Spaceflight Procedures and Operations Utilized During the Seedling Growth Experiments

The purpose of the Schedule Test is to create a protocol to be implemented during the in-flight experiment (Table 1). This test requires collaboration between the science team (the Principal Investigator or PI), the funding agency (e.g., NASA, ESA, JAXA), and the operation facilities (Norwegian User Support and Operations Center (N-USOC), NASA Telescience Support Centers, Japan Experiment Module Mission Control, etc.) controlling the hardware on board the ISS. In regard to Seedling Growth-2, the collaborative work is the PI teams, NASA, ESA, and N-USOC, which house a ground-based model of the EMCS (also termed the engineering model, (EM)) and is the facility responsible for image acquisition and control of the EMCS during the spaceflight experiment (Hellesang et al. 2005; Kiss et al. 2014).

The Schedule Test began at NASA's Ames Research Center (ARC; Mountainview, CA, USA), where surface-sterilized seeds were observed under a microscope and specially selected to increase germination rate. The seeds were selected based on morphology, specifically, color, and shape (Figure 1A–F). Seeds that appeared abnormal (Figure 1B) or premature (Figure 1C) were discarded. Mature seeds (Figure 1A) were selected for placement on gridded nitrocellulose membranes and fixed to the membrane via gum guar with the seed's micropyle facing up (Figure 2). This placement ensured that seedlings were growing in the correct orientation during the spaceflight experiment, where the lack of a gravity vector can hinder uniform orientation. The gridded membranes and seeds were then incorporated into seed cassettes (Figure 3A), and incorporated into the Experimental Containers (ECs; Figure 3B).

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Figure 3

In addition to seed selection, two germination tests of the seed stocks were conducted simultaneously at ARC. Selected surface-sterilized seeds were affixed to blotter paper and then placed in a Petri dish concurrent with the hardware build (Kiss et al., 2007). The initial germination test was conducted during the week of the build to ensure the viability of the seed stock. The second set of germination controls was saved and the seeds were imbibed concurrently with the Schedule Test to ensure that the hardware build and storage time did not cause reduced seed germination.

After conclusion of the hardware build and germination tests, the equipment was hand-carried to N-USOC (Trondheim, Norway) where the EM was used to establish in-flight procedures. The in-flight procedures were established by the PI to maximize scientific return. During this test, calibration occurred to ensure cameras were focused on the samples and seedlings were placed in a location that allowed for full view for imaging. Camera calibration is not only important for experiments that study plant growth and development in conditions of microgravity, but the imaging is also used to assess potential operational problems during the experiment and to ensure key milestones (such as proper hydration of seeds, etc.) have occurred.

In addition, illumination settings were also scheduled for the experiment at this stage. Depending on the research question and flight hardware, the light quality (red, blue, white, or infrared) and duration of illumination can be modified per the requirements of the experiment. The gravity vector (created via centrifugation) is also established during this phase. The EMCS contains two separate centrifuges that allow the experiment to be performed in a range of gravity conditions. Our most recent experiment, Seedling Growth-2, examined the effects of a continuum of gravity (from μg, to reduced g, to 1 g) on growth and tropistic movement of Arabidopsis thaliana (Arabidopsis) seedlings (Vandenbrink et al., 2016). Finally, the EMCS has the ability to monitor atmospheric conditions (percent CO₂, O₂, temperature, and humidity) that are occurring during the experiment. The Schedule Test provides information on how the experimental run affects the atmospheric conditions within the EMCS.

The Operations and Validations Test (OVT) is a trial run, or “dress rehearsal,” of the planned flight experiment (Figure 4). The OVT is described by ESA as a compact dry-run of the experiment on the ground designed to be as close to the preparation and execution of the spaceflight experiment as possible. The OVT validates that the experiment unique equipment (EUE) and flight procedures created during the Schedule Test support the experimental requirements developed by the PI. The finalized flight procedure, which includes the timing of operations and calibration of the EMCS, is utilized in this testing phase. Camera calibration, timing of image capture, centrifuge speed, and all other experiment-specific settings required by the PI are utilized during the OVT.

Figure 4

Similar to the Schedule Test, the hardware for the OVT was prepared at NASA's ARC. However, one key difference from the Schedule Test was that NASA quality assurance (QA) representatives were present to oversee all aspects of the hardware assembly (including seed selection) and the hardware build procedure. The EUE was then hand carried by NASA personnel to Norway, where N-USOC conducted the OVT. Similar to the Schedule Test, germination controls were created during the OVT hardware assembly to determine if reduction in overall seed germination had occurred as part of the hardware assembly process. The first set of controls was germinated during the seed selection phase to provide the opportunity to immediately select new seeds in the event of poor germination due to a poor seed lot. The second set of controls was germinated during the OVT to ensure hardware assembly or transport of the seeds to N-USOC did not cause a reduction in the germination of seeds within the hardware. Conclusion of the OVT resulted in a finalized set of operations that would be followed for the actual spaceflight experiment.

After a successful OVT, the final flight build takes place. Similar to the OVT, the entire process is supervised by NASA Quality Assurance personnel to ensure the procedures are followed precisely. The PI's team was responsible for seed selection and placement on the membranes, as well as the preparation of the germination controls for the experiment. These germination controls were run during the flight build, as well as during the spaceflight experiment, to determine if the hardware build or rocket launch caused a reduction in the seed's germination rate. During the spaceflight procedure, images were taken of the seedlings within the EMCS four days after the scheduled hydration event, and then compared to ground controls for reference.

Once assembly of the seeds cassettes was completed by NASA and the PI's team, the cassettes were handed over to NASA engineers for the final assembly of the EUE (Figure 3B). NASA employees then carried the finalized flight hardware to Kennedy Space Center where it was loaded onto the launch vehicle (i.e., SpaceX's Dragon/Falcon 9 launch system) in preparation for launch (Figure 3C).

An overview of the experimental procedures during our spaceflight experiment has been detailed previously (Correll and Kiss, 2005; Millar et al., 2010; Vandenbrink and Kiss, 2016). Upon arrival to the ISS in the Dragon vehicle (Figure 3D), the EUEs were unloaded and stored on the ISS until astronaut availability allowed for placement into the EMCS. Once astronauts loaded the ECs into the EMCS, initiation of the flight protocol commenced and was controlled telemetrically via N-USOC. Images of the experiment were downloaded from the ISS in near-real time to allow for monitoring of the experiment (Figure 3E). If an error occurred or command was not received by the EMCS, then operators at N-USOC were able to assess the situation and resend commands if needed.

It is important to note that obtaining access to astronauts to conduct these in-flight experiments is often difficult. Crew time on board the ISS is a limited resource, with each space agency setting weekly limits for time that can be allocated to conducting research experiments. Telemetrically controlled systems such as the EMCS or the Advanced Biological Research System (ABRS; Ferl et al., 2015; Levine et al., 2009), in addition to self-contained hardware such as Biological Research in Canisters and Petri Dish Fixation Unit (BRIC-PDFU; Kern et. al, 1999), can reduce the need for crew time to only loading and unloading operations. However, future investigators should keep in mind that access to crew time is an important consideration when planning spaceflight experiments.

Once the timeline of the experiment concluded, astronauts removed the ECs from the EMCS (Figure 3F) and removed the seedling cassettes from the hardware and placed them in a holding device termed a bandolier (Figure 3G). The cassettes were then placed in General Laboratory Active Cryogenic ISS Experiment Refrigerator (GLACIER) at −80°C download of the samples back to Earth, where they were recovered in the Pacific Ocean by SpaceX (Figure 3H) before being transferred to NASA personnel.

Post-flight operations are experiment-specific. In regards to Seedling Growth-2, samples were recovered from the Dragon capsule by SpaceX off the coast of California (Figure 3H), and returned to ARC. Samples were stored at −80°C until the PI's team arrived for sample processing (Figure 3I). Cassettes were removed from the −80°C freezer, and a syringe was used to immediately inject RNALater^®, filling the cassette and preserving the samples for RNA-seq or RT-PCR. Once in RNALater^®, tissue was transferred on dry ice to another lab without fear of RNA degradation.

In addition to tissue processing, image analysis of the photos taken during the experiment continued post-flight. Utilizing the photos taken by the EMCS, our lab identified a positive red-light phototropic response in roots and shoots of Arabidopsis seedlings (Kiss et al., 2012; Millar et al., 2010). We also found that root phototropic response to red light is positive, and inversely correlated with the magnitude of the gravity vector. In addition, our research also identified a novel blue-light phototropic response in Arabidopsis roots, and this res ponse was abolished in the presence of gravity (Vandenbrink et al., 2016). These spaceflight experiments have allowed us to better characterize plant tropisms, and have helped to detail the relationship between gravitropic and phototropic responses in Arabidopsis thaliana.