Subcellular fractionation of wheat leaf protoplasts by centrifugal elutriation

Abstract

A new method using centrifugal elutriation for subcellular fractionation of plant cells has been developed. This method takes advantage of the fact that particles sedimenting in a gravitational field can be eluted by flow against the field. A wheat protoplast homogenate was fed into an elutriation rotor spinning at high speed and the flow rate into the rotor was gradually increased. The smaller and less dense materials such as mitochondria, microbodies, endoplasmic reticulum, and cytoplasm were elutriated earlier than the larger and denser nuclei and chloroplasts. The intact chloroplasts, free of mitochondria, microbodies, endoplasmic reticulum, and cytoplasm, could be obtained within 40 min following the rupture of protoplasts. The chlorophyll-free mitochondria could be obtained within 80 min.