Isolation of giant plant protoplasts from Charophyta and higher plants
1 Isolation of giant protoplasts from Charophyta.
We have enzymatically isolated giant protoplasts from Nitella expansa Allen.
These protoplasts posses intrinsic cytoplasmic membranes and all of the contents within the cytoplasm including the complete array of chloroplasts and ectoplasm, and showed active cytoplasmic streaming as same speed as in their intact cells. Since cells underwent a conversion of shape from cylinder into sphere, the protoplasts have the north and south poles, where no streaming is observed, and the equator, where the fastest streaming is observed.
Materials and Methods
Plant material and Isolation protoplasts
An actively growing terminal bud of Nitella expansa Allen was used for source of protoplasts. Its unopened leaves were used for enzymatic digestion of cell wall to isolate theire protoplasts. Growing the plant under constant conditions is important to obtain viable protoplasts and reproducible results---->Click here for culture media.!
Enzyme treatment for digestion of cell wall.
Protoplasts were isolated as follows using a separating mesh, because any mechanical stresses such as shaking, filtering, and even gentl swirling in ordinary isolation protocols should be avoided.
.A. Terminal bud was placed in an enzyme solution consisting of 1% Cellulase Onozuka R-10, 0.05% Macerozyme Onozuka R-10, 1mM CaCl2, 1 mM NaCl, 0.1 mM KCl, and an appropriate osmotic concentration of sorbitol (pH 5.5).
B. 1 hr after the incubation with the enzyme solution.
C. 2 hr after the incubation with the enzyme solution. Only debris was left over the mesh, and isolated protoplasts were on the bottom of the vessel (D). These freshly isolated protoplasts are often not good sphere, and the cytoplasmic streaming is generally still stopping.
Click the above picture to get magnified!
Next fig shows several isolated giant protoplasts above. Generally, after 10 to 30 min protoplasts recover their active cytoplasmic streaming and become reasonably good spheres.These spherical protoplasts are suitable for electrophysiology.
<----Click the image to magnify!
Next figure shows protoplasts isolated from carrot root cells by a similar method as Nitella. These are also large enough and active enough for electrophysiology.
Electrophysiology of plant protoplasts (membrane potentials, action potentials, and comparison with their intact cells)