Development of an apparatus for monitoring protoplast isolation from plant tissues based on both dielectric and optical methods

In order to develop a method allowing objective determination of the optimal conditions for the isolation of protoplasts, the process of protoplast isolation from plant tissues was quantitatively evaluated. First, a specialized spectrophotometer cuvette (working volume = 2.0 ml) was designed for the continuous monitoring of protoplast isolation from plant tissues based on the optical method. Homogeneous mixing of tissue sections and the protoplast suspension in the cuvette was accomplished by means of a magnetic bar. The cuvette was divided into upper and lower parts by a nylon mesh. Since tissue sections in the upper part could not pass through the mesh, they did not affect the optical path in the lower part, and only isolated protoplasts were able to move freely between the two parts. At the optimal agitation speed (200 rpm), mechanical damage to protoplasts of Catharanthus roseus did not occur. Increases in the protoplast concentration during their isolation from tissue sections (leaf and petal) could be continuously monitored by measuring the optical density (O.D.), making it possible to estimate the end of protoplast isolation. Degassing treatment of the tissues markedly enhanced protoplast isolation. In order to monitor the viable protoplast concentration, a larger specialized spectrophotometer cuvette (working volume = 25 ml) was developed which enabled simultaneous measurement of the permittivity and O.D. of the suspension to be carried out during protoplast isolation. Permittivity is a measure of the viable protoplast concentration, while the O.D. shows protoplast characteristics such as color. Using this large cuvette, the time courses of protoplast isolation from leaf and petal sections were monitored and large amounts of viable protoplasts were obtained. The protoplast isolation process after degassing treatment was described by a simple first-order reaction model and the viable protoplast isolation rate was quantitatively evaluated from the rate constant (k) on the basis of permittivity changes.