Method for insertion of microelectrode into plant protoplasts
In general, insertion of microelectrodes into plant protoplasts is difficult, and therefore it is very difficult to obtain convincing measurements of membrane potentials of protoplasts. We developed an insertion method for protoplasts as follows.
1.A glass capillary electrode gently placed against the protoplast, and a short electrical impulse (5 msec) is applied. Immediately after this application, a hole in the plasma membrane develops. It is visible under low magnification of a light microscope. When concentrations of calcium ions are suitable, the hole will develop for a few seconds to reach several micrometers in diameter. This development of a hole on the surface is accompanied by movement of the layer of chloroplasts (ectoplasm) and therefore it is recognizable as a white pin hole on the green surface of the protoplast. It should be noted that the cytoplasmic streaming stops completely in just a few seconds after the application of the electrical pulse.
2. Advance the electrode tip into vacuole smoothly before closing the opening.
3. Let the electrode sit for a few minutes to allow the opening around the electrode to seal.
4. Gradually the cytoplasmic streaming recovers to original state, and the membrane potential recovers to a large negative value as in intact cells. A recorder trace of the insertion followed by changing potassium concentrations is shown in a figure below. The initial depolarization is due to generation of the action potential.
Note: If the hole keeps expanding, increase concentration of Ca 2+. If the hole closes too quickly, reduce the concentration of Ca2+. Ca2+ concentrations are an important determinant of successful insertion and meaningful results of measurements including response to ions and recording of action potentials.
Next figure shows a typical recorder trace of membrane potentials of Nitella protoplasts, using the electrical insertion method.
The action potential showing a fast raising peak is followed by a slow calcium dependent depolarization. The slow depolarization therefore appeared to be a calcium spike, and was prominent in protoplasts.
This calcium dependent development of a hole on the cytoplasmic membrane led us to induce two adjacent protoplasts to fuse electrically.->Electrofusion
We also investigated dependency on K and Ca ions and compared these electrophysiological parameters with their intact cells. The results showed that the membrane potentials of enzymatically isolated protoplasts were negative inside as their intact cells.---->More results on membrane potentials
To preparation of protoplasts. References
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Culture Media for Chara and Nitella