Name: A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses (Video) | JoVE Journal
Uploaded: 2018-01-25T15:00:00.000+00:00
Duration: 9 min 22 s
Description: Learn a simple method to isolate soybean protoplasts for transient gene expression analyses. This protocol produces large quantities of uniform cells to study regulatory mechanisms in live plant cells.

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5 minutes

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 interest and inoculate it in 20 mL Luria-Bertani (LB) liquid medium with appropriate antibiotics in a 50 mL flask.</li>
	<li>Incubate the flask at 37 &#176;C overnight on a shaker. Collect the bacteria by centrifuging the suspension at 12,000 x g at room temperature for 5 min and discarding the supernatant. Extract and purify the plasmid following the manufacturer&#39;s procedure of a plasmid prep kit.</li>
</ol>
3. Protoplast isolation
<ol>
	<li>Cut newly expanded unifoliate leaves from 10-day old soybean seedlings (Figure 1). 
	NOTE: Selection of leaves at an appropriate developmental stage is the key to the success for soybean protoplast preparation. Only use just expanded leaves at early developmental stages. As leaves mature, cell walls become harder to digest.</li>
	<li>With a fresh razor blade, remove the midrib from the unifoliate leaf and then cut the remains into 0.5-1 mm strips. 
	NOTE: Two unifoliate leaves digested in 10 mL enzyme solution will give sufficient protoplasts for more than 10 transformations.</li>
	<li>Using a pair of forceps, transfer the leaf strips immediately and gently into 10 mL of freshly prepared enzyme solution (Table 1) in a 15 mL tube. Vacuum infiltrate the leaf strips for 15 min at room temperature.</li>
	<li>Incubate the leaf strips in the enzyme solution with gentle agitation (40 rpm) under low light for 4-6 h at room temperature. Ensure that the enzyme solution turns yellow-green as protoplasts are released. Check the enzyme/protoplasts solution under the microscope (X10). 
	NOTE: The released protoplasts are spherical shaped, while the undigested cells have irregular or oval shape.</li>
	<li>Transfer the 10 mL of enzyme/protoplast solution in a 50 mL tube by gently pouring and add 10 mL of W5 solution (Table 1) at room temperature to stop the digestion. Gently invert the tube a few times. Pour gently the enzyme/protoplasts solution on a clean 75 &#181;m nylon mesh placed on top of a 50 mL tube to remove the undigested leaf tissues.</li>
	<li>Centrifuge the flow-through enzyme/protoplasts solution at 100 x g in the 50 mL tube for 1-2 min at room temperature. Gently remove the supernatant using a 10 mL serological pipette without disturbing the protoplast pellet.</li>
	<li>Resuspend the protoplasts and dilute to a concentration of 2 x 105 mL-1 in chilled W5 solution at 4 &#176;C by counting protoplast number on a hemacytometer under the microscope (x10). Keep the protoplasts on ice for 30 min.</li>
	<li>Centrifuge the suspension at 100 x g for 1-2 min at room temperature and gently remove the W5 solution using a 1 mL pipette without disturbing the protoplast pellet. Resuspend the protoplasts in MMG solution (Table 1) at a concentration of 2 x 105 mL-1 at room temperature.</li>
</ol>
4. Protoplast transformation
<ol>
	<li>Make 100 &#181;L aliquots of protoplasts (2 x 104 protoplasts at 2 x 105 mL-1) in 1.5 mL low adhesion microcentrifuge tubes using uncut 200 &#181;L pipette tips. Put one aliquot aside to serve as negative control. Add 10 &#181;L of plasmid (10-20 &#181;g) into each of the rest aliquot of protoplasts.</li>
	<li>Slowly add 110 &#181;L of freshly prepared PEG solution (Table 1) on the inner wall of the 1.5 mL microcentrifuge tube, and then gently invert and rotate the tube until the solution becomes homogeneous.</li>
	<li>Incubate the transformation mixture at room temperature for 15 min.</li>
	<li>To stop the transformation, slowly add 400 &#181;L of W5 solution to the 1.5 mL tube at room temperature and gently invert the tube until the solution becomes homogeneous. Centrifuge the tube at 100 g for 1-2 min at room temperature and discard the supernatant using a pipette.</li>
	<li>Add 1 mL of WI solution (Table 1) to the tube and resuspend by gently pipetting 1-2 times. Add 1 mL of 5% (vol/vol) sterile calf serum in each well of a 6-well tissue culture plate to coat the surface and prevent the protoplasts from sticking to the plate.</li>
	<li>After a few seconds, discard the calf serum using a pipette. Transfer the resuspended protoplasts into a well of the culture plate. Cover the plate with a lid.</li>
</ol>
5. Protoplast incubation and harvesting
<ol>
	<li>Incubate the protoplasts at room temperature for 1-2 days in the dark. 
	NOTE: We found that two-days incubation yields stronger fluorescent protein signal in general compared to one-day incubation, and that the fluorescent protein signal may last for up to 3-4 days.</li>
	<li>Transfer the protoplast solution to a 1.5 mL low adhesion microcentrifuge tube. Centrifuge the tube at 100 x g for 1-2 min at room temperature to harvest protoplasts. Remove the supernatant using a pipette and transfer 10 &#181;L protoplasts onto a glass slide.</li>
	<li>Observe fluorescent signal under fluorescence or confocal microscopy. Use non-transformed protoplasts as negative control (No signal should be observed).</li>
</ol>

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</ol>

The authors declare that they have no competing financial interests.

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><tbody><tr><td>MES</td><td>Sigma Aldrich</td><td>M8250-100G</td><td></td></tr><tr><td>Cellulase CELF</td><td>Worthington Biological Corporation</td><td>LS002611</td><td></td></tr><tr><td>Pectolyase Y-23</td><td>BioWorld</td><td>9033-35-6</td><td></td></tr><tr><td>CELLULASE &quot;ONOZUKA&quot; R-10</td><td>yakult</td><td>10g</td><td></td></tr><tr><td>MACEROZYME R-10</td><td>yakult</td><td>10g</td><td></td></tr><tr><td>Mannitol</td><td>ICN Biomedicals</td><td>152540</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher</td><td>C79-500g</td><td></td></tr><tr><td>BSA</td><td>NEB</td><td>R3535S</td><td></td></tr><tr><td>DTT</td><td>Sigma Aldrich</td><td>D5545-5G</td><td></td></tr><tr><td>NaCl</td><td>Sigma Aldrich</td><td>S7653-1kg</td><td></td></tr><tr><td>KCl</td><td>Fisher</td><td>P217-500g</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>M8266-100g</td><td></td></tr><tr><td>PEG4000</td><td>Fluka</td><td>81240</td><td></td></tr><tr><td>nylon mesh</td><td>carolina</td><td>652222N</td><td></td></tr><tr><td>Tissue Culture Plates</td><td>USA Scientific</td><td>CC7682-7506</td><td></td></tr><tr><td>Razor Blades</td><td>Fisher</td><td>12-640</td><td></td></tr><tr><td>hemacytometer</td><td>hausserscientific</td><td>1483</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>EZNA plasmid miniprep kit</td><td>Omega</td><td>D6942-01</td><td></td></tr><tr><td>GeneJET Plasmid Miniprep Kit</td><td>Thermo Scientific</td><td>K0502</td><td></td></tr><tr><td>Centrifuge 5810</td><td>eppendorf</td><td>5811000827</td><td></td></tr><tr><td>Centrifuge 5424</td><td>eppendorf</td><td>22620401</td><td></td></tr><tr><td>Jencons Powerpette Plus Pipet Controller</td><td>Jencons</td><td>14526-202</td><td></td></tr><tr><td>Zeiss 710 Confocal Microscope</td><td>Zeiss</td><td>N/A</td><td></td></tr><tr><td>Nonstick, RNase-free Microfuge Tubes, 1.5 mL</td><td>Ambion</td><td>AM12450</td><td></td></tr><tr><td>15 mL Centrifuge Tubes</td><td>Denville</td><td>C1018-P</td><td></td></tr><tr><td>50 mL Centrifuge Tubes</td><td>Denville</td><td>C1060-P</td><td></td></tr><tr><td>Newborn Calf Serum</td><td>Thermo Scientific</td><td>16010159</td><td></td></tr><tr><td>Soil</td><td>Ingram&#039;s Nursery</td><td></td><td></td></tr><tr><td>perlite</td><td>Vigoro</td><td>100521091</td><td></td></tr><tr><td>Torpedo Sand</td><td>JKS Ventures</td><td></td><td></td></tr><tr><td>LB Broth, Lennox (Powder)</td><td>Fisher</td><td>BP1427-500</td><td></td></tr></tbody></table>

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

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...

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. ...

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Research

JoVE Journal

Biology

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

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

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

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Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Here, we present a high-throughput protocol to transiently transfect millions of maize protoplasts for testing large libraries of plasmids in maize mesophyll cells.

Scalable Transfection of Maize Mesophyll Protoplasts

Here, we present a protocol for the high-efficiency production of transgenic soybean hairy roots.

Soybean Hairy Root Transformation for the Analysis of Gene Function

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

Learn a simple method to isolate soybean protoplasts for transient gene expression analyses. This protocol produces large quantities of uniform cells to study regulatory mechanisms in live plant cells.

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

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