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Review
Lunar Plant Biology—A Review of the Apollo Era
Robert J. Ferl and Anna-Lisa Paul
Abstract
Recent plans for human return to the Moon have significantly elevated scientific interest in the lunar environ-
ment with emphasis on the science to be done in preparation for the return and while on the lunar surface. Since
the return to the Moon is envisioned as a dedicated and potentially longer-term commitment to lunar explo-
ration, questions of the lunar environment and particularly its impact on biology and biological systems have
become a significant part of the lunar science discussion. Plants are integral to the discussion of biology on the
Moon. Plants are envisioned as important components of advanced habitats and fundamental components of
advanced life-support systems. Moreover, plants are sophisticated multicellular eukaryotic life-forms with
highly orchestrated developmental processes, well-characterized signal transduction pathways, and exceedingly
fine-tuned responses to their environments. Therefore, plants represent key test organisms for understanding the
biological impact of the lunar environment on terrestrial life-forms. Indeed, plants were among the initial and
primary organisms that were exposed to returned lunar regolith from the Apollo lunar missions. This review
discusses the original experiments involving plants in association with the Apollo samples, with the intent of
understanding those studies within the context of the first lunar exploration program and drawing from those
experiments the data to inform the studies critical within the next lunar exploration science agenda. Key Words:
Lunar Receiving Laboratory—Lunar regolith—Apollo—Plant biology. Astrobiology 10, 261–274.
Contents
1. Introduction 261
2. The Lunar Receiving Laboratory: Policy and Context 262
3. Apollo 11 and 12: The Initial Returns 265
4. Apollo 11–15: The Final Analyses of the Era 268
5. Summary of Apollo Era Data 270
6. Perspective on Future Lunar Plant Experiments 270
Acknowledgments 272
Abbreviations 272
References 272
1. Introduction
The return to the Moon offers a large number of op-
portunities for biological science as well as a number of
engineering driving forces relevant to biology and biological
systems. The opportunities for science largely address fun-
damental astrobiological issues:
What are the limits of terrestrial biology as it leaves Earth?
What are the biological impacts of living on a body
other than Earth?
What are the adaptive processes that occur when bi-
ology experiences conditions outside its evolutionary
history?
The engineering drivers are derived from those basic as-
trobiological issues and deal with the practical issues of
sustaining lunar explorers:
University of Florida, Interdisciplinary Center for Biotechnology Research and the Horticultural Sciences Department, Gainesville, Florida.
Online resources currently under development at http:==www.hos.ufl.edu=ferllab=Apollo%20Era%20Plant%20Biology=index.html.
ASTROBIOLOGY
Volume 10, Number 3, 2010
ªMary Ann Liebert, Inc.
DOI: 10.1089=ast.2009.0417
261
What protection must be employed where biology is
threatened beyond adaptation?
What structures or processes need to be in place to
mitigate negative biological effects of the lunar envi-
ronment?
What are the opportunities and limitations of life-support
systems that include non-human biological components?
Answers to these fundamental and application questions
are all experimentally addressable and are important to both
an informed exploration strategy and an exploration with
rich science and operations return.
As organisms for studies related to the lunar environment
and to supporting lunar exploration, plants offer unique and
valuable potential. This potential arises from the confluence
of the life-support and astrobiology agendas, and has kept
plant biology firmly within the spaceflight experiment
community. Numerous plant experiments have flown in the
Space Shuttle and International Space Station payload pro-
grams, and the following citations are only a sampling of this
research: Saunders (1968), Bucker (1974), Krikorian et al.
(1981, 1992), Kordyum et al. (1983), Guikema et al. (1994),
Kuang et al. (1996, 2000), Levine and Krikorian (1996), Brown
et al. (1997), Musgrave et al. (1997), Porterfield et al. (1997),
Adamchuk et al. (1999), Kiss and Edelmann (1999),
Nedukha et al. (1999), Sato et al. (1999), Gao et al. (2000),
Levinskikh et al. (2000), Takahashi (2000), Kern and Sack
(2001), Levine et al. (2001), Paul et al. (2001, 2005), Hoson et al.
(2003), Klymchuk et al. (2003), Clement and Slenzka (2006),
Stutte et al. (2006), Brinckmann (2007), Gilroy and Masson
(2007), Salmi and Roux (2008), Ou et al. (2009), Visscher et al.
(2009). Conclusions drawn from plant biology experiments
have highlighted biological responses to spaceflight envi-
ronments and have also illuminated engineering advance-
ments and operational advancements necessary for
conducting biological experiments in space (reviewed in
Halstead and Dutcher, 1987; Dutcher et al., 1994; Ferl et al.,
2002; Kiss et al., 2009). Moreover, plants are central parts of
bioregenerative life-support system concepts. Plants can
provide clean water through transpiration, oxygen through
photosynthesis, and food through fixation of carbon out of
the carbon dioxide expelled from human crews (reviewed in
Ferl et al., 2002). In longer-term missions, such as Mars ex-
cursions or extended lunar outposts, plant-based systems
could offer savings in the cost of transporting life-support
supplies as well as dietary variety reliability options (Drys-
dale, 2001; Drysdale et al., 2003; Czupalla et al., 2005;
Wheeler, 2009).
Plants also hold a uniquely powerful position in the his-
torical record of lunar biology experiments. Indeed, plants
were prominent test organisms during the Apollo era and
were used extensively during early evaluations of the bio-
logical impact of lunar samples. As discussed below, exper-
iments on plant interactions with lunar regolith were key to
those scientific conclusions which indicated that the returned
lunar samples were not overtly dangerous to the terrestrial
biosphere and that they did not contain toxic elements or
harmful alien life-form contaminants. Moreover, plant ex-
periments with lunar return samples showed that plants
could potentially derive nutrients from lunar regolith. But
while those Apollo-era experiments showed that terrestrial
life-forms could safely interact with the regoliths of the lunar
environment and that lunar samples were safe to bring back
to Earth, they did not answer all the necessary questions of
plant responses to growth in lunar soils.
The immediate goal of this review is to collate and contex-
tualize the experiments of the Apollo era that placed plants
directly in contact with lunar regolith. The extended goal of the
study is to illuminate what is actually known about the science
of plants grown in lunar soils and to expose what is yet un-
known. The result is hoped to be a knowledge base that would
allow the next era of lunar exploration to draw appropriately
from previous data while understanding the limitations of
those data in the design of future experiments. This review is
intended to be comprehensive in its coverage of those experi-
ments that involved interaction of Apollo lunar samples in
plant biology experiments. Occasional reference to non-plant
biology is presented for context and perspective, but treatment
of non-plant biology is not intended to be complete. While
technical reports and commentaries are used to support dis-
cussion of policy and procedure and to provide entry to liter-
ature sources (see Table 1), scientific discussion within this
paper is essentially limited to literature from peer-reviewed
sources. The presentation is largely chronological with regard
to publication dates of the studies and somewhat sequential
with regard to the Apollo missions and samples. It should be
noted, however, that some studies and many conclusions span
multiple missions and samples.
2. The Lunar Receiving Laboratory:
Policy and Context
The Lunar Receiving Laboratory (LRL) was the primary
focal point for all initial biological experiments with lunar
regolith and especially the returned samples from the early
Apollo missions. The LRL overrides most other potential foci
because it embodied the science policy, procedures, and at-
titudes that shaped all biological studies of the lunar samples
during the Apollo era (McLane et al., 1967; Kemmerer et al.,
1969; Mangus and Larsen, 2004).
The biology science policy surrounding the lunar missions
and the lunar samples was one of preventing what is com-
monly referred to as ‘‘back contamination’’—the idea that
samples returning from an extraterrestrial environment such
as the Moon might harbor alien organisms that are patho-
genic or otherwise harmful to terrestrial life-forms, or sub-
stances that could cause mutation or pathogenesis. Early in
the 1960s, the Space Science Board of the National Research
Council–National Academy of Sciences addressed the idea of
back contamination and recommended protection of terres-
trial life from the introduction of alien life-forms from the
Moon. The Interagency Committee on Back Contamination
(ICBC) was formed with representatives from the Depart-
ments of Agriculture, Health Education and Welfare, and the
Interior, as well as the National Academy of Sciences and
other agencies (Kemmerer et al., 1969; Compton, 1989; Man-
gus and Larsen, 2004). The ICBC policy formed the funda-
mental bases for processes and procedures implemented by
the scientists who handled and explored the lunar samples
from Apollo (Compton, 1989; Allton et al., 1998).
This policy is likely to have political and sociological
sources in addition to bases in sound astrobiological
concepts. Publication of the techno-novel The Andromeda
Strain in 1969 (Crighton, 1969) and the release of its movie
262 FERL AND PAUL
Table 1. NASA Technical Reports Pertaining to the Study of Plants in Lunar Regolith
Samples Returned in the Apollo Era
NASA Technical Reports Server Entry
Full abstracts can be obtained from the NASA Technical Reports Server and, in some instances, the reports themselves are available. To
access, see http:==naca.larc.nasa.gov=search.jsp then enter the Document ID.
Results of Apollo 11 and 12 quarantine studies on plants
Author(s): Horne, W.H.; Sweet, H.C.; Venketeswaran, S.; Walkinshaw, C.H.
Abstract: Botanical quarantine studies on Apollo 11 and 12 lunar soil samples effects on terrestrial plants, indicating absence
of disease generating agents
NASA Center: NASA (non Center Specific)
Publication Year: 1970
Added to NTRS: 2004-11-03
Accession Number: 71A15393; Document ID: 19710034696
Analysis of vegetable seedlings grown in contact with Apollo 14 lunar surface fines
Author(s): Walkinshaw, C.H.; Johnson, P.H.
Abstract: Study of plant seedlings treated with lunar material, grown for 14 to 21 days, and then subjected to chemical
analyses and other measurements. The purpose of the study was to determine whether plants growing in contact with…
NASA Center: Johnson Space Center
Publication Year: 1971
Added to NTRS: 2004-11-03
Accession Number: 72A35925; Document ID: 19720052259
Lunar horticulture
Author(s): Walkinshaw, C.H.
Abstract: Discussion of the role that lunar horticulture may fulfill in helping establish the life support system of an earth-
independent lunar colony. Such a system is expected to be a hybrid between systems which depend on lunar…
NASA Center: Johnson Space Center
Publication Year: 1971
Added to NTRS: 2004-11-03
Accession Number: 72A35938; Document ID: 19720052272
Histochemical and biochemical analyses of plant tissues Final report
Author(s): Halliwell, R.S.
Abstract: No abstract available
NASA Center: NASA (non Center Specific)
Publication Year: 1971
Added to NTRS: 2008-01-29
Accession Number: 71N76746; Document ID: 19710071431; Report Number: NASA-CR-115286 PDF
Technology advancements in the growth of germfree plants at the Manned Spacecraft Center
Author(s): Walkinshaw, C.H.; Wooley, B.C.; Bozarth, G.A.
Abstract: No abstract available
NASA Center: Johnson Space Center
Publication Year: 1972
Added to NTRS: 2004-11-03
Accession Number: 74N75851; Document ID: 19740077912; Report Number: MSC-06796, NASA-TM-X-70146
Apollo 12 lunar material—Effects on plant pigments
Author(s): Weete, J.D.; Walkinshaw, C.H.
Abstract: Tissue cultures of tobacco grown for 12 weeks in contact with lunar material returned by Apollo 12 contained 21 to
35 more total pigment than control tissues. This difference is due primarily to increased chlorophyll a…
NASA Center: NASA (non Center Specific)
Publication Year: 1972
Added to NTRS: 2004-11-03
Accession Number: 72A27626; Document ID: 19720043960
Apollo 12 lunar material—Effects on lipid levels of tobacco tissue cultures
Author(s): Weete, J.D.; Walkinshaw, C.H.; Laseter, J.L.
Abstract: Tobacco tissue cultures grown in contact with lunar material from Apollo 12, for a 12-week period, resulted in
fluctuations of both the relative and absolute concentrations of endogenous sterols and fatty acids. The…
NASA Center: Johnson Space Center
Publication Year: 1972
Added to NTRS: 2004-11-03
Accession Number: 72A19850; Document ID: 19720036184
Cytological studies of lunar treated tissue cultures
Author(s): Halliwell, R.S.
Abstract: An electron microscopic study was made of botanical materials, particularly pine tissues, treated with lunar
materials collected by Apollo 12 quarantine mission. Results show unusual structural changes within several …
NASA Center: NASA (non Center Specific)
Publication Year: 1972
Added to NTRS: 2004-11-03
Accession Number: 73N23054; Document ID: 19730014327; Report Number: NASA-CR-128914
(continued)
adaptation in 1971 essentially coincided with the Apollo lu-
nar sample returns. The story line deals specifically with the
return to Earth of an alien form that is devastating to life on
Earth. Certainly, lunar sample return policies from the ICBC
were in place before publication of The Andromeda Strain, but
the notion of dangerous life-forms gaining access to Earth was
prevalent and is still present in science fiction and popular
culture (Heinlein, 1951; Kneale and Cartier, 1953). Such
notions would have added pressure to the formal science
policies that were developed in the mid-1960s to define the
handling of the Apollo lunar samples and the experiments
designed to determine their impact on terrestrial biology.
Table 1. (Continued)
Lipid composition of slash pine tissue cultures grown with lunar and earth soils
Author(s): Laseter, J.L.; Weete, J.D.; Baur, P.S.; Walkinshaw, C.H.
Abstract: Lipid analyses were conducted on slash pine tissues grown in culture in the presence of lunar (Apollo 15) and earth
soils. Significant reductions in the total lipids, fatty acids, and sterol components were found in the…
NASA Center: Johnson Space Center
Publication Year: 1973
Added to NTRS: 2004-11-03
Accession Number: 74A17955; Document ID: 19740035205
Response of tobacco tissue cultures growing in contact with lunar fines
Author(s): Weete, J.D.; Walkinshaw, C.H.; Laseter, J.L.
Abstract: During the quarantine periods following each Apollo mission to the moon, various biological systems were placed
in the presence of lunar material to determine if pathogenic agents were present. Although no detrimental…
NASA Center: Johnson Space Center
Publication Year: 1973
Added to NTRS: 2004-11-03
Accession Number: 73A26483; Document ID: 19730041681
Effect of lunar materials on plant tissue culture
Author(s): Walkinshaw, C.H.; Venketeswaran, S.; Baur, P.S.; Croley, T.E.; Scholes, V.E.; Weete, J.D.; Halliwell, R.S.; Hall, R.H.
Abstract: Lunar material collected during the Apollo 11, 12, 14, and 15 missions has been used to treat 12 species of higher
plant tissue cultures. Biochemical and morphological studies have been conducted on several of …
NASA Center: Johnson Space Center
Publication Year: 1973
Added to NTRS: 2004-11-03
Accession Number: 73A26482; Document ID: 19730041680
Analytical and radio-histo-chemical experiments of plants and tissue culture cells treated with lunar and terrestrial
materials
Author(s): Halliwell, R.S.
Abstract: The nature and mechanisms of the apparent simulation of growth originally observed in plants growing in contact
with lunar soil during the Apollo project quarantine are examined. Preliminary experiments …
NASA Center: NASA (non Center Specific)
Publication Year: 1973
Added to NTRS: 2008-04-17
Accession Number: 73N30988; Document ID: 19730022256; Report Number: NASA-CR-134036
Development of germ-free plants and tissue culture
Author(s): Venketeswaran, S.
Abstract:…during this program are listed. The studies reported include: tissues cultured on various mediums, nutritional
studies, preparation of plant cultures for Apollo 15, and pine tissue cultures.
NASA Center: NASA (non Center Specific)
Publication Year: 1973
Added to NTRS: 2004-11-03
Accession Number: 73N24122; Document ID: 19730015395; Report Number: NASA-CR-128947
The lunar quarantine program
Author(s): Johnston, R.S.; Mason, J.A.; Wooley, B.C.; McCollum, G.W.; Mieszkuc, B.J.
Abstract:…of the three lunar quarantine missions, Apollo 11, 12, and 14, experienced no health problems as a result of their
exposure to lunar samples. Plants and animals also showed no adverse …
NASA Center: Johnson Space Center
Publication Year: 1974
Added to NTRS: 2004-11-03
Accession Number: 76N12688; Document ID: 19760005600
Quarantine testing and biocharacterization of lunar materials
Author(s): Taylor, G.R.; Mieszkuc, B.J.; Simmonds, R.C.; Walkinshaw, C.H.
Abstract: Quarantine testing was conducted to ensure the safety of all life on earth. The plants and animals which were
exposed to lunar material were carefully observed for prolonged periods to determine if any mutation or changes in…
NASA Center: Johnson Space Center
Publication Year: 1975
Added to NTRS: 2004-11-03
Accession Number: 76N12689; Document ID: 19760005601
264 FERL AND PAUL
It is important to note, however, that the ICBC back-
contamination policies were paralleled and in many in-
stances predated by policies and procedures for sealing and
storing lunar samples, which were driven by important and
essentially nonbiological concerns. All processes had to be
done under quarantine conditions. It was recognized that the
lunar samples themselves would be altered by exposure to
terrestrial conditions, so robust procedures were put in place
to preserve lunar samples for physical, chemical, and geo-
logical study. Nonetheless, it appears that back contamina-
tion became a dominant issue for sample return and ICBC
policies held serious sway in the development of sample-
handling procedures and in the design and funding of the
LRL, which acted as the repository for the lunar samples and
the laboratory for all initial studies with lunar regolith.
The LRL was constructed at Johnson Space Center (then
the Manned Spacecraft Center) after full-scale simulation of
procedures and was completed barely in time to accept the
Apollo 11 samples (Carter, 2001; Mangus and Larsen, 2004).
The facility was designed to house returning astronauts for
quarantine, archive and distribute lunar samples, and con-
duct initial science particularly to address back contamina-
tion before any samples were released from the facility.
Future sample handling, future lunar missions and lunar
exploration, and lunar science operations were all dependent
upon those initial studies. Given the weight of the back-
contamination issues, the LRL was designed as a complete
containment building modeled somewhat after the facility at
Fort Dietrich with a complex internal atmospheric pressure
control network and barrier system that provided two levels
of vacuum containment (Mangus and Larsen, 2004). The
LRL was also equipped with internal Class III containment
facilities as well as biological chambers as part of the build-
ing vacuum system, and experiment support equipment
specially designed to interface with the containment facilities
within the LRL (Kemmerer et al., 1969). Procedures within
the botanical laboratory of the LRL, which reflected the
containment activities of the LRL in general, were such that
personnel access was limited to a small, primary research
team. A backup research team was in place to assist with
preparation of media and plants, as well as other support,
and to ‘‘take over the laboratory if personnel were quaran-
tined due to a containment fault’’ (Walkinshaw et al., 1970;
see also Fig. 1).
From the ICBC policies and through the design and con-
struction of the LRL, plants were to be among the organisms
against which the lunar samples were to be tested (Kem-
merer et al., 1969). In part, this is likely due to the perceived
absolute need to protect the food supply and Earth’s bio-
sphere from unknown or alien life contamination. The De-
partment of Agriculture was represented on the ICBC
(Kemmerer et al., 1969; Mangus and Larsen, 2004). But it is
also likely due to the fact that plants are remarkably sensitive
to environmental stimuli, can draw elements as nutrients
from soil, and are subject to viral and bacterial pathogens
that overlap with, yet are widely different than, those of
animals or microbes.
3. Apollo 11 and 12: The Initial Returns
The successful return of Apollo 11 in July 1969 presented
the first lunar samples for analysis at the LRL. A total of 22
kilograms of lunar material was returned, including a 1 kg
contingency sample that was collected by Neil Armstrong
FIG. 1. Technician within the LRL processing germ-free plant materials within the containment cabinets. NTRS File Images,
Left: MSFC-6901319, Right: MSFC-6901321.
APOLLO ERA LUNAR PLANT BIOLOGY 265
and Buzz Aldrin very early in the lunar extravehicular ac-
tivity. Two sample return containers were sealed on the
Moon, returned to Earth in the Command and Service
Module, transferred to the mobile quarantine facility on the
carrier Hornet, and externally decontaminated for transfer to
the LRL. Within a short period of time, preliminary overall
results on sample analyses were published in Science (The
Lunar Sample Preliminary Examination Team, 1969). Pre-
liminary data indicated that microscopic examination found
no evidence of ‘‘any living, previously living, or fossil ma-
terial;’’ and an extensive biological protocol, in which a
‘‘considerable number’’ of plants were challenged with lunar
material, revealed no early ‘‘evidence of pathogenicity’’ as of
September 1969 (The Lunar Sample Preliminary Examination
Team, 1969).
The formal studies of Apollo 11 sample were published in
1970 and 1971, many appearing in the peer-reviewed litera-
ture after first being posted as NASA Technical Reports. An
issue of Science in 1970 collated many of those studies, in-
cluding several biology examinations. Chief among the early
biology studies were those that searched for evidence of
living organisms or fossils within the samples. The conclu-
sions were straightforward. Microscopic studies indicated
‘‘total absence of structure that can be interpreted as biological
in origin’’ (Barghoorn et al., 1970) and ‘‘no viable life present’’
(Oyama et al., 1970) in the Apollo 11 samples that were ex-
amined. These formal conclusions on the absence of life on
the Moon were significant and dramatic but did not elimi-
nate the idea that some thing(s) present in lunar samples
would be harmful to biology on Earth or that unrecognizable
alien, and harmful, life-forms might be present in the sam-
ples. The determination of the biological safety of the lunar
samples thus fell to slightly longer-term studies that looked
at terrestrial biology placed in contact with lunar regolith.
The initial reports on the biosafety of the lunar samples made
their way to the scientific literature in late 1970 and early
1971, again after being prepared as NASA Technical Reports
through 1970 and the latter half of 1969.
From the very first studies, it became clear that plants
were not endangered by contact with lunar samples (Walk-
inshaw et al., 1970). The methods of presenting the lunar
materials to the plants constitute a key point in this exami-
nation (Fig. 2). Lunar regolith, crushed fines and chips of
representative rocks, were presented to seeds, plants, and
tissue cultures, as well as to algal spores and cultures. In all
cases of presentation, recovery of the lunar material was a
primary goal that limited the amount of lunar material used.
For seeds and seedlings, the growth medium was a com-
mercial wood pulp product that allows for sterilization be-
forehand and efficient sample recovery after plant growth.
For plant tissue cultures and algal cultures, liquid or semi-
solid media were used. To challenge the plant materials, the
crushed lunar samples were ‘‘dusted’’ on the specimens at a
rate of 0.20–0.24 grams per individual. For seedlings with
leaves present, the lunar material was suspended in buffer
and first used to abrade the leaves before being presented to
the base of the seedling. (Note that leaf abrasion is a common
method for accelerating infections by plant pathogens.) A
FIG. 2. Dr. Charles Walkinshaw with germ-free plants grown within the LRL. Growth of gnotobiotic plants free of mi-
crobial association was a key element in preparing plants for interaction with lunar samples. NTRS File Image, S69-53894.
Color images available online at www.liebertonline.com=ast.
266 FERL AND PAUL
total of 35 species were challenged in these various tests, in
which lunar samples from both Apollo 11 and Apollo 12
were used. All the plants and cultures were germ free at the
start of the experiment to ensure no transfer of terrestrial
microbiota to the lunar materials (Walkinshaw et al., 1973b;
see also Fig. 2).
All data from this study strongly suggested that exposure
to lunar material is not harmful to plants. Seeds germinated
in the presence of lunar samples grew normally in appear-
ance, and plants exposed to, or abraded with, lunar samples
showed no evidence of ‘‘any agent capable of generating an
epiphytotic disease’’ (Walkinshaw et al., 1970) (see Fig. 3, for
example). The published data were based on visual and cy-
tological observations of the plants in the lunar tests, com-
pared to controls treated with heat-sterilized lunar material
and controls treated with heat-sterilized terrestrial materials.
Desert soils were used to treat plants as controls for Apollo
11 samples, and for the Apollo 12 samples the controls were
treated with an elemental simulant of the analyzed Apollo 11
samples. Plants not treated with any lunar simulant or ter-
restrial soils were also observed as controls.
Seed germination percentage in any of the eight species
tested was not affected by treatment samples from either
Apollo 11 or 12. In addition, for three of the four species of
seedlings germinated in the presence of the lunar samples and
grown for 15 days in the absence of added nutrient, the lunar
sample–treated seedlings attained slightly more growth than
the controls that were not treated with any minerals. Treated
cabbage seedlings clearly remained viable longer than un-
treated controls. The notion that lunar samples enhanced
growth was supported by observation of tissue cultures
treated with regolith. Lunar-treated photosynthetic cultures
of Nicotiana tobacum (tobacco), for example, were noted to stay
greener and live longer than untreated cultures. Treated algal
cultures, fern gametophytes, and bryophytes also grew better
than untreated controls. Within a single plate of geminating
fern spores, it was observed that those in contact with lunar
material were bigger and greener than those in areas of
the plate without lunar material (Walkinshaw et al., 1970). The
conclusion from these collected observations was that the
lunar samples not only had no negative effects on germination
and growth but also provided some nutritional supplement to
germinated seedlings and plant cultures (Walkinshaw et al.,
1970).
Larger plants, grown beyond the seedling stage with nu-
tritional supplement, with leaves that were abraded with the
lunar materials, were ‘‘not visibly different from unchal-
lenged controls’’ (Walkinshaw et al., 1970). Some of those
plants were observed for more than a year after treatment
without any symptoms developing. Thus, the most aggres-
sive attempts to transfer potential pathogenic material from
the lunar samples to the leaves of plants failed to reveal any
pathology (Walkinshaw et al., 1970; see also Fig. 3).
With the possibility of pathogenesis eliminated, discussion
turned to the observation of potential nutrients within the
lunar samples (Walkinshaw et al., 1970). Generally, while the
lunar materials in this study had many elements useful for
some aspects of plant nutrition, such as iron, magnesium, and
manganese, the major elements associated with plant growth
such as nitrogen, phosphorous, sulfur, and potassium were in
low amounts. And, in any case, none of these elements were in
especially water-soluble forms. The interesting and inevitable
conclusion was that the plants were conditioning the lunar
materials, releasing minerals that enhanced growth. The
seminal paper by Walkinshaw et al. both confirmed the safety
of the green terrestrial biosphere by disarming the specter of
back contamination and set the stage for the use of plants to
extract minerals and nutrients from the lunar surface (Walk-
inshaw et al., 1970), thereby opening the door for lunar agri-
culture to participate in advanced life-support concepts and
in situ utilization concepts for the Moon.
It should be noted that in none of these published exper-
iments were plants actually grown in lunar soils as the pri-
mary growth medium. The small amounts of lunar materials
available and the overt need to rule out pathogenicity meant
that the plants and cultures were grown in ‘‘contact with’’
lunar materials. All were ‘‘dusted’’ or ‘‘presented’’ with lunar
materials, while the major growth substrate was non-lunar
materials. Figure 4 shows a representative set of images
captured from archival movie films. The radish seeds can be
seen on top of the wood pulp medium. The lunar material
was sprinkled on top of the seeds and medium, then wetted
to allow the seedlings to sprout and begin development.
Animal incubations also showed no overt pathogenic ef-
fects of lunar treatments (The Lunar Sample Preliminary
Examination Team, 1969; Holland and Simmonds, 1973).
Nor did microbial cultures show inhibited growth in the
FIG. 3. A radish plant whose leaves were abraded with
lunar samples. This plant, like all others in the tests, showed
no pathogenic effects after being rubbed with lunar material.
NTRS File Image, S70-21481. Color images available online at
www.liebertonline.com=ast.
APOLLO ERA LUNAR PLANT BIOLOGY 267
presence of lunar samples from Apollo 11 (Silverman et al.,
1971). Indeed, cultures of the alga Chlamydomonas reinhardtii
(Chlamydomonas) were greener and denser when grown in
the presence of lunar samples, and pigment production
within two microorganism species was enhanced (Silverman
et al., 1971). Thus, the lack of pathology was confirmed in
every species tested; and, for at least some plants and mi-
crobes, it was likely that useful nutrients could be recovered
by biological processes acting upon lunar regolith. Moreover,
and especially given the limited contact between plants and
the small amounts of lunar materials used in the experi-
ments, plants were proven to be sensitive indicators of bio-
logical responses to lunar regolith.
Thus, by late 1970 and early 1971 it was clear that there
were no legitimate worries about back contamination from the
Moon. The implications were enormous. Strict quarantine of
the Apollo astronauts was relaxed after Apollo 14. Public
worry about alien life from the Moon disappeared in step with
reduced public interest in the Apollo program. In the wake of
these conclusions, plant biological sciences began to focus on
defining the effects, especially the potentially beneficial effects,
of growth in lunar regolith and on forwarding the concepts of
lunar agriculture (Walkinshaw, 1971).
4. Apollo 11–15: The Final Analyses of the Era
Continued analyses, published in a small series of papers
from 1971–1974, followed up on the primary conclusions of
the initial study of plants in contact with lunar samples.
These studies expanded the ideas that lunar soils could
provide beneficial nutrients to plants and that, in the process,
lunar regolith affected plant composition and pigment for-
mation without negatively impacting growth.
Samples of lunar surface fines from Apollo 14 were used
to treat a series of vegetable seeds and seedlings to test
germination, growth, and element uptake from the lunar
samples (Walkinshaw and Johnson, 1971). Seeds of six dif-
ferent vegetable species were surface sterilized and germi-
nated on sterile wood pulp medium, in the presence of small
amounts (0.22 g per 10 seedlings) of two types of Apollo 14
lunar materials, a terrestrial control, or no treatment at all.
The two lunar samples were both defined as lunar fines but
differed slightly in grain size and glass content. The terres-
trial control sample was sterilized basalt-ilmenite mixed and
ground to simulate Apollo 11 fine materials (Goldich, 1971).
Untreated controls were not supplemented with any mineral
treatments. Elemental analyses of the lunar and terrestrial
control samples showed a high correlation of the element
profiles for the added minerals.
Germinating seedlings in contact with lunar samples again
showed good germination. Fresh weight and plant height
data, after growth for a short time, indicated that for each
species one or the other lunar sample showed increased
growth compared to untreated controls, similar to the initial
studies with the Apollo 11 and 12 samples. The aerial por-
tions of the seedlings were recovered, dried, and ashed for
FIG. 4. Radish seedling challenge experiment. These images were captured from JSC archival footage from a 1971 LRL
experiment placing radish seeds in contact with lunar material and control materials. In (Aand B) the radish seeds can be
seen on top of the wood pulp medium, and the lunar material or other experimental material was sprinkled on top, then
watered (C), and set to grow (D). NASA JSC film archive, see also, for example, Walkinshaw et al. (1970). Color images
available online at www.liebertonline.com=ast.
268 FERL AND PAUL
element analyses. Most elements did not vary greatly among
the treatments; however, differences were observed in the
uptake of Al, Fe, and Ti. Uptake of Al, Fe, and Ti from the
lunar samples was similar to uptake from the terrestrial ba-
salt control. These differences in uptake from lunar or ter-
restrial control treatments were not consistent among the
lunar samples or across the species of plants tested but
clearly indicated that different plants were pulling different
elements from the treatments relative to the untreated con-
trols. The authors noted that the studies were unfortunately
limited by the amount of lunar materials available for the
experiments (Walkinshaw and Johnson, 1971). So while the
full impact of actual growth of plants in lunar soils was not
yet addressed, the growth of plants in contact with lunar
materials was confirmed, and the uptake of lunar elements
by plants was initially assessed for Apollo 14 samples.
Confirmation of plant uptake of lunar elements was ob-
tained through the use of irradiated lunar samples (Baur et al.,
1974). A sample of Apollo 11 lunar material was neutron
activated in a nuclear reactor before being added to semi-
solid agar-based Knopp’s basal medium for plant growth.
The activated lunar sample was added at a rate of 1 g per
20 ml of growth medium per jar, and each jar was used for
growth of 15 lettuce seedlings. Autoradiography of the
plants confirmed that elements from the lunar samples were
taken up by the plants. Isotopes of Mn, Co, and Sc were
clearly present in the aerial parts of the plants recovered
from the growth jars. The specific implications for those
particular elements are not entirely clear, but the funda-
mental notion that plants can absorb elements from lunar
material was solidly confirmed. In addition, elements from
the lunar samples were released into the agar medium,
which confirmed nonbiological dissolution of lunar elements
and left open several issues of the biological and nonbio-
logical forces involved in the breakdown of lunar soils (Baur
et al., 1974).
Perhaps due to the small amounts of lunar material made
available to biological science activities, all remaining pub-
lished studies of plant interactions with lunar regolith were
limited to tissue and cell culture studies. These studies were
largely extended experiments and analyses of the effects of
lunar materials on pigmentation and lipid accumulations of
tissue cultures.
Increased greening of tobacco tissue cultures was noted
in the initial studies of the Apollo 11 and 12 quarantine
samples (Walkinshaw et al., 1970). That greening phenom-
enon was repeated and quantified with additional tobacco
tissue cultures (Weete and Walkinshaw, 1972). The Apollo
12 lunar materials used to treat algal cultures (Walkinshaw
et al., 1970) were recovered, washed, and mixed with
White’s medium, and multiple cultures were started on the
media with lunar material and compared to identical cul-
tures started on medium without lunar inputs. At 3-week
intervals over a 12-week growth period, tissue samples
were recovered from the cultures and analyzed for chloro-
phyll and carotenoids. The cultures with added lunar ma-
terials contained more chlorophyll and total pigment at
each time point of the study, in keeping with the visual
determination in this and previous studies that lunar-
treated cultures were greener and stayed green longer than
cultures on medium alone. Lipid analysis of such tobacco
tissue cultures revealed that lunar-treated cultures were
higher in total sterols, including phytosterols, than were the
cultures without additions (Weete et al., 1972). Electron
microscopy analysis of lunar-treated tobacco cultures re-
vealed few differences from control cultures except an in-
creased chloroplast development that parallels the
advanced greening (Baur et al., 1973). These observations
were very consistent with previous observations of in-
creased pigmentation in some species and the notion that
elements from the lunar materials can have effects on the
metabolism of plants. Indeed, the authors noted that the
data ‘‘reflect the metabolic response by a sensitive plant
system whose environment has been altered by available
nutrients provided by lunar material’’ (Weete et al., 1972).
Data on tobacco tissue culture growth were reviewed and
summarized as indicating that treatment with lunar sam-
ples did not negatively affect culture growth but did
influence metabolic responses, such as chlorophyll pig-
mentation and sterol concentrations (Weete et al., 1973).
Tobacco cell suspension cultures showed evidence of di-
rect interaction with lunar fines when the lunar material was
added to the liquid suspension medium (Baur et al., 1973).
The suspension cells showed morphological evidence of
physiological interaction with the lunar materials in that they
presented structures consistent with high vesicular activity.
These observations were consistent with the tobacco tissue
culture experiments that suggested plant cells could take up
mineral nutrients from the lunar materials (Baur et al., 1973).
No strong differences in cell morphology were seen with
liquid suspension cultures or with the semisolid tissue cul-
tures of tobacco.
Given the lack of any evidence for back contamination,
quarantine procedures were halted after the Apollo 14 mis-
sion. The samples returned from the Apollo 15 mission in
1971 marked the apparent end of lunar samples that were
used to challenge plant growth. Even for the Apollo 15
samples, biological testing was dramatically descoped, and
no plant biological studies were published with samples
from Apollo 16 or Apollo 17.
Elemental analyses of some of the Apollo 15 samples in
comparison to data from Apollo 14 and Apollo 12 allowed
an appreciation for the range of element concentrations in the
various samples ( Johnson et al., 1972). Tissue cultures from
twelve species of plants were presented with lunar materials
in experiments similar to those previously described, with
0.2 g of material being dusted on the surface of actively
growing cultures (Walkinshaw et al., 1973a). Generally,
addition of minerals (either terrestrial control minerals or
lunar samples) enhanced culture growth compared to non-
supplemented controls. Though analytical emphases within
the study were on cytological observations, preliminary
biochemical evaluations suggested that both lunar and ter-
restrial materials had variable effects on respiration of the
cultures. For example, oxygen consumption was reduced in
Pinus elliottii (slash pine) cultures treated with lunar mate-
rials, while such reductions were not observed for other
species. Cytological observations suggested that cultures in
contact with lunar materials had increased cytoplasmic
density and showed other structural signs of increased
cellular activities, compared to cultures that received no
mineral treatments (Walkinshaw et al., 1973a). However,
slash pine cultures showed a reduction in lipids, fatty acids,
and sterols (Laseter et al., 1973). Nonetheless, the conclusions
APOLLO ERA LUNAR PLANT BIOLOGY 269
were consistent with earlier tissue culture studies: that plant
cells are not adversely affected by contact with lunar mate-
rials and that lunar regolith can supply useful nutrients
(Walkinshaw et al., 1973a).
5. Summary of Apollo Era Data
Clearly, the primary conclusion from these plant studies
with lunar samples was that back contamination was not a
problem—at no point in any of the plant studies was there
any indication that harmful alien organisms or overtly toxic
compounds were present in the returned lunar samples. This
conclusion is quite strong and comes from the observations
of many seedlings, plants, and tissue cultures in contact with
lunar materials. Any reconsideration of the experiments as-
sociated with the lunar sample returns has to view this
strong conclusion as a major scientific contribution of the era
and properly appreciate the experiments in that context.
These experiments were seminal studies conducted under
exacting conditions in extraordinary, unique, and exciting
times.
While the absence of dramatic negative impacts of lunar
contact is fundamental and profound in many senses, the
secondary conclusions from the studies reach further and
have strong implications for future lunar exploration pro-
grams. Plants were clearly sensitive to treatment with lunar
materials and demonstrated metabolic and structural re-
sponses to lunar regolith. The general findings that lunar
soils could provide nutrients that positively impact plant
growth also opened the realm of lunar agriculture and the
idea that plants can be grown in lunar soils as part of in situ
resource utilization and biological life-support systems.
However, a great deal of work remains to be done to un-
derstand the impacts lunar soils might have on plant growth.
As compelling as these results are, especially in the
context of the critical back contamination issues of the lunar
program, there remains a deep sense of science yet to be
done. In the end, and as recorded in the peer-reviewed
scientific literature, there were only three published pri-
mary studies of seeds, seedlings, and plants grown in
contact with lunar materials. In those three cases, small
amounts of lunar material were used, and the plants were
relatively large. In general, the dusting of plants or the
mixing of lunar fines with other support media makes plant
interaction with the lunar material a small part of the plant
experience. At no point were plants actually grown in lunar
samples in the way that one might imagine, with the entire
root structure growing through and in constant association
with a lunar soil. It is no accident that the wording of most
of the titles of the studies, as well as the careful discussion
within the papers, refers to growth ‘‘in contact with’’ lunar
samples—not ‘‘in’’ lunar samples. With only a small portion
of the roots, for example, interacting with the lunar mate-
rials, it is likely that plant responses to the lunar materials
were, therefore, quite attenuated due to the lack of an ex-
tensive plant=lunar soil interface. Biophysical issues, such
as root penetration of dry and variously hydrated lunar
sample types, were completely unaddressed. Thus, the
effects of actual growth within lunar soils were simply not a
part of the plant studies of the Apollo era.
So too, all experiments were conducted in the absence
of any plant-associated microbiology within the root zone.
This was done in large part to ensure that the plants did not
introduce any confounding microbial variables into the
fundamental assay for life within the returned lunar sam-
ples. But it leaves the entire area of rhizosphere lunar
biologycompletelyunexamined.Yetitwasclearthatboth
plants and microbes were capable of breaking down the
lunar regolith to minerals potentially useful for growth.
It was also clear that aqueous solutions commonly used
to support plant growth solubilized lunar samples to
some extent.
There are two relevant, yet largely unexplored, conse-
quences from the inevitable oxidation and hydration of
regolith during its initial exposure to water and oxygen
atmospheres associated with plant growth, the release of
potentially phytotoxic components, and changes in the
complement of mineral nutrients that become biologically
available under these conditions. While it has been stated
that lunar soil is stable in Earth’s atmosphere (Dabrowski
et al., 2008), it is most likely that lunar regolith is initially
reactive (Grossman et al., 1972), and the lunar glasses, with
their fine grain size and high solubility, are likely to be es-
pecially so (Whitney, 1989 and references therein). These
abundant glasses are also high in nanophase iron (Keller and
McKay, 1993), which, when exposed to oxygen, can produce
a Fe-oxide layer that may retard dissolution of Fe-bearing
minerals (Schott and Berner, 1983; Whitney, 1989). However,
it has also been suggested that exposure to water can create
micropores that significantly increase the surface area, which
in turn creates an elevated release of ions into solution
(Holmes et al., 1973). So while a soil composed of ‘‘lunar
dust’’ may yield many inorganic plant nutrients (Keller
and Huang, 1971), the complex associations among lunar
regolith geology, mineralogy, plants, microbes, and their
atmosphere were not addressed during, or subsequent to,
the Apollo era.
6. Perspective on Future Lunar Plant Experiments
A summary review in the mid-1970s commented directly
on the serious limitation of lunar samples available to bio-
logical studies and the limited experimentation that was
actually performed in the biocharacterization of lunar sam-
ples (Taylor et al., 1975). There is clear historical evidence that
the biological community in general, and plant biologists in
particular, were interested in further characterization of
plant responses to lunar regolith. However, as the Apollo
program itself was winding down, access to lunar samples
for biological studies was apparently severely reduced and
essentially curtailed. There are obscure references to some
studies after the Apollo era, in the form of the occasional
abstract or conference presentation, but no further studies
entered into the peer-reviewed literature. As the Skylab and
Space Shuttle programs activated, plant sciences turned to
space biology rather than lunar biology (Halstead and
Dutcher, 1987), while the LRL gradually converted to be-
come the center of space life sciences research at Johnson
Space Center.
Nonetheless, the Apollo era plant experiments with lunar
samples have left distinct opportunities for future research
during, and in support of, lunar exploration. From the per-
spective of advanced life-support concepts, plants can not
only survive exposure to lunar surface materials, it is likely
270 FERL AND PAUL
that plants can derive resources from lunar soils, thereby
reducing requirements for launched materials from Earth.
The full extent of plant utilization of lunar soils is unknown
and should be fully examined to determine not only how
well plants can contribute to life support on lunar bases but
also the degree to which plants can contribute to resource
recovery from regolith—all contributing to a fuller under-
standing of the interactions between lunar regolith and bi-
ology and an understanding of how each is changed by those
interactions.
Plants are exquisitely attuned to their environment and
deploy remarkable ranges of responses to changes in envi-
ronmental conditions. Hints of some of these kinds of
adaptive responses are clearly present in the plant experi-
ments with Apollo lunar samples. The sophisticated geno-
mics, proteomics, and metabolomics tools of the modern
molecular era were not available during those initial bio-
logical experiments of the Apollo era. Given the modern
approaches, and access to Apollo lunar samples, it is very
likely that a robust characterization of plant responses to
lunar regolith could be developed well in advance of lunar
sortie and outpost missions. Arabidopsis thaliana, aleading
model plant species, has an extremely well-characterized
genome, and that has led to robust molecular understand-
ing of plant metabolic responses to a tremendous range of
terrestrial environments, the kinds of baseline data that
simply did not exist in the Apollo era. Moreover, the di-
minutive size of Arabidopsis would allow full life-cycle
growth that uses less than 1 gram of lunar material per
plant as the sole growth matrix (rather than dusting or
abrading), while providing sufficient material for detailed
molecular studies. Extant lunar samples could be compared
to terrestrial breccias and modern lunar simulants to char-
acterize the expected plant responses to lunar geology (see
Fig. 5). Plants that are well characterized by forerunner
studies in Apollo samples and lunar simulants could serve
as key pioneer species in early biological studies carried out
on the surface of the Moon as part of crewed exploration or
within precursor lander missions (Gronstal et al., 2007) and
thus facilitate continuation of the tradition of using plants
within the discovery and ongoing phases of extraterrestrial
exploration.
FIG. 5. Lunar analogues. The Haughton impact crater on Devon Island in the Canadian Arctic has been used as a planetary
analogue for both Moon- and Mars-related research. The breccia hills of the Bruno Escarpment (A) are of interest as a site
analogue because of their expansive gray breccia deposits. The Arabidopsis plants shown in the bottom panels were grown in
lunar regolith simulant JSC1a (B), breccias collected from Haughton Crater (C), and are compared to growth in regular
terrestrial soil (D). All plants are the same age and received the same treatments, illustrating that even with nutrient
augmentation, a sole substrate of pure breccia fines or current lunar simulants does present various challenges to plant
growth. Photos by the authors. Color images available online at www.liebertonline.com=ast.
APOLLO ERA LUNAR PLANT BIOLOGY 271
Acknowledgments
This paper is dedicated to Charles Walkinshaw and his
colleagues of the Apollo era, whose work on plants within
the LRL and in contact with the Apollo lunar samples forms
the foundation of our current knowledge regarding lunar
plant biology. Through appreciation and recognition of that
body of science, the current exploration community will be
prepared for the next exploration encounter with the Moon.
The authors thank Dr. Walkinshaw for sharing his notes,
images, reprints, and thoughts.
The authors wish to thank the many people who have
participated in discussions of lunar plant biology or have
commented on drafts of this document, especially Andrew
Schuerger (University of Florida and KSC), Howard Levine
(NASA-KSC), Ray Wheeler (NASA-KSC), Clive Neal (Notre
Dame), Pascal Lee (Mars Institute and SETI), Gordon
Osinski (University of Western Ontario), Alain Berinstain
(Canadian Space Agency), Jen Heldman (NASA HQ and
ARC), Chris McKay (NASA-ARC) Mike Dixon (University
of Guelph), Tom Graham (University of Guelph), Matt
Bamsey (University of Guelph and CSA), and, more
broadly, the LEAG and space biology communities. Alain
Berinstain, Pascal Lee, Matt Bamsey, Tom Graham, and
Gordon Osinki were responsible for introducing the authors
to the Haughton Crater on Devon Island as a lunar analog
site and for guiding the authors through that remarkable
terrain and operations environment as participants in ac-
tivities at the Haughton Mars Project. The authors also wish
to recognize and thank the extraordinary resources of the
Lunar and Planetary Institute, the JSC Digital Image Col-
lection, the JSC History Collection at the University of
Houston–Clear Lake and the JSC History Portal. Jennifer
Ross-Nazzal, historian at JSC, was enormously helpful navi-
gating the various resources and providing connections to
dispersed archives and experts. Timothy Hinson expertly
conducted the digitization of archived footage covering
Apollo era plant activities within the LRL, including the
footage that gave rise to the images in Fig. 4.
The authors have been supported by grants NNX09
AO78G, NNX09AL96G, and NNX07AH270 from NASA to
R.J.F. and A.L.P.
Author Disclosure Statement
No competing financial interests exist.
Abbreviations
ICBC, Interagency Committee on Back Contamination;
LRL, Lunar Receiving Laboratory.
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Address correspondence to:
Robert J. Ferl
University of Florida
Interdisciplinary Center for Biotechnology Research
and the Horticultural Sciences Department
PO Box 110690
Gainesville, FL 3201-0690
E-mail: robferl@ufl.edu
Submitted 5 August 2009
Accepted 6 February 2010
274 FERL AND PAUL