CELL-CYCLE IN POTENTIALLY DEDIFFERENTIATING CEREAL MESOPHYLL PROTOPLASTS CULTURED INVITRO .2. BEHAVIOR OF THE CYTOSKELETON

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 elucidat e whether changes in the cytoskeletal patterns correlate with the abno rmal progression through the cycle observed previously. Protoplasts fr eshly isolated from mature mesophyll tissue (type B) exhibited very fe w, fragmented microtubules, whereas mesophyll protoplasts isolated fro m immature leaf tissue (type A) showed an abundant network fo microtub ules. Type B protoplasts of wheat, which were able to reenter but reca lcitrant to complete S-phase, failed to reestablish a microtubular net work. Type A wheat protoplasts capable of progressing from G1 to G2 bu t 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 proto plasts reestablished an abundant network of microtubules after about 5 days in culture. Both types of protoplasts were capable of going thro ugh mitosis. Microtubule configurations typical of mitotic cells were observed. However, several unusual features became apparent. No prepro phase bands of microtubules were found. The spindles were short and bl urred 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 w as reestablished, apparently before completion of cytokinesis. Actin m icrofilaments were found in all freshly isolated and cultured premitot ic protoplasts. They were not detected during the mitosis of oat proto plasts, but were found again in phragmoplasts. They appeared to be ass ociated in abundance with presumptive phragmosomes, even after disappe arance of phragmoplast microtubules. The cell cycle abnormalities obse rved indicate a possible cause for the cereal protoplast recalcitrance .