Potential for Spore Germination, Sporophyte Formation and Growth of Young Sporophytes of Four Fern Species from the Atlantic Forest (Brazil)

2010·6.4 MB·English

by  Rosane Hiendlmeyer

Spore
Potential for Germination, Sporophyte
Formation and Growth Young
of Sporophytes
of
Four Fern from
Species
the Atlantic Forest
(Brazil)
ROSANE HiENDLMEYER and AUREA MaRIA
RaNDI
Laboratorio de Fisiologia Vegetal, Departamento de Botanica, Universidade Federal de Santa
Catarina, 88040-900,
Florianopolis. Santa
Catarina, Brazil
The
world
fern flora contains 9,000 to 12,000 species; about 3,250 are found
American
in the continent (Tryon and
Tryon, Windisch, and
1982; 2002) 1,200
to 1,300 species are found Sehnem
in Brazil (Prado, 1997). 493
(1977) listed
species in the South of Brazil, the majority native to the Atlantic Forest.
According
"Conservation
to International" the Atlantic Forest the third
is
8%
diversity "hotspot" in the world and only
of remains
original area
its
preserved. few
In the last decades, several ferns with ornamental medicinal
or
proprieties have been
indiscriminately exploited and them
several of are
considered be endangered
to Examples some
species. include
tree ferns
belonging
the Cyatheaceae and
to Dicksoniaceae ("xaxins" "samambaia-
or
which
?us"), have been exploited commercial
for purposes, especially in the
South
of Brazil (Windisch, 2002). Dicksonia sellowiana Hook.
(Dicksoniaceae)
an endangered
is tree fern in Brazil due to the extensive harvesting in
its
IBAMA,
habitat (Sehnem, 1978, 1997) and Rumohra
adiantiformis
(Forst.)
Ching., (Dryopteridaceae) Sphaeropteris
gardneri (Hook.) R.M. Tryon, Cyathea
atrovirens (Langsd. & Domin,
Fisch.) Alsophila some
setosa Kaulf, species of
Adiantum,
Asplenium, Blechnum,
Selaginella, Marsilea, Adiantopsis and
Lycopodium
are extracted ornamental arrangements &
for (Milton Moll
1988;
Windisch,
2002).
According Pence
ex
to (2000), the situ preservation of spores, particularly of
rare species from threatened habitats, can be an important supplement
to the
maintenance
of pteridophyte The
species in the wild. conditions under which
spores are stored have a notable impact on
their viability. Generally, avoid
to
AMERICAN FERN JOURNAL: VOLUME NUMBER
100 4 (2010)
deterioration, they are stored in dry, ambient or low temperatures, although in
some cases this has resulted in loss of viability (Beri and Bir, 1993; Camloh,
1999).
The aim was
of the present study to verify the potential of germination of
spores stored under refrigeration, the percentage of sporophyte formation after
in vitro germination, and the relative growth rate (RGR) of young sporophytes
room
cultivated in a growth of four terrestrial species native from the Atlantic
Blechnum
Forest: brasiliense Desv. (Blechnaceae) an herbaceous species,
Domin
Cyathea corcovadensis (Raddi) and Cyathea delgadii Sternb. (Cyathea-
ceae) both tree ferns and Saccoloma inaequale (Kze.) Mett. (Dennstaedtiaceaej
an herbaceous fern. These species, with exception of Saccoloma inaequale, are
routinely used in Brazil as ornamental plants and are exploited indiscrimi-
nately from Saccoloma inaequale which
their habitats. a rare species, also
is
has potential as ornamental plant. Such information will be useful for
management
and conservation programs in the future.
Materials and Methods
Blechnum
brasiliense and Cyathea corcovadensis sporophylls were harvest-
ed from several plants on January 12'^ of 2003; sporophylls of Cyathea delgadii
were harvested on April 25**" 2002 and Saccoloma inaequale on March
of of
29'*' of 2003. The collections were carried out in Natural City Park Sao
Francisco de Assis, situated in Blumenau, Santa Catarina State, South Brazil,
at 26 55'15" S and 49 05'30" W. The study site a 22.29 ha natural Atlantic
is
forest fragment.
C
Sporophylls were air-dried in an oven 30 three days on paper
at for filter
in order to induce dehiscence. The spores were removed and separated
from debris by filtering through lens paper, and stored in glass jars
under ± Spores Blechnum and Cyathea
refrigeration at 7 1 C. of brasiliense
corcovadensis were kept under refrigeration for eight months before the
germination spores of Cyathea delgadii and Saccoloma inaequale were
test;
under months
kept refrigeration for six before the germination Spores
test.
10%
were surface sterilized using a (v/v) solution of commercial bleach (2% of
active chlorine) 20 min. before through paper and
for filtering sterile filter
washing several times in sterile distilled water. Spores of each species were
mL
sown
in four conical flasks containing 20 of Mohr's mineral solution as
mg
modified by Dyer supplemented with Benomyl 0.01%. About 10
(1979) of
spores were inoculated each which was plugged with two
in flask, layers of
(7X7
autoclaved transparent commercial polypropylene film cm) fixed with a
rubber band.
All tests were done in a completely randomized design. All procedures were
carried out in a laminar hood. Flasks were kept in a growth chamber (25 ± 2 C)
m"^
under a 16-hour photoperiod and photon flux density of 30 jimol s~^
provided by cool white fluorescent tubes. The irradiance was analyzed by a
PAR
LICOR
quantameter 250. of sensor (400 to 700 nm). The percentage of
abnormal spores and germination were scored daily or every two days. One
HIENDLMEYER
& RANDI: SPOROPHYTE GERMINATION AND GROWTH
from each was
slide flask prepared, with 100 spores counted on each
slide.
Germinated and
abnormal were
spores recorded microscopy
in optical (40X).
Spores were considered to be abnormal when they were empty when
totally or
they presented few
a storage grains between empty The mean
spaces.
germination time was
calculated each
for replication per treatment according
the equation:
to
where
tj is the time in days starting from day 0 and the number of spores
rij is
completing germination on day
(Labouriau, Santana and
1983; Ranal,
2004).
month
one
After
of cultivation mineral young
in solution, filamentous
gametophytes were
transferred to trays containing sterilized typic hapludult
with
soil (3 parts] addition of thermophilic organic compost The
part).
(1
thermophilic compost was
obtained from decomposition and
of vegetable
fruit
wastes
at the University of Santa Catarina. The substrate with
(soil
thermophilic compost) was CIDASC
analysis carried out in (Companhia
Integrada de Desenvolvimento
Agricola de Santa The
Catarina). substrate
pH
N
presented a low and high levels of P, K, Ca and (Table The were
trays
1).
covered with
transparent
film to avoid excessive water evaporation and
plant
dehydration. Substrate was
sterilization carried out in high power
a
microwave oven
for 20 minutes to avoid contamination with other ferns from
the spore bank. To analyze
soil the percentage of sporophyte originated from
mature gametophytes and
to obtain plants that were used growth
later in
200 mature
analysis, cordate gametophytes were
transferred to four trays (50
gametophytes
in each tray) containing the same substrate. Gametophytes were
separated from each other by cm. During
1.0 the gametophytes were
test,
watered with
water The
sterile distilled three times a week. presence
of
was
sporophytes recorded once a week. Plants were kept growth room
in a
as
described above.
When
the sporophytes of the four species were big enough be manipulated
to
cm
they were
[ca 3.0 tall), individually transplanted to small pots containing
AMERICAN VOLUME NUMBER
FERN JOURNAL:
100
4
same
the substrate used gametophyte development and were kept
for in plastic
trays covered with transparent film, in a completely randomized design (Time
1=
Ti). For the growth analysis of Blechnum brasiliense, Cyathea delgadii and
we
Saccoloma
inaequale, 20 sporophytes and growth
utilized the analysis
for
we
of Cyathea corcovadensis utilized 15 sporophytes. After 60 days of
2=
individual transplantation {Time sporophytes were count
T2), collected to
number
the of fronds and to measure the longest frond's length. Table 2 shows
the period of growth spore inoculation time and time The
after at 1 2. relative
growth rate (RGR] was estimated according Bernabe
to et (1999), as (Log^o
al.
-
-10
L2 Logio T2 -Ti where Log the base logarithm, the
La) / is Li is initial
when
leaf length the sporophytes were individually transplanted to the pots in
time and
1, L2 is the leaf length at time 2 (after 60 days of transplantation to the
individual
pots).
Data were analyzed by Windows Windows
Excel Minitab
for (Microsoft), for
and Statgraphics software. Means and standard deviation were calculated. The
Kolmogorov-Smirnov
test for goodness of for normality and the Bartlett's
fit
test for the homogeneity of variance (0.05) were applied before the analysis.
The parametric pairwise comparison among number
test the of fronds of the
four species, were done by the Duncan (5%) Muhiple Range The non-
Test.
Dunn
parametric Kruskal-Wallis followed by was employed
the
test test to
RGR
analyze the (relative growth rate) because these data did not present
residual normality or did not present homogeneity of variance (Santana and
Ranal 2004; Zar, 1996). The Student "t" test was applied to compare the
number
of fronds and the longest frond's length between Tl and T2 each
for
species,
Results
The mean germination time for the four species studied in this work ranged
from 9.9 ± 0.1 days for Blechnum brasiliense to 17.8 ± 0.1 days for Sacolloma
The ±
inaequale spores (Table percentage of germination varied from 32.0
3).
±
2.5% Cyathea corcovadensis 5.0% Cyathea
for to 77.0 for delgadii (Table 3
and Fig. la-ld) and the percentage of abnormal spores varied from 5.6 ±
HIENDLMEYER
& RANDI: SPOROPHYTE GERMINATION AND GROWTH
0.7% for Sacolloma inaequale 29.0 ± 0.7% Blechnum
to
for brasiliense
(Table
3).
All species produced sporophytes
during
the period of analysis, but the
percentage of mature gametophytes that produced sporophytes and the time
for sporophyte formation were among Blechnum
variable
species. brasiliense
presented the first sporophytes after 86 days of cuftivation and 91.5 ± 2.5% of
gametophytes produced
sporophytes 303 days The
after (Fig. 2a). first
sporophytes appeared Cyathea
in corcovadensis 117 days
after of cultivation
and ±
after 264 days of cultivation, 35.8 16.2% gametophytes produced
of
sporophytes Cyathea
(Fig. 2b). delgadii presented the sporophytes
first after
109 days and
of cultivation after 249 days of cultivation, 79.2 ± 13.5% of
gametophytes produced
sporophytes The
sporophytes
(Fig. 2c). first of
Saccoloma
inaequale appeared 78 days and
after of cuftivation after 295 days,
±
24.4%
48.4 of gametophytes produced sporophytes
(Fig. 2d).
The number
produced
of fronds ranged from Blechnum
4.5 to 7.0 for
from
brasiliense, 4.7 to 5.0 for Cyathea corcovadensis, from
5.8 to 8.5 for
Cyathea delgadii and from Saccoloma
6.0 to 9.0 for inaequale 3a
(Fig. to 3d).
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FERN JOURNAL:
100
4 (2010)
The longest frond length ranged from 3.0 to 6.9 cm for Blechnum brasiliense,
from 2.7 to 6.1cm for Cyathea corcovadensis, from 4.0 to 10cm for Cyathea
and from 9.5cm Saccoloma The
delgadii inaequale 4a
3.3 to for (Fig. to 4d).
RGR
of Saccoloma inaequale, an herbaceous fern, was statistically greater than
RGR
Cyathea was
the of corcovadensis, a tree fern, but similar to the other
species (Table The number of fronds produced two months was
4). after
among
statistically smaller for Cyathea corcovadensis but did not differ the
other species (Table
4).
Discussion
common
under method
Preservation refrigeration a of spore storage.
is
According to Pence (2000), the ex situ preservation of spores can be an
important supplement to the maintenance of pteridophyte species in the wild.
-20
Pence
(2000) stored spores of 33 species at 4 C, C, or in liquid nitrogen
and observed that spores of three species did not lose viability after more than
HIENDLMEYER
& RANDI: SPOROPHYTE GERMINATION AND GROWTH
months.
75 Eighty-two percent of Dicksonia sellowiana Hook,
spores
germinated more
than under C
after 2 years refrigeration at 10 (Filippini et
ah, 1999). Previously sterilized spores of D. sellowiana remained
viable
after
cryopreservation and
in liquid nitrogen cryopreservation seems improve
to the
germination
of the spores {Rogge
et al, 2000).
Aragon and Pangua
analyzed
(2004) the spore germination
of four
rupicolous Asplenium
taxa of adiantum-nigrum
[A. adiantum-nigrum,
L. var.
A. adiantum. nigrum
L. var. silesiacum, A. septentrionale Hoffm
subsp.
(L.)
and
septentrionale A. ruta-muraria
subsp. ruta-muraria) and
L. after
6,
1,
months
12 of storage in Eppendorf tubes on
(dry storage) or agar plates (wet
-20
C
and
storage) 20
at C, 5 C. In all cases, except for A. ruta-muraria,
germination percentage was maintained wet and
in dry storage, but in the dry
method
storage percent germination was Though
higher. the authors did not
carry out a thorough post-germination study, the plates used in the various
experiments remained chambers C
in cufture 20 months and
at for 6 the
gametophytes appeared to develop normally. They suggested that storage time
might
not affect the subsequent development of the
prothalli.
,
AMERICAN FERN VOLUME NUMBER
JOURNAL:
100 4 (2010)
b
12
^
8
n n
h
T
r
"
0 ,
, .
270 337 397
3
Days
of cultivation
12
12
1
8
8
n
n
1
0
,
0
252 312
386
4A
6
Days
of cultivation
Cyathea corcovadensis Cyathea
(b),
i),
red soil with the addition of organic
In comparison to studies done by Simabukuro et (1993) the mean
al.
Blechnum
germination time for spores of brasiliense and Cyathea delgadii
were delayed However, Simabukuro
in the present study.
et (1993)
al.
inoculated fresh unsterilized spores 25 C under continuous white The
at light.
surface sterilization could delay or reduce spore germination but necessary
is
in order to obtain higher percentages of gametophytes. Indeed, Camloh
(1993,
1999) reported that the best germination of Platycerium bifurcatum (Cav.) C.
when
Chr. occurred unsterilized spores were used, but contamination
that
always occurred 10 days Simabukuro
after of culture. et (1998) indicated
al.
that before the germination of dry-stored spores, in order to avoid the
incidence of fungal growth, there is the need to sterilize them. The differences
mean
observed in the present paper the germination time
for of B. brasiliense
and
C. delgadii could be an effect of the time and location of collection,
method
spore germination and time under
sterilization for of storage
For Blechnum and Blechnum
refrigeration. chilense (Kaulf.) Mett. cycadifo-
Thompson
lium (Colla) Sturm, the germination of spores in agar-gelled
medium
6-10
started after days of spore soaking (Perez-Garcia et 1996).
al.,
The Blechnum
germination
results of stored spores of brasiliense are similar
who
to the results of Simabukuro et al. (1993), worked with sporophytes of B.
brasiliense collected in the Biological Reserve of Moji-Guagu localized in
S.
Paulo The
State (southeast region of Brazil). data presented in this paper are in
HIENDLMEYER & RANDI: SPOROPHYTE GERMINATION AND GROWTH
b
10
10
-^
6 n.n.
u,
^
\'-
2
0
270 330 337 397
Days Days
of cultivation of cultivation
d
15 -
10
E
(cm) n
n
n.n'.
?
FL 5-
-
0
386 446
252 312
Days
of cultivation Days
of cultivation
Fig. 4. Longest frond length (FL] of Tl and T2 of Blechnum
b), Cyathea delgadii (c) and Saccoloma inaetqiia e (d) cult vated in red soil'^with the addition of
und
2 C) er a 16-h photoperiod and photon flux
accordance with similar studies in Cyathea corcovadensis (Felippe
et ah,
and
1989) C. delgadii (Randi and Felippe, 1988 Simabukuro
a, b). et al. (1998)
-12
stored previously sterilized spores of delgadii 12 months C and
C. for at
74%
observed germination days from sowing. The germination
after 7 of
Table 4. Relative growth rate (RGR) and mean number fronds per month (NF) of Blechnum
brasihense, Cvatbea corcovadensis, Cyathea delgadii, and Saccoloma inaequale cultivated in red
compound
soil with the addition of organic (3:1) in growth chamber (25 ± 2 C) under a 16-h
photoperiod and photon flux density of 30 nmol m"^ s \ is the statistic for the Bartlett test; D„„„
H
Kolmogorov-Smirnov
the the
is statistic for test, is the statistic for the Kruskal-Wallis test: sd
is
the standard deviation. Letters denote statistical differences. * Data did not show normality.
AMERICAN FERN VOLUME NUMBER
JOURNAL:
100 4
(2010)
Saccoloma
Sterilized spores of inaequale took 24 days. For spores
of related
Odontosoria
(Dennstaedtiaceae), germination took six to 15 days to reach
maximum
percentage (Granados The abnormal
et al, 2003). spores observed in
this study seems to be related with the reduction in percentage of spore
Blechnum
germination and
for brasiliense Cyathea delgadii, but seems be
to
it
not the only reason for the low germination of Cyathea corcovadensis and
Sacolloma
inaequale.
Large
differences in percentages of germination are the rule in simultaneous
experiments on
different taxa. Spore age, storage conditions and culture
conditions, have on
all a great influence fern spore germination (Camloh and
Gogala, Camloh, Camloh,
1992; 1993; 1999; Sheffield et 2001). Differences in
al.,
the time for sporophyte formation and percentages of sporophyte formation were
common
also in studies on sporophj^e development
(Sheffield et al, 2001).
In this study, the first sporophytes of Blechnum brasiliense were observed
after
months
2.9 of cultivation (86 days). Meanwhile, the sporophytes were
first
observed months
4.5 after spore inoculation for B. chilense and after 7 months for
B. cycadifolium (Perez-Garcfa The growth was
et al, 1996). of B. brasiliense
probably by
facilitated the substratum, temperature and photoperiod employed
throughout this work. This methodology can then be
utilized for the cultivation
of B. brasiliense. In fact, sporophytes of this species were transplanted to the
garden of the Department of Botany and showed normal development.
a
However,
the sporophytes of C. corcovadensis, C. delgadii and S. inaequale were
not transplanted the garden because they showed
to sensitivity to desiccation
and
require special cares during acclimatization, needing high humidity,
shadow
and
mild temperatures during Blechnum
this period. brasiliense appears
to be a plant adapted to regimes of oscillating humidity and greater light
being more
intensfty, easily acclimatized.
The
gametophytes of Sacolloma inaequale that did not produce sporophytes
A
grew
successively until the end of this study. similar observation was found
by Sakamaki and
Ino working with
(1999) Thelypteris palustris Schott
They
(Thelypteridaceae). concluded that sporophytes appeared on most
gametophytes
that reached a critical size for sporophyte formation, but some
gametophytes
that did not produce sporophytes became very and
large lived
for a long time without producing sporophytes.
Our
data for frond production by young sporophytes of Blechnum
and Cyathea
brasiliense delgadii are similar for the data found for Acrostichum
&
daneaefolium Langsd. which were ± ±
Fisch. (Pteridaceae) 1.0 0.03
to 1.3
month
0.04 fronds per (Mehltreter and Palacios-Rios, 2003; Mehltreter
et
al.,
2003). However, for adult sporophytes of several Cyatheaceae, the number of
fronds produced each year varied from 14 (Tanner, Ash,
3 to 1983; 1987;
Arens,
2001).
RGR
RGR
In this study, the of Sacolloma inaequale was greater than the of
Cyathea
corcovadensis. were
Statistically significant differences not observed
among RGR
the other species. Data for the of some angiosperms from the
Atlantic Forest, presented by Paulilo showed herbaceous
et (1993), that
al.
RGR
showed RGR
species greater than tree species. Concerning for fern