Plant Cell Rep (2002) 20:791–796
DOI 10.1007/s00299-001-0403-2
CELL BIOLOGY AND MORPHOGENESIS
J. Ambrožič-Dolinšek · M. Camloh · B. Bohanec · J. Žel
Apospory in leaf culture of staghorn fern (Platycerium bifurcatum)
Received: 17 July 2001 / Revised: 8 October 2001 / Accepted: 12 October 2001 / Published online: 11 December 2001
© Springer-Verlag 2001
Abstract The induction, origin, morphology, and
ploidy of aposporous gametophytes produced on juvenile leaves of the fern Platycerium bifurcatum (Cav.) C.
Chr. were studied. Leaf explants were grown on modified Murashige and Skoog medium with 0%, 0.01%,
0.1%, 1%, or 2% sucrose. A low sucrose concentration
(0.01%) and wounding of the adaxial side of the leaf
significantly increased the induction of aposporous gametophytes (90% of leaves produced gametophytes).
Regeneration began as a proliferation of mainly epidermal cells on both sides of the leaf; subsequent development was similar to that shown by gametophytes originating from spores. Flow cytometric analysis of sporophytes and aposporous gametophytes revealed that both
forms had the same ploidy level. On the basis of these
findings, we propose a set of conditions which regularly
and reproducibly induces apospory on most of the leaf
explants of the fern P. bifurcatum.
Keywords Platycerium bifurcatum · Staghorn fern ·
Apospory · Leaf culture · Flow cytometry
Abbreviations DAPI: 4,6-Diamidino-2-fenilindol ·
FAA: Formalin:acetic acid:alcohol
Communicated by H. Lörz
J. Ambrožič-Dolinšek (✉)
Department of Biology, PeF Maribor, Koroška 160,
2000 Maribor, Slovenia
e-mail: [email protected]
Tel.: +38-62-2293705, Fax: +38-62-2518180
M. Camloh · J. Žel
Department of Plant Physiology and Biotechnology,
National Institute of Biology, Večna pot 111,
1000 Ljubljana, Slovenia
B. Bohanec
Biotechnical Faculty,
Centre for Plant Biotechnology and Breeding, Jamnikarjeva 101,
1111 Ljubljana, Slovenia
Introduction
Apospory is a morphogenetic process that is defined as
the formation of gametophytes from sporophytic tissue
in the absence of meiosis or sporulation (Raghavan
1989; Grossniklaus et al. 2001). In fern species, various
aspects of the induction and subsequent development of
aposporous gametophytes have been studied (Sheffield
and Bell 1987; Raghavan 1989), and several factors have
been found to enhance apospory, including the detachment of organs from the parent plants, the physiological
isolation of sporophytic cells from their neighbors, contact with the moist surface of the culture medium,
juvenility of the plant material, and culture media with
either no sucrose or only low concentrations of it. During the last decade attention has been mainly directed towards the effects of growth regulators (Kwa et al. 1988,
1991) and sucrose-limiting conditions of the medium
(Materi and Cumming 1991; Materi et al. 1995). In contrast to the physical, environmental, and cultural conditions required for apospory induction, little is known
about the molecular mechanisms involved in this process. For the latter studies, a key requirement is the establishment of a simple, reproducible experimental
system in which aposporous gametophytes are induced
regularly at a high frequency.
Platycerium bifurcatum is a fern that displays a high regenerative capacity. This is evident from reports on organogenesis achieved on leaves (Camloh et al. 1994), scales
(Ambrožič-Dolinšek and Camloh 1997), and even leaf cell
suspension cultures (Teng and Teng 1997) without the
presence any exogenous growth regulators in the media. In
our previous studies, spontaneous aposporic development
was occasionally observed. However, using leaf cell suspension cultures, Teng and Teng (1997) showed that the
development of the first gametophyte clusters and, subsequently, of sporophytes share a common regeneration
route. These reports indicate that the fern P. bifurcatum has
a great potential for aposporous regeneration.
In the investigation reported here, we studied the conditions conducive to the development of aposporous ga-
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metophytes in leaf culture of P. bifurcatum, with emphasis on the sucrose content of the medium and especially
on wounding of the explant. To our knowledge, controlled wounding of the explant in combination with a
low sucrose concentration in the medium has never been
used for apospory induction in any fern species. We also
investigated the origin and morphology of aposporous
gametophytes in detail.
The appearance of antheridia and archegonia on aposporous gametophytes confirms their gametophytic nature (Takahashi 1962). However, additional evidence of
their aposporous development is based on ploidy level,
which has to be the same in the aposporous gametophytes as in the sporophytes. Therefore, we used flow
cytometric analysis to determine the ploidy of the sporophytes and aposporously formed gametophytes.
A reproducible and simple system for apospory induction at a high capacity was established for staghorn
fern through the combination of a low concentration of
sucrose in the culture medium and controlled wounding
of the explant.
Materials and methods
Results and discussion
Origin and morphology of aposporous gametophytes
All types of regenerants began to develop during the
first 20 days of culture as a proliferation of mainly epidermal cells on both sides of the leaf. The morphology
of the aposporous gametophytes was similar to that observed for gametophytes from spores (Camloh 1993;
Camloh et al. 2001), with the exception of rhizoid development and the site of antheridia development. Aposporous gametophytes at different developmental stages
and shapes were observed on the same leaf (Fig. 1a).
The early developmental stages of aposporous gametophytes consisted of filaments a few cells long. Following the division of terminal or subterminal cells, twodimensional growth began (Fig. 1b). Thereafter, the gametophytes developed into a heart-shaped form, with
marginal meristem, papillae on terminal margins, and
rhizoids on the basal end (Fig. 1c). The development of
rhizoids during the early stages of gametophyte development was rarely observed and generally only after the
gametophytes had reached the heart-shaped form. This
lag in rhizoid development during the early stages might
be explained by the nutritional connection to leaf tissue.
On the other hand, this delay could, at least partly, also
be due to the quite different origins of the two types of
gametophytes. Rhizoid development was also observed
on leaf surfaces and on the basal parts of shoots. Gametangia were first observed after 40 days of culture. They
developed primarily on the lamina of the gametophytes,
but occasionally also on the margins (Fig. 1d). Their appearance was never observed on P. bifurcatum gameto-
Fig. 1a–h The development of apospory on wounded leaves cultured on medium supplemented with 0.01% sucrose. a Different
developmental stages and shapes of aposporous gametophytes after 30 days of culture. Note the attachment of aposporous gametophytes to the leaf with one epidermal cell (arrows). Bar: 0.1 mm.
b Early developmental stage of aposporous gametophytes developed from a single cell of a leaf lamina after 30 days of culture.
Note the longitudinal divisions in the subterminal cells and the
single-cell origin (arrow). Bar: 20 µm. c Heart-shaped aposporous
gametophyte with rhizoids (r) and papillae (p) after 60 days of
culture. Bar: 0.1 mm. d Antheridia (arrowheads) developed on the
lamina or margin of aposporous gametophytes after 60 days of
culture. Bar: 0.1 mm. e Attachment of aposporous gametophytes
to the leaf with more than one epidermal cell after 60 days of culture. Bar: 0.1 mm. f Intermediates between sporophytes and gametophytes after 60 days of culture. Note the heart-shaped structure,
rhizoids (r), papillae (p), and stomata (st). Bar: 0.1 mm. g Aposporous gametophytes on wounded leaves after 40 days of culture.
Gametophytes (g), rhizoids (r), and shoots (s) were observed.
Note woundings (arrows). Bar: 1 mm. h Transversely sectioned
wounded leaf with aposporous gametophytes (g) after 30 days of
culture. Note destruction of subepidermal tissue (arrows). Epidermal cells show no, or only a few, symptoms of destruction.
Bar: 0.1 mm
▲
Juvenile plants of the fern Platycerium bifurcatum (Cav.) C. Chr.
were raised in vitro by the direct shoot initiation method without
any use of growth regulators (Camloh et al. 1994). Detached
leaves, 7–10 mm long, were placed flat on basal MS medium
(Murashige and Skoog 1962) from which adenine sulfate had been
omitted (Hennen and Sheehan 1978). The leaf base and abaxial
side of the leaf were in contact with the medium. The basal medium was supplemented with 0.8% Difco-Bacto agar and with different concentrations of sucrose (0%, 0.01%, 0.1%, 1% or 2%)
and adjusted to pH 5.7–5.8 before autoclaving. In the wounding
experiments, adaxial leaf epidermis was carefully whittled with a
scalpel so that the cuts were perpendicular to the longitudinal leaf
axis. Concentrations of 0%, 0.01%, or 2% of sucrose were used in
this experiment. Leaves with aposporous gametophytes were
transferred after 40 days to fresh medium with 0.01% of sucrose
to observe further development. Cultures were maintained at
23°±2°C under a 16-h (light)/8-h (dark) photoperiod at a light intensity of 50–80 µmol m–2 s–1 (Osram L 58W/77, Fluora).
The regeneration of aposporous gametophytes, buds, and
rhizoids was examined after 20, 30 and 40 days of culture using a
stereomicroscope. The number of regenerants was determined. At
least 30 leaves were examined per treatment.
Leaves were fixed in either FAA or glutaraldehyde, embedded
in paraplast, sectioned transversely, and stained with safranin-fast
green using conventional procedures (Gerlach 1969). Aposporous
gametophytes were fixed and stained with acetocarmine-chloral
hydrate (Edwards and Miller 1972). For scanning electron microscopy (SEM) observations, leaves were prepared as described
previously (Camloh et al. 1994) and viewed with an SEM Joel
JSM-840A.
The ploidy of gametophytes raised from spores (n), gametophytes formed aposporously (2n), and leaves from sporophytes
(2n) was assessed using flow cytometric analysis as described previously (Bohanec and Jakše 1999). Briefly, nuclei were released
by chopping the tissues in citric acid, stained with DAPI in phosphate, and measured using a PAS IIIi flow cytometer (Partec,
Münster, Germany). The relative amounts of DNA were compared
by comparing the G1 peaks of the haploid and diploid samples.
The 2×2 chi-squared test (χ2) was used for evaluating the
levels of statistical significance (P) between data obtained on medium without sucrose and data obtained on media with different
sucrose concentrations, and between data obtained from intact and
wounded leaves. All experiments were repeated twice with similar
results.
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phytes raised from spores (Camloh 2001), although it is
not uncommon in game-tophytes from spores of other
fern species (Raghavan 1989). The usual midrib arrangement of gametangia was mostly the result of competition for space and resources on the single gametophyte (Raghavan 1989). The timing of gametangia development was comparable to that of gametophytes
from spores. The aposporous gametophytes were connected to the leaf by one or more epidermal cells
(Fig. 1a, b, e). Consequently, their origin is traceable to
single cells or a group of cells, which has also been
shown for other species (Takahashi 1962; Hirsch 1976).
In addition to apospory, other types of regenerants were
always found: adventitious shoots, rhizoids, and intermediates between sporophytes and gametophytes. Such
intermediate structures bear features both of the gametophyte (heart shape, rhizoids, papillae) and of the sporophyte (stomata) (Fig. 1f).
Induction of apospory
Sucrose-limiting conditions
The first aposporous gametophytes were observed after
20 days of culture on media supplemented with 0.01% of
sucrose (data not shown). Sucrose at concentrations
above 0.01% clearly inhibited apospory after 40 days of
culture (Fig. 2a). The highest number of aposporous gametophytes was formed on medium containing 0.01%
sucrose, but the percentage of leaves with gametophytes
was still not higher than 14%. In all of the media tested,
apospory development was accompanied by the formation of rhizoids and shoots (data not shown). A sucrose
concentration of 0.01% has been shown to be optimal for
aposporic development on scales (Ambrožič-Dolinšek
and Camloh 1997), and similar results have been obtained for some other fern species (Materi and Cumming
1991). Such low concentrations of sucrose could hardly
affect apospory by nutritional value alone. In plants, sugars function as substrates for growth and also affect
changes in gene expression. Both the depletion and
abundance of carbohydrates can enhance or repress the
expression of genes and interact with plant growth regulators and other nutrients and, therefore, might be involved in development at the cell, organ, and wholeplant levels (Koch 1996). Furthermore, recent studies indicate that sugars can act as signaling molecules that
control gene expression and developmental processes in
plants (Sheen et al. 1999). Consequently, sucrose could
also affect gene expression in the experimental system
described above.
Wounding of leaves
Wounding significantly increased the potential for aposporous development in P. bifurcatum leaves. In all three
media, with and without sucrose, the percentage of
Fig. 2a–c The effects of sucrose and wounding on apospory.
a The effect of sucrose on the development of aposporous gametophytes after 40 days of culture. Statistical evaluation was carried
out between data obtained on medium without sucrose and those
with different sucrose concentrations. NS Not significant. b The
effect of wounding and sucrose on the development of aposporous
gametophytes after 40 days of culture. Statistical evaluation was
carried out between data obtained from intact and wounded leaves.
*P<0.05, ***P<0.001. c The effect of wounding and sucrose on
the number of aposporous gametophytes after 30 and 40 days of
culture. Statistical evaluation was done between data obtained on
medium without sucrose and those with different sucrose concentrations. NS Not significant, **P<0.01
leaves with aposporous gametophytes was significantly
higher on wounded leaves than on intact leaves
(Figs. 1g, 2b). This effect was already evident after
20 days of culture (data not shown), but was much more
pronounced after 40 days.
Of particular interest is our observation that on
wounded leaves grown on the medium with 0.01% sucrose as many as 90% of the leaves produced aposporous
gametophytes after 40 days of culture. A similar stimulating effect of wounding in combination with 0.01%
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Fig. 3a–c Flow cytometric analysis of fluorescence emission
from DAPI-stained nuclei measured on linear scale. Histograms
show: a gametophytic tissue from spores, b aposporous gametophytic tissue, c sporophytic leaf tissue
sucrose in the medium was observed when the number
of aposporous gametophytes was determined (Fig. 2c).
Gametophytes were visible on both sides of the leaf to
approximately the same degree, with aposporous gametophytes originating from tissue areas between cuts
(Fig. 1g). Raghavan (l989) reported that conditions conducive to apospory, in addition to other factors, essentially involve a wounding reaction of the leaf, but the
wounding reaction involved only the detachment of the
leaf from the parent plant and no special attention was
given to the process. The same is true for several reports
on the induction of apospory on variously damaged
leaves where the damage involved was mainly a result of
the isolation procedure or preparative method (for review see Sheffield and Bell 1981; Von Aderkas 1986;
Kwa et al. 1988, 1991; Raghavan 1989; Fernandez et al.
1993; Teng and Teng 1997). Rarely was any importance
given to wounding itself.
In the natural environment, wounding is a severe
stress during the life cycle of plants. Plants respond to
injury with a wound response, which is a very complex
process. For example, a variety of genes are induced
(Seo et al. 1997; Hara et al. 2000). In tissue culture,
wounding causes the release of inhibitory substances, induces the production of ethylene, and stimulates organogenesis. Furthermore, wounding results in an increase in
certain enzyme activities and may cause endogenous
growth substances to move to damaged sites on an explant (George 1994). Since many different responses result from wounding, genes regulating the induction of
gametophytic cells from the sporophytic tissue might
also be affected. On the other hand, synthesis or a
change in the location of the synthesis of growth regulators in the leaf could contribute to the wound-induced
apospory observed in the investigation described here.
Wounding promoted the formation of rhizoids and
shoots (Fig. 1g) and affected the viability of the explants.
In wounded leaves, viability decreased at sucrose concentrations of 0% and 0.01%, while at 2% sucrose no effect was observed (data not shown). Wounded leaves
with aposporous gametophytes showed characteristic
symptoms of tissue destruction (Fig. 1h). Most of the destroyed tissue was restricted to subepidermal tissue regions where both dead and living cells were present
(Fig. 1h).
Flow cytometric analysis
The ploidy level of gametophytes raised from spores,
aposporous gametophytes, and sporophytes was analyzed using the flow cytometric technique. Figure 3a
presents the relative DNA content of gametophytes originating from spores, which are haploid. It is evident that
the DNA content of aposporous gametophytes (Fig. 3b)
is double that of the haploids and is thus similar to that
in diploid sporophytes (Fig. 3c). Aposporic development
has been obtained twice in P. bifurcatum – once spontaneously on leaves (Camloh et al. 1994) and recently on
scales (Ambrožič-Dolinšek and Camloh 1997). However, in the present investigation we were able to confirm
for the first time the aposporous nature of gametophytes
using flow cytometric analysis.
In conclusion, a set of conditions which regularly and
reproducibly induce apospory on most of the leaf explants of the fern P. bifurcatum has been defined for the
first time. In addition to the juvenility of explants, the
most important factors for apospory formation are the
combination of a low sucrose concentration in the medium (0.01%) and wounding of the leaf. Such an experimental system will be useful for genetic and molecular
approaches that can yield insights into the regulation
mechanisms of apospory.
Acknowledgement This work was financially supported by the
Ministry for Education, Science and Sport of the Republic of
Slovenia.
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