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.     

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.     

Germination and elongation of flax in microgravity.

This experiment was conducted as part of a risk mitigation payload aboard the Space Shuttle Atlantis on STS-101. The objectives were to test a newly developed water delivery system, and to determine the optimal combination of water volume and substrate for the imbibition and germination of flax (Linum usitatissimum) seeds in space. Two different combinations of germination paper were tested for their ability to absorb, distribute, and retain water in microgravity. A single layer of thick germination paper was compared with one layer of thin germination paper under a layer of thick paper. Paper strips were cut to fit snugly into seed cassettes, and seeds were glued to them with the micropyle ends pointing outward. Water was delivered in small increments that traveled through the paper via capillary action. Three water delivery volumes were tested, with the largest (480 microliters) outperforming the 400 microliters and 320 microliters volumes for percent germination (90.6%) and root growth (mean=4.1 mm) during the 34-hour spaceflight experiment. The ground control experiment yielded similar results, but with lower rates of germination (84.4%) and shorter root lengths (mean=2.8 mm). It is not clear if the roots emerged more quickly in microgravity and/or grew faster than the ground controls. The single layer of thick germination paper generally exhibited better overall growth than the two layered option. Significant seed position effects were observed in both the flight and ground control experiments. Overall, the design of the water delivery system, seed cassettes and the germination paper strip concept was validated as an effective method for promoting seed germination and root growth under microgravity conditions.     

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.     

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?     

"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.     

Spaceflight hardware for conducting plant growth experiments in space: the early years 1960-2000.

The best strategy for supporting long-duration space missions is believed to be bioregenerative life support systems (BLSS). An integral part of a BLSS is a chamber supporting the growth of higher plants that would provide food, water, and atmosphere regeneration for the human crew. Such a chamber will have to be a complete plant growth system, capable of providing lighting, water, and nutrients to plants in microgravity. Other capabilities include temperature, humidity, and atmospheric gas composition controls. Many spaceflight experiments to date have utilized incomplete growth systems (typically having a hydration system but lacking lighting) to study tropic and metabolic changes in germinating seedlings and young plants. American, European, and Russian scientists have also developed a number of small complete plant growth systems for use in spaceflight research. Currently we are entering a new era of experimentation and hardware development as a result of long-term spaceflight opportunities available on the International Space Station. This is already impacting development of plant growth hardware. To take full advantage of these new opportunities and construct innovative systems, we must understand the results of past spaceflight experiments and the basic capabilities of the diverse plant growth systems that were used to conduct these experiments. The objective of this paper is to describe the most influential pieces of plant growth hardware that have been used for the purpose of conducting scientific experiments during the first 40 years of research.     

Behavior of Japanese tree frogs under microgravity on MIR and in parabolic flight.

Japanese tree frogs (Hyla japonica) were flown to the space station MIR and spent eight days in orbit during December, 1990. Under microgravity, their postures and behaviors were observed and recorded. On the MIR, floating frogs stretched four legs out, bent their bodies backward and expanded their abdomens. Frogs on a surface often bent their neck backward and walked backwards. This behavior was observed on parabolic flights and resembles the retching behavior of sick frogs on land--a possible indicator of motion sickness. Observations on MIR were carried out twice to investigate the frog's adaptation to space. The frequency of failure in landing after a jump decreased in the second observation period. After the frogs returned to earth, readaptation processes were observed. The frogs behaved normally as early as 2.5 hours after landing.     

Influence of cosmic radiation and/or microgravity on development of Carausius morosus.

Eggs of Carausius morosus were exposed to spaceflight conditions in two spaceflight missions, the German 7 day Spacelab Mission D1 and the Soviet 12.56 day Biosatellite Mission "COSMOS 1887". During spaceflight the eggs continued their development. Eggs of five different ages representing different sensitivity to radiation and different capacity to regeneration were used to investigate the influence of cosmic radiation and/or microgravity on insect development. Using the Biostack concept--eggs in monolayers sandwiched between nuclear track detectors--and the 1 g reference centrifuge of BIORACK in D1 we were able to separate effects of heavy ions of the cosmic radiation from microgravity effects and also from combined effects of these two factors in space. After retrieval, hatching rates, embryonic and larval growth kinetics and anomaly frequencies were determined. Microgravity leads to a reduced hatching rate of eggs exposed in the early stages of development. Hatching was normal in eggs which were exposed on the 1 g reference centrifuge. Hits by heavy ions caused body anomalies. The combined action of heavy ions and microgravity resulted in an unexpectedly high frequency of anomalies. These results obtained from the Spacelab Mission D1, were confirmed in an experiment onboard of COSMOS 1887. In addition to the previous analysis, embryonic development before hatching was followed which showed no major difference between flight and the ground control specimens. Since a reconfirmation of reduced hatching rates was observed in COSMOS 1887, too, the above results suggest some microgravity induced functional impairment of the hatching activity, rather than blockage in embryonic development.     

Education programme on aerospace and environmental medicine for medical faculty of Lomonosov Moscow State University.

The problems and approaches to organisation of the education process in the field of aerospace and environmental medicine for medical students are discussed. Original education developed on the basis of Russian experience in space biology and physiology, environmental medicine, aerospace medicine and medical support during spaceflight. The main goals of these programs are to acquaint students with: interaction of living organisms with natural and artificial surroundings, including space flight conditions; the physiological reactions on extreme environmental factors; basic mechanisms of human adaptation to space flight and particularly to microgravity; the current research in space medicine and new telecommunication technologies. All programs are formed in accordance with contemporary progress in life sciences and revealed a result of the interdisciplinary approach to education process.     

Signs of Müller cell gliotic response found in the retina of newts exposed to real and simulated microgravity

The effects of real and simulated microgravity on the eye tissue regeneration of newts were investigated. For the first time changes in Müller glial cells in the retina of eyes regenerating after retinal detachment were detected in newts exposed to clinorotation. The cells divided, were hypertrophied, and their processes were thickened. Such changes suggested reactive gliosis and were more significant in animals exposed to rotation when compared with desk-top controls. Later experiments onboard the Russian biosatellite Bion-11 showed similar changes in the retinas that were regenerating in a two-week spaceflight. In the Bion-11 animals, GFAP, the major structural protein of retinal macroglial cells, was found to be upregulated. In a more recent experiment onboard Foton-M3 (2007), GFAP expression in retinas of space-flown, ground control (kept at 1 g), and basal control (sacrificed on launch day) newts was quantified, using microscopy, immunohistochemistry, and digital image analysis. A low level of immunoreactivity was observed in basal controls. In contrast, retinas of space-flown animals showed greater GFAP immunoreactivity associated with both an increased cell number and a higher thickness of intermediate filaments. This, in turn, was accompanied by up-regulation of stress protein (HSP90) and growth factor (FGF2) expressions. It can be postulated that such a response of Müller cells was to mitigate the retinal stress in newts exposed to microgravity. Taken together, the data suggest that the retinal population of macroglial cells could be sensitive to gravity changes and that in space it can react by enhancing its neuroprotective function.     

Biological dosimetry in Russian and Italian astronauts.

Large uncertainties are associated with estimates of equivalent dose and cancer risk for crews of long-term space missions. Biological dosimetry in astronauts is emerging as a useful technique to compare predictions based on quality factors and risk coefficients with actual measurements of biological damage in-flight. In the present study, chromosomal aberrations were analyzed in one Italian and eight Russian cosmonauts following missions of different duration on the MIR and the international space station (ISS). We used the technique of fluorescence in situ hybridization (FISH) to visualize translocations in chromosomes 1 and 2. In some cases, an increase in chromosome damage was observed after flight, but no correlation could be found between chromosome damage and flight history, in terms of number of flights at the time of sampling, duration in space and extra-vehicular activity. Blood samples from one of the cosmonauts were exposed in vitro to 6 MeV X-rays both before and after the flight. An enhancement in radiosensitivity induced by the spaceflight was observed.     

中国载人航天工程的外部设计

在“神舟号”载人飞船工程实现了中国人往返于天地间的目的之后 ,中国应审慎地选择发展载人航天的目标。文章从中国社会对载人航天的需求出发 ,讨论了以开发利用空间微重力物质环境为目标的空间站和以发展天基航天为目标的天基航天站的外部工程系统的环境条件 ,认为中国在运载火箭、发射和回收场、测控站网方面已有较好基础 ,基本具备条件 ,运人运输器已有“神舟号”载人飞船 ,运物运输器的研制也不困难 ,但在为保障航天员在空间生活、工作的航天员系统方面和为实现载人航天工程功能和显现价值的有效载荷系统方面欠缺较多 ,需要一个研究、试验、培训和开发、演示的发展阶段     

Bioscience experiments in the German Spacelab mission D-1: introduction and summary.

The German Spacelab mission D-l was performed from 30 October through 6 November 1985. Payload operation in orbit was managed by DFVLR for the Federal Ministry of Research and Technology. The scientific program of the mission placed emphasis on microgravity research. In bioscience, the role of gravity in vital functions of biological systems was investigated, such as intracellular and intercellular interactions, developmental processes as well as regulation and adaptation in highly organized systems including human beings. In addition, the biological significance of cosmic radiation or altered zeitgeber within the complex matrix of all relevant spaceflight components were studied. Most of the experiments were accommodated in the following three payload elements: The Bioscience Experiment Package, and the ESA facilities Vestibular Sled and BIORACK. The information gained from the individual experiments will be compiled to help answer pending questions of space bioscience.     

Protein crystal growth aboard the U.S. space shuttle flights STS-31 and STS-32.

The first microgravity protein crystal growth experiments were performed on Spacelab I by Littke and John. These experiments indicated that the space grown crystals, which were obtained using a liquid-liquid diffusion system, were larger than crystals obtained by the same experimental system on earth. Subsequent experiments were performed by other investigators on a series of space shuttle missions from 1985 through 1990. The results from two of these shuttle flights (STS-26 and STS-29) have been described previously. The results from these missions indicated that the microgravity grown crystals for a number of different proteins were larger, displayed more uniform morphologies, and yielded diffraction data to significantly higher resolutions than the best crystals of these proteins grown on earth. This paper presents the results obtained from shuttle flight STS-32 (flown in January, 1990) and preliminary results from the most recent shuttle flight, STS-31 (flown in April, 1990).     

Plant growth, development and embryogenesis during Salyut-7 flight.

The growth and geotropic movements of roots and hypocotyls of lettuce have been studied on board the Salyut 7 station in a stationary position and on the centrifuge at 0.01, 0.1 and 1 g. On the centrifuge at 0.1 and 0.01 g as well as under weightlessness, the final length of hypocotyls was by 8-16% greater than in control plants on the centrifuge at 1 g. The length of roots, however, was reduced by 17% at 0.01 g and under weightlessness; at 0.1 g their growth is much the same as at 1 g. On the Earth, while growing in a vertical position, and in space at 0 < or = g, the roots and hypocotyls deviate from the longitudinal axis of the seed. Average values of deviation eagles on the Earth are always equal to zero, while this is not always the case in space, which indicates the biological effect of microgravity conditions on board a spacecraft. The threshold of geotropic sensitiveness of lettuce hypocotyls, calculated from the linear regression parameters of the dependence of the response geotropic reaction upon the value of the centrifugal force, comprised 2.9 x 10(-3) g. In the Fiton 3 micro-greenhouse under spaceflight conditions, the plants of Arabidopsis thaliana (L) Heynh have, for the first time, undergone a full cycle of individual development. The seeds sown during the flight germinated, performed growth processes, formed vegetative and generative organs and, judging by the final result, they succeeded in fecundation, embryogenesis and ripening. Despite the noted modification of growth and development of plants in space, 42% of formed seeds appeared to be valuable biologically.     

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.     

Induction of hypoxic root metabolism results from physical limitations in O2 bioavailability in microgravity.

Numerous spaceflight experiments have noted changes in the roots that are consistent with hypoxia in the root zone. These observations include general ultrastructure analysis and biochemical measurements to direct measurements of stress specific enzymes. In experiments that have monitored alcohol dehydrogenase (ADH), the data shows this hypoxically responsive gene is induced and is associated with increased ADH activity in microgravity. These changes in ADH could be induced either by spaceflight hypoxia resulting from inhibition of gravity mediated O2 transport, or by a non-specific stress response due to inhibition of gravisensing. We tested these hypotheses in a series of two experiments. The objective of the first experiment was to determine if physical changes in gravity-mediated O2 transport can be directly measured, while the second series of experiments tested whether disruption of gravisensing can induce a non-specific ADH response. To directly measure O2 bioavailability as a function of gravity, we designed a sensor that mimics metabolic oxygen consumption in the rhizosphere. Because of these criteria, the sensor is sensitive to any changes in root O2 bioavailability that may occur in microgravity. In a KC-135 experiment, the sensor was implanted in a moist granular clay media and exposed to microgravity during parabolic flight. The resulting data indicated that root O2 bioavailability decreased in phase with gravity. In experiments that tested for non-specific induction of ADH, we compared the response of transgenic Arabidopsis plants (ADH promoted GUS marker gene) exposed to clinostat, control, and waterlogged conditions. The plants were grown on agar slats in a growth chamber before being exposed to the experimental treatments. The plants were stained for GUS activity localization, and subjected to biochemical tests for ADH, and GUS enzyme activity. These tests showed that the waterlogging treatment induced significant increases in GUS and ADH enzyme activities, while the control and clinostat treatments showed no response. This work demonstrates: (1) the inhibition of gravity-driven convective transport can reduce the O2 bioavailability to the root tip, and (2) the perturbation of gravisensing by clinostat rotation does not induce a nonspecific stress response involving ADH. Together these experiments support the microgravity convection inhibition model for explaining changes in root metabolism during spaceflight.     

Embryogenesis, hatching and larval development of Artemia during orbital spaceflight.

Developmental biology studies, using gastrula-arrested cysts of the brine shrimp Artemia franciscana, were conducted during two flights of the space shuttle Atlantis (missions STS-37 and STS-43) in 1991. Dehydrated cysts were activated, on orbit, by addition of salt water to the cysts, and then development was terminated by the addition of fixative. Development took place in 5 ml syringes, connected by tubing to activation syringes, containing salt water, and termination syringes, containing fixative. Comparison of space results with simultaneous ground control experiments showed that equivalent percentages of naupliar larvae hatched in the syringes (40%). Thus, reactivation of development, completion of embryogenesis, emergence and hatching took place, during spaceflight, without recognizable alteration in numbers of larvae produced. Post-hatching larval development was studied in experiments where development was terminated, by introduction of fixative, 2 days, 4 days, and 8 days after reinitiation of development. During spaceflight, successive larval instars or stages, interrupted by molts, occurred, generating brine shrimp at appropriate larval instars. Naupliar larvae possessed the single naupliar eye, and development of the lateral pair of adult eyes also took place in space. Transmission electron microscopy revealed extensive differentiation, including skeletal muscle and gut endoderm, as well as the eye tissues. These studies demonstrate the potential value of Artemia for developmental biology studies during spaceflight, and show that extensive degrees of development can take place in this microgravity environment.     

Space experiment on behaviors of treefrog.

Japanese treefrogs (Hyla japonica) are planned to be sent to the space station MIR. Experimental system was developed to observe their behaviors under microgravity.