Oxygen requirement of germinating flax seeds.

Plant experiments in earth orbit are typically prepared on the ground and germinated in orbit to study gravity effects on the developing seedlings. Germination requires the breakdown of storage compounds, and this metabolism depends upon respiration, making oxygen one of the limiting factors in seed germination. In microgravity lack of run-off of excess water requires careful testing of water dispensation and oxygen availability. In preparation for a shuttle experiment (MICRO on STS-107) we studied germination and growth of flax (Linum usitatissimum L.) seedlings in the developed hardware (Magnetic Field Chamber, MFC). We tested between four to 32 seeds per chamber (air volume=14 mL) and after 36 h measured the root length. At 90 microliters O2 per seed (32 seeds/chamber), the germination decreased from 94 to 69%, and the root length was reduced by 20%, compared to 8 seeds per chamber. Based on the percent germination and root length obtained in controlled gas mixtures between 3.6 and 21.6% O2 we determined the lower limit of reliable germination to be 10 vol. % O2 at atmospheric pressure. Although the oxygen available in the MFC's can support the intended number of seeds, the data show that seed storage and microgravity-related limitations may reduce germination.     

Optimization of moisture content for wheat seedling germination in a cellulose acetate medium for a space flight experiment.

The Porous Tube Plant Nutrient Delivery System (PTPNDS), a hydrophilic, microporous ceramic tube hydroponic system designed for microgravity, will be tested in a middeck locker of the Space Shuttle. The flight experiment will focus on hardware operation and assess its ability to support seed germination and early seedling growth in microgravity. The water controlling system of the PTPNDS hardware has been successfully tested during the parabolic flight of the KC-135. One challenge to the development of the space flight experiment was to devise a method of holding seeds to the cylindrical porous tube. The seed-holder must provide water and air to the seed, absorb water from the porous tube, withstand sterilization, provide a clear path for shoots and roots to emerge, and be composed of flight qualified materials. In preparation for the flight experiment, a wheat seed-holder has been designed that utilizes a cellulose acetate plug to facilitate imbibition and to hold the wheat seeds in contact with the porous tube in the correct orientation during the vibration of launch and the microgravity environment of orbit. Germination and growth studies with wheat at a range of temperatures showed that optimal moisture was 78% (by weight) in the cellulose acetate seed holders. These and other design considerations are discussed.     

Ground performance of air conditioning and water recycle system for a Space Plant Box.

Researchers from 5 Japanese universities have developed a plant growth facility (Space Plant Box) for seed to seed experiments under microgravity. The breadboard model of the Space Plant Box was fabricated by assembling subsystems developed for microgravity. The subsystems include air conditioning and water recycle system, air circulation system, water and nutrient delivery system, lighting system and plant monitoring system. The air conditioning and water recycle system is simply composed of a single heat exchanger, two fans and hydrophilic fibrous strings. The strings allow water movement from the cooler fin in the Cooling Box to root supporting materials in the Plant Growth Chamber driven by water potential deficit. Relative humidity in the Plant Growth Chamber can be changed over a wide range by controlling the ratio of latent heat exchange to sensible heat exchange on the cooling fin of the heat exchanger. The transpiration rate was successfully measured by circulating air inside the Plant Growth Chamber only. Most water was recycled and a small amount of water needed to be added from the outside. The simple, air conditioning and water recycle system for the Space Plant Box showed good performance through a barley (Hordeum vulgare L.) growth experiment.     

First flight of the ASTROCULTURE (TM) experiment as a part of the U.S. Shuttle/MIR program.

A number of space-based experiments have been conducted to assess the impact of microgravity on plant growth and development. In general, these experiments did not identify any profound impact of microgravity on plant growth and development, though investigations to study seed development have indicated difficulty in plants completing their reproductive cycle. However, it was not clear whether the lack of seed production was due to gravity effects or some other environmental condition prevailing in the unit used for conducting the experiment. The ASTROCULTURE (TM) flight unit contains a totally enclosed plant chamber in which all the critically important environmental conditions are controlled. Normal wheat (Triticum aestivum L.) growth and development in the ASTROCULTURE (TM) flight unit was observed during a ground experiment conducted prior to the space experiment. Subsequent to the ground experiment, the flight unit was transported to MIR by STS-89, as part of the U.S. Shuttle/MIR program, in an attempt to determine if super dwarf wheat plants that were germinated in microgravity would grow normally and produce seeds. The experiment was initiated on-orbit after the flight unit was transferred from the Space Shuttle to MIR. The ASTROCULTURE (TM) flight unit performed nominally for the first 24 hours after the flight unit was activated, and then the unit stopped functioning abruptly. Since it was not possible to return the unit to nominal operation it was decided to terminate the experiment. On return of the flight unit, it was confirmed that the control computer of the ASTROCULTURE (TM) flight unit sustained a radiation hit that affected the control software embedded in the computer. This experience points out that at high orbital inclinations, such as that of MIR and that projected for the International Space Station, the danger of encountering harmful radiation effects are likely unless the electronic components of the flight hardware are resistant to such impacts.     

Seed-to-seed growth of Arabidopsis thaliana on the International Space Station.

The assembly of the International Space Station (ISS) as a permanent experimental outpost has provided the opportunity for quality plant research in space. To take advantage of this orbital laboratory, engineers and scientists at the Wisconsin Center for Space Automation and Robotics (WCSAR), University of Wisconsin-Madison, developed a plant growth facility capable of supporting plant growth in the microgravity environment. Utilizing this Advanced Astroculture (ADVASC) plant growth facility, an experiment was conducted with the objective to grow Arabidopsis thaliana plants from seed-to-seed on the ISS. Dry Arabidopsis seeds were anchored in the root tray of the ADVASC growth chamber. These seeds were successfully germinated from May 10 until the end of June 2001. Arabidopsis plants grew and completed a full life cycle in microgravity. This experiment demonstrated that ADVASC is capable of providing environment conditions suitable for plant growth and development in microgravity. The normal progression through the life cycle, as well as the postflight morphometric analyses, demonstrate that Arabidopsis thaliana does not require the presence of gravity for growth and development.     

Ontogeny of plants under various gravity condition.

The results of experiments performed under conditions of microgravity (MG) or under its simulation on the horizontal clinostat (HC) with the callus, seedlings of various species and embryogenic structures have revealed a definite role of gravity as an ecological factor in the processes of cytomorphogenesis, growth, and development. The transformation of differentiated somatic cells of arabidopsis seed into undifferentiated callus was not inhibited under MG, though modifications of the whole callus morphology and of mean cell and nucleus size were observed. The morphogenesis of polar structures such as root-hair bearing cells of Lactuca primary root has been shown to be modified in the course of differentiation under mass acceleration diminished below 0.1 g. Seed germination and seedling morphogenesis under MG follow their normal course, but a significant stimulation of shoot growth with no effect on primary root growth has been determined. A successful in vitro regeneration of Nicotiana tabacum plantlets from leaf cells and subsequent formation of shoots and roots on a continuously rotating HC as well as the formation of viable seeds during seed-to-seed growth of Arabidopsis plants under MG have indicated that gravity plays but a limited role in the processes of embryogenesis and organogenesis.     

Clover development during spaceflight: a model system.

The development of legume root nodules was studied as a model system for the examination of gravitational effects on plant root development. In order to examine whether rhizobial association with clover roots can be achieved in microgravity, experiments were performed aboard the KC-135 parabolic aircraft and aboard the sounding rocket mission Consort 3. Binding of rhizobia to roots and the initial stages of root nodule development successfully occurred in microgravity. Seedling germination experiments were performed in the sliding block device, the Materials Dispersion Apparatus, aboard STS-37. When significant hydration of the seeds was achieved, normal rates of germination and seedling development were observed.     

Growth protocols for etiolated soybeans germinated within BRIC-60 canisters under spaceflight conditions.

As part of the GENEX (Gene Expression) spaceflight experiment, protocols were developed to optimize the inflight germination and subsequent growth of 192 soybean (Glycine max cv McCall) seeds during STS-87. We describe a method which provided uniform growth and development of etiolated seedlings while eliminating root and shoot restrictions for short-term (4-7 day) experiments. Final seedling growth morphologies and the gaseous CO2 and ethylene levels present both on the last day in space and at the time of recovery within the spaceflight and ground control BRIC-60 canisters are presented.     

Plant reproduction systems in microgravity: experimental data and hypotheses.

Elucidation of the possibilities for higher plants to realize complete ontogenesis, from seed to seed, and to propagate by seeds in microgravity, is a fundamental task of space biology connected with the working of the CELSS program. At present, there are results of only 6 spaceflight experiments with Arabidopsis thaliana, an ephemeral plant which will flower and fruit in orbit. Morphogenesis of generative organs occurs normally in microgravity, but unlike the ground control, buds and flowers mainly contain sterile elements of the androecium and gynoecium which degenerate at different stages of development in microgravity. Cytological peculiarities of male and female sterility in microgravity are similar to those occurring naturally during sexual differentiation. Many of the seed formed in microgravity do not contain embryos. Hypotheses to explain abnormal reproductive development in microgravity are: 1) nutritional deficiency, 2) insufficient light, 3) intensification of the influence of the above-mentioned factors by microgravity, 4) disturbances of a hormonal nature, and 5) the absence of pollination and fertilization. Possible ways for testing these hypotheses and obtaining viable seeds in microgravity are discussed.     

Capillary movement of liquid in granular beds in microgravity.

A more complete understanding of the dynamics of capillary flow through an unsaturated porous medium would be useful for the development of an effective water and nutrient delivery system for the growth of plants in space. An experiment was conducted on the Mir Space Station that used an experimental cuvette called "Capillary Test Bed" to compare fluid migration under terrestrial laboratory conditions by positioning the cuvette such that the hydrostatic force is negated and on Mir under microgravity conditions. Differences in fluid migration in the cuvette were observed with migration being slower in microgravity compared with some ground control experiments.     

天宫二号碲化锌晶体生长

在天宫二号飞船综合材料实验炉六工位采用碲熔剂法生长了碲化锌晶体,生长时最高温度为800℃,以0.5mm·h-1的提拉速度向炉膛内部提拉生长晶体.飞行实验后,用相同实验参数在地面进行了对比实验.结果发现,空间样品尾部有一个非常大的橙色结晶区域（约10mm×6mm×2mm）,而地面生长样品中碲化锌晶体尺寸仅为约3mm×3mm×1mm,空间生长的碲化锌晶粒尺寸明显优于地面.空间微重力环境下,由于毛细作用,空间样品的塞子处有Te和ZnTe的外延膜生成.而地面生长的锭条在塞子处只有零星点状气相生产物.因此微重力条件有利于碲化锌晶体材料的生长.      

Genetic and physiological damage induced by cosmic radiation on dry plant seeds during space flight

Total evaluation of cosmic radiation effect with or without discrimination of individualized HZE-ion effects in dry seeds flown for 10 days on STS-9, yielded significant evidence for radiation damage in space. They depend on the biological criteria tested (seed germination, morphogenesis, embryo lethality, mutation rate) which stand for early, physiological and late genetic effects. They are also related to the radiation shielding environment in the space shuttle. Proceeding from these results three direct questions can be posed for present (LDEF-1) and future (ERA-1, D-2) experiments in space: What is the influence of cosmic radiation on cytogenetic repair and ontogenetic restitution processes? Does microgravity disorder the morphogenesis (i.e. growth and cell differentiation)? Is there an interaction between the effects of cosmic radiation and microgravity in eukaryotic plant systems?     

Effects of a hypogeomagnetic field on gravitropism and germination in soybean

Any plants grown during long-term space missions will inevitably experience an extremely low magnetic field (i.e. a hypogeomagnetic field, HGMF). It is possible that the innate adaptation of plants to the earth’s magnetic field (i.e. the geomagnetic field, GMF) would be disrupted. Effects of the HGMF on plant physiological and metabolic processes are unclear. In this study we established a hypogeomagnetic incubation system on the ground and investigated the effects of the HGMF on the gravitropism and germination of soybean seeds. The gravitropism angle, germination percentage, germination speed, water absorbance ratio, seed weight, radicle length, radicle weight, and radicle weight ratio of soybean seeds grown in the local field and the HGMF were compared. In general, the gravitropism angle in the HGMF was smaller than that in the local field when seeds were positioned before emergence in such a way that the direction of the radicle was opposite to that of gravity. The germination percentage, germination speed, and radicle weight ratio increased in the HGMF compared to the control. Our results indicate that the germination and gravitropism of soybean seeds are affected by elimination of the geomagnetic field.     

Auxin polar transport in Arabidopsis under simulated microgravity conditions — Relevance to growth and development

Activity of auxin polar transport in inflorescence axes of Arabidopsis thaliana grown under simulated microgravity conditions was studied in relation to the growth and development. Seeds were germinated and allowed to grow on an agar medium in test tubes on a horizontal clinostat. Horizontal clinostat rotation substantially reduced the growth of inflorescence axes and the productivity of seeds of Arabidopsis thaliana (ecotypes Landsberg erecta and Columbia), although it little affected seed germination, development of rosette leaves and flowering. The activity of auxin polar transport in inflorescence axes decreased when Arabidopsis plants were grown on a horizontal clinostat from germination stage, being ca. 60% of 1 g control. On the other hand, the auxin polar transport in inflorescence axes of Arabidopsis grown in 1 g conditions was not affected when the segments were exposed to various gravistimuli, including 3-dimensional clinorotation, during transport experiments. Pin-formed mutant of Arabidopsis, having a unique structure of the inflorescence axis with no flower and extremely low levels of the activity of auxin polar transport in inflorescence axes and endogenous auxin, did not continue its vegetative growth under clinostat rotation. These facts suggest that the development of the system of auxin polar transport in Arabidopsis is affected by microgravity, resulting in the inhibition of growth and development, especially during reproductive growth.     

"From seed-to-seed" experiment with wheat plants under space-flight conditions.

An important goal with plant experiments in microgravity is to achieve a complete life cycle, the "seed-to-seed experiment." Some Soviet attempts to reach this goal are described, notably an experiment with the tiny mustard, Arabidopsis thaliana, in the Phyton 3 device on Salyut 7. Normal seeds were produced although yields were reduced and development was delayed. Several other experiments have shown abnormalities in plants grown in space. In recent work, plants of wheat (Triticum aestivum) were studied on the ground and then in a preliminary experiment in space. Biometric indices of vegetative space plants were 2 to 2.5 times lower than those of controls, levels of chlorophyll a and b were reduced (no change in the ratio of the two pigments), carotenoids were reduced, there was a serious imbalance in major minerals, and membrane lipids were reduced (no obvious change in lipid patterns). Following the preliminary studies, an attempt was made with the Svetoblock-M growth unit to grow a super-dwarf wheat cultivar through a life cycle. The experiment lasted 167 d on Mir. Growth halted from about day 40 to day 100, when new shoots appeared. Three heads had appeared in the boot (surrounded by leaves) when plants were returned to earth. One head was sterile, but 28 seeds matured on earth, and most of these have since produced normal plants and seeds. In principle, a seed-to-seed experiment with wheat should be successful in microgravity.     

Seedling growth and development on space shuttle.

Young pine seedlings, and mung bean and oat seeds were flown on shuttle flights, STS-3 and STS-51F, in March, 1982 and July/August, 1985, respectively. The plant growth units built to support the two experiments functioned mechanically as anticipated and provided the necessary support data. Pine seedlings exposed to the microgravity environment of the space shuttle for 8 days continued to grow at a rate similar to ground controls. Pine stems in flight seedlings, however, averaged 10 to 12% less lignin than controls. Flight mung beans grew slower than control beans and their stems contained about 25% less lignin than control seedlings. Reduced mung bean growth in microgravity was partly due to slower germination rate. Lignin also was reduced in flight oats as compared to controls. Oats and mung beans exhibited upward growing roots which were not observed in control seedlings. Chlorophyll A/B ratios were lower in flight tissues than controls. The sealed PGCs exhibited large variations in atmospheric gas composition but the changes were similar between flight and ground controls. Ethylene was present in low concentrations in all chambers.     

Interaction of growth-determining systems with gravity

The experiments have been carried out with lettuce shoots on board the Salyut-7 orbital station, the Kosmos-1667 biological satellite and under ground conditions at 180° plant inversion. By means of the centrifuge Biogravistat-1M the threshold value of gravitational sensitivity of lettuce shoots has been determined on board the Salyut-7 station. It was found to be equal to 2.9 × 10⁻³g for hypocotyls and 1.5 × 10⁻⁴g for roots. The following results have been received in the experiment performed on board the Kosmos-1667 satellite: a) under microgravity the proliferation of the meristem cells and the growth of roots did not differ from the control; b) the growth of hypocotyls in length was significantly enhanced in microgravity; c) under microgravity transverse growth of hypocotyls (increase in cross sectional area) was significantly increased due to enhancement of cortical parenchyma cell growth. At 180° inversion in Earth's gravity root extension growth and rate of cell division in the root apical meristem were decreased. The determination of DNA-fuchsin value in the nuclei of the cell root apexes showed that inversion affected processess of the cell cycle preceeding cytokinesis.     

Differentiation of Dictyostelium discoideum vegetative cells into spores during Earth orbit in space.

We reported previously that emerged amoebae of Dictyostelium (D.) discoideum grew, aggregated and differentiated to fruiting bodies with normal morphology in space. Here, we investigated the effects of space radiation and/or microgravity on the number, viability, kinetics of germination, growth rate and mutation frequency of spores formed in space in a radiation-sensitive strain, gamma s13, and the parental strain, NC4. In gamma s13, there were hardly spores in the fruiting bodies formed in space. In NC4, we found a decrease in the number of spores, a delay in germination of the spores and delayed start of cell growth of the spores formed in space when compared to the ground control. However, the mutation frequency of the NC4 spores formed in space was similar to that of the ground control. We conclude that the depression of spore formation might be induced by microgravity and/or space radiation through the depression of some stage(s) of DNA repair during cell differentiation in the slime mold.     

Improvements in the re-flight of spaceflight experiments on plant tropisms

In order to effectively study phototropism, the directed growth in response to light, we performed a series of experiments in microgravity to better understand light response without the “complications” of a 1-g stimulus. These experiments were named TROPI (for tropisms) and were performed on the European Modular Cultivation System (EMCS), a laboratory facility on the International Space Station (ISS). TROPI-1 was performed in 2006, and while it was a successful experiment, there were a number of technical difficulties. We had the opportunity to perform TROPI-2 in 2010 and were able to optimize experimental conditions as well as to extend the studies of phototropism to fractional gravity created by the EMCS centrifuge. This paper focuses on how the technical improvements in TROPI-2 allowed for a better experiment with increased scientific return. Major modifications in TROPI-2 compared to TROPI-1 included the use of spaceflight hardware that was off-gassed for a longer period and reduced seed storage (less than 2 months) in hardware. These changes resulted in increased seed germination and more vigorous growth of seedlings. While phototropism in response to red illumination was observed in hypocotyls of seedlings grown in microgravity during TROPI-1, there was a greater magnitude of red-light-based phototropic curvature in TROPI-2. Direct downlinking of digital images from the ISS in TROPI-2, rather than the use of analog tapes in TROPI-1, resulted in better quality images and simplified data analyses. In TROPI-2, improved cryo-procedures and the use of the GLACIER freezer during transport of samples back to Earth maintained the low temperature necessary to obtain good-quality RNA required for use in gene profiling studies.     

The ASTROCULTURE(TM) flight experiment series, validating technologies for growing plants in space.

A flight experiment, ASTROCULTURE(TM)-1 (ASC-1), to evaluate the operational characteristics and hardware performance of a porous tube nutrient delivery system (PTNDS) was flown on STS-50 as part of the U.S. Microgravity Laboratory-1 mission, 25 June to 9 July, 1992. This experiment is the first in a series of planned ASTROCULTURE(TM) flights to validate the performance of subsystems required to grow plants in microgravity environments. Results indicated that the PTNDS was capable of supplying water and nutrients to plants in microgravity and that its performance was similar in microgravity to that in 1g on Earth. The data demonstrated that water transfer rates through a rooting matrix are a function of pore size of the tubes, the degree of negative pressure on the 'supply' fluid, and the pressure differential between the 'supply' and 'recovery' fluid loops. A slightly greater transfer rate was seen in microgravity than in 1g, but differences were likely related to the presence of hydrostatic pressure effects at 1g. Thus, this system can be used to support plant growth in microgravity or in partial gravity as on a lunar or Mars base. Additional subsystems to be evaluated in the ASTROCULTURE(TM) flight series of experiments include lighting, humidity control and condensate recovery, temperature control, nutrient composition control, CO2 and O2 control, and gaseous contaminant control.