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seeds per 60 mm Petri dish of each were planted on 15 mL of sterile 1/2 strength Epstein's medium (Wymer et al., 1997) supplemented with 1% (w/v) Phytagel (Sigma-Aldrich) and 10 mM sucrose, pH 5.7. The seeded dishes were then irradiated for 12 h with 70 mol m-2 s-1 white light from a fluorescent fixture (F20T12/CW; Osram Sylvania, Wilmington, MA, USA) at 22C to synchronize subsequent germination once transferred to the ISS. The irradiated dishes were then stored at 4C in the dark for ~8 h until integration into the flight hardware. Each Petri dish was assembled into a single stage Petri Dish Fixation Unit cassette (PDFU; Wells et al., 2001) under sterile conditions. The PDFU liquid injection reservoir was filled with 13 mL RNAlater solution (Life Technologies, Grand Island, NY, USA) and five PDFUs plus a HOBO data-logger (Onset Computer Corp., Bourne, MA, USA) were integrated into the Biological Research in Canister (BRIC) hardware (Wells et al., 2001), which was stored at 4C before launch to inhibit seed germination. The hardware was then transferred in a Double Cold Bag (Hutchinson and Campana, 2009), held at 4C, to the Dragon capsule of SpaceX mission CRS-4 where it was stowed 48 h before launch. Four BRICs were used for the analysis, allowing for an experimental design with four plates each of Col-0 and Ws-2 and three plates each of Ler-0 and Cvi-0. SpaceX CRS-4 launched on 21 September 2014 and docked with the ISS ~2 d later. After a further ~2 d on orbit, the BRICs were removed from the cold bag and allowed to warm to ISS ambient temperature (~24C) over 3 h. This timeline meant that the seeds were stored at 4C for 150 h from the time of integration into the BRIC hardware to removal from the cold bag on orbit. Plates were then left at ISS ambient for the seeds to germinate and seedlings to grow for 8 d. RNAlater was then injected onto the sample plates by actuating the PDFUs. Samples were allowed to fix for 12 h, then the BRICs were loaded into the Minus Eighty Laboratory Freezer on the ISS (MELFI) for sample storage. For return to Earth, the BRICs were removed from the MELFI, packed into a freezer bag (that maintained -32C throughout the return process) and loaded into the Dragon capsule. The payload splashed down on 25 October 2014 and was shipped to Kennedy Space Center, FL, USA, where the BRIC canisters were transferred to a -80C laboratory freezer. The hardware was then thawed at 4C to allow disassembly of the BRIC and the Petri dishes were de-integrated from their PDFUs. A further 10 mL of RNAlater was added to each dish, and the dishes were then frozen at -20C, packed on dry ice and shipped to Madison, WI, USA where they were stored in a -80C freezer for subsequent analysis. A duplicate set of BRICs was used for the ground control experiments. These samples were treated identically to the flight materials but were transferred to the ISS Environmental Simulator (ISSES) for the period that the flight samples were grown on board the ISS. To facilitate programming the environmental conditions of the ISSES to mimic those on the ISS, the ground controls were run with a 48 h delay to the flight experiment. After 8 d of growth in the ISSES, RNAlater was injected, with the timing and order of RNAlater actuation for each PDFU closely mirroring that of the flight protocol. The samples were left for 12 h, then the BRICs containing the sample plates were transferred to a -80C laboratory freezer. These samples were de-integrated from the hardware alongside the flight materials as described above. From this point onward, all other manipulations of the samples were identical to the flight material.\n\nBRIC-PDFU (Petri Dish Fixation Unit):  The BRIC-PDFU configuration allows biological specimens contained within 60 mm Petri dishes to be installed into polycarbonate blocks that are then installed into an aluminum box.  The polycarbonate blocks (PDFUs) provide the ability to deliver both nutrient and fixative solutions to biological specimens housed in the Petri dishes.  Important information can be gained through simple investigations in which organisms are taken to space, chemically fixed (or stabilized), and then returned for post-flight processing. The BRIC-PDFU is designed to accomplish this using a minimal amount (typically less than one hour) of astronaut crew time. Biological specimens are placed into the 60 mm Petri dishes containing agar-solidified media, although alternative approaches will be considered. Each Petri dish is then placed inside its own individually-sealed PDFU. The PDFUs are assembled and loaded with up to 17mL of either one or two fluids, such as a nutrient solution and/or a chemical fixative, in a reservoir compartment as specified by the selected investigators. Six PDFUs or five PDFUs plus one temperature data logger, depending on the investigator\u2019s requirements, are then loaded into each BRIC canister.  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