Table Of ContentAspects of Gametophyte Development
of
Dicksonia Hook
sellowiana
(Dicksoniaceae):
an Endangered
Tree Fern
Indigenous South and
to
America
Central
Claudia
Cristina L. Fiori, Maris a Santos, and Aurea M. Randi*
Department
of Botany, University of Santa Catarina, 88040-900, Florianopolis,
Santa
Catarina, Brazil
The
biome
Atlantic Forest entirely occupies three Brazilian
states: Espi'rito
Santo, Rio de and 98%
Janeiro Santa Catarina, of Parana and some
areas of 11
other Fundagao
states (IBGE, 2004;
Biodiversitas, 2006). Ferns are an
important plant group of the Brazilian According Tryon
flora. to (1970,
and Tryon and Tryon
1972)
(1982), southeastern Brazil (from Minas Gerais
to
Rio Grande do Sul) contains about 600 Some
fern species. of the Brazilian
ferns
used
are ornamental and members
as plants
of the
tree fern families
Cyatheaceae, Dicksoniaceae and
Cibotiaceae have been
indiscriminately
exploited through
the commercialization and
of pots substrate used in the
production of ornamental
plants (Windisch, For
2002). that reason, under-
*Author correspondence
for [email protected]
-
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99
3 (2009)
standing aspects of fern biology necessary for the development of methods
is
may
and management.
conservation
that assist in their
In Brazil, Dicksonia sellowiana Hook. (Dicksoniaceae) considered an
is
endangered biome (IBAMA,
species of the Atlantic Forest 1997]. occurs
It
preferentially in high humidity environments and on river banks, independent
of soil conditions (Fundagao Biodiversitas, 2006). The stem usually massive,
is
m
cm
ranging from 12-20 decumbent,
in diameter, arborescent, 10 basally
tall,
bearing dense trichomes and many which may occur from
long, fibrous roots,
the base almost the apex. occurs 1500-2500 m, sometimes up
to at ca. to
It
3500 m, or in Brazil lower elevations. occurs throughout Central America
at
It
and in South America from Venezuela to Colombia, south of Bolivia, Paraguay,
Uruguay and southeastern Brazil (Sehnem, 1978; Tryon 1970, 1972; Tryon and
known
Tryon, 1982). In Brazil, as 'xaxim' or 'xaxim bugio' and the trunks
it is
have been indiscriminately exploited through the commercialization of vases
and (Sehnem,
substrate 1978).
The
understanding of fern germination and establishment required for
is
their "ex situ" conservation. The germination of a great number of fern spores
promoted by and water and mild temperatures
light (Miller, 1968) nutrients,
is
are implicated in the growth and development of the prothallus and in
sporophyte formation (Fernandez et al, 1996, 1999). Several aspects of the
germination of D. sellowiana have been studied. Fillipini et (1999)
al.
5%
min
commercial and
sterilized spores in a solution of bleach 10
for
88%
reported that the spores of this species reached around germination at 23
± C
2 under continuous white light, seven days after sowing in liquid mineral
medium. The same under
authors reported that the spores stored refrigeration
remained viable for more than two years and reached 81.75% germination 10
days after spore sowing, which did not differ from the germination of recently
50% 36%
Under and
collected spores (Fillipini et al, 1999). irradiance, the
germination of D. sellowiana spores was delayed after 14 and 21 days,
20% 5%
compared and Higher
respectively, of culture to irradiance.
mean
percentages of germination (around 90%) and lower germination time
35%
(34 days) were observed for spores of D. sellowiana sterilized in a solution
20% 5%
of commercial bleach one hour, which germinated and sunlight;
for at
no were observed between two
statistically significant differences the light
Renner and To
treatments (Fillipini et 1999; Randi, 2004). study the
al.,
possibility of long-term spore storage of Dicksonia sellowiana for the
establishment of a germplasm bank, Rogge et (2000) stored spores in liquid
al.
nitrogen and reported that spores remained viable after being immersed in
months. Concerning gametophyte
liquid nitrogen for three patterns of
development, Nayar and Kaur (1969) described seven different types of
development homosporous works was
prothallial in the ferns. In previous
it
and
reported that germination in D. sellowiana of Vittaria type prothallic
is
development Adiantum and
of type (Perez-Garcia Fraille, 1986).
is
To
obtain sporophytes of Dicksonia sellowiana cultivated from germinated
"xaxim"
spores, Borelli et (1990) cultivated D. sellowiana in the soil of [D.
al.
and months
sellowiana) trunks observed sporophytes after six of spore sowing.
ET GAMETOPHYTE
FIORI DEVELOPMENT
AL.: OF DICKSONIA SELLOWIANA
209
They commented
that fungal contamination was
very high
in the
all
treatments. Suzuki
et al. (2005] cultivated D. sellowiana from spores and
observed
sporophytes that had been emerged
in sterilized typic hapludult
soil
(red with
soil) the addition of termophilic organic compost;
the
first
sporophyte was
frond observed 84 days
after transplantation.
The
aim
of the present study was
to observe gametophytes of Dicksonia
sellowiana grown
in different substrates
in the laboratory examine
to
morphological
aspects of gametophyte development
using microscopy
light
and
scanning
electron microscopy
in order determine
to suitable conditions
growth and
for their development.
Material and Methods
Sporophylls
of Dicksonia sellowiana were
collected from
living plants
in
August
1999 Urupema,
in a fragment
of the Atlantic Forest biome,
situated
between
27 57'25"S and 49 53'33"W, Santa
Catarina
Sporophylls
state, Brazil.
were
an oven
air-dried in at 30°C for three days on paper
filter in order
to
induce The
dehiscence. spores were removed and
separated from by
debris
fikering through
lens paper, and then were
stored under
in glass
jars
±
refrigeration
at 7 1=C.
Spores were 20%
surface-sterilized using
a (v/v) solution of commercial
bleach (2% which
active chlorine), corresponds 0.1%
to of active chlorine,
for
period min
a of 30 before through
filtering paper and washing
sterile
filter
several times with mg
About
sterile distilled water. 20
of sterilized spores were
sown
each
in of 32 conical flasks containing 20 ml of Mohr's
nutrient solution
as presented by Dyer with mg
(1979) the addition of 25 L"^ Benomyl
avoid
to
fungal contamination. The were
flasks plugged with two
layers of autoclaved
transparent commercial polypropylene x
cm)
film (7 7 fixed with rubber
bands. All the procedures were
carried out in a laminar hood. The spores were
incubated
in a 16-hour photoperiod ^moles quanta m^ ± C
(30 23
s^) at 2 in
• •
January 2002.
In February 2002, the young gametophytes medium
cultivated in liquid were
transplanted
to trays containing four types of substrates: commercial
substrate
rich in organic matter; commercial "coxim":
produced
substrate from
the
coconut
used
fiber as substitute for the soil made from the "xaxim"
[D.
sellowiana
trunks);
sterilized typic hapludult
soil (distroferric red nitosoil) and
sterilized typic hapludult
with
soil parts) addition of termophilic
(3 organic
compost
part) as described in Suzuki
(1 et al. (2005).
The
was
soil analysis carried out in Soil Laboratory CIDASC (Company
of
for
Agricultural Development
of Santa Catarina) (Table The were
trays covered
1).
with
transparent
film avoid
to excessive water evaporation and
plant
dehydration.
Substrate was
sterilization carried out in a high power
microwave
oven
for 20 minutes. The organic compost was produced
from
and
vegetable
wastes
fruit at the University of Santa Catarina. Sporophyte
emergence was
scored once a week. The mean and standard
deviation each
for
day
was
of evaluation Windows
calculated by Excel
for (Microsoft).
AMERICAN FERN
J(
(CIDASC-analysis
Tiposition
Typic
haplu,
Typic plus termc
soil
^
Specimens were collected every 15 days from the mineral solution and from
the trays containing sterilized typic hapludult soil and organic compost. For
microscopy (LM) and scanning microscopy (SEM), gametophytes
light electron
M
were fixed in 2.5% glutaraldeyde in 0.1 sodium phosphate buffer 7.2 pH.
All samples were fixed in Eppendorf tubes for 3-hr, then were centrifuged for
3 min, dehydrated with an ethanol series and stored in ethanol. The samples
LM,
were photographed with Leika-MPS 30 microscope. For the
a light
samples were mounted on glass slides with ethanol. For SEM, the cordate
96%
gametophytes were dehydrated with graded ethanol (80%, 90%, and 3
HMDS
were (hexamethyl-
times in 100%). Subsequently, they transferred to
CO2
desilasane) to substitute the critical point, avoiding cell collapse (Bozzola
and Russel, 1991). Dry samples were transferred to stubs and then were gold-
nm
coated with 20 of gold in a Baltec-CED 030. Examination was performed
with a Philips-XL 30 scanning electron microscope.
The and
spores of Dicksonia sellowiana are tetrahedral, globose the
trilete;
44-68^m and
and measure Dicksonia
surface densely granulated (Figs. 1
is 2).
and
sellowiana germination of the Vittaria type (Perez-Garcia Fraille, 1986).
is
During germination, the spores become swollen and the spore coat opens. The
first division was parallel to the equatorial axis of the spores; small
hemispheric cells were produced and gave rise to a hyaline rhizoid that does
which
not contain plastids; subsequently a spherical prothallial cell is rich in
appeared
chloroplasts 3-5).
(Figs.
Gametophytes of D. sellowiana were not able to develop in the soil rich in
organic matter. In the coxim, only filamentous gametophytes were observed
months hapludult the
after 8 of cultivation. In sterilized typic soil, first
months
sporophytes were only observed after 6 of cultivation, but in sterilized
typic hapludult with addition of termophilic organic compost, sporo-
soil
phytes were observed than months of cultivation.
after less 3
FIORI ET GAMETOPHYTE DEVELOPMENT OF
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AMERICAN FERN VOLUME NUMBER
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=
=
Figs. 9-12. 9. Laminur pliaso (idvs) Bur 50 |im. 10. Gametophyte spatulated (45 days). Bar
=
100 Cordate gametophyte Bar 200 Meristematic region with
|im. 11. (75 days). \im. 12.
=
archegonia (90 days). Bar 100 urn. ac- obconic cell amr - apical meristematic region,- ar-
,
-
archegonium, expansion,
le - lateral r rhizoid.
the development of the wings was observed 45 days spore sowing and 15
after
days after gametophyte transplantation to the sterilized typic hapludult soil
with addition of termophilic organic compost. After 75 days of spore sowing,
gametophytes presented the heart shape 9-11) and 90 days spore
(Figs. after
sowing archegonia were present 12 and The archegonia grew from
(Figs. 13).
surface cells of the apical meristem, were bottle-shaped, and presented four
rows 4-5 exposed neck and neck
of cells the canal inside (Figs. 13-16).
SEM
Antheridia were not observed in this prepared material. The gameto-
phytes did not bear trichomes.
The surface of Dicksonia sellowiana spores are granulated or reticulate with
somewhat
strands of fused spheres surrounding depressed areoles as observed
by Try on and Tryon The pattern of spore germination found
(1982). for
was and Kaur which was
Dicksonia sellowiana the Vittaria type (Nayar 1971),
by and The development
described Perez-Garcia Fraille (1986). prothallial of
Dicksonia sellowiana of the Adiantuin type, described by Nayar and Kaur
is
FIORIET GAMETOPHYTE
DEVELOPMENT
AL.: OF
DICKSONIA SELLOWIANA
:
^^^^^^^^^^^^^^^^^
and
(1969) Perez-Garcia and
Fraille
(1986). In this type of gametophyte
development,
spore
germination
results in a uniseriate, slender germ
filament
which
generally 3-7 The
is cells long. terminal and one two
cell or
cells
behind
divide
longitudinally form
it to a prothallial The
plate. division of the
terminal by
cell often the formation
is of a wall oblique
to the long axis of the
filamentous
gametophyte, and
soon second
a oblique wall
delimits a
central
obconical meristematic By
the
cell. activity of the obconical
cell, a spatulated
prothallial without
plate, trichomes, formed which
is in the apex gradually
becomes
notched. Examples
of species from
the Dicksoniaceae show
that
Adiantum
the
type
development
protallial
are Lophosoria quadripinnata
Gmel)
F. C. Chr. (Perez-Garcia and
et al, 1995) Lophosoria
(J. quadripinnata
var.contracta (Mendoza
et 1997).
al.,
The
laminar phase was
observed
after 30 days of
cultivation Dicksonia
for
sellowiana and
the heart shaped gametophytes
presented
archegonia 90 days
after spore sowing, but they did not
present
antheridia. Conversely,
Perez-
Garcia and
Fraille observed
(1986) gametophytes
of D. sellowiana
bearing
antheridia only
after 170 days
cultivation, but they did not observe
gametophytes
bearing archegonia. The same
authors analyzed gametophyte
development
only under
light microscopy and did
not analyze time
to
VOLUME NUMBER
AMERICAN FERN JOURNAL:
99 3 (2009)
sporophyte formation and percentage of sporophyte formation in different
were
amorphous gametophytes not
They found filamentous that
soils.
The gametophyte Lophosoria quadripinnata
observed in the present work. of
was spatulate 156 days of culture; antheridia were observed after 72 days,
after
270-285
but archegonia were seen only after days (Perez-Garcfa et al, 1995).
how
show
Additional analyses are necessary to sexual expression of D.
sellowiana affected by spore density in laboratory conditions.
is
predominance
of clay
In Santa Catarina State (Brazil) there is a soils,
experimental conditions
cambisoils, and nitosoils (IBGE, 2005). In the
laterites
hapluduh red nitosoil)
carried out in this study, the typic soil (distroferric
compost was most
with the addition of termophilic organic the suitable for
Dicksonia sellowiana development, and the sporophytes in this substrate
first
poor
were observed 84 days spore sowing. Atlantic Forest soils are in
after
one
nutrients and the accumulation of chemical elements in cells is of the
Fundagao
ways low-nutrient (IBGE 2004;
tropical species tolerate soil
component
fundamental of nutrient cycling,
Biodiversitas, 2006). Litterfall a
is
and the main means of transferring organic matter and mineral elements
it is
The
Moraes
back the surface (De Franga et ah, 2007; et al, 1999).
to soil
termophilic organic compost was added in order to simulate the litter in this
K
pH and
This substrate has a low and high levels of N, P, Ca. In
substrate.
when gametophytes Dicksonia
al cuhivated of
contrast, Borelli et (1990)
were
made "xaxim" sporophytes
sellowiana in the soil of trunks, the first
100%
observed only months of cultivation and the authors observed
after six
75% produced
and around gametophytes
contamination cultures of
in their
percentage of
sporophytes. Therefore, the time to sporophyte formation, the
were improved
sporophyte formation, and the prevention of contamination in
the present work. Edaphic parameters were analyzed for several fern species to
requirements and the majority of
elucidate their habitats including nutritional
and
Graves
them does sellowiana (Carlson, 1979;
prefer acidic soils, as D.
The sporophyte
Monk, Whitter and Moyroud, 1993; Ranal, 1995). time to
1982;
among Lophosoria quadripinnata
emergence quite variable fern species;
is
months
formed sporophytes 36 cultivation (Perez-Garci'a et al, 1995).
after
employed
The hapludult with added termophilic organic compost in
typic soil
work was also useful for sporophyte emergence, which was observed less than
this
work
three months after cultivation as observed in previous (Suzuki et al., 2005).
Information provided in this paper certainly will be useful for D. sellowiana
management in green houses as part of conservation strategies. Plantlets of
presented
Dicksonia sellowiana can be easily obtained following the protocol
germplasm banks
This methodology can be used for the establishment of
here.
and
gardens
with the purpose of preserving the species in botanical to
maintain genetic variability.
its
Claudia Cristina Leite Fiori thanks CAPES {Council of Improvement of University Education
Staff - Brazil) for her grant. We thank Dr. Paulo Emi'lio Lovato (UFSC) for suggestions and for
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