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><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

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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

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the African clawed frog, Xenopus, a longstanding model organism for biomedical research. Traditional breeding schemes for creating homozygous mutant lines with CRISPR/Cas9-targeted mutagenesis have several time-consuming and laborious steps. To facilitate the creation of mutant embryos, particularly to overcome the obstacles associated with knocking out genes that are essential for embryogenesis, a new method called leapfrogging was developed. This technique leverages the robustness of Xenopus embryos to &#34;cut and paste&#34; embryological methods. Leapfrogging utilizes the transfer of primordial germ cells (PGCs) from efficiently-mutagenized donor embryos into PGC-ablated wildtype siblings. This method allows for the efficient mutation of essential genes by creating chimeric animals with wildtype somatic cells that carry a mutant germline. When two F0 animals carrying &#34;leapfrog transplants&#34; (i.e., mutant germ cells) are intercrossed, they produce homozygous, or compound heterozygous, null F1 embryos, thus saving a full generation time to obtain phenotypic data. Leapfrogging also provides a new approach for analyzing maternal effect genes, which are refractory to F0 phenotypic analysis following CRISPR/Cas9 mutagenesis. This manuscript details the method of leapfrogging, with special emphasis on how to successfully perform PGC transplantation.

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

Genes essential for survival pose technical hurdles for creating mutant lines. Leapfrogging circumvents lethality by combining genome editing with primordial germ cell transplantation to create wild-type animals carrying germline mutations. Leapfrogging also permits the efficient generation of homozygous null mutants in the F1 generation. Here, the transplantation step is demonstrated.

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the ...

Genetics

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.

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.

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

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; ...

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

This article describes a protocol to efficiently derive and culture pluripotent stem cell lines from mouse embryos at the blastocyst stage.

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator

The unique traits of eusocial insects, such as social behavior and reproductive division of labor, are controlled by their genetic system. To address how genes regulate social traits, we have developed mutant ants via delivery of CRISPR complex into young embryos during their syncytial stage. Here, we provide a protocol of CRISPR-mediated mutagenesis in Harpegnathos saltator, a ponerine ant species that displays striking phenotypic plasticity. H. saltator ants are readily reared in a laboratory setting. Embryos are collected for microinjection with Cas9 proteins and in vitro synthesized small guide RNAs (sgRNAs) using home-made quartz needles. Post-injection embryos are reared outside the colony. Following emergence of the first larva, all embryos and larvae are transported to a nest box with a few nursing workers for further development. This protocol is suitable for inducing mutagenesis for analysis of caste-specific physiology and social behavior in ants, but may also be applied to a broader spectrum of hymenopterans and other insects.

Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator

Many characteristics of insect eusociality rely on within-colony communication and division of labor. Genetic manipulation of key regulatory genes in ant embryos via microinjection and CRISPR-mediated mutagenesis provides insights into the nature of altruistic behavior in eusocial insects.

The unique traits of eusocial insects, such as social behavior and reproductive division of labor, are controlled by their genetic system. To address how ...

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 ...

Preparation of Single-Somite Explants from Zebrafish Embryos

The body axis of vertebrate embryos is periodically subdivided into 3D multicellular units called somites. While genetic oscillations and molecular prepatterns determine the initial length-scale of somites, mechanical processes have been implicated in setting their final size and shape. To better understand the intrinsic material properties of somites, a method is developed to culture single-somite explant from zebrafish embryos. Single somites are isolated by first removing the skin of embryos, followed by yolk removal and sequential excision of neighboring tissues. Using transgenic embryos, the distribution of various sub-cellular structures can be observed by fluorescent time-lapse microscopy. Dynamics of explanted somites can be followed for several hours, thus providing an experimental framework for studying tissue-scale shape changes at single-cell resolution. This approach enables direct mechanical manipulation of somites, allowing for dissection of the material properties of the tissue. Finally, the technique outlined here can be readily extended for explanting other tissues such as the notochord, neural plate, and lateral plate mesoderm.

We present a protocol for isolating single somites from zebrafish embryos, the dynamics of which can be followed in culture for several hours by fluorescence time-lapse microscopy, thus providing a methodology to quantify tissue-scale shape changes at single-cell resolution.

The body axis of vertebrate embryos is periodically subdivided into 3D multicellular units called somites. While genetic oscillations and molecular ...

Isolation and Genotyping of Gastrulating Mouse Embryo for Single-Cell Sequencing

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

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, a robust pipeline was designed to perform single-cell and nuclei analysis on mouse embryos from embryonic day E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to wild-type perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, a methodology is presented designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with a same-day genotyping protocol (3 h) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. This method further includes guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate that this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula.

Author Spotlight: A Pipeline to Analyze Lineage-Specific Mutant Embryos at Single-Cell Resolution

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

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states ...

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 ...

Biology

A Method for Microinjection of Patiria minata Zygotes

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and evolution of developmental processes. The sea star, Patiria miniata, is gaining prevalence as a model system for these types of studies which were previously performed almost exclusively in the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. An advantage of these model systems is the ease of producing modified embryos in which a particular gene is up or downregulated, labeling a group of cells, or introducing a reporter gene. A single microinjection method is capable of creating a wide variety of such modified embryos. Here, we present a method for obtaining gametes from P. miniata, producing zygotes, and introducing perturbing reagents via microinjection. Healthy morphant embryos are subsequently isolated for quantitative and qualitative studies of gene function. The availability of genome and transcriptome data for this organism has increased the types of studies that are performed and the ease of executing them.

A Method for Microinjection of Patiria minata Zygotes

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.

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and ...

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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Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

A Method for Characterizing Embryogenesis in Arabidopsis

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

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator

A Simple and Effective Transplantation Device for Zebrafish Embryos

Preparation of Single-Somite Explants from Zebrafish Embryos

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

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

A Method for Microinjection of Patiria minata Zygotes