Name: A Method for Characterizing Embryogenesis in Arabidopsis (Video) | JoVE Journal
Uploaded: 2017-08-04T10:00:00.000+00:00
Duration: 10 min 24 s
Description: Learn to characterize embryo pattern formation in Arabidopsis using DIC microscopy and GFP-tagged markers. This method enables detailed study of gene function and cell identity during plant embryogenesis.

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5 minutes

We thank Dr. Jessica Habashi for critically reading of the manuscript. We thank Dr. Xianyong Sheng of the Imaging Center, College of life Sciences, Capital Normal University (Beijing, China), for performing the localization of DR5-GFP assay. This work was supported by grants from the Beijing Municipal Government Science Foundation (CIT&#38;TCD20150102) and from the National Natural Science Foundation of China (31600248).

NOTE: This protocol has three parts: 1) observing the pattern formation in wild-type Arabidopsis embryos using DIC microscopy; 2) characterizing the embryo pattern formation via the observation of marker protein expression using confocal laser scanning microscopy; and 3) determining the role of a specific gene in embryogenesis (using the naa10 mutant as an example).
1. Ovule Clearing
<ol>
	<li>Plant material preparation 
	NOTE: The protocol described here is an example on how to grow healthy plants; other ways to grow healthy plants also work.
	<ol>
		<li>Add 100 seeds to a 1.5 mL tube, and then add 1 mL of 1.5% sodium hypochlorite (mix 15 mL of 10% chlorine with 85 mL of water) to sterilize the seeds for 5 min. Invert the tube several times to ensure that the seeds and solution are well-mixed to sterilize the surface seeds completely.</li>
		<li>Force the seeds to the bottom of the tube by centrifugation for 30 s at 1,200 x g, remove the supernatant, and wash the seeds with sterile water four times at a bench to remove the residual sodium hypochlorite.</li>
		<li>Add 4.4 g of Murashige and Skoog (MS) basal salt mixture and 10 g of sucrose to 1 L of ultrapure water. Stir the mixture to dissolve the salts and sucrose. Adjust the solution to pH 5.8 with 1 M KOH, add 3 g of gelling agent, such as Phytagel, to the MS-sucrose solution, and sterilize the solution at 121 &#176;C for 15 min to make MS medium.</li>
		<li>Pour 30 mL of the MS medium into a 12-cm culture dish to generate MS medium plates.</li>
		<li>Place the sterilized seeds on the MS plate. Transfer the plate to a growth chamber and keep the plate under long-day conditions (22 &#176;C for 16 h in the light and 18 &#176;C for 8 h in the dark) after seed stratification (3 days at 4 &#176;C in the dark).</li>
		<li>After 8 days of growth, transfer the plants to soil (mix nutrient-rich soil and vermiculite at a 1:1 ratio) in a tray, cover the tray with a white, transparent lid for 5 days to avoid water loss, and leave a gap to avoid vitrification of the plants.</li>
		<li>Take the lid off the tray and cultivate the plants in a walk-in chamber under long-day conditions (see 1.1.5). Switch the light on at 6:00 am and off at 10:00 pm. After about 20 days of growth in the walk-in chamber, the plants will have flowered and produced siliques.</li>
		<li>Mark the flower buds with a thread at the position of the seed stalks in the inflorescence at 8:00 pm (do not use the first to the third flower buds; the siliques generated from these flower buds usually develop a few aborted seeds, and this may alter the proportion of normal to abnormal ovules). Define the beginning of pollination of the ovule as when the marked bud opens to flower at 8:00 am the next morning.</li>
	</ol>
	</li>
	<li>Preparation of Hoyer&#39;s solution
	<ol>
		<li>Add 24 g of chloral hydrate to a 50-mL tube and wrap the tube completely with aluminum foil (solutions of chloral hydrate are not stable under light).</li>
		<li>Next, add 9 mL of ultrapure water and 3 mL of glycerol to the above solution and shake the tube to dissolve the chloral hydrate. To make Hoyer&#39;s solution, let the tube stand overnight at room temperature to eliminate any bubbles.</li>
	</ol>
	</li>
	<li>Ovule separation and clearance
	<ol>
		<li>Attach a piece of double-sided adhesive tape to a microscope glass slide. Place a silique grown for the desired number of days after pollination (DAP) on the slide with the pseudoseptum perpendicular to the surface; this will make it easy to split the carpels (Figures 1A and 1B).</li>
		<li>Split the carpels on both sides of the pseudoseptum with a syringe needle and attach the two dissected carpels to the slide so that the ovules in the silique are visible under a stereoscope (Figures 1C and 1D).</li>
		<li>Dispense 70 &#181;L of Hoyer&#39;s solution onto the glass slide, and then moisten the tip of a pair of fine-tipped tweezers with Hoyer&#39;s solution by dipping. Using the stereoscope, move the ovules into the Hoyer&#39;s solution on the slide with the tweezers to clean the embryos. Apply a coverslip to the slide gently to avoid generating any bubbles.</li>
		<li>Put the finished slide into a box in a dark room for 2 - 12 h (the more mature an ovule is, the more time will be needed) at room temperature. Embryos at the 2/4-cell stage (1 - 2 DAP) can be observed microscopically after 2 h, while embryos between the octant and early globular stages (3 DAP) can be observed after 4 h. More mature embryos (4 - 8 DAP) can be observed after 12 h.</li>
		<li>Examine the cleared ovules using the DIC function of a microscope. Locate the embryos under a low-power field (10X) and then change to a high-power field (100X oil immersion lens) to observe the cellular pattern in the developed embryos.</li>
		<li>Examine the embryos at different developmental stages.</li>
	</ol>
	</li>
</ol>
2. Characterization of Embryo Patterning and Cell Identity via the Observation of Marker Protein Expression
<ol>
	<li>Preparation of plant materials
	<ol>
		<li>Clone the promoter and coding sequence of the marker gene or hormone response element and then fuse it with a fluorescent protein (such as GFP); next, insert the construct into the binary vector (such as pCambia1300). Here, the auxin response element DR5 is presented as an example10.</li>
		<li>Introduce the resulting DR5-GFP plasmid into the wild-type plants by Agrobacterium tumefaciens-mediated transformation11. Select the T1 plants using MS medium plates containing hygromycin (50 mg/mL of mother liquor, diluted 1:1,000). The transgenic plants will exhibit long roots and generate two rosette leaves after 8 days of growth under the conditions described in 1.1.5.</li>
		<li>Transfer the transgenic plants to soil and cultivate them as indicated in 1.1.6 and 1.1.7.</li>
		<li>Collect the T2 seeds from the independent T1 lines. Sow the seeds on MS-hygromycin plates, select the plants carrying one copy of the DR5-GFP insertion (the ratio of resistant plants to sensitive plants will be roughly 3:1), and transfer them to soil.</li>
		<li>Cultivate the T2 plants as indicated in 1.1.6 and 1.1.7.</li>
		<li>Collect the T3 seeds from the T2 plants. Sow the seeds on MS-hygromycin plates, select the homozygous transgenic plants (all of the plants from a single line will exhibit hygromycin resistance), and transfer them to soil.</li>
		<li>Mark the flower buds of the homozygous T3 plants as indicated in 1.1.8.</li>
	</ol>
	</li>
	<li>GFP signal observation
	<ol>
		<li>Mix 3 mL of glycerol with 47 mL of ultrapure water to generate a 6% glycerol solution.</li>
		<li>Attach a piece of double-sided adhesive tape to a glass microscope slide and fix a silique as indicated in 1.3.1.</li>
		<li>Split the carpels on both sides of the pseudoseptum with a syringe needle and attach the two dissected carpels to the slide under a stereoscope as indicated in 1.3.2.</li>
		<li>Dispense 30 &#181;L of 6% glycerol onto the glass slide and place all of the ovules from the dissected carpels into the solution using fine-tipped tweezers. Place a coverslip on the slide gently.</li>
		<li>As the seed coat and endosperm in each ovule can prevent the observation of GFP signals in the embryo, extract the embryo from the ovule. Tap the slide quickly and gently to extrude the embryo from the ovule (the more mature an ovule is, the less force should be applied to the slide).</li>
		<li>Examine the slide for GFP fluorescence using confocal laser scanning microscopy. Find the isolated embryos under a low-power field (10X) and record their location, then change to a high-power field (100X oil immersion lens) to observe DR5-GFP expression in the embryos by setting the excitation light source to 488 nm for detection of the GFP signal.</li>
		<li>Detect the GFP expression in the embryos to select the suitable T3 line for further analysis.</li>
	</ol>
	</li>
</ol>
3. Characterization of Embryo Pattern Formation in an Embryo-lethal Mutant via Ovule Clearance and Marker Protein Observation: Naa10 as an Example
<ol>
	<li>Embryo patterning in naa10 mutant plants
	<ol>
		<li>Prepare the plant materials as indicated in 1.1.1-1.1.7.</li>
		<li>Identify the naa10+/&#8722; plants by PCR using gene-specific primers5, and then mark the flower buds of the naa10+/&#8722; plants as indicated in 1.1.8.</li>
		<li>Perform ovule clearance and the microscopic examination of naa10+/&#8722; embryos as indicated in 1.2 and 1.3.</li>
	</ol>
	</li>
	<li>Marker protein analysis using naa10 embryos
	<ol>
		<li>Obtain suitable DR5-GFP transgenic lines using the procedure outlined in 2.1 - 2.2.</li>
		<li>Cross a homozygous DR5-GFP transgenic line with an naa10+/&#8722; plant to obtain naa10+/&#8722; plants that are homozygous for the GFP fluorescence marker at the F2 generation (identified by gene-specific PCR) to characterize naa10+/&#8722;5 and observe the GFP signal under a microscope using a homozygous DR5-GFP line.</li>
		<li>Mark the flower buds of naa10+/&#8722; plants that are homozygous for DR5-GFP as indicated in 2.1.7.</li>
		<li>Search for the GFP signal in naa10 embryos that are homozygous for DR5-GFP as indicated in 2.2.</li>
	</ol>
	</li>
</ol>

<ol><li>Jenik, P. D., Gillmor, C. S., Lukowitz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Embryonic+patterning+in+Arabidopsis+thaliana%5BTitle%5D)+AND+%22Annu+Rev+Cell+Dev+Biol%22%5BJournal%5D)">Embryonic patterning in Arabidopsis thaliana.</a> Annu Rev Cell Dev Biol. 23, 207-236 (2007).</li>
<li>Lau, S., Slane, D., Herud, O., Kong, J., Jurgens, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+embryogenesis+in+flowering+plants%3A+setting+up+the+basic+body+pattern%5BTitle%5D)+AND+%22Annu+Rev+Plant+Biol%22%5BJournal%5D)">Early embryogenesis in flowering plants: setting up the basic body pattern.</a> Annu Rev Plant Biol. 63, 483-506 (2012).</li>
<li>Wendrich, J. R., Weijers, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Arabidopsis+embryo+as+a+miniature+morphogenesis+model%5BTitle%5D)+AND+%22New+Phytol%22%5BJournal%5D)">The Arabidopsis embryo as a miniature morphogenesis model.</a> New Phytol. 199 (1), 14-25 (2013).</li>
<li>ten Hove, C. A., Lu, K. J., Weijers, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Building+a+plant%3A+cell+fate+specification+in+the+early+Arabidopsis+embryo%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Building a plant: cell fate specification in the early Arabidopsis embryo.</a> Development. 142 (3), 420-430 (2015).</li>
<li>Feng, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+N-terminal+acetylation+is+required+for+embryogenesis+in+Arabidopsis%5BTitle%5D)+AND+%22J+Exp+Bot%22%5BJournal%5D)">Protein N-terminal acetylation is required for embryogenesis in Arabidopsis.</a> J Exp Bot. 67 (15), 4779-4789 (2016).</li>
<li>Berleth, T., Jurgens, G. <a target="_blank" href="http://www.cell.com/trends/genetics/abstract/0168-9525(93)90246-E">The role of the monopteros gene in organising the basal body region of the Arabidopsis embryo.</a> Development. 118, 575-587 (1993).</li>
<li>Luo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((D-myo-inositol-3-phosphate+affects+phosphatidylinositol-mediated+endomembrane+function+in+Arabidopsis+and+is+essential+for+auxin-regulated+embryogenesis%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">D-myo-inositol-3-phosphate affects phosphatidylinositol-mediated endomembrane function in Arabidopsis and is essential for auxin-regulated embryogenesis.</a> Plant Cell. 23 (4), 1352-1372 (2011).</li>
<li>Nodine, M. D., Yadegari, R., Tax, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RPK1+and+TOAD2+are+two+receptor-like+kinases+redundantly+required+for+Arabidopsis+embryonic+pattern+formation%5BTitle%5D)+AND+%22Dev+Cell%22%5BJournal%5D)">RPK1 and TOAD2 are two receptor-like kinases redundantly required for Arabidopsis embryonic pattern formation.</a> Dev Cell. 12 (6), 943-956 (2007).</li>
<li>Ulmasov, T., Murfett, J., Hagen, G., Guilfoyle, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aux%2FIAA+proteins+repress+expression+of+reporter+genes+containing+natural+and+highly+active+synthetic+auxin+response+elements%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements.</a> Plant Cell. 9 (11), 1963-1971 (1997).</li>
<li>Friml, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efflux-dependent+auxin+gradients+establish+the+apical-basal+axis+of+Arabidopsis%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Efflux-dependent auxin gradients establish the apical-basal axis of Arabidopsis.</a> Nature. 426 (6963), 147-153 (2003).</li>
<li>Clough, S. J., Bent, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Floral+dip%3A+a+simplified+method+for+Agrobacterium-mediated+transformation+of+Arabidopsis+thaliana%5BTitle%5D)+AND+%22Plant+J%22%5BJournal%5D)">Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.</a> Plant J. 16 (16), 735-743 (1998).</li>
<li>Fiume, E., Fletcher, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+Arabidopsis+embryo+and+endosperm+development+by+the+polypeptide+signaling+molecule+CLE8%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">Regulation of Arabidopsis embryo and endosperm development by the polypeptide signaling molecule CLE8.</a> Plant Cell. 24 (3), 1000-1012 (2012).</li>
<li>Jurkuta, R. J., Kaplinsky, N. J., Spindel, J. E., Barton, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Partitioning+the+apical+domain+of+the+Arabidopsis+embryo+requires+the+BOBBER1+NudC+domain+protein%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">Partitioning the apical domain of the Arabidopsis embryo requires the BOBBER1 NudC domain protein.</a> Plant Cell. 21 (21), 1957-1971 (2009).</li>
</ol>

The authors have nothing to disclose.

Ovule clearance is a useful method for detecting cell division and assessing morphology during embryogenesis in Arabidopsis; using this technique, embryos can be observed directly under DIC microscopy5,12. The critical step for ovule clearance is step 1.3.4. The time required for ovule clearance in step 1.3.4 is variable. Embryos at the 2/4-cell stage can be clearly observed after 2 h in the Hoyer&#39;s solution, whereas they look indistinct after 12 h. ...

Embryogenesis is the earliest event in higher plant development. A mature embryo forms from a zygote through cell division and differentiation under strict genetic control1,2. Arabidopsis embryos are a useful model to study the control of morphogenesis because the sequence of cell divisions during embryogenesis follows an expected pattern3,4. However, an Arabidopsis embryo containing one to several cells is too small to be observed and analyzed. In addition, the mutation of certain genes can cause embryo lethality5, indicating the key role of those genes in embryogenesis. The characterization of pattern formation in the embryo-lethal mutants provides a basis for understanding the molecular mechanism whereby essential genes regulate embryo development.
A detailed method is described here for the characterization of embryo pattern formation in the model plant Arabidopsis by direct microscopic observation. The method involves ovule clearance followed by the microscopic observation of an embryo. The characterization of an embryo-lethal mutant is also described.
The morphology of embryos at different developmental stages can be determined directly by microscopy using the ovules that have been cleared with Hoyer&#39;s solution6,7. Such ovules exhibit a high refractive index; thus, this ovule clearing method is especially advantageous for specimens that must be observed by differential interference contrast (DIC) microscopy.
In addition, the genes that are expressed in a particular cell or a limited number of cells during embryogenesis can be used as markers to identify the cells in an embryo. Therefore, the cell identity specification during embryogenesis can be annotated by monitoring the expression of GFP-tagged marker proteins using confocal laser scanning microscopy. Conversely, observing the expression pattern of an unknown functional gene-GFP fusion during embryogenesis can provide a link between embryo patterning and the expression of that gene, and facilitate the identification of new genes that are required for embryogenesis in Arabidopsis.
The method described here can also be used to characterize the phenotype of an embryo-lethal mutant, and to determine the impact of the affected gene on embryogenesis at the cellular level. Furthermore, in combination with a comparison of the expression pattern of marker genes during embryogenesis between wild-type and mutant plants, the method can yield clues about the molecular mechanisms and signaling pathways involved in embryogenesis8,9. Indeed, the method was applied to naa10 plants, and it was found that the abnormal division of the hypophysis in the mutant may be caused by a change in the auxin distribution during embryogenesis5.

<table><tbody><tr><td>Chloral Hydrate</td><td>Sigma</td><td>15307</td><td></td></tr><tr><td>Murashige and Skoog Basal Medium</td><td>Sigma</td><td>M5519</td><td></td></tr><tr><td>Phytagel</td><td>Sigma</td><td>P8169</td><td></td></tr><tr><td>Hygromycin</td><td>Roche</td><td>10843555001</td><td></td></tr><tr><td>Growth chamber</td><td>Percival</td><td>CU36L5</td><td></td></tr><tr><td>Fluorescence microscope</td><td>Zeiss Oberkochen</td><td>Zeiss Image M2</td><td>camera: AxioCam 506 color</td></tr><tr><td>Confocal Laser Scanning Microscopy</td><td>Zeiss Oberkochen</td><td>Zeiss LSM 5</td><td>filters: BP 495 - 555 nm</td></tr><tr><td>Stereoscope</td><td>Zeiss Oberkochen</td><td>Axio Zoom V16</td><td>camera: AxioCam MRc5</td></tr><tr><td>nutrient-rich soil</td><td>KLASMANN, Germany</td><td></td><td></td></tr><tr><td>50-mL tube</td><td>Corning Inc.</td><td></td><td></td></tr><tr><td>1.5-mL tube</td><td>Corning Inc.</td><td></td><td></td></tr><tr><td>10% sodium hypochlorite solution</td><td></td><td></td><td>Domestic analytical reagent</td></tr><tr><td>sucrose</td><td></td><td></td><td>Domestic analytical reagent</td></tr><tr><td>KOH</td><td></td><td></td><td>Domestic analytical reagent</td></tr><tr><td>glycerol</td><td></td><td></td><td>Domestic analytical reagent</td></tr><tr><td>culture dish</td><td></td><td></td><td>Domestic supplies</td></tr><tr><td>vermiculite</td><td></td><td></td><td>Domestic supplies</td></tr><tr><td>glass slide</td><td></td><td></td><td>Domestic supplies</td></tr><tr><td>syringe needle</td><td></td><td></td><td>Domestic supplies</td></tr><tr><td>fine-tipped tweezers</td><td></td><td></td><td>Domestic supplies</td></tr><tr><td>super clean bench</td><td></td><td></td><td>Domestic equipment</td></tr></tbody></table>

a method for characterizing embryogenesis in arabidopsis

A Method for Characterizing Embryogenesis in Arabidopsis

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, early stage plant embryos are small and contain few cells, making them difficult to observe and analyze. A method is described here for characterizing pattern formation in plant embryos under a microscope using the model organism Arabidopsis. Following the clearance of fresh ovules using Hoyer's solution, the cell number in and morphology of embryos could be observed, and their developmental stage could be determined by differential interference contrast microscopy using a 100X oil immersion lens. In addition, the expression of specific marker proteins tagged with Green Fluorescent Protein (GFP) was monitored to annotate cell identity specification during embryo patterning by confocal laser scanning microscopy. Thus, this method can be used to observe pattern formation in wild-type plant embryos at the cellular and molecular levels, and to characterize the role of specific genes in embryo patterning by comparing pattern formation in embryos from wild-type plants and embryo-lethal mutants. Therefore, the method can be used to characterize embryogenesis in Arabidopsis.

The method described in this paper can be used to observe embryogenesis directly under a microscope, annotate the cell identity specification during embryogenesis with specific markers, and characterize the role of a particular gene during embryogenesis.
Representative results from the analysis of embryo patterning (from the elongated zygote stage to the mature walking-stick stage) in the wild-type Arabidopsis are shown...

This protocol outlines a method for observing embryogenesis in Arabidopsis via ovule clearance followed by the inspection of embryo pattern formation under a microscope.

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, ...

The overall goal of this method is to characterize the pattern formation of embryos during plant embryogenesis using the model organism. This method can help answer key questions in the plant matter field, such as embryogenesis in Arabidopsis. The main advantage of this technique is that embryo pattern formation in the model plant Arabidopsis can be characterized by the recommended operation.Demonstrating the procedure is Jinlin Feng, a graduate student from my laboratory. To begin this procedure, add 100 seeds to a 1.5 milliliter tube. Add one milliliter of sodium hypochlorite to sterilize the seeds.Invert the tube several times to ensure that the seeds and solution are well mixed. After five minutes, centrifuge the tube at 1200 Gs for 30 seconds. Remove the supernatant and wash the seeds with sterile water four times to remove any residual sodium hypochlorite.Next, add 4.4 grams of MS basal salt mixture and 10 grams of sucrose to one liter of ultra pure water. Stir to dissolve the salt and sucrose. Using potassium hydroxide at one mol, adjust the solution's pH to 5.8.Then, add three grams of gelling agent and sterilize the solution at 121 degrees Celsius for 15 minutes. Pour 30 milliliters of the resulting MS medium into a 12-centimeter culture dish. Place the sterilized seeds on the MS plate.Transfer the plate to a refrigerator, and keep it four degrees centigrade in the dark for three days to stratify the seeds. Next, keep the plate in a growth chamber under long-day conditions for eight days. Then, transfer the plants to soil in a tray.Cover the tray with a transparent white lid to prevent water loss. Leaving a small gap to avoid vitrification of the plants. After five days, remove the lid and cultivate the plants in a walk-in chamber under long-day conditions.After about 20 days of growth, use a thread to mark 10 to 15 flower buds at the position of the seed stalks and the inflorescence at 8:00 PM.Define the beginning of the ovule's pollination when the marked buds open to flower as 8:00 AM the next morning. To begin preparing the Hoyer's solution, add 24 grams of chlorhydrate to a 50 milliliter tube. Wrap the tube completely in aluminum foil, making sure the foil seals out all light.Add nine milliliters of ultra pure water, and three milliliters of glycerol. Shake the tube to dissolve the chlorhydrate. Then, let the prepared Hoyer's solution stand overnight at room temperature to eliminate any bubbles.To begin, attach a piece of double sided adhesive tape to a microscope glass slide. Place a silique, grown for the desired number of days after pollination on the side with the pseudoseptum perpendicular to the surface. Using a syringe needle, split the carpals on both sides of the pseudoseptum.Next, attach the two dissected carpals to the slide so that the ovules and the silique are visible under a stereoscope. Dispense 70 microliters of Hoyer's solution onto the glass slide. Then dip a pair of fine-tipped tweezers into the solution to moisten the tip.Using the stereoscope and tweezers, move the ovules into the Hoyer's solution to clean the embryos. After this, gently apply a cover slip to the slide, making sure not to generate any bubbles. Placed the finished slide into a box in a dark room for two to 12 hours at room temperature.After the necessary time has passed, use the microscope's DIC function to examine the cleared ovules. Locate the embryos under a low powered field, and then change to a high-power field to observe the cellular pattern in the developed embryos. Then, examine the embryos at different developmental stages.To begin, prepare the DR5 GFP plasmid and introduce it into the wild-type plants by agrobacterium to methassians mediated transformation as outlined in the text protocol. Select the T1 plants using MS medium plates containing hydrolysin. Then, transfer and cultivate the plants as outlined in the text protocol to obtain T3 plants.Using a thread, mark the flower buds of the homozygous T3 plant at the position of the seed stalks at the inflorescence at 8:00 PM.After this, mix three milliliters of glycerol with 47 milliliters of ultra pure water to generate a 6%glycerol solution. Then attach a piece of double-sided adhesive tape to a microscope glass slide. Place a T3 plant's silique on the slide with the pseudoseptum perpendicular to the surface.Using a syringe needle, split the carpals on both sides of the pseudoseptum. Attach the two dissected carpals to the slide so that the ovules and the silique are visible under a stereoscope. Next, dispense 30 microliters of 6%glycerol onto the slide.Dip a pair of fine tipped tweezers into the solution to moisten the tip. Using the stereoscope and tweezers, move the ovules into the glycerol solution. Gently place a cover slip onto the slide.Then, tap the slide quickly and gently to extrude the embryo from the ovule. After this, use confocal laser scanning microscopy to examine the slide for green fluorescent protein fluorescence. Find the isolated embryos under a low-powered field, and record their location.Change to a high powered field and set the excitation light source to 488 nanometers to observe GR5 GFP expression in the embryos. Then, detect the GFP expression, and characterize the embryo pattern formation as outlined in the text protocol. In this study, embryogenesis in Arabidopsis is observed via ovule clearance, as can be seen.After ovule clearance, the embryos at different developmental stages are distinguishable using differential interference contrast microscopy. The expression pattern of GR5 GFP during embryogenesis is then recorded under confocal laser scanning microscopy. DR5 is seen to be expressed in the root pole of the wild type globular and heart shaped embryos.DR5 is seen expressed in the embryonic cotyledon tips, and in the vasculature of mature wild-type embryos. After this, the role of naa10 in Arabidopsis during embryogenesis is examining it. The hypothesis observed to be abnormally divided, with askew and vertical division in naa10 compared to transverse asymmetric division in wild type plants.Ass seen here, oxen has nearly uniform distribution in most naa10 embryos at the globular stage, and retains a broader signal in the hypotheses compared to the wild-type embryos. These results indicate that naa10 is required for embryogenesis, and that it may be invoved via the oxen signaling pathway in Arabidopsis. While attempting this procedure, it's important to remember how to grow Arabidopsis as plant material.Following this procedure, other measures like in-situ hybridization can be performed in order to answer additional questions like expression of cell specific genes. After its development, this technique paves the way for researchers in the field of embryogenesis to explore the molecular mechanism by which specific genes mediate embryogenesis in Arabidopsis. After watching this video, you should have a good understanding of how to characterize embryogenesis in Arabidopsis.

a-method-for-characterizing-embryogenesis-in-arabidopsis

Research

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We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

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.

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

We present the procedure to assemble a seed coat bedding assay (SCBA) from Arabidopsis thaliana seeds. The SCBA was shown to be a powerful tool to explore genetically and in vitro how the endosperm controls seed germination in dormant seeds and in response to light cues. The SCBA is in principle applicable under any situation where the endosperm is suspected to influence embryonic growth.

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in 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.

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

The goal is to illustrate that the model legume Medicago truncatula can be readily utilized to investigate the regulation of early plant embryogenesis to complement the non-legume Arabidopsis model. Pod morphology is linked to zygotic embryogenesis stages and a protocol to collect embryos using tissue culture is also provided.

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

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.

Live Confocal Imaging of Developing Arabidopsis Flowers

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

We demonstrate a method to determine successful or failed fertilization on the basis of sperm nuclear morphology in Arabidopsis double fertilization using an epifluorescence microscope.

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80–100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This paper describes two phenotyping methods without the use of epidermal peels to characterize the genes controlling stomatal development. The first method demonstrates how to analyze a stomatal phenotype using a toluidine blue O-stained plant epidermis. The second method describes how to identify stomatal ligands and monitor their biological activities.

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

Here, we describe an advanced tool designed for chlorophyll biosynthesis monitoring during the early stages of Arabidopsis seedling de-etiolation. The novel methodology provides non-invasive real-time chlorophyll fluorescence imaging at high spatial and temporal resolution.

Non-invasive Assay for Chlorophyll Biosynthesis Kinetics Determination during Early Stages of Arabidopsis De-etiolation

Learn to characterize embryo pattern formation in Arabidopsis using DIC microscopy and GFP-tagged markers. This method enables detailed study of gene function and cell identity during plant embryogenesis.

A Method for Characterizing Embryogenesis in Arabidopsis

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