Isolation and osmotic relations of developing megagametophytes of Torenia fournieri

A new method is reported to isolate and handle living megagametophytes of T orenia fournieri at any developmental stage. The stages were determined usi ng right microscopy and delimited by correlating floral morphological trait s. When significant changes in the osmotic pressure were found during devel opment, enzyme solutions contained different concentrations of osmoticum. O smotic pressure is lowest in the megaspore, increases until the four-nuclea te stage and then gradually decreases until complete embryo sac formation. In enzymatic solutions containing appropriate concentrations of osmoticum, protoplasts of megaspores, two-, four-, eight-nucleate embryo sacs, egg cel ls, synergids and central cells were successfully isolated. The living prot oplasts were collected by micromanipulator, transferred into microdroplets and tested for viability.