BIOLOGICA NYSSANA 1 (1-2) ” December 2010: 77-82
Vujičić, M. et al. ” Axenically culturing the bryophytes …
th
10 SFSES • 17-20 June 2010, Vlasina lake
1 (1-2) • December 2010: 77-82
Original Article
!
Axenically culturing the bryophytes: a case study of the moss
Herzogiella seligeri (Brid.) Z. Iwats. (Plagiotheciaceae)
Milorad Vujičić*, Aneta Sabovljević, Marko Sabovljević
Institute of Botany and Garden Jevremovac, Faculty of Biology, University of Belgrade,
Takovska 43, 11000 Belgrade, Serbia
*E-mail: milorad@bio.bg.ac.rs
Abstract:
Vujičić, M., Sabovljević, A., Sabovljević, M.: Axenically culturing the bryophytes: a case study of the
moss Herzogiella seligeri (Brid.) Z. Iwats. (Plagiotheciaceae). Biologica Nyssana, 1 (1-2), December 2010:
77-82.
A moss genus Herzogiella, from the pleurocarpous family Plagiotheciaceae contains only seven species
world wide. It occurs in North, Central and South America, Europe and Asia. In Europe, only three species
occurred, namely H. seligeri, H. striatella and H. turfacea of which, the last one is threatened. With aim to
develop the methodology for protection, conservation and active propagation of H. turfacea, more commonly
distributed counterpart, H. seligeri, were taken from the National Park Fruška Gora and axenically culture
were established. The study gives overview into the problems of sterilization, in vitro establishing,
development, propagation and biology of species, as well as indices applicable to threatened counterpart.
Key words: Herzogiella seligeri, in vitro
vascular plants. Besides the problems with
bryophyte establishment in axenic culture, it is often
problem of material availability, genetic variability
of material, disposal of axenic organisms leaving on
bryophytes and low level of species biology
knowledge (e.g. D u c k e t t et al., 2004). Apart
from economic considerations of experimental work
with bryophytes, many fundamental and applicative
physiological, genetical, morphogenetic, ecological
and evolutionary, as well as other problems could be
studied more easily in bryophytes rather than in
vascular plants (S a b o v l j e v i ć et al., 2003).
Bryophytes are useful objects for the elucidation of
comlex biological processes such as apogamy,
apospory, stress-induced cellular responses in
plants, and the fusion and growth of protoplast, etc
(L a l , 1984; C o v e et al., 1997; O l i v e r and
W o o d , 1997; S h u m a k e r and D i e t r i c h ,
1998; R e s k i , 1998; W o o d et al., 2000; C v e t i ć
et al., 2005, 2009; B o g d a n o v i ć et al., 2009;
V u j i č i ć et al. 2010).
Introduction
!
Bryophytes (comprising mosses, liverworts,
hornworts and alies) are the second largest group of
higher plants after flowering plants, with estimated
15,000 species worldwide (H a l l i n g b ä c k and
H o d g e t t s , 2000). Bryophytes, although the
second largest group of terrestrial plants, received
much less attention in conservation and protection
and in comparison to vascular plants and higher
animals much less are known on their biology. They
comprise very diverse plant groups (e.g. peatmosses, latern-mosses, leafy liverworts) with quite
diverse biological characteristics (i.e. structure, size,
ecology etc).
Although culturing plant tissues and organs
under axenic conditions was firstly established and
profitably employed in bryophytes, especially
mosses (S e r v e t t a z , 1913), bryophytes did not
retain for long their rightful place as a highly
favored research object; therefore most studies of
plant morphogenesis are now being done on
77
BIOLOGICA NYSSANA 1 (1-2) ” December 2010: 77-82
Vujičić, M. et al. ” Axenically culturing the bryophytes…
Besides, axenical cultivation of bryophytes as
well as developing of methodology in propagation
of bryophytes are significant in rare species
conservation both for ex situ and reintroduction (e.g.
B a t r a et al., 2003; B i j e l o v i ć et al., 2004;
S a b o v l j e v i ć et al., 2005; R o w n t r e e and
R a m s a y , 2 0 0 5 ; 2 0 0 9 ; G o n z a l e z et al.,
2 0 0 6 ; M a l l o n et al., 2007; R o w n t r e e , 2006;
C v e t i ć et al., 2007; B r e z e a n u et al, 2008;
C h e n et al., 2009; V u j i č i ć et al., 2009,
R o w n t r e e et al., in press). This is especially
valuable for the species like bryophytes many of
which are dioecious and possibly long-lastingly in
sterile condition naturally.
Axenic culturing of bryophytes seems to be
so complicated that many investigators gave up the
attempt. However, due to possible interaction with
other organisms in non axenic conditions, sterile
culturing is necessary for certain experimental
procedures. Progress in bryophyte tissue culture has
not gone as fast as in culture of the cells of vascular
plants, and the number of cases achieved still does
not satisfy sufficiently the demands of various
research fields (F e l i x , 1994).
Like other members of the bryophyta, the
mosses are diverse haploid-dominant plants. Mosses
did not received a lot attention in chemistry research
as a source of newly and/or bioactive compounds
(S a b o v l j e v i ć and S a b o v l j e v i ć , 2008).
However, the problem for analyzing and/or certain
substance production in larger amount is often
inadequate axenical material, i.e. impossibility to
have clean material in enough amount neither to
establish bryophyte monoculture fields. One of
solution, even it seems problematic one is to
establish in vitro culture, to find the proper
developmental conditions and to propagate it for the
wanted purpose (S a b o v l j e v i ć et al., 2010)
In this study, we have focused to
pleurocarpous moss Herzogiella seligeri. The aim of
the present study was to establish stable in vitro
culture of this species and examine its development
under axenic conditions. Since up to date any
plagiothecioid moss were not cultivated axenically,
the true challenge was to establish the axenic culture
of this moss having in mind its tiny and tender
morphology and anatomy.
Material and methods
A moss H. seligeri (Bridel) Z. Iwats. (syn.
Leskea seligeri Bridel, Musc. Rec. 2(2): 47. 1801;
Dolichotheca seligeri (Bridel) Loeske; Isopterygium
seligeri (Bridel) C. Jensen; Plagiothecium
silesianum
(Weber
&
Mohr)
Schimper;
Plagiothecium seligeri
(Bridel) Lindberg;
78
Sharpiella seligeri (Bridel) Z. Iwatsuki) has been
studied. It is wide spread in contrast to its rare and
endangered counterpart H. turfacea (I r e l a n d ,
1992). H. seligeri is one of the first invading species
on the rotten logs and tree bases of all kind of trees.
The capsules are mature at the beginning of
summer. This is a species distinctive by its widespreading leaves, appearing in several rows, and its
long (2-3.5 mm), arcuate capsules.
Plants in thin mats, light- to yellowish green,
glossy. Stems to 30 × 1.5-3 mm, prostrate to
ascending, pseudoparaphyllia lacking. Leaves widespreading, ovate to ovate-lanceolate, smooth,
nondecurrent or 1-3 cells indistinctly decurrent, 12.5 × 0.5-0.9 mm, margins serrulate to serrate; cell
walls pitted at leaf base, indistinctly pitted distally,
sometimes pits lacking; median cells 30-70 × 5-7
μm; alar cells quadrate to short-rectangular,
sometimes rounded to oval and inflated, 17-48 × 1226 μm. Sexual condition autoicous. Seta light brown
to red, 1.5-2.5 cm. Capsule light brown to reddish
brown, inclined, 2-3.5 × 0.5-0.8 mm, cylindric,
strongly arcuate, when dry contracted below mouth;
operculum conic, 0.4-0.6 mm. Spores 12-22 μm.
The materials for axenic culture were
collected in Fruška Gora Mt in March 2007 on
rotten log and the voucher specimen was deposited
in the Bryophyte Collection of the Belgrade
University Herbarium (BEOU 4421).
After collection, the plants were stored in
plastic bags at +4°C, till the begging of the
experimental work. In the laboratory conditions, the
material were cleaned under dissecting microscope
from the visible mechanical impurity. The
sporophyte with mature almost ripen but unopened
capsules were separated, and the gametophyte tips
consisted of the leafy stems of ca. 10 mm longitude
were separated carefully, placed in glasses, covered
with cheese cloth, and rinsed with tap water for 30
minutes. Sporophytes and gametophyte parts were
then disinfected for 5 minutes with 3, 5, 7, 10, 13%
or 15% solution of sodium hypochlorite
(commercial bleach, NaOCl). Finally, they were
rinsed three times in sterile deionised water.
As a basal medium for establishment of in
vitro culture, we used Murashige and Skoog (1962)
(MS) medium containing Murashige and Skoog
mineral salts and vitamins, 100 mg/l inositol, 0.70%
(w/v) agar (Torlak purified, Belgrade), and 3%
sucrose and BCD medium (see S a b o v l j e v i ć et
al. 2009 for the media details).
Once, the establishment was done, and the
plants produced, the in vitro developed plant
segments (tips and protonema pieces) were used for
further developmental experiments.
BIOLOGICA NYSSANA 1 (1-2) ” December 2010: 77-82
Vujičić, M. et al. ” Axenically culturing the bryophytes …
In order to observe the influence of sucrose
and/or mineral salts on the morphogenesis of this
species, the following medium composition
combination were tested:
MS1: half strength of MS mineral salts, sugar
free;
MS2: half strength of MS mineral salts, 1.5%
sucrose;
MS3: half strength of MS mineral salts, 3%
sucrose;
MS4: MS mineral salts, sugar free;
MS5: MS mineral salts, 1.5% sucrose;
MS6: MS mineral salts, 3% sucrose;
BCD1: BCD mineral salts, 1.5% sucrose;
BCD2: BCD mineral salts, 3% sucrose;
BCD3: BCD mineral salts, sugar free;
The pH of the media was adjusted to 5.8 before
autoclaving at 114°C for 25 minutes.
The temperature and light duration varied in
combined with sets of media:
Combination C1: 16/8 hours of light to darkness,
at 25 ± 2°C.
Combination C2: 8/16 hours of light to darkness,
at 20 ± 2°C.
Combination C3: 16/8 hours of light to darkness,
at 20 ± 2°C.
Combination C4: 16/8 hours of light to darkness,
at 18 ± 2°C.
Light was supplied by cool-white fluorescent
tubes at a photon fluency rate of 47 μmol/m2s.
Cultures were subcultured for a period of 4-6 weeks.
For analysis of condition set influence to
development 10mm long apical segments
(gametophyte), spores or protonema were
transferred to various nutrient media. For each
medium composition combined with light
conditions, 40 transplants of H. seligeri were
cultivated.
The influence of tested environmental
condition was quantified after 30 days by visually
estimation of the plant habitat marked as the best
those of appearance like in nature.
grown tips, remained useless (note that the survival
does not mean axenic as well). There is rather low
probability percentage of good bleach surface
sterilization of the moss tips, without harming
plants, for establishment in vitro culture (e.g.
S a b o v l j e v i ć et al., 2003)
Surface sterilization of the sporophytes was
more successful since we choose the almost mature
but unopened capsules and did the sterilization in
various concentration of bleech for 5 minutes like
for the gametophytes. The advantage of this process
was that we did not need the capsules material itself
(so we could harmed it lethally) but the spores from
inside that should remain viable. Once, the surface
of sporophytes was sterilized, the capsules were
opened in sterile conditions and the spores were
taken out with sterile needle to the mineral salt
containing media. The success of this way starting
culture concerning sterilization of start plant
material was achieved with 100% at 10% bleach for
5 minutes. In higher concentration the sterilization
percentage remain high but the bleach started to
harm the spores quantified by spore germination
slightly decrease.
Results and discussion
The best bleach concentration for surface
sterilizing of H. seligeri was 10%. The percentage
of tested propagules, both of gametophyte tips and
sporophytes survival, decreased with concentration
increase (Fig.1). However, the bleach could not
offer proper sterilization since propagules contained
hardly disposal contamination afterwards with
fungi, algae or bacteria, or the high concentration is
lethal in high percentage (above 10%). The bleach
concentration under 10% are functional since the
propagules survived in high percentage, but not
100% of used material was axenical. Bleach
concentrations above 7% for five minutes exposure
gametophyte
sporophyte
110
100
90
80
Survival (%)
70
60
50
40
30
20
10
0
-10
0
2
4
6
8
10
12
14
16
Bleach concentration (%)
Fig 1. Percentage of surviving of gametophyte and
sporophyte parts after bleach treatment.
The attempts to establish the axenic culture
from gametophytes i.e. 10mm plant tips failed since
the concentration for surface sterilizations killed the
plant material or was not effective enough to kill the
xenic organisms on the plants and not to harm the
plants at the same time. So, even there where the
plants survive the bleach surface sterilization and
transferred to the mineral salts, it was overgrown
quickly with fungi, algae and bacterias. The try outs
to leave it until transferred plantlets overgrow the
xenic organisms, for the purpose of the use of newly
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BIOLOGICA NYSSANA 1 (1-2) ” December 2010: 77-82
Vujičić, M. et al. ” Axenically culturing the bryophytes…
kill most of the propagules and are not appropriate
for this moss gametophytes in vitro culture
establishment.
germinate 7-30 days after exposure of spores to
good conditions. In our case, it was quicker when
media contained sucrose (7-10 days) than the spore
germination on sucrose free media. Interestingly,
difference in gametophyte development was
achieved when sucrose was put by (MS1, MS4).
On BCD sugar free (BCD3), the bud
formation was noticed after a month. Bopp (1952)
explained that in native conditions protonema have
to achieved the certain size which then produce
enough amount of kinetin-like growth regulators
released in substrate. This is a trigger for bud
induction or passing from filamentous to meristemal
growth.
Buds developed rapidly into a stem which
again branched and continue growing achieving full
size and normal leaf shapes of natural plants but not
the plant shape (Figs. 4 and 5). A rather very humid
air condition of the growth-dishes favors elongation
and growth.
Figs. 2. and 3. A detail of Herzogiella seligeri
gemetophyte developed in in vitro condition
Spores were germinated on MS medium
enriched with sucrose (MS3). After releasing from
the capsules, spores germinated in relatively high
percentage (up to 100%). However, on the MS
medium enriched with sucrose they remain in the
phase of primary protonema. The subculturing to
fresh medium with sucrose was not a signal for
plantlets to pass to the next developmental stage.
The protonema is not spreading far from spore in
any media tested. The variation of light-length and
temperature condition in H. seligeri cultures did not
show significantly different behavior (Figs. 2 and
3). A set of various combination of light length,
temperature and mineral salts were tested to achieve
the bud induction and gametophyte development.
It can be concluded that in the condition when
medium contain the sugar (MS2, MS3, MS5, MS6,
BCD1, BCD2) the spore germination is stimulated,
but the gametophyte development stopped at
protonemal eventually caulonemal stage. Schoefield
(1981) stated that in most bryophytes spores
80
Figs. 4. and 5. Herzogiella seligeri gemetophyte
developed in in vitro condition
The 10mm shoot and branch tips were used
further for subculturing into new media combined
with four combination of controlled conditions of
day length and temperature.
BIOLOGICA NYSSANA 1 (1-2) ” December 2010: 77-82
Vujičić, M. et al. ” Axenically culturing the bryophytes …
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The best developed and the most similar to
the plants developed in nature were grown on MS1
and BCD3 at temperature of 18°C or 20±2°C, at
both day length. In the temperature of 25±2°C the
plants produced slightly smaller, shorter, tinny,
fragile and unbranched shoots, often developing
contamination of blue-green algae. Spore
germination was not effected by the day length and
it was similar in all temperatures.
When the plantlets tips transferred to new
media, they produced the secondary protonema, not
far from the plantlets, developing shot on them
which spread further and branching depending on
conditions of growth (Fig. 5).
Axenically culturing H. seligeri showed that
different developmental stage of this moss species
can be stimulated or stopped by various
combination of mineral nutrition, light and
temperature. The different growth condition should
be taken into account for different Herzogiella
counterpart species conservation and propagation.
Secondarily, the problems of contamination with
blue-green algae can appear but they rather do not
harm the plantlets like green algae, bacteria and
fungi do.
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