EXPERIMENT RECORD N° 9797
MULTI-TROP - Multiple-Tropism: interaction of gravity, nutrient and water stimuli for root orientation in microgravity (ASI National Contribution)
  1. 2017 • ISS "Vita" - long-duration mission
  2. 2017 • ISS Increments 53-54
Life Sciences:
  • Biology
  • Biotechnology
G. Aronne (1), V. De Micco (1), S. De Pascale (1), P. Russo (2)
(1)  
University of Naples Federico II
Department of Agricultural Sciences
Portici (Naples)
ITALY
(2)  
Liceo Scientifico Statale Filippo Silvestri
Portici (Naples)
ITALY
BACKGROUND
In order to grow healthy and functioning plants in space, it is necessary that the three plant organs (root, stem and leaf) are morphologically well developed and physiologically interactive. On Earth, roots grow into soil to anchor the plant and absorb water and nutrients. Growth direction is determined by the interaction of three stimuli: gravity (gravitropism), water (hydrotropism), and nutrients (chemotropism). The first is generally considered stronger than the others in the process of shaping the root apparatus. In microgravity, it has been proven that root apexes grow randomly without a specific direction. This phenomenon makes the development of facilities for plant cultivation in microgravity challenging.

Multiple-Tropism: Gravity, Nutrient and Water Interaction of Stimuli for Root Orientation in Microgravity (MULTI-TROP) separately evaluates the role of three stimuli - gravity, water and nutrients - on plant growth. Tropism refers to an organism directional response to an external stimulus, such as plant roots growing downward into soil in response to gravity on Earth. Previous research shows that plant roots grow randomly without specific direction in microgravity, presenting a challenge when developing facilities to cultivate plants in space.
 
AIM
The aim of the MULTI-TROP investigation is to further investigate this critical issue to define if, and to which extent, hydrotropism and/or chemotropism can direct root growth in microgravity. 
MULTI-TROP has three main goals: 
a) education, to enhance secondary school pupils’ interest in space biology; 
b) scientific, to disentangle the role of gravity from the other stimuli for root growth; and, 
c) applied, to address technical issues in the design of growth chambers. 

Results are expected to give insights for basic scientific questions on root development processes and technical proposals to develop new hardware for plant cultivation in microgravity.
 
The spaceflight experiment was conducted on the ISS in a refurbished hardware: a BIOKON container equipped with two YING-B2 experimental units (EUs) previously flown for the YING experiment supported by the European Space Agency in 2009.
 
Activities were organised in three phases: 
  • pre-flight phase, 
  • in-flight phase and 
  • post-flight phase. 
The pre-flight phase included the seed selection, according to inflight environmental conditions, and set up of the experiment container. 
The in-flight phase consisted of the seed germination and sample storage in chemical fixative. 
The post-flight phase included a ground reference experiment, processing of plant samples, data analysis and modelling.

APPLICATION OF THIS RESEARCH - IN SPACE 
For future long-term missions, astronauts will need to grow healthy, functioning plants in space. MULTI-TROP clarifies the role of gravity on root development and addresses technical issues in the design of hardware for plant cultivation in microgravity.

APPLICATION OF THIS RESEARCH - ON EARTH
Insights into basic scientific questions about root development from the MULTI-TROP investigation have the potential to lead to improvements in plant cultivation on Earth.

content mainly from NASA Space Station Research Explorer on NASA.gov
https://www.nasa.gov/mission_pages/station/research/experiments/explorer/Investigation.html?#id=7473
At launch site, carrot seeds were placed between two disks of inert substrate (one imbibed with water and the other with a disodium phosphate solution) and integrated into a hardware developed, refurbished and flight-certificated by Kayser Italia. Post-flight, a Ground Reference Experiment was performed. Root development and orientation of seedlings grown in microgravity and at 1g condition were measured through 3D-image analysis procedures after imaging with X-ray microtomography. 

Space Experiment
The spaceflight experiment was conducted on the ISS in a refurbished hardware: a BIOKON container equipped with two YING-B2 experimental units (EUs) previously flown for the YING experiment supported by the European Space Agency in 2009. Both the BIOKON and the YING-B2 units have been designed, manufactured and certified for launch by Kayser Italia. Each YING-B2 consisted of four culture chambers (CCs) each of which equipped with a fixative reservoir (Figure 4A). RNALater (Sigma-Aldrich, St. Louis, Missouri, US) was used as chemical fixative. The CCs have been implemented with a 3D-printed holder to accommodate two substrate disks (Oasis® Growing Solutions, The Netherlands) imbibed with either pure water (W) or a nutrient solution (N) (Figures 4A, B). In each CC four seeds were placed in between the two substrate disks.

Ground Reference Experiment
After the accomplishment of the experiment on the ISS, a Ground Reference Experiment (GRE) on Earth was performed using the same hardware and the same batch of the seeds used to perform the experiment on the ISS. Experiment was setup as previously reported for spaceflight experiment, using a BIOKON container and two YING-B2 EUs. To compare spaceflight experiment and GRE, we performed the control test in a growth chamber simulating the temperature regime recorded during the flight experiment and the same experiment duration. Data recorded on the ISS during the experiment showed that temperature ranged between 22 and 26°C, and was in average 24°C. The GRE was performed with two different setups obtained according to the gravity vector orientation of the Oasis disks: A) N disk at the bottom and W at the top, B) W disk at the bottom and N at the top.

Seeds of Daucus carota cv Chantenay by Franchi Sementi were used for this study. Species selection was performed applying the method of subsequent excluding criteria to a set of 50 crop species (Aronne et al., 2018). Carrot seeds showed the best adaptability to the hardware characteristics and the experimental timeline scheduled for the spaceflight experiment. Germination percentage of the seed batch was tested in dark conditions at a temperature of 22°C sowing a total of 250 seeds distributed in five petri dishes lined with wet filter paper.

also compare Reference Doc no 2 for more details:
L.G. Izzo, L.E. Romano, S. De Pascale, G. Mele, L. Gargiulo, G. Aronne, (2019), "Chemotropic vs hydrotropic stimuli for root growth orientation in microgravity", Frontiers in Plant Science10, DOI: 10.3389/fpls.2019.01547, pp. 1547.
Radicle protruded preferentially from the ventral side of the seed due to the asymmetric position of the embryo. Such a phenomenon did not prevent the achievement of MULTITROP scientific goal but should be considered for further experiments on radicle growth orientation in microgravity. The experiment conducted in space verified that the primary root of carrot shows a positive chemotropism towards disodium phosphate solution in the absence of the gravity stimulus. On Earth, the positive chemotropism was masked by the dominant effect of gravity and roots developed downward regardless of the presence/absence of nutrients in the substrate. Taking advantage of altered gravity conditions and using other chemical compounds, further studies should be performed to deepen our understanding of root chemotropic response and its interaction with other tropisms.

The MULTITROP experiment was fully successful in reaching the goal of investigating the role of hydrotropism and chemotropism in root orientation in the absence of gravity stimulus. Results contribute to the international debate on the interaction between root tropisms.

Still, further studies should be designed to investigate root chemotropism and its interaction with other tropisms by taking advantage of altered gravity conditions and using other chemical compounds.

For full details on the results, please consult, Reference Document no 2:
Izzo, L.G., Romano, L.E., De Pascale, S., Mele, G., Gargiulo, L., Aronne, G., (2019), "Chemotropic vs hydrotropic stimuli for root growth orientation in microgravity", Frontiers in Plant Science10, DOI: 10.3389/fpls.2019.01547, pp. 1547.
[1]  
G. Aronne, L.G. Izzo, L.E. Romano, S. De Francesco, V. De Micco, S. De Pascale et al., (2018), "MULTITROP: the challenge of using refurbished hardware for an educational and scientific experiment on the ISS", IAC: Proceedings of the International Astronautical Congress 2018.
[2]  
L.G. Izzo, L.E. Romano, S. De Pascale, G. Mele, L. Gargiulo, G. Aronne, (2019), "Chemotropic vs hydrotropic stimuli for root growth orientation in microgravity", Frontiers in Plant Science, 10, DOI: 10.3389/fpls.2019.01547, pp. 1547.
click on items to display

Figure 1: YING B2 experimental unit showing that each growth chamber will be filled with two different substrates. Image courtesy of the Italian Space Agency (ASI).

Figure 2: Schematic representation of the seed position and the two types of media in the growth chamber. Image courtesy of the Italian Space Agency (ASI).

Figure 3: Schematic representation of three possible final scenarios. Hypothesis 1: If chemotropism prevails on hydrotropism, roots develop only in the substrate with nutrients. Hypothesis 2: If hydrotropism prevails on chemotropism, roots grow in both types of substrates. Hypothesis 3: if gravitropism exerts a stimulus much stronger than the other two, in microgravity roots grow in any direction. Image courtesy of the Italian Space Agency (ASI).

Figure 4: Experimental setup of the MULTITROP experiment within the hardware provided by Kayser Italia. Each culture chamber is equipped with a fixative reservoir and has been implemented with two substrate disks and a substrate holder (A). The seeds were placed between two substrate disks imbibed with either pure water or nutrient solution (B).

Figure 5: Logo of the MULTI-TROP project. Image courtesy of the Italian Space Agency (ASI).

Figure 6: ASI fact sheet of the experiment in Italian language. Credit: ASI
http://eea.spaceflight.es
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Reference Document no 2; Izzo, L.G., Romano, L.E., De Pascale, S., Mele, G., Gargiulo, L., Aronne, G., (2019), "Chemotropic vs hydrotropic stimuli for root growth orientation in microgravity", Frontiers in Plant Science, 10, DOI: 10.3389/fpls.2019.01547, pp. 1547.

Figure 7: Overview of the investigation in Italian language. Credit: ASI
 
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