Evacuolation and enucleation of mesophyll protoplasts in self-generating percoll gradients

Mesophyll protoplasts from Brassica oleracea, B. napus, Nicotiana tobaccum and Solanum tuberosum were isolated and subjected to uttracentrifugation at 65000g for 30 min in percoll solutions containing various strengths of salt and osmotic stabilizing agents. After centrifugation, the self-generated percoll gradients were evaluated for their effectiveness in protoplast evacuolation and enucleation. The vacuoles, cell debris, evacuolated protoplasts and enucleated protoplasts were separated. Factors that affected evacuolation and enucleation in the percoll gradients were described. Mesophyll protoplasts produced by epidermis peeling and short enzyme incubation periods were more easily evacuolated and enucleated than those produced by leaf-slicing and long incubation periods. Lower centrifugal force at 25000g for 80 min was also successful in evacuolating and enucleating the mesophyll protoplasts. A green band that contained nearly pure evacuolated protoplasts, of which 45% were enucleated protoplasts, was obtained from the self-generated percoll gradient. Rhodamine 123 staining of mitochondria indicated that the evacuolated protoplasts were metabolically active and were capable of regenerating the vacuole and cell wall. Cell divisions were also observed when the evacuolated protoplasts were cultured.