Detecting Enzyme Variation in the Fern Genus Bommeria: An Analysis of Methodology

Abstract

Although numerous flowering plant species have been subjected to electrophoretic enzyme analysis, very few such studies have involved pteridophytes. This is largely because standard methodology typically results in little or no electro- phoretically detectable enzymatic activity in pteridophytes. The presence of large amounts of phenolic compounds in leaves of most pteridophytes suggests that the electrophoretic difficulties heretofore encountered in this group result from com- plexing of enzymes by these compounds following cellular disruption. Use of a grinding buffer containing dimethylsulfoxide and several compounds inhibitory to phenolic complexing, in combination with a grinding procedure utilizing polyvinyl- pyrrolidone and liquid nitrogen, was found to overcome the difficulties of tissue preparation that result from high concentrations of phenolic compounds. Modifi- cations of this grinding buffer and grinding procedure often resulted in reduced clarity of enzyme bands, or even a total loss of enzyme activity. In Bommeria, prep- aration of leaf tissue in liquid nitrogen and use of sodium tetraborate, sodium as- corbate, sodium meta-bisulfite, and sodium diethyldithiocarbamate were found to be of less importance than use of polyvinylpyrrolidone in obtaining active enzyme samples. We hope that discussion of our procedural data and provision of gel and electrode buffer recipes and staining schedules will stimulate further electrophoretic investigation of pteridophytes.