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>

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	<li>Jenik, P. D., Gillmor, C. S., Lukowitz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+patterning+in+Arabidopsis+thaliana.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+embryogenesis+in+flowering+plants:+setting+up+the+basic+body+pattern.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+embryo+as+a+miniature+morphogenesis+model.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+a+plant:+cell+fate+specification+in+the+early+Arabidopsis+embryo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+N-terminal+acetylation+is+required+for+embryogenesis+in+Arabidopsis.">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.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+monopteros+gene+in+organising+the+basal+body+region+of+the+Arabidopsis+embryo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=D-myo-inositol-3-phosphate+affects+phosphatidylinositol-mediated+endomembrane+function+in+Arabidopsis+and+is+essential+for+auxin-regulated+embryogenesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RPK1+and+TOAD2+are+two+receptor-like+kinases+redundantly+required+for+Arabidopsis+embryonic+pattern+formation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aux/IAA+proteins+repress+expression+of+reporter+genes+containing+natural+and+highly+active+synthetic+auxin+response+elements.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efflux-dependent+auxin+gradients+establish+the+apical-basal+axis+of+Arabidopsis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Floral+dip:+a+simplified+method+for+Agrobacterium-mediated+transformation+of+Arabidopsis+thaliana.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Arabidopsis+embryo+and+endosperm+development+by+the+polypeptide+signaling+molecule+CLE8.">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. 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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. Thus, the more mature an ovule is, the longer the time required for clearance.
In step 2.2.5, to isolate an embryo from an ovule, the amount of force applied to the slide is critical. Embryos can be crushed if too much force is applied; however, an embryo cannot be extruded from the ovule if too little force is used. In our experience, the more mature an ovule is, the less force is needed. Torpedo stage and mature embryos can be isolated directly by peeling open an ovule using a syringe needle. As some of suspensor cells might be destroyed in the isolated embryo during the isolation process, the fluorescence signal from the suspensor might be lost or incomplete. Another limitation of this method is that the embryos before the 4-cell stage were difficult to find from the extruded embryos as they are too small and shielded by ovule fragment.
The developmental stage and morphology of an embryo can be affected by the mutation of certain genes; such effects can be demonstrated by microscopy following ovule clearance. This approach will aid in the understanding of the molecular mechanism by which specific genes mediate embryogenesis5. Observing the expression pattern of an unknown functional gene-GFP fusion during embryogenesis can provide a link between the embryo patterning and the expression of that gene, and facilitate the identification of new genes that are required for embryogenesis in Arabidopsis13. Thus, the protocol described here provides a basic method for the characterization of embryogenesis in Arabidopsis.

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

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 in Figure 2. After ovule clearance, the embryos at different developmental stages could be distinguished by DIC microscopy (Figure 2). The expression pattern of DR5-GFP was recorded during embryogenesis under confocal laser scanning microscopy (Figure 3) to determine the distribution of auxin in the embryo and examine the potential role of auxin during embryogenesis in Arabidopsis.
This protocol can also be used to characterize the roles of specific genes during embryogenesis. Here, the role of Naa10 during embryogenesis in Arabidopsis was examined as an example. Abnormal division of the hypophysis was observed (askew and vertical division of the hypophysis in Naa10 compared with transverse asymmetric division of the hypophysis in wild-type plants, Figure 4). The distribution of auxin at the globular stage was nearly uniform in most Naa10 embryos and retained a broader signal in the hypophysis, as compared to the wild type embryos (Figure 5). These results indicate that Naa10 is required for embryogenesis, and that it may be involved in embryogenesis via the auxin signaling pathway in Arabidopsis.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55969/55969fig1.jpg" /> 
Figure 1: Schematic Diagram for the Dissection of Arabidopsis Silique. (A) The silique attached on the glass slide was pasted with a piece of double-sided adhesive tape. (B) The enlarged image of the box in A. The two imaginary lines represented the location to be split. (C) The enlarged image of the split silique in the box in D. (D) The image of the split silique. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55969/55969fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55969/55969fig2.jpg" /> 
Figure 2: Examination of Embryogenesis in Wild-type (Col-0) Arabidopsis by DIC Microscopy. (A) Elongated zygote. (B) A 1-cell stage embryo at 1 DAP. (C) A 2/4-cell stage embryo at 2 DAP. (D) An octant stage embryo at 3 DAP. (E) A dermatogen stage embryo at 3 DAP. (F) An early globular stage embryo at 4 DAP. (G) A late globular stage embryo at 5 DAP. (H) A heart stage embryo at 6 DAP. (I) A torpedo stage embryo at 7 DAP. (J) A walking-stick stage embryo at 8 DAP. Scale bars = 10 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55969/55969fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55969/55969fig3.jpg" /> 
Figure 3: The Expression Pattern of DR5 in Wild-type (Col-0) Arabidopsis Embryos. (a-d) GFP signals. (A-D) Merged images with bright field images. DR5 was expressed in the root pole of the wild-type globular stage (a and A, 4 DAP) and heart stage (b and B, 6 DAP) embryos. DR5 was also expressed in the embryonic cotyledon tips (c and C, 7 DAP) and in the vasculature of mature wild-type embryos (d and D, 8 DAP). Scale bars = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55969/55969fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55969/55969fig4.jpg" /> 
Figure 4: Characterization of the Role of Naa10 during Embryogenesis in Arabidopsis. Normal division of the hypophysis in a wild-type plant (A). Abnormal division of the hypophysis in Naa10 mutant plants is marked with a white line in (B) (askew division of the hypophysis) and (C) (vertical division of the hypophysis). Scale bars = 10 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55969/55969fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55969/55969fig5.jpg" /> 
Figure 5: The DR5 Expression Pattern in Globular Stage wild-type (Col-0) Arabidopsis and Naa10 Embryos. (a and b) GFP signals. (A and B) Merged images with bright field images. DR5 was expressed in the root pole of a wild-type globular stage embryo (a and A). DR5 was nearly uniformly expressed in a globular stage&#160;Naa10 embryo and retained the broader signal in hypophysis as compared to the wild type (b and B). Scale bars = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55969/55969fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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A Method for Characterizing Embryogenesis in Arabidopsis

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

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, ...

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11.1K Views.  Capital Normal University. Dit protocol beschrijft een methode voor de waarneming van de embryogenese Arabidopsis via zaadknop goedkeuring, gevolgd door de inspectie van embryo patroonformatie onder een microscoop.

Video: A Method for Characterizing Embryogenesis in Arabidopsis

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

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by pre-globular, globular, heart, torpedo and cotyledon stages. This progressive development is under the tight regulation of a complex molecular network. Harvesting sufficient early embryos at a similar stage of development is essential for investigating the cellular and molecular regulation of early embryogenesis. This is not straightforward since early embryogenesis undergoes rapid morphogenesis in a short while e.g. 8 days for Medicago truncatula to reach the early cotyledon stage. Here, we address the issue by two approaches. The first one establishes a linkage between embryo development and pod morphology in helping indicate the stage of the zygotic embryo. This is particularly based on the number of pod spirals and development of the spines. An alternative way to complement the in vivo studies is via culturing leaf explants to produce somatic embryos. The medium includes an unusual hormone combination - an auxin (1-naphthaleneacetic acid), a cytokinin (6-benzylaminopurine), abscisic acid and gibberellic acid. The different stages can be discerned growing out of the callus without dissection.

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

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.

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by ...

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. ...

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

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

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

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 most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

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

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

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

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;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.

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Biology

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

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

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

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.

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

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

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

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

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

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

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

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of dormant seeds or that of nondormant seeds irradiated by a far red (FR) light pulse. In order to further gain insight into the molecular genetic mechanisms underlying the germination repressive activity exerted by the endosperm, a &#34;seed coat bedding&#34; assay (SCBA) was devised. The SCBA is a dissection procedure physically separating seed coats and embryos from seeds, which allows monitoring the growth of embryos on an underlying layer of seed coats. Remarkably, the SCBA reconstitutes the germination repressive activities of the seed coat in the context of seed dormancy and FR-dependent control of seed germination. Since the SCBA allows the combinatorial use of dormant, nondormant and genetically modified seed coat and embryonic materials, the genetic pathways controlling germination and specifically operating in the endosperm and embryo can be dissected. Here we detail the procedure to assemble a SCBA.

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

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.

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of ...

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 ...

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

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant productivity and survival. As such, understanding the mechanisms by which stomata develop and pattern has tremendous agronomic value. This paper describes two phenotypic methods using Arabidopsis cotyledons that can be used to characterize the genes controlling stomatal development and patterning. Presented first are procedures for analyzing the stomatal phenotypes using toluidine blue O-stained cotyledons. This method is fast and reliable and does not require the use of epidermal peels, which are widely used for phenotypic analyses but require specialized training. Due to the presence of multiple cysteine residues, the identification and generation of bioactive EPF peptides that have a role in stomatal development have been challenging. Thus, presented second is a procedure used to identify stomatal ligands and monitor their biological activity by bioassays. The main advantage of this method is that it produces reproducible data relatively easily while reducing the amount of peptide solution and the time required to characterize the role of the peptides in controlling stomatal patterning and development. Overall, these well-designed protocols enhance the efficiency of studying the potential stomatal regulators, including cysteine-rich secretory peptides, which require highly complex structures for their activity.

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

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.

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant ...

A Method for Characterizing Embryogenesis in Arabidopsis

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