Key Questions
We sought out answers to these three key questions that guided our research directions:
- How can we design our experiments to derive valid knowledge?
- What are the current challenges in the field of space plant growth according to stakeholders, and how can we address these challenges?
- How are perspectives on agricultural ethics and GMOs on Earth related to our project’s proposed implementation?
Key Question 1: How can we design our experiments to derive valid knowledge?
Background
As scientists, one area in which we have to be responsible is experimental design. When designing experiments we have to consider scientific values such as validity, whether an experiment can answer the questions it is intended to answer logically.
Experts
Dr. Collin Timm
Senior Staff Scientist, Biological Sciences Group Research and Exploratory Development Department The Johns Hopkins University Applied Physics Laboratory Engineering synthetic microbiomes to complement plant phenotypes, studies plant-bacteria interactions.
Jeffrey Shipp
Staff Scientist, Biological Sciences Group Research and Exploratory Development Department The Johns Hopkins University Applied Physics Laboratory
Why we reached out
In the early brainstorming stage of our project, we were seeking feedback on the viability of our idea to implement magnetotropism in plant roots for better growth in spaceflight. We reached out to Dr. Collin Timm, senior staff scientist in the Biological Sciences Group at The Johns Hopkins University Applied Physics Laboratory (APL). As an institution, APL’s research addresses challenges in national security and space. In the Biological Sciences Group, there is expertise in plant biology as some APL scientists research the use of plants as sensors for identifying chemical and biological threats in the environment. Not being able to test our engineered plants in space poses a challenge for our project, so we discussed with Dr. Timm how we should design our experiments and hardware to validate our solution. He additionally recommended that we consult his colleague, Jeffrey Shipp, for advice on good practices for engineering plants.
Summary of Discussion about Microgravity Environment on Earth
Dr. Timm suggested we use a clinostat, a type of random positioning machine, to simulate microgravity. He provided us with an example of a clinostat that we could build (ref). We also identified a 3D clinostat from COSE instruments that we could purchase. We discussed with Dr. Timm whether the best choice was to build or buy a clinostat. One thing we had to consider was how the clinostat would fit the magnetic field generator and the plants. We asked how large can we expect the plants to get in the radial and vertical dimensions. Dr. Timm informed us that we only need to grow the Arabidopsis thaliana plant approximately 5 to 10 cm long to observe organized root growth. Furthermore, Dr. Timm told us that the best way to observe root growth is to grow the plants on agar plates and hold them up vertically. The roots grow along the surface of the solid media. With this in mind, we realized we did not need a 3D clinostat that would rotate along two axes, but only a 2D clinostat that would rotate along one axis.
How feedback impacted our project
Based on the discussion, we decided to build a 2D clinostat as part of our project. (see Contribution)
Summary of Discussion about Controls for Root Growth
We also met with Dr. Collin Timm to discuss our wetlab experimental design. We discussed what control would be necessary to understand the difference between ordered and disordered root growth. Dr. Timm recommended that we use a positive control that shows the disordered root growth phenotype under natural gravity such as a gravitropism mutant. He suggested The Arabidopsis Information Resource (TAIR) as a source to search for mutants and order seeds.
How feedback impacted our project
In response to Dr. Timm’s recommendations, we searched for starch-free mutants. Without the accumulation of starch, the statoliths will not sediment under the force of gravity, eliminating the role of statoliths in contributing to root gravitropism. Starch-free mutants should have a disordered root growth phenotype under natural gravity. We chose an ADG1 mutant (ref, ref) and a PGM mutant (ref, ref). ADG1 and PGM are key starch granule synthesis genes (ref). Without the accumulation of starch, the statoliths will not sediment under the force of gravity. In these mutants, the role of statolith in sensing gravity should be eliminated causing a disruption of the root gravitropism. We incorporated these mutants into the protocols of our natural gravity, microgravity, and gradient field experiments. (see Proposed Implementation)
Summary of Discussion about Reporter Proteins for Roots
Our experimental design involves importing ferritin into root statoliths and we would like to visualize it using a fluorescent protein. We were looking for advice on which fluorescent protein would not be at the same wavelength as autofluorescence as the roots. Particularly, we were concerned that GFP would not work because of high autofluorescence around GFP wavelength. Jeff Shipp informed us that “Autofluorescence that masks GFP is a concern where there are chloroplasts. GFP can show up very nicely in the roots.”
How feedback impacted our project
Following Shipp’s advice, we decided to use GFP as our reporter protein. (see Engineering Success)
Key Question 2: What are the current challenges in the field of space plant growth according to stakeholders, and how can we address these challenges?
Background
In order to better direct our project to solve current problems in space plant growth, we consulted with scientists who have worked with NASA’s VEGGIE II and Advanced Plant Habitat (APH) programs about what problems currently face plant growth systems.
Experts
Dr. Gioia Massa
Project Scientist NASA Kennedy Space Center Led the science team for the VEGGIE hardware validation on ISS, heads the study of fertilizer and light impacts on VEGGIE crops.
Dr. Simon Gilroy
Professor University of Wisconsin-Madison Co-chair of the NASA Plants Advanced Working Group (AWG), collaborates with NASA GeneLab on projects like the TOAST database, a transcriptome of Arabidopsis grown in space, and helps design NASA spaceflight plant experiments. Studies plant sensing and growth responses.
Dr. Richard Barker
Research Scientist University of Wisconsin-Madison Collaborates with NASA GeneLab through the Gilroy Lab, co-chair of the NASA Plants Advanced Working Group (AWG) and co-founder of The Collaborative Space Environment (CoSE).
Summary of Discussion
We learned from Dr. Gioia Massa that current work focuses on the growth of leafy greens, such as microgreens and lettuce, due to the lack of on-board cooking equipment. Fresh greens are mainly being grown to provide a psychological benefit to astronauts, both in their taste and their presence on the station.
We also learned that the medium that plants are grown in plays a crucial role in plant growth particularly in space. As such, NASA has a very strong research interest in researching mediums that efficiently provide nutrition to plants, but are also recyclable. While mediums such as agar and hydrogels make nutrients readily available to plants, they need to be reproduced once consumed. Given that a limited quantity of materials can easily be delivered to space, it is crucial that plant growth mediums are recyclable if they are to support long-term and large-scale space plant growth projects.
We learned from Dr. Massa that the revamped VEGGIE II project relies on a porous, kitty-litter-like, clay substrate called arselite (the same material that pitcher’s mounds are made of). The main goal for watering systems is to maintain an even mixture of air and water to prevent the plant from drying out or from drowning and rotting. Arselite helps provide this due to its porous nature, and it is reusable. Capillary action wicks water upward from a reservoir through fabric into this arselite with the plant. However, arselite is dense and expensive to ship into space, and it cannot be manufactured on-board. VEGGIE II also needs to be manually watered, which is labor-intensive and causes drastic cycles between high and low moisture. The APH is similar to VEGGIE II but uses an automated suction pump system to draw out excess water after waterings, which maintains the water-air balance. We also learned about a recent aeroponics experiment that relies on misting plant roots.
Drs. Simon Gilroy and Richard Barker informed us further about plant phenotypes in space and also advised some aspects of our proposed solution. They along with Dr. Massa mentioned that root growth in the VEGGIE II system is poorly understood due to the opaque arselite medium, and suggested that a transparent soil medium could aid in studying 3d root growth in a loose medium under microgravity. We learned that there is some unpublished x-ray data describing that the roots went around the rim of the pillow and were not as dense as ground controls.
They additionally advised us that simple solutions that do not require extra repairs are optimal - for example, the aeroponics project has been paused due to a broken pump, and the APH system suffers from a similar vulnerability. Additionally, they explained to us why centrifugation to simulate gravity would not be a good idea with one anecdote about the treadmills used by astronauts - at first, the treadmill was bolted to the station floor, but it was soon modified once the rotation started causing the solar panels to flex!
In regards to specifics about our project, they advised us to validate our TAIR mutants using skew assays and Lugol starch staining, corroborated with Dr. Timm and Jeffery Shipp’s recommendation to transform our starchless mutant, as it could help provide supporting evidence for the statolith model of gravitropism. They also mentioned that crops in the Brassicacae family, the one Arabidopsis belongs to, could be potential targets for our construct as they include leafy greens like turnip and cabbage that NASA also hopes to cultivate.
How feedback impacted our project
Our ultimate takeaways included designing a proposed plant pot design for use in space, a proposed plant substrate to substitute for arselite in current systems, incorporating skew assays and Lugol starch staining in our experiments, and deciding to transform starchless mutants with our ferritin construct. (see Proposed Implementation)
Based on this feedback about plant growth mediums, we decided to further analyze methods to optimize plant growth by researching the nutrient delivery abilities of several promising mediums in order to gain more insight into how we can compare promising recyclable mediums such as clay and pumice to agar (which we used in our wetlab experiments). Our dry lab team pursued this project by developing material diffusion models to study the transport of several crucial nutrients and molecules for plant growth through plant growth mediums. (see Modeling)
Key Question 3: How are perspectives on agricultural ethics and GMOs on Earth related to our project’s proposed implementation?
Background
Because our proposed implementation is to grow plants in space, which is an isolated environment, many safety and containment concerns often associated with synthetic biology implementations were eliminated. This is because our genetically modified arabidopsis would not be disturbing existing ecosystems or interact with other organisms. However, given that our project aims to make growing plants in space more viable, eventually leading to agricultural development in space that to an extent resembles agriculture on Earth, we were interested in investigating what policies and ethical principles are behind agricultural frameworks on Earth, and how our project could contribute to the establishment of ethical agricultural systems in space.
Expert
Dr. Paul B. Thompson is a Professor Emeritus of Philosophy and the W.K. Kellogg Chair in Agricultural Food and Community Ethics at Michigan State University. He engages with philosophical questions focused on agriculture and food, with particular emphasis of the role of technology and recombinant DNA on agricultural and food systems. Dr. Thompson is the author of Agricultural Ethics (1999), which explores the practical application of ethics to agriculture today. This text considers a philosophical understanding of modern challenges faced in agriculture, including the growing population and distribution, increasing yield, threats to the environment, and the role of research and technology.
Why we reached out
We reached out to discuss our perspectives on the ethics of the project and the broader scope of agricultural ethics with Dr. Thompson. Because of his familiarity with ethical, policy, and practical challenges farmers and other stakeholder in agriculture face through years of working with people in the agricultural industry, Dr. Thompson has a wealth of insight into the goals of agriculture and how policy, science, and industry structure ought to be shaped to best serve the ultimate goals of farming. We hoped to discuss Dr. Thompson’s field of study with emphasis on his insights into the ethical aspects of the safety, public opinion, and policy considerations that come with introducing genetically modified organisms in agriculture as well as how these technologies can support ethical and sustainable methods to develop and improve agricultural capabilities.
Discussion Summary
The bare minimum macronutrient composition recommended for astronauts is approximately 15% protein, 30% lipids, and 55% carbohydrates and many astronauts experience menu fatigue from the repeated food options. The main categories of food in space are: canned, dehydrated, medium moisture, natural, refrigerated, fresh, irradiated and functional food. The latter three (fresh, irradiated, and functional) are newer forms of food going through extensive testing and develop to meet commercial and recreational flights. The main limitations of space food is the presence of processed food, lack of quality advantage for refrigerated food, and limited transportation/storage space, food storage safety, menu fatigue, nutrient deficiencies, weight loss in space.
Astronauts face deficiencies in protein, vitamins (E,K, D), polyphenols, and polyunsaturated fatty acids, minerals (calcium, potassium), low elements (iron.) Iron deficiencies can lead to anemia because iron is needed to make hemoglobin. Iron is most concentrated in animal liver, seafood (clam, kelp, shrimp), egg yolk, beans, green vegetables, and fruits.
Considering all of this, it is important to have a consistent source of iron supplement for astronauts since carrying years worth of iron supplements is far more difficult than being able to grow plants in space.
On earth, arid areas and dense forests tend to have more alkaline soil, making it difficult for iron to properly be absorbed in regions. Across the earth, 1.6 billion people are anemic and several hundred million people have iron deficiency anemia. Almost two billion people are nutritionally iron deficient. Plants that are able to take up and tolerate more iron during growth may be able to help reduce the prevalence of this condition.
These studies influenced the development of our iron poisoning experiment as we wanted to see what conditions our plant would be able to tolerate as well as possibly figure out whether it did confer a significant difference in the plant’s development and growth after being added to the soil.
How feedback impacted our project
Our discussion with Dr. Thompson reaffirmed our previous belief that very few of the ethical concerns that are associated with introducing genetically engineered agricultural products on Earth would be extended to space. In particular, Dr. Thompson noted that astronauts who would consume the space food already place a great deal of faith in scientific advances and technologies that they rely on for spaceflight. As such, they have to sign disclosure forms and be educated on the technologies they will be reliant on. Plants with recombinant DNA would fall under this category, so the process to make the astronauts consuming these plants aware of what the plants are and any unique properties associated with them (such as high iron content) would be completed pre-launch.