Microtubular organization in protoplasts and cells of somatic embryo-regenerating and non-regenerating cultures of Larix

Protoplasts were isolated from both somatic embryo-regenerating and non-regenerating cultures of hybrid larch ( Larix x eurolepis Henry) and fractionated on a discontinuous Percoll density gradient, whereby a highly embryogenic protoplast fraction could be enriched. This fraction was cultured for 14 days, and the differentiating protoplasts, cells, proetmbryos and embryo-like cell clusters sampled at days 3, 5 and 14. Immunofluorescence studies showed that the microtubules became organized into parallel and helical arrays in protoplast-derived cells of the embryogenic tissue as early as day 3 in culture, at which time the protoplast-derived cells started to elongate. In most of the protoplasts from non-regenerating tissue the microtubules retained a random orientation for a longer period. Preprophase bands were observed in both lines. Mitotic spindles consisted mainly of kinetochore-associated microtubules and displayed broad polar regions at metaphase. The spindle poles contracted at anaphase, giving the spindles a pointed appearance. A difference between the two tissue lines was observed at telophase, when the phragmoplast in the non-regenerating tissue had a normal appearance, while a proportion of the phragmoplasts from the embryo-regenerating line were branched or Y-shaped. Y-shaped phragmoplasts resulted in two nucleated cells and one enucleated cell after fusion of the cell plate with the plasma membrane. The early rearrangement of cortical microtubules is an indication that organized growth is occurring but, as this phenomenon has been observed also in regenerating non-emhryogenic cells, it appears to be a doubtful indicator of the distinction between emhryogenic and non-embryogenic development.