Plant Cell Tiss Organ Cult (2006) 86:159–167
DOI 10.1007/s11240-006-9104-4
ORIGINAL PAPER
Symbiotic seed germination of Habenaria macroceratitis
(Orchidaceae), a rare Florida terrestrial orchid
Scott L. Stewart Æ Michael E. Kane
Received: 25 January 2006 / Accepted: 7 March 2006 / Published online: 7 July 2006
Springer Science+Business Media, Inc. 2006
Abstract The rapid loss of native orchid habitat
throughout ecologically important areas (e.g., Florida) has prompted researchers to develop appropriate
plans for the propagation and reintroduction of many
native orchid species. Ideally, symbiotic orchid seed
germination methods are utilized in the production of
orchid seedlings to be used in plant reintroduction
programs. In the current study we (1) describe an
efficient symbiotic seed germination protocol to
germinate seeds of the rare sub-tropical terrestrial
orchid Habenaria macroceratitis; (2) discuss the in
vitro fungal specificity demonstrated by this species;
and (3) describe the effects of three photoperiods
(0/24 h, 16/8 h, 24/0 h L/D) on in vitro symbiotic
seed germination of H. macroceratitis. Six fungal
mycobionts were isolated from both vegetative and
flowering plants of H. macroceratitis from two geographically distinct sites. Symbiotic seed germination
percent was highest (65.7%) and protocorm development was most advanced (Stage 2) when seeds
were cultured with fungal mycobiont Hmac-310.
Seeds of H. macroceratitis demonstrated a degree of
specificity toward fungal mycobionts isolated from
plants originating from the same site where seed was
S. L. Stewart (&) Æ M. E. Kane
Department of Environmental Horticulture, University of
Florida, P.O. Box 110675, Gainesville, FL 32611, USA
E-mail: sstewart@ifas.ufl.edu
Tel.: +352-392-1831
Fax: +1-352-392-1413
collected. Continual darkness (0/24 h L/D) inhibited
initial seed germination (Stage 1; 17.1%), but stimulated subsequent protocorm development (Stage 2;
53.5%). These findings will aid in developing an
efficient symbiotic seed germination protocol for the
conservation of this rare Florida terrestrial orchid,
and may prove useful in the conservation of other
sub-tropical terrestrial orchid species.
Keywords Fungal specificity Æ Mycobiont Æ
Mycorrhizae Æ Photoperiod
Abbreviations
OMA Oat meal agar
PDA Potato dextrose agar
Introduction
In nature, orchids consume endophytic mycorrhizal
fungi as a source of carbon (mycotrophy) in a parasitic association that stimulates seed germination, as
well as protocorm and seedling development (Arditti
1966; Clements 1988; Rasmussen 1995). Therefore,
the long-term survival of orchids in managed or
restored habitats requires the presence of appropriate
fungal mycobiont for seedling recruitment and plant
nutritional support (Zettler 1997a). The most efficient
way to promote this process is through the use of
symbiotic seed germination methods (Zettler 1997a,
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1997b; Clements et al. 1986; Dixon 1987). Unfortunately, few North American native orchids have been
cultured using this method; mostly species restricted
to the genera Spiranthes (Anderson 1991; Zelmer and
Currah 1997; Zettler and McInnis 1993; Stewart et al.
2003), Platanthera (Anderson 1996; Zettler and
Hofer 1998; Zettler and McInnis 1992; Sharma et al.
2003; Zettler et al. 2001; Zettler et al. 2005), and
Habenaria (Stewart and Zettler 2002). Moreover,
little is known about the identity and ecology of these
endophytic mycobionts in pure culture or in nature.
Habenaria macroceratitis Willdenow, the longhorned rein orchis, is a rare terrestrial orchid found in
central Florida, Mexico, the West Indies, and Central
America (Brown 2002; Fig. 1a, b). The orchid’s
typical habitat, rich and seasonally moist hardwood
hammocks, is becoming restricted throughout the
species’ Florida range because of land conversion to
home sites and habitat mismanagement (S.L. Stewart,
personal observation). Little information exists on the
Fig. 1 Habenaria
macroceratitis.
(a) H. macroceratitis plant
in greenhouse prior to
anthesis. (b) H.
macroceratitis inflorescence
in habitat. Scale bars=1 cm
123
Plant Cell Tiss Organ Cult (2006) 86:159–167
seed propagation of H. macroceratitis. Stewart and
Zettler (2002) provided a symbiotic seed germination
protocol using mycobionts originating from four
Florida native orchids, H. macroceratitis, H. quinqueseta (Michaux) Eaton, Spiranthes brevilabris
Lindley, and Epidendrum magnoliae Muhlenberg
var. magnoliae (syn.=E. conopseum R. Brown).
However, this study only proposed a symbiotic seed
germination protocol and did not explore the efficiency of germination using mycobionts exclusively
from H. macroceratitis. No other research exists
concerning the propagation of this species.
In the current study, an in vitro symbiotic seed
germination protocol for H. macroceratitis using six
fungal mycobionts isolated from the roots of this
species is described. Mycobionts originated from two
different sites throughout the central Florida range of
the species. A concise description and tentative
identification of the fungal mycobionts are also
provided. Finally, a brief discussion concerning the
Plant Cell Tiss Organ Cult (2006) 86:159–167
161
in vitro fungal specificity of H. macroceratitis mycobionts is included.
Materials and methods
Fungal isolation and identification
Six fungal mycobionts were chosen for in vitro
symbiotic seed germination of H. macroceratitis
(Table 1). All mycobionts were recovered from the
roots of the study species. Mycobionts were isolated
following the protocols outlined by Zettler (1997b)
and Stewart and Zettler (2002). Adult flowering and
leaf-bearing vegetative plants with intact root systems were collected, the root systems were wrapped
in sterile paper towels moistened with sterile deionized water, placed in plastic bags, stored in darkness
at ca. 10C, and transported to the laboratory (<4 h).
Root segments were detached, rinsed with cold tap
water to remove debris, and surface sterilized 1 min
in a solution containing absolute ethanol:6.00%
NaOCl:sterile deionized distilled (dd) water (1:1:1 v/
v/v). Clumps of cortical cells containing fungal pelotons were removed, placed on corn meal agar
(CMA; Sigma-Aldrich, St. Louis, MO) supplemented
with 50 mg l 1 novobiocin sodium salt (Sigma-Aldrich, St. Louis, MO), and incubated at 25C for 5 d.
Hyphal tips were excised from actively growing pelotons and subcultured onto 1/5th-strength potato
dextrose agar: 6.8 g PDA (BD Company, Sparks,
MD), 6.0 g granulated agar (BD Company, Sparks,
MD), 1 l dd water (1/5-PDA).
Fungal isolates showing cultural characteristics
similar to those orchid endophytic mycobionts previously described in the literature (Zettler 1997b;
Currah et al. 1987, 1997; Richardson et al. 1993;
Stewart et al. 2003; Zelmer et al. 1996) were
assigned a reference number and stored at 10C on
oat meal agar (OMA): 3.0 g pulverized rolled oats
(Quaker Oats, Chicago, IL), 7.0 g granulated agar,
100 mg yeast extract (BD Company, Sparks, MD),
and 1 l dd water (Dixon 1987). Mycobiont isolates
were stored until use in seed germination experiments. Mycobiont characterization and identification
followed those methods outlined by Zelmer and
Currah (1995), Currah et al. (1987, 1990, 1997), and
Zelmer et al. (1996). Hyphal and monilioid cell
characteristics were assessed using a Nikon Labophat-2 light microscope (Nikon USA, Melville, NY)
fitted with a Nikon Coolpix 4500 digital camera
(Nikon USA, Melville, NY). Staining procedures
followed those described by Schaffer and Peterson
(1993).
Seed collection
Seeds were obtained prior to dehiscence from mature
capsules on 26 September 2003. Seeds were collected
from a large (>200 flowering and vegetative plants)
population of H. macroceratitis occurring on privately owned land in Hernando County (Florida).
Immediately following collection, capsules were
dried over silica gel desiccant for 2 wks at 25C,
followed by storage at 10C in darkness for 142 d.
Prior to the initiation of symbiotic seed germination
cultures, a tetrazolium test (Lakon 1949) was conducted to assess H. macroceratitis seed viability.
Symbiotic seed germination
The effects of six fungal mycobionts (Table 1) on
symbiotic seed germination of H. macroceratitis
were evaluated. Seeds were sown according to the
procedures outlined by Stewart and Zettler (2002).
Seeds were removed from cold-dark storage, allowed
to warm to room temperature (ca. 25C), surface
sterilized 1 min in the same solution used during root
Table 1 Sources of fungal mycobionts used in the inoculation of Habenaria macroceratitis seed. All mycobionts hosted within roots
of either adult flowering or vegetative plants of study species
Isolate
Host
Collection information
Hmac-309
Hmac-310
Hmac-311
Hmac-312
Hmac-313
Hmac-314
Vegetative plant
Vegetative plant
Vegetative plant
Flowering plant
Flowering plant
Flowering plant
Collected
Collected
Collected
Collected
Collected
Collected
27
27
27
30
30
30
September
September
September
September
September
September
Identification
2003
2003
2003
2003
2003
2003
from
from
from
from
from
from
privately
privately
privately
privately
privately
privately
owned
owned
owned
owned
owned
owned
site
site
site
site
site
site
in
in
in
in
in
in
Hernando Co., FL
Hernando Co., FL
Hernando Co., FL
Sumter Co., FL
Sumter Co., FL
Sumter Co., FL
Epulorhiza
Epulorhiza
Epulorhiza
Epulorhiza
Epulorhiza
Epulorhiza
sp.
sp.
sp.
sp.
sp.
sp.
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Plant Cell Tiss Organ Cult (2006) 86:159–167
surface sterilization, and placed over the surface of a
1 cm·4 cm filter paper strip (Whatman No. 4,
Whatman International, Maidstone, UK) within a
9 cm diameter Petri plate containing ca. 25 ml OMA.
Medium pH was adjusted to 5.8 with 0.1 N HCl prior
to autoclaving at 117.7 kPa and 121C for 40 min.
Seeds were sown using a sterile bacterial inoculating
loop. Between 10 and 40 seeds were sown per plate.
Each plate was inoculated with a 1 cm3 block of
fungal inoculum, one fungal mycobiont per plate, and
a total of 8 replicate plates per mycobiont. Three
uninoculated plates served as the control. Plates were
sealed with Nescofilm (Karlan Research Products,
Santa Rosa, CA), wrapped in aluminum foil to
exclude light, and maintained in darkness (0/24 h L/
D) for 58 d at 25 – 2C. Plates were examined weekly
during dark maintenance for signs of germination or
contamination, exposing the seeds to brief (<10 min)
periods of illumination. Plates were returned to
experimental conditions after visual inspection.
After 58 d dark culture, seed germination and
protocorm development was assessed using a dissecting stereomicroscope. Germination and seedling
growth and development were scored on a scale of
0–5 (Table 2). Seed germination percentages were
based on viable seeds determined by visual inspection
with the aid of a dissection microscope. Viable seeds
were considered those seeds containing a distinct,
rounded and hyaline embryo.
Germination percentages were calculated by
dividing the number of seeds in each germination and
development stage by the total number of viable
seeds in the sample. Data were analyzed using general linear model procedures, least square means, and
Waller–Duncan at a=0.05 by SAS v 8.02 (SAS 1999).
Germination counts were arcsine transformed to
normalize variation.
Table 2 Seed germination and protocorm development in
Habenaria macroceratitis, adapted from Stewart and Zettler
(2002)
Stage
Description
0
1
No germination, viable embryo
Swelled embryo, production of rhizoid(s)
(=germination)
Continued embryo enlargement, rupture of testa
Appearance of protomeristem
Emergence of first leaf
Elongation of first leaf
2
3
4
5
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Effects of photoperiod on symbiotic seed
germination
The effects of three photoperiod treatments (0/24 h,
16/8 h, 24/0 h L/D) on symbiotic seed germination of
H. macroceratitis maintained at 25 – 3C were evaluated. Seeds were sown as previously described; with
the exception that only one fungal mycobiont was
used in all three photoperiod treatments. Mycobiont
Sbrev-266, originating from the roots of the rare
Florida terrestrial orchid Spiranthes brevilabris, was
chosen because of its effectiveness at germinating
seeds of H. macroceratitis in a previous study, as well
as seeds of other Florida terrestrial and epiphytic
orchids (Stewart and Zettler 2002; SL Stewart,
unpublished data). Illumination was provided by
General Electric F96T12 cool white fluorescent tubes
at 60.5 lmol m 2 s 1, as measured at culture level.
Plates in continual darkness were wrapped in aluminum foil to exclude light. Seeds were cultured on
OMA in 9 cm diameter Petri plates (ca. 25 ml).
Plates were sealed with one layer of Nescofilm.
Eleven replications per photoperiod treatment were
used. Seed germination and protocorm development
were scored after 96 d culture period. Germination
percentage and statistical analyses were completed as
previously outlined.
Results
Fungal mycobionts
Six fungal mycobionts were recovered from pelotons
in the roots of flowering and vegetative plants of
H. macroceratitis (Table 1; Fig. 2a, b). All six mycobionts were identified as members of the anamorphic genus Epulorhiza Moore. Only superficial
differences in cultural morphology were identified
among the group of six mycobionts. Isolates Hmac309 and Hmac-310 were cream in color after 25 d on
1/5th-strength PDA, whereas all other isolates were
ivory.
Symbiotic seed germination
Seeds began to swell within 2 wks after sowing, and
germination commenced within 5 wks. Visual contamination rate of cultures was 2%. A tetrazolium test
Plant Cell Tiss Organ Cult (2006) 86:159–167
163
Fig. 2 Epulorhiza
isolate Hmac-310,
isolated from the
roots of Habenaria
macroceratitis,
growing on 1/5thstrength potato
dextrose agar (PDA)
at 25C in 9 cm
diameter Petri plate.
(a) Whole culture
morphology at 20 d,
scale bar=1 cm. (b)
Monilioid cells
stained with acid
fuchsin at 20 d
(400·), scale
bar=15 lm
revealed H. macroceratitis seeds to be 41.4% viable,
while visual inspection revealed 52.6% viability from
the same seed lot.
All inoculated seed germinated by 58 d. An effect
of fungal mycobiont was found on the symbiotic seed
germination of H. macroceratitis. Germination after
58 d was highest when seeds were inoculated with
fungal mycobiont Hmac-310 (65.7%; Fig. 3). This
isolate not only promoted the highest final percent
germination, but also promoted Stage 2 development.
80
60
Control
Hmac-309
Hmac-310
Hmac-311
Hmac-312
Hmac-313
Hmac-314
a
ab
ab
(%)
a
b
a
ab
b
b
b
ab
ab
ab
40
b
a
20
b
ab
ab
ab
ab
ab
0
Stage 0
Stage 1
Stage 2
Developmental Stage
Fig. 3 Effects of six fungal mycobionts on percent seed
germination and protocorm development (Table 2) of Habenaria macroceratitis cultured on oat meal agar after 58 d
symbiotic in vitro culture. Histobars with the same letter are
not significantly different within stage (a=0.05)
However, no significant difference in seed germination and protocorm development was demonstrated
by Stage 2 in Hmac-310, Hmac-312, or control
treatments (65.7, 51.0, 51.5% respectively; Fig. 3).
Effects of photoperiod on symbiotic seed
germination
Seeds began to swell within 2.5 wks after sowing,
and germination commenced within 4 wks. Visual
contamination rate of cultures was 4%. Seed viabilities remained as described previously.
Seeds began germinating after 4 wks regardless of
photoperiod condition. After 96 d in culture a significant effect of photoperiod was found on the initial
symbiotic seed germination (e.g., Stage 1) of H.
macroceratitis. Seeds cultured under continual darkness (0/24 h L/D) exhibited a lower initial seed germination percentage (17.1%) than seeds cultured
under either the 16/8 h L/D or 24/0 h L/D photoperiods (37.4 and 34.4%, respectively; Fig. 4). However, protocorm development to Stage 2 was
stimulated under 0/24 h L/D conditions (53.5%;
Fig. 4), whereas development was less under both 16/
8 h L/D and 24/0 h L/D (34.6% and 34.5%, respectively; Figs. 4, 5). Symbiotic seed germination was
optimal under 16/8 h L/D photoperiods, but protocorm development was most advanced under 0/24 h
L/D photoperiod.
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Plant Cell Tiss Organ Cult (2006) 86:159–167
60
50
a
0/24 h L/D
16/8 h L/D
24/0 h L/D
b
(%)
40
b
a
30
b
b
c
b
a
20
10
0
Stage 0
Stage 1
Stage 2
Developmental Stage
Fig. 5 Photoperiodic effects (0/24 h, 16/8 h, 24/0 h L/D) on
symbiotic seed germination and protocorm development of
Habenaria macroceratitis cultured on oat meal agar with
fungal mycobiont Sbrev-266 after 96 d. Histobars with the
same letter are not significantly different within stage (a=0.05)
Discussion
Fig. 4 Photoperiodic effects (0/24 h, 16/8 h, 24/0 h L/D) on
the symbiotic germination and protocorm development of
Habenaria macroceratitis using fungal mycobiont Sbrev-266
cultured on oat meal agar after 96 d. (a) Protocorm development under 0/24 h L/D; note rhizoid development. (b)
Protocorm development under 16/8 h L/D; note lack of rhizoid
development. (c) Protocorm development under 24/0 h L/D;
note lack of rhizoid development. Scale bars=1 mm
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In vitro symbiotic seed germination is a powerful tool
for both the production of mycobiont-infected seedlings for use in plant reintroduction, and the study of
fungal specificity within the Orchidaceae. Few reports
exist concerning the in vitro symbiotic seed germination of North American terrestrial orchid species,
especially sub-tropical terrestrial species. This is only
the second report describing the successful symbiotic
seed germination of a North American Habenaria
species, and a first report of a photoperiodic effect on
symbiotic seed germination of H. macroceratitis. This
report also represents the first description of possible
fungal specificity displayed in H. macroceratitis.
Stewart and Zettler (2002) have previously reported the in vitro symbiotic seed germination of
H. macroceratitis. In their study, seeds were cultured
on oat meal agar with a fungal mycobiont originating
from the roots of H. quinqueseta, a closely related
taxa to H. macroceratitis, resulting in a maximum of
63.7% germination after 83 d. Interestingly, maximum protocorm development (Stage 4) was reported
from seeds that were cultured with a mycobiont
originating from Spiranthes brevilabris (Sbrev-266).
In Stewart and Zettler (2002), the fungal isolate
originating from H. quinqueseta was identified as
Plant Cell Tiss Organ Cult (2006) 86:159–167
belonging to the anamorphic genus Ceratorhiza
Moore, while the isolate from S. brevilabris was
identified as belonging to the anamorphic genus
Epulorhiza (Moore 1987). In the present study a
similar seed germination percentage (65.7%; Fig. 3)
was achieved; however, this percentage was achieved
in less time (58 d) than that reported in Stewart and
Zettler (2002). Additionally, all mycobionts isolated
in the current study were assignable to the anamorphic genus Epulorhiza. All six mycobionts closely
resembled E. repens (Bernard) Moore by having
comparable growth rates and slightly ovoid monilioid
cells. The slight variations in culture color seen in
these six isolates were not considered highly differential among all mycobionts (Currah et al. 1997). A
degree of specificity by the orchid for its fungal
mycobiont is likely responsible for the rapid in vitro
germination. Stewart and Zettler (2002) suggested
that H. macroceratitis was non-specific in its
requirement for a fungal mycobiont, whereas the
present study suggests that mycobionts isolated from
vegetative, and therefore possibly younger, plants of
H. macroceratitis better support in vitro symbiotic
seed germination. Fungal specificity in the Orchidaceae has been considered controversial for many
years (Curtis 1939; Hadley 1970). Differences in
orchid fungal specificity have been identified under
in vitro versus in situ conditions (Bidartondo and
Bruns 2005; Masuhara and Katsuya 1994; Taylor and
Bruns 1999; Taylor et al. 2003), and these differences
have led some to consider orchid fungal specificity as
generally low (Hadley 1970; Stewart and Zettler
2002). Nonetheless, the present study demonstrates
H. macroceratitis possesses a degree of mycobiont
specificity under in vitro conditions. This species
appears to be specific toward fungal mycobionts
isolated from plants exiting in the same population
where seed was collected since mycobionts isolated
from a distant population from the seed collection
site demonstrated lower seed germination percentages. A more complete study testing fungal mycobionts isolated from both vegetative and flowering
plants at multiple geographic sites are suggested to
further elucidate the apparent specificity found in
H. macroceratitis.
Few reports exist concerning photoperiodic effects
on the symbiotic germination of terrestrial orchids.
Typically, the seeds of terrestrial orchids germinate in
the soil, but are initially exposed to a short period of
165
illumination upon capsule dehiscence. Stewart and
Zettler (2002) reported no increase in initial symbiotic seed germination (Stage 1) when cultures of
H. macroceratitis were transferred from continuous
dark conditions to a 12/12 h L/D photoperiod; however, an increase in early protocorm development was
reported. Rasmussen and Rasmussen (1991) and
Zettler and McInnis (1994) both reported a reduction
in asymbiotic and symbiotic seed germination percentage under conditions of a dark pretreatment of
seed before light exposure in temperate terrestrial
orchid species. A similar response was found in the
current study. Initial seed germination (Stage 1) was
significantly lower under the 0/24 h L/D photoperiod
than either the 16/8 h L/D or 24/0 h L/D photoperiods (Fig. 4). However, protocorm development was
most advanced (Stage 2) under the 0/24 h L/D photoperiod (Fig. 4). As mentioned previously, this
reduction in initial seed germination percentage
may be due to the lack of an illumination pretreatment of the seed prior to sowing. Orchid seeds typically possess a hydrophobic testa that allows a seed to
remain above the soil surface where they are exposed
to the sun’s illumination. This light exposure may
only initiate nutrient mobilization and not lead
directly to seed germination (Rasmussen and
Rasmussen 1991). Interestingly, Takahashi et al.
(2000) reported no significant difference in symbiotic seed germination percentages when seeds of
H. radiata were cultured under continual darkness
(0/24 h L/D) and 24/0 h L/D conditions. This may
indicate that terrestrial orchid seed germination
response to photoperiod may be genus, or even
species specific.
The current study presents new findings on the
in vitro fungal specificity and effects of photoperiod
on the symbiotic seed germination of a rare subtropical terrestrial orchid from Florida, H. macroceratitis. The rare status of this orchid in the wild and
the threatened status of its natural habitat necessitate
the development of efficient symbiotic seed germination protocols, otherwise the species may not exist
as an independent entity in its natural habitat for long.
These data present invaluable information concerning
a previously unknown fungal specificity within populations of H. macroceratitis. This information will
be critical to future plant production and reintroduction efforts aimed at the conservation of H. macroceratitis in its natural habitat.
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Acknowledgements The authors thank Philip Kauth and Tim
Johnson (Environmental Horticulture Department, University
of Florida) for their reviews of this manuscript. Bijan Dehgan,
Ph.D. (Environmental Horticulture Department, University of
Florida) provided microscopic equipment. Appreciation is also
extended to the San Diego County Orchid Society and the
Florida Panther National Wildlife Refuge-US Fish and Wildlife
Service for providing financial support of this project. Brand
names are provided for references, the authors do not solely
endorse these particular products.
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