Table Of ContentNew
Growth
and
Care Model
Instructions of a
—
Species Lycophyte apoda
the Selaginella
Christian Schulz*
Evolution and
Biodiversity of Plants, Ruhr-University Bochum, Bochum
UniversitatstraBe 150,
Germany
44780,
Lewis B. and Dorothy Cullman Program Molecular The New
for Systematics, York Botanical
200™
New
Garden, & Southern York USA
Blvd, 10458-5126,
Damon Wm.
Anna
P. Little, Dennis Stevenson, Nowogrodzki, and
David Paquiot
Lewis B. and Dorothy Cullman Program for Molecular Systematics, The New York Botanical
200™
Garden, & Southern Blvd, New York 10458-5126, USA
with a lycophyte model
specie;
y
lytes quickly. In the case of
S.
c
lanted to They grow well
soil.
i
ath 0.15% Previcur®N
prevents
i
sm,
lycophytes, lycopods,
ferns.
apoda many
Selaginella Spring has advantages model
(L.) as a species,
including an day
85 life cycle from spore to spore (Schulz et al, 2010). The
length of the life cycle is similar to other model species, such as Ceratopteris
thalictroides Brongn., with month
(L.) a 3.5 cycle (Klekowski,
life 1970). In
work
order with model
to effectively a species, it is important to be able to
grow
quickly. In addition, high frequency
it a of fertilization desirable.
is
Many
methods
different of growing ferns have been published
Lovis,
(e.g.,
Hickok and
1968; Warne, amount known
2004), but only a small about
is
growing Methods
Selaginella. growing gametophytes and
for sporophytes of
have been
Selaginella described, but the conditions and treatments vary among
species. Bierhorst (1964) collected megaspores from around
the
soil Selagi-
nella sporophytes and placed them on wet where
paper, the spore wall
filter
opened. For
the microgametophytes, he sowed whole
microsporangia on
filter
Bold sowed
paper. (1967) spores on immersed
plaster of Paris blocks half
in
water an medium.
distilled or inorganic the plaster blocks remained
If
would known
sufficiently moist,
fertilization occur. Overall, about
little is
we
growing apoda,
S. therefore, have undertaken
this investigation present
to
how
an instruction on grow gametophytes and
to sporophytes of apoda.
S.
AMERICAN FERN VOLUME NUMBER
168 JOURNAL:
100
3 (2010)
Materials and Methods
The
specimens investigated here were collected from a naturally occurring
New
population on grounds York Garden
the of the Botanical (population
We
Moran
voucher: NY). grew specimens Nolen
Little 935, in the
St
Greenhouse (New York, USA), greenhouse Garden
in the at the Botanical of
Bochum
the Ruhr-University (Germany), and in laboratory conditions both in
New We
Bochum
York (USA) and (Germany). grew Selaginella apoda six times
through its entire life cycle. Spores were soaked in autoclaved water for
4 minutes, surface sterilized for 4 minutes, and then rinsed with autoclaved
water Benomyl
for 4 minutes. For surface sterilization, the fungicides (1-
sodium
20 mg/L), hypochlorite (0.1-0.875%), Previcur®N (propamocarb-
hydrochlorite) (0.15%), and ethanol (70%) were tested. In addition, the
antibiotic streptomycin (5-60 mg/L) was tested. Soil, sandy soil, peat soil,
autoclaved water, autoclaved water with nutrients (C-fern nutrients) and C-
fern agar (Carolina) were each used as media. The composition of these
nutrient mixtures was the same used by Klekowski
as
(1969).
Results and Discussion
many
There grow apoda
are, theoretically of course, possible protocols
to S.
under The method
different conditions. presented here one
just possibility,
is
but very fast and successful. Masses of megaspores and microspores can be
it is
collected from pots of apoda (sporophytes with cones) by placing the pots
S.
on their sides on top of a smooth collecting surface (we used a sheet protector;
Avery Denniso, CA; The number and
Brea, Fig. lA). greatest largest cones
New
summer
appeared in late and autumn (in York). The cones produced in
and summer number much
spring early are fewer in and Presumably,
shorter.
cone and, therefore, spore production are influenced by day length and
light
we
intensity, but have not investigated these parameters independently. In
volume
order to harvest a greater of spores, the plants dry one day
let for
much
during the collection, being careful not to dry the plants so that they die.
The
collected spores can be stored in dry vials at room temperature; viability
is
maintained for at least two years. Growth can be accelerated by soaking the
spores in water (or nutrient water) for 15 minutes before sowing.
Spore surface sterilization was necessary grow gametophyte
to sterile
on used
cultures agar (Fig. 1 B, C). Biological contamination rarely occurred
when
soil or autoclaved water with or without nutrients was used as a
medium; when
however, was
surface sterilization necessary using C-fern agar
as a substrate. It is important to soak the spores with autoclaved water and to
rinse the spores with autoclaved water after the treatment. Even a low
0.1% sodium
concentration used
of hypochlorite, as routinely Ceratopteris
for
spores (Hickok and Warne, 2004), killed the apoda megaspores. Sodium
S.
amount
hypochlorite prevented only a small of fungal contamination. Both
low concentrations of Benomyl and 70% ethanol also killed megaspores and
some
prevented only of the fungal contamination. In contrast, 0.15%
Previcur®N
prevented most
fungal contaminations without
killing the
megaspores.
Rarely, bacterial contamination occurred on
C-fern but
agar,
mg/L
15 streptomycin min) prevented such
contamination.
(4 In order to
prevent contamination with was remove
algae, sufficient to megaspores with
it
algae (easily observed green from
as dots) the culture.
The We
fertilization success varied with observed
substrate. the highest
fertilization success (about 95%) in autoclaved water with
nutrients. In
autoclaved water and on
C-fern agar (60-80%) was
fertilization less frequent.
On
soil, the fertilization success was moderate (about 60%: Table
1).
An
important gametophyte development
factor for temperature
(Webster,
is
The optimum
1967). temperature gametophytic development
for about 30'C.
is
Similar growth and development
of gametophytes can be obtained
22-30'C.
at
The
higher the temperature within
this range, the the gametophytes
faster
develop. Temperatures lower than 22 C
will alter or inhibit the developmental
timing. Light is not necessary for the development of the gametophytes.
Gametophytes
develop successfully under a 14h light/lOh dark cycle as well as
without any
(24h dark and
light cycle) in/on different kinds of media/
substrates.
Webster described apoda growing sandy
(1979) in finely sifted loam.
S.
Bruchmann
(1912) indicated that peat not suitable for the growth of
is
Selaginella gametoph5^es as has an inhibiting effect on denticulata
it S. (L.)
we
Spring. However, were able to grow S. apoda on peat soil (e.g., on peat
on medium
mineral
pellets), agar C-fern agar), in mineral C-fern
(e.g., (e.g.,
medium)
or in tap water. For experimental purposes, a single megaspore and a
single microspore develop very well in a 200 tube with 20 of water.
fxL |iL It is
warm
important that the gametophytes are always covered with a film of water
in order to prevent retardation of development and success
for fertilization (the
swim
spermatozoids must be able to to the egg cells). If young sporophytes are
grown
in deep water, they usually produce elongated hypocotyls.
The
optimal stage to transplant young sporophytes grown on agar IC)
(Fig.
medium when
and
or in to soil is the first three roots several leaves are evident.
medium
The optimum
mineral used gametophytes
nutrient not
for is for
medium
sporophytic growth, so transfer to a soil preferable. Soil should be
is
kept moist to wet at all times. Peat soil or sandy peat works well for S. apoda.
warm
The
plants require temperatures (15-28 and high humidity (70-
C)
100%) grow
to quickly.
The sporophytes grew very well temperatures between 15'C and
28'C.
at
The sporophytes grew higher temperatures and produced more
faster at roots
Room
and
rhizophores. temperature works fine for sporophytes. Selaginella
apoda naturally lives in partially shaded habitats without direct sun. The more
more
light the plants receive, the the leaves turn yellow/white are
(i.e.,
photobleached: Fig. ID), and the faster the cones develop. With too much
light, the plants will die. The less light the plants receive, the greener and
smaller the leaves become. The optimal habitat shady. Sporophjrtes grow
is
>
fast in peat soil, and a bit slower on agar or in (nutrient) water (n 100). If the
SCHULZ ET GROWTH OF SELAGINELLA APODA
AL.:
grow
plants in soil, it is useful to use clay pots and keep the soil wet. The
sporophytes need high humidity, otherwise the leaves dry out and the plant
The grown
will die. plants can be easily in a covered terrarium. The fungicide
0.15% Previcur®N (propamocarb-hydrochlorite) worked
well against fungal
contamination; also works well with other Selaginella species. High
it
concentrations of other fungicides can damage the
plants.
summer when
under
In or artificial light, plants are growing very
fast,
A
may
fertilizer be used. low concentration of required some
fertilizer is for
We
Selaginella species. achieved good growth and cone production with
Compo® GmbH
Orchid {3-4-5-0.9 N-P-K-micronutrience) [Comp &
fertilizer
KG, The recommended
Co. Miinster]. concentration should be applied once
a
month.
Literature Cited
Suggestions and comments on
teachi
.
Bruchmann, H. 1912. Zur Embryologie der Selaginellaceen. Flora 104:180-22'
HiCKOK, L. G. and T. R. Warne. 2004. C-Fern® Manual. Carolina.
A
Klekowski, E. 1969. Reproductive biology of the Pteridophyta. stud}
Jr. III.
J.,
Bot. Linn. Soc. 62:361-377.
J.
An
Klekowski, E. 1970. Reproductive biology of the Pteridophyta,
IV.
Jr. e^
J.,
mating systems
in Ceratopteris tbalictroides (L.) Brong. Bot. Linn. So(
J.
Lovis, D. 1968. Fern hybridists and fern hybridising. Fern hybridising
II.
J.
Leeds. Brit. Fern Gaz. 10:13-20.
W.
ScHULz, C., D. p. Little, D. Stevenson, D. Bauer, C. Moloney
the morphology, anatomy, and new mod
cycle of a
life
apoda
(L.) Spring. Int. Plant Sci. 171(7):693-712.
J.
Webster, T. R. 1967. Induction of Selaginella sporelings un
