1- Novelties in Plant Cultivation
(a) Seedlings in modules
In 1983, when the Space Transportation System-9 (STS-9), the first Spacelab Science Mission (also named STS-41-A and Spacelab) was launched on the Space Shuttle Columbia, Plant Biologists sent 4-day old sunflower seedlings to study the circumnutating (oscillations) of sunflower hypocotyls (embryonic stem of a germinating seed) under microgravity, they cultivated plants in the ground in small pots and kept inside the aluminum modules. Then these plant modules were loaded into a suitcase-like PCOC (plant carry-on container) with a temperature recorder. The removable pots with seedlings were inserted within the PCOC’s foam base. This spaceflight experiment named HEFLEX revealed that circumnutating observed under microgravity were quantitatively different from those measured in clinostat experiments.
(b) Seedlings raised in Mini containers
Subsequent Spaceflight experiments involving the studies on lentil seedlings' root growth in space (STS-61-A D1, 1985) and maize’s root growth in microgravity (1986, NASA’s Space Flight STS-61-C Columbia) used mini containers to raise seedlings from seeds in space. The mini-containers used in the STS -61-A D1 and STS-42 (International Microgravity Laboratory-1, IML-1)) were designed for the cultivation of lentils. These containers were named “lentil/roots” hardware and accommodated within the ESA (European Space Agency) Biorack unit. The size of lentil seeds is 5 mm. In the same STS-42 spaceflight experiment, gravitropic responses of the Avena sativa (oat) coleoptile under microgravity was studied using these mini containers.
(c) Minicontainers for Arabidopsis seedlings
In the subsequent spaceflight experiments, the global model plant Arabidopsis was selected because of the availability of T-DNA insertion mutants (in particular, reduced-starch mutants). More logical and efficient modifications were made in the mini containers in the STS-84 in order to use Arabidopsis in spaceflight experiments.
I drew this sketch for easy understanding and the 3-mm square gridded black membranes made of cellulose esters (non-toxic) were internally modified to accommodate almost 12.5 times smaller Arabidopsis seeds (0.4 mm). With a simple Neoprene “O” ring, a sandwich of two Whatman No. 3 filter papers and a double side stick adhesive tape to hold two mini containers. Each mini container was longitudinally divided into two chambers by a central septum (with ducts), which provided for injecting growth medium or a fixative solution. 12-14 or 24 to 28 seeds can be placed on the black membrane. 1 % (w/v) guar gum (or guaran, a galactomannan polysaccharide from guar beans extracts) glued both membranes and filter paper. The growth medium was absorbed by Whatman filter papers. The mini containers had a transparent plastic cover to allow for observations. Thus, the membrane sandwich was inventively introduced for using Arabidopsis in spaceflight experiments. To avoid microbial contamination, the sandwich assembly was prepared in the laminar air-flow cabinets. Also, a sterilized syringe was filled with 1.3 ml of sucrose-free Arabidopsis growth medium. These mini containers were efficiently used for the subsequent spaceflight experiments (STS-81 and STS-84).
(d) Seedlings in plant chambers
In the STS-65 International Microgravity Laboratory-2 (IML-2) spaceflight experiment, garden cress seedlings were germinated in plant chambers of two types. Three A-chambers and six B-chambers were mounted into the holder, and all faced the cameras at equidistance from the camera lens.
(e) Seedling cassettes with LEDs
To study the complex interaction between photo and geotropisms, Plant Space Biologists developed specifically designed seedling cassettes and used in the TROPI-I and TROPI-2 hardware (also referred EUE for experimental unique equipment) used on the European Modular Cultivation System (EMCS). The EUE consisted of five seedling cassettes with LED (light-emitting diode) lighting systems (Ref. Scheme sketched by me) and a water delivery system in an Experimental Container (EC). The red and blue LEDs were used for the phototropic stimulation phase of the experiments. Each seedling cassette had two sets of LEDs. One set of white LEDs were along the longer side of the cassette (arrow in the scheme) and the second set of red and blue LEDs on one end of the shorter sides of the cassettes (* asterisks in the scheme). This innovative design resulted in great scientific returns such as information on involvements of phytochromes under microgravity and phototropic responses in roots (STS-115 and 121 spaceflight experiments).
(f) Carbon filters and better Arabidopsis growth
In TROPI-EUE spaceflight experiments, Plant Space Biologists observed that both seed germination and plant growth were markedly affected when Arabidopsis seeds were stored in EUE for several months. When they analyzed this biocompatibility problem, they found that the polymeric film conformal coating (25-250 μm thickness) of electrical components of the EUE was the causative factor for reduced germination and seedling growth. This conformal coating was a mandatory requirement by NASA for safety to protect the electronic circuitry against moisture, dust, chemicals, and temperature extremes. By a novel and simple idea, this major problem was eliminated. Simple additions of activated carbon filter membranes to both the seedling cassettes and to the base of the EC. Nylon mesh (NM) was used to hold the carbon filters (CF) in place. These filters absorbed vaporous materials produced as a result of offgassing. This novelty resulted in producing healthy plants capable of robust tropistic responses in spaceflight experiments. This schematic diagram showed the positive influences of carbon filter membranes on Arabidopsis germination and growth. There was reduced germination and growth when there were no carbon filters in the experiment container (top image) and a moderate improvement when carbon filters were fixed only on the base of the EC (middle image). Better and vigorous seedling growth was noticed when carbon filters were placed in both the EC base and at the cassette level.
2-Novelties in TROPI-2 for better seedling germination
One of the USA Plant Space Biology Team based on the operational lessons from TROPI-1 spaceflight experiments implemented three major improvements in the subsequent TROPI-2 spaceflight experiments. In general, the seed stocks of Arabidopsis thaliana show 80 %, germination rates with very good to excellent growth. In the three runs of TROPI-1 58 %, 27 % and 10 %, respectively. Seedlings were also not vigorous (top sketch). This USA Plant Space Biology Team determined that the poor germination was due to seed storage in sealed spaceflight hardware for up to 8 months and related biocompatibility issues. They experimentally proved that reduced seed storage (less than 2 months) in space hardware prior to performing the experiments in space dramatically improved the seed germination in TROPI-2. The two runs of TROPI-2 respectively showed 89 % and 83 % of germination rates. The seedlings were healthy and vigorous (the bottom sketch of my drawing). This major novelty was attained by a simple idea of shortening seed storage time. The sucrose in the growth medium was totally avoided in TROPI-2 experiments and this reduced possible contamination and re-crystallization risks on the filter paper used in the seedling cassettes as a growth substrate. All these small and novel changes collectively resulted in a robust tropistic curvature in the TROPI-2 experiments.
3- Novelties in Bioimaging
(a) Simple camera and film roll
To understand graviperception, graviresponsiveness and gravimorphogenesis, several ground control studies were undertaken in the 1980s. In these studies, the curvature measurements (curvature degrees) were made from photographic prints, and curvature angles were measured at the root tips using a protractor. Seedlings were photographed against a dark background and illuminated with fluorescent lamps. All the photographs were taken using a macro lens on a 35-mm camera with a film roll. They observed no curvatures in starchless or reduced-starch Arabidopsis mutants.
(b) Automated video recording
In TROPI-1 experiments (for tropisms) on the European Modular Cultivation System (EMCS) had NTSC (National Television Standard Committee) analog video cameras (Sony FCBIX470) capable of recording in both visible light and near infrared (in MULTIGEN-1 around 935 nm). This camera has various digital effects in capturing motion image on still image, continuous still image and motion images on binarized still image. Automated video recording with a Sony FCB-IX470 video camera captured images with the focus of each image on half of the seedling cassette. This provided a high magnification and high-resolution view of the seedlings. High-8 video tapes were used in the NTSC format.
Each video camera was mounted on a stepper motor and can observe two ECs (experiment containers), but only one EC was visible at a time. In total, four ECs and two cameras were located on one rotor. Rotating the video camera resulted in the image up and down (along the plant growth direction). Upright camera movement with respect to plant growth direction was performed by rotating a mirror located in the light path from the EC to the video camera. Images were frame-grabbed at certain intervals and recorded either on Hi-8 mm tape (using a TeacV-80ABF NTSC recorder) or stored temporarily on a mass memory unit (MMU) and set for down-link. If continuous video observations were an experiment objective, video sequences could also be recorded of the ECs.
Most importantly, the gridded black membranes were used to get a conspicuous background contrast for video capturing of the germination and/or curvature.
(c) Direct video downlink from ISS
Even though good quality images of seedlings were obtained in TROPI-1 experiments, the use of analog video tapes caused significant delays in image processing and analysis procedures. Realizing this biocompatibility problem, a USA Plant Space Biology team modified the strategies of bio-imaging. In the subsequent TROPI-2, High-8 videotapes were eliminated. Instead, the images captured in real-time downlinked to research stations in the ground. Images were downlinked at regular time intervals. The quantifiable results on Arabi seedlings length confirmed the qualitative observations from the downlinked images in those seedlings studied from TROPI-2 were longer and better established as compared to those from TROPI-1. Two types of images were downlinked namely the full experiment container views showing the seedling cassettes to monitor the progress of the experiment and secondly the higher resolution half-cassette views from which growth and curvature were measured using the Pro Plus Image Software. Hypocotyl curvatures were measured as increments over starting points.
(d) Green Fluorescent Protein Imaging System (GIS)
During the course of successive space shuttle missions, Plant Space Biologists progressively introduced novel imaging systems. In the APEX-TAGES spaceflight experiment launched on STS-130. The GFP Imaging System (GIS) was unique spaceflight hardware specifically designed to be kept within the Advanced Biological Research System (ABRS) orbital growth chamber. The GIS had six slots that accommodate 10 sq. cm petri plates, three in an upper tier and three in a lower tier.
The middle lower tier plate is positioned directly in front of the GIS camera and collects a set of images. The imaging camera was designed to collect both white light and fluorescent images. GIP camera incorporated a GFP imaging filter as an integral component of the camera. The GIS provided 470nnm LED illumination and the GIS camera contained a long pass filter (505nm) for collecting GFP-expression images. Even though white light images (taken in the absence of the GFP excitation illumination and in the presence of the growth light) had a greenish cast, they provided a very clear record of root growth and morphology over the duration of the experiment. Images were taken every six hours and stored to an SD card housed in the GIS unit. The images were also downlinked daily to the ground.
4- Novelties in imposing g-force accelerations
(a) Onboard centrifuges
To understand the effect of gravity on plants, various gravitational accelerations were imposed in both ground and spaceflight experiments. The gravity force on Earth is 1-g (9.8 m s-2). Different terms are used to distinguish three different acceleration levels namely hypergravity (higher than 1-g), microgravity (accelerations between 10- 3 g to 10-6 g) and hypogravity (accelerations greater than 10-3g). In the early experiments, table-top clinical centrifuges, clinostats and random positioning machines were used to impose varying accelerations.
Besides microgravity, in spaceflight experiments, there was a need to use 1-g control. So, scientists innovatively designed onboard centrifuges and successfully implemented. They got novel data when they used onboard centrifuges. For instances, the roots from cress grown in microgravity were more sensitive to the stimulation time than those of roots from plants grown in a 1g centrifuge. Enhanced curvature was also found for lentil roots previously grown in microgravity compared to plants that were grown on a 1-g centrifuge. The roots from transgenic and wildtype rapeseed (Brassica napus) during microgravity had no detectable curvature after 1 hour of centrifugation at 1g, whereas roots from ground controls curved significantly after reorientation.
The onboard centrifuge in the European Modular Cultivation System (EMCS) had two centrifuges. Each centrifuge had four slots for four ECs termed as ECS (experiment container slot). Two high-resolution video cameras were affixed in the centrifuges. These cameras capture images through a polished mirror. Thus, onboard centrifuge provided an essential 1-g control to distinguish between true microgravity effects and indirect effects of spaceflight by providing 1 g-force control. These onboard centrifuges were capable of producing fractional and hypergravity up to 2-g.
(b) Centrifuge-microscope
In IML-2 mission, a German Space Biologist team studied the graviperception in the flagellate single-celled algae Euglena gracilis during the shuttle spaceflight. In a novel strategy, they integrated a modified Zeiss Axioplan microscope mounted horizontally on a rotating steel disk. The cuvette with the algal cells was mounted on the vertical object table. In the centrifuge, it was oriented radially and off-center from the rotation axis. During the experiment, the image of the swimming cells was recorded by a b/w CCD camera and stored on an NTSC video recorder. During the experimental runs, the video image was transmitted to the monitoring research station in the ground by video downlink to allow the experimenter to control and modify area selection and focus. Thus, the real-time observation of a rotating sample with a fixed camera was achieved through a centrifuge-microscope.
5- Novelties in preserving biological samples
(a) Fixation device
At the beginning of spaceflight experiments, Plant Space Biologists mainly focused on ultrastructural changes in the plant roots under microgravity. For this purpose, in the hardware design, in addition to Biorack Type I container, an additional fixation device called Biorack Type II container was used to preserve the seedlings at the end of the experiment. Glutaraldehyde prepared in buffer was filled in fixers two days prior to launching. The glutaraldehyde fixative was injected through a septum into the chambers and simultaneous release of air from the chamber into a plastic bag, also through a septum fixed the plant samples. After bringing the fixed samples into the ground laboratories, plant roots required prolonged fixation (from 19 h to several days) for the ultrastructural preservation of roots. Few researchers observed that this prolonged fixation resulted in the destruction of membrane integrity (mitochondria, plastids, ER), reduced nuclear volume and shrinkage of organelles in the cytoplasm. It was recommended to check glutaraldehyde fixative in other buffers, age of buffers and different prefixation procedures. This biocompatibility made a practical demand for an efficient sample storage system.
(b) MELFI
Until 2006, NASA cold bags were used for storing samples from spaceflight experiments. The samples were stored approximately - 35°C and freezing the samples took a long time and this resulted uncertainties in the samples. This was a major limitation. European Space Agency innovatively designed a Minus Eighty-degree Laboratory and installed in International Space Station (ISS). This MELFI allowed fast-freezing and storage of life sciences and biological samples aboard and also uniquely designed for frequent return trips. Thus, MELFI served as a world-class payload. The biological samples were stored in four identical Dewar enclosures. Each Dewar can be set to cool to below three different temperatures: –80ºC, –26ºC and +4ºC. The centralized cooling system was based on a reverse Brayton cycle using very pure nitrogen as the working fluid. The basic machine was developed under ESA’s Technology Research Program (TRP) and then modified to satisfy MELFI’s specific and stringent requirements. The Brayton expander and compressor wheels were mounted on the same shaft, running at up to 96,000 rpm and produced the cooling power at - 97ºC.
In SG2 mission (2014), a USA Plant Space Biology Team used MELFI to study the combined effects of real or simulated microgravity and red‑light photoactivation on plant root meristematic cells of Arabidopsis. Further, in both Arabidopsis cell culture experiments (BRIC-17 SpaceX-2) and the Advanced Plant Experiments 03-2 (APEX-03-2) termed as per NASA’s nomenclature Transgenic Arabidopsis Gene Expression System – Intracellular Signaling Architecture (TAGES-ISA) study on SpaceX CRS-5 mission (2015) launched to the International Space Station (ISS), MELFI was efficiently used. Based on the scientific yields from these studies, another USA Plant Space Biology Team firmly suggested that differential gene expression between wild-type and gene knock-out mutants of Arabidopsis is the measure of the quantity and character of the physiological adaptation to spaceflight. Efficient use of this cryostorage facility resulted in transcriptome and proteome studies in Arabidopsis.
(c) GLACIER
In 2007, GLACIER (General Laboratory Active Cryogenic ISS Experiment Refrigerator) was designed and developed by University of Alabama at Birmingham (UAB) Center for Biophysical Sciences and Engineering (CBSE) for NASA Cold Storage. The glacier was originally designed for use on board the Space Shuttle but is now used for storing scientific samples on ISS in the EXpedite the PRocessing of Experiments to Space Station (EXPRESS) rack and transporting samples to/from orbit via the SpaceX Dragon or Cygnus Page 10 of 10 spacecraft. GLACIER is a double middeck locker equivalent payload designed to provide thermal control between +4 °C and -180 °C. Thus, the recovery of highquality RNA from spaceflight experiments is practically possible due to novel designs of space freezers.
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