Name: Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula (Video) | JoVE Journal
Uploaded: 2015-06-09T13:00:00.000+00:00
Duration: 7 min 32 s
Description: Learn protocols for obtaining zygotic and somatic embryos in Medicago truncatula to study genetic regulation of early plant embryogenesis. This method enables gene expression analysis without tagging individual flowers.

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This research was supported by the Australian Research Council grant CEO348212 and the University of Newcastle. The assistance of Dr. Sam Zhang is acknowledged.

1. Zygotic Embryo Development
<ol>
	<li>Plant Material
	<ol>
		<li>Grow the Medicago truncatula wild type Jemalong or its near isogenic, highly re-generable genotype Jemalong 2HA13 (known as 2HA) in a glasshouse with a 14 hr photoperiod and 23&#160;&#176;C/19&#160;&#176;C day/night temperature.
		<ol>
			<li>Pierce the surface of the seed coat (with a 23 G needle) prior to sowing the seed so that water is allowed to enter the seed and soak in water overnight. Add enough water to fully cover the seed.</li>
			<li>Sow 3 seeds in each 15 cm diameter pot (total of 10 pots) in potting mixture (coarse sand, perlite, coir-peat (1:1:1) plus 5 g of Osmocote Exact Standard: slow release fertilizer) and transfer to the glasshouse. Retain the healthiest seedling after germination so there is 1 plant per pot and surround by a trellis for the plant stems to climb.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Harvesting Pods
	<ol>
		<li>Collect pods from the wild type Jemalong or 2HA to obtain the appropriate early embryo stages as first described9. 
		NOTE: The different pod stages are shown in Figure 1 and the corresponding embryo stages are shown in Figure 2.</li>
		<li>Check the different pod stages at harvest using criteria as per Table 1. Measure the time elapsed from stage 6 to help separate stages 6 and 7 (Table 2).</li>
	</ol>
	</li>
	<li>Dissecting ovules from pods and checking the embryo stage
	<ol>
		<li>Isolate ovules from pods using fine forceps alone or a stereo dissecting microscope and forceps as necessary. If required the embryo stage can be quickly checked using a standard compound microscope (Figure 3B).</li>
		<li>Prepare modified Hoyer&#39;s solution containing 7.5 g gum Arabic, 100 g chloral hydrate, 5 ml glycerol, 60 ml deionized distilled water. Dissolve by stirring for 3-5 hr or overnight.</li>
		<li>For more refined microscopy clear the dissected ovules in modified Hoyer&#8217;s solution15,16. Carefully pick up the intact ovules and place in a small volume of Hoyer&#8217;s solution (enough to cover the ovule) at room temperature until clear.</li>
		<li>View the samples with differential interference contrast (DIC) optics. Capture the images iwth a digital camera9 (Figure 2).</li>
		<li>Obtain the most uniform ovules from the central region of the pod and accumulate the required number. An example is shown in Figure 3A. Collect the ovules on ice and store at required temperature (-80 &#176;C for nucleic acids) for further analysis. 
		NOTE: For details on analysis of histological sections, transmission electron microscopy, and in situ hybridization please see papers8,9.</li>
	</ol>
	</li>
</ol>
2. Somatic Embryo Development In Vitro
<ol>
	<li>Use an M. truncatula cultivar that is able to readily form embryos in culture. For this protocol for leaf explants use 2HA plants13,17 that are 2-4 months old.</li>
	<li>Take leaflets from the latest nearly fully expanded trifoliate leaf of an elongating stem.</li>
	<li>Keep the trifoliate leaves on moist paper toweling in a culture pot to avoid dehydration. 
	NOTE: Once the trifoliate leaves are harvested they need to be utilized with minimal delay.</li>
	<li>Preparation of the culture medium
	<ol>
		<li>Use the P4 10:4:1:5 medium in agar (0.8% w:v) in 9 cm Petri dishes. 
		NOTE: The P4 10:4:1:5 medium consists of the P4 basal medium18 plus 10 &#956;M 1-naphthaleneacetic acid (NAA), 4 &#956;M 6-benzylaminopurine (BAP), 1 &#956;M abscisic acid (ABA) and 5 &#956;M gibberellic acid (GA). The use of GA + ABA accelerates embryo formation and causes substantive increases in embryo number14.</li>
		<li>Make up the the P4 basal medium in 1 L; consisting of major salts (make calcium up separately), minor salts and vitamins (Table 3), chelated iron (make up separately), casamino acids (casein hydrolysate), 30 g sucrose, 8 g agar.</li>
		<li>Store stock solutions of major salts, calcium, casamino acids, minor salts and vitamins at -20 &#176;C in suitable aliquots. Store chelated iron at 4 &#176;C.</li>
		<li>Make up the chelated iron (200x) by dissolving 7.44 g of Na2EDTA&#8226;2H2O in approximately 900 ml of deionized distilled water while stirring, then bring the solution to 98-99 &#176;C while adding 1.853 g of FeSO4&#8226;7H2O. Finally make up to 1 L when the ingredients are dissolved. Store in amber colored bottles at 4 &#176;C.</li>
		<li>Make up the P4 basal medium with the stock solutions as in Table 4. The hormones in the medium are 10 &#956;M NAA, 4 &#956;M BAP, 1 &#956;M ABA, 5 &#956;M GA (see Table 4 and Specific Materials Table). Add the NAA and BAP prior to autoclaving and add ABA and GA after autoclaving and cooling to about 55 &#176;C, using filter sterilization with a 0.22 &#181;m filter attached to a syringe. Mix well by gentle swirling.</li>
		<li>Adjust the media to pH 5.8 with 1 M KOH prior to autoclaving. Autoclave at 121 &#176;C for 20 min.</li>
		<li>After autoclaving add the filter sterilized ABA and GA, and pour approximately 25 ml of media into sterile 9 cm Petri dishes in a laminar flow hood or biohazard cabinet. Cool and let the agar set with the lid off. Then put on the lid and make sure there is no condensate on the lid. Label as required and store at 4 &#176;C (in a cardboard box to prevent lid condensate).</li>
	</ol>
	</li>
	<li>Sterilization of Leaf Tissue
	<ol>
		<li>Work in a UV-sterilized laminar flow hood or biohazard cabinet.</li>
		<li>Place leaves into the mesh ball of a previously autoclaved spring tea infuser.</li>
		<li>Immerse tea infuser containing the leaves in 70% (v:v) ethanol in a culture pot for 30 sec. 
		NOTE: For all steps in this procedure use 250 ml screw cap polycarbonate culture pots that are autoclaved.</li>
		<li>Drain and then transfer and immerse the leaf tissue in 1:8 (v:v) diluted bleach (0.5% chlorine) for 10 min. Swirl gently from time to time.</li>
		<li>Drain the bleach and transfer the tea infuser in a culture pot containing sterile deionized distilled water. Gently swirl and drain excess water. Repeat one more time with a fresh culture pot of sterile deionized distilled water.</li>
		<li>Remove leaves from the tea infuser with sterile forceps to a fresh culture pot of sterile deionized distilled water. Screw on the sterile cap and rinse by inverting and swirling. Leave leaves floating on the sterile water until ready to prepare explants.</li>
	</ol>
	</li>
	<li>Preparing and Plating Explants
	<ol>
		<li>Using sterile techniques, trim individual sterilized leaflets from the edge with a scalpel, and cut the remaining rectangle into two or three rectangular explants (8-10 x 3-5 mm) with the mid-vein in the center of each explant (Figure 5A). 
		NOTE: The size of the explant is quite important in initiating the first callusing. Carry out the cutting on sterile lids from take-away food containers.</li>
		<li>Plate 6 explants on agar-solidified culture medium in 9 cm diameter Petri dishes (Figure 5B). Plate the explants abaxial side down to maintain usual leaf orientation.</li>
		<li>Seal the Petri dishes with Parafilm (Figure 5C) stretched around the dish. The tissue is now ready for incubation.</li>
	</ol>
	</li>
	<li>Tissue Incubation
	<ol>
		<li>Incubate the plates in the dark at 28 &#176;C in a controlled temperature room or growth cabinet throughout the culture period. 
		NOTE: The explants will initiate dedifferentiation, undergo cell division, proliferation (callus formation) and embryogenesis (differentiation).</li>
		<li>Subculture the differentiating explants after 3 weeks on to fresh medium. At this subculture transfer the whole explant using a sterile stainless steel spatula and forceps, in a laminar flow hood or a biohazard cabinet. As callusing occurs and the callus exceeds the size of the largest one in Figure 5C, transfer 50% of the individual callus when subculturing to fresh medium.</li>
		<li>Observe the first embryos in about 4 weeks. While there is a degree of synchrony, collect different embryo stages over a 4-8 week incubation period (Figure 6A,B). Collect under a dissecting microscope and process according to experimental objectives.</li>
		<li>Subculture every 3 weeks and remove embryos periodically so that the explants will continue to produce embryos for 3 months.</li>
	</ol>
	</li>
</ol>

<ol><li>Gepts, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Legumes+as+a+model+plant+family.+Genomics+for+food+and+feed+report+of+the+cross-legume+advances+through+genomics+conference%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">Legumes as a model plant family. Genomics for food and feed report of the cross-legume advances through genomics conference.</a> Plant Physiol. 137 (4), 1228-1235 (2005).</li>
<li>Graham, P. H., Vance, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Legumes%3A+importance+and+constraints+to+greater+use%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">Legumes: importance and constraints to greater use.</a> Plant Physiol. 131 (3), 872-877 (2003).</li>
<li>Young, N. D., Udvardi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Translating+Medicago+truncatula+genomics+to+crop+legumes%5BTitle%5D)+AND+%22Curr.+Opin.+Plant+Biol%22%5BJournal%5D)">Translating Medicago truncatula genomics to crop legumes.</a> Curr. Opin. Plant Biol. 12 (2), 193-201 (2009).</li>
<li>Young, N. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Medicago+genome+provides+insights+into+the+evolution+of+rhizobial+symbioses%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">The Medicago genome provides insights into the evolution of rhizobial symbioses.</a> Nature. 480 (7378), 520-524 (2011).</li>
<li>Gallardo, K., Le Signor, C., Vandekerckhove, J., Thompson, R. D., Burstin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteomics+of+Medicago+truncatula+seed+development+establishes+the+time+frame+of+diverse+metabolic+processes+related+to+reserve+accumulation%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">Proteomics of Medicago truncatula seed development establishes the time frame of diverse metabolic processes related to reserve accumulation.</a> Plant Physiol. 133 (2), 664-682 (2003).</li>
<li>Verdier, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+expression+profiling+of+M.+truncatula+transcription+factors+identifies+putative+regulators+of+grain+legume+seed+filling%5BTitle%5D)+AND+%22Plant+Mol.+Biol%22%5BJournal%5D)">Gene expression profiling of M. truncatula transcription factors identifies putative regulators of grain legume seed filling.</a> Plant Mol. Biol. 67 (6), 567-580 (2008).</li>
<li>Thompson, R., Burstin, J., Gallardo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Post-genomic+studies+of+developmental+processes+in+legume+seeds%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">Post-genomic studies of developmental processes in legume seeds.</a> Plant Physiol. 151 (3), 1023-1029 (2009).</li>
<li>Wang, X. -D., Song, Y., Sheahan, M. B., Garg, M. L., Rose, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((From+embryo+sac+to+oil+and+protein+bodies%3A+embryo+development+in+the+model+legume+Medicago+truncatula%5BTitle%5D)+AND+%22New+Phytol%22%5BJournal%5D)">From embryo sac to oil and protein bodies: embryo development in the model legume Medicago truncatula.</a> New Phytol. 193 (2), 327-338 (2012).</li>
<li>Kurdyukov, S., Song, Y., Sheahan, M. B., Rose, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptional+regulation+of+early+embryo+development+in+the+model+legume+Medicago+truncatula%5BTitle%5D)+AND+%22Plant+Cell+Rep%22%5BJournal%5D)">Transcriptional regulation of early embryo development in the model legume Medicago truncatula.</a> Plant Cell Rep. 33 (2), 349-362 (2014).</li>
<li>Mansfield, S. G., Briarty, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+embryogenesis+in+Arabidopsis+thaliana.+2.+The+developing+embryo%5BTitle%5D)+AND+%22Can.+J.+Botany%22%5BJournal%5D)">Early embryogenesis in Arabidopsis thaliana. 2. The developing embryo.</a> Can. J. Botany. 69 (3), 461-476 (1991).</li>
<li>Seefried, W. F., Willman, M. R., Clausen, R. L., Jenik, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+regulation+of+embryonic+patterning+in+Arabidopsis+by+microRNAs%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">Global regulation of embryonic patterning in Arabidopsis by microRNAs.</a> Plant Physiol. 165 (2), 670-687 (2014).</li>
<li>Birnbaum, K. D., Sánchez Alvarado, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Slicing+across+kingdoms%3A+regeneration+in+plants+and+animals%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Slicing across kingdoms: regeneration in plants and animals.</a> Cell. 132 (4), 697-710 (2008).</li>
<li>Rose, R. J., Nolan, K. E., Bicego, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+development+of+the+highly+regenerable+seed+line+Jemalong+2HA+for+transformation+of+Medicagotruncatula+-+implications+for+regenerability+via+somatic+embryogenesis%5BTitle%5D)+AND+%22J.+Plant+Physiol%22%5BJournal%5D)">The development of the highly regenerable seed line Jemalong 2HA for transformation of Medicagotruncatula - implications for regenerability via somatic embryogenesis.</a> J. Plant Physiol. 155 (6), 788-791 (1999).</li>
<li>Nolan, K. E., Song, Y., Liao, S., Saeed, N., Zhang, X., Rose, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+unusual+ABA+and+GA+synergism+increases+somatic+embryogenesis%2C+facilitates+its+genetic+analysis+and+improves+transformation+in+Medicago+truncatula%5BTitle%5D)+AND+%22PloS+ONE%22%5BJournal%5D)">An unusual ABA and GA synergism increases somatic embryogenesis, facilitates its genetic analysis and improves transformation in Medicago truncatula.</a> PloS ONE. 9 (6), e99908(2014).</li>
<li>Liu, C. M., Meinke, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+titan+mutants+of+Arabidopsis+are+disrupted+in+mitosis+and+cell+cycle+control+during+seed+development%5BTitle%5D)+AND+%22Plant+J%22%5BJournal%5D)">The titan mutants of Arabidopsis are disrupted in mitosis and cell cycle control during seed development.</a> Plant J. 16 (1), 21-31 (1998).</li>
<li>Nolan, K. E., Kurdyukov, S., Rose, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+of+the+SOMATIC+EMBRYOGENESIS+RECEPTOR-LIKE+KINASE+1+%28SERK1%29+gene+is+associated+with+developmental+change+in+the+life+cycle+of+the+model+legume+Medicago+truncatula%5BTitle%5D)+AND+%22J.+Exp.+Bot%22%5BJournal%5D)">Expression of the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1 (SERK1) gene is associated with developmental change in the life cycle of the model legume Medicago truncatula.</a> J. Exp. Bot. 60 (6), 1759-1771 (2009).</li>
<li>Iantcheva, A., Vlahova, M., Atanassov, A., et al. Somatic embryogenesis from leaf explants. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aIantcheva%2C+A+%22The+Medicago+truncatula+handbook%22">The Medicago truncatula handbook</a>. Mathesius, U. , Available from:
 
 <a target="_blank" href="http://www.noble.org/MedicagoHandbook">http://www.noble.org/MedicagoHandbook</a> (2006).</li>
<li>Thomas, M. R., Johnson, L. B., White, F. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selection+of+interspecific+somatic+hybrids+of+Medicago+by+using+Agrobacterium+transformed+tissues%5BTitle%5D)+AND+%22Plant+Sci%22%5BJournal%5D)">Selection of interspecific somatic hybrids of Medicago by using Agrobacterium transformed tissues.</a> Plant Sci. 69 (2), 189-198 (1990).</li>
<li>Ochatt, S. J. Immature seeds and embryos of Medicago truncatula cultured in vitro. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aOchatt%2C+SJ+%22Plant+Embryo+Culture%3A+Methodsand+Protocols%2C+Methods+in+Molecular+Biology%22">Plant Embryo Culture: Methodsand Protocols, Methods in Molecular Biology</a>. Thorpe, T. A., Yeung, E. C. 710, Springer protocols, Humana press. 39-52 (2011).</li>
<li>Mantiri, F. R., Kurdyukov, S., Lohar, D. P., Sharopova, N., Saeed, N. A., Wang, X. D., VandenBosch, K. A., Rose, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+transcription+factor+MtSERF1+of+the+ERF+subfamily+identified+by+transcriptional+profiling+is+required+for+somatic+embryogenesis+induced+by+auxin+plus+cytokinin+in+Medicago+truncatula%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">The transcription factor MtSERF1 of the ERF subfamily identified by transcriptional profiling is required for somatic embryogenesis induced by auxin plus cytokinin in Medicago truncatula.</a> Plant Physiol. 146 (4), 1622-1636 (2008).</li>
</ol>

The authors declare that they have no competing interests.

The protocols described are relatively straight forward and allow investigation of legume embryogenesis with all the contemporary cell and molecular techniques. We recognize that there are advantages and disadvantages of both in vivo and in vitro approaches. Both allow more focus on early embryogenesis compared to culture of immature seeds19.
In the case of in vivo studies what is described is predominantly the isolation of the ovule from the pod which is ...

Legumes are the third largest family of higher plants with approximately 20,000 species and the Leguminosae (or Fabaceae) family are second to cereals in area harvested and total production1. Soybean is the third largest cultivated crop. Grain legumes provide about one-third of dietary protein and one-third of vegetable oil for human consumption2. Legumes with their N2 fixing capacity also contribute to sustainable agricultural systems. Medicago truncatula, like soybean, stores protein and oil in the cotyledons of its seeds and is a genetic and genomic legume model with considerable genetic and genomic resources3,4. While M. truncatula has enabled advances in understanding the legume-rhizobium symbiosis4 it has been increasingly employed to study legume seed biology5-7 and embryogenesis8,9. Arabidopsis embryogenesis has been extensively studied10,11 but it is a non-legume and the details of embryogenesis are not identical to Medicago8,10. Zygotic embryogenesis in M. truncatula has interesting features, with a distinctive multicellular hypophysis, an endoployploid suspensor and basal transfer cell8.
Somatic embryogenesis (SE) is commonly used for regenerating plants12. In the legume model M. truncatula, the seed line Jemalong 2HA (2HA) has been developed from the parent Jemalong to have high rates of somatic embryogenesis13. The number of embryos produced has recently been substantively increased by adding both gibberellic acid (GA) and abscisic acid (ABA) to the long established medium14. In this case GA and ABA act synergistically, which is unusual given that GA and ABA usually act antagonistically14. The embryos produced from callus develop on the surface which allows the stage of embryogenesis to be readily determined visually and readily harvested. Having near isogenic lines that are embryogenic (2HA) and non-embryogenic (Jemalong) facilitates the investigation of somatic embryogenesis and having both in vivo and in vitro systems provides different experimental possibilities.
Understanding the cellular and molecular mechanisms of embryo development is essential for understanding seed and plant development. In legumes, as in other dicotyledons, it is the cotyledons of the embryo that store the products that are used for human nutrition. Early embryogenesis involves rapid cell division, and correct embryo patterning. In approximately 8 days after fertilization, the M. truncatula embryo reaches early cotyledon stages. The morphological characterization is not exactly indicated by days after fertilization in glasshouse conditions. Thus, an efficient standardized approach to indicate the stage of developing embryos is valuable in studying the genetic regulation of early zygotic embryogenesis.
In this paper, we provide two standardized protocols to collect developing embryos for biological studies of embryogenesis in the legume model M. truncatula. The first one is to collect zygotic embryos by associating embryogenesis and pod morphology while the second is somatic embryogenesis via culturing leaf explants to provide easily accessed large embryo numbers.

<table><tbody><tr><td>P4 medium</td><td>Sigma-Aldrich</td><td></td><td>Use Sigma-Aldrich Chemicals or other analytical grade supplier</td></tr><tr><td>Major salts</td><td></td><td></td><td></td></tr><tr><td>Minor salts</td><td></td><td></td><td></td></tr><tr><td>Vitamins</td><td></td><td></td><td></td></tr><tr><td>Agar</td><td>Bacto Laboratories</td><td>214010</td><td>Bacto agar</td></tr><tr><td>Plant hormones</td><td></td><td></td><td></td></tr><tr><td>1-Naphthaleneacetic acid</td><td>Sigma-Aldrich</td><td>N0640</td><td>Dissolve in small amount of 1 M NaOH</td></tr><tr><td>Abscisic acid</td><td>Sigma-Aldrich</td><td>A1049</td><td>Dissolve in small amount of 1 M NaOH</td></tr><tr><td>6-Benzylaminopurine</td><td>Sigma-Aldrich</td><td>B3274</td><td>Dissolve in MQ water with heating and few drops 1N HCl</td></tr><tr><td>Gibberellic Acid</td><td>Sigma-Aldrich</td><td>G7645</td><td>Dissolve in small amount of ethanol</td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>Stereo dissecting microscope</td><td>Leica</td><td>MZFLIII</td><td>Or similar</td></tr><tr><td>Light microscope</td><td>Zeiss</td><td>Axiophot</td><td>Or similar, with suitable optics</td></tr><tr><td>Digital camera</td><td>Zeiss</td><td>AxioCam HRc</td><td>Or similar</td></tr><tr><td>Sterilising leaves</td><td></td><td></td><td></td></tr><tr><td>250 mL screw cap polycarbonate container with polypropylene lid</td><td>SARSTEDT</td><td>75.9922.519</td><td>Autoclavable</td></tr></tbody></table>

protocols for obtaining zygotic and somatic embryos for studying the regulation of early embryo development in the model legume medicago truncatula

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.

For zygotic embryogenesis different pod structures corresponding to the different embryo stages are shown in Figure 1A-F while the different embryo stages are shown in Figure 2A-F. By selecting pods at the same stage, samples of ovules that are quite uniform can be obtained (Figure 3A). By using RT-qPCR embryo specific genes can be readily detected and time course studies evaluated9. Some additional dissection will allow for f...

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 ...

The overall goal of the following procedures is to demonstrate how many cargo tula embryos can be obtained at different developmental stages to investigate the genetic regulation of embryogenesis in plants, this can be accomplished by linking pod morphology with the different zygotic embryo developmental stages, and by culturing leaf explan to obtain somatic embryos at the different stages. Ultimately, quantitative PCR and beta glucuronidase can be used to assess the expression of genes during embryogenesis at the specific developmental stages of interest. The advantages of these techniques are the tagging of individual flowers is not required for the in vivo experiments, and are optimized in vitro procedures, provide complimentary experimental approaches.Visual demonstration of the culture procedure is important because it allows the details of the manipulations to be more closely followed. Demonstrating the in vivo procedure will be ing Wang while Kim Nolan will be demonstrating the in vitro procedure. To facilitate zygotic embryo development, pierce the surface of the seed coats of seeds from a Monte cargo trinocular plant and soak the seeds overnight, fully covered in water.The

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The zebra finch (Taeniopygiaguttata) is a valuable model organism; however, early stages of zebra finch development  have not been extensively studied. The protocol describes how to dissect early embryos for  developmental and molecular applications.

Dissection and Downstream Analysis of Zebra Finch Embryos at Early Stages of Development

Methods that produce morphant embryos are essential to study developmental mechanisms and gene regulatory networks.  The sea star Patiria miniata is an emerging model system for these studies.  Here we present a protocol for obtaining gametes, producing cultures of embryos, and rapid microinjection of zygotes from this species.

A Method for Microinjection of Patiria minata Zygotes

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

Imaging Subcellular Structures in the Living Zebrafish Embryo

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

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

Plant morphogenic genes can be used to improve genetic transformation of recalcitrant genotypes. Described here is an Agrobacterium-mediated genetic transformation (QuickCorn) protocol for three important public maize inbred lines.

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

This paper establishes a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei.

Single-Cell RNA Sequencing of Mutant Whole Mouse Embryos: From the Epiblast to the End of Gastrulation

We describe methods to visualize nascent transcription during zygotic genome activation in single cells during early embryogenesis.

Visualizing Zygotic Genome Activation In Single Cells of Early Embryos

We describe a physical constriction method to make mini embryos from Xenopus laevis 1-cell embryos.

Physical Manipulation to Generate Xenopus Mini Embryos

Learn protocols for obtaining zygotic and somatic embryos in Medicago truncatula to study genetic regulation of early plant embryogenesis. This method enables gene expression analysis without tagging individual flowers.

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula (Video) | JoVE Journal

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

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