Preparatory tests for immunodetection of microtubules in protoplasts during IML-2

Preparatory tests to improve methods for detection of cortical microtubules (cMTs) in rapeseed protoplasts for the IML-2 experiment "TRANSFORM" were undertaken. This study is based on the results obtained in the "PROTO" experiment onboard the shuttle Discovery during an 8-day IML-1 flight in 1992. The use of free-floating protoplasts on IML-1 made it technically impossible for the astronauts to remove the glutaraldehyde fixative during the orbital period resulting in high background fluorescence which made it very difficult to detect MTs. In order to avoid this on the IML-2 mission, protoplasts will be immobilized in alginate beads. The effect of variable concentrations and fixation periods for two different fixatives on the preservation of cMTs was tested. Best results were obtained using 3.5% paraformaldehyde (PFA) for 1 hour, but 1% glutaraldehyde (GA) also gave acceptable results. The effect of low temperatures on microtubule depolymerization was also examined as freshly isolated protoplasts have to be kept at 5 degrees C for up to 20 hours pre-launch and before activation of Biorack. Only slight changes in cMT-appearance were observed at 4 degrees C indicating a minor depolymerization compared to the cMTs in non-chilled protoplasts.