Name: a simple method for isolation of soybean protoplasts and application to transient gene expression analyses
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This work was supported by the Plant Genome Research Program from the National Science Foundation (NSF-PGRP-IOS-1339388).

1. Growth of the plants
<ol>
	<li>Sow 5 - 10 soybean seeds (Williams 82) in a 13 cm pot in the greenhouse under long-day conditions (16 h light at 1,500 &#181;mol m-2 s-1) at 25 &#176;C on the custom soil mix for Soybean (the 1:1:1 ratio of soil, perlite and torpedo sand).</li>
</ol>
2. Preparation of Plasmid DNA
<ol>
	<li>Using a sterile pipette tip or toothpick, pick a single colony or frozen glycerol stock of E. coli carrying the plasmid containing the gene of...

<ol>
	<li>Melchers, G., Labib, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+hybridisation+of+plants+by+fusion+of+protoplasts.">Somatic hybridisation of plants by fusion of protoplasts.</a> Mol. Gen. Genet. 135 (4), 277-294 (1974).</li><li>Gresshoff, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+culture+of+white+clover:+callus,+suspension,+protoplast+culture,+and+plant+regeneration.">In vitro culture of white clover: callus, suspension, protoplast culture, and plant regeneration.</a> Bot. 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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=Integration+of+foreign+sequences+into+the+tobacco+plastome+via+polyethylene+glycol-mediated+protoplast+transformation.">Integration of foreign sequences into the tobacco plastome via polyethylene glycol-mediated protoplast transformation.</a> Planta. 199 (2), 193-201 (1996).</li><li>Hrazdina, G., Wagner, G. J., Siegelman, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+localization+of+enzymes+of+anthocyanin+biosynthesis+in+protoplasts.">Subcellular localization of enzymes of anthocyanin biosynthesis in protoplasts.</a> Phytochemistry. 17 (1), 53-56 (1978).</li><li>Lin, W., Wittenbach, V. 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USA. 85 (18), 6738-6742 (1988).</li><li>Cocking, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+isolation+of+plant+protoplasts+and+vacuoles.">A method for the isolation of plant protoplasts and vacuoles.</a> Nature. 187 (4741), 962-963 (1960).</li><li>Zhang, H., 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=Transgenic+rice+plants+produced+by+electroporation-mediated+plasmid+uptake+into+protoplasts.">Transgenic rice plants produced by electroporation-mediated plasmid uptake into protoplasts.</a> Plant Cell Rep. 7 (6), 379-384 (1988).</li><li>Fromm, M., Taylor, L. 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This protocol for the isolation of soybean protoplasts and the application to transient expression studies has been thoroughly tested and works very well in our laboratory. The procedures are simple and easy and require ordinary equipment and minimum cost. Our protocol yields large quantities of uniform, high quality protoplasts compared to previously reported methods34,35,36,37,<sup...

Protoplasts are plant cells that have cell walls removed. As they maintain most of features and activities of plant cells, protoplasts are a good model system to observe and evaluate diverse cellular events, and are valuable tools to study somatic hybridisation1 and plant regeneration2. Protoplasts have been also widely utilized for plant transformation3,4,5, since cell walls would otherwise block the passage of DNA into the cell. Protoplasts possess some of the physiological responses and cellular processes of intact plants, hence offering fundamental value in basic research to study subcellular protein localization6,7,8, protein-protein interactions9,10, and promoter activity11,12,13 in live cells.
The isolation of plant protoplasts was first reported in 196014 and the protocols for both isolation and transformation of protoplasts have been developed and optimized. A standard procedure of protoplast isolation involves the cutting of leaves and enzymatic digestion of cell walls, followed by separation of released protoplasts from non-digested tissue debris. Transformation strategies includes electroporation15,16, microinjection17,18, and polyethylene glycol-based (PEG)4,5,19 methods. A wide range of species have been reported successful for protoplast isolation, including Citrus20, Brassica21, Solanaceae22 and other ornamental plant families23,24. While diverse tissue types are used in various species, a system of transient expression in Arabidopsis mesophyll protoplast (TEAMP) isolated from leaves of the model plant Arabidopsis thaliana has been well established25 and widely adopted to diverse applications.
Soybean (Glycine max (L.) Merr.) is one of the most important protein and oil crops26. Unlike Arabidopsis and rice, obtaining transgenic soybean plants is known to be rather difficult and low efficiency. Agrobacterium tumefaciens-mediated infiltration has been popularly used for transient gene expression studies in the epidermal cells in tobacco27 and seedlings in Arabidopsis28,29, whereas Agrobacterium rhizogenes has been used for transformation of hairy roots in soybean30. Virus-induced gene silencing approaches have been utilized for downregulation of target genes31,32 and transient expression33 in a systemic manner. Protoplasts provide a valuable and versatile alternative to these approaches. Protoplasts can be obtained from soybean&#39;s aboveground materials and allow quick and synchronized transgene expression. However, since the initial successful isolation of soybean protoplasts in the 198334, there have been limited reports on the application of protoplasts in soybean35,36,37,38, primarily due to relatively low yields of soybean protoplasts.
Here, we describe a simple and efficient protocol for isolation of soybean protoplasts and its application for transient gene expression studies. Using young unifoliate leaves from soybean seedlings, we were able to obtain large quantities of vital protoplasts within a few hours. In addition, we have optimized a PEG-calcium-mediated transformation method that is simple and low cost to deliver DNA into soybean protoplasts with high efficiency.

<table border="0" cellpadding="0" cellspacing="0" width="723">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">MES</td>
			<td style="width:181px;">Sigma Aldrich&#160;</td>
			<td style="width:119px;">M8250-100G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellulase CELF</td>
			<td>Worthington Biological Corporation</td>
			<td>LS002611</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pectolyase Y-23</td>
			<td>BioWorld</td>
			<td>9033-35-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CELLULASE &#34;ONOZUKA&#34; R-10</td>
			<td>yakult</td>
			<td>10g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACEROZYME R-10</td>
			<td>yakult</td>
			<td>10g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mannitol</td>
			<td>ICN Biomedicals&#160;</td>
			<td>152540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2</td>
			<td>Fisher&#160;</td>
			<td>C79-500g&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td>NEB</td>
			<td>R3535S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DTT</td>
			<td>Sigma Aldrich&#160;</td>
			<td>D5545-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Sigma Aldrich&#160;</td>
			<td>S7653-1kg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>Fisher&#160;</td>
			<td>P217-500g&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgCl2</td>
			<td>Sigma Aldrich&#160;</td>
			<td>M8266-100g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PEG4000</td>
			<td>Fluka</td>
			<td>81240</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">nylon mesh</td>
			<td>carolina</td>
			<td>652222N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Tissue Culture Plates&#160;</td>
			<td>USA Scientific</td>
			<td>CC7682-7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Razor Blades</td>
			<td style="width:181px;">Fisher</td>
			<td>12-640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hemacytometer</td>
			<td>hausserscientific</td>
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		</tr>
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			<td height="21" style="height:21px;width:256px;">QIAprep Spin Miniprep Kit</td>
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			<td>27104</td>
			<td></td>
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			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:256px;">GeneJET Plasmid Miniprep Kit</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td>K0502</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Centrifuge 5810</td>
			<td style="width:181px;">eppendorf</td>
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			<td style="width:181px;">eppendorf</td>
			<td>22620401</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Jencons Powerpette Plus Pipet Controller</td>
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			<td>14526-202</td>
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		<tr height="24">
			<td height="24" style="height:25px;width:256px;">Zeiss 710 Confocal Microscope</td>
			<td style="width:181px;">Zeiss</td>
			<td style="width:119px;">N/A</td>
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		<tr height="33">
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			<td style="width:181px;">Ambion</td>
			<td style="width:119px;">AM12450</td>
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			<td height="21" style="height:21px;width:256px;">15 mL Centrifuge Tubes</td>
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			<td height="21" style="height:21px;width:256px;">50 mL Centrifuge Tubes</td>
			<td style="width:181px;">Denville</td>
			<td style="width:119px;">C1060-P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Newborn Calf Serum</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td style="width:119px;">16010159</td>
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			<td height="21" style="height:21px;width:256px;">Soil</td>
			<td style="width:181px;">Ingram&#39;s Nursery</td>
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			<td height="21" style="height:21px;width:256px;">perlite</td>
			<td style="width:181px;">Vigoro</td>
			<td style="width:119px;">100521091</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Torpedo Sand</td>
			<td style="width:181px;">JKS Ventures</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">LB Broth, Lennox (Powder)</td>
			<td style="width:181px;">Fisher</td>
			<td style="width:119px;">BP1427-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

a simple method for isolation of soybean protoplasts and application to transient gene expression analyses

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. Therefore, it is important to establish an efficient transient gene expression system in soybean. Here, we report a simple protocol for the preparation of soybean protoplasts and its application for transient functional analyses. We found that young unifoliate leaves from soybean seedlings resulted in large quantities of high quality protoplasts. By optimizing a PEG-calcium-mediated transformation method, we achieved high transformation efficiency using soybean unifoliate protoplasts. This system provides an efficient and versatile model for examination of complex regulatory and signaling mechanisms in live soybean cells and may help to better understand diverse cellular, developmental and physiological processes of legumes.

Different organs of 10-day old soybeans were tested for protoplast preparation (Figure 1) and yields were observed under the microscope (Figure 2). Cell walls from hypocotyl and epicotyl were hardly digested, and some cells stayed attached to each other (Figure 2B, 2C). In cotyledon (Figure 2D) and root (Figure 2A), cell wal...

Watch this Scientific Journal Video about A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses at JoVE.com

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

We developed a simple and efficient protocol for the preparation of large quantities of soybean protoplasts to study complex regulatory and signaling mechanisms in live cells.

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. ...

The overall goal of this method is to obtain high quality protoplast cells from soybean to examine regulatory and signaling mechanisms in live soybean cells. This method can help answer key questions in regulative networks such as what their immediate target gene is, open up transfusion factor, and what their interacting partner is, open a protein. The main advantage of this technique is art.It produces large quantities of uniform soybean protoplast cells, and it's simple and easy. Generally, individuals new to this method will struggle because it's very difficult to obtain nice protoplasts from soybean leaves unless you unifoliate at a specific stage. The selection of leaves at an appropriate developmental stage is the key to a successful soybean protoplast preparation.Cut newly expanded unifoliate leaves from 10 day old soybean seedlings. To make sure that when a sample gives a high protoplast yield, collect at least three samples of unifoliate leaves at slightly different developmental stages. With a fresh razor blade, remove the midrib from each unifoliate leaf, and then cut the remains into 0.5 to one millimeter strips.Using a pair of forceps, gently transfer the leaf strips immediately into 10 milliliters of freshly prepared enzyme solution in a 15 milliliter tube. Vacuum infiltrate the leaf strips for 15 minutes at room temperature. Incubate the leaf strips in the enzyme solution with gentle agitation under low light for four to six hours at room temperature.As protoplasts are released, the enzyme solution will turn yellow-green in color. Check the enzyme protoplast solution under the microscope at 10x magnification to select the sample with the best protoplast digestion. The released protoplasts are spherical in shape, while the undigested cells have irregular or oval shapes.To stop the digestion, gently pour the 10 milliliters of enzyme protoplast solution with the best protoplast digestion into a 50 milliliter tube, and add 10 milliliters of W5 solution at room temperature. Gently invert the tube a few times. Then, gently pour the enzyme protoplast solution into a clean 75-micron nylon mesh placed on top of a 50 milliliter tube to remove the undigested leaf tissues.Next, centrifuge the flow through enzyme protoplast solution at 100 times G for one to two minutes at room temperature. Then, use a 10 milliliter serological pipette to gently remove the supernatant without disturbing the protoplast pellet. Resuspend the protoplast in chilled W5 solution, and keep the 50 milliliter tube on ice.After counting protoplast number on a hemocytometer under the microscope, dilute to a concentration of two times 10 to the fifth protoplasts per milliliter in chilled W5 solution. Keep the protoplast on ice for 30 minutes. Centrifuge the suspension at 100 times G for one to two minutes at room temperature.Then use a one milliliter pipette to gently remove the W5 solution without disturbing the protoplast pellet. Resuspend the protoplast in MMG solution at room temperature at a concentration of two times 10 to the fifth protoplasts per milliliter. To prepare the protoplast for transformation, use uncut 200 microliter pipette tipes to make 100 microliter aliquots of protoplasts in 1.5 milliliter low adhesion microcentrifuge tubes.Set aside one aliquot to serve as a negative control. To the remaining aliquot of protoplasts, add 10 microliters of plasmid DNA. Slowly add 110 microliters of freshly prepared peg solution on the inner wall of the 1.5 milliliter microcentrifuge tube.Then gently invert and rotate the tube until the solution becomes homogeneous. Incubate the transformation mixtures for 15 minutes at room temperature. To stop the transformation, slowly add 400 microliters of W5 solution to each 1.5 milliliter tube at room temperature, and gently invert the tube until the solution becomes homogeneous.Centrifuge the tubes at 100 times G for one to two minutes at room temperature. Discard the supernatant. Add one milliliter of WI solution to each tube.Coat the well surface of a six well tissue culture plate to prevent the protoplasts from sticking to the plate. Add one milliliter of five percent sterile calf serum to each well, and after a few seconds, remove the calf serum using a pipette. Transfer the resuspended protoplasts into the wells of the culture plate, and cover the plate with a lid.Prior to harvesting, incubate the protoplasts at room temperature for two days in the dark. After two days, transfer the protoplast solution to a 1.5 milliliter low adhesion microcentrifuge tube. Centrifuge the tubes at 100 times G for one to two minutes at room temperature.Remove the supernatant using a pipette. Transfer 10 microliters of protoplast onto a glass slide. Observe fluorescent signals under fluorescence or confocal microscopy using nontransformed protoplasts as a negative control.Protoplast cells were prepared from different organs of 10 day old soybeans and observed under the microscope. Cell walls from hypocaudal and epicaudal were hardly digested. And some cells stayed attached to each other.In caudal leaden and root, cell walls were removed only in a small portion of the cells. In contrast, a large number of protoplasts were observed when unifoliate was used. The seedlings in this photo from left to right illustrate unifoliate leaves that are unexpanded, just expanded, or fully expanded.While both the unexpanded and just expanded unifoliate leaves resulted in high yields of protoplasts, the size of protoplasts from the just expanded unifoliate was more uniform than the unexpanded unifoliate. For the fully expanded unifoliate, the cell walls were still intact in most of the cells. Testing a range of different amounts of plasmid DNA suggested that larger amounts of DNA resulted in higher transformation efficiency.Confocal images of soybean protoplasts transformed with the construct that expresses GFP fused to the legume specific E1 gene, shown nuclear localization of the E1 GFP fusion protein, consistent with the previous study usng the Arabidopsis protoplast system. While attempting this procedure, it's important to remember to optimize each experimental condition empirically, such as selection of the right stage of leaf material, length of cell wall digestion time, the age of concentration, and amount of plasmid DNA for transformation. Following this procedure, other methods such as RNA and protein replication can be performed in order to answer additional questions like what genes or what proteins are regulated or unregulated?After watching this video, you should have a good understanding of how to obtain high quality soybrean protoplast cells.

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