ISOLATION AND CULTURE OF BARLEY MEGASPOROCYTE PROTOPLASTS

An efficient technique has been developed for the isolation of barley megasporocyte protoplasts at early meiotic prophase. Ovules were disse cted out of ovaries under aseptic conditions, subjected to a brief enz ymatic digestion, and then transferred to a modified Kao medium with 9 0 g/l sucrose and 20 mM CaCl2. A small incision was made with a scalpe l through the softened epi dermal cell layer of the nucellus and the m egasporocyte could then be liberated into the medium by applying gentl e pressure on the nucellus. The megasporocyte appeared to be completel y devoid of a wall and changed its in situ pyriform shape to completel y spherical when extruded into the medium. Four to nine protoplasts co uld typically be isolated per spike. Protoplasts cultured in medium de generated after a few days. Viability was dramatically improved if pro toplasts were co-cultivated with barley microspores undergoing microsp ore embryogenesis. More than half of the protoplasts were still alive after 6 days of culture, and in some cases they survived more than 12 days of culture. Fluorescence microscopy of the cultured protoplasts s tained with 4',6-diamidino-2-phenylindole (DAPI) or aniline blue revea led that the protoplasts remained uninuclear and reformed their callos e wall.