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.

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.