Isolation of protoplasts from leaves of chrysanthemum and orchardgrass
This chapter uses leaves of either chrysanthemum or orchardgrass to illustrate some aspects of protoplast technology. It investigates the influence of isolation medium, specifically the osmotic pressure, on successful procurement of viable protoplasts. When counting protoplasts one may have noticed that they were almost perfectly spherical; normally this would indicate that all of the cell wall material was digested by the enzymes. The protoplast isolation procedure often damages or kills a percentage of cells; therefore, it is advisable to assess the condition of the protoplasts. There are a number of presumptive tests to determine viability of cells. Most of these tests are based on enzyme activity or permeability of the plasmalemma to certain indicator chemicals, such as dyes or stains. Fluorescein diacetate often has been used to estimate the percentage of viable protoplasts in a preparation. This technique uses fluorescence and is very efficient — many protoplasts can be examined in a short time.