Plant Cell Tiss Organ Cult (2007) 90:313–323
DOI 10.1007/s11240-007-9270-z
ORIGINAL PAPER
Asymbiotic and symbiotic seed germination of Eulophia alta
(Orchidaceae)—preliminary evidence for the symbiotic
culture advantage
Timothy R. Johnson Æ Scott L. Stewart Æ
Daniela Dutra Æ Michael E. Kane Æ
Larry Richardson
Received: 29 March 2007 / Accepted: 7 July 2007 / Published online: 31 July 2007
Springer Science+Business Media B.V. 2007
Abstract Eulophia alta (Linnaeus) Fawcett &
Rendle seeds collected from the Florida Panther
National Wildlife Refuge (Collier County, FL;
FPNWR) were used in a screen of five asymbiotic
orchid seed germination media to determine their
effectiveness in promoting germination and protocorm development. In addition, 10 fungal isolates
collected from the roots of E. alta at sites in the
FPNWR, Highlands County (FL), and Goethe State
Forest (Levy County, FL; GSF), and a fungal isolate
from the roots of Spiranthes brevilabris collected
from GSF were screened for their effectiveness at
promoting in vitro symbiotic germination of E. alta
seeds. After 18 weeks asymbiotic culture, seeds sown
on PhytoTechnology Orchid Seed Sowing Medium
germinated to a higher percentage (87.9%) and had a
higher percentage of protocorms with developing
protomeristems (32.7%) than seeds cultured on
Knudson C, Malmgren Modified Terrestrial Orchid
Medium, ½-strength Murashige & Skoog, or Vacin &
Went Modified Orchid Medium. Significantly more
T. R. Johnson (&) S. L. Stewart D. Dutra M. E. Kane
Department of Environmental Horticulture, University of
Florida, PO Box 110675, Gainesville, FL 32611, USA
e-mail: timjohn@ufl.edu
L. Richardson
Florida Panther National Wildlife Refuge, U.S. Fish and
Wildlife Service, 3860 Tollgate Blvd., Suite 300, Naples,
FL 34114, USA
leaf-bearing protocorms were observed on
PhytoTechnology Orchid Seed Sowing Medium
(0.8%) and Vacin & Went Modified Orchid Medium
(1.3%) than other media tested. Of the fungi tested,
one fungal isolate (Ealt-396) promoted germination
to 69.0%, two isolates promoted germination to less
than 0.75% and did not support further protocorm
development, and eight isolates did not support
germination. Seeds co-cultured in darkness with
Ealt-396 grew more rapidly than asymbiotic seedlings following germination. In addition, co-cultured
(=symbiotic) seedlings continued to develop more
rapidly than asymbiotic seedlings upon transfer to 16/
8 h light/dark photoperiod. Symbiotic seed culture of
E. alta may be a more desirable method of propagation since protocorms develop more rapidly than
seeds sown on asymbiotic media. Symbiotic seedlings may be more appropriate for reintroduction to
natural areas than asymbiotic seedlings since symbiotic seedlings could serve to inoculate soils with a
germination promoting mycobiont.
Keywords Orchid Seed germination Native Conservation Terrestrial Mycorrhizae Wild coco
Abbreviations
1/51/5-strength Potato dextrose agar
PDA
½MS
½-strength Murashige & Skoog
CMA
Corn meal agar
FPNWR Florida Panther National Wildlife Refuge
123
314
dd
KC
L/D
MM
P723
TZ
VW
Plant Cell Tiss Organ Cult (2007) 90:313–323
Distilled deionized
Knudson C
Light/dark
Malmgren Modified Terrestrial Orchid
Medium
PhytoTechnology Orchid Seed Sowing
Medium
Tetrazolium
Vacin & Went Modified Orchid Medium
Introduction
Seed propagation represents the most efficient
method of propagating native terrestrial orchids
(Stewart and Kane 2006a). Symbiotic seed germination can be a cumbersome process; root samples
must be collected from which many fungi are often
isolated. Fungi must then be identified and screened
for growth promoting strains. Asymbiotic seed
germination can be a more straight forward process
since mycobionts need not be isolated to germinate
seeds of orchid taxa. However, there are circumstances when symbiotically germinated seedlings are
desired or necessary. Populations of orchids that are
established with asymbiotic seedlings remain dependent on naturally occurring fungal symbionts for
seedling recruitment (Zettler 1997b). Due to possible
ecological changes at historic orchid locales, a target
orchid species’ mycobionts may not be present at a
site if the orchid itself is not present. In these
Fig. 1 Eulophia alta.
(a) Single flower (scale
bar = 1.0 cm).
(b) Vegetative plant of
E. alta in native habitat.
(c) Eulophia alta
inflorescence (scale
bar = 4.0 cm). (d) Typical
habitat of E. alta on the
Florida Panther National
Wildlife Refuge (Collier
County, FL)
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situations, seedlings cultured symbiotically can serve
as both plant material and a source of mycobiont
inoculum for reintroduction efforts. Introducing a
compatible mycobiont into a site may facilitate the
establishment of self-sustaining populations. Additionally, symbiotic seed germination may be a more
desirable means of producing orchids if symbiotic
seedlings develop more rapidly than asymbiotic
seedlings.
Eulophia is a pantropical genus with African
affinities containing approximately 200 species
worldwide (Brown 2005). Only one species, Eulophia alta (Linnaeus) Fawcett & Rendle (common
name wild coco; Fig. 1), occurs in North America,
where it is found from southern Georgia to southern
Florida. Eulophia alta populations are typically found
in moderately wet, grass-dominated roadsides or near
the edges of forested sites dominated by live oak
(Quercus virginianus), saw palmetto (Serenoa repens), Sabal palmetto, and slash pine (Pinus eliottii).
While not currently listed as a rare species, urban
development throughout its range is threatening the
species’ current habitat. Because E. alta grows in
concentrated populations (=locally abundant), habitat
degradation at E. alta populated sites could have
long-term ramifications for the future of this species.
Common orchid taxa may serve as models for
developing reintroduction programs, which can then
be applied to threatened and endangered taxa. The
first step in this process is establishing efficient
propagation protocols to produce plants for subsequent experimentation. Little information is available
Plant Cell Tiss Organ Cult (2007) 90:313–323
concerning seed germination of Eulophia species,
and no information exists on the asymbiotic or
symbiotic seed germination requirements of E. alta.
The objectives of this research were (1) to evaluate
the potential of asymbiotic and symbiotic seed
propagation for the production of E. alta seedlings,
(2) to identify germination-promoting mycobionts,
and (3) to document the germination and seedling
development of this species. The data collected
from this study will be used to propagate plants for
further investigations of orchid reintroduction
methods.
Materials and methods
Seed source and sterilization
Eulophia alta seeds were collected from the Florida
Panther National Wildlife Refuge (FPNWR; Collier
County, FL) on 13 December 2005. Only seeds from
dehisced capsules were collected to ensure that they
were mature. Seeds were stored at 23 ± 2C over
silica gel desiccant until capsules ruptured, then
collected and stored at 10C for 15 weeks. Seeds
were transferred to a sterilized scintillation vial and
surface sterilized for 45 s in a solution containing
5 ml absolute ethanol, 5 ml 6.0% NaOCl, and 90 ml
sterile distilled deionized (dd) water. Seeds were
rinsed three times with sterile dd water after surface
sterilization. Seeds were then suspended in sterile dd
water. Solutions were removed from the vial with
sterilized Pasture pipettes that were used only once.
Asymbiotic media survey
Five nutrient media (Table 1) were assayed for their
effectiveness in promoting germination and subsequent development of E. alta seeds. All media were
prepared and modified by PhytoTechnology Laboratories, Inc. (Shawnee Mission, KS): Knudson C (KC;
Knudson, 1946), Malmgren Modified Terrestrial
Orchid Medium (MM; Malmgren, 1996), PhytoTechnology Orchid Seed Sowing Medium (P723), ½strength Murashige & Skoog (½MS; Murashige and
Skoog, 1962), and Vacin & Went Modified Orchid
Medium (VW; Vacin and Went, 1949). To standardize the sucrose and agar concentrations among media
tested, the following modifications were made to
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Table 1 Nutrient composition of germination media used in
the asymbiotic seed germination of Eulophia alta
KC
MM
P723
½MS VW
10.31 7.57
Macronutrients (mM)
Ammonium
13.82 –
5.15
Calcium
2.12
0.24
0.75
1.50
1.93
Chlorine
3.35
–
1.50
1.50
–
Magnesium
1.01
0.81
0.62
0.75
1.01
Nitrate
10.49 –
9.85
19.70 5.19
Potassium
5.19
0.55
5.01
10.02 7.03
Phosphate
1.84
0.71
0.31
0.63
Sulfate
4.91
0.92
0.71
0.86
4.92
Sodium
–
0.20
0.10
1.51
0.20
Boron
–
–
30.00
50.00 –
Cobalt
–
–
0.03
0.11
–
Copper
–
–
0.03
0.10
–
Iron
Iodine
90.00 100.00 50.00
–
–
1.20
Manganese
30.00 10.00
30.00
37.90 30.00
Molybdenum
–
–
26.00
0.52
Zinc
–
–
9.20
30.00 –
–
0.05
–
–
3.13
Micronutrients (lM)
50.00 100.00
2.50
–
Vitamins (mg/l)
Biotin
–
Casein hydrolysate –
400.00 –
–
–
Folic acid
–
0.50
–
–
–
Glycine
–
2.00
–
–
–
myo-Inositol
–
100.00 100.00
–
–
Nicotinic acid
–
–
1.00
–
–
Peptone
–
–
2000.00
–
–
Pyridoxine
–
–
1.00
–
–
Thiamine
–
–
10.00
–
–
Total N (mM)
NH4:NO3
24.31 n/a
unknown 30.01 12.76
1.32
0.52
n/a
0.52
1.46
KC—Knudson C, MM—Malmgren Modified Terrestrial
Orchid Medium, P723—PhytoTechnology Orchid Seed
Sowing Media, ½MS—½-strength Murashige & Skoog,
VW—Vacin & Went Orchid Medium
basal media: 0.8% TC1 agar was added to KC, 2.0%
sucrose was added to both MM and ½MS. Media pH
were adjusted to 5.8 using 0.1 N KOH prior to
autoclaving for 20 min at 121C and 117.7 kPa.
Sterilized media were dispensed as 30 ml aliquots
into 9 cm diameter Petri plates (Fisher Scientific,
Pittsburg, PA). Surface sterilized seeds were then
inoculated near the center of each plate using a sterile
bacterial inoculating loop before the plates were
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sealed with a single layer of Nescofilm (Karlan
Research Products, Santa Rosa, CA). Approximately
60 seeds were sown onto each plate (average seeds/
plate: 59.4). Eight replicate plates were prepared for
each treatment. Plates were stored at 22 ± 3C in
darkness for 18 weeks. Light was excluded by
wrapping plates in two layers of aluminum foil. Seeds
were exposed to short periods of light (<20 min)
during scoring. Plates were scored at two week
intervals with the aid of a dissection stereoscope.
Fungal isolation and identification
The methods of Stewart and Zettler (2002) were
modified for fungal isolation. Vegetative plants were
collected with their root systems intact, wrapped in
moist paper towel, and transported to the laboratory.
Root segments (ca. 5 cm in length) were rinsed in cold
tap water for 10 min, then surface cleansed for one
min in a solution of 5 ml absolute ethanol, 5 ml 6.0%
NaOCl, and 90 ml sterile dd water. Root segments
were macerated in Petri plates, then suspended in
sterilized molten corn meal agar (CMA; SigmaAldrich, St. Louis, MO) supplemented with 50 mg
l 1 novobiocin sodium salt (Sigma-Aldrich, St. Louis,
MO). Plates were incubated in darkness for three days
at 25C. After incubation, hyphal tips from actively
growing isolates were subcultured onto 1/5-strength
potato dextrose agar (1/5PDA): 6.8 g PDA (BD
Company, Sparks, MD), 6.0 g granulated agar (BD
Company, Sparks, MD), and 1 l dd water.
Mycobiont characterization and tentative identification followed the methods outlined by Zelmer and
Currah (1995), Currah et al. (1987, 1990, 1997), and
Zelmer et al. (1996). Hyphal and cultural morphologies were assessed visually and microscopically
using a Nikon Labophat-2 light microscope (Nikon
USA, Melville, NY). Monilioid cells were surveyed
using the microscopic equipment mentioned previously. Fungal staining procedures followed those
described by Phillips and Hayman (1970), modified
by using acid fuchsin stain (Stevens 1974).
Symbiotic fungi survey
Eleven fungal isolates (Table 2) were screened for
their effectiveness at promoting E. alta symbiotic seed
germination in vitro. All E. alta isolates were collected from two sites in Florida, and all demonstrated
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Plant Cell Tiss Organ Cult (2007) 90:313–323
characteristics resembling previously described endophytic orchid mycobionts (Currah et al. 1987, 1997;
Richardson et al. 1993; Stewart et al. 2003; Zelmer
et al. 1996; Zettler 1997a, b). An additional isolate
collected from the roots of Spiranthes brevilabris
(Sbrev-266) in Levy County, FL was also used.
Seeds were surface sterilized as previously described and sown onto the surface of a sterile
1 · 4 cm strip of Whatman No. 4 filter paper (W &
R Balston Ltd., England) (average seeds/plate: 56.6)
placed into Petri plates containing ca. 30 ml oat meal
agar (OMA; Dixon 1987). Medium was then inoculated with a 1 cm3 block of 1/5PDA containing the
actively-growing hyphae of one of the fungal isolates.
Eight replicate plates were prepared for each mycobiont treatment. Plates containing seeds but no fungus
served as the control.
Germination scoring and statistical analysis
Germination and seedling development for both the
asymbiotic and symbiotic experiments were scored
on a scale of 0–5 (Table 3; Stewart and Zettler 2002).
Percentage of seedlings in each stage was calculated
for each treatment by dividing the number of seeds in
each stage by the total number of viable seeds in each
sample. Germination and developmental data were
analyzed with SAS v 9.1 (SAS 2003) using general
linear model procedures and Waller mean separation
(a = 0.05). Data were arcsine transformed prior to
analysis to normalize variability.
Results
Asymbiotic germination
Tetrazolium (TZ) viability testing (Lakon 1949)
completed prior to experimentation indicated that
E. alta seeds collected from FPNWR were 59.7%
viable compared to observed seed germination percentages that ranged from 19.6% to 87.9% after
18 weeks culture (Fig. 2). Visible contamination was
limited to 2.5% of all replicates. No fewer than 4
replicates remained in all treatments.
Seed germination was first scored 10 weeks after
sowing (Fig. 3a). At this time, asymbiotic germination of E. alta seeds cultured in the dark (0/24 h L/D)
on P723 (56.1%) was significantly greater than seeds
cultured on KC (11.4%), MM (26.3%), ½MS (1.6%),
Plant Cell Tiss Organ Cult (2007) 90:313–323
317
Table 2 Fungi used in the symbiotic germination of Eulophia alta
Isolate
Host
Identification
Collection information
Sbrev-266
Spiranthes brevilabris
Epulorhiza repens
Collected 30 April 1999 from GSF
Ealt-385
Eulophia alta
Epulorhiza sp.
Collected 24 July 2005 from FPNWR
Ealt-386
Ealt-387
Eulophia alta
Eulophia alta
Sclerotinia sp
Epulorhiza sp.
Collected 24 July 2005 from FPNWR
Collected 24 July 2005 from FPNWR
Ealt-389
Eulophia alta
Epulorhiza sp.
Collected 24 July 2005 from FPNWR
Ealt-390
Eulophia alta
Fusarium sp.
Collected 24 July 2005 from FPNWR
Ealt-391
Eulophia alta
Fusarium sp.
Collected 24 July 2005 from FPNWR
Ealt-392
Eulophia alta
Fusarium sp.
Collected 24 July 2005 from FPNWR
Ealt-395
Eulophia alta
Epulorhiza sp.
Collected 24 July 2005 from AVON
Ealt-396
Eulophia alta
Armillaria sp.
Collected 24 July 2005 from AVON
Ealt-397
Eulophia alta
Epulorhiza sp.
Collected 24 July 2005 from AVON
GSF—Goethe State Forest (Levy County, FL), FPNWR—Florida Panther National Wildlife Refuge (Collier County, FL), AVON—
Avon Park (Highlands County, FL)
Table 3 Developmental stages of asymbiotically and symbiotically cultured Eulophia alta seeds and seedlings
Stage
Description
0
Hyaline embryo, testa intact
1
Embryo swollen, rhizoids present (=germination)
2
Continued embryo enlargement, testa ruptured
3
Appearance of protomeristem
4
Emergence of first leaf
5
Elongation of first leaf and further development
and VW (11.1%; Fig. 2). Seeds sown on P723 also
exhibited a significantly higher percentage of Stage 2
(54.7%) and Stage 3 (2.1%) protocorms than other
treatments.
After 14 weeks culture, Stage 3 protocorms were
present in all treatments; however, a significantly
higher percentage of Stage 3 protocorms (7.1%) and
germinated seeds (93.8%) were observed on P723
(Fig. 2). Seeds cultured on MM exhibited a significantly higher germination percentage (81.5%) and
percentage of Stage 2 seedlings (80.9%) than seeds
cultured on KC, ½MS, or VW. No significant difference in the percentage of Stage 3 protocorms was found
among KC, MM, or ½MS treatments. A significantly
higher percentage of Stage 3 protocorms were observed
on VW (4.5%) compared to KC, MM, and ½MS
treatments (all less than 0.5%), although more Stage 2
protocorms were observed in MM treatments than in
VW treatments (80.9% and 52.3%, respectively).
By week 18, total germination ranged from 19.6%
(½MS) to 87.9% (P723; Fig. 2). While a significantly
higher percentage of seeds cultured on MM developed to Stage 2 protocorms (85.3%) than seeds
cultured on all other media, very few protocorms
cultured on MM developed beyond Stage 3. A limited
number of protocorms developed to Stage 4 (true leaf
present) on P723 (0.8%), ½MS (0.2%), and VW
(1.3%) by week 18.
Fungal identification
Ten mycobionts were recovered from the roots of
vegetative plants of E. alta (Table 2). Six mycobionts
were identified as Basidiomycotina species, while the
remaining E. alta mycobionts were identified as
Ascomycotina species. Isolates Ealt-385, 387, 389,
395, and 397 were assigned to the anamorphic genus
Epulorhiza Moore (Moore 1987), while isolate Ealt386 was identified as a species of Sclerotinia Fckl.
and isolates Ealt-390, 391, and 392 were identified as
species of Fusarium Link ex Gray. Eulophia alta
isolate Ealt-396 was identified as a species of
Armillaria (Fr:Fr) Staude and accessioned into the
University of Alberta Microfungus Herbarium as
UAMH 10807. Isolate Sbrev-266 (UAMH 9824),
originating from the roots of the Florida terrestrial
orchid Spiranthes brevilabris, was previously identified as a strain of Epulorhiza repens (Bernard) Moore
(Moore 1987; Stewart et al. 2003). This isolate has
been shown to support the germination of epiphytic
(Zettler et al. 2007) and terrestrial orchids (Stewart
and Kane 2006b; Stewart and Zettler 2002) native to
Florida.
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318
100
(18 Weeks)
P723
KC
MM
1/2 MS
VW
D
D
80
Germination (%)
Fig. 2 Effects of culture
media on the asymbiotic
germination and seedling
development of Eulophia
alta after 10, 14, and
18 weeks in vitro culture in
dark conditions. Histobars
in each stage with the same
letter are not significantly
different (a = 0.05). KC—
Knudson C, MM—
Malmgren Modified
Terrestrial Orchid Medium,
P723—PhytoTechnology
Orchid Seed Sowing Media,
½MS—½-strength
Murashige & Skoog, VW—
Vacin & Went Orchid
Medium
Plant Cell Tiss Organ Cult (2007) 90:313–323
CD
60
C
C
B
40
B
D
20
A
A
A
AB AB
A
A
0
100
AB
AB
(14 Weeks)
D
E
D
80
Germination (%)
C
D
60
C
C
B
40
B
20
A
C
A
A AAB
0
100
D
(10 Weeks)
C
C
80
Germination (%)
B
60
D
A
40
C
20
B
A
0
Stage 0
B
A
Stage 1
Stage 2
B
AA
Stage 3
Stage 4
Stage 5
Developmental Stage
Symbiotic germination
Three fungal isolates tested (Ealt-386, Ealt-388 and
Ealt-396) supported seed germination (Stage 2), but
only Ealt-396 supported further protocorm development. The embryos in control treatments swelled
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(Stage 1; 0.35%), but did not germinate. A limited
number of seeds cultured in the presence of Ealt-386
and Ealt-388 germinated after 11 weeks of culture
(0.47% and 0.20%, respectively). Final germination
of seeds cultured in the presence of Ealt-388
(0.20%) was not significantly different than 0%. Final
Plant Cell Tiss Organ Cult (2007) 90:313–323
319
Fig. 3 Protocorms and developing seedlings of Eulophia alta.
(a) Asymbiotic protocorms of E. alta cultured 8 weeks in dark
on Vacin & Went Modified Orchid Medium (VW; scale
bar = 0.5 cm). (b) Asymbiotic seedlings of E. alta after
28 weeks culture (18 weeks in dark, followed by 10 weeks
under a 16/8 h L/D photoperiod; scale bar = 1.0 cm). (c)
Symbiotic protocorms of E. alta cultured 8 weeks in dark on
oat meal agar (OMA) with mycobiont Ealt-396 (scale
bar = 0.5 cm). (d) Seedlings of E. alta cultured symbiotically
on OMA with mycobionts Ealt-396 after 18 weeks dark
incubation followed by 10 weeks under a 16/8 L/D photoperiod (scale bars = 1.0 cm)
germination of seeds cultured with Ealt-386 was
0.75% after 18 weeks culture. Germination of seeds
cultured with Ealt-396 initiated six weeks after seeds
were sown. Maximum percent germination was
reached at this time (70.1 ± 2.6%) with 44.3 ± 2.8%
of the protocorms developing to Stage 3. By week 11,
57.2 ± 3.0% of protocorms had reached Stage 3
(Fig. 3b). Additional protocorm development was
not observed beyond week 11 until protocorms were
transferred to fresh media (Fig. 3d). Less than 1% of
E. alta seeds were observed in Stage 1 since seeds
rarely produced rhizoids before the testa was ruptured.
seedlings continued to develop and produced elongated leaves by week 28 (Fig. 3d), while asymbiotic
protocorms did not progress as rapidly. After
28 weeks culture, asymbiotic protocorms developed
into rhizominous masses (Fig. 3b). Leaves were
observed on very few asymbiotic seedlings and,
when present, were much shorter than those observed
on symbiotic seedling cultures.
Comparison of asymbiotic and symbiotic
germination methods
Seeds co-cultured with Ealt-396 had a higher percent
germination and more advanced development than
seeds cultured on asymbiotic media after 18 weeks
culture (compare Fig. 3a and 3c). Upon transfer to
fresh medium and lighted conditions, symbiotic
Discussion
The number of reports on the successful in vitro
production of North American terrestrial orchids has
been increasing (Kauth et al. 2006; Stewart and Kane
2006a, b; Stewart and Zettler 2002; Stewart et al.
2003; Zettler 1997a, b; Zettler and McInnis 1994;
Zettler et al. 2007). This trend may result from a
growing concern among conservationists that many
habitats which harbor terrestrial orchids are being
converted to residential and commercial land uses. A
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number of Eulophia species have been successfully
germinated using asymbiotic methods. Eulophia
cucullata, E. petersii, and E. streptopetala were all
cultured on MS, but only germinated after at least
three months culture (McAlister and Van Staden
1998). Seeds of Eulophia yushuiana were germinated
on various formulations of KC and MS (Weatherhead
et al. 1986). Neither of these previous reports
included precise data on the germination or subsequent growth of protocorms as is included in the
current study. This is the first report of asymbiotic
and symbiotic seed propagation of E. alta, the only
North American species of the genus.
TZ staining indicated that E. alta seed viability
was lower than observed during germination experiments. The opposite scenario has been documented
with hard-seeded orchids such as Cypripedium where
TZ staining grossly overestimated germinability
(Lauzer et al. 1994; Vujanovic et al. 2000). Differences in estimated viability and observed germinability of E. alta may be due to less than optimal
pretreatment or staining methods. These results
exemplify the importance of testing germinability of
orchid seeds and not relying on TZ staining alone as
an estimate of viability.
Asymbiotic orchid seed germination represents an
efficient means to culture a wide range of orchid taxa.
Most asymbiotic germination media contain similar
components—sugars, mineral salts, and agars. The
asymbiotic media used in this study varied greatly in
mineral salt, nitrogen, organic additives, and vitamin
compositions. Several researchers have reported that
nitrogen type and concentration can play an important role during in vitro asymbiotic orchid seed
germination (Curtis 1947; Kauth et al. 2006; Malmgren 1992, 1996; Raghavan and Torrey 1964; Spoerl
1948; Stewart and Kane 2006a). Curtis (1947)
reported that media containing peptone better supported protocorm development in Spathoglottis plicata than did media containing asparagine. Similarly,
Kauth et al. (2006) hypothesized that peptone in
P723, as used in the present study, likely helped
support the rapid germination and advanced protocorm development in the North American terrestrial
orchid Calopogon tuberoses. Interestingly, VW and
P723 supported similar percentages of Stage 2 and
Stage 4 protocorms, while considerably fewer Stage 3
protocorms were observed on VW than P723. The
reason for this is unclear, but may be linked to
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Plant Cell Tiss Organ Cult (2007) 90:313–323
nitrogen source, nitrogen availability, or enzyme
synthesis or activation within developing protocorms.
Other studies on amino acid utilization by terrestrial orchid seeds and seedlings indicate that species
vary in their ability to utilize different amino acids
(Curtis 1947; Spoerl 1948; Malmgren 1996). Organic
nitrogen may be more readily utilized by young
protocorms as available amino acids may by-pass
certain steps in the nitrogen assimilation process
(Malmgren, 1992; 1996). Alternatively, development
of protocorms cultured in the presence of inorganic
nitrogen may be postponed due to a delay in the
production of nitrate reductase until several months
after imbibition (ca. 60 days for Cattleya; Raghavan
and Torrey 1964). Currently, the optimal asymbiotic
nitrogen source and concentration during seed germination has not been determined for any orchid
species. Further investigation into the effects of
nitrogen source may be useful in improving the
asymbiotic culture of E. alta.
Both Sclerotinia and Fusarium are members of the
Ascomycotina and typically not associated with
endophytic, root-inhabiting orchid mycorrhizae.
However, Fusarium oxysporum has been isolated
from a number of other terrestrial and epiphytic
orchid taxa, including Bletia purpurea, Dendrophylax
lindenii, Encyclia tampensis, Epidendrum nocturnum,
E. stangeanum, and Platanthera praeclara (Richardson 1993; Zelmer 1994; SL Stewart, personal communication). Armillaria species have previously been
isolated from the achlorophyllus terrestrial orchid
Galeola septentrionalis (Terashita and Chuman
1989). However, the current isolation of an Armillaria species was from the heteromycotrophic orchid
E. alta. Interestingly, Armillaria species have been
shown to be effective mycobionts of both G. septentrionalis (Marxmüller 1992) and E. alta (present
study) by supporting the in vitro symbiotic seed
germination of both species. The isolation of several
strains of Epulorhiza from the roots of E. alta was
not surprising, especially given the ubiquitous distribution of Epulorhiza species throughout orchid
habitats worldwide (Zelmer 2001).
The mycobiont Epulorhiza repens (Sbrev-266),
collected from Spiranthes brevilabris in Levy
County, FL, failed to promote germination. The
inability of Sbrev-266 to support germination may
point to a degree of fungal preference in E. alta since
this strain has been useful in germinating several
Plant Cell Tiss Organ Cult (2007) 90:313–323
terrestrial and epiphytic orchid taxa (Stewart and
Kane 2006b; Stewart and Zettler 2002; Zettler et al.
2007). Furthermore, a number of Epulorhiza isolates
were obtained from the roots of E. alta and none of
these mycobionts resembled E. repens or supported
the in vitro symbiotic seed germination of this
species as well as isolate Ealt-396. Ealt-396 (Armillaria sp.) collected from a vegetative E. alta plant
was found to be more effective in promoting in vitro
symbiotic germination and further development than
control or other isolates tested. This may be further
evidence that germination of E. alta is dependent
upon infection by a preferred mycobiont or group of
mycobionts. This type of preference is not uncommon in the Orchidaceae and can be genus, species, or
site specific (McCormick et al. 2006; McKendrick
et al. 2002; Stewart and Kane 2006b, 2007; Taylor
and Bruns 1997).
Seeds co-cultured with mycobiont Ealt-396 ceased
developing after 11 weeks of culture. At this time,
necrosis became apparent in some cultures. After
18 weeks of culture in the dark, many protocorms
became too brittle to transfer to fresh medium. Since
symbiotic protocorms continued to develop into plants
when transferred to fresh medium and a 16/8 h L/D
photoperiod, the protocorm death observed earlier may
be attributed to (1) mycobiont nutrient stress resulting
in fungal pathenogenicity upon the germinated
seeds or (2) a lack of light and the protocorms’
inability to become heteromycotrophic. The line
between orchid–fungal association and fungal parasitism has been shown to be in part controlled by fungal
nutrient availability (Beyrle et al. 1991) and temperature (Rasmussen et al. 1990). The role of light on
symbiosis is less well understood. Transferring seedlings to light and fresh medium earlier may increase the
efficiency of the present symbiotic protocol.
In nature, endophytic orchid mycobionts presumably provide the essential nutrients germinating seeds
require (Cameron et al. 2006; Hadley and Purves 1974;
Rasmussen 1995). Mimicking this system in vitro
(=symbiotic seed germination) has been shown to
effectively enhance germination and protocorm development of E. alta compared to asymbiotic germination
18 weeks after seeds were sown. In addition, symbiotic
protocorms rapidly produced elongated leaves following transfer to fresh medium while asymbiotic protocorms formed rhizominous masses with only a few
short leaves (see Fig. 3b). Such a distinct difference in
321
morphology between asymbiotic and symbiotic seed
cultures has not been previously documented and
warrants further investigation. Advanced in vitro
seedling development and plant formation appears to
be reliant upon digestion of a compatible mycobiont or
uptake of a growth promoting substance provided by
mycobiont digestion (such as a plant growth regulator;
Rasmussen 1995) that is not present in the asymbiotic
orchid seed media screened in this study. However, it
can not be discounted that a fully optimized asymbiotic
seed germination protocol could begin to parallel the
efficient in vitro seed germination of E. alta. Symbiotic seed germination proved to be a more efficient
method of germinating and supporting early development of E. alta than asymbiotic germination. Although
relatively few North American orchid species have
been successfully germinated in vitro using symbiotic
germination, our results indicate that time spent
collecting, isolating, and culturing mycobionts, as well
as persistence in attempting to successfully co-culture
native orchids may prove more efficient than asymbiotic culture methods.
Zettler (1997a) concluded that if an orchid is
critically dependent on a compatible mycorrhiza for
germination, the loss of that fungus in situ will
ultimately result in the inability of that species to
establish new stands. An additional benefit of culturing orchid seeds symbiotically is that the resulting
seedlings can serve as both plant material and
inoculum for conservation efforts (Batty et al.
2006). The isolation of a suitable mycobiont for
E. alta is a promising step forward in ongoing efforts
to develop reintroduction and conservation protocols
for this species, as well as other endangered and
threatened orchid species. Continued research should
focus on improving the efficiency of E. alta symbiotic seed germination, acclimatization, and in situ
establishment methodologies to further progress in
rare orchid conservation techniques.
Acknowledgements The authors thank the Florida Panther
National Wildlife Refuge—US Fish and Wildlife Service for
providing financial and logistical support for this project.
Appreciation is also extended to Dr. Carrie Reinhardt Adams
(University of Florida) for the use of microscopic equipment,
and Dr. James Kimbrough (University of Florida) for
assistance in fungal identification. The authors would also
like to thank Philip Kauth (University of Florida) and Nancy
Philman (University of Florida) for help constructing and
revising this paper. Brand names are provided for references;
the authors do not solely endorse these particular products.
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322
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