Skagen E B, Rasmussen O S, Iversen T H
Department of Botany, University of Trondheim, Norway.
Microgravity Q. 1994 Apr;4(2):83-91.
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.
为了改进IML-2实验“TRANSFORM”中油菜原生质体皮质微管（cMTs）检测方法，进行了预备试验。本研究基于1992年在发现号航天飞机上进行的为期8天的IML-1飞行中“PROTO”实验所获得的结果。在IML-1上使用自由漂浮的原生质体，使得宇航员在轨道飞行期间从技术上无法去除戊二醛固定剂，导致背景荧光很高，这使得检测微管非常困难。为了在IML-2任务中避免这种情况，原生质体将被固定在藻酸盐珠中。测试了两种不同固定剂的不同浓度和固定时间对cMTs保存的影响。使用3.5%多聚甲醛（PFA）固定1小时获得了最佳结果，但1%戊二醛（GA）也给出了可接受结果。还研究了低温对微管解聚的影响，因为新鲜分离的原生质体在发射前和生物架激活前必须在5摄氏度下保存长达20小时。在4摄氏度下观察到cMTs外观只有轻微变化，表明与未冷藏原生质体中的cMTs相比，解聚程度较小。
