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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Legumes+as+a+model+plant+family.+Genomics+for+food+and+feed+report+of+the+cross-legume+advances+through+genomics+conference.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Legumes:+importance+and+constraints+to+greater+use.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+Medicago+truncatula+genomics+to+crop+legumes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Medicago+genome+provides+insights+into+the+evolution+of+rhizobial+symbioses.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+of+Medicago+truncatula+seed+development+establishes+the+time+frame+of+diverse+metabolic+processes+related+to+reserve+accumulation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiling+of+M.+truncatula+transcription+factors+identifies+putative+regulators+of+grain+legume+seed+filling.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-genomic+studies+of+developmental+processes+in+legume+seeds.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+embryo+sac+to+oil+and+protein+bodies:+embryo+development+in+the+model+legume+Medicago+truncatula.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+of+early+embryo+development+in+the+model+legume+Medicago+truncatula.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+embryogenesis+in+Arabidopsis+thaliana.+2.+The+developing+embryo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+regulation+of+embryonic+patterning+in+Arabidopsis+by+microRNAs.">Global regulation of embryonic patterning in Arabidopsis by microRNAs.</a> Plant Physiol. 165 (2), 670-687 (2014).</li><li>Birnbaum, K. D., S&#225;nchez Alvarado, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slicing+across+kingdoms:+regeneration+in+plants+and+animals.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+the+highly+regenerable+seed+line+Jemalong+2HA+for+transformation+of+Medicagotruncatula+-+implications+for+regenerability+via+somatic+embryogenesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+unusual+ABA+and+GA+synergism+increases+somatic+embryogenesis,+facilitates+its+genetic+analysis+and+improves+transformation+in+Medicago+truncatula.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+titan+mutants+of+Arabidopsis+are+disrupted+in+mitosis+and+cell+cycle+control+during+seed+development.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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., Mathesius, U., 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=Somatic+embryogenesis+from+leaf+explants.">Somatic embryogenesis from leaf explants.</a> The Medicago truncatula handbook. , (2006).</li><li>Thomas, M. R., Johnson, L. B., White, F. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+interspecific+somatic+hybrids+of+Medicago+by+using+Agrobacterium+transformed+tissues.">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., Thorpe, T. A., Yeung, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immature+seeds+and+embryos+of+Medicago+truncatula+cultured+in+vitro.">Immature seeds and embryos of Medicago truncatula cultured in vitro.</a> Plant Embryo Culture: Methodsand Protocols, Methods in Molecular Biology. 710, 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&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.">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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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

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...

Watch this Scientific Journal Video about Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula at JoVE.co

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.

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 ...

protocols-for-obtaining-zygotic-somatic-embryos-for-studying

Research

JoVE Journal

Developmental Biology

13.9K Views.  University of Newcastle, Callaghan, New South Wales, Australia. 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. 

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

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

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.

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal and postnatal consequences. To ensure the innocuousness of ART applications, studies in animal models are necessary. In addition, as a last step, embryo development studies require evaluation of their capacity to develop full-term healthy offspring. Here, embryo transfer to the uterus is indispensable to perform any ARTs-related experiment.
The rabbit has been used as a model organism to study mammalian reproduction for over a century. In addition to its phylogenetic proximity to the human species and its small size and low maintenance cost, it has important reproductive characteristics such as induced ovulation, a chronology of early embryonic development similar to humans and a short gestation that allow us to study the consequences of ART application easily. Moreover, ARTs (such as intracytoplasmic sperm injection, embryo culture, or cryopreservation) are applied with suitable efficiency in this species.
Using the laparoscopic embryo transfer technique and the cryopreservation protocol presented in this article, we describe 1) how to transfer embryos through an easy, minimally invasive technique and 2) an effective protocol for long-term storage of rabbit embryos to provide time-flexible logistical capacities and the ability to transport the sample. The outcomes obtained after transferring rabbit embryos at different developmental stages indicate that morula is the ideal stage for rabbit embryo recovery and transfer. Thus, an oviductal embryo transfer is required, justifying the surgical procedure. Furthermore, rabbit morulae are successfully vitrified and laparoscopically transferred, proving the effectiveness of the described techniques.

Assisted reproductive techniques (ARTs) are in continuous evaluation to improve outcomes and reduce the associated risks. This manuscript describes a minimally invasive embryo transfer procedure with an efficient cryopreservation protocol that allows the use of rabbits as an ideal animal model of human reproduction.

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and apoptosis, development and ageing, to neuronal synaptic plasticity and regeneration have been associated with Ryanodine receptors (RyRs). Despite the importance of calcium signaling, the exact mechanism of its function in early development is unclear. As an organism with a short gestational period, the embryos of Drosophila melanogaster are prime study subjects for investigating the distribution and localization of CICR associated proteins and their regulators during development. However, because of their lipid-rich embryos and chitin-rich chorion, their utility is limited by the difficulty of mounting embryos on glass surfaces. In this work, we introduce a practical protocol that significantly enhances the attachment of Drosophila embryo onto slides and detail methods for successful histochemistry, immunohistochemistry, and in-situ hybridization. The chrome alum gelatin slide-coating method and embryo pre-embedding method dramatically increases the yield in studying Drosophila embryo protein and RNA expression. To demonstrate this approach, we studied DmFKBP12/Calstabin, a well-known regulator of RyR during early embryonic development of Drosophila melanogaster. We identified DmFKBP12 in as early as the syncytial blastoderm stage and report the dynamic expression pattern of DmFKBP12 during development: initially as an evenly distributed protein in the syncytial blastoderm, then preliminarily localizing to the basement layer of the cortex during cellular blastoderm, before distributing in the primitive neuronal and digestion architecture during the three-gem layer phase in early gastrulation. This distribution may explain the critical role RyR plays in the vital organ systems that originate in from these layers: the suboesophageal and supraesophageal ganglion, ventral nervous system, and musculoskeletal system.

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Here, we describe a protocol for detection and localization of Drosophila embryo protein and RNA from collection to pre-embedding and embedding, immunostaining, and mRNA in situ hybridization.

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and ...

A Simple and Effective Transplantation Device for Zebrafish Embryos

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex developmental processes. Zebrafish embryos are ideally suited for these manipulations since they are easily accessible, relatively large in size, and transparent. However, previously developed devices for cell removal and transplantation are cumbersome to use or expensive to purchase. In contrast, the transplantation device presented here is economical, easy to assemble, and simple to use. In this protocol, we first introduce the handling of the transplantation device as well as its assembly from commercially and widely available parts. We then present three applications for its use: generation of ectopic clones to study signal dispersal from localized sources, extirpation of cells to produce size-reduced embryos, and germline transplantation to generate maternal-zygotic mutants. Finally, we show that the tool can also be used for embryological manipulations in other species such as the Japanese rice fish medaka.

Embryological manipulations such as extirpation and transplantation of cells are important tools to study early development. This protocol describes a simple and effective transplantation device to perform these manipulations in zebrafish embryos.

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex ...

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco&#39;s modified Eagle medium (HG-DMEM), &#946;-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific &#946;-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be a potential source of MSCs that differentiate into insulin-producing cells (IPCs). In this protocol, we provide detailed steps for the isolation and characterization of rat epididymal Ad-MSCs, followed by a simple, short protocol for the generation of IPCs from the same rat Ad-MSCs.

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...

En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos

The study of the cellular and molecular mechanisms underlying the development of the mammalian heart is essential to address human congenital heart disease. The development of the primitive cardiac valves involves the epithelial-to-mesenchymal transition (EMT) of endocardial cells from the atrioventricular canal (AVC) and outflow tract (OFT) regions of the heart in response to local inductive myocardial and endocardial signals. Once the cells delaminate and invade the extracellular matrix (cardiac jelly) located between the endocardium and the myocardium, the primitive endocardial cushions (EC) are formed. This process implies that the endocardium has to fill the gaps left by the delaminated cells and has to reorganize itself to converge (narrow) or extend (lengthen) along an axis. Current research has implicated the planar cell polarity (PCP) pathway in regulating the subcellular localization of the factors involved in this process. Classically, the initial phases of cardiac valve development have been studied in cross-sections of embryonic hearts or in ex vivo AVC or OFT explants cultured on collagen gels. These approaches allow the analysis of apico-basal polarity but do not allow the analysis of cell behavior within the plane of the epithelium or of the morphological changes of migrating cells. Here, we show an experimental approach that allows the visualization of the endocardium at valvulogenic regions as a planar field of cells. This experimental approach provides the opportunity to study PCP, planar topology, and intercellular communication within the endocardium of the OFT and AVC during valve development. Deciphering new cellular mechanisms involved in cardiac valve morphogenesis may contribute to understanding congenital heart disease associated with endocardial cushion defects.

En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos

Classically, the endocardium of the mouse embryonic valve primordium has been analyzed using transversal, coronal, or sagittal sections. Our novel approach for en face, two-dimensional imaging of the endocardium at valvulogenic regions allows planar polarity and cell rearrangement analysis of the endocardium during valve development.

The study of the cellular and molecular mechanisms underlying the development of the mammalian heart is essential to address human congenital heart ...