Adv. Space Res. Vol. 28, No. 4, pp.

601-606, 2001
Pergamon (C 2001 COSPAR. Published by Elsevier Science Ltd. All rights reserved
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PII: S0273-1177(01)00390-8

ONTOGENY OF PLANTS UNDER VARIOUS GRAVITY CONDITION

R. Laurinavi~ius, D. Sveg2diene, D. Raklevi~iene and P. Kenstaviriene

Institute of Botany, ZaIiuj~ e~ert{ 49, L T - 2021 Vilnius, Lithuania

ABSTRACT

The results of experiments performed under conditions of microgravity (MG) or under its simulation
on the horizontal clinostat (HC) with the callus, seedlings of various species and embryogenic structures
have revealed a definite role of gravity as an ecological factor in the processes of cytomorphogenesis,
growth, and development. The transformation of differentiated somatic cells of Arabidopsis seed into
undifferentiated callus was not inhibited under MG, though modifications of the whole callus morphology
and of mean cell and nucleus size were observed. The morphogenesis of polar structures such as root-hair
bearing cells of Lactuca primary root has been shown to be modified in the course of differentiation under
mass acceleration diminished below 0.1 g. Seed germination and seedling morphogenesis under MG
follow their normal course, but a significant stimulation of shoot growth with no effect on primary root
growth has been determined. A successful in vitro regeneration of Nicotiana tabacum plantlets from leaf
cells and subsequent formation of shoots and roots on a continuously rotating HC as well as the formation
of viable seeds during seed-to-seed growth of Arabidopsis plants under MG have indicated that gravity
plays but a limited role in the processes of embryogenesis and organogenesis.
© 2001 COSPAR. Published by Elsevier Science Ltd. All rights reserved.

INTRODUCTION
Gravity is one of the most conservative ecological factors. Its biological effects are expected to be
based on the interaction of two masses - those of the Earth and of the whole organism as well as of the
Earth and of separate parts of the organism. Examples of such interaction could be found both in most
primitive ancestors and in contemporary organisms (Bean, 1984; Merkys et al., 1989; Barlow, 1995). The
gravitropic system of spatial orientation in higher plants is the most vivid example of the employment of
this physical signal for initiation of a definite physiological reaction. The polyfunctional role of gravity in
regulating plant growth and development as well as the processes of morphogenesis and structural
polarization are often emphasized (Sinnott, 1960; Nick and Furuya, 1992; Claasen and Spooner, 1994;
Kropf, 1997; Krikorian, 1997; Hoson et al., 1997). However, it still has to be proved whether
unconditionally correct is the supposition that the structure and functions of Earth organisms, including
plants, are optimised at 9.81 m.s -2 mass acceleration, because throughout the whole course of evolution
the signal of exactly this amplitude has been the factor of selection. Results of the original experiments
performed under MG and HC conditions with callus cells, seedlings and embryogenic structures,
including both somatic and zygotic, at least partly answer the question concerning the role of gravity in
the ontogenesis of higher plants.

601
602 R. Laurinavi~ius et al.

FORMATION AND GROWTH OF CALLUS UNDER MICROGRAVITY

Gravity is a body force, therefore its effect can be expected at all levels of biological organization,
including cellular (Todd, 1989; Laurinavi6ius, 1991). There are some indications that the processes
responsible for the morphogenesis of plant organs are highly insensitive to changes in the cells, and they
are in support of a higher order of regulation rather than control at the cellular level (Schiefelbein et al.,
1997). Cell expansion is also coordinately regulated at the whole organ level by external variables such as
light, temperature and other environmental gradients as well as by internal factors, first of all hormones
(Cosgrove, 1997). Therefore it is problematic to study the impact of gravity on the cell using a whole
plant or its separate organs as experimental models.A more suitable model system is a tissue or cell
cultures in vitro.
A seed-callus experimental model of Arabidopsis has been used to study the role of gravity in the
processes of callus cell formation and growth. Callus was formed from sterilized dry seeds sown during
spaceflight on a modified solid nutrient medium of Murashige-Scoog (Negrutiu and Jacobs, 1977). The
cell and nucleus size of callus tissue was determined in histological cross-sections by microscope and
image analysis system.

A B

fresh biomass 92.4 + 30.5 mg (n = 6) fresh biomass 89.7 :k 28.0 mg (n = 6)

cell area 768.6 + 16.9 gmZ(n = 1625) cell area 644.4 + 12.6 g m 2. (n = 1683)
nucleus area 91.1 + 2.1 B m 2 (n = 1625) nucleus area 80.3 + 1 . 4 / a m 2. n = 1683)

Fig. 1. Callus ofArabidopsis thaliana formed on the ground (A) and on the Salyut 7 orbital station (B).
*The difference is significant at p < 0.05.

Microgravity has been found in principle to inhibit neither the process of transformation of
differentiated seed germ cells to undifferentiated callus cells nor the growth of callus biomass (Figure 1).
However, the consistence of callus was different: in control it was friable and soft, with larger cells and
nuclei, against denser and more solid, with smaller cells and nuclei in MG. Elimination of gravity acted as
a factor inducing alterations in the morphology of the whole callus and of single cells as well.
 Ontogeny of Plants 603

MORPHOLOGY OF TRICHOBLAST UNDER ALTERED GRAVITY

A convenient model to study the impact of gravity on the processes of morphogenesis and formation of
the polar properties at a cellular level is the trichoblasts, i.e., epidermal root-hair cells. These cells are
morphologically polarised, because a root-hair emerges at an appropriate site of the lateral wall during the
early phases of root development and epidermal cell differentiation. The shift of the root-hair initiation
site under altered mass acceleration can be considered as a proof of a possible morphogenetic role of
gravity in the subtle processes of cell polarity establishment. In the present study, the site of root-hair
insertion in the lettuce (Lactuca sativa L.) primary root-hair bearing cells dependening on the amplitude
of this force has been determined. Dry seeds located on the rotor of a speaceflight centrifuge generating
0.01, 0.1 and 1 g mass acceleration as well as on a stationary platform (microgravity conditions) were
moistened and fixed chemically after germination during 120 h on board the Salyut 7 orbital station. The
trichoblast cells located at the most apical part of roots were analysed morphometrically on an optical
microscope. The trichoblast polarity was found to change when the apically-directed centrifugal force was
diminished below 0.1 g (Table 1). At 0.01 g, particularly under MG conditions, a great part of the root
epidermal cell population possessed a root-hair located near the center of the trichoblast, while at 0.1 and
1 g it appeared to be located near the apical end of the cell. Hence, gravity really interacts with the
complex overall processes of root-hair initiation, which is supposed to be dependent on changes in plasma
membrane structure and ionic gradients, the direction of secretory vesicle transport and cell wall growth
(Miller et al., 1997). Root-hair formation modification can be induced by both the microgravity conditions
(Merkys et al., 19831) and by hormone treatments, possibly through the auxin- and ethylene-regulated
pathways (Schiefelbein et al., 1997).

Table 1. Dependence of Root-Hair Initiation Site in Trichoblasts of Lettuce Primary. Roots on the
Amplitude of Mass Acceleration

Mass acceleration, g n Distance of root-hair from apical end p

oftrichoblas% % (mean 4- SE)
Ground control, lg 128 31.3 4- 0.4 -
Centrifuge, 1 g 151 31.7 + 0.4 > 0.05
Centrifuge, 0.1 g 155 32.1 4. 0.4 > 0.05
Centrifuge, 0.01 g 160 41.8 4. 0.5 < 0.001
Microgravity 332 46.3 + 0.5 < 0.001

FORMATION OF SEEDLING AXIAL ORGANS WITHOUT GRAVITY

The gravitropic reaction which has developed and improved during the evolution of flowering plants
secures a definite orientation of the seedling main body axis and its up- or down-directed growth in
respect to the gravity vector. Due to this physiological mechanism the plant can achieve an optimal result
in the competition for light, mineral nutrition and water. Is the gravitropism a single function in plant
ontogeny dependent on gravity? To elucidate the possible morphogenetic role of this environmental
factor, a series of experiments in spaceflight (Salyut 7 and Mir orbital stations, Kosmos 1667 and Bion 10
satellites) with germinating seeds have been performed. In some of these tests a 1 g inflight centrifuge was
used as a control variant, enabling to separate the effect of microgravity from the biological impact of the
rest factors of a spaceflight.
604 R. Laurinavi~ius et al.

Table 2. Growth of Seedling Axial Organs under 1 g and Microgravity

Length of axial organs, mm (mean 4- SE)

Plant hypocotyl or coleoptile root
1g MG p 1g MG p
Lactuca 5.1 4- 0.4* 6.3 4- 0.3 < 0.05 9.5 4- 0.6* 9.4 4- 0.5 > 0.05
Lactuca 6.14-0.1" 7.04-0.1 < 0.001 17.44-0.4' 14.34-0.3 < 0.001
Lactuca 17.0 4- 0.6* 22.4 4- 0.6 < 0.001 33.7 4- 1.2' 33.9 4- 1.1 > 0.05
Lepidium -- -- -- 11.8 4- 0.6* 11.3 4- 0.2 > 0.05
Arabidopsis 8.0 + 0.2 10.0 4- 0.2 < 0.001 2.9 4- 0.2 2.7 ± 0.1 > 0.05
Hordeum 6.9 4- 0.4 9.4 :k 0.4 < 0.05 9.2 4- 0.6 9.0 4- 0.8 > 0.05

* inflight 1 g centrifuge.

In MG as in control the successful growth of germ structures and morphogenesis of seedling were
demonstrated. However, in all our experiments hypocotyls and coleoptiles grew more vigorously in MG
than at 1 g, while the length of roots was the same in most cases, except one experiment where lettuce
roots were shorter under MG (Table 2). So, the general morphological feature expressed as a root - to -
shoot ratio for the majority of MG seedlings differed from that of seedlings grown at 1 g.

SOMATIC E M B R Y O G E N E S I S ON T H E CLINOSTAT
To study the role of gravity in the somatic embryogenesis and organogenesis, regeneration of Nicotiana
tabacum L. plants from leaf segments on the vertical (VC) and on the horizontal clinostat (2 rpm) was
performed. The main requirement for the presented research is that the induction of somatic
embryogenesis and development of plantlets should be performed without the one-directed polarizing
action of gravity. Therefore the clinostats were not stopped during the whole experiment. Formation of
somatic embryos from explants was achieved after 8-9 weeks of rotation on a clinostat without changing a
modified Murashige - Skoog (1962) medium and under the influence of an appropriate dark - light cycle.

Fig. 2. Somatic embryogenesis and organogenesis of tobacco in vertical (A) and horizontal (B) clinostat.
 Ontogeny of Plants 605

At the end of experiment, 59% of VC and 64.3% o f H C explants formed 3.2 4- 0.5 (n = 16) and 3.9 4-
0.8 (n = 18) of embryogenic structures per explant, respectively. The regeneration of plantlets possessing
well-developed shoot and root systems was determined in 22.2% of VC and 28.5% of HC explants
(Figure 2). Consequently, the principal possibility of somatic embryogenesis and organogenesis without
one-directed action of gravity as a polarizing factor was demonstrated.
The presented experiment coincides with spaceflight tests, where no adverse effect was found on
carrot (Krikorian et al., 1981) and orchardgrass (Conger et al., 1998) somatic embryo induction or
development, though the number of orchardgrass embryos formed in microgravity was smaller.

SEED-TO-SEED DEVELOPMENT WITHOUT GRAVITY

The ability of higher plants to zygotic embryogenesis in weightlessness was shown in experiments with
Arabidopsis, where seedlings grown on Earth to a rosette (Musgrave et al., 1997) or a flowering (Parfenov
and Abramova, 1981) stage were transported to space and there succeeded in forming some biologically
valuable embryos and seeds, respectively. The main methodological shortcoming of these investigations is
the circumstance that test plants have spent part of their life cycle on Earth, therefore a gravitational
induction on some morphogenetic processes cannot be excluded. The only successful seed-to-seed test
under weightlessness was carried out with Arabidopsis thaliana Heynh. in a device of original
construction on board the orbital station Salyut 7 (Merkys and Laurinavi~ius, 1983, 1991). The main
result of this experiment is over 200 spaceflight-formed matured viable seeds (Figure 3).

A
O
t
seed size, m m (mean 4- SE): seed size, mm (mean + SE):
length 0.433 4- 0.004 length 0.407 + 0.003
width 0.264 4- 0.003 width 0.245 4- 0.002
length to width ratio 1.65 4- 0.02 length to width ratio 1.67 + 0.01

Fig. 3. Seeds of Arabidopsis formed under ground (A) and spaceflight (B) conditions.

From the point of view of general biology, the result is important as proving the limited role of gravity
not only in the vegetative growth, but also in spore formation, gamete development, fertilization and
embryogenesis of higher plants. The formation of zygotic embryo is supposed to be controlled mainly by
the sporophyte, the information for postembryonic development being predetermined by the structures
and metabolites already present in the embryo.
In conclusion, it could be stated that in phylogenesis the higher plants, apart from gravitropic reaction,
have not acquired any vitally important function that could not proceed without gravity. The observed
phenotypic changes of cells, embryoid structures and plants under altered gravity conditions most
probably are caused by both biological and technological reasons related with the modification of the
physical processes both inside and around the organism or cell.
606 R. Laurinavi~ius et aL

REFERENCES
Barlow, R, Gravity Perception in Plants: A Multiplicity of Systems Derived by Evolution?, Plant, Cell
and Environ., 18, 951-962, 1995.
Bean, B., Microbial Geotaxis, in: Membranes and Sensory_Transduction, eds. G. Colombetti and F. Lenci,
pp. 163-198, Plenum Press, New York 1984.
Claasen, D. E., and B. S. Spooner, Impact of Altered Gravity on Aspect of Cell Biology, Int. Rev. Cytol.,
156, 301-373 1994.
Conger, B. V., J. R. Tomaszewski, J. K. McDaniel, and A.Vasilenko, Spaceflight Reduces Somatic
Embryogenesis in Orchardgrass (Poaceae), Plant, Cell and Environ. 21, 1197-1203, 1998.
Cosgrove, D. J., Relaxation in a High-stress Environment: the Molecular Bases of Extensible Cell Walls
and Ceil Enlargement, Plant Cell, 9, 1031-1041, 1997.
Hoson, T., S. Kamisaka, I. Masuda, M. Yamashita, and B. Buchen, Evaluation of the Three-Dimensional
Clinostat as a Simulator of Weightlessness, Planta, 203, S 187-S 197, 1997.
Krikorian, A. D., F. R. Dutcher, C. E. Quinn, and F. C. Steward, Growth and Development of Cultured
Carrot Cells and Embryos under Space Flight Conditions, Adv. Space Res., 1, 117-127, 1981.
Krikorian, A. D., Space Stress and Genome Shock in Developing Plant Cells, Physiol. Plant., 98, 901-
908, 1997.
Kxopf, D. L., Induction of Polarity in Fucoid Zygotes, Plant Cell, 9, 1011-1020, 1997.
Laurinavi6ius R., Interaction Between Gravity and a Plant Cell, Experimental Biology, 4, 103-118, 1991.
Merkys, A. J., and R. S. Laurinavi6ius, Complete Cycle of Individual Development of Arabidopsis
thaliana (L.) Heynh. Plants on Board the Salyut-7 Orbital Station, Dokl. Akad. Nauk SSSR, 271,
509-512, 1983.
Merkys, A. J., and R. S. Laurinavi6ius, Development of Higher Plants under Altered Gravitational
Conditions, Advances in Space Biology and Medicine, 1, 155-181, 1991.
Merkys, A. J., R. S. Laurinavi6ius, D. V. Sveg~diene, D. E Raklevi6iene, A. V. Jarogius, et al., Evaluation
of Experiments Involving the Study of Plant Orientation and Growth under Different Gravitational
Conditions, Adv. Space Res., 9, 23-32, 1989.
Merkys, A.J., R. S. Laurinavi6ius, O. J. Rupainiene, E. K.Savi~iene, A.V. Jaro~ius, The State of Gravity
Sensors and Peculiarities of Plant Growth during Different Gravitational Loads, Adv. Space Res., 3,
211-219, 1983.
Miller, D. D., N. C. A Ruijter and A. M. C. Emons, From Signal to Form: Aspects of Cytoskeleton-
Plasma Membrane-Cell Wall Continuum in Root Hair Tips, J. Exp. Bot., 48, 1881-1896, 1997.
Murashige, T. and F. Skoog, A Revised Medium for Rapid Growth and Bioassays with Tobacco Tissue
Cultures, Physiol. Plant. 15, 473-497, 1962.
Musgrave, M. E., A. Kuang, and S. W. Matthews, Plant Reproduction during Spaceflight: Importance of
the Gaseous Environment, Planta, 203, S 177-S 184, 1997.
Negrutiu, I. and M. Jacobs, Arabidopsis thaliana as a Model System in Somatic Cell Genetics. II Cell
Suspension Culture, Plant Sci. Lett., 8, 7-15, 1977.
Nick, E, and M. Furuya, Induction and Fixation of Polarity - Early Steps in Plant Morphogenesis,
Develop. Growth Differ., 34, 115-125, 1992.
Parfenov, G. E, and V. N. Abramova, Blossoming and Maturation of Arabidopsis Seed: Experiment on
Biosatellite Kosmos 1129, Dokl. Akad. Nauk. SSSR, 256, 254-256, 1981.
Schiefelbein J. W., J. D. Masucci and H. Wang, Building a Root: the Control of Patterning and
Morphogenesis during Root Development, Plant Cell, 9, 1089-1098, 1997.
Sinnott, E. W., Plant Morphogenesis, McGraw-Hill Book Company, New York-Toronto-London, 1960.
Todd, P., Gravity-Dependent Phenomena at the Scale of the Single Cell, ASGS Bull., 2, 95-113, 1989.