Cell Cycle in Potentially Dedifferentiating Cereal Mesophyll Protoplasts Cultured in vitro II. Behaviour of the Cytoskeleton

Abstract

Summary Following up on the recent finding that mesophyll protoplasts of wheat and oats were able to reenter the cell cycle when cultured in vitro , the behaviour of the cytoskeleton was studied. The aim was to elucidate whether changes in the cytoskeletal patterns correlate with the abnormal progression through the cycle observed previously. Protoplasts freshly isolated from mature mesophyll tissue (type B) exhibited very few, fragmented microtubules, whereas mesophyll protoplasts isolated from immature leaf tissue (type A) showed an abundant network fo microtubules. Type B protoplasts of wheat, which were able to reenter but recalcitrant to complete S-phase, failed to reestablish a microtubular network. Type A wheat protoplasts capable of progressing from G1 to G2 but incapable of entering mitosis lost microtubules during culturing and eventually resembled type B protoplasts. In contrast to wheat, type A protoplasts of oats retained microtubules in culture and type B protoplasts reestablished an abundant network of microtubules after about 5 days in culture. Both types of protoplasts were capable of going through mitosis. Microtubule configurations typical of mitotic cells were observed. However, several unusual features became apparent. No preprophase bands of microtubules were found. The spindles were short and blurred and did not form distinct poles. Phragmoplast microtubules were observed, but the deposition of cell plate material seemed to be slow. The phragmoplasts disappeared and a cortical network of microtubles was reestablished, apparently before completion of cytokinesis. Actin microfilaments were found in all freshly isolated and cultured premitotic protoplasts. They were not detected during the mitosis of oat protoplasts, but were found again in phragmoplasts. They appeared to be associated in abundance with presumptive phragmosomes, even after disappearance of phragmoplast microtubules. The cell cycle abnormalities observed indicate a possible cause for the cereal protoplast recalcitrance.