Delaying Seed Germination and Improving Seedling Fixation: Lessons Learned During Science and Payload Verification Tests for Advanced Plant EXperiments (APEX) 02-1 in Space

Here we report on the science verification test (SVT) and the payload verification test (PVT) that we conducted in preparation for experiments evaluating the impact of microgravity on Arabidopsis thaliana root development and cellular structure. Hardware used for these experiments was the Advanced Biological Research System (ABRS) and Kennedy Space Center (KSC) fixation tubes (KFTs). A simple procedure to delay seed germination prior to installation on ABRS involved the construction of a metal box with a single far-red (FR) light-emitting diode (LED). The exposure of Petri dishes containing seeds (ecotype Columbia) to FR light immediately after planting and maintaining Petri dishes in the dark prevented seed germination until exposure to white light on ABRS. Additional tests revealed that germination can be delayed for up to 10 weeks with FR light treatment. Seedlings fixed in KFTs preloaded with glutaraldehyde for subsequent microscopy studies were not adequately preserved. We suspected that poor fixation was due to the extended contact of glutaraldehyde with oxygen while stored on KFTs, which likely contributed to fixative oxidation. During PVT, minor modifications to address fixation problems encountered during SVT included storing KFTs with glutaraldehyde at 4^o C in the dark, increasing glutaraldehyde concentration from 3% to 5%, and bubbling nitrogen (N₂) gas over the glutaraldehyde solution prior to loading the KFTs. These changes led to improvements in the quality of microscopic images. Lessons learned from SVT and PVT allowed us to optimize some of the preflight protocols needed to successfully implement Advanced Plant EXperiments (APEX) in space.