Protoplast isolation from Ulmusamericana L. pollen mother cells, tetrads, and microspores

Meiotic protoplasts of Ulmusamericana (American elm) are potentially valuable for producing interspecific elm hybrids through protoplast fusion. Meiotic cells (pollen mother cells, tetrads, and microspores) were incubated in either a cellulase, hemicellulase, and pectinase enzyme solution or a β-1,3-glucanase (laminarinase) solution. Respective protoplast isolation frequencies for the three meiotic cell types were 100, 50, and 10%. Exclusion staining with 0.2% Evans blue and 0.1% methyl blue suggested protoplast viability. Some of the microspore protoplasts were vacuolated, which is an important condition for cell division. Although attempts to regenerate cell walls and induce cell division were unsuccessful, these two problems may be superceded by protoplast fusion with more regenerative protoplasts. To our knowledge this is the first report of protoplast isolation from meiotic cells of a tree species.