54th Lunar and Planetary Science Conference 2023 (LPI Contrib. No. 2806)

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RICE CAN GROW AND SURVIVE IN THE MARTIAN REGOLITH WITH CHALLENGES THAT
COULD BE OVERCOMED THROUGH CONTROL OF STRESS-RELATED GENES. Peter James Icalia
Gann1,2, Abhilash Ramachandran3, Yheni Dwiningsih2, Dominic Dharwadker3, and Vibha Srivastava1,2,5 , 1Cell andMolecular Biology Program, 2Department of Crop, Soil and Environmental Sciences, University of Arkansas System
Division of Agriculture, Fayetteville, Arkansas 72701, 3Arkansas Center for Space and Planetary Sciences,
4Department of Horticulture, University of Arkansas System Division of Agriculture, Fayetteville, Arkansas 72701;
av034@uark.edu
Introduction: In-Situ Resource Utilization (ISRU)
will become increasingly important as human space
exploration progresses. Resupply missions can beexpensive, so we need practical and affordable ways
to use resources on other planetary bodies. On Mars,
there are resources such as water, regolith, light, CO2
which can be used to introduce new biomass for foodproduction [1,2]. There have been studies reported togrow Arabidopsis thaliana, Lactuca sativa, bean plants
in Martian regolith simulants such as JSC-1 andMMS-1 [1,3]. In this study we present results of rice
plants growth in MMS1.
Result and Discussion: Plants have been grown in
Martian regolith showing signs of stress response andfew underlying associated mechanisms have been
described, yet there were no efforts to regulate genes
associated to these stress responses to increasesurvivability and growth in this condition. OsSnRK1aand OsTOR encodes for signaling molecules that
regulate the reaction of rice plant to stress including
sugar starvation, salinity, drought and scarcity of soil
nutrients. Here, we demonstrate the performance ofrice in Martian regolith (MMS1), describe
differentially expressed stress-related genes and showthat perturbation of OsSnRK1a and OsTOR can
influence the ability of rice to grow from thechallenges in Martian regolith.
We initially challenged the germination and growth ofrice in MMS1, potting mix (PM) and combination ofboth. Plants grown in MMS1 have stunted growth,poor root morphology, and lower photosynthetic
activity (Figures 1a-g) which corroborates to the reportof [4] that MMS1 can be challenging for plant growth
due to poor nutrients, small amount of nitrogen andhigh pH. However, addition of any proportion of PMin MMS1 showed better growth and root
characteristics compared to 100% MMS1.
a)

b)

e)

c)

f)

d)

g)

Figure 1. Shoot and root characteristics of rice seedlings in
the different media 1 month after germination (MAG). (a)Photograph of seedlings grown in Potting Mix (PM) andMars Soil Simulant (MMS1) showing differences in shoots.
PM grown seedlings have a blue underline and orangeunderline for the seedlings from MMS1. A 12-foot ruler isprovided as reference. (b-d) Bar graphs showing the shootcharacteristics of the seedlings including shoot length, shootweight and chlorophyll content from the different growing
media used. (e-g) Bar graph showing the root characteristicsof the seedlings including root length, root number and root
weight from the different growing media used. Error bars
represent standard error of 3 biological replicates consistingof 3 seedlings each. Significance in Tukey’s multiple
comparison at  = 0.05 within a parameter is shown by small
letters.

Despite plants presenting signs of stress under MMS1,
the germination and growth observed suggests that thewater holding capacity, mineral compositions, soil pH,
particle size, other physical and chemical
characteristics of MMS1 can support the developmentof the rice plants. Moreover, shoot and root growth,and also chlorophyll content in MMS1 with PM (75%MMS1, 50% MMS1, and 25% MMS1) significantly
improved and even comparable to the control media(100% PM). Based on the development of the riceplants, all essential minerals, such as N, P, and K
content for the growth of the rice plants appear to bepresent in sufficient quantities in 100% MMS1 and
MMS1 with PM.

54th Lunar and Planetary Science Conference 2023 (LPI Contrib. No. 2806)

With the germination and growth observed in MMS1,
we incorporated magnesium chloride (Mg(ClO4)2) in
two different concentrations (0.1 and 0.3%) to closely
mimic to the actual Martian soil and challenged wild
type rice plant (WT) and two mutant lines (pNs73, andTOR) for germination and growth (Figure 2a-c).
a)b)c)

Figure 2. Shoot and root characteristics of rice seedlings in
the different media 2 weeks after germination (WAG). (a)Photograph of seedlings/seeds germinated/grown in (PM),(b) MMS1 + 0.1 % (Mg(ClO4)2) and (c) MMS1 + 0.3 %Mg(ClO4)2 . From left to right are WT, pNs73, and TOR.

All the lines germinated within two days and grow in
the PM where the WT and pNs73 showed similar
shoot and root characteristics while TOR was
significantly shorter (Figure 2a). Incorporation of
0.1% (Mg(ClO4)2) impacted germination and growth.
Germination took approximately five days.
Interestingly, pNs73 developed a shoot and root
structures in 0.1% (Mg(ClO4)2), a root was only
observed in the WT and no signs of any germination
or growth in the TOR. The ability of the pNs73 toadvance in germination and growth compared to theWT suggests that through editing the SnRK1a gene, acascade of stress-related mechanisms may havechanged and improved its response in the MMS1 soilwith (Mg(ClO4)2). However, all the three lines did not
germinate in 0.3% (Mg(ClO4)2) which indicates thathigher levels of (Mg(ClO4)2) are detrimental togermination in rice.
Our results show that in principle it is possible to growrice plants in MMS1 but levels of (Mg(ClO4)2) can be
critical to both germination and growth. Moreover, we
presented that editing SnRK1a could potentiallyprovide an approach to develop as table rice line thatcan germinate and grow in MMS1 with (Mg(ClO4)2).
Methods:
Regolith: Mojave Martian simulant (MMS1) wasused as Mars regolith simulant. We incorporated
magnesium chloride (Mg(ClO4)2) in two different
concentrations (0.1 and 0.3%) to closely mimic to the2990.pdf

actual Martian soil. As a control, we used potting mix.
We overmedicalized water to mimic water from Marsand to prevent contamination with nutrients.
Plants: The first experiment on testing germinationand growth characteristics in PM, MMS1 andcombinations of PM and MMS1 used ZHE 733. The
second setup which included testing of three different
lines on MMS1 with (Mg(ClO4)2) used Kitaake.
Experimental Designs and Observation: Small pots
were filled with 5g of soil media and demineralized
water was added to each pot. A filter was placed on thebottom of each pot to prevent soil from leaking. For
each soil type there were 3 replicate pots used. In eachpot, we positioned 3 seeds. The pots were placed in agrowth chamber in a completely randomized design.
The pots were placed on a large tray in the growthchamber.
During the experimental period, theaverage temperature was 33oC during daytime and 23o
C at night and air humidity was. Mean day time lastedfor 12 hours. The plants were watered withdemineralized water once or twice a day depending on
the evaporation rate.
Seeds were scored on germination rate and time forfirst leaf production. At the end of the experiment,
when the rice plants in control media were reaching
V3 developmental stage with three leaves; shootlength, shoot weight, chlorophyll content, rot length,
root number, and root weight were measured. And also
leaves sampling were performed for genotypiccharacteristics.
Statistical Analysis. Data were subjected to arcsine
transformation and one-way ANOVA. To determine
the significant differences, Tukey's multiplecomparison test was used. All statistical analyses wereperformed in SAS statistical software (version 9.4,SAS Institute Inc.).
Acknowledgments: We thank Jessica Kivett for thegrowth chamber support.
References:[1] Eichler, A. et al. (2021), Icarus, 354, p. 114022.
[2] R. Wheeler (2010), Gravitational and Space
Biology, Vol. 23, No. 2, pp. 25-35.
[3] Oze, C. et al. (2021), Soil Systems, 5(3), p. 37. [4]Wamelink,et al (2014). PLoS ONE 2014, 9(8),e103138.