tr>
			<td>5,000 &#181;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 ...

This method can help researches quickly and effortlessly find abnormal periods of tomato embryo development. The main advantage of this protocol is a treatment of tomato seeds with sodium hypochlorite which provides a basis for the observation of tomato embryos. Begin with the chemical preparation and harvesting of the Solanum lycopersicum fruits three to 23 days after flowering or DAF.Immediately put the fruits on ice and divide them into early, middle and late fruits, based on the days after flowering. Break the early fruit, put it on a slide and collect fresh seeds carefully with precision forceps under 1x to 4x magnification of the stereo microscope. For middle and late fruits, break the fruit and directly collect fresh seeds using precision forceps.In the conventional method, transfer the freshly collected seeds immediately into a two milliliter centrifuge tube containing 1.5 milliliters FAA fixative and place the tube on the orbital shaker at 5 rpm for four hours at room temperature. Then dehydrate the seeds in 70%95%and 100%ethanol for one hour each on an orbital shaker at 5 rpm. Once done, place the seed in the three to five drops of clearing solution present on the slide, and gently cover it with a cover slip.Use a single concave slide for middle and late seeds. Depending on the developmental stages of the seeds, incubate them at room temperature for clearing. After incubation, observe the samples with a differential interference contrast, or 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. Transfer the fresh seeds collected from the broken fruits into a two milliliter centrifuge tube containing 1.5 milliliters of disinfectant solution. Incubate the samples on an orbital shaker at 30 rpm for three to 50 minutes at room temperature.Once the innermost seed coat outline is clearly visible, discard the disinfectant solution. For middle and late seeds, transfer the seeds onto a slide. Use forceps and a dissecting needle to remove the seed mucilage under 1x to 4x magnification of the stereo microscope.Transfer the seeds back into the original centrifuge tube using forceps. Wash them five times in 1.5 milliliters of deionized water for 10 seconds each, and discard the deionized water. Next, add the double volume of the clearing solution to the seeds.Depending on the seed stage, give the intermittent vacuum treatment for zero to 50 minutes, with 10 minute intervals after each vacuum treatment of 10 minutes. After vacuum treatment, replace the clearing solution. To facilitate the clearing of seeds, place the centrifuge tube protected from light for 30 minutes to seven days at room temperature.In the case of late embryos, replace the solution daily with a fresh clearing solution, followed by subjecting the seeds to vacuum treatment for 10 minutes. Place the cleared seeds on the slide or single concave slide. Using the DIC microscope equipped with a digital camera, adjust and optimize the transmitted light brightness, DIC slider and condenser aperture in real time for each sample and capture the image.In the conventional method of clearing S.lycopersicum seeds, the dense endosperm cells blocked the visualization of early embryos at 3 and 6 DAF. The decreased permeability during the seed growth resulted in fuzzy images after 9 DAF. The seed mucilage and seed coat gradually became denser, preventing the penetration of clearing agents from 13 DAF onward.The outline of the embryo inside 14 to 19 DAF seeds was extremely blurred, Despite the extended treatment time of seven days. The internal structure in 22 DAF seeds was completely invisible. The stripped seed coat mucilage produced by the seed coat epidermal cells was prominent from 13 DAF onward.The sodium hypochlorite treatment enabled a clear identification of the inner seed coat and detachment of most of the adherent mucilage without seed damage. In the optimized clearing protocol the seeds showed satisfactory transparency in all tested developmental stages. The distinct cell layers of the seed coat at 3 DAF, distinguishable endosperm cells at 5 DAF, the stick shaped embryo at seven DAF, the globular stage at 9 DAF, and the heart stage at 11 DAF were observed.In the optimized protocol, the degree of the curl of cotyledons and shoot apical meristem was very easily captured at various stages of embryonic development. Sterilization removes seed mucilage and seed coat pigment, which enhance seed penetration and visualization. Vacuum treatment can accelerate clearing solution penetration.

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