Table Of ContentAn
Method and
Surface
Efficient for Sterilization
Sowing Fern Spores
in vitro
Wu
HuA, Chen Yuan and Chen Long-Qing*
Ping-Ting, Li-Ping,
and
Key Laboratory of Horticultural Plant Biology (Ministry of Education), College of Horticulture
Wuhan
Forestry Science, Huazhong Agricultural University, 430070, P.R. China
from spores
Surface sterilization the step for aseptic culture of ferns
is first
(Dyer, 1979). Since tissue culture widely employed as a technique for fern
is
widely used
propagation or scientific studies, spores are as a starting material. In
recent years, there have been numerous studies on in vitro culture of ferns from
and Cox These
spores (Stone, 1958; Yoroi, 1972; Kiss Kiss, 1998; et al, 2003).
researchers reported successful cultures, but admitted significant losses of
spores during the process, aseptic sowing, or due to contamination
sterilization
methods
(Warne al, 1986). Because of these problems, different of spore
et
Many
and sowing have been developed (Dyer, 1979). of these
sterilization
methods are inefficient in terms of time and spore loss (Warne et 1986).
al.,
still
we method with
Recently, established an effective for sterilization of spores a
Osmunda
funnel. The method successful with spores of japonica
filter is
Adiantum
Adiantum
Thunb., Aleuritopteris argentea Gmel., flabellulatum L.,
We
and Cyrtomium fortunei Smith (data not shown).
capillis-veneris L.
].
compared our method, here called the method, with two other widely used
filter
methods. With the packet method, spores were sterilized in filter bags/packets
method were
(Ford and Fay, 1999), while with the centrifugation the spores
and water
suspended in a sterilizing solution then harvested in sterile distilled
we
used
by (Fernandez For these comparisons,
centrifugation et 1993).
al.,
spores of Adiantam reniforme var. sinense Y. X. Lin, a rare and endemic species
China.
in
were introduced
Plant materials.—SpOTophytes of A. reniforme var. sinense
from Wanxian County along the Yangtze River in 2001 and cultivated in the
HUA
METHOD
ET OF
AL.: SPORE SURFACE AND
STERILIZATION SOWING
greenhouse Huazhong
of
Agricultural University (Wuhan,
China).
Fertile
fronds
of sporophytes
in the greenhouse were
collected and wrapped
paper
in
bags and room
dried
at temperature one week
for Then
to release spores. the
spores were
collected
in centrifuge tubes and
stored 4°C
at until used. For
this
study, spores were
stored 10 months.
for
Culture media.—Murashige
and
Skoog medium
(1962) (MS) with
1/4
strength of macronutrients were
used
for germination of spores. The medium
was supplemented 3%
with
(w/v)
of sucrose, solidified with 0.65%
(w/v) agar
and pH
adjusted cm^
to 5.8 before autoclaving 121"C and
at kg
1 1 for
^
20 min.
Spore and
sterilization sowing.—The method
was
performed
filter
in a
mg
laminar
flow hood. Three
spores were suspended and 4%
wetted with
(v/v)
Tween
min
solution
for 5 in a 1.5 ml centrifuge Suspended
tube. were
spores
collected through hmnel
a (made by
filter which was
fast filter paper), placed
on
a proper conical The tube was washed
flask.
three times with
fresh water
and
the water was poured hmnel
into the
to collect the residual spores of the
Seventy
tube. percent
(v/v) alcohol (chemical purity) was added immerge
to
the spores along
the funnel margin.
Thirty seconds ml
30
later fresh
sterile
water was
distilled continually added
to rinse the spores.
removing 4%
After
the alcohol, (w/v) sodium
hypochlorite (NaClO) 0.1%
or
(w/v) mercuric
chloride was added
(HgCla) along
the funnel margin. For
different disinfectants, the time was NaClO
sterilizing
different: (5-6 min) and
HgCl2
(2-3min). At end
the
of the
sterilizing time, fresh water
sterile distilled
was
full frlled into the funnel along
the margin,
regardless of whether
the
NaClO
HgClz
or had
solution completely
drained The
same
off or not.
operations were when
repeated twice
the dilute solution seeped from
half the
hmnel. Then
filter after the solution drained
spores were thoroughly
off, rinsed
six times with
sterile distilled water. After the sterile distilled water drained
off, the spores were rinsed with 40 ml
fresh sterile distilled water from the
paper
ftlter into a sterile container. Using
a
sterile pipette, the spore
suspension was
cm
distributed onto
culture
plates diameter
(9 Petri dishes,
containing ml
20 culture medium), which were
sealed with The
plastic
film.
sterile distilled water and
different solutions were by
transferred
transfer
with
pipette
sterilized
tips (1ml).
Ten
were made
plates each and
for treatment the experiment was conducted
The
three times. waste
of the HgCl2 was
solution and
collected adjusted
to
pH
Enough
8-10. NaaS and FeS04
were then added
to react with the HgCl^
and
produce
sediments.
After sediments formed
thoroughly, the sediments
were
and
collected
sent to the hazardous waste
disposal department
for
and
detoxification proper
disposal.
All cultures were incubated
in a controlled environment room
was
that
maintained 23±2^C
under
at m"^
a light intensity of 25 s"^ with 16/8
^il
photoperiod.
The
spore density of one drop
of spore suspension
solution from ml
a
1
transfer pipette in the method and
method
filter centrifrigation were
recorded
and means
with
standard
deviations were
(S.D.) calculated. In the packet
VOLUME NUMBER
AMERICAN FERN JOURNAL:
99 3 (2009)
method, spores were sown by directly wiping the spores on the medium, thus
was Contamination and spore germination were
spore density not scored.
examined on the 40th day after spore sowing. For germination rates, at least
random were
300 spores per plate scored.
For comparison the other two methods, the published methodologies
to
mg
(Fernandez et al, 1993; Ford and Fay, 1999) were followed using 3 of
and
spores, are not described in this paper.
methods:
Sterilization capacities of different sterilization
To methods, comparisons
test the sterilization capacities of the different
were made between different methods using different weights of spores (0.5g,
and The spores were administered into proper centrifuge tubes or
1.5g
3g).
sown
were
packed in proper papers. All spores in soil after sterilization.
filter
NaClO and
method, by were obviously bleached
In the spores sterilized
filter
was judge whether the spores were rinsed completely from the
difficult to
it
when
paper or spores drifted from the tubes poured out of the
filter if
shows were no
disinfecting solutions and rinse water. Table that there
1
between two
significant differences in spore density the disinfectants.
methods
between
However, the spore densities varied greatly the different
From method, were about 69-75 spores /drop,
the centrifugation there
tested.
number
from the method, there were about 235-248 spores/drop, and the
filter
ml was 260-272
and suspended 40 water
of spores not sterilized in directly
was by
spores/drop (Table In the packet method, sowing of spores achieved
1).
medium
wiping the spores in a swirling motion over the surface of culture
directly, so the spore density through this method was very high in the first
clump
plate and very low in the last plate. Nevertheless, the spores tended to
changed from
together on the plate. Thus, the spore densities greatly plate
first
was more
and Within the highest density
to plate across plates. a single plate,
than 3000 spores cm~^; the lowest density was less than 10 spores cm"^.
The spores from the method and packet method started to germinate 10
filter
method
days sowing, while the spores from the centrifugation started to
after
On
germination
germinate 15 days sowing. the 40th day, the highest
after
Table was obtained from the method with HgCls, which
(62.6%; see fifter
1)
NaClO
was followed by the method with (60.8%, Table
filter 1).
The germination of the packet method varied greatly. For example,
rate
when the disinfectant was HgCla ranged from 5.6% to 67%. At the highest
it
was around 20%. The
about 3000 cm"^, germination
density of rate
germination in the plate was 5.6% as there were only 287 spores in
rate last
the whole Only plates from the packet method were contaminated
plate.
(Table
1).
With increasing spore weight, different methods had different problems. For
the packet method, when the spores were more than 0.5 removing air
g,
HUA
METHOD
ET OF SPORE SURFACE AND
AL.: STERILIZATION SOWING
were taken after 40 days. PM: packet method; FM: method; CM:
filter centrifuge method.
bubbles from became
the packet very and some
difficult, spores could not be
wetted and
For when
sterilized. the centrifugation method,
the spores were
ml
0.5 1.5-2 centrifuge tubes were when
proper; the spores were 1.5-3
g,
g,
ml
1.5-2 centrifuge tubes were 5-7 ml
too small; and
centrifuge tubes
a bigger
were
centrifuge needed. For the method,
filter the spores, regardless of density,
could be completely
sterilized without any modification of the methodology.
Discussion
When
sterilizing spores via the packet method, were
the spores kept
in the
packet during
the However,
sterilizing process. the bubbles were removed
not
if
completely, the spores did not come
into contact with and
all disinfectant
this
caused an
increased contamination
rate. In addition, the sowing methodology
caused
the spores to be dispersed unevenly on the culture medium; some
plates
had spores that were clumped together and some plates had few spores were
that
spread very As resuk
far apart. a of these discrepancies, the germination
rates
and
varied confirmed
greatly, the findings of Ashcroft and
Sheffile (2000) that
spore germination
rate of ferns inhibited both high and low
is at
densities;
proper spore density important
is to fern culture.
When
sterilizing spores via the centrifuge method, the spores tended run
to off
when
pouring out the used disinfectant solution and used
sterilized distilled
water. Therefore, whole
after the few
sterilization process, spores remained,
although sown
the spores could be an even The
in density. results of testing
sterilization capacity with method show when
this that the spore weight
exceeded
and
1.5 larger centrifuge tubes were
a larger centrifuge needed.
g,
When
sterilizing spores via the method, were
filter the spores kept in the
funnel during whole
filter the sterilizing process. Thus, spore was
loss
minimal.
Besides sowing
this, the spore density could be adjusted evenly
through volume
adjusting the of the water used
sterilized to rinse off the spores
from
the
paper.
filter
Given we
these observations, conclude method
that the an
filter effective
is
way
to sterilize spores. not only simple and
is convenient, but can be used
It
to
VOLUME NUMBER
AMERICAN FERN JOURNAL:
99
3 (2009)
The
r
disinfec
whether the spores were rinsed from the centrifuge tubes and papers
off filter
thoroughly or not. However, HgCl2 not only extremely toxic to spores but
is
also to the environment. Thus, for normal in vitro culture, better to use
is
it
NaClO. HgCls might be used in cases where the spores are difficult to sterilize
become Because should be handled
or too bleached. of the toxicity of HgCla,
it
very carefully, and the waste of HgCl2 should be detoxified or sent to a waste
department
disposal for detoxification.
Literature Cited
.The
effect of spore den;
UK.
Pp. 159-168, In: Hall, R. D. (ed.). Plant cell culture protocols, 1st edn. Surrey,
New
Kiss, H. G. and Z. Kiss. 1998. Spore germination in populations of Schizaea pusilla from
J.
Jersey and Nova Scotia. International Journal of Plant Science 159:848-852.
Stone, G. 1958. The gametophyte and embryo of Polypblebium venosum (R. Br.) Copeland
I.
(Hymenophyllaceae). Australian Journal of Botany 6:183-203.
YoROi, R. 1972. Studies on spore germination and gametophyte of Japanese Hymenophyllaceae.
Science Reports of Tokyo Kyoiku Daigaku 15:81-111.
