Gravitational and Space Research

Gravitat. and Sp. Res.

<abstract><title style='display:none'>Abstract</title><p>An experimental chamber and hand-manipulated syringe apparatus were designed, tested, and utilized to assess calcium oxalate crystal yield in Terrestrial-<italic>g</italic> (1 <italic>g</italic>), micro-<italic>g</italic> (0.01 <italic>g</italic>), Lunar-<italic>g</italic> (0.16 <italic>g</italic>), and Martian-<italic>g</italic> (0.38 <italic>g</italic>). Aqueous solutions of calcium chloride (100 mM) and oxalic acid (200 mM) were mixed to precipitate calcium oxalate crystals. Gravitational differences were hypothesized to result in differences in the yield of crystal formation. These data are essential for efforts to better understand the correlation between calcium oxalate crystal formation and the production of kidney stones often associated with long-term space missions. The analyses of crystal formation produced in the micro-<italic>g</italic> (≅0.01 <italic>g</italic>) conditions of this study suggest that calcium oxalate monohydrate formation yield is slightly greater than those produced in Terrestrial-<italic>g</italic> conditions.</p></abstract>

Abstract

Department of Civil and Geomatics Engineering

Department of Civil and Environmental Engineering

Keywords

<article-title>Calcium Oxalate Crystal Yield in Various Gravity Environments</article-title>

Calcium Oxalate Crystal Yield in Various Gravity Environments

{"article-title":"Calcium Oxalate Crystal Yield in Various Gravity Environments"}

<abstract><title style='display:none'>Abstract</title><p>The objectives of this study were to evaluate transdermal scopolamine for motion sickness prophylaxis, and to evaluate off-vertical axis rotation (OVAR) as a laboratory model of motion sickness. This was a randomized, prospective, double-blind study design, set in a vestibular research laboratory. The experimental subjects consisted of 12 patients – 7 male, 5 female – ages 21 to 57, with normal auditory/vestibular function. The intervention was off-vertical axis rotation 20 degrees in the dark after administration of transdermal scopolamine or placebo. The main outcome measures were time duration of tolerated off-vertical rotation, and subjective symptom reporting during rotation at one-minute intervals on a 0–4 scale. Results were as follows: patients treated with transdermal scopolamine had statistically significant improved tolerance time to off-vertical axis rotation. Reported symptom-atology on the 0–4 subjective symptom scale was significantly improved, as compared to placebo, and was dose-dependent. Conclusions are as follows: off-vertical axis rotation is a useful modality for the evaluation of motion sickness medications. Transdermal scopolamine showed statistically significant dose-dependent effects in mitigating OVAR-induced motion sickness symptomatology and was well tolerated.</p></abstract>

Bionetics Corporation, NASA-Kennedy Space Center

University of Florida College of Medicine, UF Health–Jacksonville

Department of Neurotology Research, Legacy Health System

<article-title>Off-Vertical Axis Rotational Assessment of Transdermal Scopolamine for Motion Sickness Prophylaxis</article-title>

Off-Vertical Axis Rotational Assessment of Transdermal Scopolamine for Motion Sickness Prophylaxis

<abstract><title style='display:none'>Abstract</title><p>The endophytic fungus, <italic>Piriformospora indica</italic>, developed a subepidermal infection within <italic>Medicago truncatula</italic> at 1 <italic>g</italic> and at simulated microgravity over a period of 15 days, resulting in intracellular colonization of mature host tissue. At 1 <italic>g</italic>, <italic>P. indica</italic> inoculation affected the growth and morphology of <italic>M. truncatula</italic>, predominantly roots. Inoculated <italic>M. truncatula</italic> had a significantly greater number of roots (102%), total root length (88%), and dry root weight (25%) than non-inoculated plants. Effects on shoot morphology of <italic>P. indica</italic> inoculated <italic>M. truncatula</italic> included longer (31%) and heavier (30%) shoots, along with increased leaf surface area (98%). <italic>P. indica</italic> retained the ability to promote the growth of <italic>M. truncatula</italic> under simulated microgravity conditions upon two dimensional clinostatic rotation, significantly increasing root number by 51% and root length by 48%. These physiological and morphological changes may mitigate biotic and abiotic stresses that would otherwise limit crop productivity.</p></abstract>

Shannon Applied Biotechnology Centre, Department of Applied Science

<article-title>Mutualism Within a Simulated Microgravity Environment - <italic>Piriformospora indica</italic> Promotes the Growth of <italic>Medicago truncatula</italic></article-title>

Mutualism Within a Simulated Microgravity Environment -  Promotes the Growth of 

<abstract><title style='display:none'>ABSTRACT</title><p>Bacteria of the genera <italic>Bacillus</italic> and <italic>Staphylococcus</italic> are frequent inhabitants of the International Space Station (ISS) and represent possible opportunistic pathogens. The effect of simulated microgravity on growth and the frequency of mutation to antibiotic resistance in the model surrogate organisms <italic>Bacillus subtilis</italic> (<italic>B. subtilis</italic>) and <italic>Staphylococcus epidermidis</italic> (<italic>S. epidermidis</italic>) were investigated. The test organisms were cultivated for six days in Rotating Wall Vessel (RWV) clinostats either in the vertical (simulated microgravity) or horizontal (1 <italic>g</italic> control) orientation. Parameters measured were: optical densities (ODs); viable counts; frequencies of resistance to rifampicin (RFM); and frequencies of double resistance to RFM and trimethoprim (TMP). The results indicated that the response to simulated microgravity differed in the two microorganisms. Both <italic>B. subtilis</italic> and <italic>S. epidermidis</italic> grew to higher ODs and cell numbers in simulated microgravity. However, the frequencies of mutation, both to RFM resistance and double resistance to RFM and TMP, were observed to increase significantly in simulated microgravity-grown <italic>B. subtilis</italic> but not in <italic>S. epidermidis</italic>.</p></abstract>

ABSTRACT

Wayne L. Nicholson, Space Life Sciences Laboratory, 505 Odyssey Way, Room 201-B, Exploration Park at Kennedy Space Center, Merritt Island, FL 32953, Telephone: 321-261-3773,

Key words

<article-title>Differing Responses in Growth and Spontaneous Mutation to Antibiotic Resistance in <italic>Bacillus subtilis</italic> and <italic>Staphylococcus epidermidis</italic> Cells Exposed to Simulated Microgravity</article-title>

Differing Responses in Growth and Spontaneous Mutation to Antibiotic Resistance in  and  Cells Exposed to Simulated Microgravity

<abstract><title style='display:none'>ABSTRACT</title><p>In this report, we describe the initial colonization of environmental microorganisms associated with ISS on four different materials (Nomex, cable labeling material, printed circuit board, and aluminum), which are commonly used at the ISS. Material substrates were placed in the Russian segment of the ISS in a Target Book for 135 days. After the incubation, the Target Book was analyzed on Earth by determining colony-forming units and identifying the microorganisms by rRNA gene sequencing. The highest cell concentrations and widest biological diversity were on the polymer materials, such as Nomex and cable labeling material. Additional molecular biological identification revealed the following organisms as typical pioneer microorganisms: <italic>Staphylococcus</italic> spp., <italic>Bacillus</italic> spp., <italic>Streptococcus</italic> spp., <italic>Cladosporium</italic> spp., <italic>Sphingomonas</italic> spp., <italic>Micrococcus luteus</italic>, and <italic>Stenotrophomonas maltophilia</italic>.</p></abstract>

Ulrich Reidt, Electronics, Communications, and Intelligent Systems, Airbus Group Innovations, Department TX4, Munich, Germany, Telephone: +49 (0) 89.607-26675, Fax: +49 (0) 89.607-24001,

Department TX4 - Electronics, Communications and Intelligent Systems

<article-title>Study of Initial Colonization by Environmental Microorganisms in the Russian Segment of the International Space Station (ISS)</article-title>

Study of Initial Colonization by Environmental Microorganisms in the Russian Segment of the International Space Station (ISS)

<abstract><title style='display:none'>Abstract</title><p>The advent of the new generation of suborbital space vehicles is opening up a new and exciting realm of space science that should be of great interest to biologists. These vehicles make it possible to explore biological responses and adaptations that occur in the first few minutes of entering spaceflight and also in the first few minutes after return from space. Historically these transition stages in spaceflight have simply not been available for research, especially within human-rated vehicles. Given that complex biological responses are seldom linear over time, and that essentially all current experiments on the International Space Station (ISS) are conducted after stabilization on orbit, biologists are missing the chance to understand the pathways that lead from terrestrial existence to successful spaceflight adaptation and back. Studies conducted on suborbital spacecraft can therefore be an innovative approach to filling a substantial gap in knowledge regarding the temporal dynamics of biological responses to successful spaceflight physiological adaptation.</p></abstract>

<article-title>Suborbital Vehicles to Study Transition Adaptation to Spaceflight – Why Biologists Should Care About the New Suborbital Flight Opportunities</article-title>

Suborbital Vehicles to Study Transition Adaptation to Spaceflight – Why Biologists Should Care About the New Suborbital Flight Opportunities

<abstract><title style='display:none'>Abstract</title><p>The emerging commercial suborbital rocket industry in the U.S. presents new opportunities for research and education missions. Some companies have been publicized by the world's media and others are lower-profile. Additionally, some companies were created for the space tourism market and others have no current plans to fly humans at all. Most companies already have a Payload User's Guide published at their websites. The time for experimenters to take note of this industry is now, because in early 2014 a number of these companies were already operational or in flight test phase of their business development. When thousands of dollars, instead of millions for traditional NASA or European Space Agency (ESA) sounding rockets, are needed for a suborbital flight, many more researchers will be able to afford suborbital testing and research. In general, these rocket companies seek to provide at least three minutes of high-quality weightless test times from approximately 60 km to 100 km in altitude, and back to 60 km. Purdue University has been fortunate to have secured numerous launches for small payloads during these developmental and early operational years of the industry. Lessons from these launches include lessons in design, payload environment, procedures, launch site infrastructure, and travel preparations.</p></abstract>

<div><sec id="j_gsr-2014-0017_s_001"><div>INTRODUCTION</div><p>The next-generation commercial suborbital rocket industry (<a ref-type="bibr" href="#j_gsr-2014-0017_ref_002">Stern, 2013</a>) is presently a collection of companies that are in or nearing flight testing. Several of the companies have sought to develop experience with research customers during their flight test program, as shown in <a ref-type="fig" href="#j_gsr-2014-0017_fig_001">Figure 1</a>. Eventually it is expected that spaceports and flight providers will be experienced and well-equipped for supporting researchers at the launch sites, but at present early researchers are themselves, in effect, experiments for the spaceports and vehicle providers. The author has been fortunate to be involved in a number of these collaborations (<a ref-type="bibr" href="#j_gsr-2014-0017_ref_001">Collicott, 2011</a>) and seeks to relate lessons learned so as to benefit other early research participants. No statement below is a criticism of vehicle providers or spaceports; rustic or simplistic research infrastructure and procedures are simply the state of the industry when the most important achievement is getting the vehicle tested and flying.</p><fig id="j_gsr-2014-0017_fig_001" position="float" fig-type="figure"><label>Figure 1</label><caption><p>Launch team and two teams of researchers in the commercial suborbital industry with Armadillo's STIG-B rocket. This was after a gentle, on target, parachute landing following an Armadillo launch that aborted safely on ascent. The blue baseball hats commemorate the first licensed commercial suborbital launch at New Mexico Spaceport America on October 10, 2012.</p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="graphic/j_gsr-2014-0017_fig_001.jpg"/></fig><p>All but one of the Purdue payloads are designed and built by undergraduate aerospace engineering students in the author's class, AAE418 “Zero-gravity Flight Experiments.” Since NASA's first nationwide collegiate Reduced Gravity Student Flight Opportunity Program in 1996, students in the School of Aeronautics and Astronautics in the College of Engineering at Purdue University have designed, built, and flown 36 original student-operated parabolic aircraft flight experiments, more than 10 automated suborbital payloads, and in 2012 NASA selected this program for one student-designed experiment that was under construction for the International Space Station (ISS), but is now canceled by NASA.</p></sec><sec id="j_gsr-2014-0017_s_002"><div>G-LEVELS</div><p>Suborbital flight to 100 km does not place the payload “above the atmosphere.” Of course, there is no distinct altitude at which one is above the atmosphere, but flights in the near future will provide access to the mesosphere and lower thermosphere, up to approximately 100 km. This is above much of the atmosphere so, for example, infrared astronomers may see new opportunities as the flight path is above even more of the water vapor than a Stratospheric Observatory for Infrared Astronomy (SOFIA) aircraft flight. Others may have to accommodate the fact that aerodynamic drag remains non-zero, thereby potentially affecting microgravity physics experiments.</p><p>Drag can be estimated from a standard atmosphere, basic physics, assumed mass, and assumptions of a drag coefficient based on shape and orientation. Vehicle providers advertise approximately three minutes of coasting flight, with apogee at 100 km. Basic physics shows that, therefore, the coast begins at 60 km. The greatest drag is present at the 60 km altitude because this is where both the vehicle velocity (approximately Mach 3) and air density are the greatest. Depending on assumptions of mass and shape (drag coefficient), a range of 0.5 to 5 milli-<italic>g</italic> appears to be the range to expect. Any horizontal component of the flight velocity, which might vary significantly between the different styles of vehicles being developed for this market, will increase these <italic>g</italic>-level estimates.</p><p>Microgravity fluids experiment design can often accommodate the drag by both orienting the experiment so that this small acceleration due to drag pushes the liquid into a desired location, such as one end of a test vessel, and by modeling the fluids beforehand with the acceleration included. In general, seeking design advice or the review of one's designs from accomplished experimenters outside of one's own research group can be a productive step, particularly when seeking to accommodate imperfect zero-gravity in experiment design.</p><p>Re-orienting maneuvers of the vehicle during the mission can also create unwanted accelerations. A researcher may consider working with the vehicle provider or flight manifesting organization early in the process to assure the experiment is manifested on a mission with the necessary maneuvering, or absence of maneuvering, and perhaps also the optimal placement of the experiment within the payload section. Some maneuvers may be necessary on descent, but others may be avoidable.</p></sec><sec id="j_gsr-2014-0017_s_003"><div>VIBRATION</div><p>Design for vibration is an interesting step at this stage. Until rockets are flying full missions and such things are actually measured, the vibration spectra and amplitudes supplied, if at all, are estimates. Even experience with designing for vibrations of an orbital launch is not directly applicable because a suborbital flight requires so much less energy than an orbital launch, regardless of air-launch, horizontal take-off, or vertical launch. Current design choices to accommodate vibrations are just the common elementary steps–nylon stop nuts, Loctite, multi-strand instead of single-conductor wire, and similar. To date, in seven Armadillo launches and one Near-Space balloon-dropped glider flight (to develop upper-stratospheric parabolic flight capability), the only vibration related failure that has been encountered is a solder joint broken during the hour-long drive from a hotel in Truth or Consequences, NM, to the vertical launch facility at New Mexico Spaceport America. The experiment was resting on the floor in the back of a mid-sized SUV. No effort had been made to provide padding, so solving the problem on subsequent launches is simple – pad the floor or have someone hold the payload on their lap during the drive. This ground-transportation vibration failure suggests that the most severe vibrations that a next-gen suborbital payload may face are in the shipping from the experimenter's laboratory and delivery to the launch site. As such, care in packing the payload for air or ground shipping to the flight provider, spaceport for launch, or combined systems testing is recommended. The rocket flight may be the gentlest motion your experiment sees.</p></sec><sec id="j_gsr-2014-0017_s_004"><div>INTERFACING AND INTEGRATION</div><p>Most flight providers have at least a draft Payload User's Guide (PUG) online presently describing interfacing choices and requirements. Early experimenters may find changes are implemented as a launch date nears. That is, the early payloads are also the early experiences for the flight providers and thus, it is reasonable to expect that the providers may find important improvements will be necessary. In effect, experiences with early researchers are one part of how the providers will perfect their PUGs. In the early experiences of the Purdue program, a variety of approaches by flight providers have been encountered, presumably because of wide variations in vehicle development progress, staffing levels, and financing. Regardless, frequent communication is naturally found to be the key to effective progress. Planning for travel to the flight provider for an early interfacing visit and for a combined systems test would be wise. There has been no involvement with a payload integrating or manifesting company in the Purdue suborbital programs on which to report.</p><p>When preparing for travel to a launch, consider the drive from the launch site to the nearest hardware or home store. As rocket launches and populated areas historically have not been close together, you may need to pack more supplies, tools, spares, and similar items than you might be accustomed to taking to a parabolic aircraft flight campaign.</p></sec><sec id="j_gsr-2014-0017_s_005"><div>LAUNCH OPERATIONS</div><p>Researchers, especially preliminary researchers with a flight provider beginning commercial operations, should get long- and short-term schedule information from the flight provider and should then prepare to be adaptable. The industry is an emerging industry, not a mature industry, so expect changes as your flight provider's knowledge and experience grows with each test flight. For example, pre-launch fueling times may change; order of operations may change from one flight to the next and similar in these early days. So in these first years of operations, the procedures at a second launch may differ from those at your first launch with the same company. Thus, as with integration above, frequent communication will prevent surprises. Make sure communication is a priority on your end.</p><p>Researchers will also need to follow safety rules at a launch site. This seems obvious, but when excited for an experiment on launch day, forgetfulness can ensue. Various rules may be from the Federal Aviation Administration (FAA), insurance company, local fire safety, air traffic control, etc. Rules are not subject to alteration by your flight provider, so don’t hassle the flight provider about them. Safety briefings the day before launch are common to review safety and operational steps. Pay close attention to the researchers’ responsibilities and duties – in a launch emergency your duty is likely to stay in a bunker and not complicate things. Safety at remote sites may also include understanding if you are to supply your team's drinking water or if that is available at the launch site.</p><p>Rocket launches involve a lot of waiting on the part of the researcher. Bring plenty of things to read, manuscripts to edit, exams to grade, source code to debug, etc., as remaining calm will help you make better decisions if needed. Another step in preventing frustration is to prepare for the weather. Examine temperature extremes, which are to be expected at the launch site, and pack all proper apparel. Insist that your team members do the same.</p></sec></div>

<article-title>An Experimenter's Experiences in Early Commercial Suborbital Flight</article-title>

An Experimenter's Experiences in Early Commercial Suborbital Flight

{"article-title":"An Experimenter's Experiences in Early Commercial Suborbital Flight"}

<abstract><title style='display:none'>Abstract</title><p>The survival and transit of microorganisms in Earth's upper atmosphere is relevant to terrestrial ecology and astrobiology, but the topic is understudied due to a scarcity of suitable flight systems. We designed, built, and flew a self-contained payload, Exposing Microorganisms in the Stratosphere (E-MIST), on a large scientific balloon launched from New Mexico on 24 August 2014. The payload carried <italic>Bacillus pumilus</italic> SAFR-032, a highly-resilient spore-forming bacterial strain originally isolated from a NASA spacecraft assembly facility. Our test flight evaluated E-MIST functionality in the stratosphere, including microbiological procedures and overall instrument performance. Herein, we summarize features of the E-MIST payload, protocols, and preliminary results that indicate it is possible to conduct a tightly-controlled microbiological experiment in the stratosphere while collecting pertinent environmental data. Additional studies of this nature may permit survival models for microbes traveling through Earth's harsh upper atmosphere. Moreover, measuring the endurance of spacecraft-associated microbes at extreme altitudes may help predict their response on the surface of Mars.</p></abstract>

NASA John F. Kennedy Space Center, Engineering and Technology Directorate

University College London & Natural History Museum

<article-title>A Balloon-Based Payload for Exposing Microorganisms in the Stratosphere (E-MIST)</article-title>

A Balloon-Based Payload for Exposing Microorganisms in the Stratosphere (E-MIST)

{"article-title":"A Balloon-Based Payload for Exposing Microorganisms in the Stratosphere (E-MIST)"}

<abstract><title style='display:none'>Abstract</title><p>Biological experiments on-orbit that demonstrate the effects of gravity on plants require precise control of the initiation of plant development. Preserving seed dormancy is critical to experiments that endeavor to study the effects of the orbital environment, independent of contributions from either a normal gravity, or launch. However, spaceflight experiments are often tightly constrained with respect to the configuration of the biology and associated hardware, and it is rarely possible to launch dry seeds separated from their growth substrate. Described here are techniques established to maintain viable seeds that can remain dormant for up to a month at room temperature, and hydrated on the surface of solid, Phytagel growth medium. The configuration can also accommodate a brief (less than one minute) exposure to light during the quiescent period for quick inspection for any breaks in dormancy, and for contamination. The data presented outline the preparation of sealed, Phytagel media plates of dormant <italic>Arabidopsis thaliana</italic> seed that can be activated <italic>in situ</italic> when unwrapped and installed within a lighted growth habitat. These protocols were developed primarily for spaceflight scenarios where seeded plates must be prepared ahead of time and kept at ambient temperatures. However, these protocols can be adapted for any field application where it is desirable to transport dormant, seeded plates to a remote location where it would not be possible to prepare sterile culture plates.</p></abstract>

<div><sec id="j_gsr-2014-0018_s_001"><div>INTRODUCTION</div><p>Understanding plant growth and development on-orbit is central to humankind's space exploration agenda. The effects of microgravity on plants have been explored for several decades (<a ref-type="bibr" href="#j_gsr-2014-0018_ref_008">Paul et al., 2013a</a>; <a ref-type="bibr" href="#j_gsr-2014-0018_ref_010">Ričkienė, 2012</a>; <a ref-type="bibr" href="#j_gsr-2014-0018_ref_012">Wheeler, 2011</a>; <a ref-type="bibr" href="#j_gsr-2014-0018_ref_013">Wolverton and Kiss, 2011</a>; <a ref-type="bibr" href="#j_gsr-2014-0018_ref_014">Wyatt and Kiss, 2013</a>); however, to fully understand the effects of microgravity, it is ideal to observe differences in plants that have been developed entirely in the microgravity environment without prior exposure to Earth's 1 <italic>g</italic> force. This endeavor entails launching dormant seeds, germinating on-orbit, allowing them to grow in microgravity conditions, and comparing them to plants grown exclusively on Earth as a ground control.</p><p>One challenge in conducting such experiments in space is ensuring that the configuration is compact, lightweight, safe, and uses minimal crew time. Solid nutrient media (such as with agar or Phytagel) contained in Petri plates has been used in plant research for decades in terrestrial (<a ref-type="bibr" href="#j_gsr-2014-0018_ref_003">Barrett-Lennard and Dracup, 1988</a>; <a ref-type="bibr" href="#j_gsr-2014-0018_ref_004">Lodha and Netravali, 2005</a>) and orbital research (<a ref-type="bibr" href="#j_gsr-2014-0018_ref_007">Paul and Ferl, 2002</a>; <a ref-type="bibr" href="#j_gsr-2014-0018_ref_011">Stout et al., 2001</a>; <a ref-type="bibr" href="#j_gsr-2014-0018_ref_015">Zupanska et al., 2013</a>) by providing the necessary components required for healthy plant growth. Most importantly, this configuration is lightweight, completely contained, and allows easy access to all plant tissues – especially the roots.</p><p>Another consideration is the ease of operation for both the investigators and the astronauts on-board. Mounting an experiment to the International Space Station (ISS) can be complicated given the many conditions required for a successful launch. Hence, for the investigator, it is essential that the biological elements can withstand unpredicted changes in the launch schedule before actual lift-off and delays in operations on-orbit, as such events are common due to a myriad of technical requirements. Therefore, the ideal system should allow for a few days of flexibility without completely voiding the experiment. For example, if the investigator is interested in studying three-day-old plants, and germination is scheduled to be initiated on the ground prior to space vehicle launch, there is always a risk that upon reaching the critical time frame the plant may still be on voyage to the ISS and not accessible for any in-flight operations. Thus, the capability to send dormant seeds that can be initiated for growth at a convenient and flexible time is needed in traversing the challenging environment of spaceflight or other remote environments. An additional advantage of designing the experiment configuration with this flexibility is that the process promotes ease of operations by crew members on-board the ISS.</p><p>We have used Phytagel media plates made with commercially available 100 mm square Petri dishes and dormant <italic>Arabidopsis thaliana</italic> (arabidopsis) seed in several spaceflight and remote expedition applications with great success (<a ref-type="bibr" href="#j_gsr-2014-0018_ref_001">Abboud et al., 2013</a>; <a ref-type="bibr" href="#j_gsr-2014-0018_ref_002">Bamsey et al., 2009</a>; <a ref-type="bibr" href="#j_gsr-2014-0018_ref_009">Paul et al., 2013b</a>; <a ref-type="bibr" href="#j_gsr-2014-0018_ref_006">Paul et al. 2012</a>). The seed dormancy protocol was primarily developed for the Advanced Biological Research System/GFP imaging system (ABRS/GIS) on-board the ISS, and the comparable ground control unit installed in the Orbital Environmental Simulator (OES) at Kennedy Space Center (KSC) for the arabidopsis spaceflight experiment. However, the results of our prolific assays may be useful for other applications – not only for spaceflight scenarios with different growth payload hardware, but also whenever dormant plant biology may prove favorable. A non-spaceflight example is the deployment of plated arabidopsis to Devon Island in the Canadian high arctic with the Haughton Mars Project (e.g., <a ref-type="bibr" href="#j_gsr-2014-0018_ref_002">Bamsey et al., 2009</a>).</p></sec><sec id="j_gsr-2014-0018_s_002"><div>MATERIALS AND METHODS</div><sec id="j_gsr-2014-0018_s_002_s_001"><div>Preparation of Seeds and Plates</div><p>Two ecotypes were used for the dormancy tests: Colombia-0 (Col-0) and Wassilewskija (WS). One week before sterilization, seeds were stored in a desiccator containing <italic>Desiccant-Anhydrous Indicating Drierite</italic> (W.A. Hammond Drierite Company, stock #24001). After which, the desiccated seeds were sterilized by agitating in 70% ethanol for 10 minutes, and then thoroughly dried on sterile filter paper in a laminar flow hood (<a ref-type="fig" href="#j_gsr-2014-0018_fig_001">Figure 1A</a>). The dry sterile seeds were then stored at 4°C in sterile, screw-top microcentrifuge tubes (<a ref-type="fig" href="#j_gsr-2014-0018_fig_001">Figure 1B</a>). A planting protocol is shown in <a ref-type="fig" href="#j_gsr-2014-0018_fig_001">Figure 1</a>; a small amount (50–100 seeds; just enough to plant a few plates at a time) of dry sterile seeds are suspended in sterile water and quickly dispensed onto the surface of solid media (e.g., 0.5% MS solidified with 0.5% Phytagel) using a sterile pipette (<a ref-type="fig" href="#j_gsr-2014-0018_fig_001">Figure 1C</a>). Typically, 12–20 seeds are dispensed across the top of a single plate. A depth of 40–60 mL of Phytagel media is ideal to support growth of up to 20 arabidopsis seedlings for at least two weeks. The plates are then sealed with porous surgical Micropore® (3M) tape (<a ref-type="fig" href="#j_gsr-2014-0018_fig_001">Figure 1D</a>). The prepared plates are then immediately wrapped in Duvetyne Black-Out Fabric (Seattle Fabrics) to create a light-tight packet (<a ref-type="fig" href="#j_gsr-2014-0018_fig_002">Figure 2</a>). Each step needs to be completed quickly, so that the sequence from the time a seed comes into contact with water or the moist surface of the plate, and then secured in a dark environment, is completed in less than 10 minutes. We typically plant four plates from each small aliquot of water suspended seed, and use teams of two people (one person to plant and seal the plate in the laminar flow hood, and the other to wrap the plate in Duvetyne). The Duvetyne cloth procedure illustrated in <a ref-type="fig" href="#j_gsr-2014-0018_fig_002">Figure 2</a> covers every surface of the square Petri plate by at least two layers of cloth, and creates a light-tight, breathable package that can be stored at room temperature for several weeks.</p><fig id="j_gsr-2014-0018_fig_001" position="float" fig-type="figure"><label>Figure 1</label><caption><p>Dry sterilization and planting for dormant plates. A) Sterilized seeds drying on filter paper in a laminar flow hood. B) Collection of sterilized seeds into sterile, screw-top microcentrifuge tubes, which will be stored at 4°C until use. C) Plating sterilized seeds suspended in water across the top of a single plate with a sterile, disposable pipette. D) Plates are sealed with porous, surgical Micropore® tape and labeled.</p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="graphic/j_gsr-2014-0018_fig_001.jpg"/></fig><fig id="j_gsr-2014-0018_fig_002" position="float" fig-type="figure"><label>Figure 2</label><caption><p>Securing plates in a light-tight configuration with Duvetyne Black-Out fabric. A) A survey photograph is taken to show that the configuration of the outer label (top left of image) matches the genotype label on the plate. The plate is then wrapped by first aligning to the bottom of the cloth as shown in (B), then the plate is rolled up in the cloth (C and D), and then the sides are folded in and secured with the outer label (E). The grid shown supporting the plate and cloth in the figure is divided into one inch squares.</p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="graphic/j_gsr-2014-0018_fig_002.jpg"/></fig></sec><sec id="j_gsr-2014-0018_s_002_s_002"><div>The Dormancy Breaking Test</div><p>Two sets of plates with dormant WS or Col-0 seeds were prepared, wrapped in black cloth, and left for two, four, or six weeks at room temperature (<a ref-type="fig" href="#j_gsr-2014-0018_fig_003">Figures 3–4</a>). On the respective day, the plates were unwrapped and inspected for dormancy breakage. After inspection, they were transferred to a growth room and then evaluated for germination to ensure that the seeds remained viable. Plates were scored for the percentage of seed that held dormancy and subsequently germinated (<a ref-type="fig" href="#j_gsr-2014-0018_fig_005">Figure 5</a>).</p><fig id="j_gsr-2014-0018_fig_003" position="float" fig-type="figure"><label>Figure 3</label><caption><p>Dormancy test using Wassilewskija (WS) ecotype seeds. A) Whole plate view of dormant WS seeds after they were stored for 2, 4, and 6 weeks. All seeds remained dormant. B) Close-up view of the selected seeds corresponding to those boxed in red, in 3A. C) Exact plates from 3A that have been exposed to the light and allowed to germinate to show seed viability.</p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="graphic/j_gsr-2014-0018_fig_003.jpg"/></fig><fig id="j_gsr-2014-0018_fig_004" position="float" fig-type="figure"><label>Figure 4</label><caption><p>Dormancy test using Colombia-0 (Col-0) ecotype seeds. A) Whole plate view of dormant Col-0 seeds after they were stored for 2, 4, and 6 weeks. All seeds remained dormant. B) Close-up view of the selected seeds corresponding to those boxed in red, in 3A. C) Exact plates from 3A that have been exposed to the light and allowed to germinate to show seed viability.</p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="graphic/j_gsr-2014-0018_fig_004.jpg"/></fig><fig id="j_gsr-2014-0018_fig_005" position="float" fig-type="figure"><label>Figure 5</label><caption><p>Dormancy and germination (viability) rates of WS and Col-0 and ecotype seeds. A) Dormancy (red) and germination (blue) rates of WS seeds after they were stored for 2, 4, and 6 weeks. In WS ecotype, dormancy rates were &gt; 98% and germination rates were &gt; 94%. B) Dormancy (red) and germination (blue) rates of Col-0 seeds after they were stored for 2, 4, and 6 weeks. Dormancy rates for Col-0 were &gt; 95% and germination rates were &gt; 93%. For WS, there were between three and six replicates of each time point. Error bars are provided for each set, all standard deviations were less than 0.07. For Col-0, there were four replicates of each time point. Error bars are provided for each set, all standard deviations were less than 0.06.</p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="graphic/j_gsr-2014-0018_fig_005.jpg"/></fig></sec><sec id="j_gsr-2014-0018_s_002_s_003"><div>The Illumination Effects on Seed Dormancy Breakage</div><p>Two methods of preparing dormant WS plates were used in the illumination test. The first set of plates were planted with the dry sterilized WS seeds and exposed to far-red light (3.2 μmol m<sup>−2</sup> s<sup>−1</sup>) for 10 min (<a ref-type="bibr" href="#j_gsr-2014-0018_ref_005">Nakashima et al., 2014</a>), and were then wrapped in Duvetyne cloth and stored at 4°C. In the second set of plates, dry sterilized WS seeds were prepared as described above, wrapped in Duvetyne black out-cloth, and stored at room temperature. After one week, both sets of plates exposed to ambient room lighting (~100 μmol m<sup>−2</sup> s<sup>−1</sup>) for 30 seconds to inspect for contamination and germination, and then re-wrapped and stored at either 4°C, or at room temperature (respectively) for three additional weeks. After 4 total weeks from planting, plates from both preparation methods were scored for dormancy breakage, and then subsequently evaluated for seed viability by transferring to growth chamber (<a ref-type="fig" href="#j_gsr-2014-0018_fig_006">Figure 6</a>).</p><fig id="j_gsr-2014-0018_fig_006" position="float" fig-type="figure"><label>Figure 6</label><caption><p>Visual inspection of dormant, Duvetyne-wrapped plates: A,B,C) Plates with WS seeds were treated with Far-red light and stored at 4°C for 1 week before being unwrapped and exposed to 30 seconds of ambient room lighting, and then re-wrapped for storage at 4°C for 4 weeks. The full view of the plate three weeks after visual inspection is shown in (A), and an enlargement of the seed row is shown in (B). C) Exact plate from 5A that has been exposed to the light and allowed to germinate to show seed viability. D,E,F) Plates with WS seeds were kept at room temperature for 1 week before being unwrapped and exposed to 30 seconds of ambient room lighting, and re-wrapped for storage at room temperature for 4 weeks. The full view of the plate three weeks after visual inspection is shown in (D), and an enlargement of the seed row is shown in (E). D) Exact plate from 5A that has been exposed to the light and allowed to germinate to show seed viability.</p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="graphic/j_gsr-2014-0018_fig_006.jpg"/></fig></sec></sec><sec id="j_gsr-2014-0018_s_003"><div>RESULTS AND DISCUSSION</div><p>Arabidopsis ecotypes Col-0 and WS were evaluated for their ability to be kept in a dormant, yet viable state for greater than a month. Both ecotypes held dormancy and remained viable for six weeks (<a ref-type="fig" href="#j_gsr-2014-0018_fig_003">Figures 3</a>, <a ref-type="fig" href="#j_gsr-2014-0018_fig_004">4</a>, and <a ref-type="fig" href="#j_gsr-2014-0018_fig_005">5</a>). Virtually all (&gt; 98%) of the WS seeds remained dormant at room temperature for up to six weeks, and had a viability rate of &gt; 95% (<a ref-type="fig" href="#j_gsr-2014-0018_fig_003">Figures 3</a> and <a ref-type="fig" href="#j_gsr-2014-0018_fig_005">5</a>). Of the Col-0 plates subjected to the same regimen, &gt; 97% remained dormant, and then had an average viability rate of &gt; 93% (<a ref-type="fig" href="#j_gsr-2014-0018_fig_004">Figures 4–5</a>). However, although the Col-0 seeds had a high viability rate, the seeds that germinated after being dormant for six weeks were somewhat smaller than those that germinated after four weeks of dormancy (<a ref-type="fig" href="#j_gsr-2014-0018_fig_004">Figure 4</a>).</p><p>Due to the nature of spaceflight experiments, it is crucial to be able to inspect the quality of the prepared dormant plates before they are committed to an experiment. This step minimizes the possibility of launching plates containing seeds that have broken germination, and that may have picked up contamination during the preparations. Since the preparations of plates for spaceflight and other remote deployment experiments typically include two to three times as many plates as will actually be deployed, imperfect plates can be excluded from the set turned over for launch.</p><p>Both approaches for creating dormant arabidopsis plates discussed here can accommodate a brief (less than 30 seconds) inspection in normal room lighting (less than 180 μmoles m<sup>−1</sup>s<sup>−1</sup>) prior to deployment. A comparison of plates prepared by dry sterilization and dark, ambient (20–28°C) temperature storage, and those treated with far-red light treatment and 4°C storage (after the method of <a ref-type="bibr" href="#j_gsr-2014-0018_ref_005">Nakashima et al., 2014</a>), is shown in <a ref-type="fig" href="#j_gsr-2014-0018_fig_005">Figure 5</a>. The seeds from both methods remained dormant for three additional weeks after being exposed to ambient room light for 30 seconds one week after they were prepared. Further, greater than 95% of seeds germinated normally when they were finally unwrapped and allowed to grow in standard growth room conditions (<a ref-type="fig" href="#j_gsr-2014-0018_fig_006">Figure 6</a>). These results suggest that opening the light-tight packaging and exposing the dormant plated seeds to ambient light for less than a minute does not drastically affect the seed dormancy. Thus, the plates can be examined, scored for dormancy, and assured free of contamination before being turned over for a flight experiment, or other application where they can reside at room temperature for an extended period of time before use.</p><p>The Transgenic Arabidopsis Gene Expression System (TAGES) experiment carried out in 2009–2010 (<a ref-type="fig" href="#j_gsr-2014-0018_fig_007">Figure 7</a>) illustrates the successful use of this technique to deliver dormant arabidopsis seeds to the ISS for germination on orbit (<a ref-type="bibr" href="#j_gsr-2014-0018_ref_006">Paul et al., 2012</a>; <a ref-type="bibr" href="#j_gsr-2014-0018_ref_009">Paul et al., 2013b</a>). Dormant seeds wrapped in Duvetyne cloth and stowed under ambient (20–28°C) conditions were transported to the ISS via the space shuttle STS-129, STS-130, and STS-131. Upon arrival they were unwrapped, installed onto the ABRS/GIS hardware, and activated for growth by light stimulation. Multiple plates were used in this experiment, all of which illustrates the successful maintenance of seed dormancy and the viability of these dormant seeds (<a ref-type="fig" href="#j_gsr-2014-0018_fig_007">Figure 7</a>).</p><fig id="j_gsr-2014-0018_fig_007" position="float" fig-type="figure"><label>Figure 7</label><caption><p>Actual TAGES flight experiment on-board the ISS, documenting the successful application of this technique in delivering dormant arabidopsis seeds on-orbit. A) Visual inspection of dormant plate 4 (1B49137TK) run 1B showing dormant seeds, and no contamination. B) Identical plate 4 in <a ref-type="fig" href="#j_gsr-2014-0018_fig_006">Figure 6B</a> being harvested on ISS after 12 days of growth. C) Wrapped TAGES plate (3A19363TK). D) Identical plate in <a ref-type="fig" href="#j_gsr-2014-0018_fig_006">Figure 6C</a> shows dormant seeds, unwrapped and light-stimulated for germination onboard the ISS. The image was taken with the ABRS/GIS hardware. E,F) Gradual growth of the plant from once dormant seed shows the viability of the seed.</p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="graphic/j_gsr-2014-0018_fig_007.jpg"/></fig></sec><sec id="j_gsr-2014-0018_s_004"><div>CONCLUSIONS</div><p>Accurate characterization of plant responses to the spaceflight environment relies heavily on proper sample preparation. In this paper we described a convenient technique used to prepare sterile, dormant, and viable seeds that are well-suited for passive stowage and on-orbit activation. The material of choice for constructing the light-tight packet is Duvetyne Black-Out Fabric (Seattle Fabrics). Duvetyne is also fire retardant, and now vetted for use on the ISS. We were also able to demonstrate that dormant seeds prepared in this manner can tolerate short exposures to light in the middle of their dormancy period, which can accommodate visual inspection of the plates before they are deployed for an experiment. This feature enables investigators to check their dormant experimental plates for contamination or germinated seed, which in turn allows for the removal of compromised plates from the experimental set.</p></sec></div>

Plant Molecular and Cellular Biology Program

Interdisciplinary Center for Biotechnology Research

<article-title><italic>Arabidopsis thaliana</italic> for Spaceflight Applications–Preparing Dormant Biology for Passive Stowage and On-Orbit Activation</article-title>

 for Spaceflight Applications–Preparing Dormant Biology for Passive Stowage and On-Orbit Activation

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