Preliminary results from experiments flown on the STS-47 and STS-65 missions showed genetic abnormalities occurring in plants during space flight. Because ground-based studies indicate that water related activity can impact the integrity of chromosomes, it is possible that the results were not due necessarily to microgravity effects upon the plants, but rather to indirect effects mediated by water availability to plant cells. This experiment tested whether the cell division changes observed in the daylily embryo cultures and plantlets resulted directly from microgravity or indirectly through water availability.
It has been hypothesized that the chromosomal and nuclear abnormalities encountered in various plants exposed to space are due to a combination of factors. These include the biological status of the systems and the way in which they are grown, exposed to, and ultimately, the way in which they experience multiple stresses. The central idea is that the extent to which space-specific changes become manifest is dependent on the extent of pre-existing stresses in the system. This has been suggested in a variety of plant species grown in space, but has been particularly amenable to study using an in vitro developing daylily embryoid system.
The following questions were addressed by this experiment: Can the mitotic process and chromosome morphology in developing plant cells be predictably modified (or not) in the space environment by deliberately providing optimized and/or non-optimized environments? Can adverse alterations in osmotic status and water relations (water stress) pre-dispose cells to become damaged cytologically in the space environment?
APPROACH:
The BRIC-08 experiment flew three BRIC 100 canisters that housed 27 plastic petri dishes of daylily cells in an agar solidified medium. The BRICs were flown in the Space Shuttle Middeck on the STS-78 mission for approximately 17 days. There was no inflight manipulation of the specimens. Upon landing, 85% of the cells were chemically fixed for examination while 15% were allowed to develop. A ground control experiment was conducted in parallel in the Orbiter Environment Simulator at environmental conditions (temperature, % relative humidity, and CO2) similar to the flight samples on orbit.
To achieve different levels of "wetness" the samples were grown on either, media solidified with lower concentrations of agar (resulting in a wetter environment), or media solidified with higher concentrations of agar (resulting in a drier environment). All petri dishes contained discs of activated charcoal-impregnated filter paper on top of an identical nutrient medium, differing only in level of wetness
The embryogenic initials of daylily are most precisely viewed as very young embryos or "zygote-equivalents," i.e., they are like developing fertilized egg cells or zygotes except they are not the products of a sexual union. The quality of somatic embryo advancement is dependent upon a proper phenotype (that is, what they look like) and genotype (that is, their genetic fidelity or “closeness to type” in terms of what they started off as).
The daylily somatic embryos were completely aseptic at the outset of the experiment and the sterility of the cultures were maintained for the duration of the experiment. The canisters were flushed with 75% nitrogen, 20% oxygen and 5% carbon dioxide gas and then sealed shut so there was only minimal leakage.
Extensive post-flight analyses of mitotic activity, chromosome morphology, and embryonic development were performed on representative samples from each of the flight and ground control categories.
RESULTS:
The embryogenic daylily cells survived and progressed during the flight through the various stages of somatic embryogenesis.
The main cytological observation involved the repeated detection of a specific chromosome deletion. This deletion had been found in previous spaceflight experiments as well, leading up to this experiment in which it was hypothesized that there would be more deletions in the "wetter" petri dishes and fewer in the "drier" petri dishes. As predicted there were more signs of chromosome perturbations in the "wet" than the "dry" (5.3 % vs. 0.6%) dishes, and there were more cells in division during the postflight cell cycle in the "wet" dishes than in the "dry" dishes (3.3% vs. 1.1%). The larger, more advanced somatic embryos showed fewer and less severe chromosomal perturbations than the smaller and less developmentally advanced embryoids (0.8% vs.2.3%). While larger embryos exhibited more transient and less significant damage such as chromosomal breaks, laggards, bridges and micro-chromosomes, size was not enough to offset the production of a deletion. Since the deletion has been consistent in all materials from various space flights, this may mean, that there is an active site that responds to the space flight environment.
As has been the case in previous experiments, no ground control material showed the deletion, whereas it could be found within all forms of embryoids in space. The researchers speculate that the deletion is a significant cytological indicator of stress in daylily cultured within the space environment.
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