Hello.Today we're gonna show you a protocol that protoplasts millions of maize mesophyll cells, and transform them at high efficiency. The main steps of the protocol are first to digest and remove the plant cell wall, releasing the protoplasts, and then transform the protoplast using PEG. The big difference between this protocol and others is the scale of transformation.
Most protocols end up with between 1, 000 and 10, 000 transformed protoplasts. However, our protocol gives millions of transformed maize protoplasts. Here we have maize seedlings grown in the dark for nine to 11 days post germination.
Cut the seedlings just above soil level and place in water. Keep the plants in the dark when not in use. Select the second and third leaves of each seedling, taking care to exclude leaves with brown or damaged areas.
Select 12 to 16 leaves, and cut a centimeter off of the tips. Then cut a 10 centimeter section from each leaf. Stack leaf sections on top of each other with the basal ends together.
Clamp the bundle together at the basal end using a binder clip. Place the bundle of leaves on a piece of white paper. Cut leaf slices that are 0.5 to one millimeter wide.
It is important to cut thin consistent slices. After cutting part of the leaf bundle, transfer the slices into the enzyme solution. Don't allow the material to dry out.
Gently swirl the enzyme solution to wet the material. Place foil over the beaker to block out light. Repeat this process until the entire bundle is cut down close to the binder clip.
After cutting, the solution should look like this. Transfer the beaker of enzyme solution to a bell jar. Vacuum infiltrate for three minutes at room temperature.
Incubate on a tabletop shaker at 40 RPM at room temperature for two and a half hours. After incubation, give the solution a swirl. It should look milky, indicating a successful digestion.
Incubate on the tabletop shaker at 80 RPM at room temperature for 10 more minutes. Place the beaker with enzyme solution on ice. Stop the digestion by adding one volume of ice cold MMG.
Filter through a 40 micron cell strainer into a 50 milliliter centrifuge tube. Split the solution into aliquots of no more than 10 milliliters each. Pour into chilled round bottom glass tubes.
Centrifuge the tubes at 100 G for four minutes. Remove the supernatant, making sure not to disturb the pellet. Resuspend, then pool samples for subsequent washes.
Wash protoplasts twice with five milliliters of MMG. Spin down each time at 100 G for three minutes. The pellet should be a beautiful, bright yellow green color.
You'll be able to resuspend the pellet by gently swirling the tube. Resuspend in one milliliter of MMG, and dilute a small aliquot 10 to 20x for counting. It's important to resuspend well before aliqouting, as the protoplasts will settle quickly.
Load the diluted protoplasts into the hemocytometer. Count the protoplasts under a light microscope. A good prep won't have much excess debris floating around after washing.
Good protoplasts are circular with visible plastids. Use the cell count to calculate the total number. This prep resulted in just over 10 million protoplasts.
We recommend checking viability using fluorescein diacetate dye. Successful protoplast isolations yield the viability between 70 and 90%Mix the protoplasts with your plasmid of interest. This example uses 1 million protoplasts and 15 micrograms of DNA.
Incubate on ice for 30 minutes. Resuspend by tapping the side of the tube. Add PEG solution to reach a final concentration of 12%PEG.
Gently mix by inverting several times. Protoplasts and PEG solution are fragile, so don't shake the tube. Incubate in the dark at room temperature for 10 minutes.
Dilute with five volumes of incubation solution, and gently mix by inversion. Centrifuge the tubes at 100 G for four minutes. Wash once with one milliliter incubation buffer.
Be careful not to disturb the pellet. Spin down at 100 G for three minutes. Resuspend in incubation buffer to a concentration of 500 to 1000 cells per microliter.
Incubate in the dark overnight at room temperature. The next day, spin down at 100 G for four minutes. Wash twice with chilled incubation solution.
Spin down each time for three minutes at 100 G at room temperature. Resuspend, then load a small aliquot onto the hemocytometer for the final check. First, count the total number of protoplasts under brightfield.
Now, observe the fraction of protoplasts that fluoresce under UV to verify transfection. The protoplasts are expressing our GFP reporter. Now, let's get some metrics on transfection efficiency.
In this prep, we obtained 10 million total protoplasts, of which we transfected 1 million. The next day, we recovered 335, 000. Of those, we had a transformation rate of 30.3%This leaves us with our total of around 100, 000 protoplasts expressing GFP.
Transfection efficiency can vary. As you can see in the chart, we routinely see efficiencies between 20 and 50%among repeated preparations. This protocol allows for the collection and transformation of millions of maize mesophyll protoplasts.
One of the best parts of the protocol is its ability to scale up. By scaling the amount of volume as well as the number of protoplasts used in transformation, you can increase your transformation by more than order of magnitude from what we have shown today. To date, our largest single tube experiment started with 20 million protoplasts and 200 micrograms of DNA, and ended with 3.1 million transformed protoplasts the next day.
This high throughput protocol is especially useful when you need to test many different plasmid constructs, but collaborators have used this protocol for purposes that do not require the ability to scale up, such as designing synthetic gene circuits and maize. Thanks for watching our video. We hope it helped.
